CONTENTS

Cover

Title Page

Copyright

Preface

About the Companion Website

Chapter 1: Mouse

Introduction

Mouse Genetics and Genomics

Nomenclature

Common Inbred Strains

Genomic Considerations for the Pathologist

Anatomic Features

Bibliography for Introduction Through Anatomic Features

General References on Diseases of Mice

Infections of Laboratory Mice: Effects on Research

Bibliography for Infections of Laboratory Mice: Effects on Research

DNA Viral Infections

RNA Viral Infections

Retroelements and Retrovirus Infections

Bibliography for Viral Infections

Bacterial Infections

Bibliography for Bacterial Infections

Fungal Infections

Bibliography for Fungal Infections

Parasitic Diseases

Bibliography for Parasitic Diseases

Behavioral Disorders

Bibliography for Behavioral Disorders

Aging, Degenerative, and Miscellaneous Disorders

Bibliography for Aging, Degenerative, and Miscellaneous Disorders

Neoplasms

Bibliography for Neoplasms

Chapter 2: Rat

Introduction

Anatomic Features

Bibliography for Behavioral and Anatomic Features

General References on Diseases of Rats

DNA Viral Infections

RNA Viral Infections

Bibliography for Viral Infections

Bacterial Infections

Fungal Infections

Bibliography for Bacterial and Fungal Infections

Parasitic Diseases

Bibliography for Parasitic Diseases

Age-Related Disorders

Miscellaneous Disorders

Environmental Disorders

Drug-Related Disorders

Bibliography for Age-, Miscellaneous-, Environmental- and Drug-Related Disorders

Neoplasms

Bibliography for Neoplasms

Chapter 3: Hamster

Introduction

Anatomic Features

Bibliography for Introduction and Anatomic Features

Dna Viral Infections

Rna Viral Infections

Bibliography for Viral Infections

Bacterial and Fungal Infections

Bibliography for Bacterial and Fungal Infections

Parasitic Diseases

Bibliography for Parasitic Diseases

Nutritional and Metabolic Disorders

Diseases Associated with Aging

Environomental, Genetic, and Other Disorders

Bibliography for Noninfectious Diseases

Neoplasms

Bibliography for Neoplasms

Chapter 4: Gerbil

Introduction

Anatomic Features

Bibliography for Anatomic Features

Viral Infections

Bibliography for Viral Infections

Bacterial Infections

Bibliography for Bacterial Infections

Parasitic Diseases

Bibliography for Parasitic Diseases

Genetic, Metabolic, and Other Disorders

Bibliography for Genetic, Metabolic, and Other Disorders

Neoplasms

Bibliography for Neoplasms

Chapter 5: Guinea Pig

Behavioral, Physiologic, and Anatomic Features

Bibliography for Behavioral, Physiologic, and anatomic Features

Viral Infections

Bibliography for Viral Infections

Bacterial Infections

Bibliography for Bacterial Infections

Fungal Infections

Bibliography for Fungal Infections

Parasitic Diseases

Bibliography for Parasitic Diseases

Nutritional, Metabolic, and Toxic Disorders

Bibliography for Nutritional, Metabolic, and Toxic Disorders

Miscellaneous Disorders

Bibliography for Miscellaneous Disorders

Neoplasms

Bibliography for Neoplasms

Chapter 6: Rabbit

Introduction

Behavioral, Physiologic, and Anatomic Features

Bibliography for Behavioral, Physiologic, and Anatomic Features

DNA Viral Infections

RNA Viral Infections

Bibliography for Viral Infections

Bacterial Infections

Bibliography for Bacterial Infections

Fungal Infections

Bibliography for Fungal Infections

Parasitic Diseases

Bibliography for Parasitic Diseases

Noninfectious Gastrointestinal Disorders

Bibiliography for Noninfectious Gastrointestinal Disorders

Aging and Miscellaneous Disorders

Bibliography for Aging and Miscellaneous Disorders

Nutritional, Metabolic and Toxic Disorders

Bibliography for Nutritional, Metabolic, and Toxic Disorders

Genetic Disorders

Bibliography for Genetic Disorders

Neoplasms

Bibliography for Neoplasms

Index

End User License Agreement

List of Illustrations

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Fourth Edition

Pathology of Laboratory Rodents and Rabbits

Stephen W. Barthold, DVM, MS, PhD, Dipl. ACVP

Distinguished Professor Emeritus

Veterinary and Medical Pathology

University of California

Davis, CA

Stephen M. Griffey, DVM, PhD

Clinical Professor of Laboratory Animal Pathology

Director of the Comparative Pathology Laboratory

School of Veterinary Medicine

University of California

Davis, CA

Dean H. Percy, DVM, MSc, PhD, Dipl. ACVP

Professor Emeritus

Pathobiology Department

Ontario Veterinary College

Guelph, Ontario

This edition first published 2016 © 2016 by John Wiley & Sons, Inc.

First edition published 1993 © Iowa State University Press.

Second edition published 2001 © Iowa State University Press.

Third edition published 2007 © Blackwell Publishing Professional.

Editorial offices: 1606 Golden Aspen Drive, Suites 103 and 104, Ames, Iowa 50010, USA

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Preface

The Fourth Edition of Pathology of Laboratory Rodents and Rabbits has been extensively revised in response to reviewers' and colleagues' comments, as well as addition of new material that has arisen since the Third edition was published in 2007. In particular, the chapter on rabbits has been significantly revised and expanded. A resounding message from reviewers and colleagues was to publish this edition in color. Technology has advanced such that color images are not only possible but also cost-effective. The images in this text are nonpareil, and have been generously contributed by colleagues, gleaned from the recent literature, and derived from personal collections of the authors.

