Acrylamide Neuropathy

NeuroToxicology 23 (2002) 415±429

Acrylamide Neuropathy II. Spatiotemporal Characteristics of Nerve Cell Damage in Brainstem and Spinal Cord E.J. Lehning1, C.D. Balaban2, J.F. Ross3, R.M. LoPachin1,* 1

Department of Anesthesiology, Monte®ore Medical Center, Albert Einstein College of Medicine, Anesthesia Research-Moses 7, 111 E. 210th Street, Bronx, NY 10467, USA 2 Department of Otolaryngology, 107 Eye and Ear Institute, University of Pittsburgh, 203 Lothrop Street, Pittsburgh, PA 15213, USA 3 The Health and Environmental Safety Alliance, 442 Oliver Road, Cincinnati, OH 45215, USA Received 26 March 2002; accepted 4 June 2002

Abstract Previous studies of acrylamide (ACR) neuropathy in rat PNS [Toxicol. Appl. Pharmacol. 151 (1998) 211] and cerebellum [Neurotoxicology, 2002a] have suggested that axon degeneration was not a primary effect and was, therefore, of unclear neurotoxicological signi®cance. To continue morphological examination of ACR neurotoxicity in CNS, a cupric silver stain method was used to de®ne spatiotemporal characteristics of nerve cell body, dendrite, axon and terminal degeneration in brainstem and spinal cord. Rats were exposed to ACR at a dose-rate of either 50 mg/kg per day (i.p.) or 21 mg/kg per day (p.o.), and at selected times brains and spinal cord were removed and processed for silver staining. Results show that intoxication at the higher ACR dose-rate produced a nearly pure terminalopathy in brainstem and spinal cord regions, i.e. widespread nerve terminal degeneration and swelling were present in the absence of signi®cant argyrophilic changes in neuronal cell bodies, dendrites or axons. Exposure to the lower ACR dose-rate caused initial nerve terminal argyrophilia in selected brainstem and spinal cord regions. As intoxication continued, axon degeneration developed in white matter of these CNS areas. At both dose-rates, argyrophilic changes in brainstem nerve terminals developed prior to the onset of signi®cant gait abnormalities. In contrast, during exposure to the lower ACR dose-rate the appearance of axon degeneration in either brainstem or spinal cord was relatively delayed with respect to changes in gait. Thus, regardless of dose-rate, ACR intoxication produced early, progressive nerve terminal degeneration. Axon degeneration occurred primarily during exposure to the lower ACR dose-rate and developed after the appearance of terminal degeneration and neurotoxicity. Spatiotemporal analysis suggested that degeneration began at the nerve terminal and then moved as a function of time in a somal direction along the corresponding axon. These data suggest that nerve terminals are a primary site of ACR action and that expression of axonopathy is restricted to subchronic dosing-rates. # 2002 Elsevier Science Inc. All rights reserved.

Keywords: Toxic neuropathy; Nerve terminals; Axon degeneration; Distal axonopathy; Neurotoxicity

INTRODUCTION Acrylamide (ACR) monomer is used extensively in various chemical industries and is a well-recognized * Corresponding author. Tel.: ‡1-718-920-5054; fax: ‡1-718-515-4903. E-mail address: [email protected] (R.M. LoPachin).

neurotoxicant (US Environmental Protection Agency, 1988; Spencer and Schaumburg, 1974a,b; Tilson, 1981). Exposure of humans and laboratory animals to ACR causes ataxia and hindlimb skeletal muscle weakness (Spencer and Schaumburg, 1974a,b, 1977a,b; LeQuesne, 1980, 1985). Early morphological studies suggested that these neurological de®cits were a product of axon damage characterized by multifocal

0161-813X/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 6 1 - 8 1 3 X ( 0 2 ) 0 0 0 8 0 - 3

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swellings and eventual degeneration of long ®bers in the central and peripheral nervous systems (see reviews by Gold and Schaumburg, 2000; LoPachin and Lehning, 1994; Spencer and Schaumburg, 1976, 1978; Tilson, 1981). This damage was classi®ed as a central±peripheral distal axonopathy (Spencer and Schaumburg, 1980) and it was presumed that ACR acted at axonal sites to produce degeneration (Spencer and Schaumburg, 1978; LoPachin and Lehning, 1994). ACR is considered to be prototypical among the many neurotoxicants that produce this type of axonopathic injury (e.g. US Environmental Protection Agency, 1985, 1988). However, recent studies in rat have suggested that axon degeneration might not be a primary effect of ACR (see reviews by LoPachin et al., 2000, 2002a). Speci®cally, degeneration in peripheral nerve (sciatic, tibial, sural nerves) was restricted to a low-dose/long-term ACR intoxication paradigm (21 mg/kg per day, p.o.), i.e. a higher ACR dose-rate (50 mg/kg per day, i.p.) did not produce degeneration (Lehning et al., 1998; see also Crofton et al., 1996). A recent silver stain study of rat cerebellum (Lehning et al., 2002a) revealed that both ACR dose-rates (see above) produced progressive degeneration of Purkinje cell axons. Since this effect developed in conjunction with Purkinje cell dendrite and nerve terminal degeneration, axon argyrophilia was interpreted to be a consequence of general neuron injury. Finally, in rat forebrain, ACR intoxication at either the higher or lower dose-rate produced selective nerve terminal degeneration in many nuclei and regions (Lehning et al., 2002b). Based on a growing body of evidence (reviewed in LoPachin et al., 2000, 2002a), the role of axon degeneration in ACR neurotoxicity is now uncertain. A review of published literature suggests that nerve terminals might be an important site of action in PNS and CNS (see LoPachin et al., 2002a). However, it is noteworthy that neither nerve terminal nor axon degeneration in the CNS has been adequately documented with respect to dose-rate or time-dependent effects. As a consequence, the pathophysiological signi®cance of these and other potential neuronal sites (cell body) remains unknown. Therefore, as part of our continuing efforts to de®ne the spatiotemporal expression characteristics of neurodegeneration in CNS of ACR-intoxicated rats, we used a contemporary aminocupric silver staining method adopted from de Olmos et al. (1981, 1994) to identify degenerating neurons and their processes (dendrites, axons, nerve terminals). This paper compliments our recent cerebellar study (Lehning et al., 2002a) and focuses on argyrophilic

staining in brainstem and spinal cord of rats intoxicated with ACR at either 50 mg/kg per day (i.p.) or 21 mg/kg per day (p.o.). Compared to other silver degeneration techniques, the de Olmos method is characterized by higher sensitivity, speci®city and reproducibility, which makes it an important tool for evaluating the spatial and temporal distribution of CNS neuronal degeneration (de Olmos et al., 1994; Switzer, 2000). Silver staining also permits tracing of degenerating ®ber tracts so that the neurons of origin can be identi®ed (Balaban, 1994; de Olmos et al., 1994; Fix et al., 1996). This is a particularly important trait for the present study since we are attempting to distinguish primary or direct injury from axon degeneration that might occur secondary to perikaryal damage. Our ®ndings indicate that axon degeneration was evident in brainstem and spinal cord white matter during ACR intoxication at the lower dose-rate and was a relatively late onset effect. The dependency of axon degeneration on dose-rate suggests that it is not directly related to the neurological changes produced by ACR exposure. In contrast, certain brainstem nuclei (e.g. inferior olive, gracile and external cuneate nuclei) and spinal cord gray matter regions (e.g. layers 5±7 of the intermediate zone, layer 10) exhibited progressive nerve terminal degeneration regardless of ACR dose-rate. This provides further evidence that the nerve terminal might be a primary site of ACR action and that subsequent dysfunction and degeneration are speci®cally involved in the induction of neurological toxicity. METHODS AND MATERIALS Treatment and Neurological Evaluation of Animals All aspects of this study were in accordance with the NIH Guide for Care and Use of Laboratory Animals and were approved by the Monte®ore Medical Center Animal Care Committee. Adult male rats (Sprague± Dawley, 250±275 g; Taconic Farms, Germantown, NY) were used in this study. Rats were housed individually in polycarbonate boxes, and drinking water and Purina Rodent Laboratory Chow (Purina Mills Inc., St. Louis, MO) were available ad libitum. The animal room was maintained at approximately 22 8C and 50% humidity with a 12-h light/dark cycle. Randomly assigned groups of rats (4±6 rats per exposure group) were exposed to ACR by either i.p. injection (50 mg/kg per day) or via oral ingestion (2.8 mM in drinking water). Based on measured daily water consumption during oral exposure, rats in this group were

