Neuropathological Studies of Rats Following Multiple Exposure to Tri- Ortho -Tolyl Phosphate,Chlorpyrifos and Stress

Abstract

Adult male Long-Evans rats were exposed to 2 neurotoxic organophosphates in a setting of chronic stress, over a 63-day period. The organophosphates were tri-ortho-tolyl phosphate (TOTP) administered in 14 gavage doses of 75, 150 or 300 mg/kg, and chlorpyrifos, given in two 60 mg/kg subcutaneous exposures. Corticosterone was added to the drinking water at 400 μg/ml, to model aspects of chronic stress. These compounds/dosages were administered individually and in combination, with appropriate controls, giving rise to 16 experimental groups. The major neuropathologic change was the presence of axonal degeneration progressing to myelinated fiber degeneration, mainly in distal regions of selected fiber tracts and peripheral nerve, seen in animals sacrificed on experimental day 63. The cervical spinal cord and medullary levels of the sensory gracile fasciculus were most prominently affected. This axonopathy/fiber degeneration was TOTP dose-related at the 300 and 150 mg/kg levels. There was association of this lesion with inhibition of the enzyme neurotoxic esterase in hippocampal tissue from TOTP-treated rats. Such an association categorizes this disease process as organophosphate ester-induced delayed neuropathy. Neither chlorpyrifos nor corticosterone appeared to contribute to the neuropathic events or the enzyme inhibition. A cohort of rats was maintained on the corticosterone dosing, but without additional exposure to TOTP or chlorpyrifos, for an additional 27 days. When these rats were examined on day 90, the nerve fiber degeneration had progressed in all experimental groups administered the 300 mg/kg dose of TOTP (lower doses were not studied at the 90-day interval), although hippocampal neurotoxic esterase had returned to control values.

Introduction

Prolonged exposure to multiple chemicals is a likely event in industrialized societies, yet few studies examining the interactions of toxic chemicals, especially those eliciting injury to the nervous system, have been published (Mileson et al., 1998). In the present study, the interactions of two neurotoxic organophosphates of significance to human health were evaluated. These were the insecticide chlorpyrifos and tri-ortho-tolyl phosphate (TOTP), a congener of compounds used as lubricants and jet fuel additives (Weiner and Jortner, 1999). Both compounds have antiesterase effects, and the potential (especially TOTP) to elicit organophosphorus ester-induced delayed neuropathy (OPIDN) (Cavanagh, 1954; Richardson, 1995). Although studies have been published indicating that TOTP can exacerbate toxicities of certain other organophos-phorus cholinesterase inhibitors (Gallo and Lawryk, 1991), exposures have been short term, and none have explored the neuropathological effects.

Previous studies demonstrated that stress can enhance the toxicity of environmental chemicals such as organophosphates (Ehrich and Gross, 1983, 1986). In an effort to determine whether such a state might exist in the present study, we sought to mimic chronic stress by administering corticosterone in the drinking water over a long period of time. This regime has been noted to mirror chronic stress in rats, in particular its effect on body and thymic weights (Gannon and McEwen, 1990). Thus, this study was designed to evaluate the interactions of concurrent or sequential administration of chlorpyrifos, TOTP and corticosterone-induced stress in rats. The latter species was chosen because there exists an extensive database on its nervous system chemistry, function and structure (Paxinos, 2004). To determine neurotoxic effects, a multifaceted neurobehavioral, neurochemical and neuropathological study was undertaken. For this report, we focus on the neuropathological effects.

Materials and Methods

Experimental Design and Animals

This was a large, complex investigation of the interaction of 2 organophosphates and corticosterone in rats. There were 3 sacrifice intervals, experimental days 28, 63, and 90, each representing different toxicant exposure.

Adult male Long-Evans rats, 98–119 days old at the initiation of treatments, were used for these studies. The rats were housed singly in cages using bottles for water delivery beginning with the 7-day quarantine period before they were randomly assigned to treatment groups. Beginning at time of quarantine, they were on a 12-hour, light-dark cycle, with lights off at 7 AM. For the day 28 and day 63 sacrifices, 12 animals were included in each group for each sacrifice interval, with 4 rats/group in each of the 3 experimental blocks set up as a generalized randomized complete block design with a 2 × 2 × 4 factorial treatment structure. The latter included corticosterone in drinking water (chronic stress), chlorpyrifos 60 mg/kg, and TOTP, 75, 150, or 300 mg/kg. This resulted in 16 experimental groups, as indicated in Table 1. Dosing of organophosphorus compounds was always in the afternoon.