Also notable in this edition is the change in authorship. Dean Percy has fully retired as an active author of this text, but his significant input remains throughout the text from previous editions. This book was his dream, and its long-term success since publication of the First Edition in 1993 can be largely attributed to his vision, energy, and perseverance. Stephen Barthold stepped up to the leadership for this edition, with the considerable assistance of his friend and colleague, Stephen Griffey. Reviewers suggested a third, younger author (Dr. Griffey) to carry on the tradition of this text in future editions. Dr. Griffey, like Drs. Percy and Barthold, is a veterinary pathologist with extensive experience and expertise in laboratory animal pathology.

Delving into the literature has been a thought-provoking journey down memory lane. Many of the contributors to the laboratory animal pathology field are deceased. Unfortunately, younger members of our profession must forego the privilege of personally knowing these giants upon whose shoulders our field has been built. Discovery of new diseases continues, but not at the pace that took place in the 1970s and 1980s.

This book is dedicated to our families, our mentors, our colleagues, and our students…past, present, and future. It is especially dedicated to the subjects of this text: laboratory rodents and rabbits. They contribute enormously to biomedical and veterinary science, and deserve our respect and full support.

About the Companion Website

This book is accompanied by a companion website:

www.wiley.com/go/Barthold/pathology

The website includes–downloadable photographs and illustrations from the book.

1

Mouse

Introduction

Apart from the many inbred strains, substrains, spontaneous mutants, and outbred stocks of laboratory mice, the mouse has been, and continues to be, central to molecular genomics, with worldwide efforts continuing to knock out every functional gene in the mouse genome and define the relationship between genotype and phenotype. In addition to understanding the genome, various mouse strains and stocks, as well as the genetically engineered mouse (GEM), play critical roles in hypothesis-driven biomedical research. These trends have created rich opportunities and critical demand for comparative pathologists who are knowledgeable in mouse pathobiology. Unfortunately, the scientific literature is replete with erroneous interpretation of phenotype by scientists (as well as pathologists) lacking expertise in mouse pathology. Effective mouse pathology requires a global understanding of mouse biology, euphemistically termed Muromics (see Barthold 2002).

It is impossible for the pathologist to command in-depth knowledge of all strains, stocks, and mutant types of mice, and in many cases there is little baseline data to draw upon. Nevertheless, the mouse pathologist must be cognizant of general patterns of mouse pathology, as well as strain- and GEM-specific nuances. Recommended references (Frith & Ward, 1988; Maronpot et al. 1999; McInnes 2012; Mohr 2001; Mohr et al. 1996; Ward et al. 2000) provide thorough pictorial coverage of spontaneous mouse pathology in several common inbred strains of mice. The incidence and prevalence of strain-specific pathology are highly dependent upon genetic and environmental influences, including diet, bedding, infectious disease, age, sex, and other factors. Compared to the above-cited references, our coverage of the esoterica of spontaneous mouse pathology is relatively superficial. We herein emphasize general patterns of disease, while attempting to address important strain-, mutant-, and GEM-specific diseases when appropriate. There are a growing number of internet-accessible resources for mouse phenotyping and pathology of strains, stocks, and GEMs. A listing of these web resources is available through various sources (Bolon 2006; Brayton 2013; Fox et al. 2007, 2015). Although not specifically listed in this text, it is worth surfing through these cited websites that provide a plethora of information at multiple levels.

The unique qualities of the laboratory mouse and the precision of mouse-related research make infectious agents, even those with minimal (or no) pathogenicity, major concerns due to their potential and sometimes significant impact upon research reproducibility, including phenotype. A challenge that is unique to the mouse is the difficulty in drawing the line between commensalistic, opportunistic, or overtly pathogenic microorganisms. Since the last edition, a wide variety of immune-deficient GEMs have been created, thereby raising the status of several relatively innocuous infectious agents to the level of pathogens. Immune-deficient mice and new molecular methods of detection continue to reveal previously unrecognized mouse pathogens, such as a number of Helicobacter spp., norovirus, and most recently, astrovirus. Furthermore, the unrestricted traffic of GEMs among institutions and the pressure to reduce costs of maintenance at the expense of quality control have resulted in the re-emergence of several infectious agents that have not been seen in several decades. We, therefore, unabashedly emphasize mouse infectious diseases in this chapter. Despite advances in husbandry and diagnostic surveillance, we are reluctant to discard entities that may seem to have disappeared from laboratory mouse populations because of their likelihood of return.

Mouse Genetics and Genomics

The laboratory mouse is an artificial creation, and there is no true wild-type laboratory mouse. Furthermore, there is no such thing as normal microflora, since laboratory mice are often maintained in microbially pristine environments devoid of pathogens and opportunistic pathogens, as well as other commensal flora/fauna. Laboratory mice are largely derived from domesticated fancy mice that arose from many years of trading mouse variants among fanciers in Europe, Asia, North America, and Australia. The laboratory mouse genome is, therefore, a mosaic derived from different subspecies of the Mus musculus (house mouse) complex, including M.m. domesticus, M.m. musculus, M.m. castaneus, M.m. molossinus (a natural hybrid of M.m. musculus and M.m. castaneus), and others. The genome of M.m. domesticus is the predominant contributor to most strains of mice, but many inbred strains share a common Eve with a mitochondrial genome of M.m. musculus origin and a common Adam that contributed their Y chromosome from M.m. castaneus. In addition, there is evidence that other Mus species, outside of the M. musculus complex, have contributed to the genome of some, but not all, laboratory mouse strains. For example, the C57BL mouse genome contains a contribution from M. spretus. Perhaps the only laboratory mouse that is derived from a single M.m. domesticus species (subspecies) is the Swiss mouse. Several Mus species that are outside of the M. musculus complex, such as M. spretus, have been inbred. Thus, the laboratory mouse genome is not uniform among strains and mouse strains are not entirely within the M. musculus clade.

