Abstract
Session 1 of the 2010 STP/IFSTP Joint Symposium on Toxicologic Neuropathology, titled “Fundamentals of Neurobiology,” was organized to provide a foundation for subsequent sessions by presenting essential elements of neuroanatomy and nervous system function. A brief introduction to the session titled “Introduction to Correlative Neurobiology” was provided by Dr. Greg Hall (Eli Lilly and Company, Indianapolis, IN). Correlative neurobiology refers to considerations of the relationships between the highly organized and compartmentalized structure of nervous tissues and the functioning within this system.
The basic structural organization of the nervous system involves the central control elements (brain and spinal cord) and the communication network that sends and receives signals from outlying tissues (the peripheral and autonomic nervous systems). The nervous system is organized into functional domains based largely on contiguous signaling pathways (Bolon 2000). Functionally, these include cognitive domains concerned with complex associations, somatic domains concerned with movement and sensations, and autonomic domains that regulate internal physiology. Much complexity comes from the multitude of interconnections that overlie this basic network; in its entirety, this interconnected system may be referred to as the “connectome” (Lichtman, Livet, and Sanes 2008).
The spinal cord provides an example of basic wiring patterns in the central nervous system (CNS). Incoming signals from peripheral tissues enter the cord via the dorsal (posterior in bipeds) nerve root and may form synapses within the dorsal gray horn. Signals transmitted to the brain typically ascend the cord within tracts in the dorsal or lateral white matter. In analogous fashion, signals descending the cord from the brain run in bundles in the ventral (anterior in bipeds) white matter, form synapses within the ventral gray horn, and exit the cord via ventral nerve roots.
Among mammals the greatest variations in neural architecture occur in the cerebral cortex; however, thematic patterns are recognizable even there. For example, projections devoted to body sense and movement, vision, and hearing map to generally similar areas of cerebral cortex, although proportions of the cerebrum devoted to these various domains can vary widely between species (Bolon 2000).
With regard to factors such as blood flow, blood-brain barrier integrity, neurotransmitter composition, and cellular metabolism, considerable heterogeneity may exist among the various regional domains within a given individual and among similar brain regions in different species. Such differences may lend degrees of unpredictability with regard to neurotoxic responses across different domains of the nervous system within an individual and across species. Neurotoxicants may cause functional changes in the nervous system that may or may not have correlating structural changes (Dorman, Brenneman, and Bolon 2002; Bolon et al. 2008). Similarly, microscopic lesions may or may not be accompanied by detectable changes in nervous function (Bolon et al. 2008; Arezzo, Litwak, and Zotova forthcoming).
An important task for the toxicologic pathologist is to locate and characterize structural changes within the nervous system if they exist in the animal being examined. Because of the structural diversity and compartmentalization of the brain, considerable thought should be given to appropriate sampling and evaluation methodology, topics that received considerable attention in General Session 2: “Modern Pathology Methods for Neural Investigations” (see companion mini-review in this volume).
Given the complexity of the nervous system and the variety of manifestations of neurologic diseases and neurotoxic responses, integrated approaches to neuropathologic investigations are crucial. Following this brief and very high-level introduction addressing basic concepts of nervous system structure and biology, the invited speakers each provided a more in-depth discussion of structural neuropathology, neurobehavioral assessments, and nervous system electrophysiology. Each speaker addressed relationships between altered structure and function within the nervous system. Understanding and interacting more effectively with our scientific partners was the overarching theme of Session 1.
In the session’s first full-length presentation, titled “Overview of Neurocytology, Neurohistology, and Neurochemistry,” Dr. Robert Garman (Consultants in Veterinary Pathology, Inc., Murrysville, PA) reviewed the cytomorphologic appearances of normal and degenerating neurons utilizing images prepared from hematoxylin and eosin–stained histologic sections, as well as sections stained with the Fluoro-Jade or cupric-silver methods for neurodegeneration. Artifacts commonly encountered within histologic sections of the CNS were illustrated and discussed, including those commonly encountered in immersion-fixed brain tissue such as perivascular “retraction” spaces and cleft formation in certain regions including the granular cell layer of the dentate gyrus and the cerebellar Purkinje cell layer (Garman 1990). The dark neuron artifact, a morphologic change induced commonly by pressures exerted during the removal and handling of the unfixed brain, was particularly emphasized since it is so commonly misinterpreted as evidence of neuronal degeneration (Jortner 2006). The cytologic appearances of normal and pathologically altered glial cells were illustrated, including reactive astrogliosis, oligodendroglial intramyelinic edema, as occurs in classic triethyltin toxicity, and the infiltrative microglial responses to neurodegeneration and CNS inflammation. Some of the special stains useful in highlighting glial cells and tissue components were noted, including immunohistochemical methods for glial fibrillary acidic protein (GFAP; astrocytes) and for CD68/ED-1 (microglia) and the Luxol Fast Blue method for myelin. As the various neural cells were considered, the function and neurochemistry of a variety of neuroanatomic regions and structures that may be encountered within standard coronal sections of the rodent brain were highlighted and discussed, particularly highlighting the circumventricular organs that may not be readily recognized by all examiners.
