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Non Alcoholic Steatohepatitis 1st Edition Devanand
Sarkar Digital Instant Download
Author(s): Devanand Sarkar
ISBN(s): 9781071621271, 1071621270
Edition: 1st
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Language: english
Methods in
Molecular Biology 2455

Devanand Sarkar Editor

Non-Alcoholic
Steatohepatitis
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK

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For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
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 Non-Alcoholic Steatohepatitis

Methods and Protocols

Edited by

Devanand Sarkar
Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA, USA;
Massey Cancer Center, Virginia Commonwealth University, Richmond, VA, USA; VCU Institute of
Molecular Medicine, Virginia Commonwealth University, Richmond, VA, USA
Editor
Devanand Sarkar
Department of Human and Molecular
Genetics
Virginia Commonwealth University
Richmond, VA, USA
Massey Cancer Center
Virginia Commonwealth University
Richmond, VA, USA
VCU Institute of Molecular Medicine
Virginia Commonwealth University
Richmond, VA, USA

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-2127-1 ISBN 978-1-0716-2128-8 (eBook)
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Preface

Globally obesity is a major and significant health problem with an alarming increase in its
incidence. Obesity leads to a spectrum of metabolic disorders including cardiovascular
disease, type 2 diabetes mellitus, and non-alcoholic fatty liver disease (NAFLD). NAFLD
starts from simple steatosis or fatty liver which progressively becomes complicated with
inflammation and varying levels of fibrosis (non-alcoholic steatohepatitis or NASH), ulti-
mately leading to cirrhosis and hepatocellular carcinoma (HCC). NASH functions as the
mediating event in obesity-induced HCC. As such, there has been an increasing interest in
NASH from researchers of diverse spectra. The present volume is a comprehensive collection
of laboratory protocols used to analyze varied aspects of NASH.
We start this volume with an overview of NASH and methods for histological diagnosis
of NASH in Chapter 1. This chapter also touches upon histological characteristics of
pediatric NASH. NASH is an in vivo phenomenon. As such, a mouse model that represents
the human disease is an absolute necessity for understanding the disease pathogenesis and
evaluating treatment approaches. Chapter 2 describes the generation of a diet-induced
mouse model of NASH which resembles human disease in both phenotype and molecular
signature. Sleeping Beauty (SB) transposase-mediated somatic integration in combination
with hydrodynamic injection is a useful technique to study gene function in mouse liver. In
Chapter 3 this protocol is described for the purpose of generating an in vivo NASH model.
Hepatic lipid accumulation is the initial step in NASH. Protocol to measure hepatic
lipids, such as triglyceride and cholesterol, is described in Chapter 4. Inhibition of fatty acid
β-oxidation (FAO) in the liver contributes to steatosis, and a protocol for in vitro and in vivo
measurement of FAO is presented in Chapter 5. Chapter 6 describes the methods to analyze
in vivo VLDL and chylomicron secretion which are often dysregulated in NASH.
Lipids accumulate in hepatocytes in NASH. However, many other cell types, such as
adipocytes and macrophages, play a key role in NASH pathogenesis. In obesity, hypertro-
phied adipocytes secrete chemotactic factors which recruit pro-inflammatory macrophages.
Pro-inflammatory cytokines (PICs), released from macrophages, promote inflammation and
stimulate adipocyte lipolysis resulting in free fatty acid (FFA) release which accumulate in
hepatocytes causing steatosis. End-stage NASH is characterized by fibrosis which is regu-
lated by hepatic stellate cells. Cross-talk between all these cell types drives NASH and as such
it is necessary to isolate and culture these cells either from mouse or humans for in vitro
mechanistic studies. A collection of protocols for isolation of hepatocytes and Kupffer cells
(Chapter 7), bone marrow-derived macrophages (Chapter 8), hepatic stellate cells
(Chapter 9), and adipocytes (Chapter 10) is therefore presented. Organoid culture serves
as a useful in vitro model to study NASH. Chapter 11 describes a protocol for establishing
mouse hepatic organoid culture, and Chapter 12 describes an innovative protocol to
develop human pluripotent stem cells-derived liver organoids.
Lipid sensing nuclear receptors, such as PPAR, LXR, and FXR, are key regulators of
NASH. These receptors are ligand-dependent transcription factors. A key component of
NASH is inflammation, and PIC generation is under the control of the transcription factor
NF-κB. Assays relevant to transcription factor function and global gene regulation are
pivotal in understanding the molecular mechanism of NASH. Chapter 13 describes a
protocol for chromatin immunoprecipitation assay. Two protocols, one describing mRNA

v
vi Preface

sequencing (Chapter 14) and the other describing single-cell RNA sequencing
(Chapter 15), are described which provide information about differential gene regulation
in different cell types during NASH. Over the years genome-wide association studies
(GWAS) have identified functional roles of PNPLA3, TM6SF2, and HSD17B13 genes in
NASH. Chapter 16 describes a computational method based on next-generation sequenc-
ing to study genetics of NASH.
NASH is inherently related to obesity-associated metabolic disorders, such as insulin
resistance and type 2 diabetes mellitus. Analysis of metabolic profile at the organismal level
provides direction toward potential molecular abnormality. In Chapter 17 an indirect
calorimetry method is described to check metabolic abnormalities in NASH mice, and
Chapter 18 describes methods to analyze insulin resistance. In NASH, accumulation of fat
creates a plethora of stressful and inflammatory events resulting in cell death. Methods to
analyze lipotoxic endoplasmic reticulum stress (Chapter 19), inflammasome (Chapter 20),
and necrosis and ferroptosis (Chapter 21) are presented. The role of sphingolipids and
ceramide is increasingly being appreciated in NASH, and a lipidomic method to analyze
sphingolipids is described in Chapter 22. Another key regulator of NASH is bile acids, which
regulate the nuclear receptor FXR, and bile acid profiling in mice is presented in Chapter 23.
Finally, the volume includes a methodology for therapeutic intervention of NASH.
Gene-based therapy is being increasingly appreciated as a potential treatment option for
NASH. A protocol to generate liver-targeted nanoparticle delivering gene products, either a
plasmid or siRNA, is described in Chapter 24. Chapter 25 describes extraction of exosomes
and exosomal miRNAs from mesenchymal stem cells which can be potentially used to
interfere with molecular events contributing to NASH. Adenoviruses can easily infect
hepatocytes and serve as a useful delivery vector. In Chapter 26, a methodology to generate
adenovirus for in vitro and in vivo study is described.
NASH is a vast subject and a wide and diverse range of in vitro and in vivo experimental
procedures are needed for proper study of this disease. In this volume we endeavored to
garner both basic and advanced methodologies. We hope that these protocols will be of use
to both new and experienced investigators studying NASH. This volume will serve as an
indispensable reference on NASH for basic and clinical researchers and students.

Richmond, VA, USA Devanand Sarkar

Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Histopathological Diagnosis of Nonalcoholic
Steatohepatitis (NASH). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Shah Giashuddin and Mouyed Alawad
2 Generation of a Diet-Induced Mouse Model of Nonalcoholic
Fatty Liver Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Amon Asgharpour and Arun J. Sanyal
3 Hydrodynamic Injection for Developing NASH Model. . . . . . . . . . . . . . . . . . . . . . 31
Haichuan Wang and Xin Chen
4 Measurement of Hepatic Lipids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Sarah Forman, Patrick Tso, and Min Liu
5 Measurement of Fatty Acid Oxidation in Mammalian Cells . . . . . . . . . . . . . . . . . . 49
Wei Wang, Yibao Ma, Tianhai He, Erin Mooney,
Chunqing Guo, Xiang-Yang Wang, and Xianjun Fang
6 Measurement of In Vivo VLDL and Chylomicron Secretion . . . . . . . . . . . . . . . . . 63
Siddhartha S. Ghosh, Jing Wang, and Shobha Ghosh
7 Isolation and Culture of Mouse Hepatocytes and Kupffer Cells (KCs) . . . . . . . . . 73
Rachel Mendoza, Indranil Banerjee,
Saranya Chidambaranathan Reghupaty,
Rajesh Yetirajam, Debashri Manna, and Devanand Sarkar
8 Mouse Bone Marrow Cell Isolation and Macrophage Differentiation. . . . . . . . . . 85
Rachel Mendoza, Indranil Banerjee, Debashri Manna,
Saranya Chidambaranathan Reghupaty,
Rajesh Yetirajam, and Devanand Sarkar
9 Purification and Isolation of Hepatic Stellate Cells . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Sonia Lele, Seung Duk Lee, Devanand Sarkar,
and Marlon F. Levy
10 Isolation of the Stromal Vascular Fraction from Adipose
Tissue and Subsequent Differentiation into White
or Beige Adipocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Jared S. Farrar and Rebecca K. Martin
11 Culture of Mouse Liver Ductal Organoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Junkai Yan, Yunling Tai, and Huiping Zhou
12 Development of a Scalable Three-Dimensional Culture
of Human Induced Pluripotent Stem Cells-Derived Liver Organoids. . . . . . . . . . 131
Giuseppe Pettinato, Lev T. Perelman, and Robert A. Fisher
13 Chromatin Immunoprecipitation Assay in Primary
Mouse Hepatocytes and Mouse Liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
Simiao Xu, Yangyang Liu, and Ji Miao

vii
viii Contents

14 Analysis of Liver Responses to Non-alcoholic Steatohepatitis

by mRNA-Sequencing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Christopher D. Green, Mikhail G. Dozmorov, and Sarah Spiegel
15 Single Cell RNA Sequencing in NASH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Jana Hundertmark, Hilmar Berger, and Frank Tacke
16 Computational Pipeline for Next-Generation Sequencing
(NGS) Studies in Genetics of NASH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Adrian Salatino, Silvia Sookoian, and Carlos J. Pirola
17 Metabolic Phenotyping in Mice with NASH Using
Indirect Calorimetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
Bin Ni, Shanshan Chen, Jared S. Farrar, and Francesco S. Celi
18 Analysis of Insulin Resistance in Nonalcoholic Steatohepatitis . . . . . . . . . . . . . . . . 233
Hyunbae Kim, Deqiang Zhang, Zhenfeng Song,
Xin Tong, and Kezhong Zhang
19 Assessment of Lipotoxic Endoplasmic Reticulum (ER)
Stress in Nonalcoholic Steatohepatitis (NASH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
Gopanandan Parthasarathy and Harmeet Malhi
20 Ex Vivo Dual-Hit Method for Inflammasome Activation in Liver . . . . . . . . . . . . . 255
Debajyoti Das, Moumita Adak, and Partha Chakrabarti
21 In Vivo Analysis of Necrosis and Ferroptosis in Nonalcoholic
Steatohepatitis (NASH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
Shinya Tsurusaki, Kazuko Kanegae, and Minoru Tanaka
22 Analysis of the Sphingolipidome in NAFLD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
David Montefusco, Johana Lambert, Andrea Anderson,
Jeremy Allegood, and L. Ashley Cowart
23 Bile Acid Profiling in Mouse Biofluids and Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . 305
Bo Kong, Daniel Rizzolo, Rulaiha E. Taylor, and Grace L. Guo
24 Preparation and Characterization of a Liver Targeted,
Poly(amidoamine) Based, Gene Delivery System. . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
Kareem Ebeid, Sean M. Geary, and Aliasger K. Salem
25 Extraction of Exosomes and Exosomal miRNA from Mesenchymal
Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
Zhao Lin, Andrew Wong, and Shiekh Alam
26 Generation of Adenovirus for In Vitro and In Vivo Studies
of Hepatocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
Yangyang Liu, Simiao Xu, Yun Liu,
Yashaswini Kelagere Mayige Gowda, and Ji Miao
Correction to: Measurement of Fatty Acid Oxidation in Mammalian Cells . . . . . . . . . C1

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
Contributors

MOUMITA ADAK • Department of Zoology, Midnapore College, Midnapore, India

SHIEKH ALAM • Department of Periodontics, School of Dentistry, Virginia Commonwealth
University, Richmond, VA, USA
MOUYED ALAWAD • Department of Pathology and Lab Medicine, SUNY Downstate Health
Science Center, Brooklyn, NY, USA
JEREMY ALLEGOOD • Department of Biochemistry and Molecular Biology, Virginia
Commonwealth University, Richmond, VA, USA
ANDREA ANDERSON • Department of Biology, Virginia Commonwealth University,
Richmond, VA, USA
AMON ASGHARPOUR • Division of Gastroenterology, Hepatology and Nutrition, Department
of Internal Medicine, Virginia Commonwealth University, Richmond, VA, USA
INDRANIL BANERJEE • Department of Human and Molecular Genetics, Virginia
Commonwealth University, Richmond, VA, USA
HILMAR BERGER • Department of Hepatology and Gastroenterology, Charité—
Universit€atsmedizin Berlin, Berlin, Germany
FRANCESCO S. CELI • Department of Internal Medicine, Virginia Commonwealth
University, Richmond, VA, USA
PARTHA CHAKRABARTI • Division of Cell Biology and Physiology, CSIR-Indian Institute of
Chemical Biology, Kolkata, India
SHANSHAN CHEN • Department of Internal Medicine, Virginia Commonwealth University,
Richmond, VA, USA; Department of Biostatistics, Virginia Commonwealth University,
Richmond, VA, USA
XIN CHEN • Department of Bioengineering and Therapeutic Sciences and Liver Center,
University of California, San Francisco, CA, USA
L. ASHLEY COWART • Department of Biochemistry and Molecular Biology, Virginia
Commonwealth University, Richmond, VA, USA
DEBAJYOTI DAS • Division of Cell Biology and Physiology, CSIR-Indian Institute of Chemical
Biology, Kolkata, India
MIKHAIL G. DOZMOROV • Department of Biostatistics, Virginia Commonwealth University,
Richmond, VA, USA
KAREEM EBEID • Department of Pharmaceutical Sciences and Experimental Therapeutics,
College of Pharmacy, University of Iowa, Iowa City, IA, USA; Department of
Pharmaceutics, Faculty of Pharmacy, Minia University, Minia, Egypt; Department of
Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Manufacturing, Deraya
University, New Minia, Egypt
XIANJUN FANG • Department of Biochemistry and Molecular Biology, Virginia
Commonwealth University, Richmond, VA, USA; Massey Cancer Center, Virginia
Commonwealth University, Richmond, VA, USA
JARED S. FARRAR • Center for Clinical and Translational Research, Virginia Commonwealth
University, Richmond, VA, USA
ROBERT A. FISHER • CTI Clinical Trial & Consulting, Covington, KY, USA

