Fundamental Neuropathology for Pathologists and Toxicologists E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Fachliteratur
• Sprache: Englisch

Seitenzahl: 1711

Veröffentlichungsjahr: 2011

Beschreibung

This book offers pathologists, toxicologists, other medical professionals, and students an introduction to the discipline and techniques of neuropathology – including chemical and environmental, biological, medical, and regulatory details important for performing an analysis of toxicant-induced neurodiseases. In addition to a section on fundamentals, the book provides detailed coverage of current practices (bioassays, molecular analysis, and nervous system pathology) and practical aspects (data interpretation, regulatory considerations, and tips for preparing reports).

Leseprobe

Contents

Cover

Title Page

Copyright

Dedication

Contributors

Preface

Acknowledgments

Introduction

Part 1: Fundamentals Of Neurobiology

Chapter 1: Fundamental Neuropathology for Pathologists and Toxicologists: An Introduction

The Importance of Neurotoxicological Research

The Evolution of Toxicological Neuropathology

Requirements for Proficiency in Toxicological Neuropathology

Fundamental Principles of Toxicological Neuropathology

Concluding Remarks

References

Chapter 2: Functional Neuroanatomy

Introduction

Morphogenesis and Descriptive Anatomy of the Central Nervous System

Formation of the Central Nervous System

Ventricular System

Spinal Cord

Brain

Somatotopic Organization in the CNS

Vascular Supply

Functional Neuroanatomy

Conscious Afferents

Motor System (SE and SVE)

Afferents

Efferents

Visceral Nervous System

Integration

Limbic System

Recommended Reading

Chapter 3: Atlas of Comparative Neuroanatomy

How to Use this Atlas

Specimen Derivation and Preparation

Recommended Reading

Internet sources

Chapter 4: Principles of Comparative and Correlative Neurodevelopment and their Implications for Developmental Neurotoxicity

Introduction

Principles of Anatomical Organization in the Developing Nervous System

Early Specification of the Nervous System

Correlative Neurodevelopment

Comparative Neurodevelopment

Principles of Vertebrate Neurodevelopment

Mechanisms of Neurodevelopmental Vulnerability

Developmental Neurotoxicity: a Lifelong Menace

References

Chapter 5: Localizing Neuropathological Lesions Using Neurological Findings

Introduction

Lesion Localization Using the Syndrome Concept of Clinical Neurology

Practical Application of Neurological Syndromes by Neuropathologists and Neurotoxicologists

References

Chapter 6: Behavioral Model Systems for Evaluating Neuropathology

Introduction

Common Behavioral Tests

Models of Neurotoxicity: Behaviors and Underlying Neuropathology

Interpretation of Neurobehavioral Data

Summary

References

Chapter 7: Cognitive Assessments in Nonhuman Primates

Introduction

Modeling Specific Functions or Behaviors

Experimental Manipulations: Consequences Of Drugs, Toxicants, and Lesions

Relevance to Humans

References

Chapter 8: Impact of Aging on Brain Structure and Function in Rodents and Canines

Introduction

Human Aging and Alzheimer's disease

Animal Models of Human Aging and AD

Environmental Neurotoxicants as Potential Contributors to Neurodegenerative Disease

Summary

References

Chapter 9: Fundamentals of Neurotoxicity Detection

Introduction

Identification of the Endpoint for Neurotoxicity

Spectrum of Pathological Endpoint Detection

Study Design Principles: Location, Location, Location

Time Course for Observations of Neurotoxicity

References

Part 2: Toxicologic Neuropathology: Methodology

Chapter 10: Practical Neuropathology of the Rat andOther Species

Introduction

Specimen Preparation: Special Considerations

Collection and Preservation

Trimming and Processing

Special Stains and Techniques

Neuroanatomy

References

Chapter 11: Fluoro-Jade Dyes: Fluorochromes for the Histochemical Localization of Degenerating Neurons

Introduction

Chemical Identity

Staining Methods

Frozen Sections

Paraffin Sections

Staining Variants

Comparison of Fluoro-Jade, Fluoro-Jade B, and Fluoro-Jade C

Multiple Labeling Techniques

Comparison of Fluoro-Jade Dyes with Other Histological Markers: Advantages and Disadvantages

Specialized Applications

Discussion

Conclusions

Acknowledgments

References

Chapter 12: Histological Markers of Neurotoxicity (Nonfluorescent)

Introduction

Markers of Chemical Change

Perturbations and Inflammation

Markers of Permanent Change or Damage

Evaluation of True Degeneration or Disintegration

Evaluation of Background Staining

Artifactual Staining

References

Chapter 13: Common Histological Artifacts in Nervous System Tissues

Introduction

Retraction Spaces Around Neurons, Vessels, and Glial Cells

Dark (Basophilic) Neurons

Artifacts Involving Myelin, Axons, and Sensory Ganglion Neurons

Miscellaneous Artifacts

Acknowledgments

References

Chapter 14: High-Definition Microscopic Analysis of the Nervous System

References

Chapter 15: Stereological Solutions for Common Quantitative Endpoints in Neurotoxicology

Introduction

What is Stereology?

Generating a Representative Sample in Stereology: Systematic Uniform Random Sampling

Questionnaire in Stereology: Geometric Probes

Why Stereology? Accuracy, Precision, Variance, and Efficiency

When Stereology?

Stereological Estimators as Solutions to Common Quantitative Endpoints in Neurotoxicology

The Fractionator Principle

Optical Disector and Optical Fractionator

The Proportionator

Estimation of Total Myelinated Nerve Fiber Number and Absolute Size Distribution

Conclusions

Acknowledgments

Recommended Reading

References

Chapter 16: Anatomy and Processing of Peripheral Nerve Tissues

Introduction

Anatomy of the PNS

Sample Acquisition

Processing PNS Specimens

References

Chapter 17: Pathology Methods in Nonclinical Neurotoxicity Studies: Evaluation of Muscle

Introduction

Muscle Fiber Types

Significance of Skeletal Muscle Fiber Type in Toxicological Pathology

Skeletal Muscle Sampling

Skeletal Muscle Processing

Other Processing Techniques

Suggested Approaches to Sampling and Processing

Interpretation of Skeletal Muscle Sections

Conclusions

References

Chapter 18: In Vivo Imaging Applications for the Nervous System in Animal Models

Introduction

Nuclear Imaging: PET and SPECT in Non-Human Primate Studies

Brain Imaging in Animal Subjects

PET Imaging

MicroPET and MicroSPECT Animal Model Applications

Magnetic resonance imaging

Optical imaging

Ultrasound

Conclusions

Acknowledgments

References

Chapter 19: Cerebrospinal Fluid Analysis in Toxicological Neuropathology

Introduction

Functions of CSF and ISF in the CNS

Physiology of CSF and ISF

Composition of CSF During Health

Considerations in Sampling and Analyzing CSF

General Characteristics of CSF in Neurological Disease

Recommendations for CSF Analysis in Neurotoxicity Evaluations

References

Chapter 20: Molecular Techniques in Toxicological Neuropathology

Introduction

Factors Affecting Brain and Nerve Sample Quality

Considerations in Sampling Nervous Tissue for Molecular Analyses

Microarray Technology

Detection Methods for Gene Array Technologies

Experimental Design in Microarray Studies

Examples of Microarray Technology as Applied to Neuropathology Research

Proteomic Technologies

Techniques for Analyzing Proteins

Quantitation of Proteins

Examples of Proteomic Technology As Applied in Neuropathology

Correlation of Genomic and Proteomic Data with Biological Functions and Conventional Neuropathology Analysis

