13 Anaesthesia and Analgesia

Patricia Hedenqvist, Sweden and

Paul Flecknell, United Kingdom

CoNTENTs
Objectives....................................................................................................................................... 313
Key Factors .................................................................................................................................... 314
13.1 Pre-Anaesthetic Consideration ........................................................................................... 314
13.2 Anaesthetic Agents ............................................................................................................. 315
13.2.1 Inhalation Anaesthesia.......................................................................................... 315
13.2.1.1 Volatile Agents..................................................................................... 317
13.2.1.2 Methods of Administration.................................................................. 317
13.2.1.3 Operator Safety .................................................................................... 318
13.2.2 Injection Anaesthesia ............................................................................................ 318
13.2.2.1 Injectable Agents.................................................................................. 318
13.2.2.2 Methods of Administration.................................................................. 321
13.3 Neuromuscular Blocking Agents (NMBAs)....................................................................... 322
13.4 Assisted Ventilation ............................................................................................................ 323
13.5 Monitoring during Anaesthesia .......................................................................................... 323
13.5.1 Depth of Anaesthesia ............................................................................................ 323
13.5.2 Respiratory Function............................................................................................. 324
13.5.3 Cardiovascular Function ....................................................................................... 324
13.5.4 Maintenance of Body Temperature....................................................................... 324
13.5.5 Maintenance of Fluid and Electrolyte Balance..................................................... 325
13.6 Post-Anaesthetic Care......................................................................................................... 325
13.6.1 Warmth and Comfort ............................................................................................ 325
13.6.2 Fluid and Nutritional Support ............................................................................... 326
13.7 Analgesia ............................................................................................................................ 326
13.7.1 Evaluation of Pain ................................................................................................. 326
13.7.1.1 Physiological Parameters ..................................................................... 326
13.7.1.2 Behaviour ............................................................................................. 326
13.7.2 Drugs and Doses ................................................................................................... 327
13.7.2.1 Opioids ................................................................................................. 327
13.7.2.2 Non-Steroidal Anti-Inflammatory Agents (NSAIDs).......................... 328
13.7.2.3 Local Anaesthetic Agents .................................................................... 328
13.7.3 Methods of Delivery ............................................................................................. 329
13.8 Conclusions......................................................................................................................... 329
13.9 Questions Unresolved ......................................................................................................... 329
References...................................................................................................................................... 329

313
314 The COST Manual of Laboratory Animal Care and Use

oBjECTivEs
The use of anaesthesia and analgesia allows for surgery and other procedures to be undertaken in
laboratory animals without the distress and pain they would otherwise cause. Anaesthesia is used
in a variety of circumstances, for example when disease models are developed, for instrumentation
of animals, and for collection of research data. The aims of this section are to outline what is gen­
erally accepted as current best practise, and to provide references to more detailed descriptions of
specific techniques. Aside from the animal welfare issues mentioned above, providing anaesthesia
that is appropriate for a specific research procedure is of considerable importance if meaningful data
is to be obtained. Inappropriate selection of anaesthetic agents or failure to provide high standards of
pre-, intra-, and post-operative care can all adversely affect the quality of data obtained from research
animals. Similarly, the provision of effective post-operative analgesia is important both for reasons of
animal welfare and to reduce the potentially major confounding factors caused by unalleviated pain.
A wide range of different anaesthetic techniques is available, and although this document
seeks to promote “best practise”, this is more often dependent upon making an appropriate and
considered choice of anaesthetic, and application of good peri-operative care, rather than requir­
ing the choice of specific anaesthetic agents. Inhalation, injection or local anaesthetic techniques
may all be appropriate in particular circumstances and the choice depends on the aim, type and
length of the procedure; the animal species; and the particular research objectives.
Analgesic drugs may be used as part of a balanced anaesthetic regimen, to treat post-operative
pain or to relieve pain accompanying induced or spontaneous disease. Opioid drugs, non-steroidal
anti-inflammatory drugs (NSAIDs), and local anaesthetics may be administered separately, or in
combination, to achieve efficient pain relief.

KEy fACTors
1. Whenever possible, painful procedures should be performed under general or local
anaesthesia.
2. Stressful procedures should be performed under anaesthesia or sedation whenever
possible.
3. Physiological conditions may be more stable, and reproducible between studies, when
using inhalation anaesthesia compared with injection anaesthesia, and this may allow for a
reduction of experimental group sizes, and a consequent reduction in animal use.
4. Anaesthetic and analgesic drugs should be chosen to fit the type and length of the proce­
dure and the type and degree of pain expected.
5. Animals that may be at risk of experiencing pain should be examined and treated
appropriately.
6. If there is doubt whether an animal is experiencing pain, the animal should be given the
benefit of the doubt and treated accordingly.
7. It is not only analgesic drugs that can interfere with research objectives, but also pain itself.

13.1 PrE-ANAEsThETiC CoNsidErATioN

Before research animals are anaesthetised they should be acclimatised to the housing conditions in
the animal facility. Transport to the facility, and changes to caging, diet, environment and social group
all influence the animals’ physiology. Most of the consequent physiological changes in the cardio­
vascular, endocrine, immune and central nervous systems have returned to baseline within 7 days of
transportation (Obernier and Baldwin 2006), although some effects may persist for longer periods.
During this period of acclimatisation it is beneficial to accustom the animals to handling and to
monitor their body weight. Change in body weight is a good general indicator of health and may be
used to monitor recovery after anaesthesia and surgery. Rodents are especially likely to lose body
weight rapidly, because of their high-metabolic rate.
Anaesthesia and Analgesia 315

In contrast to many other animal species it is not necessary to withhold food from rodents and
rabbits before induction of anaesthesia, because of their inability to vomit. Withholding food from
small or young animals may even be hazardous, due to induction of hypoglycaemia. Withdrawal
of food overnight may also cause loss of body mass, a decrease in blood fatty acid concentra­
tion and changes in water intake (Hedrich and Bullock 2004; Suckow, Weisbroth, and Franklin
2006).
Sedative or analgesic agents may be administered before induction of anaesthesia in order
to reduce stress, to reduce the anaesthetic drug dose and as part of the management of post­
operative pain. Reduction of anaesthetic drug dose also reduces dose-related side effects (see
Table 13.1).
The use of pre-anaesthetic medication is often restricted to larger species, where there can
be problems providing effective manual restraint. The reduction in stress is also important,
however, and species such as rabbits that are easily stressed by handling are best sedated before
they are removed from their cage or pen (Flecknell 2009). The majority of small rodents are
anaesthetised either using an inhalational agent in an anaesthetic induction chamber, or by
using injectable agents for anaesthetic induction. Handling the animal to administer a pre-
anaesthetic sedative is therefore not a routine procedure. However, it is important to note that
some analgesic agents require 30 minutes or more to achieve full effect, so treatment with
these agents as part of pre-anaesthetic medication can ensure that more effective analgesia is
provided.
Anticholinergic agents may be administered to prevent excessive salivation, bronchial secretion
and bradycardia induced by opioids or vagal stimulation. Atropine is useful except in rabbits and
some rat strains, which have high levels of atropine esterase (Olson et al. 1994). Glycopyrrolate is a
longer-acting anticholinergic, which does not cross the blood–brain barrier or the placenta (Lemke
2007) and is not inactivated by atropine esterases.
Other pre-anaesthetic measures should include preparing procedures and equipment for intra­
operative support; for example, heating pads must be switched on ahead of time so that they reach
the required temperature. Emergency drugs must be readily accessible (e.g., antagonists to some of
the anaesthetic agents that may be used, IV fluids, adrenaline).
Finally, it is important to keep an anaesthetic record for each animal. This will help identify any
potential problems as they develop, and also aid in preparing reproducible protocols for subsequent
groups of animals.