ix
x Contributors

SARAH FORMAN • Department of Pathology and Laboratory Medicine, University of

Cincinnati College of Medicine, Cincinnati, OH, USA
SEAN M. GEARY • Department of Pharmaceutical Sciences and Experimental Therapeutics,
College of Pharmacy, University of Iowa, Iowa City, IA, USA
SHOBHA GHOSH • Department of Internal Medicine, Virginia Commonwealth University
Medical Center, Richmond, VA, USA; Hunter Holmes McGuire VA Medical Center,
Richmond, VA, USA
SIDDHARTHA S. GHOSH • Department of Internal Medicine, Virginia Commonwealth
University Medical Center, Richmond, VA, USA
SHAH GIASHUDDIN • Department of Pathology and Lab Medicine, New York Presbyterian
Brooklyn Methodist Hospital, Brooklyn, NY, USA
YASHASWINI KELAGERE MAYIGE GOWDA • Sapthagiri Institute of Medical Sciences and
Research Centre, Bangalore, India
CHRISTOPHER D. GREEN • Department of Biochemistry and Molecular Biology, Virginia
Commonwealth University, Richmond, VA, USA
CHUNQING GUO • Department of Human and Molecular Genetics, Virginia Commonwealth
University, Richmond, VA, USA
GRACE L. GUO • Department of Pharmacology and Toxicology, Ernest Mario School of
Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ, USA;
Environmental and Occupational Health Sciences Institute, Rutgers, The State University
of New Jersey, Piscataway, NJ, USA; Rutgers Center for Lipid Research, Rutgers, The State
University of New Jersey, Piscataway, NJ, USA; VA New Jersey Health Care System,
Veterans Administration Medical Center, East Orange, NJ, USA
TIANHAI HE • Department of Biochemistry and Molecular Biology, Virginia Commonwealth
University, Richmond, VA, USA
JANA HUNDERTMARK • Department of Hepatology and Gastroenterology, Charité—
Universit€atsmedizin Berlin, Berlin, Germany
KAZUKO KANEGAE • Department of Regenerative Medicine, Research Institute, National
Center for Global Health and Medicine, Tokyo, Japan
HYUNBAE KIM • Center for Molecular Medicine and Genetics, Wayne State University School
of Medicine, Detroit, MI, USA
BO KONG • Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy,
Rutgers, The State University of New Jersey, Piscataway, NJ, USA
JOHANA LAMBERT • Department of Biochemistry and Molecular Biology, Virginia
Commonwealth University, Richmond, VA, USA
SEUNG DUK LEE • Division of Transplant Surgery, Department of Surgery, Virginia
Commonwealth University, Richmond, VA, USA
SONIA LELE • School of Medicine, Virginia Commonwealth University, Richmond, VA, USA
MARLON F. LEVY • Division of Transplant Surgery, Department of Surgery, Virginia
Commonwealth University, Richmond, VA, USA
ZHAO LIN • Department of Periodontics, School of Dentistry, Virginia Commonwealth
University, Richmond, VA, USA
MIN LIU • Department of Pathology and Laboratory Medicine, University of Cincinnati
College of Medicine, Cincinnati, OH, USA
YANGYANG LIU • Division of Endocrinology, Boston Children’s Hospital, Harvard Medical
School, Boston, MA, USA; Cancer Center, Union Hospital, Tongji Medical College,
Huazhong University of Science and Technology, Wuhan, China
 Contributors xi

YUN LIU • Division of Endocrinology, Boston Children’s Hospital, Harvard Medical School,
Boston, MA, USA; Key Laboratory of Molecular Target & Clinical Pharmacology and the
State Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences & the Fifth
Affiliated Hospital, Guangzhou Medical University, Guangzhou, China
YIBAO MA • Department of Biochemistry and Molecular Biology, Virginia Commonwealth
University, Richmond, VA, USA; Alliance Pharma Inc, Malvern, PA, USA
HARMEET MALHI • Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester,
MN, USA
DEBASHRI MANNA • Department of Human and Molecular Genetics, Virginia
Commonwealth University, Richmond, VA, USA
REBECCA K. MARTIN • Department of Microbiology and Immunology, Virginia
Commonwealth University, Richmond, VA, USA
RACHEL MENDOZA • Department of Human and Molecular Genetics, Virginia
Commonwealth University, Richmond, VA, USA
JI MIAO • Division of Endocrinology, Boston Children’s Hospital, Harvard Medical School,
Boston, MA, USA
DAVID MONTEFUSCO • Department of Biochemistry and Molecular Biology, Virginia
Commonwealth University, Richmond, VA, USA
ERIN MOONEY • Department of Human and Molecular Genetics, Virginia Commonwealth
University, Richmond, VA, USA
BIN NI • Department of Internal Medicine, Virginia Commonwealth University, Richmond,
VA, USA
GOPANANDAN PARTHASARATHY • Division of Gastroenterology and Hepatology, Mayo Clinic,
Rochester, MN, USA
LEV T. PERELMAN • Center for Advanced Biomedical Imaging and Photonics, Division of
Gastroenterology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard
Medical School, Boston, MA, USA
GIUSEPPE PETTINATO • Center for Advanced Biomedical Imaging and Photonics, Division of
Gastroenterology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard
Medical School, Boston, MA, USA
CARLOS J. PIROLA • School of Medicine, Institute of Medical Research A Lanari, University of
Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina; Department of Molecular
Genetics and Biology of Complex Diseases, Institute of Medical Research (IDIM), National
Scientific and Technical Research Council (CONICET)—University of Buenos Aires,
Ciudad Autonoma de Buenos Aires, Argentina
SARANYA CHIDAMBARANATHAN REGHUPATY • C. Kenneth and Dianne Wright Center for
Clinical and Translational Research, Virginia Commonwealth University, Richmond,
VA, USA
DANIEL RIZZOLO • Department of Pharmacology and Toxicology, Ernest Mario School of
Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ, USA
ADRIAN SALATINO • School of Medicine, Institute of Medical Research A Lanari, University of
Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina; Department of Molecular
Genetics and Biology of Complex Diseases, Institute of Medical Research (IDIM), National
Scientific and Technical Research Council (CONICET)—University of Buenos Aires,
Ciudad Autonoma de Buenos Aires, Argentina
ALIASGER K. SALEM • Department of Pharmaceutical Sciences and Experimental
Therapeutics, College of Pharmacy, University of Iowa, Iowa City, IA, USA; Holden
Comprehensive Cancer Center, University of Iowa, Iowa City, IA, USA
xii Contributors

ARUN J. SANYAL • Division of Gastroenterology, Hepatology and Nutrition, Department of

Internal Medicine, Virginia Commonwealth University, Richmond, VA, USA
DEVANAND SARKAR • Department of Human and Molecular Genetics, Virginia
Commonwealth University, Richmond, VA, USA; Massey Cancer Center, Virginia
Commonwealth University, Richmond, VA, USA; VCU Institute of Molecular Medicine,
Virginia Commonwealth University, Richmond, VA, USA
ZHENFENG SONG • Center for Molecular Medicine and Genetics, Wayne State University
School of Medicine, Detroit, MI, USA
SILVIA SOOKOIAN • School of Medicine, Institute of Medical Research A Lanari, University of
Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina; Department of Clinical and
Molecular Hepatology, Institute of Medical Research (IDIM), National Scientific and
Technical Research Council (CONICET)—University of Buenos Aires, Ciudad Autonoma
de Buenos Aires, Argentina
SARAH SPIEGEL • School of Medicine and Massey Cancer Center, Richmond, VA, USA
FRANK TACKE • Department of Hepatology and Gastroenterology, Charité—
Universit€atsmedizin Berlin, Berlin, Germany
YUNLING TAI • Department of Microbiology and Immunology, Medical College of Virginia,
Virginia Commonwealth University, Central Virginia Veterans Health System, Richmond,
VA, USA
MINORU TANAKA • Department of Regenerative Medicine, Research Institute, National
Center for Global Health and Medicine, Tokyo, Japan
RULAIHA E. TAYLOR • Department of Pharmacology and Toxicology, Ernest Mario School of
Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ, USA
XIN TONG • Department of Molecular and Integrative Physiology, University of Michigan
Medical School, Ann Arbor, MI, USA
PATRICK TSO • Department of Pathology and Laboratory Medicine, University of Cincinnati
College of Medicine, Cincinnati, OH, USA
SHINYA TSURUSAKI • Department of Regenerative Medicine, Research Institute, National
Center for Global Health and Medicine, Tokyo, Japan
HAICHUAN WANG • Department of Bioengineering and Therapeutic Sciences and Liver
Center, University of California, San Francisco, CA, USA
JING WANG • Department of Internal Medicine, Virginia Commonwealth University
Medical Center, Richmond, VA, USA
WEI WANG • Department of Biochemistry and Molecular Biology, Virginia Commonwealth
University, Richmond, VA, USA
XIANG-YANG WANG • Department of Human and Molecular Genetics, Virginia
Commonwealth University, Richmond, VA, USA; Massey Cancer Center, Virginia
Commonwealth University, Richmond, VA, USA
ANDREW WONG • Department of Periodontics, School of Dentistry, Virginia Commonwealth
University, Richmond, VA, USA
SIMIAO XU • Division of Endocrinology, Tongji Hospital, Tongji Medical College, Huazhong
University of Science and Technology, Branch of the National Clinical Research Center for
Metabolic Disease, Wuhan, China; Division of Endocrinology, Boston Children’s Hospital,
Harvard Medical School, Boston, MA, USA
JUNKAI YAN • Department of Microbiology and Immunology, Medical College of Virginia,
Virginia Commonwealth University, Central Virginia Veterans Health System, Richmond,
VA, USA
 Contributors xiii

RAJESH YETIRAJAM • Department of Human and Molecular Genetics, Virginia

Commonwealth University, Richmond, VA, USA
DEQIANG ZHANG • Department of Molecular and Integrative Physiology, University of
Michigan Medical School, Ann Arbor, MI, USA
KEZHONG ZHANG • Center for Molecular Medicine and Genetics, Wayne State University
School of Medicine, Detroit, MI, USA; Department of Biochemistry, Microbiology, and
Immunology, Wayne State University School of Medicine, Detroit, MI, USA
HUIPING ZHOU • Department of Microbiology and Immunology, Medical College of Virginia,
Virginia Commonwealth University, Central Virginia Veterans Health System, Richmond,
VA, USA
 Chapter 1

Histopathological Diagnosis of Nonalcoholic

Steatohepatitis (NASH)
Shah Giashuddin and Mouyed Alawad

Abstract
Nonalcoholic steatohepatitis (NASH) is part of a spectrum of conditions collectively referred to as
nonalcoholic fatty liver disease (NAFLD). NASH/NAFLD is the most common chronic liver disease.
NASH is defined as 5% hepatic steatosis along with hepatocellular injury. Histopathological features that
indicate hepatocellular injury in NASH include ballooning degeneration, lobular inflammation, and apo-
ptotic bodies. Scoring schemes, such as the NASH Clinical Research Network (CRN), use those histo-
pathological features to grade the severity of the disease and determine a stage based on the amount of
fibrosis. Among the NAFLD spectrum, NASH has the highest risk of developing fibrosis and progressing to
liver cirrhosis. Therefore, accurate and timely diagnosis is crucial in order to initiate therapy and prevent
disease complications as well as liver-related mortality. Although several imaging modalities and laboratory
assays have been introduced to diagnose NASH, a liver biopsy remains the gold standard for diagnosing,
grading, and staging the disease.

Key words Nonalcoholic steatohepatitis (NASH), Nonalcoholic fatty liver disease (NAFLD), Hepatic
steatosis, Ballooning degeneration, NASH histopathology, NASH Clinical Research Network, NASH
cirrhosis

1 Introduction

Hepatic steatosis (HS) is the excess accumulation of fat, in the form

of triglycerides, within the cytoplasm of hepatocytes. Based on the
size of the triglyceride droplets HS is classified into two major
patterns; macrovesicular and microvesicular. Macrovesicular steato-
sis (Fig. 1) has one or up to a few medium- to large-sized triglycer-
ide droplets per hepatocyte pushing its nucleus to the periphery. In
contrast, microvesicular steatosis has numerous uniform tiny dro-
plets of lipid per hepatocyte and the nucleus is usually centrally
located. The pattern of steatosis alludes to the underlying disease
process. Macrovesicular steatosis results from dysregulation of lipid
metabolism and is largely the predominant pattern seen in fatty liver
diseases [1].

