INTERNATIONAL

REVIEW OF
NEUROBIOLOGY
VOLUME 127

SERIES EDITORS
R. ADRON HARRIS
Waggoner Center for Alcohol and Drug Addiction Research
The University of Texas at Austin
Austin, Texas, USA

PETER JENNER
Division of Pharmacology and Therapeutics
GKT School of Biomedical Sciences
King's College, London, UK

EDITORIAL BOARD
ERIC AAMODT HUDA AKIL
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NOBU HATTORI LEAH KRUBITZER
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BEAU LOTTO  A. OBESO
JOSE
MICAELA MORELLI CATHY J. PRICE
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EVAN SNYDER STEPHEN G. WAXMAN
JOHN WADDINGTON
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CONTRIBUTORS

V. Bril
Ellen and Martin Prosserman Centre for Neuromuscular Diseases, University Health
Network, University of Toronto, Toronto, ON, Canada
N.A. Calcutt
University of California, San Diego, La Jolla, CA, United States
C. Casellini
Eastern Virginia Medical School, Strelitz Diabetes and Neuroendocrine Center, Norfolk,
VA, United States
R.T. Dobrowsky
The University of Kansas, Lawrence, KS, United States
S.M. Emery
The University of Kansas, Lawrence, KS, United States
P. Fernyhough
University of Manitoba; St. Boniface Hospital Research Centre, Winnipeg, MB, Canada
O.J. Freeman
University of Manchester, Manchester, United Kingdom
N.J. Gardiner
University of Manchester, Manchester, United Kingdom
R.A. Malik
Weill Cornell Medicine-Qatar, Qatar Foundation, Education City, Doha, Qatar
C. Martin
University of Michigan Medical School, Ann Arbor, MI, United States
M.-L. Névoret
Impeto Medical Inc., San Diego, CA, United States
R. Pop-Busui
University of Michigan Medical School, Ann Arbor, MI, United States
S.M. Todorovic
School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO,
United States
A.I. Vinik
Eastern Virginia Medical School, Strelitz Diabetes and Neuroendocrine Center, Norfolk,
VA, United States
S. Yagihashi*
Hirosaki University Graduate School of Medicine, Hirosaki, Japan

*Present address: The Nukada Institute of Medical and Biological Research, 4-16 Inage-machi,
Inage-ku, Chiba, Japan. E-mail: [email protected].

xi
xii Contributors

M.A. Yorek
Iowa City Health Care System; Fraternal Order of Eagles Diabetes Research Center,
University of Iowa, Iowa City, IA, United States
D.W. Zochodne
Neuroscience and Mental Health Institute and Alberta Diabetes Institute, University of
Alberta, Edmonton, AB, Canada
PREFACE

I edited the previous version of this book—The Neurobiology of Diabetic

Neuropathy (Int. Rev. Neurobiol., volume 50), which was published in
2002. At that time the estimated number of people worldwide with diabetes
mellitus was about 170 million and the projection was that it would rise to
366 million by the year 2030. That estimate was just a little conservative.
The global number of sufferers had reached 387 million by 2014 and the
projection for 2035 is now close to 600 million (figures from the WHO
and IDF).
As Radica Pop-Busui and Catherine Martin point out, the prevalence of
neuropathy in a diabetic population is a function of disease duration and effi­
cacy of diabetes management, but a reasonable estimate is that approximately
50% of patients with diabetes will eventually develop neuropathy. So, the
topic of this book will eventually impact on approximately 300 million peo­
ple and, if the rates of morbid obesity continue to rise, that is an underesti­
mate. It represents a lot of morbidity, a lot of misery, and a huge cost to
health-care providers. Quite apart from the inherent interest and challenge
to the scientists involved, this epidemiology is reason enough for this book
to be published and, hopefully, widely read.
To put the present volume in context, we need to examine where the
2002 version left us, and to see whether the topics covered in 2015 volume
have sprung from its predecessor or whether they reflect new initiatives.
First, I have to admit that the text of the new book is not available to me
at the time of writing; I am working only from the authors’ abstracts.
In 2002 the primary metabolic progenitors for neuropathy were consid­
ered to be oxidative stress, nonenzymic glycation of proteins, and flux
through the polyol pathway, all interconnected direct consequences of
raised plasma glucose. Is glucose still viewed as the major metabolic insult
and are these three secondary dysmetabolic consequences still viewed as
important? There still seems to be a consensus that glucose remains the chief
villain of the piece, with the added possibility from Doug Zochodne, that
insulin per se may have a relevant trophic role, whose benefit is lacking
in diabetes. The three secondary villains, oxidative stress, glycation, and
polyol pathway flux, are still seen as important members of the dramatis
personae, at least as far as Paul Fernyhough is concerned.

xiii
xiv Preface

The 2002 volume went on to consider secondary targets of these

aberrations of glucose metabolism en route to neuropathy. Five such were
appraised: protein kinase C, MAP kinases, neurofilaments, apoptosis, and
nerve blood flow. These 2002 chapters and the subsequent passage of time
lead this author to the following conclusions. Alterations in activation of
protein kinase C are by-products in animal models; there is no involvement
in the development of human neuropathy. The same applies to apoptosis;
indeed evidence for its existence in the nervous system in diabetes is equiv­
ocal. On the other hand, activation of MAP kinases occurs in peripheral
nerves in both animal models and human diabetic patients. Whether this
is early enough to be a prime driver is less certain, but MAP kinases remain
an established mechanism whereby extracellular consequences of glucose
dysmetabolism can alter the phenotype of nerve and Schwann cells to pro­
mote the detriments seen in neuropathies. The arguments for an involve­
ment of neurofilaments in neuropathy were and remain strong for both
animal models and humans. Furthermore, these changes may be promoted
by MAP kinase activation. In contrast, the exquisitely careful and even­
handed analysis of nerve blood flow involvement, written by Doug
Zochodne in the 2002 book, shows that the changes depend on the animal
model and the method of measurement, and their candidacy for an early
causative factor in human sufferers is dubious.
Against this background, where does the new book take us? Soroku
Yagihashi has reviewed primary metabolic insults from raised glucose and
emphasizes the point that all these changes are well established in (at least
some) animal models, but their involvement in development of the human
condition is not proven. None of the contributors to the present volume
appear to shoot down MAP kinases or aberrant neurofilament phosphory­
lation as potential stages in the development of neuropathy. Nor do any con­
tributors appear to resurrect protein kinase C or reduced nerve blood flow
as instrumental events. We have a very interesting concept from Rick
Dobrowsky and Sean Emery, who argue that it is likely that the body
mounts defenses against the consequences of glucose toxicity and that a
potential therapeutic avenue would be to assist those defenses. This has
the significant advantage that a novel therapeutic agent, based on this pre­
mise, might have fewer side effects than agents designed to arrest events
downstream of glucose toxicity. They cite assisting heat-shock protein
70 as a feasible example to achieve neuroprotection.
Neuropathic pain is usually the earliest symptom of peripheral neurop­
athy in diabetes. It has proved to be a difficult end point for inclusion in
Preface xv

clinical trials, but we need to understand the mechanism behind it. If the
factors underlying the pain were common to those precipitating the later
neurodegeneration, then it would be a valuable therapeutic focus. An inter­
vention that had a powerful effect on neuropathic pain via its underlying
cause, rather than nonspecific analgesia, might well be useful in trials and
it would have the added advantage of ameliorating symptoms, rather than
impacting on signs, such as electrophysiology, which might be unrelated
to the condition or develop too late, when the pathology has become irre­
versible. Thus, the chapter by Slobodan Todorovic, dissecting the pain
mechanisms, is of great value to our understanding.
This new book returns to the consideration of the value of modeling the
disease and/or its components. Nigel Calcutt suggests that clinicians, as super­
intendents of failed clinical trials, are not entitled to blame inaccuracies of ani­
mal models for their failures. We are long past the notion that animal models
can holistically represent human diabetic neuropathy. Instead the focus must
be to use animals to model specific components of the dysmetabolisms inher­
ent in diabetes and to gauge their impact on the nervous system. In this
context, Mark Yorek has introduced a rat model with the defects associated
with Type II diabetes. This will give a metabolic profile that differs from the
conventional streptozotocin-diabetic rat and may include or rule out some of
the candidate metabolic precursors for neuropathy.
The same questions and constraints should be applied to experiments
based on neurons in culture. These are entirely valid as a means of tracking
subcellular changes related to glucose and its sequelae. Indeed, as Natalie
Gardiner and Ollie Freeman illustrate in their chapter, studies on nerve cul­
tures can give a uniquely clean identification of neuronal targets for glucose
toxicity. That done, the requirement is to show that these targets are features
of the human condition.
Interpretation of findings from such models demands two questions.
(1) Does the model register a deviant or exaggerated metabolic anomaly that
is relevant to human diabetes? (2) Are the direct consequences of this
dysmetabolism present in or relevant to human diabetes? The polyol path­
way serves as an excellent example. Widely studied in the rat model, the
answer to (1) is yes, but the answer to (2) is maybe, which explains the uncer­
tainty that still surrounds the efficacy of aldose reductase inhibitors. Thus,
in vitro and in vivo models are acceptable approaches to mechanisms, but
that is as far as their predictive values go. I suggest, therefore, that debates
of the “rat vs mouse” type are best left to back-stage discussions and not
dragged out in front of the audience.
xvi Preface

It follows from the above that, unless these two questions have positive
answers for an end point in a model, efficacy for a potential treatment in
animals cannot be a certain springboard for clinical trials. Again, aldose
reductase inhibitors authenticate this assertion. Thus, for many clinical trials
that have been run, their failure might be blamed on interpretation of data
from the model, at least in part.
So, we come to the issue of clinical trials. Both the old and the new
volumes have addressed this. A systematic and scientific approach to under­
standing diabetic neuropathy was kick-started in 1979 by a review published
by Rex Clements entitled “Diabetic neuropathy: new concepts of its
etiology” (Diabetes 28: 604–611), so we are not far from 50 years of research
with no therapeutic outcomes. It would not be unreasonable for the finger
of blame to be looking for targets. It might hover over clinical trials. I do not
know how many compounds have been through clinical trials for diabetic
neuropathy, but it is well into double figures, and the sad fact is that not only
have no successes emerged, but we cannot be certain that the agents tested
were failures. To generalize, the trials have been too short and we cannot be
sure that the right end points were selected. As Vera Bril points out in this
volume, trials lasting 5–8 years will be required to demonstrate clinical ben­
efits. Rayaz Malik reviews potential end points for identification of patients
at risk or for inclusion in trials. None of these is entirely satisfactory for either
purpose. Clear signs of true neuropathy probably indicate the condition is
irreversible.
There are additional problems in finding suitable treatment. A successful
agent may have to be given prophylactically for neuropathy, perhaps starting
with the first signs of background retinopathy as an indicator of susceptibility
to diabetes complications. This would mean that significant side effects
might make the treatment unacceptable for asymptomatic patients. Thus,
I can think of no more difficult challenges to pharmacologists and clinical
trial designers than diabetic neuropathy, especially when duration of trials
and numbers of patients are constrained by finance and the acceptable pace
of the return on investment. Let us hope that this book spurs the scientists to
reveal the appropriate drug targets and that the industry keeps faith enough
to rise to the challenge.
D. TOMLINSON
University of Manchester, Manchester, United Kingdom
 CHAPTER ONE

A Brief Introduction to the History

and Controversies of Clinical Trials
in Diabetic Neuropathy
N.A. Calcutt*,1, P. Fernyhough†,{
*University of California, San Diego, La Jolla, CA, United States
†
University of Manitoba, Winnipeg, MB, Canada
{
St. Boniface Hospital Research Centre, Winnipeg, MB, Canada
1
Corresponding author: e-mail address: [email protected]

The earliest clinical trials in subjects with diabetic neuropathy targeted pain
as the primary end point (Kastrup, Angelo, Petersen, et al., 1986; Kvinesdal,
Molin, Froland, et al., 1984; Rull, Quibrera, Gonzalez-Millan, et al., 1969;
Saudek, Werns, & Reidenberg, 1977). This approach recognized that pain
was a particularly disruptive feature of diabetic neuropathy and took the
pragmatic view that, in the absence of known pathogenic mechanisms, drugs
that produced pain relief in other conditions might show benefit in diabetic
patients. Sadly, the limited advances made in understanding the pathogenesis
of pain in diabetes and difficulties inherent in translating preclinical research
to therapeutic use mean that this speculative approach of almost half a cen-
tury ago is not dissimilar to many current drug development programs for
painful diabetic neuropathy. Recent progress in identifying the pathogenesis
of painful diabetic neuropathy is discussed in detail elsewhere in this volume.
It is also noteworthy that other aspects of nerve function, such as large fiber
conduction velocity and action potential amplitudes, were initially included
in clinical trials primarily to establish that any pain relief achieved by ther-
apeutic intervention was not due to general neurotoxicity of the drugs under
investigation. However, reports that glucose metabolism through the polyol
pathway contributed to large fiber nerve conduction velocity (NCV)
slowing in diabetic rodents (Tomlinson, Holmes, & Mayer, 1982) supported
the evolution of large fiber electrophysiological dysfunction into the de
facto biomarker for diabetic peripheral neuropathy. A statistically signifi-
cant improvement of conduction velocity slowing persists as the primary

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4 N.A. Calcutt and P. Fernyhough

objective of therapeutic intervention for many regulatory authorities to this

day. However, the ascendancy of such a narrow focus on large fiber function
in diabetic neuropathy has been questioned (Greene, Brown, Braunstein,
et al., 1981; Malik, 2014) and is coming under increasing scrutiny in the face
of therapies designed to primarily protect small sensory fibers. Ongoing
validation of new and sensitive methods for measuring small fiber function
and structure (Chen, Graham, Dabbah, et al., 2015) may further strengthen
the case for a more flexible, therapy-specific, approach.
Reports that glucose metabolism by aldose reductase and subsequent
nerve myo-inositol depletion caused NCV slowing in diabetic rats
(Greene, De Jesus, & Winegrad, 1975; Tomlinson et al., 1982) prompted
a number of small-scale clinical trials in subjects with diabetic neuropathy
that used NCV as an arbiter of drug efficacy. Myo-inositol supplementation
was not successful (Gregersen, Bertelsen, Harbo, et al., 1983; Salway,
Whitehead, Finnegan, et al., 1978), was not obviously pertinent in species
other than rats (Calcutt, Willars, & Tomlinson, 1988; Dyck, Sherman,
Hallcher, et al., 1980; Miwa, Kanbara, & Okuda, 1989), and has largely
faded from investigation as a contributor to the pathogenesis of diabetic neu-
ropathy. In contrast, aldose reductase inhibitors (ARIs) showed some effi-
cacy against NCV slowing (Fagius & Jameson, 1981; Judzewitsch, Jaspan,
Polonsky, et al., 1983) and sensory disorders such as pain (Jaspan, Maselli,
Herold, et al., 1983; Young, Ewing, & Clarke, 1983). These small successes
identified statistically significant improvements in NCV of around 1 m/s,
representing about 10% of the deficit but outside what was considered
meaningful when extrapolated to clinical features (Dyck & O’Brien,
1989). Even so, these results prompted a number of extensive development
and clinical trial programs that began with the ARIs sorbinil, ponalrestat
(statil), and tolrestat, as reviewed elsewhere (Oates, 2008). Improvements
of NCV in the 1 m/s range following ARI treatment continue to be
reported (Polydefkis, Arezzo, Nash, et al., 2015), despite refinement of drug
efficacy and clinical trial design.
Thioctic acid, an antioxidant also known as alpha-lipoic acid, was used in
early clinical trials (Sachse & Willms, 1980) and showed efficacy against pain
and other symptoms but not against NCV slowing. This treatment has
reemerged in the past 15 years following extensive clinical trials that were
not entirely weighted toward NCV slowing for demonstration of efficacy
(Papanas & Ziegler, 2014; Ziegler, Low, Freeman, et al., 2016). The success
of clinical trials of alpha-lipoic acid appears to depend on how efficacy is
History and Controversies of Clinical Trials in Diabetic Neuropathy 5

defined by investigators and regulatory agencies such that the opinions of

authors contributing to this volume are divided. Alpha-lipoic acid is avail-
able by prescription to treat diabetic neuropathy in Germany and oral
formulations are considered an over-the-counter nutritional supplement
in the United States but do not have FDA approval for treating diabetic
neuropathy.
Other agents used in early clinical trials included vitamin B6 as pyridox-
ine (Levin, Hanscom, Fisher, et al., 1981), which can perhaps be viewed as
a precursor to a later and recurrent therapeutic theme of testing dietary
supplements with antioxidant properties. Gangliosides, glycosphingolipid
constituents of neuronal plasma membranes, were also used (Abraham,
Abraham, & Wynn, 1984; Crepaldi, Fedele, Tiengo, et al., 1983), despite
an ill-defined mechanistic rationale. Subsequent preclinical data have
shown that gangliosides modulate content and signaling of endogenous
neurotrophic factors and their receptors (Fukuda, Fukui, Hikichi, et al.,
2015; Nishio, Fukumoto, Furukawa, et al., 2004; Vieira, de Almeida
e Silva Lima Zollner, Malaguti, et al., 2008). This places use of gangliosides
as the harbinger of a sustained interest in the development of neurotrophic
and other growth factors as a therapeutic approach for diabetic neuropathy
(Calcutt, Jolivalt, & Fernyhough, 2008). Attempts to replace diminished
endogenous neurotrophic support in diabetes or to promote growth factor-
driven neuronal survival and regrowth continue to the present. Indeed, it is
striking how many of the early therapeutic approaches have persisted in one
guise or another, with increased polyol pathway flux, oxidative stress, and
loss of neurotrophic support still being targets in recently reported clinical
trials (Kessler, Smith, Cha, et al., 2015; Polydefkis et al., 2015; Ziegler
et al., 2016). Whether this reflects an early recognition of the dominant
pathogenic mechanisms of diabetic neuropathy that has simply required
refinement of drugs, trial design, and end point measurements or else
represents an introspective intellectual stagnation is a controversy that is
frequently raised by those outside the field.
The other intervention studied during this early period of clinical trials in
diabetic neuropathy was improved glycemic control, usually by instituting
more intensive insulin regimes in type 1 diabetic subjects (Dahl-Jorgensen,
Brinchmann-Hansen, Hanssen, et al., 1986; Service, Rizza, Daube, et al.,
1985). A notable feature of such trials was that they followed subjects for
periods of months–years rather than the weeks–months that were the norm
in early drug intervention trials. Indeed, the short duration of clinical trials,
6 N.A. Calcutt and P. Fernyhough

along with low numbers of subjects, recruitment of subjects with severe and
probably irreversible neuropathy, and an inability to assess efficacy against
the proposed pathogenic mechanism (nerve polyol pathway flux, oxidative
damage, etc.), have been widely acknowledged as weaknesses that could
have limited the success of early clinical trials. Improving glycemic control
allows for a clear and well-validated readout of subject compliance and treat-
ment efficacy via HbA1C levels. The initial successes against NCV slowing
led to instigation of large multicenter clinical trials of intensive glycemic
control such as the Oslo (Amthor, Dahl-Jorgensen, Berg, et al., 1994),
DCCT (1988), and EDIC studies. These studies were careful to address trial
design concerns and emphasized standardized objective measures of nerve
function. Their influence on clinical trial design has persisted to the present.
The DCCT and EDIC trials codified many good practices for clinical
trials. These include long durations of treatment, use of multiple trial sites,
recruitment of large numbers of subjects with mild to moderate neuropathy,
implementation of standardized protocols with objective measurements, and
the tracking of subject compliance. However, their very success may also
have introduced an unhelpful rigidity to subsequent decades of preclinical
and clinical research in diabetic neuropathy, specifically in their focus on
hyperglycemia as the primary pathogenic mechanism and large fiber
NCV slowing as the primary readout of efficacy. Many of the current con-
troversies in diabetic neuropathy arise from an evolving appreciation that the
prolonged intensive insulin therapy of the DCCT was only partially effective
and that risk factors such as hypertension and dyslipidemia may contribute
independently to neuropathy. The capacity of insulin to act as a direct tro-
phic factor for peripheral nerve and the absence of C-peptide replacement
during insulin therapy may also contribute to both the apparent efficacy of
intensive glycemic control and its incomplete efficacy in studies such as the
DCCT. Further, the focus on large fiber NCV slowing as the primary read-
out of treatment efficacy may constrain use of technological advances in the
objective quantification of small fiber neuropathy to preferentially deter-
mine efficacy of agents that specifically target this aspect of diabetic
neuropathy.
The persistent failure of clinical trials of agents to treat diabetic neurop-
athy is frequently attributed to poor drug design and inappropriate animal
models that identify pathogenic mechanisms that are not pertinent to
humans. However, at the other end of the pipeline, recognition that one
size (of trial design) may not fit all (therapeutic approaches) should allow
a flexibility that embraces advances in how diabetic neuropathy is assessed
History and Controversies of Clinical Trials in Diabetic Neuropathy 7

so that both new and old therapies can be evaluated in a scientifically rational
and clinically meaningful manner.

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Sachse, G., & Willms, B. (1980). Efficacy of thioctic acid in the therapy of peripheral diabetic
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 CHAPTER TWO

Neuropathy in the DCCT/EDIC—

What Was Done Then and What
We Would Do Better Now
R. Pop-Busui1, C. Martin
University of Michigan Medical School, Ann Arbor, MI, United States
1
Corresponding author: e-mail address: [email protected]

Contents
1. Introduction 10
2. Neuropathy Outcomes Assessments 11
2.1 DCCT and EDIC Design 11
3. Complementary Assessments in EDIC 16
3.1 Evaluation of Urologic Complications 16
3.2 Gastroparesis 16
3.3 Other Evaluations in EDIC 17
4. DCCT/EDIC Findings 17
4.1 DSPN and CAN Outcomes in the DCCT and EDIC Study 17
5. Discussion 19
5.1 DCCT/EDIC and Contemporary Neuropathy Trials Design 21
References 23

Abstract
The Diabetes Control and Complications Trial (DCCT) and its epidemiological follow-up,
the Epidemiology of Diabetes Interventions and Complications (EDIC) provide impor-
tant insight on the natural history of distal symmetrical polyneuropathy and cardiovas-
cular autonomic neuropathy in patients with type 1 diabetes and on the impact of
intensive treatment of hyperglycemia on disease progression. This chapter summarizes
the design and methods used for neuropathy evaluations both in the DCCT and in
EDIC, the characteristics of the DCCT/EDIC patient population, and summarizes the
findings of the DCCT/EDIC relative to neuropathic complications of type 1 diabetes.
Lessons learned from the DCCT and EDIC experiences of longitudinal assessments of
neuropathic complications are also reviewed.

International Review of Neurobiology, Volume 127 # 2016 Elsevier Inc. 9

ISSN 0074-7742 All rights reserved.
http://dx.doi.org/10.1016/bs.irn.2016.02.020
10 R. Pop-Busui and C. Martin

1. INTRODUCTION
Diabetic neuropathies are among the most prevalent of the chronic
complications of diabetes (Boulton et al., 2005; Tesfaye et al., 2010), with
a broad spectrum of forms and clinical manifestations. The most studied in
clinical trials are distal (length-dependent) symmetrical sensorimotor poly-
neuropathy (DSPN) and cardiovascular autonomic neuropathy (CAN).
Outcome measures specific to DSPN and CAN forms were included in
the Diabetes Control and Complications Trial (DCCT) design and will
be discussed in this chapter.
The DCCT enrolled 1441 patients with type 1 diabetes between 1983
and 1989 at 28 clinical sites across United States and Canada (DCCT, 1993).
The DCCT included a primary prevention cohort and a secondary inter-
vention cohort. The primary prevention cohort had diabetes for 1–5 years
(mean 2.6 years) and no retinopathy at baseline. The secondary intervention
cohort had diabetes for 1–15 years (mean 8.7 years) and mild-to-moderate
retinopathy at baseline (DCCT, 1993). Patients were randomly assigned
to intensive (INT) or conventional (CONV) insulin therapy. Briefly, the
intensive therapy group used either insulin pumps, or three or more daily
injections of insulin, together with frequent daily self-blood glucose
monitoring in order to achieve blood glucose levels as close as possible to
normal. The conventional therapy group used one or two injections of insu-
lin daily, without routine blood glucose monitoring with a goal of minimal
symptoms related to hyper- or hypoglycemia. In 1993, after an average of
6.5 years of follow-up, HbA1c was 7.4% in the intensive group and 9.1%
in the conventional group (DCCT, 1993). The HbA1c improvement in
the intensive group was associated with significantly reduced incidence of
diabetic retinopathy, nephropathy, and neuropathy and the randomized,
clinical trial was therefore stopped (DCCT, 1993, 1995, 1998). Participants
from both the INT and CONV groups were encouraged to continue or to
adopt an intensive diabetes treatment regimen and DCCT CONV group
subjects were instructed in intensive therapy by the DCCT treatment teams.
By the end of 1993, after an average of 6.5 years of follow-up, all DCCT
participants were returned to their prior health care providers for
ongoing care.
The DCCT finding that intensive insulin therapy of type 1 diabetes to
improve glycemia profoundly reduced complications of type 1 diabetes trig-
gered fundamental changes in the management of diabetes in the United
Neuropathy in the DCCT/EDIC 11

States and worldwide. Intensive management of hyperglycemia was adopted

as the standard of care for most patients with type 1 diabetes (American
Diabetes Association, 2015).
The observational Epidemiology of Diabetes Interventions and Compli-
cations (EDIC) study began in 1994, with a majority (94%) of former DCCT
subjects agreeing to participate (Epidemiology of Diabetes Interventions and
Complications (EDIC), 1999; DCCT, 1993). The EDIC was established to
monitor the long-term effects of prior INT compared to prior CONV treat-
ment on the development and progression of more advanced complications
and of cardiovascular disease among the DCCT cohort.
Subsequent EDIC evaluations demonstrated long-term benefits of prior
intensive glycemic control on complications (DCCT, 2002; The Diabetes
Control and Complications Trial/Epidemiology of Diabetes Interventions
and Complications Research Group, 2000; Writing Team for DCCT/
EDIC, 2003) and cardiovascular disease (Nathan et al., 2005). The persistent
beneficial effect of prior intensive glucose control has been termed
“metabolic memory” (Writing Team for DCCT/EDIC, 2003).

2. NEUROPATHY OUTCOMES ASSESSMENTS

Diabetic neuropathies are one of the most prevalent chronic complica-
tions of diabetes, with multiple manifestations, consistent risk factors, and
complex pathogenetic mechanisms. The specific presentation of diabetic
neuropathy reflects the distribution and size of nerve fibers involved. By far,
the most prevalent neuropathies observed in diabetes are DSPN and diabetic
autonomic neuropathy, especially CAN (American Diabetes Association,
2015; Ang, Jaiswal, Martin, & Pop-Busui, 2014).

2.1 DCCT and EDIC Design

DSPN and CAN were evaluated both in DCCT and in EDIC. Table 1 lists
the measures obtained in DCCT and EDIC. The institutional review boards
of all participating centers approved the DCCT and EDIC study protocols,
and all participants provided written informed consent. Participation in
DSPN, CAN, and other neuropathy-related outcome assessments during
DCCT and EDIC are shown in Fig. 1.

2.1.1 Assessment of DSPN in DCCT

DSPN was assessed at DCCT baseline and after 5 years of DCCT partici-
pation and/or at the end of the DCCT study. The assessment included
12 R. Pop-Busui and C. Martin

Table 1 Neuropathy Evaluations During DCCT and During EDIC

DCCT EDIC
DSPN Symptoms of DSPN Symptoms of DSPN
evaluations Signs (neurological examination Signs (neurological examination by
by board-certified neurologist) board-certified neurologist)
Electrophysiology (median, Electrophysiology (median,
peroneal, sural nerves) peroneal, sural nerves)
Michigan Neuropathy Screening
Instrument (MNSI)
Quantitative sensory testing (VPT)
NeuroQuol
CAN Deep breathing test Deep breathing test
evaluations Valsalva maneuver Valsalva maneuver
Postural changes in blood Postural changes in blood pressure
pressure Autonomic symptom profile
Other Urologic complications
Gastroparesis (pilot only)

DCCT DCCT EDIC Y 10 EDIC Y 13/14 EDIC Y 16/17

baseline close-out UroEDIC I NeuroEDIC UroEDIC II

MNSI
DSPN DSPN Urologic
Urologic yearly
CAN CAN (ED, LUTS,
(ED, LUTS, DSPN: 1186 FSD, UI)
CAN CAN FSD, UI) CAN: 1226 CAN
(1214)

EDIC Start
1375 (94%)

1989 1993 1994 2003 2006/2007 2009/2010

Entire cohort

DCCT EDIC
Fig. 1 Timeline of main and other neuropathy evaluations in the DCCT/EDIC. CAN,
cardiovascular autonomic neuropathy; DSPN, distal symmetrical polyneuropathy;
ED, erectile dysfunction; FSD, female sexual dysfunction; LUTS, lower urinary tract
symptoms; MNSI, Michigan Neuropathy Screening Instrument; UI, urinary incontinence.

standardized medical history and physical examination by board-certified

neurologists, and electrophysiology. Nerve conduction studies were per-
formed on the dominant side and included the median (motor and sensory),
peroneal (motor), and sural (sensory) nerves using standard techniques and
Neuropathy in the DCCT/EDIC 13

specified anatomical landmarks or stimulation-to-recording electrode dis-

tances for each study of motor and sensory nerves as has been amply
described (DCCT, 1993, 1995). Absolute threshold levels for the individual
attributes were defined as the median of the upper or lower limits provided
by participating laboratories (DCCT, 1995). All participating neurologists
were required to complete protocol training and certification to assure uni-
form implementation of the study protocol across all participating sites, with
certification again required for the EDIC year 13/14 measures, including
ensuring uniform temperature match (DCCT, 1993, 1995; Martin,
Albers, & Pop-Busui, 2014). Neurologists were masked to participant treat-
ment assignment during the DCCT.
Three DSPN outcomes were defined: clinical neuropathy, nerve con-
duction study (NCS) abnormalities, and confirmed clinical neuropathy.
The diagnosis of clinical neuropathy was based on a structured history and
physical examination performed by the DCCT/EDIC-certified neurologist
and required at least two positive responses among symptoms, sensory signs,
or ankle reflexes (diminished or absent) consistent with a distal symmetrical
polyneuropathy and without causal explanation aside from diabetes (Albers
et al., 2010; Martin et al., 2014). The outcome of NCS abnormality required
an absolute abnormality of amplitude, conduction velocity, or F-wave
latency in at least two anatomically distinct nerves (DCCT, 1995). Both
clinical neuropathy and nerve conduction studies were secondary outcome
measures in DCCT (DCCT, 1993, 1995). The primary DSPN outcome of
confirmed clinical neuropathy required both the presence of clinical neuropathy
and NCS abnormalities as previously defined (Albers et al., 2010; DCCT,
1993, 1995; Martin et al., 2014).

2.1.2 Assessment of DSPN in EDIC

All the comprehensive DSPN measures performed in DCCT described ear-
lier were repeated once during the 13th and 14th years of EDIC follow-up
(the Neurology Protocol or NeuroEDIC).
A new measure, the Michigan Neuropathy Screening Instrument
(MNSI), was introduced at the start of the EDIC study and was performed
annually as a measure of DSPN before the full DCCT evaluations could be
repeated (Martin et al., 2006). The MNSI is a two-part tool that includes a
15 question self-administered symptom assessment plus a focused examina-
tion of the feet to assess skin and structural abnormalities, distal vibration per-
ception, and ankle reflexes (Feldman et al., 1994; Martin et al., 2006). The
MNSI is validated to have a good sensitivity and specificity to detect the
14 R. Pop-Busui and C. Martin

presence of DSPN (Feldman et al., 1994; Herman et al., 2012). Using the
MNSI, DSPN was defined as either an MNSI questionnaire score of
7 or
an MNSI exam score of
2.5; these scores being based on the initial vali-
dation of the MNSI instrument (Feldman et al., 1994). The MNSI evalua-
tions through EDIC year 8 suggested that the metabolic memory
phenomenon observed for retinal and renal complications, also applied to
new-onset (incident) neuropathy (Martin et al., 2006). The MNSI however,
was not designed to evaluate DSPN severity nor was it initially conceived as
a tool for longitudinal assessment of neuropathy. Further, it lacked the sen-
sitivity and specificity of the original DCCT evaluations and therefore could
not be used as a surrogate for the robust DCCT primary outcome of con-
firmed clinical neuropathy.

2.1.3 Additional DSPN Measures in EDIC

Vibration perception threshold (VPT) and a neuropathy-specific quality-of-
life instrument (NeuroQOL) were included at EDIC year 13/14
(NeuroEDIC) based on recommendations of post-DCCT era consensus
panels that recommended quantitative sensory testing and quality-of-life
measures as key components of neuropathy outcome measurement in clin-
ical trials (Boulton, Malik, Arezzo, & Sosenko, 2004; Boulton et al., 2005).
VPT was measured using a forced-choice algorithm of decreasing vibration
intensity at the dominant index finger and great toe (Martin et al., 2010).
Abnormal VPT was defined as a threshold value more than 2.5 standard
deviations above the age-adjusted mean value obtained from nondiabetic
referents (Martin et al., 2010). Quality of life was assessed using the Neuro-
QOL, an 18-item survey that addresses six specific domains (pain, lost/
reduced feeling, sensory/motor symptoms, functional, social, and emotional
experiences), as well as an assessment of overall quality of life (Vileikyte
et al., 2003).

2.1.4 Assessment of CAN in DCCT

CAN was assessed during DCCT at baseline, every 2 years and at
DCCT-end using standard cardiovascular reflex testing (CART) that
included: R-R variation to paced breathing (RRV), R-R response to
Valsalva maneuver (VR), and postural changes in blood pressure (DCCT,
1998; Martin et al., 2014; Pop-Busui et al., 2010). These tests are objective,
standardized, simple to use, and highly reproducible (Low et al., 1997;
Pop-Busui et al., 2009). All subjects were uniformly prepared for CAN
testing by instruction that included fasting, abstaining from caffeine,
Neuropathy in the DCCT/EDIC 15

tobacco, and medications the morning of testing, avoidance of vigorous

physical activity, and alcohol consumption for 48 h prior to testing and
absence of hypoglycemia prior to, and during, testing (Martin et al.,
2014). Subjects who experienced hypoglycemia after midnight (defined as
a blood glucose 50 mg/dL/2.775 mmol/L and/or signs/symptoms of
hypoglycemia) or subjects with acute illness on the day of testing were
excluded (Pop-Busui et al., 2009). Subjects with active proliferative retinop-
athy, history of laser therapy or vitrectomy, suspected (unconfirmed) prolif-
erative retinopathy, and/or no eye examination in the last 4 years were
excluded from performing the Valsalva maneuver (Pop-Busui et al.,
2009). The primary CAN outcome was defined as any of the following
criteria: abnormal R-R variation (R-R <15); R-R variation of <20 plus
a Valsalva ratio 1.5, or a decrease of >10 mmHg in diastolic blood pressure
at any point during 10 min of standing after a period of 30 min of supine rest
(postural hypotension; DCCT, 1998; Pop-Busui et al., 2010, 2009). All
CAN studies were reviewed for quality control purposes by a masked inves-
tigator at the reading center who decided if the technical quality of the
recording and conditions of the test met study criteria (Pop-Busui et al.,
2009). In the DCCT, there was a provision to confirm postural hypotension
by repeated testing with catecholamine assessment but it was rarely required
(DCCT, 1998).

2.1.5 Assessment of CAN in EDIC

All CAN evaluations described above were repeated on two occasions in
EDIC; first during EDIC years 13/14, and again during EDIC years
16/17. The catecholamine confirmation of postural hypotension was rarely
required during DCCT and it was not included in the EDIC CAN protocol
(Pop-Busui et al., 2009). Testing was performed with the Hokanson ANS
2000 device (Hokanson Inc., Bellevue, WA) and results were analyzed by a
single reader at a central reading center. During EDIC, the reproducibility of
the CAN measurements was evaluated at the reading center where a random
sample of 10% of tests were re-read, demonstrating a coefficient of reliability
between the primary and the repeat readings of 0.999 (Pop-Busui et al.,
2009). Test–retest reproducibility was also evaluated on randomly selected
subjects during EDIC with good reproducibility noted for R-R (89%) and
Valsalva ratios (91%) (Martin et al., 2014). The same DCCT CAN outcomes
were analyzed in EDIC (Pop-Busui et al., 2009). However, considering that
since DCCT-end new evidence has emerged regarding some limitations
of the categorical CAN outcomes definitions, especially related to an
16 R. Pop-Busui and C. Martin

age-related and possibly gender-related effect, new secondary CAN outcomes

in EDIC included the age- and gender-adjusted continuous R-R variation
and Valsalva ratio (Martin et al., 2014; Pop-Busui et al., 2010, 2009).
The Autonomic Symptom Profile (ASP) (Low et al., 2004), a self-
administered survey of autonomic symptoms that collects information on
symptoms of postural hypotension, gastroparesis, diarrhea, colonic atony,
genitourinary dysfunction, and hypoglycemic unawareness was added to
the battery of CAN assessments in EDIC. The ASP survey was added to bet-
ter describe the participant burden of a variety of autonomic complications
(Low et al., 2004; Pop-Busui et al., 2009).

3. COMPLEMENTARY ASSESSMENTS IN EDIC

3.1 Evaluation of Urologic Complications
The UroEDIC ancillary study was conducted at EDIC years 10 and 16 as
cross-sectional evaluations to describe the prevalence of diabetic genitouri-
nary complications that included erectile dysfunction (ED) and lower
urinary tract symptoms (LUTS) in men, and female sexual dysfunction
(FSD) and urinary incontinence (UI) in women, and to investigate relation-
ships between these and other diabetes complications (Martin et al., 2014;
Sarma et al., 2009; Wessells et al., 2011). ED was defined as an erectile func-
tion domain score of <20 using the International Index of Erectile Function
(IIEF) and by response to a single questionnaire item regarding “confidence
to get and keep an erection.” LUTS were assessed using the American Uro-
logical Association Symptom Index (AUASI) with moderate-to-severe
LUTS defined by scores >7 (Martin et al., 2014).

3.2 Gastroparesis
The DCCT did not assess gastroparesis, arguably one of the most debilitating
autonomic complications of diabetes and certainly one of the most frustrat-
ing from a clinical management perspective. In EDIC, the assessment of
gastroparesis in the entire cohort is entirely based on self-report of symptoms
obtained at annual EDIC visits. However, a formal assessment of gastric
emptying as a measure of gastroparesis was performed in 78 DCCT/EDIC
participants at EDIC year 20 using the 13C-Spirulina gastric emptying
breath test (GEBT, 223 kcal with 19.2 g carbohydrates, 12 g protein, and
10.9 g fat; AB Diagnostics, Brentwood, TN) under an investigator-initiated
Neuropathy in the DCCT/EDIC 17

investigational new drug (IND) from the Food and Drug Administration
(FDA) as described (Bharucha et al., 2015).

3.3 Other Evaluations in EDIC

Several self-report measures were also used in EDIC to evaluate the prev-
alence of hypoglycemia unawareness and use of medications to treat painful
neuropathic symptoms (Martin et al., 2014).

4. DCCT/EDIC FINDINGS
4.1 DSPN and CAN Outcomes in the DCCT and EDIC Study
DSPN (confirmed clinical neuropathy) and CAN were uncommon at
DCCT start, in part owing to the intentional exclusion of persons with
symptomatic neuropathy (DCCT, 1993; Martin et al., 2014; Pop-Busui
et al., 2010). The prevalence of confirmed clinical neuropathy increased
substantially in the conventional subjects (from 5% to 17%, P < 0.001)
and only slightly among the intensive group participants (from 7% to 9%;
DCCT, 1993, 1995; Martin et al., 2014; Pop-Busui et al., 2010). Adjusting
for the presence of confirmed DSPN at baseline, the risk reduction for inci-
dent DSPN with intensive glucose control during DCCT was 64% (95% CI,
45–76) (DCCT, 1993, 1995; Martin et al., 2014).The prevalence of CAN
almost doubled in the conventional group by DCCT-end, while remaining
static in the intensive group (DCCT, 1993, 1998). The risk reduction in
incident CAN with intensive therapy during DCCT was 45% (DCCT,
1993, 1998; Martin et al., 2014; Pop-Busui et al., 2010).
During EDIC, the HbA1c separation between former intensive and con-
ventional treatment groups observed through the DCCT quickly waned and
by the fifth year of EDIC follow-up, the HbA1c separation between treat-
ment groups completely disappeared (Martin et al., 2014; Pop-Busui et al.,
2009; Writing Team for DCCT/EDIC, 2003). Prevalence of DSPN
increased during EDIC in both groups and, despite no measureable differ-
ence in glucose control from EDIC year 5 through EDIC year 13/14, a 30%
reduction in the risk of developing confirmed DSPN by EDIC year 13/14
with prior intensive glucose control persisted (Martin et al., 2014; Pop-
Busui et al., 2010). Similar trends were observed on several NCS measures
(Albers et al., 2010; Martin et al., 2014; Pop-Busui et al., 2010). After
adjusting for NCS results at DCCT closeout, the risk reduction attenuated
to 17% (not significant), suggesting that subclinical DSPN (as documented
18 R. Pop-Busui and C. Martin

by differences in NCS at DCCT-end, but not meeting criteria for confirmed

clinical neuropathy) contributed to the finding of a persistent benefit (Albers
et al., 2010, 2007; Martin et al., 2014). Logistic regression analyses to eval-
uate the effects of glycemic control on prevalent and incident DSPN dem-
onstrated that the odds of all DSPN-related outcomes each increased per
unit (%) increase in DCCT and EDIC mean HbA1c (Albers et al., 2010;
Ang et al., 2014; Martin et al., 2014; Pop-Busui et al., 2010).
CAN prevalence also increased by EDIC year 13–14 (29% in the inten-
sive vs 35% in the conventional group), with treatment-group differences
remaining significant (Martin et al., 2014; Pop-Busui et al., 2009). After
adjusting for important covariates including age, sex, cohort assignment,
and the level of RRV at DCCT closeout, intensive glucose control during
DCCT reduced the risk of incident CAN during EDIC by 31% and of
abnormal RRV by 30% (Pop-Busui et al., 2009). The adjusted RRV was
significantly higher in the former intensive group as compared to the con-
ventional group (Pop-Busui et al., 2009). The persistent beneficial effects of
intensive glucose therapy on CAN after 13–14 years of follow-up in EDIC
were explained by the differences in the DCCT and EDIC mean HbA1c
levels between groups (Martin et al., 2014; Pop-Busui et al., 2010, 2009).
Although approximately 1/3 of DCCT/EDIC participants had CAN by
the 13th–14th year of EDIC follow-up, very few participants reported auto-
nomic symptoms (Martin et al., 2014; Pop-Busui et al., 2009). No group
differences in the prevalence of autonomic symptoms reported were
observed, supporting the notion that autonomic neuropathies may remain
largely asymptomatic, even after many years of T1D.
Among the 635 T1D men with both urologic (ED and LUTS) and CAN
evaluations at EDIC year 16/17, ED only was reported by 193 (30%), LUTS
only by 61 (10%), and ED and LUTS by 97 (15%) participants (Pop-Busui
et al., 2015). Men who developed ED and/or LUTS during EDIC had sig-
nificantly lower R-R variation and Valsalva ratio at DCCT closeout and
EDIC year 16/17 compared to those without ED or LUTS. In adjusted
analysis, participants with CAN had 2.65 greater odds of ED and LUTS
(95% CI, 1.47, 4.79) (Pop-Busui et al., 2015). Among the 580 T1D women
DCCT/EDIC participants with both urologic (FSD UI) and CAN evalu-
ations at EDIC year 16/17, 26% and 30% of women presented with FSD and
UI, respectively, and there was a significant association between measures of
CAN with FSD and UI (Hotaling et al., 2013). Additionally, in
cross-sectional analyses, both ED and LUTS in men were also associated
with DPN (Pop-Busui et al., 2014), owing perhaps to shared or overlapping
mechanisms of neuronal damage.
Neuropathy in the DCCT/EDIC 19

Epigastric fullness, used as a surrogate marker of gastroparesis in EDIC,

was reported by 8% of both INT and CONV subjects (Pop-Busui et al.,
2009). In the small pilot study that formally assessed gastric emptying, only
50% presented with normal emptying, while in 47% it was delayed and in 3%
it was rapid (Bharucha et al., 2015). The DCCT baseline HbA1c (odds ratio
[OR], 1.6; 95% confidence interval [CI], 1.1–2.3), duration of diabetes
(OR, 1.2; 95% CI, 1.01–1.3) before DCCT entry, and the mean HbA1c
during DCCT-EDIC (OR, 2.2; 95% CI, 1.04–4.5) were associated inde-
pendently with delayed gastric emptying (Bharucha et al., 2015). However,
these data have not yet been confirmed in the entire cohort.
Hypoglycemia unawareness (decreased adrenergic symptoms reported in
response to hypoglycemia) was reported by 20% of INT subjects and 25% of
CONV subjects in EDIC (p ¼ NS) (Pop-Busui et al., 2009). There were no
clinically relevant treatment-group differences in the frequency of reported
medication use to reduce neuropathic pain during EDIC (Martin et al., 2014).

5. DISCUSSION
The DCCT/EDIC has furthered the understanding of the role of glu-
cose control on development and progression of DSPN and CAN (Martin
et al., 2014; Pop-Busui et al., 2010). Differences in the incidence and prev-
alence of both DSPN and CAN reflect differences in glucose control, favor-
ing HbA1c levels that are closer to nondiabetic levels.
Confirmed clinical neuropathy increased in both intensive and conven-
tional participants by EDIC year 13–14 but treatment-group differences
were eliminated by adjusting for continuous nerve conduction variables
at the end of the DCCT (Martin et al., 2014). Among subjects without con-
firmed clinical neuropathy, former conventional subjects had worse (less
normal) electrophysiologic results at the end of the DCCT (Albers et al.,
2010, 2007; Martin et al., 2014). These subjects were already closer to
the “tipping point” for meeting confirmed clinical neuropathy criteria by
DCCT-end and this could partially explain the findings of continued differ-
ence in the development of DSPN in the intensive and conventional groups
during EDIC (Albers et al., 2010, 2007; Martin et al., 2014). Whether an
additional influence of early intensive glucose control might have been
apparent earlier during EDIC is unknown, but a persistent metabolic effect
was not required to explain the durable beneficial effects on confirmed
DSPN at EDIC year 13–14 (Albers et al., 2010, 2007; Martin et al., 2014).
In contrast, the durable benefit of intensive glucose control first observed
for retinopathy and nephropathy was also observed for CAN at EDIC year
20 R. Pop-Busui and C. Martin

13–14 and modeling that adjusted for R-R variation at DCCT closeout did
not negate the INT-associated risk reduction for development of CAN
(Martin et al., 2014; Pop-Busui et al., 2009). These findings parallel the
reported long-term benefits of prior intensive glycemic control on retinop-
athy, nephropathy (DCCT, 2002; The Diabetes Control and Complications
Trial/Epidemiology of Diabetes Interventions and Complications Research
Group, 2000; Writing Team for DCCT/EDIC, 2003), and cardiovascular
disease (Nathan et al., 2005), and are consistent with the phenomenon ter-
med “metabolic memory” (Martin et al., 2014; Pop-Busui et al., 2009;
Writing Team for DCCT/EDIC, 2003). This discordance of the impact
of DCCT treatment-group effect on longer term outcomes for DSPN
and CAN may reflect differences in susceptibility of small and large nerve
fibers to glycemic exposure (Martin et al., 2014; Pop-Busui et al., 2010).
Although both DCCT and EDIC confirmed that glycemic control is a
significant and robust predictor of neuropathy in T1D, they also show that
for most patients with T1D, current strategies for optimizing glucose control
are insufficient to fully prevent or delay the development of neuropathic
complications, as 25% of subjects in the former intensive treatment group
and 35% of subjects in the former conventional treatment group had con-
firmed DSPN at EDIC year 13–14.
Genitourinary problems associated with diabetes, with the likely excep-
tion of ED, are frequently overlooked in clinical practice and are rarely con-
sidered in the context of diabetic neuropathies. Including evaluations for the
urologic complications both in men and women as part of the UroEDIC
study thus afforded an opportunity to define the extent of genitourinary
complications of diabetes and to explore any relationships to well-defined
complications, including neuropathy (Pop-Busui et al., 2015). Our recent
findings reporting strong association between CAN, ED, and LUTS in a
large cohort of more than 600 male DCCT/EDIC participants with
T1DM demonstrate a link between CAN and more generalized autonomic
neuropathy and also suggest that CAN may predict the development of ED
and LUTS in men with long-standing T1DM (Pop-Busui et al., 2015).
Although prior experimental evidence suggested that peripheral and auto-
nomic dysfunction may be an important factor in the etiology of ED and
possibly LUTS, these findings are the first to systematically demonstrate a
link between CAN and DSPN, and ED/LUTS in a large cohort of well-
characterized men with T1DM, owing perhaps to shared or overlapping
mechanisms of neuronal damage (Pop-Busui et al., 2015).
Neuropathy in the DCCT/EDIC 21

5.1 DCCT/EDIC and Contemporary Neuropathy Trials Design

In multicenter trials, the need to systematically train, certify, and reassess
competency of staff performing tests is often overlooked, contributing to
inter- and intrasite variability over time. Yet, as demonstrated by the DCCT
and EDIC, well-standardized and validated measures of DSPN and CAN
can be successfully implemented in large, multicenter, multiyear trials
(Albers et al., 2010; DCCT, 1995, 1998; Martin et al., 2014; Pop-Busui
et al., 2010, 2009).
The DCCT and EDIC follow-up continue to add to the understanding
of diabetes-related neurologic complications. There are also, without doubt,
shortcomings and important questions that remain unanswered. Improve-
ments in technology and changes in methods for measurement of compli-
cated longitudinal assessments of well-defined or established outcomes are
required. For example, throughout DCCT and for the first 15 years of
EDIC, fundus photographs were captured on film, the film developed as
slides, and the slides then sent to a central reading center for grading. The
emergence of digital photography led to the gradual obsolescence of the
cameras which had been used throughout DCCT and EDIC (and at some
centers the cameras themselves, or parts to repair them, became unavailable).
To ensure continued photographic quality, reproducibility, and compara-
bility, a detailed and time-consuming study was performed to compare same
day, same subject film vs digital images prior to adopting digital imaging for
future EDIC studies. With regard to CAN assessments, while the testing
protocol (eg, subject preparation, 6-min supine R-R during deep breathing,
followed by 10 min of standing, followed by at least two Valsalva attempts
with standard intervals between test components and centralized analysis),
and the Hokanson, Inc. (Bellevue, WA), ANS capture devices used were
identical in both DCCT and EDIC, the methods for data capture were
not identical. Specifically, during the DCCT the changes in the R-R in
response to the cardiovascular reflex tests was recorded onto cassette tape
(sent via surface mail to the reading center), and test timing was performed
manually (stop-watch), while during EDIC a computer was used for test
timing and data capture, and files transmitted digitally to the reading center.
Indices of heart rate variability were not part of the CAN evaluations in
DCCT or in EDIC, while in a contemporary study design these measures
would likely be included.
Another area of continued interest is the role of CAN in the risk of
arrhythmia and sudden death, likely mediated through hypoglycemia.
22 R. Pop-Busui and C. Martin

While strong evidence established CAN as an independent predictor of car-

diovascular mortality in both T1D and T2D, limitations of the available tech-
nologies during DCCT and at the initial EDIC studies, direct evaluations
to document an association between CAN, hypoglycemia, and arrhythmia
were not possible. Advances in the available technologies for ambulatory
cardiac and continuous glucose monitoring will permit the EDIC study to
evaluate how changes in blood glucose levels and hypoglycemia may affect
heart rate variability and arrhythmia risk in people with and without CAN.
Advances or changes in how clinical disease states are understood and
defined may also complicate longitudinal studies such as DCCT/EDIC.
For example, the DCCT definition of confirmed clinical neuropathy, at
the time was generally well accepted as an appropriate definition for DSPN.
This outcome has received recent criticism for its lack of a comprehensive
measure of small-fiber dysfunction although, at the time the DCCT was
designed, the role and importance of small-fiber dysfunction in the progres-
sion of diabetic neuropathy was poorly understood and small-fiber assess-
ments were largely lacking from most neuropathy trials. Arguably,
measures of small-fiber neuropathy are most likely to unveil subclinical
stages of neuropathy, and are potentially most amenable to interventions
(Malik et al., 2011; Smith & Singleton, 2012). Given the natural history
of DSPN and CAN in T1D in the current era of standard of care for hyper-
glycemia and other risk factors, the design of a contemporary trial evaluating
neuropathy outcomes would certainly include measures that more directly
evaluate small fibers such as intraepidermal nerve fiber density and function,
or corneal confocal microscopy. These techniques might also allow tailored
trial designs with shorter duration, and/or smaller sample size. Although
assessments for other forms of autonomic neuropathy were not performed
in DCCT, during EDIC information on urologic complications and some
data on gastroparesis were also collected. Yet, to better understand the effects
of various risk factors and/or interventions on autonomic neuropathy,
adding validated and sensitive measures testing the urogenital, gastrointesti-
nal, or sudomotor autonomic function is necessary.
Integrating patient-specific and meaningful outcomes (for example,
quality-of-life measures, pain, sleep, and other functional assessments) that
better assess the impact of neuropathy on daily function, burden, and quality
of life should be incorporated into modern trial designs. For example, DSPN
may compromise balance in daily activities due to progressive loss of propri-
oception and later weakness, superimposed on the effect of aging, leading to
imbalance and unsteadiness in gait, with increased likelihood of a fall.
Neuropathy in the DCCT/EDIC 23

Lastly, at the time the DCCT was conducted, the limitations of exoge-
nous insulin treatment, namely the significantly greater risk of hypoglycemia
associated with INT treatment during DCCT, resulted in overall levels of
glucose control that were still not optimal. It may be that, with the use of
continuous glucose monitoring and emerging closed-loop insulin delivery
systems, normoglycemia without hypoglycemia may be reached. This
would indeed offer the opportunity to not only improve our understanding
of the impact of glycemia on neurologic outcomes, but to also examine the
effects of other risk factors including lipids and obesity.
In summary, the DSPN and CAN measures selected for the DCCT and
EDIC represent a carefully considered balance between the aims of the
study, and test validity, reproducibility, tolerability, safety, availability,
and cost. The DCCT/EDIC participants have been unbelievably generous
in their commitment to these studies, now with over 30 years of follow-up.
The EDIC study continues to evaluate the incidence, long-term outcomes,
and risk factors for neuropathic complications in a large number of well-
characterized patients with T1D and continues to demonstrate the value
of optimizing glucose control as early as possible in the course of the disease
to ameliorate the long-term effects of hyperglycemia.

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 CHAPTER THREE

The Perfect Clinical Trial

V. Bril1
Ellen and Martin Prosserman Centre for Neuromuscular Diseases, University Health Network, University of
Toronto, Toronto, ON, Canada
1
Corresponding author: e-mail address: [email protected]

Contents
1. Introduction 27
2. Study Aims: Prevent DSP, Prevent Progression, or Reverse DSP? 28
3. Selection of Study Participants 29
4. Trial Duration 31
5. Study End Points 33
6. Core Labs and Training 35
7. Intervention 35
8. Study Conduct 36
9. Streamlined Ethics and Contracts Process 37
10. Summary 37
References 38

Abstract
Multiple phase III clinical trials have failed to show disease-modifying benefits for dia-
betic sensorimotor polyneuropathy (DSP) and this may be due to the design of the clin-
ical trials. The perfect clinical trial in DSP would enroll sufficiently large numbers of
patients having early or minimal disease, as demonstrated by nerve conduction studies
(NCS). These patients would be treated with an intervention given at an effective and
well-tolerated dose for a sufficient duration of time to show change in the end points
selected. For objective or surrogate measures such as NCS and for some small fiber mea-
sures, the duration needed to show positive change may be as brief as 6–12 months, but
subsequently, trials lasting 5–8 years will be required to demonstrate clinical benefits.

1. INTRODUCTION
Multiple phase III clinical trials have lacked success in identifying
disease-modifying therapy for diabetic sensorimotor polyneuropathy
(DSP). Although numerous pharmacological agents such as neurotrophic fac-
tors, aldose reductase inhibitors, protein kinase C inhibitors, and antioxidant

International Review of Neurobiology, Volume 127 # 2016 Elsevier Inc. 27

ISSN 0074-7742 All rights reserved.
http://dx.doi.org/10.1016/bs.irn.2016.02.011
28 V. Bril

Table 1 Pharmacological Agents Tested in Phase III Trials for Disease Modification in
Diabetic Sensorimotor Polyneuropathy

Aldose reductase Sorbinil, Zenarestat, Epalrestat, ICI, Zopolrestat,

inhibitors Ranirestat
Neurotrophic factors Nerve growth factor, VEGF
Antioxidants Alpha-lipoic acid, vitamin E
Others Protein kinase C inhibitors

agents showed promising results in preclinical development and phase I and II

clinical trials, they failed in phase III clinical trials where convincing results
are critical for regulatory approval and widespread acceptance of therapeutic
benefit (Table 1). Reasons for failure at the phase III clinical trial level are con-
troversial although the most likely may be a lack of efficacy of the single
targeted intervention in a complex disorder; in other words, the selected
target may not be fundamental in the pathophysiological cascade leading to
DSP. Alternatively, deficiencies in clinical trial design may be responsible
for the repeated failures. Can we design a perfect study to overcome concerns
about trial deficiencies that may have led to previous failures to demonstrate
an effective treatment for this chronic, degenerative process?
In designing clinical trials, important elements to consider are the selec-
tion of study participants, duration of treatment, outcome measures, and
safety of patients in the study including safety of all study procedures and
the intervention. In designing the perfect clinical trial, we may ask: do
we change the outcome measures (the way we measure disease), do we
change study design, do we change expectations, and do we look for novel
biomarkers that accurately predict long-term outcomes? How should we
proceed in this field littered with the fallen corpses of clinical trials in
DSP? This chapter aims to describe what the perfect clinical trial in DSP
would be given unlimited resources and access to study participants.

2. STUDY AIMS: PREVENT DSP, PREVENT PROGRESSION,

OR REVERSE DSP?
DSP is a chronic, axonal, length-dependent polyneuropathy that
is slowly progressive and depends on duration of diabetes, glycemic control,
and other factors such has hypertension, BMI, smoking history, and
lipid profile (Dunnigan et al., 2013; Tesfaye et al., 2005). It is the most prev-
alent complication in diabetes patients that is found in 50% of subjects with
The Perfect Clinical Trial 29

long-standing diabetes and likely in more if diagnosis were based on sensitive

nerve measures (Pirart, 1978). The exact mechanisms leading to disease pro-
gression are unknown, but there is more nerve fiber loss in patients with worse
control and limited axonal regeneration in diabetes results in potentially irre-
versible axonal loss (Zochodne, 2007, 2015). Given this knowledge, it is gen-
erally considered optimal to choose patients who are early in the course of their
DSP for clinical trials in order to prevent onset or progression of neuropathy, as
reversal may be unachievable. In Type 1 patients who have diabetes duration
that can be well defined, prevention of DSP may be the optimal goal in any
given clinical trial. In Type 2 patients who may have diabetes as long as 10 years
prior to diagnosis and who often have DSP at the time of diagnosis of diabetes,
the goal of treatment may be prevention of progression rather than prevention
of DSP or even reversal of disease.

3. SELECTION OF STUDY PARTICIPANTS

The onset of Type 1 diabetes can be determined fairly accurately, and
these patients tend to develop DSP after 5 years of diabetes. So, one might
recommend limiting clinical trials to Type 1 patients with diabetes for
less than 5 years’ duration in a prevention study. Of course this approach
would limit enrollment in clinical trials mostly to children and would leave
out the 90% of patients who have Type 2 diabetes. In Type 2 diabetes
patients, the onset of diabetes is unknown and patients may have diabetes
for as long as 10 years before diagnosis. Even patients with a prediabetes state
such as impaired glucose tolerance can develop neuropathy indistinguishable
from DSP as nerve damage develops over the spectrum of glucose dys-
regulation (Singleton, Smith, & Bromberg, 2001; Sumner, Sheth, Griffin,
Cornblath, & Polydefkis, 2003). By the time that diabetes is diagnosed
in Type 2 patients, DSP can be present and advanced. Consequently, selec-
tion of Type 2 patients with early DSP cannot be determined simply by
duration of diabetes.
Patients with mild DSP can be selected on clinical or objective criteria. The
symptoms and signs of peripheral nerve disease can be summarized in scores
such as the Toronto Clinical Neuropathy Score that are related to the degree
of axonal loss (Bril & Perkins, 2002). A limitation of this approach is the degree
of variability in clinical assessment of neuropathy even when neuromuscular
experts perform the examination (Dyck et al., 2010) although the variability
when using clinical scales can be less marked (Bril, Tomioka, Buchanan,
Perkins, & mTCNS Study Group, 2009; Perkins, Olaleye, Zinman, &
Bril, 2001). The presence of clinical neuropathy based on neuropathic
30 V. Bril

symptoms, physical signs, or both, helps satisfy regulatory and diabetes com-
munity preferences for investigation of disease that is considered to be relevant
to patients. Simple clinical measures, such as the TCNS or mTCNS (Bril &
Perkins, 2002; Bril, Tomioka, et al., 2009) or the Michigan Neuropathy Score
(Herman et al., 2012) can provide a summary method of compiling the clinical
features of disease and help identify the presence of neuropathy reliably
(Zilliox, Ruby, Singh, Zhan, & Russell, 2015). Assessment of progression
may be improved with scales that are more granular and likely more sensitive
such as the mTCNS (Bril, Tomioka, et al., 2009).
Objective criteria, such as nerve conduction studies (NCS), have higher
reliability and are preferable in clinical trials (Bril et al., 1998; Dyck et al.,
2010). As progressive nerve fiber loss is the hallmark of DSP, loss of the
lower-limb sensory nerve responses shows advanced disease and selecting
patients who have a recordable sural sensory nerve response ensures that they
will have less severe neuropathy and may respond to interventions (Vinik
et al., 2005). So, all patients in clinical trials should have NCS to help select
patients and to show response to therapy when large fiber function is
expected to improve. However, DSP may be too far advanced even with
NCS in the normal range, as the healthy values for NCS parameters in
any given patient are unknown. The reference range is broad and the results
in any given patient may have declined significantly but remain in the lower
range of normal. Given unlimited resources, one might suggest that every-
one in the population have baseline NCS, so that when diabetes is diagnosed
and the NCS are repeated, one could determine the magnitude of changes
from baseline and then document the presence and severity of DSP at
an earlier stage. Naturally, a cohort of healthy volunteers would need to
have matched NCS at time points similar to those in diabetes patients to
determine the natural history of NCS in a broad population and show
the differences in diabetes subjects. Given that this approach is unrealistic
to achieve, additional means of accurately identifying disease presence
and status are required.
Identification of small fiber impairments are thought to precede
large nerve fiber loss and can help select patients for clinical trials. These
methods include evaluation of intraepidermal nerve fiber density (IENFD)
(Lauria et al., 1999), quantitative thermal thresholds (QTT) (Arezzo,
Schaumburg, & Laudadio, 1986; Yarnitsky, 1997), laser Doppler flare area
(Krishnan & Rayman, 2004), and corneal confocal nerve fiber microscopy
(CCM) (Ahmed et al., 2012; Malik et al., 2003). Tests of sudomotor
function can help supplement these studies (Casellini, Parson, Richardson,
The Perfect Clinical Trial 31

Nevoret, & Vinik, 2013). A combination of abnormal tests can reliably

identify the presence of DSP even in the setting of normal NCS (Breiner,
Lovblom, Perkins, & Bril, 2014) and these small fiber tests, particularly
IENFD, QTT, and CCM should be used for selection of subjects in clinical
trials (Breiner et al., 2014). Their use as central end points is limited by the
variability of testing, need for combination of tests for reliability, and also
the need to demonstrate that changes in these fibers will predict improvement
in all nerve fibers.
The population studied should be broad and include all racial groups and
multiple countries so that the effects of any intervention can be understood
across all people (Hotta, Kawamori, Fukuda, Shigeta, & Aldose Reductase
Inhibitor-Diabetes Complications Trial Study Group, 2012). A 60-site
study done in 20 countries or more is not unreasonable to consider for an
intervention study (Bril et al., 1998).
In summary, it is necessary to select patients with clinical evidence of
neuropathy, using NCS to document severity and including small fiber tests
for objective evidence of DSP, irrespective of whether NCS are normal or
abnormal.

4. TRIAL DURATION
The most successful clinical trial on neuropathy in diabetes to date was
the DCCT (The effect of intensive diabetes therapy on the development and
progression of neuropathy. The Diabetes Control and Complications Trial
Research Group, 1995). In this study of 1441 subjects with Type 1 diabetes,
intensive glycemic control for 5 years reduced the prevalence of neuropathy
as measured by clinical, electrophysiological, and cardiac autonomic tests, by
60% (The effect of intensive diabetes therapy on the development and
progression of neuropathy. The Diabetes Control and Complications
Trial Research Group, 1995). This study showed that the end points of
clinical evaluation, NCS, and autonomic system testing could reflect
changes in DSP and are thus valid for use in clinical trials. Also, this costly
study was funded by US governmental agencies, was planned for a duration
of 7 years, and had a very large number of subjects who were evaluated com-
prehensively with clinical examination by neurologists, autonomic system
testing, and also NCS. The DCCT showed at 5 years the benefits on
DSP of intensive compared to conventional glycemic control in Type 1
patients, so the minimum duration of any study in Type 1 patients should
32 V. Bril

be 5 years when this array of tests are the key clinical endpoints. Assessments
at intermediate time-points were not available and so shorter durations of
studies cannot be recommended. Large-scale epidemiological studies or
intervention studies without similar comprehensive evaluations of neurop-
athy did not provide strong evidence of benefits with respect to DSP, partly
because the neuropathy was not evaluated in a comprehensive manner or
because the separation of treatment groups of regular and improved control
was suboptimal (U. P. D. S. Group, 1998). Nonetheless, a separation of
vibration perception was noted at 8 years in the UKPDS study in Type 2
patients, even with these caveats. Reduction in autonomic neuropathy
has been observed with multifactorial interventions (Gaede, Vedel,
Parving, & Pedersen, 1999, Greene, Arezzo, Brown, & Zenarestat Study
Group, 1999). The experience with Type 2 patients may have differed if
different study protocols were employed. However, these studies suggest
that durations in this population need to be lengthy and perhaps as long
as 8 years or more.
The longest study funded by the pharmacology industry was a 4-year
treatment trial of an oral medication (the NATHAN study) that was not effec-
tive and did not show benefits in the main end points of the study (Ziegler et al.,
2011). The end points were similar to those in the DCCT. This outcome is
challenging to understand because evaluations in the DCCT were done at base-
line and at 5 years, but not at 4 years. We cannot know whether the reduction in
prevalence of DSP in the DCCT was linear over 5 years, or not. If the neurop-
athy assessments had been done at 4 years, then the outcome of the NATHAN
study could be compared directly to the DCCT results, but that is not the case.
Also, the study populations were different in that the NATHAN study had a
predominance of patients with Type 2 diabetes. When factors that might
have influenced the outcome were evaluated, those with a normal BMI,
normal blood pressure, and higher burden related to cardiovascular disease, dia-
betes, and neuropathy were more responsive to treatment (Ziegler, Low,
Freeman, Tritschler, & Vinik, 2016), suggesting that a multifactorial approach
when treating DSP will be required. Nonetheless, it is likely that the DCCT
would have shown some trend to less neuropathy in Type 1 diabetes subjects
at 4 years.
In summary, given the fact that DSP is a chronic, insidiously progressive
axonal neuropathy, and the results of the DCCT and UKPDS, the perfect
clinical trial would last at least 5 years in Type 1 patients and 8 years or more
in Type 2 patients.
The Perfect Clinical Trial 33

5. STUDY END POINTS

In assessing benefits of any intervention for DSP, clinical improve-
ment in disease status would be the most convincing evidence of efficacy.
The reduction in, or elimination of, symptoms and decreased signs of disease
indicate a positive response. Certain quantitative or summary scales can pro-
vide evidence of clinical benefit. The modified TCNS, the TCNS, the
MNI, and other scales can be used to show changes in DSP although some
may be more sensitive than others (Bril, Tomioka, et al., 2009; Herman et al.,
2012). A reduction in symptoms with improving objective measures of DSP
indicates improvement. However, clinical evaluation can be highly variable
necessitating the requirement for large numbers of patients who would
require careful assessment for benefit. Deficits in patients with mild disease
are mainly sensory in nature and sensory testing is the most variable on clinical
examination, so the noise level can be very high (Perkins et al., 2001).
The natural history of nerve conduction slowing in diabetes is a gradual
decline in values over many years (Gibbons et al., 2013; Partanen et al.,
1995). NCS are the best measure of peripheral nerve function and should
show separation of two treatment arms at 5 years of treatment based on
the DCCT results (D. C. A. C. T. D. R. Group, 1995). Many phase II stud-
ies have shown small changes in shorter treatment duration intervals that
have not been replicated in phase III studies and have questionable meaning.
Small changes in glycemic control or lipid profile might also have a longer
effect on NCS parameters than expected (Perkins, Dholasania, Buchanan, &
Bril, 2010). NCS should be used as an adjunct measure to confirm lack of
deterioration of nerve function if positive symptoms such as pain disappear,
to exclude the possibility that progressive disease or therapeutic interven-
tions have alleviated pain by ablating nerve function. In studies where large
nerve fiber function appears to improve by some measures such as vibration
perception threshold (VPT) or clinical examination, but NCS do not
improve (Ziegler et al., 2011), questions about the validity of the results need
to be addressed.
While large fiber function can be tracked iteratively across studies, efforts
to assess large fiber structure in clinical trials have been largely limited to sin-
gle cross-sectional studies, although the evaluation of pre- and posttreatment
sural nerve biopsy was at one time thought to provide a means to shorten the
clinical trial duration (Greene et al., 1999). The procedure is invasive and
34 V. Bril

not as objective as might be thought (Perry & Bril, 1994) without standard-
ized training of those evaluating the biopsy (Kalichman, Chalk, & Mizisin,
1999), so that large nerve fiber biopsy is not advocated at this time.
End points should include evaluation of small nerve fibers as these can be
demonstrated to be affected before NCS falls into the abnormal range
(Breiner et al., 2014; Smith, Ramachandran, Tripp, & Singleton, 2001).
The use of IENFD and other measures of epidermal and dermal nerve fibers
(such as axonal swellings and varicosities) can provide direct structural evi-
dence of nerve status (Mellgren, Nolano, & Sommer, 2013). Nerve regen-
eration with the capsaicin IENFD system can be another way to assess nerve
recovery (Polydefkis et al., 2004). So, clinical trials should include IENFD at
baseline and then at end of treatment. Sampling from the distal lower leg and
distal thigh are suggested. The procedure is invasive, but minimally so with
minor potential side effects of infection and bleeding.
Noninvasive measures of small nerve fiber function are preferable to
invasive ones, but should also supplement the invasive measure. Corneal
confocal microscopy to directly visualize small terminal trigeminal nerve
fibers in Bowman’s layer of the cornea may be useful to assess nerve regen-
eration (Ahmed et al., 2012; Malik et al., 2003). Nerve fiber length on CCM
is a surrogate measure for the longest nerve fibers, correlates with longer
nerve fiber measures (IENFD, NCS, QST), and may show changes earlier
than would be apparent in longer fibers (Hertz et al., 2011; Sivaskandarajah
et al., 2013). This end point should be measured at baseline and then every 6
months during a study lasting for a minimum of 5 years (Mehra et al., 2007).
If no change in CCM parameters is observed at earlier time points, then the
study can be terminated earlier, but if changes are apparent, then the study
should be continued for 5 years to demonstrate corresponding changes in the
longest nerve fibers and provide a convincing study result.
Quantitative sensory threshold measurements provide a numerical eval-
uation of sensory function, with thermal thresholds (QTT) indicating small
nerve fiber function and VPTs reflecting large nerve fiber function. In QTT,
cold detection thresholds (CDT) are more reliable than warming thresholds,
and so are preferable (Zinman, Bril, & Perkins, 2004). These measures
should be done in clinical trials to help evaluate how sensory nerves are func-
tioning in those with DSP. There is great variation between assessments, but
VPT and CDT are valuable end points and should be done at study start and
end (Young, Every, & Boulton, 1993; Zinman et al., 2004).
Laser Doppler flare area imaging in response to heat-mediated vasodila-
tion can provide a measure of cutaneous nerve fiber function (Krishnan &
The Perfect Clinical Trial 35

Rayman, 2004). This end point may help provide supplementary evidence
of small nerve fiber function (Krishnan, Quattrini, Jeziorska, Malik, &
Rayman, 2009).
Autonomic function tests such as sudomotor function, heart rate vari-
ability, and others can provide separate evidence of small nerve fiber
responsiveness.
Generally, all end points at baseline and end of study should have triplicate
measures to improve the accuracy of the results. The median value is used so
that one unusual value does not skew the “real” value of the parameter.

6. CORE LABS AND TRAINING

A major concern in any study is standardization of all procedures from
the clinical examination to the measurement of nerve function such as in
NCS. In order to maximize the chances of observing positive change, the
testing in the study needs to be done the same way in all patients at all sites
in the study. The degree of variability needs to be reduced to the minimum
possible so that changes are detectable, as was done for sural NCV over 5
years in DCCT (The effect of intensive diabetes therapy on the
development and progression of neuropathy. The Diabetes Control and
Complications Trial Research Group, 1995). A core lab is essential to help
control study procedures, particularly those with large technical components
that confound inexpert CROs, study monitors, and pharmaceutical staff
(Bril et al., 1998; Bril, Katzberg, et al., 2009). Such core lab functions are
essential for NCS (Bril et al., 1998), CCM, and LDI. Standardized training
sessions are necessary for the clinical examination, clinical scales, and all
other study procedures.

7. INTERVENTION
Only therapies that are effective in appropriate preclinical models at
doses that can sensibly be used in humans, allowing for interspecies allome-
tric scaling (Freireich, Gehan, Rall, Schmidt, & Skipper, 1966; Huang &
Riviere, 2014) should be advanced to human trials. In many unsuccessful
clinical trials, a study drug has been tested and found effective in preclinical
and phase II studies and then failed in phase III. This has sometimes been
related to the use of doses in the phase III study that were lower than the
phase II and subsequently proved to be less effective, as discussed for trials
of NGF in the previous edition of this volume (Apfel, 2002). The use of
36 V. Bril

interventions at effective doses is essential—the study should not be done at

all if there is a safety issue at the effective dose. The interventions tested to
date have targeted a part of the presumed pathophysiology of DSP. Multi-
level interventions may be required to successfully intervene in the cascade
that leads to DSP. Proponents of any particular disease hypothesis need to
acknowledge and accept other factors as being important in the pathway
to disease so that they can work together to bring about effective interven-
tions. In a perfect clinical trial, multiple interventions are likely to be used
together to produce a positive effect, for example, a therapy that combines
ARI, antioxidant, and axonal regenerative properties.
In addition to the specific intervention, any long-term clinical trial
would need to control factors that might contribute to DSP such as hyper-
glycemia, hypertension, hyperlipidemia, smoking, and exercise patterns
(Gaede et al., 1999; Perkins et al., 2010; Tesfaye et al., 2007). A stable reg-
imen addressing these factors needs to be established prior to the study or for
the first 3 months of the study, and then patients could be randomized to a
specific intervention.

8. STUDY CONDUCT
For a 5-year or longer clinical trial, retention of study subjects
becomes a major concern. The DCCT was successful in large part due to
the motivated population under study and the initiatives put in place by
the study coordinators: morale and team building items and activities that
engaged study participants proved key to the success of this study. For
any long duration study, such activities are necessary and should be allowed
despite the current ethical climate that focuses on perceived “coercion” of
study subjects. It is more unethical to run a study that cannot be completed
with the requisite number of subjects without constant positive reinforce-
ment and encouragement by the study staff. Small study perks (mugs,
t-shirts, baseball caps, and other items) might convince study participants
to stay the course. Adequate remuneration for study subjects’ time, coverage
of all patient study-related costs, and proper funding for sufficient numbers
of study staff so that they can spend the time necessary for subject retention
are essential. The ability to enroll subjects who live far from a study center
should be enabled by proper budgeting of participant expenses related to the
study. Newer communication tools such as videoconferencing with each
study participant using personal electronic devices might help with long-
term retention. All of the items identified in this section are part of a study
done with unlimited resources, but need to be stated explicitly to ensure that
The Perfect Clinical Trial 37

they are funded in the current research climate where all costs tend to be
minimized as much as possible, if not eliminated entirely, to the detriment
of any study.

9. STREAMLINED ETHICS AND CONTRACTS PROCESS

Increasingly, it has become more and more difficult to carry out clin-
ical research studies in the developed world due to an ever-increasing
bureaucracy in health care research. Motivated by the view that “we only
want to protect patients,” governmental agencies, ethics, and contracts per-
sonnel raise more and more barriers to clinical research. There may be a con-
flict of interest at work here as increasing the roles of these groups leads to
more employment for them. Wrangles over minor wording changes in con-
tracts, the desire to have “zero” liability in any study, and the current envi-
ronment of competitive enrollment make clinical research a herculean
endeavor. We are depending more on clinical research that is done in China,
India, and various locales in South America where the requirements appear
to differ. It will be interesting to see whether the results obtained in these
countries are applicable to different patient populations. We may have treat-
ments that do not work in local population and how can we check, if we
cannot do the clinical research in the first place? How ethical is it to take
results from clinical trials that we would not allow to take place in our
own country? In a world with perfect clinical trials, it would be sufficient
for a central ethics board to approve all study sites, and for all institutions
to accept that approval and allow the research to proceed. A standard
research contract across all academic hospitals (at least in a single country)
and agreed to by all sponsors be they governmental agencies, pharmaceutical
sponsors, or private sponsors would enable speedier clinical research and
provide results and improved care for patients.

10. SUMMARY
The perfect clinical trial in DSP would enroll sufficiently large num-
bers of patients having early or minimal disease, as demonstrated by NCS.
These patients would be treated with an intervention at an effective and
well-tolerated dose for a sufficient duration of time to show change in
the end points selected. For objective or surrogate measures such as NCS
and for some small fiber measure, this duration can be as brief as 6–12
months, but eventually longer duration trials of 5–8 years will be required
to show clinical improvement.
38 V. Bril

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 CHAPTER FOUR

An Introduction to the History and

Controversies of Animal Models of
Diabetic Neuropathy
N.A. Calcutt*,1, P. Fernyhough†,{
*University of California, San Diego, La Jolla, CA, United States
†
University of Manitoba, Winnipeg, MB, Canada
{
St. Boniface Hospital Research Centre, Winnipeg, MB, Canada
1
Corresponding author: e-mail address: [email protected]

Contents
1. Why Use Animal Models? 45
2. What Species? 46
3. What Diabetogenic Insult? 48
4. STZ Toxicity 49
5. Novel Models 50
References 50

1. WHY USE ANIMAL MODELS?

The prime value of animal models of diabetic neuropathy to biomed-
ical research has been in identifying pathogenic mechanisms that link fea-
tures of diabetes such as hyperglycemia, insulinopenia, insulin resistance,
or dyslipidemia with nerve dysfunction and damage. Diabetic animals,
and rodents in particular, are also widely used for establishing efficacy of
therapeutic interventions prior to testing in humans. The extent to which
animal models of diabetic neuropathy replicate the human condition has
been contentious almost since the advent of such models, and remains so.
Retrospective condemnation of animal models used in preclinical drug
development programs as not being predictive of drug efficacy in humans
is a not uncommon reflex response to the failure of a clinical trial that
can prevent closer examination of other flaws in the drug development pro-
cess. There are indeed many reasons why animal models of diabetic neurop-
athy cannot be faithful replicates of the human condition—not least of

#
International Review of Neurobiology, Volume 127 2016 Elsevier Inc. 45
ISSN 0074-7742 All rights reserved.
http://dx.doi.org/10.1016/bs.irn.2016.03.011
46 N.A. Calcutt and P. Fernyhough

which are their short life span (and thus exposure time to diabetes) and short
hairy legs (and thus short distance from cell body to nerve terminal).
Whether there are interspecies differences in physiological and cellular
responses to diabetes that invalidate the common animal models is more
debatable. Support for the pertinence of animal models of diabetic neurop-
athy comes from recognition that diabetic animals develop many disorders in
common with diabetic patients, including nerve conduction slowing, resis-
tance to ischemic conduction block, allodynia, loss of thermal sensation, and
reduced small sensory fiber density in the skin and cornea (Biessels et al.,
2014). Additional practical benefits of using animal models include the abil-
ity to test hypotheses by methods not permissible or practical in humans, the
lower cost of preclinical research, and the accelerated temporal progression
of neuropathy that allows longitudinal studies over months rather than years
or decades. Unfortunately, as with many model systems, the literature is
cluttered with the inappropriate use of specialized models or overly exuber-
ant interpretation of positive findings. The fidelity of animal models of
diabetic neuropathy remains an important issue that requires frequent con-
sideration, revision, and refinement.

2. WHAT SPECIES?
Diabetes is not an exclusively human disease. There are reports of
spontaneous diabetes in many long-lived species that live in close contact
with humans, including nonhuman primates (Kuhar, Fuller, & Dennis,
2013), elephants (van der Kolk et al., 2011), camelids (Cebra, 2009), dogs
(Fleeman, Rand, & Morton, 2009), and cats (O’Brien, 2002). In many cases,
the provision of unnatural diets to wild animals kept in captivity and to spe-
cies domesticated for farming or companionship is suspected as causing dia-
betes. Indeed, the rise of type 2 diabetes in the human population is being
paralleled by a similar epidemic in the domestic cat population, which is also
exposed to poor diet and limited exercise. Diabetic cats are particularly
prone to developing a peripheral neuropathy that is strikingly similar to
the human condition with nerve conduction slowing accompanied by struc-
tural damage to blood vessels, Schwann cells, and axons (Estrella et al., 2008;
Mizisin et al., 2007). These observations support use of the diabetic cat pop-
ulation as a bridge between traditional preclinical drug efficacy studies and
clinical trials so that drug efficacy can be evaluated in nerves exhibiting the
full spectrum of structural pathology. Recruitment of diabetic cats and
screening for neuropathy by veterinarians is as viable as similar recruitment
Animal Models of Diabetic Neuropathy 47

exercises for clinical trials but, in our experience, also requires the same
attention to recruitment practices such as investigator training and incentives
for both investigator and the subject (or their owner). Given the huge cost of
clinical trials, efficacy testing in diabetic cats may be useful as a cost-effective
final preclinical evaluation point in the drug development pipeline.
Some species show normal physiological features that would indicate
diabetes in humans. For example, plasma glucose levels of birds, while vary-
ing between species, are much higher (10–40 mM range) than in most mam-
mals. This is not a permanent disease condition so much as a reflection of
different physiological goals, with glucagon, not insulin, being the dominant
pancreatic hormone in chickens (Braun & Sweazea, 2008). While hypergly-
cemia and relative insulin deficiency are the hallmarks of type 1 diabetes and
implicated in most current pathogenic hypotheses, they do not appear to
adversely impact the nervous system of birds. It has been argued that such
interspecies variations may prove useful for studying how organs resist dam-
age by hyperglycemia and/or insulinopenia (Singer, 2011). The first foray by
one of us into medical research was an attempt to make chickens diabetic in
order to study the effects of hyperglycemia on the enticingly long vagus
nerve (Calcutt & Tomlinson, 1985). The approach, along with many
chickens, was ultimately doomed not only because birds are inherently
hyperglycemic, but also because it became clear that the chicken pancreas
is relatively resistant to the diabetogenic drug streptozotocin (STZ)
(Modak, Datar, Bhonde, & Ghaskadbi, 2007; Simon & Dubois, 1980).
A lifetime quest for a diabetic giraffe continues.
Experimental requirements for an adequate number of observations
to move beyond the anecdotal have restricted research in larger species to
occasional observations of the effects of diabetes on nerve from primates
(Pare et al., 2007), pigs ( Juranek et al., 2010), and dogs (Walker et al.,
2001), all of which are expensive to maintain and have yet to reveal tangible
benefits as models. By far the vast majority of studies of experimental diabetic
neuropathy have therefore been performed in rats and mice. Rodents can be
bred to consistently develop spontaneous type 1 or type 2 diabetes or else can
have diabetes induced by dietary manipulation or chemicals that ablate pan-
creatic beta cells. For many years, rats were the most widely studied species.
This was in part due to their size, which allows surgical manipulations and
yields enough tissue for crude biochemical assays. Rats were also preferred
because the dominant pathogenic mechanism under evaluation during the
1960s to 1990s was glucose flux through the polyol pathway and polyol
pathway metabolites accumulated in the nerve of diabetic rats. Mice were
48 N.A. Calcutt and P. Fernyhough

not widely studied as models for diabetic neuropathy during this period,
other than basic characterization of the db/db and ob/ob mouse models
of spontaneous type 2 diabetes, largely because it was erroneously thought
that they lacked polyol pathway activity in their nerves. Recognition that
lack of accumulation of polyol pathway intermediates in nerve was due
to distinctive enzyme kinetics that makes mice similar to humans in this
respect (Calcutt, Willars, & Tomlinson, 1988; Tomlinson, 1989) and that
polyol pathway flux can drive nerve dysfunction in mice (Calcutt,
Tomlinson, & Biswas, 1990; Ho et al., 2006; Ng et al., 1998), along with
the emergence of new technology that favors use of mice for genetic manip-
ulations, has caused the use of mouse models of both type 1 and type 2
diabetes to greatly expand over the last 10–20 years.

3. WHAT DIABETOGENIC INSULT?

The early use of pancreatectomy to produce diabetic animals for the
study of insulin secretion and diabetes was initially superseded by alloxan,
which reduces production and secretion of insulin and glucagon by damaging
pancreatic beta cells, but does not impede secretion of other pancreatic prod-
ucts. Alloxan is a prooxidant that, while having a marked effect on pancreatic
beta cells by virtue of cellular uptake by glucose transporters (Elsner, Tiedge,
Guldbakke, Munday, & Lenzen, 2002), can also damage other organs
involved in detoxification such as the liver and kidney. The first description
of neuropathy in a diabetic rodent, as implied by nerve conduction slowing,
was made in alloxan-diabetic rats (Eliasson, 1964). However, difficulties in
dose titration to ensure the induction of type 1 diabetes without producing
off-target effects or killing most of a cohort, make alloxan-induced diabetes
a notoriously difficult model. Alloxan is now only infrequently used to pro-
duce diabetes in rats or mice, although use has persisted where diabetic rabbits
are required, such as in studies of skin wound healing (Pradhan Nabzdyk et al.,
2013). Alloxan has been almost completely replaced as a diabetogenic agent by
STZ, a product of the soil bacteria Streptomyces achromogenes that was originally
developed as a therapy for pancreatic cancer. Like alloxan, STZ enters pan-
creatic beta cells via the GLUT-2 glucose transporter (Elsner, Guldbakke,
Tiedge, Munday, & Lenzen, 2000), where it causes cell death by a variety
of mechanisms, as reviewed elsewhere (Eleazu, Eleazu, Chukwuma, &
Essien, 2013). STZ replaced alloxan because it is relatively easy to dose titrate
so that insulin deficient diabetes is consistently induced without significant
damage to other organs or high mortality rates (Mansford & Opie, 1968).
Animal Models of Diabetic Neuropathy 49

Sliding scales of STZ dose have been developed for rats of different ages and
weights (Calcutt, 2004). In mice, the severity of insulinopenia and its practical
consequences such as conversion rate, survival duration, and frequency of
reversal of diabetes arising from beta cell regeneration can be tempered by
varying both STZ dose and dose frequency. A variety of protocols have been
developed that employ between 2 and 5 low doses of STZ given on consec-
utive days (Biessels et al., 2014). As STZ is inexpensive and relatively easy to
use when following established protocols, the STZ-diabetic rat and mouse
have become the dominant models of diabetic neuropathy.