Programs for Integrating “OMICS” Databases

Anatomical Correlation of Gene and Protein Expression Data Within the Brain

References

Part 3: Toxicological Neuropathology: Current Practices

Chapter 21: Evaluation of the Adult Nervous System in Preclinical Studies

Introduction

Necropsy

Trimming and Embedding

Staining

Evaluation

References

Chapter 22: Pathology Methods in Nonclinical Neurotoxicity Studies: the Developing Central Nervous System

Introduction

Designing the Developmental Neuropathology

Fixation Procedure

Brain Weight Determination

Tissue Selection and Neurohistological Processing

Sampling Receptor and Effector Organs

Trimming Procedure

Neurodevelopmental Histopathology

Linear Measurements

Second-Tier Approaches

References

Chapter 23: Neuropathological Analysis of the Peripheral Nervous System

Anatomy of the Peripheral Nervous System

Cell Structure and Function in the PNS

Types of Nerve Fibers in the Peripheral Nerves

Technical Comments on PNS Sampling

Principal Lesion Types Observed in Toxic Neuropathies

Hypertrophic Neuropathy

Pathology of Unmyelinated Peripheral Nerve Fibers

Neuronopathy

Inflammatory Lesions

Axonopathy

Myelinopathy

Proliferative and Neoplastic PNS Lesions

Recommended Reading

References

Chapter 24: Toxicological Pathology of the Retina and Optic Nerve

Introduction

Ocular Anatomy

NonNeural Structures in the Eye

Clinical Assessment of the Retina and Optic Nerve

Specimen Acquisition and Processing for Ocular Neuropathology Studies

Electron Microscopy

Quantitative analysis

Ocular Processing Recommendations for Routine Toxicological Neuropathology Studies

Principles of Ocular Toxicological Neuropathology

Categories of Lesions in Ocular Toxicological Neuropathology

References

Chapter 25: Toxicological Neuropathology of the Ear

Introduction

Anatomy and Physiology of the Inner Ear

Access of Ototoxicants to the Inner Ear

Methods for Studying the Inner Ear

Effects and Actions of Ototoxic Drugs

Classes of Ototoxic Agents

Ototoxic Interactions

Summary

References

Chapter 26: Neuropathology of the Olfactory System

Introduction

Anatomy and Function of the Olfactory Mucosa and Olfactory Tract

Preparation of the Olfactory Mucosa for Neuropathology Examination

Special Procedures for Neuropathology Evaluation of the Olfactory Mucosa

Neuropathology of the Olfactory Mucosa and Olfactory Tract: Basic Principles

Comparative Neuropathology of the Olfactory Mucosa and Olfactory Brain

Toxicological Neuropathology of the Vomeronasal Organ

References

Part 4: Applied Toxicological Neuropathology

Chapter 27: Spinal Delivery and Assessment of Drug Safety

Spinal Drug delivery

Factors Affecting the Actions of Intrathecal Agents

Preclinical Assessment of the Toxicology of Intrathecal Agents

Some Organizing Principles for Robust Preclinical Evaluations of Spinal Drug Safety

Summary

Acknowledgments

References

Chapter 28: Diagnostic Neuropathology

Introduction

Gross Examination and Tissue Collection

Basic Cellular Changes

Basic Parenchymal Changes

Lysosomal Storage Diseases

Viral Diseases

Neoplastic Disease14, 18, 29

References

Chapter 29: Toxicological Neuropathology in Medical Practice

Introduction

Common Neuropathological Changes Caused by Neurotoxicants

Basic Patterns and Mechanisms of Injury

Ischemic or Hypoxic Patterns

Mitochondrial Dysfunction

Generalized Neuronal Injury Pattern

Localized Neuronal Injury Pattern

Transmission-Related and Excitotoxic Pattern of Neuronal Injury

Immune or Inflammatory Pattern of Neuronal Injury

Free-Radical Stress Pattern of Neuronal Injury

Axonopathy Pattern of Neuronal Injury

Summary

References

Chapter 30: Toxicological Neuropathology in Veterinary Practice

Introduction

Ante Mortem Neurological Evaluations

Macroscopic Examination of the Brain and Nervous System

Harvesting the Nervous System

Trimming Neural Tissues

Tissue Embedding and the Assessment of Neuropathological Lesions

Common Artifacts

Development of Neural Lesions

Regional and Tissue Specificity

Veterinary Dietary Neurotoxicants of Note

Pyridoxine Neuropathy

References

Chapter 31: Regulatory Considerations in Toxicological Neuropathology

Introduction

Number of Animals

Tissue Sampling

Tissue Preparation

Control Groups

Qualitative Examination: Detection of Treatment-Related Effects

Dose Dependence of Treatment-Related Effects

References

Chapter 32: The Neuropathology Report and the Neuropathology Peer Review Report

Introduction

What Makes a Report a Good Report?

Structure of the Neuropathology Report

The Neuropathology Peer Review Report

References

Chapter 33: Preparation of Personnel for Performing Neuropathological Assessment

Introduction

Functional Observational Data

Necropsy Personnel Training

Histology Personnel Training

References

Chapter 34: Regulatory Guide to the Histopathological Assessment of Neurotoxicity Studies

Introduction

Resources and Methods for Development of a Regulatory Guide for Neurotoxicity Testing

Regulatory Perspective for Neurotoxicity Studies

Definitions

U.S. Environmental Protection Agency General Neurotoxicology Screening

U.S. Environmental Protection Agency Delayed Neurotoxicity Screening

U.S. Environmental Protection Agency Developmental Neurotoxicity Screening

U.S. Food and Drug Administration General Neurotoxicology Screening

U.S. Food and Drug Administration Developmental Neurotoxicity Screening

Note added in Proof

References

Chapter 35: Toxicological Neuropathology: The Next Two Decades

References

References

Appendix 1 Neural Cell Markers of Potential Utility for Toxicological Neuropathology Applications

Appendix 2 Text-Based Neuroanatomic Atlases for Toxicological Neuropathology Studies

Appendix 3 Text-Based Neurobiology References for Toxicological Neuropathologists

Appendix 4 Web-Based Neurobiology References for Toxicological Neuropathologists

Appendix 5 Toxicological Neuropathology References for Nervous System Targets

Appendix 6 Comparative and Correlative Neurobiological Parameters for the Brain

Appendix 7 Comparison of Relative Proportions in the Brain and Spinal Cord Regions of Humans and Rats

Appendix 8 Chemical Composition of Central Nervous System Tissues and Fluids

References

Index

Color Plates

Copyright © 2011 by John Wiley & Sons, Inc. All rights reserved.

Published by John Wiley & Sons, Inc., Hoboken, New Jersey.

Published simultaneously in Canada.

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Library of Congress Cataloging-in-Publication Data:

Fundamental neuropathology for pathologists and toxicologists: principles and techniques / [edited] by Brad Bolon, Mark T. Butt.

p. ; cm.

Includes bibliographical references.