13.2 ANAEsThETiC AgENTs

13.2.1 inhalation anaesthesia
The benefits of using inhalation anaesthesia are manifold; ease of delivery for induction and main­
tenance, good control over depth of anaesthesia, low levels of drug metabolism (depending upon
the agent used), limited effects on the physiology of the animal, and rapid recovery especially after
short periods of anaesthesia. Another benefit is that when oxygen is used as the carrier gas, hypoxia
during anaesthesia is prevented. The use of air alone as the carrier gas is not recommended, with the
exception of very short procedures (<10 minutes). Almost all anaesthetics reduce lung ventilation
and cardiac output, which results in poor tissue oxygenation, and therefore oxygen should be used to
a minimum of 30% of the carrier gas (McDonell and Kerr 2007). Using pure oxygen as carrier gas
renders a very high arterial blood oxygen partial pressure. More physiological levels are achieved
by mixing oxygen with air, nitrogen or nitrous oxide.
One advantage of inhalational agents is that they can be administered in a reproducible and
controlled manner. Although this is possible when using injectable agents given by the intrave­
nous route (e.g., propofol + alfentanil) this is technically difficult to accomplish in very small
animals such as rodents. The effects of administering injectable agents by the intraperitoneal (IP),
 316

TABLE 13.1
doses of Commonly used Pre-Anaesthetic and Emergency drugs in rodents and rabbits
drug mouse rat hamster guinea Pig rabbit
Anticholinergic Atropine 0.04 mg/kg SC 0.04 mg/kg SC 0.04 mg/kg SC 0.05 mg/kg SC 1–2 mg/kg SC
Glycopyrrolate 0.5 mg/kg SC 0.1 mg/kg IV, 0.5 mg/kg SC
Sedatives Medetomidine 30–100 μg/kg, SC, IP 30–100 μg/kg, SC, IP 100 μg/kg, SC, IP 0.1–0.5 mg/kg SC
Xylazine 5–10 mg/kg IP 1–5 mg/kg IP 5 mg/kg IP 2–5 mg/kg SC
Midazolam 5 mg/kg IP 5 mg/kg IP, IM 0.5–2 mg/kg IV, SC
Analgesics Buprenorphine 0.05–0.1 mg/kg SC 0.01–0.05 mg/kg SC 0.05 mg/kg SC 0.01–0.05 mg/kg SC, IV
Carprofen 5 mg/kg SC 5 mg/kg SC 1.5 mg/kg po
Antagonists Atipamezole 0.1–1 mg/kg IM, IP, SC, IV 0.1–1 mg/kg IM, SC, IV
Naloxone 0.01–0.1 mg/kg IV, IM, IP 0.01–0.1 mg/kg IV, IM
Emergency drugs Adrenaline 0.3ml/kg of 1:10,000 IV or intracardiac
Atropine 0.02 mg/kg IV or intracardiac
Lidocaine 2mg/kg IV or intracardiac

Source: Flecknell, P. A., Laboratory Animal Anaesthesia, 3rd ed., Elsevier, London, 2009.
The COST Manual of Laboratory Animal Care and Use
Anaesthesia and Analgesia 317

intramuscular (IM), or subcutaneous (SC) routes are much more variable, and hence physiological
parameters may vary much less during inhalation anaesthesia compared to most injection anaesthe­
sia regimes. This reduction in variation may substantially reduce the number of animals needed in
order to achieve identical statistical sensitivity (Chaves, Weinstein, and Bauer 2001).
The potency of volatile anaesthetics is defined as minimum alveolar concentration (MAC). It is
calculated as the concentration at which 50% of animals do not respond to a nociceptive (i.e., pain­
ful) stimulus and so is equivalent to the ED50 of the agent. To ensure most animals are unresponsive,
between 1.2 and 1.4 MAC should be given (the ED95). Higher concentrations are not usually needed,
except when inducing anaesthesia, 2.0 MAC represents a deep level of anaesthesia and in some
cases even an anaesthetic overdose (Steffey and Mama 2007). Pre-medication with tranquillisers,
sedatives and opioid drugs, reduces MAC, as does increasing age.
13.2.1.1 volatile Agents
Although several different agents have been used for laboratory animal anaesthesia, some are no
longer available as anaesthetic agents in Europe (e.g., ether and methoxyflurane), and halothane is
also no longer being supplied in many member states. Desflurane is used in medical anaesthetic
practise, but is rarely used in a research animal setting. The two agents that can be recommended
for routine use are isoflurane and sevoflurane.

13.2.1.1.1 Isoflurane
Isoflurane is a halogenated ether and the most commonly used volatile agent in the research setting
today because of its high safety and efficiency. The MAC is 1.3–1.5% for isoflurane in the adult rat
and mouse, and 2.1% in the rabbit (Steffey and Mama 2007). In 2-day-old Wistar rats, the MAC is
1.9% (Orliaguet et al. 2001). Induction of, and recovery from anaesthesia is very rapid and only 0.2%
of the inhaled isoflurane undergoes biotransformation.
Like all volatile agents, isoflurane depresses respiration in a dose-dependent fashion. Cardiac
output is preserved at clinically useful concentrations (Steffey and Mama 2007).

13.2.1.1.2 Sevoflurane
Sevoflurane has a lower blood:gas partition coefficient than isoflurane, which means that induction
and recovery are even more rapid than with isoflurane. The MAC for sevoflurane is 2.7% in the
mouse, 2.4–3% in the rat and 3.7% in the rabbit (Steffey and Mama 2007). The effects on respira­
tion and circulation are similar to those of isoflurane. Approximately 2–3% of inhaled sevoflurane
undergoes metabolism.

13.2.1.2 methods of Administration

13.2.1.2.1 Vaporiser
Calibrated vaporisers are necessary to achieve safe delivery of modern volatile anaesthetics, which
can otherwise reach dangerously high concentrations. Two main types of vaporisers can be used for
rodent and rabbit anaesthesia: traditional vaporisers, in which the carrier gas passes through the agent
to acquire the anaesthetic vapour (Hartsfield 2007), and a more recent type, which uses a syringe driver
to inject the liquid anaesthetic into the carrier gas flow (http://www.univentor.com). The latter type
allows for very low flows (50–999 ml/min) and is useful for animals with a body weight from 20 to
500 g and permits significant reduction in cost and environmental pollution. Anaesthetic vapour is then
delivered to the animal either in an anaesthetic induction chamber or via a face mask. Further details of
anaesthetic delivery systems are available in standard anaesthetic texts (Flecknell 2009).