For this study, corticosterone exposure was initiated on day 0. There were 2 courses of organophosphate administration, from day 7 to 28 and from day 42 to 63. In these, the chlorpyrifos dosing was done on days 7 and 42 (a total of 2 doses), and 7 doses of TOTP were provided over each of the 2 periods of days 14–28 and 49–63. This selection of 2 dosing sequences separated by a 2-week treatment-free period was made for several reasons. It was needed to permit recovery from the acute toxic effects of TOTP, in particular its 2 higher dosages (300 and 150 mg/kg). In addition, this design allowed there to be sufficient toxicant exposure to elicit prominent neuropathologic change.

For the day-90 sacrifice, a 2 × 2 × 2 factorial treatment structure was employed. There were eight experimental groups of the same size as in the earlier studies (Table 1). Exposures were to corticosterone, 300 mg/kg TOTP and chlorpyrifos, and relevant controls, using the schedule noted above. The 64–90-day period was one in which no organophosphates were administered, although corticosterone treatment continued.

Functional Observational Battery (FOB) and motor activity determinations (Ehrich et al., 2004) were performed, but these clinical data are only summarized in this paper. At sacrifice (days 28, 63, and 90), 6 rats of each experimental group were euthanized by CO₂ inhalation. Fresh brain tissue was collected and dissected to provide samples of the basal fore-brain, caudate putamen, cerebral cortex, and hippocampus for assays of acetylcholinesterase and neurotoxic esterase. Data from these clinical and neurochemical studies at 28 days are reported elsewhere (Ehrich et al., 2004). An additional 3 to 6 rats per group were deeply anesthetized by intraperitoneal administration of pentobarbital sodium, and then perfusion-fixed for neuropathological examination (see below). The experimental protocols were reviewed and approved by the Virginia Tech Institutional Animal Care and Use Committee.

Test Agents

Corticosterone was administered in the drinking water at 400 μg/ml w/v to provide a model of chronic stress (Gannon and McEwen, 1990). Corticosterone (>96%, Steraloids, Inc., Newport, RI) was dissolved in ethanol by sonication; water was then added to provide a final concentration of 400 μg/ml w/v corticosterone and 2.5% v/v ethanol in the drinking water. By measuring daily water consumption, rats were found to consume the following quantities of corticosterone (mg/day/rat, mean +/− SD): 28-day study, 13.51 ± 2.08; 63-day study, 9.76 ± 2.74; 90-day study, 9.54 ± 2.45. Control rats were provided with drinking water containing 2.5% v/v ethanol. Corticosterone or the vehicle control was administered throughout the 90-day duration of the study.

Chlorpyrifos (diethyl 3,5,6-trichloro-2-pyridyl phosphorothionate 99%, Chem Services, West Chester, PA in corn oil) was administered at 60 mg/kg in a single subcutaneous injection given in a volume of 1 ml/kg on days 7 and 42. This dose did not produce overt cholinergic signs. TOTP (100%, Lark Enterprises, Webster, MA in corn oil) was given by oral gavage in 2 courses (days 14–28 and 49–63), using 7 doses over 14 days. Dosages of 75, 150, and 300 mg/kg were given at 1 ml/kg. Rats not treated with the organophosphorus compounds were given 1 ml/kg of the corn oil vehicle by similar routes at the same time as organophosphorus treatments.

Esterase Assays

Neurotoxic esterase (NTE) and acetylcholinesterase activities were determined in samples of cerebral cortex, basal forebrain, hippocampus, and caudate putamen (n = 6/interval). For the present study only the hippocampal data are presented, this being a valid reflection of findings in the other regions.