ISBN 978-0-470-22733-6 (cloth)

1. Neurotoxicology 2. Nervous system–Diseases . I. Bolon, Brad. II. Butt, Mark T.

[DNLM: 1. Neurotoxicity Syndromes–diagnosis. 2. Neurotoxicity Syndromes–physiopathology

3. Models, Animal. 4. Nervous System–drug effects. 5. Neurotoxins–adverse effects. WL

140]

RC347.5F86 2011

616.8071–dc22

2010033335

oBook ISBN: 9780470939956

ePDF ISBN: 9780470939949

ePub ISBN: 9781118002230

Contributors

Ana Alcaraz, DVM, PhD, DACVP, College of Veterinary Medicine, Western University of Health Sciences, Pomona, California

Douglas C. Anthony, MD, PhD, FCAP, University of Missouri School of Medicine, Columbia, Missouri

Brad Bolon, DVM, MS, PhD, DACVP, DABT, FIATP, GEMpath, Inc., Longmont, Colorado

Rogely Waite Boyce, DVM, PhD, WIL Research Laboratories, LLC, Ashland, Ohio

Mark T. Butt, DVM, DACVP, Tox Path Specialists, LLC, Hagerstown, Maryland

Paul W. Czoty, PhD, Department of Physiology and Pharmacology, Center for the Neurobiology of Addiction Treatments, Wake Forest University School of Medicine, Winston-Salem, North Carolina

David C. Dorman, DVM, PhD, DABT, DABVT, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina

Karl-Anton Dorph-Petersen, MD, PhD, Centre for Psychiatric Research, Aarhus University Hospital, Risskov, Risskov, Denmark

Craig Fletcher, DVM, PhD, DACLAM, Division of Laboratory Animal Medicine, Department of Pathology and Laboratory Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina

Andrew Forge, BSc, MSc, PhD, Centre for Auditory Research, UCL Ear Institute, London, United Kingdom

Kathy Gabrielson, DVM, PhD, DACVP, Departments of Molecular and Comparative Pathobiology and Environmental Health Sciences, School of Medicine and Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland

Robert H. Garman, DVM, DACVP, Consultants in Veterinary Pathology, Inc., Murrysville, Pennsylvania

Mary Beth Genter, PhD, DABT, Department of Environmental Health, University of Cincinnati, Cincinnati, Ohio

Tracy Gluckman, MS, DVM, DACLAM, Northwestern University, Chicago, Illinois

Doyle G. Graham, MD, PhD, Duke–NUS Graduate Medical School, Singapore

Hans Jrgen G. Gundersen, PhD, Stereology and Electron Microscopy Research Laboratory and MIND Center, Aarhus University, Aarhus, Denmark

D. Greg Hall, DVM, PhD, DACVP, Lilly Research Laboratories, Indianapolis, Indiana

Elizabeth Head, MA, PhD, Sanders–Brown Center on Aging, Department of Molecular and Biomedical Pharmacology, University of Kentucky, Lexington, Kentucky

John W. Hermanson, BS, MS, PhD, Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York

Monty J. Hyten, HT (ASCP), Covance Laboratories, Inc., Greenfield, Indiana

Karl F. Jensen, PhD, Neurotoxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina

William H. Jordan, DVM, PhD, DACVP, Vet Path Services, Inc., Mason, Ohio

Bernard S. Jortner, VMD, DACVP, Laboratory for Neurotoxicity Studies, Virginia–Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia

Mary Jeanne Kallman, PhD, Investigative Toxicology, Covance Laboratories, Inc., Greenfield, Indiana

Wolfgang Kaufmann, Dr. med. vet., FTA Path, DECVP, Non-clinical Development--Global Toxicology, Merck Serono Research & Development, Merck KGaA, Darmstadt, Germany

Georg J. Krinke, MVDr, DECVP, Pathology Evaluations, Frenkendorf, Switzerland

Stephanie A. Lahousse, PhD, Cellular and Molecular Pathology Branch, National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina

Peter B. Little, DVM, MS, PhD, DACVP, Pathology Associates, Charles River Laboratories, Durham, North Carolina; Professor Emeritus, Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada

Lise Lyck, MSc, PhD, Human Health and Safety, DHI, Hrsholm, Denmark

Thomas J. Montine, MD, PhD, Department of Pathology, University of Washington, Seattle, Washington

Virginia C. Moser, PhD, DABT, Toxicity Assessment Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina

M. Paul Murphy, MA, PhD, Sanders–Brown Center on Aging, Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky

Michael A. Nader, PhD, Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, North Carolina

Dennis O'Brien, DVM, PhD, DACVIM (Neurology), College of Veterinary Medicine, University of Missouri, Columbia, Missouri

Anna Oevermann, Dr. med. vet., DECVP, Neurocenter, Department of Clinical Research and Veterinary Public Health, Vetsuisse Faculty, University of Bern, Bern, Switzerland

Arun R. Pandiri, BVSc & AH, MS, PhD, DACVP, Cellular and Molecular Pathology Branch, National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina

Tucker A. Patterson, PhD, Division of Neurotoxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas

Merle G. Paule, PhD, Division of Neurotoxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas

Kathleen C. Raffaele, MPH, PhD, National Center for Environmental Assessment, Office of Research and Development, U.S. Environmental Protection Agency, Washington, DC

Meg Ramos, DVM, PhD, DACVP, Drug Safety Evaluation, Allergan, Inc., Irvine, California

Christopher M. Reilly, DVM, DACVP, Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California–Davis, Davis, California

Hope Salvo, MS, RAC, SAIC-Frederick, Inc., Frederick, Maryland

Sumit Sarkar, PhD, Division of Neurotoxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas

Larry Schmued, PhD, Division of Neurotoxicology, National Center for Toxicological Research, U.S. Food and Drug Administartion, Jefferson, Arkansas

Robert C. Sills, DVM, PhD, DACVP, Cellular and Molecular Pathology Branch, National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina

Michael H. Stoffel, Prof. Dr. med. vet. habil, Department of Clinical Research and Veterinary Public Health, Vetsuisse Faculty, University of Bern, Bern, Switzerland

Robert C. Switzer III, PhD, NeuroScience Associates, Knoxville, Tennessee

Ruth Taylor, BSc, MSc, PhD, Centre for Auditory Research, UCL Ear Institute, London, United Kingdom

Beth A. Valentine, DVM, PhD, DACVP, Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, Oregon

William Valentine, PhD, DVM, DABT, DABVT, Department of Pathology, Vanderbilt University Medical Center, Nashville, Tennessee

Marc Vandevelde, Prof. Dr. med. vet., DECVN, Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Bern, Bern, Switzerland

Karen M. Vernau, DVM, MSc, DACVIM (Neurology), Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California– Davis, Davis, California

William Vernau, BVSc, BVMS, DVSc, PhD, DACVP, Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California–Davis, Davis, California

Tony L. Yaksh, PhD, Department of Anesthesiology, University of California–San Diego, La Jolla, California

Jamie K. Young, DVM, PhD, DACVP, Department of Pathology, Covance Laboratories, Inc., Greenfield, Indiana

Preface

The goal of this work was to provide, as much as possible, an essentially complete reference on the design and interpretation of studies involving toxicological neuropathology. It is a book not only for pathologists, but also for toxicologists and other scientists involved in the investigation of neurotoxicity. A series of journal articles to achieve this goal was just too fragmented an approach. A textbook was needed, and here it is.

Although there are numerous descriptions and illustrations of tissue changes, this book is not an atlas of lesions. There are other complete references, in print and online, with a plethora of images and diagnostic terms. Although the chapters contain many literature references, this book relates primarily the knowledge of many veterans of the neuropathology discipline with the hope that others can learn from our experience and mistakes. (Note: We did not specifically mention all of our mistakes.)

As with most projects that are either different or of a greater magnitude than someone is used to, the collection, assembly, and editing of this book took longer than predicted. However, the end result achieved both our vision and our goal.

Many years ago, while serving as chairman for a pathology working group for the National Toxicology Program, I (M.T.B.) pulled out my copy of Pathology of the Fischer Rat (Boorman GA, Eustis SL, Elwell MR, Montgomery CA, MacKenzie WF, eds., Academic Press, Inc., San Diego, CA, 1990). Several of the contributing authors were in the group. Each was pleased to see that the binding of my book had long ago broken down because of the number of times I had grabbed it off the shelf for a consult. It is our hope that this book will receive just as much use long before the second edition makes its way into print.

Acknowledgments

We would like to acknowledge Jake Butt and James Reickel, whose lengthy (and frequently late-night) formatting sessions provided so much assistance toward the finished product. We also thank our co-workers, who had to manage their schedules around our need to write, edit, format, and compile all the material that formed this volume.

We thank Amanda Amanullah and Jonathan T. Rose (Wiley) for their patience and support in serving as the publisher's editors of this volume, and Colin Moore (Longmont, Colorado; www.colinmoore.us) for preparing the graphic art in Chapter 4. Although not mentioned by name, we thank the many others who helped with editing, formatting, and illustrations.