Devanand Sarkar (ed.), Non-Alcoholic Steatohepatitis: Methods and Protocols,

Methods in Molecular Biology, vol. 2455, https://doi.org/10.1007/978-1-0716-2128-8_1,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

1
2 Shah Giashuddin and Mouyed Alawad

Fig. 1 Macrovesicular steatosis. This is a hematoxylin and eosin-stained histo-

logic section showing numerous hepatocytes with macrovesicular steatosis. The
black arrow points out a hepatocyte with a triglyceride droplet and peripherally
displaced nucleus

Nonalcoholic fatty liver disease (NAFLD) is an umbrella term

that outlines a broad spectrum of overlapping conditions character-
ized by macrovesicular steatosis in the absence of significant alcohol
consumption. The various forms of NAFLD are primarily distin-
guished from one another by the degree of disease severity (Fig. 2).
At the lower end of the spectrum is nonalcoholic fatty liver
(NAFL), defined by the presence of 5% HS without hepatocellu-
lar injury. This is a benign form of the disease that has a low risk of
progression to fibrosis. When active injury ensues, the lesion is
called nonalcoholic steatohepatitis (NASH); defined as 5% HS
along with hepatocellular injury principally manifesting as balloon-
ing degeneration of hepatocytes (Fig. 3), lobular inflammation, and
apoptotic bodies. Scoring schemes have adopted the aforemen-
tioned histologic features for grading NASH into various degrees
of severity. Unlike NAFL, NASH has a considerably increased rate
of progression to fibrosis. The presence of fibrosis is not required
for the diagnosis of NASH. However, the degree of fibrosis is used
for staging the disease. Cirrhosis is the highest stage of fibrosis
(stage 4) and it represents the upper end of the NAFLD spectrum
(Fig. 4) [2, 3].
 Histopathological of NASH 3

Fig. 2 Nonalcoholic fatty liver disease (NAFLD) spectrum. This diagram illustrates the progression of disease
with increasing severity from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH), and finally
to cirrhosis with the arrows indicating an overlapping disease process

Fig. 3 Ballooning degeneration of nonalcoholic steatohepatitis (NASH). This is a

hematoxylin and eosin-stained histologic section showing hepatocytes with
ballooning degeneration. The black arrows point out swollen hepatocytes with
feathery cytoplasm and clumped eosinophilic material known as Mallory-Denk
bodies

2 Pathogenesis of NAFLD

The pathogenesis of NAFLD is initiated by the accumulation of

hepatic triglycerides leading to steatosis (NAFL). This occurs due
to the increased availability of free fatty acids (FFA) in the liver that
4 Shah Giashuddin and Mouyed Alawad

Fig. 4 Progression of NAFLD histopathology. The diagram shows the histopathologic changes that occur with
the progression of NAFLD. Nonalcoholic fatty liver (NAFL) shows macrovesicular steatosis. Nonalcoholic
steatohepatitis (NASH) mainly shows hepatocyte ballooning. Cirrhosis shows bridging fibrosis and a micro-
nodular architecture

are mainly derived from peripheral lipolysis due to insulin resistance

or de novo lipogenesis due to excess carbohydrate intake. Although
the pathogenesis of NASH is not completely understood, studies
have shown that lipolysis of the stored hepatic triglycerides releases
lipotoxic intermediates such as palmitate, ceramides, and diacylgly-
cerol. Those intermediates initiate an inflammatory cascade by
activating the innate and adaptive immune systems leading to the
release of pro-inflammatory cytokines and eventual hepatocellular
injury (Fig. 5) [4].

3 Prevalence and Incidence

NAFLD is thought to be the most common chronic liver disease.

Imaging and autopsy studies estimate around 20–30% of adults in
Western countries have some form of NAFLD and about 10% of
these individuals meet the diagnostic criteria of NASH. This is
calculated to be about 2–3% of the adult population with the
progressive form of the disease and at an increased risk of
 Histopathological of NASH 5

Fig. 5 Pathogenesis of nonalcoholic steatohepatitis (NASH). This diagram shows a stepwise process of NAFLD
pathogenesis, starting from excess free fatty acids leading to hepatic triglyceride accumulation and advancing
to lipotoxic liver injury and inflammation resulting in NASH

developing cirrhosis. Similarly, the global prevalence of NAFLD is

about 25.24% with the majority of cases reported from the Middle
East and South America and the least prevalence reported from
Africa. There is a lack of data about the incidence of NAFLD in the
general population and it is believed to be less commonly reported
in Western countries [5].

4 Risk Factors

The risk of developing NAFLD is increased in individuals with

metabolic syndrome or its components, including obesity (most
common risk factor), type 2 diabetes mellitus (DM), hypertrigly-
ceridemia, and low high-density lipoprotein. Insulin resistance, one
of the main drivers of NAFLD pathogenesis, is a feature of meta-
bolic syndrome and its components. Other risk factors implicated
in the development of NAFLD include excess carbohydrate intake
either from the diet or in the form of total parental nutrition.
Surgeries that alter the bowel anatomy, such as gastrointestinal
bypasses for obesity, also increase the risk of hepatic steatosis.
NAFLD may also develop in the setting of certain drugs such as
tamoxifen, corticosteroids, amiodarone, methotrexate, and several
others. Hepatic steatosis with histologic features of NASH is seen in
some metabolic disorders such as abetalipoproteinemia and
6 Shah Giashuddin and Mouyed Alawad

Wilson’s disease. Chronic hepatitis C virus and human immunode-

ficiency virus are two main infectious risk factors for developing
hepatic steatosis [3, 6].

5 Clinical History

The most common symptoms of NAFLD include fatigue, right

upper quadrant abdominal pain, and hepatomegaly. The main lab-
oratory abnormalities include increased serum alanine aminotrans-
ferase (ALT) and aspartate aminotransferase (AST). Patients with
NASH generally have higher levels of AST and ALT. Nevertheless,
these values are not reliable because they often fluctuate and can be
normal in patients with the disease. Similarly, other available labo-
ratory assays are not sensitive nor specific for the diagnosis and
characterization of NAFLD. Imaging studies such as ultrasonogra-
phy, computed tomography, and magnetic resonance may help
detect NAFLD. However, these modalities do not make the crucial
distinction between simple steatosis and NASH. Currently, a liver
biopsy remains the gold standard for the diagnosis and characteri-
zation of NAFLD/NASH [2].

6 Natural History

Patients with NAFLD, including all its degrees, have a higher

mortality rate when compared to matched control populations.
Cardiovascular disease (CVD) has been reported to be the most
common cause of death in those patients. Liver-related mortality is
particularly increased in patients with NASH as compared to milder
forms of the disease. NASH is the second most common cause for
requiring liver transplantation. As previously mentioned, the evo-
lution of fibrosis occurs much more frequently in NASH. This is
corroborated by the fact that fibrosis is the most relevant and
independent histologic finding associated with both overall mortal-
ity and liver-related mortality as well as the need for liver transplan-
tation. Patients with advanced fibrosis or cirrhosis are also at a
higher risk of developing hepatocellular carcinoma (HCC). It is
now considered that NAFLD is the third most common cause of
HCC in the USA [3, 7].

7 Clinical Diagnosis of NAFLD

The diagnosis of NAFLD requires that (a) there is no significant

alcohol consumption, (b) there is HS by imaging or histology,
(c) there are no competing etiologies for HS, and (d) there are no
coexisting causes of chronic liver disease.
 Histopathological of NASH 7

7.1 Serologic Serologic testing in many instances uncovers some abnormalities

Evaluation that are not evident clinically either by signs/symptoms or radiolo-
gic evaluation. The most frequently found abnormalities are ele-
vated serum ferritin and autoimmune antibodies. Mildly elevated
serum ferritin is a common feature of NAFLD/NASH and it does
not necessarily indicate hepatic iron overload, although it is
thought to play a role in disease progression, particularly cirrhosis.
If serum ferritin and transferrin saturation are elevated in a patient
with suspected NAFLD, liver biopsy is warranted and genetic
hemochromatosis should be excluded. Similarly, low titers of
serum autoantibodies, particularly anti-smooth muscle and antinu-
clear antibodies, are common in patients with NAFLD/NASH.
Autoantibodies, in particular antinuclear antibody, have been
reported in up to 34% of individuals with NAFLD, but without
any clinical consequences in most of the cases, and often require a
liver biopsy to exclude autoimmune disease. Initial evaluation of
patients with suspected NAFLD should carefully consider the pres-
ence of commonly associated comorbidities such as obesity, dysli-
pidemia, insulin-resistant DM, hypothyroidism, polycystic ovary
syndrome, and sleep apnea. Other laboratory tests commonly
done in suspected NAFLD patients include alanine aminotransfer-
ase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl
transferase (GGT), as well as tests for hyperglycemia (glycated
hemoglobin [HbA1c]), insulin resistance, and lipid parameters.
Generally, ALT levels are more elevated than AST levels, and an
ALT-to-AST ratio greater than 1 is commonly seen in patients with
pre-cirrhotic NAFLD (in contrast to alcoholic liver disease). The
overall sensitivity of liver enzymes for the detection of fatty liver
disease is poor [8].

7.2 Noninvasive Noninvasive tools should be used to stratify patients as low or high
Assessment of NAFLD risk for advanced fibrosis, but a preferred sequence of testing is not
and Steatohepatitis provided in the American and Asian guidelines. The European
guideline provides a proposed diagnostic algorithm with sugges-
tions to guide referral to hepatology. In addition, it provides a
proposed follow-up strategy to monitor for disease progression
with the caveat that optimal follow-up has yet to be determined
noninvasive tools should be used to stratify patients as low or high
risk for advanced fibrosis, but a preferred sequence of testing is not
provided in the American and Asian guidelines. The European
guideline provides a proposed diagnostic algorithm with sugges-
tions to guide referral to hepatology. In addition, it provides a
proposed follow-up strategy to monitor for disease progression
with the caveat that optimal follow-up has yet to be determined.
Commonly used radiology techniques for detection of the
presence of parenchymal fat in suspected NAFLD/NASH patients
are ultrasonography, CT, or MRI. Newer types of imaging
8 Shah Giashuddin and Mouyed Alawad

modalities like magnetic resonance (MR) imaging, either by spec-

troscopy or by proton density fat fraction are an excellent noninva-
sive technique for quantifying HS and are being widely used in
NAFLD clinical trials. Tissue elastography (TE) to obtain continu-
ous attenuation parameters is a newer promising tool for quantify-
ing hepatic fat in an ambulatory setting. However, it is well
recognized that when steatosis involves less than 33% of the liver
parenchyma, visual imaging tests are not sensitive. Imaging is also
not an adequate replacement for tissue analysis for the determina-
tion of the level of disease activity, or the stage of fibrosis, in patients
with pre-cirrhotic NASH.
The Fibrosis-4 (FIB-4) score and Fibroscan help the primary
care physicians and hepatologists to estimate the amount of scar-
ring in the liver in patients with the incidental finding of steatosis on
imaging. For risk stratification, the FIB-4 scoring system uses a
combination of patient age, platelet count, AST, and ALT. A
FIB-4 score - <1.45 has a negative predictive value of over 90%
for advanced liver fibrosis of multiple etiologies; a score of >3.25
has a positive predictive value of 65% for advanced fibrosis with a
specificity of 97%. Once the patient is identified as an intermediate
or high-risk group, further workup (MR elastography or Fibros-
can) and possible liver biopsy is considered [9–16].

7.3 The Role and There is no consensus on when a biopsy should be obtained with
Indication of Liver respect to the stage of disease. Liver biopsy should be performed in
Biopsy in NAFLD those who would benefit the most from diagnosis, therapeutic
guidance, and prognostic perspectives, like in patients with
NAFLD who are at increased risk of having steatohepatitis
(SH) and/or advanced fibrosis. Several scenarios of suspected
NAFLD lead to liver biopsy. These include but are not limited to
unexplained elevations of liver enzymes, incidental findings of stea-
tosis or fibrosis on imaging studies, or incidental findings during
surgery (often bariatric surgery or cholecystectomy). The presence
of metabolic syndrome (MetS), high NAFLD fibrosis score (NFS)
or FIB-4, or liver stiffness measured by vibration-controlled tran-
sient elastography (VCTE) or MR elastography may be used for
identifying patients who are at risk for SH and/or advanced fibro-
sis. However, despite advances in clinical laboratory tests and imag-
ing studies, histologic examination of the liver biopsy remains the
gold standard for the diagnosis of NAFLD, as it allows a superior
assessment of the degree of the steatosis and liver injury (grade), as
well as fibrosis (stage), but its widespread use is limited by cost,
sampling error, and procedure-related morbidity and mortality
(Fig. 6) [6, 8, 17].
 Histopathological of NASH 9

Fig. 6 NAFLD workup algorithm. This diagram shows NAFLD workup algorithm and liver biopsy indications in
relation to clinical information, imaging studies, and laboratory studies. Abbreviations: AST: Aspartate
transaminase; ALT: Alanine transaminase; INR: international normalized ratio; CT: Computed
tomography; MRI: Magnetic resonance imaging. (Adapted from ref. 6)

8 Pathology of NAFLD and NASH

8.1 Gross Features Noncirrhotic fatty livers are typically enlarged and yellow and have a
greasy consistency. Once cirrhotic, the liver may be small or
enlarged. Fat may be observed in an irregular pattern in liver
nodules as yellow areas compared with neighboring tan nodules.
Cirrhotic livers in NAFLD may not be as firm, have the same
stroma-to-parenchyma ratio, or be as micronodular as in alcoholic
liver disease (ALD) (Fig. 7).

