چكيده لاتين :
Distal swelling and eventual degeneration of axon in the CNS and PNS have been considered to be the characteristic neuropathological effects of acrylamide (ACR) neuropathy. This study was coducted to determine the neurotoxic effects of different doses of ACR on hypothalamus of rat. To evaluate this hypothesis in hypothalamus, amino-cupric silver sataining technique of de Olmos and electron microscopic examination were conducted to define the histopathological (argyrophilia) and ultrastructural characteristic of axonal damage following ACR expo- sure in hypothalamus. For this purpose 60 adult male rats (Wistar, approximately 250 g) were selected. Rats were hosed in polycarbonate boxes as two per each. Randomly assigned groups of rats (10 rats per exposure group., total 5 exposure groups as A,B,C,D,E) were exposed to 0.5, 5,50,100 and 500 mg/kg per dayxlldays i.p. respectively. The remaining 10 rats were housed in group F as control group. Control rats received daily i.p. injections of 0.9% saline (3ml/kg). As indices of developing neurotoxicity, weight gain, gait scores and landing hindlimb foot splay (LHF) were determined. Weight gains were measured daily prior to injection. Gait scoring involved observation of spontaneous open field locomotion, included evaluations of ataxia, hopping, rearing and hind foot placement , and hind limb foot splay were determined 3-4 times per week. Gait score was assigned from 1-4. After 11 days, two rats for silver stain, and two rats for EM, were randomly selected, dissected and proper samples were collected from cerebrum, cerebellum and brain stem. Results did show no neurological behavior in groups A, B and F, whereas sever neurotoxicity was observed in group C. Rats in groups D and E died within 1-2 hours due to sever toxemia. In histopathological studies based on de Olmos technique no argyrophilic neurons or processes were observed in stained sections obtained from CNS of rats belong to groups A , B and F, while moderate to severe argyrophilic changes were
observed in different nuclei and regions of stained sections obtained from CNS of rats belong to group C. In ultrastructural studies some variations in the myelin sheet of injured axons including decompactation, interlaminar space formation, disruption of the laminar sheet, accumulation of neurofilaments, vacculation and clumping inside the axolem, and fmaly complete disappearance of laminar sheet were observed. Introduction Acrylamide (ACR) is an odorless, white crystalline solid at room temperature, water soluble and vinyl monomer that is used primarily to produce polyacrylamides with different physical and chemical properties, eg. water and waste water management (1,2,3), gell chromatography (1) and is present in certain foods that have been prepared at very high temperatures (4). ACR is a well documented neurotoxicant in both humans and laboratory animals. Subchronic low-level occupational exposure of humans to ACR produces neurotoxicity characterized by ataxia, skeletal muscle weakness and numbness of the hand and feet (1,5). Early morphological studies indicated that this neurotoxic syndrome was associated with nerve damage characterized by multifocal paranodal swellings of preterminal distal myelinated axons. These swellings contained an abundance of tubulovesicular profiles , neurofilaments, and degenerating mitochondria and, as ACR intoxication continued axonal regions below this swellings degenerated (5,6,7). Historically this type of nerve damage has been classified as a central-peripheral distal axonopathy and has been presumed to be responsible for the associated neurological defecits (8,9). However recent studies measuring multiple neurological parameters (21 mg/kg per day in drinking water versus 50 mg/kg per day ,i.p.) have indicated that ACR produced cumulative neurotoxicity. Specifically, dose-rate did not determine final magnitude of neurological deficit. Repeated daily exposure of laboratory animals ( rodents, rabbits, dogs, cats, and guinea pigs) to ACR (0.5-50 mg/kg per day) is associated with neurological signs that, in many respects, resemble the neurotoxicity occurring in humans; i.e., ataxia and skeletal muscle weakness. Early morphological studies suggested that both human and experimental neurotoxicities were mediated by Purkinjiee cell injury and by degeneration of distal axons in PNS and CNS. The neurotoxicity of ACR has been studied with respect to mammalian species and daily dose rates. The overt signs of neurotoxicity are consistent across species. In well-described rodent models, ACR intoxication at 10-50mg/kg per day produced a triad of neurological deficits; i.e., hindlimb foot splay, ataxia (open field gait abnormalities) and skeletal muscle weakness (decreased fore - and hindlimb grip strength). Assesment of neurological function over a 90 -day exposure at lower ACR dose- rates (0.05- 6.7 mg/kg per day) did not reveal evidence of neurotoxicity. This suggested that these dose - rates were non-neurotoxic. The purpose of this studt was to evaluate the effect of different doses (0.5 ,5 ,50 , 100, and 500 mg/kg per day., i.p.) of ACR on hypothalamus of rats. To define the spatiotemporal expression characteristics of neurodegeneration in hypothalamus of ACR-intoxicated rats, we used the de Olmos amino-cupric silver staining method and ultrastructural studies to identify degenerating neurons and their processes (dendrites, axons, nerve terminals). Compared to other silver degeneration techniques, the de Olmos method is characterized by higher sensitivity, specifity and reproducibility, which makes it an important tool for evaluating the spatial and temporal distribution of hypothalamic neuronal degeneration. Materials and methods the chemical substance, acrylamide monomer, used in this study, was of the best analytical grade available and obtained from Merck (99.7%) and kept at room temperature throught the study period. The solvent used was normal saline solution (0.9% ). The treatment doses used were as follow: 0.5, 5, 50, 100, 500 mg/kg bw per day i.p. with dose volume of 3 ml/kg . In this study 60 adult male rats (Wistar, ±250 grams, from Pasteure Institute) were used. Animals, after being weighted, were randomly divided into 6 groups including 5 treatment groups (A,B,C,D,E), containing 10 rats each, and 1 control group (F), containing 10 rats. Rats were housed in polycarbonate boxes, and drinking water and laboratory rodent chewing pellets were available ad libitum. The animal room was maintained at approximately 22oC and 50% humidity with a 12 h light/dark cycle. The study period was 11 days, during which the randomly assigned groups of rats (treatment groups) were exposed to neurotoxicant (ACR) at daily dosing- rates.