13.2.1.2.2 Induction Chambers

For rodents, induction of inhalation anaesthesia is most conveniently accomplished with the use of a
clear Plexiglas induction chamber. First the animal is placed in the chamber, and then the vaporiser
is turned on. The gas flow to the chamber should be sufficient to fill up the chamber within a couple
318 The COST Manual of Laboratory Animal Care and Use

of minutes, and the gas should be introduced into the bottom of the chamber (because it is denser
than air). A gas outlet is connected to the top of the chamber and connected to the exhaust system.
The base of the chamber should be covered with synthetic sheepskin bedding, or paper tissue to
soak up any urine during induction. Rodents that are housed together may be placed together in the
chamber, as long as floor space is sufficient.

13.2.1.2.3 Mask Delivery

Most inhalational agents have strong odours, and animals may not inhale them from a face mask
unless they have been sedated. Following appropriate sedation, anaesthesia can often be induced in
a controlled manner using a face mask. Rabbits will breath-hold for extended periods when exposed
to volatile agents even after sedation. Although vigorous struggling is prevented by use of sedatives,
the procedure is probably distressing for the animal and best avoided (Flecknell and Liles 1996).

13.2.1.2.4 Open Jar

For very short procedures (<1 minute), it is possible to anaesthetise mice using isoflurane in an
open jar. At normal room temperature (20°C) and pressure (1 atmosphere), 1 ml of liquid isoflurane
will evaporate to approximately 182 ml of vapour. In a 500 ml jar, 0.11 ml of liquid isoflurane will
therefore give rise to a vapour concentration of approximately 4%, which is safe for induction.
The liquid must be allowed to evaporate and mix evenly in the jar before the mouse is introduced.
After induction is complete and the mouse removed, recovery takes less than 60 seconds (personal
observation). Use of a jar must take place in a ventilated hood or on a ventilated bench, and the con­
centration of anaesthetic produced can vary considerably. If a proper calculation is not performed,
dangerously high concentration may be produced, so it is preferable to use a vaporiser to provide
controlled delivery of the anaesthetic.

13.2.1.2.5 Endotracheal Intubation

Following induction of anaesthesia, animals lose their protective coughing and swallowing reflexes,
and as a consequence may inhale material. To maintain and protect the airway, it may be advisable
to pass an endotracheal tube. Well-established techniques have been described for most species
(mouse: Hastings and Summers-Torres 1999, rat: Yasaki and Dyck 1991, rabbit: Alexander and
Clark 1980), and intubation should always be considered, especially for longer procedures (e.g.,
those lasting for more than 15–30 minutes). Intubation also allows ventilation to be assisted, and the
depressant effects of anaesthesia to be corrected. Some anaesthetic drugs depress ventilation more
than others (e.g., opioids).

13.2.1.3 operator safety

Chronic exposure to low concentrations of volatile anaesthetics may present a risk to human health.
Most health and safety agencies recommend that measures are taken to minimise exposure, and a
number of systems are available to achieve this (Hartsfield 2007).

13.2.2 injection anaesthesia

13.2.2.1 injectable Agents
13.2.2.1.1 Ketamine/Medetomidine (K/M)
Ketamine is a dissociative cyclohexamine that is an N-methyl-D-aspartic acid (NMDA) antagonist.
It produces good analgesia in non-human primates, but has less effect in rabbits and rodents, in which
it causes sedation and immobilisation, but insufficient analgesia for surgery (Green et al. 1981). Some
of the side effects are salivation, muscle rigidity and spontaneous movements, which are a disadvan­
tage if it is used as the sole anaesthetic agent. For this reason, ketamine is usually combined with a
Anaesthesia and Analgesia 319

sedative, for example, medetomidine (an alpha-2-adrenergic agonist), which improves analgesia and
muscle relaxation. The combination can be used for invasive procedures of short to medium dura­
tion in many species, including rabbits and rats.
The two compounds can be mixed in one syringe and administered IP in rodents and SC in
rabbits (Flecknell 2009). The IM injection of K/M in rabbits shows no benefit over SC administra­
tion, is seemingly more painful, and therefore best avoided (Hedenqvist et al. 2001). Respiration
is significantly reduced by K/M, and provision of supplemental oxygen is strongly recommended
(Hellebrekers et al. 1997). In rabbits K/M preserves blood pressure better than the combination
ketamine/xylazine (Henke et al. 2005).
Mice and guinea pigs do not consistently reach a plane of surgical anaesthesia when K/M is
used (Green et al. 1981; Nevalainen et al. 1989). Female mice of some strains (e.g., Swiss Webster)
may require a higher ketamine dose rate as part of the drug combination compared with male mice
(Cruz, Loste, and Burzaco 1998) whereas some female rats (e.g., Sprague-Dawley) are more sensi­
tive to K/M than males (Nevalainen et al. 1989).
The effect of medetomidine may be reversed by the administration of atipamezole (an alpha-2­
adrenergic antagonist). Female Swiss Webster mice need a higher dose of atipamezole to reverse
anaesthesia than male mice (Cruz, Loste, and Burzaco 1998). If surgery has been undertaken an
analgesic drug should be administered before reversal. Medetomidine reduces insulin concentra­
tion and increases glucose concentration. There is a decrease of blood concentration of antidiuretic
hormone and a direct effect on the kidney, so that medetomidine causes substantial fluid loss, which
should be corrected by fluid administration (Hedenqvist and Hellebrekers 2003). Since K/M also
produce cardiovascular and respiratory depression, reversal with atipamezole is strongly recom­
mended (Flecknell 2009).

13.2.2.1.2 Ketamine/Acepromazine
This combination is useful for producing surgical anaesthesia in rabbits (Flecknell 2009) but may
not produce surgical planes of anaesthesia in rodents.