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exposed to a daily dosing rate of 21 mg/kg per day. ACR exposure paradigms are discussed in terms of dose-rates based on recent toxicokinetic studies showing that dose-rate and not route (i.p. versus p.o.) was the determinant of corresponding plasma/tissue concentrations (Barber et al., 2001). As indices of developing neurotoxicity, body weight and gait scores were determined 2±3 times per week. Gait scores (range 1± 4) were based on evaluation of spontaneous open ®eld behaviors, which included levels of ataxia, hopping, rearing and hindfoot placement (for details see Lehning et al., 2002a; LoPachin et al., 2002b). For both ACR dose-rates used in the present study, groups of age-matched control rats were weighed and gait scores were determined. Control rats for the higher ACR dose-rate group received daily i.p. injections of 0.9% saline (3 ml/kg). A trained, blinded observer who was not involved in animal care or ACR exposure performed the neurological testing. Amino-Cupric Silver Staining At speci®c times during ACR intoxication (50 mg/kg per day ˆ 5, 8 and 11 days; 21 mg/kg per day ˆ 7, 14, 21, 28, 35 and 38 days), neurotoxicant-exposed rats and respective age-matched controls were heparinized (1000 units per rat i.p.) and then deeply anesthetized with pentobarbital (50 mg/kg per day; i.p.). Rats were perfused through the aorta with 0.9% saline buffer containing 2 mM cacodylate, 22 mM dextrose, 22 mM sucrose and 2 mM CaCl2 at pH 7.4. Rats were then perfused with ®xative that contained 4% paraformaldehyde, 90 mM sodium cacodylate and 115 mM sucrose (de Olmos et al., 1994; Fix et al., 1996, 2000). Brains and spinal cords were removed and stored in ®xative until embedding. Detailed descriptions of the embedding and silver stain procedures are provided in a companion publication (Lehning et al., 2002a). CNS sections were viewed on a Nikon Eclipse E400 light microscope and were photographed with a Nikon Coolpix 995 digital camera. Brainstem and spinal cord regions were identi®ed using a rat brain atlas (Paxinos and Watson, 1998). Light Microscopic Examination of Cupric Silver-Stained Slides The de Olmos amino cupric silver staining method enables speci®c impregnation of degenerating neuronal perikarya, dendrites, axons and terminal arborizations (de Olmos et al., 1994). Moreover, this technique can detect both early and progressive degenerative

417

changes in neuronal soma and processes (Balaban, 1992). At the light microscopic level, silver-impregnated, degenerating neurons and/or their processes appeared black against a pink background. Early degenerative changes appear as powder-like argyrophilia, which often progresses to a Golgi stain-like density. As the degenerative process continues, the argyrophilic neuron or process is broken into irregular, punctate debris as degenerating material is removed by macrophage activity. Speci®c criteria for somatic degeneration were a cell body-like appearance, relative size and location (e.g. spinal cord gray matter, brainstem nuclei). Generally, axon degeneration in spinal cord or brainstem white matter consisted of linearly arranged black fragments. Preterminal axon and terminal degeneration appeared as short, irregularly arranged ®ber fragments, or somewhat variably shaped punctate debris, respectively. Background levels of silver staining were established in brain and spinal cord sections from control rats (e.g. see Figs. 2A and 3A) and speci®c degenerative events were determined in silverstained sections from CNS of ACR-intoxicated rats. RESULTS Neurological Evaluation Rats intoxicated at the 50 mg/kg per day dose-rate exposure group had a mean (S.E.M.) starting body weight of 267  3 g, which declined steadily to 250  5 g at endpoint (11 days; Fig. 1A). This represents a 6% decrease relative to initial weight. Rats in the agematched control group had a similar starting body weight (270  4 g) and, during the 11 days experimental period, gained approximately 27% of original body weight (333  7 g). Thus, when compared to age-matched controls, the mean body weight of ACR-exposed rats was 73  1% of control at endpoint (Fig. 1A). ACR intoxication at the higher daily dose-rate produced progressive increases in gait scores (Fig. 1B), i.e. at endpoint (11 days), the mean (S.E.M.) score was 3:4  0:2, which represents moderate-to-severe gait abnormalities (see also Lehning et al., 2002a). Exposure of rats to the 21 mg/kg per day dose-rate was associated with a truncation of normal weight gain (Fig. 1A). Thus, age-matched control rats had a starting mean body weight of 270  4 g, which increased steadily to 451  22 g at endpoint (38 days). This represents a 67% increase in body weight during the experimental period (Fig. 1A). Rats in the ACR group had a similar starting weight (263  2 g), but gained

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Fig. 1. Effects of ACR on rat body weight (A) and gait scores (B). ACR was administered to groups of rats at a daily dose-rate of either 21 or 50 mg/kg. Spontaneous open field behavior was assessed and observations were converted to numerical values ranging from 1 to 4 (see Methods and Materials Section). Body weight and gait score data are expressed as mean  S:E:M: Weight gain for both age-matched control groups during the initial 11 days period did not differ statistically and, therefore, the corresponding data were pooled. Arrows (B) indicate tissue sample times for both ACR dosing schedules.

only 38% of their original weight, i.e. at the day 35 endpoint, intoxicated rats weighed 373  9 or 83  2% of control. Intoxication of rats at the lower dose-rate caused progressive development of gait abnormalities such that at endpoint (day 38) mean gait score was 3:6  0:1 (Fig. 1B; see also Lehning et al., 2002a). Pattern of Neurodegeneration in Brainstem and Spinal Cord The pattern of argyrophilia observed in brainstem and spinal cord of ACR-intoxicated rats was consistent with primary nerve terminal degeneration. Both dose-

rates examined in this study produced ®ne punctate nerve terminal argyrophilia and swelling in many brainstem nuclei and in all spinal cord gray matter regions (Figs. 2±4). Nerve terminal degeneration, which appeared initially in certain nuclei, preceded or coincided with the onset of signi®cant gait abnormalities. With continuing ACR intoxication, the number of involved nuclei increased as did the density of argyrophilic nerve terminals. Speci®cally, exposure of rats at the higher ACR dose-rate (50 mg/kg per day) produced a nearly pure terminalopathy in brainstem and spinal cord regions, i.e. widespread nerve terminal degeneration was present in the absence of

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Fig. 2. This presents silver-stained horizontal sections of the thoracic spinal cord region from ACR-exposed and age-matched control rats. (A) This shows a silver-stained thoracic section from an age-matched control rat of the 21 mg/kg per day  28 days exposure group. The white matter lateral funiculus and gray matter lamina 7 and intermediolateral (IML) nucleus are as indicated. The open arrows denote neurons in lamina 7. At this magnification, silver-stained spinal cord sections from control rats exhibited a background of fine, evenly distributed, black dust in nuclei and white matter regions. (B) This shows the same spinal cord area from a rat exposed to ACR at 21 mg/kg per day  28 days. The boundaries of the IML are denoted by closed arrows and the arrowhead identifies a neuron in lamina 7 (pink stained). Open arrows in gray matter of (B) indicate degenerating preterminal axons, whereas in the lateral funiculus, the open arrows denote degenerating fibers. The moderate (lamina 7) to heavy (IML) punctate argyrophilic staining represents nerve terminal degeneration in different gray matter regions. (C) This shows a section of thoracic spinal cord from a rat exposed to ACR at 50 mg/kg per day  11 days. IML boundaries are defined by closed arrows, whereas arrowheads designate neurons (pink stained). At the higher ACR dose-rate, only nerve terminal degeneration was evident as indicated by the punctate argyrophilic pattern in spinal cord gray matter regions. (D) This shows a higher magnification photomicrograph of corresponding nerve terminal argyrophilia in lamina 7 of a rat intoxicated at the 50 mg/kg per day dose rate (XII days). Calibration bar: 20 mm for (A±C); 4 mm for (D).

signi®cant argyrophilic changes in neuronal cell bodies, dendrites or axons (Figs. 2±5). Intoxication at the lower ACR dose-rate (21 mg/kg per day) caused early nerve terminal argyrophilia in both brainstem and spinal cord (Figs. 2±4) with later developing axon degeneration (Fig. 5). Perikaryal argyrophilia was not evident in either CNS area at this dose-rate (Figs. 2±4). Thus, axon degeneration in brainstem and spinal cord occurred primarily during induction of neurotoxicity at the lower ACR dose-rate and evolved after nerve terminal argyrophilia. Axon degeneration was temporally dissociated from the onset of neurological de®cits.