Finally, and most important, we gratefully and humbly acknowledge the many contributors who took time from their already too full schedules to provide chapters of this book.

Mark T. ButtBrad Bolon

Color versions of selected figures can be found online atftp://ftp.wiley.com/public/sci_tech_med/fundamental_neuropathology

Introduction

The 90-day study is over, the pathology data have been collected, and there is a problem: a microscopic change in the brain. The incidence is slightly higher in the treated groups than in the controls, or perhaps it is not present in the control groups at all. Still, the lesion does not seem like much of an issue. There were no in-life changes to suggest a problem. Brain weights were within normal limits. Even the cerebrospinal fluid was normal. No instances of seizures or abnormal ambulation were noted during cage-side observations. But was enough information gathered to make an accurate assessment? Does the lesion, which seems inconsequential to the animals, represent something that will affect the more complex lives of humans in a different and profound way? Is it possible that earlier time points need to be looked at to truly characterize the test article as “safe”? Could additional stains or the collection of morphometric data help? Did this study validate previous studies, and if not, are the differences real or due simply to variable interpretation or dissimilar terms—which, in actuality, mean the same thing? If clinical trials are able to begin, is there any way of tracking, assessing, or following this potential change in patients? These questions are what this book is about.

Diseases of the nervous system have a major impact on individuals and society. For many, perhaps for most, of these diseases we are not much closer to an effective treatment or cure than we were 10 years ago. Research moves slowly, but we need to make sure that it moves progressively and as quickly as possible. Lives are literally in the balance. We now know that Huntington's disease is a trinucleotide repeat disorder. Based on the number of repeats, we can even predict the probable progression of the disease. But we have very little to offer in terms of treatment. Using whatever tools necessary, we need to change that.

There is one nervous system. It consists of the brain and the spinal cord and the motor nerves. It is also comprised of sensory and autonomic ganglia, intraepidermal nerve fibers, and neurons that act more like endocrine glands than like traditional bipolar cell bodies. The brain is attached to the eye, to the ears, and to the pelvic ganglia, which all interact. The brain is a group of physically, chemically, and metabolically diverse groups of neurons that may look similar and may be located in generally the same place (inside the skull), but the differences end there. These groups of cells require examination. You cannot think of the brain as a liver or a kidney: one section of a brain (or even three sections) does not allow for adequate examination of the brain. The nervous system is a complex system that requires more than a lifetime to understand. This complexity is one of the central themes of this book.

This volume has been assembled to provide neuropathologists and neurotoxicologists, as well as toxicological pathologists and general toxicologists with an interest in the field, with a single resource that provides the introductory and advanced information needed to develop proficiency in the design, analysis, and interpretation of toxicologic neuropathology experiments.

Part 1 provides information on fundamental neurobiology. Since an understanding of the anatomy of the nervous system is paramount to assessment, Chapters 2 to 4 focus on neuroanatomy. Neuroanatomy is daunting, but once the anatomy is known, you can often predict where a lesion may be, based on the presenting clinical signs. That is the thrust of Chapter 5. Because the morphological evaluation of tissues is only one step toward understanding nervous system function, Chapters 6 and 7 focus on behavioral systems and cognitive assessments, and Chapter 8 deals with the effects of aging on brain structure and function. These are areas in which the neuropathologist and neurotoxicologist require general familiarity as well as specific knowledge on a study-by-study basis. Finally, Chapter 9 provides a framework for understanding the issues relevant to the design of a neurotoxicity study that will capture important morphological endpoints.

Part 2 deals primarily with methodology: how, when, and why. Chapters on practical methods of neurohistology, specialized (rapidly becoming commonplace) markers for neurotoxicity, processing and evaluation of peripheral nerves and muscle, and cerebrospinal fluid are all included, as is an essential chapter on stereology. You won't find everything you need in Part 2 (that's why second editions were invented), but it's a very good start.

Part 3 continues with more methodology, providing chapters on evaluation of the adult nervous system, the developing nervous system, the peripheral nervous system, and the ophthalmic otic and olfactory systems. The techniques described in Part 3 provide an excellent foundation for an evaluation of the nervous system.

Part 4 includes those chapters considered important to a thorough understanding of the issues facing neuropathologists and, by extension, study directors and investigators involved in running neurotoxicity studies. Direct delivery is an increasingly common means of getting various test articles, especially proteins, antibodies, lipophobic drugs, and stem cells to the central and peripheral components of the nervous system. Of those methods, spinal delivery is one of the most common, so a chapter on that delivery system and the drug safety implications is included. Other direct delivery methods, including intracerebroventricular catheters, direct parenchymal infusions, deep brain stimulation devices, and stem cell implants, were not included, due to space limitations. But through the context of intrathecal delivery, Chapter 27 does include many of the issues, real and potential, that are encountered when delivering drugs directly to the central nervous system.

The regulatory aspects of toxicologic neuropathology comprise two of the Part 4 chapters: one describes what is important to include in regulatory submissions (from the viewpoint of regulatory officials), the other provides useful suggestions for navigating the various regulatory guidelines pertinent to the conduct and interpretation of neurotoxicity studies involving pathology. Additionally in Part 4 are chapters on neuropathology in veterinary and medical practice, a chapter on diagnostic neuropathology (primarily of spontaneous diseases), and a guide to training personnel, primarily technical staff, who will be involved in the conduct of studies with neuropathological endpoints. Finally, there is a chapter on the neuropathology report. Since everyone has his or her own style and notions of what comprises a great report, this chapter is likely to be controversial. But even if only useful for stimulating debate, a chapter on reporting was needed.

Finally, Part 4 ends with a chapter on the future of neuropathology. It will be particularly interesting to dust off this first edition in 20 years and see how close the authors were in predicting changes in the dynamic field of toxicological neuropathology.

The morphological examination of the nervous system is the task of the neuropathologist. Coordinating that pathologist with all the other contributing scientists is the task of the researcher and/or study director. That is not an easy job. For all those involved in neurotoxicology in general and toxicologic neuropathology in particular, this book seeks to better inform you, to make your career even more interesting, and to provide you with an increased opportunity for success in gathering and interpreting the data necessary to make good decisions.

We hope that this book will be a boon to both experts and novices in toxicological neuropathology, and that this information will assist all those engaged in protecting the health of humans and animals from the devastating damage that can follow genetic, degenerative, physical, and toxic injury to the nervous system.

This book is dedicated to all those who have endured the joys and miseries of neuropathology:

• the mentors who struggled mightily to instill some neuropathological knowledge in us

• the colleagues who have suffered (and still suffer) our lifelong passion for neuropathology

• the many contributors that lent their time and expertise to this book

• the institutions that continue to invest research dollars to treat and cure the many devastating

diseases of the nervous system

• the professionals who have long awaited a comprehensive toxicologic neuropathology reference

• our families, who have experienced neuropathology “up close and personal” during our months

of isolation and distraction while we composed, edited, and formatted the many chapters, tables, figures, and legends that eventually resulted in this tome

We hope that the effort fulfills all your hopes and fuels your dreams.

Also, we hope that the second edition is at least several years off.

Part 1

Fundamentals of Neurobiology

Chapter 1

Fundamental Neuropathology for Pathologists and Toxicologists: An Introduction

Brad Bolon

GEMpath, Inc., Longmont, Colorado

Doyle G. Graham

Duke–NUS Graduate Medical School, Singapore

The Importance of Neurotoxicological Research

Neurotoxicology is the study of the undesirable consequences that develop in the central nervous system (CNS) or peripheral nervous system (PNS) or both after an organism is exposed to a neurotoxic agent during development or adulthood. Such agents may be exogenous materials such as chemicals contaminating the external habitat (e.g., agrochemicals, pesticides, solvents) or introduced purposely into the internal environment (i.e., drugs); metals; or peptides/proteins (e.g., microbial toxins, biopharmaceuticals). Alternatively, neurotoxic agents may be produced endogenously (e.g., ammonia, unconjugated bilirubin) during the course of certain diseases. Thus, the nervous system is likely to experience constant exposure to a range of neurotoxic agents, although in many instances the level of exposure will be insignificant.