13.2.2.1.3 Ketamine/Xylazine (K/X)

Ketamine may also be combined with the mixed alpha-1/alpha-2-adrenerigc agonist xylazine, to
achieve surgical anaesthesia in rabbits and rodents. K/X has been reported to produce surgical
anaesthesia in mice both reliably (Erhardt et al. 1984; Arras et al. 2001; Dorsch, Otto, and Hedrich
2004) and unreliably (Green et al. 1981; Buitrago et al. 2008). The differences are probably a con­
sequence of variations in mouse strain, sex and dose, as well as other factors.
Some authors recommend adding the sedative acepromazine to the K/X combination to improve
surgical anaesthesia in mice (Arras et al. 2001; Buitrago et al. 2008). Side effects caused by K/X
are hypotension, reduction of cardiac output and a reversible oedema of the cornea (Hedrich and
Bullock 2004); hypoxia is also produced and oxygen supplementation is recommended. Tissue dam­
age has been reported after IM injection of K/X in rats, marmosets, and hamsters (Davy et al. 1987;
Gaertner, Boschert, and Schoeb 1987; Smiler et al. 1990).
In guinea pigs, K/X unreliably produces surgical anaesthesia and its effectiveness may be
improved by local anaesthetic infiltration or a small dose of isoflurane. Like ketamine/medetom­
dine, K/X produces polyuria and an increase in intraocular pressure. Atipamezole administration is
recommended to reverse the effect of xylazine at the end of surgery. With or without acepromazine,
K/X has been shown to cause nerve injury when administered intramuscularly to rabbits (Beyers,
Richardson, and Prince 1991; Vachon 1999), whereas intravenous injection is effective and safe
(Green et al. 1981). Analgesia is sometimes insufficient for surgery in pigmented rabbits given K/X
and hypotension more pronounced than during K/M anaesthesia (Henke et al. 2005). In NZW rab­
bits on the other hand, surgical anaesthesia is often achieved (Green et al. 1981; Lipman, Marini,
and Erdman 1990).
320 The COST Manual of Laboratory Animal Care and Use

13.2.2.1.4 Fentanyl/Fluanisone/Midazolam
The combination of the opioid agonist (fentanyl) and the sedatives fluanisone and midazolam is per­
haps the safest and most useful alternative to K/M for producing surgical anaesthesia in rodents and
rabbits. To decrease the time to recovery, the effect of fentanyl can be reversed by administration of
a mixed opioid agonist/antagonist such as butorphanol or buprenorphine, while still retaining anal­
gesia. Fentanyl/fluanisone is sold under the trade name Hypnorm, and is at times difficult to acquire
from retailers because it is produced only in low volumes.
When combining Hypnorm with midazolam, the two must first be mixed with water for injec­
tion, otherwise crystallisation may occur. The mixture can be administered by the IP or the SC
routes in rodents. If the SC route is used, the dose needs to be approximately one-third lower than
the IP dose, because immediate hepatic metabolism does not take place.
In rabbits Hypnorm is best administered by the SC route first, and after the rabbit is sedated
(after 5–10 minutes), midazolam can be administered intravenously to effect (Flecknell 2009).

13.2.2.1.5 Propofol
Propofol is an alkylphenol that must be administered by the intravenous route to be effective, due to
a high rate of metabolism in the liver. Maintenance of anaesthesia requires continuous intravenous
infusion. After anaesthesia has been induced with a propofol bolus IV, the animal can be intubated
or placed on a face mask, and anaesthesia maintained with a volatile agent. Slow injection is neces­
sary at induction to avoid apnoea. Propofol undergoes rapid hepatic metabolism, which allows for
very fast recovery once the infusion is stopped. High doses of propofol are needed to allow invasive
procedures to be undertaken, so it is best used combined with an analgesic agent such as an opioid.
If a potent opioid is used, intubation and mechanical ventilation are necessary, because of respira­
tory depression. Attempts have been made to administer a combination of propofol and an opioid
to mice, by IP injection, but the anaesthesia produced was unreliable and associated with some
mortality (Alves et al. 2007, 2009).

13.2.2.1.6 Barbiturates
Ultra-short acting barbiturates such as thiopental, may be used to induce anaesthesia by the intrave­
nous route, to allow for maintenance of anaesthesia with volatile agents.
The medium long-acting barbiturate pentobarbital may be used to induce sleep in rodents after
IP injection, but cannot safely be used to produce surgical anaesthesia. The dose that produces
surgical planes of anaesthesia is dangerously close to the lethal dose and causes severe respiratory
and cardiovascular depression (Skolleborg et al. 1990). Pentobarbital can be useful for non-survival
surgical procedures, when administered by IV infusion and in combination with an opioid agonist
(e.g., fentanyl) to achieve good analgesia. Mechanical ventilation is necessary because of severe res­
piratory depression. Pentobarbital is not suited for survival procedures, because it causes prolonged
sedation from which there is very slow recovery. Barbiturates are not safe to use for any types of
procedures in rabbits.
The long-acting barbiturate thiobutabarbital may be administered IP to produce prolonged anaes­
thesia in rats. It may be indicated in some diabetes research, because it causes less effect on blood
glucose concentration than other anaesthetics (Hindlycke and Jansson 1992).

13.2.2.1.7 Local Anaesthetics

The use of local anaesthetics can be very beneficial to improve analgesia during and after surgery.
For example, it may prevent some sensitisation of the nociceptive pathways, resulting in less post­
operative pain (Skarda and Tranqulli 2007). The agents may be administered by infiltration of
tissue, and for regional, epidural or spinal blocks. Shorter acting (e.g., lidocaine) and longer acting
(e.g., bupivacaine) local anaesthetics are available, and the duration of action of the short-acting
agents may be increased by the addition of adrenaline. However, adrenaline use is contraindicated in
peripheral body parts (ears, nose, digits, penis), because of the risk of ischemia-induced necrosis.
Anaesthesia and Analgesia 321

The use of local anaesthesia to supplement general anaesthesia has several advantages. In old
or debilitated animals, the general anaesthetic dose can be kept to a minimum and side effects
thereby reduced. In small rodents a satisfactory plane of surgical anaesthesia is sometimes difficult
to achieve with injectable anaesthetics, and the addition of a local anaesthetic can help. Local anaes­
thetic cream (EMLA) can be used to reduce pain caused by venipuncture in rabbits, cats, dogs and
pigs (Flecknell 2009).
Maximum safe doses are similar in all species, and overdose may cause CNS and cardiovascular
toxicity.

13.2.2.2 methods of Administration

A variety of different routes can be used to administer anaesthetic agents by injection (Table 13.2).

13.2.2.2.1 Intravenous
Intravenous injection of anaesthetic agents is easily accomplished in larger species, but is more dif­
ficult in small rodents. An IV injection gives immediate effect (no absorption phase) and allows for

TABLE 13.2
dose and route of Adsministration of some of the more Commonly used Anaesthetic
Agents
dose and method of
Animal species Anaesthetic Protocol Administration specific remarks
Mouse Fentanyl/Fluanisone + 10 ml/kg IP or 5–7ml/kg SC Drugs injected IP may partly undergo
midazolam of a 1:1:2 mixture of immediate hepatic metabolism,
Hypnorm, midazolam and unlike drugs injected SC. Therefore
water for injection a higher dose may be needed for IP
than SC injection to achieve the
same effect.
Rat Fentanyl/Fluanisone + 2.7 ml/kg IP or 1.5–2 ml/kg See comment for mouse
midazolam SC of a 1:1:2 mixture of
Hypnorm, midazolam and
water for injection
Ketamine + medetomidine 60–75 mg/kg + 0.25–0.5
mg/kg IP
Guinea pig Fentanyl/Fluanisone + 8 ml/kg IP of a 1:1:2 mixture
midazolam of Hypnorm, midazolam
and water for injection
Ketamine + medetomidine 40 mg/kg + 0.5 mg/kg IP Avoid IM injection, can cause pain
and muscle damage
Hamster Fentanyl/Fluanisone + 4 ml/kg IP of a 1:1:2 mixture
midazolam of Hypnorm, midazolam
and water for injection
Ketamine + medetomidine 100 mg/kg + 0.25 mg/kg IP
Rabbit Fentanyl/Fluanisone + 0.3 ml/kg SC + 2 mg/kg SC Premedication SC with fentanyl/
midazolam or IV fluanisone is followed by IV
injection of midazolam to effect
Ketamine + medetomidine 15 mg/kg + 0.25 mg/kg SC Rapid absorption after SC
administration, no difference from
IM injection in effect or duration
Propofol 10 mg/kg injection bolus, Must be administered IV as an
0.2–0.6 mg/kg/min infusion injection or infusion due to rapid
hepatic metabolism
322 The COST Manual of Laboratory Animal Care and Use

