Nervous System

C H A P T E R

52 Nervous System Brad Bolon1, Mark T. Butt2, Robert H. Garman3, David C. Dorman4 1

The Ohio State University, Columbus, OH, USA, 2Tox Path Specialists, LLC, Frederick, MD, USA, 3 Consultants in Veterinary Pathology, Inc., Murrysville, PA, USA, 4 North Carolina State University, Raleigh, NC, USA O U T L I N E

1. Introduction 1.1. A Brief Overview of Toxicologic Neuropathology 1.2. The Toxicologic Pathologist in the Practice of Neuropathology 2. Anatomy and Function of the Nervous System 2.1. Fundamental Neurobiology 2.2. Correlative Neurobiology 2.3. Comparative Neurobiology 3. Evaluation of Neurotoxicity 3.1. Functional Assessment 3.2. Biochemical and Biomarker Evaluation 3.3. Toxicokinetics 3.4. Morphologic Evaluation 3.5. Special Techniques for Neurotoxicity Assessment 3.6. Model Systems for Neurotoxicity Research

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1. INTRODUCTION 1.1. A Brief Overview of Toxicologic Neuropathology The nervous system is the primary means by which animals interface with the external world and manage their internal systems. The highly specialized system, typically referred to by its component central and peripheral nervous system (CNS and PNS, respectively) parts, permits refined Haschek and Rousseaux’s Handbook of Toxicologic Pathology, Third Edition. http://dx.doi.org/10.1016/B978-0-12-415759-0.00052-2

4. Response to Neurotoxic Injury 4.1. Anatomic Lesions 4.2. Functional Alterations 4.3. Physiological Abnormalities 4.4. Neurochemical Changes 4.5. Background Findings and Their Implications

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5. Mechanisms of Nervous System Injury 5.1. Aberrant Cell Migration and/or Differentiation 5.2. Altered Intracellular Transport 5.3. Cell Turnover 5.4. Energy Depletion 5.5. Excitotoxicity 5.6. Macromolecular Adducts 5.7. Neurotransmission Disruption 5.8. Oxidative Damage

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6. Summary

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control of physiological responses throughout the body. Many neurotoxicants target one or more neural regions, with effects ranging from nearly undetectable to catastrophic. Identification and avoidance of neurological dysfunction secondary to xenobiotic exposure increasingly is recognized as a critical factor in safeguarding, maintaining, and improving the health of animals and man. A broad range of xenobiotics may induce neurotoxicity in humans and/or animals (Table 52.1).

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TABLE 52.1 Common Neural Targets and Neurotoxicants Site of action

Neurotoxic effect

Selected examples

Altered migration and/or differentiation

Ethanol

Neuron stem cell, neuronal

Methanol Methylazoxymethanol (MAM) Methylmercury mature cell body Cytotoxicity Alkylating agent

Doxorubicin

Energy depletion (disrupted electron transport)

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

Excitotoxicity

Domoic acid (marine algal toxin) Kainic acid (seaweed toxin) Quinolinic acid Trimethyltin

Mitotic inhibitor

Vincristine

Protein conjugation/inactivation

Methylmercury Trimethyltin

Axon

Axonopathy Central (unknown mechanism)

Clioquinol

Proximal (altered intracellular transport)

b,b0 -Iminodipropionitrile

Distal (macromolecular cross-linking)

Acrylamide Carbon disulfide n-Hexane

Synapse

Altered neurotransmission Decreased neurotransmitter release

Botulinum toxin Tetanospasmin

Persistent presence of neurotransmitters

Selective serotonin reuptake inhibitors (SSRI) St John’s wort (Hypericum perforatum)

Reduced neurotransmitter metabolism

Carbamate insecticides Monoamine oxidase inhibitors (MAOI) Organophosphorus insecticides

Termination of transmembrane ion gradients

Pyrethrin insecticides Pyrethroid insecticides Tetrodotoxin

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TABLE 52.1

Common Neural Targets and Neurotoxicantsdcont’d

Site of action

Neurotoxic effect

Selected examples

Neoplasia (mutation by DNA alkylation)

Acrylonitrile

Glia stem cell, glial

N-ethyl-N-nitrosourea (ENU) Ethylene oxide N-methyl-N-nitrosourea (MNU) astrocyte

Swelling (Alzheimer Type II cells)

Ammonia

myelinating cells Abnormal protein production e Schwann cell Isoniazid Abnormal protein production e oligodendrocyte

Lead Triethyltin

Anoxia (insufficient oxygen delivery)

Carbon monoxide

Edema e oligodendrocyte

Cuprizone Hexachlorophene

Membrane disruption

Lead Triethyltin

Metabolic disturbance

Coyotillo (Karwinskia humboldtiana)

Uncoupling oxidative phosphorylation

Bromethalin Hexachlorophene

Endothelium (capillary)

Physical penetration of bloodebrain barrier elements

Arsenic

Protein kinase dysfunction

Lead

Occupationally important neurotoxicants include agrochemicals (see Agricultural Chemicals, Chapter 42), metals (Heavy Metals, Chapter 41), and solvents. However, these same neurotoxicants also are encountered in the home or outdoor environments. Biological neurotoxins are produced by many microbes (see Mycotoxins, Chapter 39), plants (see Selected Poisonous Plants Affecting Animal and Human Health, Chapter 40), and animals such as scorpions, spiders, and vipers. Certain neurotoxicants are therapeutic agents, including many small molecules with cytotoxic (e.g., antineoplastics) or neuroactive (e.g., antidepressants, antiepileptics) effects as well as some biomolecules (e.g., TysabriÒ , a

therapy for multiple sclerosis that has been associated with progressive multifocal leukoencephalopathy; see Biopharmaceuticals, Chapter 25). Similarly, “lifestyle” or “recreational” products (e.g., alcohol, nicotine, and cocaine, to name a few) have very potent neuroactive properties. Neurotoxic agents may induce structural or functional changes affecting the CNS, PNS, or various effector organs (e.g., muscle). Structural manifestations of neurotoxicity present as anatomical changes at macroscopic, microscopic, or ultrastructural levels. Functional expressions of neurotoxicity encompass neurobehavioral, neurochemical, and neurophysiological changes. Under some conditions changes in neural function may

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lead to secondary alterations in neural structure, and neuroanatomical alterations are commonly linked to functional deficits. Certain neurological outcomes induced by chemical exposure are not deemed adverse, including desired responses to neuropharmacological treatments and transient modifications in global regional neurochemistry. In general, however, xenobiotic-induced structural changes or persistent perturbations in behavior, neurochemistry, or neurophysiology are considered to be neurotoxic effects. Reversible effects occurring at doses that could endanger performance in the workplace or that are associated with a known neurotoxicological mechanism of action are also considered adverse. Throughout recorded history, neurotoxicants have generally been discovered through epidemics of occupational or societal exposure to humans. Contemporary examples include fetal alcohol syndrome (the outcome of ethanolinduced cell death during neurogenesis following maternal binge drinking; see Embryo and Fetus, Chapter 62), Minamata disease (induced by consumption of methylmercury-contaminated fish; see Heavy Metals, Chapter 41), and solvent neurotoxicity (typically caused by acute, highlevel exposure to solvent fumes in small, poorly ventilated spaces). The incidence of neurotoxicity is unknown but potentially significant. Estimates suggest that from 3% to 28% of all commercial chemicals may be neurotoxic to some degree. The high number of potential neurotoxicants is alarming given that many chemicals in widespread use today have been incompletely characterized for neurotoxicity. Many factors predispose the nervous system to toxic insults. The most obvious such features are the complexity of the neuroanatomical connections and functional specializations as well as the limited capacity for repair, especially within the CNS. Since regions dedicated to particular tasks are intimately linked at both functional and structural levels, a localized lesion may have significant effects on more distant parts of the nervous system. Furthermore, such neural lesions may have profound effects on peripheral systems since virtually all organs and their physiological processes are directly controlled or indirectly influenced by the nervous system. The remarkable metabolic requirements of the nervous system represent another avenue by which xenobiotics can exert neurotoxic effects.

The CNS, especially the brain, is almost exclusively dependent on aerobic, glucose-dependent metabolic pathways. This high metabolic demand of the brain is sustained by approximately 15% of the total cardiac output and requires 20% of the oxygen capacity for the entire body, even though the brain accounts for only 1.5–2% of the total body weight. Elevated concentrations of polyunsaturated fatty acids and relatively low levels of antioxidant enzymes in neural tissues predispose neural cell membranes (especially myelin) to oxidative damage. The same lipid-rich structures facilitate the absorption and widespread distribution of small molecular weight, lipophilic chemicals throughout the CNS. Specific regions of the nervous system have different sensitivities to neurotoxic agents. This divergence usually results from site-specific variations in biochemistry, metabolism, and vascular supply, especially in the brain. Regenerative capacity following neurotoxic insult is another critical factor. Robust repair in the damaged PNS eventually may restore both structure and function, while limited renovation in the injured CNS even after extended periods results in mobilization of compensatory mechanisms mediated by other, less affected brain domains. Younger individuals appear to have more capacity for both anatomical (e.g., neuronogenesis and synaptic plasticity) and functional (e.g., behavioral compensation) adaptation than do older individuals, suggesting that the adult or aged CNS may be at greater risk from neurotoxic exposure. The major threat to the CNS is that widespread damage or focal injury to a domain for which compensatory mechanisms are minimal or absent will result in permanent deficits.

1.2. The Toxicologic Pathologist in the Practice of Neuropathology For their part in the non-clinical studies (and other investigational endeavors) for novel test articles and devices, toxicologic pathologists are tasked with many critical aspects of hazard identification and safety assessment. Primary roles for toxicologic pathologists are discovering and cataloguing the macroscopic and microscopic structural changes, explaining their relationship to clinical and clinical pathology abnormalities, and determining whether or not the effects

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should be considered adverse. These are arduous tasks. Although it might be optimistic, the goal of this chapter is to supply toxicologic pathologists with the basic and supplemental information needed to assess the structure of the nervous system with confidence, and to identify and interpret the significance of the morphologic changes. This chapter reviews fundamental knowledge regarding the singular anatomic and functional attributes of the nervous system, methods by which the impact of toxicants on the CNS and PNS may be investigated, common responses of damaged neural tissues, and mechanisms of action by which neurotoxicants frequently injure their target cell populations. The reader is referred to other sources for more detailed discussions of these topics (see Suggested Reading list), as the vast scope of material in this field cannot be covered comprehensively in a single chapter.

2. ANATOMY AND FUNCTION OF THE NERVOUS SYSTEM Vertebrates are preferred animal models for neurotoxicological testing because their anatomical and functional characteristics approximate those of the human nervous system. That said, both physiology and structure vary substantially among vertebrate species, thus rendering efforts to extrapolate animal data to humans (i.e., fundamental neurobiology) difficult in many instances. The knowledge base needed for credible hazard identification, medical diagnosis, and risk assessment (i.e., translational science) in neurotoxicology is aided by a thorough understanding of the relationship between structural damage and neural function (correlative neurobiology), as well as the differences that exist among species, strains, ages, or genders (comparative neurobiology).

2.1. Fundamental Neurobiology Neural Cells and Tissues NEURONS

Approximately 130 billion neurons forming 150 trillion synapses are present within the adult human brain. The neurons are the main functional elements of the CNS and PNS.

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Different neuronal populations within the CNS are distinguished by any of several features. Examples include cell shape (e.g., granule cells are round, while pyramidal neuron profiles are triangular); cell size; neurotransmitter complement (e.g., cholinergic vs dopaminergic neurons); and/or location (e.g., cortical vs deep nuclear cells). Function can also be used to classify neurons: motor (efferent) neurons carry information from the CNS to effector organs throughout the body; sensory (afferent) neurons conduct information from other organs/regions to the CNS; and interneurons connect with motor neurons or other interneurons only in their immediate vicinity. Neurons in given neural regions tend to form during the same period, while different regions exhibit unique periods of peak cell production (Figure 52.1). Once formed, neurons are not replaced to any great degree during adulthood, although small numbers of new neurons are generated during adulthood in certain brain regions, such as the dentate gyrus of the hippocampus and the subventricular zone (SVZ) bordering the lateral ventricles. Neurons generate action potentials, which are carried in neuronal processes (neurites) that receive (dendrites) or carry (axons) the resulting electrical signals. Neurons use approximately 10-fold more oxygen than do their glial neighbors, which makes them exquisitely sensitive to any insults that result in tissue hypoxia. Information flows from the processes of one neuron to an adjacent process from another neuron across small intervening gaps termed synapses. Most neural synapses have a presynaptic terminal (i.e., a specialized end of a neuronal process) that holds neurotransmitter-laden vesicles, mitochondria, and other organelles; a postsynaptic terminal that bears membrane-bound receptors for the neurotransmitters held in the presynaptic terminal; and an intervening cleft that separates the presynaptic and postsynaptic endings (Figure 52.2). Some synapses employ “electrical” pulses rather than chemical transmission to propagate the signal from the presynaptic neuron to its neighbor. Enriching experiences reinforce the structural integrity of existing synapses, and new synapses are formed to enhance commonly used circuits. Together, these phenomena are termed synaptic plasticity, and they provide the dynamic brain with a means of regular remodeling to meet

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FIGURE 52.1 Peak production of neurons occurs at distinct developmental stages for different brain regions, with neuronogenesis as a whole extending for a prolonged period during both prenatal and early postnatal life. Some domains experience two peaks, representing the critical periods for forming unique cell classes. Thus, acute/ short-term neurotoxicant exposures tend to produce targeted damage that is limited to those areas where neuronal production was peaking when the agent was delivered to the developing animal. Figure adapted from Rodier (1980) Chronology of neuron development: Animal studies and their clinical implications, Dev. Med. Child Neurol. 22, 525–545, with permission.

the shifting needs imposed by a constantly changing environment. Neurons are highly susceptible to neurotoxicants. Common responses include degeneration leading to cell necrosis, which may result from several mechanisms, and axonal destruction. Neuronal death and/or axonal degeneration in the CNS are permanent lesions. In contrast, axons in the PNS can regenerate if their original course and supporting cells remain to succor them. In some instances, toxicity may reflect a functional blockade of one or more synaptic receptors rather than destruction of the synaptic machinery. GLIA

Glia are critical elements in the normal CNS and PNS. Each CNS neuron is supported by 10–50 glial cells. Many types of glia may be found in the CNS (e.g., astrocytes, microglia, oligodendrocytes) and PNS (Schwann cells, satellite glial cells), each of which serves a distinct set of functions. Recent investigations indicate that the various classes of glia arise from different progenitor cells. Astrocytes sustain neurons and neuronal function in many fashions. During development, these cells often supply the radial glia that guide

neuronal precursors as they migrate to their proper locations. Astrocytes also provide structural and metabolic support to neurons; metabolize neurotransmitters that leak away from activated synapses (Figure 52.2); are involved in signaling; and control ionic gradients and chemical movement within the neuropil (i.e., that portion of the nervous system parenchyma which is composed mostly of cell processes [neuronal and/or glial]). Finally, astrocytes are integral components of the blood–brain barrier (BBB) and blood–nerve barrier (BNB). These “barriers” are selective gateways in the CNS and PNS, respectively, that control movement of xenobiotics, metabolites, cells, and other materials, preventing most substances from leaving the local microvasculature and gaining access to neural cells. Common astrocytic responses to neurotoxicants include swelling (when the astrocytes themselves are targeted) or hypertrophy and hyperplasia (“reactive astrogliosis”) when they multiply in an effort to repair damage in the CNS parenchyma resulting from injury and disease. Myelinating cells are another major functional class of glia. Myelin is produced by oligodendrocytes in the CNS or Schwann cells in the PNS.

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FIGURE 52.2 Neurotransmission is best understood by comprehending how synaptic structure and neurochemistry cooperate to support communication between adjacent neurons. Left (photomicrograph): In ultrastructural preparations, conventional chemical synapses appear as electron-dense plaques on the membranes of the vesicle-laden presynaptic and the organelle-poor postsynaptic terminal. Rat cerebrocortical neurons in primary culture; 4% w/v uranyl acetate followed by 0.4% w/v lead citrate. Right (schematic): The arrival of an action potential at the presynaptic terminal results in exocytosis (fusion) of some vesicles (white ovals) with the plasma membrane facing the synapse, resulting in the release of stored neurotransmitter (NT, red granules) into the synaptic cleft. The NT diffuses across the cleft to reach specific receptors (orange semi-circles) attached to membrane-spanning ion channels (orange rectangle pairs) on the postsynaptic neuron; successful NT docking with the receptor (left pair) results in opening of the ion channel and rapid influx of sodium (Naþ) , leading to membrane depolarization, while failure to bind (right pair) prevents the channel from opening. The signal from the presynaptic terminal is halted by multiple processes, including binding of NT to a presynaptic autoreceptor (blue rectangle) to inhibit further exocytosis, reuptake of NT (green rectangle pair) by the presynaptic terminal for repackaging, and removal of NT by astrocyte transporters (purple rectangles) followed by metabolic degradation and later return (yellow rectangles) of NT building blocks (triangles) to the presynaptic terminal. Source for left: Figure reproduced from Robert et al. (2012), Ultrastructural characterization of rat neurons in primary culture, Neuroscience 200, 248–260, with permission.

Each oligodendroglial cell surrounds several CNS axons, while Schwann cells encompass a single segment of a single PNS axon. Unlike post-mitotic neurons, Schwann cells and the segments of myelin that they produce are regularly lost and replaced. Regardless of its cellular

source, myelin boosts the conduction velocity of impulses within neuronal processes by serving as an insulator to accelerate propagation of the signal along the insulated fibers. This feat is accomplished because myelin prevents ion flow over the insulated portion of an axon, while

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the electrically conductive axoplasm permits the action potential to “jump” instantaneously – a phenomenon termed saltatory conduction – along the entire distance covered by a myelinating cell. The rate of propagation decelerates only when the potential reaches a node of Ranvier (i.e., where the borders of two adjacent myelinating cells abut); impulses must traverse the short nodal spans using the inherently slower process of ionic diffusion across the axonal membrane, which boosts the voltage enough to reach and depolarize the next node. The thickness of the myelin sheaths is greater for large-caliber axons tasked with carrying somatic (motor and sensory) signals at high velocities, and attenuated (often termed “unmyelinated,” though a thin myelin layer is still present) for smalldiameter axons that carry information for autonomic and certain sensory modalities at slower speeds. The usual myelinotoxic responses are degeneration and necrosis (of myelinating cells) or vacuolation due to fluid accumulation between myelin lamellae (so-called intramyelinic edema). Microglia are the resident tissue-specific macrophages of the nervous system. Despite their classification with glia (which evolve from neural precursors), microglia arise peripherally in hematopoietic organs from precursors of the monocytic lineage before migrating to the CNS to undertake immune surveillance functions. Microglia typically react to neurotoxic injury by enlarging (i.e., hypertrophy) and proliferating (i.e., hyperplasia) to enhance their phagocytic capabilities. Meninges The meninges form a tough, three-layered sheet of fibrous tissue that protects the delicate CNS tissues. The principal cell types are fibroblasts and some adipocytes, along with the elements that contribute to the vascular supply (endothelia and smooth muscle). The outer layer, or dura mater, covering the brain is anchored to the skull, and thus limits the shifting of the brain within the cranial vault. In the vertebral canal, the dura is closely applied to the spinal cord rather than attached to the vertebrae. The arachnoid mater is the thin, avascular middle layer, separated from the dura by a potential space. The inner pia mater is a highly vascular membrane that is closely adhered to the underlying

neural tissue. Together, the arachnoid and pia are referred to as the leptomeninges. Although the thick fibrous dura appears grossly to be the most impenetrable meningeal layer, the tight junctions in the arachnoid layer actually provide the most resistance to diffusion between the neuropil and potential toxicants in the blood. The meninges are fairly resistant to toxicants. CHOROID PLEXUS AND EPENDYMAL EPITHELIUM

The choroid plexus is a jumbled cluster of small arteries and thin-walled capillaries with an extensive surface area that protrudes into all four brain ventricles. The primary cell types are low cuboidal to columnar epithelial cells specialized for fluid secretion atop a modest population of stromal cells, and numerous endothelia (Figure 52.3). The choroid plexus generates cerebrospinal fluid (CSF), a low-protein filtrate of blood plasma that turns over several times a day. This fluid cushions the tightly confined brain and spinal cord from mechanical trauma; serves as a conduit for circulating many chemicals, including brain-derived hormones and metabolites; and helps to equilibrate the tissue pH. The CSF is propelled through the brain ventricles, down the central canal of the spinal cord, and ultimately to the subarachnoid space by heart-related pulsations of the choroid plexus coupled with the constant beating of apical cilia on ependymal cells lining the ventricles and central canal (Figure 52.3). The CSF finally returns to the blood via the arachnoid granulations (or villi), which protrude through the dura mater into the venous sinuses; CSF also may flow along the cranial and spinal nerves to reach lymphatic channels, especially those in the vicinity of the cribriform plate. The CSF is in intimate contact with the interstitial fluid (ISF) that bathes neurons and glia in the CNS neuropil. The ISF moves through the neural parenchyma by bulk flow, thus functioning as a channel for the distribution of certain fluids and materials (cells, drugs, hormones). The choroid plexus epithelium is the site of the organic anion transport protein in the brain. This epithelium also expresses certain xenobiotic transport proteins, including multidrug resistance protein 1 (MRP1), transferrin, and the thyroxine (T4) transport protein transthyretin, among others. The choroid plexus is altered by

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FIGURE 52.3 The choroid plexus (left image) in all four brain ventricles consists of tortuous clusters of capillaries, fed by small muscular arteries (A) and thin-walled veins (V) and covered by a low cuboidal, simple epithelium resting atop a thin layer of loose fibrous stroma. The epithelium secretes cerebrospinal fluid (CSF) while sequestering certain toxicants (e.g., heavy metals, insoluble polymers like polyethylene glycol [PEG]) before they can enter the cerebroventricular system. The ciliated ependymal lining (right image) of the brain ventricles and spinal cord central canal function to circulate CSF through the system. Lateral ventricle, adult cynomolgus monkey. Left image: formalin fixation by immersion. Right image: formalin fixation by intravascular perfusion. Both images: paraffin embedding, H&E stain.

exposure to certain toxic agents. Xenobiotic exposure can alter the function of protein transporters. Common pathological responses are vacuolation and, in some instances, degeneration with eventual sloughing of the epithelium. The ependymal cells seem to be resistant to many toxicants, but this may reflect the fact that levels of neurotoxicants do not increase in the CSF to a concentration sufficient to impact the ependyma. Anatomy At the macroscopic level, the nervous system can be separated into anatomically distinct divisions at many different levels. The most obvious

partition is between the CNS and the PNS. The CNS in turn may be parsed into the brain and spinal cord. The brain may be further subdivided into (from rostral to caudal) the olfactory bulbs and tract; forebrain (cerebral cortex, basal nuclei, corpus callosum, and hippocampus); diencephalon (thalamus and hypothalamus); midbrain (substantia nigra, rostral [superior] and caudal [inferior] colliculi, tectum, tegmentum); and hindbrain (cerebellum, pons, medulla oblongata). Each of these defined neuroanatomic structures generally has multiple subdomains, each serving a unique function. The PNS may be segregated anatomically and functionally into somatic (sensorimotor) and autonomic divisions.

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The autonomic elements can be partitioned on a functional basis into distinct parasympathetic (craniosacral) and sympathetic (thoracolumbar) divisions based upon the location of the preganglionic neuron of the particular component of the autonomic system. Brains from various vertebrates employed in neurotoxicity testing (e.g., birds, carnivores, non-human primates, rodents) exhibit significant species differences in size and macroscopic appearance. Brain weight rises rapidly as body

size grows and the phylogenetic level is raised. For instance, the adult human brain weighs approximately 1300–1400 g (representing about 1.5–2% of total body weight) while the adult rat brain weighs only 1.5–2 g (about 0.8% of total body weight). Other striking differences are the surface topography of the brain (smooth on birds and rodents, convoluted in carnivores and primates; Figure 52.4); the prominence of various cortical regions, in terms of both structure and function (Figure 52.4); and the size

FIGURE 52.4 The external landmarks of brains from adult vertebrates employed in neurotoxicity testing exhibit significant species differences. The brains of chickens and rodents (in this case, a Sprague-Dawley rat) are lissencephalic, lacking the gyri and sulci that characterize the brains of carnivores (Beagle dog) and non-human primates (cynomolgus monkey), and also humans. Other prominent differences shown here include the small cerebrum and very large optic tectum (T) of the chicken, pronounced olfactory bulbs (B) of the rodent, and progressive increase in cerebral (C) size and corresponding decrease in cerebellar (Ce) size in phylogenetically higher species.

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and location of certain spinal tracts. The arrangement of sulci and gyri in carnivores and primates varies with both age and species, and within a species it may be asymmetric between the right and left hemispheres. In the spinal cord of mammals, many white matter tracts, including the dorsal funiculus (the main tracts for transmitting most tactile and proprioceptive sensory impulses) and corticospinal pathways (which mediate cortical control of voluntary motor functions), are largest in primates, of intermediate size in carnivores, and smallest in rodents. Within the nervous system, specific structures have distinct functions. In the CNS, the dorsal regions of the spinal cord coordinate incoming (afferent) information, while the ventral cord domains provide outgoing (efferent) impulses. The motor system contains a somatic motor division, which carries efferent impulses to skeletal muscles, and a visceral motor portion, which connects to cardiac and smooth muscles. Similarly, the sensory system includes a somatic sensory division, which gathers signals from the skin and body wall regarding the external environment, and a visceral sensory domain that receives input from viscera regarding internal conditions. Neurons with similar functions are often arranged in stereotypical patterns; in large part, these blueprints are conserved across species. In the mammalian forebrain, the primary somatic centers are found in the dorsolateral cerebral cortex, with the motor area located rostral to the sensory domain (Figure 52.5). The spinal cord shows a similar functional organization, with sensory information localized to dorsal regions while motor functions are coordinated in the intermediolateral (for autonomic activities) and ventral (for somatic tasks) gray matter. The nuclei of the midbrain and brainstem serve as centers for coordinating sensory and motor signals destined for viscera as well as regulating, often at an unconscious level, numerous homeostatic functions. Electrophysiology The primary functional output of the nervous system is the action potential (Figure 52.6). These electrical pulses are produced by individual neurons following the sudden depolarization of the neuronal membrane. In resting neurons, cytoplasmic potassium ion (Kþ) levels are high

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FIGURE 52.5 Each cerebrocortical domain controls a specific body region, establishing a pattern of functional localization (termed “somatotopic”) that is conserved within and to a large extent across species. The primary somatic centers are found in the dorsolateral cerebral cortex, with the motor area located rostral to the sensory sector. Projections zones (solid colors) send axons out of the cortex to control the functions of lower neural centers, while the primary association areas (stippled colors) are located adjacent to the given projection zones for which they serve as integrating centers for incoming signals. Figure adapted from Bolon (2000) Comparative and correlative neuroanatomy for the toxicologic pathologist, Toxicol. Pathol. 28, 6–27, with permission.

inside the neuron but low outside the cell, while the cytoplasmic sodium ion (Naþ) concentrations are low inside the neuron but high outside. With depolarization, the rapid influx of Naþ ions through voltage-dependent Naþ channels produces an all-or-nothing action potential that reverses the electrical charge across the neuronal membrane. This depolarization event causes voltage-dependent Kþ channels to open and Kþ ions to rapidly leave the cell. The Naþ channels soon close, thereby halting the action potential. The energy-dependent protein Naþ/ Kþ-ATPase immediately begins to shuttle Kþ back into the cytoplasm while forcing Naþ back out. A dedicated ATP-dependent Naþ pump also helps to remove Naþ ions from the cytoplasm after an action potential, and to maintain the membrane’s “resting” potential between action potentials. The critical nature of these pumps for neuronal function is highlighted by the requirements of Naþ/Kþ-ATPase, which consumes 40% or more of all brain ATP in maintaining ionic gradients needed to sustain the electrical activity in neural tissues. Certain toxicants can interfere with conduction of the action potential. Some neurotoxic agents

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FIGURE 52.6 Conduction of impulses along neuronal processes is based on cyclic fluctuations of the cell membrane potential based on differential diffusion of sodium (Naþ [green circles]) and potassium (Kþ [red ovals]) through voltage-gated channels along actively maintained cationic gradients. The resting potential of axonal membranes (gray) at rest is –70 mV (stage 1). Arrival of an action potential that exceeds the threshold (dotted line) results in an abrupt local reversal (“spike”) of the membrane potential (stage 2) via opening of the Naþ channel (orange), which is rapidly countered (stage 3) by opening of the Kþ receptor (blue); together, these two stages span about three msec in healthy, myelinated axons. The Kþ efflux leads to a slight, transient hyperpolarization of the membrane (stage 4) that renders it refractory temporarily to the arrival or any new action potential. The profile of the Naþ channels varies from partly closed (primed for rapid opening) in the resting state to fully closed during the overshoot phase.

(e.g., saxitoxin and tetrodotoxin) inhibit Naþ/KþATPase or other enzymes needed to maintain the transmembrane ionic gradients required for initiating and propagating the action potential. Other agents (e.g., pyrethroids) act on some isoforms of voltage-sensitive calcium and chloride channels. These effects may contribute to the neurotoxicity of these molecules. Neurochemistry GENERAL PRINCIPLES OF NEUROTRANSMISSION

Action potentials traveling down an axon ultimately lead to communication between neurons.

Occasionally, such interactions occur via electrical synapses. However, in the usual setting, neurons communicate by releasing one or more chemical mediators (i.e., neurotransmitters). These mediators may interact with adjacent cells separated by a narrow synapse (i.e., classical neurotransmission), or the mediators may need to travel for considerable distances to reach their target cells (i.e., a neuroendocrine loop incorporating bloodborne hormones). A practical knowledge of regional neurochemistry is a desirable prerequisite for scientists who produce or evaluate toxicologic pathology data, as many toxicants produce their effects by altering neurotransmission.