There are over 450 inbred strains of laboratory mice that have arisen during the last century, and these strains, which were selectively inbred to pan-genomic homozygosity for purposes entirely unrelated to modern research, are the foundation upon which literally thousands of spontaneous mutants and GEMs have been built. Additional inbred strains have been developed from wild mice (M.m. castaneus, M. spretus, etc.). Furthermore, outbred mice (mostly Swiss mice) are highly homozygous and nearly inbred. In addition to historical inbreeding that may be intentional or the inadvertent result of maintaining small populations of mice, rederivation of a mouse population results in genetic bottlenecks as well. There is no such thing as a truly outbred laboratory mouse with a fully heterozygous genome representative of wild-type M. musculus, and there is no wild mouse genetic counterpart of the laboratory mouse. Recently, an octaparental Diversity Outbred (DO) mouse stock was developed from eight disparately related inbred strains of mice, but this stock is not extensively utilized. When working with mice, the pathologist must also become facile with strains, substrains, sub-substrains, hybrids, congenics, insipient congenics, coisogenics, consomics, conplastics, recombinant inbreds, recombinant congenics, spontaneous mutants, random induced (radiation, chemical, retroviral, gene trap) mutants, transgenics (random insertions), and targeted mutant mice, each with relatively unique, predictable, and sometimes unpredictable phenotypes and patterns of disease whose expression is modified by environmental and microbial variables.

The inherent value of the laboratory mouse is its inbred genome, but maintaining the genetic stability of inbred strains of mice is a challenge. Since the advent of GEMs, there has been widespread genetic mismanagement of mouse strains by investigators with considerable skill in mouse genomics but limited expertise in mouse genetics. Even with the best of intentions, continuous inbreeding leads to substrain divergence among different populations of the same parental origin due to spontaneous mutations, retrotransposon integrations, or residual heterozygosity. Genetic contamination is also a surprisingly frequent event in both commercial and academic breeding colonies of mice. Within a few generations, substrain divergence can result in significant differences in phenotype, including response to research variables. The variable genetic contributions of different origins of mice and selective inbreeding for strain characteristics, such as coat color or neoplasia, are especially important when considering retroelements, which make up 37% of the mouse genome. Retroelements are highly dynamic within the context of the inbred mouse genome. They are present in the genomes of all mammals but have become artificially important in the homozygous genome of the laboratory mouse, and in fact had much to do with development of original inbred strains of mice with unique phenotypes, especially coat color and neoplasia. It is difficult to ignore their impact on mouse pathology, and thus retroelements are discussed later in this chapter (see Section Retroelements and Retroviral Infections).

Nomenclature

The details of mouse nomenclature are beyond the scope of this book, but it is critically important that the full and correct strain, substrain, and mutant allelic or transgene nomenclature be utilized when evaluating pathology and in publications for maximal reproducibility of results. Being able to read the nomenclature of a mouse that is submitted for evaluation is critical for interpreting pathology. Guidelines for mouse nomenclature are available at the International Mouse Nomenclature home page (http://www.informatics.jax.org/mgihome/nomen/). The Mouse in Biomedical Research: History, Genetics, and Wild Mice (Fox et al. 2007), the mouse chapter in Laboratory Animal Medicine (Fox et al. 2015), and Mouse Genetics (L.M. Silver 1995) are also useful sources of information on mouse genetics, genomics, and nomenclature.

Common Inbred Strains

Among the many inbred strains, the great majority of biomedical research, including genomic research, is based on a relatively few mouse strains, including C57BL/6, BALB/c, C3H/He, 129, FVB, and outbred Swiss stocks. This is fortuitous for the pathologist, as familiarity with this relatively small list of strains provides a good basis for approaching the general pathology of mice. Despite emphasis on mouse strains, there are significant genotypic and phenotypic differences among substrains of any given strain, such as C57BL/6J versus C57BL/6N and among the various strains of 129 mice. An overview of characteristics among inbred strains has been developed by Festing (http://www.informatics.jax.org/external/festing/mouse/STRAINS.shtml) and The Mouse Phenome Database provides comprehensive information on many strains of mice (http://www.phenome.jax.org).

The reader is referred to other sources for more comprehensive information regarding the myriad possibilities of background pathology among laboratory mice (see Section General References on Diseases of Mice). This text is not intended to provide such depth of coverage, but herein provides a brief synopsis of important disease characteristics of the major strains/stocks of mice. The specific lesions are described further in later sections of this chapter.

C57BL/6 (B6) mice are the gold standard background strain for GEMs created by homologous recombination. Many mutant alleles and transgenes are backcrossed onto this strain. There are a number of other related black strains, including C57BL/10 (B10). B6 mice were initially bred for their longevity. Their melanism is manifested by their coat color, as well as melanin pigment in heart valves, splenic capsule and trabeculae, meninges, cerebral vessels, Harderian glands, and parathyroid glands. Common strain-related spontaneous diseases include hydrocephalus, hippocampal neurodegeneration, microphthalmia and anophthalmia, age-related cochlear degeneration and hearing loss, and malocclusion. B6 mice are predisposed to barbering or trichotillomania, which renders them susceptible to alopecia and staphylococcal ulcerative dermatitis. Aged B6 mice develop acidophilic macrophage pneumonia and epithelial hyalinosis, which are rapidly accelerated in B6 mice with the moth-eaten and various other mutations. B6 mice may develop late-onset amyloidosis, but this is highly dependent upon environmental and infectious factors (e.g., dermatitis). The most common B6 neoplasms are lymphoma, hemangiosarcoma, and pituitary adenoma.

BALB/c mice (BALB/c, BALB/cBy, et al.) are albinos. Mature males are rather pugilistic, requiring separate housing for particularly fractious individuals. Dystrophic epicardial mineralization of the right ventricular free wall is common, and they are prone to development of myocardial degeneration and auricular thrombosis. Corneal opacities are commonly found, and they often develop conjunctivitis, blepharitis, and periorbital abscesses. Hypocallosity (corpus callosal aplasia) is frequent, and they develop age-related hearing loss. BALB mice are remarkably resistant to spontaneous amyloidosis, in contrast to other mouse strains. The livers of normal BALB mice feature a moderate amount of hepatocellular fatty change. The most common tumors of BALB mice are pulmonary adenomas, lymphomas, Harderian gland tumors, and adrenal adenomas. Myoepitheliomas of salivary, preputial, and other exocrine glands are also relatively common in this strain.