Spectrophotometric microplate assays were used for determination of acetylcholinesterase activity in hippocampus (Correll and Ehrich, 1991) with acetylthiocholine as substrate. Neurotoxic esterase activities were also determined using a spectrophotometric microassay (Correll and Ehrich, 1991). Protein was determined using a dye-binding assay (BioRad, Richmond, CA). Esterase activities were expressed per mg protein with comparisons made to the vehicle-treated controls. For statistical analyses of these neurochemical data, the GLM procedure of the SAS system (ver. 8.0, SAS Institute Inc. Cary, NC 27513) was used to fit a linear model to test for main effects and interactions of the 3 treatment factors (stress, chlorpyrifos, and TOTP). Significant TOTP effects were further analyzed by comparing each dose to the control using Dunnett’s t-test.

Neuropathology

Three to 6 rats from each experimental group at 28, 63, and 90 days were anesthetized with intraperitoneal pentobarbital, and transcardially perfused. A 1% heparinized (using a heparin solution containing 1000 units/ml) physiological saline wash-out solution was initially administered, followed by 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer. Brain, eye, spinal cord, dorsal root ganglion, and spinal nerve roots, sympathetic trunk, and the below noted peripheral nerves were dissected from perfusion-fixed rats, and held in perfusate at 4°C, except as follows. The eye was transferred to 10% neutral buffered formalin, and the brain into a 1:1 solution of the 4% paraformaldehyde solution and phosphate buffer.

Transverse sections of brain were made at levels of the basal forebrain, median eminence, rostral pons, and mid-pons, demonstrating structures such as caudate-putamen, thalamus, hippocampus, cerebral cortex and white matter, pons, and cerebellum. These were embedded in paraffin, sectioned and stained with H&E. Brain sections were also stained with Fluoro-Jade (a fluorescent stain to detect degenerating neurons [Schmued and Hopkins, 2000]), and the in situ TUNEL reaction (Roche Molecular Biochemicals, Mannheim, Germany) to look for internucleosomal DNA fragmentation consistent with apoptosis. In addition, brain sections were immunostained for GFAP using the peroxidase-antiperoxidase method (Sternberger Monoclonals, Lutherville, MD). These preparations were examined by light microscopy, except for the Fluoro-Jade stained sections. The latter were evaluated using a Nikon E400 microscope with a Y-FL fluorescent attachment with a filter cube having an excitation filter at 450–490 nm and a barrier filter at 520 nm (Schmeud and Hopkins, 2000).

The fixed midbrain (mesencephalon) was separated from the rest of the brain and serially cross-sectioned at 40 μm thickness using a vibratome. Every fifth section throughout the oculomotor nucleus region (bilaterally) was collected from rats in groups 1, 4, 5, 8, 9, 12, 13, and 16 (Table 1), and processed for CHAT immunoreactivity (Chemicon International, Temecula, CA). These included all animals exposed to corticosterone (stress), chlorpyrifos and/or the high dose of TOTP, with appropriate controls. Digital images of these sections of the oculomotor nucleus were obtained with a Nikon ES600 microscope.

These images were transferred to a computer for neuronal counts of the CHAT-positive neurons, using the Image Processing Tool Kit 5.0 (Reindeer Graphics, Inc., Asheville, NC), a plug-in to Adobe Photoshop. For this assessment, a single individual performed blinded independent bilateral counts (6 sections/rat, 4 to 6 rats/treatment group) of immunoreactive CHAT-positive neurons. These data were combined and are expressed as mean number (± standard deviation) of CHAT-positive neurons per section for each of the groups studied by this procedure (Table 5). Statistical analysis of these data was performed by analysis of variance.

Samples of cerebral cortex and hippocampus, transverse (cross) sections of the medulla caudal to the obex, the cervical, thoracic and lumbar levels of spinal cord, and the optic, sciatic, sural, tibial and vagus nerves, and longitudinal sections of lumbar spinal nerve roots and ganglia, and sympathetic trunk were postfixed in osmium tetroxide, embedded in PolyBed epoxy resin, sectioned at 1 μm thickness, and stained with toluidine blue and safranin. These were examined by light microscopy, using qualitative and semi-quantitative analyses. The extent of myelinated nerve fiber degeneration was scored by direct microscopic study of the cross-sectioned gracile fasciculus at the caudal medullary (most distal), cervical and thoracic (progressively more proximal) levels at 63 and 90 days, using the following scoring scheme. 0- no lesions or occasional scattered swollen axons present, 1—small collections of degenerated fibers in the medial and dorsal region of the tract; 2—greater spread of lesions laterally and ventromedially; 3—more extensive dorsal involvement, also extending ventrally; 4—diffuse involvement of the tract.