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Classical neurotransmission with synaptic release of one or more neurotransmitters represents the predominant means of chemical communication for most neurons. Neurotransmitters are a diverse group numbering over 100 molecules, including such chemical classes as amines (e.g., dopamine, norepinephrine, serotonin); amino acids (e.g., g-aminobutyric acid [GABA], glutamate); polypeptides (e.g., enkephalins), and gases (e.g., nitric oxide [NO]). Many axon terminals hold more than one neurotransmitter, although each is packaged in its own class of vesicle. Following stimulation by a neighboring neurite (i.e., axon or dendrite), propagation of an action potential down the axon commonly leads to calcium (Ca2þ)-dependent neurotransmitter release at the presynaptic nerve terminal. The terminal harbors numerous prepackaged vesicles, some of which are mobilized to fuse with the presynaptic membrane (Figure 52.2). The discharged transmitters diffuse across the synaptic cleft and bind to specific membrane receptors on the postsynaptic neuron, thereby launching a postsynaptic potential. The postsynaptic neuron integrates all excitatory (depolarizing) and inhibitory (polarizing) postsynaptic potentials arriving at its many synapses over a small period of time to determine the timing and strength of its action potential. Toxicant exposures can induce neurotoxicity by disrupting neurotransmission in several fashions. Some agents may affect the levels of neurotransmitters and/or their receptors that are available at synapses. Other agents may block fusion of the neurotransmitter-laden vesicles with the membrane of the presynaptic terminal, thus preventing transmitter release. Others may block interaction of the transmitters with their receptors on the postsynaptic terminal, thus blocking the initiation of an action potential. Still other xenobiotics prevent removal of the transmitters from the synapse, thus promoting extended activity and exhaustion of the stimulated cells. ACETYLCHOLINE

Acetylcholine (ACh) is synthesized by choline acetyltransferase (ChAT) from acetyl-CoA and choline. This excitatory neurotransmitter functions at many sites in the CNS and PNS. In the vertebrate periphery, ACh participates in

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somatic motor activities via its presence at neuromuscular junctions of skeletal muscles, but cholinergic (ACh-mediated) neurotransmission also occurs at many terminals in the autonomic nervous system (ANS): all preganglionic nerve terminals, all postganglionic parasympathetic nerve terminals, the adrenal medulla (essentially a sympathetic ganglion), and the postganglionic sympathetic nerve terminals associated with sweat glands. The terminals of cholinergic neurons express one of two receptor classes. The muscarinic type (i.e., metabotropic receptors) propagates signals using G proteins and second messengers and is expressed by parasympathetic neurons. In contrast, the nicotinic type (i.e., ionotropic receptors) forms ion channels and is found at neuromuscular junctions, all ANS ganglia, in the adrenal medulla, and in the CNS. ACh is removed from the synaptic cleft by the rapid action of acetylcholinesterase (AChE), which catalyzes the hydrolysis of ACh into acetate and choline. CATECHOLAMINES

The catecholamines dopamine (DA), epinephrine (EPI), and norepinephrine (NE) serve as neurotransmitters in many CNS neurons and in the adrenal medulla. The precursor tyrosine is synthesized in the liver and then transported to catecholamine-secreting neurons, where it is converted by tyrosine hydroxylase (TH) into DA. This molecule is the neurotransmitter in some neurons, while in others DA is further processed by dopamine-b-hydroxylase (DBH) to NE. In like manner, NE may function as the final neurotransmitter, or it may be converted instead by phenylethanolamine N-methyltransferase (PNMT) into EPI. The chromaffin cells of the adrenal medulla produce both NE and EPI. Catecholamine activity in synapses is terminated by several means, including metabolism by catecholamine-O-methyltransferase (COMT) or monoamine oxidase (MAO), diffusion from the synaptic cleft, or reuptake by catecholaminespecific transporters into presynaptic terminals. The catecholamines bind to two different classes of C-protein-coupled transmembrane receptors, termed the a- and b-adrenergic receptors. These molecules usually function as excitatory mediators for critical central reflexes (e.g., respiratory stimulation) and autonomic functions (e.g., NE is the main chemical released by postganglionic

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sympathetic nerves during stressful “fight or flight” situations). Essential roles have also been confirmed for DA in controlling the CNS centers that modulate the somatic motor system and many behaviors (e.g., feelings of alertness, purpose, and reward). SEROTONIN

Serotonin (5-hydroxytryptamine [5-HT]) is another excitatory monoamine neurotransmitter in the CNS. This indoleamine is synthesized by hydroxylation and decarboxylation of L-tryptophan. Its main function involves regulating behaviors, many of which include an emotional component; examples are appetite and feeding, learning, memory, mood, motor activity, sleep, and thermoregulation. The synaptic concentration of 5-HT is controlled directly by its reuptake into the presynaptic terminal. Multiple 5-HT receptors have been identified, some using ion channels (ionotropic) while others employ G proteins (metabotropic). g-AMINOBUTYRIC ACID

GABA is the main inhibitory neurotransmitter in the vertebrate CNS but is also found in the retina. It is synthesized by the action of glutamate decarboxylase on glutamate. GABAergic neurons occur in many brain regions (particularly the basal nuclei, hippocampus, and hypothalamus); in the substantia gelatinosa (“pain center”) of the dorsal (posterior) horn in the spinal cord; and in the retina. Neurons that express GABA receptors typically have short axonal processes and function as interneurons, suggesting that their main role is to inhibit intra-hemispheric communication within the cerebrum. Impaired GABAergic transmission (e.g., by domoic acid exposure) reduces the inhibitory feedback that normally counters many excitatory pathways, which can lead to convulsions and tetany. Two GABA receptors have been described. The GABAA (ionotropic) type is a chloride (Cl) channel. Binding of GABA leads to increased Cl influx into presynaptic neurons, resulting in membrane hyperpolarization and a greater threshold to be overcome before stored neurotransmitters can be released into the synapse. The GABAB (metabotropic) variant is linked to a G protein and acts by increasing

the conveyance of Kþ out of the neuron, which also leads to hyperpolarization. GLYCINE

Glycine is an important postsynaptic inhibitory neurotransmitter in the CNS. Glycinergic neurons exist chiefly as small interneurons in the ventral (anterior) horn of the spinal cord (i.e., Renshaw cells) and the brainstem. They are proposed to function as a rheostat to dampen brainstem and spinal reflexes that involve the somatic motor system. The main glycine receptor is a Clchannel, so its activation will promote membrane hyperpolarization and reduce neuronal responsiveness. Glycine activity in the synapse is quenched by reuptake via specific transporters into presynaptic terminals and perisynaptic glial cells. The glia can release glycine, suggesting that glycine from this source may also serve as a neuromodulator. Some inhibitory synapses can simultaneously release GABA and glycine. During development, glycine may act transiently as an excitatory transmitter to help guide the maturation of CNS neurons. GLUTAMATE

Glutamate is the main excitatory neurotransmitter in the brain. Its major function is memory formation and consolidation. Most brain neurons bear one or more glutamate receptor subtypes, some of which are ionotropic and some of which are metabotropic. The receptors of key importance as neurotoxicant targets are the a-amino3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainate, and N-methyl-D-aspartic acid (NMDA) subtypes of ionotropic receptors. In general, both AMPA and NMDA receptors are present in active synapses, while the AMPA receptors are missing from “silent” synapses (which are thought to be immature). The action of glutamate is ended by its reuptake into the axon terminals and glial cells. Other signaling pathways besides GABA can impact glutamate neurotransmission. For instance, administration of cannabinoids inhibits excitatory postsynaptic currents (EPSCs) in the corpus striatum (a region in which cannabinoid receptors are highly expressed) by reducing the release of glutamate. Interestingly, the metabotropic variant of glutamate receptors exhibits synaptic cooperation with cannabinoid receptors in the CNS, acting via the production of small

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lipid messengers. Together, the two appear to play an important role in regulating the perception of pain. NON-CLASSICAL NEUROTRANSMITTERS

Many other neurotransmitters have been identified in the CNS and PNS. Essential signaling proteins include growth factors such as brainderived neurotrophic factor (BDNF) and nerve growth factor (NGF), as well as peptides like cholecystokinin (CCK), neuropeptide Y (NPY), and vasoactive intestinal peptide (VIP). These mediators typically have critical roles in modulating emotion-influenced behaviors like food consumption, learning, memory, and the stress response. Accordingly, they are widely distributed in the limbic brain and within peripheral autonomic ganglia (including the adrenal medulla). The growth factors and neuropeptides often function as blood-borne hormones rather than as synaptic neurotransmitters. Some gases, such as carbon monoxide (CO) and nitric oxide (NO), also act as mediators at nerve terminals. Given their short half-lives, gaseous messengers cannot be stored in synaptic vesicles but must be synthesized precisely when needed. Gases function by simply diffusing from one neuron into another, where they form permanent covalent linkages with intracellular molecules to modify their function. For example, CO binding to the heme iron of guanylyl cyclase (an enzyme that manufactures second messengers within neurons) results in conformational changes that activate the protein. Another means of inducing neurotoxicity is the interaction of NO, a free radical, with DNA to yield deamination sites (i.e., mutations due to free radical damage). An alternative route is excessive stimulation of poly(ADP ribose) synthetase (PARS) activity in the nucleus, which will deplete energy stores rapidly as the addition of each ADP-ribose unit represents the consumption of five high-energy phosphate bonds and PARS will add from 50 to 100 such units to each DNA-stabilizing protein (and to PARS itself) when activated. Certain cytokines are capable of serving as neuromodulators under some circumstances. For instance, interleukin-1 (IL-1) can act in the brain to enhance the release of monoamines (DA, NE, 5-HT) and influence the secretion of corticotropin-releasing hormone (CRH) from hypothalamic neurons. Similarly, tumor necrosis

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factor-alpha (TNF-a) has been shown to have a vital role in controlling interneural communication, with the signal strength regulating how the neural cells respond to injury. Cytokines can alter neural transmission in the CNS even in conditions where the primary disease is not an inflammatory process.

2.2. Correlative Neurobiology Functional Domains Brain centers coordinate the major activities of the nervous system. Primary tasks directly fulfilled by diverse cerebrocortical and subcortical brain regions include association (“thinking”), motion, and sensation. Brain centers also indirectly manage internal environmental conditions (“homeostasis”) by controlling endocrine organ function. The dorsal (anterior or superior) portions of the brain and spinal cord coordinate incoming impulses at all levels, while the ventral (posterior or inferior) regions control effector actions. Somatic portions of the CNS and PNS specify an individual’s response to external conditions, while visceral domains of the CNS and PNS modulate internal activities. The motor system includes a somatic division, which coordinates voluntary (skeletal) muscles, and a visceral division, which manages involuntary (cardiac and smooth) muscles. The sensory system is similarly partitioned into a somatic portion, which collects signals from the skin and body wall, and a visceral portion that gathers input from internal organs. The associative domains assimilate impulses arising in both subcortical centers and cerebrocortical fields, permitting integration of input from many somatic and visceral pathways. Structural Domains The simplest arrangement of the nervous system recognizes two divisions based on location: the CNS (neural elements inside the skull and vertebral column) and the PNS (nerves and ganglia [neural cell aggregates] dwelling outside the axial skeleton). The nervous system also can be split to a progressively greater extent using the region-specific anatomic margins or biochemical markers (e.g., neuronal populations expressing a given neurotransmitter, or the enzymes needed to synthesize it). In the cerebral cortex, the main functional domains (Figure 52.5) have comparable locations

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across all vertebrates, although the size of these zones varies among species. For example, in the carnivore the primary sensory and motor areas and the olfactory cortex account for 80% of the cerebral cortex, while these same zones in the primate represent less than 20% of the cortical tissue. Another telling interspecies difference in cerebrocortical anatomy is the ratio of paleocortex (which is largely controlled by the limbic system) to that of the neocortex (which participates in associative and cognitive activities). The paleocortex comprises essentially the entire cerebral cortex of the chicken, and large expanses of cortex in rodents. In contrast, the neocortex accounts for a very outsized portion of the primate cerebral cortex. Functional–Structural Correlations In general, particular neurological activities may be linked with distinct structural divisions in the CNS and PNS. Within these regions, neurons serving the same task form collections (designated nuclei in the CNS, ganglia in the PNS) that exhibit specific topographical patterns. Such stereotypical arrangements (termed somatotopic) are usually well conserved across vertebrate species. Distinct functions in the CNS are relegated to particular structures. For instance, in the mammalian cerebrum the primary somatic domains are found in the dorsal (superior) cerebral cortex, with the motor area located rostral (anterior) to the sensory region (Figure 52.5). The main associative brain is localized in the frontal cortex. White matter tracts contacting comparable fields in the two opposite cerebral hemispheres augment this integration. The spinal cord has a corresponding somatotopic organization. Sensory input from both the somatic and visceral sensory divisions enters the dorsal (posterior) gray matter, while signals from the somatic motor domain arise in the ventral (anterior) gray matter. The gray matter in the cerebrum is located superficial to the white matter, while in the spinal cord the white matter surrounds the deeper gray matter. Autonomic (visceral) functions are also relegated to particular sites. Homeostatic tasks are controlled at the subconscious level in the many CNS nuclei of the limbic system (especially the amygdala, hippocampus, and hypothalamus), midbrain, and brainstem. The visceral

sensory and motor centers are located in dispersed CNS nuclei within the midbrain and brainstem. The main autonomic component of the spinal cord is an elaboration of the visceral motor domain in the intermediolateral zone (“lateral horn”) of the thoracic region. Peripheral Nerves Normal nerves appear as narrow white trunks. They contain many parallel axons (neuronal processes) surrounded by Schwann cells (myelinating glia) and cradled in layers of connective tissue. The layer of connective tissue encircling the entire nerve is the epineurium, the connective tissue sheets that encompass bundles (or fascicles) of axons are the perineurium, and the fine connective tissue within the fascicles is the endoneurium. Other cells that can be seen within the endoneurium include endothelial cells of capillaries, fibroblasts, macrophages, and mast cells. The blood–nerve barrier (BNB) is thought to reside in the perineurium due to numerous tight junctions and specialized capillary endothelium at this site. The BNB is more permeable in some PNS structures (e.g., dorsal root ganglia, parasympathetic ganglia, and sensory and motor nerve endings), which may explain the higher vulnerability of these regions to some toxicants. Peripheral nerves have both myelinated and non-myelinated nerve fibers (Figure 52.7). In general, myelinated fibers serve somatic purposes while unmyelinated fibers fulfill visceral functions. Myelinated axons are typically 220 mm in diameter and enveloped in multiple concentric layers of myelin. The axoplasm (axonal cytoplasm) contains many types of organelles, including axoplasmic (i.e., smooth endoplasmic) reticulum, large mitochondria, and multiple cytoskeletal elements (Figure 52.8). The largest cytoskeletal constituents are the microtubules (also called neurotubules, 20 nm in diameter) and neurofilaments (the neuron-specific intermediate filament, 10 nm in diameter). In normal nerves the axonal components are evenly distributed throughout the axoplasm, but in some toxic peripheral neuropathies protein crosslinking promotes accumulation of the neurofilament proteins in the center of the axon. Microfilaments (actin aggregates, 4–6 nm in diameter) are associated with the axolemma (axonal cell membrane, 8 nm thick). Unmyelinated axons are much smaller, usually 0.2–3.0 mm in diameter.

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FIGURE 52.7 Peripheral nerves contain numerous myelinated fibers (evident as pale, large axons encircled by thick rings of dark myelin) mingled with scattered clusters (arrows) of unmyelinated fibers (seen as pale, small axons surrounded by thin myelin layers). Myelinated fibers (typically 2–20 mm in diameter) generally serve somatic functions, while unmyelinated fibers (usually 0.2–3.0 mm in diameter) fulfill visceral functions. C ¼ capillary. Crosssection of sciatic nerve from a normal (i.e., control) adult rat. Processing conditions: perfusion fixation with 4% glutaraldehyde, post-fixation by immersion in 1% osmium tetroxide, embedding in hard plastic resin, sectioning at 1 mm in transverse orientation, staining with toluidine blue. Figure reproduced from Bolon et al. (2008) Current Pathology Techniques Symposium review: Advances and issues in neuropathology, Toxicol. Pathol. 36, 871–889, with permission.

Myelin in the PNS arises from the membranes of Schwann cells. These specialized glia are of two kinds, myelinating and non-myelinating. Myelin forms when a sheet-like extension from a myelinating Schwann cell surrounds an axon many times, after which the Schwann cell cytoplasm becomes attenuated to form compact (layered) myelin (Figure 52.8). Each Schwann cell surrounds one axon, producing myelin for one internode (demarcated on each end by a node of Ranvier that does not possess myelin). The internodal length is proportional to the axonal diameter and remains relatively constant for a given fiber. Unmyelinated axons reside in pockets formed by non-myelinating Schwann cells, with multiple axons (typically 10–12) being cradled by a single Schwann cell. Abundant unmyelinated axons in a Schwann cell may indicate axonal sprouting.

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FIGURE 52.8 Electron micrograph of a normal myelinated axon (A) and the cell body of an adjacent Schwann cell showing the nucleus (N), cytoplasm (C), and many concentric layers of compact myelin (M). The axoplasm (axonal cytoplasm) is filled with evenly spaced cytoskeletal elements, including microtubules (20 nm in diameter) and neurofilaments (the neuronspecific intermediate filament, 10 nm in diameter), and contains a few large mitochondria. The cytoplasm of the Schwann cell contains abundant rough endoplasmic reticulum and many mitochondria. The endoneurium (connective tissue between adjacent nerve fibers) contains arrays of parallel collagen fibrils (all cut in cross-section). Sciatic nerve of an adult control rat. Processing conditions: perfusion fixation with 4% glutaraldehyde, post-fixation by immersion in 1% osmium tetroxide, embedding in hard plastic resin, sectioning at 600 nm in transverse orientation, staining with uranyl acetate followed by lead citrate. Image kindly provided by Dr William Valentine, Vanderbilt University, Nashville, Tennessee, USA.

Spinal Cord In cross-sections, the spinal cord consists of a central, butterfly-shaped zone of gray matter and an outer rim of white matter. The dorsal (posterior) horn neurons receive incoming

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(afferent) sensory signals, while the ventral (anterior) horn neurons distribute outgoing (efferent) motor impulses. The cervical spinal cord contains fiber tracts for both the forelimb and the hind limb and thus has a larger area in cross-section relative to the lumbar cord, which innervates only the hind limbs. The central gray matter contains neurons, axons and dendrites, glia, and capillaries. This region is divided into layers, or lamina, based on cytoarchitectural features; these laminae have distinct functions and projections, which have been described in detail in other references. In general, the cells in most dorsal horn laminae relay from superficial skin receptors to the brain (where sensation occurs) and in some instances to the adjacent spinal cord segments, where incoming data – especially for pain stimuli – are edited before being transmitted. The neurons in the intermediate zone of the thoracic cord serve as interneurons that relay signals widely throughout the CNS. The ventral horn neurons receive impulses from the vestibulospinal and reticulospinal tracts, and often project to other neurons located in adjacent spinal cord segments on both sides of the cord. The large lower motor neurons in this region are clustered

topographically into two main columns: a medial column that supplies the trunk muscles, and a lateral column that reaches the extremities (Figure 52.9). This lateral column divides into several nuclear masses that innervate distinct muscle groups in the extremities. In particular, the superficial neurons support the extensor muscles of each limb, while more deeply located cells regulate the flexors. The white matter contains multiple, bilaterally symmetrical fiber tracts parsed among the dorsal, lateral, and ventral funiculi (Figure 52.10). Tracts are named according to the direction in which impulses are conveyed, with sensory (ascending) tracts beginning with the prefix “spino-” and motor (descending) tracts ending with the suffix “-spinal.” Tracts in the dorsal funiculus do not overlap, while those in other funiculi intermingle so that only approximate locations can be defined. Nerve fibers within tracts assume specific topographic destinations based on the level at which the fibers enter or leave the cord (Figure 52.10). For instance, the dorsal funiculus contains fibers from the ipsilateral hind limb, carried in the fasciculus gracilis (the medial tract), and the ipsilateral forelimb, located in the fasciculus cuneatus (the lateral tract). Such

FIGURE 52.9 The spinal cord gray matter is arranged in a somatotopic manner. Left side: INPUT carried in descending white matter tracts is distributed to neurons in overlapping fields, with ventral neuron groups (lavender lines) handling information needed for posture and equilibrium while dorsal groups (orange lines) mediate locomotion. Right side: Similarly, OUTPUT is organized somatotopically in two planes. Neurons nearest the midline (to the left of the solid line) regulate coarse muscle movements needed for posture and equilibrium while more lateral elements provide fine muscle control needed for motion and intricate manipulations. The deep neurons (green wavy lines) innervate flexors, while the superficial components (blue speckles) regulate extensors in the same body region. Figure reproduced with minor modifications from Bolon (2000) Comparative and correlative neuroanatomy for the toxicologic pathologist, Toxicol. Pathol. 28, 6–27, with permission. III. SYSTEMS TOXICOLOGIC PATHOLOGY

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FIGURE 52.10 A schematic representation of the spinal cord to show the somatotopic organization of the major white matter tracts. Left side: The location of fibers serving specific body regions is shown for major motor (solid fields) and sensory (hatched fields) tracts. Right side: Efferent columns also are compartmentalized, with motor fibers nearest the midline (left of the dotted line) serving coarse movements needed for posture and equilibrium and lateral elements supporting fine activities needed for motion and intricate manipulations. The deep neurons (green wavy lines) innervate flexors, while the superficial components (blue speckles) regulate extensors in the same body region. Figure reproduced with minor modifications from Bolon (2000) Comparative and correlative neuroanatomy for the toxicologic pathologist, Toxicol. Pathol. 28, 6–27, with permission.

a point-to-point representation is characteristic of fiber tracts carrying discriminative sensory information that project, directly or indirectly, to the thalamus and then on to the cerebral cortex. Brainstem This region is typically defined to include the medulla oblongata caudally, the pons, and the mesencephalon (midbrain) rostrally. Its organization in each of these three divisions is generally comparable among vertebrates. Brainstem neurons form collections of nuclei that are arranged in longitudinal columns which represent the forward extensions of the similar functional areas within the spinal cord gray matter. The remaining brainstem parenchyma consists of various white matter tracts and the widely

scattered neurons that form the reticular formation. The lack of readily defined boundaries among various brainstem domains makes microscopic assessment of this region a challenge. Two primary anatomic differences distinguish functionally similar regions of parenchyma in the brainstem and spinal cord. The first trait is the shape and position of the gray matter. In contrast to the “X” conformation that surrounds the central canal of the spinal cord, the brainstem gray matter forms a “V” lining the floor of the fourth ventricle and an “O” around the mesencephalic aqueduct (of Sylvius). As in the spinal cord, brainstem sensory nuclei generally are located dorsal to those of the motor nuclei. Second, the gray matter columns in the brainstem form distinct nuclei, while those of the spinal cord are

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arranged in continuous longitudinal columns. Finally, the brainstem contains an additional gray matter column that is lacking in the spinal cord to accommodate the special visceral efferent (SVE) neurons that innervate striated muscles originating from the pharyngeal arches. The neuronal populations in the brainstem serve three main functions. The first role is to filter and integrate the input from other brain centers. Accordingly, neurons in brainstem nuclei and the reticular formation collect fibers from numerous ascending and descending pathways. The second task, coordination of autonomic activities, is undertaken by cranial nerves V and VII to XII; nuclei supplying these nerves may have two functional components (sensory and motor) or alternatively send fibers to more than one cranial nerve. Finally, many brainstem nuclei have crucial functions in regulating basal homeostasis, such as the centers that uphold involuntary cardiovascular and respiratory reflexes. The midbrain is structurally equivalent in vertebrate species. In mammals, the tectum is the dorsal portion. It is sometimes termed the corpora quadrigemina (“four bodies”), as it consists chiefly of the paired rostral (superior) and caudal (inferior) colliculi; the rostral colliculi filter signals regarding visual patterns, while the caudal colliculi modify auditory patterns. The tectum is readily visible on the dorsal surface of the rodent brain but cannot be discerned without dissection in carnivores and primates due to overgrowth of the cerebral hemispheres (Figure 52.11). The tectum of the chicken exhibits a laminar arrangement of neurons that is structurally similar to that of the mammalian cerebral cortex, indicating that the bulk of the associative activities in avians is performed in the brainstem. The tegmentum is the ventral portion of the vertebrate midbrain. It contains the majority of periaqueductal gray (PAG) matter, which acts to filter afferent and efferent signals from many brain regions before forwarding them to the cerebral cortex. In particular, the tegmentum harbors the motor nuclei of cranial nerves III, IV, and VI as well as two large somatic motor nuclei: the substantia nigra and the red nucleus. These latter two nuclei are major components of the extrapyramidal motor system. Cerebellum The cerebellum may be divided into multiple lobes using the stereotypic pattern of surface

fissures. The midline domain is referred to as the vermis, while the lateral extensions are the hemispheres. The anatomic subdivisions are closely correlated with but do not overlap exactly with specific functions and distinct molecular markers. The cerebellum is relatively large in birds and rodents (Figure 52.4). The cerebellum is readily seen from above in rodents, partially visible in carnivores, but fully hidden under the caudally protruding occipital cortices in primates (Figure 52.11). The prominence of the cerebellar hemispheres is greater in primates (Figure 52.12). From a functional perspective, the cerebellum may be divided into the corpus (or body), which coordinates muscle movements and tone, and the flocculonodular lobe, which controls equilibrium. Specific portions of the corpus are assigned to muscles located in particular body regions. Afferent (sensory) input from peripheral receptors reaches the ipsilateral cerebellum in a comparable somatotopic fashion. The functions of the right cerebellar hemisphere are modulated by the left cerebral cortex. Two classes of afferent fibers innervate the cerebellum. Climbing fibers arise only in the contralateral olive nucleus in the ventrolateral medulla oblongata before subsequently extending to the cerebellar cortex, where they cross the granular cell and Purkinje cell layers to synapse in the molecular layer on the abundant Purkinje cell dendrites (Figure 52.13). Mossy fibers ascend from neurons in the brainstem nuclei to connect with neurons in the granule cell layer. Axons from granule neurons reach the molecular layer to communicate with many neuron populations. Purkinje cells are the sole source of efferent axons from the cerebellar cortex. They supply the deep cerebellar nuclei, which in turn link to the red nucleus and thalamus. The cerebellum is not equipped with primary motor nuclei, nor does it harbor a tract that directly projects to the lower motor neurons of the spinal cord. Accordingly, all cerebellar output must first be filtered through other brain centers before reaching the muscles. Basal Nuclei These nuclear groups participate in movement control in several fashions, including an extrapyramidal motor loop (dealing with learned movements), a cognitive loop (concerned with

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FIGURE 52.11 The prominence of major regions on the external surface of mammalian brains varies considerably in species of neurotoxicological interest. In the rodent (rat), the midbrain (M) is readily visible on the dorsal surface, while the olfactory bulbs (B), olfactory tract (T), and cerebellum (partitioned as H for hemisphere and V for vermis) are relatively large. In the dog (Beagle), the cerebrum partially covers the midbrain, the olfactory bulbs and tract are moderate in size, and the vermis of the cerebellum is serpentine (arrow) rather than straight. In the non-human primate (cynomolgus monkey), the cerebrum is expanded to such a degree that the cerebellum can only be visualized from the ventral surface, the cerebellar hemispheres are much larger than the vermis, and the olfactory tract is quite small.

motor intentions), and a limbic loop (regulating the emotional aspects of motor control). The main components are the corpus striatum (the caudate nucleus along with the two lentiform

nuclei [putamen and globus pallidus]), claustrum, and subthalamic nucleus in the forebrain, and the substantia nigra in the rostral midbrain. Collectively, these nuclei relay signals to the

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FIGURE 52.12 Coronal sections through the cerebellum and pons emphasize the progressive extension of the cerebral cortex and cerebellar hemispheres as the phylogenetic tree is ascended. The expansion of these regions stems from an increase in associative and cognitive abilities (supported by the neocortex) and the rise of prehensile appendages (controlled by the cerebellar hemispheres). Species represented: rodent ¼ adult rat; carnivore ¼ adult Beagle dog; non-human primate ¼ adult cynomolgus monkey. Modified Weil stain. Whole brain sectioning and staining kindly provided by Dr Robert C. Switzer III, NeuroScience Associates, Inc., Knoxville, Tennessee, USA.

rostral portions of the ventral group of thalamic nuclei, which forward basal nuclei input to the motor cortex. The size of these nuclei is relatively larger in chickens and rodents.

signaling, including those involved in cardiovascular tone, ingestion (eating and drinking), parental care, self-preservation, sex and thermoregulation.

Diencephalon This brain division consists of the thalamus, hypothalamus, and epithalamus. The latter region is comprised of the habenular nucleus (a component of the limbic system) and the pineal gland. The thalamus is the core of the diencephalon. Its multiple nuclear groups integrate signals passing between lower brain centers and the cerebral cortex. Distinct nuclear groups within the thalamus have particular functions. Most connections are diffuse, but the ventral nuclear group receives input that is arranged in a somatotopic manner. Tracts connecting the thalamus and cerebral cortex are reciprocal (i.e., information flows in both directions between these two centers). The hypothalamus is located near the junction of all major neural pathways between the cerebral cortex and other brain centers. When directed by the cerebral cortex (often after a preliminary stop in the thalamus), the hypothalamic nuclei marshal the effector responses needed to integrate endocrine, autonomic and somatosensory functions in maintaining homeostasis. Many responses are regulated through hypothalamic

Limbic System This collection of structures comprises the most primordial part of the cerebrum. Diencephalic regions incorporated in this system include the amygdala, fornix, habenular nuclei (in the epithalamus), hypothalamus (especially the mammillary bodies), septum, and anterior thalamic nucleus. Many regions of the cerebral cortex (e.g., cingulate, medial prefrontal, and retrosplenial cortices) as well as nearby domains with distinct neuronal layering (e.g., hippocampus) also participate in this system. These cortical and diencephalic components have extensive, reciprocal cross-connections. The limbic system modulates responses that are important to emotional reactions, memory, social behaviors, and survival. Functional subdivisions may be localized to specific structures or substructures. For example, the hippocampus and parahippocampus promote self-preserving activities like feeding, fighting, and fleeing, while maternal behaviors are mediated by the cingulate gyrus. The limbic system is sometimes called the “visceral brain” because of its major influence on autonomic (especially motor) functions.

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FIGURE 52.13 Many CNS domains contain multiple, morphologically distinct neuronal populations, which in most instances are arranged in stereotypical patterns. In the cerebellum, the hemispheres consist of an outer, relatively acellular molecular layer (M); a single line of large, polygonal Purkinje cells (P); and a densely packed granular cell layer (G) hosting small, round granule cells. The spaces adjacent to the meningeal artery (A) and its radial branches (retraction artifacts following formalin fixation) are bridged by fine eosinophilic spikes representing astrocyte foot processes. The right panel is a higher magnification of a cerebellar folium, similar to that shown in the left panel, to better illustrate cytoarchitectural divergence among different neuronal classes. Young adult cat Processing conditions: formalin fixation by immersion, paraffin embedding H&E.

Cerebral Cortex This brain region may be partitioned at both the macroscopic and microscopic levels using anatomic features. The simplest divisions are defined grossly by sulci and gyri externally (Figures 52.4 and 52.11), or the locations of gray matter (nuclei) and white matter (tracts) internally (Figure 52.14). Similar separations may be made at the microscopic level using region-specific cytoarchitecture, though this often requires special stains to fully display subtle differences among neuronal populations (e.g., soma size, dendritic branching), the degree of myelination, or unique patterns of gene and protein expression. The cerebral cortex consists

of six laminae (Figure 52.15), but the clarity of these layers varies among species and also among various brain regions for a given species. The neurons from all layers in a given region of cerebral cortex are arranged (with their counterparts in the underlying thalamic nuclei) to form functionally integrated vertical columns. The fiber tracts (e.g., corpus callosum, internal capsule) that link various portions of cerebral cortex are organized in somatotopic fashion in all three dimensions both within and between the hemispheres. Two basic neuronal types may be found in the cerebral cortex. Pyramidal cells have large, triangular to polygonal profiles with large

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FIGURE 52.14 Coronal sections through the rostral cerebrum highlight the major expansion of both gray matter and white matter as the phylogenetic tree is ascended. The increases result from greatly enhanced associative and cognitive abilities (supported by the neocortex). The chicken has little if any neocortical tissue, and thus has abundant gray matter (dark blue regions) with minimal white matter (pale blue columns). The rodent (rat) has a well-defined superficial (cerebral cortex) and deep (basal nuclei) gray matter, and thus exhibits prominent white matter tracts. Carnivores (Beagle dog) and non-human primates (cynomolgus monkey) have large gyri to support the higher number of cerebrocortical neurons (especially interneurons with integrative functions) in the gray matter and even greater complement of myelinated nerve fibers (serving to boost the degree of interhemispheric connectivity). Cresyl violet stain. Whole brain sectioning and staining kindly provided by Dr Robert C. Switzer III, NeuroScience Associates, Inc., Knoxville, Tennessee, USA.

central nuclei, single prominent nucleoli, and abundant rough endoplasmic reticulum (Nissl substance) (Figures 52.15, 52.16). Their long axons project outward from the cortex to lower brain centers. In contrast, granule cells have small profiles, round or indented nuclei, and no prominent spines (Figure 52.15). Most of them function as intracortical interneurons to

link corresponding cerebral centers, so they project relatively short distances. Some granule cells in certain domains – especially cognitive centers and various somatic motor and sensory areas – may serve instead as “mirror” neurons, which fire when someone acts and also when one observes another undertaking the same action; the precise neurobiological role that these

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FIGURE 52.15 The cerebral cortex contains six layers of neurons, which are discerned by their differing cytoarchitectural features (as originally described by the German anatomist Korbinian Brodmann) and functions. The schematic diagrams (originally recorded from Nissl-stained sections of adult human brain by the Spanish anatomist and Nobel laureate Santiago Ramon y Cajal) depict vertical laminae in the visual cortex (left) and motor cortex (right), demonstrating that the pattern of layering varies among cerebrocortical regions. The various cortical layers exhibit a stereotypical complement of neuronal types and connections with other cortical and subcortical regions: neurons in laminae I (molecular layer), II (external granular layer), and III (external pyramidal layer) receive interhemispheric corticocortical afferents from the opposite layer III; neurons in lamina IV (internal granule layer) receives input from intra-hemispheric cortical and thalamic sites; neurons in lamina V (internal pyramidal layer) is the main source of projection fibers to other brain and spinal cord domains; and lamina VI (multiform or polymorphic layer) is linked to the thalamus. The photomicrograph from the motor cortex of an adult cat shows the morphologic differences that characterize the neuronal populations of laminae IV (small granular interneurons for integrating) and V (large pyramidal neurons [arrow] for projecting cortical commands). Processing conditions: formalin fixation by immersion, paraffin embedding, H&E stain.

cells play is not yet known. Granule cells are much more numerous than pyramidal cells, especially in species with large neocortices (e.g., primates) that are capable of increased associative processing (“higher thought”). Some small interneurons, but not the pyramidal neurons, may be replaced during adulthood by proliferation of neural stem cells that reside in the forebrain subventricular

zones (near the lateral ventricles) and the subgranular zones of the hippocampus. In general, such postnatal neuronogenesis feeds new neurons to the main olfactory bulb via the rostral migratory stream, but a few new neurons also may be sent to the cerebral cortex. The cerebral cortex also can be divided into regions based on evolutionary (phylogenetic)

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FIGURE 52.16 Features of cellular anatomy often are better highlighted in sections processed with cell typespecific stains rather than H&E (see Figure 52.15 for comparison). For pyramidal neurons, cell bodies are showcased by Bielchowsky’s silver stain (left image: neurons have variably darkened cytoplasm, pale yellow nuclei, and black nucleoli) and cresyl violet (right image: neurons have abundant purple Nissl substance [rough endoplasmic reticulum] in the cytoplasm, pale nuclei, and dark nucleoli). Axonal integrity is readily visible in the Bielchowsky’s stained preparation (thin black lines). Myelin sufficiency is revealed by the use of Luxol fast blue (LFB) as the counterstain for the cresyl violet-stained section (where myelin sheaths are blue). Cerebral cortex from adult control rats. Processing conditions: formalin fixation by perfusion, paraffin embedding.

traits. The archicortex is the most ancient domain. It consists of the olfactory bulbs, olfactory tracts, and olfactory cortex (the piriform lobe of mammals), and typically is of greater importance for daily functions in phylogenetically “lower” species (e.g., non-mammals, rodents). The paleocortex is the next oldest division, and includes many structures within the limbic system (e.g., hippocampus, parahippocampus, and cingulate gyrus). The neocortex is the most recent area to evolve, and consists of the frontal (rostral), parietal (dorsolateral), temporal (ventrolateral), and occipital (caudal) lobes. The paleocortex has a modest degree of associative connections, while the neocortex represents a rich source of interhemispheric linkages. The paleocortex is more prominent in lissencephalic species (those lacking gyri and sulci, such as rodents), while the neocortex is enhanced in species with gyri and sulci, especially primates. Finally, the cerebral cortex can be divided functionally into projection zones and association areas (Figure 52.5). Projection zones such as the somatic motor and somatic sensory regions send axons out of the cortex to control the functions of lower neural centers. Primary association areas are located adjacent to specific projection zones, serving to integrate signals from the contiguous

projection zone with those coming from more distant cortical regions. For example, the association areas adjacent to the primary motor and sensory projection zones repress the sensitivity to incoming afferent impulses while simultaneously decreasing efferent signals from the motor fields. Additional secondary association areas serve as regions of higher association across multiple projection zones within a hemisphere, but they act independently from any single projection zone. Regardless of their particular role as a projection zone or association area, essentially all cortical regions connect to deep subcortical centers, especially the thalamic and basal nuclei. Cortical functions develop in a stereotypical pattern. In mammals, the somatic motor cortex is always immediately rostral to the somatosensory cortex, regardless of whether these regions are separated by a sulcus (as in dogs and nonhuman primates) or the sulcus is missing (as in rodents and lagomorphs). Within a given cortical field, projections from various subcortical centers (reflecting incoming signals from specific body regions) are arranged in a somatotopic manner (Figure 52.5). The cortical regions responsible for forelimb activities develop at an earlier time both anatomically and functionally than do those that serve the hind limb, and those

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regions that monitor the upper extremities mature before those that control the lower extremities. In a similar manner, cortical regions attuned to different functions become fully operational in a reproducible sequence, with the somesthetic cortex reaching maturity in advance of the auditory cortex, which in turn develops more quickly than does the visual cortex. As with gray matter domains, the white matter tracts exhibit the general trend that more rostral structures become functional at an earlier time than do more caudal ones. Circumventricular Organs (CVOs) These six small organs are composed of numerous small capillary loops (Figure 52.17). They reside in the neural parenchyma at specific sites adjacent to the ventricular system. Five are found near the third ventricle (listed from rostral to caudal, and dorsal to ventral): the subfornical organ (SFO), vascular organ of the lamina terminalis (OVLT), pineal gland, subcommissural organ (SCO), and median eminence. In contrast, the area postrema (AP) is associated with the fourth ventricle. Except for the SCO, capillaries in these CVOs have a porous blood–brain barrier (BBB) because their endothelial linings are fenestrated. Nevertheless, these CVOs have a de facto BBB because the nearby ependyma is specialized to have few cilia and many tight junctions. Neurons are present in three CVOs (the AP, OVLT, and SFO). The pineal gland is comprised of pinealocytes, while the SCO consists of specialized ependymal cells. The median eminence is a complex region in which the axon terminals of hormone-producing neurons from the hypothalamus are intermingled with the convoluted capillaries of the hypothalamopituitary portal system. The CVOs function to modulate homeostasis (median eminence, pineal), blood pressure and water balance (AP, CP, OVLT, SFO), food aversions (AP), and rhythmic biological processes (pineal, SCO). Cranial Nerves The 12 pairs of cranial nerves (CN) are continuous with the brain. They are numbered from rostral (anterior) to caudal (posterior), according to the position of their attachments to the brain. Cranial nerves I (olfactory nerve) and II (optic nerve) are actually extensions of the brain, and as such are the only cranial nerves that are

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centrally myelinated. The remaining cranial nerves originate from the midbrain, pons, or medulla oblongata. Some cranial nerves are purely afferent (sensory), others are entirely efferent (motor), and some are mixed (both sensory and motor). Neurovascular System Blood to the brain is supplied by a series of paired radiating arteries that extend dorsally from an arterial ring located on the ventral surface of the diencephalon and rostral midbrain (the circle of Willis in humans). Three arterial pairs feed the cerebral cortices and underlying structures, while two pairs serve the cerebellum. These large vessels rest on the brain surface, while small branches enter and branch within the parenchyma. In general, the internal carotid artery supplies the rostral (anterior) and middle cerebral arteries, while the basilar artery (which generally is derived from the paired vertebral arteries) gives rise to the caudal (posterior) cerebral artery and both sets of cerebellar arteries. The blood–brain barrier (BBB) and blood– nerve barrier (BNB) serve to selectively isolate most areas of the CNS and PNS, respectively, from blood-borne xenobiotics. The structural basis of these two barriers is comprised chiefly of specialized capillary endothelial cells in which permeability is decreased by the presence of complex tight junctions, lack of fenestrae, and low endocytic activity. In the CNS, the external surfaces of these capillaries are also enveloped in astrocytic processes that appear to participate directly in the regulation, maintenance, and repair of the BBB. Materials may cross the barriers to enter neural tissues if they are small (e.g., oxygen) or can bind specific endothelial cell transport proteins (e.g., amino acids, glucose). Some BBB membrane components (e.g., P-glycoproteins) help to exclude certain xenobiotics. In general, lipophilic agents can readily penetrate the BBB, while hydrophilic agents cannot. Injurious stimuli like ischemia or toxicant exposure may cause the BBB to “open,” thereby allowing plasma constituents or even blood cells to leave vascular lumens and enter the extracellular space (i.e., vasogenic edema, hemorrhage). The extravasated fluid and protein tend to produce greater expansion in the white matter tracts, which are more loosely constructed and thus

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FIGURE 52.17 Circumventricular organs (CVOs) in the adult rodent brain (specifically rat). These six regions are the organum vasculosum of the lamina terminalis (A, outlined by arrows), subfornical organ (B), median eminence (C, bracketed by arrows), subcommissural organ (D), pineal gland (E), and area postrema (F). Neurons occur only in the CVOs in A, B, and F. The subfornical organ (B) is sometimes mistaken for a lesion (e.g., subependymal granuloma). The posterior commissure (bracketed by arrows in D) appears to be hypomyelinated because the image is from the brain of a weanling at postnatal day 21 (i.e., before myelination is complete). Processing conditions: formalin fixation by perfusion, paraffin embedding, H&E staining. Figure reproduced with minor changes from Garman (2011) Histology of the central nervous system, Toxicol. Pathol. 39, 22–35, with permission.

