Toxicologic Pathology

The methods for pathological assessment of the central nervous system and the peripheral nervous system differ, and there are also differences in the level of cellular resolution routinely achieved in screening techniques. In the PNS, plastic-embedded sections and teased nerve fibers from multiple regions provide satisfactory screening techniques; in the CNS, differential staining of paraffin sections provides the most practical screening approach. In both the CNS and PNS, the basic pathological processes are similar; these include Wallerian degeneration, distal axonal degeneration, and primary demyelination. In the future, new screening techniques based on immunocytochemical analyses and measurement of specific mRNA levels can be anticipated.

Some injuries to the developing nervous system can be detected with traditional evaluation for morphologic pathology, but many early injuries differ in character from those that are produced later in life. Such injuries arise from interference with developmental processes, rather than destruction of tissue. For example, an injury which kills neurons in the mature CNS leads to gliosis and a reduction in neuronal density, but a reduction in the number of neurons produced during development is not likely to lead to gliosis, and typically affects tissue volume rather than cell density. Some effects of developmental insults, such as misplaced and misoriented neurons, are never seen after adult injury. Functional effects reflect the role of the CNS in physiological regulation as well as in behavior. To evaluate CNS for developmental injury, it is necessary to know something about the structural and functional outcomes already recognized to result from teratogens and how these effects are related to time of exposure and time of testing.

There are many classes of chemicals widely used in a number of commercial and industrial processes having a potential to affect adversely the nervous system. Because of their relative sensitivity to some agents and general noninvasive characteristics, functional measurements of neurotoxicity are being used with greater frequency, especially at the level of hazard identification. The neurobehavioral test battery used by the National Toxicology Program (NTP) includes motor activity, fore-and hindlimb grip strength, acoustic startle response, responsiveness to an adverse thermal stimulus and general health and clinical measures (body weight, autonomic signs, tremor, convulsions). These tests have been used in nearly 40 studies involving various dosing regimens with rats and mice. The NTP battery shows several salient features of an effective screen, including the ability to differentiate known neurotoxicants from nonneurotoxicants, identify certain types of neurological sequelae or profiles of neurotoxic effects and construct dose-and time-response data in a screening context. The extent to which the NTP battery has predictive validity is still being evaluated.

In the future, quantitative techniques will probably be used in industry as part of Tier II studies for the evaluation of chemicals and drugs for their neurotoxic potential. Movement towards quantifying some structures or neuropathological changes will be made possible by advances in tissue preparation and computer technology. Emphasis will need to be placed on standardized techniques, good quality samples and sampling techniques in order to produce good quantitative data in a reasonable time. In this paper, different sampling techniques are evaluated using a cross section of rat sural nerve as the tissue for quantitative investigation.

Our philosophy is that screening tests should be applicable across species and emphasize complementarity to neuropathology. Within this context, electrophysiological tests comparable to those in human clinical neurology are powerful screening tools. For example, while histopathologic evaluation of the cochlea for ototoxicity is difficult, evoked potential audiometry is fast and easy. In this instance, one might routinely screen for deficits in auditory function, and reserve morphologic techniques for a characterization role rather than one of discovery. Lesions of neurons, axons and myelin are, however, readily assessed by light microscopy. A suitable combination of functional and morphologic screening tests, therefore, enhances the ability to discover neurotoxicity, and these data of ten are ideal for generation of refined hypotheses for subsequent characterization studies.

The peripheral nervous system and the central nervous system (CNS) are comprised of assemblies of neurons that communicate via electrical and chemical signals. Different disease processes selectively affect specific populations of neurons and/or specific cell functions (i.e., “selective vulnerability” of neurons is a principal determinant of phenotypes of disease). New cellular and molecular biological approaches have begun to clarify some of the mechanisms of selective cell injury in human diseases and their animal models. Following a brief review of the normal biology of nerve cells, we use illustrations drawn from studies of experimental and human diseases to discuss the mechanisms of structural/chemical abnormalities that occur in a variety of neuronal disorders.

Techniques currently used in the assessment of structural and/or functional damage to the peripheral auditory system are summarized. Two histological approaches are described: one which allows light microscopic evaluation of all structures of the auditory periphery, and a second which concentrates on the sensory cells and their innervation. The latter technique allows electron microscopic analysis of selected regions after a thorough light microscopic survey. Two electrophysiological methods are described as well: a single-fiber approach which provides detailed information about cochlear condition at all frequency locations and a simpler and faster evoked-potential approach which is well suited to screening for cochlear changes. The correlations between structural and functional changes arc described using examples from studies of acoustic injury of the inner ear.

