Study On Acrylamide-induced Synaptic Damage And Mechanism In Rats

ObjectiveAcrylamide (Acrylamide, ACR) is a water-soluble vinyl monomer, that is a whitecrystalline solid at room temperature, mainly used for the production ofpolyacrylamide, widely used in drinking water purification, sewage treatment, cosmetics, dyes, ore processing, paper production, biological experiments (such as gel chromatography and gel electrophoresis), etc. The consumption of a high temperature cooking food may also leadto certain amount of acrylamide intake; animal experiments show that ACR exposure is through the respiratory tract, skin, mucous membranes and many other ways, causing neurotoxicity, reproductive toxicity, genotoxicity and carcinogenicity (International Center for Research on Cancer IARC, 1994 has listed it as "possible human carcinogen" 2A), and other health damages, while onlyneurotoxicity induced by acrylamide has been proved by human pandemic epidemiological survey results. ACR occupational poisoning cases frequently occured in exposed workers in many fields such as polyacrylamidesynthesis, sewage treatment and construction grouting operation. Patients have significant neurological injury symptoms, such as weakness, ataxia, peripheral hyperalgesia, vibration and position sense loss, etc.This study was aimed to explore the abnormal changes of central nervous system induced by acrylamide in rats, including neurotransmitter, pathological changes, distributionof synaptic vesicles, protein expression of Synapsin I, P-Synapsin I and brain-derived neurotrophic factor (BDNF), and to investigate the mechanism of acrylamide-induced subacute neurotoxicity of the central nervous system.Methods1. SD male rats were randomly divided into 11 days test group (control group, 30mg/kg ACR group,50mg/kg ACR group) and 21 days test group (control group,15mg/kg ACR group,30mg/kg ACR group).2. Rats were weighed and recorded everyday after the start of the experiment.3. General behavior and abnormal signs were observed and recordedafter the start of the experiment,gait scores were noted at day 7th and 11th of the 11 days test group,7th,14th and 21th of the 21 days test group.4. At the end of the exposure, prepared the cerebral cortex and cerebellum of rats carefully, measured the levels of Glu and GAB A using LC-MS/MS method.5. The morphology of rat cerebral cortex and cerebellum was observed by light microscopy, ultrastructure of nerve injury was observed by electron microscopy, and the distribution of the synaptic vesicles was quantitative analyzed.6. The distribution of Synapsin I, P-Synapsin Iin rat cerebral cortex and cerebellum wasdetermined using immunohistochemistry method.7. Changes of Synapsin I, P-Synapsin I, BDNF protein expression in rat cerebral cortex and cerebellum were observed using Western blot.Results1. Weight of rats treated with 50mg/kg ACR was decreased significantly compared with the control group and30mg/kg ACR treatment group at the end of the test (P<0.05). Weight of rats treated with 30mg/kg ACR was decreased significantly compared with the control group and 15mg/kg ACR treatment group at the end of the test (P<0.05).2. Gait scores of rats treated with 50mg/kg ACR was increased significantly compared with the control group and30mg/kg ACR treatment group at the end of the test (P<0.05). Gait scores of rats treated with 30mg/kg ACR was increased significantly compared with the control group and15mg/kg ACR treatment group at the end of the test (P<0.05).3. The level of Glutamate in cerebral cortex and cerebellum was decreased significantly at 30mg/kg group at the end of 21 d exposure period and 50mg/kg group at the end of 11d exposure period compared with the control group (P<0.05).4. ACR subacute exposure can cause neurons condensation, nuclear membrane recessed, glial cell edema, BBB structural damage, mitochondrial damage, polysome depolymerization and rough endoplasmic reticulum and Golgi apparatus dilation of central nervous system in rat.5. The number of synapses in cerebral cortex and cerebellum of rats treated with 30mg/kg ACR was decreased significantly compared with the control group at the end of the test (P<0.05), and the number of synapses in cerebral cortex and cerebellum of rats treated with 50mg/kg ACR was decreased significantly compared with the control group and30mg/kg ACR treatment group at the end of the test (P<0.05).6. The AZD (active zone distance) and NND(nearest neighbor distance)of synaptic vesiclesin cerebral cortex and cerebellum of rats treated with 30mg/kg ACR was increased significantly compared with the control group at the end of the test (P<0.05), and the AZDand NNDof synaptic vesiclesin cerebral cortex and cerebellum of rats treated with 50mg/kg ACR was increased significantly compared with the control group and30mg/kg ACR treatment group at the end of the test (P<0.05).7. ACR subacute exposure altered the distribution ratio of synaptic vesicles in RRP(readily releasable pool) and RP(reserve pool), trend test results showed that the RRP synaptic vesicles proportion of the cerebral cortex and cerebellum increased and RP synaptic vesicles proportion of the cerebral cortex and cerebellum decreased in a dose-dependentmanner(P<0.01).8. Immunohistochemistry results showed that Synapsin I and P-Synapsin I immunoreactive products were brown particles and the distribution were the same in rat cerebral cortex:positive particles distributed around the neurons, bring out the contours of the neuronal cell bodies. Synapsin I and P-Synapsin I immunoreactive products were brown particles and the distribution were the same in rat cerebellum:positive particles distributed around the Purkinje cells in the Purkinje cell layer, extending to the molecular layer and brought out the contours of dendritic branching, granular layer was also visible of the distribution of immunoreactive products.9. The protein expressionof Synapsin I andP-Synapsin Iin cerebral cortex and cerebellum of rats treated with 30mg/kg ACR was decreased significantly compared with the control group at the end of the test (P<0.05), and the protein expressionof Synapsin I andP-Synapsin I in cerebral cortex and cerebellum of rats treated with 50mg/kg ACR was decreased significantly compared with the control group and30mg/kg ACR treatment group at the end of the test (P<0.05). The protein expression of brain-derived neurotrophic factor (BDNF) induced by ACR subacute exposure waslightly increasedin cerebral cortex and cerebellum of rats.Conclusion1. The level reduction of Glu in cerebral cortex and cerebellum may be one of the mechanisms of ACR-induced subacute neurotoxicity.2. ACR subacute exposure increased the AZD and NND of synaptic vesicles and decreased the distribution ratio of synaptic vesicles in RRPin cerebral cortex and cerebellum.3. ACR subacute exposure decreasedthe protein expressionof Synapsin I andP-Synapsin Iin cerebral cortex and cerebellum, which explained the AZD and NND increase and the distribution ratiodecrease of synaptic vesicles in RRP. ACR subacute exposureincreasedbrain-derived neurotrophic factor (BDNF) protein expressionto a certain extentin cerebral cortex and cerebellum, which may be a protective factor to the central nervous system in short term.