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have greater compliance than does gray matter. In addition, astrocyte processes contributing to the BBB also may swell, and inadequate functioning of the sodium and potassium pump in the astrocytic cell membrane may lead to intracellular (“cytotoxic”) edema. The mechanisms promoting this edema response are incompletely understood, but local release of mediators (e.g., pro-inflammatory cytokines) appears to play a significant role. Choroid Plexus and Ependyma Small tufts of choroid plexus occur in all brain ventricles as balls of tortuous capillaries enveloped in a thin, loose connective tissue matrix and an outer layer of low cuboidal epithelium (Figure 52.3). While representing only about 1% of the total brain weight, the folded surface of the four choroid plexii accounts for approximately 50% of the BBB surface area in the brain. These structures produce cerebrospinal fluid (CSF) by passive filtration and active secretion. These primary tasks are accompanied by a secondary role as a depot for concentrating certain materials (including many toxicants, especially heavy metals like cadmium, lead, manganese, and mercury) to keep them from entering the CSF and/or accumulating in the brain parenchyma. Ependymal cells line the four ventricles and mesencephalic aqueduct in the brain as well

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as the spinal cord central canal (Figure 52.3). These low columnar cells are specialized to prevent leakage of CSF into the periventricular neuropil. In addition, apical cilia serve to circulate the CSF from its points of formation throughout other portions of the ventricular system. Myelination Production of myelin to surround axons does not occur at the same time in all parts of the nervous system, but instead develops progressively (Figure 52.18). In the cerebrum, sizeable white matter tracts and commissures are the first to be insulated, followed in sequence by the major subcortical centers (e.g., thalamic and basal nuclei) and finally the cerebral cortex. Myelination in the cortex occurs first in projection zones before spreading later to the association areas. In the spinal cord, cervical tracts are myelinated prior to those in the lumbar region. Fiber tracts that carry similar information tend to myelinate at the same stage of development. The myelination process is not fully accomplished for some weeks after birth in rodents, and for years in human infants. If viewed at high resolution by light microscopy (i.e., in resin-embedded sections in the range of 1 mm in thickness) or transmission electron microscopy, myelin sheaths in the central

FIGURE 52.18 Schematic representation of the human cerebrum demonstrating the progressive zonal maturation of myelin during early brain development. Myelin forms earliest in projection fields for major somatic (motor and sensory) functions and then spreads sequentially to the association areas. Comparable patterns exist in other mammals. Figure reproduced with minor modifications from Bolon (2000) Comparative and correlative neuroanatomy for the toxicologic pathologist, Toxicol. Pathol. 28, 6–27, with permission.

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nervous system appear relatively thin (with respect to the surrounded axon) when compared to myelin sheaths in the peripheral nervous system. This anatomic variation is of importance when assessing normal myelination and myelin alterations (e.g., demyelination, remyelination) in the central nervous system. Myelin is particularly sensitive to neurotoxicants during two stages of development. The first is a period of rapid glial cell proliferation (i.e., the first two postnatal weeks in rodents). The second is the time of active myelination, the peak of which occurs on approximately postnatal day 20 in rodents. Therefore, the location and extent of myelinotoxic insults will vary with the timing of the exposure.

2.3. Comparative Neurobiology Vertebrate species share considerable similarities in neural anatomy, chemistry, and function. Indeed, for a particular species and strain, these features – when adjusted for variations due to age, gender, or size – are essentially identical among all individuals. This high degree of concordance is reflected in the utility of stereotaxic atlases as means of providing accurate, standardized coordinates to define the usual positions of brain structures (see Suggested Reading list for a selection of such volumes). However, the precise margins are often shifted to a minor degree due to subtle discrepancies in the location and/or size of a given neuroanatomic structure even in age-, sex-, and weightmatched individuals. The primary mammalian species of neurotoxicological importance may be segregated into three classes using reproducible structural criteria. These classes are Rodent (including true rodents like mice and rats but also rabbits [which are lagomorphs]), Carnivore (cat and dog), and Primate (monkeys and humans). The Primate category can be further subdivided into modestly fissured platyrrhine (e.g., capuchin [Cebus sp.], marmoset [Callithrix jacchus], squirrel monkey [Saimiri sciureus]) and more highly fissured catarrhine (e.g., baboon [Papio sp], cynomolgus [Macaca fascularis], rhesus [Macaca mulatta]), (chimpanzee [Pan troglodytes], human [Homo sapiens]) groups.

Structural Divergence among Species Region-specific anatomic variations relative to the pattern defined for a generic vertebrate nervous system are common across all species. These adjustments are generally more sizeable in the CNS as the structure of the PNS (other than nerve caliber and length) is equivalent among species used for neurotoxicity testing. In the spinal cord, two primary structural features are greatly divergent among species. First, the amount of white matter visible in a typical cross-section rises more quickly than does the quantity of gray matter as the size of the animal increases. This appearance reflects the larger surface area and the enhanced axonal supply needed to adequately innervate it. Because the increase is primarily in sensory fibers needed to innervate the digits, the extent of white matter amplification is more prominent in the proprioceptive and tactile tracts of the dorsal funiculus, particularly in Carnivores and Primates. The second major structural trait in the spinal cord is the arrangement and size of the principal spinal motor tracts that control voluntary motor activities. The two main mammalian pathways are the corticospinal tracts (which transmit cortical signals) and the rubrospinal tract (which supplies subcortical signals). The primary corticospinal tract in Carnivores and Primates is in the lateral funiculus, while the functionally identical conduit in Rodents is found in the dorsal funiculus. The proportion of total white matter filled by the two corticospinal tracts expands progressively as one ascends the phylogenetic scale, accounting in Carnivores for about 10% and in Primates for between 20% (in non-human species) to 30% (in humans) of fibers. The rubrospinal tract (connecting the red nucleus to spinal cord motor neurons) is much smaller in Primates than in Rodents and Carnivores. In the brainstem, several structures exhibit species-dependent anatomic attributes. The most visible is the enlargement of the lateral portion of the inferior olivary nucleus in Carnivores, and even more so in Primates. This phenomenon results from more connections between this cell group and the greatly expanded cerebellar hemispheres in these species. The ascending (i.e., sensory) white matter tracts, such as the medial lemniscus and the spinothalamic tract, that traverse the brainstem are also

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much bigger in Carnivores and Primates due to the increased number of fibers required to form fine somatotopic connections to subcortical sensory centers in these species. With respect to the cerebellum, an animal’s movements and posture are well correlated with the relative size and shape of this structure. The main organizing principle for the mammalian cerebellum is that species like Primates with highly developed limbs capable of substantial independent movement have much larger cerebellar hemispheres (Figures 52.11, 52.12). The cranial lobe, which processes sensory data from the limbs, is expanded laterally to a higher degree in Primates. However, certain regions of this lobe (e.g., lingula and parafloccular lobule) are more developed in animals with long tails, especially if they are prehensile. The parafloccular lobule in particular is fairly large in Rodents, Carnivores, and monkeys but is reduced to insignificance in humans. The middle cerebellar lobe is small in both Rodents and Carnivores, but the same structure (especially the lateral portions) is much larger in Primates. The vermis on the caudal cerebellar surface is long and tortuous in Carnivores but fairly straight in Rodents and Primates (Figure 52.11). The Primate cerebellum is equipped with larger connections to the cortex and more discrete cerebellar nuclei relative to those of Rodents and Carnivores. The corpus striatum (i.e., a major component of the basal nuclei) appears as a single body in Rodents but is separated in Carnivores and Primates into discrete caudate and lentiform (i.e., putamen plus globus pallidus) nuclei by passage of the internal capsule. The higher degree of regional specialization in primates dictates that subcortical centers in these species are much more dependent on maintaining their functional connections with other brain regions. The corollary is that severing these links (e.g., by toxicants, trauma, or vascular accidents) will greatly, and often permanently, disrupt neurological function in Primates but may cause minimal and/or transient clinical effects in Rodents. The same trend toward better retention of function by less specialized domains may be observed in some human patients. In particular, unilateral lesions in the human motor cortex will be followed by more rapid and complete improvement if the injury is localized to areas that control leg function rather than arm

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function. This outcome is hypothesized to reflect the ability of reflexes controlled by spinal cord motor neurons to promote effective (albeit abnormal) leg movements in the presence of reduced cortical interactions. In the cerebral cortex, the surface area is not proportional to brain size even though the brain weight of vertebrates is closely correlated to the total body weight. Instead, the cortical surface area increases as a square function. The quantity of gray matter also builds as a square function, while the amount of white matter expands as a cubic function. Therefore, the more convoluted brain surfaces of large-brained animals (especially Primates) also have relatively more white matter (Figure 52.14). The sizes of the cerebral hemispheres are slightly unequal for almost all individuals of most species, although the relationship, if any, to hemisphere-specific functions (e.g., left-sided speech centers in humans) is unclear. Some structural differences are visible in the cerebral cortex even though the basic features are similar across vertebrate species. The most obvious example is the variable appearance of the boundary landmarks between major brain regions (Figure 52.4 and Figure 52.11). The Rodent pattern is that the midbrain is evident dorsally between the occipital cortex and the cranial lobe of the cerebellum, while the midbrain is covered by the cerebral cortex in Carnivores and Primates. The olfactory brain (i.e., olfactory bulbs with associated tract and cortex) of Rodents and (to a lesser extent) Carnivores is quite pronounced relative to the comparable Primate structures (Figure 52.11), which is understandable given the minimal utility of the olfactory sense in primates. The surface area of the frontal cortex in the dog and human is similar, with both extending from the rostral margin of the brain to about midway through the parietal region. However, in proportion to the dog, the human has a larger parietal cortex and smaller occipital and temporal cortices because much more cerebral mass is allocated to controlling fine motor and sensory tasks (parietal-based) associated with the digits than to acuity in discriminating input to either the auditory (temporal-based) or visual (occipital-based) domains. Another gross divergence in neuroanatomy among species is the extent of folding in the cerebral cortex and, to a lesser degree, the

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cerebellum. Each species bears a specific pattern of cerebrocortical sulci and gyri, which can differ in each hemisphere. For instance, neither gyri nor sulci are evident in Rodents (lissencephaly) and embryos of larger mammalian species, but both sulci and gyri are prominent and plentiful in Carnivores and especially in Primates during the late fetal through adult stages of development (Figures 52.4, 52.11, and 52.14). The greater cerebrocortical folding in the surface tissues of the Primate brain results almost wholly from organ enlargement rather than the addition of evolutionarily new circuits and functions. Even so, Primate brains vary substantially in the degree of fissuration, as is readily apparent by comparing the platyrrhine (modestly fissured; e.g., marmoset, squirrel monkey) and catarrhine (more highly fissured; e.g., baboon, cynomolgus, rhesus, chimpanzee, human) patterns of surface indentations. Of relevance to neurotoxicity testing, New World monkeys (e.g., marmoset, squirrel monkey) typically have smaller brains equipped with fewer and shallower sulci than do either Old World monkeys (e.g., baboon, cynomolgus, and rhesus) or humans. Cerebrocortical regions with similar cytoarchitectural characteristics are generally accepted to serve equivalent functions. Rodents, Carnivores, and Primates follow the trend that afferent pathways relaying a particular class of information (e.g., visual input) will produce cortical neurons with similar cytoarchitectonic features even though the stereotactic coordinates at which the neurons are found within the cortex are different. Therefore, functional areas in the cerebral cortex are more constrained by their connectivity to various subcortical centers than to any given gross structural landmarks. The main functional regions of cerebral cortex occupy similar positions among all vertebrate species, but their extent diverges broadly (Figures 52.4 and 52.11). For instance, the combined surface area inhabited by the olfactory, motor, and sensory cortices is approximately 80% in Carnivores but less than 20% in Primates. In contrast, this ratio is reversed for the associative areas adjacent to neocortical projection zones in these two species, while little cerebral cortex in Rodents (and rabbits) is dedicated to neocortical or associative area functions. This divergence appears to reflect the greatly heightened importance of interneuron connections in Primates,

especially for the primary somatic motor and sensory domains. These same somatic functions reside at a more rostral position in Rodents and Carnivores (specifically cats), which reduces the amount of rostral cortex dedicated to the frontal “lobe” in these two species. The foremost functional difference among mammalian cerebral cortices is the ratio of paleocortex (which largely serves limbic functions) to neocortex (which engages chiefly in associative and cognitive tasks). The cortex of Rodents contains large expanses of paleocortex, while the prominence of the neocortex is expanded progressively in species with higher phylogenetic standings. Structural and Functional Divergence among Strains Regional neuroanatomy generally exhibits more modest variations among different Primate and Carnivore species. Clear-cut examples include differences in brain size and the extent of cerebrocortical folding among platyrrhine (modestly fissured) and catarrhine (more highly fissured) species of Primates. In contrast, strain-related disparities in Rodents are numerous. Principal instances include discrepancies in behavior (especially in motor-based tasks and stereotyped actions), neurochemical composition (hormone production, regional neurotransmitter levels), and sensitivity to xenobiotics. The impact of Rodent strain on such differences changes with the endpoint being assessed. Investigators engaged in designing neurotoxicity tests would be well advised to check the literature and, for unusual endpoints or new techniques, to conduct appropriate pilot experiments to verify that the procedures are effective, the personnel adequately trained, and the choice of species and strain are suitable to undertake the proposed study. Structural and Functional Divergence between Sexes Many anatomic and functional attributes of the nervous system can be impacted by the subject’s sex. Structural differences include the size of various brain regions (e.g., males have a larger amygdala and hypothalamus – especially the sexually dimorphic nucleus – in many species, including humans); cell density of selected cell populations (e.g., greater oligodendrocyte numbers in the corpus callosum and spinal cord

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white matter in male rodents); and the size of white matter tracts (larger in males). Common sex-linked neurochemical variations in mammalian species relate to region-specific production of neurotransmitters and hormones, many of which show diurnal fluctuations. Such differences have been associated with the quantities and cycling of gonad-derived sex hormones. Structural and Functional Divergence with Age Neural anatomy, chemistry, and function all undergo extensive changes over relatively short periods of time during gestation as well as the first few postnatal days (rodents and rabbits), months (carnivores), or years (primates). Mammalian species all undergo a neural “growth spurt” in which myriad neurons and glia are generated and white matter tracts are myelinated. The timing of this growth peak varies, occurring before birth in such precocious species as guinea pigs, near birth for Carnivores and Primates, and shortly after birth for Rodents. The blood–brain barrier (BBB) is incompletely formed in developing animals, while levels of neurotransmitters as well as their receptors and synthetic enzymes gradually rise over time. Developing animals often have inactive or minimally active detoxification pathways, thereby enhancing their susceptibility to xenobiotic-induced neurotoxicity. Senescent animals also exhibit age-related changes in brain anatomy, chemistry, and function. Obvious examples of relevance to toxicologic neuropathology studies include the progressive cerebrocortical atrophy (a consequence of neuronal loss) in long-lived species, and the more porous nature of the BBB in aged animals relative to that of mature but not senescent adults.

3. EVALUATION OF NEUROTOXICITY Success in neurotoxicity studies generally depends on careful experimental design. One critical aspect is that such studies must be built with a clear question in mind. In most cases, this requirement means that experimental subjects should be as nearly identical as possible except for a single variable, such as a manipulation (e.g., genotype or treatment) or physiological state (e.g., age, genetic background, sex).

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Neuropathology examination is an established “gold standard” for neurotoxicity testing as observed structural damage is adverse by definition. However, this diagnostic approach is slow and invasive. Therefore, recent neurotoxicological research has focused on finding more rapid, less invasive, and less laborious means for identifying neurotoxic risks (Table 52.2), with the choice of methods being dictated by the kinds of neurotoxic effects that the investigators hope to observe (Table 52.3). Some modalities are performed during life, such as neurological evaluations and behavioral testing. Others are typically confined to the post-mortem setting, such as macroscopic and microscopic assessment. A few can be used during life as well as after death. The current section provides a brief overview of the principal means of evaluating the nervous system for toxicant-induced effects. However, the toxicologic pathology focus of this Handbook dictates that the majority of this portion will review pathology techniques.

3.1. Functional Assessment In all cases, the toxicologic neuropathologists and neurotoxicologists should consider that functional assessments often lack specificity for the nervous system. Systemic disease with secondary disruption of the nervous system (e.g., hepatic encephalopathy) may incite profound functional changes in the absence of primary neurotoxic effects. Accordingly, the results of functional tests must be interpreted within the context of data from other neurotoxicity screening methods (e.g., neuropathology) as well as information regarding the health of other systems. Therefore, neurotoxicity testing remains an exercise in systems biology (i.e., a “whole animal” approach to evaluation, rather than emphasis only on the nervous system proper). Neurological (Clinical) Evaluation Clinical examination of neural function during life often reveals signs that can be used to localize lesions to particular neuroanatomic structures with reasonable confidence. Neurological examinations are performed on individuals and are devised to probe the integrity of all major neural functions. Distinct clinical syndromes have been defined for six large brain regions (cerebrum, diencephalon, mesencephalon, brainstem,

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Flow Chart for Toxicologic Neuropathology Endpoints in Conventional Non-Clinical Toxicity Testing

I. Gross examination e for general toxicity studies (Tier I “screens”) and dedicated neurotoxicity studies (Tier II investigations) External assessment e brain, spinal cord (sometimes not performed for survey studies), peripheral nerve Organ weight e brain Internal evaluation e brain (on coronal slabs, approximately 0.5e2 cm thick depending on brain size) II. Light microscopic evaluation A. Central nervous system 1. Technical considerations a. Formalin fixation e neutral buffered 10% (3.7% formaldehyde, approximate pH 7.4) i. Immersion for general toxicity studies (Tier I) ii. Intravascular perfusion for dedicated neurotoxicity studies (Tier II) b. Paraffin embedding e sections at 4e8 mm c. Conventional stains i. General architecture e hematoxylin and eosin (H&E) ii. Neuronal features e cresyl violet iii. Axonal integrity e silver stains (Bielchowsky’s of Bodian’s) stains iv. Myelin integrity e Luxol fast blue (LFB) d. Special stains i. Neuronal degeneration e amino cupric silver (frozen sections required) or Fluoro-JadeÒ ii. Gliosis 1. Astrocytic reaction e glial fibrillary acidic protein (GFAP) immunohistochemistry 2. Microglial reaction e ionized calcium binding adaptor molecule 1 (Iba1) immunohistochemistry 2. Regions to consider for routine sampling a. Forebrain e cerebral cortex, basal nuclei, corpus callosum, olfactory bulb (rodents only) b. Diencephalon e hippocampus, internal capsule, thalamus, hypothalamus c. Midbrain e red nucleus, substantia nigra, rostral and caudal colliculi (tectum), tegmentum d. Hindbrain e cerebellum, pons, medulla oblongata e. Spinal cord e cervical, thoracic, lumbar (emphasizing intumescences; cross and longitudinal orientations) B. Peripheral nervous system 1. Technical considerations a. Fixation i. Formalin e initial fixative ii. Osmium tetroxide (OsO4), 1% e post-fixation (to stabilize myelin so that it is retained while processing) b. Embedding i. Paraffin e sections at 4 mm (suitable for screening) ii. Plastic e sections at 1 mm (typically required for detailed evaluation) c. Conventional stains e General architecture e H&E (paraffin) or toluidine blue (plastic) d. Sample orientation e cross and longitudinal orientations 2. Regions to consider for sampling a. Sciatic nerve (proximal trunk) b. Tibial, peroneal, or sural nerves (distal branches) c. Dorsal root ganglia (especially those supplying sciatic nerve) d. Cranial nerves (if indicated by neurological findings) e. Autonomic nerves and ganglia (if indicated by neurological findings)

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TABLE 52.2

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Flow Chart for Toxicologic Neuropathology Endpoints in Conventional Non-Clinical Toxicity Testingdcont’d

III. Special procedures e generally for dedicated neurotoxicity testing A. Ultrastructural assessment e typically requires special study 1. Technical considerations a. Fixation e neutral buffered 10% formalin (3.7% formaldehyde, approximate pH 7.4) i. Intravascular perfusion preferred ii. Perfusate should include glutaraldehyde at 1% to 4% b. Embedding e hard plastic resin to permit thin sectioning (< 1 mm) c. Stains i. Uranyl acetate ii. Lead citrate 2. Regions to consider for sampling e based on light microscopy results B. Immunohistochemistry for additional markers 1. Neurons a. Tau b. Cell type-specific markers (e.g., neurotransmitters) c. Functional markers (e.g., tyrosine hydroxylase [TH] for catecholamine-synthesizing neurons) 2. Glia a. Oligodendroglia e myelin basic protein (MBP) b. Schwann cells e S100 c. Microglia e lectin histochemistry for Griffonia simplicifolia, isolectin B4 3. Blood vessels a. Presence e endothelial markers (e.g., Factor VIII-related antigen) b. Permeability e detection of extravasated horseradish peroxidase (HRP) or immunoglobulin (Ig) 4. Miscellaneous a. Leukocyte markers b. Degenerating cells (apoptosis) e anti-caspase-3 immunohistochemistry, or equivalent c. Proliferating cells e anti-bromodeoxyuridine (BrdU) immunohistochemistry, or equivalent C. Enzyme histochemistry 1. Cytochrome oxidase e energy metabolism in neurons 2. Reporter genes e lacZ (bacterial b-galactosidase), or equivalent, at sites of engineered gene expression D. Quantitative procedures 1. Morphometry e linear or areal measurements in two dimensions 2. Stereology e volumetric measurements or object counts in three dimensions

cerebellum, and vestibular system); five spinal cord domains (upper cervical [C1–C5], cervicothoracic [C6–T2], thoracolumbar [T3–L3], lumbosacral [L4–S2], and caudal [Cd1–Cdx]); and the PNS. Additional syndromes have been identified that affect multiple neural regions, or that cannot be localized. In most cases, these syndromes result from causes other than xenobiotic exposure. However, a few syndromes can be linked routinely to specific agents, or sometimes an entire class (usually heavy metals, pesticides, and solvents). Toxicant-related brain syndromes most commonly result from lesions localized to a few

CNS sites: cerebrum (following systemic exposure), cerebellum (systemic delivery), or brainstem (intrathecal injection). Cerebral syndrome reflects damage to the cerebral cortex or, rarely, the subcortical relay centers and presents as an altered mental state (e.g., apathy, aggression, depression, disorientation, or hyperexcitability) with gait or postural abnormalities (e.g., continual pacing or head pressing; Figure 52.19). Toxic causes of cerebral syndrome include heavy metals (e.g., lead, mercury), some mycotoxins (e.g., fumonisin B1), and certain solvents (e.g., hexachlorophene). Cerebellar syndrome is indicated by abnormal motion and posture, typically

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Potential Endpoints of Neurotoxicity

I. Anatomic (structural) indices e appropriate for mature and developing individuals A. Macroscopic changes 1. Gross abnormalities e brain, spinal cord, peripheral nerve 2. Weight alteration e brain B. Microscopic changes 1. General changes in architecture 2. Lesions (especially regional destruction of the parenchyma or specific neuronal populations) 3. Repair reactions (enhanced numbers of reactive astrocytes and/or microglia) 4. Altered patterns of molecular expression (cell type-specific and functional markers) II. Behavioral and clinical indices A. Overt clinical signs of neurotoxicity B. Effector endpoints e changes (increases or decreases) in: 1. Learning, memory, and attention 2. Motor activity 3. Schedule-controlled behavior (rate or temporal patterning) C. Sensory endpoints e altered sensations of sight, smell, sound, or taste D. Developmental deficits 1. Change in quality 2. Shift in timing a. Late onset of behavioral landmarks b. Premature onset of behaviors associated with senescence III. Neurochemical indices A. Neurotransmission shifts e changes in: 1. Neurotransmitter synthesis, release, uptake, and/or degradation 2. Signal transduction pathways (especially second messenger generation) 3. Synaptic activity of acetylcholinesterase (AChE) or neuropathy target esterase (NTE) B. Markers of neurotoxicity e during maturity or development 1. Neuronal responses a. Heat shock proteins (may be upregulated by damage) b. Neurotransmitter levels 2. Glial reactions a. Astrocytes e glial fibrillary acidic protein (GFAP) immunohistochemistry b. Microglia e ionized calcium binding adaptor molecule 1 (Iba1) immunohistochemistry IV. Neurophysiological indices e changes in: A. Electroencephalographic (EEG) pattern e shift in pattern B. Nerve conduction e altered amplitude, velocity, or refractory period C. Sensory-evoked potential e changed amplitude or latency

in the presence of normal mental status, and can be induced by toxic agents that target the cerebellum: heavy metals (e.g., methyl mercury and trimethyltin [TMT]); rodenticides (e.g., bromethalin); solvents (e.g., hexachlorophene), and toxins (e.g., plants of the genus Solanum). Brainstem syndrome arises from lesions in the pons and/or medulla oblongata and presents as altered mental status (e.g., coma or depression); abnormal movement with normal reflexes;

respiratory anomalies; and dysfunction of multiple cranial nerves (V–XII). Neurotoxic causes of brainstem syndrome include local (intracerebroventricular, intracisternal, intrathecal) delivery of small molecule drugs and systemic delivery of organophosphates. In the spinal cord, syndromes affecting all five domains have been observed after xenobiotic exposure, but local delivery of the xenobiotic usually is necessary to produce the effect.

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Behavioral Evaluation GENERAL PRINCIPLES OF NEUROBEHAVIORAL TESTING

FIGURE 52.19 Neurotoxicity often presents with clinical signs indicating that specific portions of the nervous system have been damaged. This Doberman Pinscher dog is exhibiting the postural abnormality known as head pressing, indicating that the lesion is localized to the cerebral cortex or possibly a subcortical relay center (i.e., cerebral syndrome). The cause was hyperammonemia due to hepatic encephalopathy. This image was provided by Dr Billy C. Ward and obtained from the Noah’s Arkive database of veterinary pathology lesions: http://www.vet.uga.edu/vpp/noahsarkive/na.php; image no. 4724.

The functional observational battery (FOB) is a standard collection of simple, non-invasive, clinical endpoints designed to identify overt neurotoxic effects in rodents as well as larger animal species. The observations typically proceed from the least manipulative tests (e.g., home cage observations without handling) to the most manipulative methods (e.g., a basic neurological examination to assess various reflexes and reactivity to nociceptive stimuli) in order to reduce the influence of handling on subsequent behavior. The main advantage of the FOB is that the endpoints are acquired rapidly and at relatively low cost. The disadvantage is that behavioral deficits following toxicant exposure cannot be linked to damage in specific neural regions without further testing by different means (e.g., microscopic examination of the many neural structures that might contribute to the abnormal response).