The potential scope of toxicant-induced neuropathology is immense. Each year in the United States, industries manufacture about 85,000 chemicals and register another 2000 to 3000 new compounds.1 Approximately 3 to 5% of chemicals (between 2500 and 5000 entities) are estimated to be neurotoxic to some degree.2 This estimate has serious implications for human, animal, and environmental health, because up to two-thirds of high-production-volume chemicals (those made yearly in quantities exceeding 1 million pounds) have never been tested sufficiently for neurotoxic potential.3 The recognition that neurological dysfunction is a major occupational hazard for adults4, 5 and a common congenital occurrence in children6 has engendered a wide-ranging global effort to identify and eliminate possible sources of neural damage—principally, sources of neurotoxicant exposure.

Neurotoxicity can present as aberrations in neural structure (i.e., toxicological neuropathology) or function (including altered behavior, biochemistry, cognition, or impulse conduction), or both.7–11 All structural changes and any persistent functional deficits associated with xenobiotic exposure are judged to be neurotoxic because such effects cannot be countered by the meager regenerative capabilities of the CNS.12 Reversible functional deficits linked to a recognized neurotoxicological mechanism (e.g., outright neurodegeneration or exaggerated neuropharmacological activity) or that might jeopardize occupational health (for adults) or scholastic performance (especially for children) are also considered to be neurotoxic manifestations. The current “best practice” in conducting risk assessments for potential neurotoxicants is to integrate all available structural and functional evidence in reaching a verdict.9, 13, 14 Nevertheless, the permanence of toxicant-induced structural changes in the CNS typically leads regulators to place more emphasis on morphological data rather than on behavioral or biochemical alterations to determine reference doses for managing neurotoxic risk.15 Therefore, a comprehensive toxicological neuropathology evaluation is and will remain a critical element of the risk assessment process for novel xenobiotics.16

The catastrophic outcome of neurotoxic damage to affected persons, and the strain placed on the resources (money, time) of their immediate caretakers and the societal entities that must often fund chronic health care, has led to the expanded use of neurotoxicity endpoints as major criteria for assessing the risks posed by exposure to xenobiotics.17 This approach is a direct result of two factors. First and foremost, an unfortunate aspect of human history from ancient times through the twentieth century is that the neurotoxic effects of many agents [e.g., ethanol, n-hexane, lead, mercury, polychlorinated biphenyls (PCBs)] have been identified first in humans.18 Second, exposure to potential neurotoxicants remains a common feature of human existence. Slightly less than a third of all high-volume industrial chemicals can elicit neurotoxic syndromes in the workplace.19 Similarly, many drugs [antiepileptics (e.g., valproic acid), antineoplastics (e.g., vincristine)] can induce neurotoxic sequelae as an undesirable side effect.18, 20, 21 Thus, a primary goal of current neurotoxicological research is to prospectively recognize the neurotoxic potential of novel compounds in laboratory animals rather than to discover it retrospectively after epidemics of neurotoxicity in humans.

The Evolution of Toxicological Neuropathology

People have exhibited an interest in fundamental neuroscience for millennia (Table 1).22, 23 Initial neurobiology investigations concentrated on gross anatomical characterization of the CNS and its PNS projections as well as the clinical detection and treatment of diseases affecting the nervous system. Neurohistological evaluations were first undertaken in a piecemeal sense early in the eighteenth century, and more systematic assessments of discrete neural regions were begun in the 1840s. These early studies were organized as descriptive studies of the normal nervous system anatomy. The first neuropathology reports examined neuroanatomical alterations resulting from physical disruption (e.g., Wallerian degeneration in transected axons, first described in 1850) rather than toxicant-mediated neural damage. This emphasis reflected the close alliance between neuropathology and clinical neurology in the European (mainly German) medical schools in which neuropathological research was formalized in the modern era.

Table 1 Selected Historical Landmarks in the Evolution of Toxicological Neuropathology.

Source: Adapted in part from Chudler.22

DateEventca. 1700 b.c.e.First written record about the nervous systemca. 1000 b.c.e.First written treatise describing surgical treatments for some neurological disorders (Al-Zahrawi, also known as Abulcasis or Albucasis)ca. 500 b.c.e.First descriptions of nervous system dissection (cranial and sensory nerves) (Alcmaion of Crotona)ca. 80First description linking lead exposure to neurological disease (Dioscorides)1549Publication of De Cerebri Morbis, an early book devoted to neurological disease (Jason Pratensis)1660–1700First publications dedicated to neuroanatomy: Cerebri Anatome (Thomas Willis, 1664), Neurographia Universalis (Raymond Vieussens, 1684) and The Anatomy of the Brain (Humphrey Ridley, 1695)1684First record of a special preservation technique for neural tissue (boiling oil as a hardening agent, by Raymond Vieussens)1717First description of the nerve fiber in cross section (Anton van Leeuwenhoek)1760Initial demonstration that cerebellar damage affects motor coordination (Arne-Charles Lorry)1766Earliest scientific description of the cerebrospinal fluid (Albrecht von Haller)1810–1825First functional–structural correlates for many CNS regions are defined1836Neuron nucleus and nucleolus first differentiated by microscopy (Gabriel Gustav Valentin)Myelinated and unmyelinated axons are discerned (Robert Remak)1837Cerebellar neurons and their processes first investigated (Jan Purkinje)1838Myelin-forming cells in the peripheral nervous system described (Theodor Schwann)1842Spinal cord anatomy first studied in serial sections (Benedikt Stilling)1844First illustration provided of the six cerebrocortical layers (Robert Remak)1850Initial experimental investigation of axonal degeneration (Augustus Waller)1859The term neuroglia is coined (Rudolph Virchow)1861Functional localization in the cerebral cortex is described (Paul Broca)1865Axons and dendrites are first differentiated (Otto Friedrich Karl Deiters)1873First work on the silver nitrate method to enhance neuronal contrast (Camillo Golgi)1878Regular interruptions in the peripheral nerve myelin are first appreciated (Louis-Antoine Ranvier)1884Granular endoplasmic reticulum is discriminated in neurons (Franz Nissl)1889Nerve cells are proposed to be independent functional elements (Santiago Ramón y Cajal)1891The lumbar puncture (spinal tap) is developed (Heinrich Quinke)Journal of Comparative Neurology is founded1897Formaldehyde is employed as a brain fixative (Ferdinand Blum)1906First description of Alzheimer's disease (Alois Alzheimer)Nobel Prize in Physiology or Medicine awarded to Camillo Golgi and Santiago Ramón y Cajal for their work on neural cytoarchitecture1921Microglia described (Pío del Río-Hortega)1929Correlation between nerve fiber size and function is identified (Joseph Erlanger and Herbert Spencer Gasser)1949National Institute of Mental Health (NIMH) is launched at the U.S. National Institutes of Health (NIH)1950National Institute of Neurological Disorders and Stroke (NINDS) is established at the NIH1959Methylmercury from industrial effluent identified as the cause of a neurotoxicity epidemic in humans and feral cats living in villages lining Minamata Bay in Japan1961International Brain Research Organization (IBRO) is formed as an independent, nongovernmental organization1964Methylnitrosourea (MNU) identified as a relatively selective model neurocarcinogen in rats1968Neurotoxic potential of polychlorinated biphenyls (PCBs) is first recognized in Japan among people who have ingested rice oil that was contaminated during manufacturing1969Society for Neuroscience (SfN) is founded in the United States1973Fetal alcohol syndrome (FAS) is coined as the term for a distinct pattern of craniofacial (including brain), limb, and cardiovascular defects in children born to alcoholic mothers19821-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) shown to be the etiology of Parkinsonism in young illicit drug users who used an improperly synthesized bootleg version of an opioid analgesic1990“Decade of the Brain” is declared in the United States by presidential proclamation

Human interest in toxicology also dates from antiquity.24 The impact of widespread neurotoxicity on the advance of civilization became clear with the rise of industrialization in medieval and Renaissance Europe, when chronic exposure to lead and mercury represented a substantial occupational hazard to members of many professions (alchemists, goldsmiths, hatters, and millworkers, to name a few). Toxicological inquiry progressed in fits and starts during the nineteenth and early twentieth centuries before ultimately evolving into the hypothesis-driven applied science that exists today. The intermittent progress in toxicology stemmed from its expansive approach to experimentation; the field grew from a synthesis of most other basic biological and chemical disciplines, and at its inception the numbers of people with the time and money to excel in such diverse intellectual arenas were few.