Spatiotemporal analysis of the data (see subsequent sections) suggested that degeneration began at the nerve terminal and then moved as a function of time in a rostral direction along the corresponding axon. Spatiotemporal Distribution of Argyrophilia in Brainstem and Spinal Cord ACR Intoxication at the 50 mg/kg per Day Dose-Rate ACR intoxication at the higher daily dose-rate produced widespread, progressive degeneration of nerve

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Fig. 3. This presents silver-stained horizontal sections of the brainstem gracile nucleus from ACR-exposed and age-matched control rats. (A) This shows a silver-stained section from an age-matched control rat of the 21 mg/kg per day  28 days exposure group. The open arrows denote gracile neurons. (B and C) These show the gracile nucleus of rats exposed to ACR at either 21 mg/kg per day  28 days or 50 mg/kg per day  11 days, respectively. As is evident by the punctate argyrophilic staining pattern in these figures, the gracile nucleus exhibited substantial nerve terminal degeneration regardless of ACR dosing-rate. (D) This shows a higher magnification photomicrograph of corresponding nerve terminal argyrophilia in gracile nucleus of a rat intoxicated at the 50 mg/kg per day dose-rate (11 days). Calibration bar: 50 mm for (A±C); 4 mm for (D).

terminals in brainstem and spinal cord. Prior to the emergence of gait abnormalities (day 5; gait score ˆ 1:1  0:0; Fig. 1B), silver-impregnated nerve terminals were evident in many brainstem nuclei and regions, e.g. mesencephalic trigeminal nucleus, parabrachial complex, central gray area of the pons (Table 1). This effect was selective for nerve endings since neither somal, dendritic nor axonal degeneration was present in brainstem of these ACR-exposed rats. Also at the day 5 endpoint, argyrophilic changes were not present in spinal cord gray or white matter (Table 2). The appearance of slight gait abnormalities on day 8 of exposure (Fig. 1B; gait score ˆ 2:3  0:1) was associated with an increased incidence of argyrophilic nerve terminals in those nuclei originally affected (Table 1). Terminal degeneration was also evident in

additional nuclei that were unaffected at the earlier endpoint (day 5), e.g. gracile, cuneate and hypoglossal nuclei (Table 1). Other argyrophilic neuronal (i.e. dendrite, cell body or axon) changes were not evident in brainstem at the day 8 time-point. Rats exposed for 11 days to the higher ACR doserate exhibited severe gait impairment (Fig. 1B; gait score ˆ 3:4  0:2). In these animals, the spatial distribution of nerve terminal degeneration in brainstem did not change relative to day 8 data, i.e. additional nuclei were not affected (Table 1). However, the density of argyrophilic nerve terminals increased in all affected nuclei and regions (Table 1). Heavy terminal degeneration was noted in the brainstem mesencephalic trigeminal nucleus, parabrachial complex, central gray area of the pons and the periaquaductal

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Table 1 Density of nerve terminal degeneration in brainstem nuclei of rats intoxicated with ACR at the 50 mg/kg per day dose-ratea Region

Day 5

Gracile nucleus Cuneate nucleus External cuneate nucleus Vagus dorsal motor nucleus Solitary tract nucleus

0.0 0.0 0.0 0.0 1.3

Hypoglossal nucleus Inferior olivary complex Mesencephalic trigeminal nucleus Parabrachial complex Reticular formation Raphe nuclei

0.0  0.0 1.5  0.6 3.0  0.8

1.3  0.6 2.5  0.5 3.8  0.5

1.8  0.5 3.8  0.5 4.8  0.5

3.5  0.5 1.5  0.5 1.3  1.2

3.8  0.5 2.8  0.5 2.8  0.5

5.0  0.0 3.3  0.3 3.5  0.5

Tegmental nuclei Pons central gray Periaqueductal gray Inferior colliculus Superior colliculus

1.5 3.3 3.0 1.3 1.5

2.8 3.5 4.0 1.8 3.0

3.3 5.0 5.0 2.0 4.0

    

    

Day 8 0.0 0.0 0.0 0.0 0.5

0.5 0.5 0.0 0.6 0.6

1.7 0.9 1.8 1.3 2.5

    

    

Day 11 0.6 0.5 0.5 0.6 0.6

0.5 0.6 0.0 1.0 0.0

3.3 2.2 3.0 1.8 3.8

    

    

0.5 0.6 0.0 0.5 0.5

0.3 0.0 0.0 0.0 0.0

a Data are expressed as mean (S.E.M.) density of argyrophilic nerve terminals in different brainstem nuclei and regions. The density of degeneration was rated according to the following scaleÐ0: none, 1: rare, 2: occasional, 3: slight, 4: moderate or 5: heavy. ``Day'' represents duration of ACR exposure at the 50 mg/kg per day dose-rate. Degenerating neurons or their processes were not found in silver-stained brainstem sections from age-matched control rats (see Figs. 3 and 4).

Fig. 4. This presents silver-stained horizontal sections of the inferior olivary complex from brainstem of ACR-exposed and age-matched control rats. (A) This shows a silver-stained section from an age-matched control rat of the 21 mg/kg per day  28 days exposure group. (B and C) These show the inferior olivary complex of rats exposed to ACR at either 21 mg/kg per day  28 days or 50 mg/kg per day  11 days, respectively. Note that exposure to the lower ACR dose-rate (B) produces punctate nerve terminal degeneration that is restricted to the ventrolateral corner of the dorsal inferior olive (delineated by closed arrows), whereas terminal damage was evident in all olivary nuclei of rats

gray (Table 1). Notable degeneration was also evident in the superior colliculus, solitary tract nucleus, inferior olivary complex (Fig. 4) and in the reticular formation, tegmental and raphe nuclei (Table 1). At the day 11 endpoint, nerve terminals remained the selective targets since other argyrophilic neuronal changes were not observed in brainstem (Figs. 3 and 4). Although many brainstem regions sustained nerve terminal damage during intoxication at the higher dose-rate, several nuclei remained unaffected, e.g. lateral reticular, spinal trigeminal, facial, cochlear and red nuclei (data not shown). In spinal cord gray matter, nerve terminal degeneration was widespread at the day 11 endpoint (Fig. 2; Table 2). At all spinal cord levels (cervical±sacral), terminal degeneration was heaviest in laminae 5±9 and area 10 (Table 2). Axon degeneration in spinal white matter was rare (Fig. 5), with the exception of modest axonal argyrophilia …3:0  0:6† in the ventromedial area of the upper intoxicated at the higher dose-rate (C). AbbreviationsÐIOD: dorsal nucleus of the inferior olive; IOPr: principal nucleus of the inferior olive; IOM: medial nucleus of the inferior olive. Calibration bar: 50 mm for (A±C).