A practical definition of behavior is the net neural output in response to global sensorimotor integration in the nervous system. Thus, behavioral alterations are a useful indicator of neurotoxicant exposure since behaviors reflect the coordinated summation of neural output over large expanses of a complex neural net. Behavioral endpoints are generally non-invasive, so they can be used to assess subjects regularly over the entire course of a study. Furthermore, each individual can be tested prior to the start of an experiment so that a baseline response can be gathered. Potential disadvantages of such techniques are that interindividual variation in behavioral responses often is quite broad. Furthermore, functional deficits may be avoided if compensatory behaviors are deployed, which can greatly reduce or even eliminate the ability to detect subtle behavioral changes associated with neurotoxicity. Behavioral endpoints commonly employed in neurotoxicity bioassays include acoustic startle, motor activity, tests of learning and memory, and the FOB. Tests of simple reflex behaviors tend to be specific for damage to one or a few neural regions but have fairly low sensitivity as they typically are affected by relatively few neurotoxicants. In contrast, measurements of complex behaviors (e.g., learning, memory, motor activity) are less specific as they simultaneously survey many functions. Thus, a battery of tests encompassing both simple and complex behaviors often is preferred for identifying neurotoxic hazards. At the present time, a consensus has not been reached regarding selection of a “standard” battery of behavioral tests to assess chemically induced cognitive, motor, and sensory dysfunction in animal models. ACOUSTIC STARTLE RESPONSE

The acoustic startle response assesses a sensoryevoked motor reflex that depends on a complex neural circuit spanning multiple neural domains (afferent arm ¼ cochlea, cranial nerve VIII, cochlear nucleus, and caudal [inferior] colliculus; efferent branch ¼ reticular nucleus in midbrain, reticulospinal tract, ventral (anterior) horn large motor neurons, and somatic motor nerves to the limb muscles). Neurotoxic effects in brain regions distant from the primary startle pathway also

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may alter the response. For example, the startle amplitude is increased by damage to the olfactory bulbs, frontal cortex, brachium of the caudal colliculus, reticular formation, septal area, periaqueductal gray matter, and the median raphe nuclei. In contrast, acoustic startle activity is depressed by lesions of the locus coeruleus. Assessment of these sites using histopathological or other approaches is often required in order to interpret an abnormal startle reflex. The test typically is performed over a 10- to 15minute period by placing an animal in a small box, stimulating it intermittently with a tone or touch (e.g., puff of air), and recording the amount of movement using automated piezoelectric sensors. The startle response can be measured in most laboratory animal species and at most ages, making it a suitable option for developmental tests and comparative (i.e., translational) studies. This test can be used as a rudimentary means for assessing learning and memory, as the startle reflex shows habituation, modification, and sensitization over time by prior associative learning. MOTOR ACTIVITY

Motor activity is represented by several behaviors (e.g., ambulation, grooming, rearing, sniffing) that involve coordinated involvement of sensory, motor, and associative processes. Motor activity testing often is performed in a novel environment using an automated detection system. Rodents may exhibit a substantial diurnal cyclicity in their level of spontaneous motor activity that must be considered when designing test batteries and interpreting data. Motor activity changes may result from CNS and/or PNS damage. Since these behaviors reflect the sum of central and peripheral pathways required for motor function, no specific neural domain can be implicated when activity levels are altered. The nature of the motor dysfunction can provide important clues regarding potential target sites within the nervous system. Some neurotoxicants increase motor activity. This elevation may be transient, presumably as a result of enhanced neurotransmitter release (e.g., toluene, xylene), or the boost may be permanent if specific brain regions are destroyed (e.g., hippocampal toxicity by trimethyltin). Heightened motor activity is observed most often with agents that can interact with either the cholinergic or dopaminergic systems (e.g., amphetamine, cocaine). Conversely, motor activity

is reduced following exposure to certain classes of neurotoxic agents, such as heavy metals (lead, tin, and mercury) and many pesticides (e.g., carbamates, chlorinated hydrocarbons, organophosphates, and pyrethroids). Other agents from different classes decrease motor activity by sharing a particular neurotoxic mechanism (e.g., acrylamide, carbon disulfide, and methyl-n-butyl ketone crosslink axonal proteins in peripheral nerves). COGNITIVE TESTING

Tests of learning and memory are employed to evaluate associative capabilities in laboratory animals. The single-trial passive avoidance procedure is widely used for this purpose. The general approach is to place a rat in the lighted side of an automated box with dark and lit compartments. A mild electrical shock to the foot is used to train the animal to ignore its instinctive inclination to move into the dark chamber for a specified length of time (e.g., 60 seconds). Several trials may be conducted per animal in each session to assess learning, followed by a subsequent re-test to assess memory. Schedule-controlled operant behavior (SCOB) involves a preliminary motivational procedure (e.g., food deprivation) followed by intermittent reinforcement (e.g., a food pellet reward) to maintain the behavior. These tests measure learning in the context of sensory, motor, and cognitive integration. The primary endpoints are alterations in the response rate or frequency as well as the temporal pattern in which responses occur. Training times are fairly short, which permits studies to be conducted in a timely fashion. The baseline performance of each animal usually serves as the gauge against which subsequent performances are compared. INTERPRETING BEHAVIORAL TOXICITY DATA

Interpretation of behavioral data in neurotoxicity risk assessment requires considerable care. The data sets produced by behavioral testing may be poorly correlated to those acquired by other means of neural assessment, such as neuropathology. The usual explanation for the discrepancy is that many behavioral deficits are acute and/or transient (i.e., minutes to a few hours), resulting from a biochemical disruption such as altered membrane excitability or neurotransmission. In general, such acute functional

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changes often cannot be linked with specific structural lesions. Longer-lasting behavioral alterations may invoke corresponding neuropathological abnormalities. The other common means of explaining the poor correlation between behavioral and neuropathological data is to recognize that conventional tissue sampling strategies do not permit examination of all brain regions that participate in complex behaviors. Electrophysiological Evaluation The raison d’eˆtre for neural cells and tissues is to generate and conduct electrical currents. Measurements of electrical activity may be made at the level of individual cells in a research setting (e.g., patch-clamp, voltage-clamp, and sharp electrode techniques), but for the purposes of toxicologic neuropathology the typical electrophysiological methods used during life are electroencephalography (EEG), electromyography (EMG), electroretinography (ERG), and nerve conduction. These techniques measure the cumulative synchronized electrical activity (voltage fluctuations) of all neurons or axons with a shared orientation within a given circuit; this activity may be spontaneous or purposely induced by presentation of some relevant stimulus (i.e., an “evoked” potential). Recordings are captured over a short period (generally 10–20 minutes) using non-invasive or minimally invasive electrodes at the ends or spaced at intervals along the circuit. The typical electrophysiological procedure employed in animals in neurotoxicity studies is the nerve conduction study (NCS). This method evaluates the function of motor and sensory nerves in the PNS, commonly using nerve conduction velocity (NCV) as the principal screen of nerve integrity. The NCS may probe motor nerves by stimulating the root of the nerve near the vertebral column (i.e., the sciatic notch) and then measuring the time it takes for the impulse to propagate peripherally to reach a recording electrode near an effector organ – typically a skeletal muscle on the distal hind limb (e.g., dorsal surface of the foot). Alternatively, a NCS may assess sensory nerve function by measuring the time it takes for a stimulus applied to a purely sensory branch of nerve (e.g., the distal portion of a digit) to travel proximally to the root (i.e., near the vertebral column). The time required for impulses to travel from the

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stimulating electrode to the recording electrode is termed the latency, and is measured in milliseconds (ms). The size of the response is the amplitude, which usually is measured in millivolts (mV) in motor nerves but microvolts (mV) in sensory nerves. Toxicant-induced damage to axons or myelin in the PNS would be expected to slow the NCV in multiple limbs, likely for both the motor and sensory fibers.

3.2. Biochemical and Biomarker Evaluation Recent neurotoxicological research has focused on finding more rapid, less invasive, and less laborious means for identifying neurotoxic risks. Discovery and validation of novel chemical and molecular biomarkers has been a particularly active area of inquiry in this regard. Clinical Pathology Alterations in peripheral blood samples (e.g., whole blood, plasma, serum) typically are uninformative in terms of diagnosing neurotoxic changes, but cerebrospinal fluid (CSF) analysis may be of use in certain instances. Collection of CSF is much less invasive and injurious to the CNS parenchyma than acquiring a tissue sample. Typical analytes include various biochemical tests (especially protein concentrations, but also levels of neurotransmitters and their metabolites) and the evaluation of cell numbers and morphology. In general, CSF collection in rodents is confined to satellite groups of animals as the anesthesia protocols required for this endpoint may impact other parameters. The utility of CSF as a substrate stems from its link to interstitial fluid (ISF), which is the principal fluid constituent in the brain and spinal cord within the parenchyma immediately surrounding neurons and glia. Analysis of CSF has reasonable sensitivity but low specificity as an indicator of CNS health, as the range of aberrant changes in this fluid abnormalities is narrow relative to the many possible neurological diseases (especially if the evaluation is limited to determining total and differential cell counts and the protein concentration). Diseases localized to the meninges and neuropil adjacent to the ventricular system (the “CSF compartment”) usually generate greater changes in CSF parameters than do diseases in the deep parenchyma (the “ISF compartment”). Since most neurotoxic lesions reside primarily in

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the deep neuropil, properties of CSF samples from individuals with neurotoxic conditions generally are within normal limits. Neurochemistry Neurochemical measurements are common endpoints in neurotoxicological studies. Such tests not only provide evidence that an agent has altered one or more neurobiological parameters but also point toward specific mechanisms of neurotoxic action. Neurotransmitter systems are quintessential neurochemical biomarkers. The importance of neurotransmitters during development (as morphogens; see Embryo and Fetus, Chapter 62) and adulthood (as signaling molecules) stems directly from the need to maintain their appropriate synthesis, release, and removal to support normal function of the CNS and PNS. Neurotransmitter systems may be evaluated in many fashions. Chemical assays (e.g., chromatography or microdialysis to define transmitter levels) and molecular biological techniques (e.g., northern analysis of RNA levels, western analysis or enzyme-linked immunosorbent assays [ELISA] for peptide transmitters, receptors, and signaling cascade proteins) can directly detect specific elements contributing to the function of a given transmitter system. The measurements reveal the quality (presence or absence) and quantity of particular elements within a given pathway, but do not directly assess the functional integrity of the system. Thus, increases or decreases in neurotransmitter levels induced by neuroactive agents are not necessarily indicative of a neurotoxic effect unless they are accompanied by other functional (e.g., neurobehavioral, neurophysiological) or structural (i.e., neuropathological) aberrations. Microdialysis techniques coupled with chemical analyses provide one ante mortem method of assessing neurotransmitter function in animals. Specialized neuroimaging approaches (e.g., positron emission tomography [PET] and single photon emission computed tomography [SPECT]) have been increasingly used to assess neurotransmitter function in animals and people (see In Vivo Small Animal Imaging: A Comparison with Gross and Histopathologic Observations in Animal Models, Chapter 9). Glial fibrillary acidic protein (GFAP) expression is another frequent biomarker for CNS

damage, including that caused by neurotoxicants. Accumulation of GFAP results from astrocyte hypertrophy; cell expansion requires enhanced production of all cell constituents, and GFAP is chief among them as the major intermediate filament protein of astrocytes. Biochemical assays (e.g., radioimmunoassay, sandwich ELISA, and western analysis) or immunohistochemical stains (see below) indicating an increase in GFAP levels are a sensitive, simple approach for confirming the presence of a CNS response to prior damage. The cause of the change usually cannot be defined without other tests (e.g., histopathology), and sometimes not even then as GFAP expression may rise in the absence of histopathological changes. Therefore, in adult rodents, stand-alone increases in GFAP above control levels may be indicative of neurotoxicity if a history of exposure is known. An important consideration in interpreting such increases is that GFAP levels will also be higher following injurious events other than neurotoxic damage (e.g., increased corticosteroid levels, normal aging). Interpretation remains problematic when faced with decreased GFAP levels in adults or any alterations of GFAP expression in developing animals. Enzyme histochemistry is another common means for detecting changes in biochemical and metabolic pathways that might serve as biomarkers for neurotoxic lesions and their mechanisms. An advantage of these procedures is that they measure not only the presence of the enzyme but also whether or not the protein is functional. Enzyme histochemical tests that are commonly used in toxicity studies include visualization of extravasated horseradish peroxidase as a probe for xenobiotic-induced damage to neurovascular endothelium, and assessment of acetylcholinesterase (AChE) inhibition in fluids and tissues as an indicator of exposure to organophosphate or carbamate insecticides. Toxicogenomics In many cases the initial neurological changes that might be detected following xenobiotic exposure will not be macroscopic and microscopic structural lesions or functional abnormalities, but rather reflect molecular, neurochemical, and/or ultrastructural (i.e., submicroscopic) effects. Innovative technological platforms now permit the easy acquisition of complex data

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sets from neural tissues. Toxicogenomics (see The Application of Toxicogenomics to the Interpretation of Toxicologic Pathology, Chapter 11) is a key investigational strategy that currently is evolving to play a more regular role in neurotoxicity assessment. The basic principle of this approach is that the expression pattern of a constituent of interest can be probed qualitatively or quantitatively in homogenates of normal and diseased tissue samples to determine a molecular signature that may be used in diagnosing the presence and/or progression of a given condition. These methods provide high-throughput production of “omics” data at the cellular level with molecular or nanometer resolutions that permit exploration of the pathways responsible for maintaining health as well as the abnormalities that must develop to initiate and sustain disease. The application of various “omics” disciplines, including assessments of DNA (genomics), messenger RNA (transcriptomics) or protein (proteomics) responses, is increasingly used to determine early steps in the pathogenesis of neurotoxicity. Conceivably, such items as intercellular networks (connectomics) also might be adapted for this purpose. The “omics” platforms serve two primary roles in neurotoxicological research. The first is to assess variations in the molecular signature among different target cell populations (e.g., neurons vs astrocytes, granule vs pyramidal neurons). The second purpose is to examine molecular pathways that play roles in xenobiotic responses that might be used to diagnose or treat particular health conditions (e.g., receptors capable of interacting with antidepressant drugs, signaling cascades impacted by exposure to drugs of abuse). Other roles undoubtedly will be added as this technology matures as a means for neurotoxicological exploration. Standard strategies for the current generation of toxicogenomic studies rely on differential gene and protein expression. Common methods for detecting such effects are microarrays as well as two-dimensional gel electrophoresis and mass spectrometry. Technical details of these methods are beyond the scope of this chapter, but references in the Suggested Reading section can provide more depth in this regard. “Omics” techniques are quite sensitive to the quality of the starting material, so tissues must be collected as rapidly as possible after death to prevent artifactual molecular degradation in

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neural cells (especially in the deep CNS parenchyma) that arise due to post mortem acidosis, dehydration, hypoglycemia, and hypoxia. In general, samples should be collected fresh and frozen at 80 C until analysis, but molecular data can often be obtained from fixed or fixed and previously embedded samples as long as the fixation length was relatively short and the blocks were not subjected to extreme temperature fluctuations during their time in storage. Where known in advance, the experimental design can accommodate parallel collection of fresh tissue for “omic” (and other neurochemical) endpoints and fixed tissues for conventional neuropathology evaluation. Tissues should also be collected with a consideration for defining the precise location within the nervous system and accurately determining the sampling time. An additional factor that is critical for success is an appropriate sampling strategy, which typically rests on isolating the correct cell population for analysis (see Statistical Assessment of Toxicologic Pathology Studies, Chapter 30). Cell harvesting may be done by gross dissection of large tissue blocks where regional homogeneity at the macroscopic level is evident. However, the differential expression of various genes at the cellular level invariably results in a readout in which the expression data represent an “average” from multiple neural cell types. The alternative approach is to use laser capture microdissection (LCM), a more laborious and low-throughput undertaking that permits sampling of a defined cell population (see Special Techniques in Toxicologic Pathology, Chapter 7).

3.3. Toxicokinetics Neurotoxicological effects depend on multiple factors, among the most important of which are the concentration of agent reached at the target site and the duration of exposure. As with other systems, xenobiotic disposition in the nervous system is governed collectively by the absorption, distribution, metabolism, and excretion (ADME) of the agent (see Pharmacokinetics and Toxicokinetics, Chapter 2). In many respects, however, unique characteristics of the CNS and PNS modulate the ability of toxicants introduced into the body to reach neural targets. The most well-known example is the existence of tight blood–brain

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and blood–nerve barriers that limit the entry of many compounds into the CNS and PNS, respectively; in general, lipophilic agents pass freely across these barricades, while protein-bound and water-soluble compounds are excluded. Passage for certain molecules into the CNS is enhanced by transport systems, such as those in the choroid plexus that transfer many metals. Sequestration within the CNS (due to site-specific chemical binding to tissue macromolecules) and activation of non-toxic prodrugs (e.g., conversion of 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine [MPTP] to the toxic metabolite 1-methyl-4-phenylpyridinium [MPPþ] in dopaminergic neurons of the substantia nigra) are other crucial determinants, especially in instances where xenobiotic metabolites mediate neurotoxicity. The limited metabolic capacity of neural cells, especially terminally differentiated neurons, for xenobiotics often delays their elimination. Toxicokinetic factors represent an essential aspect of neurotoxic risk assessment. Interspecies extrapolation requires detailed characterization of the many physiological parameters that dictate xenobiotic disposition into and within the CNS. The best means of achieving this ideal usually is to generate toxicokinetic data for a given agent in multiple species (including humans, where feasible). Common methods for evaluating the likelihood that an agent will reach or selectively accumulate in the CNS include in vitro assessments of the molecule’s relative preference for lipid-rich tissues (e.g., oil or homogenized brain matter) vs water-rich compartments (e.g., water or blood); direct measurements of the materials entry into the CNS by quantifying levels in fluid samples (e.g., CSF or microdialysis aliquots) or tissue specimens (typically harvested post mortem); and autoradiography to visualize accumulation of the radiolabeled compound in various neural domains. In most cases, specific measurement of the parent compound and/or major metabolites is not undertaken in neural tissues unless other data (e.g., neurological abnormalities, evidence of neural accumulation in autoradiography studies) indicate that the material actually penetrates into the CNS or PNS. In instances of inhalation exposure, the olfactory bulbs and tract should be considered exceptions to this latter rule based on their close proximity to the nasal passages and the short, direct conduit to them provided

by the axons of the olfactory mucosa. A number of chemicals and metals have been shown to reach the brain via the olfactory route, including inhaled nanoparticles (see Nanoparticulates, Chapter 43).

3.4. Morphologic Evaluation Neuropathological changes observed by macroscopic or microscopic means after exposure to a xenobiotic are considered by definition as adverse events. This section describes the options for morphological endpoints that might be used to evaluate toxicant-induced structural changes in the nervous system, placing them in the context of conventional study design considerations and existing regulatory guidance. Toxicant-induced changes in neural structure may be prominent at the organ level. One form of gross abnormality is altered brain weight, which typically is assessed at necropsy immediately after organ removal or in fixed organs prior to tissue trimming. Measurement of organ weight is a common quantitative albeit indiscriminate means for identifying large changes that affect considerable expanses of the brain. In general, altered absolute brain weights (which usually present as decreases in neurotoxicity studies) are deemed to be a biologically significant marker of neurotoxic damage. Macroscopic changes related to neurotoxicant exposure include specific lesions, such as regions of brain degeneration (Figure 52.20) and neoplasms, as well as alterations in the dimensions (length, area, or volume) of a major region (Figure 52.21). Many neuropathology changes produced by toxicant exposure are visible only at the light microscopic level. The type of histopathological abnormality will depend on multiple factors. Perhaps the most important is the age of the lesion. Acute cell death typically presents as shrunken cells with hypereosinophilic cytoplasm and dark, condensed or fragmented nuclei (i.e., classic “red dead” neurons; Figure 52.22). In contrast, more chronic lesions often result in cell loss or neuropil cavitation (i.e., the end-stage lesion following liquefactive necrosis), typically with an increase in the number of GFAP-positive reactive astrocytes in the immediate vicinity (Figure 52.23). Another important determinant of the final histological pattern is the time during life when the exposure occurred. Different brain regions

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(Figure 52.1), so transient exposures during different periods of life will elicit divergent outcomes. Third, the distribution of lesions among various neural cell populations often depends on cell-specific factors. Examples include the rich synaptic beds in the cerebral and cerebellar cortices and hippocampus, which makes these three domains common targets for toxicants, and the preferential susceptibility of dopaminergic neurons in the substantia nigra due to their ability to concentrate and metabolize MPTP.

FIGURE 52.20 Macroscopic appearance is an essential tool for identifying neurotoxic lesions. Upper: Relative to a control animal (left), a rat that had inhaled carbonyl sulfide (500 ppm for 6 h/day for 2 weeks) developed bilaterally symmetrical encephalomalacia (arrows) affecting the frontoparietal cerebral cortex. Lower: Severe nigropallidal encephalomalacia (arrows) presents as well-demarcated, bilaterally symmetrical zones of pallor (cream-colored with a thin brown rim, where the adjacent gray matter is light brown) in the affected basal nuclei of this horse that had consumed yellow star thistle (Centaurea solstitialis) for an extended period. Source for Upper: Figure reproduced from Sills et al. (2004) Contribution of magnetic resonance microscopy in the 12-week neurotoxicity evaluation of carbonyl sulfide in Fischer 344 rats, Toxicol. Pathol. 32, 501–510, with permission. Source for Lower: This image was provided by Dr Billy C. Ward and obtained from the Noah’s Arkive database of veterinary pathology lesions: http://www.vet.uga. edu/vpp/noahsarkive/na.php; image no. 4942.

have unique windows of vulnerability based on when they undergo their peak of neuron production and differentiation during development

Study Design Toxicant-induced structural lesions in the CNS and PNS may be assessed as a stand-alone diagnostic problem or as an organized, prospective investigation of neurotoxic potential. In general, diagnostic neuropathology is undertaken in a clinical practice setting (e.g., medical or veterinary medical office) to ascertain whether or not an agent can be associated with damage to one or more neural structures. The objective of such diagnostic studies is to facilitate the return to full neurological function, or at the least save the patient’s life, so morphological assessment of the nervous system generally is confined to non-intrusive imaging modalities like computed tomography (CT) or magnetic resonance imaging (MRI) (see In Vivo Small Animal Imaging: A Comparison of Gross and Histopathologic Observations in Animal Models, Chapter 9) or minimally invasive techniques like peripheral nerve biopsy. In contrast, neural evaluation in regulatory-type non-clinical studies is done either as one aspect of a general screen for toxicity to all organ systems (i.e., “general toxicity studies”) or to provide a comprehensive examination of structural damage in multiple nervous system domains (i.e., “dedicated neurotoxicity studies”). The different objectives of these studies warrant a smaller neural tissue list for general studies relative to the expanded tissue battery used for studies devoted mainly to a detailed neuropathology analysis. It is impossible to define a single duration of exposure that is suitable for all neurotoxicants, as different agents elicit a fluctuating spectrum of lesions that peak in incidence and degree at various times after exposure. When known in advance, the timing of necropsies should be arranged to occur when toxicant-induced neural lesions are at their peak, keeping in mind that

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FIGURE 52.21 Macroscopic alterations in the dimensions (length, area, or volume) of major neural structures are another important means for identifying neurotoxic damage. In this case, pronounced cerebral hypoplasia leads to exposure of the caudal olfactory bulbs and entire midbrain in a 21-day-old Wistar rat following maternal treatment with the neuroteratogen methylazoxymethanol (MAM, given at 30 mg/kg body weight by intraperitoneal injection on gestational day 15). Images kindly provided by Dr Wolfgang Kaufmann, Merck KGaA, Darmstadt, Germany.

many agents exhibit multiple peaks characterized by distinct kinds of lesions. This principle is well illustrated by the excitotoxic agent kainic acid, injection of which causes neuronal necrosis in 1–4 days but synaptic terminal disintegration between 4 and 14 days. In many cases, neurotoxicity studies are designed to recapitulate specific requirements listed in regulatory guidelines (see Suggested Reading). Testing requirements are relatively specific for dedicated neurotoxicity studies but permit a fair degree of discretion for general toxicity studies. Tissue Fixation Diagnostic samples (e.g., nerve biopsies) and neural tissues for general toxicity studies generally are fixed by immersion in neutral buffered 10% formalin (NBF, a 3.7% formaldehyde solution, approximate pH 7.4). This choice is dictated by historical and practical considerations. NBF is available in bulk from many vendors. The small size of the formaldehyde molecule permits it to penetrate dense neural tissues easily, while the reactive aldehyde group promotes rapid tissue fixation. Nervous system samples fixed in this manner may be processed routinely along with samples from other organs, thus reducing the cost and labor required to prepare tissue blocks for histological sectioning.

For specialized neuropathology studies, the preferred choice for preparing neural tissues is intravascular perfusion because it offers the best preservation of neural morphology in all CNS and PNS structures (Figure 52.24). Perfusion introduces fixative deep into the nervous system, thus speeding fixation, and it prevents the induction of many artifacts that tend to arise when unfixed CNS tissues are manipulated during necropsy. This method permits the use of fixative mixtures that include paraformaldehyde (essentially formaldehyde but without the stabilizing agents added to commercial formalin formulations) and/or glutaraldehyde. These agents are typically employed at strengths of 1–4%. Combinations of formaldehyde and glutaraldehyde are sometimes referred to as McDowell-Trumps or Karnovsky’s fixatives. Glutaraldehyde is a sine qua non for preserving subcellular organelles for ultrastructural analysis. This molecule is a more effective crosslinking agent since it has two aldehyde moieties, but its length greatly slows tissue penetration. Furthermore, glutaraldehyde alone or in combination with formaldehyde enhances the stabilization of lipid-rich membranes, especially myelin. Perfusion fixation typically is conducted at pressures ranging from 70 to 120 mmHg (i.e., the peak intravascular pressure during systole). Other

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FIGURE 52.23 More chronic neurotoxicant-induced lesions often are best visualized by demonstrating cell type-specific markers in reactive glial cells. The multifocal zones of increased brown staining in this rat cerebellum reveal reactive astrocytes with increased expression of glial fibrillary acidic protein (GFAP) well after exposure to an unidentified neurotoxicant led to acute neuronal death. Processing conditions: formalin fixation by intravascular perfusion, paraffin embedding, anti-GFAP immunohistochemistry with hematoxylin counterstain. FIGURE 52.22 Acute neuronal death following neurotoxicant exposure typically presents as classic “red dead” neurons. Relative to adjacent cells with normal features, necrotic neurons (circles delineate representative examples) have shrunken, spiky profiles; hypereosinophilic cytoplasm; dark, condensed or fragmented nuclei; and are sometimes bordered by clear retraction spaces. Cerebral cortex (upper) and cerebellum (lower) of a rat exposed to an unspecified neurotoxicant. Processing conditions: formalin fixation by intravascular perfusion, paraffin embedding, H&E staining.

critical aspects of perfusion fixation – buffering capacity, osmolarity, pH, and temperature – have been discussed in greater detail in other publications (see Suggested Reading for details). Following perfusion fixation, neural tissues are removed from the carcass. In general, they are post-fixed by immersion for an additional period in NBF. Specimens slated for detailed analysis of myelin and/or electron microscopy analysis are often post-fixed a second time in 1% osmium tetroxide (OsO4) to further stabilize lipid-rich membranes. Fixation in OsO4 must be carried out in a chemical hood because osmium vapors

readily bind and discolor the corneal surface and skin. Even in paraffin-embedded specimens of nerve, osmium post-fixation greatly enhances a pathologist’s ability to evaluate myelin sheaths. Immersion-fixed neural tissues often exhibit artifacts associated with handling at necropsy and/or suboptimal processing. Examples include dark neurons (Figure 52.24), myelin bubbling, and neuropil vacuolation (all of which are discussed in more detail below). Their presence does not preclude evaluation of neural structures, but greater care must be taken to avoid falsepositive results (i.e., interpretation of artifactual changes as genuine lesions) and false-negative outcomes (i.e., the inability to detect or properly construe modest lesions that may be camouflaged by more widespread artifactual changes). Many molecular pathology procedures may be performed in NBF-fixed tissues, but certain immunohistochemical (IHC) techniques and polymerase chain reaction (PCR) often require harvesting of unfixed tissue. Intravascular perfusion with ice-cold, buffered physiological saline may decrease the core temperature enough to lessen post-mortem autolysis until the specimens

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can be removed and frozen. In some instances, reduced-strength fixatives (e.g., 1% or 2% formaldehyde) may be used to provide better tissue preservation while retaining the ability to retrieve labile antigens, although a pilot study is recommended to ensure that the altered fixation protocol will support the desired endpoints. In terms of fixative choice, it is best to start with prior knowledge of the required endpoints, then preserve the necessary tissues accordingly.

FIGURE 52.24 Neural cell architecture is greatly improved if fixation occurs by intravascular perfusion (upper) rather than immersion (lower). Perfused tissue features neurons with readily distinguished nuclear and cytoplasmic detail as well as improved contrast between neurite (pale eosinophilic) and myelin (darker eosinophilic) elements. Immersed tissue exhibits several artifactual changes that result from delayed penetration of the fixative. First and foremost, clusters of “dark neurons” may be identified by the blending of their darkly amphophilic nuclei and cytoplasm, and often by the presence of a twisted (corkscrew-shaped) and thickened dendrite; a typical error by inexperienced individuals is to interpret dark neurons as evidence of toxicant-induced neuronal necrosis. Second, neurons often are encircled by clear retraction spaces. Third, neurons (often small pyramidal cells or stellate cells) may be associated with small, oval, clear vacuoles near the periphery (arrows), which are thought to be swollen astrocyte processes. Finally, the contrast between structures in the neuropil (i.e., the expanse of myelinated neurites between cell bodies) often is minimal. Cerebral cortex of adult rats. Processing conditions: paraffin embedding, H&E staining.

Brain Weights Two major advantages accrue to brain weight as an endpoint for recognizing neurotoxicity. First, brain weight is inherently quantitative and objective, which removes a potentially major source of bias in its acquisition and interpretation. Second, weights may be acquired at necropsy by minimally trained personnel, thereby providing a fast and simple indication that a pronounced neurotoxic event has occurred. Brain weights may be taken at necropsy on fresh (i.e., unfixed) or perfusion-fixed organs, or after some period of fixation. Either option is considered acceptable, as long as weights for all subjects in a given study are taken from organs treated in the same fashion. The handling needed for weighing can induce several artifacts in some brain regions, so in dedicated neuropathology studies separate cohorts of animals for each treatment group are included for taking brain weights and performing histopathological assessments. The key to acquiring reproducible brainweight data sets is to ensure that all organs are harvested in a consistent manner. For example, the olfactory bulb of rodents comprises 6% to 7% of the brain weight, so this region should always be included or excluded when building a study data set. Similarly, the brain should be separated from the spinal cord at a standard position. We typically use a visible external landmark, such as the obex (the point on the dorsal medulla oblongata at which the fourth ventricle closes to become the central canal of the spinal cord), as a visual marker for where to make this separating cut.

=

Figures reproduced from Bolon and Butt (2012) Fixation and processing of CNS tissue, in Encyclopedia of Neurological Sciences, 2nd Ed. (in press) Academic Press, San Diego with permission.

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Tissue Collection and Trimming Neural tissues are prone to artifacts if handled roughly or excessively during necropsy. For instance, prompt removal of the brain from the cranial vault, even shortly after intravascular perfusion with fixative, can increase the propensity for artifactual cell shrinkage and increased amphophilia (i.e., able to bind acidic and basic dyes) that are the hallmark features of “dark” neurons (Figure 52.24). A better technique for more pristine brain microanatomy is to perfuse the subject with fixative followed by careful removal of the skullcap and submersion of the entire head (with brain left in place) into fresh fixative for at least 24 hours before removal. If the brain is removed rather than fixed in situ, immediate immersion into a suitable fixative is preferred relative to delays associated with additional handling (e.g., for weighing). In general, toxicologic neuropathology requires sampling of the CNS and PNS at multiple sites. The minimal approach to sampling often is a matter of institutional preference. For example, some common brain sampling schemes for general toxicity studies are three to five full coronal (“transverse”) brain sections, or one para-sagittal (“longitudinal”) section and three to five coronal hemi-sections. Dedicated neurotoxicity studies typically collect double to triple this number of sections. We generally trim the brain and spinal cord using free-hand transverse sections, in which planes for sample acquisition are defined using reproducible external or internal anatomic landmarks alone, as this approach is fast and reliable for screening studies. However, brain molds (i.e., acrylic or metal matrices with multiple parallel slots) may be used if desired when all organs from the test group are of suitable size to fit the apparatus (typically true for adults but not juveniles). The advantage of this latter “egg-slicing” approach, especially when using fixed brains, is that the slabs can be made quite thin by placing blades in adjacent slots of the mold. Other planes of section than the transverse (coronal) orientation are preferred by some practitioners for screening brain. Examples include using a single midsagittal (i.e., longitudinal) section, and combining a single mid-sagittal section with three to five coronal hemi-sections. We advocate examining the spinal cord and peripheral nerves using both longitudinal (either para-sagittal or oblique) and

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transverse sections, as damage to nerve fibers in these organs often is best showcased in the longitudinal orientation. The keys to proper sampling are to make sure that critical sites serving major functions and that are targeted by known toxicants are taken. In general, these sites include (listed alphabetically) the basal nuclei, cerebellum, cerebral cortex (multiple regions), hippocampus, hypothalamus, medulla oblongata, midbrain, olfactory bulb, pons, and thalamus in the brain; three segments of spinal cord (cervical, thoracic, and lumbar); peripheral nerve; and the eye (for retina) with attached optic nerve (i.e., cranial nerve II). Additional sites may be taken if warranted by other factors (e.g., the presence of neurological signs or a known tendency for agents with a given structure or mechanism to target a specific neural domain). Organs should be sampled so that the resulting tissue sections are relatively homologous (i.e., exhibit the same structural landmarks) among animals both within and across studies. When planning a sampling scheme for a given study, we typically arrange our collection strategy starting in the CNS and work to the PNS. In general, we also work from rostral to caudal for the CNS, and from proximal to distal in the PNS, to ensure that nothing is missed when sampling. Neurohistological Procedures In general, CNS and PNS samples are embedded in paraffin. The advantage of this approach is that neural tissues can be processed along with other organs. Some regulatory guidelines require that PNS tissues instead be embedded in plastic (usually glycol methacrylate [GMA]) or resin (e.g., epon, Spurr’s). This process allows tissues to be sectioned at thicknesses of 1–2 mm rather than the standard 4–8 mm used for paraffin, which can yield improved resolution of fine architectural detail during light microscopic evaluations, especially for peripheral nerves (Figure 52.7). However, in our experience this increase in resolution is of significance only for dedicated neurotoxicity studies where the neural tissues were fixed by perfusion and post-fixed in osmium tetroxide to permit a more systematic evaluation. For immersion-fixed neural samples used in general toxicity studies, paraffin is a suitable embedding medium for screening purposes.

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The histological analysis of neural tissues generally is performed in a tiered manner. The examination starts with sections stained using hematoxylin and eosin (H&E), which permits the evaluation of cytoarchitectural features. Where needed, pathologists exercising their professional judgment will request serial sections stained using special neurohistological methods to reveal additional detail. Such special stains illuminate particular cell populations or probe potential processes that have been shown to be vulnerable to toxicant-induced disruption. Neurons are commonly highlighted using a Nissl stain (e.g., cresyl violet [Figure 52.16], thionine) to detect RNA bound to the rough endoplasmic reticulum (“Nissl substance”). Reduced Nissl staining indicates that neuronal function has been impaired. In our experience, the Nissl stains show the fine cellular details of more neuronal populations than does a simple H&E stain. The health of myelinating cells is

examined using methods that label intact myelin, such as Luxol fast blue (LFB, a histochemical method) or myelin basic protein (MBP, a marker protein localized using an immunohistochemical procedure). Neurons and myelin can be visualized in the same section by combining stains (e.g., cresyl violet with LFB [Figure 52.16]). Toxicant-induced neuronal death and/or axonal degeneration can be demonstrated using a number of special procedures, such as fluorescent (e.g., Fluoro-JadeÒ ) and metallic enhancement (e.g., amino cupric silver) stains that collect preferentially in damaged cells (Figure 52.25). Sites of neural injury can also be defined using special techniques to reveal glial responses (e.g., enhanced GFAP expression to demonstrate reactive astrocytes) to prior neuronal injury. Our long collective experience in toxicologic neuropathology has shown us that labor, money, and time frequently are saved by designing

FIGURE 52.25 Special neurohistological techniques to highlight small numbers of degenerating neurons within larger populations of unaffected cells are a standard strategy for speeding the microscopic evaluation of brain tissue. Dark neuron artifact is less likely to present an interpretive problem when using these procedures. While affected neurons are relatively indistinct in H&E-stained sections even at high magnifications (see Figure 52.22), dead cells are easily differentiated at low and intermediate magnifications using amino cupric silver (left; degenerating neurons and processes are black) or Fluoro-JadeÒ (right; damaged cells are bright green). The amino cupric silver procedure requires frozen tissue, while the Fluoro-Jade method can be performed on routinely processed (i.e., formalin-fixed, paraffin-embedded) specimens. Cerebral cortex from adult rats given unidentified neurotoxicants.