4. STZ TOXICITY
Concerns are intermittently raised that the cachexia that can occur in
rodents treated with high doses of STZ makes them poor models, particu-
larly for behavioral studies such as those using nociceptive testing to assess
sensory function. Such concerns can be mitigated by appropriate dose titra-
tion of STZ that produces hyperglycemia without the extreme
insulinopenia that underlies cachexia, or by trace insulin replacement
(Calcutt, 2004), along with reporting of performance in rotarod or other
tests. High doses of STZ can also produce direct and acute nephrotoxicity
in mice (Breyer et al., 2005) and direct STZ neurotoxicity has been evoked
to explain tactile allodynia in STZ-injected animals that did not develop
overt hyperglycemia. However, STZ-induced insulinopenia that was not
sufficient to produce overt hyperglycemia but contributes to the pathogen-
esis of allodynia is a more likely explanation (Romanovsky, Cruz, Dienel, &
Dobretsov, 2006). A number of other observations also appear to argue
against meaningful neurotoxic effects of STZ:
• STZ enters cells via the GLUT-2 transporter, which is not found in
peripheral nerve.
• STZ is rapidly cleared from the body, disassociating it from the indices of
neuropathy that can take weeks to emerge.
• Insulin replacement therapy to normalize blood glucose levels and
started days after STZ delivery prevents onset of indices of neuropathy.
• Coinjection of STZ and 6-O-methyl glucose, which competitively
inhibits STZ-uptake by GLUT-2 transporters, produces neither diabetes
nor neuropathy as measured by NCV slowing and loss of heat-induced
pain sensation (Davidson et al., 2009).
• Indices of neuropathy in STZ-injected animals are replicated in other
models of type 1 and type 2 diabetes.
50 N.A. Calcutt and P. Fernyhough

The wealth of reference data already collected means that STZ-diabetic

rodents are likely to remain the dominant model of diabetic neuropathy.
However, all data obtained in STZ-injected rodents should be interpreted
with appropriate caution and the provenance of newly identified disorders
validated using groups cotreated with 6-O-methyl glucose or insulin. Fur-
ther, given the possibility that the pathogenesis of diabetic neuropathy may
differ in type 1 and type 2 diabetes, STZ-diabetic rodents may best be
deployed in combination with models of type 2 diabetes to evaluate whether
new disorders and therapies are pertinent to both forms of the disease.

5. NOVEL MODELS
The neuropathy phenotype of commonly used rodent models of dia-
betes has recently been evaluated under the aegis of Neurodiab and the NIH.
The resulting consensus statement is comprehensive and may be viewed
elsewhere (Biessels et al., 2014) so need not be replicated in this volume.
We have therefore taken the opportunity to invite colleagues to instead con-
sider alternative approaches to modeling diabetic neuropathy. A number of
such models, particularly models of type 2 diabetes and metabolic syndrome,
are currently undergoing characterization. The review “Alternatives to the
STZ-Diabetic Rodent” by Yorek is therefore a particularly opportune con-
tribution. Moreover, the pertinence of neuronal and glial cell cultures to
model the toxic impact of hyperglycemia, insulinopenia, or other diabetes-
associated insults is perhaps even more controversial than use of animal
models of diabetic neuropathy, with the advantage of selective manipulation
of the local environment offset by the disadvantage of lack of intercellular
and physiological interactions. The chapter “Can Diabetic Neuropathy
Be Modeled In Vitro?” by Gardiner eloquently addresses these trade-offs
to make a case that, within reason, we can indeed “do it in a dish.”

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Calcutt, N. A. (2004). Modeling diabetic sensory neuropathy in rats. Methods in Molecular

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Diabetologia, 43, 1528–1533.
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 CHAPTER FIVE

Can Diabetic Neuropathy

Be Modeled In Vitro?
N.J. Gardiner1, O.J. Freeman
University of Manchester, Manchester, United Kingdom
1
Corresponding author: e-mail address: [email protected]

Contents
1. Introduction 54
2. Diabetic Neuropathy 56
3. The Somatosensory Nervous System 56
4. Can We Model Diabetic Neuropathy In Vitro? 62
4.1 Choice of Cells 63
4.2 Choice of Stimuli 68
4.3 Choice of Output Measure 69
4.4 Looking Forward to a Better In Vitro Model of Diabetic Neuropathy 76
5. Conclusions 79
Acknowledgments 79
References 79

Abstract
Diabetic neuropathy is a common secondary complication of diabetes that impacts on
patient's health and well-being. Distal axon degeneration is a key feature of diabetic
neuropathy, but the pathological changes which underlie axonal die-back are incom-
pletely understood; despite decades of research a treatment has not yet been identified.
Basic research must focus on understanding the complex mechanisms underlying
changes that occur in the nervous system during diabetes. To this end, tissue culture
techniques are invaluable as they enable researchers to examine the intricate mecha-
nistic responses of cells to high glucose or other factors in order to better understand
the pathogenesis of nerve dysfunction. This chapter describes the use of in vitro models
to study a wide range of specific cellular effects pertaining to diabetic neuropathy
including apoptosis, neurite outgrowth, neurodegeneration, activity, and bioenergetics.
We consider problems associated with in vitro modeling and future refinement such as
use of induced pluripotent stem cells and microfluidic technology.

International Review of Neurobiology, Volume 127 # 2016 Elsevier Inc. 53

ISSN 0074-7742 All rights reserved.
http://dx.doi.org/10.1016/bs.irn.2016.02.004
54 N.J. Gardiner and O.J. Freeman

1. INTRODUCTION
Diabetic neuropathy affects approximately half of all patients with dia-
betes mellitus (Dyck et al., 1993). It can be a debilitating condition that
impacts on patient’s health and quality of life and presents a substantial
expense for health-care providers (Hex, Bartlett, Wright, Taylor, &
Varley, 2012). Patients typically present with sensory symptoms arising in
a “glove and stocking” distribution, with feet commonly affected before
hands. Sensory symptoms are divided into the “positive”: neuropathic pain,
paraesthesia, and dysesthesia; and the “negative”: loss of sensation, decreased
nerve conduction velocity, and nerve dysfunction. Autonomic and motor
neuropathies also occur, and increasing evidence suggests that this secondary
complication of diabetes extends to the central nervous system (CNS). Neu-
rons show a distal–proximal pattern of injury, with the longest axons show-
ing the worst pathology.
Diabetic neuropathy is multifactorial in etiology and many pathogenic
mechanisms have been described (Fernyhough & Calcutt, 2010;
Fernyhough, Roy Chowdhury, & Schmidt, 2010; Freeman et al., 2016;
Obrosova, 2002, 2003; Zochodne, 2014). Despite this, treatment options
are currently limited to glycemic control and/or symptomatic treatment
and are inadequate. Experimental rodent models of diabetes have long been
utilized to assess behavioral, electrophysiological, and morphological deficits
in diabetic neuropathy and to test the efficacy of novel therapeutics. These
models will be considered elsewhere in this volume. The sad fact stands that,
despite many decades of research, a treatment for diabetic neuropathy that
significantly reverses or ameliorates the progression of the disease has not
been identified. The search for efficacious drugs to better treat neuropathic
pain, prevent axon degeneration, and/or promote nerve regeneration is
therefore an urgent clinical need.
Basic research in diabetic neuropathy must focus on understanding
the complex mechanisms underlying the changes that occur in the ner-
vous system. To this end, tissue and cell culture techniques are valuable
as they enable researchers to dissect out the intricate mechanistic responses
of cells to high glucose or other exogenous factors. The crucial benefit
afforded by an in vitro over an in vivo approach is the ability to precisely
control and manipulate the environment (Table 1), thereby allowing
study of a wide range of specific cellular events ranging from survival to
death; neurite outgrowth to neurodegeneration; excitation to inhibition;
Can Diabetic Neuropathy Be Modeled In Vitro? 55

Table 1 Advantages and Disadvantages to Consider When Selecting an In Vitro

Modeling Approach for Diabetic Neuropathy
In Vitro Techniques
Advantages Tight control of the extracellular environment
Ability to study specific cell responses to individual exogenous
stimuli
Allows real-time imaging and recording of complex intracellular
pathways and events
Ability for high-throughput screening of drugs
Fewer ethical issues than using in vivo models
Disadvantages Simplistic
Fails to recapitulate the complex in vivo environment
Variable purity of primary cultures
Acutely dissociated primary cells will be in a regenerative rather
than homeostatic mode
Inability to test therapeutics on functional changes
Translation—may not mimic the clinical condition as well as
experimental models of diabetes

subcellular bioenergetics; and axonal transport. While in vitro systems

are by their nature simplistic, and findings should only be extrapolated
to the clinical condition following validation in other more complex
models, data can be used to better understand the pathogenesis of nerve
dysfunction and to identify potential therapeutic mechanisms to inform
in vivo studies.
Pathogenic mechanisms associated with diabetes have been investigated
using a range of in vitro techniques such as dissociated neuronal and/or
glial cell culture, cocultures, and organotypic explants. This chapter will
guide the reader through the choices of cell types and culture system to
consider and will also highlight phenotypic responses of cells to hypergly-
cemia and other stressors to emphasize the diversity and usefulness of
in vitro models to study diabetic neuropathy. We have focussed on work
that can only currently be done in vitro but will also consider the important
caveats for in vitro modeling approaches (Table 1) and the exciting new
technologies and techniques that may allow better modeling of a “nerve
in a dish.”
56 N.J. Gardiner and O.J. Freeman

2. DIABETIC NEUROPATHY
The first symptoms of diabetic neuropathy typically arise in the
somatosensory aspect of the peripheral nervous system (PNS). The skin is
innervated by peptidergic and nonpeptidergic unmyelinated afferents that
form free axon terminals in the epidermis, and myelinated afferents that ter-
minate at specialized mechanoreceptors (Fig. 1) (Hilliges, Wang, &
Johansson, 1995; Owens & Lumpkin, 2014). Distal processes of sensory
nerves degenerate in both clinical and experimental diabetic neuropathy
(Kennedy & Zochodne, 2000, 2005; Tomlinson & Gardiner, 2008;
Yasuda et al., 2003), and degeneration occurs at a faster rate than rein-
nervation, leading to loss of sensation. This degeneration, along with other
structural changes in the nerve such as axonal dystrophy, Schwann cell
pathology, paranodal demyelination, and endoneurial microangiopathy,
coincides with electrophysiological changes and sensory dysfunction
(Tomlinson & Gardiner, 2008). Autonomic and motor neuropathies are less
well characterized than sensory neuropathies, possibly reflective of the lower
incidence of motor and visceral autonomic neuropathy in patients with dia-
betes (Dyck et al., 1993; Spallone et al., 2011) and the comparative ease of
studying the somatosensory system in both humans and experimental
models of diabetes. Since the interpretation of in vitro experiments requires
knowledge of the context in which different cell populations function, this
chapter will open with a brief overview of the development, maturation,
and structure of the PNS.

3. THE SOMATOSENSORY NERVOUS SYSTEM

The PNS is divided into the somatic (sensory and motor neurons) and
autonomic (sympathetic, parasympathetic, and enteric neurons) nervous
systems and links peripheral tissues with the brain and spinal cord compo-
nents of the CNS. PNS efferents include motor neurons and autonomic
neurons, whose cell bodies in the ventral horn of the spinal cord or auto-
nomic ganglia extend axons to neuromuscular junctions in skeletal muscle
and internal organs, respectively. The cell bodies of sensory neurons reside
within the dorsal root ganglia (DRG) in the intervertebral foramen adjacent
to the vertebral column, and in the trigeminal ganglia. Sensory neurons are
pseudo-unipolar neurons and extend an axon which bifurcates, with one
branch projecting to the CNS and the other to peripheral target tissue.
Can Diabetic Neuropathy Be Modeled In Vitro? 57

Fig. 1 The peripheral nervous system. (A) The cell bodies of most peripheral sensory
neurons are located in the dorsal root ganglia (DRG). The motor neuron cell bodies
within the ventral horn of the spinal cord extend efferents via the ventral roots to join
the peripheral nerve and terminate in specialized neuromuscular junctions. (B) Sensory
neurons are a heterogeneous population, express different receptors and phenotypic
markers, and respond to different neurotrophic factors. Peripheral sensory afferents ter-
minate as free nerve endings or in specialized sensory receptors such as Pacinian cor-
puscles (PC) in the skin (C). Central branches are directed to the spinal cord and through
collateral branches to the dorsal column nuclei (DCN). The electron micrograph
(D; kindly provided by Prof. David Tomlinson, University of Manchester) shows the inter-
nal organization of a peripheral nerve. Large-diameter sensory and motor axons (AX) are
surrounded by compacted myelin (m), while groups of small-diameter unmyelinated
sensory axons (ax) are grouped in Remak bundles (arrows). Endoneurial ECM, collagen
fibrils (c) surround Schwann cell basement membranes (arrowheads) of both Remak
bundles and myelinated axon/Schwann cell units. Reprinted from Gardiner, N. J.
(2011). Integrins and the extracellular matrix: Key mediators of development and regener-
ation of the sensory nervous system. Developmental Neurobiology, 71, 1054–1072, with
permission from John Wiley and Sons © 2011.

Sensory afferents are capable of transducing a wide variety of modalities,

such as touch, temperature, pain, and itch, and transmit this information
to the CNS. They are highly diverse in somal size, axon diameter, and neu-
rochemistry. Schwann cells are the principal glial cell of the PNS and play a
58 N.J. Gardiner and O.J. Freeman

central role in the homeostasis of the endoneurial environment (Mirsky

et al., 2002). Axons in the peripheral nerve and spinal roots are tightly asso-
ciated with myelinating or nonmyelinating Schwann cells and enclosed
within discrete basement membranes (Fig. 1).
Both Schwann cells and sensory neurons arise from a migratory popula-
tion of neural crest cells (NCCs) derived from the dorsal surface of the neural
tube (Harris & Erickson, 2007). These NCCs migrate ventrally to regions
proximal to the neural tube and there differentiate into the DRG and cells
of the PNS (Bronner-Fraser, Stern, & Fraser, 1991; Sanes, 1983). The two
main intermediate cells involved in the generation of Schwann cells from
NCCs are the Schwann cell precursor, typically found in rat nerves at
embryonic (E) day 14/15 (mouse: E12/13), and the immature Schwann cell,
present from E17 (mouse E15; Jessen & Mirsky, 1997, 2005). These associ-
ate with developing axons to form a cooperative relationship with each cell
promoting survival and differentiation of the other (Jessen & Mirsky, 1997).
Immature Schwann cells integrate signals derived from the axon and the
basement membrane to differentiate along a myelinating or nonmyelinating
fate. Axonal-derived β1 neuregulin (NRG1) binds to the glial Erbb3
receptor to regulate development of Schwann cells. Assignment of the
myelinating/nonmyelinating Schwann cell–axon phenotype is completed
within the early postnatal period when Schwann cells send cytoplasmic
processes into axon bundles, a process known as radial sorting (Chen &
Strickland, 2003; Jessen & Mirsky, 1997; Mirsky et al., 2002). Large-
diameter axons produce high levels of NRG1 type III, which induces
Schwann cell precursors to differentiate into myelinating Schwann cells,
while small-diameter axons produce lower levels and associated Schwann
cell precursors therefore differentiate into nonmyelinating Schwann cells
(Taveggia et al., 2005).
There are two distinct periods of sensory neuron generation, starting
around E10–13 in mouse and E9–14 in rat (Fig. 2). Large-diameter TrkC-
positive sensory neurons fated to become proprioceptors or low-threshold
mechanoreceptors are formed first, followed by a second period in which
small-diameter TrkA-expressing neurons are generated (Lawson & Biscoe,
1979; Marmigere & Ernfors, 2007; Marmigere et al., 2006). The factors
underlying the initial differentiation of sensory neurons into subpopulations
are coupled to the discrete spatial and temporal expression of extracellular
matrix (ECM) and neuronal integrins, a family of ECM receptors
(Gardiner, 2011; Guan, Puthenveedu, & Condic, 2003). The brain-specific
homeobox/POU domain protein 3A gene (Brn3) and Neurogenin 1 and 2
Can Diabetic Neuropathy Be Modeled In Vitro? 59

E9.5
1st wave 2nd wave 3rd wave
Premigratory
Multipotent

Sox10+

Sensory
biased Multipotent
Sox10+ Krox20+
Ngn2+ Sox10+ Sox10+
Migratory

SN committed
+
Sox10
+
Ngn2
+
Foxs1

Multipotent Glia
SN committed
Dorsoventral axis

−
Sox10
Postmigratory

+
Foxs1
+ Sox10 Glia?
SN committed?
Brn3a+ +
Sox10
+
Ngn1 SN committed
+
Brn3a −
Sox10
+
Ngn1
+
Brn3a
+
Foxs1

-E10.5–E11.5 Runx3
+
Runx1
+
+ +
TrkC TrkA

-E14.5 -P0–P30
Runx3
downregulation
+ + +
TrkB TrkB TrkC
+ + +
TrkC Ret Runx3 + Ret
+
TrkA −
Ret
+/– TrkA
+
Runx1
+ +
TrkB Ret
Peptidergic Nonpeptidergic
Mechanoreceptors Proprioceptors neurons neurons
No or low RUNX3 RUNX3 No RUNX1 RUNX1
Fig. 2 Genetic control of development of the somatosensory nervous system. In the
first wave of neurogenesis, SOX10+ cells migrate and begin to express neurogenin 2
(Ngn2). Ngn2+ cells subsequently commit to a sensory neuronal (SN) fate and express
forkhead transcription factor (Foxs1+) during migration. These postmigratory neurons
then begin to express Brn3a and form large sensory neurons (TrkC+ and Runx3+) at
an early developmental stage. In contrast, cells with little or no Ngn2 remain uncommit-
ted throughout migration. Postmigratory cells in the DRG then start to express Brn3a,
Ngn1, and Foxs1. They either produce TrkC+ or TrkA+ populations of sensory neurons by
(Continued)
60 N.J. Gardiner and O.J. Freeman

are important factors in controlling neurogenesis (Lallemend & Ernfors, 2012;

Marmigere & Ernfors, 2007; Marmigere et al., 2006). The transcription factor
Runx1 is expressed by TrkA-positive, but not TrkC-positive neurons at early
embryonic stages (Fig. 2; Chen et al., 2006; Kramer et al., 2006; Marmigere
et al., 2006). Sensory neurons must make connections with their peripheral
target tissues, the source of neurotrophins, to survive (Silos-Santiago et al.,
1995). During the early postnatal period, around half of the small-diameter
neurons downregulate Runx1 and retain TrkA (Molliver, Radeke,
Feinstein, & Snider, 1995; Molliver & Snider, 1997; Molliver et al., 1997).
The remaining neurons retain Runx1, lose TrkA, and begin to express
Ret, the transmembrane signaling component of the receptor for glial cell
line-derived neurotrophic factor (GDNF) family members (Bennett,
Averill, Clary, Priestley, & McMahon, 1996; Molliver et al., 1997). Large-
diameter trkC-positive neurons, which constitute approximately 30% of
the sensory neuron population in the adult DRG, have large-diameter mye-
linated axons (Aβ fibers) that transmit low-threshold tactile and proprioceptive
information to the CNS and respond to neurotrophin (NT)-3. Small- to
medium-diameter sensory neurons have either thinly myelinated or unmy-
elinated axons (Aδ and C-fibers), mechanosensing, thermal and/or nocicep-
tive functions and are divided into either trkA-positive, peptidergic neurons
that respond to NGF or nonpeptidergic, Ret receptor-positive neurons that
respond to the GDNF family of neurotrophic factors (Averill, McMahon,
Clary, Reichardt, & Priestley, 1995; Bennett et al., 1996; Karchewski,
Kim, Johnston, McKnight, & Verge, 1999; McMahon, Armanini, Ling, &
Phillips, 1994; Wright & Snider, 1995).

Fig. 2—Cont'd expressing Runx3 or Runx1, respectively. A third wave of neurogenesis

arises from cells expressing Sox10 and Krox20 which contribute mainly to the
RUNX1/TrkA+ population of neurons and to glia. Cells from the first wave that main-
tain Runx3 expression maintain TrkC expression and become proprioceptors, whereas
cells that lose or reduce Runx3 expression may express TrkB and TrkC, TrkB alone,
RET alone, or RET and TrkB to become mechanoreceptive neurons. The small neurons
which maintain Runx1 expression become nonpeptidergic small-diameter neurons
(Ret+/TrkA
/Runx1+ cells), while downregulation of Runx1 fates neurons toward
an NGF-responsive peptidergic phenotype (TrkA+/Ret+/
/Runx1
). E, embryonic day;
P postnatal day. Reprinted from Marmigere, F., & Ernfors, P. (2007). Specification and
connectivity of neuronal subtypes in the sensory lineage. Nature Reviews Neuroscience,
8, 114–127, with permission from Macmillan Publishers Ltd © 2007.
Can Diabetic Neuropathy Be Modeled In Vitro? 61

It has long been acknowledged that there is overlap between phenotypic

markers and neurotrophic factor responsiveness. Categorizing adult sensory
neurons into three populations (Fig. 1) based on their neurochemistry
(expression of specific ion channels neurotransmitters and neurotrophin
receptors) or cytoskeletal components (Lawson, 1995; Lawson, Perry,
Prabhakar, & McCarthy, 1993; McMahon et al., 1994), while convenient,
is simplistic. Recent advances in genomic and single-cell sorting techniques
have enabled a much more thorough expression profiling of rodent
somatosensory neurons (Thakur et al., 2014; Usoskin et al., 2015).
A comprehensive unbiased transcriptome analysis of mouse sensory neurons
using single-cell RNA-seq (Usoskin et al., 2015) revealed not three, but
eleven distinct subpopulations: three low-threshold mechanoreceptive,
two proprioceptive, and six principal types of thermosensitive, itch-
sensitive, low-threshold mechanosensitive, and nociceptive neurons
(Usoskin et al., 2015). This highlights the diversity and complexity of adult
sensory neurons.
Large-diameter axons are ensheathed by a specialized plasma membrane
of the myelinating Schwann cells that wraps in concentric rings and com-
pacts around the axon. Small-diameter unmyelinated axons are sorted into
Remak bundles, which can contain over 20 axons isolated from each other
by processes of a nonmyelinating Schwann cell and enclosed within a base-
ment membrane (Murinson & Griffin, 2004; Murinson, Hoffman,
Banihashemi, Meyer, & Griffin, 2005). Schwann cells are important for
the proper formation of nodes of Ranvier (Salzer, Brophy, & Peles,
2008) and localization of ion channels within the node (Feinberg et al.,
2010). Schwann cells are not purely structural however, and they contribute
toward the trophic support of axons. For example, signaling from axonal
neuregulin-1 to Schwann cells (Chen et al., 2003) and Schwann cell to axon
transfer of ciliary-derived neurotrophic factor (Calcutt, Muir, Powell, &
Mizisin, 1992) is critical for healthy axonal function. Schwann cells regulate
myelin sheath parameters (Michailov et al., 2004; Taveggia et al., 2005) and
axon cytoskeletal components (Kirkpatrick & Brady, 1994). Schwann cells
play critical roles in mediating successful axon regeneration. After a periph-
eral nerve injury, dedifferentiated Schwann cells remove distal myelin and
axon debris, form bands of Büngner that provide ECM-rich pathways for
regenerating axons, neurotrophic support, and then remyelinate regenerated
axons (Allodi, Udina, & Navarro, 2012). There is also a developing literature
describing the role of Schwann cells in the metabolic support of peripheral
62 N.J. Gardiner and O.J. Freeman

axons. For example, it has been demonstrated that direct impairment of

Schwann cell mitochondrial function leads to neuropathy in mice (Viader
et al., 2011, 2013). In addition, Schwann cells have the ability to transfer
ribosomes and mRNA to axons (Court, Hendriks, MacGillavry,
Alvarez, & van Minnen, 2008), implying that Schwann cells and axons have
extensive functional coupling. The PNS is closely associated with a wide
range of other cell and tissue types: keratinocytes, immune cells, and endo-
thelial cells of blood vessels to name but a few. PNS structure and function
also depends upon association with a complex architecture of ECM proteins,
modification of which in disease can all impact upon neuronal phenotype
and nerve function.

4. CAN WE MODEL DIABETIC NEUROPATHY IN VITRO?

Tissue culture paradigms have long been used alongside animal models
of disease or nerve injury to study physiological and pathological mecha-
nisms underlying neuronal phenotype. The use of cell culture significantly
reduces the legitimate ethical problems associated with using in vivo models
of nerve injury or disease. Russell and Burch’s ethical framework for the
humane use of animals in scientific research (reviewed in Tannenbaum &
Bennett, 2015) describes “The Three R’s” (Replacement, Reduction,
and Refinement). Culture techniques using cells or tissues derived from ani-
mals are regarded as “relative replacements” and so offer a valuable ethical
advantage.
Tissue culture allows researchers the exquisite advantage of a carefully
controlled extracellular environment in which to examine phenotypic
responses of neurons/glia to exogenous stimuli and to elucidate intracellular
mechanisms underlying neurite outgrowth, bioenergetics, and cell death.
But can one realistically expect to model such a complex, multifactorial dis-
order as diabetic neuropathy in a dish? Is simply adding high concentrations
of glucose and/or limiting the extracellular insulin concentration of neurons
good enough? Is it best to use homogenous well-characterized cell lines, dis-
sociate primary cultures of neurons and glia, or use tissue explants which pre-
serve cellular diversity, interactions, and overall tissue structure? Should we
use embryonic or adult tissue? Should we use tissue from control animals or
tissue from animals with experimental diabetes? What should our endpoint
measures be? These questions represent a significant challenge for the
in vitro biologist.
Can Diabetic Neuropathy Be Modeled In Vitro? 63

4.1 Choice of Cells

One must first decide whether to utilize primary tissue culture or an immor-
talized cell line and how to maintain them under optimized conditions that
best replicate the in vivo environment.

4.1.1 Immortalized Cell Lines

Tumor-derived cell lines including SH-SY5Y, NTERA-2, and particularly
PC12 cells, a line derived from a pheochromocytoma of the rat adrenal
medulla, have been used extensively in basic neurophysiological research.
These cell lines grow continuously as undifferentiated cells but can be
stimulated with compounds such as retinoic acid, phorbol esters, cAMP,
or NGF to differentiate into neuronal-like cells that develop long pro-
cesses expressing neuronal markers such as βIII-tubulin, synaptophysin,
microtubule-associated protein-2 (MAP2), and NeuN. Immortalized cell
lines have also been generated from sensory neurons of the DRG. For exam-
ple, the 50B11 rat embryonic DRG cell line can be differentiated by
forskolin to extend processes that express nociception-specific receptors
(Chen, Mi, Haughey, Oz, & Hoke, 2007) such as trkA or GFRα1/α2,
and respond to both NGF and GDNF by extending these processes
(Bhattacherjee, Liao, & Smith, 2014). Since cells are generated from
DRG isolated from E14.5 rats prior to the in vivo influence of Runx1
(Fig. 2), this coexpression of diverse neurotrophin receptors is not surprising
and 50B11 cells most accurately represent immature sensory neurons.
Schwann cell lines have also been established from Schwannomas, by trans-
fection of Schwann cells with oncogenes or through spontaneous immortal-
ization. ITMS32 cells from adult ICR mice show distinct proliferative
Schwann cell phenotypes but do not myelinate, whereas IFRS1 cells can
myelinate sensory neurons in coculture models (Sango, Yanagisawa,
Takaku, Kawakami, & Watabe, 2011).
Immortalized cell lines are useful for electrophysiological, molecular,
and biochemical studies and for high-throughput screening of drugs, which
requires a large number of cells. They have distinct advantages over pri-
mary cultures, notably their ability to proliferate and provide an unlimited
supply of a homogeneous population of cells and their relative ease of
transfection. There is also no need to cull animals and dissect each culture,
which represents considerable time saving and ethical advantages.
However, since they do not replicate the heterogeneity of adult neuron
phenotypes, caution must always be taken when interpreting data, and
64 N.J. Gardiner and O.J. Freeman

findings should be validated in primary cell culture before being extrapo-

lated to the in vivo situation.

4.1.2 Primary Tissue Culture

Primary tissue culture includes the isolation and maintenance of either
whole tissue organotypic explants/slices or the dissociation of tissue by
mechanical and/or enzymatic means to produce a cell monolayer. If cells
of the primary culture are proliferative, as are Schwann cells, they can be
maintained and passaged as a secondary culture or cell line (Kaewkhaw,
Scutt, & Haycock, 2012). Cells can then be maintained in optimized con-
ditions to mimic the in vivo nerve environment. The use of acutely disso-
ciated primary neuron and glial cultures expanded considerably following
identification and subsequent widespread availability of specific neuro-
trophic factors and extracellular components (eg, laminin) for use as a sub-
stratum to support survival and enable growth of distinct neuron populations
(Lindsay, 1988). While preparation and culture of primary neurons is
undoubtedly challenging, DRG, sympathetic ganglia, peripheral nerve, hip-
pocampus, and cortex can all be extracted from chicken, mouse or rat
embryos, neonates, or adult animals. Excellent protocols exist in the litera-
ture describing methodologies for successfully culturing neuronal and
Schwann cells. Dissociated adult rat and mouse sensory neurons have been
widely utilized in a range of molecular, physiological, and live-cell imaging
studies to investigate mechanisms of growth cone dynamics and neurite out-
growth, neurotrophin responsiveness and signal transduction pathways,
programmed cell death, axonal transport, electrophysiology, calcium signal-
ing, and mitochondrial function.
While embryonic and neonatal neurons require serum or exogenous
growth factors to survive, adult mammalian neurons can be maintained
for long periods in the absence of trophic support (Gavazzi, Kumar,
McMahon, & Cohen, 1999; Lindsay, 1988). Adult sensory neuron cultures
reflect the broad diversity of neuronal populations and show predicted neu-
ritogenic population responses to exogenous growth factors when plated on
laminin (Fig. 3; Gardiner et al., 2007). In contrast, fewer neurons respond to
neurotrophin stimulation when plated on fibronectin and the response is sig-
nificantly less than expected, given the distribution of neurotrophin recep-
tors in DRG (Bennett et al., 1996; Gardiner et al., 2005; Gavazzi et al., 1999;
McMahon et al., 1994). This difference perhaps reflects the normal associ-
ation of axons with ECM components in vivo. In peripheral nerve, laminin
is localized to the internal surfaces of the Schwann cell basement membranes
Can Diabetic Neuropathy Be Modeled In Vitro? 65

Fig. 3 Neurotrophin-stimulated neurite outgrowth from adult rat dissociated neurons

plated on laminin (LM) and fibronectin (FN). Dissociated adult rat sensory neurons were
plated on FN or LM treated with GDNF (50 ng/mL, B, F), NGF (10 ng/mL, C, G), and NT-3
(50 ng/mL, D, H), or untreated (A, E). For all parameters of growth assessed, LM was sig-
nificantly better than FN at supporting neurite outgrowth. Fewer neurons extended
neurites in response to neurotrophins when plated on FN (I, mean % neurite-bearing
cells  S.D.). Those that were extended were significantly shorter than those grown
on LM (J, mean length of longest neurite  S.D.) and less dense (total neurite density
was quantified by a series of concentric circles overlaid onto an image of a neuron, the
number of times neurites crossed each circle was measured; K, mean number of
crosspoints per cell  S.D., n ¼ 4 independent cultures). Reprinted from Gardiner, N. J.,
Moffatt, S., Fernyhough, P., Humphries, M. J., Streuli, C. H., & Tomlinson, D. R. (2007).
Preconditioning injury-induced neurite outgrowth of adult rat sensory neurons on fibronec-
tin is mediated by mobilisation of axonal α5 integrin. Molecular and Cellular Neurosci-
ences, 35, 249–260, © 2007 with permission from Elsevier.

that ensheath axons. Fibronectin is more diffuse, being localized along exter-
nal surfaces of endoneurial tubes and the perineurium. This distinction high-
lights the need to optimize the environment of the culture systems to that of
the cell being studied.
Neurons do not naturally exist in simple two-dimensional monolayers,
but occupy a complex 3-dimensional space closely associated with multiple
cell types and ECM in an environment that is constantly adapting and
66 N.J. Gardiner and O.J. Freeman

changing. Axon terminals in vivo constantly undergo dynamic collateral

branching with terminal fields being remodeled according to local environ-
mental factors or activity. A primary cell monolayer is therefore a useful but
highly simplistic approach to modeling. The limitation of studying dissoci-
ated and purified neurons in isolation can be addressed using organotypic
cultures obtained from dorsal root or sympathetic ganglia to maintain tissue
structure, neuronal–glial interaction, and a three-dimensional environment.
For this approach, ganglia are removed from adult or embryonic animals, cut
into small pieces, with or without attached nerve stumps, and embedded in
collagen or Matrigel™ with defined medium, treated with exogenous fac-
tors and cultured for several days. Axons extend from the isolated tissue into
the surrounding matrix and Schwann cells proliferate and migrate radially
(Fig. 4). This system is not without its limitations, as lack of blood supply
and density of tissue mass can lead to hypoxia and cell death in the center
of the explant. It is also difficult to use these cultures to investigate specific
cellular effects other than measuring Schwann cell migration and axon
extension distances. Future development of self-assembled aggregates of
cells of the PNS could act as a bridge between traditional two-dimensional
PNS-derived cell monolayers and in vivo models. Bioengineering
approaches have already been taken with cells of the CNS, which can be
maintained in culture for several weeks to enable neural network maturation

Fig. 4 High glucose inhibits Schwann cell migration. Bright-field micrographs from
adult wild-type (WT) mouse DRG explants cultured for 8 days in media containing
NGF (20 ng/mL) with (A, B) 10 or 60 mM glucose (glc) or (C) 60 mM mannitol (man) show
evidence of reduced Schwann cell migration (demarcated with dashed line) in high glu-
cose. Reprinted from Gumy, L. F., Bampton, E. T., & Tolkovsky, A. M. (2008). Hyperglycaemia
inhibits Schwann cell proliferation and migration and restricts regeneration of axons and
Schwann cells from adult murine DRG. Molecular and Cellular Neurosciences, 37,
298–311, © 2008 with permission from Elsevier.
Can Diabetic Neuropathy Be Modeled In Vitro? 67

(Chwalek, Tang-Schomer, Omenetto, & Kaplan, 2015; Hopkins,

DeSimone, Chwalek, & Kaplan, 2015). Such cortical spheroids formed from
primary postnatal rat cortex contain neurons and glia networks that are elec-
trically active with excitatory and inhibitory synapses (Dingle et al., 2015).
Other disadvantages of primary neuronal culture approaches include the
low yield, due to neurons being postmitotic, and the labor-intensive nature
of collecting neurons. Purity of cultures can vary, especially of those derived
from adult animals, as ganglia consist of both sensory neuron cell bodies and
nonneuronal cells (Schwann cells, satellite glia, and fibroblasts). Further-
more, isolation of cell bodies by necessity involves nerve injury (axotomy)
so that cultured primary neurons switch from a homeostatic “housekeeping”
in vivo phenotype to an injury-response “regenerative” phenotype
(Smith & Skene, 1997). However, while these caveats must be accounted
for in experimental design and interpretation, the use of primary neurons
offers the distinct and valuable advantage that one can isolate DRG to com-
pare phenotypic changes in mature adult sensory neurons obtained from
control and diabetic rodents.

4.1.3 Induced Pluripotent Stem Cells

The continued failure of translation of drugs from the bench to the clinic
highlights the need for better model systems that predict clinical efficacy.
Interspecies differences between rodents commonly used as sources for pri-
mary cultures and human patients may be one underlying cause of this.
A better in vitro model of neuropathy may in the future be provided through
induced pluripotent stem cell (iPSC) technology. iPSCs are particularly use-
ful for the study of hereditary diseases as one can obtain a skin biopsy from
human patients, isolate fibroblasts, reprogram them into pluripotent cells via
the insertion of four genes (oct3/4, sox2, klf4, c-myc; Takahashi et al., 2007),
and then differentiate them into many different cell types. Published proto-
cols describe differentiation into sensory neurons (Chambers et al., 2012)
and Schwann cells (Liu et al., 2012). It is also possible to compare phenotypic
changes between cells from genetically defined individuals to gain insight
into pathogenic mechanisms. For example, iPSCs have been used to define
transcriptional and functional changes induced in human motor neurons by
mutant SOD1 (Kiskinis et al., 2014). While using iPSC-derived neurons/
glia to study disease is not without limitations (Sandoe & Eggan, 2013), this
technology has the potential to transform our future understanding of the
pathogenesis of diabetic neuropathy.
68 N.J. Gardiner and O.J. Freeman

4.2 Choice of Stimuli

Diabetic neuropathies, which include both focal mononeuropathies and
diffuse polyneuropathies, have a multifactorial etiology. Many patho-
genic mechanisms have been suggested including hyperglycemia, micro-
angiopathy and subsequent hypoxia, increases in polyol flux through the
aldose reductase pathway leading to sorbitol and fructose accumulation,
oxidative stress, NAD(P)-redox imbalances, mitochondrial dysfunction, cal-
cium dysregulation, dyslipidemia, glycation of intracellular and extracellular
proteins, and also reduced trophic support from insulin and other factors
(Fernyhough & Calcutt, 2010; Fernyhough et al., 2010; Freeman et al.,
2016; Obrosova, 2002, 2003; Zochodne, 2014). Intersection and synergy
between these multiple pathways leads to progressive pathology. Primary
cultures of peripheral neurons and glia have been widely used to investigate
and compare effects of hyperglycemia, dyslipidemia, or stressors such as loss
of insulin signaling and other trophic support.
The majority of studies to date that have used in vitro models of diabetic
neuropathy have investigated the effects of raised glucose on cells using con-
centrations ranging from 10 to 200 mM. Immediate considerations for such
a study should include whether to use cells derived only from control
rodents, thereby allowing assessment of acute pathogenic mechanisms, or
to also include cells from diabetic rodents that may have developed an altered
metabolic phenotype in response to prolonged hyperglycemia. Similarly,
since insulin has direct effects on sensory neurons (Huang et al., 2003;
Huang, Verkhratsky, & Fernyhough, 2005), it is important to consider
whether insulin, under its guise as a trophic factor, should be present in cul-
ture media and at what concentration. For example, cells derived from a
control adult rodent will have matured in an environment of 4–8 mM glu-
cose and fluctuating insulin, whereas those from a type 1 diabetic rodent may
have spent months adapting to an environment with little insulin and excess
glucose. This can make the choice of what constitutes control and experi-
mental conditions quite complex. It is worth noting that the vast majority of
published cell culture studies that used neurons but were unconcerned with
studying diabetes employed basal glucose concentrations (20–30 mM in
DMEM) that would be considered hyperglycemic by those studying diabe-
tes in vitro and in vivo. Glucose levels of 50 mM+ have been used in some
in vitro studies but are unlikely to be of pathophysiological relevance (see
below) and 20–50 mM glucose represents more realistic levels that occur
in the blood of poorly controlled diabetic rodents. Direct modeling of
human diabetes requires external glucose in the 7–20 mM range.
Can Diabetic Neuropathy Be Modeled In Vitro? 69

The value of in vitro techniques can be illustrated by studies that

investigated the hypothesized role of intracellular oxidative stress as a path-
ogenic mechanism downstream of hyperglycemia by application of pro-
oxidants such as hydrogen peroxide and diethyl maleate, which binds to
glutathione, thereby compromising the glutathione redox cycle (Purves
et al., 2001). Other potentially toxic products of hyperglycemia, including
lipid peroxidation products such as 4-hydroxynonenal (Zherebitskaya,
Akude, Smith, & Fernyhough, 2009) and reactive dicarbonyl intermediates,
including glyoxal, methylglyoxal, and glycolaldehyde (Radu, Dumitrescu,
Mustaciosu, & Radu, 2012), have also been directly applied in vitro. Hyper-
lipidemia has been modeled using oxidized LDLs (Vincent et al., 2009) or
long chain fatty acids (such as palmitate, linoleate, and oleate; Hinder et al.,
2014). Another less precise, but perhaps more pertinent, approach has
been to use serum or plasma from diabetic patients or rats and nondiabetic
controls as a direct challenge to neurons in vitro (Bierhaus et al., 2012;
Ristic, Srinivasan, Hall, Sima, & Wiley, 1998; Srinivasan, Stevens, Sheng,
Hall, & Wiley, 1998). The complexity of diabetes presents an enormous
challenge for the in vitro neurobiologist to select an appropriate stimulus
for the cultured cells to mimic the diabetic milieu and this is a significant
limitation to in vitro modeling of diabetes.

4.3 Choice of Output Measure

The cellular responses most amenable to study in vitro include cell
survival or death, proliferation and migration, and neurite initiation and
extension. Live-cell imaging and electrophysiological approaches may also
be applied.

4.3.1 Life or Death?

A simple output measure for neurodegeneration in vitro is to assess
cell death. Methods to measure death include the trypan blue exclusion
assay, the lactate dehydrogenase release assay, and the MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduc-
tion assay. Quantification of apoptosis-related events such as cleavage of
caspases and detection of DNA fragmentation by terminal deoxynucleotidyl
transferase dUTP nick end labeling can also be used. The response of neurons
to high glucose differs depending on their developmental stage (Fig. 2).
Studies using cultures of sensory neurons derived from E15 rat DRG
and already maintained in 20 mM+ glucose found that exposure to further
excess glucose induced apoptosis, oxidative stress, and cell death through
70 N.J. Gardiner and O.J. Freeman

a mitochondrial-dependent pathway (Russell et al., 2002; Vincent, Olzmann,

Brownlee, Sivitz, & Russell, 2004; Vincent, Russell, Low, & Feldman, 2004).
In contrast, studies using cultures of adult sensory neurons found no evidence
of glucose-induced apoptosis (Gumy, Bampton, & Tolkovsky, 2008; Huang
et al., 2003; Purves et al., 2001; Zherebitskaya et al., 2009). DRG explants or
isolated adult sensory neurons cultured in serum-free medium containing
NGF, and 10–60 mM glucose showed no evidence of hyperglycemia-
induced DNA fragmentation, cyt-c release from mitochondria, or activation
of caspase-3 (Gumy et al., 2008). Apoptosis of sympathetic neurons from
superior cervical ganglion (SCG) in response to high glucose has been
described in both embryonic (Russell & Feldman, 1999) and adult neurons
(Semra, Smith, & Lincoln, 2004), and decreased viability of adult celiac/
superior mesenteric ganglia (CG/SMG) neurons relative to SCG neurons
was observed at 100 mM glucose (Semra et al., 2004).
It has been suggested that a reason for these discrepancies is the develop-
mental difference in the neurotrophic requirements of embryonic and adult
DRG neurons. During early sensory neuron development all three Trk
receptors and p75 receptors are expressed and embryonic DRG neurons
depend on neurotrophins for survival. As sensory neurons mature, they
develop mechanisms to prevent apoptosis (Vogelbaum, Tong, & Rich,
1998). If neurons are harvested from embryos during the developmental
period of programmed cell death, they will require neurotropic support in
order to survive in culture (Snider & Silos-Santiago, 1996). The differential
apoptotic response of embryonic and adult cells to high glucose may there-
fore simply reflect developmental stage. Indeed, embryonic sensory neurons
cultured for several weeks with neurotrophic support will mature and then
do not show high glucose-induced apoptosis (Yu, Rouen, & Dobrowsky,
2008). Adult DRG explants cultured for 8 days in 60 mM glucose also
showed no evidence of Schwann cell apoptosis (Gumy et al., 2008), but con-
flicting evidence exists regarding the neonatal Schwann cell response
(Delaney, Russell, Cheng, & Feldman, 2001; Gumy et al., 2008). Since dra-
matic apoptosis does not occur in the sensory or autonomic ganglia of dia-
betic rodents (Cheng & Zochodne, 2003; Kamiya, Zhang, & Sima, 2006;
Kamiya, Zhangm, & Sima, 2005; Schmidt, 2001) and neuronal cell loss does
not appear to underlie the distal axon pathology, studying in vitro apoptotic
responses of neurons to high glucose is difficult to relate to clinical pathology.

4.3.2 Altered Bioenergetics and Oxidative Stress

Oxidative stress is a common phenomenon in many theories of the patho-
genesis of diabetic neuropathy (Obrosova, 2002). Early studies in cultured
Can Diabetic Neuropathy Be Modeled In Vitro? 71

endothelial cells identified formation of reactive oxygen species by increased

electron donation to the mitochondrial electron transport chain under
hyperglycemic conditions, and overload of oxidative defense mechanisms
was presented as a pivotal event linking glucotoxic mechanisms of pathogen-
esis of diabetic complications (Brownlee, 2001, 2005; Nishikawa et al.,
2000). Although support of such a unifying mechanism of diabetic
complications has waned, metabolic dysfunction and oxidative stress
are considered to be key contributors to the pathogenesis of diabetic
neuropathy (Chowdhury, Smith, & Fernyhough, 2013; Fernyhough,
2015; Fernyhough & Calcutt, 2010; Fernyhough & McGavock, 2014;
Obrosova, 2002). Intracellular calcium flux, mitochondrial dynamics, and
oxidative stress are all highly amenable to confocal imaging in vitro, using
cell-permeable fluorescent dyes. These dyes can provide real-time measure-
ment of fluctuations of intracellular Ca2+ concentration, mitochondrial
membrane potential, or increased reactive oxygen species in response to
stimuli such as high glucose, or to compare responses of cells obtained from
control and diabetic rats. For example, mitochondrial membrane polariza-
tion status in sensory neurons from age-matched control or diabetic rats was
assessed using tetramethyl rhodamine methyl ester (Zherebitskaya et al.,
2009) and rhodamine 123 (Huang et al., 2003) with reactive oxygen species
detected using MitoSOX™ red (Akude et al., 2011). Using these dyes, it has
been demonstrated that the mitochondrial inner membrane potential is
depolarized in adult sensory neurones cultured from STZ-diabetic rats
and that this could be prevented by systemic treatment with low doses of
insulin or NT-3 (Huang et al., 2003, 2005). Recently, these imaging studies
have been complemented by in vitro work using the Seahorse Biosciences
XF Analyzer to assess and compare the mitochondrial function of dissociated
sensory neurons obtained from control or STZ-diabetic rats (Chowdhury
et al., 2013). By measuring oxygen consumption rate in cultured cells in
response to specific inhibitors, one can examine various functions of the
electron transport chain (Akude et al., 2011; Chowdhury et al., 2013). These
studies provide excellent examples of how in vivo and in vitro analyses can
work in tandem. Many of the studies described above utilize primary cul-
tures of DRG neurons derived from animals with STZ diabetes and thus
benefit from using an in vivo model as the diabetes-stimulus, but utilize
in vitro methods to probe the specific mechanistic changes.
Evidence of bioenergetic dysfunction in diabetic neuropathy has been
expanded with the use of “-omic” technologies, in which in vitro studies
play a central role. Stable isotope labeling of amino acids in cell culture is
an in vitro method of proteomic analysis which has been performed on
72 N.J. Gardiner and O.J. Freeman

mitochondrial extracts of DRG neurons from STZ-diabetic rats (Akude

et al., 2011) and control neonatal Schwann cells cultured in high glucose
(Zhang, Yu, et al., 2010). In neurons derived from the DRG of
22-week-STZ-diabetic rats and healthy controls, there was a significant
downregulation of proteins related to oxidative phosphorylation and the tri-
carboxylic acid (TCA) cycle in diabetes (Akude et al., 2011). In contrast, in
Schwann cells derived from the sciatic nerve of neonatal rats and cultured in
high glucose for 2, 6, or 16 days, there was an upregulation of oxidative
phosphorylation and the TCA cycle under hyperglycemia at all time points
(Zhang, Yu, et al., 2010). Use of purified cell culture allows detailed exam-
ination of differential cellular responses to stimuli, rather than a global
mixed-cell tissue response, and therefore enables cell-specific mechanisms,
theories of pathogenesis, and therapeutic targets to be identified.

4.3.3 Neuronal Hyperexcitability

Altered expression of ion channels and increased activity of sensory afferents
are likely to contribute to neuropathic pain and nerve dysfunction in diabe-
tes. For example, dysregulated expression of genes for sodium channels
Na(v)1.3, Na(v)1.6, Na(v)1.8, and Na(v)1.9 has been described in the rat
DRG in experimental diabetes (Craner, Klein, Renganathan, Black, &
Waxman, 2002). Patch-clamp recordings and electrophysiological studies
have been performed on the whole excised DRG, nerve, or dissociated neu-
rons in vitro. Current-clamp recordings from cultured mouse DRG neurons
have characterized the effects of exogenous methylglyoxal on sensory neu-
ron excitability (Bierhaus et al., 2012). In another recent study, STZ-
induced diabetes was associated with increased TTX-S and TTX-R current
densities, while current densities of both transient and sustained components
of potassium current in sensory neurons in L4/5 DRG were decreased but
could be ameliorated by treating rats with docosahexaenoic acid (Heng, Qi,
Yang, & Xu, 2015). These studies and others highlight the increase of sen-
sory neuron excitability in diabetes. Another in vitro modeling approach
compared responses of isolated skin–nerve preparations from control or dia-
betic rats and found an increase in low-frequent ongoing activity of C-fibers
from diabetic animals compared to control rats (Fuchs, Birklein, Reeh, &
Sauer, 2010). After baseline recordings, the skin was superfused with media
aerated with 97.5% N2/2.5% CO2 instead of normal carbogen for 30 min to
model hypoxia. The response to hypoxia was significantly increased in fibers
recorded from diabetic animals compared to control rats in either 2.5 or
25 mM glucose, suggesting that fibers from diabetic rats were more sensitive
Can Diabetic Neuropathy Be Modeled In Vitro? 73

to hypoxic acidosis (Fuchs et al., 2010). Such detailed electrophysiological

studies can only be performed in vitro due to the ability to control the extra-
cellular environment.

4.3.4 Axonal Degeneration and Regeneration

Distal axon retraction or pruning and axonal dystrophy are both hallmark
features of clinical and rodent models of diabetic neuropathy (Kennedy &
Zochodne, 2000, 2005; Polydefkis et al., 2004; Yasuda et al., 2003). Sensory
and autonomic axons degenerate before motor neurons in both clinical and
experimental diabetes (Wilson & Wright, 2014; Zochodne, 2014) and neu-
rodegeneration is first evident in the long sensory axons, notably those
innervating feet and hands. Reinnervation of distal target tissues can either
occur by regeneration of damaged axons or by collateral branching of
undamaged axons. However, these processes are impaired in diabetes,
and there is a significant reduction in the number of regenerating axons
observed in clinical nerve biopsies indicating a reduction in intrinsic growth
capacity of sensory neurons in diabetes (Bradley et al., 1995) and/or devel-
opment of an unsupportive extracellular environment in diabetes. STZ-
induced diabetic rats show impaired axonal regeneration following focal
nerve injury, and the ability of nerves to regenerate is inversely proportional
to the duration of diabetes (Bisby, 1980; Ekstrom & Tomlinson, 1989;
Kennedy & Zochodne, 2000). This all culminates in axonal degeneration
occurring at a faster rate than regeneration, and permanent loss of end-target
innervation. The need to protect against diabetes-associated axon degener-
ation and promote regeneration are therefore major clinical needs.
A cell culture approach to studying the mechanisms underlying impaired
axon regeneration in diabetes is relatively easy to employ as neurons can be
stimulated to extend neurites by different agonists/antagonists/stressors and
neurite initiation, elongation, and density measures are amenable for quan-
tification in short-term cultures (see Fig. 3). Given the diversity in axonal
morphology (particularly in primary cell culture), it is imperative that the
researcher conducts analysis in a blinded and randomized fashion to remove
bias. One can evaluate changes in neurite morphology, and efficacy of ther-
apeutics, before taking studies forward to in vivo studies. Distal axon degen-
eration is more problematic to model in vitro, although recent advances in
compartmentalized microfluidic systems mean that it is now easier to sepa-
rate axon terminals from the cell body and these systems have been used in
models of chemotherapy-associated neuropathy to study axonal degenera-
tion in similar “die-back” neuropathies (see Fig. 5; Yang, Siddique,
74 N.J. Gardiner and O.J. Freeman

Fig. 5 Paclitaxel-induced axonal degeneration. DRG neurons were added to micro-

fluidic devices and cultured for 5–7 days, for axons to grow through the channels into
the other chamber. Paclitaxel (25 ng/mL) was then applied to either the soma or neurite
chamber. Axonal degeneration was detected after local axonal application (A ¼ before
paclitaxel; B ¼ after paclitaxel) but not when paclitaxel was applied to the cell body com-
partment (C ¼ before paclitaxel; D ¼ after paclitaxel). Reprinted from Yang, H., Siddique,
R., Hosmane, S., Thakor, N., & Ho €ke, A. (2009). Compartmentalized microfluidic culture plat-
form to study mechanism of paclitaxel-induced axonal degeneration. Experimental Neu-
rology, 218, 124–128, © 2009 with permission from Elsevier.

Hosmane, Thakor, & Hoke, 2009). We envisage that this system will soon
be employed to generate a “diabetes-induced” model of distal axonopathy
(see Section 4.4).
Immortalized cell lines, especially PC12 cells, have long been utilized in
regeneration studies and are useful tools to study neurite outgrowth since cells
differentiated with NGF extend neurite-like processes that can be easily mea-
sured. While neurite outgrowth appears reduced in cells cultured in high
glucose conditions, apoptosis is also often described (Koshimura, Tanaka,
Murakami, & Kato, 2002; Lelkes, Unsworth, & Lelkes, 2001; Sharifi,
Mousavi, Farhadi, & Larijani, 2007), meaning that caution should be exerted
in translating results to the in vivo situation. The ease of use of immortalized
Can Diabetic Neuropathy Be Modeled In Vitro? 75

cell lines has enabled the development of rapid high-throughput assays, pri-
marily exploited by the pharmaceutical industry (Radio, Breier, Shafer, &
Mundy, 2008), to quantify the effects of compounds on neuritogenesis.
High-throughput assay systems treat cells in multiwell plates with com-
pounds, then automatically capture images using fluorescent or bright-field
microscopy, and quantify neurite extension. This unbiased automated
approach to assess neurite outgrowth is useful for rapid drug screening and
toxicological assessment and may be used to identify compounds with regen-
erative properties using novel compounds or existing drug libraries
(Vincent & Feldman, 2008). However, using immortalized cell lines or
embryonic neurons for the study of regenerative pathways is simplistic,
due to the heterogeneous nature of adult neurons, as discussed above. There-
fore it is preferable to use dissociated adult neurons, with their mature com-
plement of neurotrophic factor receptors, for neurite outgrowth assays.
Following an initial quiescent period, adult sensory neurons in culture
exhibit two distinct forms of neurite outgrowth: an early arborizing form that
can be enhanced by addition of neurotrophic factors such as NGF, NT-3, or
GDNF (Fig. 3), and a later elongating regenerative growth mode which
depends on novel gene transcription (Diamond, Foerster, Holmes, &
Coughlin, 1992; Gavazzi et al., 1999; Kimpinski, Campenot, & Mearow,
1997; Smith & Skene, 1997). The regenerative responses of cells exposed
directly to high glucose and diabetes-associated cell stressors have been assessed
using a number of culture paradigms, including comparisons between cells
derived from either control or diabetic rodents. Indeed, reduced intrinsic
regenerative capacity of neurons may be most evident using tissue derived
from diabetic rats. Work from the Fernyhough laboratory clearly illustrates
a difference in susceptibility of neurons from STZ-diabetic rats to metabolic
stress (Akude et al., 2011; Akude, Zherebitskaya, Roy Chowdhury,
Girling, & Fernyhough, 2010; Zherebitskaya et al., 2009, 2012).
While treatment of dissociated sensory neurons from control or diabetic
rats with elevated glucose does not impair neurite outgrowth per se, neurites
of neurons from diabetic rats displayed abnormal swelling and beading along
the axon that immunostained for 4-HNE adducts indicative of lipid perox-
idation (Zherebitskaya et al., 2009). This suggests that high glucose induces
oxidative stress and dysmorphology in axons of adult sensory neurons, but
only those isolated from diabetic rats. Direct exposure of control adult rat
sensory neurons to 4-HNE at concentrations as low as 3 μM caused a sig-
nificant reduction in neurite outgrowth and similar changes to axonal mor-
phology (Akude et al., 2010). Similar studies have been performed using
dissociated CG/SMG neurons, which have a slower regenerative response
76 N.J. Gardiner and O.J. Freeman

in vitro than SCG neurons and were more sensitive to high glucose than
SCG neurons in that exposure to high glucose caused a significant decrease
in neurite-bearing cells from CG/SMG but not SCG (Semra et al., 2004).
While hyperglycemia can impact neurite initiation and morphology, neurite
length is not altered by up to 45 mM glucose in adult mouse DRG explants,
although supraphysiological glucose levels (60 mM) had some effect (Gumy
et al., 2008). There was also no effect on the density of growth cones at any
glucose level tested, indicating that exposure to supraphysiological glucose
did not reduce neurite initiation, but impaired rate of neurite extension.
These experiments also revealed a striking inhibition of Schwann cell migra-
tion from explants in high glucose (Fig. 4) that was confirmed using a
Matrigel™ drop assay of isolated neonatal Schwann cells in 30 mM glucose
(Gumy et al., 2008). The proliferation of Schwann cells thus appears partic-
ularly sensitive to elevated glucose concentrations, which could impact on
regenerative capacity in diabetic neuropathy.
Another key aspect to the regenerative deficit in diabetic neuropathy is an
unsupportive extracellular environment, both in terms of neurotrophic support
from other cells and the ECM (Bradley, King, Muddle, & Thomas, 2000;
Calcutt, Jolivalt, & Fernyhough, 2008; Gardiner, 2011; Kennedy &
Zochodne, 2005; King, Llewelyn, Thomas, Gilbey, & Watkins, 1989;
Yasuda et al., 2003). The ECM of peripheral nerve plays a key role in supporting
axonal regeneration, and attachment of neurons to ECM is an important phase
for successful neurite outgrowth in culture (Duran-Jimenez et al., 2009). Dis-
sociated sensory neurons from adult diabetic mice show reduced adhesion to
laminin, type I and IV collagens, and fibronectin (Sango, Horie, Okamura,
Inoue, & Takenaka, 1995), possibly through altered expression of ECM recep-
tors. ECM proteins are glycated in the peripheral nerve in diabetes (Duran-
Jimenez et al., 2009), and exogenous glycation of purified ECM proteins using
glucose, glyceraldehyde, or methylglyoxal inhibited neurite outgrowth from
neuroblastoma cells (Federoff, Lawrence, & Brownlee, 1993), dissociated neo-
natal (Luo, King, Lewin, & Thomas, 2002) and adult sensory neurons
(Duran-Jimenez et al., 2009; Radu et al., 2012), and adult DRG explants
(Ozturk, Sekeroglu, Erdogan, & Ozturk, 2006). Reducing AGE formation
in the peripheral nerve and target tissues may thereby diminish the inhibitory
environment and enable reinnervation of target tissue.

4.4 Looking Forward to a Better In Vitro Model of Diabetic

Neuropathy
The ultimate aim of all in vitro models of neuropathy should be to create a
system that best mimics the in vivo microenvironment but still affords the
Can Diabetic Neuropathy Be Modeled In Vitro? 77

advantage of exquisite control of the cellular environment. Peripheral neu-

rons in vivo extend long axons whose terminals may be over a meter away
from their soma so that distinct regions of a neuron may face different
metabolic demands and extracellular environments. Conventional mono-
layer or explant cultures cannot effectively model this and do not allow
the researcher to control and manipulate different microenvironments.
A compartmentalized approach to neuronal cell culture was first developed
by Robert Campenot in 1977. His system consisted of three chambers sep-
arated by a fluid-impermeable barrier so that the composition of media in
each chamber could be individually manipulated. “Campenot Chambers”
were first used to study the subcellular actions of NGF, and it was found that
neurites from rat sympathetic neurons would preferentially grow into cham-
bers containing NGF (Campenot, 1977). This system has now been widely
used to study axonal transport and regeneration as well as to isolate axons
from cell bodies for molecular and biochemical analysis. A disadvantage of
the Campenot chamber is that creation of the barrier is tricky and leakage
between chambers is common. Consequently, this pioneering technique
has been developed and modified over the past decades and many elegant
devices now exist in which isolated neurons are introduced into one com-
partment and axons grow into another. One example is the recent develop-
ment of microfluidic platforms that consist of one or more chambers
separated by thin “microgrooves.” Microfluidic devices can be bonded to
glass slides to create a three-dimensional fluid-filled microenvironment.
Neuron cell bodies are plated in one chamber and treated with growth factors
so that neurites extend through the microgrooves to the other chamber.
Since cell somata cannot pass through the microgrooves and hydrostatic pres-
sure is present between chambers, this system can spatially and fluidically iso-
late cell bodies from axons. One can thus begin to probe the specific effects of
exogenous chemicals/factors on the axon or soma compartments. Studies
have used these chambers with live-cell imaging methodologies to study axo-
nal transport of NGF (Zhang, Osakada, et al., 2010), calcium imaging
(Tsantoulas et al., 2013), and genesis and conduction of action potentials
in sensory axons using patch-clamp methodology (Tsantoulas et al., 2013).
Compartmentalized culture systems have been used with models of
HIV- and chemotherapy-associated neuropathy to probe whether distal
axon degeneration is due to local axonal damage or originates from the neu-
ronal cell body (Melli, Jack, Lambrinos, Ringkamp, & Hoke, 2006; Melli,
Keswani, Fischer, Chen, & Hoke, 2006; Silva, Wang, Wang, Ravula, &
Glass, 2006; Yang et al., 2009). The potential of such systems will be of obvi-
ous interest to researchers modeling diabetic neuropathy in vitro,
78 N.J. Gardiner and O.J. Freeman

particularly if combined with coculture methods. It has long been possible to

drive myelination of dissociated sensory neurons by their long-term
coculture with Schwann cells and supplementation with ascorbic acid
(Eldridge, Bunge, Bunge, & Wood, 1987). In such a system, Schwann cells
and neurites can interact and mature, as illustrated by the formation of nodes
of Ranvier (Zhang et al., 2012). A microfluidic-based coculture system
could be used to investigate the interactions of Schwann cells and other per-
tinent cells such as keratinocytes in modulating axonal function. Combining
such techniques with real-time imaging to monitor biochemical, electro-
physiological, and morphological changes (for example, see Fig. 6;
Tsantoulas et al., 2013) may move us closer to modeling a “nerve in a dish.”

Fig. 6 Sensory neuron–keratinocyte coculture using a microfluidic system. (A) Neonatal

rat keratinocytes immunostained with the keratinocyte marker cytokeratin 5 (CK5) and
differentiation marker cytokeratin 10 (CK10). (B) Dissociated neonatal rat sensory neu-
rons (immunostained with β3-tubulin+, seeded into left DRG compartment) extend
axons into the keratinocytes (CK5+)-containing compartment. (D) Coculture with
keratinocytes did not alter regeneration of sensory axons—with no evident differences
in DiO tracer uptake between cultures grown with or without keratinocytes. Micro-
fluidics can be used to study a wide variety of phenotypes, including regeneration, axo-
nal transport, and activity. For example, (C) shows Ca2+ imaging of the soma in response
to capsaicin stimulation of axonal compartment  keratinocytes. Reprinted from
Tsantoulas, C., Farmer, C., Machado, P., Baba, K., McMahon, S. B., & Raouf, R. (2013). Probing
functional properties of nociceptive axons using a microfluidic culture system. PLoS One, 8,
e80722, © 2013, an open-access article distributed under the terms of the Creative Com-
mons Attribution License.
Can Diabetic Neuropathy Be Modeled In Vitro? 79

5. CONCLUSIONS
Can we model diabetic neuropathy in vitro? The answer is perhaps
“no, or at least not in its entirety.” However, we can now model many
aspects of neuropathy effectively. The in vitro models used to study the
pathophysiological responses to hyperglycemia, reduced insulin signaling,
hyperlipidemia, and other stressors, while simplistic, do enable us to predict
how neurons may behave in more complex in vivo environments. They also
enable us to investigate subcellular responses and dissect out mechanistic
changes that contribute to the pathogenesis of the disease, test neuro-
protective strategies, and develop high-throughput drug discovery screens
for drugs to promote axonal regeneration. As such, they will remain a
valuable component to the neurobiologists’ toolbox. Future refinement
of models, including use of iPSCs and the ability to challenge different
neuronal compartments using microfluidic devices, will hopefully enable
us to discover underlying mechanisms that predispose the distal axon to
degeneration in diabetic neuropathy and improve treatment options.

ACKNOWLEDGMENTS
The authors would like to extend thanks to the Juvenile Diabetes Research Foundation,
Diabetes UK and the Medical Research Council, UK for funding their research over the
past years. We apologize to the many authors whose work we were not able to cite due
to space constraints. We thank the editors Paul Fernyhough and Nigel Calcutt for their
helpful comments.

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 CHAPTER SIX

Alternatives to the
Streptozotocin-Diabetic Rodent
M.A. Yorek1
Iowa City Health Care System, Iowa City, IA, United States
University of Iowa, Iowa City, IA, United States
Fraternal Order of Eagles Diabetes Research Center, University of Iowa, Iowa City, IA, United States
1
Corresponding author: e-mail address: [email protected]

Contents
1. Introduction 90
2. Rodent Models of Obesity 91
2.1 High-Fat Fed Sprague-Dawley Rats and C57Bl6/J Mice 91
2.2 Zucker Rats 92
3. Rodent Models of Type 2 Diabetes 93
3.1 Zucker Diabetic Fatty Rats 93
3.2 Spontaneously Diabetic Torii Rat 94
3.3 Zucker Diabetic Sprague-Dawley Rat 94
3.4 Goto–Kakizaki Rat 95
3.5 BioBreeding Zucker Diabetic Rat 96
3.6 Otsuka Long-Evans Tokushima Fatty Rat 96
3.7 Ob/ob and db/db Mice 97
3.8 Tsumura Suzuki Obese Diabetes Mouse 98
3.9 Combined High-Fat Fed, Low-Dose Streptozotocin Models of
Type 2 Diabetes 98
3.10 Streptozotocin–Nicotinamide Rat 101
4. Rodent Models of Type 1 Diabetes 101
4.1 Spontaneously Hypertensive Rat 101
4.2 BioBreeding/Worcester Rat 102
4.3 Ins2Akita Mouse 102
4.4 Nonobese Diabetic Mouse 103
5. Other Animal Models 103
6. Conclusions 103
References 104

Abstract
The study of diabetic neuropathy has relied primarily on the use of streptozotocin-
treated rat and mouse models of type 1 diabetes. This chapter will review the creation
and use of other rodent models that have been developed in order to investigate the
contribution of factors besides insulin deficiency to the development and progression

#
International Review of Neurobiology, Volume 127 2016 Elsevier Inc. 89
ISSN 0074-7742 All rights reserved.
http://dx.doi.org/10.1016/bs.irn.2016.03.002
90 M.A. Yorek

of diabetic neuropathy as it occurs in obesity, type 1 or type 2 diabetes. Diabetic periph-

eral neuropathy is a complex disorder with multiple mechanisms contributing to its
development and progression. Even though many animal models have been devel-
oped and investigated, no single model can mimic diabetic peripheral neuropathy
as it occurs in humans. Nonetheless, animal models can play an important role in
improving our understanding of the etiology of diabetic peripheral neuropathy and
in performing preclinical screening of potential new treatments. To date treatments
found to be effective for diabetic peripheral neuropathy in rodent models have failed
in clinical trials. However, with the identification of new endpoints for the early detection
of diabetic peripheral neuropathy and the understanding that a successful treatment
may require a combination therapeutic approach there is hope that an effective treat-
ment will be found.

1. INTRODUCTION
For years the standard animal model for the study of diabetic neurop-
athy has been the streptozotocin-treated rodent. In rats depending on the
species, age, and delivery, a single dose of streptozotocin ranging from
40 to 75 mg/kg is usually sufficient to destroy enough β cells to cause an
insulin-deficient form of diabetes (Rees & Alcolado, 2005; Tesch &
Allen, 2007). Such rats fail to gain weight and often lose weight unless
supported with a low-dose insulin treatment regime, which can allow such
animals to be maintained for extended periods of time in a hyperglycemic
state (Calcutt, 2004). In mice, the dose of streptozotocin required to create
a model of type 1 diabetes is generally higher than that used for rats, with
single doses ranging from 100 to 200 mg/kg (Rees & Alcolado, 2005;
Tesch & Allen, 2007). More recently, multiple low dosing of streptozo-
tocin has become the preferred method to induce an insulin-deficient
form of diabetes in mice (O’Brien, Sakowski, & Feldman, 2014; Rees &
Alcolado, 2005; Tesch & Allen, 2007). Using the latter approach creates a
type 1 diabetic mouse model that is more stable in regard to maintaining
their initial weight and will even gain weight compared to mice treated with
a single high dose of streptozotocin (personal observation). The potential for
high doses of streptozotocin to cause nonspecific effects on nerve and kidney
has for many years been a criticism of this type 1 diabetic model, even though
studies have shown that neurotoxicity is not the cause of slowing of nerve
conduction velocity or changes in thermal nociception in streptozotocin-
treated diabetic rats or mice (Davidson et al., 2009; Wiese, Matsushita,
Lowe, Stokes, & Yorek, 1996). Nonetheless, investigators seeking the
ultimate animal model to mimic the human development and progression
Alternatives to the Streptozotocin-Diabetic Rodent 91

of diabetic neuropathy have developed additional models in order to deci-

pher the complex pathogenesis of neuropathy in obesity and types 1 and 2
diabetes. The purpose of this chapter is to review some of the characteristics
of these other rodent models of diabetes and diabetic neuropathy.
Peripheral neuropathy is a multifaceted complication of diabetes that fre-
quently leads to foot ulcers and may progress to limb amputations (Kim,
Kim, & Yoon, 2012). Even though it is the most common complication
of diabetes, the only recommended clinical treatment is good glycemic
control which, at best, only delays the onset and slows progression
(Callaghan, Little, Feldman, & Hughes, 2012; Figueroa-Romero, Sadidi, &
Feldman, 2008). Diabetic peripheral neuropathy has been described by some
investigators to be a disease of the vasculature leading to nerve ischemia and
altered nerve function (Cameron, Cotter, Archibald, Dines, & Maxfield,
1994; Cameron, Cotter, Dines, et al., 1994; Cameron, Cotter, & Low,
1991; Nukada & Dyck, 1984). Other investigators have proposed that diabetic
peripheral neuropathy is caused by a combination of metabolic defects associ-
ated with an increased flux of glucose through the aldose reductase pathway
leading to a defect in Na+/K+-ATPase activity and an alteration of signal
transduction pathways in the nerve (Cameron, Cotter, Dines, & Love,
1992; Cotter, Dines, & Cameron, 1993). Additional pathologic contributors
to diabetic peripheral neuropathy have been reported to include increased
formation of advanced glycation endproducts, reduced neurotrophic support,
and increased inflammatory and oxidative stress (Pop-Busui, Sima, & Stevens,
2006; Sima, 2006). These and likely other mechanisms, which are reviewed in
detail elsewhere in this volume, cause damage to neurons, Schwann cells, and
the vasculature. Ultimately, relentless damage to the nerve complex and
surrounding vasculature leads to diabetic peripheral neuropathy. Given the
complex etiology of diabetic peripheral neuropathy, creating an animal model
that mimics all the pathologies and developmental pattern of neuropathy seen
in human diabetic subjects is probably an impossible task. Therefore, investi-
gators have focused on gaining what information they can from the multiple
animal models that have been created before determining whether there is
some translational value in these data.

2. RODENT MODELS OF OBESITY

2.1 High-Fat Fed Sprague-Dawley Rats and C57Bl6/J Mice
Feeding rats or mice a high fat or Western diet to induce obesity has recently
become a more common approach to creating models of prediabetes
(Coppey, Davidson, Lu, Gerard, & Yorek, 2011; Davidson, Coppey,
92 M.A. Yorek

Calcutt, Oltman, & Yorek, 2010; Groover et al., 2013; Nowicki, Kosacka,
Serke, Bluher, & Spanel-Borowski, 2012; O’Brien, Sakowski, et al., 2014;
Obrosova et al., 2007; Yorek et al., 2015). There have been many different
formulations of these diets, but in general they cause excess weight gain, raise
circulating lipid levels, and cause insulin resistance. Our studies have shown
that rats fed a high-fat (45% kcal) diet rapidly gained weight, became hyp-
erinsulinemic, and developed insulin resistance in 4–6 weeks (unpublished
data). However, fasting blood glucose and hemoglobin A1C levels were
not increased (Davidson et al., 2010; Davidson, Coppey, Dake, & Yorek,
2011; Davidson, Coppey, Kardon, & Yorek, 2014). In 12 weeks the rats
developed sensory neuropathy, as indicated by slowing of sensory nerve
conduction velocity and onset of thermal hypoalgesia (Davidson et al.,
2010; Davidson, Coppey, Dake, et al., 2011; Davidson, Coppey, Kardon,
et al., 2014). C57Bl6/J mice fed a high-fat diet also develop a prediabetes
phenotype including insulin resistance, impaired glucose utilization, fasting
hyperglycemia, and sensory neuropathy (Coppey et al., 2011; Yorek et al.,
2015). In our studies, diet-induced obese mice, like their rat counterparts,
had a normal motor nerve conduction velocity after 12 weeks of a high-fat
diet. Others have reported both motor and sensory neuropathy in C57Bl6/J
mice fed a high-fat diet for 16 weeks (Obrosova et al., 2007) or longer
(Anderson, King, Delbruck, & Jolivalt, 2014). Reasons for these differences
could include the type of high-fat diet used or study duration. It has been
reported that exercise can alleviate some of the neuropathic deficits associ-
ated with diet-induced obesity (Groover et al., 2013). C57Bl6/J mice have
been commonly used by investigators. However, it should be noted that
other strains of mice are also used and may respond differently to high-
fat-containing diets. For example, Swiss Webster mice develop insulin resis-
tance and cognitive impairment but do not gain weight or show indices of
peripheral neuropathy (Anderson et al., 2014). Human subjects determined
to have impaired glucose tolerance have also been reported to develop a
sensory neuropathy (Asghar et al., 2014; Kannan et al., 2014; Papanas,
Vinik, & Ziegler, 2011; Papanas & Ziegler, 2012; Smith & Singleton, 2008).

2.2 Zucker Rats

The Zucker (fa/fa) rat is the best-known and most widely used rat model of
genetic obesity. The fa mutation was discovered by Zucker and Zucker
(Kava, Greenwood, & Johnson, 1990) in crosses between Sherman and
Merck stock M rats (13M strain). Animals homozygous for the fa allele
Alternatives to the Streptozotocin-Diabetic Rodent 93

become noticeably obese by 3–5 weeks of age, and by 14 weeks of age their
body composition is over 40% lipid. Obese Zucker rats do not become
hyperglycemic, but are hyperlipemic, hypercholesterolemic, hyperinsuli-
nemic, and develop adipocyte hypertrophy and hyperplasia (Alderson
et al., 2003; Bray, 1977; Kurtz, Morris, & Pershadsingh, 1989). The Zucker
rat has also occasionally been used in investigations of obesity-associated
noninsulin-dependent diabetes mellitus. In our studies with this model,
we found that vascular and neural dysfunction developed at a slower rate
in obese Zucker rats than in the Zucker diabetic fatty (ZDF) rat model of
type 2 diabetes (see later). In both models, vascular impairment preceded
slowing of motor nerve conduction velocity, which occurs at 12–14 weeks
of age in ZDF rats and 32 weeks of age in obese Zucker rats (Oltman et al.,
2005).

3. RODENT MODELS OF TYPE 2 DIABETES

3.1 Zucker Diabetic Fatty Rats
There is a wide variety of models duplicating various pathologies associated
with type 2 diabetes. In rats and mice the most commonly used to study
neuropathy are those that lack a functional leptin receptor (ZDF rat, spon-
taneously diabetic Torii (SDT) rat, and db/db mouse) or are leptin defi-
cient (ob/ob mouse) (Sasase, Yokoi, Pezzolesi, & Shinohara, 2015). The
ZDF rat is recognized as a standard model for metabolic syndrome and type
2 diabetes and has been extensively used. It develops obesity and hyper-
glycemia at 8–10 weeks of age due to the leptin-receptor deficit and a
second mutation that developed in a colony of obese Zucker rats that was
isolated through inbreeding (Peterson, Shaw, Neel, Little, & Eichberg,
1990). The ZDF rat has been used to study pain mechanisms associated
with early diabetic neuropathy (Gao & Zheng, 2014; Lirk et al., 2012,
2015; Otto, Wyse, Cabot, & Smith, 2011). It has also been used to examine
effect of various treatments on diabetic neuropathic endpoints (Li et al.,
2006; Lupachyk, Watcho, Hasanova, Julius, & Obrosova, 2012; Lupachyk,
Watcho, Obrosov, Stavniichuk, & Obrosova, 2013; Oltman et al., 2008;
Oltman, Davidson, Coppey, Kleinschmidt, & Yorek, 2009). We found that
monotherapy treatment of ZDF diabetic fatty rats once complications had
developed using different classes of drugs for vascular and neural dysfunction
did not achieve expected efficacy levels suggesting a complex etiology that
includes multiple mechanisms (Oltman et al., 2008).
94 M.A. Yorek

3.2 Spontaneously Diabetic Torii Rat

The SDT rat is an inbred strain of Sprague-Dawley rat that has been
established as a nonobese model of type 2 diabetes (Sasase et al., 2013). Male
SDT rats show high plasma glucose levels by 20 weeks of age with pancreatic
islet histopathology. Prior to the onset of diabetes, glucose intolerance with
hypoinsulinemia is observed (Sasase et al., 2013). SDT rats develop profound
complications including retinopathy, nephropathy, and neuropathy (Sasase
et al., 2013). However, another group has reported that the time of onset of
glucosuria is different between male and female SDT rats with male rats
demonstrating a 100% incidence of diabetes at 40 weeks of age, while it
is only 33% for female rats (Shinohara et al., 2000). In order to shorten
the period of development of hyperglycemia in the SDT rat, a second
SDT type 2 diabetic rat model was created by introducing the fa allele
of the Zucker fatty rat into the SDT rat genome. This created a new
obese model of type 2 diabetes with both male and female SDT fatty rats
showing overt obesity, hyperglycemia, and hyperlipidemia at an early age
(Kemmochi et al., 2013). Diabetic peripheral neuropathy including
decreased nerve conduction velocity and a decrease in sural nerve fibers
has been reported in this model (Yamaguchi et al., 2012).

3.3 Zucker Diabetic Sprague-Dawley Rat

As discussed earlier, masking the effects of leptin by either decreasing the
leptin receptor or eliminating leptin production has been used to create
models of obesity-related type 2 diabetes. One concern for the applicability
of the ZDF rats to humans is the recessive homozygous mutation in the
leptin receptor (fa) that causes loss of function and induces severe hyperpha-
gia (Davis, Cain, Banz, & Peterson, 2013; Reinwald, Peterson, Allen, &
Burr, 2009). Leptin has been shown to have an array of effects that may
influence development of the metabolic syndrome (Beltowski, 2012; Gade,
Schmit, Collins, & Gade, 2010; Kaur, 2014; Ricci & Bevilacqua, 2012). Thus,
loss of leptin signaling in the ZDF rat makes it a less than ideal model for
complications of type 2 diabetes in humans, who generally do not have a
leptin-receptor deficit. These concerns led to the creation of the Zucker
diabetic Sprague-Dawley (ZDSD) rat (Davis et al., 2013; Peterson et al.,
2015; Reinwald et al., 2009). The ZDSD rat was developed by crossbreeding
the Charles River Laboratory diet-induced obese rat (Sprague-Dawley-
derived) with lean ZDF / rats. Selective inbreeding produced animals
with a predisposition to obesity and a propensity to develop overt diabetes
Alternatives to the Streptozotocin-Diabetic Rodent 95

between 15 and 21 weeks of age with nutritional intervention (Reinwald

et al., 2009). Importantly, ZDSD rats have an intact leptin-signaling pathway
and more modest accumulation of body fat compared to ZDF rats (Reinwald
et al., 2009). We obtained ZDSD rats with the objective of characterizing
endpoints associated with diabetic neuropathy in comparison to age matched
Sprague-Dawley rats (Davidson, Coppey, Holmes, et al., 2014). We found
that chronic hyperglycemia in ZDSD rats caused vascular and neural dysfunc-
tion similar to that documented in other diabetic rat models and consistent
with the development of diabetic neuropathy (Coppey, Davidson, Dunlap,
Lund, & Yorek, 2000, Coppey, Gellett, Davidson, Dunlap, & Yorek,
2002; Davidson et al., 2010; Oltman et al., 2005; Terata et al., 1999). The
one exception was that chronic hyperglycemia in ZDSD rat did not cause
a decrease in intraepidermal nerve fibers (IENF) in the skin, as has been
reported for other rat models of diabetes. There was, however, a decrease
in epidermal Langerhans cells. Overall, we found that the ZDSD rat is an easily
maintained animal model for type 2 diabetes that spontaneously develops
hyperglycemia upon dietary manipulation. This was followed by the devel-
opment of diabetic neuropathy, including loss of nerves in the subepithelial
layer of the cornea and decreased corneal function. Since determination of
corneal nerve density and function are noninvasive surrogate markers for dia-
betic neuropathy (Quattrini et al., 2007), the ZDSD rat with its functional
leptin pathway may be a good model for preclinical testing of treatments
for diabetic neuropathy.

3.4 Goto–Kakizaki Rat

The Goto–Kakizaki (GK) rat is considered one of the best animal models of
nonobese type 2 diabetes (Akash, Rehman, & Chen, 2013) as they exhibit
many characteristics in common with diabetic patients. The GK rat sponta-
neously develops type 2 diabetes due to the complex interaction of multiple
mechanisms that include the presence of several susceptibility genes, gesta-
tional metabolic impairment inducing an epigenetic programming of the
offspring pancreas and the major insulin target tissues, and environmental-
induced loss of β-cell differentiation due to chronic exposure to hypergly-
cemia/hyperlipidemia, inflammation, and oxidative stress (Portha, Giroix,
Tourrel-Cuzin, Le-Stunff, & Movassat, 2012). Investigations using GK rats
have demonstrated a number of diabetes-related neuronal defects in this
model compared to the Wistar rats that are commonly used as controls.
GK rats have impaired glucose tolerance and progressive insulinopenia
96 M.A. Yorek

and go on to develop peripheral nerve abnormalities in the absence of overt

hyperglycemia (Murakawa et al., 2002). Onset of hyperglycemia worsens
the development/progression of peripheral neuropathy in GK rats. Several
studies have demonstrated that sustained improvement in hyperglycemia can
improve diabetic peripheral neuropathy in GK rats (Ueta et al., 2005; Wada
et al., 1999). Recently, Wang, Gao, Yin, and Yu (2012) demonstrated that
diabetic neuropathy also occurs in the cornea of type 2 diabetic GK rats.
They concluded that defects in the sensory nerve and/or tear film may con-
tribute to diabetic keratopathy and delayed wound healing in diabetic cor-
neas. Their finding that subepithelial corneal nerve loss in GK rats was more
prominent in the central cornea than in the limbal region is similar to our
own findings using the high-fat fed, low-dose streptozotocin-treated rat
model as described later (Davidson, Coppey, Kardon, et al., 2014; Wang
et al., 2012).

3.5 BioBreeding Zucker Diabetic Rat

The BioBreeding Zucker diabetic rat (BBZDR)/Wor rat was created by
breeding the insulin-resistant Zucker fatty rat into the inbred Bio-
Breeding/Worcester (BB/Wor) strain in order to introduce the defective
leptin receptor gene (Tirabassi et al., 2004). BBZDR/Wor rats develop
hyperglycemia at about 7 weeks of age and become obese with elevated tri-
glyceride and cholesterol levels. Lipid levels can be further increased by feed-
ing a diet containing high fat and sucrose. These rats are insulin resistant with
hyperinsulinemia and develop microvascular- and macrovascular-related
complications including retinopathy, neuropathy, nephropathy, and coro-
nary artery disease with hypertension (Gao & Zheng, 2014; Tirabassi
et al., 2004). With regard to neuropathy, the diabetic BBZDR/Wor rat
shows a slowly progressive nerve conduction defect accompanied by mild
myelinated fiber atrophy, mild changes of the node of Ranvier, and signif-
icant segmental demyelination and Wallerian degeneration (Sima et al.,
2000). These changes were much less dramatic than those observed in
the type 1 spontaneously diabetic BB/Wor rat maintained with the same
degree of hyperglycemia (Kamiya, Murakawa, Zhang, & Sima, 2005;
Sima et al., 2000).

3.6 Otsuka Long-Evans Tokushima Fatty Rat

OLETF rats, when fed a diet with or without sucrose, were hyperglycemic
compared to control rats (Kamenov, Higashino, Todorova, Kajimoto, &
Alternatives to the Streptozotocin-Diabetic Rodent 97

Suzuki, 2006). At 10 months of age motor nerve conduction velocity and

thermal nociception were significantly decreased, and all parameters deteri-
orated further when OLETF rats received a sucrose-supplemented diet.
Other studies with sucrose-fed OLETF rats have demonstrated a reduction
in sciatic nerve blood flow and decreased Na+/K+-ATPase activity in the
sciatic nerve (Nakamura et al., 2001).

3.7 Ob/ob and db/db Mice

The most common mouse models of type 2 diabetes are those with the ob/
ob or db/db mutations. These mice are deficient in leptin (ob/ob) or lack a
functional leptin receptor (db/db). They are also obese, insulin resistant and
develop spontaneous hyperglycemia. Studies by Grote et al. (2013) demon-
strated that insulin resistance occurred in muscle, liver, and sciatic nerve.
Characterization of peripheral diabetic neuropathy in ob/ob mice revealed
that after 9–13 weeks of hyperglycemia motor and sensory nerve conduction
velocity are decreased, and mice have thermal hypoalgesia, tactile allodynia,
and a significant loss of IENF (Drel et al., 2006; O’Brien, Hur, et al., 2014).
Treating ob/ob mice with an aldose reductase inhibitor or with per-
oxynitrite decomposition catalysts improved diabetic peripheral neuropathy
endpoints (Drel et al., 2006; Vareniuk et al., 2007) suggesting that activation
of the aldose reductase pathway as well as increased oxidative stress contrib-
ute to the development and progression of diabetic peripheral neuropathy in
the ob/ob mouse.
The leptin-receptor deficiency present in db/db mice has been crossed
into a number of background strains allowing for studies of multiple path-
ological conditions (Sullivan et al., 2007). The db/db mouse, like the ZDF
rat, is obese and insulin resistant. It develops a severe hyperglycemia and
peripheral neuropathy with decreased nerve conduction velocity and mor-
phological changes in peripheral nerves (Cho et al., 2014; Norido, Canella,
Zanoni, & Gorio, 1984; Nowicki et al., 2012; Pande et al., 2011;
Robertson & Sima, 1980; Shi et al., 2013; Sima & Robertson, 1978). In
a study comparing the effect of hyperglycemia in 4-month-old C57BL6
(control), ob/ob, and db/db mice it was found that myelin thickness was
significantly reduced in small, medium-sized, and large axons of db/db mice
compared with control C57Bl6 mice (Nowicki et al., 2012). In contrast,
only large fibers showed a decrease in myelin sheath thickness in ob/ob
mice, while the number of nonmyelinated nerve fibers was lower in ob/
ob mice than in db/db mice. A thickened basal lamina of Schwann cells also
98 M.A. Yorek

occurred only in ob/ob mice. The basement membrane of endoneural

microvessels was thickened in both ob/ob and db/db mice. Thus, fundamen-
tal differences in some endpoints of diabetic neuropathology exist between
ob/ob and db/db mice.

3.8 Tsumura Suzuki Obese Diabetes Mouse

Other mouse models of obesity and spontaneous type 2 diabetes include
the Tsumura Suzuki Obese Diabetes (TSOD) mouse (Lizuka et al., 2005).
The male mice exhibit polydipsia and polyuria at about 2 months of age
followed by hyperglycemia and hyperinsulinemia. Sensory and motor neu-
ropathy deficits can be observed at 12 and 14 months of age, respectively, with
weakness of front and hind paws at about 17 months of age (Lizuka et al.,
2005). At this time, light microscopic and electron microscopic examination
of the sciatic nerve shows a decrease in the density of nerve fibers as well as
degenerative changes of myelinated fibers. Interestingly, a study by Kawada
et al. (2010) showed that TSOD mice even after 18 months of age did not
develop any form of cardiac dysfunction.