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Fig. 5. (A and B) These show the gracile fasciculus from the cervical spinal cord region of rats exposed to ACR at either 50 mg/kg per day  11 days (A) or 21 mg/kg per day  28 days (B). Open arrows in (B) denote degenerating axons. (C and D) These show the spinal trigeminal tract from the brainstem of rats exposed to ACR at either 50 mg/kg per day  11 days (C) or 21 mg/kg per day  28 days (D). Closed arrows in (D) denote degenerating axons. These figures demonstrate that the lower ACR dose-rate produces abundant axon degeneration in spinal cord (B) and brainstem (D) white matter, whereas intoxication at the higher dose-rate does not cause degeneration in white matter of these CNS regions (A and C). Calibration bar in (D): 10 mm for (A±D).

thoracic cuneate fasciculus (data not shown). Thus, intoxication of rats at the higher ACR dose-rate (50 mg/kg per day) produced selective and progressive degeneration of nerve terminals in many brainstem and spinai cord regions. ACR Intoxication at the 21 mg/kg per Day Dose-Rate Exposure to the lower ACR dose-rate produced early, speci®c nerve terminal degeneration with subsequent axonal degeneration in rat brainstem and spinal cord. In preclinical (i.e. gait score ˆ 1:1  0:1, Fig. 1B) rats exposed to ACR for 7 days, no argyrophilic changes were evident in these CNS region (Tables 2±5). In slightly affected rats (day 14; gait score ˆ 2:0  0:1; Fig. 1B), nerve terminal degeneration was apparent in several brainstem regions, especially the gracile nucleus, medial portion of the cuneate

nucleus and in the dorsal portion of the external cuneate (Table 3). Argyrophilic axons, dendrites or soma were not evident in either brainstem or spinal cord of these rats. By day 21, when rats in the lower exposure group displayed moderate gait impairment (gait score ˆ 2:8  0:2; Fig. 1B), the density of nerve terminal argyrophilia increased in those nuclei affected at the earlier time point (i.e. day 14). Also at day 21, the spatial distribution of nerve terminal degeneration broadened among brainstem nuclei (Table 3) and occasional axon degeneration was noted in several brainstem white matter tracts (Table 4). As intoxication with the lower ACR dose-rate continued (day 28), gait abnormalities worsened signi®cantly (gait score ˆ 3:3  0:1; Fig. 1B) and the incidence of nerve terminal and axon degeneration progressed (Tables 3 and 4, respectively; see also Figs. 3 and 4). Additional brainstem nuclei such as the spinal trigeminal, pontine

E.J. Lehning et al. / NeuroToxicology 23 (2002) 415±429 Table 2 Density of nerve terminal degeneration in spinal cord gray matter of ACR-intoxicated ratsa Region

21 mg/kg per day

50 mg/kg per day

Cervical enlargement L1-4 L5-7 L8-9 Area 10

0.0 3.3 3.0 2.3

   

0.0 0.6 0.0 0.6

3.0 3.3 3.3 4.7

   

0.3 0.6 0.7 0.6

Mid-thoracic L1-4 L5-7 L8-9 Area 10 IML nucleus Clarke's nucleus

1.7 3.7 2.7 3.0 2.0 3.7

     

0.6 0.5 0.6 0.9 0.0 0.6

2.0 3.3 1.3 2.3 3.7 2.7

     

0.9 0.6 0.5 0.6 1.0 0.6

Lumbar enlargement L1-4 L5-7 L8-9 Area 10

2.0 3.3 3.0 3.0

   

0.0 0.6 0.0 0.0

2.3 3.7 3.3 4.3

   

0.7 0.9 0.6 0.4

Sacral L1-4 L5-7 L8-9 Area 10

1.7 3.7 1.7 3.7

   

0.6 0.4 0.6 0.9

2.3 3.7 3.3 3.7

   

0.6 0.4 0.6 0.0

a Data are expressed as mean (S.E.M.) density of argyrophilic nerve terminals in different gray matter regions of rat spinal cord. The density of degeneration was rated according to the following scaleÐ0: none, 1: rare, 2: occasional, 3: slight, 4: moderate or 5: heavy. Rats were exposed to ACR at 50 mg/kg per day  5 or 11 days or 21 mg/kg per day  14 or 28 days. No argyrophilic changes were evident at day 5 of the higher doserate or day 14 of the lower dose-rate. Degenerating neurons or their processes were not found in silver-stained spinal cord sections from agematched control rats (Fig. 2). AbbreviationsÐL: laminae; IML: intermediolateral nucleus.

reticular and subcoeruleus reticular nuclei also exhibited nerve terminal degeneration (Table 3). It is important to note that, when compared to the higher doserate, intoxication at the 21 mg/kg per day dose-rate affected a smaller number of brainstem nuclei and in some instances only speci®c regions of nuclei were involved. For example, the entire inferior olivary complex (Fig. 4C) and the cuneate nucleus (Table 1) exhibited nerve terminal damage during ACR exposure at the higher dose-rate. In contrast, the lower dose-rate affected speci®c portions of these nuclei, i.e. the ventrolateral portion of the inferior olive (Fig. 4B) and the medial portion of the cuneate (Table 3). Moreover, although both dose-rates produced nerve terminal degeneration in several common brainstem nuclei (e.g. gracile, cuneate, inferior olive), the 50 mg/kg per day dose-rate affected additional nuclei such as the periaquaductal gray and solitary tract nucleus (compare

423

Tables 1 and 3). In spinal gray matter, widespread nerve terminal silver impregnation was evident at the day 28 endpoint (Table 2; Fig. 2B). The pattern of affected gray matter regions was similar to that of the higher ACR dose-rate (Table 2), i.e. terminal degeneration was heaviest in layers 5±9 and area 10. Axon degeneration in spinal white matter also occurred on day 28 and was heaviest in the gracile fasciculus and in the pyramidal and rubrospinal tracts (Table 5; Fig. 5B). Beyond day 28 of ACR intoxication at the lower doserate gait disturbances did not change signi®cantly, i.e. day 35 gait score ˆ 3:5  0:2; day 38 gait score ˆ 3:6  0:1 (Fig. 1B) and, correspondingly, the magnitude of nerve terminal and axon degeneration in brainstem regions remained constant (Tables 3 and 4, respectively). DISCUSSION We have suggested that axon degeneration was a secondary effect related to duration of ACR exposure (see reviews by LoPachin et al., 2000, 2002a). However, supporting evidence was primarily derived from studies in PNS tissues (e.g. sciatic, tibial and sural nerve; Lehning et al., 1998) and it was possible that at higher dose-rates axonopathy was primarily expressed in CNS (O'Shaughnessy and Losos, 1986; Burek et al., 1980; Yoshimura et al., 1992). Therefore, to determine the spatiotemporal expression characteristics of degenerating neuronal somata, dendrites, terminals and axons in brain and spinal cord of ACR-intoxicated rats, we used a contemporary silver stain technique. In this publication, we report our ®ndings from analyses of silver-stained sections from brainstem and spinal cord. Results show that intoxication of rats at the higher dose-rate (50 mg/kg per day) produced nerve terminal degeneration in brainstem and spinal cord. This effect was speci®c for terminals since, argyrophilic changes in other neuronal components in brainstem, spinal cord (this study) or dorsal root ganglion (Lehning et al., unpublished) were not evident at any time during intoxication at the higher dose-rate. This suggests that nerve terminal degeneration was not secondary to general neuron injury. Argyrophilic terminals appeared in several brainstem nuclei prior to the onset of neurological dysfunction. As ACR intoxication continued, the intensity and scope of nerve terminal damage in brainstem nuclei progressed in parallel with advancing gait abnormalities. Manifestation of maximal neurological effects (severe gait abnormalities) on day 11 of the higher ACR dosing paradigm coincided with

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Table 3 Density of nerve terminal degeneration in brainstem nuclei of rats intoxicated with ACR at the 21 mg/kg per day dose-ratea Region

Day 7

Day 14

Day 21

Day 28

Day 35

Day 38

Gracile nucleus Cuneate nucleusb External cuneate nucleusc Nucleus X Spinal trigeminal nucleusd

0.0 0.0 0.0 0.0 0.0

    

0.0 0.0 0.0 0.0 0.0

3.0 3.0 2.0 1.7 0.0

    

0.0 0.0 1.7 0.5 0.0

4.0 3.3 3.7 3.7 0.0

    

0.0 0.6 0.6 0.6 0.0

5.0 4.0 5.0 4.3 2.3

    

0.0 0.0 0.0 0.6 0.6

4.7 3.7 4.7 4.3 2.3

    

0.6 0.6 0.6 0.6 0.6

5.0 4.0 5.0 5.0 2.0

    

0.0 0.0 0.0 0.0 0.0

Principal sensory trigeminald Mesencephalic trigeminal Inferior olivary complexe Lateral reticular nucleus Intermediate reticular nucleus Lateral paragigantocellular ret