8.2 Microscopic Liver biopsy in a suspected NAFLD case should be evaluated to

Features confirm or exclude the diagnosis and assess the severity of the
disease. A generalized approach for histopathological evaluation
of liver biopsies in suspected NAFLD cases is to assess all of the
10 Shah Giashuddin and Mouyed Alawad

Fig. 7 Gross and microscopic spectrum of NAFLD. This pictorial chart shows gross and histologic images of a
normal liver compared to livers in the various forms of NAFLD. The top row shows the cut surface of the liver
parenchyma on gross examination. Note the yellowish-brown discoloration of livers in NAFLD and the
irregularity and loss of smooth texture with progression to fibrosis. The middle row shows hematoxylin and
eosin stains demonstrating macrovesicular steatosis around a central vein (second column from left),
hepatocyte ballooning in steatohepatitis (third column from left), and micronodular cirrhosis (fourth column
from left). The bottom row shows trichrome stains to evaluate for fibrosis. Trichrome stains collagen blue, note
the bridging fibrosis of micronodular cirrhosis (fourth column from left)

relevant features: (a) degree of steatosis, (b) presence or absence of

hepatocyte injury (ballooning degeneration), (c) lobular inflamma-
tion, and (d) fibrosis.
The current practice guidelines from AASLD (American Asso-
ciation for the Study of Liver Diseases) recommends reporting the
grade (necroinflammatory “activity”) and stage (location of abnor-
mal/increased collagen deposition, that is, “fibrosis”) following
clinically useful scoring systems.
Several histological scoring systems for NAFLD have been
practiced clinically in the USA; two were reported by Matteoni
and Brunt in 1999, and then a semiquantitative scoring system
was developed by the National Institutes of Health-sponsored
Nonalcoholic Steatohepatitis Clinical Research Network (NASH
CRN) in 2005. The NASH CRN system describes the NAFLD
activity score (NAS), which is a composite score of steatosis, lobular
inflammation, hepatocyte ballooning, and fibrosis (disease stage).
The following diagnostic categories for NAFLD have been utilized
by the NASH CRN: (Table 1)
 Histopathological of NASH 11

Table 1
NASH Clinical Research Network (CRN) semiquantitative scoring system

NASH CRN Scoring System

Lobular Inflammation Hepatocyte

Steatosis
(counted in x 20 fields) Ballooning

Score Criteria Score Criteria Score Criteria

0 <5% 0 None 0 None

1 5-33% 1 < 2 foci 1 Few

2 34-66% 2 2-4 foci 2 Many

3 >66% 3 >4 foci

NAFLD activity score (NAS)

NAS total range (0-8):
Steatosis (0-3) + Lobular inflammation (0-3) + Hepatocyte Ballooning (0-2)

Fibrosis Staging

Stage Criteria

0 None
Mild (zone 3 perisinusoidal fibrosis that may only
1a
be only be identified on a trichrome stain)
Moderate (zone 3 perisinusoidal fibrosis that is
1b
easily identified on a hematoxylin and eosin stain)
1c Portal/periportal fibrosis only
Zone 3 perisinusoidal fibrosis + Portal/periportal
2
fibrosis
3 Bridging fibrosis

4 Cirrhosis
Adapted and modified from ref. 19
12 Shah Giashuddin and Mouyed Alawad

(a) Not NAFLD (<5% steatosis, by definition);

(b) NAFL, not NASH (5% steatosis, with or without lobular and
portal inflammation);
(c) Borderline steatohepatitis, zone 3 or zone 1 (most, but not all
criteria for SH present, with accentuation of steatosis or injury
in zone 3 or zone 1, respectively); and.
(d) Definite steatohepatitis (all criteria present, including steatosis,
hepatocellular ballooning, and lobular inflammation).
The NAS (NAFLD Activity Score) is widely recognized and has
been implemented primarily for the use in clinical research. The
system uses the unweighted scores of steatosis (scores 0–3), inflam-
mation (scores 0–3), and hepatocellular injury reflected by balloon-
ing degeneration (scores 0–2) to obtain an overall score from
0 to 8.
It is important to understand that the NAS (NAFLD score) is
not a substitute for histologic evaluation, including the crucial
distinction between NASH and simple steatosis. Moreover, NAS
cannot be used to predict the risk of progressive fibrosis, likely
because it is composed of unweighted contributions of steatosis
relative to inflammation and hepatocellular injury to the total score
(0–8). Nonetheless, some clinicians have found the NAS useful in
their practice because it has been reported that a NAS of 5 or more
correlates with NASH, whereas a NAS of less than 3 essentially
excludes NASH [18–20].

8.3 Steatosis In normal liver parenchyma, there is a minimal amount of fat

accumulation (<5%) present and it is not considered abnormal.
The significance of this finding is unknown and not considered
pathologic or diagnostic for steatosis. Considering the morpho-
logic appearance and the size of the lipid droplets within the cyto-
plasm of the hepatocytes, steatosis is subtyped as macrovesicular or
microvesicular. Microvesicular steatosis is characterized by numer-
ous, tiny, relatively uniform lipid vacuoles that create a bubbly
appearance of the hepatocytes. Pure diffuse microvesicular steatosis
is rare and is usually a result of abnormalities in mitochondrial and
peroxisomal-β oxidation. The most commonly seen clinical entities
with pure microvesicular steatosis are acute fatty liver of pregnancy
in adults and Reye’s syndrome in children. Histologically, micro-
vesicular steatosis is commonly seen as a patchy finding in the
background of macrovesicular steatosis of NAFLD or ALD.
Macrovesicular steatosis is present in nearly all of the NAFLD
cases and is a required histologic finding for the definitive diagnosis
for NASH. Macrovesicular steatosis is characterized by large lipid
droplets occupying the cytoplasm, displacing the nucleus to the
periphery (large droplet macrovesicular steatosis), or multiple small
lipid droplets of variable size occupying the cytoplasm with the
 Histopathological of NASH 13

nucleus maintaining its central location (small-droplet macrovesi-

cular steatosis). A common pitfall is to mistake small-droplet
macrovesicular steatosis for microvesicular steatosis. In adult
NAFLD, steatosis commonly begins in zone 3 hepatocytes, which
contrasts to zone 1 steatosis in viral hepatitis C or pediatric cases of
NAFLD.
Current sophisticated imaging quantification studies have
shown that the involvement of 5% or less hepatocytes by steatosis
is normal (within the reference range) in the American population.
Steatosis of at least 5% or more is, therefore, the minimum criterion
for a histopathological diagnosis of NAFLD. Additionally, zonal
distribution (around central vein, zone 3 predominance vs. around
portal tracts, zone 1 predominance) is a clue to determine the
etiology (adult NAFLD vs. pediatric and steatosis secondary to
HCV, respectively). By light microscopy, clinically significant stea-
tosis can range from mild (5–33%, grade 1) to moderate (34–66%,
grade 2) or severe (>66%, grade 3). Overestimation of steatosis is a
common clinical problem and can be resolved by a consensus
opinion from an expert pathologist [17].

8.4 Steatohepatitis The minimal criteria for steatohepatitis are the presence of steatosis
(>5%), hepatocellular ballooning, and lobular inflammation
(Fig. 7). Hepatocyte ballooning is considered a manifestation of
significant cell injury. Histologically, ballooning degeneration cor-
responds to swelling of the hepatocyte with round contour, and
cytoplasm appears reticulated, rarified, or flocculant quality. The
cytoplasm of the ballooned hepatocytes often contains clumps of
eosinophilic ropey material known as Mallory-Denk bodies
(MDBs) or Mallory hyaline, which are composed of hyperpho-
sphorylated misfolded intermediate filaments, ubiquitin, and
ubiquitin-binding protein P62. It is important to remember that
the MDB can be present in non-ballooned hepatocytes. Although
required for the diagnosis of steatohepatitis, ballooning degenera-
tion occurs in other diseases, like chronic cholestasis, in which it is
usually confined to periportal hepatocytes (zone 1) and known as
feathery degeneration or pseudoxanthomatous change. Histologic
evaluation of ballooning degeneration is further complicated by
significant intra-observer and inter-observer variability.
Despite the diagnostic challenge, it is crucial to identify hepa-
tocyte ballooning and MDB because they are associated with an
increased rate of progression to fibrosis. The underlying pathoge-
netic mechanisms that result in ballooning in NASH are not fully
understood [1, 2, 8, 17].

8.5 Lobular Steatosis is commonly associated with chronic mixed inflammatory

Inflammation infiltrates in hepatic lobules. These inflammatory infiltrates are
patchy and usually mild and composed of mostly lymphocytes and
rare neutrophils and plasma cells. Neutrophils when predominant
14 Shah Giashuddin and Mouyed Alawad

and surrounding a ballooning hepatocyte, known as “satellitosis,”

are more frequently seen in alcohol-induced liver injury and less
common in NASH. Another potential misleading finding is “surgi-
cal hepatitis” where neutrophils are present as clusters in lobules
and perivenular location. Surgical hepatitis is a known consequence
of surgery, anesthesia, or organ manipulation, and is frequently
seen when the liver biopsies are obtained during cholecystectomy
or bariatric surgery. The recommended approach to prevent surgi-
cal hepatitis is to obtain the intraoperative liver biopsy before the
procedure and manipulation of the liver. During the microscopic
evaluation of lobular inflammation, pathologists should pay careful
attention to the location and composition of the inflammatory
infiltrates. Eosinophils, neutrophils, and lipogranulomas should
not be considered in the assessment of lobular inflammation.
Another very common histologic finding is the portal inflamma-
tion, accompanied by mixed lymphocytic and mononuclear cell
infiltrates; currently, there are four clinical scenarios in which portal
inflammation can be noted in NAFLD/NASH: (a) Pediatric
NAFLD, (b) severe NASH in adults and children, (c) post-
treatment for NASH, and (d) concurrent liver disease in NAFLD.
A mild to moderate portal chronic inflammation showed significant
correlations with steatosis, ballooning, and advanced fibrosis in
adults, and with portal/periportal fibrosis in children. There were
no correlations with lobular inflammation scores, laboratory mar-
kers of liver injury, or presence of autoantibodies [1–3, 6, 8, 17].

8.6 Other These are non-essential for the diagnosis of NAFLD/NASH, yet
Histopathologic commonly encountered histologic findings during the evaluation
Features of NAFLD of liver biopsies. Those are: Mallory-Denk bodies, apoptosis and
hepatocyte necrosis, ductular reaction, megamitochondria, iron
deposition, glycogenated nuclei, and glycogenosis [3, 8, 17].

9 Fibrosis and Architectural Remodeling: The Potential Complications of NASH

Similar to other chronic liver diseases, the potential complication of

NAFLD/NASH is the development of aberrant collagen deposi-
tion, the “fibrosis,” and it is not currently considered a requirement
for the diagnosis of NASH. The distinctive pattern of fibrosis seen
in NAFLD/NASH is pericellular and perisinusoidal (“chicken
wire”), which results from abnormal deposition of collagen and
extracellular matrix in the space of Disse. Pericellular fibrosis, in the
early stage, usually begins in zone 3 of the hepatic lobule and may
or may not be associated with features of steatosis and/or steato-
hepatitis. In the NASH CRN/NAS scoring system, this early peri-
cellular fibrosis is detectable by trichrome stain (stage 1a) and when
it is detectable by routine hematoxylin and eosin (H&E) stain, it is
 Histopathological of NASH 15

stage 1b. When chronic liver injury continues, with the remodeling
of liver parenchyma, pericellular fibrosis progresses to portal fibrous
expansion, periportal fibrosis, bridging fibrosis, and eventually, the
development of cirrhosis. Bridging fibrosis, in many cases can be
seen in a central-central, central-portal, or portal-portal pattern.
NAFLD-associated cirrhosis is usually macronodular or mixed
micro-and macronodular. In an established case of cirrhosis, active
features of steatohepatitis and perisinusoidal fibrosis may not be
appreciated histologically (Table 1) [1–3, 6, 8, 17].
The pathology committee of the National Institute of Diabetes
and Digestive and Kidney Diseases (NIDDK) sponsored the NASH
Clinical Research Network (CRN) which developed and validated a
feature-based scoring system for the spectrum of lesions of
NAFLD. The NASH CRN scoring system maintained the features
of the Brunt schema for grading, but the staging system was mod-
ified. The system includes the lower end of the histological spec-
trum of NAFLD, as represented by 5% steatosis for minimum
criterion for NAFLD. The fibrosis score is a modification of the
previously proposed “Brunt” score to separate delicate (requires
trichrome stain to visualize) and dense (identified by routine H&E
stain) zone 3 perisinusoidal fibrosis (1a and 1b, respectively), and to
document non-bridging, portal-only fibrosis (1c). It is worth men-
tioning here that, the numeric value of the NAFLD activity score is
not intended to be a diagnostic aid for NASH and should be used
to assess the activity of the disease once the diagnosis of NASH has
been established by qualitative, non-numeric parameters. The diag-
nosis and grading are separate exercises with distinct purposes, and
the NAFLD activity score is formulated chiefly for comparison of
histologic changes to assess the therapeutic efficacy of various treat-
ment strategies [1–3, 6, 8, 17, 19].

10 Pathology of Pediatric NAFLD/NASH

NAFLD in the pediatric population is more attributed to genetic

risk and increased penetrance and sensitivity to environmental fac-
tors. Obesity/overweight, various metabolic syndromes (MetS),
fatty acid oxidation defects, lysosomal storage disease, and peroxi-
somal disorders are compounding factors in addition to those
considered for adults. Low serum titers of autoantibodies are
often present in children with NAFLD, but higher titers, particu-
larly in association with higher serum aminotransferases, high glo-
bulins, or high total protein to albumin ratio, should prompt a liver
biopsy to exclude autoimmune hepatitis (AIH) and related autoim-
mune disorders.
Histopathology of pediatric NAFLD can differ from that found
in adults by: (a) pronounced hepatocellular injury/steatohepatitis;
(b) less or no ballooning or Mallory–Denk bodies; (c) less lobular
16 Shah Giashuddin and Mouyed Alawad

Fig. 8 Microscopic differences between adult and pediatric NAFLD/NASH. This pictorial chart shows histologic
sections of NAFLD/NASH in an adult case compared to a pediatric case (top row: hematoxylin and eosin stains;
bottom row: trichrome stains). Note the portal-based lesion in pediatric NAFLD/NASH showing increased
macrovesicular steatosis in zone 1 of the liver along with marked portal and periportal inflammation

inflammation; (d) few or no polymorphonuclear leukocytes; and

(e) increased portal tract inflammation and fibrosis (Fig. 8).
Growing experience with pediatric NASH shows the findings
may more closely resemble adult histology than in early published
series. Distinct from the typical zone 3 injury seen in adults,
NAFLD/NASH in children is typically characterized by diffuse,
marked, macrovesicular, hepatocellular steatosis or zone 1, peripor-
tal steatosis, portal inflammation, and portal fibrosis in the absence
of ballooning. Ballooning degeneration is not an obvious feature. It
is worthwhile mentioning that many affected children with
advanced fibrosis demonstrate this pattern of injury [1–3, 6, 8,
17, 21].