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neurotoxicity experiments so that a battery of neurohistological stains is produced automatically as the first tier of the neuropathology examination (e.g., H&E, Fluoro-JadeÒ , and antiGFAP), if only for the high-dose treatment group and the negative control cohort to start. The reason for this choice is that potential neurotoxic lesions of a subtle character can be more readily identified and rapidly evaluated if sections have already been produced and stained in advance. Furthermore, the special stains often reveal changes that were not readily apparent on the H&E-stained sections. That said, we must reiterate that a systematic evaluation (i.e., an adequate number of sections from multiple CNS and PNS regions) generally is more

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important for hazard identification and risk assessment than the use of special stains. Morphometry The quantification of samples that are acceptable for performing measurements in two dimensions (2D) – for example, nerve-fiber diameters in cross-sections of nerves or linear measurements of structures in the brain as is commonly performed in developmental neurotoxicity studies – is a conceptually simple but moderately laborious means for providing objective data during neuropathology analysis. Common 2D means for quantification include linear (e.g., lengths, widths, heights, perimeters) and area assessments. Such measurements can

FIGURE 52.26 Morphometric measurements offer an important means of confirming and quantifying potential toxicant-induced differences in the dimensions (length or thickness, area, volume) of CNS regions. In this example, the thickness of the cerebral cortex appeared to be attenuated (left) in some but not all overtly normal, near-term (gestational day [GD] 17.5) mouse fetuses carried by dams that had inhaled methanol (15 000 ppm for 6 h/day during neurulation [GD 7–9]) relative to the thicknesses of room air-exposed control fetuses (middle). The validity of this equivocal qualitative finding was explored by measuring the thicknesses of the entire cerebral cortex and its layers (denoted by letters, which are defined in Table 52.4) using an ocular micrometer. The location of the trimming plane is shown by the thick black vertical line in the schematic diagram (upper right), while the site where measurements were taken in sections from this level is within the box (lower right). The anterior commissure is denoted by an asterisk. Processing conditions: Immersion fixation in Bouin’s solution, paraffin embedding, H&E staining. Figures reproduced with minor modifications from Bolon et al. (1994) Methanol-induced neural tube defects in mice: Pathogenesis during neurulation, Teratology 49, 497–517, with permission.

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TABLE 52.4 Quantitative Morphometric Data can Detect Subtle Differences in Toxicant-Disrupted Neural Development Methanol-exposed fetuses Control fetuses

All litters

Litters lacking dysraphism

Numbers of litters

24

16

6

Numbers of fetuses

56

39

14

Neuroepithelium (N)

98.5  1.3

108.8  2.1**

107.1  2.5**

Intermediate cortex (I)þ Subventricular plate (S)

229.8  3.3

190.9  3.7**

193.6  3.1**

Cortical plate (C)

129.6  1.4

127.3  2.3

128.2  4.0

Cortical layer 1 (O)

30.2  0.6

22.9  0.6**

23.4  1.2**

Total thickness

488.1  3.9

449.9  5.9**

452.3  7.4**

Thickness of frontal cortex layers in brains of grossly normal, near-term (gestational day [GD 17.5]) fetal mice following maternal inhalation of methanol (15 000 ppm for 6 h/day) during the developmental stage of neurulation (GD 7–9). The location of the cerebral cortical region that was evaluated is depicted in Figure 52.27. Values represent means  standard error (SEM), in mm (for n ¼ 2 or more fetuses per litter, chosen at random). Double asterisks denote a significant difference, P  0.05, relative to age-matched controls by analysis of variance and Scheffe’s F-test (for details, see Statistical Assessment of Toxicologic Pathology Studies, Chapter 30). Table reproduced from Bolon et al. (1994) Methanol-induced neural tube defects in mice: Pathogenesis during neurulation, Teratology 49, 497–517, with permission.

be acquired at multiple levels (see Figure 52.26 and Table 52.4). Examples include macroscopic lengths (e.g., greatest mid-axial length of the dorsal cerebral cortex) and areas (e.g., area of the dorsal profile of the cerebellar hemispheres) of major brain regions; thicknesses or areas of particular domains on histologic sections (e.g., height of the cerebral cortex, width of the basal nuclei); and counts of small structures (e.g., cells in a given nucleus, axonal density). To be valid, such measurements must be performed on relatively homologous samples (e.g., tissue sections with comparable internal landmarks), and ideally using a coded (“blinded” or “masked”) analysis in which the person doing the measurement does not know the treatment history for the specimens. Quantification of objects in three-dimensional (3D) structures, such as neuron counts in dorsal root ganglia or in the brain nuclei, requires stereological investigations (see below). Investigation design is exceedingly important for this category of specialized morphometric analysis. Particular emphasis must be placed on randomly but systematically examining the entire structure,

with a sampling parameter that ensures that each and every object of interest has, at the beginning of the study, an equal opportunity of being counted/measured. Teased Fiber Preparations Isolated nerve fibers, consisting of a single axon and its myelin sheath obtained from a peripheral nerve, can be evaluated for lesions over long distances (typically 0.5–1 cm). Details of the teasing procedure are beyond the scope of this chapter, but, in brief, nerves slated for teasing are fixed by immersion in an aldehyde, separated into individual nerve bundles using forceps, and then post-fixed in 1% OsO4 to further stabilize and darken the lipid-rich membranes. Individual myelinated axons are teased apart in cedarwood oil under a stereomicroscope using forceps, mounted on slides, dried, and cover-slipped. Features evident for inspection include myelin integrity, the appearance of the nodes of Ranvier, and the internodal distance, which can be used to discriminate between degenerative and regenerative processes affecting the axon and the myelin.

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3.5. Special Techniques for Neurotoxicity Assessment Given the importance of neurotoxicity as a health hazard, considerable effort is ongoing to further refine the battery of tests that is available to diagnose neurotoxic conditions in the clinical setting and/or to identify potential neurotoxicants among newly developed and existing agents to which humans and animals are exposed. Morphological changes again represent the gold standard for optimizing these evolving procedures. Non-Invasive Imaging The multiple imaging modalities that have been developed in the last two decades exhibit great promise as tools for toxicologic neuropathology (see In Vivo Small Animal Imaging: A Comparison with Gross and Histopathologic Observations in Animal Models, Chapter 9). Major advantages include their ability to be used repeatedly during life, thus permitting observations of lesion progression over time, and the capacity to translate both the technology and the findings directly to use in human patients that have been exposed to neurotoxicants. The main disadvantage of noninvasive imaging as applied to animal models is insufficient structural resolution at the microscopic level, but recent technical advances permit assembly of 3D images at fairly high resolutions (down to 20 mm). The remaining drawback is that such high-resolution images require very expensive equipment. Imaging modalities may be used to examine anatomic features or functional attributes, or to correlate functional changes to structural lesions. Technologies that provide anatomic data – computed tomography (CT), magnetic resonance imaging (MRI) and microscopy (MRM), and ultrasound (US) – form images using distinct endogenous properties of different cells and tissues to delineate the margins between structures. Platforms that yield functional data – optical imaging, positron emission tomography (PET), and single photon emission computed tomography (SPECT) – define features of a specific biochemical or metabolic pathway within a given set of structures. In particular, PET and SPECT are powerful means of probing neural responses as they depend on systemic delivery of appropriate radiolabeled tracers with selective accumulation in various CNS structures. Examples of

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tracers for neuro-imaging applications include [18F]-fluorodeoxyglucose ([18F]-FDG), a glucose analog that accumulates selectively in highly active cells such as neurons, and [11C]-raclopride, a dopamine analog that can be used to explore brain domains that support motor activities. A series of two-dimensional (2D) images is acquired over a few minutes and then reassembled into a “z-stack” using an automated algorithm to produce a 3D representation showing where the tracers have localized, albeit at low resolution. A more precise determination of the anatomic localization of the tracers is achieved by aligning the functional data sets from PET and SPECT in register with high-resolution 3D images from CT or MRI. Further details on the principles and practical application of all these modalities may be found in In Vivo Small Animal Imaging: A Comparison with Gross and Histopathologic Observations in Animal Models, Chapter 9. Stereology Precise and unbiased quantitative assessment of the distribution, orientation, number, shape, or size (i.e., volume) of objects in three dimensions (3D) is an increasingly important approach for identifying neurotoxic agents. The attraction of this strategy is that 3D stereological methods are quite sensitive to abnormalities in the number of a given class of structures (e.g., neurons, neuronal processes), which generally is not the case for macroscopic and microscopic (including 2D morphometric) procedures that form the basis of conventional neuroanatomic examinations for toxicant-induced neuropathology. The primary disadvantage when using stereology is that the processing and analysis is a cumbersome, lowthroughput approach. Recent improvements in stereological theory, sampling protocols, digital imaging systems, and automated analytical software have overcome this difficulty, substantially enhancing the efficiency and speed with which such data can be acquired from animals (see Stereological Principles and Sampling Procedures for Toxicologic Pathologists, Chapter 8). Another disadvantage remains, which is the inability to easily translate stereological methodology from animal subjects to human patients. Nonetheless, stereology will see increasing use in neurotoxicity testing when 3D quantitative data will be preferable to either 2D quantitative (i.e., morphometric) or qualitative analyses.

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Stereology generally is undertaken in neurotoxicity testing in two settings. The first is as a retrospective (post hoc) method if a sufficient weight of evidence has been generated to suggest that numbers of neural cells or neural cell processes have been modified following exposure to a xenobiotic. In this context, stereology can identify sites of vulnerability that were not evident using routine macroscopic and microscopic evaluations. The second scenario is as a prospective procedure when an agent or a structurally related compound has a known propensity to impact neural structure or function. Here, stereology helps to better define the potential for subtle neural defects. Structures that are selected most commonly for stereological analysis are domains that are common sites for neurotoxic damage, such as the cerebral cortex, corpus callosum, basal nuclei (especially the corpus striatum), hippocampus, and cerebellum in the CNS, and various ganglia and somatic nerve trunks in the PNS. In general, stereology in the toxicologic neuropathology setting focuses on two tasks. The first is obtaining cell counts, typically of neurons, using either an optical disector/fractionator or a physical disector method. The other is providing volume estimates using a Cavalieri-based procedure. For example, a physical disector technique can be undertaken at a given CNS site by counting approximately 100 objects in 25–50 fields within 6–8 serial, 3 mm to (more commonly) 10 mm thick disectors (where a disector is a set of two adjacent sections, and objects are counted only if they are present in one section). Production of the disector sets takes about 5 hours per rodent brain: 4 hours for serial sectioning and 1 hour for systematic uniform random sampling (SURS) at 400. In contrast, the optical disector approach usually is undertaken on thick (40þ mm) frozen sections, where the disector is a pair of adjacent focal planes (as defined in the z [i.e., up-and-down] axis). While slow relative to standard neuropathology evaluation, the discriminative power of quantitative neuroanatomical analysis increases as one moves from gross assessments (e.g., brain weight measurement) to simple microscopic endpoints (e.g., acquisition of areal or linear dimensions) to enumeration of specific objects (stereology). The theoretical basis and practical application

of stereology to toxicologic pathology endpoints may be explored more fully in Stereological Principles and Sampling Procedures for Toxicologic Pathologists, Chapter 8.

3.6. Model Systems for Neurotoxicity Research As with other systems of the body, the normal biology and consequences of toxic injury in the CNS and PNS may be investigated using various model systems. The most common options are in vivo and in vitro experiments in living tissues (see below). In Vivo Studies Testing in living subjects, whether animal or human, is a common means by which new neurotoxic agents are identified and information needed for risk management is gathered. The appeal of in vivo models is founded on the ability to more easily extrapolate such data to protect the health of human populations. Neurotoxicity testing is based on the premise that any adverse anatomic, biochemical, or functional effects that develop in the nervous system of animals after exposure to a particular agent would also be likely to occur in exposed humans, in a qualitative sense if not in a quantitative fashion. Accordingly, neurotoxicity bioassays are designed to discover target sites (i.e., cell populations or organ subregions), elucidate dose–response relationships, catalog similarities and differences in sensitivity among species, and determine mechanisms of toxicant action. Another important aspect of neurotoxicity testing is to define any special attributes of certain classes of individuals who are likely to have nervous systems that are particularly vulnerable to neurotoxic agents. Examples of such classes are developing animals (as neural cells and circuits are established over an extended period [Figure 52.1]) and senescent organisms (who theoretically have less functional reserve available to compensate for neurotoxic episodes). The bulwark of conventional hazard identification and risk assessment in vivo involves toxicity studies in young adult rats. Animals typically are enrolled on studies at 2–3 months of age, and exposures occur over standard lengths of time (usually 4, 13, or 26 weeks).

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The neural evaluation may occur as a survey of selected CNS and PNS functions and structures in conjunction with surveys of other major body systems (see Pathology in Non-Clinical Drug Safety Assessment, Chapter 24). Such general toxicity studies are arranged to screen for pronounced effects in major neural regions using a stereotypical battery of endpoints: in-life observations, collection of gross findings and the brain weight at necropsy, and histopathological assessment of a few critical CNS and PNS regions (Table 52.2). If adverse neural events do not appear to be present using these measures, additional neurotoxicity testing generally is not performed. However, enhanced neurotoxicity testing is undertaken if an agent either impacts a neurological parameter (anatomic especially, but also functional) or has a molecular form that suggests a structure-activity relationship (SAR) to known neurotoxicants. Such enhanced testing adds additional endpoints to the screen (e.g., expanded behavioral testing, electrophysiology, neurochemical measurements, and sections for neurohistological evaluation) to probe the operational integrity of the nervous system more fully. Developmental neurotoxicity testing (DNT) is a related but separate strategy that examines the special susceptibility of the immature nervous system. Again, rats are the preferred species for testing due to their fecundity and short gestational period. In general, pregnant animals are exposed to a test article from gestational day 6 (the approximate time at which the rat blastocyst implants in the uterine wall) through postnatal day 11 (the approximate end of major neuronal production in the rat) or postnatal day 21 or 22 (the conclusion of neuronal and glial expansion as well as initial circuit generation and myelination). Since the main purpose of DNT is to examine the nervous system, the battery of endpoints is broader than that used for CNS and PNS screening in general toxicity studies for adult rats. A special feature of DNT that must be remembered when designing the statistical analysis is that the appropriate experimental unit is the litter, since the dam is the individual that has been exposed to the agent (see Statistical Assessment of Toxicologic Pathology Studies, Chapter 30). Although rats are the most common test species for evaluations of neurotoxicity,

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considerable difficulties may arise in selecting the most suitable species in terms of predicting specific effects in humans. In most cases, at least one animal species will mirror the types of neurotoxic effects seen in people, while in other species tested the changes may be different in kind, altered in magnitude, or absent altogether. For example, organophosphate-induced delayed neuropathy (OPIDN) develops in humans and hens, while rodents are relatively insensitive to this response. The fact that different species react in diverse ways to the same agent is probably due to species-specific variations in neural anatomy (especially barriers), developmental timing, pharmacokinetics (both inside and outside the nervous system), and/or inherent pharmacodynamic differences. Since the extent and importance of these interspecies discrepancies often is incompletely or even totally unknown, any neurotoxic effect seen in animals is presumed to portend a risk to humans even though effects seen in animals may not be the same as those produced in humans. Consequently, a clear understanding of the pathogenesis and the mechanism(s) of neurotoxicant action that lead to neural damage are critical to produce the best risk assessment decisions (see Risk Assessment, Chapter 31). Genetically engineered models (usually mice) comprise a growing resource for in vivo neurotoxicity testing and defining mechanisms of action. One advantage of these models is that they have been altered to express a human gene, and thus may serve as a “humanized” model for probing the importance of that gene to the progression or amelioration of neurotoxic events. For instance, several lines of transgenic and knockout mice have been engineered to recapitulate the biochemical, behavioral, and neuropathological lesions of Alzheimer’s disease (AD), a neurodegenerative condition that is currently attributed to accumulation of the neurotoxic b-amyloid protein fragment near neurons in the associative domains of the cerebral cortex (see Genetically Engineered Animals in Product Discovery and Development, Chapter 12). The neurotoxicity analysis of such models typically should be magnified beyond conventional functional and neuropathology tests to include one or more additional

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techniques targeted to particular elements (established or hypothetical) that are specific to the pathway that involves the engineered gene. For instance, for the Alzheimer’s-like mouse models, a common approach is special staining to highlight the distribution and size of plaques (extra-neuronal deposits of amyloid) in the cerebral cortex and hippocampus. The plaque burden then can be estimated semi-quantitatively (minimal, mild, moderate, or marked, numbers, or none) or quantitatively (by actually measuring plaque numbers or dimensions). The inclusion of additional endpoints is important in choosing among the various transgenic models of AD, as one model develops the functional and structural lesions typical of AD in humans while other models exhibit behavioral deficits or pathological changes, but not both. Invertebrate animal models (e.g., the hydra [Hydra vulgaris] or the nematode Caenorhabditis elegans) have seen increasing use in mechanistic neurotoxicology studies. A substantial benefit when using these platforms is that their simple nervous systems have been mapped in detail, so neurotoxicant-induced structural and cellular changes can be evaluated with relative ease. An added advantage of these models is that simple behavioral measurements can be included to probe the functional implications of xenobiotic exposure. In Vitro Experiments The complex anatomic (including barriers), functional, and compensatory capabilities of the intact nervous system often muddle efforts to interpret neurotoxicologic studies in experimental animals. Therefore, less complicated in vitro models have been developed to investigate specific mechanisms of neurotoxicological relevance. In vitro neural systems include whole embryos (including in ovo exposure of chicken eggs as well as isolated mammalian embryos), whole organs (e.g., dorsal root ganglia), explants (e.g., brain slices), neural micromass or dissociated primary cell cultures, and immortal neural cell lines. The usual donors for primary specimens (cells, micromass samples, tissue slices, and whole embryos) are mice or rats; in the hands of an experienced technician, a single donor can supply sufficient material to test

multiple agents or doses. Immortal neural cell lines have been derived from a number of species, including humans. Incorporation of in vitro models offers several advantages for mechanistic neurotoxicological research. While in vivo tests are unsuitable for screening large numbers of agents, in vitro tests can be employed to comprehensively screen the potential toxicity of numerous materials toward specific cell types of the brain in short periods of time. In vitro models are easily defined and reproduced. Potential neurotoxicants or their metabolites may be studied individually or in combination for direct neurotoxic effects on specific cell populations, including those of human origin. Potential antagonists or other protective agents (including addition of cell homogenates from other organs, like liver, to provide metabolic capacity to the system) may be tested as modifiers of an agent’s neurotoxicity. Various chemical and molecular parameters (e.g., enzyme activity, gene expression, neurotransmitter release, receptor binding assays) are common endpoints for many in vitro studies, as they are easily collected and quantified. However, “gross” and histological changes, as well as special stains for cellular function, can be used to perform a “neuropathological” examination of in vitro models. Neurotoxicant classes that have been studied extensively using in vitro methodologies include biotoxins, heavy metals, pesticides, pharmaceuticals, and solvents. In Silico Investigations Computer modeling based on in vivo and/or in vitro data is proving to be a promising new tool for defining novel mechanisms of neurotoxicity and identifying possible neurotoxic agents. In silico simulations have been used chiefly to explore structure–activity relationships (SAR) between known neurotoxicants and molecules of unknown neurotoxic potential, and to examine the interaction of prototypic neurotoxicants and particular target molecules. An extension of such studies has been to deliberately design drugs that retain their efficacy but have greatly reduced neurotoxicity. A detailed discussion of this topic is outside the scope of this chapter, so interested researchers are referred to the Suggested Reading list to find additional resources on this topic.

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4. RESPONSE TO NEUROTOXIC INJURY

4. RESPONSE TO NEUROTOXIC INJURY The ability of toxic agents to disrupt neural activities depends on many parameters. These factors typically are specific to given entities, such as particular regions or cell populations or subcellular structures. This section describes the most common responses of the nervous system to toxic injury. Frequent artifactual changes are defined where relevant so that they will not be misinterpreted as evidence of neurotoxicity.

4.1. Anatomic Lesions In general, structural damage that manifests in the nervous system may be divided using one of two fundamental approaches. The first is to catalog anatomic changes resulting from injury to a specific cell population (e.g., neuron, myelinating element) or cell part (e.g., axon, synapse). The terms applied to such conditions are commonly designated in broad terms using the name of the targeted cell or cell part followed by the suffix “-opathy.” The second option is to diagnose alterations based on the class of lesion that they represent (inflammation, neoplasia, etc.). These basic categories of neural lesions (Table 52.1) are briefly described below. More detailed consideration of individual lesions and their possible mechanisms may be found by perusing the Suggested Reading list. Cell Type-Specific Lesions NEURONOPATHIES

Certain toxicants attack specific groups of neurons, usually residing within the CNS. The lesions have been characterized chiefly using rats, but humans typically are vulnerable as well. For instance, 3-acetylpyridine irreversibly destroys the (inferior) olivary nuclei, thus inducing cerebellar ataxia by decimating the climbing fibers leading to the cerebellum. The neurotoxic effects of MPTP are confined to the dopaminergic neurons of the substantia nigra, resulting in cell death in humans and rats (with humans representing the far more sensitive species). The NMDA antagonist MK-801 (dizocilpine maleate) induces peracute (within hours) vacuole formation in the neurons of the caudal cingulate/retrosplenial cortex of rats. A constellation of toxins has been linked to the induction

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of amyotrophic lateral sclerosis/Parkinsonismdementia complex (ALS-PDC) in Chamorro natives of Guam, with the expanded range of symptoms being attributed to variable mixtures of toxicants that act individually to target neurons in the cerebral cortex, the substantia nigra, and the spinal cord. One of the key toxins thought to be responsible for ALS-PDC is bmethylamino-L-alanine (BMAA), a product of cyanobacteria that live symbiotically with cycad trees; this agent contaminates flour from cycad seeds and is bioconcentrated in tissues of seedeating bats, two staples of the Guamanian diet. In many instances, the sensitivity of particular neuronal groups to toxic agents first is discerned via their distinctive patterns of functional alterations and/or neurochemical changes rather than by morphological analysis. The basic neuropathology findings associated with a neuronopathy depend on the length of time during which the toxic injury has been taking place. Degeneration is considered by some to represent a more acute change, as the major features of the affected cells are readily distinguished despite the presence of minimal to marked abnormalities of certain subcellular structures. For instance, granule neurons in the caudal cingulate/retrosplenial cortex targeted by MK-801 clearly contain fine cytoplasmic vacuoles (often termed the Olney lesion after its discoverer) within hours of exposure (Figure 52.27), but their nuclei and cell membranes are intact. Neuronal vacuolation is caused by expansion of intra-neuronal cytoplasm or membrane-bound organelles as a consequence of retained fluid or metabolic by products. In principle, degenerative changes may be reversed if exposure to the toxicant ceases in time and/or the dose was below the lethal threshold. In practice, however, damaged neurons often progress to irreversible necrosis (Figure 52.28). The hallmark features of toxicant-induced neuronal necrosis are contraction of the cell (leaving a clear, pericellular halo); condensation and/or fragmentation of the nucleus; and loss of the Nissl substance, leading to hypereosinophilic cytoplasm. In particular, the latter feature is the classic trait of the “red dead” necrotic neuron (Figures 52.22 and 52.28). Genuine neurodegenerative lesions often harbor an array of dying cells, with features ranging from acutely injured to dead and disintegrating.

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FIGURE 52.27 Peracute neuronal degeneration may present initially as clear, round, cytoplasmic vacuoles (arrows) in affected cells. In this case, the change is evident in granule neurons within the retrosplenial cortex of an adult rat 6 hours after it had been given MK801, an excitotoxic antagonist of the N-methyl-D-aspartate (NMDA) glutamate receptor. This pattern is often termed the “Olney lesion” after its discoverer, American neuropathologist John Olney. Interpretations of this lesion range from a transient perturbation to a precursor of neuronal necrosis to an artifact of aldehyde fixation. Processing conditions: formalin fixation by intravascular perfusion, paraffin embedding, H&E staining.

When present in minimal numbers, special stains commonly are employed to selectively highlight dead neurons. The usual choices for this purpose are methods like Fluoro-JadeÒ (performed on formalin-fixed, paraffin-embedded tissue sections) and amino cupric silver stains (done in thick frozen sections of unfixed tissue) (Figure 52.25). With time, dead neurons may attract complements of microglia that help to remove the cellular debris in a process termed neuronophagia (Figure 52.29). The end-stage lesion of extensive necrosis within an affected population is cell depletion. Neuronal heterotopia (or ectopia) is a distinct category of neuronal lesion. Instead of cell loss, the fundamental change is a developmental disturbance leading to abnormal migration and/or terminal differentiation. A common presentation for ectopic neurons is to see clusters of cells located in atypical sites, leading to disruption of the normal arrangement of neurons. The misplaced cells exhibit the typical features of the neural site at which they normally would have been found. The most frequent locations for these changes are the cerebral cortex,

FIGURE 52.28 Acute neuron degeneration (“neuronal necrosis”) presents as cells with shrunken, condensed (i.e., pyknotic) or fragmenting (i.e., karyorrhectic) nuclei surrounded by brightly eosinophilic cytoplasm. These “red dead” granule neurons in the retrosplenial cortex were evident 24 hours after treating an adult rat with the excitotoxic agent MK-801, an antagonist of the N-methyl-D-aspartate (NMDA) glutamate receptor. Processing conditions: formalin fixation by intravascular perfusion, paraffin embedding, H&E staining.

FIGURE 52.29 Satellitosis of small glial cells around toxicant-damaged neurons typically indicates either a final attempt by oligodendrocytes to support degenerating but still living cells or early efforts by microglia to begin scavenging debris from dead cells. This image, from an adult ox exposed to an organophosphorus insecticide, represents the microglial response. Processing conditions: formalin fixation by immersion, paraffin embedding, H&E staining. This image was kindly provided by Dr Patrick Nation, Animal Pathology Services Ltd, Edmonton, Alberta, Canada and obtained from the Noah’s Arkive database of veterinary pathology lesions: http://www.vet.uga.edu/vpp/ noahsarkive/na.php; image no. 4134.

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FIGURE 52.30 Neuronal heterotopiae (ectopia) results from defective migration and/or differentiation of neuronal precursors following developmental exposure to a neuroteratogen. Left: This 5.5-month-old, neurologically normal NSG mouse (i.e., non-obese diabetic [NOD] scid gamma, or NOD.Cg-Prkdcscid Il2rgtm1Wjl/ SzJ) received whole-body irradiation on postnatal day 1 and developed multiple major abnormalities in the laminar organization of the cerebellar cortex: retained external granule cell clusters in the molecular layer (arrows), displacement of numerous Purkinje cells into the granule cell layer and foliar white matter, and reduced thickness and many fewer neurons for the granular cell layer. Right: This age-matched C57BL6/J control mouse exhibits the normal organization of the cerebellar cortex. The presence of a persistent focus of external granule cells under the meninges (arrow) is a common incidental finding in mice. Processing conditions: formalin fixation by immersion, paraffin embedding, H&E staining. This specimen was kindly provided by Dr Stefan Niewiesk, The Ohio State University, Columbus, Ohio, USA.

hippocampus, and cerebellum (Figure 52.30); any layer or multiple layers may be affected. Minor heterotopiae often are seen as isolated cell clusters in macroscopically unaffected brains, while major lesions frequently occur together with overt brain malformations like hydrocephalus or microcephaly. Aberrant anatomic development often but not always is accompanied by functional alterations. Alcohols like ethanol, methanol, and methylazoxymethanol (MAM) are potent disruptors of neural cell migration. Neuronal storage diseases result from accumulation of materials in affected cells. Many storage diseases result from inborn genetic defects that lessen or prevent production of an enzyme required for normal metabolism. In the absence of the functional enzyme, its substrates accumulate within organelles (e.g., Golgi apparatus, lysosomes) or the cytoplasm. Toxic causes of neuronal storage diseases include cationic amphophilic drugs, which lead to phospholipidosis, and chronic ingestion of swainsonine (the toxic principle in locoweed). Lesions in mild

(usually acute) cases of neurotoxicant-induced neuronal storage disease may be reversible if the exposure is halted, but severe (chronic) lesions usually progress to full-blown cell engorgement (Figure 52.31) and eventually to neuronal degeneration. AXONOPATHIES

Other toxicants do not attack neurons, but rather damager their processes. These lesions may develop within the CNS, where they are typically confined to specific tracts, or the PNS. In many instances, neurotoxicants with this capability were first discovered following occupational exposures in humans. Three basic lesion patterns can develop. The first is primary axonopathy, which is produced when the axon itself is the major site of damage. Examples include agents that crosslink macromolecules in distal axons, like acrylamide, carbon disulfide, and n-hexane. The fiber proximal to the xenobiotic-induced lesion will remain intact, while the fiber distal to the

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FIGURE 52.31 Drug-induced phospholipidosis (DIPL) is a neuronal storage disorder in which multi-lamellar (“myeloid”) bodies fill the cytoplasm of large neurons after chronic ingestion of a cationic amphophilic drug (CAD). Left: Typical light microscopic (LM) lesions are pallor and swelling of the cell body. Hippocampus field CA1 of an adult rat given an undeclared CAD for an indeterminate period. Processing conditions for LM: formalin fixation by immersion, paraffin embedding, H&E staining. Right: Transmission electron microscopy (TEM) is needed to view the myeloid bodies (evident as spaces partially or completely filled with electron-dense, laminated whorls or cores). Cytoplasm of a neuron from the medulla oblongata of a Beagle dog gavaged with the CAD posaconazole (30 mg/kg for 52 weeks). Processing conditions for TEM: formalin fixation by immersion, other steps specified as “conventional” but not detailed. The TEM image is reprinted from Cartwright et al. (2009) Phospholipidosis in neurons caused by posaconazole, without evidence for functional neurologic effects, Toxicol. Pathol. 37, 902–910, with permission.

lesion will disintegrate since it has lost its connection to the neuron. The primary changes affecting the distal axon are fragmentation followed by the formation of elliptical digestion chambers that hold cellular debris (Figure 52.32). This appearance should be referred to as Wallerian-like degeneration, as it resembles Wallerian degeneration, but the genuine Wallerian lesion is a consequence of physical (e.g., trauma) rather than chemical transection of the nerve. Fragmentation may begin within hours of axonal injury, but in practice degeneration may require a day or more to appreciate with the light microscope. Over time, macrophages of hematogenous origin (often termed gitter cells) are drawn to the site to phagocytize the axonal fragments (Figure 52.33). The next pattern is secondary axonopathy, which occurs when the capacity of the neuronal body to export materials is compromised below the minimal level needed to sustain its axon and/or nerve terminal. For instance,

b,b0 -iminodipropionitrile (IDPN) exposure induces a proximal axonopathy because it interferes with slow axonal transport. Continued inability by the neuronal cell body to supply its processes will result in gradual progression of the degenerative changes to more proximal portions of the axon (termed “dying back”); bipolar primary sensory neurons having both central and peripheral axons may exhibit this change in both processes simultaneously. Morphologic features of the neuronal body and proximal axon may remain morphologically normal even as the changes in the distal axon grow in their extent and severity. The third pattern is neuroaxonal dystrophy, which is characterized by axonal expansion (termed spheroids). In general, widespread neuroaxonal dystrophy is more common as an inherited or acquired neurodegenerative disease in humans and animals than as a consequence of toxicantinduced damage, although a few agents (e.g.,

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FIGURE 52.32 Primary axonopathies resulting from xenobiotic exposure begin as axonal degeneration (here evident as a swollen fiber [D]) before progressing to axonal disintegration with collection of debris and phagocytic macrophages (“gitter” cells) within dilated spaces (termed “digestion chambers” [C]) confined within the original fiber tract. Advanced lesions (A) result in removal of the myelinating cells as well, leaving a nearly empty fiber tract bordered by the remnant basal lamina (visible as hypereosinophilic lines by H&E). In peripheral nerves, proliferating Schwann cells (S) will fill fiber tracts with an intact basal lamina to provide a bed through which the regenerating axon will extend. In most instances, small (so-called “unmyelinated”) axons (arrows) are not affected by toxicants. Left: Unspecified peripheral nerve of an adult rat given acrylamide for an extended period. Processing conditions: formalin fixation by immersion, paraffin embedding, H&E staining. Right: Sural nerve of a human patient who developed acute ascending polyradiculoneuropathy after combined intravenous and intrathecal chemotherapy to treat leukemia. Processing conditions: formalin fixation by immersion (presumptive), epon embedding, toluidine blue staining (presumptive). The right figure is reprinted from Rison (2008) Ascending sensory motor polyradiculoneuropathy with cranial nerve involvement following administration of intrathecal methotrexate and intravenous cytarabine in a patient with acute myelogenous leukemia: a case report,” Cases J. 1, 255, with permission.

aluminum, methylmercury) can produce spheroids as one element of their pathological presentation. These enlarged fibers appear as large, fairly homogeneous, elliptical swellings by light microscopy, which are shown to harbor a tangled web of cytoskeletal elements and cellular organelles by electron microscopy. The swellings are eosinophilic when stained with H&E and black when viewed in silver-impregnated sections (Figure 52.34), and they are much larger in

diameter than adjacent “normal” axons. The proposed pathogenesis is a disturbance in retrograde axonal transport that results in progressive accumulation of less flexible materials at points of axonal constriction (i.e., the nodes of Ranvier, which are the gaps formed between the edges of adjacent myelinating cells). Regeneration of PNS axons can occur once the axonal damage has been cleared provided the endoneurial tube is intact. The Schwann cells

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FIGURE 52.33 Destruction of large expanses within the nervous system results in liquefactive necrosis, in which the degenerating lipid-rich cells and cell processes are reduced to fluid and then removed by an influx of myriad macrophages (termed “gitter” cells). Upper: Optic nerve malacia in an adult rat, with gitter cells collecting in the cavity. This lesion arises as an iatrogenic (experimenter-incited) infarct induced by traumatic blood collection from the retro-orbital venous plexus. White vacuoles in the lower right periphery of the nerve are fiber tracts in which the axons have been lost. Lower: Gitter cells have the characteristic traits of large, histiocyte-derived phagocytes: large, oval to reniform (kidney-shaped), often eccentric nuclei with abundant, pale eosinophilic, often vacuolated cytoplasm and distinct cell borders. These cells were congregating in a cystic cerebral lesion in a non-human primate. Processing conditions: formalin fixation by immersion, paraffin embedding, H&E staining.

proliferate to form solid columns of cells (termed bands of Bu¨ngner) that reach from the site of the axonal lesion to its distal termini. Subsequently, the proximal axon will grow distally into these Schwann cell columns, which guide the axon as it travels out to re-establish its link with the sites that it originally innervated. The regenerative process in the axon requires a transient boost in macromolecular synthesis (mainly proteins) in the neuronal body. This need is accommodated by dispersion of the Nissl substance and translocation of the nucleus to the cell periphery, a change termed central chromatolysis. This repair response in the neuronal cell body is termed the axonal reaction, although it is very common to observe axonal degeneration without seeing any associated changes in the neuronal cell body. Regeneration of axons proceeds at the same velocity as slow axonal transport, and thus is limited to several millimeters a day. Accordingly, full functional recovery takes weeks to months for distant effector organs, especially in larger species. In general, axonal regeneration of CNS axons is ineffective. These axonal changes may be recognized in sections stained routinely with H&E, but in many instances special procedures are undertaken to provide additional contrast. The techniques may highlight the axon itself, either by silver impregnation (e.g., Bielschowsky’s [Figure 52.34] or Bodian’s stains) or by immunohistochemical (IHC) labeling to detect an axonal marker (e.g., neurofilament protein [NFP], which is one constituent of the cytoskeleton). Alternatively, the procedure may label the myelin sheath using a histochemical stain (e.g., LFB) that stains lipid, an IHC method to localize a myelin marker (e.g., myelin basic protein [MBP]), or metal enhancement to increase the contrast between the axon and its sheath (e.g., post-fixation in OsO4). These myelin-detecting procedures may serve as effective means to indirectly highlight primary axonal damage (Figure 52.35). A definitive means of differentiating between the axon and myelin as the primary target site is to isolate individual fibers in teased preparations. Axonal lesions are characterized by the absence of the fiber with multifocal attenuation of the Schwann cell profile over long distances, while primary myelin damage leads to segmental loss of the myelinating cell with retention of the naked but intact axon (Figure 52.36).