Thus, toxicological neuropathology represents the modern-era intersection of three scientific fields: basic neurobiology, applied toxicology, and pathology. The rise of toxicological neuropathology was delayed until the first decades of the twentieth century because it required the advent of both technical advances in neuroanatomical handling and processing techniques (Table 1) and the availability of well-trained scientists versed in the fundamental concepts of all three disciplines. These prerequisites were clearly attained by 1906, as indicated by the publication in that year of a detailed neuropathological description of presenile neurodegeneration by Alois Alzheimer as well as the presentation of the Nobel Prize in Physiology or Medicine to Camillo Golgi and Santiago Ramón y Cajal for their studies of nervous system cytoarchitecture. The subsequent founders of toxicological neuropathology built on these accomplishments by developing significant expertise in morphological pathology, dedicating decades of research to defining the experimental conventions and procedures used in modern toxicological neuropathology investigations, and familiarizing ever greater numbers of colleagues with these conventions and procedures (via their numerous publications and many graduate students).

In this regard, two scientists in particular served as major role models for the growth of toxicological neuropathology during the mid-twentieth century, ultimately influencing several generations of modern toxicological neuropathologists (including the careers of the two authors). One person was John B. Cavanagh, a British physician and professor who devised many of the routine morphological approaches used to evaluate toxicants (especially metals, pesticides, and solvents) associated with occupational neurotoxicity.25–30 The other founder was Adalbert Koestner, a German veterinarian and professor at several U.S. universities whose interests ranged from the morphology and mechanisms of mutagen-induced neural neoplasms to the potential utility and safety of food additives and novel neurotherapeutics.31–34 Modern investigations in toxicological neuropathology have since evolved to incorporate many other innovative neuropathology endpoints in addition to the traditional morphological techniques (Table 2). Nevertheless, the methods pioneered by these two men and others are—and will remain—the foundation of toxicologic neuropathology investigations in the foreseeable future.

Table 2 Morphological Techniques Used in the Modern Practice of Toxicological Neuropathology.

TestType of Neuropathology DataGross evaluationIdentifies overt lesions within neural tissues (via subjective gross examination of surface and internal features)May provide a crude quantitative estimate of large-scale cell loss (via organ weights or linear or areal morphometric measurements)Light microscopyIdentifies region-specific vulnerability and susceptible cell populations [routine stains such as H&E, Fluoro-Jade (for neuronal degeneration), and anti-GFAP (for reactive astrocytes in affected regions)]Characterizes the nature, location, and quantity of macromolecules, and provides insights into neurotoxic mechanisms [special histochemical, immunohistochemical, and molecular methods, especially if employed in conjunction with such specialized microscopy methods as laser capture microdissection (LCM)]Electron microscopyIdentifies subcellular targets of neurotoxicity and provides an indication of the metabolic state of the nervous system (transmission electron microscopy)Addresses the subcellular distribution of xenobiotics (specialized autoradiographic and immunoelectron microscopy techniques, elemental composition analysis)Noninvasive imagingAllows in vivo assessment of neuroanatomic integrity [computed tomography (CT), magnetic resonance imaging (MRI) and microscopy (MRM), ultrasound (US)]Permits in vivo investigation of region-specific neurochemistry and function, offering insights into mechanisms of neurotoxicity [optical imaging, positron emission tomography (PET), single photon-emission computed tomography (SPECT)]

Requirements for Proficiency in Toxicological Neuropathology

More than for any of the other subdisciplines of toxicology or pathology, competent practitioners of toxicological neuropathology must have the appropriate educational and work-related experiences to succeed. Advanced theoretical and practical training in neurobiology and experience in neuropathology will appreciably enhance the pathologist's ability to recognize abnormalities in neural tissues.35 Proficiency as a toxicological neuropathologist requires comprehension at multiple levels of biological organization (e.g., whole animal, cellular, biochemical, and molecular) and the ability to integrate this information with basic medical tenets to formulate differential diagnoses as well as to identify and characterize etiologies and mechanisms of neural disease. Acceptable “entry-level” proficiency in toxicological neuropathology requires that a person have expertise in (1) comparative and correlative aspects of normal neuroanatomy and neurophysiology, (2) causes and mechanisms of major background and neurotoxicant-induced diseases of humans and common laboratory animal species, and (3) principal techniques used for evaluating neuropathological changes (e.g., gross dissection, light and electron microscopy, immunocytochemistry, advanced in situ molecular methods, and morphometry). Therefore, the most direct means of acquiring sufficient expertise in toxicological neuropathology is to complete a clinical degree in either medicine or veterinary medicine and then pursue postgraduate training in either diagnostic pathology (e.g., a residency) or toxicological pathology (such as an advanced research degree or a clinical fellowship) in a program that specializes in nervous system investigations. Fundamental research in the field can also be done by Ph.D. biologists with in-depth training in a relevant discipline (e.g., comparative pathology, neurotoxicology) as long as the focus emphasizes an integrative strategy for nervous system assessment (i.e., investigating questions at the whole animal, organ, cellular, and biochemical/molecular levels, as necessary) rather than a reductionist approach (e.g., limited to cellular or molecular studies).

In current practice, however, general toxicological pathologists and toxicologists must often undertake their own instruction in toxicological neuropathology. Such exposure is usually acquired via self-study or through mentored on-the-job experience, and may be gained in several fashions. The most straightforward way is to study standard references in the field, and in allied biological disciplines (see Appendixes 2, 3, 4, and 5). Indeed, people engaged in toxicological neuropathology on a regular basis will require ready access to many of these references, particularly to neuroanatomy atlases (Appendix 2), in order to undertake meaningful analyses of neurotoxicant-induced lesions. Two other paths are to find Web sites (Appendix 4) or to read classical literature reports related to specific research questions. In the authors' experience, however, the two latter routes are suitable only if one has sufficient prior familiarity with the field for efficient and effective sifting of many possible citations to find those that are most useful. Thus, we recommend that generalists tasked with learning toxicological neuropathology spend the effort, money, and time to understand the relationship between various neural structures and functions (a correlative approach), and to do so across species (a comparative approach).36

Fundamental Principles of Toxicological Neuropathology

The complexity of the nervous system is a key factor in its vulnerability to toxicant insult.37 Moreover, these same structural and functional intricacies render even the simplest assessments quite challenging.13, 38 Success in research in toxicological neuropathology thus requires strict adherence to a few fundamental principles. In the remainder of the chapter we list these basic concepts and suggest some practical steps to implement them in toxicological neuropathology research. These principles and practices are described in much greater detail in later chapters.