C3H/He mice are agouti mice that are blind due to rd1 mutation (Pde6brd1) and are also prone to corneal opacities and hearing loss later in life. They frequently develop focal myocardial and skeletal mineralization and myocardial degeneration. C3H/HeJ mice develop alopecia areata as they age. They are susceptible to exogenous murine mammary tumor virus (MMTV)-induced mammary tumors and develop a relatively high incidence of mammary neoplasia later in life due to endogenous MMTV. Other relatively common tumors include hepatocellular tumors.

129 mice rank high in the panoply of mousedom as the most frequent source of embryonic stem (ES) cells, from which most targeted mutant mice are derived. The 129 mouse is not a single strain, and in fact 129 is represented by 16 recognized strains and substrains. This is due to accidental and intentional genetic contamination of the original 129 strain by various laboratories. Thus, the designation 129 is followed by P, S, T, or X, and other designations, in addition to substrain determinants. Genetic differences between the targeting construct and the ES cells can significantly influence efficiency of homologous recombination. The differences among 129 mice are not subtle, with variation in coat color, behavior, and other characteristics, including patterns of pathology. Hypocallosity is relatively common in many 129 mice. 129 mice, like B6 mice, are prone to pulmonary proteinosis and epithelial hyalinosis. Megaesophagus occurs in some types of 129 mice. Blepharitis and conjunctivitis are common in 129P3 mice. 129/Sv mice are renown for development of testicular teratomas (aka embryonal carcinomas). Other common neoplasms in 129 mice are lung tumors, Harderian gland tumors, ovarian tumors, and hemangiosarcomas.

FVB/N mice are inbred Swiss mice that gained popularity for creation of transgenic mice in an inbred genetic background. They are blind due to homozygosity of rd1 allele (Pde6brd1) and prone to seizures. Many lines of FVB mice develop persistent mammary hyperplasia and hyperplasia or adenomas of prolactin-secreting cells in the anterior pituitary, but mammary tumors are rare (unless through transgenesis). Common neoplasms include tumors of lung, pituitary, Harderian gland, liver, lymphomas, and pheochromocytomas.

NOD mice are inbred Swiss mice that were selectively bred for cataracts, and during that process were found to develop type 1 diabetes (nonobese diabetes (NOD)). This strain develops a number of other autoimmune disorders that are genetically determined at multiple loci. Notably, they have functional defects in macrophage and dendritic cell function, NK cells, NKT cells, regulatory CD4+CD25+ cells, and are C5a deficient. Their susceptibility to diabetes is highest when they are maintained in relatively germ-free environments, and is much lower in conventional environments. The NOD strain was genetically modified through backcrossing to create a xenotransplant host that is globally defective in NK cells, macrophage and dendritic cells (NOD characteristics), T and B cells (Prkdcscid), and IL-2-receptor γ (IL-2rγtm1Wjl). The resultant strain, NOD.CgPrkdcscidIL2rγtm1Wjl/SvJ (NSG) has become the optimal host for xenogeneic transplants, particularly human stem cell and T-cell engraftment. As a result, graft versus host disease (GVHD) arises in engrafted mice, characterized by human T-cell infiltration of skin, liver, intestine, lungs, and kidneys (see discussion of GVHD in Section Aging, Degenerative, and Miscellaneous Disorders). Because of their global immunodeficiency, mice of this strain are uniquely susceptible to opportunistic infections.

Outbred Swiss mice are all closely related derivatives of a small gene pool of founder animals that were inbred for many generations in various laboratories before outbreeding, primarily by commercial vendors. Outbred Swiss mice are often erroneously considered wild-type for comparison with inbred mice. As noted previously, they are far from outbred and differ genetically from inbred mice. Many, but not all, Swiss mouse stocks have retinal rd1 degeneration (homozygous recessive), reflecting their high degree of homozygosity. Swiss mice are particularly prone to amyloidosis, which is a major life-limiting disease. They develop a variety of incidental lesions, and the most common tumors are lymphomas, pulmonary adenomas, liver tumors, pituitary adenomas, and hemangiomas/sarcomas, among others.

Genomic Considerations for the Pathologist

Having stressed the importance of strain and substrain, it is notable that the mouse genomic community does not utilize a single strain of mouse, and when they do use a similar strain, it is often a different substrain. GEMs are created in a variety of ways, including random mutagenesis (chemical mutagenesis, radiation, random transgenesis, gene trapping, retroviral transgenesis) and targeted mutagenesis (homologous recombination). Issues relevant to the pathologist with the most common means of creating GEMs, random transgenesis and targeted mutations, are discussed below.

Random insertion of transgenes is accomplished through pronuclear microinjection of zygotes with ectopic DNA (transgenes). This has generally been achieved using hybrid zygotes of 2 inbred parental strains, outbred Swiss mice, or from inbred Swiss FVB/N mice to take advantage of hybrid vigor to compensate for the trauma of microinjection and facilitate the process of microinjection by providing large pronuclei. Transgenes become randomly integrated throughout the genome, often in tandem repeats, so that each pup within a litter arising from microinjected zygotes is hemizygous for the transgene, but is genetically distinct from its littermates. The degree of transgene expression (phenotype) varies with the location of the transgene within the genome. Each founder line of the same transgene represents a unique and nonreproducible genotype and, therefore, phenotype. Transgenes tend to be genetically unstable, and copies may be lost in subsequent generations, resulting in ephemeral phenotypes. Transgene insertions can also lead to unanticipated altered function of genes through insertional mutagenesis, or regulation by flanking genes within the area of insertion. Unanticipated phenotypes, such as immunodeficiency or other effects, can therefore occur. The use of hybrids or outbred mice as founders requires selective inbreeding to attain a useful model. This can be circumvented by using inbred founders, such as FVB/N mice. Maintaining the transgene on an outbred genetic background or incompletely backcrossed background poses problems with uncontrolled modifier and compensatory genes that may unpredictably influence phenotype.