3.9 Combined High-Fat Fed, Low-Dose Streptozotocin

Models of Type 2 Diabetes
A rodent model for type 2 diabetes that I have found to be useful and easy
to work with is the high-fat fed, low-dose streptozotocin-treated rat, or
mouse (Gao & Zheng, 2014; Skovso, 2014). The rationale behind this
approach is that the high-fat diet renders the rodent insulin resistant, and
the low dose of streptozotocin destroys enough of the β cells to induce
a constant state of hyperglycemia.
Reed et al. (2000) were the first to use the high-fat fed streptozotocin-
treated rat as a model for type 2 diabetes. Sprague-Dawley rats were fed a
high-fat diet for 7 weeks and then treated with streptozotocin (50 mg/
kg). These rats were hyperglycemic, insulin resistant, and sensitive to the
glucose-lowering effects of metformin and troglitazone. Others have also
found this model to be suitable for testing agents for the treatment of type
2 diabetes (Ding et al., 2005; Gaikwad, Viswanad, & Ramarao, 2007; Reed
et al., 2000; Srinivasan, Viswanad, Asrat, Kaul, & Ramarao, 2005; Wang
et al., 2009; Zhang, Lv, Li, Xu, & Chen, 2008; Zhu, Peng, Liu,
Zhang, & Li, 2010). It should be noted that the literature contains some var-
iability in the insults used to create this model. A study by Wang, Li,
Liu, Liu, and Sun (2011) examined parameters for creating the high-fat
Alternatives to the Streptozotocin-Diabetic Rodent 99

diet streptozotocin–diabetic rat model and found that duration of high-fat

diet, dosage of streptozotocin, and the age of rats were the most important
factors for establishing this model. Generally the high-fat diet is consistent at
40–50% calories as fat for a period of 4–8 weeks (Gao & Zheng, 2014).
However, the dose of streptozotocin used ranges from 20 to 50 mg/kg
(Reed et al., 2000; Wang et al., 2011). It has been our experience using rats
that were 12 weeks of age at the onset of the study, that after 8 weeks on
a high-fat diet, 20 mg/kg streptozotocin resulted in a low success rate
for inducing hyperglycemia. When we used 40 mg/kg streptozotocin,
our mortality rate was high and rats began dying 2–4 weeks following
the injection of streptozotocin. With 30 mg/kg streptozotocin our success
rate was over 90%, and the mortality rate was less than 5%. Rats do not
require insulin treatment to maintain weight, unlike streptozotocin-induced
type 1 diabetic rat models (Davidson, Coppey, Holmes, et al., 2011;
Davidson, Kleinschmidt, Oltman, Lund, & Yorek, 2007). An important
item to remember is that the potency of streptozotocin can vary widely
between vendors and between different lots purchased from the same ven-
dor. We agree with Reed et al. (2000) that the high-fat fed streptozotocin–
diabetic rat models late stage type 2 diabetes in patients. The diabetes in these
rats is analogous to the development of human type 2 diabetes when the
decline in hyperinsulinemia is not able to compensate for insulin resistance
and hyperglycemia occurs (Reed et al., 2000).
My laboratory was the first to characterize diabetic neuropathy in the
high-fat fed low-dose streptozotocin-treated rat (Davidson, Coppey,
Holmes, et al., 2011). In our studies, Sprague-Dawley rats were fed a high-
fat diet for 8 weeks before treating with streptozotocin. Our initial study
examined vascular and neural endpoints at 16 and 24 weeks after the onset
of hyperglycemia and high-fat diet, respectively (Davidson, Coppey,
Holmes, et al., 2011). Compared to high-fat fed rats we found that following
induction of hyperglycemia glucose utilization was further impaired. At the
end of the study period, the diabetic rats weighed about the same as control
rats, insulin and leptin levels were near control values, and they were hyper-
lipidemic. Furthermore, we found that at the end of the study period weight
of the epididymal fat pad was significantly less in the high-fat streptozotocin–
diabetic rats compared to high-fat fed rats. However, epididymal fat pad
weight in the high-fat streptozotocin–diabetic rats was still greater than con-
trol rats. Serum insulin and leptin levels were significantly decreased in high-
fat streptozotocin–diabetic rats compared to high-fat fed rats but similar to
control rats. Two notable differences between high-fat fed rats and high-fat
100 M.A. Yorek

streptozotocin–diabetic rats were that in diabetic rats motor nerve conduc-

tion velocity was significantly decreased and superoxide levels and
nitrotyrosine staining were increased in epineurial arterioles (Davidson
et al., 2010; Davidson, Coppey, Holmes, et al., 2011). This indicates that
oxidative stress in the microvasculature is increased by hyperglycemia. In
epineurial arterioles from high-fat fed rats superoxide levels and
nitrotyrosine staining were not increased and motor nerve conduction
velocity not significantly impaired (Davidson et al., 2010). Sensory nerve
conduction velocity, thermal nociception, and intraepidermal nerve fiber
profiles were impaired in both high-fat fed rats and high-fat
streptozotocin–diabetic rats. Interestingly, acetylcholine- and CGRP-
mediated vascular relaxations of epineurial arterioles were impaired to a sim-
ilar degree in high-fat fed rats and high-fat streptozotocin–diabetic rats. It
was expected that because of the increase in superoxide and nitrotyrosine
staining in epineurial arterioles from high-fat streptozotocin–diabetic rats
compared to high-fat fed rats that vascular relaxation to acetylcholine would
be impaired to a greater degree in high-fat streptozotocin–diabetic rats. This
suggests that mechanisms in addition to oxidative stress contribute signifi-
cantly to impairment of vascular function in epineurial arterioles from high-
fat fed rats and high-fat streptozotocin–diabetic rats. More recent studies
have revealed loss of corneal nerves and decrease in corneal nerve sensitivity
in diet-induced obese rats and high-fat streptozotocin–diabetic rats
(Davidson, Coppey, Kardon, et al., 2014).
We have also used the high-fat diet low-dose streptozotocin approach to
create a mouse model of type 2 diabetes (Yorek et al., 2015). Unlike rats,
feeding C57Bl6/J mice a high-fat diet causes an elevated level of fasting
blood glucose (Coppey et al., 2011; Davidson, Coppey, Holmes, et al.,
2011). However, hyperglycemia in the high-fat fed mouse is modest and
not associated with an increase in hemoglobin A1C levels. Moreover, blood
glucose was not increased in the fed state. Using a low dose of streptozotocin
with a high-fat fed C57Bl/6J mouse creates a higher level of blood glucose,
which is present in both the fasted and fed state. This type 2 diabetic mouse
model has been previously used to examine pharmacological interventions,
vascular biology and atherosclerosis, cardiomyocyte hypertrophy, and β-cell
function (Bansal et al., 2012; Lin et al., 2015; Lv et al., 2010; Mali et al.,
2014; Ullevig, Zhao, Zamora, & Asmis, 2011; Wang, Hsu, Lin, & Chen,
2014; Xue, Ding, & Liu, 2010; Zhu et al., 2014). In our studies comparing
the effect of diet-induced obesity, type 1 and type 2 diabetes on neuropathy,
we found the severity of hyperglycemia was significantly different between
Alternatives to the Streptozotocin-Diabetic Rodent 101

the types 1 and 2 diabetic mouse models (Yorek et al., 2015). However,
motor and sensory nerve conduction velocity, thermal and mechanical sen-
sitivity, and intraepidermal nerve fiber density in the skin were all impacted
similarly in diet-induced obesity and types 1 and 2 diabetic mice. Loss of
corneal nerves in the subepithelial layer and penetrating the corneal epithe-
lium occurred more rapidly in the diet-induced obesity mice and type 2 dia-
betic mice compared to type 1 diabetic mouse. This suggests that
hyperglycemia is not the only factor contributing to nerve fiber loss in
the cornea and that loss of intraepidermal and corneal nerve fibers is medi-
ated by different factors and/or occurs at different rates.
Overall, these studies demonstrate that the high-fat fed low-dose
streptozotocin-treated rodents are a good animal model for preclinical
studies for discovery and evaluation of new treatments for diabetic neurop-
athy especially in relation to changes in nerve structure in the skin and
cornea.

3.10 Streptozotocin–Nicotinamide Rat

For this model the rat is treated with a dose of streptozotocin generally used
to create type 1 diabetes followed by nicotinamide (Sharma et al., 2012;
Szkudelski, 2012). Nicotinamide is a poly(ADP-ribose) polymerase inhibi-
tor and has been shown to reverse neurological and neurovascular deficits in
streptozotocin–diabetic rats (Negi, Kumar, Kaundal, Gulati, & Sharma,
2010; Stevens et al., 2007). When nicotinamide is used in combination with
streptozotocin, it protects β cells from the effects of streptozotocin and
the result is a model of type 2 diabetes (Szkudelski, 2012). In this model
the severity of diabetes strongly depends on the doses of streptozotocin
and nicotinamide given to the animal (Szkudelski, 2012). It has been
reported that the streptozotocin–nicotinamide-treated rat develops neurop-
athy (Sharma et al., 2012).

4. RODENT MODELS OF TYPE 1 DIABETES

4.1 Spontaneously Hypertensive Rat
Use of streptozotocin has been the most often used approach to induce type
1 diabetes in rodents. However, inducing type 1 diabetes in Sprague-
Dawley or Wistar rats does not cause hypertension, which is an independent
risk factor for neuropathy in diabetic patients. Streptozotocin has therefore
been used to induce type 1 diabetes in spontaneously hypertensive rats
102 M.A. Yorek

(Gregory, Jolivalt, Goor, Mizisin, & Calcutt, 2012; Sanada et al., 2015) to
study the combined effects of diabetes and hypertension on peripheral
neuropathy.

4.2 BioBreeding/Worcester Rat

There are several genetic rat and mouse models that have been used to rep-
licate type 1 diabetes. The BB/Wor rat has been stated to model human
insulin-dependent diabetes mellitus (Whalen, Mordes, & Rossini, 2001).
This rat requires daily insulin treatment to survive. It has been reported that
motor and sensory nerve conduction velocities are decreased after 4 and
6 weeks of diabetes and continue to decline for up to 9 months (Kamiya,
Zhang, & Sima, 2009). Myelinated sural nerve fibers show progressive
decreases in fiber number and size. Other deficits include thermal
hyperalgesia, loss of content of neuropeptides in dorsal root ganglia, and
reduced endoneurial blood flow (Kamiya, Zhang, & Sima, 2004; Stevens,
Zhang, Li, & Sima, 2004). Interestingly, Sima and colleagues have demon-
strated that many of the neurological deficits caused by diabetes in the
BB/Wor rat can be prevented/reversed with C-peptide treatment
(Kamiya et al., 2004; Sima et al., 2001; Stevens et al., 2004; Zhang
et al., 2001).

4.3 Ins2Akita Mouse

The heterozygous Ins2Akita mouse spontaneously develops insulin-
dependent diabetes, including hyperglycemia, hypoinsulinemia, polydipsia,
and polyuria, which is more severe in males than females. The Akita mouse
has been widely used to introduce a diabetic background into other mouse
genetic models. For instance, crossing the Akita mouse with the apoE
( / ) mouse created a mouse model used for studying the effect of diabetes
on atherosclerosis (Jun, Ma, & Segar, 2011). The C57BL/6-Akita mouse
has been proposed as a nonobese model for type 2 diabetes (Yaguchi,
Nagashima, Izumi, & Okamoto, 2003) and has been used to examine the
effect of diabetes on the central nervous system. Studies using the Akita
mouse for determination of the effect of diabetes on autonomic neuropathy
have shown that the Akita mouse develops abnormal cardiac function, with a
loss in nerve density (Yang & Chon, 2011). In regard to peripheral
neuropathy, 16-week-old Ins2Akita mice develop sensory neuropathy inclu-
ding reduced nerve conduction velocity, thermal and mechanical hypoalgesia,
and tactile allodynia (Drel et al., 2011). Motor nerve conduction velocity
Alternatives to the Streptozotocin-Diabetic Rodent 103

showed a nonsignificant trend to slowing, whereas others (de Preux Charles

et al., 2010) have reported a decrease in motor nerve conduction velocity in
Ins2Akita mice at 12 weeks of age.

4.4 Nonobese Diabetic Mouse

The nonobese diabetic (NOD) mouse is susceptible to the development
of autoimmune diabetes but also multiple other autoimmune diseases
(Bour-Jordan et al., 2013). Over 20 susceptibility loci linked to diabetes have
been identified in NOD mice (Bour-Jordan et al., 2013), and the strain has
been used extensively to cross the diabetic background onto other mice
(Gross et al., 2008). Invasive insulitis, seen in NOD mice, causes early sympa-
thetic islet neuropathy, but it remains to be determine whether this is respon-
sible for the impaired glucagon secretion (Taborsky et al., 2009). In addition to
the sympathetic neuropathy developed by the NOD mouse, it has also been
reported to develop sensory neuropathy with hypoalgesia and correction by
use of a peroxynitrite decomposition catalyst (Obrosova et al., 2005).

5. OTHER ANIMAL MODELS

This chapter has focused primarily on rodent models of obesity and
diabetes and peripheral neuropathy. A number of other animal models
have also been used successfully in the study of the effect of diabetes on
peripheral and autonomic neuropathy. These include guinea pigs, rabbits,
cats, dogs, pigs, and monkeys (Cohen, Tesfamariam, Weisbrod, & Zitnay,
1990; Estrella et al., 2008; Islam, 2013; Mesangeau, Laude, & Elghozi,
2000; Mizisin et al., 2007; Morgan, Vite, Radhakrishnan, & Hess, 2008).
However, ethical, practical, and financial costs concerns will likely continue
to limit the extent to which these other models are used.

6. CONCLUSIONS
In 1997, Dr. Tomlinson coauthored a review article titled “Does neu-
ropathy develop in animal models?” (Hounsom & Tomlinson, 1997). Inves-
tigators have been addressing this question for the past 18 years. In the
abstract of that paper the authors wrote in reference to diabetic neuropathy,
“currently the cornerstone of treatment lies with the maintenance of
euglycemia and development of effective treatments for diabetic neuropathy
is urgently needed.” The same statement could be made today. The only
accepted treatment for diabetic neuropathy remains good glycemic control,
104 M.A. Yorek

but it is now clear that this is ineffective, especially in patients with type 2
diabetes (Callaghan et al., 2012). Hounsom and Tomlinson further noted
that animal models have been developed to investigate the pathogenesis
of diabetic neuropathy and evaluate potential therapeutic agents. However,
no model is perfect and no one would suggest that diabetic rodents can
replicate the human condition fully. Since this article was written, animal
models, including those discussed earlier, continue to be used to investigate
and identify potential new treatments for diabetic neuropathy. However,
translation of findings to humans with diabetic peripheral neuropathy has
continued to fail. Many articles have been written to rationalize these fail-
ures. We know that diabetic neuropathy is a complex disease with mul-
tiple etiologies. It is my belief that animal models of diabetes and diabetic
neuropathy can still play a role in discovery of a treatment. However, this
treatment will likely be a combination therapy that will delay progression
and induce nerve repair when used in combination with glycemic control.
It should also be accepted that the preclinical efficacy of any treatment be
tested in several animal models utilizing multiple endpoints. A consensus
statement regarding the phenotyping of rodent models for diabetic peri-
pheral neuropathy was published following a joint meeting of the Diabetic
Neuropathy Study Group of EASD (Biessels et al., 2014). Following
these guidelines will help in standardizing studies coming from multiple
laboratories.

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 CHAPTER SEVEN

An Introduction to the History and

Controversies of the Pathogenesis
of Diabetic Neuropathy
P. Fernyhough*,†,1, N.A. Calcutt{
*University of Manitoba, Winnipeg, MB, Canada
†
St. Boniface Hospital Research Centre, Winnipeg, MB, Canada
{
University of California, San Diego, La Jolla, CA, United States
1
Corresponding author: e-mail address: [email protected]

Early studies into mechanisms of diabetic peripheral neuropathy focused pri-

marily on hyperglycemia-driven pathways. Central to this approach was
strong, hypothesis-driven work showing that excessive glucose flux through
the aldose reductase pathway (aka the polyol pathway) generated metabolic
disturbances leading to impaired nerve conduction velocity and reduced
axonal caliber (Tomlinson & Gardiner, 2008). Diverse inhibitors of aldose
reductase (ARIs) were developed that blocked accumulation of the pathway
intermediates sorbitol and fructose in diabetic rats and prevented or reversed
indices of diabetic neuropathy (Oates, 2008). Unfortunately, subsequent
clinical trials failed to demonstrate efficacy acceptable to regulatory agencies.
A variety of reasons have been cited for this failure, ranging from drug design
issues such as toxic side effects and poor efficacy at blocking AR activity in
humans to trial design issues such as performing short duration trials in sub-
jects with severe degenerative neuropathy. With hindsight it is also clear that
endpoints used to assess efficacy in these clinical trials may have been sub-
optimal. For example, structural integrity of distal regions of small sensory
fibers was not assessed but can now be measured in skin biopsies or by cor-
neal confocal microscopy. Moreover, the main physiological endpoint used
in both preclinical and subsequent clinical studies, large myelinated fiber
conduction slowing, develops within weeks of onset of diabetes in animal
models and may not accurately replicate the slowly developing NCV deficit
in humans with diabetic neuropathy where segmental demyelination and
fiber loss contribute. An additional area of interest when discussing ARIs

#
International Review of Neurobiology, Volume 127 2016 Elsevier Inc. 115
ISSN 0074-7742 All rights reserved.
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116 P. Fernyhough and N.A. Calcutt

is the true site of action. AR has been localized to Schwann cells of myelin-
ated fibers, epineurial endothelial cells, and axons of sympathetic neurons,
but a clear explanation as to how excessive polyol pathway activity drives
distal axonal damage in small sensory fibers remains to be fully developed
(Jiang, Calcutt, Ramos, & Mizisin, 2006). Yagihashi revisits some of these
issues in his contribution entitled “Glucotoxic Mechanisms and Related
Therapeutic Approaches.”
The clinical failure of ARIs has not, in our opinion, denigrated the
central idea that excessive glucose flux through the polyol pathway is a key
pathogenic process in diabetic neuropathy. As ARI-related research has
stalled, various research groups have explored putative downstream conse-
quences of hyperglycemia, primarily in endothelial cell cultures, to study
the impact of excessive glucose metabolism and polyol pathway flux in
augmenting ischemia and oxidative stress (Figueroa-Romero, Sadidi, &
Feldman, 2008; Nishikawa, Edelstein, & Brownlee, 2000). The role of ische-
mia due to hyperglycemia-induced impaired nerve blood flow remains
a major source of controversy. Diabetic neuropathy is classified by many as
a microvascular disease. However, the pathology of nerve damage in diabetes
does not reflect an ischemic process. For example, in ischemic nerve damage
large fibers are preferentially targeted, which does not appear to be the case in
diabetic neuropathy (Fujimura, Lacroix, & Said, 1991). In the contribution
“Sensory Neurodegeneration in Diabetes: Beyond Glucotoxicity” by
Zochodne, the inconsistencies in the vascular hypothesis as a cogent explana-
tion of nerve damage in diabetes are briefly discussed.
Oxidative stress clearly occurs in the nerve of diabetic rodents and
diverse antioxidants are protective in various animal models of the disease
(Figueroa-Romero et al., 2008). Unfortunately, clinical trials of antioxidants
in diabetic neuropathy have been underwhelming, although some support
has been gained from use of agents such as alpha lipoic acid that includes
antioxidant activity among its properties (Ziegler et al., 2011). The caveats
separating successful preclinical studies from demonstrable clinical efficacy,
as discussed above for ARI, may equally apply to antioxidants with the addi-
tional problem that the financial incentive for drug development in this area
is limited. A much more cohesive understanding of the multiple sources
of ROS in nerve tissue is also required. For example, endothelial cells
may indeed produce excessive ROS via high glucose flux-driven aberrant
overactivity of the proximal aspect of the mitochondrial electron transport
chain (Nishikawa, Edelstein, Du, et al., 2000). However, it is becoming rec-
ognized that neurons and glia have distinct energy demands and produce
History and Controversies of the Pathogenesis of Diabetic Neuropathy 117

ATP by operating flexible regulation of the relative levels of glycolysis and

oxidative phosphorylation (Halim et al., 2010). Indeed, in adult sensory
neurons maintained under hyperglycemic conditions the flux of electrons
via the electron transport system is significantly diminished and this is
accompanied by downregulation of the mitochondrial biogenesis pathway
(Chowdhury et al., 2010; Zhang, Zhao, Blagg, & Dobrowsky, 2012). This
reduced electron transport capacity of sensory neurons in diabetes is also
associated with lowered ROS production by the mitochondria—the oppo-
site of what happens in endothelial cells maintained under high glucose
concentration (Akude et al., 2011). It appears that hyperglycemia, or
nutrient stress, drives neurons toward relying on glycolysis for energy pro-
duction and diminishes the contribution of mitochondria (Chowdhury,
Dobrowsky, & Fernyhough, 2011; Chowdhury, Smith, & Fernyhough,
2013). Thus, excess substrate, as provided by diabetes, appears to impact
mitochondrial function and ROS formation in a cell-specific manner.
Interesting headway has been made in identifying mechanisms for the
generation of other potentially neurotoxic molecules. For example, diabe-
tes is also associated with dyslipidemia and studies suggest that certain forms
of oxidized lipoproteins are also toxic to sensory neurons and peripheral
nerve function (Vincent, Hayes, et al., 2009; Vincent, Hinder, Pop-
Busui, & Feldman, 2009). The original competing hypothesis to polyol
pathway overactivity was hyperglycemia-driven nonenzymatic glycation
of proteins leading to their dysfunction and aberrant turnover (Vlassara,
Brownlee, & Cerami, 1981). This has subsequently expanded into an
appreciation that extracellular advanced glycation end products can also
produce adverse intracellular effects, including toxic ROS formation, via
receptor-mediated signaling (Thornalley, 2002). Modification of ion chan-
nels by intermediates of glucose metabolism such as methylglyoxal or by
glycosylation of ion channels has also been explored, primarily in the con-
text of diabetes-induced changes to neuronal excitability that can produce
loss of function prior to degeneration or instability and excess activity
(Bierhaus et al., 2012; Orestes et al., 2013). The chapter by Todorovic
“Painful Diabetic Neuropathy: Prevention or Suppression?” introduces
pathways of aberrant modification of ion channels as part of the complex
etiology of pain in diabetic neuropathy. Investigation of glucotoxic mech-
anisms of nerve degeneration and pain in diabetes over the past 10 years has
greatly expanded sites for potential therapeutic intervention. While this is
encouraging, the plethora of plausible mechanisms and apparently effective
interventions identified in animal models does beg the inconvenient
118 P. Fernyhough and N.A. Calcutt

question of how so many highly selective interventions can be so successful

when the majority of other pathogenic mechanisms remain operational.
An alternative approach to intervening against the apparently complex
etiology of diabetic neuropathy has been to side-step the issue of hypergly-
cemia and its heterogeneous pathogenic mechanisms and to instead focus
on repairing the nervous system. This began with the discovery of endog-
enous neurotrophic factors and their use to enhance in-built regenerative
pathways that can repair nerve damage in diabetes (Calcutt, Jolivalt, &
Fernyhough, 2008). This approach was initially presented as exogenous
replacement of lost endogenous trophic support, and in some cases loss
of trophic support was shown to be downstream of aldose reductase activity
(Brewster, Fernyhough, Diemel, Mohiuddin, & Tomlinson, 1994;
Mizisin, Calcutt, DiStefano, Acheson, & Longo, 1997). Neurotrophic fac-
tors such as nerve growth factor (NGF), ciliary neurotrophic factor, and
neurotrophin-3 were extensively studied using in vitro and in vivo models
(Mizisin, Vu, Shuff, & Calcutt, 2004; Tomlinson, Fernyhough, & Diemel,
1997). Unfortunately, as discussed in an earlier volume (Apfel, 2002), a
flawed clinical trial with NGF failed to meet its goals and interest in this
approach diminished. In parallel, the role of insulin and insulin-like growth
factors as neurotrophic factors for sensory and sympathetic neurons was
introduced by Douglas Ishii, with the appealing implication that hypergly-
cemia was not the sole pathogenic insult in diabetes (Ishii, 1995). The non-
glucotoxic components of diabetic neuropathy are being increasingly
recognized, and the chapter “Sensory Neurodegeneration in Diabetes:
Beyond Glucotoxicity” by Zochodne reviews the latest findings demon-
strating that insulin is a very effective treatment for preventing various
molecular and structural defects associated with diabetic neuropathy inde-
pendent of glycemic regulation. This chapter also introduces other novel
glucose-independent pathways that drive nerve regeneration in diabetes,
including signal transduction routes involving the HSP-27, PTEN, and
Rb1 proteins. Dobrowsky in chapter “Promoting Neuronal Tolerance
of Diabetic Stress: Modulating Molecular Chaperones” summarizes the
state of play with regard to mobilization of the heat shock response to
repair protein damage caused by diabetes and subsequently enhance nerve
recovery. The novel pathways to encourage nerve repair introduced in
these chapters provide promising new avenues for drug discovery that
operate outside the need to manipulate glucose and thus may offer a com-
plimentary approach to the fine-tuning of glycemic control and blockade
of glucotoxic mechanisms.
History and Controversies of the Pathogenesis of Diabetic Neuropathy 119

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 CHAPTER EIGHT

Glucotoxic Mechanisms and

Related Therapeutic Approaches
S. Yagihashi1,2
Hirosaki University Graduate School of Medicine, Hirosaki, Japan
2
Corresponding author: e-mail address: [email protected]

Contents
1. Introduction 122
2. Glucose-Metabolizing Pathways Relevant to DPN 124
3. Polyol (AR or Sorbitol–Fructose) Pathway 125
3.1 Metabolic Sequelae After Increased Flux Through the Polyol Pathway 125
3.2 Studies in Transgenic and Knockout Mice 128
3.3 Effects of AR Inhibition 129
3.4 Clinical Application of ARIs 130
3.5 Polyol Pathway in Ischemia/Reperfusion Injury 131
4. Nonenzymatic Glycation and AGEs 131
4.1 Glycation of Proteins in Diabetes 131
4.2 Glycation and AGE in DPN 132
4.3 Transgenic and Knockout Mice Studies 134
4.4 Clinical Application of Antiglycation Agents 134
5. Oxidative Stress 135
5.1 Production of Oxidative Stress by Hyperglycemia 135
5.2 Oxidative Stress and Diabetic Neuropathy 136
5.3 Effects of Antioxidants on Diabetic Neuropathy 137
6. PKC Activity 138
7. Glycosylation 139
7.1 The Hexosamine Pathway 139
7.2 Other Glycosylation Events 140
8. Conclusion 141
Acknowledgments 141
References 141

Abstract
Neuropathy is the earliest and commonest complication of diabetes. With increasing
duration of diabetes, frequency and severity of neuropathy are worsened. Long-term

1
Present address: The Nukada Institute of Medical and Biological Research, 4-16 Inage-machi,
Inage-ku, Chiba, Japan. E-mail: [email protected].

International Review of Neurobiology, Volume 127 # 2016 Elsevier Inc. 121

ISSN 0074-7742 All rights reserved.
http://dx.doi.org/10.1016/bs.irn.2016.03.006
122 S. Yagihashi

hyperglycemia is therefore implicated in the development of this disorder. Nerve tissues

require glucose energy to function and survive. Upon excessive glucose entry into the
peripheral nerve, the glycolytic pathway and collateral glucose-utilizing pathways are
overactivated and initiate adverse effects on nerve tissues. During hyperglycemia, flux
through the polyol pathway, formation of advanced glycation end-products, produc-
tion of free radicals, flux into the glucosamine pathway, and protein kinase C activity
are all enhanced to negatively influence nerve function and structure. Suppression of
these aberrant metabolic pathways has succeeded in prevention and inhibition of
the development of neuropathy in animal models with diabetes. Satisfactory results
were not attained, however, in patients with diabetes and further clinical trials are
required. In this review, the author summarizes the hitherto proposed theories on
the pathogenesis of diabetic neuropathy related to glucose metabolism and future
prospects for the effective treatment of neuropathy.

1. INTRODUCTION
There is a long history of research to clarify the pathogenetic mech-
anism of diabetic polyneuropathy (DPN), but it remains unsettled. Since the
prevalence of DPN is primarily dependent on the duration of diabetes and
the degree of blood glucose control, long-term metabolic aberration is con-
sidered to be a major cause of DPN (Pirart, 1978). A large prospective study
disclosed that hyperglycemia, duration of diabetes, hypertension, hyperlip-
idemia, and smoking are significant risk factors for the development of DPN
in patients with type 1 diabetes (Tesfaye et al., 2005). Indeed, the 10-year
follow-up of patients with type 1 diabetes by the Diabetic Complication
and Control Trial (DCCT) demonstrated that meticulous blood glucose
control by intensive insulin therapy suppressed the incidence of DPN
(DCCT Research Group, 1993). The effects of tight glycemic control per-
sisted for a further 8 years after the termination of the trial as legacy effects
termed glucose or metabolic memory (Martin et al., 2006). Although the
role of hyperglycemia in driving the progression of DPN in type 2 diabetes
is less clear (UKPDS Group, 1998), continuous lowering of glycated hemo-
globin below 7% for 6 years was found to suppress the deterioration of DPN
in patients with type 2 diabetes (Ohkubo et al., 1995). Despite the ample
epidemiological data, there remains no clear-cut explanation why long-term
hyperglycemia leads to DPN.
It is known that the most distal portions of somatic nerves are preferen-
tially affected in diabetes. Both anatomical and biochemical characteristics of
Glucotoxicity and Diabetic Neuropathy 123

the peripheral nervous system seem to contribute to the distal and sensory
predominant nerve lesions in diabetes (Dyck et al., 1984; Dyck, Lais,
Karnes, O’Brien, & Rizza, 1986). Structurally, the sensory neurons extend
extremely long axons from their cell bodies in the dorsal root ganglia
(DRG). The relative imbalance in this architecture between the soma
and axon can result in insufficient supply of nerve nutrients and energy from
the cell body to the distal processes. The anatomic organization of the vas-
cular supply to the peripheral nerve may also impose negative impacts on
the peripheral nerve because of the relative paucity of endoneurial blood
vessels and their lack of autoregulation (Kihara, Schmelzer, et al., 1991;
Smith, Kobrine, & Rizzoli, 1977). Such a vascular system likely renders
the peripheral nerve ischemic, resulting in severe pathology when ischemia
is further exaggerated by vascular occlusion (Nukada, McMorran, Baba,
Ogasawara, & Yagihashi, 2011), though ischemic diabetic nerve is tempo-
rarily resistant to nerve conduction failure (Low, Schmelzer, & Ward,
1986). Endoneurial microangiopathic changes characteristic of diabetes
may further augment nerve damage due to increased permeability and
ischemia (Dyck & Giannini, 1996; Malik et al., 2005; Thrainsdottir
et al., 2003). Features such as the strong expression of aldose reductase
(AR) in peripheral nerve and the abundance of interstitial collagen impli-
cate the polyol pathway and glycation as etiological factors in DPN.
Glucose metabolism in the peripheral nerve is not completely under-
stood. Peripheral nerves express the GLUT-1 glucose transporter at the
perineurial and endothelial blood:nerve barriers (Stark, Carlstedt,
Cullheim, & Risling, 2000), and glucose uptake into nerve is therefore insu-
lin independent so that endoneurial glucose concentrations will follow
plasma glucose concentrations. Under normoglycemic conditions, the
majority of glucose entering the cytoplasm is used by mitochondria to pro-
duce adenosine triphosphate (ATP) through the tricarboxylic acid (TCA)
cycle, downstream from the glycolytic pathway. In neurons, synthesized
proteins, cytoplasmic organelles, cargoes of microtubules and neuro-
filaments, as well as neurotransmitters, are all transported in an energy-
requiring process to the distal nerve endings. It has been proposed that if
the energy production in the axon is deficient then the distal portion is first
affected by the insufficient supply of these materials (Scott, Clark, &
Zochodne, 1999; Yagihashi, Kamijo, & Watanabe, 1990). Additionally,
hyperglycemia makes glucose available for other uses and perturbed glyco-
lytic pathways may lead to impaired nerve function and disruption of
124 S. Yagihashi

nerve structure. In this review, I will list the collateral glycolytic pathways
operating downstream of hyperglycemia and discuss their possible implica-
tion in the onset and development of DPN.

2. GLUCOSE-METABOLIZING PATHWAYS RELEVANT

TO DPN
There are a number of metabolic pathways that operate collaterally to
glycolysis that may contribute to development of DPN as a downstream
consequence of hyperglycemia (Fig. 1).
(1) The polyol pathway uses AR to produce sorbitol, which is then
converted to fructose by sorbitol dehydrogenase (SDH) (Gabbay,
1975; Kinoshita, 1990). AR has a relatively low affinity for D-glucose

Fig. 1 Glycolytic pathways in hyperglycemia proposed for the cause of diabetic com-
plications. In normoglycemia, intracellular glucose undergoes glycolysis and enters the
TCA cycle in mitochondria to produce ATP. Hyperglycemia induces an increased flux
through the polyol pathway in endothelial and Schwann cells, nonenzymatic glycation
with AGE formation, superoxide production in endothelial cell mitochondria, increased
activity of protein kinase C, and enhancement of the hexosamine pathway. These com-
plicated glycolytic pathways are proposed to jointly lead to functional and structural
abnormalities encountered in diabetic complications. It should be of note, however,
that this theory is mainly built from data on vascular complications in diabetes, and
not uniformly applied to neuropathy. For example, reactive oxygen species production
in mitochondria is reduced in diabetic neurons (see text).
Glucotoxicity and Diabetic Neuropathy 125

so this metabolic process is enhanced only when glucose concentrations

are high in the cytoplasm.
(2) Hyperglycemia leads to activation of the glycation or advanced glycation
end-products (AGEs) pathway. Excessive glucose attaches to the amino
base of proteins in a nonenzymatic manner, resulting in the production
of AGEs (Brownlee, 1992; Yamagishi, Nakamura, & Imaizumi, 2005).
AGE then binds with the receptor for AGE (RAGE), giving rise to
negative influences on cells and tissues.
(3) Activation of oxidative stress pathways that produce reactive oxygen
species (ROS), in part through enhanced mitochondrial respiration
in endothelial cells, is considered to be a major pathway for vascular
and neural tissue damage in diabetes (Baynes & Thorpe, 1999;
Brownlee, 1992; Leinninger, Edwards, Lipshaw, & Feldman, 2006).
(4) Activity of the PKC pathway is augmented by hyperglycemia. With
increased production of diacylglycerol, the protein kinase C (PKC)-β
isoform was shown to be activated and to impair vascular function with
presumed downstream consequences for neuronal function (Geraldes &
King, 2010; King & Brownlee, 1996).
(5) Glycosylation: recent studies have introduced the glucosamine pathway as
a potentially causative factor in DPN (Hardivill
e & Hart, 2014; James
et al., 2002). With excessive entry of glucose into the cell, a glucose-
attached amine, termed glucosamine (GlcN), accumulates. Since the
glucose modification of amine groups is mediated by the enzyme
glutamine-fructose-6-phosphate amidotransferase (GFAT) (synonym;
glucosamine-6-phosphate synthase), this process is distinct from the
process of nonenzymatic glycation. Other glycosylation events and
glucose-derived products such as methylglyoxal (MG) have also been
implicated in aspects of diabetic neuropathy.
I will now deal in more detail with the processes outlined in each pathway as
listed earlier.

3. POLYOL (AR OR SORBITOL–FRUCTOSE) PATHWAY

3.1 Metabolic Sequelae After Increased Flux Through
the Polyol Pathway
A complete mechanism of how the polyol pathway is involved in develop-
ment of diabetic neuropathy remains elusive. Sorbitol is an important
osmoregulator and does not readily permeate the cell membrane. Accumu-
lation of sorbitol therefore causes increased osmotic pressure within the cell
126 S. Yagihashi

Fig. 2 Implication of polyol pathway in the pathogenesis of diabetic neuropathy. With

excessive flux of polyol pathway, accumulation of sorbitol as osmolytes elicits increased
osmotic pressure within the cytoplasm, resulting in depletion of other osmolytes such
as myo-inositol, taurine, and adenosine. Since myo-inositol and taurine are important
substrates for energy production of phosphatidylinositol, downstream metabolic aber-
ration results in insufficient energy production with reduced activity of protein kinase
C-α and Na,K-ATPase activity (1). Conversion of glucose to sorbitol by aldose reductase
requires consumption of coenzyme of NADPH which is also utilized for the production
of reduced glutathione. Therefore, depletion of reduced glutathione results in excessive
free radical formation (2). On the other hand, the conversion from sorbitol to fructose
induces redox changes of NADH/NAD (3), which in turn increases phosphatidic acid and
diacylglycerol, resulting in activation of protein kinase C-β (4). Oxidative stress also acti-
vates poly(ADP)-ribose polymerase (PARP), which causes nuclear DNA damage, caspase
activation, and cell death. It also causes inhibition of GAPDH to increase the level of glyc-
eraldehyde, leading to AGE formation and PKC-β activation (5).

(Fig. 2). The original osmotic theory proposed that increased polyol path-
way flux during hyperglycemia caused intracellular hyperosmolarity by
accumulation of sorbitol, resulting in the expansion of cells and ultimately
cell lysis (Gabbay, 1975; Kinoshita, 1990). However, although increased
sorbitol concentrations were demonstrated in nerves of persons with diabe-
tes, there was no evidence of nerve edema, swollen cells, or correlation with
nerve fiber pathology (Dyck et al., 1980, 1988).
Greene and his associates proposed the poor energy utilization theory as a
surrogate of the osmotic theory (Greene, Lattimer, & Sima, 1987; Greene,
Sima, Stevens, Feldman, & Lattimer, 1992). With accumulation of sorbitol,
other osmolytes including myo-inositol, taurine, and adenosine are depleted
Glucotoxicity and Diabetic Neuropathy 127

in the cytoplasm. In turn, myo-inositol deficiency results in depletion of

phosphatidylinositol, poor production of ATP, and reduced activities of
Na,K-ATPase and protein kinase C (PKC)-α. This theory was criticized
due to the fact there was no clear myo-inositol depletion in nerves of persons
with diabetes (Dyck et al., 1980, 1988). However, total nerve myo-inositol
levels may not necessarily be low in the setting of increased polyol flux with
critical decreases occurring in biologically active subpools (Simmons,
Kern, Winegrad, & Martin, 1986; Winegrad, 1987). The precise relation-
ship between the polyol flux and energy utilization in diabetes is still open
to question.
Subsequent studies have suggested that adverse effects of increased polyol
pathway flux are mediated by increased oxygen radicals and poor nitric
oxide (NO) production (Greene, Stevens, Obrosova, & Feldman, 1999;
Obrosova, 2009; Pop-Busui, Sima, & Stevens, 2006). Conversion of glucose
to sorbitol depletes the coenzyme nicotinamide adenine dinucleotide phos-
phate (NADPH). NADPH is used for the production of reduced glutathi-
one, a major scavenger of free radicals, and is also required for the production
of NO during the conversion of arginine to citrulline. Excessive polyol
pathway flux and subsequent exhaustion of NADPH can therefore cause
increased free radicals and poor production of NO (Stevens et al., 1994)
so that tissues suffer from free radical injury and impaired vascular function
due to NO depletion.
The second half of polyol pathway, involving conversion of sorbitol to
fructose by SDH, also elicits redox changes when driven to excess, with
an exaggerated ratio of NADH/NAD leading to lower oxygen levels,
similar to the condition of hypoxia. These conditions have been proposed
as a pseudohypoxia that accounts for early neural and vascular dysfunction
in diabetes (Ido et al., 2010; Williamson et al., 1993). However, studies
with inhibitors of SDH that block the second part of the polyol pathway
while allowing flux through AR to proceed have not shown consistent effi-
cacy against neuropathy (Schmidt et al., 2001). Conversely, feeding animals
galactose, an alternative hexose substrate for AR which converts it to
galactitol (dulcitol), produces a significant slowing of nerve conduction
and nerve fiber atrophy (Calcutt, Tomlinson, & Biswas, 1990; Nukada,
Dyck, Low, Lais, & Sparks, 1986) that parallels aspects of neuropathy
reported in streptozotocin (STZ)-induced diabetes. However, some down-
stream aspects of peripheral nerve metabolism in galactose-fed animals are
opposite to those reported in STZ-diabetic rats. For example, nerve from
galactose-fed rats shows endoneurial oedema and increased Na,K-ATPase
128 S. Yagihashi

activity (Lambourne, Tomlinson, Brown, & Willars, 1987), whereas

Na,K-ATPase activity is widely reported as being reduced in STZ-diabetic
rats. Exaggerated flux through the entire polyol pathway may be necessary
to precipitate the full repertoire of disorders associated with diabetic
neuropathy.

3.2 Studies in Transgenic and Knockout Mice

We have constructed transgenic mice overexpressing human AR (hAR-Tg)
in the search for a cause of DPN. hAR-Tg mice develop severe neuropathy
when they were fed galactose (Yagihashi et al., 1996). Thus, without hyper-
glycemia or insulin deficiency, increased flux through the first part of the
polyol pathway caused peripheral nerve dysfunction and myelinated fiber
changes that are similar to findings in diabetic animals. The study was also
extended to STZ-induced diabetes, and there was more severe nerve conduc-
tion slowing and reduced Na,K-ATPase activity in diabetic hAR-Tg mice
with an enhanced accumulation of sorbitol and fructose, compared with
nontransgenic diabetic mice, despite comparable levels of hyperglycemia
(Yagihashi et al., 2001). The functional decay in nerve was accompanied
by structural changes and alterations of neuropeptide expression in DRG
(Uehara, Yamagishi, Otsuki, Chin, & Yagihashi, 2004). Concurrently, dia-
betic hAR-Tg mice showed a reduction of PKC activity in the endoneurium
with decreased membranous expression of PKC-α and a relative increase
in the expression of the PKC-β isoform (Yamagishi, Uehara, Otsuki &
Yagihashi, 2003). These neuropathic changes in diabetic hAR-Tg mice were
markedly improved by the AR inhibitor (ARI), fidarestat.
The pertinence of AR activity to diabetic neuropathy in rodents was fur-
ther established by studies in AR-deficient mice made diabetic by STZ
(Ho et al., 2006). In this model, glutathione levels were preserved and
peripheral nerve function and structure remained normal, despite protracted
hyperglycemia. These studies convincingly confirm that AR contributes to
the development of DPN. In contrast, mice lacking the second enzyme of
the polyol pathway, SDH, continued to develop nerve conduction slowing
during STZ-induced diabetes that matched that of STZ-diabetic wild-type
mice, despite exaggerated sorbitol accumulation (Ng et al., 1998). Together,
these data place focus on exaggerated flux through AR as being the major
pathogenic component of polyol pathway overactivity that contributes to
the nerve conduction deficit during hyperglycemia, rather than simple
sorbitol accumulation.
Glucotoxicity and Diabetic Neuropathy 129

3.3 Effects of AR Inhibition

Early studies showed that inhibition of polyol pathway flux by an ARI
succeeded in protection of peripheral nerve function, structure, and axonal
transport in rats with experimental diabetes (Tomlinson, Moriarty, &
Mayer, 1984; Yagihashi, Kamijo, Ido, & Mirrlees, 1990). Subsequent studies
demonstrated that oxidative and nitrosative stress contributed tissue damage in
STZ-diabetic rats (Obrosova, Drel, et al., 2005) and animals fed with a high-
fat diet (Obrosova et al., 2007) and that an ARI was protective, suggesting
that these potentially toxic stressors were downstream of polyol pathway flux.
AR inhibition also suppressed oxidative stress-induced poly(ADP-ribose)
polymerase (PARP) activation, which was closely associated with a neuro-
pathic phenotype in diabetic animals (Obrosova, Pacher, et al., 2005). Inter-
estingly, a potential pathogenic contribution of the SDH component of the
polyol pathway has recently been reemphasized by studies of the relationship
between enhanced polyol pathway flux and Sirtuin 2 expression (Schartner,
Chowdhury, Smith, & Fernyhough, 2015). Sirtuin is an NAD-dependent
protein deacetylase that participates in diverse biological processes such as cell
cycle control, genomic integrity, cell differentiation, metabolic networks, and
aging. Sirtuin 2 expression is reduced in DRG of diabetic animals. Using
DRG neuron cultures derived from adult rodents and grown under a high
glucose concentration, it was demonstrated that blockade of the polyol path-
way using an SDH inhibitor restored Sirtuin 2 expression, optimized neurite
outgrowth, and augmented mitochondrial function.
Hyperglycemia-induced exaggerated flux through the polyol pathway
also induces structural changes in autonomic nerves as indicated by axonal
dystrophy and a reduction of synapses (Schmidt, 2002). AR inhibition sig-
nificantly reduced the frequency of dystrophies, whereas treatment with an
SDH inhibitor had no protective effect, indicating specific involvement of
the first segment of the polyol pathway in structural damage to autonomic
nerves (Schmidt et al., 2001; Schmidt, Plurad, Sherman, Williamson, &
Tilton, 1989). This finding is contradictory to the reported beneficial effects
of an SDH inhibitor on vascular dysfunction in diabetes (Ido et al., 2010;
Williamson et al., 1993). The effects of polyol inhibition therefore appear
to be different between neural and vascular tissues and could be accounted
for by a difference in the tissue distribution of the two enzymes. We, and
others, have reported that AR was highly expressed in Schwann cells of
somatic nerves (Kasajima, Yamagishi, Sugai, Yagihashi, & Yagihashi,
2001) and in axons of autonomic nerves ( Jiang, Calcutt, Ramos, &
130 S. Yagihashi

Fig. 3 Topographic difference in the localization of the enzymes that catalyze polyol
pathway. Aldose reductase locates in the endoneurium, mainly Schwann cells and
the wall of epineurial artery. On the other hand, sorbitol dehydrogenase (SDH)
expresses mainly in the wall of epineurial artery, but not much in the endoneurium.
Such difference may contribute to the distinct downstream of metabolic signals such
as protein kinase C and redox changes. Cited from Kasajima, H., Yamagishi, S., Sugai,
S., Yagihashi, N., & Yagihashi, S. (2001). Enhanced in situ expression of aldose reductase
in peripheral nerve and renal glomeruli in diabetic patients. Virchows Arch, 439, 46–54.

Mizisin, 2006), whereas SDH was more highly expressed in vascular tissues
(Fig. 3). Such anatomical differences in the localization of polyol pathway
enzymes may also be involved in the differential effects of diabetes on
PKC activity between nerve and vascular tissues, where it is increased in vas-
cular tissues and decreased in neural tissues (Yamagishi et al., 2003).

3.4 Clinical Application of ARIs

Based on preclinical studies, many ARIs have been developed. In early trials,
patients treated with ARI (sorbinil or zenarestat) for a year showed increased
nerve fiber density in the sural nerve biopsy specimens compared to the
untreated group (Greene, Arezzo, & Brown, 1999; Sima et al., 1988).
Unfortunately, safety concerns of sorbinil and zenarestat did not allow the
approval of the compounds for clinical applications. The majority of other
ARI clinical trials have been unsuccessful in meeting efficacy requirements
for regulatory approval. Factors contributing to this lack of success include
dose efficacy, short trial durations, and inappropriate patient selection. Cur-
rently, epalrestat (ONO2235) is the only approved ARI, and it is licensed
only in Japan. Epalrestat was approved after a 3-month double-blinded
trial, which showed improvement of symptoms and nerve conduction
(Goto, Hotta, Shigeta, Sakamoto, & Kikkawa, 1995). Further clinical studies
confirmed that epalrestat treatment significantly suppressed progressive
nerve conduction slowing over a 3-year period (Hotta et al., 2006, 2008)
Glucotoxicity and Diabetic Neuropathy 131

and appearance of negative symptoms such as sensory loss (Baba, Kimura,

Suda, Yagihashi, & Aomori Diabetic Study (ADNS) Group, 2006). In a
long-term follow-up, positive symptoms such as pain or paresthesia were
not influenced by ARI (Baba et al., 2006). The ARI effects on nerve con-
duction were most marked in patients with early neuropathy and exhibiting
modestly elevated levels of glycated hemoglobin (Hotta et al., 2006). The
ARI ranirestat has also been shown to produce modest, but statistically sig-
nificant, improvements in peroneal motor nerve conduction velocity, again
in patients with relatively mild neuropathy (Bril & Buchanan, 2006). In a
more recent study, however, comparison of its effects with a placebo group
of well-controlled diabetic subjects faced challenges to obtain significant dif-
ferences (Polydefkis et al., 2015).
Although these studies confirmed the critical role of AR in contributing
to the development of DPN, it seems to be clear from the clinical experience
of ARI trials that the polyol pathway is unlikely to be the only causative fac-
tor in DPN.

3.5 Polyol Pathway in Ischemia/Reperfusion Injury

Emerging evidence suggests that AR is strongly implicated in ischemia/
reperfusion injury in nondiabetic conditions (Ramasamy & Goldberg,
2010). In experimental studies, ARI alleviated the pathological lesions
observed during infarction of the heart (Iwata, Matsuno, Nishinaka,
Persson, & Yabe-Nishimura, 2006; Ramasamy & Goldberg, 2010), the brain
(Yeung et al., 2010), and the retina (Cheung, Lo, So, Chung, & Chung,
2007). Our study also found that acute kidney injury caused by renal ischemia
(Yagihashi et al., 2010) or experimental septicemia (Takahashi et al., 2012)
was rescued by ARI. Because diabetic nerves are susceptible to ischemia/
reperfusion injury (Nukada et al., 2011), it is possible that ischemia/reperfu-
sion is also involved in the progression or exacerbation of DPN and that ARI
might be effective in treating this disorder.

4. NONENZYMATIC GLYCATION AND AGEs

4.1 Glycation of Proteins in Diabetes
Increased nonenzymatic glycation of proteins is considered to be a
major contributor to a variety of diabetic complications including
micro- and macroangiopathy (Brownlee, 1992; Yamagishi et al., 2005).
132 S. Yagihashi

In a hyperglycemic environment, glucose is attached nonenzymatically to

amino residues of proteins forming glucose adducts as initial glycation
metabolites that progress from Schiff base to Amadori products
(Thornalley, 2002). These products are unstable and likely to transform
to MG or 3-deoxyglucosone (3DG) as intermediate glycation products,
both of which are toxic to neural tissues (Kikuchi et al., 1999; Sekido
et al., 2004) (Fig. 1). Increased levels of MG, an intermediate glycation
product also arising from intermediates of glycolysis, have also been asso-
ciated with pain and hyperalgesia in diabetic subjects (Bierhaus et al.,
2012). MG has been reported to bind to the Nav1.8 sodium channel of
nociceptive neurons. However, others have emphasized that relative activ-
ity of glyoxalase I, the degrading enzyme of MG, may be the critical
parameter ( Jack, Ryals, & Wright, 2011). Intermediate glycation products
may also crosslink together, forming large molecules of AGEs. AGE depo-
sition has been found in nerves of diabetic patients and animal models of
diabetes. Every component of peripheral nerve tissue is affected including
axoplasm, Schwann cells, endoneurial vessels, and stromal collagen
(Sugimoto, Nishizawa, Horiuchi, & Yagihashi, 1997). Glycation of
tubulin or neurofilament is suggested to trigger disturbed axoplasmic flow
of these proteins leading to axonal atrophy and degeneration (McLean,
Pekiner, Cullum, & Casson, 1992; Ryle, Leow, & Donaghy, 1997;
Williams, Howarth, Devenny, & Bitensky, 1982). Glycated laminin,
fibronectin, and collagen are also invoked as contributing to the impairment
of nerve regeneration reported in diabetic animals (Duran-Jimenez et al.,
2009). Thus, glycation alters the basic structure of tissue components, resulting
in pathologic alterations of the peripheral nerve, with the intensity of AGE
deposition detected by carboxymethyllysine immunoreactions correlating
well with reduced myelinated fiber density (Sugimoto et al., 1997;
Sugimoto, Yasujima, & Yagihashi, 2008).

4.2 Glycation and AGE in DPN

In diabetes, serum concentrations of AGE are elevated, and clearance is dis-
turbed in cases with renal insufficiency. In vascular endothelial cells, AGE
binds with its RAGE, resulting in activation of a downstream cascade
including NADPH oxidase with subsequent release of free radicals and
activation of the transcription factor, nuclear factor-κB (NF-κB)
(Haslbeck et al., 2005) (Fig. 4). RAGE is expressed by many cells in
Glucotoxicity and Diabetic Neuropathy 133

Fig. 4 Biological reactions elicited by the binding of AGE with RAGE in endothelial cells
and neural tissues. AGE exerts activation of NADPH oxidase to release oxygen radicals
after binding with RAGE. Concurrently, nuclear factor-κB (NF-κB) is transferred into the
nuclei to promote gene activation. This results in cellular dysfunction and activation of
cell death signals.

peripheral nerve including vascular endothelial cells, Schwann cells, and

neurons (Wada & Yagihashi, 2005). Therefore, high AGE in situ may elicit
adverse effects on peripheral nerve tissues by signaling through RAGE.
Indeed, embryonic sensory neurons underwent functional degeneration
and apoptosis when exposed to AGE via production of oxidative stress
(Vincent, McLean, Backus, & Feldman, 2005), while endoneurial endo-
thelial cells exposed to exogenous AGE show high expression of
NF-κBp65 together with swollen and vacuolar changes at the ultrastruc-
tural levels (Nishizawa et al., 2010). AGE-induced toxicity has also been
reported in vivo, as animals given exogenous AGE developed vascular dys-
function and a neuropathy resembling that caused by diabetes, with
reduced nerve Na,K-ATPase activity, fiber size, and conduction velocity
(Nishizawa et al., 2010).
The association of AGE formation and neuropathy has been tested using
agents that inhibit AGE formation. Inhibition of AGE accumulation by ami-
noguanidine or OPB9135 was found to suppress the development of
134 S. Yagihashi

neuropathy, confirming the pathogenic role of AGE in DPN (Wada et al.,

2001; Yagihashi, Kamijo, Baba, Yagihashi, & Nagai, 1992). The ameliora-
tion of neuropathy by aminoguanidine was also associated with improve-
ment of nerve blood flow (Kihara, Weerasuriya, & Low, 1991).
However, effects of aminoguanidine should be interpreted with caution,
since it is also a selective inhibitor of inducible nitric oxide synthase
(Corbett & McDaniel, 1996).

4.3 Transgenic and Knockout Mice Studies

Animals transgenic for RAGE were developed to clarify the biological con-
sequences of AGE/RAGE reactions (Inagi et al., 2006; Yamamoto et al.,
2001). RAGE overexpression by endothelial cells induced characteristic
pathology in the kidney in diabetes (Yamamoto et al., 2001). Over-
expression of RAGE in vascular endothelial cells also produced a more
severe neuropathy phenotype after induction of diabetes by STZ when
compared to nontransgenic diabetic mice (Wada & Yagihashi, 2005). In
contrast, diabetic mice lacking the RAGE gene were protected against
the development of neuropathy and showed enhanced regeneration after
nerve injury ( Juranek et al., 2013). These findings support the role of
AGE/RAGE in the functional and structural development of DPN.

4.4 Clinical Application of Antiglycation Agents

The potential pathogenic role of AGE may extend to subjective symptoms
of diabetic neuropathy as sensory loss in diabetic subjects was closely related
to the increased concentrations of AGE and its downstream signals of NF-κB
(Bierhaus et al., 2004). Aminoguanidine was tested as an antiglycation agent
in a clinical trial in which the primary target was nephropathy. It was initially
reported to be effective at inhibiting nephropathy, as indicated by a reduc-
tion in proteinuria, but no significant improvement was found at the study
end (Bolton et al., 2004). Information for neuropathy is not available for
aminoguanidine. Benfotiamine, a vitamin B1 derivative with AGE clearing
and antioxidant properties, has shown some efficacy on neuropathy symp-
tom scores, most prominently on pain scores, in patients with diabetes fol-
lowing studies of 3–6 weeks duration (Haupt, Ledermann, & K€ opcke, 2005;
Stracke, Gaus, Achenbach, Federlin, & Bretzel, 2008). The combination of
benfotiamine with vitamins B6 and B12 also improved nerve conduction in
diabetic subjects following 9 months of treatment (Stracke, Lindemann, &
Glucotoxicity and Diabetic Neuropathy 135

Federlin, 1996). However, since these studies used a rather small number of
subjects, more definitive trials are required.
Other agents that intervene against AGE accumulation or the biological
reactions downstream of AGE/RAGE interaction have also been devel-
oped, including pyridoxamine/vitamin B6 (Metz, Alderson, Thorpe, &
Baynes, 2003), AGE breakers (Vasan, Foiles, & Founds, 2003), and soluble
RAGE (Nagai, Shirakawa, Ohno, Moroishi, & Nagai, 2014), although at
present there is no information regarding the effects of these agents against
diabetic neuropathy in clinical trials.

5. OXIDATIVE STRESS
5.1 Production of Oxidative Stress by Hyperglycemia
The generation of free radicals due to increased flux of glucose through
glycolysis has been proposed as a major contributor to diabetic complications,
including DPN. This hypothesis was originally raised by the short-term
experiments mostly on in vitro endothelial cells and is still not confirmed
in neural tissues. During hyperglycemia, increased mitochondrial oxidation
of NADH and FADH2 leads to excessive formation of superoxide ions,
a form of ROS (Brownlee, 2005) (Fig. 5). Reduced expression of
manganese–superoxide dismutase also contributes to production of ROS
(Boyle, Newsom, Janssen, Lappas, & Friedman, 2013; Santos, Tewari,
Goldberg, & Kowluru, 2011). Thus, in endothelial cells superoxide produc-
tion, possibly via enhanced mitochondrial electron transport, could induce
DNA injury, which in turn drives increased expression of poly(ADP)-ribose
polymerase (PARP) to repair DNA damage. Activated PARP inhibits the
action of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Obrosova,
Drel, et al., 2005). Consequently, upstream glycolytic metabolites before
GAPDH will be increased in the cytoplasm, which in turn induces
enhancement of four glycolytic pathways during hyperglycemia (Baynes &
Thorpe, 1999; Brownlee, 1992). However, recent studies do not support
the oxidative stress having a mitochondrial origin in sensory neurons in
experimental diabetes (Akude et al., 2011; Zherebitskaya, Akude, Smith, &
Fernyhough, 2009). These authors found reduced electron transport in mito-
chondria which was associated with reduced superoxide production, which
makes sense since less electron flux equates with less electron leakage. The
Dobrowsky group also detected reductions in the rates of electron transport
in adult sensory neurons exposed to hyperglycemia or derived from diabetic
mice (Urban et al., 2012; Zhang, Zhao, Blagg, & Dobrowsky, 2012). Studies
136 S. Yagihashi

Fig. 5 Mitochondrial electron transport and superoxide production. For the production
of ATP NADH is first oxidized to NAD with release of electrons at the site of complex I of
inner mitochondrial membrane. At the site of complex II, conversion of succinate to
fumarate is mediated with FADH2 conversion to FAD. Then, electrons are directly trans-
ferred at the complex III from coenzyme Q (Co-Q) to cytochrome C (Cyt-C). If this step is
augmented by hyperglycemia in endothelial cells or blocked by drugs such as actino-
mycin D, superoxide radical formation is enhanced. Recent data suggest that this
theory is only applicable to vascular tissues, but not neurons (see text). Modified from
Brownlee, M. (2005). The pathobiology of diabetic complications: A unifying mechanism.
Diabetes, 54, 1615–1625.

in cultured Schwann cells showed no augmentation of superoxide under high

glucose concentration (Zhang et al., 2010). These studies in neuronal cells sug-
gest a diversity in the role of mitochondria and contribution to oxidative stress
in various tissues.

5.2 Oxidative Stress and Diabetic Neuropathy

There are numerous data that have shown oxidative stress-induced tissue
injury in the peripheral nerve in experimental diabetes, though its origin
is still controversial (Baynes & Thorpe, 1999; Brownlee, 1992;
Rabbani & Thornalley, 2015; Thornalley, 2002; Yagihashi, Mizukami, &
Sugimoto, 2011). Based on this background, attempts have been made to
inhibit DPN with antioxidants. In particular, α-lipoic acid has been used
for suppression of oxidative stress in experimental diabetic rats and found
Glucotoxicity and Diabetic Neuropathy 137

to protect nerve conduction, nerve blood flow, and nerve structure

(Cameron, Cotter, Archibald, Dines, & Maxfield, 1994; Cameron,
Cotter, & Maxfield, 1993; Kishi et al., 1999).
Obrosova et al. (2004) demonstrated that PARP-deficient mice
exhibited no nerve conduction delay or impaired nerve blood flow in
STZ-induced diabetes. They further demonstrated that inhibition of PARP
significantly prevented the development of DPN by suppression of oxidative
stress (Lupachyk, Shevalye, Maksimchyk, Drel, & Obrosova, 2011). In
diabetic rats, hyperglycemia stimulated NO production and increased per-
oxynitrate by superoxide reaction, which impaired function in epineurial
and endoneurial arterioles (Obrosova, Drel, et al., 2005). Conversely, deg-
radation of peroxynitrates ameliorated the nerve conduction delay or pain
sensation in diabetic mice. Since NO or peroxynitrate cannot be measured
directly because of their instability, nitrotyrosine as nitrate or peroxynitrate-
derived metabolites of NO were used as markers of nitrosylation of proteins,
or biochemical marker of nitrosative stress (Ilnytska et al., 2006).
It is interesting that a small amount of insulin, which does not alter
systemic blood glucose levels, was shown to improve the impaired
mitochondrial membrane potential and delayed nerve conduction in
STZ-diabetic rats (Chowdhury et al., 2010; Huang et al., 2003). Impaired
signaling from adenosine monophosphate-activated protein kinase is also
found to associate with mitochondrial dysfunction and neuropathy in
STZ-diabetic rats, in which reduced production of ROS in mitochondria
was characteristic in DRG (Akude et al., 2011; Roy Chowdhury et al.,
2012). Thus, the derivation and possible role of oxidative stress in diabetic
neuropathy seems to be different from vascular tissues and should be inter-
preted with caution.

5.3 Effects of Antioxidants on Diabetic Neuropathy

α-Lipoic acid is the most extensively studied antioxidant in humans with dia-
betic neuropathy and is marketed in Germany (Ziegler & Gries, 1997; Ziegler,
Reljanovic, Mehnert, & Gries, 1999). In experimental animal models, posi-
tive effects of α-lipoic acid were found on nerve blood flow, conduction
velocity, and peripheral nerve structure (Cameron, Cotter, Horrobin, &
Tritschler, 1998; Kishi et al., 1999; Low, Nickander, & Tritschler, 1997;
Stevens, Obrosova, Cao, Van Huysen, & Greene, 2000). A short-term clinical
study reported that α-lipoic acid alleviated neuropathic symptoms in patients
138 S. Yagihashi

with diabetes (Ametov et al., 2003), and this finding was replicated in the
NATHAN study of 4 years duration (Ziegler et al., 2011). However, the
results concerning efficacy against nerve conduction slowing and other objec-
tive endpoints were inconclusive due to minimal progression of neuropathy
over the study period.

6. PKC ACTIVITY
PKC plays a role in maintaining normal nerve function, and its aber-
rant regulation may contribute to the pathogenesis of DPN (Way, Katai, &
King, 2001). PKC has several isoforms from α, β, γ, δ, and so on. Under
ambient hyperglycemia, glucose is converted to glyceraldehydes-3-
phosphate and phosphatidic acid, which in turn changes into diacylglycerol
that serves as a substrate of PKC-β. Vascular tissues in diabetes thus show
increased PKC activity, leading to increased permeability and dysfunction
(Geraldes & King, 2010). An inhibitor of PKC-β was found to be effective
for correction of vascular abnormalities in diabetic animals (King &
Brownlee, 1996). Based on the premise that microangiopathy in nerve
may contribute to development of neuropathy, preclinical and clinical trials
of PKC-β inhibitor were conducted. Experimental studies demonstrated
beneficial effects of a PKC-β inhibitor on peripheral nerve dysfunction
and nerve blood flow in diabetic rats (Cameron & Cotter, 2002). However,
the alterations of PKC activity are complex in nerves and their supportive
vascular systems, in part due to the fact that the major enzymes of collateral
glycolytic pathways are different between these two tissues (Fig. 3) (Kasajima
et al., 2001). Such nonuniform tissue composition might explain inconsis-
tent findings regarding PKC activity in diabetic nerves. Thus, Nakamura
et al. (1999) did not find any significant change of PKC activity in homog-
enates of whole peripheral nerve from STZ-diabetic rats, although a PKC-
β-specific inhibitor improved nerve conduction slowing and nerve blood
flow. In contrast, in our studies on STZ-induced diabetic mice, we separated
the peripheral nerve tissues into endoneurium and epineurium for the mea-
surement of PKC activity, the latter of which was rich in microvessels
(Yamagishi et al., 2003). We found that the former showed decreased
PKC activity with significantly decreased expression of the membranous
PKC-α isoform, whereas the latter showed increased PKC activity with
enhanced expression of the PKC-β isoform. This increase of PKC activity
in the epineurial compartment is consistent with reports in other systemic
vascular tissues, while the decrease in the endoneurial compartment is
Glucotoxicity and Diabetic Neuropathy 139

consistent with a report that Schwann cells exposed to high glucose in vitro
show reduced expression of PKC-α and reduced PKC activity (Kamiya
et al., 2003). Given these findings, the application of PKC-β-specific inhib-
itor would be expected to be a useful for the treatment of diabetic vascular
complications, but not any disorders related to reduced endoneurial expres-
sion of PKC-α. A 6-month clinical trial of the PKC-β-specific inhibitor
Ruboxistaurin in subjects with DPN showed that it increased skin blood
flow and mitigated some sensory symptoms but was without effect on objec-
tive measures of neuropathy such as large-fiber nerve conduction velocity
slowing, reduced small sensory fiber nerve density in the epidermis, or
impaired autonomic function (Casellini et al., 2007).

7. GLYCOSYLATION
7.1 The Hexosamine Pathway
Under conditions of elevated intracellular glucose and flux into glycolysis,
excess fructose 6-phosphate is converted to glucosamine 6-phosphate
(GlcN-6-phosphate) by glutamine-fructose-6-phosphate amidotransferase
(GFAT). GlcN-6-phosphate is then modified to UDP-N-acetylglucosamine
(UDP-GlcNAc) (Du et al., 2000; Issad, Masson, & Pagesy, 2010) (Fig. 6).
UDP-GlcNAc has an affinity for cell membrane or nucleic acid or transcrip-
tion factors and excessive modification of these substrates by posttranslational
modification of serine and threonine residues (termed O-GlcNAcylation) can
perturb cellular functions and may contribute to both the pathogenesis of
type 2 diabetes by promoting insulin resistance and also to the complications
of diabetes (Peterson & Hart, 2016). Evidence is strongest for a role of the
hexosamine pathway in predominantly vascular complications such as reti-
nopathy and nephropathy. For example, inhibition of GFAT suppresses
expression of genes for transforming growth factor (TGF)-α and TGF-β1
gene and also the activation of plasminogen activator inhibitor-I (PAI-I).
As increased expression and activity of these factors are known to be induced
by hyperglycemia, the hexosamine pathway is therefore implicated in vascular
complications of diabetes. UDP-GlcNAc modification of Akt activation sites
in vascular endothelial cells also reduced eNOS activity. Further, GFAT in
vascular smooth muscle cells is activated during hyperglycemia and muscle
proteins are excessively modified by UDP-GlcNAc (Heath et al., 2014). In
contrast to the plentiful data in vascular tissues, it is not yet entirely clear what
kinds of peripheral nerve proteins may be modified by the hexosamine path-
way during diabetes, although there is emerging evidence that imbalances in
140 S. Yagihashi

Fig. 6 Hexosamine (synonym of glucosamine) pathway as a route to diabetic

complications. This pathway is activated in hyperglycemia. As a consequence,
N-acetylglucosamine (GlcNAc) is attached with proteins by O-glycosylation (O-GlcNAc
modification), resulting in suppression of competitive phosphorylation. It is proposed
that O-GlcNAc modification is implicated in the beta-cell dysfunction or insulin action,
or endothelial dysfunction. Glucosamine is popular as a dietary supplement and
metabolized by hexokinase, but the downstream metabolism is not known.

the O-GlcNAcylation state of nervous system proteins is associated with neu-

rodegenerative disease (Hart, 2014). In experimental studies, it was shown that
GlcN was toxic to neuronal cells, eliciting apoptosis by induction of death
signals (Lim et al., 2010). This process involved oxidative stress and was
inhibited by an antioxidant of N-acetyl-cysteine. The contribution of the
hexosamine pathway and dysregulation of O-GlcNAcylation to diabetic
neuropathy remains to be explored.

7.2 Other Glycosylation Events

Posttranslational glycosylation of the asparagine residues of neuronal pro-
teins such as the T-type calcium channel (Cav3.2) can influence their func-
tion and therefore membrane stability and axonal excitability. There is
recent evidence that aberrant glycosylation of neuronal ion channels during
hyperglycemia may contribute to painful diabetic neuropathy (Todorovic,
2015), and this currently evolving hypothesis is covered in detail elsewhere
in this volume.
Glucotoxicity and Diabetic Neuropathy 141

8. CONCLUSION
It is well established that long-term hyperglycemia contributes to the
genesis and development of DPN. However, the process is complicated and
the precise mechanisms of how high glucose affects the assorted cell types
within a nerve are still mysterious. Epidemiological studies suggest that many
factors are responsible for DPN, and the targeting of a single collateral path-
way may not be sufficient for the protection and suppression of DPN.
Experimental studies have identified multiple pathways that appear to be
involved in the functional and structural alterations in the peripheral nerves
in animal models of diabetes and there may yet be other undiscovered path-
ways. As the clinical signs and symptoms of DPN differ from case to case,
the dominant pathogenic mechanisms may also vary based on individual
genotype and phenotype. If this is the case, in addition to meticulous blood
glucose control, it will be important to identify which collateral pathway is
most relevant to specific clinical manifestations. Future investigations should
perhaps explore in more detail the relationships between each collateral
pathway and specific clinical manifestations of DPN.

ACKNOWLEDGMENTS
The author appreciates the previous collaborators who contributed to the published works
from the author’s laboratory. The author also is grateful for the research funding from the
Japanese government of the Ministry of Science, Culture, Education, and Sports, the
Ministry of Health and Welfare, and the Juvenile Diabetes Foundation International,
New York.
Duality of Interest: There is no conflict of interest for the author regarding the content of
this review.

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 CHAPTER NINE

Sensory Neurodegeneration in
Diabetes: Beyond Glucotoxicity
D.W. Zochodne1
Neuroscience and Mental Health Institute and Alberta Diabetes Institute, University of Alberta,
Edmonton, AB, Canada
1
Corresponding author: e-mail address: [email protected]

Contents
1. Terminals at Risk: Sensory Neurodegeneration in Diabetes 152
2. Not Necessarily a Microvascular Disease 154
3. Altered Insulin Signaling 156
4. Ongoing Growth: Other Forms of Support 160
4.1 C-Peptide 160
4.2 Glucagon-Like Peptide-1 161
4.3 Heat-Shock Proteins 162
5. Neurons “on Edge” 162
6. Diabetes, Neurons, and Epigenetics 165
7. A Regeneration Strategy 169
8. Conclusions 174
Acknowledgments 174
References 174

Abstract
Diabetic polyneuropathy in humans is of gradual, sometimes insidious onset, and is
more likely to occur if glucose control is poor. Arguments that the disorder arises chiefly
from glucose toxicity however ignore the greater complexity of a unique neurodegen-
erative disorder. For example, sensory neurons regularly thrive in media with levels of
glucose at or exceeding those of poorly controlled diabetic persons. Also, all of the link-
ages between hyperglycemia and neuropathy develop in the setting of altered insulin
availability or sensitivity. Insulin itself is recognized as a potent growth, or trophic factor
for adult sensory neurons. Low doses of insulin, insufficient to alter blood glucose levels,
reverse features of diabetic neurodegeneration in animal models. Insulin resistance, as
occurs in diabetic adipose tissue, liver, and muscle, also develops in sensory neurons,
offering a mechanism for neurodegeneration in the setting of normal or elevated insulin
levels. Other interventions that “shore up” sensory neurons prevent features of diabetic
polyneuropathy from developing despite persistent hyperglycemia. More recently evi-
dence has emerged that a series of subtle molecular changes in sensory neurons can be

International Review of Neurobiology, Volume 127 # 2016 Elsevier Inc. 151

ISSN 0074-7742 All rights reserved.
http://dx.doi.org/10.1016/bs.irn.2016.03.007
152 D.W. Zochodne

linked to neurodegeneration including epigenetic changes in the control of gene

expression. Understanding the new complexity of sensory neuron degeneration may
give rise to therapeutic strategies that have a higher chance of success in the clinical
trial arena.

1. TERMINALS AT RISK: SENSORY

NEURODEGENERATION IN DIABETES
Persons that develop diabetic polyneuropathy (DPN) may have
underlying type 1 or type 2 diabetes mellitus (DM). With rare exceptions,
DPN develops gradually and insidiously, targeting the distal terminals of the
sensory neurons first (Zochodne, Ramji, & Toth, 2008). This usually trans-
lates into damage to the longest sensory neurons, innervating the toes.
Newer approaches, however, have also identified early nerve terminal
retraction in shorter neurons, such as trigeminal axons that innervate the
cornea, assessed by corneal confocal microscopy (Malik, Kallinikos,
Abbott, et al., 2003). Most persons experiencing symptoms of DPN first
report tingling, pain, or loss of sensation in their toes. With time, and par-
ticularly if glucose control is poor, neuropathy advances with sensory loss
involving more of the extremities and even in the central chest, the terminal
zones of the intercostal sensory nerves. Epidermal biopsies have confirmed
that distal extremity loss of sensory axon terminals is greater than that
observed at more proximal sites, such as the thigh (Polydefkis, Hauer,
Griffin, & McArthur, 2001). This pattern of onset, shared with other
chronic polyneuropathies, bespeaks a unique pattern of gradual neu-
rodegeneration with initial manifestations in distal sensory territories.
The gradual loss of distal terminals during chronic diabetes is not accom-
panied, to our knowledge, by early or substantial loss of parent sensory neu-
rons in dorsal root ganglia (DRGs). While recent investigations into DRG
pathology in human diabetes are lacking, original work suggested a lack of
significant parent neuron damage (Greenbaum, Richardson, Salmon, &
Urich, 1964; Schmidt, Dorsey, et al., 1997). In several animal models,
despite loss of distal footpad epidermal axons, neuron numbers in DRGs
are preserved (Kamiya, Zhangm, & Sima, 2005; Zochodne, Verge,
Cheng, Sun, & Johnston, 2001). While not discussed in this chapter, similar
preservation of autonomic primary neurons also appears to be the case
(Schmidt et al., 1998; Schmidt, Dorsey, et al., 1997). Autonomic dysfunc-
tion, a separate form of diabetic targeting, does offer many parallels to
Sensory Neurodegeneration in Diabetes 153

sensory loss and often accompanies it. Collectively, these findings have indi-
cated that neurons do not experience classical apoptosis early in diabetes and
that substantial dropout is not a feature of the disease. Thus, acute hypergly-
cemia or “glucotoxicity” in humans does not appear to lead to early and
massive neuronopathy. Overall, this information is important news for
DPN since it indicates that once DM is reversed by therapy or islet trans-
plantation, preserved parent neurons are available to reestablish connections
with terminals. Improvement in the sensory deficits of DPN may be
possible.
Why chronic DM exhibits this topographical distribution of change has
generated considerable thought. That it is a generalized feature of neu-
rodegeneration is supported by similar, albeit more rapid changes in ALS,
where motor terminals are retracted before the loss of anterior horn cells.
Several ideas are relevant to understanding this form of neuronal alteration.
Loss of distal terminals in models accompanies early key, but nonlethal alter-
ation of the perikaryal (cell body) gene output and protein synthesis (Cheng,
Kobayashi, Martinez, et al., 2015). For example, declines in the gene expres-
sion of neurofilament polymer subunits in the cell body accompany parallel
declines of similar magnitude in neurofilament investment of distal axons
(Scott, Clark, & Zochodne, 1999). A decline in neurofilaments, part of
the key internal structural lattice of the axon, within distal nerves accom-
panies distal axon atrophy. However, declines in neurofilament investment
probably do not completely account for a number of other features of DPN,
such as conduction velocity slowing. In mice lacking axonal neurofilaments,
diabetes caused accelerated conduction abnormalities, indicating a separate
alteration in membrane excitability (Zochodne, Sun, Cheng, & Eyer, 2004).
Since axons themselves are now recognized to translate proteins, declines in
perikaryal mRNAs targeted toward distal axonal ribosomes may similarly
connect subtle changes in cell body function with the behavior and later
retraction of terminals (Willis & Twiss, 2006). Three additional mechanisms
might account for distal axon targeting by diabetes. First, the intimate relation-
ship between Schwann cells (SCs) and axons, including the exchange of ribo-
somes, illustrates an important codependence (Court, Hendriks, MacGillavry,
Alvarez, & van Minnen, 2008). Axons support SCs with mitogenic molecules
such as neuregulin and CGRP, whereas SCs supply extracellular basement
membrane molecules that support axon growth and trophic factors that are
taken up and signal axons and their parent neurons. Second, considerable
attention has been devoted toward explaining DPN as a mitochondrio-
pathy with neuronal energy failure arising from oxidative stress and other
154 D.W. Zochodne

mechanisms (Bennett, Doyle, & Salvemini, 2014; Fernyhough & McGavock,

2014; Ma, Farmer, Pan, et al., 2014; Ma, Pan, Anyika, Blagg, & Dobrowsky,
2015; Sivitz & Yorek, 2010). Mitochondrial dysfunction may be important
in the development of chemotherapy-related polyneuropathies and there
are specific alterations documented in heredity motor and sensory poly-
neuropathies, such as mutations in mitofusin-2, a mitochondrial fusion
protein. Why distal mitochondria, transported from the cell body, would
be selectively targeted is uncertain and why patients with diabetes do not
exhibit myopathy, a more common target of mitochondriopathies, is also
unexplained. Despite these questions, important connections between mito-
chondria and axon viability may be critical to understanding axonopathy.
Third, distal targeting of nerve terminals in diabetes may occur because
of their demands to undergo ongoing growth and plasticity. In the skin, nor-
mal shedding and egress of keratinocytes are part of a microenvironment that
is constantly changing and remodeling. To maintain their role within the
skin, and perhaps other structures, persistent growth and branching are
required. For example, epidermal nerve terminals express higher levels of
GAP43, a growth protein, than do “stable” more proximal axons
(Cheng, Guo, Martinez, Singh, & Zochodne, 2010). Local trophic interac-
tions between the skin and these terminals may be quite active and include
roles for NGF, HGF, and others.
Overall, the neurodegenerative phenotype of sensory neurons in diabe-
tes is unique and is probably paralleled by autonomic neurons and later by
motor neurons. Attribution of this phenotype to glucotoxicity alone is prob-
lematic, given that at least in the short term, adult sensory neurons grown in
hyperglycemic (20–30 mmol/L or higher) conditions fare remarkably well
with no evidence of neuronal death or terminal retraction (Huang, Price,
Chilton, et al., 2003; Singh, Singh, Krishnan, et al., 2014). While toxicity
has been described at higher concentrations of glucose (Russell, Sullivan,
Windebank, Herrmann, & Feldman, 1999), these studies reflect conditions
not experienced chronically by patients, if at all.

2. NOT NECESSARILY A MICROVASCULAR DISEASE

DPN continues to be listed as a “microvascular” complication of DM,
despite equivocal evidence for this description. Part of the confusion arises
because DPN prevalence correlates in human epidemiological studies with
that of cardiovascular risk factors including atherosclerosis (Tesfaye,
Chaturvedi, Eaton, et al., 2005). These correlations however do not prove
Sensory Neurodegeneration in Diabetes 155

cause and effect. Against the “microvascular” hypothesis is evidence that

DPN can develop in children without long-term disease of their blood ves-
sels, in as short a period as 3 months after the development of DM (Rundles,
1945). There has also been no evidence that specific cardiovascular interven-
tions blunt DPN prevalence in humans. The selective involvement of
peripheral nerves, especially sensory axons, is unexplained by this hypothesis
since vasculopathy would be more likely to target more ischemia-prone tis-
sues such as brain and spinal cord. Peripheral nerves have a redundant vas-
cular supply and only relatively severe ischemia leads to axonal degeneration
or alterations in nerve function (Schmelzer, Zochodne, & Low, 1989).
Nerve blood vessels are likely to exhibit enough structural change to make
human nerves ischemic only later in the disease; the role of microangiopathy
in the development of DPN remains controversial. Finally the more recent
identification of loss of corneal nerves seems incompatible with an ischemic
etiology in this anatomical distribution (Tavakoli, Quattrini, Abbott,
et al., 2010).
Some older experimental models, largely in rat, have identified reduc-
tions in nerve blood flow (NBF) in DPN (Cameron, Cotter, & Low,
1991). This literature was extensively reviewed in a previous edition of
this text, concluding that there is significant uncertainty of their impor-
tance (Zochodne, 2002). Reductions in NBF do not appear in all
models of DM and some long-term models in rats had normal NBF
(Zochodne & Ho, 1992). A large number of papers linking improvement
in electrophysiological indices of DPN with better NBF have offered an
unrealistic palette of potential vascular treatments for the disorder from
simple vasodilators to cannabinoids. Independent actions on axons and
blood vessels could not be excluded in most of this work, and key phys-
iological variations, including potential changes in nerve temperature,
could account for improved nerve conduction in some. Thus, technical
issues such as lack of temperature control of preparations, reliance on single
nonquantitative measures of NBF, inappropriate intraneural recording
electrodes, and other factors have been problematic in the literature.
Morphological studies of microvessels in diabetic models with DPN
have not identified loss of vessels or attenuation of vessel calibers
(Zochodne & Nguyen, 1999). Instead, our own group has identified rises
in luminal caliber or evidence of angiogenesis (Zochodne & Nguyen,
1999). Finally, declines in whole nerve trunk blood flow do not adequately
explain distal terminal retraction or selective involvement of sensory or
autonomic axons.
156 D.W. Zochodne

In humans with advanced DPN, reductions in nerve vascular transit

times and nerve hypoxia have been reported (Newrick, Wilson,
Jakubowski, Boulton, & Ward, 1986; Tesfaye, Harris, Jakubowski, et al.,
1993). However, these patients had evidence of advanced disease, reflecting
parallel microvascular and nerve disease. In patients undergoing nerve
biopsy for research purposes in a DPN trial, direct measures of blood flow
did not identify declines in NBF (Theriault, Dort, Sutherland, & Zochodne,
1997). Indeed, NBF measures tended toward higher levels in patients who
proved to have more severe DPN, as evidenced by fiber loss and recordings
of sensory nerve action potentials.
Despite the difficulties in linking the development, or cause of DPN
with initial changes in NBF or microvessels, there is clear evidence that
microangiopathy does eventually develop in DM. A variety of epineurial
and endoneurial alterations have been carefully described in human nerve
biopsy samples, most often in patients with well-established DM and
DPN (Korthals, Gieron, & Dyck, 1988; Malik, Newrick, Sharma, et al.,
1989; Malik, Veves, Masson, et al., 1992; Yasuda & Dyck, 1987). Taken
together, it is likely that microangiopathy develops in parallel with early
perikaryal and axon changes in DM and both become prominent in later
disease. Functional microvascular changes in the properties of vasa nerv-
orum have been identified in models and may emerge, for example, after
nerve injury when rises in blood flow are expected (Kennedy &
Zochodne, 2002). The balance of these changes suggests impaired mecha-
nisms of vasodilatation (Thomsen, Rubin, & Lauritzen, 2002).

3. ALTERED INSULIN SIGNALING

Disentangling intimate connections between glycemic control and
insulin administration in trials, such as DCCT, is difficult (The Diabetes
Control and Complications Trial Research Group, 1993). While patients
with better control of hyperglycemia fared better after 5 years, they were
also exposed to more physiological and often larger doses of insulin. The
robust trophic actions of insulin on neurons, independent of its glycemic
actions, have been recognized for several decades. For example, Frazier
and colleagues (Frazier, Angeletti, & Bradshaw, 1972) described structural
and possible evolutionary parallels between insulin and nerve growth factor
(NGF) in 1972. Fernyhough, Tomlinson, and colleagues subsequently
described dose-dependent rises in neurite outgrowth of adult sensory
Sensory Neurodegeneration in Diabetes 157

neurons exposed to insulin in vitro (Fernyhough, Willars, Lindsay, &

Tomlinson, 1993). Insulin receptors are expressed in most sensory neurons
and at nodes of Ranvier (Brussee, Cunningham, & Zochodne, 2004;
Sugimoto, Murakawa, & Sima, 2002; Sugimoto, Murakawa, Zhang,
Xu, & Sima, 2000; Xu et al., 2004) (Fig. 1). Insulin also acts as a growth
factor in vivo following nerve injury. Small systemic doses of insulin
improved the reinnervation of foot interosseous endplates by motor axons
after sciatic nerve transection, increased numbers of regenerating myelinated
axons, and enhanced recovery of mouse hindpaw function (Xu et al., 2004).
In separate work, low-dose intrathecal administration of insulin also
improved structural and functional indices of distal axon regeneration
(Toth et al., 2006). Collectively these findings establish a major role for
insulin trophic support of neurons and axons during regeneration.
Given all of these observations, it is plausible that sensory neurode-
generation is related to altered trophic support from deficits of insulin sig-
naling. In type 1 DM, there is absence of the insulin ligand. While these
patients receive insulin supplementation, its administration may have been
inadequate in dose and nonphysiological, given the intermittent regimens
many diabetic persons use. In type 2 DM, circulating insulin levels may
be normal, elevated, or later lowered. However, these patients usually have
insulin resistance involving muscle, liver, or adipose tissue. In type 2 DM
models, high doses of insulin fail to reverse the DPN phenotype. We
now recognize however that neurons, like muscle, liver, and fat, may be
resistant to the trophic properties of insulin, a concept first proposed
by Singh et al. (Grote, Morris, Ryals, Geiger, & Wright, 2011; Kim,
McLean, Philip, & Feldman, 2011; Singh et al., 2009; Singh, Xu,
McLaughlin, et al., 2012). This is important because it indicates that
DPN might develop as a result of the downstream failure of insulin to
activate growth pathways that support neurons, including their require-
ment for constant terminal remodeling. Grote et al. noted that ob/ob mice
with leptin deficiency and type 2 DM had blunted Akt activation with
insulin and IGF-1 and had elevated levels of JNK, a mediator of insulin
resistance in other tissues (Grote et al., 2013). Singh et al. (2012) noted that
neurons exposed to high doses of insulin, even briefly, failed to respond to
subsequent exogenous insulin. Moreover, chronic low-dose insulin expo-
sure similarly blunted subsequent challenges of insulin to support growth
signaling. Potential mechanisms for neuronal insulin resistance, with some
evidence supporting them, have included upregulated levels of GSK-3β,
a known “brake” on axon growth, with downregulated pGSK-3β and
158 D.W. Zochodne

Fig. 1 Insulin directly ligates receptors on sensory neurons. In (A) sensory neurons and
axons in the lumbar dorsal root ganglia (DRG) of rats are labeled with an antibody
directed against IRβ, a subunit of the insulin receptor (IR) (bar ¼ 200 μm). In (B) DRG neu-
rons are labeled by FITC-labeled insulin that attached to the cell surface. The panel to
the right is the identical section viewed under light microscopy (bar ¼ 20 μm). Labeled
insulin was delivered by intrathecal injection and improved phenotypic features of
Sensory Neurodegeneration in Diabetes 159

lowered levels of pAkt. Exogenous insulin also downregulated expression of

its own receptor, IRβ. Additional work has noted that insulin resistance in
neurons may be linked to IRS2 serine phosphorylation (Grote et al., 2011).
In addition to problems of inadequate or nonphysiological dosing and
resistance, insulin may have difficulty accessing neurons because of the
blood–brain barrier so that CSF insulin levels may be different from the
blood (Folli, Bonfanti, Renard, Kahn, & Merighi, 1994). To address this
possibility, we administered low doses of insulin intrathecally, using chronic
catheters, in diabetic rats (Brussee et al., 2004). This route and dosing of
insulin did not influence systemic glucose levels. Intrathecal insulin offered
dose-dependent improvements in motor and sensory conduction velocities.
It corrected axonal atrophy and improved epidermal innervation of the foot-
pads. A control insulin infusion, otherwise identical, into the subcutaneous
skin of the back of the diabetic rat did not improve neuropathy. The findings
are particularly remarkable given this evidence that “central” administration,
at the level of the spinal fluid that accesses DRG, was able to correct elec-
trophysiological and structural changes in distal axons. This is evidence of a
robust connection between perikaryal function and axons. In additional
work, we showed that intrathecal administration of antiinsulin antibody,
an intervention designed to sequester CSF insulin, generated conduction
abnormalities and axonal atrophy in nondiabetic rats, resembling the
changes of DM (Brussee et al., 2004). Dosing with antialbumin antibody
in identical doses had no impact. This work indicated that ongoing, or ambi-
ent insulin signaling through its elaboration in spinal fluid that bathes DRG
within root sleeves, is important for the ongoing maintenance of sensory
neurons. Interruption of this support elicits neuropathic abnormalities.
As an alternative route to direct intrathecal infusion, intranasal adminis-
tration may be an option for administering low doses to access DRG neu-
rons (Reger, Watson, Green, et al., 2008). This route, tested in human
Alzheimer’s disease, allows CSF access of the peptide and there is evidence
it may improve DPN (C. de la Hoz & D. Zochodne, unpublished data;
Francis, Martinez, Liu, et al., 2011; Toth et al., 2007).

experimental diabetic neuropathy. In (C) dissociated adult rat sensory neurons are
triple labeled with neurofilament antibody (red; gray in the print version), FITC-labeled
insulin (green; gray in the print version) added to the media, and DAPI (blue; gray in the
print version) illustrating that insulin is taken up by neurons and labels both the cyto-
plasm and nucleus of sensory neurons. Reproduced with permission from the American
Diabetes Association and Brussee, V., Cunningham, F. A., & Zochodne, D.W., 2004. Direct
insulin signaling of neurons reverses diabetic neuropathy. Diabetes, 53, 1824–1830.
160 D.W. Zochodne

The earlier work has established that insulin ligation of receptors at the
level of the perikaryon, or cell body influences the behavior of the entire
neuronal tree, repairing damage in distal axon segments. There is additional
evidence that distal axons also express the insulin receptor. During injury
and regeneration, receptors were identified on regrowing axons just beyond
the injury site (Xu et al., 2004). Singhal, Cheng, Sun, and Zochodne (1997)
injected low subhypoglycemic doses of insulin around sciatic nerves of rats
with experimental diabetes and compared their function with contralateral
limb sciatic nerves exposed to carrier. Despite ongoing evidence of hyper-
glycemia, and progressive DPN in the opposite limb, nerves exposed to
local insulin had improvements in electrophysiology and had a rise in the
number of small myelinated axons. Dermal and epidermal axons also express
the insulin receptor and mRNAs for downstream insulin transduction
pathways, IRS-1 and IRS-2, are found in skin. As discussed earlier, this
supports the idea that this “normal” population of axons is in a growth state,
constantly remodeling to keep apace of keratinocyte turnover. Guo, Kan,
Martinez, and Zochodne (2011) injected small dermal doses of insulin,
insufficient to alter systemic glucose levels into the hind paw of mice with
chronic DM of 5 months duration. The contralateral paws in the same
mice were injected with carrier. Local insulin, but not carrier, unilaterally
improved epidermal innervation of the hind paw over a surprisingly
short period of within 1 week. Improvements occurred in both type 1
and 2 diabetic mice. These findings support the concept that ongoing
plasticity of skin innervation offers opportunities for short-term manipula-
tion of regrowth to improve neurological function. Chen, Calcutt, and col-
leagues (Chen et al., 2013) have also demonstrated similar short-term
plasticity, over 4 weeks, of nerve fibers in the subbasal plexus of cornea
in rats. DM in this model was associated with a progressive decline in
corneal innervation, whereas local administration of insulin over the cornea
prevented loss of corneal axons.