In the session’s second major presentation, titled “Functional Assays,” Dr. Virginia Moser (U.S. EPA, Research Triangle Park, NC) discussed neurobehavioral and pathological evaluations of the nervous system as complementary components of basic research, as well as for toxicity testing of pharmaceutical and environmental chemicals. Behavior is a gross measure of integrated neural function that may be evaluated at many different levels and with a variety of tests. Common procedures for behavioral toxicity testing were reviewed, including initial screening studies designed to determine whether and at what doses a drug or chemical acts on the nervous system (e.g., the Irwin screen and functional observational batteries) and more complex, higher-tier assessments of neuromotor function (e.g., rotarod testing) and of cognition (e.g., simple water mazes and passive avoidance tests) that are designed to more completely characterize and quantify the behavioral effects and dose-response relationships (Slikker et al. 2005).
Examples of chemical-specific neurobehavioral-pathological correlations were discussed (e.g., cerebellar ataxia in 3-Acetyl pyridine treated mice, trimethyl tin toxicity in rats, and carbon disulfide inhalation in rats) to illustrate approaches to the interpretation and integration of neuropathologic and behavioral outcomes. Dr. Moser emphasized that correlations between behavioral changes and morphologic nervous system lesions have been identified in some but not all cases of neurotoxic injury. Potential explanations for situations in which behavioral changes are observed without recognition of morphologic lesions include effects on receptors and/or neurotransmitter release, as these often occur without inducing histologic changes (Dorman, Brenneman, and Bolon 2002). Timing of the histopathologic assessments may be critical, since instances in which behavioral changes precede structural changes are recognized (Lehning et al. 2003; Moser et al. 1992). While neuropathologic assessments provide insight into morphologic changes in neurons and other neural cells, behavioral and physiological methods evaluate the functional consequences of disrupted neuronal communications. Behavioral testing has an advantage over conventional measures of morphologic pathology in that it permits repeated evaluations over time to characterize the onset, progression, duration, and reversibility of a neurotoxic injury within a single animal. The underlying causes of some behavioral alterations are understood in certain cases, but many do not have known direct associations with specific neuropathologic processes. However, studies of genetically manipulated mice have provided considerable information into the genetic and phenotypic bases of both normal and altered behaviors.
The final presentation of the session, titled “Electrophysiologic Correlates of Structural Neuropathology,” was given by Dr. Joe Arezzo (Albert Einstein College of Medicine, Bronx, NY). Consideration of ways in which electrophysiology adds to and complements structural neuropathologic assessments, as well as areas in which these two disciplines diverge, were key topics explored in the session. Common types of electrophysiologic measures were discussed, including nerve conduction velocity, evoked potentials, compound muscle action potentials, transcranial magnetic motor stimulation, and electroencephalography (EEG).
Strengths of electrophysiologic assessments were noted. The methods (1) are noninvasive and can be repeated over time in the same animal to monitor the progression or recovery of functional decline; (2) are objective, specific for and focused on particular aspects of nerve function; (3) are reliable, with low coefficient of variation for repeated measures; (4) are identical to methods that are available in clinical settings; and (5) allow detection and characterization of true recovery of neural function. Examples of the last point were given from preclinical toxicology studies of nerve lesions that had returned to histologically normal appearance but remained functionally deficient at the end of nondosing recovery periods. In contrast, for many nervous functions, electrophysiologic assessments can provide a true determination of the degree of recovery of function. Limitations of electrophysiology were also discussed: (1) the various testing modalities generally reflect the activity of a subset of neurons within the structure being assessed, (2) certain regions (e.g., the autonomic nervous system, the association cortex, the fornix) are not readily assessed by these methods, and (3) there are instances in which assessed function is normal in spite of the demonstrated presence of morphologic lesions. A principal strength of electrophysiologic measures is their ability to detect functional deficits that may precede overt histopathologic lesions. A key limitation is that many measures reflect activity in only a subset of available neurons (e.g., large-diameter myelinated axons for nerve conduction velocity).
Several examples of the application of electrophysiologic assessments to a variety of neuropathologic conditions were addressed, including neuropathies, myelinopathies, axonopathies, and central excitotoxicity conditions. Additionally, EEG can be used to recognize preseizure activity in animals. Prior to testing, EEG screening may identify specific animals that have a preexistent increased risk for seizures, allowing deliberations as to whether the animal should be placed on the study. During the course of a study, EEG testing may identify onset of test article–related changes in brain function that could later culminate in a seizure.
Histopathologic assessments of nervous system structural abnormalities were recognized as foundational in neuropathology, the “granddaddy of measures.” Strengths include the high degree of validation, the ability to provide unquestionable evidence of abnormality, and a high degree of sensitivity to some induced changes. However, sensitivity for recognition of injury tends to be most acute during active phases of lesion development (e.g., when inflammatory changes or cell debris are present in tissue sections) and may be diminished at later times (e.g., when evidence of injury is marked by loss of cell processes or modestly decreased cellularity). Appropriate tissue sampling and sectioning are obviously pivotal factors in the sensitivity of histopathologic assessments. Focal lesions are missed when they do not appear in the histologic slides. Dying back neuropathies were offered as particularly illustrative of the significance of tissue sampling for histology: the sciatic nerve is the peripheral nerve typically sampled in preclinical toxicology safety studies but will not reflect the lesions developing in more distal sites. Finally, absence of active histologic findings is often incorrectly considered to represent lesion recovery.
In summary, General Session 1 of the STP/IFSTP 2010 Joint Symposium on Toxicologic Neuropathology addressed interrelationships of altered function and altered structure within the nervous system and disciplines for assessing these endpoints. The fundamental message of the session was that neuropathology and neural function testing (e.g., behavior testing, electrophysiology) are complementary approaches that, when used together, provide a comprehensive evaluation of the presence, onset, progression, and possible recovery of neurotoxic injuries.