The discipline of mouse genomics has lent itself to incredible precision through homologous recombination, with the ability to alter not only specific genes but also gene function at specific time points during development or life stage, create tissue-specific gene alterations, gain of function, loss of function, and targeted integration of transgenes that allow customized development of mouse models of human disease that would not ordinarily arise within the context of the indigenous mouse genome. Targeted mutant mice are often created in one of several types of 129 ES cells, and once germline transmission has been effected, the 129-type mutant mouse is usually backcrossed to a more utilitarian mouse strain, such as B6. Full backcrossing to congenic status requires 3–4 years, which is seldom fulfilled. In constructs that require cre-lox technology, mutant mice are further crossed with cre transgenic mice, which may be of another strain, substrain, or stock background. Thus, despite superb precision in altering a gene of interest, the rest of the mouse's genome can remain highly heterogeneous, which defeats the inherent value of the GEM for research, or at least limits its full potential.

ES cells, and the mutations that they carry, are most often derived from one of the 129-type mouse strains, and ES cells become mice through the generation of chimeric progeny. Insufficient backcrossing, with retention of 129 characteristics, may result in erroneous assumptions about the phenotype of the targeted gene. There is considerable genetic variation among different 129 ES cell lines, which can be a potential problem for comparing phenotypes of the same gene alteration among different 129 ES cell-derived mice. The process of creating chimeric mice, which is an essential step involving microinjection of 129 ES cells into a recipient blastocyst, has consequences. Most ES cell lines are male (XY), but blastocysts are either male or female. Hermaphroditism is quite common in chimeric mice arising from XY and XX cells. XX/XY chimeras are usually phenotypically male, but may have testicular hypoplasia and lower fertility. XX/XY chimeras may also have cystic Muellerian duct remnants, an ovary and a testis, and/or ovotestes. In addition to gonadal teratomas that are inherent in many 129 mouse strains, extragonadal teratomas arising from 129 cells in chimeric mice can develop in perigenital regions and the midline.

Because of the highly inbred nature of laboratory mice, experimental mutation of many genes often leads to embryonic or fetal death that precludes evaluation of phenotype in adult mice. Thus, pathologists are being increasingly called upon to familiarize themselves with fetal development and evaluate developmental defects. Fetal pathology is beyond the scope of this text, but the reader can access several excellent sources of information (see Kaufman 1995; Kaufman and Bard 1999; Rossant and Tam 2002; Ward et al. 2000). Embryonic/fetal viability is most often influenced by abnormalities in placentation, liver function, or cardiovascular function (including hematopoiesis). Particular attention should be paid to these factors. Depending upon genetic background, lethality can vary. Gene expression, and therefore circumvention of events such as embryonic lethality, can be controlled temporally and quantitatively by tissue-specific promoters with drug-regulated transcription systems and with cre/lox deletion, in which cre recombinase can be controlled with transcription techniques. Temporal and quantitative control of transgenes poses unique challenges to pathologists when evaluating phenotype.

In addition to predicted phenotypes, GEMs often manifest unique pathology that is not present in parental strains. Genetic constructs are usually inserted into the genome with a promoter to enhance expression, to target expression within a specific tissue, or to conditionally express the transgene, but promoters can affect phenotype as much as the gene of interest. Promoters are seldom totally tissue-specific and can impact upon other types of tissue. Conversely, overexpression of transgenes, regardless of their nature, can result in abnormalities in normal cell function. Tumors, particularly malignant tumors of mesenchyme, including hemangiosarcomas, lymphangiosarcomas, fibrosarcomas, rhabdomyosarcomas, osteosarcomas, histiocytic sarcomas, and anaplastic sarcomas, are frequent spontaneous lesions in transgenic mice that are relatively rare in parental strains of mice. Lymphoreticular tumors, which are quite common in parental strains of mice, reach epic proportions in GEMs. In some cases, relatively rare forms of lymphoma, such as marginal zone lymphomas, arise frequently in GEMs. Tumor phenotypes found in transgenic mice bearing myc, ras, and neu are distinctive and found only in mice with these transgenes. Many gene alterations have specifically targeted immune response genes, but others have unintentional effects upon immune response. When the immune responsiveness of the mouse is altered, opportunistic pathogens become an important factor in phenotype. Phenotypes have been known to disappear when mutant mice are rederived and rid of their adventitious pathogens.

Consequently, the pathologist must be cognizant of general mouse pathology, strain-related patterns of spontaneous pathology, infectious disease pathology, developmental pathology, comparative pathology (to validate the model), methodology used to create the mice, predicted outcomes of the gene alteration (including effects of the promoter), potential but unexpected outcomes of the gene alteration, and Mendelian genetics. The pathologist must also resist temptation to overemphasize a desired phenotype, underemphasize an undesired phenotype, or proselytize a phenotype as a model for human disease when it isn't. There is no better person to be the gatekeeper of reality in the world of functional genomics than the comparative pathologist.

Anatomic Features

The laboratory mouse has several unique characteristics, and there are vast differences in normal anatomy, physiology, and behavior among different strains of mice, many of which represent abnormalities arising from homozygosity of recessive or mutant traits in inbred mice.

Integumentary System

The history of the laboratory mouse is steeped in selective breeding for variation in coat color and consistency, with many defined mutants. Hair growth occurs in cyclic waves, beginning cranially and progressing caudally. Examination of mouse skin mandates awareness of the growth cycle and location examined. Melanin pigment is restricted to the hair follicular epithelium and hair shaft, with minimal pigmentation of the interfollicular epidermis. Thus, newborn mice, regardless of their ultimate coat color, are uniformly pink until hair growth begins.