The medullary cross-section contained most distal levels of the gracile fasciculus bilaterally, including fibers within caudal aspects of the nucleus gracilis (Fugisawa and Shiraki, 1978). In descriptions that follow, this is termed the terminal (or medullary) level of the fasciculus. Lesions were so subtle at 28 days (Figure 1A) that these were assessed by direct microscopic counts of swollen myelinated axons in terminal levels of the gracile fasciculus. In addition, the severity of the peripheral neuropathy was assessed in cross-sections of the sural nerves by direct microscopic counts of degenerating fibers. All semiquantitative evaluations were done in a blinded fashion.

Samples of eye (mid-saggital section of the globe), liver, kidney, adrenal, lung, and heart taken from perfusion-fixed rats were embedded in paraffin, sectioned at 6 μm thickness, stained with H&E and examined by light microscopy.

Results

There was significant TOTP dose-associated inhibition of hippocampal NTE and acetylcholinesterase activities at post-dosing day 28 (Ehrich et al., 2004). As an example, exposure to TOTP at the 300 mg/kg level inhibited NTE to 42 ± 1% and acetylcholinesterase to 49% ± 12% of control values (Ehrich et al., 2004). Chlorpyrifos exposure also inhibited acetyl-cholinesterase. Data for activities of these enzymes in hip-pocampal tissue on days 63 and 90 are presented in Table 2. On day 63 (following two courses of toxicant exposure), there was enhanced TOTP dose-related inhibition of neurotoxic esterase ( p < 0.0001), whether administered alone or in combination with chlorpyrifos and/or corticosterone. Both TOTP and chlorpyrifos significantly inhibited hippocampal acetyl-cholinesterase activity ( p < 0.0001) (Table 2). TOTP did this in a dose-related fashion. In contrast to neurotoxic esterase, activity of acetylcholinesterase was inhibited by chlorpyrifos alone ( p < 0.0001) (Table 2). By day 90, after a 27-day period without exposure to organophosphates, the hippocampal neurotoxic esterase activity recovered, but that of acetyl-cholinesterase remained depressed, although to a lesser degree (Table 2).

Clinical findings from FOB testing are to be reported in detail elsewhere (this has been done in part in Ehrich et al., 2004), but some major findings (with causative agents) are briefly summarized here. During the course of the study, these included decreased body weight (TOTP, corticosterone [stress]), diminished grip strength (TOTP, corticosterone [stress], chlorpyrifos), decreased ability to remain on a ro-torod (TOTP, corticosterone [stress], chlorpyrifos), when compared to negative controls (group 1, Table 1). Interactions of these agents often enhanced the clinical effects.

Acute toxicity was noted during the first (days 14–28) and second courses (days 46–63) of TOTP administration. This was a TOTP dose-associated condition, present at the 300 mg/kg level, and to a lesser degree at 150 mg/kg, as it was not observed following chlorpyrifos administration on days 7 or 42. Clinical signs included weight loss, tremor, dehydration and recumbancy, necessitating supportive treatment (maintenance of hydration and body temperature). Mortality was noted in the 63-day study, with loss of 9/55 rats at the 300 mg/kg dosage and 5/52 at 150 mg/kg, with replacement of these in later blocks of the study.

The major neuropathological change was axonopathy progressing to myelinated fiber degeneration, best defined in the 1-μm-thick, toluidine blue and safranin-stained sections of the epoxy resin embedded tissue. These lesions were noted in medulla, spinal cord and peripheral nerves. At 28 days (after a single course of chlorpyrifos/TOTP exposure) these consisted of occasional swollen axons restricted to the terminal (medullary) level of the gracile fasciculus (Figure 1A). This modest change was noted in 4/6 rats given the 300 mg/kg dose of TOTP plus chlorpyrifos and 1/5 given this dosage of TOTP alone. It was not seen in animals exposed to TOTP and corticosterone (data was originally reported by Ehrich et al., 2004).