In order to demonstrate the types of artifacts commonly seen in inadequately fixed central nervous system (CNS) tissues, rat brains were fixed by either perfusion or immersion in 10% neutral buffered formalin. Perfused brains were either removed immediately or left in situ for 1 hr prior to removal. Some of the brains destined for immersion fixation were first allowed to sit for either one-half or 1 hr in either room air or in an isotonic saline solution. Other brains were subjected to various handling procedures immediately after removal from the cranial vault in order to simulate the types of trauma which commonly occur during routine removal/dissection. Commonly observed artifacts included the presence of basophilic (dark) neurons, retraction spaces around neurons, vessels, and glial cells, displacement of the neuropil, and neuropil vacuolar change. The results of the fixation and handling procedures utilized in the collection of these brains substantiate the well documented fact that the least degree of artifact will be seen in brains fixed by perfusion and left in situ for a reasonable period of time (several hours) prior to removal.

Male and female Fischer 344 rats, 30 weeks of age, were examined for neuropathologic changes after a 13-week inhalation neurotoxicologic study. Tissues were preserved by whole-body perfusion with 1.5% glutaraldehyde/4% formaldehyde solution. An extensive set of neural tissues was embedded in paraffin, sectioned, and stained with hematoxylin and eosin, luxol fast blue/periodic acid-Schiff/Thematoxylin, Seviwr-Munger silver, and cresyl echt violet. Lesions in the central and peripheral nervous system were comparable between sexes and between control and treated animals. Bilateral swollen axons were present in the medial aspect of the nucleus gracilis adjacent to the area postrema. Occasional swollen axons also were observed in the dorsal and ventral funiculi of the spinal cord. Degeneration of individual nerve fibers was present in the trapezoid body, vestibular nerve root, trigeminal nerve, cerebellar peduncles, and the funiculi of the spinal cord. Individual nerve fiber degeneration also was present in the spinal nerve roots, sciatic and tibial nerves. Nerve fiber degeneration was characterized by myelin disruption and degeneration, vacuoles and axonal fragmentation. Similar spontaneous neuropathology may be encountered in rats from other subchronic neurotoxicologic studies and must be differentiated from treatment-related toxicity.

A novel type of intoxication in Canada in 1987 was traced to consumption of cultivated mussels contaminated with the excitotoxin domoic acid. Studies carried out in rats and monkeys revealed that parenterally administered domoic acid induces in rats neuroexcitatory phenomena culminating in seizures. Monkeys respond with gagging, emesis and less clearly evident seizure activity. CNS damage consisting of dendrotoxic and gliotoxic edema and nerve cell degeneration occurs in structures of the limbic system and the retina in both species. CNS lesion distribution similarities in animals treated with domoic acid or kainic acid suggest that these excitotoxins share a common pathogenesis mediated by glutamic acid, a putative endogenous excitatory neurotransmitter.

Nerve edema is a common response to the nerve injury seen in many peripheral neuropathies and is an important component of Wallerian degeneration. However, independent pathologic effects of nerve edema that aggravate or induce nerve injury extend the role of edema beyond that of an epiphenomenon of injury. New insights into the mechanism and impact of nerve edema come largely from animal models. In the following review, we discuss the cause and consequences of nerve edema with particular reference to en-doneurial fluid pressure and its relevance to the nerve microenvironment. Experimental models of nerve edema include conditions with increased vascular permeability such as lead poisoning, experimental allergic neuritis, and murine globoid leukodystrophy. Increased perineurial permeability induced by local anesthetics and neurolytic drugs can also induce nerve edema sufficient to increase endoneurial fluid pressure. Both perineurial and vascular permeability are increased after damage induced by crush, freeze, or laser injury. One of the most important forms of nerve edema is induced by external compression; the significance of this change is that edema has local compressive effects that persist after the external pressure has been relaxed. Nerve edema and increased endoneurial fluid pressure also occur in conditions in which vascular permeability appears to be unchanged such as experimental diabetic neuropathy and in hexachlorophene intoxication. In both of these conditions, reduced nerve blood flow has been demonstrated in rats and is viewed as a consequence of increased endoneurial fluid pressure. Whatever its mechanism, endoneurial edema has important structural and functional consequences for nerve fibers. A clear understanding of the underlying pathology of the nerve microenvironment may provide useful insights into treatment of clinical neuropathies.