0.0 0.0 0.0 0.0 0.0 0.0

     

0.0 0.0 0.0 0.0 0.0 0.0

0.0 2.0 0.0 0.0 0.0 0.0

     

0.0 0.0 0.0 0.0 0.0 0.0

0.0 2.0 2.7 2.7 0.0 2.3

     

0.0 0.6 0.6 0.6 0.0 0.6

2.3 4.3 5.0 4.7 2.7 4.3

     

0.6 0.6 0.0 0.6 0.6 0.6

2.3 4.0 4.3 4.0 2.0 4.0

     

0.6 0.0 0.6 0.0 0.0 0.0

3.0 4.0 4.3 4.0 2.0 4.0

     

0.0 0.0 0.6 0.0 0.0 0.0

Pontine reticular nucleus Subcoeruleus reticular nucleus Spf lat parabrachial Superficial gray sup colliculus Optic N layer sup colliculus

0.0 0.0 0.0 0.0 0.0

    

0.0 0.0 0.0 0.0 0.0

0.0 0.0 2.3 0.0 0.0

    

0.0 0.0 0.6 0.0 0.0

0.0 0.0 3.7 1.7 1.7

    

0.0 0.0 0.6 0.6 0.6

3.3 3.7 4.3 3.3 2.3

    

0.6 0.6 0.6 0.6 0.6

3.7 3.7 4.7 3.0 2.3

    

0.6 0.6 0.6 1.0 0.6

3.7 3.7 5.0 2.5 2.5

    

0.6 0.6 0.0 0.7 0.7

a

Data are expressed as mean (S.E.M.) density of argyrophilic nerve terminals in different brainstem nuclei and regions. The density of degeneration was rated according to the following scaleÐ0: none, 1: rare, 2: occasional, 3: slight, 4: moderate or 5: heavy. ``Day'' represents duration of ACR exposure at the 21 mg/kg per day dose-rate. Degenerating neurons or their processes were not found in silver-stained brainstem sections from age-matched control rats (see Figs. 3 and 4). AbbreviationsÐlateral paragigantocellular ret: lateral paragigantocellular nucleus of the reticular formation; spf lat parabrachial: superficial (dorsolateral portion) lateral parabrachial nuclear complex; superficial gray sup colliculus: superficial gray layer of the superior colliculus; optic nerve layer sup coll: optic nerve layer of the superior colliculus. b Medial portion of the cuneate affected. c Dorsal portion of the external cuneate affected. d Dorsal third of these nuclei were affected. e Ventrolateral corner of the medial and dorsal inferior olive nuclei affected.

moderate-to-heavy nerve terminal degeneration in many brainstem nuclei and spinal cord gray matter regions. The lower ACR dose-rate also produced initial, selective nerve terminal degeneration, although this effect was associated with later developing axon degeneration. Nerve terminal damage occurred commensurate to the onset of neurological de®ciencies, and as intoxication at the lower dose-rate progressed, terminal damage and neurological dysfunction increased correspondingly. Axon degeneration, however, was

delayed relative to the initiation of terminal argyrophilia and changes in gait. The dissociation from neurotoxicity and differential dose-rate expression support our contention that axon degeneration is of secondary neurotoxicological importance. Results of the present study indicate that, irrespective of ACR dosing conditions, nerve terminal degeneration was the initial neuropathologic event in brainstem and spinal cord. These ®ndings con®rm and extend results from earlier morphological studies

Table 4 Density of axon degeneration in brainstem white matter tracts of rats intoxicated with ACR at the 21 mg/kg per day dose-ratea Tract

Day 7

Spinal trigeminal tract Sensory trigeminal tract Probst's tract Solitary tract Br superior colliculus

0.0 0.0 0.0 0.0 0.0

a

    

0.0 0.0 0.0 0.0 0.0

Day 14

Day 21

Day 28

Day 35

Day 38

0.0 0.0 0.0 0.0 0.0

0.0 0.0 1.3 2.3 0.0

2.3 3.3 3.3 3.0 2.0

3.7 3.0 3.0 3.3 1.3

3.7 3.0 3.0 3.5 1.5

    

0.0 0.0 0.0 0.0 0.0

    

0.0 0.0 0.6 0.6 0.0

    

0.6 0.6 0.6 0.0 0.0

    

0.6 1.0 0.0 0.6 0.6

    

0.6 0.0 0.0 0.7 0.7

Data are expressed as mean (S.E.M.) density of argyrophilic axons in different brainstem tracts. The density of degeneration was rated according to the following scaleÐ0: none, 1: rare, 2: occasional, 3: slight, 4: moderate or 5: heavy. ``Day'' represents duration of ACR exposure at the 21 mg/kg per day doserate. Degenerating neurons or their processes were not found in silver-stained brainstem sections from age-matched control rats (see Figs. 3 and 4). AbbreviationsÐsensory trigeminal: sensory root of the trigeminal nerve; Probst's tract: mesencephalic trigeminal tract; Br superior colliculus: brachium of the superior colliculus.

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425

Table 5 Density of axon degeneration in spinal cord white matter of rats after 28 days of dosing at the 21 mg/kg per day dose-ratea Region

Cervical

Thoracic

Lumbar

Sacral

Gracile fasiculus Cuneate fasiculus Lateral funiculus Pyramidal tract Rubrospinal tract Ventral funiculus

5.0 2.3 2.7 4.3 3.3 3.0

2.7 2.0 3.0 3.7 4.0 2.7

2.7 ± 1.7 3.0 3.7 1.3

3.0 ± 1.0 4.0 4.7 1.0

     

0.0 0.6 0.6 0.6 0.6 1.0

     

0.6 0.0 0.0 0.6 0.0 0.6

 0.6    

1.2 0.0 0.6 0.6

 0.6    

1.0 0.0 0.6 1.0

a Data are expressed as mean (S.E.M.) density of argyrophilic axons in different gray matter regions of rat spinal cord. The density of degeneration was rated according to the following scaleÐ0: none, 1: rare, 2: occasional, 3: slight, 4: moderate or 5: heavy. Rats were exposed to ACR at 21 mg/kg per day  14 or 28 days. No argyrophilic changes were evident at day 14 of this dose-rate. Degenerating neurons or their processes were not found in silver-stained spinal cord sections from age-matched control rats (Fig. 2).

of brainstem and spinal cord. Prineas (1969) reported that nerve terminal swelling and degeneration in cat CNS (spinal cord gray matter, gracile nucleus) were early consequences of ACR intoxication (10 mg/kg per day). Subsequent studies by Ghetti et al. (1973) and Cavanagh (1982a) also identi®ed early nerve terminal degeneration in spinal cord gray matter, gracile nucleus and superior colliculus of ACR-intoxicated animals. Electrophysiological studies of synaptic transmission in spinal cord of ACR-exposed cats determined that a signi®cant nerve terminal defect developed prior to the onset of neurological signs (Lowndes et al., 1978a,b; Goldstein and Lowndes, 1979, 1981; Goldstein, 1985; De Rojas and Goldstein, 1987). Electrophysiological and morphological studies in PNS of ACR-intoxicated animals also demonstrated early nerve terminal changes that were purported to be related to developing neurological changes (DeGrandchamp et al., 1990; DeGrandchamp and Lowndes, 1990; Tsujihata et al., 1974; see review by LoPachin et al., 2002a). Therefore, several lines of evidence suggest that ACR intoxication is associated with primary nerve terminal dysfunction and eventual degeneration. Expression of this terminalopathy in different nervous tissues might be the major neuropathologic event mediating autonomic and somatic de®cits that are characteristic of ACR neurotoxicity (see subsequent sections). Whether nerve terminal damage is caused by a direct or indirect effect of ACR is presently unknown. It is possible that reduced perikaryal synthesis and/or transport of nerve terminal components impairs transmission and, due to gradual depletion of materials, leads to degeneration (Cavanagh, 1964, 1979; Sickles et al., 1996; Stone et al., 2000). However, we have found that distal tibial axons from rats intoxicated at the higher ACR doserate were structurally intact and exhibited normal energy production, ion distribution and Na‡/K‡ATPase activity and enzyme content (reviewed in