At 63 and 90 days there were prominent lesions in the cross-sectioned gracile fasciculus. As noted above, these rats had 2 courses of chlorpyrifos and TOTP exposure (days 7–28 and 42–63). The 90-day animals also had a subsequent 27-day respite from such organophosphate exposure, although corticosterone dosing persisted. The neuropathological changes were qualitatively similar in all affected experimental groups, and were most severe in terminal levels of the gracile fasciculus, but sometimes extended proximally (in a diminishing fashion) to cervical or thoracic regions (Figures 1B, C). These alterations consisted of myelinated axonal swelling with pallor or hyperchromatic staining (Figure 1D). In addition, many axons underwent collapse, with associated disorganized myelin sheaths indicative of advancing Wallerian-like degeneration (Figure 1D). Axonal loss and associated gliosis were noted in severely affected regions of the gracile fasciculus. The axonopathy was at times represented by massively, swollen dystrophic neurites, some of which demonstrated remnants of attenuated myelin sheaths (Figure 1E). Such massively swollen axons were more prominent in the high-dose TOTP rats on day 90 than on day 63, and were most frequent in terminal levels of the gracile fasciculus.

Scores reflecting the extent of lesions in cross-sections of the terminal (medullary) level of the gracile fasciculus for each treatment group are recorded in Table 3. This indicates that at 63 days, the greatest involvement is seen in rats receiving multiple exposures of the 300 mg/kg TOTP dose regardless of other concurrent treatments. Thus, prominent changes were noted in rats dosed with TOTP at the 300 mg/kg level alone (group 4), as well as in those also administered chlorpyrifos (group 8), corticosterone (group 12), or chlorpyrifos and corticosterone (group 16) (Table 3). Lesions of diminished incidence and intensity were noted in rats receiving the next lowest TOTP dose (150 mg/kg), in particular animals dosed with TOTP plus chlorpyrifos (group 7), TOTP plus corticosterone (group 11) and those receiving all 3 compounds (group 15) (Table 3).

At 90 days, only animals exposed to the 300 mg/kg level of TOTP alone (group 4), or in combination with chlorpyrifos (group 8), corticosterone (group 12) or both (group 16) were examined, along with appropriate controls (groups 1, 5, 9, 13–Tables 1, 3). Myelinated fiber degeneration in the terminal (medullary) gracile fasciculus was still prominent in animals given the high dose of TOTP (Table 3). In these groups, the fiber degeneration was qualitatively similar to that seen on day 63. There was a modest increase in lesion intensity in rats dosed with TOTP alone or in combination with other compounds (groups 4, 8, 12, 16, Table 3) over the 63- to 90-day period. In both the 63- and 90-day intervals, fiber degeneration of a lesser extent was often seen in more proximal levels (cervical, thoracic) of the tract in affected animals, and was proportional to severity of the medullary change (Figure 1C). Relative to day 63, these more proximal lesions were enhanced at 90 days. In cross-sections of the spinal cord and medulla, the medial-dorsal regions of the gracile fasciculus were most prone to contain degenerating fibers, at any level and interval examined (Figures 1B, C).

Peripheral nerve lesions consisted of axonopathy progressing to Wallerian-like myelinated fiber degeneration (Figure 1F), including bands of Büngner. This was seen in the sciatic, tibial and sural (Table 4) nerves, but spared the spinal nerve roots and the fibers and neurons of the dorsal root ganglia. The intensity of change in peripheral nerve, as measured by counts of actively degenerating sural nerve fibers, had a similar dose relationship to that in the gracile fasciculus of the central nervous system (Tables 3, 4). However, the peripheral changes were less prominent. As in the central nervous system, peripheral nerve fiber degeneration was most extensive in any group receiving the 300 mg/kg dose of TOTP, either alone or in combination with other agents (groups 4, 8, 12, 16–Table 4). The lesions had greater prominence at 90 days as compared to 63 days (Table 4). Comparison of lesions in tibial (distal) and sciatic (proximal) levels of peripheral nerve indicated greater degeneration in the former, especially at 90 days. At both 63 and especially at 90 days some thinly myelinated regenerating fibers were noted.