4. ONGOING GROWTH: OTHER FORMS OF SUPPORT

4.1 C-Peptide
Novel routes of insulin administration may offer feasible and potent therapy
for patients in clinical trials. Indeed a pilot Phase I trial of intranasal insulin in
type 1 diabetic subjects has been completed to evaluate safety (Korngut,
Mawani, Francis, et al., 2013). Work is ongoing. The concept of insulin
resistance does add a cautionary note to relying on the insulin peptide alone
Sensory Neurodegeneration in Diabetes 161

for chronic therapy in diabetic patients. These patients may have already
been exposed to daily systemic injection or they may exhibit systemic insulin
resistance. In patients with established DPN, often first presenting to
clinicians, reversing DPN has usually not been possible and features of
DPN persist despite improvements in glucose control. Experimentally, it
also appears difficult to reverse features of DPN in models with established
disease (Kan, Guo, Singh, Singh, & Zochodne, 2012). For all of these
reasons, moving downstream of insulin, or identifying approaches to sup-
plement its actions may be important. For example, extensive work on
C-peptide, the normally cleaved bridging peptide of proinsulin, suggests that
it benefits DPN through an insulin-sensitizing action. C-peptide has evi-
dence of benefit in models and early human trials (Ekberg et al., 2003;
Kamiya, Zhang, Ekberg, Wahren, & Sima, 2006; Zhang et al., 2001).

4.2 Glucagon-Like Peptide-1

GLP-1 is an incretin peptide, released by the intestine following meal inges-
tion (Baggio & Drucker, 2007). Its actions include enhancing insulin secre-
tion and sensitivity, of importance in type 2 DM. However, GLP-1 also has
direct actions in the nervous system that include trophic actions, impacts
on CNS function, and protection from pyridoxine neuropathy (Perry,
Holloway, Weerasuriya, et al., 2007). Along with work of two independent
groups, we identified impacts of GLP-1 in experimental DPN (Himeno,
Kamiya, Naruse, et al., 2011; Jolivalt, Fineman, Deacon, Carr, & Calcutt,
2011; Kan et al., 2012). Exendin-4, isolated from Heloderma suspectum, is a
GLP-1 agonist that is resistant to dipeptidyl peptidase-4 cleavage allowing
a longer half-life. GLP-1 receptors are widely expressed in DRG sensory
neurons and exendin-4 had direct trophic actions on adult sensory
neurons in vitro, increasing their outgrowth. In models of both experi-
mental type 1 and type 2 DM, exendin-4 offered benefits among estab-
lished indices of DPN including motor and sensory conduction slowing
and mechanical and thermal sensory loss. While the impact was incomp-
lete and did not reverse all facets of the disorder (for example, sensory
conduction and mechanical sensation in type 2 DM were not reversed),
the improvements differed from those offered by systemic insulin. For some
of these indices of DPN, normalizing glucose levels with chronic insulin
infusions was unhelpful, whereas exendin-4 provided benefits. Overall,
these findings indicated that GLP-1 agonism, like insulin, may improve
DPN through direct signaling actions on neurons, but that its actions
may be complementary.
162 D.W. Zochodne

4.3 Heat-Shock Proteins

Heat-shock protein (HSP) 27 is a molecular chaperone that helps to refold
denatured proteins and inhibits apoptosis. Its levels rise in sensory neurons of
experimental DM (Zochodne et al., 2001). Korngut, Ma, Martinez, et al.
(2012) studied experimental type 1 DM in mice with overexpression of
a human HSP27 transgene in their peripheral neurons. In comparison to
wild-type littermates, these mice had attenuation of some of the features
of DPN: loss of footpad thermal sensation, mechanical allodynia, loss of
epidermal innervation, and slowing of sensory conduction velocity. Pro-
tection was more pronounced in female diabetic mice compared to
males. RAGE, NFκB, and activated caspase-3 nuclear expression, features
of neuronal dysfunction in DM within sensory neurons, were also attenu-
ated. Collectively, these data suggested that upregulation of the HSP27
pathway in sensory neurons protects them from DPN. Since the impact
of this approach was incomplete, its actions may need to supplement
that of other strategies. The differential impact on female DM mice is
unexplained and raises the possibility that gender may have important influ-
ences on the development of DPN. Work in the Dobrowsky laboratory has
demonstrated that a small molecule modulating another HSP, HSP70,
improved the behavioral, electrophysiological, structural, and bioenergetic
abnormalities in experimental DPN (Ma et al., 2014).

5. NEURONS “ON EDGE”

In chronic DM sensory neurons might be characterized as being
“under stress” without frank cell loss. Unfortunately this is a vague term,
usually reserved for a cell at acute risk of apoptotic cell death. In the case
of chronic diabetes, several indicators suggest abnormal physiology,
although not necessarily imminent demise. DRG sensory neurons express
cleaved caspase-3, including nuclear localization (Cheng & Zochodne,
2003). A large literature has linked activated caspase-3 expression as equiv-
alent to apoptosis, a virtual cellular “executioner.” However, other markers
indicate that the neurons in this long-term model of diabetes were not under
imminent threat. Nuclear fragmentation was not a feature of their archi-
tecture, TUNEL labeling was negative in neurons, and careful three-
dimensional unbiased counts of neurons using the “physical dissector”
methodology did not identify neuron dropout, all analyzed in long-duration
DM models (12 months in rats). Caspase-3 localization in neurons included
Sensory Neurodegeneration in Diabetes 163

a presence in initial axon segments. Schmidt and colleagues, in humans and

other models of DPN, have characterized these segments as also harboring
prominent neurofilament inclusions (Schmidt, Beaudet, Plurad, & Dorsey,
1997; Schmidt, Dorsey, et al., 1997; Schmidt & Scharp, 1982). Fernyhough
and colleagues have identified specific sites of oxidative stress that target
axons unevenly (Zherebitskaya, Akude, Smith, & Fernyhough, 2009).
While caspase-3 is not usually considered as among the mediators of local-
ized axonal degeneration, mislocalized caspase-3 could potentially offer this
kind of role. It is not known whether caspase-3 inhibitors have a potential
role in modifying the course of DPN.
PARP (poly (ADP-ribose) polymerase) is a DNA repair molecule
downstream of caspase-3 activation. Excessive activation of PARP leads to
neuronal death from excessive consumption of ATP. In chronic experimental
DPN of rats, we identified rises in the intensity of both nuclear and cytoplas-
mic PARP. Cytoplasmic expression was not prominent in nondiabetic con-
trols (Cheng & Zochodne, 2003). Similar to caspase-3, unusual localization to
initial axon segments was also present. Upregulation of PARP in neurons,
however, has not been widely accepted. For example, a separate group did
not identify its upregulation in chronic type 1 BB/Wor rats (Kamiya,
Zhang, & Sima, 2006; Kamiya et al., 2005). Despite these discrepancies,
PARP inhibitors have been noted to protect diabetic mice from DPN. PARP
may also be expressed in nerve microvessels, and its inhibition has been
thought to provide benefit through inhibition of microangiopathy
(Obrosova, Drel, Pacher, et al., 2005; Obrosova, Li, Abatan, et al., 2004).
Chronic hyperglycemia leads to the production and deposition of
advanced glycation endproducts (AGEs), irreversible products of non-
enzymatic glycation of proteins (Brownlee, Cerami, & Vlassara, 1988).
AGEs can be detected in the circulation, the nervous system, and other
tissues. Receptors for AGEs (RAGE) are also expressed in the nervous sys-
tem and specifically in sensory neurons. Moreover, RAGE expression rises
in experimental DM and RAGE-null mice appear to be protected from
DPN (Cameron, Gibson, Nangle, & Cotter, 2005; C. de la Hoz &
D. Zochodne, unpublished data; Toth et al., 2004). AGE–RAGE interac-
tions may therefore have a role in generating neuronal stress and dysfunc-
tion. However, like the difficulties described around PARP, the exact
role of AGE–RAGE signaling is unclear in the nervous system. For example,
inhibiting this pathway in adult sensory neurons in vitro paradoxically
impaired adult neuron outgrowth (Saleh, Smith, Tessler, et al., 2013).
Inhibitors of AGE formation such as aminoguanidine, metformin, and
164 D.W. Zochodne

n-phenacylthiazolium bromide (Reddy & Beyaz, 2006) and scavenging of

circulating AGEs by soluble RAGE infusion have all been proposed as ther-
apy for DPN and diabetes complications generally. In the case of ami-
noguanidine, its actions are nonspecific and widespread beyond AGE–
RAGE disruption and its use has been associated with significant side effects
in humans (Thornalley, 2003).
AGE–RAGE signaling alters neuronal function through the NFκB path-
way. NFκB is a transcription factor that is sensitive to varied forms of cellular
“stress” (Ahn & Aggarwal, 2005) and signals through its subunits p50 and
RelA (p65) that are normally inhibited by cytoplasmic IκB. Degradative
phosphorylation of IκB allows divergent p50 and RelA signaling in the
nucleus. Downstream, this pathway participates in both neuroprotection
and neurodegeneration, and its activity may be attenuated by DM
(Fernyhough, Smith, Schapansky, et al., 2005; Massa, Xie, Yang, et al.,
2006; Purves & Tomlinson, 2002). Expression studies have also identified
rises in NFκB expression in experimental DM (Kan et al., 2012; Korngut
et al., 2012; Toth et al., 2004). Therefore, like PARP and AGE–RAGE,
NFκB may have varied roles in neuronal survival and DPN.
Nitrergic stress has been listed together with oxidative stress as a mech-
anism of diabetic complications. This is usually considered to arise from per-
oxynitrite toxicity, a by-product of the interaction of nitric oxide (NO) and
superoxide. In the peripheral nervous system, all three nitric oxide synthase
(NOS) isoenzymes are identified: nNOS (“neuronal” NOS) in subsets of
sensory neurons, eNOS (“endothelial” NOS) classically described in vascu-
lar endothelial cells but also localized to neurons, and iNOS (“inducible” or
inflammatory NOS) found in macrophages and other inflammatory cells.
NO may contribute to diverse actions in the peripheral nervous system
including pain, local vasodilatation, and regeneration (Levy, Hoke, &
Zochodne, 1999; Levy, Tal, Hoke, & Zochodne, 2000; Zochodne,
Levy, Zwiers, et al., 1999). Mice lacking iNOS, for example, have impaired
regeneration linked to abnormal clearance of the products of axonal degen-
eration, a requirement for subsequent axonal growth (Levy, Kubes, &
Zochodne, 2001). In experimental DM, “fingerprints” of NO toxicity were
identified in neurons evidenced by nitrotyrosine deposition. There were
chronic rises in overall NOS enzymatic activity not accompanied by specific
changes in mRNA or immunohistochemical localization of any of the three
isoforms. To address the role of NOS isoforms in experimental DM, it was
shown that mice lacking iNOS had protection from experimental DPN but
those lacking nNOS did not (Vareniuk, Pacher, Pavlov, Drel, & Obrosova,
Sensory Neurodegeneration in Diabetes 165

2009; Vareniuk, Pavlov, & Obrosova, 2008). A peroxynitrite decomposi-

tion catalyst was also protective (Drel, Pacher, Vareniuk, et al., 2007).

6. DIABETES, NEURONS, AND EPIGENETICS

A puzzling aspect of sensory neurodegeneration in DM is the range of
apparently disconnected phenotypic features that characterize DPN. These
include loss of sensory terminal innervation in the epidermis that does not
always correlate with uniform and “across the board” behavioral loss of sen-
sation. There are seemingly unrelated electrophysiological changes that
include slowing of motor and sensory conduction velocity, the development
of resistance to ischemic conduction failure and axonal atrophy. Moreover,
these phenotypic features may vary among models and patients. In some
models, the behavior abnormalities involve heightened sensitivity with
hyperalgesia and allodynia, respectively, rather than loss of sensation. Sim-
ilarly, on the molecular side, a range of mRNA changes have been observed
in differing models, depending on the duration of DM. These include loss of
structural protein mRNAs such as neurofilament subunits, and tubulin, loss
of growth molecules such as GAP43, rises in injury and stress-related mol-
ecules discussed earlier that include NFκB, caspase-3, RAGE, and others,
alterations in ion channels (reviewed in Zochodne, 2014), and additional
rises in insulin receptor mRNAs. All of these alterations suggest an overrid-
ing shift in gene expression that may link the varied manifestations of the
disorder. How this might arise however is uncertain. One possibility is that
epigenetic alterations of neurons might be layered onto this diverse array of
abnormalities. This additional layer of control could be a secondary phe-
nomenon or could represent a primary initial alteration fundamental to
the disease.
miRNAs are a key element of epigenetic control. Precursor nucleotides
that eventually form miRNAs are exported from the nucleus and processed
to form small single-chain endogenous noncoding nucleotides of 19–23 base
pair length. These nucleotides then associate with the RISC (RNA-induced
silencing complex), a protein complex that cleaves mRNA. miRNAs thus
interfere with normal translation through mRNA cleavage but also by bind-
ing to mRNAs to impair their translation. A wide family of miRNAs target
mRNAs with diverse impacts both in the targets of each miRNA and in
overlap of the number of miRNAs that may target a single relevant mRNA.
To explore the role of posttranscriptional RNA silencing in the sensory neu-
rons of mice with longstanding and chronic DM, we characterized type 1,
166 D.W. Zochodne

STZ mice untreated for 5 months. These mice exhibit key phenotypic
features of DPN including motor and sensory conduction slowing, loss of
sensation to thermal and mechanical stimuli, and loss of epidermal innerva-
tion. GW/P bodies are ultrastructural cytoplasmic complexes that contain
key elements of RISC. Their expression may identify neurons under
“stress.” We noted that GW/P expression was heightened in the sensory
neurons of mice with DM, indicating overt structural evidence of altered
mRNA processing as a feature of this chronic disease (Cheng et al., 2015).
In the same cohort of diabetic mice and littermates, we examined 28,869
DRG mRNAs for differential expression of at least a 1.5-fold change in
DM samples and within these identified 261 mRNAs that included
91 upregulated and 170 downregulated. Of these 24 achieved a statistical
difference between diabetics and nondiabetics of p < 0.05 (5 down and
19 up). Almost all coded for proteins of unknown function in sensory
neurons or diabetes. For example, one upregulated molecule, CWC22, pro-
vided a valuable lead to analyze spliceosome function in diabetic sensory
neurons, work in progress that offers new ideas about specific molecular
deficits in diabetic sensory neurons (Kobayashi, Cheng, de la Hoz, &
Zochodne, 2015). We further explored concurrent changes in miRNAs,
their supraregulatory companions. As in the case of mRNAs, we identified
a number of differentially expressed miRNAs in DRGs from mice with
chronic DM and DPN. Of 1042 examined, there were 19 altered that
included 12 downregulated and 7 upregulated high-abundance miRNAs.
Additionally, there were 123 low-abundance miRNAs altered including
56 downregulated and 67 upregulated. Focusing on miRNAs in the
high-abundance group, we studied expression of mmu-let-7i, which was
downregulated by 39% in diabetic DRGs, a change also confirmed by
qRT-PCR. mmu-let-7i was an interesting miRNA to consider first, given
widely divergent impacts on over 900 targets. To begin with, we assessed
whether this miRNA was expressed in neurons, rather than DRG satellite
cells or vessels. By in situ hybridization, we noted striking expression in most
sensory neurons of the DRG (Fig. 2). To determine whether alterations in
mmu-let-7i might be associated with an overall phenotype, given its diver-
gent actions, we studied the impact of sensory neuron transfection with
an exogenous mmu-let-7i mimic in vitro. The approach was associated
with robust trophic actions including an increase in neurite outgrowth
and branching. Collectively these findings suggested that the downregu-
lation of mmu-let-7i in chronic diabetic DRG neurons might impair overall
supportive or trophic mechanisms.
Fig. 2 Epigenetic miRNAs are expressed in sensory neurons and influence their growth
properties. In (A), in situ hybridization images of normal mouse DRGs identify mmu-
let-7i expressed in sensory neurons at lower (left) and higher power (right) (bar ¼ 100
and 50 μm, respectively). A control image without label is below. In (B) are illustrated
confocal images of preinjured (axotomized) adult rat sensory neurons harvested from
lumbar DRG and grown in the presence of control carrier solution (top images) or a
mimic molecule of mmu-let-7i for 20 h and stained with antineurofilament antibody
(bar ¼ 100 μm). Reproduced with permission from Cheng, C., Kobayashi, M., Martinez, J. A.,
et al. (2015). Evidence for epigenetic regulation of gene expression and function in chronic
experimental diabetic neuropathy. Journal of Neuropathology and Experimental Neurology,
74, 804–817.
168 D.W. Zochodne

Identifying the specific and relevant target genes responsible for a growth
phenotype in sensory neurons is challenging. A comparison of parallel micro-
arrays, in the same diabetic and nondiabetic mouse cohorts, examining statis-
tically significant changes in mRNAs due to diabetes with four of our altered
miRNAs identified some predicted changes. For example, these included
upregulation of connective tissue growth factor, upregulation of dual-
specifity phosphatase 1, upregulation of F3 and TXNIP (a thioredoxin-
interacting protein that may mediate oxidative stress), a small rise in insulin
receptor, and downregulation of CACNG4, a subunit of the calcium L-type
channel. Most have unclarified relationships to sensory neuron function
to date. Two previous reports have linked abnormalities in calcium
channels to experimental diabetes (Hall, Sima, & Wiley, 1995; Voitenko,
Kruglikov, Kostyuk, & Kostyuk, 2000). These predicted changes were only
based on four high-abundance miRNA changes, suggesting that much more
extensive interactions occur than we explored in this work. However, none
of the apparent alterations mediated by these mRNAs offered clear or
established candidates for growth enhancement.
The next step in addressing the relevance of miRNA changes in chronic
DM is understanding whether these changes represent an epiphenomenon
of the disease or whether they effect critical changes in neuron function.
Given confirmation that mmu-let-7i was downregulated in DRGs with
clear impacts on sensory neuron biology, we tested whether supplementing
neurons with exogenous mmu-let-7i, in the form of a mimic molecule,
might alter the DPN phenotype. The approach to this was not trivial given
that access of the nucleotide to sensory perikarya would be key to its poten-
tial actions. A viral vector approach to transfection may be a potent option
for stable replenishment of mmu-let-7i. However, with a thought toward
downstream human translation, we tested a nonviral approach that involved
intranasal administration of an mmu-let-7i mimic molecule. To test whether
miRNAs might access the CSF and DRGs through their root sleeves by this
approach, we first administered an unrelated miRNA by intranasal
delivery—a plant species miRNA–Arabidopsis thaliana miR171, not present
in mammals. After administration for 6 days, miR171 was detected in both
the olfactory bulb and the lumbar DRG of mice indicating access to the CSF
through this route. Next we administered a mimic mmu-let-7i by the
intranasal route and confirmed that in nondiabetic mice, daily dosing for
1 week could raise mmu-let-7i levels as tested in DRG by qRT-PCR.
Given the evidence that a nonconventional route of miRNA delivery
had the potential to access the CNS and spinal spaces including DRG root
Sensory Neurodegeneration in Diabetes 169

sleeves, we tested its impact on diabetic mice. Using mice with 5-month
duration DM and well-established indices of DPN, we administered intra-
nasal mmu-let-7i mimic over 6 days and retested after a further 3 weeks. The
rationale was that alterations in gene expression effected by mmu-let-7i
might require at least 2–3 weeks to alter epidermal innervation and other
features of DPN. Intranasal mmu-let-7i, but not control treatment,
improved impaired diabetic mechanical sensitivity to the levels of mice
without DM and improved loss of thermal sensation, both accompanied
by an improvement in epidermal innervation. Motor and sensory conduc-
tion velocities were also improved by the mmu-let-7i mimic. Additional
work examined whether knockdown of a second separate, but in this case,
elevated miRNA mmu-341, in diabetic mice might also impact DPN. In
contrast to mmu-let7i, mmu-341 was the most robustly upregulated
miRNA detected in our array survey. In this case contrary knockdown
with an miRNA anti-miR also improved features of DPN, although the
results were less robust, influencing only sensory conduction and thermal
sensation. Taken together, however, these findings identified a striking
impact of nucleotide delivery on structure, electrophysiology, and behavior
in diabetic mice.
Epigenetic manipulation of a range of mRNA targets altered in DM is an
attractive “single bullet” that might be available to repair neuropathic dam-
age. Albeit promising, particularly in the use of nonviral delivery, the
approach needs independent confirmation. The overall possibilities linked
to manipulating gene alterations in neurological disease using intranasal
access to the CSF are also enormous, but require verification. Despite these
caveats, the observations that relatively short-term interventions, presum-
ably targeting altered but not lost parent neurons, might allow recovery from
deficits that have developed chronically over several months are exciting.
There are other facets of epigenetic regulation in DM that may be equally
important, including targeting of SCs, also key targets of DM, that are not
discussed in this review.

7. A REGENERATION STRATEGY
The axon terminals of adult sensory neurons might be considered as
being in a permanent and ongoing state of growth. Within the epidermis,
the constant movement, replenishment, and shedding of keratinocytes
require that axons adapt and remodel to retain their roles. Indeed, the tra-
jectories of epidermal axons, while well spaced, are highly irregular, with
170 D.W. Zochodne

twists and turns that suggest ongoing growth. More direct evidence arises
from their prominent expression of growth proteins such as GAP43 and
the finding that interventions, including simple and noninvasive steps such
as hair clipping, can dramatically influence the density of innervation
(Cheng et al., 2010).
In chronic DM with DPN, epidermal terminals are retracted, requiring
regrowth to restore their innervation. Collateral sprouting, distinct from
regenerative sprouting, allows reinnervation of denervated territories by
invasion from intact neighbor axons. Work by Diamond and colleagues
(Diamond, Coughlin, Macintyre, Holmes, & Visheau, 1987; Diamond,
Holmes, & Coughlin, 1992) has shown that skin collateral sprouting, but
not regenerative sprouting, is NGF dependent. Collateral sprouting does
occur in DM (Theriault, Dort, Sutherland, & Zochodne, 1998), but this
form of recovery generally may not be sufficiently robust to completely
restore sensory fidelity. With this caveat in mind, retracted axons in DM
may be called upon to regrow into their previous territories, forestalling
ingrowth by neighbors. Taken together, strategies that support both forms
of neurological recovery may be required.
In DM, there are other considerations that make regenerative strategies
important. Patients with DM are disposed to develop single nerve lesions, or
focal neuropathies such as carpal tunnel syndrome or ulnar neuropathy at the
elbow (Zochodne, 2007). A more exacting clinical terminology thus refers
to diabetic neuropathies, recognizing a range of additional complications
involving the peripheral nervous system. Focal neuropathies are common
and disabling. Considering the range of neurological complications identi-
fied, DM imposes a “double hit,” a degenerative polyneuropathy, or DPN,
but also a deficit in regenerative capacity. For example, in carpal tunnel syn-
drome, surgical decompression of the entrapped nerve at the wrist and sub-
sequent recovery has a less satisfactory outcome in DM.
Mechanisms for impaired diabetic nerve regeneration have been consid-
ered extensively and have included impaired neurotrophic support, micro-
angiopathy that renders the regenerative microenvironment unsupportive,
local oxidative stress, impaired macrophage clearance, accelerated retrograde
loss of neurons, polyol flux, mitochondrial dysfunction, nonenzymatic gly-
cosylation of basement membrane regeneration scaffolds, SC dysfunction,
and others (Kennedy & Zochodne, 2005). New molecular approaches to
coax greater plasticity and regrowth out of reluctant adult neurons have
highlighted the potential roles of targeting their intrinsic growth mecha-
nisms. These operate downstream of growth factor signals and may be final
Sensory Neurodegeneration in Diabetes 171

arbiters of growth cone behavior. Several “brakes” to regrowth fall within

the class of “tumor suppressors,” molecules that help block uncontrolled
oncogenic growth. The first example in this category is PTEN (phosphatase
and tensin homolog deleted on chromosome 10), a tumor suppressor mol-
ecule that inhibits PI3K–pAkt signaling. The latter is a critical growth path-
way for neurons and its inhibition or “braking” attenuates growth. Unlike
oncogenesis however, neurons are characterized by poor growth. Persistent
expression of PTEN in neurons, despite injury and regenerative demands, is
an example of a counterproductive barrier to growth. In the intact adult ner-
vous system, limitations of growth behavior may be important in protecting
fine and detailed connections, especially in the CNS. After injury however,
PTEN may attenuate growth and recovery. In adult sensory neurons,
inhibition or knockdown of PTEN was associated with robust neurite
outgrowth (Christie, Webber, Martinez, Singh, & Zochodne, 2010). Out-
growth was most prominent in preinjured neurons, a remarkable finding.
Given that preexisting injury acts as a “conditioning lesion” that ramps
up regenerative behavior, further acceleration from PTEN knockdown
indicates a synergistic action beyond preconditioning. Regrowth at this level
has not previously been described. Following nerve transection in adult rats,
PTEN inhibition or knockdown increased the outgrowth of adult axons
from the proximal stump. However, PTEN expression in sensory neurons
is uneven, with very prominent expression in small caliber nonpeptidergic
IB4 neurons. These neurons have been linked with significantly slower
growth properties, additional evidence of a close link between PTEN
expression and regenerative potential (Guo, Singh, & Zochodne, 2014;
Tucker, Rahimtula, & Mearow, 2006). Since this class of neurons also
innervates the skin, PTEN’s role in diabetic epidermal regrowth may be
important.
PTEN is upregulated in diabetic sensory neurons (Singh et al., 2014;
Fig. 3). Not only do individual neurons appear to have more intense expres-
sion, but also higher expression neurons seem to be more widely distributed
in the DRG. Emphasizing the potential importance of PTEN action,
in vitro adult sensory neurons from mice with chronic DM that were
harvested and analyzed in culture retained the capacity for increased growth
following PTEN knockdown. In mice with chronic DM and established
indices of DPN, we studied the impact of PTEN knockdown on recovery
from superimposed focal nerve injuries (Fig. 3). Nonviral siRNA delivery at
the site of crush injury was associated with ipsilateral DRG PTEN mRNA
knockdown at the DRG and spinal cord with evidence that the nucleotide
172 D.W. Zochodne

Fig. 3 PTEN, a phosphatase that acts as a “brake” on regeneration, is expressed in adult

sensory neurons, upregulated in diabetes. PTEN knockdown improves regeneration. In
(A) Western immunoblots are illustrated that are labeled with PTEN (beta-actin control
band) in wild type and diabetic dorsal root ganglia before (basal, Day 0) 3 and 6 days
after sciatic nerve injury. Expression was analyzed in 3-month type 1 diabetic mice with
or without nerve injury and 12-week-old type 2 diabetic (db/db) mice. In (B) are illus-
trated examples of adult sensory neurons from wild-type, streptozotocin (STZ)-induced
type 1 diabetic, and db/db, type 2 diabetic dorsal root ganglia labeled with PTEN (green;
gray in the print version). Note that PTEN is expressed in a larger number of neurons
with brighter luminosity in diabetics (bar ¼ 100 μm). In (C) and (D) are illustrated the
impact of PTEN siRNA on in vivo regeneration of axons following injury. Analysis is
carried out in nondiabetic mice and mice with type 1 diabetes (3 months) treated
for an additional 1 month (28 days). In (C) are representative images of semithin
sections of regenerating distal tibial nerves (10 mm from the injury site) from wild-type
and diabetics 21 days after sciatic nerve injury with and without PTEN siRNA. Diabetic
Sensory Neurodegeneration in Diabetes 173

was retrogradely transported along axons to inhibit gene expression. PTEN

knockdown improved the recovery of motor compound muscle action
potentials, conduction velocities of regenerating motor and sensory axons,
repopulation of myelinated axons distal to the injury site, and reinnervation
of the hind paw skin. Taken together the findings demonstrated not only an
active inhibitor role of PTEN in diabetic axon regrowth but also a novel
approach to unilaterally effect alterations in gene expression of injured
nerves.
The RhoA–ROK (RhoA kinase) pathway is an example of an intrinsic
regenerative “brake” that causes growth cone retraction. It operates through
one of several mechanisms including phosphorylation of cofilin that inter-
feres with actin turnover in growth cones (Ng & Luo, 2004), inhibition of
myosin phosphatase that increases myosin ATPase activity (Luo, Jan, & Jan,
1997), and direct phosphorylation of myosin (Giniger, 2002) converging on
increased central actin bundle contractility and stability to collapse growth
cones (Jin, Guan, Jiang, et al., 2005). RhoA and ROK are expressed in
peripheral sensory neurons and ROK inhibition increased the outgrowth
of neurites in primary neuronal cultures. ROK inhibition also increased
regrowth of axons in vivo following a nerve transection (Cheng,
Webber, Wang, et al., 2008). This pathway has not been explored in DPN.
Additional pathways, also yet to be explored in DM, may offer further
targets for influencing nerve regeneration. In our laboratory, more recent
work has marshaled evidence that a second tumor suppressor molecule,
retinoblastoma 1 (Rb1), may operate as a regenerative brake, similar to
PTEN and RhoA, to peripheral neuron regrowth (Christie, Krishnan,
Martinez, et al., 2014). Rb1 inhibits divergent transcriptional signals by
binding to E2F. It is widely expressed in sensory neurons, and its knock-
down in vitro and in vivo increases axon outgrowth. Rb1 may mediate
its actions through the PPARγ pathway.

animals displayed regeneration deficits with significantly fewer and smaller caliber
axons regenerating 3 weeks after injury. PTEN inhibition partially rescued the deficit
(bar ¼ 50 μm). In (D) are representative immunohistochemically labeled (PGP 9.5)
images of footpads (*external surface of the skin) from wild type and diabetics with
and without PTEN siRNA indicating newly regenerating sensory afferents in the epider-
mis. Reinnervation was reduced in diabetics with few reinnervating axons crossing
into the epidermis and improved with PTEN siRNA (bar ¼ 100 μm). Reproduced with
permission from Singh, B., Singh, V., Krishnan, A., et al. Regeneration of diabetic axons is
enhanced by selective knockdown of the PTEN gene. Brain, 137, 1051–1067.
174 D.W. Zochodne

8. CONCLUSIONS
Exploiting novel molecular approaches to support sensory neurons in
chronic DM may offer a palette of therapeutic targets. These are urgently
required in a field that has suffered from a series of failed clinical trials. Mov-
ing beyond “microvascular” ideas might include approaches to offer direct
neuronal insulin signaling through new delivery routes, adding GLP-1
agonism to the mix and supporting neurons with HSP27 or inhibitors of
caspase-3, PARP, or AGE–RAGE signaling. New epigenetic understand-
ing of the pathogenesis of this complex disorder is warranted. Finally, new
strategies that support the regenerative potential of peripheral neurons dam-
aged by DM may be essential to restore neurological function in patients.

ACKNOWLEDGMENTS
The work highlighted in this review was supported by operating grants from the Canadian
Institutes of Health Research (FRN184584), the Canadian Diabetes Association (OG-3-12-
3669), the Juvenile Diabetes Research Foundation, and the National Institutes of Health
(NIDDK), USA. The author has been supported by the Alberta Heritage Foundation for
Medical Research and the University Hospital Foundation, Edmonton. The Zochodne
laboratory is supported by the Neuroscience and Mental Health Institute, Alberta
Diabetes Institute, Division of Neurology and Department of Medicine, University of
Alberta.

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 CHAPTER TEN

Promoting Neuronal Tolerance

of Diabetic Stress: Modulating
Molecular Chaperones
S.M. Emery, R.T. Dobrowsky1
The University of Kansas, Lawrence, KS, United States
1
Corresponding author: e-mail address: [email protected]

Contents
1. Introduction 182
2. Molecular Chaperones 183
3. Extracellular Hsp70 185
3.1 Import, Export, and Neuronal Support 185
3.2 Immunomodulation, Inflammation, and Oxidative Stress 186
4. Intracellular Hsp70 189
4.1 Chaperone Functions 189
4.2 Oxidative Stress 190
4.3 Inflammation 194
4.4 Insulin Sensitivity, JNK, and c-Jun 195
5. DPN and Modulating Hsp70 197
6. Concluding Remarks 201
Acknowledgments 201
References 201

Abstract
The etiology of diabetic peripheral neuropathy (DPN) involves an interrelated series of
metabolic and vascular insults that ultimately contribute to sensory neuron degenera-
tion. In the quest to pharmacologically manage DPN, small-molecule inhibitors have
targeted proteins and pathways regarded as “diabetes specific” as well as others whose
activity are altered in numerous disease states. These efforts have not yielded any
significant therapies, due in part to the complicating issue that the biochemical
contribution of these targets/pathways to the progression of DPN does not occur with
temporal and/or biochemical uniformity between individuals. In a complex, chronic
neurodegenerative disease such as DPN, it is increasingly appreciated that effective
disease management may not necessarily require targeting a pathway or protein
considered to contribute to disease progression. Alternatively, it may prove sufficiently
beneficial to pharmacologically enhance the activity of endogenous cytoprotective
pathways to aid neuronal tolerance to and recovery from glucotoxic stress. In pursuing

International Review of Neurobiology, Volume 127 # 2016 Elsevier Inc. 181

ISSN 0074-7742 All rights reserved.
http://dx.doi.org/10.1016/bs.irn.2016.03.001
182 S.M. Emery and R.T. Dobrowsky

this paradigm shift, we have shown that modulating the activity and expression of
molecular chaperones such as heat shock protein 70 (Hsp70) may provide translational
potential for the effective medical management of insensate DPN. Considerable evi-
dence supports that modulating Hsp70 has beneficial effects in improving inflamma-
tion, oxidative stress, and glucose sensitivity. Given the emerging potential of
modulating Hsp70 to manage DPN, the current review discusses efforts to characterize
the cytoprotective effects of this protein and the benefits and limitations that may arise
in drug development efforts that exploit its cytoprotective activity.

1. INTRODUCTION
Over 300 million individuals worldwide are estimated to be affected
by diabetes, with projections that exceed half a billion people by 2030
(Whiting, Guariguata, Weil, & Shaw, 2011). About half of these individuals
will develop diabetic peripheral neuropathy (DPN). DPN symptoms can be
quite varied and manifest in the extremities as weakness/numbness (insensate
DPN) or painful tingling, burning, or lancinating sensations (painful DPN).
Though the painful aspects of DPN can be quite debilitating, the loss of sen-
sation can lead to life-altering amputations by exacerbating the seriousness of
ulcerations that occur in 15% of patients. In fact, diabetes is the leading cause
of nonaccidental lower extremity amputations. Current therapies manage
painful symptoms with anticonvulsants, antidepressants, or opiates. How-
ever, pharmacologic approaches to manage insensate DPN have not realized
any significant clinical success. To this point, management of glycemic con-
trol is our most effective option in ameliorating the onset and severity of
DPN symptoms, but this approach is more effective for Type 1 than Type
2 diabetics (Callaghan, Cheng, Stables, Smith, & Feldman, 2012).
The recurrent hyperglycemia central to the development of DPN results
in a host of biochemical and metabolic changes. Elevated blood glucose
levels can increase its flux through the polyol and hexosamine pathways,
activate protein kinase C (PKC), increase poly(ADP-ribose) polymerase
activity, and enhance formation of advanced glycation endproducts
(AGE), which activate AGE receptors (RAGE) to heighten oxidative stress
(Brownlee, 2001; Edwards, Vincent, Cheng, & Feldman, 2008; Farmer,
Li, & Dobrowsky, 2012). Though each pathway is likely to independently
contribute to the metabolic alterations that underlie DPN, current theories
suggest that the progression of DPN is strongly related to a convergence of
these metabolic disturbances on promoting oxidative stress (Obrosova et al.,
Modulating Hsp70 to Treat DPN 183

2005; Stavniichuk et al., 2011; Vareniuk et al., 2007) and mitochondrial dys-
function (Chowdhury, Smith, & Fernyhough, 2013; Farmer et al., 2012).
Additionally, inflammatory signaling, a well-accepted component of Type
2 diabetes, is also fed by oxidative stress and can play a role in exacerbating
DPN through cytokine production, immune cell infiltration, and further
elevation of oxidative stress (Wei et al., 2009; Wellen, 2005; Wright,
Scism-Bacon, & Glass, 2006).
Classic approaches to pharmacologically manage DPN have assumed
that targeting a single biochemical insult is sufficient to alter the interwoven
molecular cascade that culminates in the onset of symptomatic sensory
neuropathy and the eventual dying back of distal axons. If DPN arose from
damage to a single cell population from a single biochemical insult, this
approach may be highly fruitful. However, the natural history of the
disorder develops over decades due to damage to vascular, glial, and neu-
ronal cell populations that are not necessarily affected synchronously by
hyperglycemic stress. Indeed, despite the current focus on minimizing
oxidative stress, antioxidant therapies have also met with limited success
in treating DPN, though the use of α-lipoic acid may have some benefit
(Garcia-Alcala et al., 2015; Ziegler et al., 2006, 2011). Thus, realigning our
thinking toward pharmacologic approaches to address this problem may
prove beneficial.
A rather unexplored and clinically unappreciated alternative approach
for managing DPN is to deemphasize that it is necessary to topple a bio-
chemical kingpin to destroy the downstream network of molecular minions
whose slavings contribute to disease progression. On the other hand, cells
have evolved endogenous mechanisms to tolerate stress. Pharmacologic
upregulation of these pathways may facilitate recovery from glucotoxicity
and/or enhance the efficacy of agents that target direct pathogenetic mech-
anisms of DPN. Growing evidence suggests that modulating endogenous
cytoprotective responses by targeting the activity and expression of molec-
ular chaperones such as heat shock protein 90 (Hsp90) and Hsp70 may
achieve this purpose.

2. MOLECULAR CHAPERONES
Heat shock proteins are divided into families defined by their molec-
ular size: Hsp110, Hsp100, Hsp90, Hsp70, Hsp60, Hsp40, and small Hsps
(Kim, Hipp, Bracher, Hayer-Hartl, & Hartl, 2013; Saibil, 2013). This
184 S.M. Emery and R.T. Dobrowsky

review will focus primarily on the Hsp90 and Hsp70 proteins that can be
found in the endoplasmic reticulum (ER), mitochondria, cytosol, and extra-
cellularly (Becker & Craig, 1994; Haas & Wabl, 1983; Hightower &
Guidon, 1989; Leustek, Dalie, Amir-Shapira, Brot, & Weissbach, 1989).
Both Hsp90 and Hsp70 have isoforms that are constitutively expressed or
induced by thermal, ischemic, oxidative, and metabolic stressors, as well as
by exercise (Beckmann, Lovett, & Welch, 1992; Krause et al., 2007;
Richard, Kaeffer, & Thuillez, 1996; Yang et al., 1996). Under physiologic
conditions Hsp90 binds and inhibits the transcription factor heat shock fac-
tor 1 (HSF1). In response to the accumulation of damaged and/or unfolded
proteins, HSF1 is released from this complex, undergoes trimerization, and
is phosphorylated before being translocated to the nucleus (Neef, Jaeger, &
Thiele, 2011; Vihervaara & Sistonen, 2014). Upon binding to heat shock
elements, HSF1 upregulates an array of inducible chaperones including
forms of Hsp70 (Hsp70.1 and Hsp70.3) and Hsp90 (Hsp90α), as well as
antioxidant proteins (Benarroch, 2011). Hsp70 can help refold unfolded
proteins, prevent inappropriate protein interactions, manage protein aggre-
gate formation, and direct the degradation of damaged or dysfunctional pro-
teins. Importantly, the efficacy of modulating chaperones is not limited to
neurodegenerative diseases driven by the formation of protein aggregates.
Hsp70 has also been shown to downregulate inflammatory signaling,
decrease oxidative stress, and improve mitochondrial function in DPN
and other conditions whose etiology is not linked to the formation of pro-
tein aggregates (Ianaro et al., 2003; Jones, Voegeli, Li, Chen, & William
Currie, 2011; Li, Ma, Zhao, Blagg, & Dobrowsky, 2012; Ma, Pan,
Anyika, Blagg, & Dobrowsky, 2015; Madden, Sandstrom, Lovell, &
McNaughton, 2008; Saibil, 2013; Zhang, Zhao, Blagg, & Dobrowsky,
2012). However, an important aspect of Hsp70 biology that must be con-
sidered in moving forward in therapy development is how modulating the
intra- vs extracellular pools of Hsp70 may affect disease progression.
Opposing effects have been demonstrated for intracellular Hsp70
(iHsp70) vs extracellular Hsp70 (eHsp70) in multiple studies (Krause
et al., 2015; Rodrigues-Krause et al., 2012). Extracellular Hsps were first
described as glia–axon transfer proteins when giant squid axons were sub-
jected to heat stress (Tytell, Greenberg, & Lasek, 1986). Later studies spe-
cifically identified Hsp70 as one of the glia–axon transfer proteins
(Hightower & Guidon, 1989). More recently, eHsp70 has been shown in
blood where it acts as a paracrine factor released from dendritic, neuronal,
and other cell types (De Maio, 2011). eHsp70 is associated with immune
Modulating Hsp70 to Treat DPN 185

system activation and inflammatory/oxidative stress conditions (Whitham &

Fortes, 2008) and has been hypothesized to act as a warning to the immune
system for combating infections (Campisi, Leem, & Fleshner, 2003).
Conversely, iHsp70 acts to suppress immune activation, in line with it also
promoting antiinflammatory and antioxidant actions (Ianaro et al., 2003;
Johnson & Fleshner, 2006). Elevated eHsp70 expression and decreased
iHsp70 expression are linked to diabetes, obesity, and insulin resistance
(Chung et al., 2008; Kurucz et al., 2002; Rodrigues-Krause et al., 2012).
Considering these conflicting actions, this review will focus on differences
in the mechanistic actions of extracellular vs intracellular Hsp70 proteins and
what roles their pharmacological manipulation might have in therapies
for DPN.

3. EXTRACELLULAR Hsp70
3.1 Import, Export, and Neuronal Support
Hsp70 export was unknowingly first characterized in heat-treated giant
squid axon when protein levels of a heat shock-like transferrin protein
increased in the axoplasm (Tytell et al., 1986). This transferrin protein
was specifically identified as Hsp70 when heat-treated rat embryonic cell
cultures (presumably fibroblasts) were shown to release Hsp70 into the
media, independent of an increase in cell death (Hightower & Guidon,
1989). Glial eHsp70 secretion has also been confirmed in models of heat-
treated glioblastoma cells (Guzhova et al., 2001).
The export mechanisms behind eHsp70 secretion are still not well
understood since Hsp70 lacks a secretory signal sequence and inhibiting
the secretory pathway has no effect on the release of eHsp70
(Hightower & Guidon, 1989). Some evidence has pointed toward an innate
ability of the protein to traverse the cell membrane (Multhoff, 2007), lipid
raft-mediated lysosomal release (Hunter-Lavin et al., 2004), and secretory-
like granule excretion (Evdonin et al., 2006). However, the largest collec-
tion of evidence points to exosome-dependent trafficking; heat-shocked
peripheral blood mononuclear cells increased eHsp70 levels and produced
Hsp70-loaded exosomes (Lancaster & Febbraio, 2005). Though further
study is needed to fully understand this Hsp70 export mechanism, exosomal
eHsp70 release has been demonstrated in other models including colon
and pancreatic cancer cell lines (Gastpar et al., 2005) as well as breast and
leukemic cancer cells (Bausero, Gastpar, Multhoff, & Asea, 2005).
186 S.M. Emery and R.T. Dobrowsky

Once excreted eHsp70 can be absorbed and utilized by neighboring

cells, typically for the purpose of cell support (Guzhova et al., 2001). The
neuroblastoma cell line LAN-5 showed lower levels of cytotoxicity from
both heat shock and staurosporine treatment after internalizing exogenously
administered Hsp70 (Guzhova et al., 2001). These cytoprotective effects can
likewise translate to nonneoplastic neuronal models. Injection of recombi-
nant human Hsp70 into G93A mutant Cu/Zn superoxide dismutase
(SOD1) mice helped preserve motor neurons resulting in a delayed onset
of amyotrophic lateral sclerosis (ALS) symptoms (Gifondorwa et al.,
2007). In vitro and ex vivo experiments using a trophic factor-deprived
environment and sciatic nerve axotomy, respectively, confirm eHsp70
decreased motor neuron death (Robinson et al., 2005; Tidwell,
Houenou, & Tytell, 2004). Exogenously administered Hsp70 also preserved
viability of dorsal root ganglia (DRG) sensory neurons after sciatic nerve
axotomy (Tidwell et al., 2004). Though the exact mechanism for cellular
protection is not known, eHsp70 may act as iHsp70 upon internalization
(Turturici, Sconzo, & Geraci, 2011).

3.2 Immunomodulation, Inflammation, and Oxidative Stress

In contrast to the effects of iHsp70, the actions of receptor-mediated eHsp70
are typically immune stimulating, proinflammatory, and promote oxidative
stress (Krause et al., 2015). For example, multiple studies have identified
eHsp70 as mediating an initial inflammatory response after tissue injury
(Kovalchin et al., 2006; Senf, Howard, Ahn, Ferreira, & Judge, 2013).
Senf et al. (2013) reported that cardiotoxin injections into the tibialis ante-
rior (TA) muscles of Hsp70 knockout mice induced severe muscle injury
and delayed recovery when compared to control animals. Hsp70 expression
vectors electroporated into TA muscles restored recovery time after injury
by reinvigorating the initial postinjury inflammatory response. Extracellular
Hsp70 release after TA injury was then shown to be integral to restoring the
early inflammatory response when cardiotoxin was injected into TA muscle
with and without recombinant Hsp70. The exogenously administered
Hsp70 increased macrophage and neutrophil infiltration and resultant cyto-
kine production to control levels in almost all measures (Senf et al., 2013).
This study summarizes eHsp70’s attributes as a chemoattractant, immune
cell activator, and proinflammatory mediator, which essentially allows it
to act as an immunomodulatory agent.
Focusing first on chemoattraction, macrophages, neutrophils, and natu-
ral killer (NK) cells directly respond to eHsp70 chemotaxic signaling (Horn
Modulating Hsp70 to Treat DPN 187

et al., 2007; Ortega, Hinchado, Martin-Cordero, & Asea, 2009; Senf et al.,
2013). However, eHsp70 can also indirectly increase chemotaxis of immune
cells by stimulating T-cell-mediated beta-chemokine release of Rantes and
macrophage inflammatory protein-1β, which drive infiltration of numerous
innate and adaptive immune components (Lehner et al., 2000). Once B- and
T-lymphocytes have infiltrated the inflammatory site, they may use eHsp70
itself as a chemoattractant. In vitro experiments with and without heat shock
have demonstrated lymphocyte export of eHsp70 (Hunter-Lavin et al.,
2004). Similarly, increases in macrophage phagocytosis and neutrophil
microbicide activity have been reported after exogenous Hsp70 administra-
tion (Kovalchin et al., 2006; Ortega et al., 2006). eHsp70 also prompted
proinflammatory cytokine release from epithelial, innate immune, and adap-
tive immune cells, which may explain why eHsp70 is associated with inflam-
matory cytokine production in multiple mouse and human studies (Asea et al.,
2000, 2002; Basu, Binder, Ramalingam, & Srivastava, 2001; Chase et al.,
2007; Dvoriantchikova, Santos, Saeed, Dvoriantchikova, & Ivanov, 2014;
Dybdahl et al., 2005; Huang, Wang, Chen, Wang, & Zhang, 2013; Qiao,
Liu, & Li, 2008). Collectively, these data support a strong capacity of eHsp70
to initiate and escalate an immune-mediated inflammatory response.
A growing list of receptors seem to mediate the immunomodulatory
actions of eHsp70 by evoking a host of proinflammatory cytokines
(IL-1β, IL-6, IL-8, IL-12, TNF-α) through a CD14-dependent, Toll-like
receptor (TLR) 2/4 mechanism (Asea et al., 2000, 2002; Chase et al.,
2007; Dvoriantchikova et al., 2014; Dybdahl, 2001). TLR2 has also been
linked to eHsp70 stimulation of neutrophil chemotaxis (Ortega et al.,
2009). Similarly, CD94, a C-type lectin receptor, has been demonstrated
to mediate eHsp70 stimulation of IFN-γ release from NK cells (Gross,
Hansch, Gastpar, & Multhoff, 2003). However, a separate report indicated
that this response required coculturing the NK cells with dendritic cells that
produce NK G2D ligand after eHsp70 treatment (Qiao et al., 2008). The
differences in these studies might be clarified by their use of patient-derived
NK cells vs the YT leukemic NK cell line. CD40 was determined to be nec-
essary for mycobacterial-Hsp70-driven beta-chemokine release and human
eHsp70 was demonstrated to interact with CD40 (Lehner et al., 2000). The
CD40-eHsp70-dependent effects were expanded to include internalization
of eHsp70–peptide complexes for antigen presentation in macrophages
(Becker, 2002; Lehner et al., 2000; Wang et al., 2001). Surprisingly, an anal-
ogous study identified CD91, not CD40, as managing antigen presentation
in macrophages and dendritic cells through the MHC class I molecule by
188 S.M. Emery and R.T. Dobrowsky

an eHsp70–peptide complex (Basu et al., 2001). However, Theriault,

Mambula, Sawamura, Stevenson, and Calderwood (2005) demonstrated
that Hsp70 has little capacity to directly bind any of the receptors mentioned
earlier. Instead, they showed a robust ability for eHsp70 to bind LOX-1, a
class E scavenger receptor that collects modified lipoproteins such as oxi-
dized low-density lipoprotein. The authors hypothesize that eHsp70 inter-
acts directly with LOX-1 and then indirectly utilizes CD14, CD40, CD91,
CD94, and TLR2/4 to coordinate its effects (Theriault et al., 2005). This
hypothesis is supported by eHsp70’s LOX-1-dependent ability to modulate
antigen cross-presentation in dendritic cells (Delneste et al., 2002). With the
exception of Theriault et al. (2005), one limitation of these studies is that
they utilized antagonism and correlation to confirm the involvement of
these receptors in eHsp70 signaling. Becker (2002) and Wang et al.
(2001) are the exceptions, as both studies showed specific binding of fluo-
rescently labeled eHsp70 after CD40 transfection in HEK293 and COS-7
cells, respectively. However, these data are in direct contradiction to
Theriault et al. (2005) who showed a lack of fluorescent Hsp70 binding
in HEK293 cells after CD40 transfection. Unfortunately, the authors were
unable to reconcile the differences between these earlier results indicating
more work is still needed to clarify how eHsp70 may interact with putative
receptor proteins.
Downstream of eHsp70 binding, NF-κB has been implicated in
neutrophil chemotaxis, bronchial epithelial cell-mediated inflammation,
LPS-induced liver injury, monocyte cytokine production, and ischemia–
reperfusion neurotoxic inflammation (Krause et al., 2015). Multiple lines
of evidence support NF-κB involvement in these eHsp70-dependent pro-
cesses: NF-κB nuclear translocation, Iκ-Bα phosphorylation, Bay 11-7082
and parthenolide antagonism, and NF-κB activity measured by various
reporter assays (Asea et al., 2000, 2002; Chase et al., 2007;
Dvoriantchikova et al., 2014; Huang et al., 2013; Ortega et al., 2009).
PKC has also been implicated in the immunomodulatory action of eHsp70
based on antagonism by polymixin B (Dvoriantchikova et al., 2014). How-
ever, polymixin B has not been shown to inhibit eHsp70-dependent NF-κB
activation, which suggests PKC and NF-κB may function as separate signal-
ing pathways (Asea et al., 2000). One obvious gap in this research is that all
evidence for eHsp70 activation of NF-κB signaling has evaluated CD14 and
TLR 2/4 as the intermediary receptors. Work is still needed to investigate
downstream eHsp70 signaling after LOX-1 binding and what role CD40,
CD91, and CD94 might play in that process.
Modulating Hsp70 to Treat DPN 189

4. INTRACELLULAR Hsp70
4.1 Chaperone Functions
The molecular chaperone activity of intracellular heat shock proteins has
been extensively characterized (De Maio, 2011). iHsp70 is an integral com-
ponent of this chaperone system and the complex cochaperone interactions
and molecular mechanisms that facilitate this process have been described in
numerous reviews (Becker & Craig, 1994; Kim et al., 2013; Saibil, 2013;
Stetler et al., 2010). A major function of this chaperone activity is to main-
tain intracellular proteostasis by assisting nascent peptide folding, refolding of
unfolded proteins, preventing inappropriate protein interactions, managing
protein aggregate formation, and degrading dysfunctional proteins (Saibil,
2013). These activities have particular relevance in neurodegenerative
diseases, which are often associated with aggregates of misfolded proteins,
including Alzheimer’s disease, Parkinson’s disease, ALS, Huntington’s
disease, and others (Turturici et al., 2011). It is believed that postmitotic
neurons affected in these pathologies are particularly vulnerable due to an
inability to diminish misfolded protein concentrations through cell division
(Muchowski & Wacker, 2005). Consequently, enhancing iHsp70 action
might prove a viable therapeutic approach to treating these pathologies.
In vitro, iHsp70 increases the solubility and microtubule interaction of
the tau protein, as well as early-stage aggregation of Aβ that leads to amyloid
plaque formation in Alzheimer’s disease (Dou et al., 2003; Evans, Wisen, &
Gestwicki, 2006). However, managing inappropriate protein interactions is
only part of the solution to minimizing misfolded protein stress. If a protein
cannot be refolded to a functional state, then it must be degraded to prevent
accumulation.
SOD1 is a cytosolic free radical scavenger protein that is integral to man-
aging oxidative stress and mutant SOD1 promotes mitochondrial dysfunc-
tion that contributes to the onset of familial ALS (Tan, Pasinelli, & Trotti,
2014). CHIP is a C-terminal Hsp70-interacting protein that facilitates
ubiquitination and degradation of misfolded iHsp70 client proteins like
mutant SOD1. Mutant SOD1 is also degraded by chaperone-mediated
autophagy where regulator proteins such as Bcl-2-associated athanogene
3 (BAG-3) and Hsp22 direct iHsp70’s chaperone functions to identify, traf-
fic, and present misfolded proteins for autophagic degradation (Kalmar,
Lu, & Greensmith, 2014). This orchestrated removal of dysfunctional pro-
tein is crucial for neurological homeostasis, as well as metabolic disorders
190 S.M. Emery and R.T. Dobrowsky

such as diabetes mellitus. In particular, Type 2 diabetes has been specifically

linked to ER stress, which results from misfolded proteins accumulating
in the ER. Glucose-regulated protein 78, the ER-specific Hsp70 family
member, is a well-known mediator of the unfolded protein response
(UPR) to manage ER stress (Kalmar et al., 2014; Lee & Ozcan, 2014).
However, there is also evidence that iHsp70-mediated cytosolic protein
degradation facilitates stabilization of ER stress in addition to the UPR
(Goeckeler & Brodsky, 2010).

4.2 Oxidative Stress

A significant portion of misfolded or dysfunctional proteins are caused by
oxidative stress. Although the chaperone functions of iHsp70 are essential
to managing intracellular damage directly, iHsp70 can also indirectly miti-
gate disturbances in proteostasis due to a potent ability to manage oxidative
stress. In the human premonocytic U937 cell line, heat shock protects from
H2O2-driven mitochondrial membrane depolarization and structural
changes (Polla et al., 1996). Similarly, Hsp70-overexpressing transgenic ani-
mals have curtailed age-associated oxidative stress (Broome et al., 2006) and
multiple cell lines are protected from ROS-mediated cytotoxicity via heat
treatment and/or iHsp70 overexpression (Bellmann, Jaattela, Wissing,
Burkart, & Kolb, 1996; Bellmann et al., 1995; Jaattela & Wissing, 1993).
Multiple mechanisms have been linked to mediating these protective effects.
For example, the superoxide-generating NADPH oxidases (NOX) are neg-
atively regulated by iHsp70. In diabetic sensory neurons, NOX2 mRNA
overexpression was reversed in an Hsp70-dependent manner by treatment
with the C-terminal Hsp90 inhibitor, KU-596 and this correlated with an
improvement in DPN (see Section 5) (Ma et al., 2015). Further, in vascular
smooth muscle cells, coimmunoprecipitation and Hsp70 knockdown stud-
ies demonstrate iHsp70 trafficking of NOX4 regulates degradation of the
protein (Bocanegra, Manucha, Pena, Cacciamani, & Valles, 2010; Gil
Lorenzo, Bocanegra, Benardon, Cacciamani, & Valles, 2014). NOX
isoforms are a major source of nonmitochondrial ROS and modulating
Hsp70 in a mouse model of Type 2 diabetes decreased nonmitochondrial
oxygen respiration, indirectly suggesting an effect on decreasing the activity
of NOX isoforms (Ma et al., 2014).
Much of total cellular ROS production does not require NOX activity
and Hsp70 management of these non-NOX sources requires intervention
with antioxidant enzymes such as SODs. In Hsp70 knockout mice, a
Modulating Hsp70 to Treat DPN 191

connection between Hsp70 and SOD1 expression was surmised since cere-
brum homogenates from these animals showed a marked decrease in SOD1
protein levels and activity (Choi et al., 2005). Interestingly, this regulation
may not be unidirectional, since SOD1-overexpressing transgenic mice
have elevated levels of Hsp70 mRNA following cerebral ischemia
(Kondo et al., 1996). Nevertheless, iHsp70 seems to have a capacity to mod-
ulate SOD1 expression. Evidence for Hsp70 regulation of SOD2 is not so
clear-cut, however. Hsp70 knockout mice also have decreased SOD2 activ-
ity, but these mice do not show changes in SOD2 protein expression levels
(Choi et al., 2005). This difference between SOD2 activity and expression
may be explained by two observations. First, iHsp70 has been found to traf-
fic SOD2 to the mitochondria (Afolayan et al., 2014) and second, SOD2
requires additional processing in the mitochondria before it becomes fully
functional (Candas & Li, 2014). As a result, genetic deletion of iHsp70
would prevent SOD2 from being efficiently localized to the mitochondria
for activation. Consistent with this premise, pharmacological induction of
Hsp70 in hyperglycemically stressed rat embryonic sensory neurons
increased mitochondrial SOD2 expression, as determined by quantitative
mass spectrometry (Zhang et al., 2012).
Another major role iHsp70 plays in managing oxidative stress involves
supporting proper mitochondrial function. Mitochondria are a major source
of ROS production within the cell, having a concentration 5- to 10-fold
higher than in the cytosol (Cadenas & Davies, 2000). Left unchecked, intra-
cellular stress can overload or impair the mitochondrial machinery, decrease
protein import, and ultimately result in mitochondrial dysfunction and sig-
nificant increases in oxidative stress (Baseler et al., 2011; Tomlinson &
Gardiner, 2008). For instance, hyperglycemic stress of embryonic sensory
neurons decreased the translation of numerous mitochondrial proteins
and mitochondrial oxygen consumption while increasing superoxide levels.
However, increasing iHsp70 levels with a C-terminal Hsp90 inhibitor
decreased superoxide production and improved mitochondrial bioenerget-
ics (Zhang et al., 2012). Further studies from our lab have demonstrated that
pharmacologically modulating Hsp70 has a beneficial effect on mitochon-
drial bioenergetics (Ma et al., 2014; Urban et al., 2012; Zhang et al.,
2012). Interestingly, RNA-Seq analysis indicated that the mitochondrial
transcriptome was unaltered by pharmacological modulation of Hsp70 in
diabetic mice, despite an improvement in mitochondrial bioenergetics.
Therefore iHsp70’s protection of mitochondrial bioenergetics may not be
significantly mediated through gene transcription (Ma et al., 2015).
192 S.M. Emery and R.T. Dobrowsky

iHsp70-dependent influx of mitochondrial proteins may offer an explana-

tion for at least part of this mitochondrial bioenergetics protection
(Afolayan et al., 2014).
Only 1% of mitochondrial proteins are encoded by mitochondrial DNA,
the remaining 99% must be imported after nuclear transcription (Schmidt,
Pfanner, & Meisinger, 2010; Wiedemann, Frazier, & Pfanner, 2004). Hsp70
was first shown to bind and facilitate translocation of proteins to mitochon-
dria in yeast (Deshaies, Koch, Werner-Washburne, Craig, & Schekman,
1988; Murakami, Pain, & Blobel, 1988). Translocases of the outer mito-
chondrial membrane (Tom) form a complex that is composed of various
receptors and Tom40, an import pore formed which most nuclear-
transcribed proteins must traverse (Schmidt et al., 2010; Wiedemann
et al., 2004). Tom70 acts as the mitochondrial receptor for iHsp70 to facil-
itate mitochondrial protein import (Young, Hoogenraad, & Hartl, 2003).
After transit through the outer membrane, the preprotein that iHsp70
was transporting is pulled through the inner mitochondrial membrane using
translocases of the inner mitochondrial membrane (Tim). Tim50 helps tran-
sition from the outer mitochondrial membrane to the inner, sending the
preprotein to a channel formed by Tim23 and Tim17 (Schmidt et al.,
2010; Wiedemann et al., 2004). Mitochondrial Hsp70 is anchored to
Tim44 and together they act as the major components of an ATP-dependent
motor that pulls the preprotein into the inner mitochondrial space
(Krimmer, Rassow, Kunau, Voos, & Pfanner, 2000). Once inside, the
preprotein is processed to facilitate proper mitochondrial function
(Schmidt et al., 2010; Wiedemann et al., 2004).
Mitochondrial dysfunction is only one mechanism by which intracellular
stress can drive oxidative stress. Another can be seen in the interaction
between hyperglycemia- and thioredoxin (Trx)-interacting protein
(TXNIP). It has been speculated that TXNIP plays a role in regulating
insulin-independent glucose transport into tissues (Singh, 2013). This is
largely based on an observation that TXNIP can regulate glucose transporter
expression and localization (Wu et al., 2013). However, prolonged hyper-
glycemic stress can drive persistent activation of TXNIP, as was shown in the
retina of Type 1 diabetic rats (Devi et al., 2012). TXNIP’s most well-
characterized function is as an inhibitor of the antioxidant- and thiol-
reducing protein, Trx, and extended inhibition of Trx by TXNIP drives
oxidative stress (Schulze et al., 2004). Further exacerbating the situation,
prolonged TXNIP activation can also increase inflammatory signaling,
although it is unclear if these oxidative and inflammatory events occur
Modulating Hsp70 to Treat DPN 193

sequentially or in parallel (Devi et al., 2012; Lane, Flam, Lockey, &

Kolliputi, 2013). Recent work from our lab has shown that Hsp70 may play
a role in affecting the expression of TXNIP. RNA-seq analysis of mRNA
from DRG of Type 1 diabetic mice showed a twofold elevated expression of
TXNIP expression (Ma et al., 2015) and treating diabetic mice with the
chaperone modulator, KU-596 (see Section 5), suppressed TXNIP expres-
sion to control levels (Fig. 1A). Moreover, this suppression was Hsp70
dependent as TXNIP mRNA levels remained elevated (1.4-fold) in
DRG from diabetic Hsp70 KO mice that were treated with KU-596
(Fig. 1B). Though it remains unclear whether Hsp70 inhibited TXNIP
overexpression directly or by alleviating the initial hyperglycemic/oxidative

Fig. 1 Modulating Hsp70 with KU-596 decreased the expression of txnip in diabetic DRG
in an Hsp70-dependent manner. Wild-type (A) and Hsp70 KO (B) mice were rendered
diabetic with streptozotocin and after 12 weeks of diabetes, treated with vehicle or
weekly doses of KU-596 at 20 mg/kg for 4 weeks. mRNA was harvested from the lumbar
DRG and used for RNA-Seq analysis. Shown are representative alignments to the mouse
txnip gene from nondiabetic, diabetic, and diabetic animals that received KU-596 ther-
apy. Dark blue (black in the print version) bars indicate sequences that mapped to the
forward strand, while dark red (dark gray in the print version) bars are sequences that
mapped to the reverse strand of the eight exons of the txnip gene. See Ma et al., 2015 for
additional details.
194 S.M. Emery and R.T. Dobrowsky

stress insult, these results suggest that Hsp70 attenuates intracellular oxidative
stress induced by diabetes, which correlates with an improvement in clini-
cally relevant measures of DPN.

4.3 Inflammation
Another major aspect of iHsp70 biology is its ability to regulate inflamma-
tory signaling. Heat shock prevents TNF-α production after ischemic stress
in the renal tubular epithelial cell line LLC-PK1 and in rat liver after induc-
tion of sepsis by cecal ligation and puncture (CLP; Chen, Kuo, Wang, Lu, &
Yang, 2005; Meldrum, 2003). In vivo, Hsp70 overexpression suppresses
increased expression of IL-6 and TNF-α induced by LPS, as well as elevation
of IL-6 and cytokine-induced neutrophil chemoattractant-1 in rat blood
after CLP (Dokladny, Lobb, Wharton, Ma, & Moseley, 2010; Weiss
et al., 2007). Each of these studies reported that an Hsp70-dependent sup-
pression of NF-κB activation mediated the antiinflammatory action. Using
DNA binding and nuclear localization, numerous studies have supported
this claim with heat shock (Feinstein et al., 1996), liposomal Hsp70 delivery
(Meldrum, 2003), and Hsp70 overexpression (Feinstein, Galea, & Reis,
1997). Mechanistically, this effect may be due to Hsp70-dependent decrease
in degradation of the NF-κB inhibitor (IκBα), which binds NF-κB to
inhibit its nuclear localization (Dokladny et al., 2010; Feinstein et al.,
1997; Wong, Ryan, & Wispe, 1997). Precipitating this action is Hsp70’s
ability to inhibit phosphorylation of the IκB kinase (IKK). IKK phosphor-
ylates IκBα, causing it to dissociate from NF-κB and be degraded. In the
absence of IKK phosphorylation, this process is blocked (Chan, Ou,
Wang, & Chan, 2004; Chung et al., 2008; Weiss et al., 2007). Immunopre-
cipitation studies demonstrate that Hsp70 associates with the IKK complex,
which hinders the IKKβ subunit of the complex from being integrated
(Weiss et al., 2007). Additional studies support Hsp70 binding to IKKγ
(NEMO), which prevents its trimerization and function as a structural com-
ponent of the IKK complex (Agou et al., 2002; Chen et al., 2005; Ran
et al., 2004).
A second Hsp70 mechanism for hindering NF-κB activation has also
been described since Hsp70 stabilized phosphorylated and ubiquitinated
IκBα. This interaction prevented the degradation of IκBα without affecting
the IKK complex (Weiss et al., 2007). Additionally, a separate report sug-
gests a third mechanism where Hsp70 directly upregulates IκBα expression
to suppress NF-κB activation (Wong, Ryan, Menendez, & Wispe, 1999).
Modulating Hsp70 to Treat DPN 195

Taken together, these studies outline a highly redundant system where

iHsp70 can prevent NF-κB activation by manipulating IκB. Further, two
studies also suggest that Hsp70 can also prevent NF-κB activation without
altering IκBα concentrations. Overexpression of Hsp70 in C6 rat glioma
cells did not prevent LPS-induced IκBα degradation, despite suppression
of p65 NF-κB subunit nuclear localization. The authors speculated that
in certain situations, Hsp70 may instead prevent NF-κB nuclear localization
by either competing for the nuclear pore or binding NF-κB after its initial
dissociation from IκB (Feinstein et al., 1997). A later study replicated this
finding using liposomal delivery of Hsp70 (Meldrum, 2003) and
coimmunoprecipitation of Hsp70 with the p65 subunit of NF-κB, which
supports the viability of this hypothesis (Chen et al., 2005). Clearly, this
body of work demonstrates that NF-κB can be intimately regulated by
iHsp70. However, as activation of the NF-κB pathway in sensory neurons
has been shown to ameliorate DPN (Saleh, Roy Chowdhury, et al., 2013;
Saleh, Schapansky, et al., 2013), it may be counterproductive to invoke
therapies which may upregulate both the NF-κB pathway and iHsp70 in
neurons.

4.4 Insulin Sensitivity, JNK, and c-Jun

In Type 2 diabetes, iHsp70 may be able to improve insulin resistance and
blood glucose levels. Mice fed a high-fat diet show both insulin resistance
and glucose intolerance compared to normal chow-fed animals. Transgenic
animals overexpressing Hsp70 show no insulin resistance and markedly
improved glucose tolerance when fed the high-fat diet (Henstridge,
Bruce, et al., 2014; Henstridge, Whitham, & Febbraio, 2014). The actions
of Hsp70 overexpression were attributed to decreased phosphorylation of
c-jun N-terminal kinase (JNK), a known regulator of insulin sensitivity.
Heat treatment and pharmacological induction of Hsp70 also inhibited
JNK phosphorylation (Chung et al., 2008; Henstridge, Bruce, et al.,
2014). Similarly, Hsp70 overexpression and heat shock suppressed JNK1
activity in NIH 3T3 cells after ultraviolet light stress. JNK1 and Hsp70
can physically interact and this prevented JNK1 phosphorylation and
suppressed c-jun transactivating activity (Park, Lee, Huh, Seo, & Choi,
2001). However, JNK regulation is not the only mechanism by which
iHsp70 can modulate targets downstream of JNK.
C-jun is emerging as a key negative regulator of myelination (Arthur-
Farraj et al., 2012; Hutton et al., 2011; Parkinson et al., 2008) and altering
196 S.M. Emery and R.T. Dobrowsky

its expression via modulation of iHsp70 may offer an attractive approach to

treat the segmental demyelination that occurs in human DPN, as well as in
various inherited neuropathies. In myelinated DRG explants, induction of
Hsp70 by treatment with the C-terminal Hsp90 modulator KU-32
inhibited c-jun/phospho-c-jun expression and decreased the extent of
neuregulin-induced demyelination (Fig. 2A and C–E). Though KU-32
did not decrease the phosphorylation of JNK and ERK, inhibiting the

Fig. 2 Modulating Hsp70 with KU-32 decreased neuregulin-induced c-jun expression

and demyelination in an Hsp70-dependent manner. Myelinated DRG explants were pre-
pared from wild-type or Hsp70 KO mice. The cultures were treated with vehicle or 1 μM
KU-32 overnight prior to inducing demyelination with 200 ng/mL neuregulin 1. Cultures
were stained for myelin basic protein and the degeneration of the myelinated segments
was quantified (A and B). ***p < 0.001 vs control; p ^ < 0.01 vs NRG1. Other cultures were
prepared for immunoblot analysis of c-jun and the effects of the treatments on c-jun
and phospho-c-jun expression quantified (C–E). **p < 0.01 vs control in WT and
Hsp70 KO; p ^ < 0.05 vs NRG1 in WT only. Modified with permission from Li, C., Ma, J., Zhao,
H., Blagg, B. S., & Dobrowsky, R. T. (2012). Induction of heat shock protein 70 (Hsp70) pre-
vents neuregulin-induced demyelination by enhancing the proteasomal clearance of c-jun.
ASN Neuro, 4, e00102. ASN Neuro published by Portland Press.
Modulating Hsp70 to Treat DPN 197

proteasome with MG132 prevented the decrease in c-jun expression. These

data support that iHsp70 facilitates the proteasomal degradation of c-jun to
attenuate demyelination (Fig. 2) (Li et al., 2012). Importantly, the necessity
of iHsp70 in preventing the increase in c-jun expression and demyelination
was validated using myelinated DRG explants prepared from Hsp70 KO
mice. Although neuregulin treatment promoted a robust induction of
c-jun in these cultures, treatment with KU-32 was unable to block this
increase and prevent demyelination, as was observed in myelinated neuronal
cultures prepared from wild-type mice (Fig. 2B–E). Similarly, adenovirus-
mediated reexpression of Hsp70 in the Hsp70 KO neurons was sufficient to
decrease c-jun induction following neuregulin treatment. Since trans-
criptomic analysis of mRNA from sural nerve of patients with progressive
DPN showed an increase in a gene subnetwork centered on c-jun (Hur
et al., 2011), it is tempting to speculate that iHsp70 modulation of c-jun sig-
naling may benefit later stages of progressive DPN. Unfortunately, diabetic
rodent models do not recapitulate the extensive segmental demyelination
that is observed in human DPN, but ongoing work using animal models
of demyelinating neuropathies is examining whether decreasing c-jun
expression by modulating iHsp70 may improve myelinated fiber function.

5. DPN AND MODULATING Hsp70

Clearly, a large body of evidence supports the concept that modulating
Hsp70 may have both beneficial and deleterious effects (Fig. 3). Indeed
overexpression of iHsp70 is associated with various forms of cancer and
its downregulation can increase sensitivity to chemotherapeutics (Lianos
et al., 2015). eHsp70 may contribute to the development of experimental
autoimmune encephalitis, an animal model of multiple sclerosis (Mansilla
et al., 2014). Furthermore, elevated eHsp70 levels may contribute to inflam-
mation in obese, Type 2 diabetic patients, ketoacidotic Type 1 diabetics
(Oglesbee, Herdman, Passmore, & Hoffman, 2005; Rodrigues-Krause
et al., 2012) and play a role in the progression of diabetes based on its ability
to induce β-cell dysfunction and death (Krause et al., 2014). On the other
hand, increasing iHsp70 expression in skeletal muscle improved insulin
resistance and decreased metabolic parameters of Type 2 diabetes (Chung
et al., 2008; Henstridge, Bruce, et al., 2014). The chaperone balance
hypothesis gives context to these observations by proposing that a high ratio
of [eHsp70]:[iHsp70] is associated with promoting chronic inflammatory
responses, while a low [eHsp70]:[iHsp70] ratio is more antiinflammatory
198 S.M. Emery and R.T. Dobrowsky

Fig. 3 Summary of potential impact of iHsp70 and eHsp70 on hyperglycemia-mediated

dysfunction. (I) Hyperglycemia manipulates multiple metabolic pathways to induce both
intra- and extracellular stresses to the cell. These events converge at three points affecting
mitochondrial dysfunction, oxidative stress, and proinflammatory signaling to facilitate
the progression of DPN. (II) iHsp70 inhibits hyperglycemia-induced dysfunctions through
multiple mechanisms including mitochondrial protein shuttling, up/downregulation of
anti/prooxidant proteins, respectively, repair or degradation of damaged and misfolded
protein, and inhibition of proinflammatory signaling via NF-κB. (III) eHsp70 may play
a complicated role since if internalized, it can bolster iHsp70 stores and actions while
external binding to one of the multiple purported receptors can antagonize the action
of iHsp70 by initiating or exacerbating NF-κB-mediated inflammatory signaling.

(Krause et al., 2015). If this hypothesis proves to be true, then attempts to

alter the progression of DPN by modulating Hsp70 may benefit from exam-
ining this relationship. This begets the question, what are our options for
modulating Hsp70?
Modulating Hsp70 to Treat DPN 199

Though genetic approaches to downregulate or overexpress Hsp70 pro-

vide an avenue to modulate its activity in animal models of disease, these
approaches have not been translated to humans. Similarly, no selective
small-molecule inhibitors (or activators) of inducible Hsp70 exist that can
be translated to the clinic (Evans, Chang, & Gestwicki, 2010; Lianos
et al., 2015). This difficulty is partially attributed to the fact that five cellular
Hsp70 family members share 86–99% homology (Daugaard, Rohde, &
Jaattela, 2007). However, there are viable small-molecule therapies that
can indirectly modulate Hsp70 levels by targeting Hsp90 or HSF1.
Hsp90 is required for the folding of many proteins into their final con-
formation and is the master regulator of the heat shock response. Numerous
compounds have been developed that target either the N- or C-terminal of
the protein to alter its enzymatic activity or interaction with cochaperones
(Miyata, Nakamoto, & Neckers, 2013; Peterson & Blagg, 2009; Walton-
Diaz et al., 2013). Since many Hsp90 client proteins are also oncoproteins,
N-terminal inhibitors may serve as chemotherapeutics by promoting client
protein degradation. As chemotherapeutics, one attribute of N-terminal
Hsp90 inhibitors is their selectivity for inhibiting Hsp90 and promoting cli-
ent protein degradation in cancerous vs noncancerous cells (Chiosis et al.,
2003; Kamal et al., 2003; Luo, Rodina, & Chiosis, 2008). However, induc-
tion of client protein degradation and cytotoxicity often occurs at drug con-
centrations that also induce the heat shock response. Unfortunately, an
increase in chaperone expression can antagonize the compound’s cytotoxic
potential by facilitating oncoprotein refolding, narrowing the drug’s effec-
tive therapeutic window (Peterson & Blagg, 2009). On the other hand, an
increase in chaperone synthesis may be useful for treating neurodegenerative
diseases since N-terminal Hsp90 inhibitors have been shown to limit or
decrease tau aggregation in models of Alzheimer’s disease (Dickey et al.,
2007; Luo et al., 2007), huntingtin protein in motor neurons (Waza
et al., 2005), α-synuclein in Parkinson’s disease (Ebrahimi-Fakhari,
Saidi, & Wahlster, 2013), and peripheral myelin protein 22 in Schwann cells
from a mouse model of Charcot Marie Tooth 1A (Chittoor-Vinod, Lee,
Judge, & Notterpek, 2015). However, it is still necessary to dissociate the
degradation of client proteins from stimulation of the heat shock response
since client protein degradation could lessen the neuroprotective effective-
ness of the drugs.
Over the past 5 years we have identified novologues as a class of
C-terminal Hsp90 inhibitors derived from novobiocin that induce Hsp70
in the absence of significant client protein degradation (Kusuma et al.,
200 S.M. Emery and R.T. Dobrowsky

2012; Urban et al., 2010). KU-32 and KU-596 are two such compounds
that were mentioned above and these drugs improved multiple clinical
indices of DPN in models of both Type 1 and Type 2 diabetes, in an
Hsp70-dependent manner (Ma et al., 2014, 2015; Urban et al., 2010,
2012). Similarly, an N-terminal Hsp90 inhibitor has proven beneficial in
treating diabetic nephropathy and the physiologic improvement in renal
function may also be Hsp70 dependent (Lazaro et al., 2015). As neither
of these diabetic complications has an etiology that centers on formation
of any one protein aggregate, clearly modulating Hsp70 may prove effica-
cious toward treating complex metabolic complications of diabetes.
Although all these compounds were shown to increase iHsp70, their effects
on eHsp70 levels were not examined. Consistent with many of the studies
described in Section 4, novologues diminished the level of oxidative stress,
improved mitochondrial bioenergetics, and decreased an array of genes in
DRG that were associated with inflammation (Ma et al., 2014, 2015;
Urban et al., 2012; Zhang et al., 2012). The improvement of DPN and
decrease in inflammation by KU-596 therapy was also associated with a
transcriptomic profile that was predicted to inhibit the NF-κB pathway
in DRG of diabetic wild-type mice but not diabetic Hsp70 KO mice
(Ma et al., 2015). These actions would be inconsistent with enhancing sys-
temic levels of eHsp70, but it is possible that a more localized exchange of
eHsp70 between Schwann cells and sensory neurons may contribute to the
dependence of drug effectiveness on Hsp70.
It is interesting that increasing Hsp70 in skeletal muscle using the HSF1
activator, BGP-15, improved fasting glucose and insulin signaling in obese
mice (Henstridge, Bruce, et al., 2014; Henstridge, Whitham, et al., 2014).
However, novologues improved DPN in an Hsp70-dependent manner
without correcting any metabolic parameters in models of both Type 1
and Type 2 diabetes. Similarly, the N-terminal Hsp90 inhibitor and
geldanamycin derivative (DMAG) improved diabetic nephropathy without
effecting metabolic measures of diabetes: weight loss, blood glucose, and
HbA1c (Lazaro et al., 2015). The reason for the difference between these
compounds is curious, but may lie in BGP-15 being a more direct activator
of HSF1. It is encouraging that no adverse effects were reported from a short
28-day clinical trial in patients given 200 or 400 mg BGP-15 daily, which
may be consistent with minimal drug effect on increasing eHsp70 activity
(Literáti-Nagy et al., 2009). As treatment of diabetic complications such
as DPN will require prolonged drug administration, it will be important
to assess any effects on eHsp70 as novel modulators of Hsp70 move forward
Modulating Hsp70 to Treat DPN 201

clinically. Fortunately, eHsp70 should be easy to monitor in a clinical trial

and this will provide some insight into whether this pool of the protein
correlates with adverse, presumably inflammatory, side effects.

6. CONCLUDING REMARKS
In summary, results in animal models and small clinical trials clearly
provide compelling proof of concept that modulating molecular chaperones
and particularly iHsp70 provides a viable approach to improve insulin resis-
tance and diabetic complications. Although we are not putting forth the pre-
mise that directly targeting proteins and pathways that are linked to the
pathophysiological progression of DPN is a less than fruitful approach to dis-
ease management, it seems that altering any one pathway may be insufficient
to treat the human disease. A similar fate may befall an approach that only
modulates iHsp70 to treat DPN. However, since modulating Hsp70
improves oxidative stress, mitochondrial function, and to some extent
inflammation, increasing the ability of neurons and Schwann cells to tolerate
ongoing diabetic stress may facilitate the action of additional agents or
improve the ability of glycemic control to slow the rate of onset and dimin-
ish the severity of DPN, especially in Type 2 diabetic patients.

ACKNOWLEDGMENTS
This work was supported by Grant DK095911 from the National Institute of Diabetes,
Digestive, and Kidney Diseases to R.T.D. and a postdoctoral fellowship from the
Pharmaceutical Research and Manufacturers of America Foundation to S.M.E.

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 CHAPTER ELEVEN

Painful Diabetic Neuropathy:

Prevention or Suppression?
S.M. Todorovic1
School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, United States
1
Corresponding author: e-mail address: [email protected]

Contents
1. Introduction 212
2. Nociceptive Ion Channels Control Neuronal Excitability 213
3. Alterations of Voltage-Gated and Ligand-Gated Ion Channels in Sensory Neurons
in Animal Models of Type 1 Diabetes 215
4. Alterations of CaV3.2 T-Type Channels in Nociceptive Sensory Neurons in Animal
Models of Type 2 Diabetes with Painful PDN 217
5. Posttranslational Modification of Pronociceptive Ion Channels in PDN 217
6. Diminished Inhibitory Drive in the Spinal Cord May Also Contribute to Painful PDN 221
7. Conclusions 221
Acknowledgments 222
References 223

Abstract
Pain-sensing sensory neurons (nociceptors) of the dorsal root ganglia (DRG) and dorsal
horn (DH) can become sensitized (hyperexcitable) in response to pathological condi-
tions such as diabetes, which in turn may lead to the development of painful peripheral
diabetic neuropathy (PDN). Because of incomplete knowledge about the mechanisms
underlying painful PDN, current treatment for painful PDN has been limited to some-
what nonspecific systemic drugs that have significant side effects or potential for abuse.
Recent studies have established that several ion channels in DRG and DH neurons are
dysregulated and make a previously unrecognized contribution to sensitization of pain
responses by enhancing excitability of nociceptors in animal models of type 1 and type
2 PDN. Furthermore, it has been reported that targeting posttranslational modification
of nociceptive ion channels such as glycosylation and methylglyoxal metabolism can
completely reverse mechanical and thermal hyperalgesia in diabetic animals with
PDN in vivo. Understanding details of posttranslational regulation of nociceptive chan-
nel activity may facilitate development of novel therapies for treatment of painful PDN.
We argue that pharmacological targeting of the specific pathogenic mechanism rather
than of the channel per se may cause fewer side effects and reduce the potential for
drug abuse in patients with diabetes.

#
International Review of Neurobiology, Volume 127 2016 Elsevier Inc. 211
ISSN 0074-7742 All rights reserved.
http://dx.doi.org/10.1016/bs.irn.2016.03.005
212 S.M. Todorovic

1. INTRODUCTION
Diabetes mellitus is a chronic, multifactorial disease characterized by
hyperglycemia. The World Health Organization estimates that 220 million
people worldwide currently suffer from diabetes. Diabetes mellitus type 1
(formerly called insulin-dependent diabetes or juvenile diabetes) is a form
of diabetes that results from autoimmune destruction of insulin-producing
β-cells in the pancreas. In contrast, type 2 diabetes (formerly called
noninsulin-dependent or adult-onset diabetes), the most common form
of diabetes, results from insulin resistance and is most often associated with
obesity. Peripheral diabetic neuropathy (PDN) is the most frequent compli-
cation of diabetes, and it has been estimated that 60–70% of diabetic patients
will develop PDN symptoms with prevalence increasing with the duration
of diabetes mellitus (Centers for Disease Control and Prevention, 2011).
The prevalence of painful PDN is often underestimated, but a recent large
community-based study with more than 15,000 patients with diabetes
reported painful symptoms in about one-third of all participants (Abbott,
Malik, van Ross, Kulkarni, & Boulton, 2011). These patients can suffer from
both stimulus-evoked painful episodes, including hyperalgesia (exaggerated
pain experience upon presentation of noxious stimuli) and/or allodynia
(normally nonnoxious stimuli that may evoke pain sensation), as well as
spontaneous pain in the form of burning or tingling. Interestingly, these
painful pathologies in diabetics usually occur in parallel with degeneration
of peripheral nerves (Abbott et al., 2011). Eventually, these painful symp-
toms usually subside as the disabling pain is replaced by complete loss of sen-
sation (insensate PDN). Both intractable pain and loss of sensation have
significant adverse effects on quality of life measures. Unfortunately, current
treatment options are unable to reverse these symptoms and are often asso-
ciated with serious side effects. For example, CaV2.2 (N-type) subtypes of
high-voltage-activated (HVA) calcium channels and their regulatory sub-
unit α2δ are considered a major cellular target for the anticonvulsants
gabapentin and pregabalin, which are commonly used to relieve diabetes-
induced pain in clinics. However, more than 50% of patients using
gabapentin or pregabalin experience side effects, such as excessive sedation,
ataxia, dizziness, euphoria, and weight gain, all of which limit their clinical
use (Edwards, Vincent, Cheng, & Feldman, 2008). Although opioids are
partially effective for treatment of painful PDN, they are not suitable for
chronic use since their long-term use is associated with serious side effects
including constipation, urinary retention, impaired cognitive function,
Painful Diabetic Neuropathy: Prevention or Suppression? 213

respiratory depression, and issues associated with tolerance and addiction.