LoPachin et al., 2000, 2002a). Moreover, although brainstem synaptosomes prepared from ACR-intoxicated rats were defective with respect to neurotransmitter release, no corresponding changes in protein content (e.g. GAP-43, SNAP-25, synaptotagmin) were found (LoPachin et al., 2002c). These ®ndings suggest that anterograde delivery is normal and are, therefore, inconsistent with the possibility that ACR inhibits cell body synthesis and/or axon transport of nerve terminal components. Instead, we have proposed that ACR adducts presynaptic cysteine-containing proteins (e.g. SNAP-25, NSF) that are critically involved in the formation of SNARE (soluble N-ethylmaleimide fusion protein receptor) complexes. These core complexes mediate membrane fusion processes such as neurotransmission and plasmalemmal turnover (Benfenati et al., 1999; Futterman and Banker, 1996; Lin and Scheller, 2000). Theoretically, ACR adduction of these proteins could impair synaptic function and promote nerve terminal degeneration (see hypothesis paper by LoPachin et al., 2002a). In contrast to nerve terminal damage, axon degeneration was observed only during subchronic ACR intoxication. Like previous studies (Ghetti et al., 1973; Spencer and Schaumburg, 1974b, 1976), degeneration was abundant in spinal cord white matter (e.g. gracile fasciculus) and in several brainstem tracts (e.g. spinal trigeminal tract) of rats intoxicated at the lower ACR dose-rate (21 mg/kg per day). However, we found that axon degeneration was not prevalent in either brain region of rats exposed to the higher exposure rate. These CNS ®ndings correspond to earlier PNS observations (Lehning et al., 1998), i.e. axon degeneration was found in tibial, sural and sciatic nerves during subchronic exposure (21 mg/kg per day), whereas intoxication at the higher dose-rate (50 mg/ kg per day) did not cause peripheral axon degeneration. Furthermore, in both CNS (this study) and PNS

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(Lehning et al., 1998) of rats intoxicated at the lower ACR dose-rate, axon degeneration was delayed relative to the onsets of nerve terminal damage and neurological dysfunction. This indicates that the functional signs of ACR neurotoxicity can be produced in absence of axon degeneration in either the PNS or CNS. Considered together, these data suggest that axonopathy is not a necessary component of the pathophysiological process induced by ACR. Spatiotemporal analysis suggests that the degenerative reaction is initiated in the nerve terminal and subsequently progresses in a retrograde direction. For example, the gracile nucleus in brainstem receives primary afferent input (terminals) from peripheral sensory neurons, the axonal projections of which travel in the gracile fasciculus of the spinal white matter (Leong and Tan, 1987; Ueyama et al., 1994). Our ®ndings indicate that the gracile nucleus of rats intoxicated at the lower doserate exhibited signi®cant nerve terminal degeneration at day 14, a time-point when axon degeneration in the gracile fasciculus was not evident. Similarly, the external cuneate nucleus also receives synaptic input from peripheral sensory afferent ®bers that ascend in the cuneate fasciculus (LaMotte et al., 1991; Campbell et al., 1974). Nerve terminal degeneration in this nucleus occurred at day 14 in the absence of axon degeneration in the cuneate fasciculus. Like these observations, Cavanagh (1982a,b) described initial swelling and argyrophilia of motor and sensory nerve terminals in skeletal muscle (fore- and hindlimb, tongue and masseter) of ACR-exposed rats (30 mg/kg per day  21 days). As intoxication progressed, preterminal axon degeneration developed and spread back into the corresponding intramuscular nerve and eventually into the main nerve trunk. These spatiotemporal relationships suggest that degeneration begins in the terminal and, as neurotoxicant exposure continues, the degenerative reaction spreads in a somal direction along the corresponding preterminal axon. Regardless of the present ®ndings, previous neuropathological research has provided substantial evidence for axon degeneration in spinal cord and brainstem white matter of ACR-intoxicated animals (Prineas, 1969; Ghetti et al., 1973; Spencer and Schaumburg, 1977b). However, it is important to note that these early studies focused on intoxication at lower ACR dose-rates (reviewed in LoPachin et al., 2000, 2002b) and that we have con®rmed axon degeneration at these rates. Our observation that ®ber degeneration is not a component of intoxication at higher ACR dose-rates suggests that this effect is of secondary neurotoxicological signi®cance.

Regardless of ACR dose-rate, many brainstem and spinal cord regions exhibited moderate-to-heavy nerve terminal degeneration. Although not determined in the present study, it is likely that this level of afferent damage impacts the overall functional output of affected nuclei. Consequently, damage to speci®c nuclei in the brainstem and spinal cord might play a signi®cant role in the somatosensory, somatomotor and autonomic dysfunction that characterizes ACR neurotoxicity (Spencer and Schaumburg, 1974a,b; LeQuesne, 1980, 1985; Gold and Schaumburg, 2000). For example, the cuneate and gracile brainstem nuclei were signi®cantly affected during exposure to both ACR dose-rates. In rat, these nuclei receive nerve terminals from somatosensory afferents primarily mediating cutaneous touch from the upper and lower portions of the body, respectively (Tracey and Waite, 1995; Beck, 1981; Ueyama et al., 1994). Also in the brainstem, the external cuneate nucleus exhibited substantial nerve terminal damage regardless of dose-rate. The external cuneate is the relay nucleus for the ascending cuneospinocerebellar tract which brings proprioceptive information from skeletal muscles of the forelimbs and upper body to the cerebellum (Beck, 1981; Campbell et al., 1974). In spinal cord, the dorsal nucleus of Clarke was signi®cantly affected by both ACR dose-rates. The principal synaptic input to this nucleus is primary afferents from dorsal root ganglion (DRG) neurons in the mid-thoracic, lumbar and sacral regions (Tracey, 1995). Clarke's nucleus is the initial relay nucleus in the dorsal spinocerebellar tract that carries proprioceptive information from the lower body, hindlimbs and tail to the cerebellum (Tracey, 1995). These ®ndings in conjunction with evidence for structural and functional damage to peripheral somatic sensory receptors (e.g. Pacinian corpuscle, muscle spindle; Schaumburg et al., 1974; Spencer and Schaumburg, 1977a; Sumner and Asbury, 1974; Lowndes et al., 1978a,b), suggest that somatosensory processing in ACR-intoxicated animals is impaired signi®cantly by damage to associated nerve terminals in PNS and CNS. Both ACR dose-rates also caused nerve terminal damage in the spinal intermediolateral cell column (IML nucleus) which contains preganglionic sympathetic neurons. This spinal nucleus receives afferent input from the hypothalamic paraventricular nucleus and provides efferent output to various organs under autonomic control (Jansen et al., 1995). Autonomic de®ciencies such as megaesophagus and changes in circulatory, baroreceptor and bladder control have been noted previously in ACR-intoxicated animals (Abelli et al., 1991; Satchell and McLeod,

E.J. Lehning et al. / NeuroToxicology 23 (2002) 415±429

1981; Satchell, 1990; Ralevic et al., 1991). Defective peripheral nerve terminal function has been implicated as a possible basis for these de®ciencies (Post and McLeod, 1977; Munch et al., 1994; Ralevic et al., 1991). Our data suggest that ACR-induced visceral problems also involve impaired central synapses in the autonomic nervous system. Finally, nerve terminal degeneration was evident in the intermediate (laminae 5±7) and ventral (laminae 8±9) regions of the spinal cord gray matter. Much of this degeneration is probably related to descending motor input from the rubrospinal, corticospinal and vestibulospinal tracts that terminate on interneurons and motoneurons in the gray matter (Antal et al., 1992; Tracey, 1995; Zemlan et al., 1979). By modulating the activity of spinal motoneurons that innervate the axial and limb musculature, these descending systems in rat in¯uence posture and movement (Tracey, 1995). Therefore, the ataxia and muscle weakness that characterize ACR neurotoxicity might be mediated by synaptic damage to upper motoneurons (this study) and by well-documented degeneration at the NMJ (e.g. DeGrandchamp et al., 1990; DeGrandchamp and Lowndes, 1990; Tsujihata et al., 1974). Clearly, additional experimentation is necessary to determine the functional status of these and other CNS nuclei and regions in ACR-intoxicated laboratory animals. Nonetheless, these data suggest that ACR causes sensory, motor and autonomic dysfunction by damaging peripheral and central synapses in respective nervous systems. SUMMARY The present study has shown that, regardless of doserate, nerve terminal degeneration was an early consequence of ACR intoxication. In contrast, we found that axon degeneration occurred only during intoxication at a lower dose-rate. The same pattern of nerve terminal and axon degeneration has been ascribed to the peripheral neuropathy induced by ACR (reviewed in LoPachin et al., 2000, 2002a). Our study of rat forebrain (Lehning et al., 2002b) has shown that both ACR dose-rates produced early nerve terminal degeneration in certain nuclei of the thalamus, hippocampus and basal ganglion. Axon degeneration in forebrain white matter was not evident in rats intoxicated at either dose-rate. In cerebellum, it appears that ACR causes early Purkinje neuron injury regardless of dose-rate (Lehning et al., 2002a). A growing body of evidence, therefore, suggests that ACR neurotoxicity is mediated by primary injury to cerebellar Purkinje cells and to