Principle 1: Learn the lingo. As with many technical fields, neurotoxicological research has developed a jargon that is typically the unique domain of experts in the field. It goes without saying that a solid knowledge of this nomenclature is a mandatory prerequisite to achieving proficiency as either a toxicological neuropathologist or a neurotoxicologist.

A topic that has caused some confusion is the difference between the naming conventions for neural structures in humans (and nonhuman primates) and other animals. The misunderstanding arises from the dissimilar body orientations of these species. Primates are bipedal, with a nervous system arranged along a vertical (upright) axis, whereas other laboratory animals commonly employed in toxicological neuropathology research are quadrupeds having a horizontal nervous system axis. These divergent body carriages dictate different naming conventions for neural structures in primates and other vertebrates (Table 3). To avoid confusion, publications and reports that describe toxicological neuropathology findings should invoke the correct nomenclature for the species being investigated. A compromise that can be applied when naming neural structures in animals is to include the medical (nomina anatomica) term for the structure in parentheses behind the veterinary (nomina anatomica veterinaria) term. For example, the superior cervical ganglion in humans should be designated in animals as either the cranial cervical ganglion (the recognized term) or the cranial cervical ganglion (superior cervical ganglion). Descriptive anatomical terms should be used rather than eponyms (e.g., mesencephalic aqueduct in preference to aqueduct of Sylvius) when identifying neural structures to promote clarity in communication of neuropathology findings across all species.

Table 3 Species-Specific Directional Nomenclature for Designating Neural Structures.

BipedQuadrupedDirection(Humans, Nonhuman Primates)(Carnivores, Lagomorphs, Rodents)UpSuperiorDorsalDownInferiorVentralFrontCranial (outside the skull)Cranial (outside the skull)Anterior (inside the skull)Rostral (inside the skull)BackPosteriorCaudal

Principle 2: Responses are restricted. Neuropathological lesions resulting from neurotoxicant exposure have been implicated in acute21, 39 and delayed18, 40–43 neurodegeneration, neuronal heterotopia,44 and neural neoplasia.32, 45, 46 The same lesion generally is elicited by many structurally different neurotoxicants, because these agents often act via a common molecular mechanism (e.g., peripheral axonopathy as a consequence of cytoskeletal cross-linking following exposure to n-hexane or carbon disulfide47). Therefore, the pathologist who is able reliably to discern a few basic lesions (Table 4) in neural tissue is reasonably well equipped to participate in toxicological neuropathology assessment.

Table 4 Fundamental Structural Alterations in Neural Tissues from Toxicological Neuropathology Studies.

Cell TypeLesion TypePreferred Method of Neuropathology AnalysisaNeuronCell deathLight microscopy of specially stained sections (Fluoro-Jade, silver impregnation)Cell lossLight microscopy of specially processed sections [IHC for cell type–specific markers (e.g., enzymes or neurotransmitters)]Morphometric measurements of specific regions on tissue sectionsStereological counts of specific cell populationsCell displacement (ectopia)Light microscopy of routinely stained sections (H&E) or sections processed to reveal cell type–specific markersAbnormal neurite conformationLight microscopy of specially stained sections (Fluoro-Jade, silver impregnation)Altered axonal sizeLight microscopy of specially stained sections (IHC for cell type–specific cytoskeletal markers, silver stains)GliaNumerical changesLight microscopy of specially processed sections (IHC for cell type–specific markers)Myelin amount/integrityLight microscopy of specially stained sections (Luxol fast blue, IHC for cell type–specific markers)a. H&E, hematoxylin and eosin; IHC, immunohistochemistry.

Considerable care must be taken when investigating chronic neural diseases, as the damage to the principal target cell population may elicit secondary changes in other parts of the affected cells (e.g., central chromatolysis of the neuron cell body after transection of its axon) and/or in nearby groups of healthy cells (e.g., Schwann cells, which proliferate as a normal response to degeneration and dissolution of their associated axon).48, 49 The extent of the secondary repair processes may substantially exceed the reaction by the primary target cells, especially if the long-standing neural disease has already obliterated the target cells. The complete absence of a defined cell population may be obvious [e.g., selective loss of neurons in specific CA (cornu ammonis domains of the hippocampus)], but more often it is quite subtle and may easily be missed if more complex structures are evaluated by subjective estimates of cell number rather than objective quantification (e.g., reduction in neuronal numbers within the layers of the cerebral cortex). Special immunohistochemical procedures to detect markers specific for reactive astrocytes or activated microglia (Appendix 1) are often needed to detect neuronal lesions reliably, as expression of these glial markers is typically elevated in regions where neuronal degeneration has occurred.

Principle 3: Some sectors are selectively sensitive. Certain neural structures are much more susceptible to injury induced by many etiological agents, including neurotoxicants. Perhaps the most important attribute of toxicological neuropathologists and neurotoxicologists is their knowledge of the basic lesion patterns that can develop following neurotoxicant exposure.

“Hot spots” for neurotoxic damage can arise from many different factors.37 One mechanism of enhanced regional susceptibility is the intricacy of the neural circuitry in a given structure. The more complex interconnections and dense synaptic beds that are characteristic of the cerebral cortex, hippocampus, and cerebellum render these regions quite sensitive to neurotoxic insult, particularly in periods of rapid cell proliferation during development.50, 51 Another factor leading to differential vulnerability of various neuron populations is the markedly high metabolic rate of the brain. This organ consumes disproportionate shares of the total cardiac output and blood-borne oxygen supply (approximately 15% and 20%, respectively) even though the brain mass represents only about 2% of the total body mass.52 This tremendous metabolic rate makes the brain as a whole especially vulnerable to neurotoxicants that disrupt intracellular energy production.53 That said, zonal variations in basal metabolic rate among neuronal populations predispose certain brain regions [especially gray matter (nuclei) of the thalamus, mammillary bodies, periaqueductal and periventricular brain stem, and cerebellar vermis] to toxicant-induced injury, above and beyond the level of vulnerability for the bulk of the brain.25, 26 An important ancillary consideration is that dependence on a high rate of oxidative metabolism rate is a property also shared by the heart. Toxicants that injure the brain often injure the heart, and vice versa, so that in many instances (e.g., cyanide toxicity) it is difficult to distinguish a primary neurotoxic event from neural damage that results from primary cardiac toxicity. A third factor contributing to augmented regional vulnerability is the existence of cell type–specific neurochemical machinery. A biochemical example of such compartmentalization is the selective sensitivity of dopaminergic neurons to toxicants such as 6-hydroxydopamine54 and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP),55 both of which result in selective degeneration of dopaminergic neurons. A related biochemical route leading to higher susceptibility results from variations in chemical composition among cells; the extensive lipid content of neural cell membranes, especially myelin, provides an abundant target for the oxidizing actions of certain neurotoxicants.56 Enhanced regional vulnerability may also reflect disparities in local blood flow and repair mechanisms. The efficiency of most biochemical, metabolic, and reparative processes decreases with age, which can further magnify zonal differences in neural tissue sensitivity to neurotoxicants.

Principle 4: What gets wrecked depends on when it gets whacked. As in other organs, toxicant-induced damage in the nervous system occurs only if the agent reaches a target cell population at a time when those cells are vulnerable. Such critical periods of sensitivity to toxicant exposure have been well documented in the developing nervous system following exposure to many different chemicals. For example, grossly evident malformations of the neuraxis happen in mouse embryos only if exposure occurs during neurulation,57–59 which is the stage at which the cranial neuropore closes to form the brain primordium. As the brain continues to evolve during late gestation, each nucleus has one or two other critical periods for neuron production; a brief toxicant exposure during region-specific neurogenesis can thus decimate a structure engaged in its peak effort at neuronal production while causing minimal or no disruption in nearby quiescent regions.60–62 Critical periods for some neuronal populations and processes extend well after birth,61, 63 including neuronal and glial expansion and migration, axonogenesis, synaptogenesis, and myelin formation over the first several years of postnatal life in human infants.64–66

Principle 5: When assessing acute lesions in neurons, “red and dead” is the real deal. In our experience, the majority of neurotoxicant-induced lesions in neurons are the outcome of primary degeneration. The main evidence for such a process is often the presence of dead and dying neurons, usually in clusters or dispersed throughout a given brain region. These degenerating cells exhibit a characteristic constellation of changes dominated by cytoplasmic hypereosinophilia in conjunction with either pyknosis (condensation and shrinkage) or karyorrhexis (fragmentation) of the nucleus. Such disintegrating neurons are typically termed acidophilic neurons, eosinophilic neurons, or “red dead” neurons (Chapter 13, Figure 2C and D).