Furthermore, topical applications of therapeutic creams with lidocaine
and capsaicin are inconsistently effective in patients with painful PDN
and cause unpleasant side effects like numbness and burning, respectively.
This underscores the fact that new mechanism-specific therapeutic treat-
ments must be considered that could be more effective and possibly better
tolerated in diabetic patients.
Pain is often a result of the activation of neurons (nociceptors) that are
activated following injury of peripheral tissue such as the skin and internal
organs. This form of pain, called nociceptive pain, is self-protective and typ-
ically evokes actions aimed at limiting or avoiding further tissue damage.
Nociceptive pain is typically responsive to treatments with conventional
painkillers, such as nonsteroidal antiinflammatory drugs or opiates. In con-
trast, neuropathic pain results from primary dysfunction of peripheral noci-
ceptive, as well as nonnociceptive nerves and/or components of the central
nervous system (central pain). Neuropathic pain typically outlasts the initial
stage of injury and frequently leads to debilitating disorders that are not ame-
nable to conventionally available drug therapies. It is generally accepted that
pathologies associated with neuropathic pain result from increased excitabil-
ity of peripheral nerves and/or pain pathways in central nervous system
(CNS). For example, nociceptors can become inappropriately sensitized
by various mechanisms in response to local or systemic metabolic conditions
or peripheral tissue injury associated with diabetes. Multiple pathogenic
mechanisms, such as the formation of intracellular advanced glycation end
products (AGE), release of inflammatory cytokines, increased glucose
metabolism by the polyol pathway, and oxidative stress, may contribute
to the impaired function of sensory neurons in animals with PDN. How-
ever, preclinical and clinical studies aiming to target several of these mech-
anisms while simultaneously ensuring proper blood glucose control have
been unable to provide definite and complete pain relief (Edwards et al.,
2008; Gooch & Podwall, 2004). This incomplete pain reversal strongly sug-
gests that other molecular mechanisms may be involved in hyperglycemia-
induced alterations of function of nociceptive sensory neurons.

2. NOCICEPTIVE ION CHANNELS CONTROL NEURONAL

EXCITABILITY
Several studies conducted in recent years have reported on the plas-
ticity of various ion channels expressed in nociceptive sensory neurons,
also known as dorsal root ganglion (DRG) neurons, which play a critical
214 S.M. Todorovic

role in modulating overall cellular excitability (reviewed by Calcutt, 2013;

Todorovic & Jevtovic-Todorovic, 2011; Todorovic & Jevtovic-Todorovic,
2014). These findings are both important and relevant, since increased excit-
ability of sensory neurons is believed to contribute directly to the develop-
ment and maintenance of painful symptoms, including hyperalgesia,
allodynia, and spontaneous pain. Studies have shown that the Cav3.2 iso-
form of the T-type voltage-gated calcium channel (T-channel) is heavily
expressed in DRG cells and the dorsal horn (DH) of the spinal cord and plays
a distinct role in supporting pathological pain in animal models of both type
1- and type 2-induced PDN (Jacus, Uebele, Renger, & Todorovic, 2012;
Jagodic et al., 2007; Latham et al., 2009). T-type channels were first discov-
ered in preparations of acutely dissociated sensory neurons of small size
(Carbone & Lux, 1984). The characteristic biophysical features, including
activation by small membrane depolarization, relatively fast and voltage-
dependent inactivation kinetics, as well as slow deactivation kinetics upon
channel closing following maximal activation, make them ideally suited
to control cellular excitability of sensory neurons. Relatively soon after their
initial discovery, it was indeed demonstrated that T-channels control
subthreshold excitability of DRG cells by lowering the threshold for action
potential generation in a subpopulation of acutely dissociated rat DRG cells
of medium size (White, Lovinger, & Weight, 1989). By convention, it is
accepted that most of the smaller diameter DRG cells (cell soma < 31 μm)
are putative nociceptors, while medium-size DRG cells (cell soma
32–45 μm) can be either nociceptive or nonnociceptive sensory neurons.
Most acutely dissociated small DRG cells expressing T-currents are sensitive
to the algogene compound from hot peppers, capsaicin, and have the elec-
trical properties of classically described nociceptors with wide action poten-
tials (Nelson et al., 2007). T-channels are also present in a subpopulation of
medium-sized acutely dissociated sensory neurons that are either putative
nociceptors, since they bear the IB4 (isolectin B4) antigen (Jagodic et al.,
2007), or that subserve touch sensation mediated by specific DRG cell type
called D-hair mechanoreceptors (Dubreuil et al., 2004; Shin, Martinez-
Salgado, Heppenstall, & Lewin, 2003). Furthermore, the CaV3.2 isoform
of T-channels is a critical regulator of cellular excitability of nociceptive
DRG neurons termed “T-rich” cells that are also responsive to capsaicin
and are IB4 positive (Nelson, Joksovic, Perez-Reyes, & Todorovic,
2005). Prominent CaV3.2 T-currents in “T-rich” and medium-size DRG
cells generate visible after-depolarizing potentials and high-frequency
burst firing along with small depolarizations of the membrane potential.
In addition, amplification of subthreshold membrane depolarizations via
Painful Diabetic Neuropathy: Prevention or Suppression? 215

CaV3.2 T-channels typically decreases the threshold for action potential fir-
ing in DRG cells that express T-channels.

3. ALTERATIONS OF VOLTAGE-GATED AND

LIGAND-GATED ION CHANNELS IN SENSORY
NEURONS IN ANIMAL MODELS OF TYPE 1 DIABETES
Several studies have demonstrated upregulation of CaV3.2 currents
in both small- and medium-size DRG cells in animal models of painful
PDN in type 1 diabetes. For example, in diabetic Bio-Bred/Worchester
(BB/W) rats, a model for autoimmune-mediated type 1 diabetes,
upregulation of T-channel current density of about threefold was observed
in a mixed population of small- and medium-size DRG cells after 8 months
in a diabetic state (Hall, Sima, & Wiley, 1995). The current–voltage rela-
tionship in these cells was not different between control, healthy rats, and
experimental diabetic rats. Similarly, studies with streptozotocin (STZ)-
induced diabetic neuropathy, which used electrophysiological recordings
from acutely isolated IB4-positive medium- and small-size DRG cells,
found that T-current amplitude was upregulated about twofold (Cao,
Byun, Chen, & Pan, 2011; Jagodic et al., 2007; Khomula et al., 2013).
In addition, these three studies have shown that biophysical properties
of T-channels were altered in DRG cells from diabetic animals, with a sig-
nificant depolarizing shift in steady-state inactivation curves. As a result of
this shift, more T-channels were available at physiological membrane
potentials, and this increased propensity for burst firing in DRG cells from
diabetic rats as compared with healthy ones (Jagodic et al., 2007; Khomula
et al., 2013).
Additional studies have documented upregulation of pronociceptive ion
channels such as voltage-gated sodium channels, particularly the NaV1.7 and
NaV1.8 isoforms (Bierhaus et al., 2012; Hoeijmakers, Faber, Merkies, &
Waxman, 2014), purinergic P2X3 receptors (Xu, Li, Liu, & Huang,
2011), and the vanilloid family of ligand-gated channels, especially transient
receptor potential vanilloid 1, TRPV1 (Brederson, Kym, & Szallasi, 2013),
within sensory neurons in animal models of diabetes. Given the prominent
role of TRP channels in sensory transduction, it is reasonable to assume that
dysregulation of capsaicin-gated TRPV1 channels may also contribute to
abnormal pain signaling in different subpopulations of DRG cells from rats
with STZ-induced PDN. For example, Hong and Wiley found using
immunohistochemistry that membrane expression of TRPV1 protein in rats
was increased in myelinated A-type fiber DRG cells, while it was decreased
216 S.M. Todorovic

in small unmyelinated C-type nociceptive DRG cells as a result of diabetes

(Hong & Wiley, 2005). In contrast, a study by Pabbidi and colleagues dem-
onstrated increased expression of TRPV1 protein and greater whole-cell
current in small DRG cells in both STZ-induced and transgene-mediated
type 1 painful PDN in mice (Pabbidi, Cao, Parihar, Pauza, &
Premkumar, 2008; Pabbidi, Yu, et al., 2008). Consistent with increased
function of TRPV1 in DRG cells expressing T-currents, capsaicin-gated
currents were increased in medium-size (Jagodic et al., 2007) and small
DRG cells (Khomula et al., 2013; Shankarappa, Piedras-Renterı́a, &
Stubbs, 2011) from STZ-induced diabetic rats with painful PDN. This
observation underscores the importance of possible cross talk between noci-
ceptive ligand and voltage-gated ion channels that control calcium signaling
in DRG cells. Increased activity of both channel types in concert could con-
tribute to cellular hyperexcitability and consequently to hyperalgesia and
allodynia in PDN. Pabbidi and colleagues reported that STZ may have
had a direct effect on expression of TRPV1 channels in DRG cells that
was independent of hyperglycemia and hence may result from the direct
toxicity of this compound (Pabbidi, Cao, et al., 2008; Pabbidi, Yu, et al.,
2008). However, present evidence demonstrates that this is not the case with
DRG CaV3.2 T-channels. A causal relationship between hyperglycemia in
STZ-induced PDN in rats and expression of T-type currents in DRG cell
was effectively documented since upregulation of T-currents closely
followed development of hyperglycemia and was completely reversed in
parallel with diabetic hyperalgesia reversal upon chronic insulin treatments
(Messinger et al., 2009). An interesting report has documented that forced
exercise in diabetic STZ-treated rats prevented the depolarizing shift in
steady-state inactivation of T-currents in small DRG cells in parallel with
improvement of pain symptoms of PDN in vivo (Shankarappa et al.,
2011). This is important given that these findings could potentially be trans-
lated in human medicine. Furthermore, in vivo knockdown of the CaV3.2
isoform of T-channels in DRG cells using antisense oligonucleotides
reversed upregulation of T-currents in small DRG cells and diabetic
hyperalgesia in STZ-treated rats (Messinger et al., 2009; Obradovic et al.,
2014). Finally, as proof of concept, it was reported that when compared
to wild-type littermates, diabetic STZ-treated knockout mice lacking the
CaV3.2 isoform of T-channels failed to develop thermal and mechanical
hyperalgesia despite the prominent hyperglycemia (Latham et al., 2009).
Taken together, these studies have firmly established that the CaV3.2 iso-
form of T-channels in DRG cells is required for the development and main-
tenance of painful PDN in rats and mouse models of type 1 diabetes.
Painful Diabetic Neuropathy: Prevention or Suppression? 217

4. ALTERATIONS OF CaV3.2 T-TYPE CHANNELS IN

NOCICEPTIVE SENSORY NEURONS IN ANIMAL
MODELS OF TYPE 2 DIABETES WITH PAINFUL PDN
Morbid obesity may be accompanied by type 2 diabetes and painful
PDN that is manifested by mechanical/thermal allodynia and hyperalgesia.
Importantly, clinical studies have documented that painful PDN is at least
twofold more common in patients with type 2 PDN than in patients
with type 1 PDN (Edwards et al., 2008). Hence, the function of CaV3.2
T-channels in DRG cells and its possible relationship with abnormal pain
perception in PDN was investigated in leptin-deficient morbidly obese
ob/ob mice (Latham et al., 2009). It was found that ob/ob mice developed
significant mechanical and thermal pain hypersensitivity early in life that
coincided with the onset of hyperglycemia (maximal at the age of 10–16
weeks) and was reversed with timely administered insulin therapy that
was initiated before diabetes fully developed. These disturbances were
accompanied by significant biophysical and biochemical modulation of
T-channels in DRG neurons as indicated by about a twofold increase in
the amplitude of T-currents and about a threefold increase in expression
of channel mRNA. The most prevalent isoform of T-channels expressed
in nociceptive DRG cells, Cav3.2, was the most strongly affected. More-
over, systemic injections of (3β,5α,17β)-17-hydroxyestrane-3-carbonitrile
(ECN), a neuroactive steroid compound and selective T-channel antago-
nist, provided dose-dependent alleviation of neuropathic thermal and
mechanical hypersensitivity in diabetic ob/ob mice. This study indicates that
selective pharmacological antagonism of T-channels can potentially be an
important novel therapeutic approach for the management of pain associated
with PDN. Thus, it appears that in both type 1 and type 2 painful PDN,
metabolic abnormalities leading to hyperglycemia may be causative events
that eventually lead to upregulation of DRG T-current, increased cellular
excitability of nociceptors, and consequently hyperalgesia and allodynia.

5. POSTTRANSLATIONAL MODIFICATION OF
PRONOCICEPTIVE ION CHANNELS IN PDN
Aberrant regulation of function of nociceptive ion channels in sensory
neurons via posttranslational modification and ensuing hyperexcitability
may be factors contributing to painful symptoms of PDN. One of the most
prevalent forms of posttranslational modification of proteins is glycosylation,
218 S.M. Todorovic

which involves the attachment of sugar molecules to a protein. Glycosyla-

tion regulates protein conformation and activity, is involved in protein pro-
tection from proteolytic degradation, and plays an important role in protein
trafficking and secretion (Moremen, Tiemeyer, & Naim, 2012). However,
very little is known about the possible involvement of glycosylation in the
sensitization of nociceptive responses in the setting of diabetic hyperglyce-
mia. Unsurprisingly, it has been reported that diabetes results in a nearly
threefold increase in peripheral nerve glycosylation as measured in tissue
homogenates from diabetic rats and dogs (Vlassara, Brownlee, & Cerami,
1981). Hence, it is plausible that aberrant and/or excessive glycosylation
of certain susceptible proteins in peripheral nerves may play a role in the
pathogenesis of PDN. Unfortunately, the identity of these glycosylated pro-
teins and their precise role in the pathogenesis of diabetes-induced nerve
dysfunction remains elusive.
Protein glycosylation frequently involves extracellular asparagine resi-
dues. Thus, it is plausible to propose that as glucose levels rise in sensory neu-
rons, as a consequence of diabetic hyperglycemia, excessive glycosylation
may become a maladaptive process and lead to dysfunction of certain ion
channels that control cellular excitability. Prevailing literature has focused
on protein glycation in a diabetic environment, a process that typically
involves intracellular accumulation of AGE that alter protein function by
modifying intracellular arginine and lysine residues (Jack & Wright, 2012;
Obrosova, 2009). However, decreasing production of AGE in vivo has
inconsistently and incompletely reversed pain symptoms in animal models
of diabetes and in patients with PDN (Edwards et al., 2008). Hence, it is
possible that other parallel signaling mechanisms, such as glycosylation,
may be involved in hyperglycemia-induced alterations of nociceptive ion
channel function in sensory neurons.
Upregulation of CaV3.2 (but not CaV3.1 or CaV3.3) transcripts in DRG
tissues from ob/ob mice can account at least partially for increased T-current
density in small DRG cells, but it does not account for kinetic alterations of
T-currents (Latham et al., 2009). Thus, it was considered that these channels
in DRGs from diabetic ob/ob mice must be additionally modified by other
mechanisms including glycosylation. This was recently addressed by using
patch-clamp recordings from small DRG and recombinant human embry-
onic kidney (HEK-293) cells, immunohistochemistry, biochemical studies,
as well as in vivo pain studies using ob/ob mice (Orestes et al., 2013). First, it
was found that, in addition to increased current density, T-currents in small
DRG cells exhibit marked speeding of macroscopic current kinetics in
Painful Diabetic Neuropathy: Prevention or Suppression? 219

diabetic ob/ob mice. The glycosylation inhibitor neuraminidase completely

reversed upregulation and kinetic alterations of CaV3.2 in small DRG cells
from diabetic ob/ob mice, while it had a much smaller effect on T-currents
in healthy wild-type mice. Intraplantar (i.pl.) injections of neuraminidase
directly into peripheral receptive fields of diabetic ob/ob mice were able
to reverse completely the mechanical and thermal hyperalgesia. To test
whether the antihyperalgesic effect of neuraminidase in ob/ob mice is
related to its effects on T-currents, the selective T-channel blocker ECN
was injected and completely reversed thermal and mechanical hyperalgesia
in ob/ob mice, while subsequent i.pl. injections of neuraminidase in paws
did not further decrease pain thresholds. Hence, these studies indicate that
glycosylation inhibitors like neuraminidase may be used to normalize sen-
sory transmission in DRG cells and peripheral nerves in animals with
painful PDN.
Two recent independent studies have reported that glycosylation of crit-
ical extracellular asparagine residues of CaV3.2 channels causes kinetic alter-
ations, upregulation of T-currents, as well as an increase in membrane
expression of T-channels (Orestes et al., 2013; Weiss, Black, Bladen,
Chen, & Zamponi, 2013). Molecular studies using site-directed mutagenesis
identified conserved extracellular asparagine residues, most notably
N192 and N1466, as important regulators of CaV3.2 current kinetics and
channel membrane expression, respectively. The involvement of N192
was supported by patch-clamp recordings that demonstrated slower current
kinetics in an N192Q CaV3.2 mutant, with apparently normal membrane
expression. Interestingly, T-currents in HEK-293 cells transfected with an
N1466Q CaV3.2 mutant could not be consistently recorded, and imaging
studies showed minimal membrane expression of this mutant (Orestes
et al., 2013). Thus, different glycosylation sites in CaV3.2 channels may have
distinct functional roles. Surprisingly, authors of both studies could not
record T-currents from N271Q Cav3.2 channels despite their presence in
the cell membrane. It remains possible that N271Q channels trafficked to
the membrane are nonfunctional. In contrast to the finding of Orestes
et al., Weiss and colleagues found that asparagine N192 serves as a regulator
of CaV3.2 channel membrane expression and asparagine N1466 as a regu-
lator of channel kinetics (Weiss et al., 2013). It is possible that different levels
of glucose in cell culture could have contributed to the different findings
between the studies. Glycosylated products have larger molecular mass,
meaning that glycosylation or the enzymatic removal of these groups from
the channel protein will induce a shift in the migration of proteins bands
220 S.M. Todorovic

resolved by gel-shift analysis and visualized by Western blotting. The study

by Orestes and colleagues took advantage of this and directly demonstrated,
using gel-shift analysis, that recombinant CaV3.2 channels are, indeed,
glycosylated within domain I of the channel protein. However, levels of
glycosylation of native CaV3.2 channels in sensory neurons in diabetic ani-
mals remain to be determined in future studies. Finally, neither of these two
studies examined the role of neuraminidase or other agents that can modu-
late glycosylation upon membrane excitability of nociceptors in animals
with PDN.
Several other in vitro studies have reported that glycosylation may mod-
ulate properties of other pronociceptive voltage-gated ion channels. For
example, it was reported that in embryonic DRG neurons, neuraminidase
alters steady-state inactivation of voltage-gated sodium channels (Tyrrell,
Renganathan, Dib-Hajj, & Waxman, 2001). This study did not find any
effects of neuraminidase on voltage-gated sodium channels in small DRG
cells from healthy adult animals. However, it remains to be determined in
future studies if neuraminidase could modulate voltage-gated sodium chan-
nels in nociceptive DRG cells from diabetic animals. Future extensive elec-
trophysiological studies could also be expanded to involve examination of
other ligand-gated channels (eg, TRPV1) that are crucial for the control
of cellular excitability of DRG cells from diabetic animals that might also
be modulated by glycosylation (Cohen, 2006; Jing et al., 2012; Pertusa,
Madrid, Morenilla-Palao, Belmonte, & Viana, 2012).
In addition to glycosylation of nociceptive ion channels, posttranslational
modification of the voltage-gated sodium channels, specifically Nav1.8
isoform and transient receptor potential channel A1 (TRPA1), via
methylglyoxal, an endogenous reactive metabolite of glucose, has been
demonstrated in DRG neurons in animals with STZ-induced diabetes
(Andersson et al., 2013; Bierhaus et al., 2012; Eberhardt et al., 2012).
High plasma concentrations of methylglyoxal correlate well with occur-
rence of painful symptoms in patients with PDN (Bierhaus et al., 2012).
Furthermore, methylglyoxal depolarizes putative nociceptive DRG neu-
rons and induces hyperexcitability of these cells thus facilitating action
potential firing and in vivo administration of methylglyoxal evoked thermal
and mechanical hyperalgesia. In contrast to glycosylation, above-mentioned
studies have demonstrated that this form of posttranslational modification
of Nav1.8 channels and TRPA1 channels is via modification of intracel-
lular lysine and arginine-binding sites (Bierhaus et al., 2012; Eberhardt
et al., 2012).
Painful Diabetic Neuropathy: Prevention or Suppression? 221

Finally, upregulation of HVA calcium currents in sensory neurons in

STZ-treated diabetic rats via G-proteins may underlie their diminished
responsiveness to opioids (Hall, Liu, Sima, & Wiley, 2001; Hall, Sima, &
Wiley, 1996).

6. DIMINISHED INHIBITORY DRIVE IN THE SPINAL CORD

MAY ALSO CONTRIBUTE TO PAINFUL PDN
Activation of nociceptors can trigger a lasting increase in the excitabil-
ity and synaptic efficacy of DH neurons in a phenomenon termed central
sensitization, which in turn contributes to many pain disorders in humans
including: neuropathic pain, fibromyalgia, and osteoarthritis (Woolf,
2011). This can be manifested as prolonged and/or exaggerated response
to painful stimuli (hyperalgesia), a reduction in threshold for pain (allodynia),
and/or receptive field expansion that enables input from noninjured tissues
to produce pain (secondary hyperalgesia). Hence, drugs like gabapentin and
pregabalin that reduce central facilitation of DH neurons by diminishing
glutamate-mediated excitatory drive, via binding to the α2δ ancillary sub-
unit of voltage-gated calcium channels, are among the most effective treat-
ments for hyperalgesia and allodynia in PDN. Unfortunately, in many
patients (about 50%) these drugs induce sedation, making them unsuitable
for long-term therapy (Gooch & Podwall, 2004). Furthermore, it is well
documented that promotion of inhibitory drive mediated via
γ-aminobutyric acid (GABA) activation in the DH to activate inhibitory
inputs is also beneficial in alleviating neuropathic pain symptoms in various
animal models of peripheral nerve injury (Sivilotti & Woolf, 1994; Woolf,
2011). Surprisingly, the possibility that spinal GABAergic system may be
involved in painful PDN has not been extensively studied. In a very inter-
esting recent study, Lee-Kubli and Calcutt (2014) have found diabetic rats
have impaired spinal GABAA receptor inhibitory function arising from
reduced spinal potassium chloride cotransporter, which collectively leads
to spinal disinhibition of pain responses. Hence, designing therapies that
aim to restore impaired GABAergic function in spinal cord may be a novel
approach to the treatment of painful PDN.

7. CONCLUSIONS
It is likely that multiple signaling pathways targeting nociceptive ion
channels may work in concert to promote hyperexcitability of sensory
222 S.M. Todorovic

neurons and contribute to hyperalgesia, allodynia, and spontaneous pain in

patients with PDN. This chapter, and a recent commentary (Todorovic,
2015), summarize how different pronociceptive ion channels expressed in
peripheral nerve terminals in skin, cell somas in DRG, and central terminals
of sensory neurons may work together with spinal GABA-mediated dys-
function to increase sensitization of pain responses under diabetic
conditions.
It is hoped that future efforts in targeting metabolic pathways such as
glycosylation and/or methylglyoxal formation may lead to novel and safer
pain therapies in patients with diabetes. We posit that a therapeutic strategy
to suppress pain by normalizing nociceptive channel function will be
advantageous to that of direct channel blockade because, by correcting
the pathology of PDN at its source, this approach should avoid the mul-
titude of unintended side effects that are associated with current therapies.
We envision that specific targeting of dysregulated biochemical pathways
that regulate function of nociceptive ion channels may be developed into
a novel, effective, and safe mechanistic-based therapeutic approach. This
may be achieved by specifically targeting peripheral axons of nociceptors
using therapeutic ointments and creams and/or applying drugs that reverse
cellular hyperexcitability directly onto DRGs using fluoroscopy-guided
epidural injections in pain clinics. Alternatively, systemically administered
drugs may be used, although this will likely increase the risk of side effects.
Most patients with painful PDN ask for medical help well after painful
symptoms have developed. It is an attractive alternative approach to
attempt to identify the population at risk for painful PDN using screening
test for glycosylated and methylglyoxal-modified proteins in patients with
PDN. This would allow physicians to initiate treatments before sensitiza-
tion of peripheral and central pain responses is fully developed. To our
knowledge, this topic is controversial, and a recent clinical study failed
to find correlation between serum methylglyoxal levels and symptoms of
peripheral and cardiovascular autonomic neuropathy (Hansen et al.,
2015). However, studies like this remain an important area of future
investigations.

ACKNOWLEDGMENTS
Our research is supported by American Diabetes Association National Award for
Basic Research 7-09-BS-190 (to S.M.T.) and research funds from the Department of
Anesthesiology at the University of Virginia, Charlottesville, VA, as well as Department
of Anesthesiology at the University of Colorado Anschutz Medical Campus, Aurora, CO.
Painful Diabetic Neuropathy: Prevention or Suppression? 223

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 CHAPTER TWELVE

New Directions in Diabetic

Neuropathy: Evolution or
Extinction?
P. Fernyhough*,†,1, N.A. Calcutt{
*University of Manitoba, Winnipeg, MB, Canada
†
St. Boniface Hospital Research Centre, Winnipeg, MB, Canada
{
University of California, San Diego, La Jolla, CA, United States
1
Corresponding author: e-mail address: [email protected]

Contents
1. NCV as an Endpoint in Animal Models 229
2. Treatment Paradigms 230
3. Insanity: Doing the Same Thing Over and Over Again and Expecting
a Different Result 231
References 232

The earlier chapters “Neuropathy in the DCCT/EDIC” by Pop-Busui and

“The Perfect Clinical Trial” by Bril explained the value of long clinical
trials (5 or more years) to enable any therapeutic effect against progressive
diabetic neuropathy to be detected. These calculations were based primarily
on findings from the Diabetes Control and Complications Trial (DCCT),
where intensive insulin therapy significantly protected nerve conduction
velocity (NCV) in type 1 diabetic patients (DCCT, 1993, 2002). Because
of the slow degradation of NCV at a rate that has historically been of
approximately 0.5–1.0 m/s per annum, a trial of this duration was required
to see separation between untreated and treated groups given the study
group sizes and data variability. NCV clearly provides an objective measure
that gives information about the current status of large myelinated nerve
fiber dysfunction and is predictive of its progression. Thus, it is a strongly
validated measure of large fiber neuropathy that can detect effective ther-
apeutic intervention.

#
International Review of Neurobiology, Volume 127 2016 Elsevier Inc. 229
ISSN 0074-7742 All rights reserved.
http://dx.doi.org/10.1016/bs.irn.2016.03.009
230 P. Fernyhough and N.A. Calcutt

1. NCV AS AN ENDPOINT IN ANIMAL MODELS

Drugs entered into clinical trial against diabetic neuropathy have,
to date, been selected primarily based upon their ability to prevent NCV
slowing in rodent models of diabetes. A potential problem with this approach
is the applicability of such preclinical efficacy studies to the human setting. The
NCV deficit in animal models usually develops within the first month of onset
of type 1 diabetes (Eliasson, 1964; Greene, De Jesus, & Winegrad, 1975). The
acute disruption of metabolism causes rapid changes in endoneurial blood flow
and other parameters that negatively impact nerve conduction such as
Schwann cell function, nodal function, and axonal function (Hounsom &
Tomlinson, 1997; Sima, 2004; Yagihashi, Yamagishi, & Wada, 2007). These
acute alterations in cell biology and function are preventable and rapidly
reversible by many interventions and occur months before any structural dete-
rioration of large myelinated nerve fibers is detectable. It remains to be
unequivocally proven that these early deficits in nerve function are causal fac-
tors in subsequent structural deterioration of large myelinated nerve fibers.
The other proboscidean in this particular parlour is that structural deterioration
of large nerve fibers is a very subtle affair in diabetic rodents. In STZ-diabetic
rats, where the majority of preclinical drug studies have been performed, there
is minimal overt degeneration of large myelinated nerve fibers, no demyelin-
ation, and little axonal pathology (Kalichman, Dines, Bobik, & Mizisin, 1998;
Zochodne, Verge, Cheng, Sun, & Johnston, 2001). The only established and
widely reproduced sign of a degenerative process is impaired maturation of
axonal diameter and later axonal dwindling (Jakobsen, 1976a, 1976b). As dis-
cussed earlier in this volume, alternative models that develop a large-fiber neu-
ropathology that reflects the human disease, such as cats, are available but have
not yet been widely used (Mizisin et al., 2007; Mizisin, Shelton, Burgers,
Powell, & Cuddon, 2002). At present, there is little evidence to suggest that
positive effects on NCV deficits in animal models of diabetic neuropathy pro-
vide any true mechanistic insight into the degenerative process occurring in
human diabetic neuropathy or have predictive value for efficacy when trans-
lated to clinical use. It may well be that the preclinical pipeline that has fed
clinical trials has always been broken, or at least is not correctly aligned.

2. TREATMENT PARADIGMS
The majority of preclinical studies have used a prevention paradigm.
Drugs are applied at the beginning of the study and forestall development
New Directions in Diabetic Neuropathy 231

of deficits in NCV. In some longer-term studies this approach has also

prevented onset of reduced axonal caliber of large myelinated fibers
(Calcutt et al., 2003, 1999; Kato, Mizuno, Makino, Suzuki, & Yagihashi,
2000). However, the ability of an intervention to correct established struc-
tural deterioration of large myelinated fibers has rarely, if ever, been evalu-
ated. It is therefore not surprising that drugs have shown minimal effects in
phase 2 and phase 3 clinical trials with patients exhibiting established neu-
ropathy that is likely accompanied by large-fiber degeneration and loss.
Indeed it is plausible that the statistically significant, but sadly unimpressive,
1.0–1.5 m/s improvements in NCV reported in recent well-designed and
executed clinical trials (Bril, Hirose, Tomioka, & Buchanan, 2009;
Wahren, Foyt, Daniels, & Arezzo, 2016) represent the maximal effect that
can be achieved against large-fiber dysfunction when the bulk of the NCV
deficit in humans may reflect established structural pathology that is unre-
sponsive to the intervention. To date, the drugs under investigation have
not demonstrated efficacy against pertinent indices of neuropathy in appro-
priate animal models.

3. INSANITY: DOING THE SAME THING OVER AND OVER

AGAIN AND EXPECTING A DIFFERENT RESULT
After 30+ years of failed clinical trials, we owe it to those with diabetic
neuropathy, and those likely to face it in the future, to review current
approaches to developing therapies for diabetic neuropathy. Fortunately,
we may not have to completely reinvent the wheel, just adjust our
approaches, biases, and inertia. The measurement of NCV is a valid and use-
ful endpoint in clinical trials for diabetic neuropathy. However, it need not
be the central component upon which any decision on success or failure is
made. Indeed, the perceived failure of a recent clinical trial of C-peptide that
could not separate improvement of NCV in treated subjects vs those treated
with placebo but did report statistically significant improvement of vibration
perception (Wahren et al., 2016) highlights the absurdity of focusing on
NCV as the dominant biomarker for neuropathy while ignoring the patient.
As the authors of the above study noted, measuring large-fiber NCV may
not even reflect other more relevant aspects of large-fiber sensory function.
The chapters “Alternative Quantitative Tools in the Assessment of Diabetic
Peripheral and Autonomic Neuropathy” by Vinik and “Wherefore Art
Thou O Treatment for Diabetic Neuropathy?” by Malik describe interest-
ing and, for us, compelling arguments for using measures of small-fiber
neuropathy, both sensory and autonomic, that allow us to reassess our model
232 P. Fernyhough and N.A. Calcutt

of the preclinical drug screen as a precursor to clinical trials. Perhaps the most
pertinent development in recent years is that studies in rodent models of
diabetes are now using an array of small-fiber endpoints that replicate the
clinical condition to test ideas on etiology and screen new therapies
(Beiswenger, Calcutt, & Mizisin, 2008; Christianson, Riekhof, & Wright,
2003; Christianson, Ryals, Johnson, Dobrowsky, & Wright, 2007;
Davidson, Coppey, Holmes, & Yorek, 2012a, 2012b). Loss of epidermal
fibers, loss of sweat gland innervation, loss of thermal (hot and cold) sensa-
tion, and loss of corneal nerve fiber density all occur in both diabetic animals
and humans (Arezzo, Schaumburg, & Laudadio, 1986; Breiner, Lovblom,
Perkins, & Bril, 2014; Hossain, Sachdev, & Malik, 2005; Kennedy,
Wendelschafer-Crabb, & Johnson, 1996; Liu et al., 2015; Malik et al.,
2003; Zinman, Bril, & Perkins, 2004). Other advantages of working with
small-fiber endpoints arise from reports that onset of structural change
can be detected early in the course of disease in both diabetic animals
and humans (Azmi et al., 2015; Christianson et al., 2003, 2007; Mehra et
al., 2007; Petropoulos et al., 2015; Quattrini et al., 2007; Smith,
Ramachandran, Tripp, & Singleton, 2001). Corneal nerve fiber loss, which
can be detected by confocal microscopy, can be viewed noninvasively so as
to allow frequent iterative assessment before and after therapeutic interven-
tions in diabetic rodents and humans (Chen et al., 2013; Tavakoli et al.,
2013). It should also be remembered that the aspects of diabetic neuropathy
that most trouble patients reflect sensory and autonomic dysfunction. Once
fully validated, the introduction of measures of small-fiber nerve pathology
and dysfunction as primary goals and endpoints in clinical trials of treatments
for diabetic neuropathy may allow shorter, less costly, trials that have better
chances of gaining regulatory approval and improving the lives of patients.

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 CHAPTER THIRTEEN

Alternative Quantitative Tools

in the Assessment of Diabetic
Peripheral and Autonomic
Neuropathy
voret†
A.I. Vinik*,1, C. Casellini*, M.-L. Ne
*Eastern Virginia Medical School, Strelitz Diabetes and Neuroendocrine Center, Norfolk, VA, United States
†
Impeto Medical Inc., San Diego, CA, United States
1
Corresponding author: e-mail address: [email protected]

Contents
1. Introduction 236
2. Diabetic Peripheral Neuropathy 238
2.1 Clinical Presentation 238
2.2 Diagnosis 240
2.3 Falls Risk in Older Adults with Diabetes 241
3. Diabetic Autonomic Neuropathy 245
3.1 Clinical Manifestations 245
3.2 Cardiovascular Autonomic Neuropathy 247
3.3 Prevention and Reversibility of Autonomic Neuropathy 251
4. Measuring Diabetic Neuropathy: Established and Innovative Approaches 253
4.1 QOL Measures 253
4.2 Clinical Assessment Tools 256
4.3 Objective Measurements 256
5. Concluding Remarks 270
References 271

Abstract
Here we review some seldom-discussed presentations of diabetic neuropathy, includ-
ing large fiber dysfunction and peripheral autonomic dysfunction, emphasizing the
impact of sympathetic/parasympathetic imbalance. Diabetic neuropathy is the most
common complication of diabetes and contributes additional risks in the aging adult.
Loss of sensory perception, loss of muscle strength, and ataxia or incoordination lead to
a risk of falling that is 17-fold greater in the older diabetic compared to their young
nondiabetic counterparts. A fall is accompanied by lacerations, tears, fractures, and
worst of all, traumatic brain injury, from which more than 60% do not recover. Auto-
nomic neuropathy has been hailed as the “Prophet of Doom” for good reason. It is con-
ducive to increased risk of myocardial infarction and sudden death. An imbalance in the

International Review of Neurobiology, Volume 127 # 2016 Elsevier Inc. 235

ISSN 0074-7742 All rights reserved.
http://dx.doi.org/10.1016/bs.irn.2016.03.010
236 A.I. Vinik et al.

autonomic nervous system occurs early in the evolution of diabetes, at a stage when
active intervention can abrogate the otherwise relentless progression. In addition to
hypotension, many newly recognized syndromes can be attributed to cardiac auto-
nomic neuropathy such as orthostatic tachycardia and bradycardia. Ultimately, this con-
stellation of features of neuropathy conspire to impede activities of daily living,
especially in the patient with pain, anxiety, depression, and sleep disorders. The resulting
reduction in quality of life may worsen prognosis and should be routinely evaluated and
addressed. Early neuropathy detection can only be achieved by assessment of both
large and small- nerve fibers. New noninvasive sudomotor function technologies
may play an increasing role in identifying early peripheral and autonomic neuropathy,
allowing rapid intervention and potentially reversal of small-fiber loss.

1. INTRODUCTION
Neuropathy is the most common and troublesome complication of
diabetes mellitus, leading to the greatest morbidity and mortality and
resulting in a huge economic burden for diabetes care (Holzer et al.,
1998; Vinik, Mitchell, et al., 1995) (Fig. 1). It is the most common form
of neuropathy in the developed countries of the world, accounts for more
hospitalizations than all the other diabetic complications combined, and is

Clinical impact of aging and DPN

DPN

Painful Impairment
Neuropathic
neuropathic disability
deficits
symptoms handicap

Mortality
Foot ulcers
Ataxia charcot
weakness
Cost
$37B

Quality of Falls Infection

life 12,000,000/y (skin, bone)

Fractures Surgery
Cost lacerations amputation
$200B traumatic brain 96,000/y
injury

Fig. 1 The clinical and financial implications of aging and diabetic peripheral neurop-
athy (DPN).
Alternative Neuropathy Assessments 237

responsible for 50–75% of nontraumatic amputations (Caputo, Cavanagh,

Ulbrecht, Gibbons, & Karchmer, 1994; Holzer et al., 1998). Diabetic neu-
ropathy represents a set of clinical syndromes that affect distinct regions of
the nervous system, single or combined. It may be silent and go undetected
while exercising its ravages or it may present with clinical symptoms and
signs that, although nonspecific and insidious, mimic those seen in many
other diseases. Diabetic neuropathy is, therefore, diagnosed by exclusion.
Unfortunately neither endocrinologists nor nonendocrinologists are rou-
tinely trained to recognize the condition and, even when neuropathy is
symptomatic, less than one-third of physicians recognize the cause or discuss
this with their patients (Herman & Kennedy, 2005).
The true prevalence of diabetic neuropathy is not known and reports
vary in their estimates from 10% to 90% in diabetic patients, depending
on the criteria and methods used to define neuropathy (Caputo et al.,
1994; Dyck et al., 1993; Holzer et al., 1998; Young, Boulton, MacLeod,
Williams, & Sonksen, 1993). Twenty-five percent of patients attending a
diabetes clinic volunteered symptoms and 50% were found to have neurop-
athy after a simple clinical test such as the ankle jerk or vibration perception
test, while almost 90% tested positive in more sophisticated evaluations of
autonomic or peripheral nerve function (Vinik, 1999). Neurologic compli-
cations occur equally in type 1 and type 2 diabetes mellitus and in the various
forms of acquired diabetes (Dyck et al., 1993). The major morbidity asso-
ciated with neuropathy is foot ulceration, the precursor of gangrene and
limb loss. Neuropathy increases the risk of amputation 1.7-fold, 12-fold if
there is deformity (itself a consequence of neuropathy), and 36-fold if there
is a history of previous ulceration (Armstrong, Lavery, & Harkless, 1998). In
2010, 73,000 amputations were performed on diabetic patients in the
United States, yet up to 75% are preventable (Caputo et al., 1994). Globally
there is an amputation every 30 s. Diabetic neuropathy also has a tremen-
dous impact on patients’ quality of life (QOL), predominantly by causing
weakness, ataxia, and incoordination predisposing to falls and fractures
(Vinik et al., 2005). Once autonomic neuropathy sets in, life can become
quite dismal and the mortality rate approximates 25–50% within 5–10 years
(Levitt, Stansberry, Wychanck, & Vinik, 1996; Rathmann, Ziegler, Jahnke,
Haastert, & Gries, 1993).
Diabetes results in a variety of peripheral neuropathy syndromes for
which there is no universally accepted classification. These syndromes
are generally subdivided into focal/multifocal neuropathies, including dia-
betic amyotrophy, and symmetric polyneuropathies, including diabetic
238 A.I. Vinik et al.

sensorimotor polyneuropathy (DSPN). The latter is the most common type,

affecting about 30% of diabetic patients in hospital care and 25% of those in
the community (Sadosky, McDermott, Brandenburg, & Strauss, 2008;
Vinik, Ullal, Parson, & Casellini, 2006; Ziegler, Papanas, Vinik, & Shaw,
2014). The Toronto Diabetic Neuropathy Expert Group defined DSPN
as a symmetrical, length-dependent sensorimotor polyneuropathy attribut-
able to metabolic, and microvascular alterations as a result of chronic hyper-
glycemia exposure (diabetes) and cardiovascular risk covariates (Tesfaye
et al., 2010). Its onset is generally insidious and without treatment the course
is chronic and progressive. Loss of small fiber-mediated sensation results in
the loss of thermal and pain perception, whereas large fiber impairment
results in loss of touch and vibration perception. Sensory fiber involvement
may also result in “positive” symptoms, such as paresthesias and pain,
although up to 50% of neuropathic patients are asymptomatic. DSPN can
be associated with concurrent involvement of the autonomic nervous sys-
tem (ANS) that rarely causes severe symptoms (Boulton, Malik,
Arezzo, & Sosenko, 2004; Ziegler, 2008) but in its cardiovascular form is
associated with at least a threefold increased risk for mortality (Vinik,
Maser, & Ziegler, 2010, 2011; Vinik & Ziegler, 2007).

2. DIABETIC PERIPHERAL NEUROPATHY

2.1 Clinical Presentation
Neuropathy is frequently missed in diabetic patients due to absence of symp-
toms or lack of appropriate diagnostic scrutiny. The clinical presentation
varies depending on the effort placed into finding it. A population survey
reported that 30% of type 1 and 36–40% of type 2 diabetic patients experi-
enced neuropathic symptoms (Harris, Eastman, & Cowie, 1993). Several
studies have also suggested that impaired glucose tolerance (IGT) may lead
to polyneuropathy, with IGT reporting rates in patients with chronic idio-
pathic polyneuropathies of 25–62% (Novella, Inzucchi, & Goldstein, 2001;
Singleton, Smith, & Bromberg, 2001a, 2001b; Sumner, Sheth, Griffin,
Cornblath, & Polydefkis, 2003; Ziegler et al., 2014). Studies using skin
and nerve biopsies have shown a progressive reduction in peripheral nerve
fiber density from the time of diagnosis of diabetes or even in earlier predi-
abetic stages (Dyck, Lais, Karnes, O’Brien, & Rizza, 1986; Pittenger et al.,
2004). Sensory symptoms are more prominent than motor symptoms
and usually involve the lower limbs and include pain, paresthesiae,
Alternative Neuropathy Assessments 239

hyperesthesiae, deep aching, burning, and sharp stabbing sensations similar

to, but less severe than, those described in acute sensory neuropathy. In addi-
tion, patients may experience negative symptoms such as numbness in the
feet and legs that presage painless foot ulcers and subsequent amputations
if the neuropathy is not promptly recognized and treated. Unsteadiness is
also frequent, due to abnormal proprioception and muscle sensory function
(Cavanagh, Simoneau, & Ulbrecht, 1993; Katoulis et al., 1997). Alterna-
tively, some patients may be completely asymptomatic and signs may be only
discovered by a detailed neurological examination.
The natural history of neuropathy syndromes in diabetes separates them
into two very distinctive entities, namely those which progress gradually
with increasing duration of diabetes and those which remit, usually
completely. DSPN and autonomic neuropathies generally progress, while
mononeuropathies, radiculopathies, and acute painful neuropathies,
although manifesting severe symptoms, are short lived and tend to recover
(Watkins, 1993). Progression of neuropathy is related to glycemic control in
both type 1 and type 2 diabetes (DCCT Research Group, 1993, 1995). It
appears that the most rapid deterioration of nerve function occurs soon after
the onset of type 1 diabetes; then within 2–3 years there is a slowing of pro-
gression. In type 2 diabetes, slowing of nerve conduction velocity (NCV)
may be one of the earliest neuropathic abnormalities and is often present
at diagnosis (Ziegler, Cicmir, Mayer, Wiefels, & Gries, 1988).
On physical examination a symmetrical stocking-like distribution of
sensory abnormalities in both lower limbs is usually seen. In more severe
cases hands may also be involved. All sensory modalities can be affected,
particularly vibration, touch, and position perceptions that are indicative
of large Aα/β fiber damage and pain, with abnormal heat and cold temper-
ature perception that is indicative of small thinly myelinated Aδ and unmy-
elinated C-fiber damage. Deep tendon reflexes may be absent or reduced,
especially in the lower extremities. Mild muscle wasting may be seen but
severe weakness is rare and should raise the question of a possible
nondiabetic etiology (Boulton et al., 2004; Sinnreich, Taylor, & Dyck,
2005). Most patients show a “mixed” neuropathy with both large and small
nerve fiber damage and DSPN is also frequently accompanied by auto-
nomic neuropathy, which will be described in more detail below. It is
important to remember that all patients with DSPN are at increased risk
of neuropathic complications such as foot ulceration and Charcot’s
neuroarthropathy.
240 A.I. Vinik et al.

2.2 Diagnosis
The diagnosis of DSPN should rest on the findings of the clinical and neu-
rological examinations, with consideration of the presence of neuropathic
symptoms (positive and negative, sensory, and motor) and signs (sensory def-
icit, allodynia and hyperalgesia, motor weakness, and absence of reflexes).
According to the American Academy of Neurology report on the case def-
inition of distal symmetric polyneuropathy, there is good evidence that:
(A) Symptoms alone have poor diagnostic accuracy in predicting the pres-
ence of polyneuropathy,
(B) Signs are better predictors than symptoms,
(C) Multiple signs are better predictors than a single sign, and
(D) Relatively simple examinations are as accurate as complex scoring sys-
tems (England et al., 2005).
The most recent position statement of the American Diabetes Association
recommends that all patients with diabetes be screened for neuropathy at
diagnosis in type 2 diabetes and 5 years after diagnosis in type 1 diabetes.
Neuropathy screening should be repeated annually and must include sensory
examination of the feet and ankle reflexes (American Diabetes Association,
2015). The basic neurological assessment comprises the general medical and
neurological history to exclude other potential causes of neuropathy, inspec-
tion of the feet, and neurological examination of sensation. As for all patients
with distal symmetric polyneuropathy (England et al., 2009), screening lab-
oratory tests may be considered in selected patients with DSPN, with serum
B12 with its metabolites and serum protein immunofixation electrophoresis
being those with the highest yield of abnormalities (England et al., 2009).
The neurological examination should focus on the lower extremities and
always include an accurate foot inspection for deformities, ulcers, fungal
infection, muscle wasting, hair distribution or loss, and the presence or
absence of pulses. Footwear should also be inspected at every visit. Sensory
modalities should be assessed using simple handheld devices (Table 1)
(Haanpaa et al., 2009). The most sensitive measure is the vibration detection
threshold, although sensitivity of 10-g Semmes-Weinstein monofilament
to identify feet at risk varies between 86% and 100% (Armstrong, Lavery,
Vela, Quebedeaux, & Fleischli, 1998; Kumar et al., 1991). Combinations
of more than one test have more than 87% sensitivity in detecting DSPN
(Boulton et al., 2005; Vinik, Suwanwalaikorn, et al., 1995). Longitudinal
studies show that these simple tests are good predictors of foot ulcer risk
(Abbott et al., 2002). Knee and ankle reflexes should also be tested
Alternative Neuropathy Assessments 241

Table 1 Examination—Bedside Sensory Tests

Associated Sensory
Sensory Modality Nerve Fiber Instrument Receptors
Vibration Aβ (large) 128 Hz Ruffini corpuscle
Tuning fork mechanoreceptors
Pain (pinprick) C (small) Neurotips Nociceptors for pain
and warmth
Pressure Aβ, Aα (large) 1 and 10 g Pacinian corpuscle
Monofilament
Light touch Aβ, Aα (large) Wisp of cotton Meissner’s corpuscle
Cold Aδ (small) Cold tuning fork Cold thermoreceptors

(Boulton, Gries, & Jervell, 1998; Boulton et al., 2005) and assessment of
joint position and motor power may be indicated.
The following findings should alert the physician to consider causes for
DSPN other than diabetes and cause referral for a detailed neurological
work-up: (1) pronounced asymmetry of the neurological deficits; (2) predom-
inantly motor deficits, mononeuropathy, or cranial nerve involvement;
(3) rapid development or progression of the neuropathic impairments; (4) pro-
gression of the neuropathy despite optimal glycemic control; (5) symptoms
from the upper limbs; and (6) family history of nondiabetic neuropathy.

2.3 Falls Risk in Older Adults with Diabetes

2.3.1 Falls and Aging
The older adult is often faced with a myriad of health-related issues and fall-
ing is a major concern for persons over 65 years of age. The outcomes of a fall
extend from short-term health problems (lacerations, fractures, and trau-
matic brain injury) to long-term consequences (decline in muscle strength,
increased fatigue, and heightened fear of falling) that reduce physical activity
(Stevens, Corso, Finkelstein, & Miller, 2006). In 2010, the Centers for
Disease Control reported that 2.3 million older adults were treated in emer-
gency departments for nonfatal fall-related injuries. Furthermore, falls and
fall-related consequences have been reported to be the leading cause of
injury-related death and hospitalization in adults over 65 years of age.
A staggering one-third of persons this age who fall are likely to suffer one
242 A.I. Vinik et al.

of these adverse events within a given year. Given the range of health issues
that can arise following a fall, prevention should be the first course of action.
The key to preventing falls is identification of persons at risk and
implementing appropriate interventions. This requires recognizing factors
that can lead to increased risk of falling. However, despite our understanding
of the seriousness of falls and the high cost of medical care, identifying a few
critical factors that are strongly predictive of falls in high-risk populations is
challenging, partly because over 400 factors are linked with falls in adult
populations (Close, Lord, Menz, & Sherrington, 2005). Something as simple
as an individual’s perception of threat around them when they move (often
referred to as a “fear of falling”) can be a significant health issue. Nearly
13 million (36%) of American adults aged 65 years or more have been found
to be moderately or very afraid of falling, illustrating that developing a fear of
suffering an adverse event is strongly linked with actual falls (Boyd &
Stevens, 2009).

2.3.2 Risk of Falling Is Increased for Older Adults with Type 2 Diabetes
Persons with type 2 diabetes are at increased risk of falling compared to
healthy adults of a similar age. A combination of age (>65 years) and diabetes
further increases the risk of falling 17-fold (Cavanagh, Derr, Ulbrecht,
Maser, & Orchard, 1992; Pijpers et al., 2012) due to factors such as sensory
neuropathy, declining cognitive function, and use of multiple prescription
medications (Close et al., 2005; Peron & Ogbonna, 2015; Schwartz et al.,
2002, 2008). The likelihood of suffering a fall increases dramatically with
increasing age and/or the emergence of type 2 diabetes, with risk being
increased significantly by the presence of diabetes alone (Clark, Lord, &
Webster, 1993; Close et al., 2005; Lord, 1996; Lord & Clark, 1996;
Pickering et al., 2007; Robinovitch et al., 2000; Sosnoff, Motl, &
Morrison, 2013; Sosnoff et al., 2011). Older persons with diabetes must con-
tend with both age-related declines in balance control, muscle strength,
walking ability, and proprioception (Morrison, Colberg, Mariano,
Parson, & Vinik, 2010; Morrison, Colberg, Parson, & Vinik, 2012;
Schwartz et al., 2002, 2008; Wallace et al., 2002) and health-related issues
associated with diabetes (Berlie & Garwood, 2010; Maurer, Burcham, &
Cheng, 2005; Richardson & Hurvitz, 1995; Tilling, Darawil, & Britton,
2006). Indeed, the additional range of potential risk factors in anyone
with diabetes is extensive, covering neuropathy, visual deficits, loss of coor-
dination, cognitive impairment, autonomic dysfunction with orthostatic
hypotension, tachycardia, bradycardia, pain, poor lower body function,
Alternative Neuropathy Assessments 243

high-body mass index, cardiovascular syncope, vestibular dysfunction, fron-

tal cortex dysfunction, and use of various medications, all of which may
interact and can have an additive effect (Close et al., 2005; Morrison
et al., 2010, 2012; Volpato, Leveille, Blaum, Fried, & Guralnik, 2005;
Volpato, Maraldi, & Fellin, 2010). Thus, the older person with diabetes typ-
ically has a significantly greater risk of suffering a fall when compared to a
healthy adult of similar age (Berlie & Garwood, 2010; Maurer et al.,
2005; Pijpers et al., 2012).

2.3.3 Neuropathy Is Strongly Linked to Falling

Declining sensory and motor function arising from neuropathy is a major
contributing factor to the overall increase in fall risk factor for persons with
diabetes (Boucher, Teasdale, Courtemanche, Bard, & Fleury, 1995;
Richardson & Hurvitz, 1995; Uccioli et al., 1995; Volpato et al., 2005;
Witzke & Vinik, 2005). Consequences extend to decrements in balance
and altered walking function (Chiles et al., 2014; Resnick et al., 2002,
2000; Strotmeyer et al., 2008), which are obvious mediators for increased
falls risk (Morrison et al., 2010; Pittenger, Mehrabyan, et al., 2005;
Richardson & Hurvitz, 1995; Vinik, Strotmeyer, Nakave, & Patel, 2008;
Witzke & Vinik, 2005). The ability to optimally control one’s balance is
essential for mobility, avoidance of disability, and preservation of indepen-
dence in older people (Colberg et al., 2005). The complexity of the balance
system makes localization of the problem difficult since the abnormality may
occur in one or more of the sensory sites or in the motor system. A thorough
evaluation of the sensory-motor systems affecting balance is required to
arrive at a diagnosis and to create a platform to provide a menu for treatment
and management choices.
Since neuropathy progression follows a distal-to-proximal gradient, the
effects of neuropathy on strength and balance are most evident at the ankles
and feet and loss of nerve function can have dramatic implications for both
standing and walking tasks (Colberg et al., 2005). For example, diabetic per-
sons with sensory deficits in the feet can exhibit increased postural motion
and slower gait speed (Chiles et al., 2014; Resnick et al., 2002, 2000;
Strotmeyer et al., 2008) with increased stride time variability (Corriveau
et al., 2000; Fioretti, Scocco, Ladislao, Ghetti, & Rabini, 2010; Lafond,
Corriveau, & Prince, 2004; Lalli et al., 2013; Resnick et al., 2002;
Simoneau, Ulbrecht, Derr, Becher, & Cavanaugh, 1994; Turcot, Allet,
Golay, Hoffmeyer, & Armand, 2009; Uccioli et al., 1995). Their impact
is further magnified when the task is made more difficult, as when walking
244 A.I. Vinik et al.

or standing on irregular surfaces (Richardson & Hurvitz, 1995; Richardson,

Thies, & Ashton-Miller, 2008; Richardson, Thies, DeMott, & Ashton-
Miller, 2005). In addition, there is slowing of the reaction time, loss of
the ability to prevent progression to a fall after its initiation, and dorsiflexion
weakness (Strotmeyer et al., 2009), which increase the susceptibility to trip-
ping on loose rugs, carpets, and minor variations in step height. Although
older adults with diabetes are a heterogeneous group, ranging from fit
and healthy to frail with many comorbidities and functional disabilities,
poorly controlled diabetes with polyuria escalates falls risk simply because
of the number of visits to, and urgency to use, the bathroom (Berlie &
Garwood, 2010; Maurer et al., 2005; Volpato et al., 2005, 2010).

2.3.4 Can Risk of Falling Be Reversed?

Reversal of falls risk depends first and foremost on screening and
identification of the candidate at risk. Fig. 2 shows an algorithm that iden-
tifies candidates and matches them to the intervention most likely to
succeed. Numerous studies have shown the benefits of various balance/
exercise programs in reducing falls risk in healthy older persons (Barnett,
Smith, Lord, Williams, & Baumand, 2003; Howe, Rochester, Jackson,

Individual at risk of falling

Falls risk screening

Risk factors Decreased Diminished Fear of falling, Impaired Polypharmacy

identified by strength, slow balance balance cognitive
screening reactions control, slow confidence ability/visual
reactions problem

Potential targeted interventions

Structured CDC Steadi Cognitive Adjust BP,

exercise, instruction, training, diabetes, pain,
balance training medical nutrition cognitive antidepressant
eg, yoga Tai therapy therapy, visual meds
chi, aerobic correction

Fig. 2 A clinical algorithm for the evaluation of and targeted intervention for the aging
patient at risk of falling.
Alternative Neuropathy Assessments 245

Banks, & Blair, 2007; Peterson et al., 2009; Robinovitch et al., 2000;
Sherrington, Lord, & Herbert, 2003) and studies have reported that
targeted interventions can improve balance and walking ability and also
reduce falls risk in diabetes (Allet et al., 2009; Morrison et al., 2010,
2012). Structured balance training can lead to improvements in posture
and/or gait function (Allet et al., 2010; Morrison et al., 2010, 2012) as well
as general gains from physical activity like faster reaction times, improve-
ments in sensory perception and lower limb strength, and better sympa-
thetic/parasympathetic balance (Colberg, 2006; Colberg, Stansberry,
McNitt, & Vinik, 2002; Herriott, Colberg, Parson, Nunnold, & Vinik,
2004; Morrison et al., 2010, 2012). Interestingly, recent studies have
reported that exercise can also lead to improvements in neuropathy symp-
toms, including increased nerve fiber branching (Kluding et al., 2012) and
improved sensory responses in the lower limbs (Balducci et al., 2006).

3. DIABETIC AUTONOMIC NEUROPATHY

Diabetic autonomic neuropathy (DAN) is among the least recognized
and understood complications of diabetes despite its significant negative
impact on survival and QOL in people with diabetes (Vinik & Erbas,
2001). It is also a major source of increased cost of caring for the diabetic
patient. The two divisions of the ANS—the parasympathetic and the sym-
pathetic nervous systems—work in balanced opposition to control the heart
rate, the force of cardiac contraction, the dilatation and constriction of blood
vessels, the contraction and relaxation of smooth muscle in the digestive and
urogenital systems, and the secretions of glands and pupillary size. Diabetes
can cause dysfunction of any or every part of the ANS, leading to a wide
range of disorders.
Reported prevalence of DAN can vary. For example, in a community-
based study the prevalence of DAN, as defined by one or more abnormal
heart rate variability (HRV) test results, was 16.7% (Neil, Thompson,
John, McCarthy, & Mann, 1989). If stricter criteria were used (ab-
normalities present in at least three of six autonomic function tests),
the prevalence was 16.8% for individuals with type 1 diabetes and
22.1% for individuals with type 2 diabetes (Ziegler, Gries, Spuler, &
Lessmann, 1992).

3.1 Clinical Manifestations

The ubiquitous distribution of the ANS renders virtually all organs sus-
ceptible to dysfunction (Table 2) so that clinical manifestations frequently
246 A.I. Vinik et al.

Table 2 Symptoms and Signs Associated with Diabetic Autonomic Neuropathy

Cardiovascular Autonomic
Neuropathy Gastrointestinal Urogenital
Resting tachycardia Gastroparesis Bladder dysfunction
• Nausea • Frequency
Abnormal blood pressure • Bloating • Urgency
regulation • Loss of appetite • Nocturia
• Nondipping • Early satiety • Hesitancy
• Reverse dipping • Postprandial • Weak stream
vomiting • Dribbling
Orthostatic hypotension/ • Brittle diabetes • Urinary
tachycardia/bradycardia incontinence
• Lightheadedness Esophageal • Urinary retention
• Weakness dysfunction
• Faintness • Heartburn Male sexual
• Dizziness • Dysphagia for dysfunction
• Visual impairment solids • Erectile dysfunction
• Syncope • Decreased libido
Diabetic diarrhea • Abnormal
(All with standing) • Profuse and ejaculation
watery diarrhea
• Fecal Female sexual
incontinence dysfunction
• Alternates with • Decreased sexual
constipation desire
• Increased pain
Constipation during intercourse
• Decreased sexual
arousal
• Inadequate
lubrication

occur concurrently, but with inconsistent patterns. A patient diagnosed

with diabetes should be suspected of having at least subclinical dis-
turbances of the ANS, which can occur within a year of diagnosis in type
2 diabetic patients and within 2 years in type 1 diabetic patients (Pfeifer
et al., 1984). Clinical symptoms generally do not occur until long after
the onset of diabetes. The most important etiologic factors that have been
associated with DAN are poor glycemic control, diabetes duration, age,
female sex, and higher body mass index (BMI). Of patients with symp-
tomatic autonomic dysfunction, 25–50% die within 5–10 years of diag-
nosis (Ewing, Boland, Neilson, Cho, & Clarke, 1991; Rathmann et al.,
1993) and the 5-year mortality rate in patients with DAN is three
times higher than in diabetic patients without autonomic involvement
Alternative Neuropathy Assessments 247

(O’Brien, McFadden, & Corrall, 1991). Leading causes of death in

patients with either symptomatic or asymptomatic DAN are heart dis-
ease and nephropathy while DAN is also an independent risk factor
for stroke.

3.2 Cardiovascular Autonomic Neuropathy

Perhaps one of the most overlooked of all serious complications of diabetes
is cardiovascular autonomic neuropathy (CAN) (Maser, Lenhard, &
DeCherney, 2000). CAN results from damage to autonomic nerve fibers
that innervate the heart and blood vessels that causes abnormalities in heart
rate control and vascular dynamics. CAN has been linked to resting tachy-
cardia, postural hypotension, exercise intolerance, enhanced intraoperative
or perioperative cardiovascular liability, increased incidence of asymptom-
atic ischemia, myocardial infarction (MI), and decreased rate of survival after
MI. The Diabetes Control and Complications Trial (DCCT) found that
1.65% had abnormal HRV at baseline for less than 5 years duration of
diabetes, rising to 6.2% among those with less than 9 years but more than
5 years duration of diabetes, and 12.2% by 9 years or more (Diabetes
Control and Complications Trial Research Group, 1998). CAN may be
present at diagnosis and prevalence increases with age, duration of diabetes,
and poor glycemic control. CAN also cosegregates with distal symmetric
polyneuropathy, microangiopathy, and macroangiopathy. Age, diabetes,
obesity, and smoking are risk factors for HRV in type 2 diabetes. Hemoglo-
bin A1c, hypertension, distal symmetrical polyneuropathy, retinopathy, and
exposure to hyperglycemia were all shown to be risk factors for developing
CAN in type 1 diabetes (Witte et al., 2005; Ziegler et al., 2004, 2006).
CAN may be associated with abnormalities in left ventricular (LV) sys-
tolic, and particularly diastolic, function in the absence of cardiac disease in
diabetic patients. Echocardiographic studies have shown a significant corre-
lation of the severity of CAN with reduced peak diastolic filling rate and
with an augmented atrial contribution to diastolic filling as assessed by
Doppler echocardiography. CAN is also associated with left ventricular dia-
stolic dysfunction (LVDD) at rest, both in patients with long-term type 2
and type 1 diabetes. LVDD may progress to heart failure, mainly with pre-
served LV systolic function (diastolic heart failure). The pathophysiology of
LVDD includes delayed relaxation, impaired LV filling, and/or increased
stiffness. In patients with CAN, vagal impairment can lead to a relative
predominance of sympathetic activity in the sympathovagal balance. Sym-
pathetic overactivity stimulates the renin–angiotensin–aldosterone system
248 A.I. Vinik et al.

and increases heart rate, stroke volume, and peripheral vascular resistance,
thus contributing to LV dysfunction. Such sympathetic hyperactivity, in
combination with regional myocardial sympathetic denervation, has
been shown to lead to diminished coronary blood flow reserve and diastolic
dysfunction in diabetic patients with early microangiopathy (Dinh et al.,
2011; Sacre et al., 2010; Vinik & Erbas, 2013).

3.2.1 Diagnosis of CAN

There are simple bedside tests to diagnose CAN using HRV, responses to
breathing, the Valsalva maneuver, and standing. Functional abnormalities
and imbalance between the sympathetic and parasympathetic nervous sys-
tem are discerned with respiratory modulation of different frequency oscil-
lations in HRV.

3.2.2 Cardiovascular Symptoms and Signs

CAN is identified by a number of presentations:

3.2.2.1 Resting Tachycardia

Resting tachycardia and a fixed heart rate are characteristic late findings in
diabetic patients with vagal impairment. Resting heart rates of 90–100 bpm
and occasional heart rate increments up to 130 bpm occur. A fixed heart rate
that is unresponsive to moderate exercise, stress, or sleep indicates almost
complete cardiac denervation (Ewing & Clarke, 1986).

3.2.2.2 Exercise Intolerance

Autonomic dysfunction impairs exercise tolerance, reduces response in heart
rate and blood pressure, and blunts increases in cardiac output in response to
exercise. Diabetic patients who are likely to have CAN should be tested for
cardiac stress before undertaking an exercise program. Patients with CAN
need to rely on their perceived exertion, not heart rate, to avoid hazardous
levels of intensity of exercise (Albers, Krichavsky, & Balady, 2006; Colberg,
Swain, & Vinik, 2003; Vinik & Erbas, 2002, Vinik, Erbas, & Pfeifer, 2003).

3.2.2.3 Intraoperative Cardiovascular Liability

Hemodynamic changes occur during surgery for individuals without diabe-
tes. Perioperative cardiovascular morbidity and mortality are increased
two- to threefold in patients with diabetes. Compared with nondiabetic sub-
jects, diabetic patients undergoing general anesthesia may experience a
greater degree of decline in heart rate and blood pressure during induction
Alternative Neuropathy Assessments 249

of anesthesia and less of an increase after tracheal intubation and extubation.

There is also an association between CAN and more severe intraoperative
hypothermia that results in decreased drug metabolism and impaired wound
healing.

3.2.2.4 Orthostatic Hypotension

Orthostatic hypotension is defined as a fall in blood pressure (>20 mmHg
for systolic or >10 mmHg for diastolic) in response to postural change, from
supine to standing. In patients with diabetes, orthostatic hypotension is usu-
ally due to damage to the efferent sympathetic vasomotor fibers, particularly
in the splanchnic vasculature. In addition, there is a decrease in cutaneous,
splanchnic, and total vascular resistance. Normally, in response to postural
change there is an increase in plasma norepinephrine. For individuals with
orthostatic hypotension, there may be a reduction in this response relative
to the fall in blood pressure. Diminished cardiac acceleration and cardiac
output, particularly in association with exercise, may also be present. Patients
with orthostatic hypotension typically present with lightheadedness and pre-
syncopal symptoms. Many patients, however, remain asymptomatic despite
significant falls in blood pressure.

3.2.2.5 Silent Myocardial Ischemia/Cardiac Denervation Syndrome

The presence of both symptomatic and asymptomatic coronary artery disease
is increased in diabetic patients and subclinical neuropathy is an important
cause of silent ischemia in patients with diabetes (Airaksinen & Koistinen,
1992; Marchant, Umachandran, Stevenson, Kopelman, & Timmis, 1993).
Features of MI in patients with CAN are silence, cough, nausea and vomiting,
dyspnea, tiredness, and ECG changes. Reduced appreciation for ischemic
pain can impair timely recognition of myocardial ischemia or infarction,
thereby delaying appropriate therapy. Silent ischemia in diabetic patients
may either result from CAN, from autonomic dysfunction attributable to cor-
onary artery disease itself, or from both. In the Framingham Study, the rates
of unrecognized MI’s were 39% in diabetic patients and 22% in nondiabetic
subjects, but the difference was not significant (Margolis, Kannel, Feinleib,
Dawber, & McNamara, 1973). In a survey from the National Registry of
Myocardial Infarction, of 434,877 patients presenting with MI, 33% did
not have chest pain. Among those presenting without chest pain, 32% had
diabetes vs 25.4% in the group with chest pain (Canto et al., 2000). Thus,
patients with CAN warrant careful attention and cardiovascular autonomic
250 A.I. Vinik et al.

function testing may be an important component in the risk assessment of

diabetic patients with coronary artery disease.

3.2.2.6 Increased Risk of Mortality

A number of studies have shown a 2.3-fold increased risk of CAN in diabetic
patients showing a prolonged QT interval, leading to speculation that CAN
might also predispose to malignant ventricular arrhythmias and to sudden
death from cardiac arrest caused by torsades de pointes, as in long QT syn-
drome. Furthermore, symptomatic CAN at 5 years of diabetes predicted
mortality at 10 years, even after adjusting for conventional cardiovascular
disease (CVD) risk factors. HRV was found to be an independent predictor
of all-cause mortality during a period of 9 years (Wirta, Pasternack,
Mustonen, & Laippala, 1997). Moreover, impaired autonomic function is
associated with increased all-cause and cardiovascular mortality so that
CAN in patients already at risk (diabetes, hypertension, or history of
CVD) may be especially hazardous (Gerritsen et al., 2001). Results from
the ACCORD trial confirmed the association of CAN and mortality as indi-
viduals in this trial with baseline CAN were 1.55–2.14 times as likely to die
as individuals without CAN. Furthermore, CAN in the presence of periph-
eral neuropathy was the highest predictor of CVD mortality and combining
indexes of autonomic dysfunction have been shown to be associated with
the risk of mortality (Lykke, Tarnow, Parving, & Hilsted, 2008; Maser,
Mitchell, Vinik, & Freeman, 2003; Ziegler et al., 2008).

3.2.2.7 Increased Mortality After MI

Mortality rates after an MI are higher for diabetic than nondiabetic patients.
A simple bedside test that measured 1-min HRV during deep breathing was
a good predictor of all-cause mortality for 185 patients (17.8% with diabetes)
after a first MI (Stewart, Medow, & Montgomery, 2003).

3.2.2.8 CAN and Sudden Death

Sudden, unexpected deaths occur among subjects with CAN. One potential
cause may be severe but asymptomatic ischemia, which can induce lethal
arrhythmias. QT prolongation may also predispose individuals to life-
threatening cardiac arrhythmias and sudden death. Male patients with
impaired HRV had a higher corrected QT prolongation than males without
this complication (Veglio, Borra, Stevens, Fuller, & Perin, 1999).
Alternative Neuropathy Assessments 251

3.2.3 Impact of CAN on Diabetes Management

The question of more intensive therapy and reduction in cardiovascular
complications was addressed for people with type 2 diabetes of long duration
in three studies: ACCORD (Buse et al., 2007), ADVANCE (Patel et al.,
2008), and VADT (Duckworth et al., 2009). Although the three studies uti-
lized different patient cohorts, with varying durations of diabetes and had
different treatment regimens, the results indicated that intensive glucose
control did not reduce CVD events. Recent analyses of the ACCORD trial
examined whether the effects of CAN or self-reported history of neuropathy
at baseline could have been a contributor to the higher mortality risk in the
intensive glycemic arm (272,273). The paradox was that higher HbA1C was
associated with higher hypoglycemic rates within both intensive and stan-
dard treatment groups. The HR for hypoglycemia by each 1% higher
updated average HbA1C was 1.15 for the intensive and 1.76 for the standard
and hypoglycemia doubled the risk of death with intensification of glycemic
control. This is in stark contrast with the DCCT study, where rates of hypo-
glycemia increased with lowering of the blood glucose. It was not the hypo-
glycemia which killed the patients but rather the predictive capacity of
hypoglycemia for sudden death!

3.3 Prevention and Reversibility of Autonomic Neuropathy

Restoration of autonomic balance is possible and has been shown following
lifestyle changes, increased physical activity, β-adrenergic blockers, aldose
reductase inhibitors, ACE inhibitors, angiotensin receptor blockers, and
potent antioxidants such as α-lipoic acid. Obesity in humans has been asso-
ciated with autonomic dysfunction and increased sympathetic activity
(Lambert, Sari, et al., 2010; Lambert, Straznicky, Lambert, Dixon, &
Schlaich, 2010; Piestrzeniewicz, Luczak, Lelonek, Wranicz, & Goch,
2008; Straznicky et al., 2009). Furthermore, several studies have shown that
weight loss improves HRV measures and autonomic imbalance (Casellini
et al., 2015; Karason, Molgaard, Wikstrand, & Sjostrom, 1999; Maser,
Lenhard, Irgau, & Wynn, 2007; Nault et al., 2007; Perugini et al., 2010;
Ravussin, 2010; Ziegler et al., 2015).
There are exciting new prospects for pathogenesis-oriented intervention
to reverse established CAN (Vinik et al., 2011). Strict glycemic control
(DCCT Research Group, 1995) and a stepwise progressive management
of hyperglycemia, lipids, and blood pressure as well as the use of antioxidants
(Ziegler & Gries, 1997) and ACE inhibitors (Athyros et al., 1998) reduce the
252 A.I. Vinik et al.

odds ratio for autonomic neuropathy (Gaede, Vedel, Parving, & Pedersen,
1999). The EDIC study, a longitudinal cohort follow-up study for the
DCCT in which patients with type 1 diabetes were randomized to conven-
tional or intensive glycemic control, demonstrated persistent beneficial
effects of past glucose control on microvascular complications despite the
loss of glycemic separation in type 1 diabetic patients (DCCT EDIC
Research Group, 2003). During the EDIC follow-up, CAN progressed
in both treatment groups, but the incidence and prevalence of CAN
remained lower in the formerly intensive group than in the formerly con-
ventional group, despite similar levels of glycemic control during EDIC. To
minimize the development of autonomic neuropathy, intensive glucose
control of type 1 diabetes should therefore be started as early as possible
(Pop-Busui et al., 2009). However, while glycemic control with a reduction
of HbA1c from 9.5 to 8.4 improved HRV in those patients with mild auto-
nomic abnormalities, this was not so in type 1 diabetics with advanced auto-
nomic abnormalities (Burger, Weinrauch, D’Elia, & Aronson, 1999).
The effects of glycemic control in type 2 diabetics are less definite. The VA
Cooperative Study showed no difference in the prevalence of autonomic neu-
ropathy after 2 years of intensive glycemic control in type 2 diabetic patients
(Azad et al., 1999). In the Steno-2 Study, where people with type 2 diabetes
received intensive multifactorial treatment that targeted hyperglycemia,
hypertension, dyslipidemia, and microalbuminuria, along with secondary pre-
vention of CVD with aspirin, the approach reduced autonomic dysfunction
by 63%. The glucose-lowering agents appeared to have the least effect when
compared with antihypertensive treatment, lipid-lowering agents, aspirin, and
vitamin–mineral supplements (Gaede et al., 1999). In addition, a survey of
evidence from clinical trials shows that early identification of autonomic neu-
ropathy permits timely initiation of therapy with the antioxidant alpha-lipoic
acid, which slows or reverses progression of CAN (Ziegler et al., 1997).
Early ACE inhibition or angiotensin receptor blockade improved both
DAN and LVDD after 1 year of treatment in asymptomatic patients with long-
term diabetes, with the combination being slightly better than monotherapies
and auguring well for the patient with established CAN (Didangelos et al.,
2006). Fluvastatin treatment also improves cardiac sympathetic neuropathy
in the diabetic rat heart, in association with attenuation of increased cardiac
oxidative stress (Matsuki et al., 2010). On the other hand, selective
COX-2 inactivation confers protection against sympathetic denervation dur-
ing experimental diabetes by reducing intramyocardial oxidative stress and
inflammation (Kellogg, Converso, Wiggin, Stevens, & Pop-Busui, 2009).
Alternative Neuropathy Assessments 253

Therefore, statins and COX-2 inactivation might help to attenuate cardiac

sympathetic dysfunction. It has also been shown that early mortality is a func-
tion of loss of beat-to-beat variability with MI. This can be reduced by 33%
with acute administration of insulin (Malmberg, Norhammar, Wedel, &
Ryden, 1999). Kendall, Rooney, Smets, Salazar Bolding, and Robertson
(1997) reported that successful pancreas transplantation improves epinephrine
response and normalizes hypoglycemia symptom recognition in patients with
long-standing diabetes and established autonomic neuropathy. Burger et al.
(1999) showed that a reversible metabolic component of CAN exists in
patients with early CAN. There are, therefore, exciting new prospects for
pathogenesis-oriented intervention (Vinik et al., 2011).

4. MEASURING DIABETIC NEUROPATHY: ESTABLISHED

AND INNOVATIVE APPROACHES
A summary of the different diagnostic tools available to the physician
for the evaluation of diabetic neuropathies is provided in Table 3.

4.1 QOL Measures

A number of instruments have been developed and validated to assess QOL.
The NeuroQoL measures patients’ perceptions of the impact of neuropathy
and foot ulcers (Vileikyte et al., 2003). The Norfolk QOL questionnaire is a
validated tool addressing specific symptoms and the impact of large, small,
and autonomic nerve fiber functions. When tested in 262 subjects differ-
ences between DSPN patients and both diabetic and healthy controls were
significant (P < 0.05) for all item groupings (small fiber, large fiber, and auto-
nomic nerve function; symptoms; and activities of daily living (ADL)). The
nerve fiber-specific domains in particular have been shown to correlate with
objective measures of nerve function (Vinik et al., 2005; Vinik, Hayes,
Oglesby, & Vinik, 2004; Vinik, Paulson, Ford-Molvik, & Vinik, 2008).
Total QOL scores correlated with total neuropathy scores. The ADL, total
scores, and autonomic scores were also greater in diabetic controls compared
to healthy controls (P < 0.05), suggesting that diabetes per se impacts some
aspects of QOL (Vinik et al., 2005). The tool has been translated into 60 lan-
guages and has served as an endpoint in trials of diabetic neuropathy (Boyd,
Casellini, Vinik, & Vinik, 2011; Vinik et al., 2014). It captured 6600 patients
with previously undiagnosed neuropathy in a screening of 25,000 patients in
Romania (Veresiu et al., 2015) and recently served as a tool to demonstrate
safety in a study of topical capsaicin (Vinik, Perrot, et al., 2015).
Table 3 Performance Range of Tools for the Diagnosis of Peripheral Neuropathy
Test Sensitivity Specificity Gold Standard Used in Study Study Subject Population
Large fiber
10 g Monofilament (Dros, 41–93% 68–100% NCS Unselected diabetic (DM)
Wewerinke, Bindels, & van Weert, patients
2009)
Vibration perception (Lai, Ahmed, 20–26% 88–89% NCS Neurology outpatient
Bollineni, Lewis, & Ramchandren, clinic
2014)
Vibration perception (Martin et al., 75–87% 51–62% Clinical signs and symptoms; Adult type 1 diabetic
2010) abnormal NCS; or both patients (T1D)
Ankle reflexes (Shehab et al., 2012) 91.5% 67.4% NCS Type 2 diabetes patients
(T2D)
Ankle reflexes (Taksande, Ansari, 72.22–78.95% 43.42–46.77% NCS Unselected DM patients
Jaikrishnan, & Karwasara, 2011)
EMG—sural-nerve amplitude 77% 73% IENFD < 5.4 fibers/mm Patients with painful
(Ebadi, Perkins, Katzberg, Lovblom, polyneuropathy and
& Bril, 2012) normal NCS
EMG—sural and peroneal 59–73% 53–78% Neuropathy Disability Score DM patients and healthy
(Gibbons, Freeman, & Veves, 2010) (NDS) > 2 controls
Small fiber
Pinprick (Neurotip®) 50% 50% N/A—all patients with diabetic Patients with diabetic foot
(Nather et al., 2011) foot
Pinprick (Neurotip®) (Paisley, 91.8% 41% NDS
6 Unselected DM patients
Abbott, van Schie, & Boulton, 2002)
QST: CDT foot and HP foot 62–65% 63–65% IENFD < 5.4 fibers/mm Patients with painful
(Ebadi et al., 2012) polyneuropathy and
normal NCS
QST: CDT, CP, HDT, HP foot 67–89% 29–72% NDS > 2 DM patients and healthy
(Gibbons et al., 2010) controls
IENFD (Smith, Lessard, Reyna, 63% 63% Utah early neuropathy scale
4 Healthy controls and
Doudova, & Singleton, 2014) suspected distal
neuropathy
IENFD (England et al., 2009) 45–90% 95–97% Unknown: American Academy Healthy controls and
of Neurology evidence-based established
review polyneuropathy
LDI flare (Ebadi et al., 2012) 54% 54% IENFD < 5.4 fibers/mm Patients with painful
polyneuropathy and
normal NCS
LDI flare (Vas & Rayman, 2013b) 75–77% 85–90% NDS
3 Healthy controls and DM
patients
Sudoscan (Casellini, Parson, 78% 92% NIS-LL Healthy controls and DM
Richardson, Nevoret, & Vinik, 2013) patients
Sudoscan (Selvarajah et al., 2015) 87.5% 80.0% Toronto Consensus DPN Healthy controls and T1D
definition patients
CDT, cooling detection threshold; CP, cold pain; EMG, electromyography; HDT, heat detection threshold; HP, heat pain; LDI flare, laser Doppler flare imaging; NIS-
LL, Neuropathy Impairment Score of the Lower Limbs.
256 A.I. Vinik et al.

4.2 Clinical Assessment Tools

Clinical assessment should be standardized and conducted using validated,
sufficiently reproducible, scores for both the severity of symptoms and
the degree of neuropathic deficits. Tools include the Michigan Neuropathy
Screening Instrument (MNSI) (Feldman et al., 1994); the Neuropathy
Symptom Score (NSS) for neuropathic symptoms and the Neuropathy Dis-
ability Score (NDS) or the Neuropathy Impairment Score (NIS) for neuro-
pathic deficits. These questionnaires are useful for patient follow-up and to
assess response to treatment. The neurological history and examination
should be performed initially and then with all subsequent visits. Minimum
criteria for the clinical diagnosis of neuropathy according to the NSS and
NIS are: (1) moderate signs with or without symptoms or (2) mild signs with
moderate symptoms. However, this means that the exclusive presence of
neuropathic symptoms without deficits is not sufficient to diagnose DSPN.
Therefore, early stages of DSPN or a painful small-fiber neuropathy with or
without minimal deficits can only be verified using more sophisticated tests
such as quantitative sensory testing (QST). QST enables more accurate assess-
ment of sensory deficits, including those related to small-fiber function, by
applying controlled and quantified stimuli, and standardized procedures. Mul-
tiple studies have proven the value of QST measures in the detection of sub-
clinical small-fiber neuropathy, the assessment of progression of neuropathy,
and the prediction of risk of foot ulceration (Abbott, Vileikyte, Williamson,
Carrington, & Boulton, 1998; Dyck, Dyck, Larson, O’Brien, & Velosa, 2000;
Vinik, Suwanwalaikorn, et al., 1995; Yarnitsky & Sprecher, 1994). QST stan-
dardized measures of vibration and thermal perception thresholds are probably
an effective tool in diabetic neuropathy (Shy et al., 2003) and also play an
important role in multicenter clinical trials as primary efficacy endpoints.

4.3 Objective Measurements

4.3.1 Nerve Conduction Studies
An atypical pattern of presentation of symptoms or signs, such as the pres-
ence of relevant motor deficits, an asymmetrical or proximal distribution, or
rapid progression, always requires referral for electrodiagnostic testing.
Furthermore, in the presence of such atypical neuropathic signs and symp-
toms other forms of neuropathy should be sought and excluded. After diag-
nosis, slowing of NCV generally progresses at a steady rate of approximately
1 m/s/year and the level of impairment is positively correlated with duration
of diabetes. Although most studies have documented that symptomatic
Alternative Neuropathy Assessments 257

patients are more likely to have slower NCVs than patients without
symptoms, these do not relate with the severity of symptoms. In a long-term
follow-up study of type 2 diabetes patients (Partanen et al., 1995), electro-
physiologic abnormalities in the lower limb increased from 8% at baseline to
42% after 10 years with a decrease in sensory and motor amplitudes
(indicating axonal destruction) being more pronounced than NCV slowing.
Using objective measures of sensory function such as the vibration percep-
tion threshold test, the rate of decline in function has been reported as 1–2
vibration units/year. However, the most recent clinical studies suggest
that there is a decline in this rate of progression. The advent of therapeutic
lifestyle change and the use of statins and ACE inhibitors likely contribute
to slowed progression of neuropathy and have drastically changed the
requirements for placebo-controlled studies (Casellini et al., 2007). It is also
important to recognize that diabetic neuropathy is a disorder wherein the
prevailing abnormality is loss of nerve fibers and that this is reflected by a
reduction in electromyogram amplitudes, not conduction velocity. There-
fore, changes in NCV may not be an appropriate means of monitoring
progress or deterioration of nerve function. Furthermore, small, unmyelin-
ated nerve fibers are affected early in diabetes and are not reflected in NCV
studies. Other methods of measuring neuropathy that do not depend on
NCV, such as QST (see earlier), or skin biopsy with quantification of
intraepidermal nerve fibers (IENF), are necessary to identify these patients
(Pittenger et al., 2004; Pittenger, Simmons, et al., 2005; Sinnreich
et al., 2005).

4.3.2 Skin Biopsy

The importance of the skin biopsy as a diagnostic tool for DSPN is increas-
ingly being recognized as it allows investigation of small caliber sensory neu-
rons including somatic unmyelinated IENF, dermal myelinated nerve fibers,
and autonomic nerve fibers (Lauria, Bakkers, et al., 2010; Lauria, Hsieh,
et al., 2010; Pittenger, Mehrabyan, et al., 2005). Since small fibers are
impacted early in the course of diabetic neuropathy (Vinik, Casellini, &
Nevoret, 2015), it is imperative to catch onset prior to further progression,
when treatment and reversibility may be limited. For diagnostic purposes a
3-mm punch skin biopsy at the distal leg is recommended and quantification
of the linear density of IENF in at least three 50 μm thick sections per biopsy.
Tissue is viewed by bright-field immunohistochemistry or immunofluores-
cence using protein gene product 9.5 antibodies (Joint Task Force of the
EFNS and the PNS, 2010). Quantification of IENF density appeared more
258 A.I. Vinik et al.

sensitive than sensory nerve conduction or sural-nerve biopsy in diagnosing

small-fiber neuropathy. Use of skin biopsies has also led to the recognition of
the small nerve fiber syndrome as part of IGT and the metabolic syndrome so
that when patients present with the “burning foot or hand syndrome,” an
evaluation for glucose tolerance and the metabolic syndrome becomes man-
datory. Therapeutic life style changes can result in nerve fiber regeneration,
reversal of the neuropathy, and alleviation of symptoms (Smith et al., 2006).

4.3.3 Corneal Confocal Microscopy

Corneal confocal microscopy (CCM) is a noninvasive technique used to
detect small nerve fiber loss in the cornea, which correlates with both
increasing neuropathic severity and reduced IENF in diabetic patients
(Quattrini et al., 2007; Tavakoli et al., 2010). This novel technique of
real-time mapping permits an area of 3.2 mm2 to be mapped with a total
of 64 theoretically nonoverlapping single 400 μm2 images (Zhivov, Blum,
Guthoff, & Stachs, 2010). In type 1 diabetes subjects who received a pan-
creas transplantation, CCM was able detect nerve regrowth in the cornea
before any improvement of IENF (Tavakoli et al., 2013). However, it is
as yet unclear whether improvements in CCM parameters predict improve-
ments in peripheral neuropathy or patient-oriented outcomes such as pain,
disability, and QOL. Furthermore, the performance of CCM as an early bio-
marker of nerve regeneration in patients with type 2 diabetes receiving other
interventions has not yet been demonstrated (Shtein & Callaghan, 2013).