No lesions were noted in the brain stained by the H&E, GFAP, or TUNEL and Fluoro-Jade procedures. It must be noted that these sections did not include the region most prone to fiber degeneration, the caudal medulla, since that level had been reserved for epoxy resin embedding. In addition, no lesions were seen in sections of epoxy resin-embedded hippocampus and cerebral cortex. Counts of CHAT positive neurons in the oculomotor nucleus revealed no treatment-related effects on this neuronal population in rats exposed to corticosterone, chlorpyrifos, and the 300 mg/kg dosage of TOTP, alone or in combination at 63 and 90 days ( p > 0.05, Table 5). Similar results were shown for these groups at 28 days (Ehrich et al., 2004).

Extraneural lesions included TOTP-dose related focal hepatocellular necrosis, and atrophy of adrenal cortex associated with corticosterone exposure. Rats dosed with TOTP at 300 mg/kg levels and dying prior to the 63-day sacrifice had prominent hepatocellular necrosis. Since they died spontaneously, these rats were not perfusion-fixed, and thus not included as part of the neuropathological study in this report. The liver lesions were minimal in rats sacrificed on day 63, and only 25% of high-dose rats were affected. An exception was group 16, where 2/3 rats had moderate lesions. By day 90 these lesions had resolved. In corticosterone-exposed rats, the adrenal cortical atrophy involving the zonae fasciculata and reticularis was prominent on day 63, and had advanced by day 90, regardless of other treatments.

Discussion

This was a complex study in which the neuropathological effects and interactions of 2 neurotoxic organophosphates and a model of chronic stress were assessed in rats. The primary neurotoxic agent was noted to be TOTP, which elicited distal axonopathy progressing to Wallerian-like myelinated fiber degeneration in a dosage-associated fashion following 2 courses of treatment. Only selected fiber populations were noted to be affected, specifically distal regions of the sensory gracile fasciculus and peripheral nerves. There did not appear to be an effect of concurrent exposure to a second toxicant, chlorpyrifos, and/or stress (corticosterone in the drinking water) on the TOTP-associated fiber degeneration. However, this conclusion may not be absolute, due to the small size of some experimental groups and the absence of the full complement of groups examined on day 90 (see later and Tables 3, 4).

One interesting feature of the neuropathy was the prominence of fiber degeneration in the central and peripheral nervous systems at 90 days after 27 days without additional exposure to organophosphates, during which brain neurotoxic esterase activity had largely returned to control values (Table 2). In all of the 300 mg/kg dosage groups (groups 4, 8, 12, 16–Tables 3, 4), the lesions at 90 days were somewhat more prominent than at 63 days, even though organophosphate exposure had terminated. While it is known that the central nervous system post-toxicant exposure lesions of OPIDN resolve very slowly, those in peripheral nerve regenerate quite readily (Veronesi, 1984; Jortner et al., 1989). In the present study, fiber degeneration was still active in both regions. A striking lesion at 63 and especially at 90 days was the presence of massively swollen degenerating axons in the terminal gracile fasciculus (Figure 1E). These were considered to represent an advanced stage of the axonopathy, termed axonal dystrophy (Fujisawa and Shiraki, 1978; Jellinger, 1973). Dystrophic axons may represent a non-specific response to chronic, non-lethal neuronal injury, and are seen as aging-related events in terminal fibers of the gracile fasciculus in man and other animals (Fujisawa and Shiraki, 1978; Jellinger, 1973). These massive swellings are associated with intra-axonal accumulations of smooth membranes, tubular and cisternal structures, altered mitochondria and electron dense material (Jellinger, 1973).

The primacy of distal axonopathy of long myelinated fibers in OPIDN is demonstrated in the gracile fasciculus lesions. On days 63 and 90 we noted prominent fiber degeneration in distal/terminal regions of the tract, most prominent in dorsal and medial regions. Such regions are occupied by longer fibers, since the gracile fasciculus contains ascending primary collaterals of sensory dorsal root ganglion neurons having a medial to lateral organization based upon the caudal-rostral sequence of origin (tail, hindlimb, trunk, forelimb) (Davidoff, 1989; Tracy, 2004).