dosing to effect according to an individual response, unlike the situation with IM, SC or IP injec­
tion. Continuous intravenous infusion of short-acting drugs such as propofol, sufentanil or remifen­
tanil enable accurate control over anaesthetic depth by adjustment of the infusion rate. Establishing
intravenous access is also beneficial if emergency drugs need to be administered.
An IV injection/infusion also allows for rapid buffering of anaesthetic solutions that are acidic
(ketamine) or alkaline (barbiturates), which, if injected by another route, may give rise to tissue dam­
age and pain (Smiler et al. 1990; Branson 2001). Propofol can cause local pain upon intravenous injec­
tion, but the pain is minimised by premedication with an opioid or alpha-2-agonist (Branson 2001).

13.2.2.2.2 Intramuscular
This route of administration may be used in rabbits and larger species, but should be avoided in
rodents, because of the risk of tissue damage. An IM injection of K/X may cause tissue damage and
pain even in larger species (Davy et al. 1987; Gaertner, Boschert, and Schoeb 1987; Smiler et al.
1990; Beyers, Richardson, and Prince 1991).

13.2.2.2.3 Intraperitoneal
This injection route is commonly used in rodents because intravenous access may be difficult and
they have a small muscle mass. The peritoneal cavity is richly vascularised and drug uptake is rapid
after injection of small volumes. Part of the injected solution will be transported via the hepatic
portal system to the liver before reaching the systemic blood circulation, which results in high first
pass hepatic metabolism. A risk with IP injections is that part or all of the injected solution may be
deposited in the gut or intra-abdominal fat and not be effective.
Some anaesthetic agents such as tribromoethanol or chloral hydrate cause inflammation and pain
upon IP injection and therefore are best avoided (Vachon et al. 2000; Lieggi et al. 2005).

13.2.2.2.4 Subcutaneous
Subcutaneous administration of anaesthetics can be an alternative to IM or IP injection. The absorp­
tion rate of small volumes is often not very different between the routes and SC injection is seem­
ingly less stressful or painful than IM injection in rabbits (Hedenqvist, Roughan, and Flecknell
2000). Anaesthetics that may be administered subcutaneously include ketamine/medetomidine in
rabbits and rodents and fentanyl/fluanisone/midazolam in rats. The SC dose of the latter combina­
tion is approximately one-third of the IP dose (personal observations).

13.3 NEuromusCuLAr BLoCKiNg AgENTs (NmBAs)

The NMBAs work by binding to the nicotinic acetylcholine receptor at the neuromuscular junc­
tion and block nerve conduction, thereby paralysing the animal (Martinez 2007). For ethical
reasons, the use of NMBAs should be restricted to procedures for which they are absolutely
necessary. They should not be used to increase muscle relaxation on a routine basis or to prevent
the animal from breathing against the respirator. Greater muscle relaxation can be achieved by
increasing the anaesthetic depth, and the respiratory drive can be depressed by hyperventilating
the animal. Unlike the case in humans, animals are generally easy to intubate without the use
of NMBAs.
If NMBAs need to be used, a number of steps must be taken to eliminate the risk of animals being
insufficiently anaesthetised during paralysis. It is advisable to use doses of NMBAs that only partly
paralyse the animal and thus still allow for movement in response to nociceptive stimulation. It is also
advisable to allow the effects to subside before administering additional doses, in order to evaluate
anaesthetic depth using withdrawal reflexes at regular intervals. Animals also need to be monitored
for changes in blood pressure and heart rate, and inhalation and infusion devices should be equipped
with alarms to indicate lack of function. Respiratory function must also be monitored (e.g., pulse
oximetry and end tidal CO2). Further, a familiar anaesthetic regimen that allows for a stable plane
Anaesthesia and Analgesia 323

of anaesthesia should be used and stability must be established before the NMB is administered.
Examples of agents available for use in laboratory animals are provided in Table 13.3.

13.4 AssisTEd vENTiLATioN

It might be necessary to artificially ventilate an animal to support respiration and maintain near nor­
mal physiological function, particularly during long-term anaesthesia. Assisted ventilation may also be
needed during imaging, to synchronise respiratory movements with data collection (see Chapter 12).
If the animal is to recover consciousness, an endotracheal tube needs to be inserted, whereas for
non-recovery procedures, a tracheostomy can be carried out. The animal can then be connected
to a suitable ventilator. Tubes and intubation techniques must be refined and optimised to avoid
trauma to the delicate structures in the pharynx and trachea and to minimise dead-space. The
ventilator must be capable of delivering the volumes and frequencies appropriate for the species.
During intermittent positive pressure ventilation (IPPV) air is forced into the thorax. This causes a
positive pressure in the thorax in contrast to the negative pressure during spontaneous inhalation,
which normally supports venous filling of the heart. Artificial ventilation therefore also affects the
circulatory system. This effect can be reduced by allowing a long expiratory phase, and short period
for inspiration, with an inspiration:expiration ratio of 1:3–1:4.
It is not necessary to use NMB agents in order to mechanically ventilate an animal, although a
case can sometimes be made for their use during non-invasive imaging (Chapter 12). If an animal
breathes spontaneously, the mechanical ventilator rate should be increased to produce moderate
hypocapnia. The animal will usually stop making respiratory efforts at this point. The rate can then
be reduced slowly, until normocapnia (measured using a capnograph) is achieved, if necessary.

13.5 moNiToriNg duriNg ANAEsThEsiA

Anaesthesia poses a risk to the animal’s vital functions and consequently some form of monitor­
ing must take place. Vital signs that need to be monitored include respiration, circulation, body
temperature, acid–base status, kidney function and level of anaesthesia. In brief, low-risk proce­
dures, monitoring may be simple, whereas long-duration, invasive and high-risk procedures require
more sophisticated monitoring.