11 Conclusion

Clinical history (i.e., history of alcohol abuse, severe malnutrition,

or drug history) is the key to the assessment of liver biopsies
showing steatosis and steatohepatitis, particularly with the NAS
system. Important examples where clinical history proves to be
indispensable include a severely malnourished patient or a patient
with a long history of alcohol use who may have severe steatosis
with steatohepatitis; in such cases, the use of the NAS system would
 Histopathological of NASH 17

be completely inappropriate. Many histologic features of steatohe-

patitis are mainly due to some medications. Hepatocyte ballooning
degeneration is an appropriate example in this scenario. It is cru-
cially important to pay attention to smaller details (i.e., zone 3 vs.
zone 1 involvement and the composition of inflammatory cell
infiltrate where neutrophils and eosinophils should not be consid-
ered and many more). In equivocal cases, the pathologist should
mention in the report whether there is insufficient or unclear
clinical history. The NAS system for scoring steatosis, ballooning,
lobular inflammation, and fibrosis is useful and allows a brief and
transparent conception of the severity of disease to the clinician but
does not replace the histologic evaluation and discrete reporting of
the presence or absence of steatohepatitis [17].

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 Chapter 2

Generation of a Diet-Induced Mouse Model of Nonalcoholic

Fatty Liver Disease
Amon Asgharpour and Arun J. Sanyal

Abstract
The obesity epidemic is driving the increased prevalence of nonalcoholic fatty liver disease (NAFLD)
globally. The more aggressive subtype of NAFLD, nonalcoholic steatohepatitis (NASH), can lead to
progressive disease and ultimately lead to cirrhosis, liver cancer, and death. There are many unmet needs
in the field of NAFLD including understanding of molecular mechanisms driving disease, natural history,
risk for liver cancer, and most importantly FDA approved therapeutics. Animal models serve as a tool to aid
in answering some of these questions. Here, we describe the diet-induced animal model of NAFLD
(DIAMOND), a mouse model with many characteristics that mimic human NASH.

Key words Nonalcoholic fatty liver disease (NAFLD), Nonalcoholic steatohepatitis (NASH), Hepa-
tocellular carcinoma (HCC), Mouse model, Liver fibrosis, DIAMOND

1 Introduction

Nonalcoholic fatty liver disease, NAFLD, is a common liver disease

that affects about a quarter of the world population [1]. NAFLD is
intimately linked to the metabolic syndrome that consists of obe-
sity, insulin resistance, hypertension, and dyslipidemia. The preva-
lence of obesity is increasing worldwide and NAFLD has followed a
similar trend [2, 3]. Furthermore, large-scale epidemiological stud-
ies link obesity and being overweight as risk factors for hepatocel-
lular carcinoma (HCC) [4], with NAFLD as the liver manifestation
of the metabolic syndrome. Most individuals that have NAFLD
have a nonalcoholic fatty liver, NAFL, previously called “simple
steatosis” as there is >5% hepatic steatosis without significant his-
tological activity (hepatocyte ballooning and inflammation). Non-
alcoholic steatohepatitis, NASH, is the more aggressive subtype of
NAFLD with hepatic steatosis, >5%, hepatocyte ballooning and

Conflict-of-Interest: Both authors own equity in SanyalBio.

Devanand Sarkar (ed.), Non-Alcoholic Steatohepatitis: Methods and Protocols,

Methods in Molecular Biology, vol. 2455, https://doi.org/10.1007/978-1-0716-2128-8_2,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

19
20 Amon Asgharpour and Arun J. Sanyal

lobular inflammation that can lead to progressive liver fibrosis,

cirrhosis, and HCC [5]. NASH is estimated to affect 3–6% of the
U.S. population and can progress to cirrhosis in 20% of cases
[6]. The growing incidence and prevalence of NASH cirrhosis has
seen a parallel growth in the need for liver transplantation in the
USA for NASH, becoming the number one indication for liver
transplantation in females [7]. There are currently no FDA
approved medications to treat patients with NAFLD, but weight
loss has the propensity to achieve resolution of NASH and can
reverse/resolve fibrosis [8]. Unfortunately, many patients cannot
attain adequate weight loss to achieve these goals. There are a
multitude of ongoing clinical trials in the NASH space but there
is a critical need to further understand the natural history, mechan-
isms, and drivers of this disease process so that realistic intervention
and medications can be utilized to treat those afflicted by this
disease.
In order to gain ground on our numerous unmet needs in the
NASH space, preclinical models of NASH are leveraged to facilitate
our understanding. There are many preclinical models, with mouse
models being some of the most widely used. No model perfectly
mimics the human state, but an ideal animal model would share
disease drivers and features concordant with human NASH. Phe-
notypically they would exhibit weight gain leading to obesity, insu-
lin resistance, and dyslipidemia. On liver histology they would
demonstrate steatosis, lobular inflammation, hepatocyte balloon-
ing, Mallory-Denk bodies, pericellular fibrosis, cirrhosis, and possi-
ble HCC (Fig. 1). For there to be a diagnosis of NASH there needs
to be steatosis, lobular inflammation, and hepatocyte ballooning
present on histology but the degree of each parameter can vary.
Fibrosis staging is independent of a NASH diagnosis as NASH can
be present with no fibrosis to cirrhosis. Alterations in diet, genetics,
and usage of toxins are often utilized to achieve these desired
characteristics. One must exhibit caution when deciding which
mouse model to use as they all have their own strengths and
weaknesses. It is important to choose a model that is best suited
to answer the scientific question.
Some models sacrifice phenotypic features aligned with human
NAFLD to achieve histological features that mimic human NASH.
An example is the methionine and choline deficient, MCD, diet
where, on histology, there is progressive fibrosis and steatosis but
animals lose weight and are not insulin-resistant [9–11]. Other
models use physiologically relevant diets with high fat, high choles-
terol, and high fructose corn syrup to illicit NASH with progressive
fibrosis [12, 13]. To help condense the timeline of disease progres-
sion some models have made use of toxins such as carbon tetra-
chloride to induce fibrosis [14, 15] and diethylnitrosamine to
induce tumors [16, 17]. In humans, certain medications such as
methotrexate are capable of causing a fatty liver with fibrosis, but
 Diet-Induced Mouse Model of NAFLD 21

Fig. 1 Schema of NAFLD rodent models that use various diets, genetic mutations, and/or toxins to achieve
phenotypic and histologic features of human NAFLD/NASH

liver injury in this setting is more aligned with drug-induced liver

injury as opposed to NAFLD [18]. Toxins are not implicated in the
majority of NASH in humans and there use in preclinical models
could serve as potential confounders when compared to human
disease. Genetically engineered mice can also be used to induce
disease relevant drivers such as endoplasmic reticulum stress via a
mutation in urokinase plasminogen activator (MUP-uPA mice) in
combination with a high fat diet to precipitate NASH with disease
progression [19]. However, genetically altered mice usually have
the induced mutation in at least all hepatocytes, if not other or all
cell types, while in humans these mutations typically only exist in
tumor cells and not surrounding tissue [20]. Mutations in human
NASH have relevancy, and genes that increase susceptibility,
PNPLA3 and TM6SF2, and reduce susceptibility, HSD17B13,
were described using genome wide association studies [21–23],
but implementation of these mutations in preclinical models is
not widely used.
This chapter will focus on the diet-induced animal model of
NAFLD, DIAMOND, that utilizes a stable isogenic strain of
C57BL6/J and S129S1/svlmJ mice with nearly 60% of genes
arising from the C57BL6/J background. These mice do not con-
tain any known genetic mutations and the model does not
22 Amon Asgharpour and Arun J. Sanyal

Fig. 2 DIAMOND mouse liver histology after 52 weeks of Western diet with high-fructose water solution. (a)
H&E stain with blue arrow pointing to macrovesicular steatosis. Black arrow pints to a foci of lobular
inflammation. (b) H&E stain at a higher magnification with black arrow pointing to a ballooned hepatocyte
with Mallory-Denk bodies. (c) Picrosirius Red staining with red color indicating areas of fibrosis

incorporate usage of potentiating toxins. If mice are given a stan-

dard diet they exhibit no features of NAFLD and are not obese
having normal weight, do not demonstrate insulin resistance and
have normal liver histology. If these mice are given a high fat diet
with physiologically relevant cholesterol: 42% calories from fat,
0.1% cholesterol and fructose/glucose: 23.1 g/L d-fructose and
18.9 g/L d-glucose in drinking water they develop progressive
NAFLD leading to NASH with advanced fibrosis and HCC. DIA-
MOND mice develop obesity, insulin resistance, dyslipidemia, and
elevations in aminotransferases, all aligned with human NASH.
Histologic examination of mouse livers shows macrovesicular stea-
tosis after 4 weeks and progression to steatohepatitis with inflam-
mation, hepatocyte ballooning, and Mallory-Denk body formation
after 8–16 weeks (Fig. 2). Pericellular liver fibrosis, stage 1 fibrosis,
begins at 16 weeks and progresses to stage 3 and early stage
4 fibrosis by 52 weeks of the diet. HCC develops in approximately
90% of mice by 32–52 weeks and mice have evidence of multiple
liver lesions. All mice with evidence of HCC have at least well-
differentiated HCC, 40% have poorly differentiated HCC, and 25%
have hepatic adenomas. Some hepatic adenomas have foci of HCC
within them seen on histology. Furthermore, the transcriptomic
sequence of non-HCC liver tissue in DIAMOND mice demon-
strated concordance with human NASH at both 8 weeks and
52 weeks of diet with oxidative stress, activation of unfolded pro-
tein response, activation of inflammatory, apoptotic, and fibrogenic
pathways [13]. At a transcriptomic level, HCC in DIAMOND mice
was related to the S1 or S2 subclasses of human HCC, false discov-
ery rate (FDR): 0.013 and 0.014, respectively [13, 24]. While no
model is a perfect representation of the human state, the above
features make the DIAMOND model suitable to help answer many
potential questions in the NASH space. Limitations of this model
include the duration needed for histologic NASH with fibrosis,
 Diet-Induced Mouse Model of NAFLD 23

8–16 weeks, and HCC development, 32–52 weeks. Furthermore,

the mixed genetic background makes it less amenable to genetic
alterations such as knocking-out or over expressing genes of inter-
est to investigate biologic pathways.

2 Material

2.1 Mice A unique, isogenic mouse strain derived from a C57BL/6J and
129S1/SvImJ background, B6/129, is created and maintained
with inbreeding (see Note 1). The strain is started by mating the
F2, second filial generation (Jackson Stock # 101045), with one
another. After 20 generations derived from brother-sister mating,
an isogenic strain is developed from the original F2 strain of mice
(see Note 1). Pure C57BL/6J, B6, or 129S1/SvImJ, S129, are
purchased from Jackson Laboratory and used as controls.

2.2 Diet 1. Western diet: 42% calories from fat, 0.1% cholesterol (Harlan).
2. Standard chow diet (Harlan).
3. High fructose-glucose drinking water: 23.1 g/L d-fructose
+18.9 g/L d-glucose.

2.3 Glucose and 1. Accu-Chek Compact plus glucometer (Roche Diagnostics)

Insulin Tolerance with test strips.
Tests (GTT, ITT) 2. Scalpel or scissors for tail nick to obtain blood.
3. Dextrose.
4. Insulin.
5. Sterile Phosphate Buffered Saline (PBS).

2.4 Blood Collection 1. Heparinized micro-hematocrit capillary tubes (Fisher

(See Note 2) Technology).
2. Eppendorf tubes.
3. Refrigerated centrifuge.
4. Bucket with ice.
5. Advia 1800 Chemistry System (Siemens).
6. Mouse Adiponectin/Acrp30 Quantikine ELISA Kit (R&D
Systems).
7.
80  C freezer.

2.5 Tissues Sample 1. Liquid Nitrogen.

Collection 2. Necropsy instruments: scissors and forceps.
3. Anesthetics, inhaled or injectable, according to animal use
protocol (see Note 3).
4. Cryotubes with cardboard boxes to place the tubes in for
storage (see Note 4).
24 Amon Asgharpour and Arun J. Sanyal

5. 10 ml plastic containers prefilled with 10% formalin (see

Note 5).
6. RNAlater (ThermoFisher) placed in Eppendorf tubes.
7. Scale.
8. Camera.
9. Ruler.
10. 70% Ethanol.
11. Glass bead sterilizer to clean necropsy instruments (Braintree
scientific).

2.6 Histological 1. Hematoxylin and eosin (H&E) stain.

Analysis 2. Trichrome stain.
3. Sirius red stain.
4. Aperio system (Leica).

3 Methods

3.1 Mice and Diets 1. Breed/purchase mice to generate animals of the desired num-
ber and sex. Record date of birth.
2. Feed male mice, 8–12 weeks of age (see Note 6) ad libitum
Western diet with high fructose-glucose drinking water [12]
(see Note 7).
3. Feed control mice a standard chow diet with normal, tap water.
4. House all mice in a 12 h light–12 h dark cycle in a 21–23  C
facility and euthanize at varying time points following initiation
of dietary intervention.
5. Weigh mice weekly and record results.

3.2 Glucose and GTT and ITT are performed several days from each other on the
Insulin Tolerance same mice (see Note 8).
Tests (GTT, ITT)
1. Fast mice overnight.
2. Measure baseline blood glucose levels in tail-vein blood using
an Accu-Chek Compact plus glucometer. For the tail nick, a
small cut is made at the tip of the tail so that a drop of blood can
be obtained for the glucometer.
3. Inject glucose in the form of 1 mg dextrose/g body weight in
sterile PBS intraperitoneally.
4. Measure blood glucose prior to, and 15, 30, 60, 90, and
120 min after glucose injection [25]. “Milk” the tail to obtain
a drop of blood to place on the glucose strip for a reading.
Record glucose levels.
 Diet-Induced Mouse Model of NAFLD 25

5. For the insulin tolerance test, fast mice for 4 h and intraperito-
neally inject with 0.75 U/kg body weight regular insulin [25].
6. Measure blood glucose levels prior to, and 30 and 60 min after
insulin injection.

3.3 Blood Collection 1. Collect blood via retro-orbital bleeding (see Note 9) using
and Analysis heparinized micro-hematocrit capillary tubes into untreated
Eppendorf tubes and place on ice for temporary storage until
necropsy is completed.
2. Centrifuge blood samples for 15 min at 1500  g at 4  C.
3. Collect the serum and store at
80  C for later analysis.
4. Measure alanine aminotransferase (ALT) and aspartate amino-
transferase (AST) in serum samples using an Advia 1800
Chemistry System (see Notes 10 and 11).
5. Perform lipid analysis including determination of triglycerides,
cholesterol, and calculated low-density lipoprotein levels in
serum samples using the Advia 1800 Chemistry System (see
Notes 12–14).
6. Serum adiponectin is measured using mouse Adiponectin/
Acrp30 Quantikine ELISA Kit.