We failed to detect fiber degeneration and associated responses in the brain, even with the use of Fluoro-Jade and immunostaining for GFAP, except for that found in the caudal medulla. This is consistent with another study of OPIDN in the rat using silver degeneration staining, a sensitive measure of neuronal degeneration, which failed to demonstrate alterations in the brain above the medulla (Lehning et al., 1996). However, Itoh et al. (1985) did see minimal fiber degeneration in pons and cerebellum of rats administered multiple dosages of TOTP. More sensitive species, such as the ferret and chicken, often have lesions of OPIDN in cerebellum and brainstem (Cavanagh, 1954; Tanaka and Bursian, 1989; Tanaka et al., 1991). In our hands, the semithin sections from epoxy resin embedded caudal medulla, spinal cord, and peripheral nerve were quite sensitive in detecting fiber degeneration. These preparations allow critical, high resolution study by light microscopy. Except for the medulla and small samples of hippocampus and cerebral cortex, this approach was not used for the brain, possibly contributing to the negative findings rostral to the nucleus gracilis.

Lesions in the gracile fasciculus and peripheral nerve induced by TOTP in a dose- and time-related fashion and their evolution in association with toxicant-related inhibition of hippocampal NTE are indicative of OPIDN. The latter is manifest by distal axonopathy in large, long myelinated fibers which progresses to Wallerian-like degeneration (Cavanagh, 1954). The precise pathogenesis of this toxic neuropathy is unclear, but there is an association with antecedent inhibition and aging of NTE (Ehrich and Jortner, 2001). NTE inhibition is due to irreversible binding of pentavalent organophosphorus compounds to NTE, a membrane-associated carboxylesterase found in nervous and other tissues. However, a mechanistic pathway between inhibited NTE and axonal degradation has not been demonstrated. A number of toxicant-induced pathogenetic events have been demonstrated in nervous tissue in the evolving neuropathy. These include altered axonal transport, alteration of membrane lipids (especially gangliosides) and enhanced phosphorylation of axonal cytoskeletal elements (Carrington and Abou-Donia, 1985; Moretto et al., 1987; Lapadula et al., 1991, 1992; Bush et al., 1995; Gupta et al., 1997).

We noted changes indicative of axonopathy progressing to myelinated fiber degeneration and axonal collapse with myelin debris. These are replicas of lesions previously noted in OPIDN of rats and in more susceptible species, such as the hen (Cavanagh, 1954; Veronesi, 1984; Padilla and Veronesi, 1985; Jortner and Ehrich, 1987; Dyer et al., 1992; Inui et al., 1993). Corticosterone, chlorpyrifos, or a combination of these had no qualitative effect on what was believed to be the TOTP-induced lesions. The lesions in our study are consistent with other examples of this entity in rats (Veronesi, 1984; Padilla and Veronesi, 1985; Veronesi et al., 1986; Inui et al., 1993), both in nature and to a large degree in distribution. As regards the latter, while some studies noted more widespread change in spinal cord white matter tracts, the gracile fasciculus was the major site of fiber degeneration (Veronesi et al., 1986). Other work, in agreement with ours, demonstrates it to be the sole detected central nervous system site of injury in rats (Inui et al., 1993; Lehning et al., 1996).

Most multiple-dose, long-term studies of OPIDN feature sacrifice of animals shortly after cessation of toxicant administration (Prentice and Majeed, 1984; Veronesi, 1984). Itoh et al. (1985) performed a multiple dose study of tri-ortho-cresyl phosphate (identical to TOTP) in Sprague–Dawley rats, using a biweekly subcutaneous 600 mg/kg dose for six weeks. At sacrifice on postdosing days 60 and 160, axonopathy which appeared to progress to myelinated fiber degeneration, was noted in distal and terminal gracile fasciculus and in peripheral nerve, along with some neuronal loss. There was no indication of progression or repair of these lesions in the 100 days between the neuropathological assessments. Although our postdosing interval was only 27 days, we did observe progression of nerve fiber lesions in that period. It is well known that single neurotoxic doses of organophosphates in susceptible species such as the chicken give rise to nerve fiber degeneration after a latent period of 1 week or more (Cavanagh, 1954; Jortner and Ehrich, 1987). Our finding is consistent with such events occurring after cessation of multiple exposures, and confirms the USEPA policy of requiring such a post-dosing interlude in regulatory studies (USEPA, 1998).