13.5.1 DePth oF anaesthesia

The depth of anaesthesia may be described as light, medium or deep. Light anaesthesia (immobil­
ity) involves loss of consciousness, as indicated by loss of the righting reflex. Muscular tone and
response to noxious stimulation are gradually lost with increasing depth, together with a number
of reflexes, forming a pattern that differs between animal species as well as the anaesthetic agents

TABLE 13.3
Neuromuscular Blocking drugs for use in small Laboratory Animals
mouse rat guinea Pig rabbit
Alcuronium — — — 0.1–0.2 mg/kg IV
Atracurium — — — —
Pancuronium — 2 mg/kg IV 0.06 mg/kg IV 0.1 mg/kg IV
Tubocurarine 1 mg/kg IV 0.4 mg/kg IV 0.1–0.2 mg/kg IV 0.4 mg/kg IV
Vecuronium — 0.3 mg/kg IV —

Source: Flecknell, P. A., Laboratory Animal Anaesthesia, 3rd ed., Elsevier, London 2009.
324 The COST Manual of Laboratory Animal Care and Use

used. Before surgery is undertaken, reactions to noxious stimulation such as toe-pinch (rat), ear-
pinch (rabbit), or tail-pinch (mouse) must be absent. At a surgical plane of anaesthesia, noxious
stimulation should cause only minimal changes in respiratory rate, heart rate or blood pressure
(typically less than 10–15%).
Parameters and measurements that may help indicate the level of anaesthesia are the degree of mus­
cle relaxation, the pattern and depth of respiration, and the heart rate and blood pressure. A change to
marked abdominal movements with each breath signals a deeper level of anaesthesia in rodents. With
lighter levels of anaesthesia, respiration rate, heart rate and blood pressure usually increase.

13.5.2 resPiratory Function

Respiratory function can be assessed by clinical observation of the respiratory rate and pattern.
With severe respiratory depression, the skin and mucous membranes turn visibly cyanotic (blue
or purple). Blood oxygenation, a function of respiration, can be monitored more accurately with a
pulse-oximeter or by arterial blood gas analysis.
A pulse-oximeter measures the oxygenation of haemoglobin in arterial blood by placing a sen­
sor across a capillary bed (e.g., across a digit, or the tongue, tail, or ear). In an awake healthy ani­
mal breathing room air, haemoglobin saturation is >95%. During anaesthesia, the aim is to keep
oxygenation levels over 90%, which can usually be achieved by providing supplemental oxygen.
Specialist instruments suitable for use even in very small animals are now available. Although a
pulse-oximeter enables the degree of oxygenation to be assessed, no indication of carbon dioxide
concentration or pH is obtained. Accurate measures of all three parameters can be obtained using
a blood gas analyser, but this requires sampling of arterial blood. A good measure of blood and
tissue carbon dioxide concentration is provided by measuring respiratory end tidal CO2 concentra­
tion using a capnograph. Further details of monitoring devices can be found in standard veterinary
anaesthetic text books (e.g., Flecknell 2009).

13.5.3 carDioVascular Function

Measurements of heart rate and rhythm, blood pressure, ECG and capillary refill time are helpful
in assessing cardiovascular function. Heart rate can be measured by palpating a peripheral pulse, or
palpating the heart in small animals. Bradycardia may be caused by anaesthetic overdosage, opioids,
alpha-2-agonists, vagal stimulation, hypothermia or hypoxia (Haskins 2007). Parasympatholytic
agents (atropine, glycopyrrolate) may be used to correct bradycardia due to opioids or vagal stimu­
lation. Tachycardia can be caused by hypovolemia and hyperthermia, or be a response to surgi­
cal stimulation in lightly anaesthetised animals. A simple estimate of peripheral perfusion can be
obtained from the capillary refill time. The gums are usually the most accessible site, and the refill
of capillaries following blanching by digital pressure can be observed in most larger species. In
normal animals, following blanching by pressing with a finger, the mucous membranes regain their
normal colour in less than a second. If refill is significantly delayed (> 1 second), it indicates poor
peripheral tissue perfusion and possible circulatory failure.
Mean arterial blood pressure can be measured invasively or non-invasively, and should be main­
tained above 60–70 mm Hg, to ensure adequate tissue oxygenation and renal perfusion. Hypotension
can be caused by hypovolemia, poor cardiac output, or vasodilation and hypertension by hyperther­
mia, renal failure, and light planes of anaesthesia.
The electrical activity of the heart can be measured non-invasively using an ECG. It should be
remembered that ECG records the electrical activity of the heart and not mechanical performance.

13.5.4 maintenance oF boDy temPerature

Anaesthesia induces heat loss by reducing metabolism and muscular activity and interference with
hypothalamic thermostatic mechanisms. Many anaesthetics also cause peripheral vasodilatation,
Anaesthesia and Analgesia 325

further increasing heat loss. Body temperature falls much faster in small animals than in large
animals, because of their larger body surface to body weight ratio. Clipping fur, disinfecting the
skin, contact with cold surfaces, opening of body cavities, and injection of cold fluids are actions
that contribute to the development of hypothermia. Hypothermia may lead to over-dose of anaes­
thetic agent and prolonged recovery, because a drop in body temperature reduces anaesthetic need.
Blood pressure and cardiac output fall during hypothermia, whereas peripheral vascular resistance
increases (Branson 2001). Severe hypothermia may lead to cardiac failure caused by ventricular
fibrillation or cardiac arrest.
To prevent hypothermia, warming must be initiated as soon as the anaesthetic drugs start to take
effect. Rodents are best placed in a heating chamber (26–28°C) after administration of injectable
anaesthetics, or on a heating pad after induction of inhalation anaesthesia. Heating must be contin­
ued during anaesthesia and recovery, until the animal is fully awake. At the same time care must be
taken not to burn or overheat the animal. Best practise is to use a thermostatically regulated heat­
ing pad, which may be connected to a rectal thermometer. In any event, body temperature must be
monitored throughout anaesthesia.

13.5.5 maintenance oF FluiD anD electrolyte balance

Fluid and electrolyte imbalances can arise during and after anaesthesia and surgery. Body fluids
may be lost as a result of haemorrhage and evaporation from the surgical site or expired air (Seeler
2007). Fasting before surgery and reduction of food and water intake after surgery additionally
threaten homeostasis. The use of alpha-2-adrenergic agonists (e.g., medetomidine) contributes to
loss of body fluids by causing diuresis and polyuria (Meyer and Fish 2008). Imbalances can occur
in the tissues as well as the vascular space and need to be counteracted to maintain cardiovascular
stability and proper tissue perfusion.
Fluids may be administered orally, subcutaneously, intraperitioneally, and in emergency, intra­
venously. Lactated Ringer’s solution is an isotonic balanced electrolyte solution that may be used
for maintenance or replacement purposes, and is better suited than isotonic saline (0.9%), which
generally does not restore the animal’s electrolyte requirements (Seeler 2007). For minor surgery,
administration of 2.5 ml/kg/h of fluid SC or IV is recommended, whereas for severely traumatic
procedures 10–15 ml/kg/h can be estimated. For blood loss, an extra volume of up to five times the
estimated volume lost should be administered.