3.4 Liver Sample 1. Weigh mice and then expose to inhaled isoflurane.
Collection and 2. Collect blood from mice prior to euthanizing.
Processing
3. Euthanize mice by cervical dislocation.
4. Perform a laparotomy with visual inspection of all abdominal
organs.
5. Obtain a picture next to a ruler for scale.
6. Remove the liver in toto from the abdominal cavity and weigh
with a picture of the liver taken next to a ruler for scale.
7. It is convenient to include a label in the picture for easy identi-
fication at a later time.
8. Note the appearance of the liver surface, its color and weight.
9. Document the number of visible tumors, defined by gross
masses apparent at the liver surface, with or without hemor-
rhage, and their size.
10. Section the liver in a sagittal plane and count the number of
tumors within the liver.
11. Place a portion of the liver in separately labeled plastic 10 ml
containers of 10% formalin for later histologic processing and
analysis.
12. When tumors are present, they are dissected from the remain-
ing liver and are placed into separate cryotubes and snap frozen
in liquid nitrogen.
26 Amon Asgharpour and Arun J. Sanyal

13. For histologic analysis of tumors, masses are taken with sur-
rounding non-tumor parenchymal tissue and placed in 10 ml
containers prefilled with 10% formalin.
14. The remaining liver is aliquoted into several cryotubes that
either contain RNAlater or are simply snap frozen in liquid
nitrogen.
15. Other tissue of interest (kidneys, intestine, stool, etc.) may be
obtained at this time.
16. Necropsy tools are cleaned between mice with 70% ethanol and
a glass bead sterilizer.
17. Snap frozen samples are moved to the
80  C freezer for
storage.
18. Samples in RNAlater can be stored at 4  C in a refrigerator for
up to a month until processed.

3.5 Histological 1. Liver samples in formalin are processed for basic histology with
Analysis (See Note 15) hematoxylin and eosin (H&E) stains.
2. The presence of steatosis and type of steatosis, micro- and
macrovesicular, as well as its distribution are noted (Fig. 2).
3. Steatohepatitis is defined by the presence of steatosis, inflam-
mation, and hepatocellular ballooning [26, 27], as per the
FLIP algorithm [28].
4. The severity of steatosis, lobular inflammation, and hepatocel-
lular ballooning are scored using the NASH-Clinical Research
Network (CRN) criteria [29]. Specifically, the amount of stea-
tosis (percentage of hepatocytes containing fat droplets) is
scored as 0: <5%, 1: 5–33%, 2: >33–66%, and 3: >66%. Hepa-
tocyte ballooning is classified as 0: none, 1: few or 2: many
cells/prominent ballooning. Foci of lobular inflammation are
scored as 0: no foci, 1: <2 foci per 200 field, 2: 2–4 foci per
200 field, and 3: >4 foci per 200 field. The NAFLD activity
score (NAS) is computed from the grade of steatosis, inflam-
mation, and ballooning.
5. Slides are trichrome stained for fibrosis analysis.
6. Fibrosis is scored as stage F0: no fibrosis, stage F1a: mild, zone
3, perisinusoidal fibrosis, stage F1b: moderate, zone 3, perisi-
nusoidal fibrosis, stage F1c: portal/periportal fibrosis, stage
F2: perisinusoidal and portal/periportal fibrosis, stage F3:
bridging fibrosis, and stage F4: cirrhosis.
7. Quantitative analysis of fibrosis is performed by morphometry
from digitalized Sirius Red-stained sections using the Aperio
system after tuning the threshold of fibrosis detection under
visual control.
 Diet-Induced Mouse Model of NAFLD 27

8. For ease, F1a, F1b, and F1c fibrosis are all scored as 1. The
results are expressed as the percent collagen proportional area
(CPA) [30].
9. Human NASH liver samples are used to compare main histo-
logical features with DIAMOND mice and belong to an
archive of human NASH samples which are scored [29].
10. Histological classification of tumor is based on usual criteria.
11. Adenoma is defined as a nodular proliferation of normal hepa-
tocytes retaining a trabecular organization, well-differentiated
HCC include cytological and architectural abnormalities such
as major trabecula thickening, loss of sinusoidal barrier, pseu-
doacinar formation, and isolated arteries.
12. Poorly differentiated HCC are characterized by major cytolog-
ical abnormalities such as significant increase in nuclear/cyto-
plasmic ratio, architectural disorganization with loss of
trabecular organization.

4 Notes

1. For our experiments, B6/129 mice were initially a gift from

Dr. Sandra Erickson of University of California San Francisco,
CA and derived from mice with C57BL/6J and 129S1/SvImJ
backgrounds. With progressive generations derived from
brother-sister mating, the hepatic phenotype of NAFLD devel-
ops with increasing consistency following initiation of a high fat
and carbohydrate diet. DIAMOND mice are commercially
available through Metabaxis laboratories.
2. Blood is obtained prior to mice being euthanized.
3. Isoflurane is used in a chamber in our experiment, but other
anesthetics can be used.
4. We found it best to pre-label all cryotubes prior to euthanizing
animals. Large Styrofoam containers filled with liquid nitrogen
are used to house cardboard boxes (with small holes on the
bottom) so that once specimens were placed in cryotubes they
can be snap frozen upon placement into the cardboard boxes.
5. Plastic containers prefilled with formalin are purchased from
VWR, but formalin can be obtained separately and used to fill
plastic containers for histology. It is important to make sure
that the tissue is submerged in formalin.
6. Male mice are usually more susceptible to the development
NASH and HCC, reproducing sex differences found in
human patients. Mouse strains have variable susceptibility to
the development of NASH and HCC.
28 Amon Asgharpour and Arun J. Sanyal

7. The Western diet food pellets will oxidize much quicker than
chow diet and the mice will not want to eat the Western diet
pellets if they are old. It is best to change the food at least twice
a week and keep the Western diet stored in a refrigerator until
ready to use. The drinking water should also be changed at least
weekly.
8. It is convenient to use separate cages for each mouse while
performing GTT and ITT.
9. Cardiac puncture or facial vein bleeding can also be used.
However, retro-orbital bleeding is the preferred method.
Blood is collected before cervical dislocation.
10. Both ALT and AST measurements utilize the Alanine Amino-
transferase, ALTP5P, Aspartate Aminotransferase, ASTP5P,
method, respectively, using pyridoxal-50 -phosphate and the
addition of α
ketoglutarate [31].
11. The concentration of NADH is measured by its absorbance at
340/410 nm. The decrease in absorbance values reflects utili-
zation of NADH and is thus proportional to AST or ALT
activity in their respective reactions.
12. For triglyceride measurement, the method is based on the
Fossati three-step enzymatic reaction with a Trinder endpoint
[32]. The single-reagent procedure quantitates the total trigly-
cerides including the mono and diglycerides and the free glyc-
erol fractions.
13. The method for cholesterol measurement is based on the
determination of Δ4-cholestenone after enzymatic cleavage of
the cholesterol ester by cholesterol esterase, conversion of
cholesterol by cholesterol oxidase, and subsequent measure-
ment by the Trinder reaction of the hydrogen peroxide
formed [33].
14. The calculated low-density lipoprotein is determined by sub-
tracting the determined high-density lipoprotein and one-fifth
of the triglycerides measured from the total cholesterol [34].
15. Histological analysis is performed by a pathologist well-versed
in liver histology, NASH, and HCC. Hematoxylin and eosin,
H&E, stained slides, trichrome stained slides, and picrosirius
red-stained slides can be used. H&E stained slides will provide
all histologic information except for fibrosis which is assessed
by trichrome stain or picrosirius red stain. These are very
standard stains, but the difficulty lies in the interpretation of
the NAS components and determining the stage of fibrosis.
 Diet-Induced Mouse Model of NAFLD 29

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3. Younossi Z, Anstee QM, Marietti M et al high-fat diet and multiple administration of
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et al (2003) Overweight, obesity, and mortality 16. Park EJ, Lee JH, Yu GY et al (2010) Dietary
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(2018) The diagnosis and management of non- 17. Hui L, Bakiri L, Mairhorfer A et al (2007)
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 Chapter 3

Hydrodynamic Injection for Developing NASH Model

Haichuan Wang and Xin Chen

Abstract
The hydrodynamic tail vein injection (HTVi) is a technique that is used to deliver plasmid genes into live
mice or rats. The HTVi leads to the in vivo transfection of exogenous DNA primarily in the liver, serving as
a reliable approach of establishing animal models for the study of liver diseases. The nonalcoholic steato-
hepatitis (NASH) is liver inflammation and damage resulting from an accumulation of fat in the liver. With
the rising prevalence of obesity worldwide, NASH is becoming an increasingly common health problem.
The pathogenesis of NASH is a multi-step process involving complicated pathways. The molecular
mechanisms of NASH remain poorly understood. Here, we describe the use of HTVi to establish animal
models for the study of NASH.

Key words Hydrodynamic tail vein injection, Nonalcoholic steatohepatitis, Mouse, Rat

1 Introduction

The nonalcoholic steatohepatitis (NASH) is liver inflammation and

damage resulting from an accumulation of fat in the liver. NASH
was first defined in 1980 by Ludwig et al. among patients who have
fat build up in the livers without excessive alcohol use [1]. Subse-
quently, both the pathological and clinical features of NASH have
been defined. The consensus of NASH pathophysiological change
is the inflammatory insult which occurs in the fatty liver. Increased
free fatty acids and hepatic lipid peroxidation as well as the presence
of peripheral insulin resistance are associated with NASH patients
[2]. Because of the rising prevalence of obesity worldwide, NASH is
becoming an increasingly common health issue [3]. However, the
underlying mechanisms of NASH development are complicated
and remain unclear.
During the past decades, genetic studies have revealed crucial
signaling pathways involved in nonalcoholic fatty liver disease
(NAFLD) and NASH [4, 5]. Recently, high-throughput genomic
studies, including microarrays, array-based comparative genomic
hybridization, and deep sequencing, in combination with

Devanand Sarkar (ed.), Non-Alcoholic Steatohepatitis: Methods and Protocols,

Methods in Molecular Biology, vol. 2455, https://doi.org/10.1007/978-1-0716-2128-8_3,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

31
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GENESIS!
 By R. R. WINTERBOTHAM

Renzu was mad, certainly! From Venus' lifeless

clay he dreamed of moulding a mighty race; a
new Creation, with himself as God!

[Transcriber's Note: This etext was produced from

Planet Stories Summer 1941.
Extensive research did not uncover any evidence that
the U.S. copyright on this publication was renewed.]
The unreal silence of outer space closed in about The Traveler. In
front of the huge atom-powered space rocket hung the sun's
dazzling disc and behind the pale, silver face of the earth echoed the
light. Captain Vic Arlen was a god in the heavens; Dave McFerson,
the engineer, was a demi-god. And what was Harry Renzu? It was
hard to call a great scientist a devil.
There was Gheal—neither god nor devil, only a poor, hideous, half-
human slave that had been brought with Renzu to the earth from a
previous expedition to Venus. Captain Arlen quit trying to classify
himself and his passengers. They were neither gods nor devils. Not
even men, taking the group as a whole.
An ominous chill seemed to reach through the beryllium hull of the
ship from outer space, caressing Arlen's backbone. A faint cry
sounded in the passageway that led to the sleeping quarters behind
the control room.
The captain tripped the controls into neutral. The acceleration was
complete and from now until the braking rockets were fired, the
craft would follow its carefully calculated orbit.
Again came the cry, a groan of pain and a moaning sob. The captain
strode into the passage.
"Gheal!" he called, recognizing the Venusian's hoarse voice. "Gheal!
What's the matter?"
A repetition of the cry was the only answer. The passageway was
open, but the sobs seemed to be coming from the cabin of Harry
Renzu, the scientist who had chartered the moon rocket for his
second expedition to Venus.
The captain paused before the cabin door, listening. The cry came
again and he pushed open the door.
The hideous Venusian was on the floor, looking upward with his two
light-sensitive eye-glands at Renzu, who stood over him with an
upraised cane.
Gheal's rubbery, lipless mouth was agape, revealing his long, sharp
teeth. He had raised one of his long, rope-muscled arms to catch the
descending blow. His hairless, leathery body trembled slightly with
pain.
"You dumb, dim-witted chunk of Venusian protoplasm!" Renzu
snarled as he brought the cane crashing over the monster's
shoulders. "When I want a thing done, I want it done!"
Arlen pushed into the room and seized Renzu's arm before the
scientist could strike again.
"Hold on, Renzu!" Arlen commanded, pushing the scientist back and
seizing the cane. "Lay off! Can't you treat this miserable wretch with
decency?"
Renzu's face flushed angrily. His deep-set eyes burned with fury.
"This is none of your affair!" Renzu snapped. "Go back to your
business of running this ship. I didn't hire you to run my business."
"This may be your expedition," Arlen replied stubbornly, "but while
we're in space, I'm the captain of this ship and my orders are to be
obeyed. My orders are to give this Venusian beast humane
treatment."
A whimpering sob broke from the throat of the brute on the floor.
Renzu sullenly twisted his arm loose from the captain's grasp. He
appeared more calm now.
"You are right, Arlen," he said. "Your orders are to be obeyed. But
you aren't a scientist. You don't know Gheal. He's not like the
animals we know on the earth. He has to be beaten."
"Not while we're in space. I won't stand for it."
"You can't stand in the way of science, Arlen. I shall whip Gheal, if I
deem it necessary." Renzu ended his words with a suggestive snap
of his fingers in Gheal's direction. The monster cringed into a corner
of the stateroom.
"Come with me, Gheal," Arlen ordered, beckoning to the monster.
The creature, seeming to understand, rose to his feet and followed
Arlen out of the door.