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FIGURE 52.34 Axonal expansions termed “spheroids” are the characteristic feature of toxicant-induced neuroaxonal dystrophies. The proposed pathogenesis is reduced axonal transport leading to expanding snarls of cytoskeletal proteins lodged at the narrow nodes of Ranvier. The dilated axons are large, fairly homogeneous, elliptical swellings (arrows) which are eosinophilic when stained with H&E (left) and black when highlighted by silver stains (right; Bielchowsky’s method). Processing conditions: formalin fixation by immersion (left) or intravascular perfusion (right), paraffin embedding. SYNAPTOPATHIES

The nervous system houses trillions of synapses, with each neuron playing host to many thousands of them. Their minute size prevents synapses from being observed in standard light microscopy sections. Nonetheless, ultrastructural lesions can be observed in synapses following exposure to certain types of neurotoxicants. One major change in experimental neurotoxicity reports is altered membrane maintenance and turnover in axonal terminals (Figure 52.37). Lesions may affect either the presynaptic or the postsynaptic elements. Agents capable of altering membrane structure include chloromycetin, which targets cerebrocortical motor neurons; diphenylhydantoin (DPH), which impacts cerebellar neurons; and 3,4-methylenedioxymethamphetamine (MDMA), which transforms monoaminergic neurons in the corpus striatum. Common anomalies include thickening of the synaptic membranes or formation of membranous inclusions in the axon terminals. The progressive nature of the inclusions that characterize the DPH-induced lesion suggests that their pathogenesis begins with accretion of interconnected tubules in gradually expanding axon terminals, followed by the build-up of inclusions and the eventual formation of axonal spheroids. Ultimately, the affected neurons undergo coagulation necrosis, seemingly as a consequence of

excessive synaptic degeneration. The membranous inclusions in the MDMA-injured cells also contain ubiquitin, which provides added confirmation that this finding is an outcome of significant damage to cell membranes. Other ultrastructural lesions induced by neurotoxicants have also been observed as precursors of subsequent degenerative changes within CNS synapses. For instance, adult chickens treated with tri-ortho-cresyl phosphate (TOCP) develop massive swelling of synaptic vesicles in the axon terminals of axo-somatic synapses within the gray matter of the spinal cord ventral horn as early as 1 day after exposure. Similarly, simultaneous incubation with amyloid-beta (Ab), a protein fragment of amyloid precursor protein (APP) thought to be responsible for the neural lesions in advanced Alzheimer’s disease, and ryanodine induces mitochondrial swelling and a severe reduction in the numbers of small synaptic vesicles and synapse-anchored proteins like synaptophysin and actin in rat cerebrocortical synaptosomes. Depletion of actin at the synapses can be reversed by incubation with caspase inhibitors, suggesting that extended abnormalities of synaptic structure can invoke the apoptotic pathway. MYELINOPATHIES

Toxicant-induced damage to myelin can develop within the CNS or PNS, depending on

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FIGURE 52.35 Myelin integrity as well as the contrast between axons and their myelin sheaths are both improved by metal enhancement using post-fixation in osmium tetroxide (OsO4). Relative to a control nerve with its many large fibers with uniform axonal diameters and thick myelin sheaths (left), nerves with severe primary neuropathy exhibit substantial loss of large fibers and attenuation of their myelin sheaths (right). Unspecified nerve from an adult rat given an unidentified neurotoxicant. In comparison, small-caliber (“unmyelinated”) fibers are relatively spared. Processing conditions: formalin fixation and OsO4 post-fixation by immersion, paraffin embedding, H&E staining. This image was kindly provided by Dr William Valentine, Vanderbilt University, Nashville, Tennessee, USA.

whether the population of injured cells is the oligodendrocytes or the Schwann cells. From a practical standpoint, however, the more important consideration usually is to define the manner in which the lesion is incurred. Primary demyelination is produced when myelin sheaths are the primary target of the insult. In such instances, the axons are not harmed, and accordingly are preserved intact within the disintegrated myelin sheath. The classic agent responsible for inducing this effect is tellurium. The typical appearance of white matter tracts or nerves with primary demyelination is swelling of the affected sheath segments (where each segment is the product of a single cell) with formation of myelin bubbles (also

termed balloons or blebs). These bubbles may be distinguished from digestion chambers produced in axonopathies because the bubbles surround continuous (i.e., viable) albeit shrunken axons in close association with myelin debris and macrophages. Primary demyelinating insults generally result in a fairly diffuse distribution of white matter damage, which is readily detected in conventional tissue sections that have been processed to demonstrate myelin in white matter tracts (Figure 52.38). A variant of primary myelinopathy is myelin edema, resulting from fluid accumulation within the myelin sheaths. Hexachlorophene is the prototypic neurotoxicant that induces this lesion. Myelin edema can occur in both the CNS and

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FIGURE 52.36 Teased fiber preparations offer a definitive means of differentiating between the axon and the myelin sheath as the target site for neurotoxicant activity. Axonal degeneration (upper strand; unidentified neurotoxicant) leads to axonal loss (arrows) with secondary attenuation of the myelin sheaths at multiple points over extended distances. In contrast, primary myelin damage (lower strand; 1% disulfiram in the diet for 4 weeks) results in segmental loss of the myelin sheath (i.e., the length supported by a single Schwann cell [located between the two arrowheads]) while leaving the axon intact. Unaffected fibers (middle strand) have smooth continuous myelin sheaths interrupted only occasionally by nodes of Ranvier (asterisk). Isolated fibers from caudal (posterior) tibial nerves of adult rats. Processing conditions: glutaraldehyde fixation by intravascular perfusion followed by osmium tetroxide post-fixation by immersion. These photographs were provided by Dr William Valentine, Vanderbilt University, Nashville, Tennessee, USA. The bottom two images have been reproduced with minor modifications from Tonkin et al, (2000) Disulfiram produces a non-carbon disulfide-dependent Schwannopathy in the rat, J. Neuropathol. Exp. Neuro.l 59: 786–797, with permission.

PNS, and usually appears as vacuoles within myelin-rich regions in the absence of myelin degeneration. The volume expansion caused by the increased fluid leads to splitting of the myelin lamellae. Mild cases may be reversible to some extent, but pronounced or chronic lesions typically will lead to secondary axonal damage. Secondary demyelination results from irreversible degradation of a myelin sheath following the loss of its axon. The microscopic appearance of myelin bubbles in this scenario includes loss of the axon, indicating that the myelin disintegration merely follows the prior axonal damage. This conversion of myelin lipoprotein to fully degraded fat typically takes about 2 weeks. Regeneration of myelin sheaths generally is confined to the PNS, and can occur fairly rapidly since it arises from cell precursors already in

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place at the lesion site. In the PNS, each Schwann cell envelops a single axon, so damage to the Schwann cell will lead to segmental loss of myelin (Figure 52.36) that is more severe at the level of the spinal nerve roots (i.e., where the axons are of larger caliber and thus are encompassed by longer and thicker myelin sheaths). As remyelination proceeds, each gap is filled by a greater number of Schwann cells producing a series of shorter segments with thinner sheaths than were present in the original. GLIOPATHIES

In many respects, non-myelinating glia are more resistant to neurotoxic insult than their neuronal and myelin-producing neighbors. Injured glial cells typically respond by swelling. In most instances, the affected elements are astrocytes. Lesions of minimal to moderate extent are often reversible if the cause of the change is removed. The usual appearance of swollen astrocytes is that of cell expansion and/or vacuolation (Figure 52.39), the outcome of fluid or material accumulating in the cytoplasm or a membranebound organelle. The change occurs most frequently in the brain, and presents most prominently in the gray matter. Microscopically, the parenchyma of this region is riddled with small holes, some of which appear to compress nearby neurons. In general, the effect exhibits bilateral symmetry. The formation of Type II astrocytes embodies an unusual variant of astrocyte swelling. This cytotoxic response results from nuclear expansion following exposure to elevated levels of nitrogenous waste products (the chief of which is thought to be ammonia); thus, these elements represent a secondary toxic response to primary hepatic disease. Affected astrocytes have swollen clear nuclei with very thin rims of marginated heterochromatin, sometimes with enlarged nucleoli, and indistinct cytoplasm (Figure 52.40). These cells tend to accumulate in the neocortex, basal nuclei, and hippocampus. GLIAL REACTIONS TO NEURAL INJURY

Glia play prominent roles in the repair response following toxicant-induced damage to other populations of neural cells. The main responders are the non-myelinating cells of the CNS – astrocytes (i.e., true glia) and microglia (i.e. the bone marrow-derived resident

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FIGURE 52.37 Synaptic pathology induced by neurotoxicants in either presynaptic or the postsynaptic terminals must be evaluated by transmission electron microscopy (TEM). These electron micrographs compare synaptic and axonal morphology in the molecular layer of the CA3 region in the hippocampus from control (A, C) and lead (Pb)exposed (B, D) Wistar rats at 21 (A, B) and 30 (C, D) days of age, following maternal Pb ingestion (0.2% Pb acetate administered in the drinking water) during postnatal days 1 to 21. (A, C) Synapses (arrows) in control rats have many vesicles and mitochondria in their presynaptic terminals, and the opposing membranes of the presynaptic and postsynaptic cells are equally electron-dense. (B, D) Exposure to Pb has markedly reduced synaptic numbers but has not affected synaptic structure. Older Pb-exposed rats (D) also exhibit swollen dendrites and dense granules (Pb deposits) within the cytoplasm of glial cell processes. Processing conditions: immersion fixation in 4% glutaraldehyde followed by 1% osmium tetroxide, embedding in epoxy resin, sectioning at 70 mm, staining with uranyl acetate and Pb citrate. Scale bar ¼ 500 nm. These images have been reproduced with minor modifications from Rahman et al. (2012) Overactivation of hippocampal serine/threonine protein phosphatases PP1 and PP2A is involved in leadinduced deficits in learning and memory in young rats, NeuroToxicology 33, 370–383, with permission.

histiocytes of the CNS) – but oligodendrocytes and Schwann cells also participate in such efforts. Hypertrophy and hyperplasia are the main reactions by CNS glia to neural damage. The generic term for this change is gliosis, which implies enhanced production of multiple cell lineages, but cell type-specific increases (e.g., astrocytosis,

microgliosis) are also possible. The presentations and functions served by these reactions are quite distinct. Astrocytosis occurs to fill or encompass fairly large damaged regions in the CNS, usually produced as a consequence of neuronal or pancellular necrosis. Reactive (i.e., Type I) astrocytes are recognized in H&E-stained sections as large, stellate cells with large, pale nuclei and scant to

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FIGURE 52.38 Distinct patterns of neurotoxicant-induced white matter vacuolation result from the common mechanism of intramyelinic edema. Such lesions are particularly prominent in major white matter tracts in the deep cerebellum (left panels) and cerebrum, although the neuropil is also affected (right panels). Findings typically are bilaterally symmetrical. The pathogenesis is progressive intramyelinic accumulation of fluid, which may be seen at the light microscopic level as clefts in the myelin sheaths (lower left panel) or as vacuoles (lower right panel). The different pathogenic mechanisms between these two similar but morphologically distinct patterns of edema are highlighted by comparing their causes and progressions: the two left panels show lesions in adult rats caused by acute exposure to triethyltin (TET), while the two right panels demonstrate lesions in young rats exposed for approximately 2 months starting on postnatal day 4 to vigabatrin (an anti-epileptic GABAergic drug that suppresses neuronal activity) as described in Walzer et al. (2011). Oral toxicity of vigabatrin in immature rats: Characterization of intramyelinic edema, NeuroToxicology 32, 963–974, with permission [NOTE: Toxicant doses were not specified.] Processing conditions: formalin fixation by intravascular perfusion, paraffin embedding, H&E staining.

modest amounts of pale eosinophilic cytoplasm (Figure 52.41); they may also be seen in IHCstained material via their increased expression of cytoskeletal proteins like GFAP (Figure 52.41) and vimentin. A variant of reactive cell termed the gemistocytic astrocyte may be observed in some CNS lesions as many plump, brightly eosinophilic cells supporting numerous processes (Figure 52.41). The functional basis for the gemistocytic transformation is not clear. Microgliosis results from proliferation of the resident “immune” elements in the CNS, usually

in response to more localized neuronal insults. When activated, microglia can perform both surveillance (e.g., antigen presentation) and effector (e.g., phagocytosis) functions. Reactive microglia are identified in H&E-stained tissue as small, elongate, sometimes twisted nuclei near damaged CNS neuropil (Figure 52.42). Special stains may be used to raise their visibility, such as IHC labeling for ionized calcium-binding adaptor molecule 1 (Iba1 [Figure 52.42]) or lectin histochemistry to detect the carbohydrate marker Griffonia simplicifolia.

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FIGURE 52.39 Vacuolation of the brain gray matter often results from peracute swelling of astrocytes. This finding commonly presents as variably sized but large, oval to round, clear spaces located adjacent to capillaries and/or neurons (with both changes evident here). Globus pallidus (a basal nucleus) of an adult rat following exposure to an unspecified pharmaceutical agent. Processing conditions: formalin fixation by immersion, paraffin embedding, H&E staining.

The response by reactive oligodendrocytes is termed satellitosis as the myelinating cells aggregate around degenerating neurons, presumably in an effort to support their survival and repair. Reactive oligodendrocytes are evident in H&E-stained sections as partial to complete rings of cells encircling abnormal neurons (Figure 52.43). In general, oligodendrocytes are the least reactive population of glia. Pathologists must be careful in avoiding undue haste in interpreting the relevance of satellite cells near neurons, as neoplastic leukocytes in a perineuronal location may mimic the appearance of oligodendrocytes (Figure 52.43). Global Classifications of Toxicant-Induced Neural Lesions Several kinds of toxicant-induced abnormalities that damage the nervous system recapitulate similar processes that occur in non-neural tissues elsewhere in the body. In general, these lesions are categorized as non-proliferative or proliferative based on the prominence of cell division as a mechanism for enhancing the degree of neural damage. The remainder of this section reviews the basic features of the major lesion types in both these categories. NON-PROLIFERATIVE LESIONS

FIGURE 52.40 Alzheimer’s Type II astrocytes represent an unusual reaction to hyperammonemia. Affected cells are swollen and have open, pale, nuclei with thin rims of marginated chromatin and pale cytoplasm (arrows), while normal astrocytes have pale basophilic nuclei with clumped chromatin and indistinct cytoplasm (inset). Cerebral cortex of a dog with experimental hepatic encephalopathy. Processing conditions: formalin fixation by immersion, paraffin embedding, H&E staining. The figure was kindly provided by Dr Michael D. Norenberg, University of Miami, Miami, Florida, USA.

Developmental disturbances as a class are an important outcome following exposure to many developmental neurotoxicants (Table 52.1). The most common gross abnormalities are neural tube defects (NTDs). The NTDs generally result from enhanced cytotoxicity during the period of neurulation, in which the neural folds of the planar embryo progressively elevate and then fuse to form the neural tube (the precursor to the CNS). The spectrum of NTDs ranges from complete non-fusion of the brain (anencephaly [absence of the brain], exencephaly [exposure of the brain]) and/or spinal cord (spina bifida) to smaller foci of non-fusion in which a small amount of neural tissue (e.g., encephalocele or myelocele) or only the meninges (meningocele) protrudes from the defect (see Embryo and Fetus, Chapter 62, for a more detailed discussion and figures). Other classes of toxicant-induced developmental defects include microcephaly (presenting as a reduction in cerebrocortical size; Figure 52.21),

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FIGURE 52.41 Reactive astrocytes adopt one of several recognizable forms in response to toxicant-induced neural damage. Substantial areas of neuronal loss or parenchymal necrosis (left, upper and lower) result in hypercellularity due to astrocyte hypertrophy and hyperplasia. In most cases, reactive astrocytes have basophilic, oval to round, often eccentric nuclei with abundant cytoplasm but indistinct cell margins; however, cell borders can be seen when the astrocytes are located in regions undergoing liquefactive necrosis (left, lower cells denoted by arrows). In certain settings, reactive astrocytes acquire a “gemistocytic” phenotype (right, upper) in which the cytoplasm expands greatly and becomes hypereosinophilic. A common means of determining reactive astrocyte numbers is to evaluate the distribution of glial fibrillary acidic protein (GFAP), as reactive astrocytes express this protein in large amounts in their hypertrophied bodies and thickened processes (right, lower). All images except the upper right are of brain sections from adult rats; the upper right photomicrograph is of the primary visual cortex of a macaque monkey after chronic methylmercury intoxication. The lower right micrograph is from the CA1 hippocampal sector of an adult rat necropsied after a grand mal seizure. Processing conditions: formalin fixation (immersion in all panels but upper right, in which intravascular perfusion was employed); paraffin embedding; H&E staining (all panels except lower right, in which anti-GFAP immunohistochemistry was used).

cerebellar hypoplasia (affecting the entire organ or just the hemispheres; Figure 52.44), and neuronal heterotopiae (Figure 52.30). These lesions result from toxicant exposure well after neural tube closure. The former two changes reflect excessive death and reduced formation of neural circuits, while the heterotopiae generally stem from aberrant migration of newly made neurons and/or defective differentiation of the

radial glia that serve to guide migrating neurons to their appropriate positions. Hydrocephalus results from dilation of one or more channels within the cerebroventricular system. The change is observed most commonly in the lateral ventricles (Figure 52.45). The presentation often includes reduced thickness of the overlying brain parenchyma due to increased pressure from the accumulated CSF;

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FIGURE 52.42 Reactive microglia also are prominent players in the glial response to neurotoxicant-induced damage in the brain. Upper: Compared to the small, round to oval, isolated nuclei of resting microglia (arrow), activated microglia have elongated, serpentineor spindle-shaped nuclei and commonly form small nodules at sites of parenchymal damage. H&E. Lower: Reactive microglia can be highlighted using immunohistochemistry to detect the cell type-specific marker ionized calcium binding adaptor molecule 1 (Iba1; done here with a hematoxylin counterstain). Globus pallidus of a rat exposed to an unspecified neurotoxicant.

the reduction arises by hypoplasia of neural cells if the lesions begins during development, but usually represents atrophy if the change starts after birth. Hydrocephalus is an end-stage lesion that serves as the final common outcome for many different etiologies. In the toxicologic neuropathology setting, hydrocephalus in immature subjects frequently occurs as a compensatory mechanism to fill space within the cranial vault left vacant by xenobiotic-induced reductions in neural cell

numbers in the cerebrum. In contrast, in adults the usual cause is CSF blockade by occlusion of the mesencephalic aqueduct (of Sylvius) by a space-occupying mass such as an abscess (a possible sequel to immunosuppressive therapy or placement of an intrathecal catheter for xenobiotic delivery) or neoplasm (an occasional finding in rodent lifetime carcinogenicity bioassays). Infarcts (colloquially termed “strokes” in human patients) are characterized by regional necrosis of a neural region, usually within the brain or spinal cord. The cause is interruption of the blood flow to a particular domain as a consequence of blockage or rupture in a larger artery or vein. The absence of blood flow leads to ischemia of all cells within the region supplied by the affected vascular arcade; neurons are most sensitive to ischemia due to their very high basal metabolic rates. A transient flow disruption typically will culminate in neuronal necrosis, which will appear as a reduction or absence of these cells in an otherwise intact region of parenchyma (Figure 52.46). In many instances, the numbers of astrocytes and microglia (Figure 52.46) will be enhanced in regions where the neurons used to reside, serving as a lasting marker of prior neuronal damage. Persistent interference with the blood flow will decimate all cells within the domain, which will finally lead to liquefactive necrosis of the affected parenchyma. Eventual removal of the necrotic debris by activated microglia and macrophages recruited from the blood (gitter cells) will leave a cyst that typically has a marginal zone enriched with fibrous (intensely GFAP-positive) astrocytes (i.e., a glial scar; Figure 52.41). The site and cause of the infarct rarely are seen on gross or histopathologic examination. Potential toxic causes of infarcts usually are confined to intravascular thrombosis secondary to endothelial cell damage and/or activation of the clotting cascade. Agents that can produce these circulatory injuries include bacterial endotoxins or certain chemicals. Inflammatory lesions result from damage to the neural parenchyma in the CNS and/or PNS by accumulations of astrocytes and/or microglia in conjunction with circulating leukocytes (of one, a few, or all classes). Because leukocyte infiltrates can occur in the absence of tissue damage (Figure 52.47), the concept of “inflammation”

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FIGURE 52.43 Satellitosis of oligodendrocytes (left image, unspecified location in the brain) around toxicantdamaged neurons takes place in a final attempt to prevent degenerating cells from dying. The encircling cells have very dark, basophilic, round nuclei and may (or may not) exhibit the peri-nuclear halo that is typical of their nonreactive counterparts within white matter tracts. Differential diagnoses for peri-neuronal round cell aggregates include reactive microglia (Figures 52.29 and 52.42) and neoplastic lymphocytes (right image). In general, microglia collect near intact but dead or disintegrating cells and exhibit the typical elongated nuclei of reactive cells. In contrast, the lymphoma cells tend to encircle viable large neurons (as shown here, in a dorsal root ganglion) and display a moderate degree of cellular atypia. Species: rat. Processing conditions: formalin fixation by immersion, paraffin embedding, H&E staining.

should only be invoked when other evidence of neural injury is evident along with the leukocytes; such features may include neural-specific changes like axonal and/or myelin degeneration or gliosis, or they may be more general findings like edema, hemorrhage, necrosis, and vascular congestion. Neural inflammation as a primary toxic response typically results from direct placement of a drug delivery apparatus into the CNS (Figure 52.47), especially if coupled with delivery of a potentially irritating test article. It may also occur as a secondary consequence of agents that incite disease processes (e.g., immune dysfunction, neural damage) in which the barriers protecting the nervous system are breached and the injured cells and newly exposed neural antigens become targets. Examples of this latter class include experimental allergic encephalitis in rodents, and perhaps some instances of multiple sclerosis in humans.

Several pigments may be observed within the CNS, either as reactions to administration of a neurotoxic agent or as an incidental finding. Lipofuscin accumulation occurs within the cytoplasm of large CNS neurons (mainly pyramidal cells), astrocytes, and oligodendrocytes via the gradual accretion of cell degradation by products. The autofluorescent material represents the phospholipid-rich residue of autophagosomal lysosomes produced in cells having substantial lipid peroxidation in their membranes. The microscopic appearance of the granules generally is faint yellow-brown in H&E-stained sections, pink in periodic acid-Schiff (PAS)stained sections, and dark blue to purple in LFB-stained sections (Figure 52.48). In many instances, lipofuscin is stored as a spontaneous change in aged animals, more so for non-human primates and humans and, to a lesser extent, carnivores than for rodents. These spontaneous

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FIGURE 52.44 Cerebellar hypoplasia is a common developmental defect following gestational exposure to a neuroteratogen. In this case, progressive genesis of the cerebellar folia (especially IX and X) was attenuated in Sprague-Dawley rats exposed to all-trans retinoic acid (RA, given at 2.5 mg/kg orally to pregnant dams on gestational days 11, 12, and 13). PND ¼ postnatal day. Processing conditions: 4% paraformaldehyde fixation by intravascular perfusion, cryo-protection in 30% sucrose, frozen sectioning at 20 mm, toluidine blue staining. Scale bars ¼ 200 mm. Figure adapted from Coluccia et al. (2008) Gestational all-trans retinoic acid treatment in the rat: Neurofunctional changes and cerebellar phenotype, Neurotoxicol. Teratol. 30, 395–403, with permission.

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FIGURE 52.45 Hydrocephalus can develop as a spontaneous background lesion (as here) or as a sequel to toxicant-induced neural lesions. In this instance, the markedly thin cerebral cortex is plastered against the inner surface of the calvarium, and the massively expanded lateral ventricles have fused into a single giant cavity. Prototypical mechanisms for producing toxicantassociated hydrocephalus are reduced cerebral thickness, with compensatory dilation of the lateral ventricles, or obstruction due to focal space-occupying necrotic or neoplastic lesions (commonly along the mesencephalic aqueduct [of Sylvius]) with fluid accumulation behind the blockage. Cross-section through the caudal forebrain and skull of a 1-month-old C57BL/6 “transgenic adenocarcinoma of the mouse prostate” (TRAMP) mouse noted to exhibit substantial doming of the cranium and cognitive deficits. This image was kindly provided by Drs Lisa Berman-Booty and Krista La Perle, The Ohio State University, Columbus, Ohio, USA.

deposits elicit little if any cytotoxicity in the affected cells. In contrast, lipofuscin has been reported to accumulate in rodents given some neurotoxic agents, including alcohol and lead, and in these cases its accumulation may be associated with cytotoxicity. Deposition of hemosiderin within the CNS represents another form of pigment associated with certain neurotoxic treatments. The pattern of deposition depends on the nature of the exposure. For example, Ab – the endogenous toxicant

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FIGURE 52.46 Neural infarcts (i.e., “strokes”) result from ischemia-associated regional necrosis, usually affecting the brain or spinal cord. As demonstrated convincingly here in the hippocampal CA3 field, metabolically active neurons are the most sensitive elements. Oxygen-starved cells will die and disintegrate, and the adjacent neuropil (composed primarily of neuronal processes) will become rarified (upper image; H&E). Given sufficient time, reactive glia will accumulate at the edges of the infarct to scavenge the liquefying debris (lower image; immunohistochemical method to detect the microglia-specific marker ionized calcium binding adaptor molecule 1 [Iba1], done here with a hematoxylin counterstain). Brain section from a rat model of cardiopulmonary (CPR) resuscitation. Processing conditions: formalin fixation by immersion, paraffin embedding.

responsible for cerebral amyloid angiopathy (CAA) in Alzheimer’s disease – has been linked to scattered microhemorrhages in perivascular tissues (Figure 52.48) of affected brain regions of APP-transgenic animal models as well as human patients. In contrast, larger foci of hemorrhage may be evident at sites where

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FIGURE 52.47 Leukocyte aggregates within the nervous system may collect at various sites, especially within the loose connective tissues of the choroid plexus and the meninges (shown here). (A, B) This vehicle-treated control animal had a modest mixed infiltrate of lymphocytes and eosinophils, underscoring the fact that small collections of leukocytes may collect in the brain in the absence of active inflammation. Direct intrathecal delivery of idursulfase at 3 mg/dose (C, D) or 100 mg/dose (E, F) resulted in more pronounced infiltration of leukocytes, but the inflammatory response was reversible once protein instillation was stopped. Scale bars, 200 mm (right column). Processing conditions: formalin fixation by immersion, paraffin embedding, H&E staining. Reproduced from Felice et al. (2011) Safety evaluation of chronic intrathecal administration of idursulfase-IT in cynomolgus monkeys, Toxicol. Pathol. 39, 879–892, with permission. III. SYSTEMS TOXICOLOGIC PATHOLOGY

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FIGURE 52.48 Pigment deposition in the nervous system occurs in several toxicant-induced processes and as a spontaneous background finding (as here). Upper: Large pyramidal neurons in the cerebral cortex of this dog harbor large deposits of lipofuscin, a byproduct of ongoing cell organelle degradation that gradually accumulates over time. These deposits appear faint yellow-brown by H&E, pink with periodic acid-Schiff (PAS), dark blue to purple by LFB, and black using Sudan black (shown here). Lower: Phagocytes near small blood vessels in the thalamus of this rat are packed with brown hemosiderin granules, indicating prior episodes of local microhemorrhage. H&E. Processing conditions: formalin fixation by immersion, paraffin embedding.

implantation of a direct delivery device has produced microtrauma to the blood vessels coursing through the neuropil. Very small deposits of pigment, presumably hemosiderin, in the Purkinje cell layer can be a hint that there was a prior loss of cells from this neuronal population.

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Storage diseases may occur as a consequence of neurotoxicant exposure, when the active agent reduces or halts the activity of an enzyme required for normal metabolism. The prototypic toxicant-induced storage disease is produced by swainsonine, an alkaloid toxin fabricated by plants of the genera Astragalus, Oxytropis, and Swainsona (e.g., locoweed) that inhibits amannosidase, a protein involved in the hydrolysis of N-linked glycosides. Affected neurons are observed to have many cytoplasmic vacuoles (representing swollen Golgi bodies) by light microscopy. A variant of xenobiotic-induced storage disease is drug-induced phospholipidosis (DIPL), in which phospholipids accumulate in multi-lamellar (“myeloid”) bodies within the cytoplasm (Figure 52.31). Agents associated with this lesion are classed chemically as cationic amphophilic small molecules, and they represent essential drugs for treating many indications: angina treatments, antidepressants, antimalarials, and cholesterol-lowering agents. The usual neural cells targeted by these drugs are large neurons, such as those in dorsal root ganglia and the spinal cord gray matter. In contrast to traditional storage diseases, DIPL results from selective uptake and/or retention of the undigested xenobiotic substrate in lysosomes rather than from enzyme inhibition. Confirmation that a molecule can produce DIPL requires transmission electron microscopy, so in nearly all cases analysis for this finding is limited to experimental animals. PROLIFERATIVE LESIONS

An infrequent but confirmed consequence of xenobiotic exposure is neurocarcinogenesis, or the induction of neoplasms in the CNS and/or PNS. Most neural neoplasms, including the bulk of xenobiotic-induced lesions, in laboratory animals and humans are glial tumors arising in the CNS. These glial neoplasms may evolve from a single cell lineage (e.g., astrocytoma, oligodendroglioma) or from multiple lines (e.g., mixed gliomas, which contain both astrocytic and oligodendroglial elements). In general, chemically-induced glial tumors in experimental animals do not evolve into the aggressive malignancies (e.g., glioblastoma multiforme) characteristic of the most serious lesions observed in human patients.

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Neural cancers originating from other CNS cell types also have been described occasionally after exposure to xenobiotics, albeit rarely. These entities include medulloblastoma, a primitive neuroectodermal tumor (PNET) of the cerebellum; granular cell tumors (in rodents) and meningiomas of the meninges; and ependymomas (typically occurring in the spinal cord central canal). Interested readers are referred to reviews in the list of Suggested Reading for details on differentiating among various types of CNS tumors. In the PNS, neural tumors most commonly arise from Schwann cells only or from Schwann cells in combination with endoneurial and perineurial fibroblasts that form the connective tissue which parses axons into fascicles within nerves. Traditionally, these neoplasms have been designated as Schwannomas (benign or malignant) and neurofibromas (for benign lesions)/neurofibrosarcomas (for malignant), respectively, but recently some workers have preferred to combine them into a single class as nerve sheath tumors (benign or malignant). The PNS axons themselves cannot give rise to neural tumors since they merely represent cell processes, and not cells. However, PNS cell bodies in various ganglia (including the adrenal medulla) can serve as the source of peripheral tumors of neural origin. Chemically-induced PNS tumors usually will possess features of Schwannomas (i.e., originating only from Schwann cells). Differential diagnosis of neural tumors is complicated by two main factors. The first is the difficulty in discriminating between reactive and neoplastic neural cells, particularly in the PNS. Schwann cells proliferate extensively as a regenerative response to any form of damage, so any connective tissue neoplasm that encompasses a nerve is likely to include a substantial proportion of activated Schwann cells intermingled with the neoplastic tissue. A second factor is that tumor cells often express unusual complements of proteins. Such altered signatures are particularly evident for malignant neoplasms, which may produce greatly reduced amounts of typical marker proteins or manufacture multiple markers that are characteristic of an immature pluripotent cell. From a practical perspective, an increase in the number of neoplasms following xenobiotic exposure is an adverse event regardless of the cell of origin, so the

main focus should be on discriminating between reactive and neoplastic lesions. Agents that act as neurocarcinogens include alkylating chemicals, radiation, and certain viruses. In general, exposures leading to carcinogenicity occur during development (e.g., by transplacental or neonatal exposure) or during young adulthood. The same agent (e.g., N-ethyl-Nnitrosourea [ENU]) can produce increases in the incidences of glial and non-glial neoplasms in the CNS. Some neural tumors only develop following direct introduction of the toxicant into the CNS (e.g., choroid plexus carcinomas following intracerebroventricular injection), indicating the importance of barrier systems (e.g., the BBB and BNB) in protecting neural tissues from blood-borne toxic agents. In the PNS, tumors of peripheral nerve origin must be distinguished from neuromas, which are focal, non-neoplastic lesions that form at the site of a local traumatic injury (commonly transection with displacement or removal of the distal trunk). Neuromas represent the disordered proliferation of elongating proximal axons that failed to find their former pathways and thus are unable to re-establish connectivity with their effector organ. The presence of numerous axons is a diagnostic feature of neuromas, as Schwannomas contain few if any axons. Toxicants cannot induce neuromas, as axons cannot become neoplastic.