13.6 PosT-ANAEsThETiC CArE

13.6.1 warmth anD comFort
In survival studies, care must be taken to continue warming the animal until it has fully recovered
from anaesthesia. For rodents, incubation chambers that can hold cages are preferable. Larger
animals should be placed in rooms with increased temperature and covered with blankets. If
the effect of any of the anaesthetic drugs can be reversed with an antagonist, recovery will be
more rapid. Care must always be taken to provide analgesia if any post-procedure pain can be
expected.
Rodents should not be placed directly on sawdust or wood shaving bedding during recovery,
but in a cage with paper or tissue towels or synthetic sheep skin (e.g., Drybed). Once the animal
becomes ambulatory it may be returned to its home cage, and if needed, food should be placed
on the cage floor. To facilitate food intake, food pellets may be crushed and soaked in water
and presented in a petri dish on the cage floor. Soft paper may also be placed in the home cage.
Animals that are housed in familiar groups should be returned to the group after recovery. A
familiar surrounding is helpful in reducing fear and stress, as are subdued lighting and low noise
levels.
326 The COST Manual of Laboratory Animal Care and Use

13.6.2 FluiD anD nutritional suPPort

Animals that are dehydrated need supportive fluids given either by mouth or subcutaneously, intra­
peritoneally, or intravenously. Hypoglycemia should be corrected with glucose administered orally
or IP (rodents) or dextrose solutions IV. Fluids should always be warmed to body temperature before
being administered.
Rabbits have very sensitive intestinal tracts and may suffer from ileus (gut stasis) if eating does
not resume soon after recovery from anaesthesia. If the rabbit does not eat voluntarily, it must be
fed with a liquidised food replacement (e.g., critical care formula for rodents and rabbits) through
a syringe. Canned baby food such as mashed carrots or mashed banana may also be fed in small
amounts to stimulate appetite. Animals of other species need nutritional support if eating is not
resumed soon after recovery. The need for (additional) analgesic treatment should always be con­
sidered in animals that lack appetite.

13.7 ANALgEsiA
Alleviating pain reduces suffering, which is one of the most important aims when working with
laboratory animals. Analgesic treatment in conjunction with surgery not only reduces suffering but
improves recovery and reduces morbidity and mortality. Surgery is associated with neuroendocrine,
metabolic, and immune alterations resulting from tissue damage, anaesthesia, and psychological
stress (Shavit, Fridel, and Beilin 2006). Preoperative administration of analgesics provides more
effective pain relief and may reduce the anaesthetic dose needed (Flecknell 2009). For the most
effective pain relief, different drugs may be combined, for example, an opioid, a NSAID, and a local
nerve block during an intervention, followed by repeated opioid and NSAID administration in the
immediate postoperative period, and finally NSAID administration alone when pain is less severe.
For doses of the most commonly used analgesic drugs see Table 13.4.

13.7.1 eValuation oF Pain

13.7.1.1 Physiological Parameters
Physiological responses generally arise from changes in the sympatho-adrenal and the
hypothalamic–pituitary–adrenal systems. Changes in heart and respiration rate, blood pressure
and stress hormone concentrations in blood may indicate the presence of pain (Kent and Molony
2009). However, many of these changes may also occur in response to handling, eating, exercise
and a number of stress factors. The link between pain scores and heart rate or respiratory rate was
shown to be weak in dogs after orthopaedic surgery (Holton et al. 1998). Plasma cortisol/cortisone
is elevated after surgery in calves and lambs, and is reversed by analgesic treatment (Kent, Molony,
and Robertson 1993; Stafford et al. 2002), but appears to show a ceiling effect that suggests that the
assessment of the relative severity of intense pain cannot be achieved. The association with chronic
pain in sheep is low (Ley, Livingston, and Waterman 1991; Ley et al. 1994). In mice, faecal corticos­
terone concentrations were elevated after vasectomy and could be reduced by meloxicam treatment
(Wright-Williams et al. 2007). Factors that may limit the value of corticosterone measurements
for assessment of pain include individual variation, circadian changes, age and breed effects, and
a variety of alternative stressors both pleasurable and stressful that activate the HPA-system (Kent
and Molony 2009). These measures are useful only as research tools, because they do not allow
rapid assessment of animals and administration of analgesic to control pain effectively.

13.7.1.2 Behaviour
Both spontaneous and evoked behaviour may be useful to assess pain in animals. Spontaneous
behaviours include changes in posture, activity and vocalisation and evoked behaviours include
reactions to handling and threshold testing to mechanical, chemical and thermal stimulation
Anaesthesia and Analgesia 327

(Kent and Molony 2009). Ongoing pain has been shown to elicit pain-related behaviour, which is
species-specific and procedure-related. Rats, for example, show an increased frequency of back-
arching and writhing after abdominal surgery (Roughan and Flecknell 2001) and similar behaviours
can be observed in mice (Wright-Williams et al. 2007) and rabbits (Leach et al. 2009). Guarding
of injured areas may also be present, for example guarding the hind foot after sciatic nerve lesion
(Bennett and Xie 1988). These behaviours are reduced in frequency by treatment with analgesic
drugs, which indicates that they may be related to pain.
Measurements of body weight and food and water intake have been proposed as indicators of
post-operative pain and the efficacy of analgesic therapy (Liles et al. 1998). These latter measures
are objective, but they are retrospective measures and so cannot be used to modify analgesic therapy
for a particular animal. They can, however, be used as a simple measure of post-operative recovery,
and as a means of adjusting future analgesic regimens for similar animals undergoing similar surgi­
cal procedures.
Unfortunately, there are few well-described and fully validated pain assessment techniques for
laboratory animals. Those schemes that have been described (e.g., Roughan and Flecknell 2003)
relate to particular types of surgery, so in many circumstances pain is difficult to assess. It is impor­
tant to appreciate that the signs of pain in many animals are subtle, and quite difficult to detect, even
by an experienced observer. It is therefore safest to assume that some pain will be present after any
surgical procedure, and that analgesics will be required. Initial dosing with any of the analgesics
described above will rarely cause undesirable side effects, and the positive effects on recovery from
the procedure provide a strong justification for their routine use. What is also difficult, however, is
to determine how long analgesic treatment should be continued. Prolonged treatment with opioids,
especially when these are given at high doses, can have detrimental effects including reduction in
food and water intake. At present, the following advice is offered:

1. After any major surgical procedure (e.g., laparotomy, thoracotomy, craniotomy), administer
at least one dose of opioid analgesic (e.g., buprenorphine), combined with a single dose of
NSAID. Less invasive procedures (e.g., vessel cannulation) probably require only a single
dose of opioid or a single dose of NSAID.
2. Assess the animals as carefully as possible. Spend time assessing their normal behaviour
pre-operatively, so that post-operative changes in behaviour can be identified. Monitor
body weight and food and water consumption. If animals are failing to gain weight after 24
hours, administer a second dose of analgesic and if this produces an improvement, adopt
this for all future procedures of this type.
3. Regularly review pain assessment and pain management schemes so that they can be
updated as new information is published.