Two decades of research with resorptive neurocarcinogens firmly established the high potency of methyl and ethylnitrosourea (MNU and ENU) as neurocarcinogens, particularly in rats. There are significant differences in susceptibility to these agents among species. There are also differences among age groups. Fetuses are between 50 to 100 times more susceptible than adult rats. One single iv inoculation of 20-50 mg/kg ENU into pregnant rats may produce neurogenic tumors in 100% of the of fspring. The tumors produced by these compounds have been well characterized morphologically, biologically, biochemically and histochem-ically. Tumors produced by both compounds are mostly gliomas and neurinomas (Schwannomas), however, clear differences exist between ENU and MNU produced neoplasms. Transplacental exposure to ENU generally results in a high number of anaplastic neurinomas and mostly differentiated gliomas (astrocytomas, oligodendrogliomas or mixed gliomas). In contrast, multiple exposures of adult rats to MNU result in a moderate number of mostly differentiated neurinomas and a high number of anaplastic gliomas. Tumors usually start out as well differentiated oligodendrogliomas or astrocytomas. As they grow larger, they become more mixed and anaplastic. In contrast to spontaneous gliomas in old rats, MNU and ENU-induced astrocytomas can be readily identified with well established biomarkers such as the S100 protein and particularly GFAP (glial fibrillary acidic protein). Neurinomas are also strongly positive for S100 protein. No reliable markers exist for oligodendrogliomas. Neurogenic tumors induced by MNU or ENU, as well as derived cell lines and clones from such tumors, have been successfully used as models for neurocarcinogenesis and therapeutic screening. Extensive research has been directed toward the molecular basis of neoplastic transformation (initiation) of neuroepithelial target cells by N-nitroso-compounds. Selective alkylation of DNA has been recognized as a crucial factor of initiation. More recently, host factors (growth and differentiation factors) have been recognized as prospective reverse transformation agents affecting tumor progression.

Although the morphology of neural tumors induced in rats by N-ethyl-N-nitrosourea (NEU) and related alkylating agents has been extensively investigated, their histogenesis and the molecular basis of malignant transformation are still largely unknown. This review gives an account of the interaction of neurocarcinogenic agents with cellular DNA, the possible role of promutagenic O6-alkyldcoxyguanines and their deficient repair by the cerebral O6-alkylguanine-DNA alkyltransferase. A new experimental model is described in which neural tumors are induced in fetal brain transplants. Pregnant rats received a single iv dose of NEU (50 mg/kg) on the 14th day of gestation. One day later, suspensions were prepared from the fetal forebrain and stereotactically injected into the caudoputamen of adult rats. After additional exposure to NEU of the host animals 8 days and 9 weeks post transplantation, all rats developed brain tumors within the neural graft. Histopathologically, all neoplasms were classified as oligodendrogliomas. Other neoplasms typically induced by NEU transplacentally (astrocytomas, mixed gliomas, ependymomas) were absent. The selective induction of oligodendrogliomas indicates that neoplastic transformation in the nervous system can occur in a differentiated glial cell or a precursor cell committed to oligodendrocytic differentiation, and that transformation of a pluripotential stem cell is not necessary. Transplacental exposure of the donor fetuses to NEU alone, i.e., without additional postgrafting exposure, did not produce brain tumors in any of the experimental animals indicating that in the microenvironment of fetal brain transplants the multistep development of gliomas requires additional mutational events. Malignant schwannomas perinatally induced by NEU carry a point mutation in the transmembrane domain of the neu gene. The mode of oncogene activation in NEU-induced CNS gliomas has not yet been elucidated. We have used cerebral grafting techniques to study the effects of known oncogenes on the developing nervous system, taking advantage of efficient gene transfer by replication-defective retroviral vectors and of the extraordinary capacity of fetal CNS to differentiate in and fully integrate with the host brain. Rats carrying transplants exposed in vitro to the polyoma medium T-antigen developed endothelial hemangiomas in the graft which of ten led to fatal cerebral hemorrhage within 13-50 days after transplantation. Introduction of the viral src gene caused astrocytic and mesenchymal tumors after latency periods of 2-6 months. Following infection of fetal donor cells with a vector encoding the v-myc oncogene, only a single embryonal CNS tumor was observed whereas exposure to v-H-ras produced a low incidence of gliomas. The combined expression of the oncogenes v-H-ras and v-myc resulted in the rapid induction of multiple malignant neoplasms, indicating a potent complementing effect of these genes in the brain. These results demonstrate that oncogene transfer into fetal brain cells constitutes a challenging new model to identify the effects of transforming genes on the various cellular conponents of the CNS.

The eye is an isolated unit but with a potentially high degree of sensitivity to toxic substances. The multiplicity of types of reaction to injury reflects the unique anatomical, physiological and biochemical features of the eye. The following are examples of such: The albino rat is not a good model for retinal toxicity because of problems of phototoxic retinopathy, the absence of pigment within the pigment epithelial layer and the high incidence of spontaneous retinal pathologies; The ocular toxicity of a compound cannot be anticipated from its chemical structure; Pharmacological side effects are similar between species, and are predictive for man; Mechanisms of ocular toxicity are poorly understood.

In a recent text by Grant (3), approximately 2,800 substances are listed as having some type of untoward effect on ocular tissue. The kinds of substances that may cause toxic damage/effects range from carbolic acid (2) to oxygen (1). It is the purpose of this communication to briefly review ocular toxicology as investigated in laboratory animals