427

nerve terminals in the PNS and CNS. Axon degeneration occurs in the PNS and CNS during induction of subchronic neurotoxicity by lower daily ACR doserates. Consequently, the role of axon degeneration in the production of neurotoxicity is uncertain. ACKNOWLEDGEMENTS Research presented in this manuscript was supported by a grant (to R.M.L.) from the National Institute of Environmental Health Sciences (RO1 ES03830-16) and by funds provided by the Procter and Gamble Co., Cincinnati, OH. REFERENCES Abelli L, Ferri GL, Astolfi M, Conte B, Geppetti P, Parlani M, Dahl D, Polak JM, Maggi CA. Acrylamide-induced visceral neuropathy: evidence for the involvement of capsaicin-sensitive nerves of the rat urinary bladder. Neuroscience 1991;41:311± 21. Antal M, Sholomenko GN, Moschovakis AK, Storm-Mathisen J, Heizmann CW, Hunziker W. The termination pattern and postsynaptic targets of rubrospinal fibers in the rat spinal cord: a light and electron microscopic study. J Comp Neurol 1992;325: 22±37. Balaban CD. The use of selective silver degeneration stains in neurotoxicology. In: Isaacson RL, Jensen KF, editors. The vulnerable brain and environmental risks: malnutrition and hazard assessment, vol. 1. New York: Plenum Press; 1992. p. 223±38. Barber DS, Hunt JR, Ehrich MF, Lehning EJ, LoPachin RM. Metabolism, toxicokinetics and hemoglobin adduct formation in rats following subacute and subchronic acrylamide dosing. Neurotoxicology 2001;22:341±53. Beck CHM. Mapping of forelimb afferents to the cuneate nuclei of the rat. Brain Res Bull 1981;6:503±16. Benfenati F, Onofri F, Giovedi S. Protein±protein interactions and protein modules in the control of neurotransmitter release. Phil Trans R Soc Lond 1999;354:243±57. Burek JD, Albee RR, Beyer JE, Bell TJ, Carreon RM, Morden DC, Wade CE, Hermann EA, Gorzinski SJ. Subchronic toxicity of acrylamide administered to rats in drinking water followed by up to 144 days of recovery. J Environ Pathol Toxicol 1980; 4:157±82. Campbell SK, Parker TD, Welker W. Somatotopic organization of the external cuneate nucleus in albino rats. Brain Res 1974;77: 1±23. Cavanagh JB. The significance of the dying-back process in experimental and human neurological disease. Int Rev Exp Pathol 1964;3:219±67. Cavanagh JB. The dying back process. Arch Pathol Lab Med 1979;103:659±64. Cavanagh JB. The pathokinetics of acrylamide intoxication: a reassessment of the problem. Neuropath Appl Neurobiol 1982a; 8:315±36.

428

E.J. Lehning et al. / NeuroToxicology 23 (2002) 415±429

Cavanagh, J.B., Mechanisms of axon degeneration in three toxic neuropathies: organophosphorus, acrylamide and hexacarbon compared. In: Smith WT, Cavanagh JB, editors. Recent advances in neuropathology. New York: Churchill Livingstone; 1982b. p. 213±42. Crofton KM, Padilla S, Tilson HA, Anthony DC, Raymer JH, MacPhail RC. The impact of dose rate on the neurotoxicity of acrylamide: the interaction of administered dose, target tissue concentrations, tissue damage, and functional effects. Toxicol Appl Pharmacol 1996;139:163±76. DeGrandchamp RL, Lowndes HE. Early degeneration and sprouting at the rat neuromuscular junction following acrylamide administration. Neuropathol Appl Neurobiol 1990;16:239±54. DeGrandchamp RL, Reuhl KR, Lowndes HE. Synaptic terminal degeneration and remodeling at the rat neuromuscular junction resulting from a single exposure to acrylamide. Toxicol Appl Pharmacol 1990;105:422±33. de Olmos JS, Ebbesson SOE, Heimer L. Silver methods for the impregnation of degenerating axoplasm. In: Heimer L, Robards MJ, editors. Neuroanatomical tract-tracing methods. New York: Plenum Press; 1981. p. 117±70. de Olmos JS, Beltramino CA, de Lorenzo de Olmos S. Use of an amino-cupric silver technique for the detection of early and semiacute neuronal degeneration caused by neurotoxicants, hypoxia and physical trauma. Neurotoxicol Teratol 1994;16: 545±61. De Rojas TC, Goldstein BD. Primary afferent terminal function following acrylamide: alterations in the dorsal root potential and reflex. Toxicol Appl Pharmacol 1987;88:175±82. Fix AS, Ross JF, Stitzel SR, Switzer RC. Integrated evaluation of central nervous system lesions: stains for neurons, astrocytes and microglia reveal the spatial and temporal features of MK801-induced neuronal necrosis in the rat cerebral cortex. Toxicol Pathol 1996;24:291±304. Fix AS, Stitzel SR, Ridder GM, Switzer RC. MK-801 neurotoxicity in cupric silver-stained sections: lesion reconstruction by threedimentional computer image analysis. Toxicol Pathol 2000;28: 84±90. Futterman AH, Banker GA. The economics of neurite outgrowthÐ the addition of new membrane to growing axons. Trends Neurosci 1996;19:144±9. Ghetti B, Wisneiwski HM, Cook RD, Schaumburg HH. Changes in the CNS after acute and chronic acrylamide intoxication. Am J Pathol 1973;70:78A. Gold BG, Schaumburg HH. Acrylamide. In: Spencer PS, Schaumburg HH, Ludolph AC, editors. Experimental and clinical neurotoxicology. 2nd ed. New York: Oxford University Press; 2000. p. 124±32. Goldstein BD. Acrylamide neurotoxicity: altered spinal monosynaptic response to quipazine, a serotonin agonist in cats. Toxicol Appl Pharmacol 1985;78:436±44. Goldstein BD, Lowndes HE. Spinal cord defect in the peripheral neuropathy resulting from acrylamide. Neurotoxicology 1979; 1:75±87. Goldstein BD, Lowndes HE. Group Ia primary afferent terminal defect in cats with acrylamide neuropathy. Neurotoxicology 1981;2:297±312. Jansen ASP, Van Nguyen X, Karpitskiy V, Mettenleiter TC, Loewy AD. Central command neurons of the sympathetic nervous system: basis of the fight or flight response. Science 1995;270:644±6.