The captain took the Venusian forward into the control room, where
he daubed the welts on the creature's naked shoulders with arnica.
McFerson, easy-going, but dependable old spaceman, watched the
operation critically. Gheal winced as the arnica touched his skin. He
squirmed and tried to resist.
"Hold on a minute, Cap," McFerson said. "Look at the right shoulder,
where you put the arnica; it's red and inflamed."
"So it is, but arnica ought to help."
"Look at the left shoulder, where you haven't put any arnica."
"Great guns! It's almost healed!"
"I'd say maybe arnica wasn't the best treatment."
Captain Arlen corked the bottle and put it aside. "Gheal looks like a
man. Sometimes he acts like a man. Yet he's entirely different most
of the time.
"I've been watching him, Cap. I somehow get the idea that Gheal
finds it unhandy, most of the time, to be built like a man."
The captain laughed. He took Gheal's arm and held it up. "Look at
that. Good, human bones, but the body of a monster. I wish you
could talk, Gheal. I wish you could tell us more about yourself. Why
are you almost a man yet the farthest point south?"
Gheal uttered a sort of deep-throated growl.
"Renzu says you can be vicious—that you're a killer at heart. Renzu
said one of your kind killed Jimmy Brooks on the first expedition. You
don't look like a killer. Brooks was a big man. You'd have a hard time
killing him."
Gheal's sight-glands stared from Arlen to McFerson.
Arlen laughed and patted Gheal's hairless head and pointed to a
built-in seat in the corner.
"You're welcome to stay here as long as you don't bother us," he
said.
Gheal shuffled uneasily and whimpered, but he did not go to the
seat. Instead, he turned and moved toward the door. The creature
looked ridiculous, clad as he was only in a pair of Renzu's discarded
trousers, which had been rolled at the bottom to fit his stubby legs.
At the door the Venusian hesitated and glanced back at the captain.
Then he slowly turned and shuffled down the passageway.
"Hey you!" Captain Arlen shouted. "Come back here!"
Gheal did not stop. He was striding to Renzu's room. He pushed
open the door.
A fear for Renzu's safety rushed into the captain's mind. He ran after
the creature and entered Renzu's cabin. But as he opened the door
he gasped in astonishment.
Gheal was crawling into a corner of the room, while Renzu stood
nearby laughing.
"You see, Arlen," smiled Renzu, "I'm his master. He recognizes my
authority and no one else's. He would not desert me, no matter how
I treated him."
Renzu picked up the cane that Arlen had tossed on the bunk a few
minutes before. As the scientist shook the stick at Gheal, Arlen
thought he saw a look of satisfaction creep into the creature's face.
"Just the same," Arlen said, "I can't stand your beating him. He may
enjoy it. He may be a masochist at heart, but I won't stand for it."
"Your mind is provincially human, Arlen," said Renzu. "When you
look at Gheal you see the product of an entirely different evolution.
You see a creature without emotions, without ethics. He's devoid of
every terrestrial feeling, especially gratitude. He may even hate you
for taking his side against me."
There was a trace of bitterness in Renzu's voice.
"I wouldn't be too sure, Renzu," Arlen said. "If the laws of physics
apply on Venus, as well as the earth, why couldn't biological and
psychological laws apply there also. Even the lowest of creatures
show understandable reactions on earth. Why not on Venus?"
"Because Gheal has been made differently," Renzu said, with a
repulsive grin.

Hour by hour Captain Arlen watched Venus grow in size. The planet
expanded from a glowing crescent to the size of the moon as seen
from the earth; soon it floated large in space, filling half the sky
ahead of the ship, a billowing, fluffy ball of shining clouds. Its
surface was entirely obscured by its misty atmosphere.
Arlen began braking the ship and he called Renzu into the control
room for a conference on where to pierce the cloud blanket.
Renzu, huge and muscular, overdid himself in graciousness as he
greeted Arlen in the control room. The scientist seemed to radiate
exaltation and he strained himself to appear congenial.
The man was excited, Arlen decided, for Arlen himself was thrilled at
the prospect of adventure, of seeing strange sights on a strange
planet. But the reaction was different in Arlen. Where Renzu swelled
and swaggered, Arlen looked dreamily into the clouds ahead.
"I'm bringing the ship around to the sunward side," Arlen said. "It's
best to land about noon—that is the noon point. The planet turns
once in thirty hours and that will give us a little more than seven
hours of daylight to orient ourselves after the landing."
Renzu nodded in agreement. All this had been threshed out before.
"Very well," he said, "but it is best that you pierce the clouds at
about forty-five degrees north latitude. There's ocean there that
nearly circles the planet and there's fewer chances of running into
mountains beneath the clouds. Once we're through the cloud belt,
we'll have no difficulty. The clouds are three or four miles above the
surface and there's plenty of room to maneuver beneath them."
Arlen twisted the valves and the deceleration became uncomfortably
violent. Renzu's first trip had determined the existence of a
breathable atmosphere on the surface of Venus, although the cloud
belt was filled with gases given off by Venusian volcanoes, and many
of these gases were poisonous to man.
In a few minutes the rocket ship stood off just above the cloud belt.
McFerson checked the landing mechanism and made his final report
to the captain. Arlen checked the gravity gauge, which now would
be used as an altimeter during the blind flying in the Venusian
clouds.
"Okay!" Captain Arlen called.
"Okay!" echoed McFerson.
The Traveler nosed downward into the rolling clouds. A whistling
whine arose as the craft struck the atoms of the atmosphere.
Repulsion jets set up their thunder and the landing operation began.
The ship settled slowly through the clouds. The mist completely
obscured everything outside the craft and Arlen flew blind, trusting
his meteor detection devices to warn him of mountain peaks, which
he feared despite Renzu's assurance that there were no high ranges
at this latitude.
At last the craft dropped through the wispy canopy to float serenely
over a calm ocean which bulged upward toward them in the solar
flood tide.
To the northwest was a dim coastline. High mountains were faintly
visible against the horizon.
"Perfect!" said Renzu. "That is my continent—our destination. Sail
toward it."
The ship zoomed toward the land at the comparatively slow speed of
five hundred miles an hour. In a few minutes it was decelerating
again, with the continent before them.
The high mountain range clambered up from a narrow plain that
skirted the sea. This plain was sandy, a desert waste, but Renzu
indicated it was the spot for the landing.
Arlen brought The Traveler down gently alongside a broad stream
that emptied into the sea. When the dust of the landing cleared
away, he looked with dumbfounded amazement at the Venusian
scene.
As far as his eyes could see were barren rocks and sand: there were
no trees, no grass, no signs of life. The planet was as sterile as an
antiseptic solution. Even seaweed and mosses were missing from the
seashore.
"Maybe you know what you're doing, Renzu," Arlen said, "but it
looks to me as if you've directed us to the edge of a desert."
"'Tain't no small desert, either," chimed McFerson.
"My dear Arlen," Renzu replied, cracking his lips in another of his
irritating smiles, "this is one of the most fertile spots on the entire
planet. You must remember, Venus is much different from the
earth."
Immediately after the landing all hands, including Renzu, were busy
with the routine duties that the expedition required. Gheal was given
simple tasks, such as unpacking boxes of equipment to be used by
the expedition, but the Venusian seemed to attend to these in a
preoccupied manner. He worked in sort of a daze, frequently
whimpering like a sick dog, and turning his globular eyes from time
to time out of the porthole at the landscape of his native planet.
"He's homesick," McFerson suggested to Arlen. "But look! What's he
got in his hand?"
It was a long white bar of metal. Arlen quickly seized the bar and
examined it. It was pure silver. Gheal had been unpacking a box
crammed with silver bars of assorted lengths and thicknesses,
ranging from the size of small wire up to rods half an inch thick and
a foot or more in length. A fortune in silver had been transported to
Venus.
"Well, that's Renzu's business, not mine," Arlen decided.
He returned to his duties. There was much to do: the engines had to
be recharged, preparatory to a quick takeoff, should conditions arise
to make the planet untenable for earthmen.
Tests of the soil revealed utter sterility of all forms of life. It was
baffling. Some sort of bacteria should have been in the soil, even
though the place was only a desert.
Arlen opened the arms chest and issued small but powerful atomic
disintegrators to McFerson, Renzu and himself. He did not give Gheal
one of the weapons, for Gheal did not appear to have the skill
necessary to operate it. His uncanny ignorance was so obvious.
The disintegrators were simple magnetic mechanisms capable of
collapsing atoms of atmosphere and sending the resultant force of
energy in a directed stream toward a target. Fire from disintegrators
could melt large rocks almost instantly and it could destroy any living
creature known to man.
Renzu strapped his weapon at his side and turned to Arlen.
"I'm going outside for a walk with Gheal," he said. "Gheal seems
nervous and uneasy. Perhaps his actions are due to his return to his
native land. A walk might make him happier, in his own peculiar
way."
Arlen nodded and went back to the control room to talk to
McFerson. He found the engineer looking out of a porthole.
"Look!" McFerson said, pointing out the porthole.
Trudging along the beach, carrying the case containing the silver
rods, were Renzu and Gheal. The Venusian was walking with
difficulty, but as he faltered, Renzu would kick him unmercifully and
force him on.
"The devil!" Captain Arlen said. "He doesn't dare beat Gheal when
he knows I'm watching."
McFerson shook his head.
"Maybe he's right, treating Gheal that way," he said. "After all, Renzu
is a scientist and he knows more about Gheal than we do. Maybe
he's right in saying beating is the only treatment Gheal understands.
Besides, I don't know if I trust Gheal. Since we've landed he's acted
like a tiger in a cage. Gheal's a Venusian and Venusians are
supposed to have murdered Renzu's partner on the first expedition."
"But even the worst creature on earth—except man, perhaps—
doesn't kill without a reason. And even man sometimes has a
reason, when apparently he hasn't."
Darkness descended rapidly on Venus and Renzu did not return. The
two spacemen decided it was unnecessary to stand guard and
turned in. Renzu knew how to operate the space locks from the
outside of the ship and could enter when he returned. Gheal, whose
clumsy fingers were too unwieldly even to operate a disintegrator
gun, would not be able to operate the locks, nor would any creature
like him.
It was still dark when Arlen awakened. The long, fifteen-hour
Venusian night was completed and still Renzu had not returned.
The captain awakened McFerson. They ate a light breakfast and did
minor chores on the ship until daylight suddenly lighted the
landscape.
"Do you suppose we ought to look for them? Maybe Gheal went
haywire. Maybe something's happened."
Arlen considered. Renzu was armed, while Gheal was not. Renzu
claimed complete mastery over the Venusian, yet something might
have happened to give Gheal the upper hand. Not that Renzu didn't
deserve it.
"I'll go outside and look around," Arlen said.
Arlen stepped through the locks. The warm Venusian air was
invigorating. He took a deep breath.
A shuffling sound behind him caused the captain to turn. There,
rounding the end of the ship was a creature, fully naked, staring at
him with gland-like eyes and baring his teeth in a vicious snarl.
"Gheal!" Arlen cried. "Gheal! Where's Renzu?"
The creature did not reply. Instead, it advanced slowly with a
shuffling crouch, stretching his arms menacingly toward Arlen.

Arlen's hand went to his disintegrator. The creature resembled

Gheal, but it did not act like Gheal. The captain's eyes swept over
the animal again. No, it wasn't Gheal. There were differences. It was
another of Gheal's race.
Arlen hesitated to kill the creature. If there were a tribe of the
creatures in the vicinity, such an act would arouse enmity. It would
lead to complications that would endanger Renzu, who was away
from the ship. Yet, Arlen could not be sure what reaction would
follow a slaying. Renzu had said that Venusians had no emotions, in
the sense that man has them. But Gheal certainly had been nostalgic
on the day before. That at least was understandable in a human
sense.
Arlen leveled his pistol. Suddenly another figure appeared.
A low-voiced whine sounded as the second figure darted forward.
It was the real Gheal. He was still wearing Renzu's trousers.
The first Venusian turned. He hesitated stupidly, undecided whether
to continue his charge toward Arlen, or to meet the foe who came
from behind. Finally, the beast apparently decided that Arlen was the
most tempting.
The animal sprang at the captain.
Arlen held his gun ready to fire, but the Venusian had acted with a
swiftness that belied his clumsy appearance. Before Arlen could fire,
a heavy, rubbery arm crashed down on his skull. A meteor shower
seemed to flash through Arlen's brain, and then darkness closed in
about him as he tumbled to the sandy beach.
Arlen opened his eyes. He had no way of telling how long he had
lain on the ground. On Venus one never sees the sun; daylight
appears and daylight fades, but there is no way of telling the time of
day from the position of the sun overhead.
The captain's head ached as he lifted himself from the ground. He
shook his head to clear away the haze and he stretched his arms to
rise. His fingers struck something leathery and cold.
There at his side lay the Venusian monster who had attacked him. A
wave of nausea swept over him as he saw the lifeless body horribly
mutilated and torn. The sandy soil of the beach was torn with the
struggle that had taken place.
Arlen forgot his aching head at he examined the dead Venusian. His
disintegrator had not slain the Venusian; clearly Gheal had done the
job.
"So Gheal came to my rescue!" Arlen exclaimed. "Renzu must have
been wrong. These Venusians do have gratitude."
His eyes saw something else as they traveled over the body.
Protruding from the body was a silver rod. Gingerly Arlen tried to
pull the rod from the animal's body, but it would not budge. Was it a
weapon?
Arlen saw other rods sticking from the animal, covered with blood.
All of them seemed firmly set in the body of the Venusian.
Arlen looked behind him. The locks of the space ship were open. He
moved wearily to the door and stuck his head inside.
"McFerson!" he called.
There was no answer.
Arlen entered the ship. He carried his disintegrator in his hand.
Venusians might have entered the ship ahead of him. Lights were
still burning in the living quarters, but McFerson was gone.
Arlen moved on; he searched each cabin, but there was no sign of
McFerson, until he reached the control room. There furniture had
been overturned, instruments smashed, and a pool of blood lay on
the floor.
Gheal had done this. Arlen was sure that no other Venusian could
have entered the ship and crept up on McFerson without arousing
suspicion. McFerson's disintegrator lay on the floor beside the pool
of blood, indicating that McFerson had grown suspicious too late.
The gun had not been discharged.
The first thing Arlen had to do was to protect himself from further
attack. He drew his own gun and closed the outer locks. The next
thing would be to decide what had happened and what to do.
Renzu probably had suffered the same fate as McFerson, Arlen
decided. He was alone, in a strange world, face to face with a race
of mankilling monsters. The only thing in his favor was that one of
these monsters had befriended him. But how long and how far could
Arlen trust this friendship?
There was, however, a chance that McFerson or Renzu still might be
living. He had to know for sure about this before he did anything
else. And the only way to learn was to investigate.
He left the ship, carefully closing the locks and fastening them
behind him. He found many tracks leading away from the ship, along
the banks of the stream that flowed from the mountains.
From among the tracks he picked out Renzu's bootprints. There
were tracks of Gheal going away, coming back, and going away
again. He distinguished the two sets of Gheal's prints leading toward
the mountains by the fact that one set was more deeply imprinted in
the moist sand than the other. Gheal had been carrying McFerson's
body.
But what was this? There was another set of tracks coming toward
the space ship. They were not Gheal's prints, for they were three
toed. Gheal had five toes. Gheal and the creature who had attacked
Arlen were different—one had three, the other five toes.
Gheal might not have rescued Arlen out of gratitude after all. A
natural enmity might have existed between the two races of
Venusians. Arlen's rescue might have been an accident.
Arlen studied. There was something else that fitted into the picture.
If he could fit it correctly, he would have the answer. Somehow,
now, he doubted if Gheal had rescued him out of gratitude; yet, he
doubted if the rescue had been purely accidental.
Arlen returned to the space ship and loaded a haversack with food.
He was going into the mountains to get to the bottom of the
mystery. He scribbled a note and left it in the control cabin in case
Renzu or McFerson returned; if either were alive.