4.3.4 Contact Heat Evoked Potentials

Contact heat-evoked potentials (CHEPS) have been studied in healthy
controls, newly diagnosed diabetic patients, established diabetic patients,
and patients with the metabolic syndrome. It appears that CHEPS is
capable of detecting small-fiber neuropathy in the absence of other indices
and that CHEPS correlates with quantitative sensory perception and
objective tests of small-fiber function such as the cooling detection threshold
and cold pain (Parson, Nguyen, Boyd, & Vinik, 2009). CHEPS is there-
fore a useful diagnostic tool for the evaluation of small nerve fiber
function in neuropathic patients (Chen, Niddam, & rendt-Nielsen, 2001;
Itskovich, Fei, & Harkins, 2000; Opsommer, Masquelier, & Plaghki,
1999). In the past, laser-evoked potentials were used but left undesirable
thermal damage to intact nerve fibers and surrounding healthy tissue.
Long-term assessment using this modality is not practical for quality patient
Alternative Neuropathy Assessments 259

care (Bromm & Treede, 1991). CHEPS, with its rapidly heating thermode
(70°C/s), allows for repeated assessments without the risk of long-term
damage to the area of interest. Studies supporting reproducibility of CHEPS
after 6 months (Ruscheweyh, Emptmeyer, Putzer, Kropp, & Marziniak,
2013), especially in areas of interest (Kramer et al., 2012), lend further sup-
port for its use in repeated clinical assessments.
Various groups, including ours, have performed comparison studies using
CHEPS in neuropathic populations, and CHEPS intrapeak amplitudes (IA)
have proven to be a distinguishable marker differentiating neuropaths from
nonneuropaths (Atherton et al., 2007; Chao et al., 2010; Parson et al., 2013;
Wong & Chung, 2011). CHEPS IA also correlates with other assessments of
small nerve fibers, including skin-flare response and IENF density (Atherton
et al., 2007; Chao, Hsieh, Tseng, Chang, & Hsieh, 2008; Chao et al., 2010).
Assessment of structural changes may miss early nerve dysfunction in the
presence of normal IENF density. However, CHEPS provides a sensitive
(76.4%) and specific (80.6%) tool in assessing nerve fiber loss in the dorsal foot
with IENF as a standard for comparison, while providing a functional eval-
uation (Casanova-Molla, Grau-Junyent, Morales, & Valls-Sole, 2011).
In a comparison of diabetic and nondiabetic patients, CHEPS IAs of the
lumbosacral region, dorsal, and volar forearm negatively correlated with
HbA1c (Parson et al., 2013). Diabetic neuropaths also tend to have CHEPS
responses of longer latencies and lower amplitudes (Parson et al., 2013)
(Chao et al., 2010). Upon closer examination of patients with DSPN,
CHEPS responses tend to be higher in those with pain than those without
(Parson et al., 2013), which has led to further investigation of the tool for
studying pain models and assessing intervention.
The versatility of CHEPS to study function in conjunction with other
devices, such as fMRI and magnetoencephalography, further adds to its
value in providing a more global evaluation of future therapeutics and
broadening our understanding of the mechanism of pain (Gopalakrishnan,
Machado, Burgess, & Mosher, 2013; Kramer, Jutzeler, Haefeli, Curt, &
Freund, 2015; Shenoy et al., 2011). Thus, there is a great need to create
a standard for CHEPS testing and normative values. Endeavors by Dutch
(Lagerburg et al., 2015) and Taiwanese groups (Chen et al., 2006) to create
such values should further accelerate its validation in diabetic neuropathy
and its various subtypes. However, future normative values must be
obtained from a more universally diverse cohort to truly create a robust stan-
dard with meaningful clinical significance.
260 A.I. Vinik et al.

4.3.5 LDI Flare

Vasodilation induced by both acetylcholine iontophoresis (ACh Ionto) and
the laser Doppler flare technique (LDIFT) can be used to measure small-
fiber function. When local anesthetic is applied, axonal transport but not
ACh Ionto is inhibited, thus hyperemic flow induced by Ach Ionto is
not neurogenic. By contrast, the hyperemia induced by heating the skin
to 44°C is blocked by local anesthetic and therefore reflects C-fiber func-
tion. Krishnan and Rayman demonstrated that the ratio of hyperemic area
to stimulus area was significantly reduced by local anesthesia in the LDIFT
[ratio (mean  SD): 2.33  0.67 with local anesthesia; 6.84  1.33 without
local anesthesia; P < 0.0001], but not in the ACh Ionto group
(2.61  0.57 with local anesthesia; 2.67  1.27 without local anesthesia),
confirming that LDIFT measures small-fiber function (Green,
Krishnan, & Rayman, 2009). The same authors showed that this measure
of C-fiber function was more sensitive than QST using the Case IV device
(Krishnan & Rayman, 2004).
A modified laser Doppler imager method (mLDIf ) uses 47°C (vs 44°C),
producing larger flares than the older method for assessing C-fiber function
in foot skin and making it quicker and better suited for clinical use. It has also
been validated for assessing C-fiber function in the clinical setting (Vas &
Rayman, 2013a). Rayman et al. evaluated a rapid, low-cost, point-of-care
nerve conduction device (POCD; NC-stat®jDPNCheck™) for the assess-
ment of DPN and compared it with mLDIf in 162 patients with diabetes and
80 healthy controls. SNCV measured with the POCD correlated signifi-
cantly with mLDIf in both control and diabetic subjects (r < 0.90 and
r ¼ 0.78, respectively) as did SNAP (r ¼ 0.88 and r ¼ 0.73, respectively)
(Sharma, Vas, & Rayman, 2015). Sharma and Rayman also presented the
results of a longitudinal 1-year study of the technique in type 1 and 2 diabetes
at Neurodiab 2014 (Sopron, Hungary—September 24, 2014) (Tucker,
2014). At year 1, the LDI-flare size compared to baseline was reduced by
6.8% in the type 1 diabetes patients and by 5.9% in the type 2 patients,
but just 0.84% among the healthy controls (P < 0.001 for both diabetes
groups vs controls). Among the type 1 diabetes patients, the percent reduc-
tion was significantly greater among the 56 with microangiopathy compared
with the 24 without microangiopathy (8.7% vs 4.6%; P < 0.0001). How-
ever, there was no significant difference between the 49 type 2 diabetes
patients with microangiopathy and the 33 without (4.9% vs 6.0%;
P ¼ 0.06). The differences in findings between the type 1 and type 2 diabetes
patients can be explained on the grounds of differences in the pathogenesis of
Alternative Neuropathy Assessments 261

small-fiber dysfunction between the two groups. As Sharma commented,

“While type 2 diabetes is more related to metabolic syndrome, where insulin
resistance plays an important part, the same cannot be said of type 1, where
glycemic control plays an important role in the genesis of small-fiber
neuropathy.” There was a highly significant correlation between triglycer-
ides and LDI-flare size in both the controls and the diabetes patients (both
types) at baseline (P ¼ 0.008 and 0.001, respectively) and at year 1 (P ¼ 0.01
and 0.004, respectively). The change in triglycerides also correlated with the
change in LDI-flare size (P ¼ 0.008 and 0.009, respectively), supporting
increasing evidence to suggest that triglycerides play an important role in
the pathogenesis of diabetic polyneuropathy. This relationship is so far
shown to be independent of glycemic control in various cross-sectional
studies. Change in small-fiber function as measured by LDI flare did not cor-
relate with BMI, other lipid indices, or blood pressure. Neuropathy disabil-
ity scores, sural-nerve amplitude, and sural-nerve conduction velocity did
not change over the study year, nor did they correlate with changes in
HbA1c, lipids, or blood pressure suggesting that the technique may have dis-
tinct advantages in evaluating prediabetic neurovascular dysfunction.
Neurovascular dysfunction studies conducted by ourselves in lean con-
trols, obese controls, relatives of patients with IGT, IGT, and type 2 diabetes
show that skin perfusion response to occlusion and raising the ambient tem-
perature from 32°C to 44°C may be the single most sensitive tool for
detecting abnormalities in IGT and obesity but even more so in relatives
of patients with diabetes (Vinik, Nevoret, Casellini, & Parson, 2013)
(Fig. 3). A study comparing new techniques of quantification of small-fiber
structure and function with LDI flare would be of great interest.

4.3.6 Assessment of Autonomic Neuropathy

Since many conditions affect the ANS, and autonomic neuropathy is not
unique to diabetes, the diagnosis of DAN rests with establishing the diagno-
sis and excluding other causes (Table 4). The best-studied diagnostic
methods, for which there are large databases and evidence to support their
use in clinical practice, relate to the evaluation of cardiovascular reflexes. In
addition, the evaluation of orthostasis is fairly straightforward and is readily
done in clinical practice, as is the establishment of the cause of gastrointes-
tinal symptoms and erectile dysfunction. The combination of cardiovascular
autonomic tests with sudomotor function tests may allow a more accurate
diagnosis of DAN (England et al., 2009; Vinik et al., 2013). Table 5 presents
the diagnostic tests that would be applicable to the diagnosis of CAN. These
262 A.I. Vinik et al.

160
Lean controls, n = 19
140
Obese controls, n = 13
120 Relatives, n = 19
Skin perfusion units

IGT, n = 14
100 Type 2 diabetes, n = 22

80

60

40

20

0
Occlusion 32° C 44° C
Minutes
Fig. 3 Abnormalities in heat-mediated vasodilation of hairy skin in obese controls, rel-
atives of subjects with diabetes, impaired glucose tolerance (IGT), and type 2 diabetes
subjects, as compared with lean controls (Vinik et al., 2013).

Table 4 Differential Diagnosis of Diabetic Autonomic Neuropathy

Clinical Manifestations Differential Diagnosis
Cardiovascular Cardiovascular disorders
Resting tachycardia, exercise Idiopathic orthostatic hypotension, multiple
intolerance system atrophy with Parkinsonism, orthostatic
Orthostatic tachycardia and tachycardia, hyperadrenergic hypotension
bradycardia syndromes Shy-Drager syndrome
Cardiac denervation, painless Panhypopituitarism
myocardial infarction Pheochromocytoma
Orthostatic hypotension Hypovolemia
Intraoperative and perioperative Congestive heart disease
cardiovascular instability Carcinoid syndrome

tests can be used as a surrogate for the diagnosis of DAN in any system since it
is generally rare to find involvement of any other division of the ANS in the
absence of cardiovascular autonomic dysfunction. For example, if one enter-
tains the possibility that the patient has erectile dysfunction due to DAN,
then prior to embarking upon a sophisticated and expensive evaluation of
erectile status, a measure of heart rate and its variability in response to deep
breathing would—if normal—exclude the likelihood that the erectile dys-
function is a consequence of disease of the ANS. The cause thereof would
have to be sought elsewhere. Similarly it is extremely unusual to find
gastroparesis secondary to DAN in a patient with normal cardiovascular
autonomic reflexes.
Alternative Neuropathy Assessments 263

Table 5 Diagnostic Tests of Cardiovascular Autonomic Neuropathy

Test Method/Parameters
Time and frequency evaluation of >100 Beats/min is abnormal. With the
resting heart rate beat-to-beat heart patient at rest and supine (no overnight
rate variationa and response to deep coffee or hypoglycemic episodes),
breathing breathing 6 breaths/min, heart rate
monitored by EKG or ANSCORE
device, a difference in heart rate of
>15 beats/min is normal and
<10 beats/min is abnormal, R-R
inspiration/R-R expiration >1.17. All
indices of HRV are age dependentb.
SDNN and RMSSD.
Heart rate response to standinga During continuous EKG monitoring, the
R-R interval is measured at beats 15 and
30 after standing. Normally, a tachycardia
is followed by reflex bradycardia. The
30:15 ratio is normally >1.03.
Heart rate response to Valsalva The subject forcibly exhales into the
maneuvera mouthpiece of a manometer to 40 mmHg
for 15 s during EKG monitoring. Healthy
subjects develop tachycardia and peripheral
vasoconstriction during strain and an
overshoot bradycardia and rise in blood
pressure with release. The ratio of longest
R-R shortest R-R should be >1.2.
Spectral analysis of heart rate variation, Series of sequential R-R intervals into its
very low-frequency power (VLFP various frequent components. It defines
0.003–0.04), and high-frequency two fixed spectral regions for the low-
power (HFP 0.15–0.40 Hz) frequency and high-frequency measure.
Systolic blood pressure response to Systolic blood pressure is measured in the
standing supine subject. The patient stands and the
systolic blood pressure is measured after
2 min. Normal response is a fall of
<10 mmHg, borderline is a fall of
10–29 mmHg, and abnormal is a fall of
>30 mmHg with symptoms.
Diastolic blood pressure response to The subject squeezes a handgrip
isometric exercise dynamometer to establish a maximum.
Grip is then squeezed at 30% maximum for
5 min. The normal response for diastolic
blood pressure is a rise of >16 mmHg in
the other arm.
Continued
264 A.I. Vinik et al.

Table 5 Diagnostic Tests of Cardiovascular Autonomic Neuropathy—cont'd

Test Method/Parameters
EKG QT/QTc intervals spectral The QTc (corrected QT interval on EKG)
analysis with respiratory frequency should be <440 ms. VLF peak
(sympathetic dysfunction) LF peak
(sympathetic dysfunction) HF peak
(parasympathetic dysfunction) LH/HF
ratio (sympathetic imbalance).
Neurovascular flow Using noninvasive laser Doppler measures
of peripheral sympathetic responses to
nociception.
Sudorimetry Use of Sudoscan to evaluate the
electrochemical skin conductance
response to low voltage stimulation
applied to the skin of the volar surfaces of
the hands and feet measured as electrical
skin conductance (ESC)
a
These can now be performed quickly (<15 min) in the practitioners’ office, with a central reference
laboratory providing quality control and normative values. LF, VLF, HF—low-, very low-, and
high-frequency peaks on spectral analysis. These are now readily available in most cardiologist’s practice.
b
Lowest normal value of E/I ratio: Age 20–24:1.17, 25–29:1.15, 30–34:1.13, 35–30:1.12, 40–44:1.10,
45–49:1.08, 50–54:1.07, 55–59:1.06, 60–64:1.04, 65–69:1.03, and 70–75:1.02.

4.3.7 Sudorimetry
Small unmyelinated C- and Aδ-fibers in the periphery subserve somatic and
autonomic nerve functions such as warm, cold, and pain perception as well
as innervating sweat glands. The gold standard for their evaluation has been
skin biopsy and quantification of IENF density, an invasive procedure
(Pittenger et al., 2004; Vinik et al., 2013). On the other hand, the evaluation
of sweating has the appeal of quantifiable noninvasive determination of the
integrity of the ANS. Sudomotor nerves are thin unmyelinated C-fibers that
are primarily cholinergic at the ganglion although, epinephrine, norepi-
nephrine, vasoactive intestinal peptide, atrial natriuretic peptide, calcitonin
gene-related polypeptide, galanin, ATP, and substance P have also been
localized to periglandular nerves (Freeman & Chapleau, 2013). A variety
of techniques have capitalized on quantifying sweating (Illigens &
Gibbons, 2009) or the innervation of sweat glands (Gibbons, Illigens,
Wang, & Freeman, 2009; Wang & Gibbons, 2013) to capture the impact
of disordered regulation on structure and function of sweat glands as surro-
gates for the underlying autonomic dysfunction.
Alternative Neuropathy Assessments 265

Sudorimetry, or sudomotor function testing, is the science of measuring

the function of sweat gland innervation and is unique among the autonomic
function tests in that it evaluates the peripheral sympathetic system but relies
principally on cholinergic postganglionic neurotransmission. The Neuropad
is an indicator test for the detection of sudomotor dysfunction that assesses
plantar sweat production by means of a color change from blue to pink. The
patch contains the complex salt anhydrous cobalt-II-chloride. In the pres-
ence of water, this salt absorbs water molecules, normally changing its color
from blue to pink. If the patch remains completely or partially blue within
10 min, the result is considered abnormal (Papanas et al., 2005).
Recently, a newer sudomotor function technology has become available
and adds a new tool at point of care when autonomic testing had been pre-
viously restricted to specialized neurological laboratories. The Sudoscan®,
an instrument capable of detecting chloride ion flux in response to a very
low current, is an objective, noninvasive, and quantitative sudomotor func-
tion test with promising sensitivity and specificity in the investigation of
small-fiber function in DSPN (Casellini et al., 2013; Gin, Baudoin,
Raffaitin, Rigalleau, & Gonzalez, 2011; Smith, Lessard, Reyna,
Doudova, & Singleton, 2014; Yajnik, Kantikar, Pande, & Deslypere,
2012) and has shown correlation to the severity of pain symptoms
(Casellini et al., 2013; Selvarajah et al., 2015; Smith et al., 2014). The entire
evaluation takes only 2 min and can be done in an ambulatory setting. The
unique qualities of sudomotor function tests are that they may yield diagnos-
tic information not only of autonomic dysfunction but also enhance the
assessment of the small somatosensory nerves (Boger et al., 2012;
Thaisetthawatkul, Fernandes Filho, & Herrmann, 2013). An excellent
review on sudomotor function tests can be read elsewhere (Illigens &
Gibbons, 2009).
Sudoscan™ technology is based on different electrochemical principles
(reverse iontophoresis and chronoamperometry) to measure sudomotor
function than prior technologies, affording it a much more practical and pre-
cise performance profile for routine clinical use with potential as a research
tool. The device consists of a desktop computer connected to two pairs of
large surface stainless steel electrodes: one pair for application of the palms
and the other for the soles. The subject places both hands and feet simulta-
neously on the designated electrodes and a painless scanning process ensues
over 2–3 min, during which a low DC voltage (1–4 V) is incrementally
applied to the electrodes, with the left and right electrodes serving alterna-
tively as cathode and anode. At voltages under 10 V, the stratum corneum is
266 A.I. Vinik et al.

electrically insulating whereas the sweat glands consist of a cellular bilayer

and therefore can transmit electrically charged ions to the skin surface and
the electrodes by reverse iontophoresis. The current of sweat-derived chlo-
ride ions generated in response to the incremental voltage applied can be
quantified and reflects sweat gland function and, by inference, functional
innervation. This chloride ion current is reported as electrochemical skin
conductance (ESC)—the inverse of resistance—measured in microSiemens
(μS). The palms and soles are assessed due to their very high density of sweat
glands and the frequency of small-nerve degeneration. By alternating the left
and right extremities as anode and cathode, each extremity is evaluated sep-
arately during the 3 min scan, and asymmetry between the extremities can
be calculated.

4.3.7.1 Clinical Diagnostic Evaluation Using Sudomotor Function

The clinical diagnostic accuracy of Sudoscan™ has been evaluated for
DSPN, peripheral small-fiber neuropathy, and autonomic neuropathy
against other standard methods. Casellini et al. (2013) examined 83 type 1
and 2 diabetic patients, both with and without DSPN, and compared them
to 210 healthy controls. Diabetic patients with DSPN had significantly
worse ESCs of both feet and hands than patients without DSPN or healthy
controls. Somewhat surprisingly, ESCs correlated significantly with clinical
neuropathy scores of sensory, motor, and reflex function, somatic (QST and
NCV studies) and autonomic (quantitative autonomic function testing)
measures of DSPN suggesting that the measure captures both somatic and
autonomic nerve function. Using a Neuropathy Impairment Score of the
Lower Limb (NIS-LL) > 2 as the gold standard for the diagnosis of neurop-
athy, the sensitivity and specificity of ESC measured in the feet for the diag-
nosis of DSPN were 78% and 92%, respectively (Fig. 4). The investigators
further determined correlations between sudorimetry and somatic and auto-
nomic function and found strong correlations between ESC and both
somatic and autonomic functions with sudorimetry scores being able to sep-
arate painful from nonpainful DSPN.
The value of ESC has been confirmed by others. Smith et al. (2014) stud-
ied 55 patients with suspected idiopathic or diabetes-related small-fiber neu-
ropathy and 42 controls to evaluate the diagnostic performance of ESC.
Using an abnormal Utah Early Neuropathy Score (UENS
4) as the diag-
nostic gold standard, ESC of feet and hands were significantly lower than
those of controls, regardless of the etiology of small-fiber neuropathy. More
importantly, ESC and IENF density had similar diagnostic performance in
detecting small-fiber neuropathy. Mirroring the findings of Casellini et al.
Alternative Neuropathy Assessments 267

Sudoscan

Symmetry

Conductances Autonomic
measured on neuropathy
hands and feet (LF HRV model)

Fig. 4 Display of electrochemical skin conductance (ESC) results for the assessment
of sudomotor function. Numeric ESC scores are provided in microSiemens and visually
displayed on a scale of normative ranges. Asymmetry represents the percent difference
in ESC measures between left and right limbs. The cardiac neuropathy risk score (pCN) is
calculated from the measured ESC and patient characteristics.

(2013), ESC in the feet had a very high negative predictive value (83%), and
significantly correlated with both symptoms and signs assessed using the
MNSI and the Utah early neuropathy symptom scale. ESC measurement
may also be a simple tool for early identification of peripheral autonomic
neuropathy and useful in screening for subclinical CAN. Some of the major
studies completed using ESC measurement in a clinical diagnostic capacity
are summarized in Table 6.

4.3.7.2 Validation of ESC Robustness

For ESC measurement to be clinically meaningful, widely applicable thresh-
olds for normal and abnormal sudomotor function must be determined and
high reproducibility of results demonstrated. Initially, a large cohort of
healthy subjects (432 females, 177 males) indicated that there was almost
no drop-off in normal sudomotor function from age 21 to 80 and no sexual
dimorphism. However, data from population studies in different countries
indicate that normative values for sudomotor function as quantified by ESC
varies by ethnicity so that diagnostic thresholds are needed to address these
differences (Table 7). Reproducibility of testing has also been performed
under various stress scenarios including hyperglycemia, CVD, and pre-
and postexercise and coefficients of variation for ESC ranged from 7% to
15% for hands and 4–10% for feet ESC. Comparison between tests
Table 6 Feet ESC Diagnostic Accuracy vs Established Clinical Tools
Study Diagnostic Variable Comparison Sensitivity (%) Specificity (%) PPV (%) NPV (%)
Casellini et al. (2013) DPN NIS-LL 78.34 92.38 74.6 93.72
Selvarajah et al. (2015) DPN AAN guidelines 87.5 76.2 N/A N/A
for DPN
Selvarajah et al. (2015) CAN—diabetes 5 Cardiac 65 85 N/A N/A
autonomic reflex
tests
Smith et al. (2014) Distal symmetric UENS 77 67 59 83
polyneuropathy
Yajnik et al. (2012) DPN VPT 73 62 N/A N/A
Eranki et al. (2013) DPN VPT
15 V 82 55 N/A N/A
Yajnik et al. (2013) CAN—diabetes 3 Cardiac 92 49 N/A N/A
autonomic reflex
tests
Ozaki et al. (2011) Diabetic kidney eGFR, ACR 94 78 81 93
disease
ACR, albumin creatinine ratio; CAN, cardiac autonomic neuropathy; CARTs, cardiac autonomic reflex tests; DPN, diabetic peripheral neuropathy; eGFR, estimated
glomerular filtration rate; NIS-LL, neuropathy impairment score-lower limbs; PPV, positive predictive value; NPV, negative predictive value; UENS, Utah Early Neu-
ropathy Score; VPT, vibration perception threshold.
Alternative Neuropathy Assessments 269

Table 7 Electrochemical Skin Conductance (ESC) Zones for Normal Sudomotor

Function (Green), Moderate Sudomotor Dysfunction (Yellow), and Severe Sudomotor
Dysfunction (Red) as Defined by Racial Background
Hands ESC Hands ESC Hands ESC Feet ESC Feet ESC Feet ESC
Red Yellow Green Red Yellow Green
Race Zone Zone Zone Zone Zone Zone
Caucasian 0−0 40−60 60−100 0−50 50−70 70−100
African 0−30 30−50 50−100 0−40 40−60 60−100
American
Ancestry
Other 0−40 40−60 60−100 0−40 40−60 60−100
(Asian,
Indian)
All numeric values in microSiemens (μS). Reproducibility testing was performed under various stress
scenarios as well as normal conditions including hyperglycemia, cardiovascular disease, and pre- and post-
exercise; coefficients of variation ranged from 7% to 15% for hands ESC and 4% to 10% for feet ESC.
Comparison between tests performed twice on three different devices demonstrated that for feet ESC the
mean coefficient of variation for repeatability was 2.8  1.6% in healthy volunteers (HV) and 6.9  6.3%
in patients with type 2 DM (PDT2), while coefficient of variation for reproducibility were 3.1  1.5%
and 6.9  6.3%, respectively (submitted for publication).
Data from Casellini, C. M., Parson, H. K., Richardson, M. S., Nevoret, M. L., & Vinik, A. I. (2013).
SUDOSCAN, a noninvasive tool for detecting diabetic small fiber neuropathy and autonomic dysfunc-
tion. Diabetes Technology & Therapeutics, 15, 948–953; Smith, A. G., Lessard, M., Reyna, S., Doudova,
M., & Singleton, J. R. (2014). The diagnostic utility of SUDOSCAN for distal symmetric peripheral
neuropathy. Journal of Diabetes and Its Complications, 28, 511–516; Freedman, B. I., Smith, S. C., Bagwell,
B. M., Xu, J., Bowden, D. W., & Divers, J. (2015). Electrochemical skin conductance in diabetic kidney
disease. American Journal of Nephrology 41, 438–447.

performed twice on three different devices demonstrated that, for feet ESC,
the mean coefficient of variation for repeatability was 2.8  1.6% in healthy
volunteers and 6.9  6.3% in patients with type 2 diabetes while coefficient
of variation for reproducibility were 3.1  1.5% and 6.9  6.3%, respectively
(submitted for publication).

4.3.7.3 Sudomotor Function as a Tool to Measure Progression and Regression

of Disease
One of the most difficult and frustrating tasks in the study of peripheral neu-
ropathy has been to measure disease progression and response to therapeutic
intervention with an easy, objective, and accurate tool. Several research stud-
ies have now demonstrated that ESC may be a clinically meaningful tool in
measuring neuropathy progression and regression. In a cohort of 52 type 1 and
115 type 2 diabetic patients followed for 1 year with no change in their gly-
cemic control, type 2 patients treated without insulin showed a decrease in
hand and foot ESC, while a significant increase was observed in those
270 A.I. Vinik et al.

Table 8 Change Between Baseline and 12-Month Follow-Up in Weight, Waist, VO2max,
and ESC Risk Score Value in 154 Women Included in a Lifestyle Intervention Program
(Raisanen, Eklund, Calvet, & Tuomilehto, 2014)
Without Follow- Low Weekly
Up of Training Activitya High Weekly
Level (n 5 72) (n 5 62) Activityb (n 5 20)
Mean SD Mean SD Mean SD p
Change in weight (kg) 0.9 3.4 1.6 4.0 3.3 4.8 NS
Change in waist (cm) 2.1 4.7 2.4 4.5 3.6 5.9 NS
Change in estimated +0.5 0.9 +0.8 0.9 +1.1 1.2 NS
VO2max (METs)
Change in hand ESC (μS) +5.0 8.4 +3.0 9.4 +8.4 12.3 0.043
Change in foot ESC (μS) +5.6 8.9 +4.9 8.9 +10.8 12.8 0.024
Change in ESC risk score (%) 5.1 5.3 4.7 6.4 8.5 6.8 0.027
a
Less than 150 min of moderate activity and 75 min of high activity.
b
More than 150 min of moderate activity or 75 min of high activity; moderate activity 3–7 METs, high
activity > 7 METs.

receiving insulin. Type 1 patients taking insulin therapy also had a nonsignif-
icant increase in their feet ESC (Calvet, Dupin, Winiecki, & Schwarz, 2013).
Raisanen et al. (2014) demonstrated that a 12-month lifestyle intervention
could improve small-fiber function in metabolic syndrome. Among 154
female participants with the lowest fitness level at baseline, those performing
the highest level of weekly activity showed the greatest improvement in ESC,
which was more pronounced than the changes in weight, waist circumfer-
ence, or VO2max (Table 8). These, and other ongoing studies, indicate that
ESC may provide information on the status of small nerve fiber function that
may not otherwise be known to or measurable by the physician.

5. CONCLUDING REMARKS
Diabetic neuropathy is the most common complication of diabetes
and contributes additional risks in the aging adult. For eons, the focus has
been on pain and loss of sensory perception leading to development of foot
ulcers and amputations. There is now recognition that a broader view of
neuropathy needs to be taken. Loss of sensory perception, loss of muscle
strength, and ataxia or incoordination lead to an increased risk of falling
Alternative Neuropathy Assessments 271

17-fold greater in the older diabetic than their young nondiabetic counter-
parts. A fall is accompanied by lacerations, tears, fractures and, worst of all,
traumatic brain injury from which more than 60% do not recover. There
clearly is an increased need for early recognition of people at risk and to
embark on preventive measures. Autonomic neuropathy has been hailed
as the “Prophet of Doom” (Vinik et al., 2011) for good reason. It is condu-
cive to increased risk of MI and sudden death. It is therefore critical that we
identify people with cardiac autonomic neuropathy and embark on preven-
tive strategies. It also has been recognized that an imbalance in the ANS
occurs early in the evolution of diabetes at a stage when active intervention
can abrogate the relentless progression. Many new and emerging syndromes
can be attributed to CAN such as orthostatic tachycardia and bradycardia in
addition to hypotension. While pain is a common and debilitating accom-
paniment of neuropathy, providers have failed to recognize the importance
of the accompanying sleep deprivation, anxiety, and depression which, if
unattended, compromise appropriate management of pain. Ultimately the
constellation of features of neuropathy conspires to reduce QOL and
ADL. Thus, meticulous evaluation of DSPN and DAN in the aging diabetic
patient at diagnosis and then on a yearly basis is critical, in addition to falls
risk assessment and determination of autonomic integrity. Early neuropathy
detection can only be achieved by assessment of small-nerve fibers. New
noninvasive sudomotor function technologies may play an increasing role
in identifying early peripheral and autonomic neuropathy, allowing rapid
intervention and potentially prevention or reversal of small-fiber loss.

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 CHAPTER FOURTEEN

Wherefore Art Thou, O Treatment

for Diabetic Neuropathy?
R.A. Malik1
Weill Cornell Medicine-Qatar, Qatar Foundation, Education City, Doha, Qatar
1
Corresponding author: e-mail address: [email protected]

Contents
1. The Problem 288
2. What Can We Do? 291
3. Diagnostic Tests are Not Necessarily Good Surrogate End Points 293
4. Clinical Trials in Diabetic Neuropathy 294
4.1 Glycemic Control 295
4.2 Pancreas Transplantation 297
4.3 α-Lipoic Acid 298
4.4 Aldose Reductase Inhibitors 298
4.5 Vitamin B 299
4.6 Angiotensin-Converting Enzyme Inhibitors 299
4.7 Protein Kinase C Activation 300
4.8 C-Peptide 300
4.9 Actovegin 301
4.10 Disease Modification 301
4.11 Nerve Growth Factor 302
4.12 Other Growth Factors 303
4.13 Erythropoietin 303
4.14 Vitamin D 304
4.15 Angiotensin Axis 305
5. Can We Ever Succeed? 306
References 309

Abstract
As of March 2016, we continue to advocate the diagnosis of diabetic neuropathy using a
simple foot examination or monofilament, which identifies only those with severe neu-
ropathy and hence risk of foot ulceration. Given the fact that the 5-year mortality rate of
diabetic patients with foot ulceration is worse than that of most common cancers, surely
we should be identifying patients at an earlier stage of neuropathy to prevent its pro-
gression to a stage with such a high mortality? Of course, we lament that there is no
licensed treatment for diabetic neuropathy. Who is to blame? As researchers and carers,
we have a duty of care to our patients with diabetic neuropathy. So, we have to look
forward not backwards, and move away from our firmly entrenched views on the design

International Review of Neurobiology, Volume 127 # 2016 Elsevier Inc. 287

ISSN 0074-7742 All rights reserved.
http://dx.doi.org/10.1016/bs.irn.2016.03.008
288 R.A. Malik

and conduct of clinical trials for diabetic neuropathy. Relevant organizations such as
Neurodiab, the American Diabetes Association and the Peripheral Nerve Society have
to acknowledge that they cannot continue to endorse a bankrupt strategy. The FDA
needs an open and self-critical dialogue with these organizations, to give pharmaceu-
tical companies at least a fighting chance to deliver effective new therapies for diabetic
neuropathy.

I have not failed, I've just found 10,000 ways that won't work
attr. Thomas Edison

1. THE PROBLEM
As the doctors say of a wasting disease, to start with it is easy to cure but difficult to
diagnose; after a time... it becomes easy to diagnose but difficult to cure.
Niccolo Machiavelli

Diabetic neuropathy affects at least 50% of patients with diabetes during their
lifetime. The human and economic burden of diabetic neuropathy and its
consequences in the form of painful neuropathy, foot ulceration, and ampu-
tation are considerable for patients and healthcare systems. This is true in
both in the developed (Abbott, Carrington, Ashe, et al., 2002; Boulton,
Vileikyte, Ragnarson-Tennvall, et al., 2005) and particularly in the devel-
oping world (Riaz et al., 2014) where the consequences of disability are
grave due to loss of employment and hence livelihood—not just for the
patient, but the whole family. The life expectancy of diabetic patients with
a foot ulcer (14.4 years) or Charcot foot (13.9 years) is markedly reduced
compared to a normative U.K. population (van Baal, Hubbard, Game,
et al., 2010). Moreover, by the time, a patient with diabetes has developed
a foot ulcer their 5-year mortality is greater than lymphoma (14%), breast
(10.6%), prostate (10%), and bladder (22%) cancer (Armstrong,
Wrobel, & Robbins, 2007; Marshall, Webb, Hall, et al., 2016) and is much
higher than colon cancer (11%), non-Hodgkin’s (31%), and Hodgkin’s lym-
phoma (14%) (Al-Hamadani, Habermann, Cerhan, et al., 2015). Indeed, it is
comparable to feared cancers such as lung cancer (48%) (Field, Duffy,
Baldwin, et al., 2016; Malvezzi, Carioli, Bertuccio, et al., 2016) (Fig. 1).
Furthermore, according to the World Health Organization and a recent sys-
tematic subnational analysis for the Global Burden of Disease in China, the
three leading causes of death in the world are ischemic heart disease, stroke,
and chronic obstructive pulmonary disease (COPD) (Zhou, Wang, Zhu,
et al., 2016). The 5-year mortality after a myocardial infarct (28%)
(Bata, Gregor, Wolf, et al., 2006), stroke (41.7%) (Hankey, Jamrozik,
Wherefore Art Thou, O Treatment for Diabetic Neuropathy? 289

Fig. 1 Five-year mortality of common cancers compared to diabetic patients with foot
ulceration.

Fig. 2 Five-year mortality for the three leading causes of death in the world compared
to patients with diabetic foot ulceration.

Broadhurst, et al., 2000), and COPD (22%) (Martinez, Mannino, Jaimes,

et al., 2015) are, in each case, lower than in patients with diabetes and a foot
ulcer (Walsh, Hoffstad, Sullivan, & Margolis, 2015) (Fig. 2).
It is instructive to compare the consequences of diabetic neuropathy with
other complications of diabetes. A recent population-based survey of
414,523 people with diabetes found that 20,737 (5%) developed foot ulcers
and that the 5-year mortality of these patients was 42.2%. Even after con-
trolling for major risk factors for mortality in this population the fully
adjusted hazard ratio (HR) for death were 2.48, suggesting a unique and
290 R.A. Malik

as yet unidentified relationship between diabetic neuropathy and mortality

(Walsh et al., 2015). In contrast, in a prospective (7.9 years of follow-up)
study of 2237 patients with type 1 diabetes, the age- and sex-adjusted
HRs of all-cause mortality were 1.45 (95% CI 0.71–2.96) and 4.16
(1.96–8.84) in patients with nonproliferative and proliferative retinopathy
at baseline. After adjustment for cardiovascular risk factors, the association
with nonproliferative retinopathy and proliferative retinopathy became
nonsignificant (van Hecke, Dekker, Stehouwer, et al., 2005). For diabetic
nephropathy, the FIELD study of 9795 patients with T2DM reported that
the risk of total CVD events when comparing patients with an estimated
GFR (eGFR) of
90 mL min1 1.73 m2 was increased (HR [95% CI]
1.14 [1.01–1.29] for eGFR 60–89 mL min1 1.73 m2 and 1.59 [1.28–1.98]
for eGFR 30–59 mL min1 1.73 m2; P < 0.001). Microalbuminuria and
macroalbuminuria also increased the total CVD event rate (HR 1.25
[1.01–1.54] and 1.19 [0.76–1.85] P ¼ 0.001 for trend) independent of eGFR
(
90 mL min1 1.73 m2), respectively. However, in multivariable analy-
sis, 77% of the effect of eGFR and 81% of the effect of albumin:creatinine
ratio were accounted for by a low HDL-cholesterol and elevated blood
pressure (Drury, Ting, Zannino, et al., 2011). We readily deploy retinal
screening and microalbuminuria testing to detect the earliest phases of
diabetic retinopathy and nephropathy. Yet for neuropathy, we recommend
the monofilament and foot examination, tests which can only identify the
“high risk diabetic foot” (Mayfield & Sugarman, 2000).
In 2016 we have no FDA approved treatment for diabetic neuropathy
and a graveyard of potential therapies. Many explanations, such as the wrong
types of patients, severity of neuropathy, and inadequate trial duration have
been suggested for the singular lack of progress in developing an effective
treatment for diabetic neuropathy (Malik, 2014b). The ultimate test of
the usefulness of a drug or device depends on the determination of out-
comes. These have to be quantified reliably, have relevance to the underly-
ing disease process, and should be tested in randomized clinical trials (RCTs)
that use appropriate numbers and types of patients and duration of treatment.
The purported gold standard end points (Tesfaye, Boulton, Dyck, et al.,
2010) of symptoms and signs of diabetic neuropathy (Dyck, Overland,
Low, et al., 2010) combined with neurophysiological testing (Dyck,
Albers, Wolfe, et al., 2013; Litchy, Albers, Wolfe, et al., 2014) perform inad-
equately even in the hands of the “experts,” and yet continue to be advo-
cated as primary end points in clinical trials (Dyck, 2014).
Professional organizations continue to endorse consensus guidance, which
by default only allows minor incremental change or no change at all.
Wherefore Art Thou, O Treatment for Diabetic Neuropathy? 291

The FDA will carry out due process and endorse the low-risk consensus
view. The consequence of this approach has been failure after failure of clin-
ical trials in diabetic neuropathy, so that the field has the unenviable record
of a 100% failure rate. When future trials fail, and they will, investigators will
readily blame “big pharma” for enrolling the wrong types of patients, too
few patients, or choosing an inadequate study duration or the wrong end-
point. And thus the cycle continues.

2. WHAT CAN WE DO?

We need to learn to measure what we value, not value what we can easily measure
Marcus Aurelius

Because we have previously not been able to adequately quantify small

fibers, the default position, and hence consensus, has been to ignore them.
Techniques that quantify small fiber function, such as quantitative sensory
testing, have been shown to be reliable (Dyck, Argyros, Russell, et al.,
2014) and newer techniques that quantify small fiber structure, present
intuitively attractive end points for clinical trials of diabetic neuropathy,
especially for assessing early repair (Malik, 2014a). Two such techniques
are the visual quantification of intraepidermal nerve fibers through skin
biopsy (Fig. 3) (Lauria, Bakkers, Schmitz, et al., 2010; Lauria, Hsieh,

Fig. 3 Skin biopsies immunostained for neuronal marker PGP 9.5 from a healthy subject
(A) and a patient with severe DPN (B). Note the almost complete depletion of IENFD (red
(gray in the print version) arrows) and only very small nerve fiber profiles (blue (dark gray
in the print version) arrows) scattered in the epidermis and a reduction of the subepi-
dermal nerve plexus (yellow (light gray in the print version) arrows) in (B). Original mag-
nification  200, scale bar ¼ 100 μm.
292 R.A. Malik

Fig. 4 Corneal confocal microscopy images of the sub-basal nerve plexus from a control
(A), a patient without DPN (B), and a patient with DPN (C) showing the reduction in cor-
neal nerves in those with DPN. Red (gray in the print version) arrows indicate main nerve
fibers; yellow (white in the print version) arrows indicate branches.

Johansson, et al., 2010; Lauria, Morbin, Lombardi, et al., 2003) and corneal
confocal microscopy (CCM), which allows noninvasive in vivo imaging of
corneal nerves (Fig. 4) (Malik, Kallinikos, Abbott, et al., 2003). Indeed a
robust body of data, which is conveniently ignored, shows that small fiber
damage occurs in patients with early diabetic neuropathy (Bitirgen,
Ozkagnici, Malik, et al., 2014; Malik, 2014a; Ziegler, Papanas, Zhivov,
et al., 2014) and subjects with IGT (Asghar, Petropoulos, Alam, et al.,
2014; Singleton, Smith, & Bromberg, 2001; Smith, Ramachandran,
Tripp, et al., 2001). Of clinical relevance to the hard end point sought by
the FDA (foot ulceration), an equally strong body of data shows that small
cutaneous fibers release an array of neuropeptides, which are key to the pro-
cess of wound healing (Ashrafi, Baguneid, & Bayat, 2015; Laverdet, Danigo,
Girard, et al., 2015) and hence repair after foot ulceration.
While estimation of IENF density (IENFD) by skin biopsy offers an
objective means of quantifying small fiber pathology, it is an invasive and
costly procedure that does not allow for the same nerves to be visualized
repeatedly (Lauria, Bakkers, et al., 2010; Lauria, Hsieh, et al., 2010;
Lauria, Morbin, Lombardi, et al., 2003). Our previous NIH and JDRF
funded studies have shown that CCM can quantify early small fiber axonal
damage in diabetic neuropathy (Petropoulos, Alam, Fadavi, et al., 2014),
reliably (Petropoulos, Manzoor, Morgan, et al., 2013), with high sensitivity
and specificity (Petropoulos et al., 2014). CCM has a better sensitivity and
comparable specificity for the diagnosis of diabetic neuropathy (Chen,
Graham, Dabbah, et al., 2015) and correlates with IENF loss (Quattrini,
Tavakoli, Jeziorska, et al., 2007). We have also shown that CCM can be
deployed in children with type 1 diabetes, that corneal nerve loss is evident
in young people with T1DM without retinopathy (Szalai, Deak, Modis,
Wherefore Art Thou, O Treatment for Diabetic Neuropathy? 293

et al., 2016), and that it predicts incident neuropathy in type 1 diabetes

(Pritchard, Edwards, Russell, et al., 2015) in those with poorer glycemic
control and lower HDL (Dehghani, Pritchard, Edwards, et al., 2016).
The potential of corneal nerve loss as a surrogate end point for diabetic neu-
ropathy has been further established by showing that it detects nerve regen-
eration in diabetic patients undergoing simultaneous pancreas and kidney
transplantation (Tavakoli, Mitu-Pretorian, Petropoulos, et al., 2013) and
in patients with sarcoid (van Velzen, Heij, Niesters, et al., 2014) and diabetic
(Brines, Dunne, van Velzen, et al., 2014) neuropathy treated with the novel
peptide, ARA 290. These latter studies showed nerve repair within 6 months
and 28 days respectively, over which time all other currently endorsed end
points, such as neurological deficits, quantitative sensory testing, and neuro-
physiology remained unchanged. Furthermore, we have recently published a
large normative reference database (Tavakoli, Ferdousi, Petropoulos, et al.,
2015) and developed an automated image analysis algorithm to enable rapid
and objective quantification of corneal nerve morphology (Dabbah,
Graham, Petropoulos, et al., 2011). Views on the role and importance of
small fibers in neuropathy and their utility in clinical trials of diabetic neu-
ropathy have to change. Unless we incorporate a measure of small fiber dam-
age and repair as a surrogate end point in clinical trials of diabetic neuropathy,
we will continue to fulfil the self-fulfilling prophecy of failure after failure.

3. DIAGNOSTIC TESTS ARE NOT NECESSARILY GOOD

SURROGATE END POINTS
An army of philosophers would not be sufficient to change the nature of error and
to make it truth
Averroes

In 2010, the Toronto consensus panel separated diabetic peripheral neurop-

athy (DPN) into typical and atypical groups (Tesfaye et al., 2010). Typical
DPN is “a symmetrical, length-dependent sensorimotor polyneuropathy
attributable to metabolic, and microvessel alterations as a result of chronic
hyperglycemia exposure and cardiovascular risk covariates.” Atypical vari-
ants of diabetic neuropathy differ in onset, course, manifestations, associa-
tions, and putative mechanisms and are likely to be associated with pain
and/or dysautonomia. It is therefore important to identify different subtypes
of DPN to ensure homogeneity of patients recruited into trials.
The presentation of diabetic neuropathy can be variable and may include
pain, which is reported by approximately one-third of patients with diabetes,
regardless of associated neurological deficit (Abbott, Malik, van Ross, et al.,
294 R.A. Malik

2011). The classical description is of an unremitting burning pain that is

characteristically worse at night, with a gradual distal-to-proximal progres-
sion of symptoms in a glove-and-stocking distribution. Overt motor deficits
in the form of muscle wasting and weakness were typically thought to occur
in more advanced neuropathy (Andersen, 2014). However, our recent study
suggests that weakness and muscle atrophy may occur at an earlier stage
(Almurdhi, Reeves, Bowling, et al., 2016). Measures of strength or size
of muscles could potentially be incorporated as end points in clinical trials
of early neuropathy, as these measures are sensitive to change and have clin-
ical relevance.
Various tools have been validated to assess neurological impairment in
diabetic neuropathy. These include the Michigan Neuropathy Screening
Instrument (MNSI) and Neuropathy Disability Score (NDS). Indeed,
because of the wide use and ease of administering these tools, they were
incorporated into a number of intervention trials of glycemic control in type
2 diabetes. To date, they have all singularly failed to show a significant
improvement in neuropathy in studies such as ACCORD and VADT trials
(Calles-Escandon, Lovato, Simons-Morton, et al., 2010; Duckworth,
Abraira, Moritz, et al., 2009). A Cochrane review has concluded that
“improved glycemic control does not improve neuropathy in type 2
diabetes” (Callaghan, Little, Feldman, et al., 2012). Is this really the conclu-
sion that we should be drawing? Or should we be addressing the major defi-
ciency in these trials: namely use of the wrong end points?
Pain-specific questionnaires have also been utilized in order to quantify
painful symptoms. The available instruments include the Brief Pain Inven-
tory and the Neuropathic Pain Questionnaire (NPQ), McGill Pain
Questionnaire, and visual analog scales (VAS). Additionally, neuropathy-
specific quality-of-life measures, such as the NeuroQoL and the Norfolk
Quality of Life Scale, may play a role in identifying patient-important factors
in neuropathy (Javed, Alam, & Malik, 2015a).

4. CLINICAL TRIALS IN DIABETIC NEUROPATHY

They sent forth men to battle, But no such men return; And home, to claim their
welcome, Come ashes in an urn
Aeschylus

There have been many clinical trials in subjects with diabetic neuropathy.
As discussed earlier, their failure may derive from many factors ranging
Wherefore Art Thou, O Treatment for Diabetic Neuropathy? 295

from incorrect choice of the presumed pathogenic mechanism to poor

trial design, so that the data should be viewed with all of these caveats
in mind.

4.1 Glycemic Control

The Eurodiab Insulin-Dependent Diabetes Mellitus (IDDM) study found
that suboptimal glycemic control was associated with the development of
DPN in a large cohort of 3000 people with T1DM. 1172 patients with
type 1 diabetes were assessed for neuropathy at baseline (1989–91) and at
follow-up (1997–99), with a mean follow-up period of 7.3  0.6 years.
A standardized protocol for the evaluation of DPN included clinical evalu-
ation, QST, and autonomic-function tests. A number of factors were found
to be independently associated with the incidence of neuropathy including
the duration of diabetes and change in HbA1c (Tesfaye, Chaturvedi, Eaton,
et al., 2005). Additionally, the incidence of neuropathy was associated with
potentially modifiable cardiovascular risk factors, including body mass index,
smoking, and hypertension, as well as raised blood triglyceride levels
(Tesfaye et al., 2005). Other observational studies have also emphasised
the impact of poor glycemic control on the development and progression
of DPN (Herman, Aubert, Engelgau, et al., 1998; Oyibo, Prasad,
Jackson, et al., 2002).
Whether or not an improvement in glycemic control can prevent or
delay progression of clinical neuropathy in patients with type 1 diabetes
has been formally assessed in two key studies. The Stockholm Diabetes
Intervention Study was initiated in 1982, at which time 48 patients were
randomized to intensive insulin and 54 to standard insulin and followed
for 7.5 years. While there was a positive effect on nerve conduction there
was no change in symptoms or in vibration and thermal thresholds
(Reichard, Nilsson, & Rosenqvist, 1993). In the landmark Diabetes Control
and Complications Trial (DCCT), clinical neuropathy was defined as an
abnormal neurologic examination and either abnormal nerve conduction
in at least two peripheral nerves or unequivocally abnormal autonomic-
nerve testing. Details of the DCCT trial are discussed elsewhere in this vol-
ume. Intensive therapy reduced the appearance of neuropathy after 5 years
by 69% in the primary-prevention cohort and by 57% in the secondary-
intervention cohort (The Diabetes Control and Complications Trial
Research Group, 1993, 1995). Of note, this was a 1400-patient multicentre
study and took 5 years to show an effect on patients with type 1 diabetes who
296 R.A. Malik

had no neuropathy at baseline. The apparent success of the DCCT has been
used to highlight the need for large numbers of patients without neuropathy
at baseline who are followed for at least 5 years using a combination of clin-
ical neurological examination, neurophysiology, and autonomic-function
testing in clinical trials of therapies for DPN.
In type 2 diabetes, the evidence of a benefit of improved glycemic con-
trol on neuropathy is less clear—or is it? The United Kingdom Prospective
Diabetes Study (Stratton, Adler, Neil, et al., 2000) reported a significant risk
reduction in amputation for every 1% reduction in mean HbA1c (Stratton
et al., 2000) and a lower rate of impaired VPT with intensive therapy versus
standard therapy. However, this effect only became significant after 15 years,
with no significant advantage observed at 3, 6, 9, and 12 years. What does
this mean for trials that incorporate vibration perception threshold as a pri-
mary or secondary end point over 1–2 years?
Intensive glycemic control in type 2 diabetes has been shown to reduce
microvascular complications in a 6-year randomized, prospective study
(Ohkubo, Kishikawa, Araki, et al., 1995). Does this mean that the minimum
duration of trials should be 6 years? Boussageon, Bejan-Angoulvant,
Saadatian-Elahi, et al. (2011) conducted a meta-analysis of seven trials
involving over 34,000 patients and did not find a reduction in the incidence
of DPN in patients managed through intensive glycemic control. Further-
more, a Cochrane review of 17 randomized trials concluded that tight gly-
cemic control prevented neuropathy in type 1 diabetes, driven principally by
the DCCT findings, but that it had no benefit in type 2 diabetes (Callaghan,
Little, et al., 2012). It should be noted that these meta-analyses have signif-
icant limitations as the targeted glucose levels, therapeutic strategies, out-
come measures, trial design, and duration of follow-up differ between
studies. The principal culprit for demonstrating a lack of benefit is, however,
that the majority of these studies utilized crude neuropathy end points such
as a monofilament and foot exam and vibration perception.
The VA Cooperative study of type 2 diabetes reported no difference in
the prevalence of autonomic neuropathy between the intensive and standard
therapy arms at 2 years (Duckworth et al., 2009). In contrast, the Steno-2
Trial reported that an intervention that integrated glucose control and
multiple cardiovascular risk factor management reduced the prevalence of
cardiac autonomic neuropathy but not unsurprisingly showed no benefit
for somatic neuropathy, as assessed using vibration perception thresholds
in patients with type 2 diabetes and microalbuminuria even after 7.8 years
of follow-up (Gaede, Lund-Andersen, Parving, et al., 2008).
Wherefore Art Thou, O Treatment for Diabetic Neuropathy? 297

4.2 Pancreas Transplantation

The only known therapy to restore insulin secretion in response to feedback
mechanisms in patients with diabetes is pancreas transplantation (Fiorina &
Secchi, 2007; Tavakoli & Liong, 2012). Differing lengths of time for
improvement in DPN have been reported in patients treated by pancreas
transplantation. Fioretto, Steffes, Sutherland, et al. (1998) found that the
reversal of DPN was only evident 10 years after transplantation. Agudo,
Valls-Solé, Recasens, et al. (2002) reported an increase in action potential
amplitude and conduction velocity at 3 months and 1 year after transplanta-
tion in a small study involving 26 patients. In the controlled study by
Kennedy, Navarro, Goetz, et al. (1990), an improvement in sensory and
motor function 12 and 24 months after pancreas transplantation was reported
in a cohort of 61 patients with type 1 diabetes, but neurophysiological
and autonomic-function tests did not demonstrate recovery. Navarro,
Sutherland, and Kennedy (1997) reported similar findings in 115 subjects
with type 1 diabetes who underwent pancreas transplantation with an
improvement in composite scores assessing motor and sensory neuropathy
but only a slight improvement in autonomic function after a longer 10-year
follow-up. In contrast, Boucek, Havrdova, Voska, et al. (2008) assessed
IENFD in 18 subjects following pancreas and kidney transplantation and
found that only three patients showed an improvement, suggesting that in
some patients DPN may reach a nonreversible stage. It should be noted that
all these studies used different outcome measures to assess DPN, with variable
follow-up periods. Hence, it is likely that the improvement in DPN reported
in the literature depends entirely on the endpoint used to assess DPN. Indeed,
in a contemporary cohort of 15 patients (Mehra, Tavakoli, Kallinikos,
et al., 2007), we found no change in electrophysiology; quantitative sensory
testing and IENFD, whereas CCM parameters improved significantly
12 months after transplantation (Tavakoli, Mitu-Pretorian, et al., 2013).
While pancreas transplantation may represent the most effective method
for restoring normoglycemia, its use is limited to patients in end-stage kid-
ney disease or, less frequently, to patients with type 1 diabetes and
unpredictable hypoglycemia. This is due to the restricted and unpredictable
availability of suitable organs, complications of surgery and the risks accom-
panying long-term immunosuppression. Recently, islet-cell transplantation
has been considered as a less invasive alternative for suitable patients with
diabetes. Del Carro, Fiorina, Amadio, et al. (2007) reported marked
improvement in neurophysiology with no change in skin biopsies.
298 R.A. Malik

4.3 α-Lipoic Acid

Hyperglycemia results in increased production of reactive oxygen species
due to the autooxidation of excess glucose and a failure of antioxidant mech-
anisms (Kaur, Pandhi, & Dutta, 2011). These oxygen free radicals mediate
endothelial dysfunction by inhibiting nitric oxide, leading to ischemic nerve
damage. α-Lipoic acid (ALA) is a free-radical scavenger that is suggested to
alleviate this oxidative stress. It is licensed for the treatment of DPN in Ger-
many and was found to be well tolerated and efficacious in the management
of painful DPN, when administered parenterally. Indeed, Ziegler, Nowak,
Kempler, et al. (2004) reported a clinically significant improvement in the
symptoms of DPN after administration of a 600-mg daily dose of ALA over
3 weeks in their meta-analysis of four placebo-controlled trials. However in
the more recently published NATHAN 1 study, a 4-year multicenter ran-
domized controlled trial comparing ALA to placebo, Dyck, Norell,
Tritschler, et al. (2007) found no improvement in neurophysiology,
QST, and composite neuropathy scores. Unfortunately, the primary end
point failed to deteriorate significantly in placebo-treated subjects,
highlighting the lack of decline of placebo-treated subjects in recent trials
of human diabetic neuropathy (Dyck et al., 2007). In a study of 44 partici-
pants with type 1 diabetes randomized to antioxidant treatment with allo-
purinol, ALA, and nicotinamide, or placebo over 24 months, there was
no change in symptoms, signs, electrophysiology and intraepidermal nerve
fiber density, or cardiovascular autonomic reflex testing (Pop-Busui,
Stevens, Raffel, et al., 2013). In a recent meta-analysis from China, 1410
participants in 18 RCTs demonstrated a significant benefit on median
and peroneal nerve conduction velocities in those given lipoic acid, prosta-
glandin E1, and methylcobalamin compared to prostaglandin E1 and meth-
ylcobalamin alone. However, in these studies, there were only 30–100
patients in each arm, the drugs were administered intravenously, there
was no placebo group and some of the studies were <14 days duration
(Jiang, Li, Wang, et al., 2015).

4.4 Aldose Reductase Inhibitors

Aldose reductase is the rate-limiting enzyme in the polyol pathway for glu-
cose metabolism (Callaghan, Cheng, Stables, et al., 2012) and hyperglycemia
increases flux through this pathway leading to reduced production of the
vasodilator nitric oxide and eventually perpetuating ischemic nerve injury.
Aldose reductase inhibitors (ARIs) initially showed an improvement in
nerve conduction velocity and some improvement in myelinated nerve fiber
Wherefore Art Thou, O Treatment for Diabetic Neuropathy? 299

density and regenerative clusters in sural nerve biopsies (Dyck, Zimmerman,

Vilen, et al., 1988; Greene, Arezzo, & Brown, 1999; Sima, Bril, Nathaniel,
et al., 1988), but failed consistently in subsequent phase III clinical trials
(Boulton, Malik, Arezzo, et al., 2004). There are also concerns over toxicity
and given the limited efficacy this has precluded their use in patients
(Boulton, Kempler, Ametov, et al., 2013). Epalrestat is the only ARI which
is licensed in Japan and India based on limited randomized controlled trial
data (Goto, Hotta, Shigeta, et al., 1995). Indeed, a Cochrane collaboration
review of 32 trials comprising 4970 participants found no overall benefit of
ARIs in DPN (Chalk, Benstead, & Moore, 2007).

4.5 Vitamin B
Benfotiamine is a fat-soluble derivative of thiamine that has been shown in
animal models to inhibit three major pathways implicated in oxidative stress
and vascular dysfunction in diabetes: the advanced glycation end-product
pathway, the hexosamine pathway, and the protein kinase C (PKC)–
diacylglycerol pathway (Hammes, Du, Edelstein, et al., 2003). Although
the multimodal effects of benfotiamine present an attractive option for
treating DPN, clinical trials are unclear about its efficacy. A randomized
placebo-controlled trial (Stracke, Gaus, Achenbach, et al., 2008) of 165
patients reported an improvement in patient-reported symptoms in the
per-protocol arm of the study, although there was no improvement in
the intent-to-treat group compared to placebo. Furthermore there was an
improvement in symptoms but no change in deficits between the placebo
and treatment arms of the study (Stracke et al., 2008). In a multicenter, ran-
domized, double-blind, placebo-controlled trial of 214 patients with type 2
diabetes randomly assigned to 24 weeks of treatment with either L-meth-
ylfolate calcium 3 mg, methylcobalamin 2 mg, and pyridoxal-50 -phosphate
35 mg, or placebo the primary end point, not unsurprisingly, VPT did not
change, while the secondary end points of the Neuropathy Total Symptom
Score (NTSS-6) and Short Form 36 (SF-36), did (Fonseca, Lavery, Thethi,
et al., 2013). In a recent “real-world” study, 544 patients reported a mean
reduction of 35% in NTSS-6 scores after 12 weeks on a medical food product
containing L-methylfolate-methylcobalamin-pyridoxal-5-phosphate (Trippe,
Barrentine, Curole, et al., 2016).

4.6 Angiotensin-Converting Enzyme Inhibitors

The development and progression of nephropathy, retinopathy, and
neuropathy are closely related. Angiotensin-converting enzyme (ACE)
300 R.A. Malik

inhibitors delay progression of both nephropathy and retinopathy. We

investigated the effect of ACE inhibition on diabetic neuropathy and found
an improvement in neurophysiology after 12 months of treatment with the
ACE inhibitor trandolapril in 41 normotensive patients with DPN, though
there was no change in autonomic function compared to placebo (Malik,
Williamson, Abbott, et al., 1998). In the DEMAND study, 200 patients with
type 2 diabetes showed a significant reduction in the odds ratios for periph-
eral neuropathy at 3 years between the calcium channel blocker Manidipine
and ACE inhibitor delapril vs delapril alone vs placebo: 0.45 (0.24–0.87;
P ¼ 0.017) and 0.52 (0.27–0.99; P ¼ 0.048) (Ruggenenti, Lauria, Iliev,
et al., 2011). Neuropathy was assessed using symptoms, deficits, autonomic
function, QST, and neurophysiology.

4.7 Protein Kinase C Activation

Free radicals generated by the hyperglycemia-induced activation of PKC are
thought to play a role in the pathogenesis of DPN by altering vascular per-
meability and causing vasoconstriction. The PKC inhibitor ruboxistaurin
has been evaluated in clinical trials (Brooks, Delaney-Robinson,
Molyneaux, et al., 2008; Vinik, Bril, Kempler, et al., 2005). A recent sys-
tematic review (Bansal, Badhan, Gudala, et al., 2013) of six randomized con-
trolled trials concluded that ruboxistaurin offered no benefit in the treatment
of DPN.

4.8 C-Peptide
C-peptide deficiency is an important contributing factor to the characteristic
functional and structural abnormalities of peripheral nerves (Sima, 2003).
C-peptide binds to cell membranes, resulting in stimulation of endothelial
nitric oxide synthase (eNOS) and Na+, K+-ATPase (Wahren, Ekberg, &
Jornvall, 2007). In the Joslin 50-Year Medallist Study (Sun, Keenan,
Cavallerano, et al., 2011), protection from complications (retinopathy,
nephropathy, and neuropathy) was thought to be due to the presence of
enriched protective factors against microvascular complications. In two
double-blind, placebo-controlled studies in type 1 DM patients, C-peptide
replacement or placebo was given with the patients’ regular insulin therapy
(Ekberg, Brismar, Johansson, et al., 2003, 2007). Sensory NCV assessed in
the sural nerve showed a significant improvement. A recent large phase III
trial has reported no significant improvement in nerve conduction param-
eters compared to placebo, although it should be noted that the placebo arm
Wherefore Art Thou, O Treatment for Diabetic Neuropathy? 301

showed an unexpected increase in nerve conduction that paralleled the

anticipated treatment effect. As C-peptide, but not placebo, improved
vibration perception, the authors suggested that large fiber conduction
velocity did not adequately reflect large fiber-mediated sensory function
(Wahren, Foyt, Daniels, et al., 2016).

4.9 Actovegin
Actovegin is a hemoderivative produced from calf blood by ultrafiltration
that exerts insulin-like activity, stimulating glucose transport, pyruvate
dehydrogenase, and glucose oxidation (Ziegler, Movsesyan, Mankovsky,
et al., 2009). In a multicenter randomized placebo-controlled double-blind
trial, 567 patients with type 2 diabetes received 20 intravenous infusions of
Actovegin (2000 mg day1) (n ¼ 281) or placebo (n ¼ 286) once daily
followed by three tablets of Actovegin (1800 mg day1) or placebo three
times daily for 140 days. TSS (P ¼ 0.0003), VPT (P ¼ 0.017), and NIS-LL
sensory function (P ¼ 0.021) improved significantly (Ziegler et al., 2009).
However, this is the only trial of Actovegin and comprehensive clinical trials
are needed to confirm its benefits in the treatment of DPN (Boulton
et al., 2013).

4.10 Disease Modification

Erratic blood glucose control has been associated with DPN and it has been
proposed that stable glycemic control may be essential for treating neurop-
athy. Previously (Callaghan, Little, et al., 2012), it has been demonstrated
that continuous subcutaneous insulin infusion (CSII) may achieve near-
normal glycemia in people with type 1 diabetes and also lead to an improve-
ment in neurophysiology and painful symptoms. In a recent study by Azmi,
Ferdousi, Petropoulos, et al. (2015), there was evidence of corneal nerve
regeneration but no impact on QST or neurophysiology in the CSII cohort
compared to subjects on a multiple daily injection insulin regimen. This was
despite comparable HbA1c over 2 years of follow-up.
Dyslipidemia has been linked with DPN and Wiggin, Sullivan, Pop-
Busui, et al. (2009) showed that elevated triglyceride levels correlate with
a reduction in myelinated fiber density. Fried, Forrest, Ellis, et al. (2001)
have suggested that lowering blood lipid levels may prevent or alleviate
the symptoms of DPN. Smith et al. reported that a decrease in triglyceride
levels in a cohort of patients with impaired glucose tolerance managed
through lifestyle interventions was associated with an increase in IENFD.
Furthermore, a recent 12-week double-blind, placebo-controlled study of
302 R.A. Malik

rosuvastatin showed that there was an improvement in neurological deficits,

symptoms, and nerve conduction parameters of DPN after 12 weeks
(Hernández-Ojeda, Román-Pintos, Rodrı́guez-Carrı́zalez, et al., 2014).
The significant reduction in minor amputations in the FIELD study for
patients with T2DM receiving fenofibrate points to an important role for
triglycerides or alternative mechanisms of benefit for the PPARα agonists
(Rajamani, Colman, Li, et al., 2009).

4.11 Nerve Growth Factor

Nerve growth factor (NGF) was first isolated in 1952 by Rita Levi-
Montalcini and identified as a protein that induces the growth of nerves,
for which she received the Nobel prize in Physiology or Medicine in
1986 (Shelton, 2014). Tanezumab, is a monoclonal antibody, completely
humanized against NGF, that binds to NGF with high affinity and selectiv-
ity, thereby blocking the NGF–TrKA interaction, inhibiting signaling
through sensory and sympathetic neurones and pain (Hirose, Kuroda, &
Murata, 2016). One could argue that it is potentially one of the few successes
to be born out of the failure of the NGF trials in diabetic neuropathy (Apfel,
Kessler, Adornato, et al., 1998). In the pivotal phase III study of recombinant
human NGF (rhNGF), which enrolled 1019 patients with diabetic neurop-
athy, the primary end point was NIS-LL (neurological exam assessing mus-
cle strength, reflexes, and sensory testing) and the secondary end points were
vibration perception, cold perception, and heat pain thresholds (Apfel,
2002). It is not clear why one would evaluate large myelinated fiber function
to assess the efficacy of a drug that targets small fibers. Not surprisingly, the
study failed and perhaps prematurely or ill advisedly, Genentech stopped fur-
ther trials. However, during development of rhNGF, one of the trouble-
some side effects of treatment by injection was small fiber-mediated
neuropathic pain. This resulted in a massive dose deescalation to amounts
which in hindsight were probably nonefficacious (Shelton, 2014). In a
recent efficacy and safety study, a single 20 mg dose of subcutaneous
Tanezumab showed a significant reduction in pain scores in patients with
diabetic neuropathy (Bramson, Herrmann, Carey, et al., 2015). A logical
concern for such studies is that an anti-NGF approach could potentially
destroy c-fibers. In a safety study of patients with osteoarthritis who were
administered Tanezumab: “Neurological safety was evaluated via a compos-
ite score made up of nerve conduction attributes and heart rate variability
with deep breathing and intraepidermal nerve fiber density.” Choosing to
evaluate nerve conduction, a large myelinated fiber measure and indeed
Wherefore Art Thou, O Treatment for Diabetic Neuropathy? 303

heart rate variability, a measure of parasympathetic function as a safety mea-

sure for a drug, which might harm unmyelinated c-fibers or sympathetic
fibers appears to be looking in the wrong place. Indeed, although not sig-
nificant at 24 weeks, IENFD was reduced in the group given 10 mg of
Tanezumab (Brown, Herrmann, Goldstein, et al., 2014). It has been
suggested that the development of postural hypotension will be an adequate
marker of harm, but this will only occur with severe loss of sympathetic
fibers. This appears to be a clear case of where one should employ sensitive,
fiber-type-specific end points such as skin biopsy and CCM to detect early
c-fiber degeneration.

4.12 Other Growth Factors

Verheyen, Peeraer, Lambrechts, et al. (2013) have suggested that targeting
pathogenetic pathways implicated in DPN by utilizing vascular endothelial
growth factor (VEGF) and VEGF-derived peptides may be an option for
future therapies. However, a recent clinical trial of VEGF in DPN was
stopped due to lack of efficacy (Ropper, Gorson, Gooch, et al., 2009).
Hepatocyte growth factor (HGF) is a multifunctional, mesenchyme-derived
cytokine with potent neurotrophic, angiogenic, and antiapoptotic effects.
These combined neurotrophic and angiogenic properties of HGF ought
to make it an ideal candidate as a disease-modifying treatment of diabetic
neuropathy. Yet two clinical trials have been undertaken with pain as a pri-
mary end point. In small numbers of patients, both show efficacy after intra-
muscular injections of plasmid DNA expressing two different isoforms of
HGF (VM202). In the first study 36 patients showed a significant reduction
in mean VAS from baseline by 47.2% (P ¼ 0.002) at 6 months and by 44.1%
(P ¼ 0.005) at 12 months (Ajroud-Driss, Christiansen, Allen, et al., 2013). In
a larger follow-up study of 84 patients receiving 8 mg VM202 per leg, there
was a significant reduction in the mean pain score at 3 but not at 6 and 9
months and there was no improvement in the MNSI, monofilament testing,
or IENFD at 6 months (Kessler, Smith, Cha, et al., 2015). These disparate
data sets raise the question of whether to pursue pain as a primary end point,
or to consider alternative end points to show disease modification? Viromed,
the sponsors of VM202, have chosen to pursue a further trial with pain as a
primary end point. Is this not a case of the tail wagging the dog?

4.13 Erythropoietin
Erythropoietin (EPO) is produced in situ by cells under stress and has
been found to antagonize the production of proinflammatory molecules
304 R.A. Malik

and promote tissue healing. While EPO has been found to ameliorate
experimental DPN, its use is limited by serious adverse effects, in partic-
ular increased thrombotic risk (Brines et al., 2014). ARA 290, a
nonhematopoietic peptide designed from the structure of EPO, selectively
interacts with the EPO receptors that mediate tissue protection. In a recent
phase II trial to evaluate the activity of ARA 290 in type 2 diabetes and
DPN, ARA 290 (4 mg) or placebo were self-administered subcutaneously
daily for 28 days and the subjects followed for an additional month without
further treatment. Neuropathic symptoms were found to improve signifi-
cantly in the ARA 290 group. Additionally, a significant improvement in
corneal nerve morphology was found using CCM in patients randomized
to the ARA 290 arm of the trial relative to placebo. ARA 290 remains in
development for DPN and type 2 diabetes (Brines et al., 2014).

4.14 Vitamin D
There is an association between vitamin D deficiency and pain in the gen-
eral population (Hirani, 2012). A mechanistic link between vitamin D and
pain has been supported by a recent study reporting that nociceptive cal-
citonin gene-related peptide (CGRP)-positive neurons have a distinct
vitamin D phenotype with hormonally regulated ligand and receptor levels
(Tague & Smith, 2011). Vitamin D deficiency results in increased numbers
of axons containing CGRP. In cultured neurons, vitamin D receptor
(VDR) expression is increased in growth cones and sprouting appears
to be regulated by VDR-mediated rapid response signaling pathways
(Tague, Clarke, Winter, et al., 2011). NGF is known to be depleted in
experimental diabetes (Hellweg, Wohrle, Hartung, et al., 1991) and in
a study of patients with diabetic neuropathy, NGF immunostaining on
skin keratinocytes correlated with skin axon–reflex vasodilation, a measure
of small fiber neuropathy (Anand, Terenghi, Warner, et al., 1996). In an
experimental study, NGF expression was maintained in sciatic nerves of
diabetic animals treated with a vitamin D analog (CB1093). Similarly
Tacalcitol, active vitamin D3, induces NGF production in human epider-
mal keratinocytes (Fukuoka, Sakurai, Ohta, et al., 2001). Treatment with
vitamin D3 has been shown to reduce demyelination in a cuprizone
experimental model of demyelination (Wergeland, Torkildsen, Myhr,
et al., 2011) and in a separate spinal cord compression model it has been
shown to induce axonal regeneration (Bianco, Gueye, Marqueste,
et al., 2011).
Wherefore Art Thou, O Treatment for Diabetic Neuropathy? 305

Several recent observational studies in patients with diabetes have dem-

onstrated a significant association between vitamin D deficiency and paraes-
thesia and numbness (Soderstrom, Johnson, Diaz, et al., 2012), neurological
deficits and electrophysiology (Alamdari et al., 2015; Shehab, Al-Jarallah,
Mojiminiyi, et al., 2012), and parasympathetic dysfunction (Maser,
Lenhard, & Pohlig, 2015). Furthermore, a recent systematic review and
meta-analysis of 1484 patients with type 2 diabetes demonstrated a highly
significant association (odds ratio 2.68) between vitamin D deficiency and
the development of DPN (Lv, Zhao, Gong, et al., 2014). A more detailed
study using electrophysiology and Douleur Neuropathique 4 (DN4) scores
has shown that serum levels of vitamin D are significantly reduced while
serum vitamin D-binding protein (VDBP) and vitamin D receptor
(VDR) levels are comparable between diabetic patients with and without
peripheral neuropathy (Celikbilek, Gocmen, Tanik, et al., 2015). Recently,
the FIELD multinational study has shown that vitamin D deficiency was
present in 50% of 9795 patients with type 2 diabetes and it predicted micro-
vascular outcomes (Herrmann, Sullivan, Veillard, et al., 2015).
Few studies have investigated the potential therapeutic benefits of vitamin
D therapy. An open-label prospective study in 51 patients with type 2 diabetes
and painful neuropathy showed that 2000 IU of cholecalciferol daily for 3
months resulted in an 50% decrease in the Visual Analog Score (VAS) (Lee &
Chen, 2008). More recently, in a placebo-controlled study of 112 patients
with type 2 diabetes randomized to 50,000 IU of Cholecalciferol once weekly
for 8 weeks, there was a significant increase in 25OHD and an improvement in
the Neuropathy Symptom Score, but no change in the Neurological Disabil-
ity Score or neurophysiology (Shehab, Al-Jarallah, Abdella, et al., 2015). We
have recently shown a significant improvement in three separate measures of
painful diabetic neuropathy following the administration of a single intramus-
cular dose of 600,000 IU of vitamin D3 (Basit et al., 2016).

4.15 Angiotensin Axis

The angiotensin II type 2 receptor (AT2R) axis has been found to play a
substantial role in promoting nociceptive signaling by stimulating hyper-
excitability and persistent ectopic firing of first-order sensory neurones. Rice
et al. have demonstrated that EMA401, a novel small molecule AT2R antag-
onist, provides significant pain relief in postherpetic neuralgia in a novel
phase II randomized, placebo-controlled trial of 183 patients (Rice,
Dworkin, McCarthy, et al., 2014). This drug remains in development as
a therapeutic agent for neuropathic pain of different etiologies.
306 R.A. Malik

5. CAN WE EVER SUCCEED?

Every generation needs a new revolution
Thomas Jefferson

A number of different therapeutic approaches that target the various patho-

genetic mechanisms of diabetic neuropathy have been the subject of clinical
trials (Javed, Alam, & Malik, 2015b). These treatments aim to favorably
impact the underlying pathophysiological abnormalities encountered in
DPN by targeting different elements in the pathways leading to neurovascular
dysfunction (Fig. 5). While numerous pathogenetic treatments have shown
promise in experimental and early phase II studies, a recurring theme has been
the lack of “translation” in phase III clinical trials. One may question the rel-
evance of experimental findings, particularly short-term studies in animal
models, which predominantly show functional and hence easily reversible
deficits, rather than structural pathology. To address this, representatives
from NIDDK, JDRF, and the Diabetic Neuropathy Study Group of EASD
met to develop a set of consensus criteria for the phenotyping of rodent models
of diabetic neuropathy (Biessels, Bril, Calcutt, et al., 2014) in relation to:
• Different rodent models of diabetes (type 1/2 diabetes, IGT/
prediabetes).
• Characteristic alterations in nerve structure.
• Electrophysiological assessments of nerve function.
• Behavioral assessments of nerve function.
• The role of biomarkers in disease phenotyping.

Fig. 5 Integrating pathophysiology of diabetic neuropathy and the sites of action of

pathogenetic drugs (ARIs, aldose reductase inhibitors; ACE, angiotensin-converting
enzyme; AGE, advanced glycation end products).
Wherefore Art Thou, O Treatment for Diabetic Neuropathy? 307

It was agreed that neuropathy in rodents should be defined as the presence of

statistically different values between diabetic and control animals in 2 of 3
assessments:
• Nocifensive behaviour (paw withdrawal response to heat or von Frey
monofilament).
• Nerve conduction velocities (sensory and motor).
• Nerve structure (nerve axon count, epidermal nerve fiber density,
corneal nerve fiber density).
The salient fact remains that at present there are no FDA approved medica-
tions for the treatment of diabetic neuropathy (Malik, 2014b). One has to
ask why no pathogenetic treatment for DPN has achieved regulatory
approval. This is not a new question and indeed was addressed following
the huge investment, and subsequent serial failure, of the ARI program dur-
ing the 1980–90s, where virtually every large pharma house invested mul-
timillions in large phase III clinical trials, all of which were deemed as failed.
A Cochrane review found thirty-two randomized controlled trials of ARIs
involving 879 treated participants and 909 controls with adequate data.
There was no overall significant difference between the treated and control
groups in neurological function (13 studies), nerve conduction parameters
(27 studies), or foot ulceration (1 study) (Chalk et al., 2007). The authors
concluded: “Any future clinical trials of ARIs should be restricted to
compounds proven to have substantial biological or preclinical advantages
over previously tested agents.” Surely that is the wrong message!
Pfeifer and Schumer (1995) reviewed the outcomes and limitations
of previous clinical trials and suggested future directions. They noted that
there was improvement of nerve function associated with some short-term
clinical trials addressing a number of possible etiologic pathways (improved
glucose control, high myoinositol diet, ARIs, gamma-linolenic acid). An
improvement in nerve morphometry had been demonstrated in some
short-term clinical trials with ARIs (Dyck et al., 1988; Greene et al.,
1999; Sima et al., 1988), perhaps attesting to the potential for using morpho-
logical measures as end points in clinical trials of diabetic neuropathy.
However, the use of serial sural nerve biopsies is not something that can
be advocated for clinical trials of diabetic neuropathy, given their highly
invasive nature.
In 2002, Ziegler and Luft suggested that, until the mid-1990s, trials were
hampered by a generally poor design, short follow-up, and by being limited
to patients with advanced DPN (Ziegler & Luft, 2002). Therefore, an
adequately designed RCT in diabetic neuropathy must consider:
308 R.A. Malik

• The type and stage of neuropathy.

• Homogeneity of the study population (type 1/2/LADA).
• Relevant surrogate end points (not just neurophysiology).
• Intermediate clinical end points.
• Ultimate clinical outcomes (foot ulceration and amputation).
• Quality of life.
The trial should also take into account the natural history of the disease in
order to adequately power the study in terms of the sample size and study
duration. Furthermore, the reproducibility of the surrogate and intermediate
end points and their clinical relevance and applicability to all patients with
diabetes needs to be considered. They (Ziegler & Luft, 2002) further
suggested that trials should focus preferentially on:
• Patients with mild or moderate neuropathy.
• Be undertaken over 3–5 years.
• Aim to slow or prevent, rather than reverse diabetic neuropathy.
• Use end points that have clinical and prognostic significance.
Tesfaye, Tandan, Bastyr, et al. (2007) reported on changes in signs, symp-
toms, and quantitative vibration testing in the placebo-treated arms of two
randomized controlled trials of ruboxistaurin in DPN. They suggested that
to demonstrate deterioration in these measures in any placebo-treated DPN
group, studies of >12 months were needed. In the same year, Dyck et al.
examined the challenges of selecting appropriate end points for clinical
trials by examining data from the placebo-treated groups of two large inter-
vention trials as well as the Rochester Diabetic Neuropathy Study (Dyck
et al., 2007). They concluded that studies were failing to demonstrate
efficacy due to:
• A strong placebo effect for symptoms and signs.
• Measurement noise.
• Because DPN may progresses more gradually than previously thought
due to concomitant treatment with ACE inhibitors (Malik et al.,
1998; Reja, Tesfaye, Harris, et al., 1995) and lipid-lowering
(Calles-Escandon et al., 2010; Davis, Yeap, Davis, et al., 2008;
Rajamani et al., 2009) therapies.
Despite these analyses, we continue to enroll small numbers of heteroge-
neous patients in trials of short duration (1–2 years maximum), using the
wrong end points such as pain for a trial of a disease modifier or large fiber
nerve conduction when the underlying benefit is to small fibers. Why does
this occur? Because investors want a quick return for their money and have
the naı̈ve and sometimes ill-advised hope that a quick 12–20-week pain trial
Wherefore Art Thou, O Treatment for Diabetic Neuropathy? 309

will be positive for a drug that is supposed to repair nerves, or at the very least
limit further degeneration. Trials are also constrained by current FDA
guidance, which has not changed for 20 years, and continues to endorse
symptoms and signs, QST, and neurophysiology. In my opinion, future
clinical trials in patients with diabetic neuropathy must minimally:
• Enrol patients with mild or moderate neuropathy.
• Account for the progression rate of neuropathy in the placebo cohort.
• Use surrogate end points of small fiber repair, where pertinent to the
therapeutic approach.
• Operate in a regulatory environment that accepts small fiber repair as a
desirable primary end point.
We can continue to go from failure to failure, or we can have the courage to
question how we diagnose and conduct clinical trials in diabetic neuropathy
in order to make a difference for future generations of patients with diabetic
neuropathy. Many potentially efficacious therapies may well have been
aborted prematurely and need to be reconsidered using the correct end
points.

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INDEX

Note: Page numbers followed by “f ” indicate figures, and “t” indicate tables.