13.7.2 Drugs anD Doses

13.7.2.1 opioids
Opioids provide the most effective way of controlling pain caused by trauma or surgery. Pure
mu opioid agonists (morphine, fentanyl, sufentanil, alfentanil) can be used intra-operatively as
part of the anaesthetic regime and post-operatively by repeated injections (morphine) or IV infu­
sion (fentanyl, sufentanil). These drugs may depress respiration, so mechanical ventilation is often
necessary during anaesthesia and sometimes oxygen supplementation must be provided during
recovery. Pure opioid agonists have no ceiling effect, which means that the higher the dose, the
more effective the pain relief. Side effects (sedation, respiratory depression and reduced gastroin­
testinal peristalsis) are more severe with administration of pure agonists such as morphine than
after partial opioid agonists such as buprenorphine or butorphanol, but these latter drugs do have
a ceiling effect.
328 The COST Manual of Laboratory Animal Care and Use

TABLE 13.4
Analgesics for use in small Laboratory Animals. Note that These are only suggestions
Based on Clinical Experience and the Limited Published data which is Available. dose
rates should be Adjusted depending upon the Clinical response of the Animal
Analgesic mouse rat hamster guinea Pig rabbit
Buprenorphine 0.05–0.1mg/kg 0.05mg/kg SC 0.1mg/kg SC 0.05mg/kg SC 0.01–0.05mg/kg SC
SC 8–12 hourly 8–12 hourly 8–12 hourly 8–12 hourly 6–12 hourly
Carprofen 5mg/kg SC uid 5mg/kg SC uid 4mg/kg SC uid 1.5mg/kg per os uid
Meloxicam 5mg/kg SC uid 1mg/kg SC uid 0.3mg/kg SC uid 0.6–1mg/kg SC uid
Ketoprofen 5mg/kg uid SC 5mg/kg uid SC 3mg/kg uid SC
Morphine 2.5mg/kg SCor 2.5mg/kg SC or 2–5mg/kg SC or 2–5mg/kg SC or IM
IM 4 hourly IM 4 hourly IM 4 hourly 4 hourly

Source: Data adapted from Flecknell and Waterman-Pearson, 2000

Note: uid = once daily

Buprenorphine has a slow onset of action and reaches its peak about 60 minutes after SC injec­
tion (Dobromylskyj et al. 2000). The duration of action is relatively long, 6–8 hours, which is ben­
eficial when treating postoperative pain. If administered before induction of anaesthesia, it reduces
the need for isoflurane by approximately 20%. If a pure opioid agonist (e.g., fentanyl) is used as part
of the anaesthetic regime, buprenorphine should not be administered beforehand, but may instead
be used to reverse the effects of the pure agonist and provide postoperative pain relief. This will
reduce the time to recovery.
Care must be taken if administering buprenorphine before K/M anaesthesia in rats, because this
has been shown to increase mortality (Hedenqvist, Roughan, and Flecknell 2000) and it is advisable
to administer this analgesic during recovery from this anaesthetic regime. For doses see table 13.4.

13.7.2.2 Non-steroidal Anti-inflammatory Agents (NsAids)

Non-steroidal, anti-inflammatory drugs are useful for treating mild to moderate post-operative
pain. For more effective pain control, they can be combined with an opioid and/or a local anaes­
thetic drug. The following NSAIDs have been shown to have positive effects on behaviour and
recovery after surgery in rodents: ibuprofen (Hayes et al. 2000), ketoprofen and carprofen (Cabre
et al. 1998; Roughan and Flecknell 2001), meloxicam (Roughan and Flecknell 2004), and flunix­
ine meglumine (Stewart and Martin 2003). Carprofen has found widespread use for post-operative
pain control due to its long-lasting effect (24 hours) as well as low gastric and renal toxicity.
Meloxicam has a similar duration of action and is palatable to rodents and non-human primates
(Flecknell 2009). For doses see table 13.4.

13.7.2.3 Local Anaesthetic Agents

Local anaesthetics block sodium channels and thereby nerve conduction. Sensory nerves are more
sensitive than motor nerves to their effect. Local anaesthetics may be administered topically on the
skin, mucous membranes, eyes and ears, infiltrated into tissues by injection, deposited in the area
of a nerve (local block), in body cavities or in the epidural or subarachnoidal space (epidural or
spinal block). Lidocaine has a shorter duration of action, which may be increased by the addition
of adrenaline. The combination with adrenaline must not be used in the peripheral body parts (e.g.,
tail, toes, ears), or irreversible ischemic damage may be caused. Bupivacaine has a longer duration
of action than lidocaine, because it is more lipophilic in nature. The maximum doses are similar in
all species (4 mg/kg for lidocaine and 2 mg/kg for bupivacaine). Toxicity results in CNS and cardiac
effects. For doses see table 13.4.
Anaesthesia and Analgesia 329

13.7.3 methoDs oF DeliVery

Even though administration of ibuprofen in the drinking water has been shown to be effective
in mice after surgery (Hayes et al. 2000), this route of administration cannot be recommended.
Animals often have highly variable water consumption after a surgical procedure and intake of a
drug is very unpredictable. The more pain the animal experiences, the less it may drink. In addi­
tion, rodents may only drink during the dark phase of the photoperiod, so there may be a prolonged
period during which no analgesic is ingested. Opioids and NSAIDs may be administered orally
using a syringe, or mixed with palatable food (e.g., fruit jelly) and ingested voluntarily. Voluntary
ingestion is likely to be more successful if the food or jelly has been presented before the surgical
procedure. The dose must be adjusted accordingly to account for the first pass hepatic effect that
occurs after oral administration. Generally, more reproducible and reliable results are obtained by
administering analgesics by injection.

13.8 CoNCLusioNs
Standards of laboratory animal anaesthesia have increased dramatically over the last decade.
Research workers are now more aware of the potential interactions between different anaesthetic
agents and their animal models. They are also more aware of the problems that poor anaesthetic
practise can cause, and the need to maintain high standards of peri-operative care. Anaesthetic regi­
mens are now more often reported in more detail in the materials and methods sections of papers,
and are also being published as short papers in specialist journals. These trends should be encour­
aged, since successful establishment of an animal model developed in another laboratory often
requires careful attention to all aspects of the research protocol, including the anaesthetic methodol­
ogy. As validated pain scoring systems become established for different species, and efficacy data
for different analgesics is obtained, pain management will improve.

13.9 QuEsTioNs uNrEsoLvEd

Despite the improvements that have been made in anaesthetic practise, older anaesthetic agents
such as barbiturates are still widely used—sometimes appropriately, but in many instances they
would be better replaced with more modern anaesthetic agents. It is essential that research workers
consider the anaesthetic and peri-operative care procedures as an integral part of their research
protocol.
Post-operative analgesic use is becoming more widespread, but the appropriate use of these
agents is still limited by our poor ability to assess pain accurately in many laboratory species.
Concerns related to the potential side effects and interactions of analgesics with particular research
protocols need to be addressed by a careful and critical review of the relevant literature. In many
instances, potential effects are likely to be of significance only when analgesics are administered at
high-dose rates, for prolonged periods of time (Flecknell 2009).

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