Hematology and Hematopoeitic System

Mouse hematology has been recently reviewed (Everds 2007). Strain-specific data and comparisons among inbred mouse strains are available through the Mouse Phenome Database. Recommended approaches to evaluation of GEMs with hematological phenotypes are also available (Car and Eng 2001). Mouse erythrocytes are small, with a high reticulocyte count, moderate polychromasia, and anisocytosis. Lymphocytes are the predominant circulating leukocyte and constitute approximately three-fourths of the total differential count. Mature male mice have significantly higher granulocyte counts than do female mice. Peripheral blood granulocytes tend to be hypersegmented, and band cells are rare, except when mice have chronic suppurative infections. Granulocytes in tissues and bone marrow often have ring-shaped nuclei (Fig. 1.1). Ring-shaped nuclei can be visualized as early as the progranulocyte stage in bone marrow, spleen, and liver, and only rarely can be found in peripheral blood. They also occur in cells of the monocytic lineage. Mice have circulating basophils, but they are extremely rare. Mice possess a very large platelet mass, due to high platelet numbers and relatively low mean volume, although some platelets can be as large as erythrocytes. The spleen is a major hematopoietic organ throughout life in the mouse, and hematopoiesis is found in the liver up to weaning age but may return in adults during disease states. Hepatic hematopoiesis can be misconstrued as inflammation. Hematopoiesis remains active in long bones throughout life.

Fig. 1.1 Ring-shaped nuclei (arrow) of myeloid progenitor cells in the bone marrow of a normal mouse.

Respiratory System

Cross sections of the nose reveal prominent vomeronasal organs, which are important in pheromone sensing and are frequent targets of viral attack. Virus-associated vomeronasal and olfactory rhinitis in neonatal mice can result in failure to suckle. Respiratory epithelium may contain eosinophilic secretory inclusions (hyalinosis), which are especially obvious in B6 and 129 mice. The lungs have a single left lobe and 4 right lobes. Cartilage surrounds only the extrapulmonary airways in mice, rats, and hamsters. Thus, primary bronchi are extrapulmonary. Respiratory bronchioles are short or nonexistent. Cardiac muscle surrounds major branches of pulmonary veins and should not be misconstrued as medial hypertrophy. Bronchus-associated lymphoid tissue is normally present only at the hilus of the lung, except in hamsters. Lymphoid accumulations are present on the visceral pleura of mice, within interlobar clefts. These are organized lymphoid structures that are contiguous with the underlying lung tissue and are similar to milkspots in the peritoneum. Although not a normal finding, focal intra-alveolar hemorrhage is a consistent agonal finding in lungs of mice, regardless of the means of euthanasia. As in other species, focal subpleural accumulation of alveolar macrophages (alveolar histiocytosis) is common (see Rat chapter 2, alveolar histiocytosis).

Gastrointestinal System

Mice are coprophagic, with approximately one-third of their dietary intake being feces. Stomach contents will reflect this behavior. Incisive foramina, located posterior to the upper incisors, communicate between the roof of the mouth and the anterior nasal cavity. Incisors grow continuously, but cheek teeth are rooted. Mice have no deciduous teeth, and their incisors are pigmented due to deposition of iron beneath the enamel layer. One of several sexual dimorphisms in the mouse is found in the salivary glands. The submandibular salivary glands in sexually mature males are nearly twice the size as females and parotid salivary glands are also larger. Male submandibular glands have increased secretory granules in the cytoplasm of serous cells (Fig. 1.2). These glands undergo similar masculinization in pregnant and lactating females. The intestine is simple. Gut-associated lymphoid tissue (Peyer's patches) is present in both the small and large intestine. Paneth cells occupy crypt bases in the small intestine. These specialized enterocytes have prominent eosinophilic cytoplasmic granules (Fig. 1.3), which are larger in mice than in other laboratory rodents. Pregnant and lactating mice have noticeably thickened bowel walls due to physiological mucosal hyperplasia. Mice have a very short (1–2 mm) rectum, which is the terminal portion of the large bowel that is not enveloped in serosa. Because of this feature, mice are prone to rectal prolapse, especially if they have colitis.

Fig. 1.2 Submandibular (submaxillary) salivary gland from an adult male mouse. Note the prominent secretory granules (arrow) in the cytoplasm of epithelial cells.

Fig. 1.3 Ileal mucosa of a mouse illustrating the distinct cytoplasmic granules within Paneth cells at the base of the crypts.

The intestine of neonatal mice has several unique features. Neonatal small intestinal enterocytes are vacuolated and may contain eosinophilic inclusions due to the presence of the apical–tubular system, which is involved in uptake of macromolecules (Fig. 1.4). It disappears as the intestine undergoes maturation. The neonatal mouse bowel has very shallow crypts of Leiberkuhn populated with mitotically inactive stem cells and very long villi that are populated with terminally differentiated, absorptive epithelium. Intestinal cell turnover kinetics are slow in the neonate, making neonates highly vulnerable to acute cytolytic viruses. Turnover kinetics accelerate with acquisition of microflora and dietary stimuli.

Fig. 1.4 Enteric mucosa of a neonatal mouse, illustrating the vacuolated appearance of villus enterocytes.

The liver of mice has variable lobation. Polyploidy is common in mouse liver cells. Hepatocytes frequently display cytomegaly, anisokaryosis, polykarya, and karyomegaly (Fig. 1.5). Cytoplasmic invagination into the nucleus is frequent, giving the appearance of nuclear inclusion bodies (Fig. 1.6). Hematopoiesis normally occurs in the infant liver (Fig. 1.7) but wanes by weaning age, although islands of myelopoiesis or erythropoiesis can be found in hepatic sinusoids of older mice, particularly in disease states (Fig. 1.8). Hepatocytes frequently contain cytoplasmic fat vacuoles. Some strains, such as BALB mice, normally have diffuse hepatocellular fatty change, resulting in grossly pallid livers, compared with the mahogany-colored livers of other mouse strains.