LaMotte CC, Kapadia SE, Shapiro CM. Central projections of the sciatic, saphenous, median, and ulnar nerves of the rat demonstrated by transganglionic transport of choleragenoidHRP (B-HRP) and wheat germ agglutinin-HRP (WGA-HRP). J Comp Neurol 1991;311:546±62. Lehning EJ, Persaud A, Dyer KR, Jortner BS, LoPachin RM. Biochemical and Morphologic characterization of acrylamide peripheral neuropathy. Toxicol Appl Pharmacol 1998;151:211± 21. Lehning EJ, Balaban CD, Ross JF, Reid MA, LoPachin RM. Acrylamide neuropathy. I. Spatiotemporal characteristics of nerve cell damage in rat cerebellum. Neurotoxicology 2002a; 23:397±416. Lehning EJ, Balaban CD, Ross JF, LoPachin RM. Acrylamide neuropathy. III. Spatiotemporal characteristics of nerve cell damage in rat forebrain. Neurotoxicology 2002b [submitted for publication]. Leong SK, Tan CK. Central projections of rat sciatic nerve fibres as revealed by Ricinus communis agglutinin and horseradish peroxidase tracers. J Anat 1987;154:15±26. LeQuesne PM. Acrylamide. In: Spencer PS, Schaumburg HH, editors. Experimental and clinical neurotoxicology. 1st ed. Baltimore (MD): Williams and Wilkins; 1980. p. 309±25. LeQuesne PM. Clinical and morphological findings in acrylamide toxicity. Neurotoxicology 1985;6:17±24. Lin RC, Scheller RH. Mechanisms of synaptic vesicle exocytosis. Annu Rev Cell Dev Biol 2000;16:19±49. LoPachin RM, Lehning EJ. Acrylamide-induced distal axon degeneration: a proposed mechanism of action. Neurotoxicology 1994;15:247±60. LoPachin RM, Lehning EJ, Opanashuk LA, Jortner BS. Rate of neurotoxicant exposure determines morphologic manifestations of distal axonopathy. Toxicol Appl Pharmacol 2000;167:75± 86. LoPachin RM, Ross JF, Lehning EJ. Nerve terminals as the primary site of acrylamide action: a hypothesis. Neurotoxicology 2000a [in press]. LoPachin RM, Ross JF, Reid ML, Dasgupta S, Mansukhani S, Lehning EJ. Neurological evaluation of chemically-induced neuropathies: acrylamide and 2,5-hexanedione. Neurotoxicology 2002b [in press]. LoPachin RM, Schwarcz AI, Gaughan CL, Mansukhani S, Das S. Acylamide impairs synaptosomal neurotransmitter uptake and release through protein thiol interactions. Neurotoxicology 2002c [submitted for publication]. Lowndes HE, Baker T, Michelson LP, Vincent-Ablazey M. Attenuated dynamic responses of primary endings of muscle spindles: a basis for depressed tendon responses in acrylamide neuropathy. Ann Neurol 1978a;3:433±7. Lowndes HE, Baker T, Cho E-S, Jortner BS. Position sensitivity of de-efferented muscle spindles in experimental acrylamide neuropathy. J Pharmacol Exp Theor 1978b;205:40±8. Munch G, Lincoln J, Maynard KI, Belai A, Burnstock G. Effects of acrylamide on cotransmission in perivascular sympathetic and sensory nerves. J Autonom Nerv Syst 1994;49:197±205. O'Shaughnessy DJ, Losos GJ. Comparison of central and peripheral nervous system lesions caused by high-dose shortterm and low-dose subchronic acrylamide treatment in rats. Toxicol Pathol 1986;14:389±94. Paxinos, G., Watson, C., The rat brain in stereotaxic coordinates. 4th ed. New York: Academic Press; 1998.

E.J. Lehning et al. / NeuroToxicology 23 (2002) 415±429 Post EJ, McLeod JG. Acrylamide autonomic neuropathy in the cat. Part 2. Effects on mesenteric vascular control. J Neurol Sci 1977;33:375±85. Prineas J. The pathogenesis of dying-back polyneuropathies. Part II. An ultrastructural study of experimental acrylamide intoxication in the cat. J Neuropath Exp Neurol 1969;28:598±621. Ralevic V, Aberdeen JA, Burnstock G. Acrylamide-induced autonomic neuropathy of rat mesenteric vessels: histological and pharmacological studies. J Autonom Nerv Syst 1991;34: 77±88. Satchell PM. Baroreceptor dysfunction in acrylamide axonal neuropathy. Brain 1990;113:167±76. Satchell PM, McLeod JG. Megaesophagus due to acrylamide neuropathy. J Neurol Neurosurg Psychiatr 1981;44:906±1003. Schaumburg HH, Wisniewski HM, Spencer PS. Ultrastructural studies of the dying-back process. I. Peripheral nerve terminal and axon degeneration in systemic acrylamide intoxication. J Neuropath Exp Neurol 1974;33:260±84. Sickles DW, Brady ST, Testino A, Friedman MA, Wrenn RW. Direct effect of the neurotoxicant acrylamide on kinesin-based microtubule motility. J Neurosci Res 1996;46:7±17. Spencer PS, Schaumburg HH. A review of acrylamide neurotoxicity. Part I. Properties, uses and human exposure. Can J Neurol Sci 1974a;1:151±69. Spencer PS, Schaumburg HH. A review of acrylamide neurotoxicity. Part II. Experimental animal neurotoxicity and pathologic mechanisms. Can J Neurol Sci 1974b;1:170±92. Spencer PS, Schaumburg HH. Central±peripheral distal axonopathyÐthe pathology of dying-back polyneuropathies. In: Zimmerman H, editor. Progress in neuropathology. New York: Grune & Stratton 3; 1976. p. 253±76. Spencer PS, Schaumburg HH. Ultrastructural studies of the dyingback process. III. The evolution of experimental peripheral giant axonal degeneration. J Neuropath Exp Neurol 1977a;36:276± 99. Spencer PS, Schaumburg HH. Ultrastructural studies of the dyingback process. IV. Differential vulnerability of PNS and CNS fibers in experimental central±peripheral distal axonopathy. J Neuropath Exp Neurol 1977b;36:300±20. Spencer PS, Schaumburg HH. Pathobiology of neurotoxic axonal degeneration. In: Waxman SG, editor. Physiology and pathobiology of axons, New York: Raven Press; 1978. p. 265± 82.

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Spencer PS, Schaumburg HH. Classification of neurotoxic disease: a morphological approach. In: Spencer PS, Schaumburg HH, editors. Experimental and clinical neurotoxicology. Baltimore: Williams and Wilkins; 1980. p. 92±9. Stone JD, Peterson AP, Eyer J, Sickles DW. Neurofilaments are nonessential elements of toxicant-induced reductions in fast axonal transport: pulse labeling in CNS neurons. Neurotoxicology 2000; 21:447±58. Sumner A, Asbury AK. Acrylamide neuropathy: selective vulnerability of sensory fibers. Trans Ann Neurol Assoc 1974; 99:78±83. Switzer RC. Application of silver degeneration stains for neurotoxicity testing. Toxicol Pathol 2000;28:70±83. Tilson HA. The neurotoxicity of acrylamide: an overview. Neurobehav Toxicol Teratol 1981;3:445±61. Tracey DJ. Ascending and descending pathways in the spinal cord. In: Paxinos G, editor. The rat nervous system. New York: Academic Press; 1995. p. 689±704. Tracey DJ, Waite PME. Somatosensory system. In: Paxinos G, editor. The rat nervous system. New York: Academic Press; 1995. p. 67±80. Tsujihata M, Engel AG, Lambert EH. Motor end-plate fine structure in acrylamide dying-back neuropathy: a sequential morphometric study. Neurology 1974;24:849±56. Ueyama T, Houtani T, Ikeda M, Sato K, Sugimoto T, Mizuno N. Distribution of primary afferent fibers projecting from hindlimb cutaneous nerves to the medulla oblongata in the cat and rat. J Comp Neurol 1994;341:145±58. Toxic Substance Control Act Testing Guidelines. vol. 50. Federal register, 40 CFR, Part 798, Subpart G. no. 188, Washington (DC): Office of Toxic Substances, US Environmental Protection Agency; 1985. Preliminary assessment of health risks from exposure to acrylamide. Washington (DC): Office of Toxic Substances, US Environmental Protection Agency; 1988. Yoshimura S, Imai K, Saitoh Y, Yamaguchi H, Ohtaki S. The same chemicals induce different neurotoxicity when administered in high doses for short term or low doses for long term to rats and dogs. Mol Chem Neuropathol 1992;16:59±84. Zemlan FP, Kow LM, Morrell JI, Pfaff DW. Descending tracts of the lateral columns of the rat spinal cord: a study using the horseradish peroxidase and silver impregnation techniques. J Anat 1979;128:489±512.