The captain followed the stream into a deep-walled canyon opening

into the mountains. A short distance from the ship he found Gheal's
discarded trousers, indicating beyond a doubt that the Venusian had
come this way after Arlen had been knocked unconscious in the
sand.
A mile or so farther on he saw a print where Gheal had placed
McFerson on the ground. Then, a thrill of gratitude swept over Arlen,
another set of boot prints appeared on the trail. McFerson was not
dead. He was walking.
The daylight was fading and Arlen realized he would not have much
more time to follow the tracks without the aid of his flashlight. The
walls of the gorge were almost perpendicular now and nearly a mile
high on each side of the stream. The river boiled and churned over
the barren rocks, but its movement was the only animation of the
scene. Nowhere were there signs of life, excepting the footprints on
the trail.
At last the trail forked upward from the stream, following a narrow
ledge of rock along the canyon wall. The footprints of the slain
Venusian now were wide apart and deeply imprinted in the sand,
indicating that the creature had run rapidly down the path.
"He probably spotted our ship landing and headed toward us right
away," muttered Arlen. "His presence outside the craft may have
been what made Gheal so uneasy yesterday. Gheal sensed an
enemy near at hand." But this didn't seem to be the answer, either.
Beyond the next curve the canyon walls slid back and the ledge
widened into a gentle slope leading to the top of the canyon. As
Arlen climbed over the rim he found himself on a plateau.
It was dark now, but the place was lighted by a huge campfire not
far away. Huddled around the campfire were four figures. In the still
air of the night, Arlen heard guttural grunts of Venusians and above
these tones he heard the sharp voice of Harry Renzu issuing
commands to these alien beasts.
Arlen crept forward and concealed himself behind a rock. There were
three Venusians. He saw something else, too. McFerson, his head
swathed in bandages, was sitting in the shadow of a huge square
stone.
Arlen watched. He could not hear Renzu's words and he moved
forward to obtain a better view, when his hand sank into a sticky
mass of slime.
"Ugh!" he grunted in disgust, lifting his hand.
It was covered with a thick, viscous jelly. It was sticky and as he
turned his flashlight on the stuff he saw that it was colorless and
translucent. It was not a plant or an animal. It did not move, it was
cold, and had no structure, nor roots.
Shielding his light so that it could not be seen from the campfire,
Arlen examined the ground around him. There were other small
pools of the stuff in the hollows of rocks and in thick masses on the
ground.
The captain examined the material more closely. It looked strangely
familiar, and some of the text-book science he had learned in college
came back to him. He remembered examining stuff like this once
under a microscope. It was not petroleum, but something vastly
different—something that was synonymous with life.
It was protoplasm!

Vic Arlen gasped.

"Protoplasm! Inanimate protoplasm!"
He forgot he had been nauseated by the slime a moment before and
began to examine the stuff closely. Of course, it was protoplasm, it
couldn't be anything else. Vic Arlen had studied it. He knew. Nothing
could hold water granules in suspension in exactly the same way;
nothing had the same baffling construction.
But there was a question: scientists admitted life could not exist
without protoplasm, but could protoplasm exist without life?
In living protoplasm, death alters the structure. But other processes
than life could, conceivably, preserve the stability of the substance.
This would explain the existence of inanimate protoplasm on Venus.
And why didn't inanimate protoplasm exist on the earth? Arlen
thought for a moment and had the answer for that too. Animal life
lives on protoplasm, as well as being protoplasm itself. Animate
protoplasm can reproduce its kind, but the inanimate kind can
neither fight back nor replace its losses. The inanimate protoplasm
on the earth had disappeared with the appearance of the first animal
life. The coming of the first microbes had caused it to "decay."
If protoplasm existed on the face of Venus it meant there were no
bacteria, no germs of any sort—no life!
How could Arlen explain Gheal without evolution from the simple to
the complex? Was evolution working differently on Venus? Again
Arlen had run up a blind alley.
The campfire cast a flickering red glow against the clouds. In spots
above the skies were tinted with other glows from the craters of
Venusian volcanoes. It was not absolutely dark, but it was far from
being as light as a moonlit night on the earth.
Arlen crept closer to the scene. He could see the Venusians plainly
now. Two of them had three toes, while one had five. The five-toed
one was Gheal.
Renzu stood before them, grasping his cane. He would make sharp
commands and the Venusians would rise. If they disobeyed, he
would strike them with the cane. They would shriek with pain. At
last these maneuvers ceased and Renzu turned to McFerson.
"They have to be taught everything," he said. "They have no reflex
actions, no emotions, no instinct—nothing that the lowest creatures
on earth may have. Yet they have everything that makes those
things in the creatures of the earth."
McFerson did not reply. He was watching with staring eyes; eyes
filled with horror.
Renzu reached behind a rock. He drew what appeared to be a
human skeleton from the shadow. As Arlen looked a second time, he
saw that it was not a human skeleton, but an imitation built of the
silver rods and wires that Renzu had transported to Venus. The truth
was dawning on Arlen, but it was unnecessary now, for Renzu was
explaining.
"I have created life, McFerson. I have moulded a human likeness out
of protoplasm and fitted it over bones of silver. An electrical device I
have made starts the biological processes going and the protoplasm,
working with chemical exactitude, reforms itself into glands, organs,
muscles and nerves. The product is a beast, inferior to man but
superior to the highest animal on earth, except that he is totally
devoid of such things as reflexes, instincts, emotions and other
survival psychological processes."
As he spoke, Renzu was moulding some of the protoplasm over the
framework of bones. Arlen understood now why the silver rods had
protruded from the Venusian he had found on the beach. Those
pieces of silver had been the creature's bones.
"I made four of the creatures on my previous expedition. Brooks
helped me construct three of them, including the creature that
attacked and killed Arlen on the beach. I made Gheal myself. Gheal
was a masterpiece. He was almost, but not quite human. That is
why I took him to earth with me."
"You're inhuman, Renzu!" McFerson managed to say. "You're less
human than Gheal!"
"Gheal was more human than you think, McFerson. Brooks, you
know, was killed by one of his creations. The same monster that
killed Arlen accounted for him. Yet that monster, in some ways, was
above average. At least he had the beginnings of an instinct. He
wanted to kill. After Brooks was killed, I used his bones for Gheal's
skeleton."
Arlen stared in speechless horror and amazement.
"And that isn't all. I'm going to use Arlen's bones for a creature more
human than Gheal. Perhaps, McFerson, your bones may be used for
something greater still. I will make other men, and women, from
silver wire and protoplasm, and create a race of Venusians that will
bring life to this planet. Think of a planet that has evolution
beginning with man and ending with something greater than man
has ever dreamed. And I, McFerson, will be the god of this race!"
McFerson tried to rise, but Gheal rose with a low throated growl, and
the spaceman sank back on the ground.

Renzu had finished moulding the protoplasm over the silver bones.
With the help of one of the Venusians he lifted the still form into the
air and placed it carefully inside the stone behind McFerson.
The stone had been hollowed to form a rock sarcophagus.
Arlen saw in the firelight that electric wires ran from a small battery
beside the box.
Renzu touched the switch.
There was a flash of blinding light and sparks flew over the box.
Then Renzu turned off the current and opened the sarcophagus. He
worked rapidly with his hands and then stepped back, holding his
cane before him.
From the box emerged another Venusian. A replica of Gheal's three-
toed companions.
For a moment the creature stood motionless, staring from the sight
glands at his surroundings. Renzu struck the monster sharply with
his cane. The brute moved. Again Renzu struck and the creature
moved. At last it seemed to understand, after Renzu struck it
repeatedly. The beast got out of the box.
Renzu belabored his creation unmercifully with the cane, each
movement had to be directed.
"They have to be taught everything," Renzu said. "They understand
nothing but pain. I have to beat instincts and reflexes into their
dumb brains, for they have no inherited ones."
That also explained why Renzu was a complete master over Gheal.
The Venusian depended on Renzu for everything.
So interested was Arlen watching Renzu train the newly made
Venusian, that the captain did not hear the scrape of a leathery hide
on the rocks behind him. He was unaware of the danger until a ropy
cord of some vile, repulsive tentacle seized him, pulled him off his
feet to the ground and dragged him toward the camp fire.
The rays of the firelight revealed Arlen's captor: a serpent as large
as a python which held him in the crushing folds of its body as it
moved deliberately toward Renzu.
Renzu was amazed at the sight of Arlen.
"I thought you were dead!" he gasped.
"No," Arlen said. "Your creation didn't quite succeed in killing me."
Renzu smiled. "But I see that you did bring your fine bones to me
after all!" He struck the serpent sharply with his cane and the
monster released his grip on Arlen. "The animal that caught you,
captain, was one of our first experiments. It was by charging a string
of protoplasm with electricity, that we discovered that we could
make it live. The result was the pseudo-python, who makes a good
watchdog, if nothing else. It's entirely harmless, since it feeds
entirely on inanimate protoplasm. Unfortunately for Brooks, it was
this creature that caught him and held him while No. 3—the
Venusian—killed him."
"It was deliberate murder," said Arlen.
"Perhaps terrestrial law would define it as murder," Renzu said. "But
here on Venus there is no law. It was a scientific experiment."
"And you will murder McFerson and me?"
"I need your skeletons. They will be a fine heritage for future races
of Venusians. Think how you and McFerson will be glorified in
Venusian mythology."
Renzu's eyes were glowing in the firelight with madness. Arlen
looked at the hideous Venusians, seated nearby, watching idiotically.
It was diabolical!
"Now comes an important decision. Shall I use you, or McFerson,
first?"

McFerson closed his eyes.

"The man's insane, Cap!"
Arlen looked about him. The python was nearby, coiled neatly beside
a rock, ready to spring if he tried to escape.
One of the Venusians rose and threw some shale on the fire. It was
crude petroleum shale. An idea came to Arlen. If he could put out
the fire, he might be able to escape in the darkness.
Then Arlen remembered. His disintegrator was still in his pocket.
Renzu, interested in his experiment, had forgotten to search him,
believing perhaps that Arlen had been disarmed in the attack on the
beach.
Arlen was tempted to use the weapon now, and to blast Renzu and
his hideous tribe of monsters out of existence. But to kill a man
without giving him a chance was not Arlen's way of doing things.
The Venusians, too, now had a right to live. Had they attacked, Arlen
would not have hesitated to kill, but Arlen realized that the only
vicious Venusian was dead. Perhaps Renzu himself had taught that
single Venusian how to kill.
"McFerson," spoke Arlen, "are you all right? Did Gheal hurt you?"
"He bloodied my nose and knocked me out," McFerson said. "He
didn't mean to harm me. Gheal really is gentle as a kitten."
"I think I will use your bones first, Arlen," said Renzu. "You may sit
down beside McFerson. I may as well warn you that there is no
chance of escape. The python guards the only way back and my
Venusians enjoy the creation of another of their kind. They won't let
a chance to see it be spoiled."
Renzu began filling some woven baskets with the inanimate
protoplasm as Arlen sat down beside his companion.
"Could you run for it, if I knocked out the campfire?" Arlen asked.
"I can run, but how will you knock out the fire?"
Vic Arlen acted quickly. His hand brought the disintegrator out of his
pocket and he fired straight into the center of the campfire. The
atomic blast instantly consumed the inflammable material in the fire
and the plateau was dark.
"Run!" Arlen cried. "And look out for the python."
Arlen sprang forward. He heard a leathery scrape ahead of him. It
was the serpent. He dodged back. Suddenly from behind came a
hoarse cry.
Arlen turned, ready to blast the Venusian that had shouted. But the
Venusian did not attack. Instead, it darted forward, and with a flying
leap it sprang upon the python. A roar came from the Venusian's
throat.
It was Gheal. Arlen would have recognized the voice anywhere.
The faint glow from the volcanoes showed him the edge of the
plateau.
Renzu was screaming behind him and he heard the pad-pad of the
running feet of the three remaining Venusians. But Arlen was clear
and McFerson was running beside him.
Arlen took his flashlight from his pocket and used it to follow the
narrow ledge down the mountain into the canyon. Behind the two
men, sounds of pursuit grew fainter.
"We're safe," Arlen said, slackening his pace. "Renzu won't follow us
as long as he knows we're armed."
"He's armed, too," McFerson said.
"He wants our bones too badly to use a disintegrator on us," Arlen
laughed.