Fig. 1.5 Polykarya and megalokarya, indicative of polyploidy, are commonly found in the liver and increase with age and disease states.

Fig. 1.6 Cytoplasmic invagination into the nucleus of a hepatocyte, a common finding in rodents that has been misinterpreted as viral inclusions.

Fig. 1.7 Liver from a newborn mouse. There are numerous hematopoietic cells in the sinusoidal regions.

Fig. 1.8 Liver from an adult mouse with suppurative pyelonephritis, illustrating marked hepatic myelopoiesis.

Genitourinary System

Female mice have a large clitoris, or genital papillus, with the urethral opening near its tip, which is located anterior to the vaginal orifice. Females that develop in utero between male fetuses are somewhat masculinized, reflected by an increased anogenital distance and behavior. Tissues of the adult uterine wall are normally infiltrated with eosinophils, which wax and wane cyclically and disappear during pregnancy. Eosinophils increase in number in response to semen. Mice have hemochorial placentation. Males have large redundant testes that readily retract into the abdominal cavity through open inguinal canals, particularly when the mice are picked up by the tail. Both sexes have well-developed preputial (or clitoral) glands, and males have conspicuous accessory sex glands, including large seminal vesicles, coagulating glands, and prostate. Ejaculation results in formation of a coagulum, or copulatory plug. This frequently occurs agonally. Coagulum can be found in urinary bladder or urethra as a normal incidental finding at necropsy (Fig. 1.9) and must not be misconstrued as a calculus or obstruction. However, copulatory plugs can and do cause obstructive uropathy. Sexual maturity in males results in several sexual dimorphic features, including larger kidneys, larger renal cortices, larger cells in proximal convoluted tubules, larger renal corpuscles, and cuboidal epithelium lining the parietal layer of Bowman's capsule, resembling tubular epithelium (Fig. 1.10). This is not absolute, since some glomeruli of male mice are surrounded by flat epithelium and some glomeruli of female mice are surrounded by cuboidal epithelium. Mice are endowed with relatively large numbers of glomeruli per unit area, compared with other species, such as the rat. Mice have a single, long renal papilla that extends into the upper ureter. Proteinuria is also normal in mice, with highest levels in sexually mature male mice. Major contributors to proteinuria in male mice are mouse urinary proteins, which function as pheromones. In particular, MUP-1 is highly antigenic and a major cause of occupational allergies among animal handlers.

Fig. 1.9 Copulatory plug in the urinary bladder of a male mouse. The presence of ejaculate coagulum is common in the urethra and bladder as an agonal finding, although antemortem ejaculation may result in urinary obstruction.

Fig. 1.10 Renal cortex from an adult male mouse, illustrating the cuboidal epithelium lining the parietal surface of Bowman's capsule.

Endocrine System

The mouse adrenal gland has several notable features. The adrenals of male mice tend to be smaller and have less lipid than those of females. Accessory adrenals, either partial or complete, are very common in the adrenal capsule or surrounding connective tissue. The zona reticularis of the adrenal cortex is not discernible from the zona fasciculata. Proliferation of subcortical spindle cells, with displacement of the cortex, is common in mice of all ages (Fig. 1.11). The function of these cells is not known. A unique feature of the mouse adrenal is the X zone of the cortex, which surrounds the medulla. The X zone is composed of basophilic cells and appears in mice around 10 days of age. When males reach sexual maturity and females undergo their first pregnancy, the X zone disappears. The X zone disappears gradually in virgin females. During involution, the X zone undergoes marked vacuolation in females (Fig. 1.12) but not in males. Residual cells accumulate ceroid. Pancreatic islets are highly variable in size, including giant islets that can be confused with hyperplasia or adenomas.

Fig. 1.11 Subcapsular spindle cell proliferation in the adrenal gland of a normal adult mouse. This change is common, but its significance is not known.

Fig. 1.12 Vacuolating degeneration of the involuting X zone at the corticomedullary junction of the adrenal gland of an adult female mouse.

Skeletal System

Bones of mice, like those of rats and hamsters, do not have Haversian systems, and ossification of physeal plates with age is variable and incomplete, depending upon mouse genotype.

Lymphoid System

Rodents do not have tonsils, but have nasal-associated lymphoid tissue (NALT). Germinal centers are not well defined in lymph nodes. The thymus does not involute in adults. Hassall's corpuscles are indistinct. Islands of ectopic parathyroid tissue may be encountered in the septal or surface connective tissue of the thymus, and, conversely, thymic tissue may occur in thyroid and parathyroid glands. Epithelial-lined cysts are also common. The splenic red pulp is an active hematopoietic site throughout life (Fig. 1.13). During disease states and pregnancy, increased hematopoiesis can result in splenomegaly. Lymphocytes tend to accumulate around renal interlobular arteries, salivary gland ducts, urinary bladder submucosa, and other sites, increasing with age. These sites are often involved in generalized lymphoproliferative disorders. Melanosis of the splenic capsule and trabeculae is common in melanotic strains of mice (Fig. 1.14). This must be differentiated from iron (hemosiderin) pigment (Fig. 1.15), which tends to accumulate in the red pulp as mice age, particularly in multiparous females. Mast cells can be frequent in the spleen of some mouse strains, such as A strain mice.

Fig. 1.13 Spleen from an adult mouse, illustrating the large numbers of hematopoietic cells, including megakaryocytes, in the sinusoids, a common finding throughout life.

Fig. 1.14 Splenic melanosis in a melantotic (C57BL) mouse. Note the patches of pigmented capsule.

Fig. 1.15 Iron pigment (hemosiderin) in the spleen of an adult female mouse (Perl's stain).