A B
ACE inhibitors, 299–300 BioBreeding/Worcester rat, 102
Actovegin, 301 BioBreeding Zucker diabetic rat
Advanced glycation endproducts (AGEs) (BBZDR)/Wor rat, 96
clinical application, antiglycation agents,
134–135 C
DPN, 132–134, 133f Calcitonin gene-related peptide (CGRP)-
glycation of proteins, 131–132 positive neurons, 304
sensory neurodegeneration, diabetes, Campenot Chambers, 76–77
163–164 Cardiovascular autonomic neuropathy
transgenic and knockout mice studies, (CAN). See also Neuropathy
134 DAN
AGE–RAGE signaling, 164 diabetes management, 251
Aldose reductase inhibition (ARI) diagnosis, 248
DPN, 115–116 exercise intolerance, 248
growth factor, 298–299 heart rate variation (HRV), 247
NCV, 4 increased risk, mortality, 250
Aldose reductase (AR) pathway. intraoperative cardiovascular liability,
See Polyol (aldose reductase 248–249
(AR)/sorbitol–fructose) pathway left ventricular diastolic dysfunction
α-lipoic acid, 298 (LVDD), 247–248
Altered bioenergetics and oxidative stress, myocardial infarction (MI), increased
70–72 mortality due to, 250
Altered insulin signaling orthostatic hypotension, 249
Alzheimer’s disease, 159 resting tachycardia, 248
dermal and epidermal axons, 160 silent myocardial ischemia/cardiac
DRG, 159 denervation syndrome, 249–250
sensory neurons, receptors, 156–157, 158f sudden death, 250
type 1 DM, 157–159 diagnostic tests, 261–262, 263t
type 2 DM, 157–159 Caspase-3 expression, 162–163
Angiotensin II type 2 receptor (AT2R), 305 CaV3.2 T-type channels, 217
Apoptosis. See Cell death Cell death, 69–70
Arabidopsis thaliana miR171, 168 C-peptide, 160–161, 300–301
Axonal degeneration and regeneration
“die-back” neuropathies, 73–74 D
4-HNE, 75–76 Diabetic autonomic neuropathy (DAN)
immortalized cell lines, 74–75 CAN
paclitaxel-induced axonal degeneration, diabetes management, 251
73–74, 74f diagnosis, 248
regenerative responses, 75 exercise intolerance, 248
reinnervation, distal target tissues, 73 heart rate variation (HRV), 247
unsupportive extracellular environment, 76 increased risk, mortality, 250

319
320 Index

Diabetic autonomic neuropathy (DAN) CGRP-positive neurons, 304

(Continued ) DN4 scores, 305
intraoperative cardiovascular liability, EPO, 303–304
248–249 HGF, 303
left ventricular diastolic dysfunction Neuropathy Symptom Score, 305
(LVDD), 247–248 NGF, 302–303
myocardial infarction (MI), increased tanezumab, 302–303
mortality due to, 250 VEGF, 303
orthostatic hypotension, 249 high-risk diabetic foot, 289–290
resting tachycardia, 248 IENF density (IENFD), 292–293
silent myocardial ischemia/cardiac in vitro modeling approach
denervation syndrome, 249–250 advantages and disadvantages, 54, 55t
sudden death, 250 altered bioenergetics and oxidative
clinical manifestations, 245–247, 246t stress, 70–72
prevention and reversibility, autonomic axonal degeneration and regeneration,
neuropathy, 251–253 73–76, 74f
Diabetic neuropathy Campenot Chambers, 76–77
animal models cell death, 69–70
alloxan, 48–49 DRG neurons, 70
novel models, 50 immortalized cell lines, 63–64
species, 46–48 iPSC, 67
STZ toxicity, 49–50 microgrooves, 76–77
value of, 45–46 neuronal hyperexcitability, 72–73
assessment tools, 294 NGF, 76–77
atypical variants, 293 primary tissue culture, 64–67,
classical description, 293–294 65–66f
clinical trials relative replacements, 62
ACE inhibitors, 299–300 Schwann cells, 77–78
actovegin, 301 sensory neuron–keratinocyte
ARI, 298–299 coculture, 77–78, 78f
C-peptide, 300–301 stimuli, choice of, 68–69
designed RCT, 307–308 multivariable analysis, 289–290
dyslipidemia, 301–302 randomized clinical trials (RCTs), 290
efficacy studies, 308 risk factors, 289–290
erratic blood glucose control, 301 sensory symptoms, 54
glycemic control, 295–296 skin biopsies, 291–292, 292f
α-lipoic acid, 298 somatosensory nervous system
pancreas transplantation, 297 cell and tissue types, 61–62
phase III clinical trials, 306–307, 306f genetic control, development, 58–60,
PKC activation, 300 59f
trial design, 308–309 large-diameter axons, 61–62
vitamin B, 299 neural crest cells (NCCs), 58
corneal confocal microscopy (CCM) PNS, 56–58, 57f
images, 291–292, 291f TrkA, 58–60
five-year mortality, 288–289, 289f treatment, 54
foot ulcer, 288–289 Diabetic peripheral neuropathy (DPN)
growth factors advanced disease, 156
AT2R, 305 ARI, 115–116
Index 321

clinical assessment tools, 256 transcription factor heat shock factor 1

clinical impact, aging, 236–237, 236f (HSF1), 184
clinical presentation, 238–239 nerve blood flow (NBF), 155
contact heat-evoked potentials (CHEPS), nerve conduction studies, 256–257
258–259 neuropathy, 243–244
corneal confocal microscopy (CCM), 258 nonglucotoxic components, 118
development, 182–183 oxidative stress, 116–117
diagnosis, 240–241, 241t pathogenetic mechanisms, 183
differential diagnosis, 261–262, 262t pharmacologic approaches, 182
eHsp70 prevalence, 237
immunomodulation, inflammation, Prophet of Doom, 270–271
and oxidative stress, 186–188 QOL measures, 253–255, 254t
import, export, and neuronal support, reversal, falls, 244–245, 244f
185–186 risk of, 237
modulating Hsp70, 197–198 ROS production, 116–117
endogenous neurotrophic factors, 118 sensory fiber involvement, 238–239
epineurial and endoneurial alterations, skin biopsy, 257–258
156 sudorimetry
falls and aging, 241–245 clinical diagnostic evaluation, 266–267,
focal/multifocal neuropathies, 237–238 267f, 268t
glucotoxic mechanisms, 122 electrochemical skin conductance
hyperglycemia-driven nonenzymatic (ESC), 265–266
glycation, 117–118 gold standard, evaluation, 264
iHsp70 neuropad, 265
animal models, 195–197 progression and regression, disease,
chaperone functions, 189–190 269–270, 270t
c-jun, 195–197, 196f Sudoscan®, 265
c-jun N-terminal kinase (JNK), 195 validation, ESC robustness, 267–269,
inflammation, 194–195 269t
oxidative stress, 190–194 symptoms, 182
inflammatory signaling, 182–183 type 2 diabetes, 242–243
laser Doppler flare technique (LDIFT), Diabetic sensorimotor polyneuropathy
260–261, 262f (DSP)
microangiopathy, 154–155 axonal process, 28–29
microvascular complication, 154–155 clinical neuropathy, 29–30
modulating Hsp70 DCCT, 31–32
beneficial and deleterious effects, disease progression, 28–29
197–198, 198f identification, small fiber impairments,
BGP-15, 200–201 30–31
C-terminal Hsp90 inhibitors, IENFD, 34
199–200 large fiber structure, 33–34
N-terminal Hsp90 inhibitor, 199–200 laser Doppler flare area imaging, 34–35
oncoproteins, 199 NATHAN study, 32
molecular chaperones NCS, 30
eHsp70, 184–185 pharmacological agents, 27–28, 28t
heat shock proteins, 183–184 prevention, 28–29
Hsp90 and Hsp70, 184 quantitative sensory threshold
iHsp70, 184–185 measurements, 34
322 Index

Diabetic sensorimotor polyneuropathy G

(DSP) (Continued ) γ-aminobutyric acid (GABA), 221
small nerve fiber function, 34 Gastroparesis, 16–17
type 1 and type 2 diabetes, 29 Glucagon-like peptide-1 (GLP-1), 161
Diminished inhibitory drive, spinal cord, Glucose-metabolizing pathways, 124–125,
221 124f
Distal (length-dependent) symmetrical Glucotoxic mechanisms
sensorimotor polyneuropathy anatomical and biochemical
(DSPN) characteristics, 122–123
DCCT, 11–13 DPN, 122
EDIC, 11 glucose-metabolizing pathways,
Dorsal root ganglion (DRG) neurons 124–125, 124f
altered insulin signaling, 159 glycosylation
in vitro modeling approach, 70 hexosamine pathway, 139–140, 140f
nociceptive ion channels, 213–215 T-type calcium channel (Cav3.2), 140
posttranslational modification, 218–219 nonenzymatic glycation and AGEs
voltage-gated and ligand-gated ion clinical application, antiglycation
channels, 215 agents, 134–135
Douleur Neuropathique 4 (DN4) scores, DPN, 132–134, 133f
305 glycation of proteins, 131–132
Dyslipidemia, 301–302 transgenic and knockout mice studies,
134
E normoglycemic conditions, 123–124
Endothelial NOS (eNOS), 164–165 oxidative stress
Epidemiology of Diabetes Interventions and antioxidant effects, 137–138
Complications (EDIC) diabetic neuropathy and, 136–137
additional measures, 14 production of, 135–136, 136f
assessment PKC activity, 138–139
CAN, 15–16 polyol (AR/sorbitol–fructose) pathway
DSPN, 11 AR inhibition (ARI), 129–130, 130f
DCCT, 11 clinical application, 130–131
gastroparesis, 16–17 ischemia/reperfusion injury, 131
self-report measures, 17 metabolic sequelae, 125–128, 126f
urologic complications, evaluation, 16 transgenic and knockout mice, 128
Epigastric fullness, 19 Glycemic control
Erythropoietin (EPO), 303–304 DCCT, 295–296
Extracellular Hsp70 (eHsp70) HbA1c, 296
immunomodulation, inflammation, and incidence of, 295
oxidative stress NCV, 5–6
chemotaxic signaling, 186–187 Steno-2 trial, 296
LOX-1, 187–188 Glycosylation
NF-κB, 188 hexosamine pathway, 139–140, 140f
receptors, 187–188 T-type calcium channel (Cav3.2), 140
tibialis anterior (TA) muscles, 186 Goto–Kakizaki (GK) rat, 95–96
import, export, and neuronal support, Growth factors, diabetic neuropathy
185–186 AT2R, 305
modulating Hsp70, 197–198 CGRP-positive neurons, 304
molecular chaperones, 184–185 DN4 scores, 305
Index 323

EPO, 303–304 paclitaxel-induced axonal

HGF, 303 degeneration, 73–74, 74f
Neuropathy Symptom Score, 305 regenerative responses, 75
NGF, 302–303 reinnervation, distal target tissues, 73
tanezumab, 302–303 unsupportive extracellular
VEGF, 303 environment, 76
Campenot Chambers, 76–77
H cell death, 69–70
Heat-shock protein (HSP) 27, 162 DRG neurons, 70
Heat shock proteins, 183–184 immortalized cell lines, 63–64
Hepatocyte growth factor (HGF), 303 iPSC, 67
Hexosamine pathway, 139–140, 140f microgrooves, 76–77
High-fat fed Sprague–Dawley rats and neuronal hyperexcitability, 72–73
C57Bl6/J mice, 91–92 NGF, 76–77
Hypoglycemia primary tissue culture
heart rate variability and arrhythmia risk, disadvantages, 67
21–22 high glucose, 65–67, 66f
unawareness, 19 neurotrophin-stimulated neurite
outgrowth, 64–65, 65f
I Schwann cells, 64
Immortalized cell lines, 63–64 relative replacements, 62
Induced pluripotent stem cell (iPSC), 67 Schwann cells, 77–78
Inducible/inflammatory NOS (iNOS), sensory neuron–keratinocyte coculture,
164–165 77–78, 78f
Ins2Akita mouse, 102–103 stimuli, choice of, 68–69
Intracellular Hsp70 (iHsp70)
animal models, 195–197 M
chaperone functions, 189–190 Metabolic memory, 19–20
c-jun, 195–197, 196f Microgrooves, 76–77
c-jun N-terminal kinase (JNK), 195 Mitochondrial bioenergetics, 191–192
inflammation, 194–195 Modulating Hsp70
modulating Hsp70, 197–198 beneficial and deleterious effects,
molecular chaperones, 184–185 197–198, 198f
oxidative stress BGP-15, 200–201
mitochondrial function, 191–192 C-terminal Hsp90 inhibitors, 199–200
NADPH oxidases (NOX), 190 N-terminal Hsp90 inhibitor, 199–200
SODs, 190–191 oncoproteins, 199
translocases of the outer mitochondrial Molecular chaperones
membrane (Tom), 192 eHsp70, 184–185
TXNIP, 192–194, 193f heat shock proteins, 183–184
In vitro modeling approach Hsp90 and Hsp70, 184
advantages and disadvantages, 54, 55t iHsp70, 184–185
altered bioenergetics and oxidative stress, transcription factor heat shock factor 1
70–72 (HSF1), 184
axonal degeneration and regeneration Multiple phase III clinical trials
“die-back” neuropathies, 73–74 core labs and training, 35
4-HNE, 75–76 designing, 28
immortalized cell lines, 74–75 DSP
324 Index

Multiple phase III clinical trials (Continued ) DSPN assessment, 11–13

axonal process, 28–29 EDIC study, 11
clinical neuropathy, 29–30 insulin therapy, 10–11
DCCT, 31–32 primary prevention cohort, 10
disease progression, 28–29 secondary intervention cohort, 10
identification, small fiber impairments, EDIC
30–31 additional measures, 14
IENFD, 34 CAN assessment, 15–16
large fiber structure, 33–34 DSPN assessment, 11
laser Doppler flare area imaging, 34–35 gastroparesis, 16–17
NATHAN study, 32 self-report measures, 17
NCS, 30 urologic complications, evaluation, 16
pharmacological agents, 27–28, 28t epigastric fullness, 19
prevention, 28–29 genitourinary problems, 20
quantitative sensory threshold HbA1c separation, 17–18
measurements, 34 hypoglycemia
small nerve fiber function, 34 heart rate variability and arrhythmia
type 1 and type 2 diabetes, 29 risk, 21–22
intervention, 35–36 unawareness, 19
limitation, 27–28, 28t limitations, 23
streamlined ethics and contracts metabolic memory, 19–20
process, 37 multicenter trials, 21
study conduct, 36–37 multiple phase III clinical trials, 29–30
outcomes assessments, 12f, 12t
N prevalence of, 17
Nerve conduction studies (NCS), 30 RRV, 18
Nerve conduction velocity (NCV) Neuropathy Symptom Score, 305
advantages, 231–232 NF-κB activation, 194–195
animal models, 230 Nitrergic stress, 164–165
ARI, 4 Nociceptive ion channels
DCCT and EDIC trials, 6 characteristic biophysical features,
gangliosides, 5 213–215
glycemic control, 5–6 DRG neurons, 213–215
measurement, 231–232 Nociceptive pain, 213
nerve myo-inositol depletion, 4 Nonenzymatic glycation and AGEs
polyol pathway, 3–4 clinical application, antiglycation agents,
thioctic acid, 4–5 134–135
treatment paradigms, 230–231 DPN, 132–134, 133f
Nerve growth factor (NGF) glycation of proteins, 131–132
growth factors, 302–303 transgenic and knockout mice studies,
in vitro modeling approach, 76–77 134
Neuronal hyperexcitability, 72–73 Nonobese diabetic (NOD) mouse, 103
Neuronal NOS (nNOS), 164–165
Neuropathy O
CAN, 10 Obesity, streptozotocin-diabetic rodent
confirmed clinical neuropathy, 19 high-fat fed Sprague–Dawley rats and
DCCT C57Bl6/J mice, 91–92
CAN assessment, 14–15 Zucker (fa/fa) rat, 92–93
Index 325

Ob/ob and db/db mice, 97–98 metabolic sequelae, 125–128, 126f

Otsuka long-Evans Tokushima fatty transgenic and knockout mice, 128
(OLETF) rat, 96–97 Poly (ADP-ribose) polymerase (PARP), 163
Oxidative stress Posttranslational modification
antioxidant effects, 137–138 CaV3.2 channels, 219–220
diabetic neuropathy and, 136–137 conserved extracellular asparagine
DPN, 116–117 residues, 219–220
production of, 135–136, 136f DRG, 218–219
forms of, 217–218
P neuraminidase, 220
Painful peripheral diabetic neuropathy protein glycosylation, 218
(PDN) TRPA1, 220
CaV3.2 T-type channels, 217 Primary tissue culture
diabetes mellitus, 212–213 disadvantages, 67
diminished inhibitory drive, spinal cord, high glucose, 65–67, 66f
221 neurotrophin-stimulated neurite
nociceptive ion channels outgrowth, 64–65, 65f
characteristic biophysical features, Schwann cells, 64
213–215 Protein kinase C (PKC) activity, 138–139
DRG neurons, 213–215
nociceptive pain, 213 R
posttranslational modification Receptors for AGEs (RAGE) signaling,
CaV3.2 channels, 219–220 163–164
conserved extracellular asparagine Regeneration strategy, sensory
residues, 219–220 neurodegeneration
DRG, 218–219 collateral sprouting, 170
forms of, 217–218 focal neuropathy, 170
neuraminidase, 220 impaired diabetic nerve regeneration,
protein glycosylation, 218 170–171
TRPA1, 220 phosphatase and tensin homolog deleted
prevalence, 212–213 on chromosome 10 (PTEN),
symptoms, 212–213 170–173, 172f
therapeutic strategy, 222 retinoblastoma 1 (Rb1), 173
treatment, 212–213 RhoA kinase pathway, 173
voltage-gated and ligand-gated ion RNA-induced silencing complex (RISC),
channels 165–166
DRG cells, 215 R–R variation to paced breathing
NaV1.7 and NaV1.8 isoforms, (RRV), 18
215–216
STZ-treated knockout mice, 215–216 S
TRPV1, 215–216 Schwann cells, 64, 77–78
Pancreas transplantation, 297 Sensory neurodegeneration, diabetes
PKC activation, 300 acute hyperglycemia/glucotoxicity,
Polyol (aldose reductase (AR)/ 152–153
sorbitol–fructose) pathway AGE–RAGE signaling, 164
AR inhibition (ARI), 129–130, 130f AGEs, 163–164
clinical application, 130–131 altered insulin signaling
ischemia/reperfusion injury, 131 Alzheimer’s disease, 159
326 Index

Sensory neurodegeneration, diabetes Somatosensory nervous system

(Continued ) cell and tissue types, 61–62
dermal and epidermal axons, 160 genetic control, development, 58–60, 59f
DRG, 159 large-diameter axons, 61–62
sensory neurons, receptors, 156–157, neural crest cells (NCCs), 58
158f PNS, 56–58, 57f
type 1 DM, 157–159 TrkA, 58–60
type 2 DM, 157–159 Spontaneously diabetic Torii (SDT) rats, 94
Arabidopsis thaliana miR171, 168 Spontaneously hypertensive rat, 101–102
caspase-3 expression, 162–163 Streptozotocin-diabetic rodent
distal axon targeting, 153–154 dosage of, 90–91
DPN obesity
advanced disease, 156 high-fat fed Sprague–Dawley rats and
epineurial and endoneurial alterations, C57Bl6/J mice, 91–92
156 Zucker (fa/fa) rat, 92–93
microangiopathy, 154–155 peripheral neuropathy, 91
microvascular complication, 154–155 treatment, 103–104
nerve blood flow (NBF), 155 type 1 diabetes
DRGs, 152–153 biobreeding/Worcester rat, 102
eNOS, 164–165 Ins2Akita mouse, 102–103
epidermal biopsies, 152 NOD mouse, 103
epigenetic manipulation, 169 spontaneously hypertensive rat, 101–102
iNOS, 164–165 type 2 diabetes
loss of structural protein mRNAs, 165 BBZDR/Wor rat, 96
miRNAs, 166, 167f combined high-fat fed, low-dose,
mmu-let-7i, 168–169 98–101
neurofilament, declines in, 153–154 GK rat, 95–96
nitrergic stress, 164–165 Ob/ob and db/db mice, 97–98
nNOS, 164–165 OLETF rat, 96–97
ongoing growth SDT rats, 94
C-peptide, 160–161 streptozotocin–nicotinamide rat, 101
GLP-1, 161 TSOD mouse, 98
HSP 27, 162 ZDF rats, 93
PARP, 163 ZDSD rat, 94–95
phenotype, 154 Streptozotocin–nicotinamide rat, 101
regeneration strategy Sudomotor function testing.
collateral sprouting, 170 See Sudorimetry
focal neuropathy, 170 Sudorimetry
impaired diabetic nerve regeneration, clinical diagnostic evaluation, 266–267,
170–171 267f, 268t
phosphatase and tensin homolog electrochemical skin conductance (ESC),
deleted on chromosome 10 (PTEN), 265–266
170–173, 172f gold standard, evaluation, 264
retinoblastoma 1 (Rb1), 173 neuropad, 265
RhoA kinase pathway, 173 progression and regression, disease,
RISC, 165–166 269–270, 270t
Sensory neuron–keratinocyte coculture, Sudoscan®, 265
77–78, 78f validation, ESC robustness, 267–269, 269t
Index 327

T combined high-fat fed, low-dose, 98–101

Tanezumab, 302–303 GK rat, 95–96
The Diabetes Control and Complications Ob/ob and db/db mice, 97–98
Trial (DCCT) OLETF rat, 96–97
assessment SDT rats, 94
CAN, 14–15 streptozotocin–nicotinamide rat, 101
DSPN, 11–13 TSOD mouse, 98
EDIC study, 11 ZDF rats, 93
insulin therapy, 10–11 ZDSD rat, 94–95
multiple phase III clinical trials, 31–32
NCV, 6
V
primary prevention cohort, 10
Vascular endothelial growth factor (VEGF),
secondary intervention cohort, 10
303
Transient receptor potential channel A1
Vitamin B, 299
(TRPA1), 220
Voltage-gated and ligand-gated ion channels
Transient receptor potential vanilloid 1
DRG cells, 215
(TRPV1), 215–216
NaV1.7 and NaV1.8 isoforms, 215–216
Tsumura Suzuki obese diabetes (TSOD)
STZ-treated knockout mice, 215–216
mouse, 98
TRPV1, 215–216
Type 1 diabetes
biobreeding/Worcester rat, 102
Ins2Akita mouse, 102–103 Z
NOD mouse, 103 Zucker diabetic fatty (ZDF) rats, 93
spontaneously hypertensive rat, 101–102 Zucker diabetic Sprague–Dawley (ZDSD)
Type 2 diabetes rat, 94–95
BBZDR/Wor rat, 96 Zucker (fa/fa) rat, 92–93
CONTENTS OF RECENT VOLUMES

Volume 37 Section V: Psychophysics, Psychoanalysis,

and Neuropsychology
Section I: Selectionist Ideas and Neurobiology
Phantom Limbs, Neglect Syndromes, Repressed
Selectionist and Instructionist Ideas in Memories, and Freudian Psychology
Neuroscience V. S. Ramachandran
Olaf Sporns
Neural Darwinism and a Conceptual Crisis
Population Thinking and Neuronal Selection: in Psychoanalysis
Metaphors or Concepts? Arnold H. Modell
Ernst Mayr
A New Vision of the Mind
Selection and the Origin of Information Oliver Sacks
Manfred Eigen
INDEX
Section II: Development and Neuronal
Populations
Morphoregulatory Molecules and Selectional
Dynamics during Development
Volume 38
Kathryn L. Crossin Regulation of GABAA Receptor Function and
Gene Expression in the Central Nervous System
Exploration and Selection in the Early Acquisition
A. Leslie Morrow
of Skill
Esther Thelen and Daniela Corbetta Genetics and the Organization of the Basal
Ganglia
Population Activity in the Control of Movement
Robert Hitzemann, Yeang Olan, Stephen Kanes,
Apostolos P. Georgopoulos
Katherine Dains, and Barbara Hitzemann
Section III: Functional Segregation and
Structure and Pharmacology of Vertebrate
Integration in the Brain
GABAA Receptor Subtypes
Reentry and the Problem of Cortical Integration Paul J. Whiting, Ruth M. McKernan, and Keith
Giulio Tononi A. Wafford
Coherence as an Organizing Principle of Cortical Neurotransmitter Transporters: Molecular
Functions Biology, Function, and Regulation
Wolf Singerl Beth Borowsky and Beth J. Hoffman
Temporal Mechanisms in Perception Presynaptic Excitability
Ernst P€oppel Meyer B. Jackson
Section IV: Memory and Models Monoamine Neurotransmitters in Invertebrates
and Vertebrates: An Examination of the Diverse
Selection versus Instruction: Use of Computer
Enzymatic Pathways Utilized to Synthesize and
Models to Compare Brain Theories
Inactivate Biogenic Amines
George N. Reeke, Jr.
B. D. Sloley and A. V. Juorio
Memory and Forgetting: Long-Term and Gradual
Neurotransmitter Systems in Schizophrenia
Changes in Memory Storage
Gavin P. Reynolds
Larry R. Squire
Physiology of Bergmann Glial Cells
Implicit Knowledge: New Perspectives on
Thomas Müller and Helmut Kettenmann
Unconscious Processes
Daniel L. Schacter INDEX

329
330 Contents of Recent Volumes

Volume 39 Calcium Antagonists: Their Role in

Neuroprotection
Modulation of Amino Acid-Gated Ion Channels A. Jacqueline Hunter
by Protein Phosphorylation
Stephen J. Moss and Trevor G. Smart Sodium and Potassium Channel Modulators:
Their Role in Neuroprotection
Use-Dependent Regulation of GABAA Tihomir P. Obrenovich
Receptors
Eugene M. Barnes, Jr. NMDA Antagonists: Their Role in
Neuroprotection
Synaptic Transmission and Modulation in the Danial L. Small
Neostriatum
David M. Lovinger and Elizabeth Tyler Development of the NMDA Ion-Channel
Blocker, Aptiganel Hydrochloride, as a Neuro-
The Cytoskeleton and Neurotransmitter protective Agent for Acute CNS Injury
Receptors Robert N. McBurney
Valerie J. Whatley and R. Adron Harris
The Pharmacology of AMPA Antagonists
Endogenous Opioid Regulation of Hippocampal and Their Role in Neuroprotection
Function Rammy Gill and David Lodge
Michele L. Simmons and Charles Chavkin
GABA and Neuroprotection
Molecular Neurobiology of the Cannabinoid Patrick D. Lyden
Receptor
Mary E. Abood and Billy R. Martin Adenosine and Neuroprotection
Bertil B. Fredholm
Genetic Models in the Study of Anesthetic Drug
Action Interleukins and Cerebral Ischemia
Victoria J. Simpson and Thomas E. Johnson Nancy J. Rothwell, Sarah A. Loddick, and Paul
Stroemer
Neurochemical Bases of Locomotion and Ethanol
Stimulant Effects Nitrone-Based Free Radical Traps as Neuro-
Tamara J. Phillips and Elaine H. Shen protective Agents in Cerebral Ischemia and Other
Pathologies
Effects of Ethanol on Ion Channels Kenneth Hensley, John M. Carney, Charles
Fulton T. Crews, A. Leslie Morrow, Hugh A. Stewart, Tahera Tabatabaie, Quentin Pye,
Criswell, and George Breese and Robert A. Floyd
INDEX Neurotoxic and Neuroprotective Roles of Nitric
Oxide in Cerebral Ischemia
Volume 40 Turgay Dalkara and Michael A. Moskowitz
Mechanisms of Nerve Cell Death: Apoptosis or A Review of Earlier Clinical Studies on Neuro-
Necrosis after Cerebral Ischemia protective Agents and Current Approaches
R. M. E. Chalmers-Redman, A. D. Fraser, Nils-Gunnar Wahlgren
W. Y. H. Ju, J. Wadia, N. A. Tatton, and
INDEX
W. G. Tatton
Changes in Ionic Fluxes during Cerebral Ischemia
Tibor Kristian and Bo K. Siesjo Volume 41
Techniques for Examining Neuroprotective Section I: Historical Overview
Drugs in Vitro
Rediscovery of an Early Concept
A. Richard Green and Alan J. Cross
Jeremy D. Schmahmann
Techniques for Examining Neuroprotective
Section II: Anatomic Substrates
Drugs in Vivo
Mark P. Goldberg, Uta Strasser, and Laura The Cerebrocerebellar System
L. Dugan Jeremy D. Schmahmann and Deepak N. Pandya
Contents of Recent Volumes 331

Cerebellar Output Channels Olivopontocerebellar Atrophy and Friedreich’s

Frank A. Middleton and Peter L. Strick Ataxia: Neuropsychological Consequences of
Bilateral versus Unilateral Cerebellar Lesions
Cerebellar-Hypothalamic Axis: Basic Circuits and
Therese Botez-Marquard and Mihai I. Botez
Clinical Observations
Duane E. Haines, Espen Dietrichs, Gregory Posterior Fossa Syndrome
A. Mihailoff, and E. Frank McDonald Ian F. Pollack
Section III. Physiological Observations Cerebellar Cognitive Affective Syndrome
Jeremy D. Schmahmann and Janet C. Sherman
Amelioration of Aggression: Response to
Selective Cerebellar Lesions in the Inherited Cerebellar Diseases
Rhesus Monkey Claus W. Wallesch and Claudius Bartels
Aaron J. Berman
Neuropsychological Abnormalities in Cerebellar
Autonomic and Vasomotor Regulation Syndromes—Fact or Fiction?
Donald J. Reis and Eugene V. Golanov Irene Daum and Hermann Ackermann
Associative Learning Section VI: Theoretical Considerations
Richard F. Thompson, Shaowen Bao, Lu Chen,
Cerebellar Microcomplexes
Benjamin D. Cipriano, Jeffrey S. Grethe,
Masao Ito
Jeansok J. Kim, Judith K. Thompson,
Jo Anne Tracy, Martha S. Weninger, and Control of Sensory Data Acquisition
David J. Krupa James M. Bower
Visuospatial Abilities Neural Representations of Moving Systems
Robert Lalonde Michael Paulin
Spatial Event Processing How Fibers Subserve Computing Capabilities:
Marco Molinari, Laura Petrosini, and Liliana Similarities between Brains and Machines
G. Grammaldo Henrietta C. Leiner and Alan L. Leiner
Section IV: Functional Neuroimaging Studies Cerebellar Timing Systems
Richard Ivry
Linguistic Processing
Julie A. Fiez and Marcus E. Raichle Attention Coordination and Anticipatory Control
Natacha A. Akshoomoff, Eric Courchesne, and
Sensory and Cognitive Functions
Jeanne Townsend
Lawrence M. Parsons and Peter T. Fox
Context-Response Linkage
Skill Learning
W. Thomas Thach
Julien Doyon
Duality of Cerebellar Motor and Cognitive
Section V: Clinical and Neuropsychological
Functions
Observations
James R. Bloedel and Vlastislav Bracha
Executive Function and Motor Skill Section VII: Future Directions
Learning
Mark Hallett and Jordon Grafman Therapeutic and Research Implications
Jeremy D. Schmahmann
Verbal Fluency and Agrammatism
Marco Molinari, Maria G. Leggio, and Maria
C. Silveri
Classical Conditioning Volume 42
Diana S. Woodruff-Pak Alzheimer Disease
Mark A. Smith
Early Infantile Autism
Margaret L. Bauman, Pauline A. Filipek, and Neurobiology of Stroke
Thomas L. Kemper W. Dalton Dietrich
332 Contents of Recent Volumes

Free Radicals, Calcium, and the Synaptic Vesicle Recycling at the Drosophila Neuromuscu-
Plasticity-Cell Death Continuum: Emerging lar Junction
Roles of the Trascription Factor NFκB Daniel T. Stimson and Mani Ramaswami
Mark P. Mattson
Ionic Currents in Larval Muscles of Drosophila
AP-I Transcription Factors: Short- and Long- Satpal Singh and Chun-Fang Wu
Term Modulators of Gene Expression in the Brain
Development of the Adult Neuromuscular
Keith Pennypacker
System
Ion Channels in Epilepsy Joyce J. Fernandes and Haig Keshishian
Istvan Mody
Controlling the Motor Neuron
Posttranslational Regulation of Ionotropic Gluta- James R. Trimarchi, Ping Jin, and Rodney
mate Receptors and Synaptic Plasticity K. Murphey
Xiaoning Bi, Steve Standley, and Michel Baudry
Heritable Mutations in the Glycine, GABAA, and
Nicotinic Acetylcholine Receptors Provide New
Insights into the Ligand-Gated Ion Channel
Volume 44
Receptor Superfamily Human Ego-Motion Perception
Behnaz Vafa and Peter R. Schofield A. V. van den Berg
INDEX Optic Flow and Eye Movements
M. Lappe and K.-P. Hoffman
The Role of MST Neurons during Ocular Track-
Volume 43 ing in 3D Space
K. Kawano, U. Inoue, A. Takemura, Y. Kodaka,
Early Development of the Drosophila Neuromus-
and F. A. Miles
cular Junction: A Model for Studying Neuronal
Networks in Development Visual Navigation in Flying Insects
Akira Chiba M. V. Srinivasan and S.-W. Zhang
Development of Larval Body Wall Muscles Neuronal Matched Filters for Optic Flow
Michael Bate, Matthias Landgraf, and Mar Ruiz Processing in Flying Insects
Gómez Bate H. G. Krapp
Development of Electrical Properties and Synaptic A Common Frame of Reference for the Analysis
Transmission at the Embryonic Neuromuscular of Optic Flow and Vestibular Information
Junction B. J. Frost and D. R. W. Wylie
Kendal S. Broadie
Optic Flow and the Visual Guidance of
Ultrastructural Correlates of Neuromuscular Locomotion in the Cat
Junction Development H. Sherk and G. A. Fowler
Mary B. Rheuben, Motojiro Yoshihara, and
Stages of Self-Motion Processing in Primate
Yoshiaki Kidokoro
Posterior Parietal Cortex
Assembly and Maturation of the Drosophila Larval F. Bremmer, J.-R. Duhamel, S. B. Hamed, and
Neuromuscular Junction W. Graf
L. Sian Gramates and Vivian Budnik
Optic Flow Analysis for Self-Movement
Second Messenger Systems Underlying Plasticity Perception
at the Neuromuscular Junction C. J. Duffy
Frances Hannan and Yi Zhong
Neural Mechanisms for Self-Motion Perception
Mechanisms of Neurotransmitter Release in Area MST
J. Troy Littleton, Leo Pallanck, and Barry R. A. Andersen, K. V. Shenoy, J. A. Crowell,
Ganetzky and D. C. Bradley
Contents of Recent Volumes 333

Computational Mechanisms for Optic Flow Epilepsy-Associated Plasticity in gamma-

Analysis in Primate Cortex Amniobutyric Acid Receptor Expression,
M. Lappe Function and Inhibitory Synaptic Properties
Douglas A. Coulter
Human Cortical Areas Underlying the Perception
of Optic Flow: Brain Imaging Studies Synaptic Plasticity and Secondary Epileptogenesis
M. W. Greenlee Timothy J. Teyler, Steven L. Morgan, Rebecca
N. Russell, and Brian L. Woodside
What Neurological Patients Tell Us about the Use
of Optic Flow Synaptic Plasticity in Epileptogenesis: Cel-
L. M. Vaina and S. K. Rushton lular Mechanisms Underlying Long-Lasting
Synaptic Modifications that Require New Gene
INDEX
Expression
Oswald Steward, Christopher S. Wallace, and Paul
F. Worley
Volume 45 Cellular Correlates of Behavior
Mechanisms of Brain Plasticity: From Normal Emma R. Wood, Paul A. Dudchenko, and Howard
Brain Function to Pathology Eichenbaum
Philip. A. Schwartzkroin
Mechanisms of Neuronal Conditioning
Brain Development and Generation of Brain David A. T. King, David J. Krupa, Michael
Pathologies R. Foy, and Richard F. Thompson
Gregory L. Holmes and Bridget McCabe
Plasticity in the Aging Central Nervous System
Maturation of Channels and Receptors: Conse- C. A. Barnes
quences for Excitability
Secondary Epileptogenesis, Kindling, and
David F. Owens and Arnold R. Kriegstein
Intractable Epilepsy: A Reappraisal from the Per-
Neuronal Activity and the Establishment of spective of Neuronal Plasticity
Normal and Epileptic Circuits during Brain Thomas P. Sutula
Development
Kindling and the Mirror Focus
John W. Swann, Karen L. Smith, and
Dan C. McIntyre and Michael O. Poulter
Chong L. Lee
Partial Kindling and Behavioral Pathologies
The Effects of Seizures of the Hippocampus of the
Robert E. Adamec
Immature Brain
Ellen F. Sperber and Solomon L. Moshe The Mirror Focus and Secondary Epileptogenesis
B. J. Wilder
Abnormal Development and Catastrophic
Epilepsies: The Clinical Picture and Relation to Hippocampal Lesions in Epilepsy: A Historical
Neuroimaging Review
Harry T. Chugani and Diane C. Chugani Robert Naquet
Cortical Reorganization and Seizure Generation Clinical Evidence for Secondary Epileptogensis
in Dysplastic Cortex Hans O. Luders
G. Avanzini, R. Preafico, S. Franceschetti,
Epilepsy as a Progressive (or Nonprogressive
G. Sancini, G. Battaglia, and V. Scaioli
“Benign”) Disorder
Rasmussen’s Syndrome with Particular Refer- John A. Wada
ence to Cerebral Plasticity: A Tribute to Frank
Pathophysiological Aspects of Landau-Kleffner
Morrell
Syndrome: From the Active Epileptic Phase to
Fredrick Andermann and Yuonne Hart
Recovery
Structural Reorganization of Hippocampal Marie-Noelle Metz-Lutz, Pierre Maquet, Annd De
Networks Caused by Seizure Activity Saint Martin, Gabrielle Rudolf, Norma Wioland,
Daniel H. Lowenstein Edouard Hirsch, and Chriatian Marescaux
334 Contents of Recent Volumes

Local Pathways of Seizure Propagation in Neurosteroids and Behavior

Neocortex Sharon R. Engel and Kathleen A. Grant
Barry W. Connors, David J. Pinto, and Albert
Ethanol and Neurosteroid Interactions in the
E. Telefeian
Brain
Multiple Subpial Transection: A Clinical A. Leslie Morrow, Margaret J. VanDoren, Rebekah
Assessment Fleming, and Shannon Penland
C. E. Polkey
Preclinical Development of Neurosteroids as
The Legacy of Frank Morrell Neuroprotective Agents for the Treatment of
Jerome Engel, Jr. Neurodegenerative Diseases
Paul A. Lapchak and Dalia M. Araujo
Clinical Implications of Circulating Neurosteroids
Andrea R. Genazzani, Patrizia Monteleone,
Volume 46 Massimo Stomati, Francesca Bernardi, Luigi
Cobellis, Elena Casarosa, Michele Luisi, Stefano
Neurosteroids: Beginning of the Story
Luisi, and Felice Petraglia
Etienne E. Baulieu, P. Robel, and M. Schumacher
Neuroactive Steroids and Central Nervous System
Biosynthesis of Neurosteroids and Regulation of
Disorders
Their Synthesis
Mingde Wang, Torbj€orn Bäckstr€om, Inger
Synthia H. Mellon and Hubert Vaudry
Sundstr€om, G€oran Wahlstr€om, Tommy Olsson,
Neurosteroid 7-Hydroxylation Products in the Di Zhu, Inga-Maj Johansson, Inger Bj€orn, and
Brain Marie Bixo
Robert Morfin and Luboslav Stárka
Neuroactive Steroids in Neuropsychopharma-
Neurosteroid Analysis cology
Ahmed A. Alomary, Robert L. Fitzgerald, Rainer Rupprecht and Florian Holsboer
and Robert H. Purdy
Current Perspectives on the Role of Neu-
Role of the Peripheral-Type Benzodiazepine rosteroids in PMS and Depression
Receptor in Adrenal and Brain Steroidogenesis Lisa D. Griffin, Susan C. Conrad, and Synthia
Rachel C. Brown and Vassilios Papadopoulos H. Mellon
Formation and Effects of Neuroactive Index
Steroids in the Central and Peripheral Nervous
System
Roberto Cosimo Melcangi, Valerio Magnaghi, Volume 47
Mariarita Galbiati, and Luciano Martini
Introduction: Studying Gene Expression in Neu-
Neurosteroid Modulation of Recombinant and ral Tissues by in Situ Hybridization
Synaptic GABAA Receptors W. Wisden and B. J. Morris
Jeremy J. Lambert, Sarah C. Harney, Delia Belelli,
Part I: In Situ Hybridization with Radiolabelled
and John A. Peters
Oligonucleotides
GABAA-Receptor Plasticity during Long-Term In Situ Hybridization with Oligonucleotide
Exposure to and Withdrawal from Progesterone Probes
Giovanni Biggio, Paolo Follesa, Enrico Sanna, Wl. Wisden and B. J. Morris
Robert H. Purdy, and Alessandra Concas
Cryostat Sectioning of Brains
Stress and Neuroactive Steroids Victoria Revilla and Alison Jones
Maria Luisa Barbaccia, Mariangela Serra, Robert
Processing Rodent Embryonic and Early Postnatal
H. Purdy, and Giovanni Biggio
Tissue for in Situ Hybridization with
Neurosteroids in Learning and Memory Processes Radiolabelled Oligonucleotides
Monique Vallee, Willy Mayo, George F. Koob, and David J. Laurie, Petra C. U. Schrotz,
Michel Le Moal Hannah Monyer, and Ulla Amtmann
Contents of Recent Volumes 335

Processing of Retinal Tissue for in Situ Molecular Modeling of Ligand-Gated Ion

Hybridization Channels: Progress and Challenges
Frank Müller Ed Bertaccini and James R. Trudel
Processing the Spinal Cord for in Situ Hybridiza- Alzheimer’s Disease: Its Diagnosis and
tion with Radiolabelled Oligonucleotides Pathogenesis
A. Berthele and T. R. T€olle Jillian J. Kril and Glenda M. Halliday
Processing Human Brain Tissue for in Situ DNA Arrays and Functional Genomics in
Hybridization with Radiolabelled Neurobiology
Oligonucleotides Christelle Thibault, Long Wang, Li Zhang, and
Louise F. B. Nicholson Michael F. Miles
In Situ Hybridization of Astrocytes and Neurons INDEX
Cultured in Vitro
L. A. Arizza-McNaughton, C. De Felipe, and
S. P. Hunt
Volume 49
In Situ Hybridization on Organotypic Slice
What Is West Syndrome?
Cultures
Olivier Dulac, Christine Soufflet, Catherine Chiron,
A. Gerfin-Moser and H. Monyer
and Anna Kaminski
Quantitative Analysis of in Situ Hybridization
The Relationship between encephalopathy and
Histochemistry
Abnormal Neuronal Activity in the Developing
Andrew L. Gundlach and Ross D. O’Shea
Brain
Part II: Nonradioactive in Situ hybridization Frances E. Jensen
Nonradioactive in Situ Hybridization Using Alka- Hypotheses from Functional Neuroimaging
line Phosphatase-Labelled Oligonucleotides Studies
S. J. Augood, E. M. McGowan, B. R. Finsen, Csaba Juhász, Harry T. Chugani, Ouo Muzik,
B. Heppelmann, and P. C. Emson and Diane C. Chugani
Combining Nonradioactive in Situ Hybridization Infantile Spasms: Unique Sydrome or General
with Immunohistological and Anatomical Age-Dependent Manifestation of a Diffuse
Techniques Encephalopathy?
Petra Wahle M. A. Koehn and M. Duchowny
Nonradioactive in Situ Hybridization: Simplified Histopathology of Brain Tissue from Patients with
Procedures for Use in Whole Mounts of Mouse Infantile Spasms
and Chick Embryos Harry V. Vinters
Linda Ariza-McNaughton and Robb Krumlauf
Generators of Ictal and Interictal Electroencepha-
INDEX lograms Associated with Infantile Spasms: Intra-
cellular Studies of Cortical and Thalamic Neurons
M. Steriade and I. Timofeev
Cortical and Subcortical Generators of Normal
Volume 48 and Abnormal Rhythmicity
David A. McCormick
Assembly and Intracellular Trafficking of GABAA
Receptors Eugene Role of Subcortical Structures in the Pathogenesis
Barnes of Infantile Spasms: What Are Possible Subcortical
Mediators?
Subcellular Localization and Regulation of
F. A. Lado and S. L. Moshe
GABAA Receptors and Associated Proteins
Bernhard Lüscher and Jean-Marc Fritschy D1 What Must We Know to Develop Better
Dopamine Receptors Therapies?
Richard Mailman Jean Aicardi
336 Contents of Recent Volumes

The Treatment of Infantile Spasms: An Evidence- Volume 50

Based Approach
Mark Mackay, Shelly Weiss, and O. Carter Part I: Primary Mechanisms
Snead III How Does Glucose Generate Oxidative Stress In
ACTH Treatment of Infantile Spasms: Mecha- Peripheral Nerve?
nisms of Its Effects in Modulation of Neuronal Irina G. Obrosova
Excitability
Glycation in Diabetic Neuropathy: Characteris-
K. L. Brunson, S. Avishai-Eliner, and
tics, Consequences, Causes, and Therapeutic
T. Z. Baram
Options
Neurosteroids and Infantile Spasms: The Paul J. Thornalley
Deoxycorticosterone Hypothesis
Part II: Secondary Changes
Michael A. Rogawski and Doodipala
S. Reddy Protein Kinase C Changes in Diabetes: Is the
Concept Relevant to Neuropathy?
Are there Specific Anatomical and/or Transmitter
Joseph Eichberg
Systems (Cortical or Subcortical) That Should Be
Targeted? Are Mitogen-Activated Protein Kinases
Phillip C. Jobe Glucose Transducers for Diabetic Neuropathies?
Tertia D. Purves and David R. Tomlinson
Medical versus Surgical Treatment: Which Treat-
ment When Neurofilaments in Diabetic Neuropathy
W. Donald Shields Paul Fernyhough and Robert E. Schmidt
Developmental Outcome with and without Apoptosis in Diabetic Neuropathy
Successful Intervention Aviva Tolkovsky
Rochelle Caplan, Prabha Siddarth, Gary
Nerve and Ganglion Blood Flow in Diabetes:
Mathern, Harry Vinters, Susan Curtiss,
An Appraisal
Jennifer Levitt, Robert Asarnow, and
Douglas W. Zochodne
W. Donald Shields
Part III: Manifestations
Infantile Spasms versus Myoclonus: Is There a
Connection? Potential Mechanisms of Neuropathic Pain in
Michael R. Pranzatelli Diabetes
Nigel A. Calcutt
Tuberous Sclerosis as an Underlying Basis for
Infantile Spasm Electrophysiologic Measures of Diabetic Neu-
Raymond S. Yeung ropathy: Mechanism and Meaning
Joseph C. Arezzo and Elena Zotova
Brain Malformation, Epilepsy, and Infantile
Spasms Neuropathology and Pathogenesis of Diabetic
M. Elizabeth Ross Autonomic Neuropathy
Robert E. Schmidt
Brain Maturational Aspects Relevant to Patho-
physiology of Infantile Spasms Role of the Schwann Cell in Diabetic Neuropathy
G. Auanzini, F. Panzica, and S. Franceschetti Luke Eckersley
Gene Expression Analysis as a Strategy to Under- Part IV: Potential Treatment
stand the Molecular Pathogenesis of Infantile
Polyol Pathway and Diabetic Peripheral
Spasms
Neuropathy
Peter B. Crino
Peter J. Oates
Infantile Spasms: Criteria for an Animal Model
Nerve Growth Factor for the Treatment of
Carl E. Stafstrom and Gregory
Diabetic Neuropathy: What Went Wrong, What
L. Holmes
Went Right, and What Does the Future Hold?
INDEX Stuart C. Apfel
Contents of Recent Volumes 337

Angiotensin-Converting Enzyme Inhibitors: Diabetes, the Brain, and Behavior: Is There a

Are there Credible Mechanisms for Beneficial Biological Mechanism Underlying the Association
Effects in Diabetic Neuropathy? between Diabetes and Depression?
Rayaz A. Malik and David R. Tomlinson A. M. Jacobson, J. A. Samson, K. Weinger,
and C. M. Ryan
Clinical Trials for Drugs Against Diabetic Neu-
ropathy: Can We Combine Scientific Needs With Schizophrenia and Diabetes
Clinical Practicalities? David C. Henderson and Elissa R. Ettinger
Dan Ziegler and Dieter Luft
Psychoactive Drugs Affect Glucose Transport and
INDEX the Regulation of Glucose Metabolism
Donard S. Dwyer, Timothy D. Ardizzone,
and Ronald J. Bradley

Volume 51 INDEX

Energy Metabolism in the Brain

Leif Hertz and Gerald A. Dienel
Volume 52
Neuroimmune Relationships in Perspective
The Cerebral Glucose-Fatty Acid Cycle: Evolu-
Frank Hucklebridge and Angela Clow
tionary Roots, Regulation, and (Patho) physio-
logical Importance Sympathetic Nervous System Interaction with the
Kurt Heininger Immune System
Virginia M. Sanders and Adam P. Kohm
Expression, Regulation, and Functional Role of
Glucose Transporters (GLUTs) in Brain Mechanisms by Which Cytokines Signal the Brain
Donard S. Dwyer, Susan J. Vannucci, Adrian J. Dunn
and Ian A. Simpson
Neuropeptides: Modulators of Immune
Insulin-Like Growth Factor-1 Promotes Neu- Responses in Health and Disease
ronal Glucose Utilization During Brain Develop- David S. Jessop
ment and Repair Processes
Brain–Immune Interactions in Sleep
Carolyn A. Bondy and Clara M. Cheng
Lisa Marshall and Jan Born
CNS Sensing and Regulation of Peripheral
Neuroendocrinology of Autoimmunity
Glucose Levels
Michael Harbuz
Barry E. Levin, Ambrose A. Dunn-Meynell, and
Vanessa H. Routh Systemic Stress-Induced Th2 Shift and Its Clinical
Implications
Glucose Transporter Protein Syndromes
Ibia J. Elenkov
Darryl C. De Vivo, Dong Wang, Juan M. Pascual,
and Yuan Yuan Ho Neural Control of Salivary S-IgA Secretion
Gordon B. Proctor and Guy H. Carpenter
Glucose, Stress, and Hippocampal Neuronal
Vulnerability Stress and Secretory Immunity
Lawrence P. Reagan Jos A. Bosch, Christopher Ring, Eco J. C. de Geus,
Enno C. I. Veerman, and Arie V. Nieuw
Glucose/Mitochondria in Neurological
Amerongen
Conditions
John P. Blass Cytokines and Depression
Angela Clow
Energy Utilization in the Ischemic/Reperfused
Brain Immunity and Schizophrenia: Autoimmunity,
John W. Phillis and Michael H. O’Regan Cytokines, and Immune Responses
Fiona Gaughran
Diabetes Mellitus and the Central Nervous
System Cerebral Lateralization and the Immune System
Anthony L. McCall Pierre J. Neveu
338 Contents of Recent Volumes

Behavioral Conditioning of the Immune System Section V: Neurodegenerative Disorders

Frank Hucklebridge
Parkinson’s Disease
Psychological and Neuroendocrine Correlates of L. V. P. Korlipara and A. H. V. Schapira
Disease Progression
Huntington’s Disease: The Mystery Unfolds?
Julie M. Turner-Cobb
Åsa Petersen and Patrik Brundin
The Role of Psychological Intervention in Mod-
Mitochondria in Alzheimer’s Disease
ulating Aspects of Immune Function in Relation
Russell H. Swerdlow and Stephen J. Kish
to Health and Well-Being
J. H. Gruzelier Contributions of Mitochondrial Alterations,
Resulting from Bad Genes and a Hostile Envi-
INDEX
ronment, to the Pathogenesis of Alzheimer’s Disease
Mark P. Mattson

Volume 53 Mitochondria and Amyotrophic Lateral Sclerosis

Richard W. Orrell and Anthony H. V. Schapira
Section I: Mitochondrial Structure and Function
Section VI: Models of Mitochondrial Disease
Mitochondrial DNA Structure and Function
Models of Mitochondrial Disease
Carlos T. Moraes, Sarika Srivastava, Ilias
Danae Liolitsa and Michael G. Hanna
Kirkinezos, Jose Oca-Cossio, Corina van Waveren,
Markus Woischnick, and Francisca Diaz Section VII: Defects of β Oxidation Including
Carnitine Deficiency
Oxidative Phosphorylation: Structure, Function,
and Intermediary Metabolism Defects of β Oxidation Including Carnitine
Simon J. R. Heales, Matthew E. Gegg, and John Deficiency
B. Clark K. Bartlett and M. Pourfarzam
Import of Mitochondrial Proteins Section VIII: Mitochondrial Involvement in Aging
Matthias F. Bauer, Sabine Hofmann, and Walter
The Mitochondrial Theory of Aging: Involve-
Neupert
ment of Mitochondrial DNA Damage and Repair
Section II: Primary Respiratory Chain Disorders Nadja C. de Souza-Pinto and Vilhelm A. Bohr
Mitochondrial Disorders of the Nervous System: INDEX
Clinical, Biochemical, and Molecular Genetic
Features
Volume 54
Dominic Thyagarajan and Edward Byrne Unique General Anesthetic Binding Sites Within
Distinct Conformational States of the Nicotinic
Section III: Secondary Respiratory Chain Disorders
Acetylcholine Receptor
Friedreich’s Ataxia Hugo R. Ariaas, William, R. Kem, James
J. M. Cooper and J. L. Bradley R. Truddell, and Michael P. Blanton
Wilson Disease Signaling Molecules and Receptor Transduction
C. A. Davie and A. H. V. Schapira Cascades That Regulate NMDA Receptor-
Mediated Synaptic Transmission
Hereditary Spastic Paraplegia
Suhas. A. Kotecha and John F. MacDonald
Christopher J. McDermott and Pamela J. Shaw
Behavioral Measures of Alcohol Self-Administration
Cytochrome c Oxidase Deficiency
and Intake Control: Rodent Models
Giacomo P. Comi, Sandra Strazzer, Sara Galbiati,
Herman H. Samson and Cristine L. Czachowski
and Nereo Bresolin
Dopaminergic Mouse Mutants: Investigating
Section IV: Toxin Induced Mitochondrial
the Roles of the Different Dopamine Receptor
Dysfunction
Subtypes and the Dopamine Transporter
Toxin-Induced Mitochondrial Dysfunction Shirlee Tan, Bettina Hermann, and Emiliana
Susan E. Browne and M. Flint Beal Borrelli
Contents of Recent Volumes 339

Drosophila melanogaster, A Genetic Model System Gene Therapy for Mucopolysaccharidosis

for Alcohol Research A. Bosch and J. M. Heard
Douglas J. Guarnieri and Ulrike Heberlein
INDEX
INDEX

Volume 56
Volume 55
Behavioral Mechanisms and the Neurobiology of
Section I: Virsu Vectors For Use in the Nervous Conditioned Sexual Responding
System Mark Krause
Non-Neurotropic Adenovirus: a Vector for Gene NMDA Receptors in Alcoholism
Transfer to the Brain and Gene Therapy of Neu- Paula L. Hoffman
rological Disorders
P. R. Lowenstein, D. Suwelack, J. Hu, X. Yuan, Processing and Representation of Species-Specific
M. Jimenez-Dalmaroni, S. Goverdhama, and Communication Calls in the Auditory System of
M.G. Castro Bats
George D. Pollak, Achim Klug, and Eric E. Bauer
Adeno-Associated Virus Vectors
E. Lehtonen and L. Tenenbaum Central Nervous System Control of Micturition
Gert Holstege and Leonora J. Mouton
Problems in the Use of Herpes Simplex Virus as a
Vector The Structure and Physiology of the Rat Auditory
L. T. Feldman System: An Overview
Manuel Malmierca
Lentiviral Vectors
J. Jakobsson, C. Ericson, N. Rosenquist, and Neurobiology of Cat and Human Sexual Behavior
C. Lundberg Gert Holstege and J. R. Georgiadis

Retroviral Vectors for Gene Delivery to Neural INDEX

Precursor Cells
K. Kageyama, H. Hirata, and J. Hatakeyama
Section II: Gene Therapy with Virus Vectors for Volume 57
Specific Disease of the Nervous System
Cumulative Subject Index of Volumes 1–25
The Principles of Molecular Therapies for
Glioblastoma
G. Karpati and J. Nalbatonglu
Volume 58
Oncolytic Herpes Simplex Virus
J. C. C. Hu and R. S. Coffin Cumulative Subject Index of Volumes 26–50

Recombinant Retrovirus Vectors for Treatment

of Brain Tumors
N. G. Rainov and C. M. Kramm Volume 59
Adeno-Associated Viral Vectors for Parkinson’s Loss of Spines and Neuropil
Disease Liesl B. Jones
I. Muramatsu, L. Wang, K. Ikeguchi, K-i
Schizophrenia as a Disorder of Neuroplasticity
Fujimoto, T. Okada, H. Mizukami, Y. Hanazono,
Robert E. McCullumsmith, Sarah M. Clinton, and
A. Kume, I. Nakano, and K. Ozawa
James H. Meador-Woodruff
HSV Vectors for Parkinson’s Disease
The Synaptic Pathology of Schizophrenia: Is
D. S. Latchman
Aberrant Neurodevelopment and Plasticity to
Gene Therapy for Stroke Blame?
K. Abe and W. R. Zhang Sharon L. Eastwood
340 Contents of Recent Volumes

Neurochemical Basis for an Epigenetic Vision of Oct-6 Transcription Factor

Synaptic Organization Maria Ilia
E. Costa, D. R. Grayson, M. Veldic, and
NMDA Receptor Function, Neuroplasticity, and
A. Guidotti
the Pathophysiology of Schizophrenia
Muscarinic Receptors in Schizophrenia: Is There Joseph T. Coyle and Guochuan Tsai
a Role for Synaptic Plasticity?
INDEX
Thomas J. Raedler
Serotonin and Brain Development
Monsheel S. K. Sodhi and Elaine Sanders-Bush
Volume 60
Presynaptic Proteins and Schizophrenia
Microarray Platforms: Introduction and Applica-
William G. Honer and Clint E. Young
tion to Neurobiology
Mitogen-Activated Protein Kinase Signaling Stanislav L. Karsten, Lili C. Kudo, and Daniel
Svetlana V. Kyosseva H. Geschwind
Postsynaptic Density Scaffolding Proteins at Experimental Design and Low-Level Analysis of
Excitatory Synapse and Disorders of Synaptic Microarray Data
Plasticity: Implications for Human Behavior B. M. Bolstad, F. Collin, K. M. Simpson,
Pathologies R. A. Irizarry, and T. P. Speed
Andrea de Bartolomeis and Germano Fiore
Brain Gene Expression: Genomics and Genetics
Prostaglandin-Mediated Signaling in Schizophrenia Elissa J. Chesler and Robert W. Williams
S. Smesny
DNA Microarrays and Animal Models of Learning
Mitochondria, Synaptic Plasticity, and and Memory
Schizophrenia Sebastiano Cavallaro
Dorit Ben-Shachar and Daphna Laifenfeld
Microarray Analysis of Human Nervous System
Membrane Phospholipids and Cytokine Interac- Gene Expression in Neurological Disease
tion in Schizophrenia Steven A. Greenberg
Jeffrey K. Yao and Daniel P. van Kammen
DNA Microarray Analysis of Postmortem Brain
Neurotensin, Schizophrenia, and Antipsychotic Tissue
Drug Action Károly Mirnics, Pat Levitt, and David A. Lewis
Becky Kinkead and Charles B. Nemeroff
INDEX
Schizophrenia, Vitamin D, and Brain
Development

Alan Mackay-Sim, François FEron, Darryl Eyles, Volume 61
Thomas Burne, and John McGrath
Section I: High-Throughput Technologies
Possible Contributions of Myelin and Oligoden-
Biomarker Discovery Using Molecular Profiling
drocyte Dysfunction to Schizophrenia
Approaches
Daniel G. Stewart and Kenneth L. Davis
Stephen J. Walker and Arron Xu
Brain-Derived Neurotrophic Factor and the
Proteomic Analysis of Mitochondrial Proteins
Plasticity of the Mesolimbic Dopamine Pathway
Mary F. Lopez, Simon Melov, Felicity Johnson,
Oliver Guillin, Nathalie Griffon, Jorge Diaz,
Nicole Nagulko, Eva Golenko, Scott Kuzdzal,
Bernard Le Foll, Erwan Bezard, Christian Gross,
Suzanne Ackloo, and Alvydas Mikulskis
Chris Lammers, Holger Stark, Patrick Carroll, Jean-
Charles Schwartz, and Pierre Sokoloff Section II: Proteomic Applications
S100B in Schizophrenic Psychosis NMDA Receptors, Neural Pathways, and Protein
Matthias Rothermundt, Gerald Ponath, and Volker Interaction Databases
Arolt Holger Husi
Contents of Recent Volumes 341

Dopamine Transporter Network and Pathways Neuroimaging Studies in Bipolar Children and
Rajani Maiya and R. Dayne Mayfield Adolescents
Rene L. Olvera, David C. Glahn, Sheila
Proteomic Approaches in Drug Discovery
C. Caetano, Steven R. Pliszka, and Jair C. Soares
and Development
Holly D. Soares, Stephen A. Williams, Peter Chemosensory G-Protein-Coupled Receptor
J. Snyder, Feng Gao, Tom Stiger, Christian Rohlff, Signaling in the Brain
Athula Herath, Trey Sunderland, Karen Putnam, Geoffrey E. Woodard
and W. Frost White
Disturbances of Emotion Regulation after Focal
Section III: Informatics Brain Lesions
Antoine Bechara
Proteomic Informatics
Steven Russell, William Old, Katheryn Resing, The Use of Caenorhabditis elegans in Molecular
and Lawrence Hunter Neuropharmacology
Jill C. Bettinger, Lucinda Carnell, Andrew
Section IV: Changes in the Proteome by Disease
G. Davies, and Steven L. McIntire
Proteomics Analysis in Alzheimer’s Disease: New
INDEX
Insights into Mechanisms of Neurodegeneration
D. Allan Butterfield and Debra Boyd-Kimball
Proteomics and Alcoholism
Volume 63
Frank A. Witzmann and Wendy N. Strother Mapping Neuroreceptors at work: On the Defini-
tion and Interpretation of Binding Potentials after
Proteomics Studies of Traumatic Brain Injury
20 years of Progress
Kevin K. W. Wang, Andrew Ottens,
Albert Gjedde, Dean F. Wong, Pedro Rosa-Neto,
William Haskins, Ming Cheng Liu, Firas
and Paul Cumming
Kobeissy, Nancy Denslow, SuShing Chen, and
Ronald L. Hayes Mitochondrial Dysfunction in Bipolar Disorder:
From 31P-Magnetic Resonance Spectroscopic
Influence of Huntington’s Disease on the Human
Findings to Their Molecular Mechanisms
and Mouse Proteome
Tadafumi Kato
Claus Zabel and Joachim Klose
Large-Scale Microarray Studies of Gene Expres-
Section V: Overview of the Neuroproteome
sion in Multiple Regions of the Brain in Schizo-
Proteomics—Application to the Brain phrenia and Alzeimer’s Disease
Katrin Marcus, Oliver Schmidt, Heike Schaefer, Pavel L. Katsel, Kenneth L. Davis, and Vahram
Michael Hamacher, AndrÅ van Hall, and Helmut Haroutunian
E. Meyer
Regulation of Serotonin 2C Receptor PRE-
INDEX mRNA Editing By Serotonin
Claudia Schmauss
The Dopamine Hypothesis of Drug Addiction:
Volume 62 Hypodopaminergic State
Miriam Melis, Saturnino Spiga, and Marco Diana
GABAA Receptor Structure–Function Studies:
A Reexamination in Light of New Acetylcholine Human and Animal Spongiform Encephalopa-
Receptor Structures thies are Autoimmune Diseases: A Novel Theory
Myles H. Akabas and Its supporting Evidence
Bao Ting Zhu
Dopamine Mechanisms and Cocaine Reward
Aiko Ikegami and Christine L. Duvauchelle Adenosine and Brain Function
Bertil B. Fredholm, Jiang-Fan Chen, Rodrigo
Proteolytic Dysfunction in Neurodegenerative
A. Cunha, Per Svenningsson, and Jean-Marie Vaugeois
Disorders
Kevin St. P. McNaught INDEX
342 Contents of Recent Volumes

Volume 64 Mechanistic Connections Between Glucose/

Lipid Disturbances and Weight Gain Induced by
Section I. The Cholinergic System Antipsychotic Drugs
John Smythies Donard S. Dwyer, Dallas Donohoe, Xiao-Hong
Section II. The Dopamine System Lu, and Eric J. Aamodt
John Symythies Serotonin Firing Activity as a Marker for Mood
Section III. The Norepinephrine System Disorders: Lessons from Knockout Mice
John Smythies Gabriella Gobbi

Section IV. The Adrenaline System INDEX

John Smythies
Section V. Serotonin System
John Smythies Volume 66
INDEX Brain Atlases of Normal and Diseased Populations
Arthur W. Toga and Paul M. Thompson
Neuroimaging Databases as a Resource for
Scientific Discovery
Volume 65 John Darrell Van Horn, John Wolfe, Autumn
Insulin Resistance: Causes and Consequences Agnoli, Jeffrey Woodward, Michael Schmitt, James
Zachary T. Bloomgarden Dobson, Sarene Schumacher, and Bennet Vance

Antidepressant-Induced Manic Conversion: Modeling Brain Responses

A Developmentally Informed Synthesis of the Karl J. Friston, William Penny, and Olivier David
Literature Voxel-Based Morphometric Analysis Using Shape
Christine J. Lim, James F. Leckman, Christopher Transformations
Young, and AndrEs  Martin Christos Davatzikos
Sites of Alcohol and Volatile Anesthetic Action on The Cutting Edge of f MRI and High-Field
Glycine Receptors f MRI
Ingrid A. Lobo and R. Adron Harris Dae-Shik Kim
Role of the Orbitofrontal Cortex in Rein- Quantification of White Matter Using Diffusion-
forcement Processing and Inhibitory Tensor Imaging
Control: Evidence from Functional Magnetic Hae-Jeong Park
Resonance Imaging Studies in Healthy Human
Perfusion f MRI for Functional Neuroimaging
Subjects
Geoffrey K. Aguirre, John A. Detre, and Jiongjiong
Rebecca Elliott and Bill Deakin
Wang
Common Substrates of Dysphoria in Stimulant
Functional Near-Infrared Spectroscopy: Potential
Drug Abuse and Primary Depression: Therapeutic
and Limitations in Neuroimaging Studies
Targets
Yoko Hoshi
Kate Baicy, Carrie E. Bearden, John Monterosso,
Arthur L. Brody, Andrew J. Isaacson, and Edythe Neural Modeling and Functional Brain Imaging:
D. London The Interplay Between the Data-Fitting and Sim-
ulation Approaches
The Role of cAMP Response Element–Binding
Barry Horwitz and Michael F. Glabus
Proteins in Mediating Stress-Induced Vulnerability
to Drug Abuse Combined EEG and fMRI Studies of Human
Arati Sadalge Kreibich and Julie A. Blendy Brain Function
V. Menon and S. Crottaz-Herbette
G-Protein–Coupled Receptor Deorphanizations
Yumiko Saito and Olivier Civelli INDEX
Contents of Recent Volumes 343

Volume 67 Let’s Talk Together: Memory Traces Revealed by

Cooperative Activation in the Cerebral Cortex
Distinguishing Neural Substrates of Heterogeneity Jochen Kaiser, Susanne Leiberg, and Werner
Among Anxiety Disorders Lutzenberger
Jack B. Nitschke and Wendy Heller
Human Communication Investigated With Mag-
Neuroimaging in Dementia netoencephalography: Speech, Music, and
K. P. Ebmeier, C. Donaghey, and N. J. Dougall Gestures
Prefrontal and Anterior Cingulate Contributions Thomas R. Kn€osche, Burkhard Maess, Akinori
to Volition in Depression Nakamura, and Angela D. Friederici
Jack B. Nitschke and Kristen L. Mackiewicz Combining Magnetoencephalography and Func-
Functional Imaging Research in Schizophrenia tional Magnetic Resonance Imaging
H. Tost, G. Ende, M. Ruf, F. A. Henn, and Klaus Mathiak and Andreas J. Fallgatter
A. Meyer-Lindenberg Beamformer Analysis of MEG Data
Neuroimaging in Functional Somatic Syndromes Arjan Hillebrand and Gareth R. Barnes
Patrick B. Wood Functional Connectivity Analysis in
Neuroimaging in Multiple Sclerosis Magnetoencephalography
Alireza Minagar, Eduardo Gonzalez-Toledo, James Alfons Schnitzler and Joachim Gross
Pinkston, and Stephen L. Jaffe Human Visual Processing as Revealed by
Stroke Magnetoencephalographys
Roger E. Kelley and Eduardo Gonzalez-Toledo Yoshiki Kaneoke, Shoko Watanabe, and Ryusuke
Kakigi
Functional MRI in Pediatric Neurobehavioral
Disorders A Review of Clinical Applications of
Michael Seyffert and F. Xavier Castellanos Magnetoencephalography
Andrew C. Papanicolaou, Eduardo M. Castillo,
Structural MRI and Brain Development Rebecca Billingsley-Marshall, Ekaterina Pataraia,
Paul M. Thompson, Elizabeth R. Sowell, Nitin and Panagiotis G. Simos
Gogtay, Jay N. Giedd, Christine N. Vidal, Kiralee
M. Hayashi, Alex Leow, Rob Nicolson, Judith INDEX
L. Rapoport, and Arthur W. Toga
Neuroimaging and Human Genetics
Georg Winterer, Ahmad R. Hariri, David Volume 69
Goldman, and Daniel R. Weinberger Nematode Neurons: Anatomy and Anatomical
Neuroreceptor Imaging in Psychiatry: Theory and Methods in Caenorhabditis elegans
Applications David H. Hall, Robyn Lints, and Zeynep Altun
W. Gordon Frankle, Mark Slifstein, Peter Investigations of Learning and Memory in
S. Talbot, and Marc Laruelle Caenorhabditis elegans
INDEX Andrew C. Giles, Jacqueline K. Rose, and
Catharine H. Rankin
Neural Specification and Differentiation
Volume 68 Eric Aamodt and Stephanie Aamodt
Fetal Magnetoencephalography: Viewing the Sexual Behavior of the Caenorhabditis elegans
Developing Brain In Utero Male
Hubert Preissl, Curtis L. Lowery, and Hari Eswaran Scott W. Emmons
Magnetoencephalography in Studies of Infants The Motor Circuit
and Children Stephen E. Von Stetina, Millet Treinin, and David
Minna Huotilainen M. Miller III
344 Contents of Recent Volumes

Mechanosensation in Caenorhabditis elegans Volume 71

Robert O’Hagan and Martin Chalfie
Autism: Neuropathology, Alterations of the
GABAergic System, and Animal Models
Christoph Schmitz, Imke A. J. van Kooten, Patrick
Volume 70 R. Hof, Herman van Engeland, Paul H. Patterson,
and Harry W. M. Steinbusch
Spectral Processing by the Peripheral Auditory
The Role of GABA in the Early Neuronal
System Facts and Models
Development
Enrique A. Lopez-Poveda
Marta Jelitai and Emı´lia Madarasz
Basic Psychophysics of Human Spectral
GABAergic Signaling in the Developing
Processing
Cerebellum
Brian C. J. Moore
Chitoshi Takayama
Across-Channel Spectral Processing
Insights into GABA Functions in the Developing
John H. Grose, Joseph W. Hall III, and Emily Buss
Cerebellum
Speech and Music Have Different Requirements Mo´nica L. Fiszman
for Spectral Resolution
Role of GABA in the Mechanism of the Onset of
Robert V. Shannon
Puberty in Non-Human Primates
Non-Linearities and the Representation of Ei Terasawa
Auditory Spectra
Rett Syndrome: A Rosetta Stone for Understand-
Eric D. Young, Jane J. Yu, and Lina
ing the Molecular Pathogenesis of Autism
A. J. Reiss
Janine M. LaSalle, Amber Hogart, and Karen
Spectral Processing in the Inferior Colliculus N. Thatcher
Kevin A. Davis
GABAergic Cerebellar System in Autism: A Neu-
Neural Mechanisms for Spectral Analysis in the ropathological and Developmental Perspective
Auditory Midbrain, Thalamus, and Cortex Gene J. Blatt
Monty A. Escabı´ and Heather L. Read
Reelin Glycoprotein in Autism and Schizophrenia
Spectral Processing in the Auditory Cortex S. Hossein Fatemi
Mitchell L. Sutter
Is There A Connection Between Autism,
Processing of Dynamic Spectral Properties of Prader-Willi Syndrome, Catatonia, and GABA?
Sounds Dirk M. Dhossche, Yaru Song, and
Adrian Rees and Manuel S. Malmierca Yiming Liu
Representations of Spectral Coding in the Human Alcohol, GABA Receptors, and Neuro-
Brain developmental Disorders
Deborah A. Hall, PhD Ujjwal K. Rout
Spectral Processing and Sound Source Effects of Secretin on Extracellular GABA and
Determination Other Amino Acid Concentrations in the Rat
Donal G. Sinex Hippocampus
Hans-Willi Clement, Alexander Pschibul, and
Spectral Information in Sound Localization
Eberhard Schulz
Simon Carlile, Russell Martin, and Ken McAnally
Predicted Role of Secretin and Oxytocin in the
Plasticity of Spectral Processing
Treatment of Behavioral and Developmental
Dexter R. F. Irvine and Beverly A. Wright
Disorders: Implications for Autism
Spectral Processing In Cochlear Implants Martha G. Welch and David A. Ruggiero
Colette M. McKay
Immunological Findings in Autism
INDEX Hari Har Parshad Cohly and Asit Panja
Contents of Recent Volumes 345

Correlates of Psychomotor Symptoms in Autism Shared Susceptibility Region on Chromosome 15

Laura Stoppelbein, Sara Sytsma-Jordan, and Leilani Between Autism and Catatonia
Greening Yvon C. Chagnon
GABRB3 Gene Deficient Mice: A Potential Current Trends in Behavioral Interventions for
Model of Autism Spectrum Disorder Children with Autism
Timothy M. DeLorey Dorothy Scattone and Kimberly R. Knight
The Reeler Mouse: Anatomy of a Mutant Case Reports with a Child Psychiatric Exploration
Gabriella D’Arcangelo of Catatonia, Autism, and Delirium
Jan N. M. Schieveld
Shared Chromosomal Susceptibility Regions
Between Autism and Other Mental Disorders ECT and the Youth: Catatonia in Context
Yvon C. Chagnon index Frank K. M. Zaw
INDEX Catatonia in Autistic Spectrum Disorders: A Med-
ical Treatment Algorithm
Volume 72 Max Fink, Michael A. Taylor, and Neera
Ghaziuddin
Classification Matters for Catatonia and Autism in
Children Psychological Approaches to Chronic Catatonia-
Klaus-Jürgen Neumärker Like Deterioration in Autism Spectrum Disorders
Amitta Shah and Lorna Wing
A Systematic Examination of Catatonia-Like
Clinical Pictures in Autism Spectrum Disorders Section V: Blueprints
Lorna Wing and Amitta Shah Blueprints for the Assessment, Treatment, and
Catatonia in Individuals with Autism Spectrum Future Study of Catatonia in Autism Spectrum
Disorders in Adolescence and Early Adulthood: Disorders
A Long-Term Prospective Study Dirk Marcel, Dhossche, Amitta Shah, and Lorna
Masataka Ohta, Yukiko Kano, and Yoko Nagai Wing

Are Autistic and Catatonic Regression Related? A INDEX

Few Working Hypotheses Involving GABA,
Purkinje Cell Survival, Neurogenesis, and ECT
Dirk Marcel Dhossche and Ujjwal Rout
Volume 73
Psychomotor Development and Psychopathology
Chromosome 22 Deletion Syndrome and
in Childhood
Schizophrenia
Dirk M. J. De Raeymaecker
Nigel M. Williams, Michael C. O’Donovan, and
The Importance of Catatonia and Stereotypies in Michael J. Owen
Autistic Spectrum Disorders
Characterization of Proteome of Human Cere-
Laura Stoppelbein, Leilani Greening, and Angelina
brospinal Fluid
Kakooza
Jing Xu, Jinzhi Chen, Elaine R. Peskind,
Prader–Willi Syndrome: Atypical Psychoses and Jinghua Jin, Jimmy Eng, Catherine Pan,
Motor Dysfunctions Thomas J. Montine, David R. Goodlett, and
Willem M. A. Verhoeven and Siegfried Tuinier Jing Zhang
Towards a Valid Nosography and Psychopathol- Hormonal Pathways Regulating Intermale and
ogy of Catatonia in Children and Adolescents Interfemale Aggression
David Cohen Neal G. Simon, Qianxing Mo, Shan Hu,
Carrie Garippa, and Shi-Fang Lu
Is There a Common Neuronal Basis for Autism
and Catatonia? Neuronal GAP Junctions: Expression, Function,
Dirk Marcel Dhossche, Brendan T. Carroll, and and Implications for Behavior
Tressa D. Carroll Clinton B. McCracken and David C. S. Roberts
346 Contents of Recent Volumes

Effects of Genes and Stress on the Neurobiology of Artistic Changes in Alzheimer’s Disease
Depression Sebastian J. Crutch and Martin N. Rossor
J. John Mann and Dianne Currier
Section IV: Cerebrovascular Disease
Quantitative Imaging with the Micropet Small-
Stroke in Painters
Animal Pet Tomograph
H. Bäzner and M. Hennerici
Paul Vaska, Daniel J. Rubins, David L. Alexoff,
and Wynne K. Schiffer Visuospatial Neglect in Lovis Corinth’s Self-
Portraits
Understanding Myelination through Studying its
Olaf Blanke
Evolution
Rüdiger Schweigreiter, Betty I. Roots, Art, Constructional Apraxia, and the Brain
Christine Bandtlow, and Robert M. Gould Louis Caplan
INDEX Section V: Genetic Diseases
Neurogenetics in Art
Alan E. H. Emery
Volume 74 A Naı̈ve Artist of St Ives
Evolutionary Neurobiology and Art F. Clifford Rose
C. U. M. Smith
Van Gogh’s Madness
Section I: Visual Aspects F. Clifford Rose
Perceptual Portraits Absinthe, The Nervous System and Painting
Nicholas Wade Tiina Rekand
The Neuropsychology of Visual Art: Conferring Section VI: Neurologists as Artists
Capacity
Anjan Chatterjee Sir Charles Bell, KGH, FRS, FRSE
(1774–1842)
Vision, Illusions, and Reality Christopher Gardner-Thorpe
Christopher Kennard
Section VII: Miscellaneous
Localization in the Visual Brain
Peg Leg Frieda
George K. York
Espen Dietrichs
Section II: Episodic Disorders
The Deafness of Goya (1746–1828)
Neurology, Synaesthesia, and Painting F. Clifford Rose
Amy Ione
INDEX
Fainting in Classical Art
Philip Smith
Migraine Art in the Internet: A Study of 450
Contemporary Artists
Klaus Podoll
Volume 75
Introduction on the Use of the Drosophila Embry-
Sarah Raphael’s Migraine with Aura as Inspiration
onic/Larval Neuromuscular Junction as a Model
for the Foray of Her Work into Abstraction
System to Study Synapse Development and
Klaus Podoll and Debbie Ayles
Function, and a Brief Summary of Pathfinding
The Visual Art of Contemporary Artists with and Target Recognition
Epilepsy Catalina Ruiz-Cañada and Vivian Budnik
Steven C. Schachter
Development and Structure of Motoneurons
Section III: Brain Damage Matthias Landgraf and Stefan Thor
Creativity in Painting and Style in Brain- The Development of the Drosophila Larval Body
Damaged Artists Wall Muscles
Julien Bogousslavsky Karen Beckett and Mary K. Baylies
Contents of Recent Volumes 347

Organization of the Efferent System and Structure ID, Ego, and Temporal Lobe Revisited
of Neuromuscular Junctions in Drosophila Shirley M. Ferguson and Mark Rayport
Andreas Prokop
Section II: Stereotaxic Studies
Development of Motoneuron Electrical Proper-
Olfactory Gustatory Responses Evoked by
ties and Motor Output
Electrical Stimulation of Amygdalar Region in
Richard A. Baines
Man Are Qualitatively Modifiable by Interview
Transmitter Release at the Neuromuscular Junction Content: Case Report and Review
Thomas L. Schwarz Mark Rayport, Sepehr Sani, and Shirley M. Ferguson
Vesicle Trafficking and Recycling at the Neuro- Section III: Controversy in Definition of Behav-
muscular Junction: Two Pathways for Endocytosis ioral Disturbance
Yoshiaki Kidokoro
Pathogenesis of Psychosis in Epilepsy. The
Glutamate Receptors at the Drosophila Neuromus- “Seesaw” Theory: Myth or Reality?
cular Junction Shirley M. Ferguson and Mark Rayport
Aaron DiAntonio
Section IV: Outcome of Temporal Lobectomy
Scaffolding Proteins at the Drosophila Neuromus-
Memory Function After Temporal Lobectomy for
cular Junction
Seizure Control: A Comparative Neuropsy chi-
Bulent Ataman, Vivian Budnik, and Ulrich Thomas
atric and Neuropsychological Study
Synaptic Cytoskeleton at the Neuromuscular Shirley M. Ferguson, A. John McSweeny, and Mark
Junction Rayport
Catalina Ruiz-Cañada and Vivian Budnik
Life After Surgery for Temporolimbic Seizures
Plasticity and Second Messengers During Synapse Shirley M. Ferguson, Mark Rayport, and Carolyn
Development A. Schell
Leslie C. Griffith and Vivian Budnik
Appendix I
Retrograde Signaling that Regulates Synaptic Mark Rayport
Development and Function at the Drosophila Neu-
Appendix II: Conceptual Foundations of Studies
romuscular Junction
of Patients Undergoing Temporal Lobe Surgery
Guillermo Marques and Bing Zhang
for Seizure Control
Activity-Dependent Regulation of Transcription Mark Rayport
During Development of Synapses
INDEX
Subhabrata Sanyal and Mani Ramaswami
Experience-Dependent Potentiation of Larval
Neuromuscular Synapses
Christoph M. Schuster
Volume 77
Regenerating the Brain
Selected Methods for the Anatomical Study of
David A. Greenberg and Kunlin Jin
Drosophila Embryonic and Larval Neuromuscular
Junctions Serotonin and Brain: Evolution, Neuroplasticity,
Vivian Budnik, Michael Gorczyca, and Andreas and Homeostasis
Prokop Efrain C. Azmitia
INDEX
Therapeutic Approaches to Promoting Axonal
Regeneration in the Adult Mammalian Spinal Cord
Volume 76 Sari S. Hannila, Mustafa M. Siddiq, and Marie
T. Filbin
Section I: Physiological Correlates of Freud’s
Evidence for Neuroprotective Effects of Antipsy-
Theories
chotic Drugs: Implications for the Pathophysio-
The ID, the Ego, and the Temporal Lobe logy and Treatment of Schizophrenia
Shirley M. Ferguson and Mark Rayport Xin-Min Li and Haiyun Xu
348 Contents of Recent Volumes

Neurogenesis and Neuroenhancement in the Patho- Schizophrenia and the α7 Nicotinic Acetylcholine
physiology and Treatment of Bipolar Disorder Receptor
Robert J. Schloesser, Guang Chen, and Husseini Laura F. Martin and Robert Freedman
K. Manji
Histamine and Schizophrenia
Neuroreplacement, Growth Factor, and Small Jean-Michel Arrang
Molecule Neurotrophic Approaches for Treating
Cannabinoids and Psychosis
Parkinson’s Disease
Deepak Cyril D’Souza
Michael J. O’Neill, Marcus J. Messenger, Viktor
Lakics, Tracey K. Murray, Eric H. Karran, Philip Involvement of Neuropeptide Systems in Schizo-
G. Szekeres, Eric S. Nisenbaum, and Kalpana phrenia: Human Studies
M. Merchant Ricardo Cáceda, Becky Kinkead, and Charles
B. Nemeroff
Using Caenorhabditis elegans Models of Neuro-
degenerative Disease to Identify Neuroprotective Brain-Derived Neurotrophic Factor in Schizo-
Strategies phrenia and Its Relation with Dopamine
Brian Kraemer and Gerard D. Schellenberg Olivier Guillin, Caroline Demily, and Florence
Thibaut
Neuroprotection and Enhancement of Neurite
Outgrowth With Small Molecular Weight Com- Schizophrenia Susceptibility Genes: In Search of a
pounds From Screens of Chemical Libraries Molecular Logic and Novel Drug Targets for a
Donard S. Dwyer and Addie Dickson Devastating Disorder
Joseph A. Gogos
INDEX
INDEX

Volume 78
Neurobiology of Dopamine in Schizophrenia
Olivier Guillin, Anissa Abi-Dargham, and Marc Volume 79
Laruelle
The Destructive Alliance: Interactions of
The Dopamine System and the Pathophysiology Leukocytes, Cerebral Endothelial Cells, and the
of Schizophrenia: A Basic Science Perspective Immune Cascade in Pathogenesis of Multiple
Yukiori Goto and Anthony A. Grace Sclerosis
Alireza Minagar, April Carpenter, and J. Steven
Glutamate and Schizophrenia: Phencyclidine,
Alexander
N-methyl-D-aspartate Receptors, and Dopamine–
Glutamate Interactions Role of B Cells in Pathogenesis of Multiple
Daniel C. Javitt Sclerosis
Behrouz Nikbin, Mandana Mohyeddin Bonab,
Deciphering the Disease Process of Schizophrenia:
Farideh Khosravi, and Fatemeh Talebian
The Contribution of Cortical GABA Neurons
David A. Lewis and Takanori Hashimoto The Role of CD4 T Cells in the Pathogenesis of
Multiple Sclerosis
Alterations of Serotonin Transmission in
Tanuja Chitnis
Schizophrenia
Anissa Abi-Dargham The CD8 T Cell in Multiple Sclerosis: Suppressor
Cell or Mediator of Neuropathology?
Serotonin and Dopamine Interactions in Rodents
Aaron J. Johnson, Georgette L. Suidan, Jeremiah
and Primates: Implications for Psychosis and Anti-
McDole, and Istvan Pirko
psychotic Drug Development
Gerard J. Marek Immunopathogenesis of Multiple Sclerosis
Smriti M. Agrawal and V. Wee Yong
Cholinergic Circuits and Signaling in the Patho-
physiology of Schizophrenia Molecular Mimicry in Multiple Sclerosis
Joshua A. Berman, David A. Talmage, and Lorna Jane E. Libbey, Lori L. McCoy, and Robert
W. Role S. Fujinami
Contents of Recent Volumes 349

Molecular “Negativity” May Underlie Multiple Detection of Cortical Lesions Is Dependent on

Sclerosis: Role of the Myelin Basic Protein Family Choice of Slice Thickness in Patients with
in the Pathogenesis of MS Multiple Sclerosis
Abdiwahab A. Musse and George Harauz Ondrej Dolezal, Michael G. Dwyer, Dana
Horakova, Eva Havrdova, Alireza Minagar, Srivats
Microchimerism and Stem Cell Transplantation in
Balachandran, Niels Bergsland, Zdenek Seidl,
Multiple Sclerosis
Manuela Vaneckova, David Fritz, Jan Krasensky,
Behrouz Nikbin, Mandana Mohyeddin Bonab, and
and Robert Zivadinov
Fatemeh Talebian
The Role of Quantitative Neuroimaging
The Insulin-Like Growth Factor System in
Indices in the Differentiation of Ischemia from
Multiple Sclerosis
Demyelination: An Analytical Study with Case
Daniel Chesik, Nadine Wilczak, and Jacques De
Presentation
Keyser
Romy Hoque, Christina Ledbetter, Eduardo
Cell-Derived Microparticles and Exosomes in Gonzalez-Toledo, Vivek Misra, Uma Menon,
Neuroinflammatory Disorders Meghan Kenner, Alejandro A. Rabinstein,
Lawrence L. Horstman, Wenche Jy, Roger E. Kelley, Robert Zivadinov, and
Alireza Minagar, Carlos J. Bidot, Alireza Minagar
Joaquin J. Jimenez, J. Steven Alexander,
and Yeon S. Ahn HLA-DRB1*1501, -DQB1*0301, -DQB1*0302,
-DQB1*0602, and -DQB1*0603 Alleles Are
Multiple Sclerosis in Children: Clinical, Diagnos- Associated with More Severe Disease
tic, and Therapeutic Aspects Outcome on MRI in Patients with Multiple
Kevin Rostásy Sclerosis
Robert Zivadinov, Laura Uxa, Alessio Bratina,
Migraine in Multiple Sclerosis
Antonio Bosco, Bhooma Srinivasaraghavan, Alireza
Debra G. Elliott
Minagar, Maja Ukmar, Su yen Benedetto, and
Multiple Sclerosis as a Painful Disease Marino Zorzon
Meghan Kenner, Uma Menon, and
Glatiramer Acetate: Mechanisms of Action in
Debra Elliott
Multiple Sclerosis
Multiple Sclerosis and Behavior Tjalf Ziemssen and Wiebke Schrempf
James B. Pinkston, Anita Kablinger, and Nadejda
Alekseeva Evolving Therapies for Multiple Sclerosis
Elena Korniychuk, John M. Dempster,
Cerebrospinal Fluid Analysis in Multiple Eileen O’Connor, J. Steven Alexander, Roger
Sclerosis E. Kelley, Meghan Kenner, Uma Menon, Vivek
Francisco A. Luque and Stephen L. Jaffe Misra, Romy Hoque, Eduardo C. Gonzalez-
Toledo, Robert N. Schwendimann, Stacy Smith,
Multiple Sclerosis in Isfahan, Iran
and Alireza Minagar
Mohammad Saadatnia, Masoud Etemadifar,
and Amir Hadi Maghzi Remyelination in Multiple Sclerosis
Divya M. Chari
Gender Issues in Multiple Sclerosis
Robert N. Schwendimann and Nadejda Trigeminal Neuralgia: A Modern-Day Review
Alekseeva Kelly Hunt and Ravish Patwardhan
Differential Diagnosis of Multiple Sclerosis Optic Neuritis and the Neuro-Ophthalmology of
Halim Fadil, Roger E. Kelley, and Eduardo Multiple Sclerosis
Gonzalez-Toledo Paramjit Kaur and Jeffrey L. Bennett
Prognostic Factors in Multiple Sclerosis Neuromyelitis Optica: New Findings on
Roberto Bergamaschi Pathogenesis
Dean M. Wingerchuk
Neuroimaging in Multiple Sclerosis
Robert Zivadinov and Jennifer L. Cox INDEX
350 Contents of Recent Volumes

Volume 80 Manuela Vaneckova, David Fritz, Jan Krasensky,

and Robert Zivadinov
Epilepsy in the Elderly: Scope of the Problem
Ilo E. Leppik The Role of Quantitative Neuroimaging
Indices in the Differentiation of Ischemia from
Animal Models in Gerontology Research
Demyelination: An Analytical Study with Case
Nancy L. Nadon
Presentation
Animal Models of Geriatric Epilepsy Romy Hoque, Christina Ledbetter, Eduardo
Lauren J. Murphree, Lynn M. Rundhaugen, and Gonzalez-Toledo, Vivek Misra, Uma Menon,
Kevin M. Kelly Meghan Kenner, Alejandro A. Rabinstein, Roger
Life and Death of Neurons in the Aging E. Kelley, Robert Zivadinov, and Alireza Minagar
Cerebral Cortex HLA-DRB1*1501, -DQB1*0301,-DQB1
John H. Morrison and Patrick R. Hof *0302,-DQB1*0602, and -DQB1*0603 Alleles
An In Vitro Model of Stroke-Induced Epilepsy: Are Associated with More Severe Disease Out-
Elucidation of the Roles of Glutamate and come on MRI in Patients with Multiple Sclerosis
Calcium in the Induction and Maintenance of Robert Zivadinov, Laura Uxa, Alessio Bratina,
Stroke-Induced Epileptogenesis Antonio Bosco, Bhooma Srinivasaraghavan,
Robert J. DeLorenzo, David A. Sun, Robert E. Alireza Minagar, Maja Ukmar, Su yen Benedetto,
Blair, and Sompong Sambati and Marino Zorzon

Mechanisms of Action of Antiepileptic Drugs Glatiramer Acetate: Mechanisms of Action in

H. Steve White, Misty D. Smith, and Karen Multiple Sclerosis
S. Wilcox Tjalf Ziemssen and Wiebke Schrempf
Epidemiology and Outcomes of Status Epilepticus Evolving Therapies for Multiple Sclerosis
in the Elderly Elena Korniychuk, John M. Dempster,
Alan R. Towne Eileen O’Connor, J. Steven Alexander,
Roger E. Kelley, Meghan Kenner, Uma Menon,
Diagnosing Epilepsy in the Elderly
Vivek Misra, Romy Hoque, Eduardo C. Gonzalez-
R. Eugene Ramsay, Flavia M. Macias, and
Toledo, Robert N. Schwendimann, Stacy Smith,
A. James Rowan
and Alireza Minagar
Pharmacoepidemiology in Community-Dwelling
Remyelination in Multiple Sclerosis
Elderly Taking Antiepileptic Drugs
Divya M. Chari
Dan R. Berlowitz and Mary Jo V. Pugh
Trigeminal Neuralgia: A Modern-Day Review
Use of Antiepileptic Medications in Nursing Homes
Kelly Hunt and Ravish Patwardhan
Judith Garrard, Susan L. Harms, Lynn E. Eberly,
and Ilo E. Leppik Optic Neuritis and the Neuro-Ophthalmology of
Multiple Sclerosis
Differential Diagnosis of Multiple Sclerosis
Paramjit Kaur and Jeffrey L. Bennett
Halim Fadil, Roger E. Kelley, and Eduardo
Gonzalez-Toledo Neuromyelitis Optica: New Findings on
Pathogenesis
Prognostic Factors in Multiple Sclerosis
Dean M. Wingerchuk
Roberto Bergamaschi
INDEX
Neuroimaging in Multiple Sclerosis
Robert Zivadinov and Jennifer L. Cox
Detection of Cortical Lesions Is Dependent on Volume 81
Choice of Slice Thickness in Patients with
Epilepsy in the Elderly: Scope of the Problem
Multiple Sclerosis
Ilo E. Leppik
Ondrej Dolezal, Michael G. Dwyer, Dana
Horakova, Eva Havrdova, Alireza Minagar, Srivats Animal Models in Gerontology Research
Balachandran, Niels Bergsland, Zdenek Seidl, Nancy L. Nadon
Contents of Recent Volumes 351

Animal Models of Geriatric Epilepsy Outcomes in Elderly Patients With Newly

Lauren J. Murphree, Lynn M. Rundhaugen, Diagnosed and Treated Epilepsy
and Kevin M. Kelly Martin J. Brodie and Linda J. Stephen

Life and Death of Neurons in the Aging Recruitment and Retention in Clinical Trials of
Cerebral Cortex the Elderly
John H. Morrison and Patrick R. Hof Flavia M. Macias, R. Eugene Ramsay, and
A. James Rowan
An In Vitro Model of Stroke-Induced Epilepsy:
Elucidation of the Roles of Glutamate and Treatment of Convulsive Status Epilepticus
Calcium in the Induction and Maintenance of David M. Treiman
Stroke-Induced Epileptogenesis Treatment of Nonconvulsive Status Epilepticus
Robert J. DeLorenzo, David A. Sun, Robert Matthew C. Walker
E. Blair, and Sompong Sambati
Antiepileptic Drug Formulation and Treatment
Mechanisms of Action of Antiepileptic Drugs in the Elderly: Biopharmaceutical Considerations
H. Steve White, Misty D. Smith, and Barry E. Gidal
Karen S. Wilcox
INDEX
Epidemiology and Outcomes of Status Epilepticus
in the Elderly
Alan R. Towne

Diagnosing Epilepsy in the Elderly Volume 82

R. Eugene Ramsay, Flavia M. Macias, Inflammatory Mediators Leading to Protein Mis-
and A. James Rowan folding and Uncompetitive/Fast Off-Rate Drug
Pharmacoepidemiology in Community-Dwelling Therapy for Neurodegenerative Disorders
Elderly Taking Antiepileptic Drugs Stuart A. Lipton, Zezong Gu, and Tomohiro
Dan R. Berlowitz and Mary Jo V. Pugh Nakamura