This change must be distinguished from dark neuron artifact (Chapter 13, Figure 2A and B). Dark neurons indisputably embody the most common CNS artifact encountered by neuropathologists. Unfortunately, dark neuron artifacts have often mistakenly been judged by inexperienced pathologists, toxicologists, and neuroscientists to be evidence of neurodegeneration, and has been reported as such in the neuroscience and neurotoxicology literature.67 Such reports have misidentified artifacts as neurotoxic injury, with subsequent unnecessary regulatory and public health alarm. Dark neuron artifact is usually observed with larger cells, such as the pyramidal neurons in the cerebral cortex and motor neurons in the spinal cord, and is characterized by darkly stained cytoplasm (especially intense in the apical dendrite) and nucleoplasm and shrunken cell bodies. If dark neurons represent the only visible alteration in sections of neural tissue, it is generally safe to interpret the change as artifactual. The main exception to this rule is for studies designed specifically to detect hyperacute neuron damage, as the earliest evidence of incipient neurodegeneration is transient cytoplasmic basophilia (Chapter 13), but even here later time points can be used to verify the nonartifactual nature of the alteration. Any practicing neuropathologist knows that dark neurons are readily produced by even mild trauma to the tissue before fixation is achieved, possibly as a consequence of localized ischemia, hypoglycemia, and excitatory neurotoxicity.68 The simplest prospective way to avoid misinterpretation of dark neurons is to ensure that the neural tissues are fixed properly before they are handled and processed (Chapter 10). A post hoc means of distinguishing genuine lesions from dark neuron artifact is to process a serial section of each sample to reveal reactive astrocytes or activated microglia using immunohistochemical markers (Appendix 1), either or both of which may collect in areas where true neurodegeneration has transpired.

Principle 6: Make “special” stains part of your routine. The workhorse stain for screening most organs for toxicant-induced lesions is hematoxylin and eosin (H&E). This stain works well in the brain but is not suitable for detecting the entire spectrum of neurotoxic lesions that is ordinarily induced in neural tissue. The most readily recognized lesion in H&E-stained neural sections are neoplasms, but such sizable masses are an infrequent consequence of toxicant exposure. The more common toxicant-induced neural lesions—especially neuronal degeneration, myelin disruption, and glial hypertrophy/hyperplasia—may be recognized on H&E-stained sections, but the low contrast between the affected cells and the adjacent neuropil makes such evaluations relatively laborious and prone to false-negative errors.

The cure for this difficulty is to expand the menu of routine procedures that are used to screen neural tissues for neurotoxic lesions to include certain special stains. For most hypothesis-driven animal studies, the H&E-stained section should automatically be accompanied by serial sections processed to reveal degenerating neurons (e.g., Fluoro-Jade; Chapter 11) and reactive astrocytes [e.g., antiglial fibrillary acidic protein (GFAP); Chapters 10 and 21]. Inclusion of these two additional methods as a matter of course when conducting prospective neurotoxicity studies rather than waiting to request them based on the outcome of the examination using the H&E-stained section will substantially shorten the length of the analytical phase, because these two “special” procedures greatly simplify the neuropathologist's efforts to identify lesions, especially subtle ones. The choice regarding whether or not to include these additional stains in a diagnostic neuropathology setting can be left to the discretion of the pathologist.

Principle 7: Seeing is believing, but don't believe everything you see. Neuropathologists and neurotoxicologists have been educated to possess built-in biases to detect toxicant-induced alterations in cells. However, not all structural changes observed in neural tissues after exposure to potential neurotoxicants during a carefully controlled neurotoxicity study are the result of exposure to that agent.

We have seen several spurious causes of neurological dysfunction in toxicant-treated individuals which had nothing to do with the test agent. One example is spinal cord trauma and paralysis in incompletely restrained rabbits, which can kick so hard when handled that they fracture their vertebral column. A second instance is the incidental occurrence at necropsy of widespread neuronal necrosis in the cerebral cortex, hippocampus, and thalamus of some transgenic mice generated on the FVB genetic background. This spontaneous lesion has been attributed to intermittent seizure activity69 rather than toxicant exposure, as the identical finding is evident in untreated control animals that have the same genetic background; the high susceptibility of FVB mice to chemically induced seizures indicates that great care will be required to confirm that neurodegenerative changes of this nature are truly related to toxicant exposure rather than to background neural overactivity. Finally, we have observed rodents treated with a known neurotoxicant to develop disorientation and ataxia as a sequel to acute bacterial meningitis. The point of these anecdotes is that the toxicological neuropathologist cannot set aside fundamental diagnostic skills when analyzing neural tissues from toxicant-exposed individuals.

Principle 8: Don't limit yourself to the pathology perspective. Although toxicant-induced neuroanatomical changes are often emphasized by regulators in managing neurotoxic risk,15 reliance solely on neuropathological changes to identify neurotoxicants can be misleading. Some well-known neurotoxicants induce profound functional changes in the absence of recognizable structural alterations.9, 10 Some classic instances include chlorinated hydrocarbons (e.g., dieldrin), pyrethroids, and strychnine, all of which incite excessive synaptic excitation but no neuropathology, as well as barbiturates, lithium, and organic solvents (e.g., xylene), which cause neuronal depression in the absence of neuromorphological changes. On the other hand, clinical observation of functional deficits can signal the presence of subtle structural lesions. An example is the ability of early reductions in hindlimb grip strength and later progression to paralysis to indicate the presence of a distal axonopathy.

Furthermore, neurological signs in a toxicant-exposed individual are not necessarily evidence of direct neurotoxicity. Anorexia and associated weight loss in rodents are associated with many behavioral changes, including increased motor activity and escape behaviors, decreased hindlimb grip strength, and cognitive learning deficits.70–72 In like manner, chemically induced injury to some extraneural organs (especially the kidney and liver) can lead to the induction of secondary neurological dysfunction via the accretion of unprocessed neurotoxic waste products. The classic example of this scenario is hepatic encephalopathy, in which severe liver damage permits ammonia accumulation in the blood and brain and ultimately disrupts many CNS metabolic pathways (especially in astrocytes) and glutamatergic excitatory neurotransmission.73, 74 Similarly, renal failure leads to uremic encephalopathy following increased circulating levels of many amino acids and protein metabolites. These examples again underscore the importance of integrating all available structural and functional evidence in reaching a conclusion regarding the risk posed by a potential neurotoxicant.9, 13, 14, 16

Principle 9: Carry on with care. In practice, screening studies for neurotoxicity generally administer high doses of test agent to a small-animal species (typically, rats) over relatively short periods of time, assuming that the data gained in the exercise can be extrapolated from high doses to low and from animals to humans. This approach has worked reasonably well but is obviously not perfect, as a number of neurotoxicants have been detected first by epidemic intoxications in humans.18

Efforts at extrapolation among species are complicated by the large divergence in responsiveness following neurotoxicant exposure. For example, MPTP depletes nigrostriatal dopaminergic cells in humans and nonhuman primates, eliminates nigrostriatal synaptic terminals in mice, but has a minimal impact on comparable structures in the rat.39