4.2. Functional Alterations Neurological and behavioral signs of neurotoxicity are well correlated among animals and humans, which is to be expected given the many close parallels in neural function across species. Accordingly, regulatory agencies generally consider any distinguishable change in one or more behavioral parameters as adverse – whether increased or decreased in degree, and/or accelerated or delayed in onset – until proven otherwise by demonstration of a non-neural explanation. Such functional indices may differ in extent but usually do not diverge in quality among strains of a given species. Functional abnormalities may appear before, together with, or in the absence of identifiable neuropathology. Furthermore, compensatory adaptations can ameliorate or even eliminate

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functional abnormalities over time, often beginning within hours to days of the initial evidence of dysfunction. Restoration is especially common when the cause of the change is an acute, transient pharmacological effect (e.g., altered cell excitability or neurotransmission). In contrast, behavioral deficits are more likely to persist and be accompanied by neuropathological lesions following prolonged exposures. Histopathological evaluation may not discern lesions associated with the induction of substantial behavioral abnormalities if the affected structures are small (i.e., the injury is highly localized) or the damage is diffuse (e.g., hepatic encephalopathy). Different batteries of functional tests are used to evaluate CNS and PNS lesions. Neuron-associated dysfunction typically arises from abnormalities in multiple CNS domains, so patterns of behavioral deficits often stem from the inability to perform complex behaviors that require successful integration among associative, motor, and sensory centers. For example, 3-acetylpyridine (3-AP) destroys the (inferior) olivary nuclei in the rat brainstem, thereby depleting the climbing fibers that supply the cerebellar cortex with connections for coordinating voluntary movements. Clinical aberrations caused by 3-AP include ataxia (uncoordinated motion), disequilibrium (altered righting), and paresis (weakness of voluntary muscles). In a similar fashion, trimethyl tin (TMT) preferentially removes neurons in various limbic system elements such as the amygdala, hippocampus, and piriform cortex, leading to a pattern of limbic-mediated deficits like hyperactivity and impaired learning and memory. Toxicant-induced functional deficits in the PNS most often reflect axonal, myelin, and/or synaptic lesions located in either the CNS or PNS. Initial functional alterations associated with such damage are aberrant body or limb positioning, attenuated muscle strength, gait disruptions, and/or abnormal (e.g., burning, tingling) or reduced sensation in peripheral organs. In healthy individuals, such tasks require integration of afferent (incoming) and efferent (outgoing) motor and sensory pathways in the spinal cord, but they are not absolutely dependent on further control by higher brain centers. Deficits begin first and progress most rapidly in the most distal sites (e.g., a “stocking and glove”

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distribution affecting the digits) that are supplied by the longest, and thus the most vulnerable, axons. The hind limbs often are affected first and most severely, since the axons in these trunks are longer than those supplying the forelimbs. With time and persistence of the toxic agent, the functional deficits tend to increase in severity and progress proximally to affect the motion and perceptions in effector organs located closer to the CNS. Many developmental neurotoxicants are first identified via their capacities to induce CNS dysfunction. Such behavioral consequences often occur at doses below those required to induce other indicators of developmental neurotoxicity (e.g., neuropathology). In addition, the doses that elicit developmental neurotoxicity typically engender minimal toxicity in adults. Some agents produce temporary behavioral effects only during the early stage of development, while others induce apparently transient dysfunction that re-emerges later in life.

4.3. Physiological Abnormalities Neurotoxic agents can produce several physiological alterations that may or may not be associated with anatomic and functional abnormalities. Electrophysiological disturbances often manifest as dampened and delayed transmission of action potentials along nerve trunks during tests of conduction velocity. Such disturbances closely track the behavioral and clinical deficits observed using functional assays. In our experience, electrophysiological abnormalities typically start in advance of structural lesions. Similarly, functional recovery can begin before normal structure has been restored. Both these phenomena are typical of PNS structures that can regenerate, such as olfactory neuroepithelium and peripheral nerves. Another physiological abnormality produced by some xenobiotics (e.g., bacterial toxins) is enhanced porosity of the BBB. A more open neurovascular barrier allows blood-borne materials better access to the neural parenchyma and reduces fluid regulation capabilities in the CNS. This effect can be demonstrated graphically using large molecules (e.g., dyes, immunoglobulins, India ink) that normally cannot cross the capillary walls to reach the CNS (Figure 52.49).

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4.5. Background Findings and Their Implications

FIGURE 52.49 Enhanced porosity of the blood–brain barrier (BBB) may be confirmed by administering exogenous agents (e.g., Evans blue dye, India ink) or detecting endogenous protein (here, direct immunohistochemistry to localize extravasated immunoglobulin G [IgG]). The staining intensity, which correlates with the extent of IgG leakage, is highest in the region of the median eminence, one of the circumventricular organs (CVOs; see Figure 52.17) with a porous blood– brain barrier. Species: rat. Processing conditions: formalin fixation by immersion, frozen section.

4.4. Neurochemical Changes Abnormalities in the concentrations of various neurochemicals, especially neurotransmitters, are a frequent response to neurotoxicant exposure. The effect may be measured for a single molecule, or multiple elements from several chemical classes may be altered. In our experience, the nature of the neurochemical change must be determined empirically. Furthermore, great care must be taken to define the significance of the neurochemical shift. For example, in some cases the altered chemical may play a direct role in the pathogenesis of a neurotoxic condition, while in others the modification may be a secondary outcome of neurotoxicity that plays no part in initiating or sustaining the neurotoxic episode. Similarly, measurements of neurochemical aberrations in homogenates taken from multiple regions of the CNS will provide an “average” value for the measured analyte across the sample that will obscure the relevance of more substantial abnormalities in discrete regions. The interested reader is referred to other works (see Suggested Reading) for details regarding neurochemical alterations as a response to neurotoxicant exposure.

Certain incidental changes are frequently misidentified as neuropathological lesions by inexperienced researchers. The alterations described here are common findings in vertebrate brains. Identified artifacts should be not reported in the pathology data set. However, systematic distribution of an artifact, where one dose group only is involved, may be recognized and noted as a comment to the organ/tissue at the discretion of the study pathologist. “Dark neuron” artifact in the brain is the most problematic spontaneous change, as it is misinterpreted as neuronal degeneration with distressing frequency. This artifact occurs more regularly in certain brain regions, particularly the cerebral cortex (Figure 52.24), hippocampus, Purkinje cell layer of the cerebellum, and large pyramidal neurons of many brainstem nuclei and the spinal cord. Affected neurons typically have darkly amphophilic nuclei and cytoplasm, a twisted (corkscrew-shaped) and thickened dendrite, and are contracted so that the shrunken cell is surrounded by a clear retraction space with irregular borders. All affected neurons have comparable features, in contrast to genuine foci of neuronal degeneration in which injured cells exhibit a range (i.e., early to advanced) of neurodegenerative stages. A common presentation for dark neuron artifact is an asymmetric, focal column of affected cells extending from the deep layers of the cerebral cortex down to the hippocampus. This appearance is consistent with manipulation of inadequately fixed CNS tissue, leading to localized ischemia and metabolic abnormalities in neurons exposed to superficial pressure. Induction of excitotoxicity post mortem may be involved in the pathogenesis of “dark neurons,” as administration of glutamate antagonists (MK-801, 6-cyano-7-nitroquinoxaline-2,3-dione [CNQX], etc.) at necropsy has been suggested to reduce their numbers. The usual method for mitigating this artifact is to fix by perfusion and, if time and space permit, to follow this procedure by an additional period of in situ post-fixation by immersion following removal of the calvarium. “Myelin bubble” artifact generally involves large myelinated fibers of the spinal cord white matter tracts and PNS major trunks. The finding

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FIGURE 52.50 “Myelin bubble” artifact is readily appreciated in this markedly affected sciatic nerve section from a control mouse. The finding presents as clear, dilated fiber tracts (alone or in chains) surrounding an intact axon in the absence of any Schwann cell or phagocytic reaction. When mild, only a few fiber tracts may be affected, so the change often is recognized more easily in longitudinal sections. The mechanism is unclear but is thought to involve insufficient and/or delayed fixation. Processing conditions: formalin fixation by immersion, paraffin embedding, H&E staining.

presents as clear, localized, elliptical spaces surrounding an intact axon; they may occur in isolation or in chains (Figure 52.50). The change often is confined to one or a few axons, so it is more readily recognized in longitudinal sections than coronal ones. The main differential diagnosis for this change is axonal degeneration, a true neurotoxic lesion that presents as a string of digestion chambers that contain fragmented axonal or myelin debris (sometimes along with phagocytic gitter cells). Myelin bubble artifacts tend to develop in immersion-fixed, paraffinembedded specimens. The mechanism is unknown, but the suggested pathogenesis is manipulation of unfixed tissue leading to fluid accumulation within the myelin. “Vacuolation” artifact can take several forms in the CNS (Figure 52.51). Diffuse vacuolation may be observed if fixation is delayed. However, this circumstance is unlikely in experimental studies, and can be managed by careful scheduling of tissue harvesting for diagnostic cases. A more frequent variant in CNS white matter is focally extensive collections of large vacuoles in densely myelinated tracts of the cerebrum,

FIGURE 52.51 Vacuolation artifact assumes several forms in white matter tracts of the central nervous system (demonstrated here in the rat cerebellum). Upper: A common presentation in deep white matter tracts is focally extensive collections of large vacuoles with irregular margins; in some cases, the vacuoles contain pale, homogeneous material (i.e., “Buscaino bodies”). These vacuoles are more extensive if tissues are retained in 70% alcohol for an extended period – such as retention in the alcohol bath on an automated tissue processor over the weekend – possibly as a consequence of excessive lipid extraction. Lower: Markedly swollen astrocyte processes may be responsible for substantial perineuronal vacuolation. The adjacent neurons typically are unaffected or may exhibit the early stages of dark neuron artifact (arrows; compare to neurons with genuine degenerative changes [Figures 52.22 and 52.28] and advanced dark neuron features [Figure 52.24]). Such astrocytic swelling is likely a consequence of hypoxiaassociated energy depletion and the inability to actively sustain transmembrane ion gradients and fluid balance during the delay before fixation begins. Processing conditions: formalin fixation by immersion, paraffin embedding, H&E staining.

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cerebellum, and brainstem. The vacuoles may have irregular borders and be filled with pale, homogeneous material (i.e., “Buscaino bodies”), or have smooth margins and be empty. In our experience, these vacuoles are more extensive if tissues are retained in 70% alcohol for an extended period – such as holding in the alcohol bath stage on an automated tissue processor over the weekend – possibly as a consequence of excessive lipid extraction. Another form of vacuolar artifact in the CNS results from swollen organelles in particular cell types, especially astrocytes. These may occur as small clear holes in the cytoplasm or as larger, clear vacuoles in the astrocytic processes adjacent to shrunken neurons or blood vessels. The main toxicant-induced differential to this artifact is intramyelinic edema, in which the myelin sheaths encircling intact axons are disrupted by small to large vacuoles (Figure 52.38). Genuine vacuolar lesions typically exhibit a bilaterally symmetrical distribution and are fairly widespread, while the artifactual changes are often limited to one or a few locations. Axonal “spheroids,” or focally enlarged axons, are occasionally encountered in white matter tracts of the brainstem and spinal cord as a spontaneous change in older animals. Animals from all treatment groups may be affected, including the negative control cohort, and no relationship to xenobiotic dose is evident. A common site for this finding is the cranial portion of the dorsal funiculus, which is the terminal point for the ascending sensory tracts in the spinal cord. Spheroids also can result from neurotoxicant exposure (Figure 52.34), but in such cases their number is higher and their numbers usually exhibit a dose–response relationship. Melanin occurs as a normal pigment in certain sites within the CNS. This location should not be surprising, since melanocytes are derived from precursors of neural crest origin. Typical areas for deposition are the meninges in pigmented mice (Figure 52.52) and certain sheep breeds, or the neurons of the substantia nigra, the locus coeruleus, and certain other basal nuclei in primates, including humans. The neuronal form, termed neuromelanin, is minimal at birth but accumulates over time. The meningeal melanin has not been linked to a specific function, nor is its extent altered by exposure to neurotoxicants. The proposed tasks for

FIGURE 52.52 Melanin pigment is a common incidental background finding in the meninges of mouse strains with pigmented hair. Cerebral cortex of a control C57BL6/J mouse. Processing conditions: formalin fixation by immersion, paraffin embedding, H&E staining.

neuromelanin are protective, such as storage depots for oxyradicals resulting from metabolism of monoamine neurotransmitters (e.g., dopamine and norepinephrine) or residual products of autophagy. This role is supported by the ability of neuromelanin to selectively sequester the neurotoxic metabolite MPPþ, a monoamine analog. A more active function for neuromelanin is suggested by the loss of pigmented neurons in basal nuclei in concordance with the onset of certain neurodegenerative diseases. The ancillary tissues within the CNS are host to two incidental changes. The first is focal fibrosis of the meninges, which appears as a localized accumulation of fibroblasts and collagen (Figure 52.53). The second is occasional small aggregates of leukocytes in the choroid plexus and, to a lesser extent, near meningeal blood vessels. These foci usually contain mature lymphocytes, mainly of the T-cell lineage, and they are seemingly present to fulfill a surveillance function for the acquired arm of the immune system. Mast cells scattered within the perineurium are a frequent finding in large nerve trunks of the PNS. In general, none of these changes is impacted by neurotoxicant exposures. In contrast, the presence of granulocyte and/or macrophage aggregates in neural tissues is associated with an immediate response by the innate immune arm to a noxious

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FIGURE 52.53 Meningeal fibrosis is an infrequent background finding in species of neurotoxicological significance. Distinguishing features include thickening of the meninges, collagen deposition, and a variable increase in fibroblast numbers. Small nodular lesions may slightly compress the underlying parenchyma, but they will not invade. Cerebral cortex of a control mouse (unspecified strain). Processing conditions: formalin fixation by immersion, paraffin embedding, H&E staining.

stimulus, though usually one caused by irritation (i.e., implantation of a direct delivery device) or bacterial contamination.

5. MECHANISMS OF NERVOUS SYSTEM INJURY Neurotoxicants have been shown to produce their effects in many different fashions. Basic mechanisms may target any of several neural elements: particular structures, specific cell types, or certain molecular pathways. The precise nature of the resulting neuropathological lesions will be dictated by the mode of action. Accordingly, the remainder of this section will briefly review basic mechanisms of neurotoxicity using prototypical neurotoxicants (Table 52.1).

5.1. Aberrant Cell Migration and/or Differentiation The typical lesion resulting from abnormal migration and terminal differentiation of neuronal precursors is heterotopiae (Figure 52.30), but the mechanisms by which neurotoxicants produce this change are incompletely understood. Experiments with avian and rodent embryos suggest that damage to neuronal stem cells, radial glial (which guide neuronal precursors to their appropriate positions), or both

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may be factors. Several hypotheses have been advanced to explain the defects. One alternative is that pluripotent stems cells may be destroyed before they can produce enough daughter cells to populate one or more waves of neuronal migration. This effect may be confined to the neuroepithelium of the neurulating embryo, or it may also impact partially differentiated structures such as ganglia (which are derived from neural crest cells). A second option is that the spatial and/or temporal expression of critical morphogens (growth factors, neurotransmitters, etc.) is disrupted on one or more lineages of neural cell precursors. This consequence may lead to several kinds of functional abnormalities in the affected cells: misdirected migration, reduced movement in the proper direction, and/or incorrectly timed terminal differentiation. In most cases, neurons will seek – and usually succeed, at least in part – to connect with their normal cellular targets. Nonetheless, behavioral or other neurological deficits may be observed since the resulting fiber tracts typically will be too long and insufficiently wired to support normal neural activities.

5.2. Altered Intracellular Transport Toxic agents that disrupt the transfer of essential macromolecules from the cell body to its distant processes will produce degeneration in outlying structures. This finding is typical of axonopathies, in which blocked slow axonal transport results in chemical rather than physical transection of the axon. Axonotoxic chemicals generally induce this effect by promoting the formation of covalent crosslinks between macromolecules (e.g., neurofilaments). The resulting disordered filamentous masses will lodge at axonal constriction points like the nodes of Ranvier. The axon distal to the plug, including the presynaptic terminal, will starve and eventually disintegrate as a primary lesion, to be followed in time by secondary degeneration of the local Schwann cells. The longest axons (e.g., those in long tracts in the spinal cord white matter and in PNS trunks) are affected first. Regeneration can occur in PNS axons if the insult is transient, but affected CNS axons cannot be restored. The neuron generally will survive since the supply

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of nutrients to its metabolically active body remains intact, although chronic exposure to certain agents (e.g., b,b0 iminodipropionitrile [IDPN]) can induce an irreversible loss of CNS motor neurons.

5.3. Cell Turnover Chemical exposure in the rat has been linked to heightened incidences of glial neoplasms (mainly in the deep cerebrum), granular cell tumors (cerebral and cerebellar meninges, chiefly on the dorsal midline), and malignant reticulosis (cerebral and cerebellar meninges). These tumors are thought to evolve from populations of partially committed, oligopotent stem cells that retain the capacity for low-level cell division throughout adulthood. The specific sites are the cerebral subventricular zone for the glial masses, or fibroblasts or facultative phagocytes in the meninges. These cell populations offer targets for genotoxic carcinogens (discussed in Section 5.6, on macromolecular adducts), but cell proliferation is a necessary requirement by which genetic mutations will become fixed in the genome. Cell proliferation occurs in corresponding sites in adult rodents, monkeys, and humans. Abnormal cell turnover also is an important factor in neurotoxicity. During development, increased or decreased programmed cell death at an inappropriate time will either reduce or prevent neuronal processes from correctly connecting with their target cells. Such disruptions typically will perturb anatomic and functional maturation. Similarly, toxicants can alter postnatal neuronogenesis in the brains of mature individuals, which can impact behavioral and cognitive abilities (e.g., learning, memory).

5.4. Energy Depletion Reduced availability of energy stores within highly active neural cells, especially neurons, is a common predisposing factor to cell degeneration and eventual cell loss in many brain regions. The mechanism of action involves the capacity of such neurotoxicants to interfere with enzymes in the electron transport chain by which the mitochondria replenish ATP; in the absence of this energy storage molecule, neurons lose their ability to sustain their ionic gradients and thus fail to produce action potentials. The neurotoxic

moiety may reach the target neurons by a circuitous route. For example, MPTP is converted to the toxic metabolite MPPþ by perisynaptic astrocytes in a two-step reaction that requires the enzyme monoamine oxidase B (MAO-B). Next, dopaminergic neurons in the substantia nigra selectively sequester MPPþ using transporters for the reuptake of monoamine neurotransmitters. Finally, MPPþ enters the mitochondria, where it quenches the activity of reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase, the first enzyme in the electron transport chain. Other agents (e.g., bromethalin, hexachlorophene) damage myelinating cells by uncoupling mitochondrial oxidative phosphorylation, which leads to fluid accumulation in the interlamellar space (Figure 52.38).

5.5. Excitotoxicity Excitotoxic lesions are thought to result from imbalances in reciprocal feedback between neuronal populations that produce the excitatory neurotransmitter glutamate and the inhibitory transmitter GABA. For example, trimethyltin (TMT) increases glutamate release in the hippocampus while reducing the synthesis and reuptake of GABA and glutamate. Enhanced excitation results in persistent influx of ions across the neuronal plasma membrane, including entry of Ca2þ. The rising Ca2þ tide finally floods the neuronal cytoplasm and propels the cell to begin a death spiral. The initial lesion of punctate vacuolation in neurons, consistent with swelling of intracytoplasmic tubular organelles (Figure 52.27), occurs within hours of injury and may progress to full-fledged necrosis (Figure 52.28) over 1–2 days. The vulnerability of various neuronal populations to TMT-induced excitotoxicity is affected by many factors. For example, mice are more sensitive to TMT than rats. However, the lesion pattern in the hippocampus diverges in these two species; mice preferentially develop dentate lesions with little CA cell involvement, while the converse is true in rats. The sensitivity of the CA neurons is age-dependent in rats, with lesions developing only after the hippocampal pyramidal cells mature (i.e., after postnatal day 7). This phenomenon highlights the need to evaluate neurotoxicity under various conditions

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when seeking to identify and characterize new neurotoxic agents.

5.6. Macromolecular Adducts Known neurocarcinogens in rats (e.g., acrylonitrile, ethylene oxide, nitrosoureas) chiefly produce glial neoplasms following prenatal or prolonged postnatal exposure. These agents are all potent DNA-alkylating chemicals, indicating that the likely mechanism of neural cell initiation is the formation of DNA adducts leading to mutations in critical genes. The deep cerebral location of the tumors suggests that the target populations are likely to be retained progenitor cells in the cerebral periventricular zone or subcortical white matter. It is not known whether or not neurotoxicant exposure is responsible for the preponderance of glial tumors in humans, or which chemical(s) or critical developmental periods in people might be most vulnerable to neurocarcinogenic toxicants.

5.7. Neurotransmission Disruption Neurotoxic agents that block synaptic neurotransmission can induce profound neurological dysfunction in the absence of major structural lesions. Classic presentations include altered contraction of skeletal muscles in certain body regions or all major muscle groups, abnormal cognitive abilities, and/or dysfunction of the autonomic nervous system (ANS). Affected subjects can recover if vital functions (e.g., breathing, nutrient intake) can be maintained until the affected synaptic elements are restored. Disruption of synaptic function occurs in several ways. Decreased Neurotransmitter Release Attenuated release of neurotransmitters is responsible for botulism and tetanus, both of which are caused by toxins made by bacteria of the genus Clostridium as metabolic by products when grown under anaerobic conditions. Botulism develops in fish, birds, and mammals following bacterial colonization of the digestive tract or deep wounds, or by ingestion of the preformed toxin in contaminated food. Botulinum toxin serves as a protease to degrade docking molecules needed for fusion of synaptic vesicles with the presynaptic membrane of the axon terminal. In the absence of vesicle fusion, acetylcholine (ACh) is not

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released and the postsynaptic membrane is not stimulated. Skeletal muscles cannot contract, so flaccid paralysis ensues (e.g., limberneck, the manifestation of botulism in waterfowl). In contrast, tetanus arises when bacteria within infected puncture wounds generate tetanospasmin, which is taken up at neuromuscular junctions in the body periphery and then transported to the CNS by retrograde axonal transport. Tetanospasmin also functions as a protease that thwarts vesicle docking, but affected synapses fail to discharge the inhibitory transmitters GABA and glycine. The removal of feedback control that modulates the excitatory signals to skeletal muscles results in the characteristic presentation of tetany. Persistent Neurotransmitter Activity Sustained neurotransmission is another recognized route to synaptic neurotoxicity. This effect can be produced in several fashions, and typically occurs in association with CNS neurons. One means is to increase neurotransmitter release from presynaptic terminals. This approach explains the efficacy of mirtazapine (RemeronÒ ), a tetracyclic antidepressant that acts as an a2adrenergic blocker to promote the discharge of serotonin (5HT) and norepinephrine (NE). A second, more common way of maintaining neurotransmitter levels in the synapse is to reduce the rate at which transmitters are removed. Inhibition of neurotransmitter reuptake is the desired pharmacological response of several selective 5HT reuptake inhibitor (SSRI) antidepressants, such as fluoxetine (ProzacÒ ), paroxetine (PaxilÒ ), and sertraline (ZoloftÒ ). However, self-medication with two such drugs and/or related pro-serotonergic agents (e.g., amphetamines, monoamine oxidase inhibitors [MAOI], some nutraceuticals [e.g., St John’s wort, Hypericum perforatum], and certain opioids) can overstimulate organs with high numbers of 5HT receptors (e.g., CNS, ANS, and gastrointestinal tract), resulting in the potentially lethal “serotonin syndrome.” Some agents concurrently lessen reuptake of two neurotransmitters: dopamine (DA) and NE – the effect of bupropion (WellbutrinÒ ); or 5HT and NE – duloxetine (CymbaltaÒ ) and venlafaxine (EffexorÒ ). St John’s wort is thought to inhibit the uptake of DA, 5HT, and NE, although the exact mechanism remains unclear.

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Reduced Neurotransmitter Metabolism A third strategy is to decrease the degradation of neurotransmitters. This effect is produced by treatment with MAOIs, where the two MAO isoforms are bound to the outer mitochondrial membrane of most cells, including neurons and astrocytes; this enzyme promotes the oxidative deamination of DA, 5HT, and NE. Similarly, inhibition of synaptic acetylcholinesterase (AChE) by exposure to carbamate or organophosphate (OP) insecticides prevents the hydrolytic removal of ACh from synapses in the CNS and PNS as well as at neuromuscular junctions. The OPs irreversibly phosphorylate the serine residue at the active site of AChE, so any recovery must await the synthesis of new enzyme (a matter of several days). Administration of pralidoxime (2-pyridine aldoxime methyl chloride [2-PAM]) early after OP exposure can reduce the extent of neurotoxicity by hydrolyzing the OP-serine bond that inactivates AChE. In contrast, the bond between a carbamate and AChE is reversible, with the hydrolytic removal over several hours leading to the gradual restoration of AChE activity. Termination of Transmembrane Ionic Gradients Some agents affect neurotransmission by altering the character of the action potential. The maintenance of normal ionic gradients across the charged membranes of neuronal processes is critical for sustaining the ability to conduct impulses. Neurotoxicants may disrupt the flow of a single ion, or they may impact multiple currents. For example, the type I pyrethroid insecticides allethrin and tetramethrin prolong Naþ influx, lessen the peak of the Naþ flow, and decrease the steady-state Kþ efflux, apparently by modulating resting (i.e., closed) Naþ channels so that they open more slowly than normal. In contrast, tetrodotoxin blocks the extracellular pores of Naþ channels, preventing the inward flow of Naþ ions, and thus prevents propagation of any nascent action potential. Neurotoxic effects on ion channels often occur in the absence of structural lesions. Unknown Impact on Neurotransmission The pathogenesis for the delayed neurotoxicity in OPIDN is unclear, but presumably stems from abnormal propagation of neural impulses. Certain OP classes (e.g., phosphoramidates and

phosphonates) incite central–peripheral distal axonopathy (“dying back polyneuropathy”) in association with phosphorylation of a second enzyme, neuropathy target esterase (NTE). This molecule is an integral membrane protein in all neurons. Structurally, NTE is unrelated to AChE and other major serine esterases. The physiological role for NTE during adulthood is unknown; in development, NTE appears to control interactions between neurons and glia. Agents that cause OPIDN are postulated to bind covalently to the NTE active site, thereby leading to a toxic gain of function.

5.8. Oxidative Damage Gradual accumulation of free radical-mediated damage to cellular macromolecules is a well-recognized pathway by which toxic agents can produce cell lethality. In the nervous system, the usual cellular targets are neurons due to their high metabolic rates and correspondingly outsized exposure to oxygen. Within neurons, oxidative damage has been postulated to distort critical genes, cell and organelle membranes, or signaling molecules. The role of oxidative stress has been examined in the neurotoxicity of copper, iron, manganese (Mn), rotenone, and other redox-active chemicals. Certain toxicants can become concentrated in susceptible neurons, thereby extending the period during which oxidative injury can occur. For example, Mn is localized in the basal nuclei of primates, including humans, apparently due to efficient blood-borne delivery by the ironcarrying protein transferrin and/or the strong affinity that Mn has for neuromelanin deposits in certain primate nuclei (e.g., substantia nigra). One proposed pathogenesis of Mn-induced neurotoxicity is oxidation of DA. The degree of DA modification may be heightened synergistically in neurons with elevated intracellular concentrations of iron (Fe), which is a potent oxidizing agent in its own right. Rodents are much less susceptible to Mn neurotoxicity as their basal nuclei neurons do not contain neuromelanin, and are thus unable to selectively sequester Mn. Loss of antioxidant molecules in the nervous system is an indirect way of promoting damage by oxygen free radicals. For example, reduced glutathione (GSH) is concentrated in the neuropil

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and white matter tracts of the CNS, especially astrocytes, but is expressed in high levels in only a few neuronal populations (e.g., cerebellar granule and Purkinje cells, sensory neurons in dorsal root ganglia). Thus, administration of the oxidizing agent 1,3-dinitrobenzene (DNB) injures astrocytes to a greater extent than neurons. Vascular Impairment Increased vascular permeability (dysoria) resulting from damage to the BBB and/or BNB is a common mechanism employed by several neurotoxicants. In this setting, the microvasculature is the primary target. Nonetheless, once the barriers have been breached, other CNS cells may experience direct toxic effects of their own. For example, lead (Pb) neurotoxicity is associated with altered vessel diameters (constricted or narrowed); mural changes (endothelial swelling, necrosis); and thrombosis in CNS capillaries – especially those in the cerebrum and cerebellum. The neuropil near damaged vessels is edematous as a consequence of fluid and protein extravasation. Nearby neurons (cerebrocortical pyramidal cells and cerebellar Purkinje cells) are necrotic, and in chronic lesions are associated with regional accumulation of reactive astrocytes. At least some instances of neuronal death may arise from direct Pb-induced toxicity to neurons since dead cells have been observed in CNS regions that lacked evidence of vascular damage. The primary changes induced by Pb in PNS trunks are Wallerian-like axonal degeneration and segmental demyelination mainly affecting motor nerves. Multiple Mechanisms Some neurotoxic agents appear to cause neural damage by several mechanisms at once. Hexachlorophene induces massive intramyelinic edema by uncoupling oxidative phosphorylation from ATP synthesis in oligodendroglia and by altering GABA concentrations in CNS neurons. Methylmercury produces profound neuronal necrosis in many brain regions by disrupting BBB permeability, altering neural cell metabolism, inhibiting synthesis of RNA and protein, and promoting oxidation and denaturation of cell proteins and membranes. Ethanol decreases DNA, RNA, and protein levels in neurons and astroglia, and also incites

apoptosis of neural cell precursors during development. In our experience, many agents elicit multiple chemical and molecular changes that can be linked to the induction or progression of neurotoxicity, and only careful attention to defining the appropriate study design can permit conclusions to be drawn regarding the primary mechanism of neurotoxic action.

6. SUMMARY The regimented arrangement of anatomic and chemical attributes within the CNS and PNS dictates the functional responses by which individuals maintain homeostasis and interacts with their external environment. These same stereotypical features are responsible for the patterns of neurotoxic changes produced by numerous xenobiotics. The susceptibility of the CNS is further enhanced by the extraordinary metabolic needs and limited capacity for repair. Taken together, these characteristics render the possibility of neurotoxic damage among the greatest challenges in navigating the world around us, and in bringing new products to market. The means for identifying new neurotoxic hazards as well as assessing and managing the risk posed by exposure to such agents are evolving as technical advances increase the affordability, speed, and utility of more detailed neuropathology assessments. The current reliance on limited batteries of functional (e.g., behavior, functional observational battery) and structural (e.g., gross lesions, brain weight, histopathology) endpoints for screening is a reasonable first-tier approach for use with general toxicity studies. More extensive investigations are the norm for second-tier instances where neurotoxicity has been observed or is predicted to be likely based on prior observations. Over time, we predict that current third-tier approaches, like non-invasive imaging to follow disease progression during life and more extensive “omics” evaluations to explore molecular mechanisms responsible for neurotoxicity, will be used on a more regular basis. The next decade or two hold considerable promise for a substantially improved understanding of neurobiology in both health and disease, and the knowledge gained using various toxicants to probe the CNS and PNS will be instrumental in this renaissance.

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SUGGESTED READING Introduction Abou-Donia, M.B., 1992. Neurotoxicology. CRC Press, Boca Raton, FL. Bearer, C.F., 2001. Developmental neurotoxicity: Illustration of principles. Pediatr. Clin. North Am. 48, 1199–1213. Blain, P.G., Harris, J.B., 1999. Medical Neurotoxicology: Occupational and Environmental Causes of Neurological Dysfunction. Oxford University Press, New York, NY. Bolon, B., Bradley, A., Garman, R., Krinke, G., 2011. Useful toxicologic neuropathology references for pathologists and toxicologists. Toxicol. Pathol. 39, 234–239. Cannon, J.R., Greenamyre, J.T., 2011. The role of environmental exposures in neurodegeneration and neurodegenerative diseases. Toxicol. Sci. 124, 225–250. Chang, L.W., 1994. Principles of Neurotoxicology, 9th ed. Marcel Dekker, Inc., New York, NY. Dorman, D.C., 2000. An integrative approach to neurotoxicology. Toxicol. Pathol. 28, 37–42. Lowndes, H.E., Reuhl, K.R., 1999. Comprehensive Toxicology, vol. 11. Nervous System and Behavioral Toxicology. Elsevier, New York, NY. Slikker Jr., W., Chang, L.W., 1998. Handbook of Developmental Neurotoxicology. Academic Press, San Diego, CA. Spencer, P.S., Schaumburg, H.H., 1980. Experimental and Clinical Neurotoxicology, 1st ed. Williams & Wilkins, Baltimore, MD. Spencer, P.S., Schaumburg, H.H., Ludolph, A.C., 2000. Experimental and Clinical Neurotoxicology, 2nd ed. Oxford University Press, New York, NY. Tilson, H.A., Mitchell, C.L., 1992. Neurotoxicology. Raven Press, New York, NY. Tilson, H.A., Harry, G.J., 1999. Neurotoxicology, 2nd ed. Taylor & Francis, Philadelphia, PA.