Other Anatomic Features

The brain and spinal cord are larger in mature male mice compared to females. Melanosis occurs in the anteroventral meninges of the olfactory bulbs, optic nerves, parathyroid glands, heart valves, and spleens of melanotic mouse strains, such as B6 mice. Foci of cartilage or bone can be found within the base of the aorta. These foci are not an os cordis but rather occur within the wall of the aorta. Mice have 3 pectoral and 2 inguinal pairs of mammary glands, with mammary tissue enveloping much of the subcutis, including the neck. Mammary tissue can be found immediately adjacent to salivary glands, which is especially apparent during lactation. Nipple development is hormonally regulated in mice, and nipples are quite small in males. Mammary tissue of males totally involutes during development. Remarkably, virgin female mice can be induced to lactate by the presence of other females nursing litters. Mammary glands normally involute between pregnancies, but they do not involute in multiparous FVB mice, due to a tendency to develop hyperplasia of prolactin-producing cells and pituitary adenomas. Brown fat is prominent as a subcutaneous fat pad over the shoulders and is also present in the neck, axillae, and peritoneal tissue.

Immunologic Idiosyncracies

Neonatal mice are globally immunodeficient. Different components of the innate and acquired immune response subsequently evolve at differing rates, depending upon genetic background. Although mice are generally immunocompetent at weaning, they are not fully so until 6–12 weeks of age. Neonates depend upon acquisition of maternal antibody to protect them during early life. Maternal IgG is transferred in utero through Fc yolk sac receptors, and postnatally through IgG receptors in the small intestine, which actively acquire immunoglobulin up to 2 weeks of age. Milk-borne IgA is also important in protecting suckling mice, but neither IgA nor IgM are absorbed. Passive immunity is a critical component in understanding the outcome of viral infections in mouse populations. Epizootic infections can be devastating in naïve populations of neonates, but once the infection becomes enzootic within a population, maternal antibody protects suckling mice during their period of age-related vulnerability. Maternal antibody generally persists in the serum of pups for about 6 weeks.

The immune response can vary considerably among different strains of mice. An often-cited feature is the Th1-Th2 polarized T-cell response, in which BALB/c mice tend to respond to antigenic stimuli with a Th2 skewed response and B6 mice with Th1 skewed responses. This is far from absolute, but there seems to be truth in the concept that B6 mice deal more efficiently with viral infections. B6, B10, SJL, and NOD mice have their own unique immunoglobulin isotype, IgG2c, in lieu of, but distinct from, IgG2a. IgG2c is not an allelic variant of IgG2a, since in these strains the IgG2a gene is completely absent, and in IgG2a-positive strains, the IgG2c gene is absent. This may impact accurate measurements of humoral responses. The mouse genome possesses approximately 40 histocompatibility loci, and the major histocompatibility (MHC) loci are located on chromosome 17 within the MHC complex, known as the H-2 complex. Each inbred strain of mouse has a defined H-2 haplotype, or combinations of alleles, which are well-recognized determinants of strain-specific immune responses, including responses to infectious disease. Because of the inbred nature of laboratory mouse strains, H-2 haplotype is a singularly important strain characteristic.

Various stressors, including dehydration, hypothermia, and acute infections, may result in massive corticosteroid-induced lymphocytic apoptosis. This is accompanied by generalized lymphoid depletion and transient nonspecific alterations of immune responsiveness. This is especially apparent in the thymus and is a frequent and rapid onset lesion in water bottle accidents, when mice become hypothermic or dehydrated. Recently rederived and xenobiotic mice have lymphoid hypoplasia, accompanied by functional hyporesponsiveness.

Genetic engineering has given rise to many immunologic mutants of mice, and other naturally arising immune mutants have also been popularized, such as nude (T-cell-deficient), SCID (B- and T-cell-deficient), and beige (NK cell-deficient) mice. The preeminent immunodeficient mouse is the NSG mouse, discussed above. Immunodeficient mice must never be considered to be simply missing a single functional component of the immune system, since they typically have compensatorily activated innate and acquired immune responses compared to wild-type. Homozygous immunodeficient inbred mouse mutants that are progeny of the heterozygous (immunocompetent) parental matings or through embryo transfer into immunocompetent recipients can acquire functional immunoglobulin-secreting B cells from their immunocompetent dams. They can also acquire functional B cells postnatally through foster nursing. The chimeric cells may remain functional for at least several months.

Less obvious and often overlooked immunologic idiosyncrasies also exist among common inbred strains. All strains of adult male mice manifest a sexual dimorphism in which serum levels of both C4 and C5 are higher than in females, and male SJL mice have a significantly higher level of C5 compared to males of other strains. In addition, inadvertent consequences have arisen from inbreeding and selection for other characteristics. One such common defect is a 2 base pair gene deletion in the 5th component of complement (C5). This mutation results in C5 deficiency in many inbred strains of mice, including AKR, SWR, DBA/2J, A/J, A/HeJ, NOD, and RF, among others. SJL mice are NK cell-deficient. NOD mice have multiple immune defects (see above). Substrain divergence due to spontaneous acquisition of mutations can give rise to novel new substrain phenotypes, such as the LPS unresponsiveness of C3H/HeJ and C57BL/10ScN mice, which is attributed to a mutation of toll-like receptor 4 (TLR4). All strains of mice lack functional TLR10 due to genetic disruption by a retroviral insertion. CBA/CaN (CBA/N), but not other CBA mice, have an X-linked defect in humoral immunity, with impaired maturation of B cells, diminished immunoglobulin production, and impaired T-independent immune responses. Thus, knowledge of specific strain and substrain characteristics greatly improves the understanding of responses to experimental variables.

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General References on Diseases of Mice

This text has used the following references extensively as sources of information. Many of these citations have multiple authors embedded within, but for the sake of space, individual authors within review books are not cited. Various sections of this chapter refer back to these basic general references, rather than repeat them for each section.

Brayton, C. (2007) Spontaneous diseases in commonly used mouse strains. In: The Mouse in Biomedical Research. Diseases, 2nd edn (eds. J.G. Fox, S.W. Barthold, M.T. Davisson, C.E. Newcomer, F.W. Quimby, & A.L. Smith), pp.