Use of Antiepileptic Medications in Nursing Innate Immunity and Protective Neu-

Homes roinflammation: New Emphasis on the Role of
Judith Garrard, Susan L. Harms, Lynn E. Eberly, Neuroimmune Regulatory Proteins
and Ilo E. Leppik M. Griffiths, J. W. Neal, and P. Gasque
Glutamate Release from Astrocytes in Physiolog-
Age-Related Changes in Pharmacokinetics:
ical Conditions and in Neurodegenerative Disor-
Predictability and Assessment Methods
ders Characterized by Neuroinflammation
Emilio Perucca
Sabino Vesce, Daniela Rossi, Liliana Brambilla, and
Factors Affecting Antiepileptic Drug Pharmacoki- Andrea Volterra
netics in Community-Dwelling Elderly
The High-Mobility Group Box 1 Cytokine
James C. Cloyd, Susan Marino,
Induces Transporter-Mediated Release of
and Angela K. Birnbaum
Glutamate from Glial Subcellular Particles
Pharmacokinetics of Antiepileptic Drugs in Elderly (Gliosomes) Prepared from In Situ-Matured
Nursing Home Residents Astrocytes
Angela K. Birnbaum Giambattista Bonanno, Luca Raiteri, Marco
Milanese, Simona Zappettini, Edon Melloni,
The Impact of Epilepsy on Older Veterans Marco Pedrazzi, Mario Passalacqua, Carlo
Mary Jo V. Pugh, Dan R. Berlowitz, and Tacchetti, Cesare Usai, and Bianca Sparatore
Lewis Kazis
The Role of Astrocytes and Complement System
Risk and Predictability of Drug Interactions in the in Neural Plasticity
Elderly Milos Pekny, Ulrika Wilhelmsson, Yalda
Rene H. Levy and Carol Collins Rahpeymai Bogestål, and Marcela Pekna
352 Contents of Recent Volumes

New Insights into the Roles of Metalloproteinases Differential Modulation of Type 1 and Type 2
in Neurodegeneration and Neuroprotection Cannabinoid Receptors Along the Neuroimmune
A. J. Turner and N. N. Nalivaeva Axis
Sergio Oddi, Paola Spagnuolo, Monica Bari,
Relevance of High-Mobility Group Protein Antonella D’Agostino, and Mauro Maccarrone
Box 1 to Neurodegeneration
Silvia Fossati and Alberto Chiarugi Effects of the HIV-1 Viral Protein Tat on Central
Neurotransmission: Role of Group I Meta-
Early Upregulation of Matrix Metalloproteinases botropic Glutamate Receptors
Following Reperfusion Triggers Neuro-
Elisa Neri, Veronica Musante, and Anna Pittaluga
inflammatory Mediators in Brain Ischemia in Rat
Diana Amantea, Rossella Russo, Micaela Gliozzi, Evidence to Implicate Early Modulation of Inter-
Vincenza Fratto, Laura Berliocchi, G. Bagetta, leukin-1β Expression in the Neuroprotection
G. Bernardi, and M. Tiziana Corasaniti Afforded by 17β-Estradiol in Male Rats Under-
gone Transient Middle Cerebral Artery Occlusion
The (Endo)Cannabinoid System in Multiple Olga Chiappetta, Micaela Gliozzi, Elisa Siviglia,
Sclerosis and Amyotrophic Lateral Sclerosis
Diana Amantea, Luigi A. Morrone, Laura
Diego Centonze, Silvia Rossi, Alessandro Berliocchi, G. Bagetta, and M. Tiziana Corasaniti
Finazzi-Agrò, Giorgio Bernardi, and Mauro
Maccarrone A Role for Brain Cyclooxygenase-2 and Prosta-
glandin-E2 in Migraine: Effects of Nitroglycerin
Chemokines and Chemokine Receptors: Multi- Cristina Tassorelli, Rosaria Greco, Marie Therese
purpose Players in Neuroinflammation
Armentero, Fabio Blandini, Giorgio Sandrini, and
Richard M. Ransohoff, LiPing Liu, and Astrid Giuseppe Nappi
E. Cardona
The Blockade of K+-ATP Channels has Neuro-
Systemic and Acquired Immune Responses in protective Effects in an In Vitro Model of Brain
Alzheimer’s Disease Ischemia
Markus Britschgi and Tony Wyss-Coray
Robert Nisticò, Silvia Piccirilli, L. Sebastianelli,
Neuroinflammation in Alzheimer’s Disease and Giuseppe Nisticò, G. Bernardi, and N. B. Mercuri
Parkinson’s Disease: Are Microglia Pathogenic
Retinal Damage Caused by High Intraocular
in Either Disorder?
Pressure-Induced Transient Ischemia is Prevented
Joseph Rogers, Diego Mastroeni, Brian Leonard,
by Coenzyme Q10 in Rat
Jeffrey Joyce, and Andrew Grover
Carlo Nucci, Rosanna Tartaglione, Angelica
Cytokines and Neuronal Ion Channels in Health Cerulli, R. Mancino, A. Spanò, Federica Cavaliere,
and Disease Laura Rombolà, G. Bagetta, M. Tiziana
Barbara Viviani, Fabrizio Gardoni, and Marina Corasaniti, and Luigi A. Morrone
Marinovich
Evidence Implicating Matrix Metalloproteinases
Cyclooxygenase-2, Prostaglandin E2, and Micro- in the Mechanism Underlying Accumulation of
glial Activation in Prion Diseases IL-1β and Neuronal Apoptosis in the Neocortex
Luisa Minghetti and Maurizio Pocchiari of HIV/gp120-Exposed Rats
Rossella Russo, Elisa Siviglia, Micaela Gliozzi,
Glia Proinflammatory Cytokine Upregulation as a
Diana Amantea, Annamaria Paoletti,
Therapeutic Target for Neurodegenerative
Laura Berliocchi, G. Bagetta, and
Diseases: Function-Based and Target-Based
M. Tiziana Corasaniti
Discovery Approaches
Linda J. Van Eldik, Wendy L. Thompson, Neuroprotective Effect of Nitroglycerin in a
Hantamalala Ralay Ranaivo, Heather A. Behanna, Rodent Model of Ischemic Stroke: Evaluation
and D. Martin Watterson of Bcl-2 Expression
Rosaria Greco, Diana Amantea, Fabio Blandini,
Oxidative Stress and the Pathogenesis of Neuro-
Giuseppe Nappi, Giacinto Bagetta, M. Tiziana
degenerative Disorders
Corasaniti, and Cristina Tassorelli
Ashley Reynolds, Chad Laurie, R. Lee Mosley, and
Howard E. Gendelman INDEX
Contents of Recent Volumes 353

Volume 83 Seizures in Pregnancy: Diagnosis and

Management
Gender Differences in Pharmacological Response Robert L. Beach and Peter W. Kaplan
Gail D. Anderson
Management of Epilepsy and Pregnancy:
Epidemiology and Classification of Epilepsy: An Obstetrical Perspective
Gender Comparisons Julian N. Robinson and Jane Cleary-Goldman
John C. McHugh and Norman Delanty
Pregnancy Registries: Strengths, Weaknesses, and
Hormonal Influences on Seizures: Basic Bias Interpretation of Pregnancy Registry Data
Neurobiology Marianne Cunnington and John Messenheimer
Cheryl A. Frye
Bone Health in Women With Epilepsy: Clinical
Catamenial Epilepsy Features and Potential Mechanisms
Patricia E. Penovich and Sandra Helmers Alison M. Pack and Thaddeus S. Walczak
Epilepsy in Women: Special Considerations for Metabolic Effects of AEDs: Impact on Body
Adolescents Weight, Lipids and Glucose Metabolism
Mary L. Zupanc and Sheryl Haut Raj D. Sheth and Georgia Montouris
Contraception in Women with Epilepsy: Pharma- Psychiatric Comorbidities in Epilepsy
cokinetic Interactions, Contraceptive Options, W. Curt Lafrance, Jr., Andres M. Kanner, and
and Management Bruce Hermann
Caryn Dutton and Nancy Foldvary-Schaefer
Issues for Mature Women with Epilepsy
Reproductive Dysfunction in Women with Epi- Cynthia L. Harden
lepsy: Menstrual Cycle Abnormalities, Fertility,
and Polycystic Ovary Syndrome Pharmacodynamic and Pharmacokinetic Interac-
Jürgen Bauer and Deirdre Cooper-Mahkorn tions of Psychotropic Drugs with Antiepileptic
Drugs
Sexual Dysfunction in Women with Epilepsy: Andres M. Kanner and Barry E. Gidal
Role of Antiepileptic Drugs and Psychotropic
Medications Health Disparities in Epilepsy: How Patient-
Mary A. Gutierrez, Romila Mushtaq, and Glen Oriented Outcomes in Women Differ from Men
Stimmel Frank Gilliam

Pregnancy in Epilepsy: Issues of Concern INDEX

John DeToledo
Teratogenicity and Antiepileptic Drugs: Potential
Mechanisms Volume 84
Mark S. Yerby
Normal Brain Aging: Clinical, Immunological,
Antiepileptic Drug Teratogenesis: What are the Neuropsychological, and Neuroimaging Features
Risks for Congenital Malformations and Adverse Maria T. Caserta, Yvonne Bannon, Francisco
Cognitive Outcomes? Fernandez, Brian Giunta, Mike R. Schoenberg,
Cynthia L. Harden and Jun Tan
Teratogenicity of Antiepileptic Drugs: Role of Subcortical Ischemic Cerebrovascular Dementia
Pharmacogenomics Uma Menon and Roger E. Kelley
Raman Sankar and Jason T. Lerner
Cerebrovascular and Cardiovascular Pathology in
Antiepileptic Drug Therapy in Pregnancy I: Alzheimer’s Disease
Gestation-InducedEffectsonAEDPharmacokinetics Jack C. de la Torre
Page B. Pennell and Collin A. Hovinga
Neuroimaging of Cognitive Impairments in
Antiepileptic Drug Therapy in Pregnancy II: Fetal Vascular Disease
and Neonatal Exposure Carol Di Perri, Turi O. Dalaker, Mona K. Beyer,
Collin A. Hovinga and Page B. Pennell and Robert Zivadinov
354 Contents of Recent Volumes

Contributions of Neuropsychology and Neuro- GluK1 Receptor Antagonists and Hippocampal

imaging to Understanding Clinical Subtypes Mossy Fiber Function
of Mild Cognitive Impairment Robert Nisticò, Sheila Dargan, Stephen
Amy J. Jak, Katherine J. Bangen, Christina M. Fitzjohn, David Lodge, David E. Jane, Graham
E. Wierenga, Lisa Delano-Wood, Jody Corey- L. Collingridge, and Zuner A. Bortolotto
Bloom, and Mark W. Bondi
Monoamine Transporter as a Target Molecule
Proton Magnetic Resonance Spectroscopy in for Psychostimulants
Dementias and Mild Cognitive Impairment Ichiro Sora, BingJin Li, Setsu Fumushima, Asami
H. Randall Griffith, Christopher C. Stewart, Fukui, Yosefu Arime, Yoshiyuki Kasahara, Hiroaki
and Jan A. den Hollander Tomita, and Kazutaka Ikeda
Application of PET Imaging to Diagnosis Targeted Lipidomics as a Tool to Investigate
of Alzheimer’s Disease and Mild Cognitive Endocannabinoid Function
Impairment Giuseppe Astarita, Jennifer Geaga, Faizy Ahmed,
James M. Noble and Nikolaos Scarmeas and Daniele Piomelli
The Molecular and Cellular Pathogenesis The Endocannabinoid System as a Target for
of Dementia of the Alzheimer’s Type: An Novel Anxiolytic and Antidepressant Drugs
Overview Silvana Gaetani, Pasqua Dipasquale, Adele
Francisco A. Luque and Stephen L. Jaffe Romano, Laura Righetti, Tommaso Cassano,
Alzheimer’s Disease Genetics: Current Status and Daniele Piomelli, and Vincenzo Cuomo
Future Perspectives
GABAA Receptor Function and Gene Expression
Lars Bertram
During Pregnancy and Postpartum
Frontotemporal Lobar Degeneration: Insights Giovanni Biggio, Maria Cristina Mostallino, Paolo
from Neuropsychology and Neuroimaging Follesa, Alessandra Concas, and Enrico Sanna
Andrea C. Bozoki and Muhammad U. Farooq
Early Postnatal Stress and Neural Circuit Underly-
Lewy Body Dementia ing Emotional Regulation
Jennifer C. Hanson and Carol F. Lippa Machiko Matsumoto, Mitsuhiro Yoshioka,
and Hiroko Togashi
Dementia in Parkinson’s Disease
Bradley J. Robottom and William J. Weiner Roles of the Histaminergic Neurotransmission
Early Onset Dementia on Methamphetamine-Induced Locomotor Sen-
Halim Fadil, Aimee Borazanci, Elhachmia Ait Ben sitization and Reward: A Study of Receptors
Haddou, Mohamed Yahyaoui, Elena Korniychuk, Gene Knockout Mice
Stephen L. Jaffe, and Alireza Minagar Naoko Takino, Eiko Sakurai, Atsuo Kuramasu,
Nobuyuki Okamura, and Kazuhiko Yanai
Normal Pressure Hydrocephalus
Glen R. Finney Developmental Exposure to Cannabinoids
Causes Subtle and Enduring Neurofunctional
Reversible Dementias Alterations
Anahid Kabasakalian and Glen R. Finney Patrizia Campolongo, Viviana Trezza, Maura
INDEX Palmery, Luigia Trabace, and Vincenzo Cuomo

Neuronal Mechanisms for Pain-Induced Aver-

sion: Behavioral Studies Using a Conditioned
Place Aversion Test
Volume 85 Masabumi Minami

Involvement of the Prefrontal Cortex in Problem Bv8/Prokineticins and their Receptors: A New
Solving Pronociceptive System
Hajime Mushiake, Kazuhiro Sakamoto, Naohiro Lucia Negri, Roberta Lattanzi, Elisa Giannini,
Saito, Toshiro Inui, Kazuyuki Aihara, and Jun Michela Canestrelli, Annalisa Nicotra,
Tanji and Pietro Melchiorri
Contents of Recent Volumes 355

P2Y6-Evoked Microglial Phagocytosis Neurotrophic and Neuroprotective Actions of

Kazuhide Inoue, Schuichi Koizumi, Ayako Kataoka, an Enhancer of Ganglioside Biosynthesis
Hidetoshi Tozaki-Saitoh, and Makoto Tsuda Jin-ichi Inokuchi
PPAR and Pain Involvement of Endocannabinoid Signaling in the
Takehiko Maeda and Shiroh Kishioka Neuroprotective Effects of Subtype 1 Meta-
botropic Glutamate Receptor Antagonists in
Involvement of Inflammatory Mediators in Neu-
Models of Cerebral Ischemia
ropathic Pain Caused by Vincristine
Elisa Landucci, Francesca Boscia, Elisabetta Gerace,
Norikazu Kiguchi, Takehiko Maeda, Yuka
Tania Scartabelli, Andrea Cozzi, Flavio Moroni,
Kobayashi, Fumihiro Saika, and Shiroh Kishioka
Guido Mannaioni, and Domenico E.
Nociceptive Behavior Induced by the Endogenous Pellegrini-Giampietro
Opioid Peptides Dynorphins in Uninjured Mice:
NF-kappaB Dimers in the Regulation of
Evidence with Intrathecal N-ethylmaleimide
Neuronal Survival
Inhibiting Dynorphin Degradation
Ilenia Sarnico, Annamaria Lanzillotta, Marina
Koichi Tan-No, Hiroaki Takahashi, Osamu
Benarese, Manuela Alghisi, Cristina Baiguera,
Nakagawasai, Fukie Niijima, Shinobu Sakurada,
Leontino Battistin, PierFranco Spano, and Marina
Georgy Bakalkin, Lars Terenius, and Takeshi
Pizzi
Tadano
Oxidative Stress in Stroke Pathophysiology:
Mechanism of Allodynia Evoked by Intrathecal
Validation of Hydrogen Peroxide Metabolism as a
Morphine-3-Glucuronide in Mice
Pharmacological Target to Afford Neuroprotection
Takaaki Komatsu, Shinobu Sakurada,
Diana Amantea, Maria Cristina Marrone, Robert
Sou Katsuyama, Kengo Sanai, and Tsukasa
Nisticò, Mauro Federici, Giacinto Bagetta,
Sakurada
Giorgio Bernardi, and Nicola Biagio Mercuri
(–)-Linalool Attenuates Allodynia in Neuropathic
Role of Akt and ERK Signaling in the Neuro-
Pain Induced by Spinal Nerve Ligation in
genesis following Brain Ischemia
C57/Bl6 Mice
Norifumi Shioda, Feng Han, and Kohji Fukunaga
Laura Berliocchi, Rossella Russo, Alessandra
Levato, Vincenza Fratto, Giacinto Bagetta, Shinobu Prevention of Glutamate Accumulation and
Sakurada, Tsukasa Sakurada, Nicola Biagio Upregulation of Phospho-Akt may Account for
Mercuri, and Maria Tiziana Corasaniti Neuroprotection Afforded by Bergamot Essential
Oil against Brain Injury Induced by Focal Cerebral
Intraplantar Injection of Bergamot Essential Oil
Ischemia in Rat
into the Mouse Hindpaw: Effects on Capsaicin-
Diana Amantea, Vincenza Fratto, Simona Maida,
Induced Nociceptive Behaviors
Domenicantonio Rotiroti, Salvatore Ragusa,
Tsukasa Sakurada, Hikari Kuwahata, Soh
Giuseppe Nappi, Giacinto Bagetta, and
Katsuyama, Takaaki Komatsu, Luigi A. Morrone,
Maria Tiziana Corasaniti
M. Tiziana Corasaniti, Giacinto Bagetta,
and Shinobu Sakurada Identification of Novel Pharmacological Targets
to Minimize Excitotoxic Retinal Damage
New Therapy for Neuropathic Pain
Rossella Russo, Domenicantonio Rotiroti, Cristina
Hirokazu Mizoguchi, Chizuko Watanabe, Akihiko
Tassorelli, Carlo Nucci, Giacinto Bagetta, Massimo
Yonezawa, and Shinobu Sakurada
Gilberto Bucci, Maria Tiziana Corasaniti, and
Regulated Exocytosis from Astrocytes: Physiolog- Luigi Antonio Morrone
ical and Pathological Related Aspects
INDEX
Corrado Calı`ı´, Julie Marchaland, Paola Spagnuolo,
Julien Gremion, and Paola Bezzi
Glutamate Release from Astrocytic Gliosomes
Volume 86
Under Physiological and Pathological Conditions Section One: Hybrid Bionic Systems
Marco Milanese, Tiziana Bonifacino, Simona EMG-Based and Gaze-Tracking-Based Man–
Zappettini, Cesare Usai, Carlo Tacchetti, Machine Interfaces
Mario Nobile, and Giambattista Bonanno Federico Carpi and Danilo De Rossi
356 Contents of Recent Volumes

Bidirectional Interfaces with the Peripheral Section Four: Brain-Machine Interfaces and
Nervous System Space
Silvestro Micera and Xavier Navarro Adaptive Changes of Rhythmic EEG Oscillations
in Space: Implications for Brain–Machine
Interfacing Insect Brain for Space Applications
Interface Applications
Giovanni Di Pino, Tobias Seidl,
G. Cheron, A. M. Cebolla, M. Petieau,
Antonella Benvenuto, Fabrizio Sergi, Domenico
A. Bengoetxea, E. Palmero-Soler, A. Leroy, and
Campolo, Dino Accoto, Paolo Maria Rossini,
B. Dan
and Eugenio Guglielmelli
Validation of Brain–Machine Interfaces During
Section Two: Meet the Brain
Parabolic Flight
Meet the Brain: Neurophysiology
Jose del R. Millán, Pierre W. Ferrez, and Tobias
John Rothwell
Seidl
Fundamentals of Electroencefalography, Magne-
Matching Brain–Machine Interface Performance
toencefalography, and Functional Magnetic
to Space Applications
Resonance Imaging
Luca Citi, Oliver Tonet, and Martina Marinelli
Claudio Babiloni, Vittorio Pizzella, Cosimo Del
Gratta, Antonio Ferretti, and Gian Luca Romani Brain–Machine Interfaces for Space
Applications—Research, Technological Devel-
Implications of Brain Plasticity to Brain–Machine
opment, and Opportunities
Interfaces Operation: A Potential Paradox?
Leopold Summerer, Dario Izzo, and Luca Rossini
Paolo Maria Rossini
INDEX
Section Three: Brain Machine Interfaces, A New
Brain-to-Environment Communication Channel
An Overview of BMIs
Francisco Sepulveda Volume 87
Neurofeedback and Brain–Computer Interface: Peripheral Nerve Repair and Regeneration
Clinical Applications Research: A Historical Note
Niels Birbaumer, Ander Ramos Murguialday, Bruno Battiston, Igor Papalia, Pierluigi Tos, and
Cornelia Weber, and Pedro Montoya Stefano Geuna
Flexibility and Practicality: Graz Brain–Computer Development of the Peripheral Nerve
Interface Approach Suleyman Kaplan, Ersan Odaci, Bunyami Unal,
Reinhold Scherer, Gernot R. Müller-Putz, and Bunyamin Sahin, and Michele Fornaro
Gert Pfurtscheller
Histology of the Peripheral Nerve and Changes
On the Use of Brain–Computer Interfaces Out- Occurring During Nerve Regeneration
side Scientific Laboratories: Toward an Applica- Stefano Geuna, Stefania Raimondo, Giulia Ronchi,
tion in Domotic Environments Federica Di Scipio, Pierluigi Tos, Krzysztof Czaja,
F. Babiloni, F. Cincotti, M. Marciani, S. Salinari, and Michele Fornaro
L. Astolfi, F. Aloise, F. De Vico Fallani, and
Methods and Protocols in Peripheral Nerve
D. Mattia
Regeneration Experimental Research:
Brain–Computer Interface Research at the Part I—Experimental Models
Wadsworth Center: Developments in Noninva- Pierluigi Tos, Giulia Ronchi, Igor Papalia,
sive Communication and Control Vera Sallen, Josette Legagneux, Stefano Geuna, and
Dean J. Krusienski and Jonathan R. Wolpaw Maria G. Giacobini-Robecchi
Watching Brain TV and Playing Brain Ball: Methods and Protocols in Peripheral Nerve
Exploring Novel BCL Strategies Using Real– Regeneration Experimental Research: Part
Time Analysis of Human Intercranial Data II—Morphological Techniques
Karim Jerbi, Samson Freyermuth, Lorella Minotti, Stefania Raimondo, Michele Fornaro, Federica Di
Philippe Kahane, Alain Berthoz, and Jean-Philippe Scipio, Giulia Ronchi, Maria G. Giacobini-
Lachaux Robecchi, and Stefano Geuna
Contents of Recent Volumes 357

Methods and Protocols in Peripheral Nerve Enhancement of Nerve Regeneration and

Regeneration Experimental Research: Part III— Recovery by Immunosuppressive Agents
Electrophysiological Evaluation Damien P. Kuffler
Xavier Navarro and Esther Udina
The Role of Collagen in Peripheral Nerve
Methods and Protocols in Peripheral Nerve Repair
Regeneration Experimental Research: Part IV— Guido Koopmans, Birgit Hasse, and
Kinematic Gait Analysis to Quantify Peripheral Nektarios Sinis
Nerve Regeneration in the Rat
Gene Therapy Perspectives for Nerve Repair
Luı´s M. Costa, Maria J. Simões, Ana C. Maurı´cio
Serena Zacchigna and Mauro Giacca
and Artur S.P. Varejão
Use of Stem Cells for Improving Nerve
Current Techniques and Concepts in Peripheral
Regeneration
Nerve Repair
Giorgio Terenghi, Mikael Wiberg, and
Maria Siemionow and Grzegorz Brzezicki
Paul J. Kingham
Artificial Scaffolds for Peripheral Nerve
Transplantation of Olfactory Ensheathing Cells
Reconstruction
for Peripheral Nerve Regeneration
Valeria Chiono, Chiara Tonda-Turo, and
Christine Radtke, Jeffery D. Kocsis, and Peter
Gianluca Ciardelli
M. Vogt
Conduit Luminal Additives for Peripheral
Manual Stimulation of Target Muscles has
Nerve Repair
Different Impact on Functional Recovery after
Hede Yan, Feng Zhang, Michael B. Chen, and
Injury of Pure Motor or Mixed Nerves
William C. Lineaweaver
Nektarios Sinis, Thodora Manoli, Frank Werdin,
Tissue Engineering of Peripheral Nerves Armin Kraus, Hans E. Schaller, Orlando
Bruno Battiston, Stefania Raimondo, Pierluigi Tos, Guntinas-Lichius, Maria Grosheva, Andrey
Valentina Gaidano, Chiara Audisio, Anna Scevola, Irintchev, Emanouil Skouras, Sarah Dunlop, and
Isabelle Perroteau, and Stefano Geuna Doychin N. Angelov
Mechanisms Underlying The End-to-Side Nerve Electrical Stimulation for Improving Nerve
Regeneration Regeneration: Where do we Stand?
Eleana Bontioti and Lars B. Dahlin Tessa Gordon, Olewale A. R. Sulaiman, and
Adil Ladak
Experimental Results in End-To-Side
Neurorrhaphy Phototherapy in Peripheral Nerve Injury:
Alexandros E. Beris and Marios G. Lykissas Effects on Muscle Preservation and Nerve
Regeneration
End-to-Side Nerve Regeneration: From the
Shimon Rochkind, Stefano Geuna, and
Laboratory Bench to Clinical Applications
Asher Shainberg
Pierluigi Tos, Stefano Artiaco, Igor Papalia, Ignazio
Marcoccio, Stefano Geuna, and Bruno Battiston Age-Related Differences in the Reinnervation
after Peripheral Nerve Injury
Novel Pharmacological Approaches to Schwann
Urosˇ Kovacˇicˇ, Janez Sketelj, and Fajko
Cells as Neuroprotective Agents for Peripheral
F. Bajrovic´
Nerve Regeneration
Valerio Magnaghi, Patrizia Procacci, and Neural Plasticity After Nerve Injury and
Ada Maria Tata Regeneration
Xavier Navarro
Melatonin and Nerve Regeneration
Ersan Odaci and Suleyman Kaplan Future Perspective in Peripheral Nerve
Reconstruction
Transthyretin: An Enhancer of Nerve
Lars Dahlin, Fredrik Johansson, Charlotta
Regeneration
Lindwall, and Martin Kanje
Carolina E. Fleming, Fernando Milhazes Mar,
Filipa Franquinho, and Mónica M. Sousa INDEX
358 Contents of Recent Volumes

Volume 88 Cocaine-Induced Breakdown of the Blood–Brain

Barrier and Neurotoxicity
Effects Of Psychostimulants On Neurotrophins: Hari S. Sharma, Dafin Muresanu, Aruna Sharma,
Implications For Psychostimulant-Induced and Ranjana Patnaik
Neurotoxicity
Francesco Angelucci, Valerio Ricci, Gianfranco Cannabinoid Receptors in Brain:
Spalletta, Carlo Caltagirone, Aleksander A. Mathe, Pharmacogenetics, Neuropharmacology, Neu-
and Pietro Bria rotoxicology, and Potential Therapeutic
Applications
Dosing Time-Dependent Actions of Emmanuel S. Onaivi
Psychostimulants
Intermittent Dopaminergic Stimulation causes
H. Manev and T. Uz
Behavioral Sensitization in the Addicted Brain
Dopamine-Induced Behavioral Changes and and Parkinsonism
Oxidative Stress in Methamphetamine-Induced Francesco Fornai, Francesca Biagioni, Federica
Neurotoxicity Fulceri, Luigi Murri, Stefano Ruggieri,
Taizo Kita, Ikuko Miyazaki, Masato Asanuma, Antonio Paparelli
Mika Takeshima, and George C. Wagner
The Role of the Somatotrophic Axis in
Acute Methamphetamine Intoxication: Brain Neuroprotection and Neuroregeneration of the
Hyperthermia, Blood–Brain Barrier, Brain Addictive Brain
Edema, and morphological cell abnormalities Fred Nyberg
Eugene A. Kiyatkin and Hari S. Sharma
INDEX
Molecular Bases of Methamphetamine-Induced
Neurodegeneration
Jean Lud Cadet and Irina N. Krasnova
Volume 89
Involvement of Nicotinic Receptors in Metham-
Molecular Profiling of Striatonigral and
phetamine- and MDMA-Induced Neurotoxicity:
Striatopallidal Medium Spiny Neurons: Past, Pre-
Pharmacological Implications
sent, and Future
E. Escubedo, J. Camarasa, C. Chipana,
Mary Kay Lobo
S. Garcı´a-Rates, and D.Pubill
BAC to Degeneration: Bacterial Artificial
Ethanol Alters the Physiology of Neuron–Glia
Chromosome (Bac)-Mediated Transgenesis for
Communication
Modeling Basal Ganglia Neurodegenerative
Antonio González and Gines M. Salido
Disorders
Therapeutic Targeting of “DARPP-32”: Xiao-Hong Lu
A Key Signaling Molecule in the Dopiminergic
Behavioral Outcome Measures for the Assessment
Pathway for the Treatment of Opiate Addiction
Supriya D. Mahajan, Ravikumar Aalinkeel, of Sensorimotor Function in Animal Models of
Jessica L. Reynolds, Bindukumar B. Nair, Movement Disorders
Donald E. Sykes, Zihua Hu, Adela Bonoiu, Sheila M. Fleming
Hong Ding, Paras N. Prasad, and Stanley The Role of DNA Methylation in the Central
A. Schwartz Nervous System and Neuropsychiatric Disorders
Pharmacological and Neurotoxicological Actions Jian Feng and Guoping Fan
Mediated By Bupropion and Diethylpropion Heritability of Structural Brain Traits: An
Hugo R. Arias, Abel Santamarı´a, and Syed F. Ali Endo-phenotype Approach to Deconstruct
Schizophrenia
Neural and Cardiac Toxicities Associated With
Nil Kaymaz and J. Van Os
3,4-Methylenedioxymethamphetamine
(MDMA) The Role of Striatal NMDA Receptors in Drug
Michael H. Baumann and Richard Addiction
B. Rothman Yao-Ying Ma, Carlos Cepeda, and Cai-Lian Cui
Contents of Recent Volumes 359

Deciphering Rett Syndrome With Mouse Genet- Part III—Transcranial Sonography in other
ics, Epigenomics, and Human Neurons Movement Disorders and Depression
Jifang Tao, Hao Wu, and Yi Eve Sun
Transcranial Sonography in Brain Disorders with
INDEX Trace Metal Accumulation
Uwe Walter
Transcranial Sonography in Dystonia
Volume 90 Alexandra Gaenslen
Part I: Introduction Transcranial Sonography in Essential Tremor
Heike Stockner and Isabel Wurster
Introductory Remarks on the History and Current
Applications of TCS VII—Transcranial Sonography in Restless Legs
Matthew B. Stern Syndrome
Jana Godau and Martin Sojer
Method and Validity of Transcranial Sonography
in Movement Disorders Transcranial Sonography in Ataxia
David Školoudı´k and Uwe Walter Christos Krogias, Thomas Postert and Jens Eyding
Transcranial Sonography—Anatomy Transcranial Sonography in Huntington’s Disease
Heiko Huber Christos Krogias, Jens Eyding and Thomas Postert
Transcranial Sonography in Depression
Part II: Transcranial Sonography in Parkinsons Milija D. Mijajlovic
Disease
Transcranial Sonography in Relation to SPECT Part IV: Future Applications and Conclusion
and MIBG Transcranial Sonography-Assisted Stereotaxy and
Yoshinori Kajimoto, Hideto Miwa and Tomoyoshi Follow-Up of Deep Brain Implants in Patients
Kondo with Movement Disorders
Diagnosis of Parkinson’s Disease—Transcranial Uwe Walter
Sonography in Relation to MRI Conclusions
Ludwig Niehaus and Kai Boelmans Daniela Berg
Early Diagnosis of Parkinson’s Disease INDEX
Alexandra Gaenslen and Daniela Berg
Transcranial Sonography in the Premotor Diag-
nosis of Parkinson’s Disease
Stefanie Behnke, Ute Schroder and Daniela Berg Volume 91
Pathophysiology of Transcranial Sonography Sig- The Role of microRNAs in Drug Addiction:
nal Changes in the Human Substantia Nigra A Big Lesson from Tiny Molecules
K. L. Double, G. Todd and S. R. Duma Andrzej Zbigniew Pietrzykowski
Transcranial Sonography for the Discrimination of The Genetics of Behavioral Alcohol Responses in
Idiopathic Parkinson’s Disease from the Atypical Drosophila
Parkinsonian Syndromes Aylin R. Rodan and Adrian Rothenfluh
A. E. P. Bouwmans, A. M. M. Vlaar, K. Srulijes,
Neural Plasticity, Human Genetics, and Risk for
W. H. Mess AND W. E. J. Weber
Alcohol Dependence
Transcranial Sonography in the Discrimination of Shirley Y. Hill
Parkinson’s Disease Versus Vascular Parkinsonism
Using Expression Genetics to Study the Neurobi-
Pablo Venegas-Francke
ology of Ethanol and Alcoholism
TCS in Monogenic Forms of Parkinson’s Disease Sean P. Farris, Aaron R. Wolen and Michael
Kathrin Brockmann and Johann Hagenah F. Miles
360 Contents of Recent Volumes

Genetic Variation and Brain Gene Expression in Neuroimaging of Dreaming: State of the Art and
Rodent Models of Alcoholism: Implications for Limitations
Medication Development Caroline Kusse, Vincenzo Muto, Laura Mascetti,
Karl Bj€ork, Anita C. Hansson and Luca Matarazzo, Ariane Foret, Anahita Shaffii-Le
W. olfgang H. Sommer Bourdiec and Pierre Maquet
Identifying Quantitative Trait Loci (QTLs) and Memory Consolidation, The Diurnal Rhythm of
Genes (QTGs) for Alcohol-Related Phenotypes Cortisol, and The Nature of Dreams: A New
in Mice Hypothesis
Lauren C. Milner and Kari J. Buck Jessica D. Payne
Glutamate Plasticity in the Drunken Amygdala: Characteristics and Contents of Dreams
The Making of an Anxious Synapse Michael Schredl
Brian A. Mccool, Daniel T. Christian, Marvin
Trait and Neurobiological Correlates of Individ-
R. Diaz and Anna K. Läck
ual Differences in Dream Recall and Dream
Ethanol Action on Dopaminergic Neurons in Content
the Ventral Tegmental Area: Interaction with Mark Blagrove and Edward F. Pace-Schott
Intrinsic Ion Channels and Neurotransmitter
Consciousness in Dreams
Inputs
David Kahn and Tzivia Gover
Hitoshi Morikawa and Richard
A. Morrisett The Underlying Emotion and the Dream: Relat-
ing Dream Imagery to the Dreamer’s Underlying
Alcohol and the Prefrontal Cortex
Emotion can Help Elucidate the Nature of
Kenneth Abernathy, L. Judson Chandler and John
Dreaming
J. Woodward
Ernest Hartmann
BK Channel and Alcohol, A Complicated Affair
Dreaming, Handedness, and Sleep Architecture:
Gilles Erwan Martin
Interhemispheric Mechanisms
A Review of Synaptic Plasticity at Purkinje Neu- Stephen D. Christman and Ruth E. Propper
rons with a Focus on Ethanol-Induced Cerebellar
To What Extent Do Neurobiological Sleep-
Dysfunction
Waking Processes Support Psychoanalysis?
C. Fernando Valenzuela, Britta Lindquist and
Claude Gottesmann
Paula A. Zflmudio-Bulcock
The Use of Dreams in Modern Psychotherapy
INDEX
Clara E. Hill and Sarah Knox
INDEX

Volume 92
The Development of the Science of Dreaming Volume 93
Claude Gottesmann
Underlying Brain Mechanisms that Regulate
Dreaming as Inspiration: Evidence from Religion, Sleep-Wakefulness Cycles
Philosophy, Literature, and Film Irma Gvilia
Kelly Bulkeley
What Keeps Us Awake?—the Role of Clocks and
Developmental Perspective: Dreaming Across the Hourglasses, Light, and Melatonin
Lifespan and What This Tells Us Christian Cajochen, Sarah Chellappa and Christina
Melissa M. Burnham and Christian Conte Schmidt
REM and NREM Sleep Mentation Suprachiasmatic Nucleus and Autonomic Nervous
Patrick Mcnamara, Patricia Johnson, Deirdre System Influences on Awakening From Sleep
McLaren, Erica Harris,Catherine Beauharnais and Andries Kalsbeek, Chun-xia Yi, Susanne E.
Sanford Auerbach la Fleur, Ruud m. Buijs, and Eric Fliers
Contents of Recent Volumes 361

Preparation for Awakening: Self-Awakening Vs. Volume 95

Forced Awakening: Preparatory Changes in the
Pre-Awakening Period Introductory Remarks: Catechol-O-Methyl-
Mitsuo Hayashi, Noriko Matsuura and transferase Inhibition–An Innovative Approach
Hiroki Ikeda to Enhance L-dopa Therapy in Parkinson’s Dis-
ease with Dual Enzyme Inhibition
Circadian and Sleep Episode Duration Influences Erkki Nissinen
on Cognitive Performance Following the Process
of Awakening The Catechol-O-Methyltransferase Gene: its
Robert L. Matchock Regulation and Polymorphisms
Elizabeth M. Tunbridge
The Cortisol Awakening Response in Context
Angela Clow, Frank Hucklebridge and Distribution and Functions of Catechol-O-
Lisa Thorn Methyltransferase Proteins: Do Recent Findings
Change the Picture?
Causes and Correlates of Frequent Night Awak- Timo T. My€ohänen and Pekka T. Männist€o
enings in Early Childhood
Amy Jo Schwichtenberg and Beth Goodlin-Jones Catechol-O-Methyltransferase Enzyme: Cofactor
S-Adenosyl-L-MethionineandRelatedMechanisms
Pathologies of Awakenings: The Clinical Problem Thomas Müller
of Insomnia Considered From Multiple Theory
Levels Biochemistry and Pharmacology of Catechol-
Douglas E. Moul O-Methyltransferase Inhibitors
Erkki nissinen and Pekka T. Männisto
The Neurochemistry of Awakening: Findings
from Sleep Disorder Narcolepsy The Chemistry of Catechol-O-Methyltransferase
Seiji Nishino and Yohei Sagawa Inhibitors
David A. Learmonth, László E. Kiss, and Patrı´cio
INDEX Soares-da-Silva
Toxicology and Safety of COMT Inhibitors
Kristiina Haasio

Volume 94 Catechol-O-Methyltransferase Inhibitors in Pre-

clinical Models as Adjuncts of L-dopa Treatment
5-HT6 Medicinal Chemistry Concepció Marin and J. A. Obeso
Kevin G. Liu and Albert J. Robichaud
Problems with the Present Inhibitors and a Rele-
Patents vance of New and Improved COMT Inhibitors in
Nicolas Vincent Ruiz and Gloria Oranias Parkinson’s Disease
5-HT6 Receptor Charactertization Seppo Kaakkola
Teresa Riccioni Catechol-O-Methyltransferase and Pain
5-HT6 Receptor Signal Transduction: Second Oleg Kambur and Pekka T. Männist€o
Messenger Systems INDEX
Xavier Codony, Javier Burgueño, Maria Javier
Ramı´rez and Jose Miguel Vela
Electrophysiology of 5-HT6 Receptors
Volume 96
Annalisa Tassone, Graziella Madeo, Giuseppe The Central Role of 5-HT6 Receptors in Modu-
Sciamanna, Antonio Pisani and Paola Bonsi lating Brain Neurochemistry
Lee A. Dawson
Genetic Variations and Association
Massimo Gennarelli and Annamaria Cattaneo 5-HT6 Receptor Memory and Amnesia: Behav-
ioral Pharmacology – Learning and Memory
Pharmacokinetics of 5-HT6 Receptor Ligands
Processes
Angelo Mancinelli
Alfredo Meneses, G. Perez-Garcı´a, R. Tellez,
INDEX T. Ponce-Lopez and C. Castillo
362 Contents of Recent Volumes

Behavioral Pharmacology: Potential Antidepres- Peripheral and Central Mechanisms of Orofacial

sant and Anxiolytic Properties Inflammatory Pain
Anna Wesołowska and Magdalena Jastrzbska- Barry J. Sessle
Wisek
The Role of Trigeminal Interpolaris-Caudalis
The 5-HT6 Receptor as a Target for Developing Transition Zone in Persistent Orofacial Pain
Novel Antiobesity Drugs Ke Ren and Ronald Dubner
David Heal, Jane Gosden and Sharon Smith
Physiological Mechanisms of Neuropathic Pain:
Behavioral and Neurochemical Pharmacology of The Orofacial Region
5-HT6 Receptors Related to Reward and Koichi Iwata, Yoshiki Imamura, Kuniya Honda and
Reinforcement Masamichi Shinoda
Gaetano Di Chiara, Valentina Valentini and
Neurobiology of Estrogen Status in Deep Cranio-
Sandro Fenu
facial Pain
5-HT6 Receptor Ligands and their Antipsychotic David A Bereiter and Keiichiro Okamoto
Potential
Macroscopic Connection of Rat Insular Cortex:
Jørn Arnt and Christina Kurre Olsen
Anatomical Bases Underlying its Physiological
5-HT6 Receptor Ligands as Antidementia Drugs Functions
Ellen Siobhan Mitchell Masayuki Kobayashi
Other 5-HT6 Receptor-Mediated Effects The Balance Between Excitation And Inhibition
Franco Borsini And Functional Sensory Processing in the
Somatosensory Cortex
INDEX
Zhi Zhang and Qian-Quan Sun
INDEX

Volume 97 Volume 98
Behavioral Pharmacology of Orofacial Movement
An Introduction to Dyskinesia—the Clinical
Disorders
Spectrum
Noriaki Koshikawa, Satoshi Fujita and Kazunori
Ainhi Ha and Joseph Jankovic
Adachi
L-dopa-induced Dyskinesia—Clinical Presenta-
Regulation of Orofacial Movement: Dopamine
tion, Genetics, And Treatment
Receptor Mechanisms and Mutant Models
L.K. Prashanth, Susan Fox and Wassilios
John L. Waddington, Gerard J. O’Sullivan and
G. Meissner
Katsunori Tomiyama
Experimental Models of L-DOPA-induced
Regulation of Orofacial Movement: Amino Acid
Dyskinesia
Mechanisms and Mutant Models
Tom H. Johnston and Emma L. Lane
Katsunori Tomiyama, Colm M.P. O’Tuathaigh,
and John L. Waddington Molecular Mechanisms of L-DOPA-induced
Dyskinesia
The Trigeminal Circuits Responsible for
Gilberto Fisone and Erwan Bezard
Chewing
Karl-Gunnar Westberg and Arlette Kolta New Approaches to Therapy
Jonathan Brotchie and Peter Jenner
Ultrastructural Basis for Craniofacial Sensory
Processing in the Brainstem Surgical Approach to L-DOPA-induced
Yong Chul Bae and Atsushi Yoshida Dyskinesias
Tejas Sankar and Andres M. Lozano
Mechanisms of Nociceptive Transduction and
Transmission: A Machinery for Pain Sensation Clinical and Experimental Experiences of
and Tools for Selective Analgesia Graft-induced Dyskinesia
Alexander M. Binshtok Emma L. Lane
Contents of Recent Volumes 363

Tardive Dyskinesia: Clinical Presentation and Homeostatic Control of Neural Activity:

Treatment A Drosophila Model for Drug Tolerance and
P.N. van Harten and D.E. Tenback Dependence
Alfredo Ghezzi and Nigel S. Atkinson
Epidemiology and Risk Factors for (Tardive)
Dyskinesia Attention in Drosophila
D.E. Tenback and P.N. van Harten Bruno van Swinderen
Genetics of Tardive Dyskinesia The roles of Fruitless and Doublesex in the Control
Heon-Jeong Lee and Seung-Gul Kang of Male Courtship
Brigitte Dauwalder
Animal Models of Tardive Dyskinesia
S.K. Kulkarni and Ashish Dhir Circadian Plasticity: from Structure to Behavior
Lia Frenkel and Marı´a Fernanda Ceriani
Surgery for Tardive Dyskinesia
Stephane Thobois, Alice Poisson and Philippe Learning and Memory in Drosophila: Behavior,
Damier Genetics, and Neural Systems
Lily Kahsai and Troy Zars
Huntington’s Disease: Clinical Presentation and
Treatment Studying Sensorimotor Processing with Physiol-
M.J.U. Novak and S.J. Tabrizi ogy in Behaving Drosophila
Johannes D. Seelig and Vivek Jayaraman
Genetics and Neuropathology of Huntington’s
Disease: Huntington’s Disease Modeling Human Trinucleotide Repeat Diseases
Anton Reiner, Ioannis Dragatsis and Paula Dietrich in Drosophila
Zhenming Yu and Nancy M. Bonini
Pathogenic Mechanisms in Huntington’s Disease
Lesley Jones and Alis Hughes From Genetics to Structure to Function: Explor-
ing Sleep in Drosophila
Experimental Models of HD And Reflection on
Daniel Bushey and Chiara Cirelli
Therapeutic Strategies
Olivia L. Bordiuk, Jinho Kim and Robert J. Ferrante INDEX
Cell-based Treatments for Huntington’s Disease
Stephen B. Dunnett and Anne E. Rosser
Volume 100
Clinical Phenomenology of Dystonia
Structural Properties of Human Monoamine
Carlo Colosimo and Alfredo Berardelli
Oxidases A and B
Genetics and Pharmacological Treatment of Claudia Binda, Andrea Mattevi and
Dystonia Dale E. Edmondson
Susan Bressman and Matthew James
Behavioral Outcomes of Monoamine Oxidase
Experimental Models of Dystonia Deficiency: Preclinical and Clinical Evidence
A. Tassone, G. Sciamanna, P. Bonsi, G. Martella Marco Bortolato and Jean C. Shih
and A. Pisani
Kinetic Behavior and Reversible Inhibition of
Surgical Treatment of Dystonia Monoamine Oxidases—Enzymes that Many
John Yianni, Alexander L. Green and Tipu Want Dead
Z. Aziz Keith F. Tipton, Gavin P. Davey and
Andrew G. McDonald
INDEX
The Pharmacology of Selegiline
Kálmán Magyar
Volume 99 Type A Monoamine Oxidase Regulates Life and
Seizure and Epilepsy: Studies of Seizure- Death of Neurons in Neurodegeneration and
disorders in Drosophila Neuroprotection
Louise Parker, Iris C. Howlett, Zeid M. Rusan and Makoto Naoi, Wakako Maruyama,
Mark A. Tanouye Keiko Inaba-Hasegawa and Yukihiro Akao
364 Contents of Recent Volumes

Multimodal Drugs and their Future for Abnormalities in Metabolism and Hypothalamic–
Alzheimer’s and Parkinson’s Disease Pituitary–Adrenal Axis Function in Schizophrenia
Cornelis J. Van der Schyf and Werner J. Geldenhuys Paul C. Guest, Daniel Martins-de-Souza,
Natacha Vanattou-Saifoudine, Laura W. Harris
Neuroprotective Profile of the Multitarget Drug
and Sabine Bahn
Rasagiline in Parkinson’s Disease
Orly Weinreb, Tamar Amit, Peter Riederer, Immune and Neuroimmune Alterations in Mood
Moussa B.H. Youdim and Silvia A. Mandel Disorders and Schizophrenia
Roosmarijn C. Drexhage, Karin Weigelt, Nico van
Rasagiline in Parkinson’s Disease
Beveren, Dan Cohen, Marjan A. Versnel, Willem
L.M. Chahine and M.B. Stern
A. Nolen and Hemmo A. Drexhage
Selective Inhibitors of Monoamine Oxidase Type
Behavioral and Molecular Biomarkers in Transla-
B and the “Cheese Effect”
tional Animal Models for Neuropsychiatric
John P.M. Finberg and Ken Gillman
Disorders
A Novel Anti-Alzheimer’s Disease Drug, Ladostigil: Zoltán Sarnyai, Murtada Alsaif, Sabine Bahn,
Neuroprotective, Multimodal Brain-Selective Agnes Ernst, Paul C. Guest, Eva Hradetzky,
Monoamine Oxidase and Cholinesterase Inhibitor Wolfgang Kluge, Viktoria Stelzhammer and
Orly Weinreb, Tamar Amit, Orit Bar-Am and Hendrik Wesseling
Moussa B.H. Youdim
Stem Cell Models for Biomarker Discovery in
Novel MAO-B Inhibitors: Potential Therapeutic Brain Disease
Use of the Selective MAO-B Inhibitor PF9601N Alan Mackay-Sim, George Mellick and Stephen
in Parkinson’s Disease Wood
Mercedes Unzeta and Elisenda Sanz
The Application of Multiplexed Assay Systems for
INDEX Molecular Diagnostics
Emanuel Schwarz, Nico J.M. VanBeveren,
Paul C. Guest, Rauf Izmailov and
Volume 101 Sabine Bahn
General Overview: Biomarkers in Neuroscience Algorithm Development for Diagnostic Bio-
Research marker Assays
Michaela D. Filiou and Christoph W. Turck Rauf Izmailov, Paul C. Guest, Sabine Bahn and
Emanuel Schwarz
Imaging Brain Microglial Activation Using
Positron Emission Tomography and Translocator Challenges of Introducing New Biomarker Prod-
Protein-Specific Radioligands ucts for Neuropsychiatric Disorders into the
David R.J. Owen and Paul M. Matthews Market
The Utility of Gene Expression in Blood Cells for Sabine Bahn, Richard Noll, Anthony Barnes,
Diagnosing Neuropsychiatric Disorders Emanuel Schwarz and Paul C. Guest
Christopher H. Woelk, Akul Singhania, Josue Toward Personalized Medicine in the Neuropsy-
Perez-Santiago, Stephen J. Glatt and Ming chiatric Field
T. Tsuang Erik H.F. Wong, Jayne C. Fox, Mandy
Proteomic Technologies for Biomarker Studies in Y.M. Ng and Chi-Ming Lee
Psychiatry: Advances and Needs Clinical Utility of Serum Biomarkers for Major
Daniel Martins-de-Souza, Paul C. Guest, Psychiatric Disorders
Natacha Vanattou-Saifoudine, Laura W. Harris Nico J.M. van Beveren and Witte
and Sabine Bahn J.G. Hoogendijk
Converging Evidence of Blood-Based Biomarkers
The Future: Biomarkers, Biosensors, Neu-
for Schizophrenia: An update
roinformatics, and E-Neuropsychiatry
Man K. Chan, Paul C. Guest, Yishai Levin,
Christopher R. Lowe
Yagnesh Umrania, Emanuel Schwarz, Sabine Bahn
and Hassan Rahmoune SUBJECT INDEX
Contents of Recent Volumes 365

Volume 102 Neurotrophic Factors and Peptides on the Whole

Body Hyperthermia-Induced Neurotoxicity:
The Function and Mechanisms of Nurr1 Action in Modulatory Roles of Co-morbidity Factors and
Midbrain Dopaminergic Neurons, from Develop- Nanoparticle Intoxication
ment and Maintenance to Survival Hari Shanker Sharma, Aruna Sharma, Herbert
Yu Luo M€ossler and Dafin Fior Muresanu
Monoclonal Antibodies as Novel Neurotherapeutic Alzheimer’s Disease and Amyloid: Culprit or
Agents in CNS Injury and Repair Coincidence?
Aruna Sharma and Hari Shanker Sharma Stephen D. Skaper
The Blood–Brain Barrier in Alzheimer’s Disease: Vascular Endothelial Growth Factor and Other
Novel Therapeutic Targets and Nanodrug Angioglioneurins: Key Molecules in Brain
delivery Development and Restoration
Hari Shanker Sharma, Rudy J. Castellani, Mark Jose Vicente Lafuente, Naiara Ortuzar, Harkaitz
A. Smith and Aruna Sharma Bengoetxea, Susana Bulnes and Enrike G. Argandoña
Neurovascular Aspects of Amyotrophic Lateral INDEX
Sclerosis
Maria Carolina O. Rodrigues, Diana G.
Hernandez-Ontiveros, Michael K. Louis, Alison
Volume 103
E. Willing, Cesario V. Borlongan, Paul R. Sanberg, Lost and Found in Behavioral Informatics
Júlio C. Voltarelli and Svitlana Garbuzova-Davis Melissa A. Haendel and Elissa J. Chesler
Quercetin in Hypoxia-Induced Oxidative Stress: Biological Databases for Behavioral Neurobiology
Novel Target for Neuroprotection Erich J. Baker
Anand Kumar Pandey, Ranjana Patnaik, Dafin
A Survey of the Neuroscience Resource Land-
F. Muresanu, Aruna Sharma and Hari Shanker
scape: Perspectives from the Neuroscience Infor-
Sharma
mation Framework
Environmental Conditions Modulate Neuro- Jonathan Cachat, Anita Bandrowski, Jeffery S. Grethe,
toxic Effects of Psychomotor Stimulant Drugs Amarnath Gupta, Vadim Astakhov, Fahim Imam,
of Abuse Stephen D. Larson, and Maryann E. Martone
Eugene A. Kiyatkin and Hari Shanker Sharma
The Neurobehavior Ontology: An Ontology for
Central Nervous Tissue Damage after Hypoxia Annotation and Integration of Behavior and
and Reperfusion in Conjunction with Cardiac Behavioral Phenotypes
Arrest and Cardiopulmonary Resuscitation: Georgios V. Gkoutos, Paul N. Schofield, and
Mechanisms of Action and Possibilities for Robert Hoehndorf
Mitigation
Ontologies for Human Behavior Analysis and
Lars Wiklund, Cecile Martijn, Adriana Miclescu,
Their Application to Clinical Data
Egidijus Semenas, Sten Rubertsson and Hari
Janna Hastings and Stefan Schulz
Shanker Sharma
Text-Mining and Neuroscience
Interactions Between Opioids and Anabolic Kyle H. Ambert and Aaron M. Cohen
Androgenic Steroids: Implications for the
Development of Addictive Behavior Applying In Silico Integrative Genomics to
Fred Nyberg and Mathias Hallberg Genetic Studies of Human Disease: A Review
Scott F. Saccone
Neurotrophic Factors and Neurodegenerative
Diseases: A Delivery Issue SUBJECT INDEX
Barbara Ruozi, Daniela Belletti, Lucia Bondioli,
Alessandro De Vita, Flavio Forni, Maria Angela Volume 104
Vandelli and Giovanni Tosi
Cross Species Integration of Functional Genomics
Neuroprotective Effects of Cerebrolysin, a Experiments
Combination of Different Active Fragments of Jeremy J. Jay
366 Contents of Recent Volumes

Model Organism Databases in Behavioral Rho Signaling and Axon Regeneration

Neuroscience L. McKerracher, Gino B. Ferraro, and Alyson
Mary Shimoyama, Jennifer R. Smith, E. Fournier
G. Thomas Hayman, Victoria Petri, and
Neuron-Intrinsic Inhibitors of Axon Regenera-
Rajni Nigam
tion: PTEN and SOCS3
Accessing and Mining Data from Large-Scale Xueting Luo and Kevin K. Park
Mouse Phenotyping Projects INDEX
Hugh Morgan, Michelle Simon, and Ann-Marie
Mallon

Bioinformatics Resources for Behavior Studies Volume 106

in the Laboratory Mouse
Carol J. Bult Neurotrophic Factors and the Regeneration of
Adult Retinal Ganglion Cell Axons
Using Genome-Wide Expression Profiling to Alan R. Harvey, Jacob Wei Wei Ooi, and Jennifer
Define Gene Networks Relevant to the Study Rodger
of Complex Traits: From RNA Integrity to
Network Topology MBS: Signaling Endosomes and Growth Cone
M.A. O’Brien, B.N. Costin, and M.F. Miles Motility in Axon Regeneration
Michael B. Steketee and Jeffrey L. Goldberg
Genetic and Molecular Network Analysis of
Behavior Intrinsic Mechanisms Regulating Axon Regener-
Robert W. Williams and Megan K. Mulligan ation: An Integrin Perspective
Richard Eva, Melissa R. Andrews, Elske H.P.
Large-Scale Neuroinformatics for In Situ Hybrid- Franssen, and James W. Fawcett
ization Data in the Mouse Brain
Lydia L. Ng, Susan M. Sunkin, David Feng, The Role of Serotonin in Axon and Dendrite
Chris Lau, Chinh Dang, and Michael J. Hawrylycz Growth
Ephraim F. Trakhtenberg and Jeffrey L. Goldberg
Opportunities for Bioinformatics in the Classifica-
tion of Behavior and Psychiatric Disorders Inflammatory Pathways in Spinal Cord Injury
Elissa J. Chesler and Ryan W. Logan Samuel David, Juan Guillermo Zarruk, and Nader
Ghasemlou
SUBJECT INDEX
Combinatorial Therapy Stimulates Long-Distance
Regeneration, Target Reinnervation, and Partial
Volume 105 Recovery of Vision After Optic Nerve Injury in
Mice
Optic Nerve Disease and Axon Pathophysiology Silmara de Lima, Ghaith Habboub, and Larry I.
Alireza Ghaffarieh and Leonard A. Levin Benowitz
Role of Electrical Activity of Neurons for From Bench to Beside to Cure Spinal Cord
Neuroprotection Injury: Lost in Translation?
Takeshi Morimoto Andreas Hug and Norbert Weidner
Molecular Control of Axon Growth: Insights SUBJECT INDEX
from Comparative Gene Profiling and High-
Throughput Screening
Murray G. Blackmore
Gatekeeper Between Quiescence and Differentia-
Volume 107
tion: p53 in Axonal Outgrowth and Neurogenesis Neuromodulation: A More Comprehensive Con-
Giorgia Quadrato and Simone Di Giovanni cept Beyond Deep Brain Stimulation
Clement Hamani and Elena Moro
Cyclin-Dependent Kinase 5 in Axon Growth and
Regeneration Computational Models of Neuromodulation
Tao Ye, Amy K. Y. Fu, and Nancy Y. Ip Christopher R. Butson
Contents of Recent Volumes 367

Neurophysiology of Deep Brain Stimulation Bone Marrow Mesenchymal Stem Cell Trans-
Manuela Rosa, Gaia Giannicola, Sara Marceglia, plantation for Improving Nerve Regeneration
Manuela Fumagalli, Sergio Barbieri, and Alberto Priori Júlia Teixeira Oliveira, Klauss Mostacada, Silmara
de Lima, and Ana Maria Blanco Martinez
Neurophysiology of Cortical Stimulation
Jean-Pascal Lefaucheur Perspectives of Employing Mesenchymal Stem
Cells from the Wharton’s Jelly of the Umbilical
Neural Mechanisms of Spinal Cord Stimulation
Cord for Peripheral Nerve Repair
Robert D. Foreman and Bengt Linderoth
Jorge Ribeiro, Andrea Gartner, Tiago Pereira,
Magnetoencephalography and Neuromodulation Raquel Gomes, Maria Ascensão Lopes,
Alfons Schnitzler and Jan Hirschmann Carolina Gonçalves, Artur Varejão, Ana Lúcia
Luı´s, and Ana Colette Maurı´cio
Current Challenges to the Clinical Translation of
Brain Machine Interface Technology Adipose-Derived Stem Cells and Nerve Regener-
Charles W. Lu, Parag G. Patil, and Cynthia A. ation: Promises and Pitfalls
Chestek Alessandro Faroni, Giorgio Terenghi, and
Adam J. Reid
Nanotechnology in Neuromodulation
Russell J. Andrews The Pros and Cons of Growth Factors and Cyto-
kines in Peripheral Axon Regeneration
Optogenetic Neuromodulation
Lars Klimaschewski, Barbara Hausott, and Doychin
Paul S. A. Kalanithi and Jaimie M. Henderson
N. Angelov
Diffusion Tensor Imaging and Neuromodulation:
Role of Inflammation and Cytokines in Peripheral
DTI as Key Technology for Deep Brain
Nerve Regeneration
Stimulation
P. Dubový, R. Jancˇálek, and T. Kubek
Volker Arnd Coenen, Thomas E. Schlaepfer, Niels
Allert, and Burkhard Mädler Ghrelin: A Novel Neuromuscular Recovery Pro-
moting Factor?
DBS and Electrical Neuro-Network Modulation
Raimondo Stefania, Ronchi Giulia, Geuna Stefano,
to Treat Neurological Disorders
Pascal Davide, Reano Simone, Filigheddu Nicoletta,
Amanda Thompson, Takashi Morishita, and
and Graziani Andrea
Michael S. Okun
Neuregulin 1 Role in Schwann Cell Regulation
Neuromodulation in Psychiatric Disorders
and Potential Applications to Promote Peripheral
Yasin Temel, Sarah A. Hescham, Ali Jahanshahi,
Nerve Regeneration
Marcus L. F. Janssen, Sonny K. H. Tan, Jacobus
Giovanna Gambarotta, Federica Fregnan, Sara
J. van Overbeeke, Linda Ackermans, Mayke
Gnavi, and Isabelle Perroteau
Oosterloo, Annelien Duits, Albert F. G. Leentjens,
and LeeWei Lim Extracellular Matrix Components in Peripheral
Nerve Regeneration
Ethical Aspects of Neuromodulation
Francisco Gonzalez-Perez, Esther Udina, and
Christiane Woopen
Xavier Navarro
SUBJECT INDEX
SUBJECT INDEX
Volume 108
Tissue Engineering and Regenerative Medicine:
Volume 109
Past, Present, and Future The Use of Chitosan-Based Scaffold to Enhance
António J. Salgado, Joaquim M. Oliveira, Albino Regeneration in the Nervous System
Martins, Fábio G. Teixeira, Nuno A. Silva, Sara Gnavi, Christina Barwig, Thomas Freier,
Nuno M. Neves, Nuno Sousa, and Rui L. Reis Kirsten Haarstert-Talini, Claudia Grothe, and
Stefano Geuna
Tissue Engineering and Peripheral Nerve Recon-
struction: An Overview Interfaces with the Peripheral Nerve for the Con-
Stefano Geuna, S. Gnavi, I. Perroteau, trol of Neuroprostheses
Pierluigi Tos, and B. Battiston Jaume del Valle and Xavier Navarro
368 Contents of Recent Volumes

The Use of Shock Waves in Peripheral Nerve The Neuropathology of Neurodegeneration with
Regeneration: New Perspectives? Brain Iron Accumulation
Thomas Hausner and Antal Nógrádi Michael C. Kruer
Phototherapy and Nerve Injury: Focus on Muscle Imaging of Iron
Response Petr Dusek, Monika Dezortova, and Jens Wuerfel
Shimon Rochkind, Stefano Geuna, and Asher
The Role of Iron Imaging in Huntington’s Disease
Shainberg
S.J.A. van den Bogaard, E.M. Dumas, and
Electrical Stimulation for Promoting Peripheral R.A.C. Roos
Nerve Regeneration
Lysosomal Storage Disorders and Iron
Kirsten Haastert-Talini and Claudia Grothe
Jose Miguel Bras
Role of Physical Exercise for Improving Post-
Manganese and the Brain
traumatic Nerve Regeneration
Karin Tuschl, Philippa B. Mills, and Peter T. Clayton
Paulo A.S. Armada-da-Silva, Cátia Pereira,
SandraAmado, and António P. Veloso Update on Wilson Disease
Aggarwal Annu and Bhatt Mohit
The Role of Timing in Nerve Reconstruction
Lars B. Dahlin An Update on Primary Familial Brain Calcification
R.R. Lemos, J.B.M.M. Ferreira, M.P. Keasey,
Future Perspectives in Nerve Repair and
and J.R.M. Oliveira
Regeneration
Pierluigi Tos, Giulia Ronchi, Stefano Geuna, and INDEX
Bruno Battiston
INDEX
Volume 111
Volume 110 History of Acupuncture Research
Yi Zhuang, Jing-jing Xing, Juan Li, Bai-Yun Zeng,
The Relevance of Metals in the Pathophysiology of and Fan-rong Liang
Neurodegeneration, Pathological Considerations
Effects of Acupuncture Needling with Specific
Kurt A. Jellinger
Sensation on Cerebral Hemodynamics and
Pantothenate Kinase-Associated Neurodegener- Autonomic Nervous Activity in Humans
ation (PKAN) and PLA2G6-Associated Neuro- Kouich Takamoto, Susumu Urakawa, Kazushige
degeneration (PLAN): Review of Two Major Sakai, Taketoshi Ono, and Hisao Nishijo
Neurodegeneration with Brain Iron Accumula-
Acupuncture Point Specificity
tion (NBIA) Phenotypes
Jing-jing Xing, Bai-Yun Zeng, Juan Li, Yi Zhuang,
Manju A. Kurian and Susan J. Hayflick
and Fan-rong Liang
Mitochondrial Membrane Protein-Associated
Acupuncture Stimulation Induces Neurogenesis
Neurodegeneration (MPAN)
in Adult Brain
Monika Hartig, Holger Prokisch, Thomas Meitinger,
Min-Ho Nam, Kwang Seok Ahn, and Seung-Hoon
and Thomas Klopstock
Choi
BPAN: The Only X-Linked Dominant NBIA
Acupuncture and Neurotrophin Modulation
Disorder
Marzia Soligo, Stefania Lucia Nori, Virginia Protto,
T.B. Haack, P. Hogarth, A. Gregory, P. Prokisch,
Fulvio Florenzano, and Luigi Manni
and S.J. Hayflick
Acupuncture Stimulation and Neuroendocrine
Neuroferritinopathy
Regulation
M.J. Keogh, C.M. Morris, and P.F. Chinnery
Jung-Sheng Yu, Bai-Yun Zeng, and
Aceruloplasminemia: An Update Ching-Liang Hsieh
Satoshi Kono
Current Development of Acupuncture Research
Therapeutic Advances in Neurodegeneration with in Parkinson’s Disease
Brain Iron Accumulation Bai-Yun Zeng, Sarah Salvage, and
Giovanna Zorzi and Nardo Nardocci Peter Jenner
Contents of Recent Volumes 369

Acupuncture Therapy for Stroke Patients Animal Models Recapitulating the Multifactorial
Xin Li and Qiang Wang Origin of Tourette Syndrome
Simone Macrı`, Martina Proietti Onori, Veit
Effects of Acupuncture Therapy on
Roessner, and Giovanni Laviola
Alzheimer’s Disease
Bai-Yun Zeng, Sarah Salvage, and Peter Jenner Neuroendocrine Aspects of Tourette Syndrome
Davide Martino, Antonella Macerollo, and
Acupuncture Therapy for Psychiatric Illness
James F. Leckman
Karen Pilkington
Clinical Pharmacology of Dopamine-Modulating
Acupuncture for the Treatment of Insomnia
Agents in Tourette’s Syndrome
Kaicun Zhao
Sabine Mogwitz, Judith Buse, Stefan Ehrlich, and
Acupuncture for the Treatment of Drug Veit Roessner
Addiction
Clinical Pharmacology of Nondopaminergic
Cai-Lian Cui, Liu-Zhen Wu, and Yi-jing Li
Drugs in Tourette Syndrome
Acupuncture Regulation of Blood Pressure: Andreas Hartmann
Two Decades of Research
Antiepileptic Drugs and Tourette Syndrome
John C. Longhurst and Stephanie Tjen-A-Looi
Andrea E. Cavanna and Andrea Nani
Effect and Mechanism of Acupuncture on
Clinical Pharmacology of Comorbid Obsessive–
Gastrointestinal Diseases
Compulsive Disorder in Tourette Syndrome
Toku Takahashi
Valeria Neri and Francesco Cardona
INDEX
Clinical Pharmacology of Comorbid Attention
Deficit Hyperactivity Disorder in Tourette
Syndrome
Volume 112 Renata Rizzo and Mariangela Gulisano
Emerging Treatment Strategies in Tourette
An Introduction to the Clinical Phenomenology
Syndrome: What’s in the Pipeline?
of Tourette Syndrome
Davide Martino, Namrata Madhusudan, Panagiotis C. Termine, C. Selvini, G. Rossi, and
U. Balottin
Zis, and Andrea E. Cavanna
Tics and Other Stereotyped Movements as Side
Functional Neuroanatomy of Tics
Effects of Pharmacological Treatment
Irene Neuner, Frank Schneider, and N. Jon Shah
Marcos Madruga-Garrido and Pablo Mir
Functional Imaging of Dopaminergic Neurotrans-
INDEX
mission in Tourette Syndrome
Bàrbara Segura and Antonio P. Strafella
Nondopaminergic Neurotransmission in the
Pathophysiology of Tourette Syndrome
Volume 113
Patrick T. Udvardi, Ester Nespoli, Autism Spectrum Disorder and the Cerebellum
Francesca Rizzo, Bastian Hengerer, and Esther B.E. Becker and Catherine J. Stoodley
Andrea G. Ludolph
Contribution of Long Noncoding RNAs to
Reinforcement Learning and Tourette Syndrome Autism Spectrum Disorder Risk
Stefano Palminteri and Mathias Pessiglione Brent Wilkinson and Daniel B. Campbell
Genetic Susceptibility and Neurotransmitters in Identifying Essential Cell Types and Circuits in
Tourette Syndrome Autism Spectrum Disorders
Peristera Paschou, Thomas V. Fernandez, Susan E. Maloney, Michael A. Rieger, and Joseph
Frank Sharp, Gary A. Heiman, and D. Dougherty
Pieter J. Hoekstra
Connecting Signaling Pathways Underlying
Pharmacological Animal Models of Tic Communication to ASD Vulnerability
Disorders Stephanie Lepp, Ashley Anderson, and Genevieve
Kevin W. McCairn and Masaki Isoda Konopka
370 Contents of Recent Volumes

MET Receptor Tyrosine Kinase as an Autism Mechanisms of Ictogenesis

Genetic Risk Factor Thomas Blauwblomme, Premysl Jiruska, and Gilles
Yun Peng, Matthew Huentelman, Christopher Huberfeld
Smith, and Shenfeng Qiu
Seizure Termination
Transcriptional Dysregulation of Neocortical Frederic Zubler, Andreas Steimer, Heidemarie Gast,
Circuit Assembly in ASD and Kaspar A. Schindler
Kenneth Y. Kwan
Epileptic Focus and Alteration of Metabolism
Motor Skill in Autism Spectrum Disorders: A Jakub Otáhal, Jaroslava Folbergrová, Richard
Subcortical View Kovacs, Wolfram S. Kunz, and Nicola Maggio
Leanne Chukoskie, Jeanne Townsend, and Marissa
Modern Techniques of Epileptic Focus
Westerfield
Localization
Orchestration of Neurodevelopmental Programs Lukas Martinkovic, Hrvoje Hecimovic, Vlastimil
by RBFOX1: Implications for Autism Spectrum Sulc, Radek Marecek, and Petr Marusic
Disorder
From Treatment to Cure: Stopping Seizures,
Brent R. Bill, Jennifer K. Lowe, Christina T.
Preventing Seizures, and Reducing Brain Propen-
DyBuncio, and Brent L. Fogel
sity to Seize
Immune Dysregulation in Autism Spectrum Ivan Pavlov and Stephanie Schorge
Disorder
INDEX
Elaine Y. Hsiao
Autism Susceptibility Genes and the Transcrip-
tional Landscape of the Human Brain
Shingo Miyauchi and Irina Voineagu Volume 115
INDEX Environmental Alterations of Epigenetics Prior to
the Birth
Volume 114 Chiao-Ling Lo and Feng C. Zhou
Transgenerational Epigenetics and Brain Disorders
Modern Concepts of Focal Epileptic Networks
Nadia Rachdaoui and Dipak K. Sarkar
Premysl Jiruska, Marco de Curtis, and John G.R.
Jefferys The Epigenetic Landscape of Alcoholism
Harish R. Krishnan, Amul J. Sakharkar,
Neocortical Focus: Experimental View
Tara L. Teppen, Tiffani D.M. Berkel, and
Igor Timofeev, Sylvain Chauvette, and
Subhash C. Pandey
Sara Soltani
Epigenetic Regulatory Mechanisms in Stress-
Malformations of Cortical Development and
Induced Behavior
Neocortical Focus
Sumana Chakravarty, Salil Saurav Pathak,
Heiko J. Luhmann, Werner Kilb, and Hans
Swati Maitra, Nitin Khandelwal, Karisetty
Clusmann
Bhanu Chandra, and Arvind Kumar
Limbic Networks and Epileptiform Synchroniza-
Epigenetics of Schizophrenia: An Open and Shut
tion: The View from the Experimental Side
Case
Charles Behr, Margherita D’Antuono, Shabnam
David P. Gavin and Christina Floreani
Hamidi, Rochelle Herrington, Maxime Levesque,
Pariya Salami, Zahra Shiri, Rüdiger K€ohling, and Epigenetic Mechanisms in Autism Spectrum
Massimo Avoli Disorder
Adrian Zhubi, Edwin H. Cook, Alessandro
Limbic Networks: Clinical Perspective
Guidotti, and Dennis R. Grayson
Aylin Y. Reid and Richard J. Staba
MicroRNAs and Ethanol Toxicity
Modern Concepts of Seizure Modeling
Rajesh C. Miranda
Christophe Bernard, Sebastien Naze, Timothee
Proix, and Viktor K. Jirsa INDEX
Contents of Recent Volumes 371

Volume 116 Cerebellar Mechanisms of Learning and Plasticity

Revealed by Delay Eyelid Conditioning
IntroductiontoSequencing the Brain Transcriptome Michael D. Mauk, Wenke Li, Andrei Khilkevich,
Robert Hitzemann, Priscila Darakjian, Nikki and Hunter Halverson
Walter, Ovidu Iancu, Robert Searles, and
Shannon McWeeney Cerebellar Long-Term Potentiation: Cellular
Mechanisms and Role in Learning
Analysis Considerations for Utilizing RNA-Seq to Giorgio Grasselli and Christian Hansel
Characterize the Brain Transcriptome
Christina Zheng, Sunita Kawane, Daniel Bottomly, The Ontogeny of Associative Cerebellar Learning
and Beth Wilmot John H. Freeman

Data Integration and Reproducibility for High- INDEX

Throughput Transcriptomics
Michael Mooney and Shannon McWeeney
Coexpression and Cosplicing Network Volume 118
Approaches for the Study of Mammalian Brain Neuroimmune Mechanisms of Alcohol and Drug
Transcriptomes Addiction
Ovidiu Dan Iancu, Alexander Colville, Priscila Changhai Cui, David Shurtleff, and R. Adron
Darakjian, and Robert Hitzemann Harris
Splicing in the Human Brain Neuroimmune Pathways in Alcohol Consump-
Ammar Zaghlool, Adam Ameur, Lucia Cavalier, tion: Evidence from Behavioral and Genetic
and Lars Feuk Studies in Rodents and Humans
Understanding Complex Transcriptome Dynam- Gizelle Robinson, Dana Most, Laura B. Ferguson,
ics in Schizophrenia and Other Neurological Dis- Jody Mayfield, R. Adron Harris, and
eases Using RNA Sequencing Yuri A. Blednov
Xi Wang and Murray J. Cairns Fetal Alcohol Spectrum Disorders and
The Central Role of Noncoding RNA in the Neuroimmune Changes
Brain Paul D. Drew and Cynthia J.M. Kane
Boris Guennewig and Antony A. Cooper Role of Microglia in Regulation of Ethanol Neu-
Genetics of Gene Expression in CNS rotoxic Action
Robert W. Williams and Ashutosh K Pandey Lucy Chastain and Dipak K. Sarkar

Transcriptomic Changes in Brain Development Functions of the Chemokine Receptor CXCR4

Allissa A. Dillman and Mark R. Cookson in the Central Nervous System and Its Regulation
by μ-Opioid Receptors
Gene Expression in the Addicted Brain Bradley Nash and Olimpia Meucci
Zhifeng Zhou, Mary-Anne Enoch, and David
Goldman Discovery of a Novel Site of Opioid Action at the
Innate Immune Pattern-Recognition Receptor
RNA-Seq Reveals Novel Transcriptional Reor- TLR4
ganization in Human Alcoholic Brain Jonathan Henry W. Jacobsen, Linda R. Watkins,
Sean P. Farris and R. Dayne Mayfield and Mark R. Hutchinson
INDEX Neuroimmune Basis of Methamphetamine
Toxicity
Jennifer M. Loftis and Aaron Janowsky
Marijuana Use Brain Immune Mechanisms
Volume 117 Guy A. Cabral and Melissa Jamerson
Learning-Induced Structural Plasticity in the Interactions of HIV and Drugs of Abuse: The
Cerebellum Importance of Glia and Host Genetic Factors
Hiroshi Nishiyama Kurt F. Hauser and Pamela E. Knapp
372 Contents of Recent Volumes

Neuroimmune Basis of Alcoholic Brain Damage Adenosine Receptors and Huntington’s Disease
Fulton T. Crews and Ryan P. Vetreno Chien-fei Lee and Yijuang Chern
Converging Actions of Alcohol on Liver and Adenosine Receptors and Epilepsy: Current Evi-
Brain Immune Signaling dence and Future Potential
Gyongyi Szabo and Dora Lippai Susan A. Masino, Masahito Kawamura, Jr., and
David N. Ruskin
Opportunities for the Development of
Neuroimmune Therapies in Addiction Adenosine Receptor Control of Cognition in
Lara A. Ray, Daniel Roche, Keith Heinzerling, and Normal and Disease
Steve Shoptaw Jiang-Fan Chen
Use of Addictive Substances and NeuroHIV Adenosine Receptors in Cerebral Ischemia
Sulie L. Chang, Kaitlyn P. Connaghan, Yufeng Alessia Melani, Anna Maria Pugliese, and Felicita
Wei, and Ming D. Li Pedata
INDEX Roles of Adenosine and its Receptors in Sleep–
Wake Regulation
Zhi-Li Huang, Ze Zhang, and Wei-Min Qu
Volume 119 Involvement of Adenosine A2A Receptors in
Adenosine Receptor Neurobiology: Overview Depression and Anxiety
Jiang-Fan Chen, Chien-fei Lee, and Yijuang Chern Koji Yamada, Minoru Kobayashi, and Tomoyuki
Kanda
Adenosine Receptor PET Imaging in Human
Brain The Adenosine Neuromodulation System in
Masahiro Mishina and Kiich Ishiwata Schizophrenia
Daniel Rial, Diogo R. Lara, and Rodrigo A. Cunha
An Overview of Adenosine A2A Receptor Antag-
onists in Parkinson’s Disease INDEX
Peter Jenner
Mode of Action of Adenosine A2A Receptor Volume 120
Antagonists as Symptomatic Treatment for Par-
The Story of “Speed” from “Cloud Nine” to
kinson’s Disease
Brain Gain
Akihisa Mori
Andrew Lees, Katrin Sikk, and Pille Taba
Adenosine Receptors and Dyskinesia in
Amphetamine-Type Stimulants: The Early His-
Pathophysiology
tory of Their Medical and Non-Medical Uses
Masahiko Tomiyama
Nicolas Rasmussen
Clinical/Pharmacological Aspect of Adenosine
Miracle or Menace?
A2A Receptor Antagonist for Dyskinesia
Mike Jay
Tomoyuki Kanda and Shin-ichi Uchida
Psychostimulants: Basic and Clinical Pharmacology
Interaction of Adenosine Receptors with Other
Andrew C. McCreary, Christian P. Müller, and
Receptors from Therapeutic Perspective in Par-
Małgorzata Filip
kinson’s Disease
Nicolas Morin and Thérèse Di Paolo Epigenetic Mechanisms of Psychostimulant-
Induced Addiction
Effects of the Adenosine A2A Receptor Antagonist
Anti Kalda and Alexander Zharkovsky
on Cognitive Dysfunction in Parkinson’s Disease
Shin-ichi Uchida, Takako Kadowaki-Horita, and Experimental Models on Effects of Psycho-
Tomoyuki Kanda stimulants
Sulev Kõks
Clinical Nonmotor Aspect of A2A Antagonist in
PD Treatment Neurologic Complications of Psychomotor Stim-
Masahiro Nomoto, Masahiro Nagai, and Noriko ulant Abuse
Nishikawa Juan Sanchez-Ramos
Contents of Recent Volumes 373

Neurobehavioral Sequelae of Psychostimulant Comparative Proteomics for the Evaluation

Abuse of Protein Expression and Modifications in
Atbin Djamshidian Neurodegenerative Diseases
Antonio Conti and Massimo Alessio
Neuropsychiatric Adverse Effects of Amphet-
amine and Methamphetamine INDEX
Jaanus Harro
“Addicted to Euphoria”: The History, Clinical
Presentation, and Management of Party Drug Volume 122
Misuse
Jenny Bearn and Matthew O’Brien Utility of Autoantibodies as Biomarkers for
Diagnosis and Staging of Neurodegenerative
“Natural Amphetamine” Khat: A Cultural Tradi- Diseases
tion or a Drug of Abuse? Cassandra DeMarshall, Abhirup Sarkar, Eric P.
Nilesh B. Patel Nagele, Eric Goldwaser, George Godsey, Nimish K.
Methcathinone “Kitchen Chemistry” and Perma- Acharya, and Robert G. Nagele
nent Neurological Damage Metabolomics of Neurodegenerative Diseases
Katrin Sikk and Pille Taba Alejandro Botas, Hannah Moore Campbell,
“Legal Highs” – An Emerging Epidemic of Novel Xu Han, and Mirjana Maletic-Savatic
Psychoactive Substances Parkinson’s Disease: In Vivo Brain Metabolomics
Jolanta B. Zawilska by MRS
Psychostimulants and Artistic, Musical, and Liter- Mario Rango
ary Creativity Recent Advances and Applications of Met-
Iain Smith abolomics to Investigate Neurodegenerative
Opium as a Literary Stimulant: The Case of Diseases
Samuel Taylor Coleridge Clara Ibáñez, Alejandro Cifuentes,
Neil Vickers and Carolina Simó

INDEX Lipidomics of Human Brain Aging and

Alzheimer’s Disease Pathology
Alba Naudı´, Rosanna Cabre, Mariona Jove,
Victoria Ayala, Hugo Gonzalo, Manuel
Portero-Otı´n, Isidre Ferrer, and
Volume 121 Reinald Pamplona
Alzheimer’s Disease: Genomics and Beyond INDEX
Fuhai Song, Guangchun Han,
Zhouxian Bai, Xing Peng, Jiajia Wang, and
Hongxing Lei
The Potential of Proteomics in Understanding
Volume 123
Neurodegeneration Unifying Mechanism of Controlling Kir3 Chan-
Ramavati Pal, Jan Petter Larsen, and Simon Geir nel Activity by G Proteins and Phosphoinositides
Moller Diomedes E. Logothetis, Rahul Mahajan,
Scott K. Adney, Junghoon Ha, Takeharu Kawano,
Proteomics Approach to Identify Biomarkers in
Xuan-Yu Meng, and Meng Cui
Neurodegenerative Diseases
Annapurna Nayak, Gregory Salt, Sunil K. Verma, The Roles of Gβγ and Gα in Gating and Regula-
and Uday Kishore tion of GIRK Channels
Nathan Dascal and Uri Kahanovitch
Uncovering Neurodegenerative Protein Modifi-
cations via Proteomic Profiling RGS Redundancy and Implications in GPCR–
Xavier Gallart-Palau, Aida Serra, GIRK Signaling
and Siu Kwan Sze Craig A. Doupnik
374 Contents of Recent Volumes

Structural Insights into GIRK Channel Function The Role of Depression in the Uptake and Main-
Ian W. Glaaser and Paul A. Slesinger tenance of Cigarette Smoking
Janet Audrain-McGovern, Adam M. Leventhal,
Localization and Targeting of GIRK Channels in
and David R. Strong
Mammalian Central Neurons
Rafael Luján and Carolina Aguado Part IV: Parkinson’s Disease
Nicotine and Nicotinic Receptor Drugs: Potential
GIRK Channel Plasticity and Implications for
for Parkinson’s Disease and Drug-Induced Move-
Drug Addiction
ment Disorders
Ezequiel Marron Fernandez de Velasco,
Maryka Quik, Tanuja Bordia, Danhui Zhang, and
Nora McCall, and Kevin Wickman
Xiomara A. Perez
GIRK Channels: A Potential Link Between
Part V: Alzheimer’s Disease
Learning and Addiction
Nicotinic Cholinergic Mechanisms in Alzheimer’s
Megan E. Tipps and Kari J. Buck
Disease
Behavioral and Genetic Evidence for GIRK Jianxin Shen and Jie Wu
Channels in the CNS: Role in Physiology, Path-
INDEX
ophysiology, and Drug Addiction
Jody Mayfield, Yuri A. Blednov, and R. Adron
Harris
INDEX Volume 125
The Endocannabinoid Signaling System in the
CNS: A Primer
Volume 124 Cecilia J. Hillard
Evidence for a Role of Adolescent Endo-
Part I: Introductory Chapter
cannabinoid Signaling in Regulating HPA Axis
Neuronal Nicotinic Acetylcholine Receptor Stress Responsivity and Emotional Behavior
Structure and Function and Response to Nicotine Development
John A. Dani Tiffany T.-Y. Lee and Boris B. Gorzalka
Part II: Schizophrenia The Endocannabinoid System and Its Role in
The Role of Nicotine in Schizophrenia Regulating the Intrinsic Neural Circuitry of the
Robert E. Featherstone and Steven J. Siegel Gastrointestinal Tract
Samantha M. Trautmann and Keith A. Sharkey
Neuronal α7 Nicotinic Receptors as a Target for
the Treatment of Schizophrenia Endocannabinoid Mechanisms Influencing Nausea
Tanya L. Wallace and Daniel Bertrand Martin A. Sticht, Erin M. Rock, Cheryl L.
Limebeer, and Linda A. Parker
Role of the Neuregulin Signaling Pathway in
Nicotine Dependence and Co-morbid Disorders Endocannabinoid Regulation of Neuroendocrine
Miranda L. Fisher, Anu Loukola, Jaakko Kaprio, Systems
and Jill R. Turner Jeffrey G. Tasker, Chun Chen, Marc O. Fisher,
Xin Fu, Jennifer R. Rainville, and Grant L. Weiss
Effective Cessation Strategies for Smokers with
Schizophrenia The Role of the Brain’s Endocannabinoid System
A. Eden Evins and Corinne Cather in Pain and Its Modulation by Stress
Louise Corcoran, Michelle Roche, and David P. Finn
Part III: Mood Disorders
Role of the Brain’s Reward Circuitry in Depres- Endocannabinoid Signaling in Motivation,
sion: Transcriptional Mechanisms Reward, and Addiction: Influences on
Eric J. Nestler Mesocorticolimbic Dopamine Function
Claudia Sagheddu, Anna Lisa Muntoni, Marco
Nicotine Addiction and Psychiatric Disorders
Pistis, and Miriam Melis
Munir Gunes Kutlu, Vinay Parikh, and
Thomas J. Gould INDEX
Contents of Recent Volumes 375

Volume 126 Genes and Alcohol Consumption: Studies with

Mutant Mice
Considerations in the Evaluation of Potential J. Mayfield, M.A. Arends, R.A. Harris, and
Efficacy of Medications for Alcohol and Drug Y.A. Blednov
Use Disorders: An Editorial
M. Egli, D.A. White, and J.B. Acri Gene Targeting Studies of Hyperexcitability and
Affective States of Alcohol Withdrawal in
A Pressing Need for Pharmacotherapy Develop- Rodents
ment to Treat Drug Addiction: An Editorial from G.D. Greenberg and J.C. Crabbe
a Legal Perspective
B. Andraka-Christou Abstinence-Conflict Model: Toward an Optimal
Animal Model for Screening Medications Pro-
Identification of Treatment Targets in a Genetic moting Drug Abstinence
Mouse Model of Voluntary Methamphetamine J.A. Peck
Drinking
T.J. Phillips, J.R.K. Mootz, and C. Reed Prairie Voles as a Model to Screen Medications for
the Treatment of Alcoholism and Addictions
Screening Medications for the Treatment of A.E. Ryabinin and C.M. Hostetler
Cannabis Use Disorder
L.V. Panlilio, Z. Justinova, J.M. Trigo, and Animal Models for Medication Development and
B. Le Foll Application to Treat Fetal Alcohol Effects
S. Barron, A. Hawkey, L. Fields, and
How can we Improve on Modeling Nicotine J.M. Littleton
Addiction to Develop Better Smoking Cessation
Treatments? Using In Vitro Electrophysiology to Screen Med-
M. Shoaib and Y. Buhidma ications: Accumbal Plasticity as an Engram of
Alcohol Dependence
An Animal Model of Alcohol Dependence R. Renteria, Z.M. Jeanes, R.A. Mangieri,
to Screen Medications for Treating Alcoholism E.Y. Maier, D.M. Kircher, T.R. Buske, and
H.C. Becker and M.F. Lopez R.A. Morrisett
A Genetic Animal Model of Alcoholism for The Zebrafish, a Novel Model Organism for
Screening Medications to Treat Addiction Screening Compounds Affecting Acute and
R.L. Bell, S. Hauser, Z.A. Rodd, T. Liang, Chronic Ethanol-Induced Effects
Y. Sari, J. McClintick, S. Rahman, and S. Tran, A. Facciol, and R. Gerlai
E.A. Engleman
INDEX
Animal Models and the Development of Vaccines
to Treat Substance Use Disorders
O. Ohia-Nwoko, T.A. Kosten, and C.N. Haile