Anatomy and Function of the Nervous System General References Abbott, N.J., 2004. Evidence for bulk flow of brain interstitial fluid: Significance for physiology and pathology. Neurochem. Int. 45, 545–552. Abbott, N.J., Patabendige, A.A., Dolman, D.E., Yusof, S.R., Begley, D.J., 2010. Structure and function of the blood–brain barrier. Neurobiol. Dis. 37, 13–25. Alloway, K.D., Pritchard, T.C., 2007. Medical Neuroscience, 2nd ed. Hayes Barton Press, Raleigh, NC. Arie¨ns Kappers, C.U., Huber, G.C., Crosby, E.C., 1967. The Comparative Anatomy of the Nervous System of Vertebrates, Including Man. Hafner, New York, NY. Bayer, S.A., Altman, J., 2002. The Spinal Cord from Gestational Week 4 to the 4th Postnatal Month, Atlas of Human Nervous System Development, vol. 1. CRC Press, Boca Raton, FL.

Bayer, S.A., Altman, J., 2004. The Human Brain During the Third Trimester, Atlas of Human Nervous System Development, vol. 2. CRC Press, Boca Raton, FL. Bayer, S.A., Altman, J., 2005. The Human Brain During the Second Trimester, Atlas of Human Nervous System Development, vol. 3. CRC Press, Boca Raton, FL. Bayer, S.A., Altman, J., 2006. The Human Brain During the Late First Trimester, Atlas of Human Nervous System Development, vol. 4. CRC Press, Boca Raton, FL. Bayer, S.A., Altman, J., 2008. The Human Brain During the Early First Trimester, Atlas of Human Nervous System Development, vol. 5. CRC Press, Boca Raton, FL. Bloom, F.E., Bjo¨rklund, A., Ho¨kfelt, T., 1997. The Primate Nervous System, Part I, Handbook of Chemical Anatomy, vol. 13. Elsevier, Amsterdam. Bloom, F.E., Bjo¨rklund, A., Ho¨kfelt, T., 1998. The Primate Nervous System, Part II, Handbook of Chemical Anatomy, vol. 14. Elsevier, Amsterdam. Bloom, F.E., Bjo¨rklund, A., Ho¨kfelt, T., 1999. The Primate Nervous System, Part III, Handbook of Chemical Anatomy, vol. 15. Elsevier, Amsterdam. Bolon, B., 2000. Correlative and comparative neuroanatomy for the toxicologic pathologists. Toxicol. Pathol. 28, 6–27. Butler, A.B., Hoods, W., 1996. Comparative Vertebrate Neuroanatomy: Evolution and Adaptation. Wiley-Liss, New York, NY. Clancy, B., Darlington, R.B., Finlay, B.L., 2001. Translating developmental time across mammalian species. Neuroscience 105, 7–17. Cooper, J.R., Bloom, F.E., Roth, R., 2012. The Biochemical Basis of Neuropharmacology, 8th ed. Oxford University Press, New York, NY. Daube, J.R., Rubin, D.I., 2009. Clinical Neurophysiology, 3rd ed. Oxford University Press, New York, NY. de Lahunta, A., Glass, E., 2009. Veterinary Neuroanatomy and Clinical Neurology, 3rd ed. Saunders (Elsevier), St Louis, MO. Fawcett, J.W., Rosser, A.E., Dunnett, S.B., 2001. Brain Damage, Brain Repair. Oxford University Press, New York, NY. Felten, D.L., Jo´zefowicz, R.F., 2003. Netter’s Atlas of Human Neuroscience. Icon Learning Systems, Teterboro, NJ. FitzGerald, M.J.T., Gruener, G., Mtui, E., 2007. Clinical Neuroanatomy and Neuroscience, 5th ed. Saunders (Elsevier), Philadelphia, PA. Garman, R.H., 2011. Histology of the central nervous system. Toxicol. Pathol. 39, 22–35. Hagan, C.E., Bolon, B., Keene, C.D., 2011. Nervous System. In: Treuting, P.M., Dintzis, S.M. (Eds.), Comparative Anatomy and Histology: A Mouse and Human Atlas. Academic Press (Elsevier), San Diego, CA, pp. 339–394. Herschkowitz, N., Kagan, J., Zilles, K., 1997. Neurobiological bases of behavioral development in the first year. Neuropediatrics 28, 296–306. Herschkowitz, N., Kagan, J., Zilles, K., 1999. Neurobiological bases of behavioral development in the second year. Neuropediatrics 30, 221–230.

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Jenkins, T.W., 1972. Functional Mammalian Neuroanatomy. Lea & Febiger, Philadelphia, PA. Jortner, B.S., 2006. The return of the dark neuron. A histological artifact complicating contemporary neurotoxicologic evaluation. Neurotoxicology 27, 628–634. Kaas, J.H., 2009. Evolutionary Neuroscience. Academic Press (Elsevier), San Diego, PA. Kandel, E.R., Schwartz, J.H., Jessell, T.M., 2000. Principles of Neural Science, 4th ed. McGraw-Hill, New York, NY. Kempermann, G., 2006. Adult Neurogenesis. Oxford University Press, New York, NY. Nestler, E.J., Hyman, S.E., Malenka, R.C., 2008. Molecular Neuropharmacology: A Foundation for Clinical Neuroscience, 2nd ed. McGraw-Hill, Columbus, OH. Paxinos, G., 1995. The Rat Nervous System, 2nd ed. Academic Press, San Diego, CA. Paxinos, G., 2004. The Rat Nervous System, 3rd ed. Academic Press, San Diego, CA. Peters, A., Palay, S.L., Webster, H.D., 1991. The Fine Structure of the Nervous System: Neurons and Their Supporting Cells, 3rd ed. Oxford University Press, New York, NY. Robert, F., Cloix, J.F., Hevor, T., 2012. Ultrastructural characterization of rat neurons in primary culture. Neuroscience 200, 248–260. Rodier, P.M., 1980. Chronology of neuron development: Animal studies and their clinical implications. Dev. Med. Child Neurol. 22, 525–545. Rousseaux, C.G., 2008. A review of glutamate receptors I: Current understanding of their biology. J. Toxicol. Pathol. 21, 25–51. Sanes, D.H., Reh, T.A., Harris, W.A., 2006. Development of the Nervous System, 2nd ed. Academic Press (Elsevier), San Diego, CA. Scheffler, B., Walton, N.M., Lin, D.D., Goetz, A.K., Enikolopov, G., Roper, S.N., Steindler, D.A., 2005. Phenotypic and functional characterization of adult brain neuropoiesis. Proc. Natl. Acad. Sci. U.S.A. 102, 9353–9358. Schmahmann, J.D., Pandya, D.N., 2006. Fiber Pathways of the Brain. Oxford University Press, New York, NY. Siegel, G.J., Albers, R.W., Brady, S., Price, D., 2006. Basic Neurochemistry: Molecular, Cellular, and Medical Aspects, 7th ed. Academic Press (Elsevier), San Diego, CA. Squire, L.R., Berg, D., Bloom, F., du Lac, S., 2008. Fundamental Neuroscience, 3rd ed. Academic Press (Elsevier), San Diego, CA. Young, P.A., Young, P.H., Tolbert, D.L., 2008. Basic Clinical Neuroscience, 2nd ed. Lippincott Williams & Wilkins, Baltimore, MD.

Neuroanatomical Atlases (Recent) Adrianov, O.S., Mering, T.A., 2010. Atlas of the Canine Brain. NPP Books, Arlington, MA. Altman, J., Bayer, S.A., 1995. Atlas of Prenatal Rat Brain Development. CRC Press, Boca Raton, FL.

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Alvarez-Bolado, G., Swanson, L.W., 1996. Developmental Brain Maps: Structure of the Embryonic Rat Brain. Elsevier, New York, NY. Ashwell, K.W.S., Paxinos, G., 2008. Atlas of the Developing Rat Nervous System, 3rd ed. Academic Press (Elsevier), San Diego, CA. Bellairs, R., Osmond, M., 2005. Atlas of Chick Development, 2nd ed. Academic Press (Elsevier), San Diego, CA. Franklin, K.B.J., Paxinos, G., 2007. The Mouse Brain in Stereotaxic Coordinates, 3rd ed. Academic Press (Elsevier), San Diego, CA. Jacobwitz, D.M., Abbott, L.C., 1998. Chemoarchitectonic Atlas of the Developing Mouse Brain. CRC Press, Boca Raton, FL. Mai, J.K., Paxinos, G., Voss, T., 2007. Atlas of the Human Brain, 3rd ed. Academic Press (Elsevier), San Diego, CA. Mazzioatta, J.C., Toga, A.W., Frackowiak, R.S.J., 2000. Brain Mapping: The Systems. Academic Press, San Diego, CA. Morin, L.P., Wood, R.I., 2001. A Stereotaxic Atlas of the Golden Hamster Brain. Academic Press, San Diego, CA. O’Rahilly, R.R., Mu¨ller, F., 2006. The Embryonic Human Brain: An Atlas of Developmental Stages, 3rd ed. Wiley-Liss, Wilmington, DE. Palazzi, X., 2011. The Beagle Brain in Stereotaxic Coordinates. Springer Verlag, New York, NY. Palazzi, X., Bordier, N., 2008. The Marmoset Brain in Stereotaxic Coordinates. Springer Verlag, New York, NY. Palkovits, M., Brownstein, M.J., 1988. Maps and Guide to Microdissection of the Rat Brain. Elsevier, New York, NY. Paxinos, G., Mai, J.K., 2004. The Human Nervous System, 2nd ed. Academic Press, San Diego, CA. Paxinos, G., Watson, C., 2007. The Rat Brain in Stereotaxic Coordinates, 6th ed. Academic Press (Elsevier), San Diego, CA. Paxinos, G., Ashwell, K.W.S., To¨rk, I., 1994. Atlas of the Developing Rat Nervous System, 2nd ed. Academic Press, San Diego, CA. Paxinos, G., Halliday, G., Watson, C., Koutcherov, Y., Wang, H., 2007. Atlas of the Developing Mouse Brain at E17.5, P0, and P6. Academic Press (Elsevier), San Diego, CA. Paxinos, G., Huang, X.-F., Petrides, M., Toga, A.W., 2008a. The Rhesus Monkey Brain in Stereotaxic Coordinates, 2nd ed. Academic Press (Elsevier), San Diego, CA. Paxinos, G., Watson, C., Carrive, P., Kirkcaldie, M., Ashwell, K., 2008b. Chemoarchitectonic Atlas of the Rat Brain, 2nd ed. Academic Press (Elsevier), San Diego, CA. Puelles, L., Martinez de-la-Torre, M., Paxinos, G., Watson, C., Martinez, S., 2007. The Chick Brain in Stereotaxic Coordinates. Academic Press (Elsevier), San Diego, CA. Schambra, U., 2008. Prenatal Mouse Brain Atlas. Springer Verlag, New York, NY. Swanson, L.W., 2003. Brain Maps: Structure of the Rat Brain, 3rd ed. Academic Press, San Diego, CA. Toga, A.W., Mazzioatta, J.C., 2000. Brain Mapping: The Systems. Academic Press, San Diego, CA. Wu, J., Bowden, D.M., Dubach, M.F., Robertson, J.E., 2000. Primate Brain Maps: Structure of the Macaque Brain. Elsevier, New York, NY.

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Wulliman, M.F., Rupp, B., Reichert, H., 1996. Neuroanatomy of the Zebrafish Brain: A Topological Atlas. Birkha¨user Verlag, Basel, Switzerland.

Evaluation of Neurotoxicity General References Bolon, B., Bradley, A., Butt, M., Jensen, K., Krinke, G., 2011. Compilation of international regulatory guidance documents for neuropathology assessment during Nonclinical general toxicity and specialized neurotoxicity studies. Toxicol. Pathol. 39, 92–96. Chang, L.W., Slikker Jr., W., 1995. Neurotoxicology Approaches and Methods. Academic Press, San Diego, CA. Claassen, V., 1994. Neglected Factors in Pharmacology and Neuroscience Research. Elsevier, New York, NY. Cory-Slechta, D.A., 2005. Studying toxicants as single chemicals: does this strategy adequately identify neurotoxic risk? Neurotoxicology 26, 491–510. Crofton, K.M., Mundy, W.R., Lein, P.J., Bal-Price, A., Coecke, S., Seiler, A.E., Knaut, H., Buzanska, L., Goldberg, A., 2011. Developmental neurotoxicity testing: recommendations for developing alternative methods for the screening and prioritization of chemicals. ALTEX 28, 9–15. Eisenbrandt, D.L., Allen, S.L., Berry, P.H., Classen, W., Bury, D., Mellert, W., Millischer, R.J., Schuh, W., Bontinck, W.J., 1994. Evaluation of the neurotoxic potential of chemicals in animals. Food Chem. Toxicol. 32, 655–669. Kaufmann, W., Bolon, B., Bradley, A., Butt, M., Czasch, S., Garman, R.H., George, C., Gro¨ters, S., Krinke, G., Little, P., McKay, J., Narama, I., Rao, D., Shibutani, M., Sills, R., 2012. Proliferative and non-proliferative lesions of the rat and mouse central and peripheral nervous systems. Toxicol. Pathol. 40, 87S–157S. Mattsson, J.L., Eisenbrandt, D.L., Albee, R.R., 1990. Screening for neurotoxicity: complementarity of functional and morphologic techniques. Toxicol. Pathol. 18 (1 Pt 2), 115–127. Organisation for Economic Co-operation and Development (OECD), 1995. Guideline 418: Delayed Neurotoxicity of Organophosphorus Substances Following Acute Exposure http://miranda.sourceoecd.org/vl=63915458/cl=18/nw=1/ rpsv/cw/vhosts/oecdjournals/1607310x/v1n4/contp1-1. htm; (last accessed January 4, 2013). Organisation for Economic Co-operation and Development (OECD), 1997. Guideline 424: Neurotoxicity Study in Rodents http://miranda.sourceoecd.org/vl=63915458/cl=18/nw=1/ rpsv/cw/vhosts/oecdjournals/1607310x/v1n4/contp1-1. htm; (last accessed January 4, 2013). Organisation for Economic Co-operation and Development (OECD), 2007. Guideline 426, Developmental Neurotoxicity Study http://miranda.sourceoecd.org/vl=63915458/ cl=18/nw=1/rpsv/cw/vhosts/oecdjournals/1607310x/ v1n4/contp1-1.htm; (last accessed January 4, 2013). Sette, W.F., MacPhail, R.C., 1992. Qualitative and quantitative issues in assessment of neurotoxic effects. In: Tilson, H.A.,

Mitchell, C. (Eds.), Neurotoxicology. Traven Press, Ltd., New York, NY, pp. 345–361. US Environmental Protection Agency (EPA), 1998a. Health Effects Test Guidelines: OPPTS 870.6200. Neurotoxicity Screening Battery http://www.epa.gov/ocspp/pubs/frs/ publications/Test_Guidelines/series870.htm; (last accessed January 4, 2013). US Environmental Protection Agency (EPA), 1998b. Health Effects Test Guidelines: OPPTS 870.6300. Developmental Neurotoxicity Study http://www.epa.gov/ocspp/pubs/ frs/publications/Test_Guidelines/series870.htm; (last accessed January 4, 2013). Wang, C., Slikker Jr., W.J., 2011. Developmental Neurotoxicology Research: Principles, Models, Techniques, Strategies, and Mechanisms. John Wiley & Sons, Hoboken, NJ.

Functional Assessment Broxup, B., Robinson, K., Losos, G., Beyrouty, P., 1989. Correlation between behavioral and pathological changes in the evaluation of neurotoxicity. Toxicol. Appl. Pharmacol. 101, 510–520. Clark, D.L., Boutros, N.N., Mendez, M., 2005. The Brain and Behavior: An Introduction to Behavioral Neuroanatomy, 2nd ed. Cambridge University Press, Malden, MA. Cory-Slechta, D.A., Crofton, K.M., Foran, J.A., Ross, J.F., Sheets, L.P., Weiss, B., Mileson, B., 2001. Methods to identify and characterize developmental neurotoxicity for human health risk assessment. I: Behavioral effects. Environ. Health Perspect. 109 (Suppl. 1), 79–91. Crawley, J.N., 2007. What’s Wrong With My Mouse? Behavioral Phenotyping of Transgenic and Knockout Mice, 2nd ed. Wiley-Liss, New York, NY. Dobbs, M.R., 2009. Clinical Neurotoxicology: Syndromes, Substances, Environments. Saunders. (Elsevier), Philadelphia, PA. Gerber, G.J., O’Shaughnessy, D., 1986. Comparison of the behavioral effects of neurotoxic and systemically toxic agents: How discriminatory are behavioral tests of neurotoxicity? Neurobehav. Toxicol. Teratol. 8, 703–710. Lorenz, M.D., Kornegay, J.N., 2004. Handbook of Veterinary Neurology, 4th ed. Saunders (Elsevier), St Louis, MO. Weiss, B., O’Donoghue, J.L., 1994. Neurobehavioral Toxicity. Analysis and Interpretation. Raven Press, New York, NY.

Biochemical and Biomarker Evaluation Jain, K.K., 2010. The Handbook of Biomarkers. Humana Press, Totowa, NJ. Johannessen, J.N., 1993. Markers of Neuronal Injury and Degeneration. New York Academy of Sciences, vol. 679, New York, NY [Ann. N.Y. Acad. Sci.]. O’Callaghan, J.P., 1993. Quantitative features of reactive gliosis following toxicant-induced damage to the CNS. Ann. N.Y. Acad. Sci. 679, 195–210.

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Rosenberg, G.A., 1990. Brain Fluids and Metabolism. Oxford Unversity Press, New York, NY. Slikker Jr., W., Bowyer, J.F., 2005. Biomarkers of adult and developmental neurotoxicity. Toxicol. Appl. Pharmacol. 206, 255–260. Vernau, W., Vernau, K.M., Bolon, B., 2011. Cerebrospinal fluid analysis in toxicological neuropathology. In: Bolon, B., Butt, M.T. (Eds.), Fundamental Neuropathology for Pathologists and Toxicologists: Principles and Techniques. John Wiley & Sons, Hoboken, NJ, pp. 271–283.

Toxicokinetics Dorman, D.C., Allen, S.L., Byczkowski, J.Z., Claudio, L., Fisher, E.J., Fisher, J.W., Harry, G.J., Li, A.A., Makris, S.L., Padilla, S., Sultatos, L.G., Mileson, B., 2001. Methods to identify and characterize developmental neurotoxicity for human health risk assessement. III: Pharmacokinetic and pharmacodynamic considerations. Environ. Health Perspect. 109 (Suppl. 1), 101–111. Lipscomb, J.C., Ohanian, E.V., 2007. Toxicokinetics and Risk Assessment. Informa Healthcare USA, New York, NY.

Morphological Evaluation Bolon, B., Butt, M., 2011. Fundamental Neuropathology for Pathologists and Toxicologists: Principles and Techniques. John Wiley & Sons, Hoboken, NJ. Bolon, B., Garman, R., Jensen, K., Krinke, G., Stuart, B., 2006. A “best practices” approach to neuropathologic assessment in developmental neurotoxicity testing – for today. Toxicol. Pathol. 34, 296–313. Bolon, B., Anthony, D.C., Butt, M., Dorman, D., Green, M.V., Little, P., Valentine, W.M., Weinstock, D., Yan, J., Sills, R., 2008. Current pathology techniques symposium review: Advances and issues in neuropathology. Toxicol. Pathol. 36, 871–889. Bondy, S.C., 1985. Especial considerations for neurotoxicological research. Crit. Rev. Toxicol. 14, 381–402. Cassella, J., Hay, J., Lawson, S., 1997. The Rat Nervous System: An Introduction to Preparatory Techniques. John Wiley & Sons, New York, NY. de Groot, D.M., Bos-Kuijpers, M.H., Kaufmann, W.S., Lammers, J.H., O’Callaghan, J.P., Pakkenberg, B., Pelgrim, M.T., Waalkens-Berendsen, I.D., Waanders, M.M., Gundersen, H.J., 2005. Regulatory developmental neurotoxicity testing: A model study focussing on conventional neuropathology endpoints and other perspectives. Environ. Toxicol. Pharmacol. 19, 745–755. Dorman, D.C., Bonnefoi, M., Morgan, K.T., 1995. Enzyme histochemical methods and techniques. In: Chang, L.W., Slikker Jr., W. (Eds.), Neurotoxicology Approaches and Methods. Academic Press, San Diego, CA, pp. 67–79. Downing, A.E., 1992. Neuropathological histotechnology. In: Prophet, E.B., Mills, B., Arrington, J.B., Sobin, L.H.Q. (Eds.), Laboratory Methods in Histotechnology. American Registry of Pathology, Washington, DC, pp. 81–104. Dyck, P.J., Thomas, P.K., 2005. Peripheral Neuropathy, 4th ed. Saunders (Elsevier), Philadelphia, PA.

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Eisenbrandt, D.L., Mattsson, J.L., Albee, R.R., Spencer, P.J., Johnson, K.A., 1990. Spontaneous lesions in subchronic neurotoxicity testing of rats. Toxicol. Pathol. 18 (1 Pt 2), 154–164. Eisenbrandt, D.L., Allen, S.L., Berry, P.H., Classen, W., Bury, D., Mellert, W., Millischer, R.J., Schuh, W., Bontinck, W.J., 1994. Evaluation of the neurotoxic potential of chemicals in animals. Food Chem. Toxicol. 32, 655–669. Fiala, J.C., Spacek, J., Harris, K.M., 2002. Dendritic spine pathology: Cause or consequence of neurological disorders? Brain Res. Rev. 39, 29–54. Fix, A.S., Garman, R.H., 2000. Practical aspects of neuropathology: A technical guide for working with the nervous system. Toxicol. Pathol. 28, 122–131. Fix, A.S., Ross, J.F., Stitzel, S.R., Switzer 3rd, R.C., 1996. Integrated evaluation of central nervous system lesions: Stains for neurons, astrocytes and microglia reveal the spatial and temporal features of MK-801-induced neuronal necrosis in the rat cerebral cortex. Toxicol. Pathol. 24, 291–304. Fix, A.S., Stitzel, S.,R., Ridder, G.M., Switzer 3rd, R.C., 2000. MK-801 neurotoxicity in cupric silver-stained sections: Lesion reconstruction by 3-dimensional computer image analysis. Toxicol. Pathol. 28, 84–90. Garman, R.H., 2003. Evaluation of large-sized brains for neurotoxic endpoints. Toxicol. Pathol. 31, 32–43. Garman, R.H., Fix, A.S., Jortner, B.S., Jensen, K.F., Hardisty, J.F., Claudio, L., Ferenc, S., 2001. Methods to identify and characterize developmental neurotoxicity for human health risk assessment. II: Neuropathology. Environ. Health Perspect. 109 (Suppl. 1), 93–100. Jortner, B.S., 1982. Selected aspects of the anatomy and response to injury of the chicken (Gallus domesticus) nervous system. Neurotoxicology 3, 299–310. Krinke, G.J., 1989. Neuropathologic screening in rodent and other species. J. Am. Coll. Toxicol. 8, 141–146. Krinke, G.J., Classen, W., Rauch, M., Weber, E., 1997. Optimal conduct of the neuropathology evaluation of organophosphorus induced delayed neuropathy in hens. Exp. Toxicol. Pathol. 49, 451–458. Krinke, G.J., Vidotto, N., Weber, E., 2000. Teased-fiber technique for peripheral myelinated nerves: Methodology and interpretation. Toxicol. Pathol. 28, 113–121. Krinke, G.J., Classen, W., Vidotto, N., Suter, E., Wu¨rmlin, C.H., 2001. Detecting necrotic neurons with fluoro-jade stain. Exp. Toxicol. Pathol. 53, 365–372. Love, S., Louis, D.N., Ellison, D.W., 2008. Greenfield’s Neuropathology, 8th ed. Hodder Arnold, London, UK. Pardo, I.D., Garman, R.H., Weber, K., Bobrowski, W.F., Hardisty, J.F., Morton, D., 2012. Technical guide for nervous system sampling of the cynomolgus monkey for general toxicity studies. Toxicol. Pathol. 40, 624–636. Rao, D.B., Little, P.B., Malarkey, D.E., Herbert, R.A., Sills, R.C., 2011. Histopathological evaluation of the nervous system in National Toxicology Program rodent studies: A modified approach. Toxicol. Pathol. 39, 463–470.

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Sills, R.C., Morgan, D.L., Herr, D.W., Little, P.B., George, N.M., Ton, T.V., Love, N.E., Maronpot, R.R., Johnson, G.A., 2004. Contribution of magnetic resonance microscopy in the 12-week neurotoxicity evaluation of carbonyl sulfide in Fischer 344 rats. Toxicol. Pathol. 32, 501–510. Summers, B.A., Cummings, J.F., DeLahunta, A., 1995. Veterinary Neuropathology. Mosby, St Louis, MO. Switzer 3rd, R.C., 2000. Application of silver degeneration stains to neurotoxicity testing. Toxicol. Pathol. 28, 70–83. Switzer 3rd, R.C., Lowry-Franssen, C., Benkovic, S.A., 2011. Recommended neuroanatomical sampling practices for comprehensive brain evaluation in nonclinical safety studies. Toxicol. Pathol. 39, 73–84. Toga, A.W., 1997. Brain-mapping neurotoxicity and neuropathology. Ann. NY. Acad. Sci. 820, 1–13. Weber, K., Garman, R.H., Germann, P.G., Hardisty, J.F., Krinke, G., Millar, P., Pardo, I.D., 2011. Classification of neural tumors in laboratory rodents, emphasizing the rat. Toxicol. Pathol. 39, 129–151. Whitney, K.M., Schwartz Sterman, A.J., O’Connor, J., Foley, G.L., Garman, R.H., 2011. Light microscopic sciatic nerve changes in control beagle dogs from toxicity studies. Toxicol. Pathol. 39, 835–840.

Special Techniques for Neurotoxicity Assessment Arora, A., Neema, M., Stankiewicz, J., Guss, Z.D., Guss, J.G., Prockop, L., Bakshi, R., 2008. Neuroimaging of toxic and metabolic disorders. Semin. Neurol. 28, 495–510. Bagnell, R., Langaman, C., Madden, V., Suzuki, K., 1995. Ultrastructural methods for neurotoxicology and neuropathology. In: Chang, L.W., Slikker Jr., W. (Eds.), Neurotoxicology Approaches and Methods. Academic Press, San Diego, CA, pp. 81–98. Coggeshall, R.E., Lekan, H.A., 1996. Methods for determining numbers of cells and synapses: A case for more uniform standards of review. J. Comp. Neurol. 364, 6–15. de Groot, D.M., Hartgring, S., van de Horst, L., Moerkens, M., Otto, M., Bos-Kuijpers, M.H., Kaufmann, W.S., Lammers, J.H., O’Callaghan, J.,P., Waalkens-Berendsen, I.D., Pakkenberg, B., Gundersen, H.G., 2005. 2D and 3D assessment of neuropathology in rat brain after prenatal exposure to methylazoxymethanol, a model for developmental neurotoxicty. Reprod. Toxicol. 20, 417–432. Diemer, N.H., 1982. Quantitative morphological studies of neuropathological changes. Part 1. Crit. Rev. Toxicol. 10, 215–263. Hyman, B.T., Gomez-isla, T., Irizarry, M.C., 1998. Stereology: A practical primer for neuropathology. J. Neuropathol. Exp. Neurol. 57, 305–310. Xie, Z., Yang, D., Stephenson, D., Morton, D., Hicks, C., Brown, T., Bocan, T., 2010. Characterizing the regional structural difference of the brain between tau transgenic (rTg4510) and wild-type mice using MRI. Med. Image Comput. Comput. Assist. Interv. 13 (Pt 1), 308–315.

Yang, D., Xie, Z., Stephenson, D., Morton, D., Hicks, C.D., Brown, T.M., Sriram, R., O’Neill, S., Raunig, D., Bocan, T., 2011. Volumetric MRI and MRS provide sensitive measures of Alzheimer’s disease neuropathology in inducible Tau transgenic mice (rTg4510). Neuroimage 54, 2652–2658.

Model Systems Bolon, B., Dorman, D.C., Bonnefoi, M.S., Randall, H.W., Morgan, K.T., 1993. Histopathologic approaches to chemical toxicity using primary dissociated neural cells grown in chamber slides. Toxicol. Pathol. 21, 465–479. Coecke, S., Eskes, C., Gartlon, J., Kinsner, A., Price, A., van Vliet, E., Prieto, P., Boveri, M., Bremer, S., Adler, S., Pellizzer, C., Wendel, A., Hartung, T., 2006. The value of alternative testing for neurotoxicity in the context of regulatory needs. Environ. Toxicol. Pharmacol. 21, 153–167. Dow, G.S., Koenig, M.L., Wolf, L., Gerena, L., LopezSanchez, M., Hudson, T.H., Bhattacharjee, A.K., 2004. The antimalarial potential of 4-quinolinecarbinolamines may be limited due to neurotoxicity and cross-resistance in mefloquine-resistant Plasmodium falciparum strains. Antimicrob. Agents Chemother. 48 2624–232. Harry, G.J., Billingsley, M., Bruinink, A., Campbell, I.L., Classen, W., Dorman, D.C., Galli, C., Ray, D., Smith, R.A., Tilson, H.A., 1998. In vitro techniques for the assessment of neurotoxicity. Environ. Health. Perspect. 106 (Suppl. 1), 131–158.

Nervous System Responses to Neurotoxic Injury Abbott, N.J., Revest, P.A., Romero, I.A., 1992. Astrocyteendothelial interaction: physiology and pathology. Neuropathol. Appl. Neurobiol. 18, 424–433. Cartwright, M.E., Petruska, J., Arezzo, J., et al., 2009. Phospholipidosis in neurons caused by posaconazole, without evidence for functional neurologic effects. Toxicol. Pathol. 37 (7), 902–910. Coluccia, A., Belfiore, D., Bizzoca, A., Borracci, P., Trerotoli, P., Gennarini, G., Carratu` MR.Colucia, A., 2008. Gestational all-trans retinoic acid treatment in the rat: Neurofunctional changes and cerebellar phenotype. Neurotoxicol. Teratol. 30, 395–403. Di Monte, D.A., Royland, J.E., Irwin, I., Langston, J.W., 1996. Astrocytes as the site for bioactivation of neurotoxins. Neurotoxicology 17, 697–703. Felice, B.R., Wright, T.L., Boyd, R.B., Butt, M.T., Pfeifer, R.W., Pah, J., Ruiz, J.A., Heartlein, M.W., Calias, P., 2011. Safety evaluation of chronic intrathecal administration of idursulfase-IT in cynomolgus monkeys. Toxicol. Pathol. 39, 879–892. Garman, R.H., 1990. Artifacts in routinely immersion fixed nervous tissue. Toxicol. Pathol. 18, 149–153. Holtcamp, W., 2012. The emerging science of BMAA: Do cyanobacteria contribute to neurodegenerative disease? Environ. Health Perspect. 120, A110–A116.

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Janzer, R.C., 1993. The blood–brain barrier: Cellular basis. J. Inherit. Metab. Dis. 16, 639–647. Johansson, B.B., 1990. The physiology of the blood-brain barrier. Adv. Exp. Med. Biol. 274, 25–39. LoPachin, R.M., Aschner, M., 1999. Neuronal-glial interactions as potential targets of neurotoxicant effect. In: Tilson, H.A., Mitchell, C. (Eds.), Neurotoxicology. Taylor and Francis, Philadelphia, PA, pp. 53–80. Rahman, A., Khan, K.M., Al-Khaledi, G., Khan, I., Al-Shemary, T., 2012. Overactivation of hippocampal serine/threonine protein phosphatases PP1 and PP2A is involved in leadinduced deficits in learning and memory in young rats. Neuro toxicology 33, 370–383. Rison, R.A., 2008. Ascending sensory motor polyradiculoneuropathy with cranial nerve involvement following administration of intrathecal methotrexate and intravenous cytarabine in a patient with acute myelogenous leukemia: A case report. Cases J. 1, 255. Tonkin, E.G., Erve, J.C., Valentine, W.M., 2000. Disulfiram produces a non-carbon disulfide-dependent Schwannopathy in the rat. J. Neuropathol. Exp. Neurol. 59, 786–797. Walzer, M., Bekersky, I., Wanaski, S., Collins, S., Jortner, B., Patterson, R., Garman, R., Sagar, S., Tolbert, D., 2011. Oral

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toxicity of vigabatrin in immature rats: Characterization of intramyelinic edema. Neurotoxicology 32, 963–974.

Mechanisms of Nervous System Injury Aschner, M., Costa, L.G., 2005. The Role of Glia in Neurotoxicity, 2nd ed. CRC Press, Boca Raton, FL. Jortner, B.S., 2000. Mechanisms of toxic injury in the peripheral nervous system: neuropathologic considerations. Toxicol. Pathol. 28, 54–69. Lester, D.S., Slikker Jr., W., Lazarovici, P., 2002. Site-Selective Neurotoxicity. Taylor & Francis, London, UK. Nicklas, W.J., Saporito, M., Basma, A., Geller, H.M., Heikkila, R.E., 1992. Mitochondrial mechanisms of neurotoxicity. Ann. N.Y. Acad. Sci. 648, 28–36. Philbert, M.A., Billingsley, M.L., Reuhl, K.R., 2000. Mechanisms of injury in the central nervous system. Toxicol. Pathol. 28, 43–53. Rodier, P.M., 2004. Environmental causes of central nervous system maldevelopment. Pediatrics 113 (Suppl. 4), 1076–1083. Sayre, L.M., Perry, G., Smith, M.A., 2008. Oxidative stress and neurotoxicity. Chem. Res. Toxicol. 21, 172–188.

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