Effects Of Acrylamide Exposure On Anterior Horn Motor Neurons Of Spinal Cord,Sciatic Nerve And Gastrocnemius Motor Endplate In Rats

The toxicity of acrylamide(ACR)is well known,including: neurotoxicity,carcinogenicity,genotoxicity,reproductive toxicity and genetic toxicity.The general population mainly through dietary intake,while occupational population through occupational environment.One of the most important toxic effect of ACR on humans and animals is neurotoxic effect,especially on peripheral nerves.Sensory and motor dysfunction,such as skeletal muscle weakness,weight loss,and ataxia,can occur in both experimental animal models and people exposed to the substance over time.ACR mainly ACTS on nerve endings and exerts a neurotoxic effect by impairing synaptic transmission.Therefore,it is of certain practical significance to pay attention to the damage of ACR to peripheral nerves.Studies have shown that among the symptoms in rats exposed to ACR,skeletal muscle weakness in both hind limbs is particularly obvious.As an important nerve of the lower limbs,the sciatic nerve is the most sensitive to toxic effects.Meanwhile,the gastrocnemius muscle is innervated by the sciatic nerve branch and plays an important role in the movement of the hind limbs.The motor endplate(MEP)of the gastrocnemius muscle is a crucial structureto maintain the normal motor function of the skeletal muscle.Also the sciatic nerve is composed of the axons of anterior horn motor neurons of central nervous system(CNS),and its functional state is necessarily closely related to the functional state of the neurons.Therefore,in this study,MEP of gastrocnemius muscle,sciatic nerve and spinal cord of corresponding segments(lumbosacral enlargement)were selected to detect the changes in tissue morphology and the expression of related proteins,so as to explore the possible causes of skeletal muscle weakness induced by ACR poisoning in rats,and provide experimental basis for the treatment of ACR poisoning in clinical practice.Method:1.Animal model establishment: 32 SPF grade adult male SD rats were randomly divided into control group(0mg/kg),low(9mg/kg),medium(18mg/kg)and high(36mg/kg)dose groups,with 8 rats in each group.ACR was transfected by gavage for a total of 21 days.During the period,the body weight was weighed on the next day,the dosage was adjusted,and the behavioral test(gait score and landing foot opening distance)was conducted once a week.On the 22 nd day,the samples were taken,and the wet weight of the gastrocnalis muscle was weighed.2.To detect the toxic effects of ACR on the anterior horn motor neurons of the spinal cord in rats: the morphological changes of the anterior horn motor neurons of the spinal cord in rats were observed by HE staining;Nissl staining was used to observe the changes of nissl body content in spinal cord of rats;The ultrastructural changes of the synapses of the anterior horn motor neurons of spinal cord in rats were observed by transmission electron microscopy;The expressions of AchE,CGRP and GAP43 were detected by IHC and WB.3.To detect the toxic effects of ACR on the sciatic nerve of rats: changes in the structure of the sciatic nerve in rats were observed by HE staining;Transmission electron microscope was used to observe the ultrastructural changes of the myelin lamina of the sciatic nerve in rats;LFB staining was used to observe the expression of myelin in the sciatic nerve of rats;IHC and WB were used to detect the expression of MBP and MAG in the sciatic nerve of rats.4.To detect the toxic effect of ACR on motor endplate of gastrocnemius in rat : changes in rat muscle fiber structure were observed by HE staining;The morphological changes of nerve endings and MEP in rats were observed by gold chloride staining;The content of acetylcholinesterase in gastrocnemius muscle was detected by acetylcholinesterase staining;Expression of AchE and CGRP in gastrocnemius muscle was detected by IHC and WB.Result:1.Behavioral changes in rats: after ACR exposure,the weight of rats began to increase from the third week,and the growth rate was negatively correlated with the exposure dose.The weight of rats in the high-dose group began to decrease,and the comparison between the groups showed statistical differences of different degrees.The gait score results showed that the gait score of the control group was always 1 point,while the gait score of the low,medium and high dose groups increased and showed a dose-dependent effect.The comparison between the groups from the second week showed statistical difference.The results of landing foot opening distance showed that from the second week,the high-dose group was significantly wider than the other groups(P<0.01).From the third week,there were significant widths in the medium dose group compared with the control group and the low dose group(P<0.01),and there were also widths in the low dose group compared with the control group (P<0.01).2.Changes in the toxicity of motor neurons in the anterior horn of the spinal cord: HE results showed that after ACR exposure,the cell bodies of motor neurons were distributed sparsely,and atrophy and staining were observed.At the same time,the range of nissl staining became smaller(P<0.01),the color became lighter,the intracellular nissl bodies were scattered,and the cell volume became smaller.The results of transmission electron microscopy showed that the number of synapses in the anterior horn of the spinal cord in the high-dose group was less,the structure was not clear,the color was light,the synaptic vesicles and the release of transmitters was reduced.The expressions of AchE,CGRP and GAP43 in the low,medium and high dose groups were decreased in a dose-dependent way compared with the control group by IHC and WB tests(P<0.01).3.Changes in sciatic nerve toxicity: HE results showed that after ACR exposure,the myelin sheath in the low and medium dose groups showed varying degrees of swelling and the axon structure was changed.In the high-dose group,medullary nerve fibers were disordered,myelin sheath structure was not clear,and axons disappeared.Transmission electron microscopy showed that after exposure,cavities were formed in the myelin lamellar layer,and the axonal mutation occurred.The organelles of schwann cells were dissolved and collagen fibers proliferated.LFB myelin staining(eosin staining)showed that after exposure,myelin structure became thinner and thinner,and staining became lighter and uneven.IHC and WB detection showed that,compared with the control group,MBP and MAG expressions in the low,medium and high dose groups decreased with the increase of toxic dose(P<0.01).4.Changes in the toxicity of the motor endplate of gastrocnemius: the results of wet weight of the gastrocneum showed that,the moderate dose group was significantly reduced when compared to the control group(P<0.05),and the high-dose group was the most significant reduction(P<0.01).The results of HE showed that the myofibrosis was atrophic and became thinner,and the arrangement was dense,with the phenomenon of nuclear aggregation.The higher the dose,the more obvious it was.Gold chloride staining showed that after poisoning,the peripheral nerve branches became smaller and thinner,the nerve fiber structure became shorter and thinner, the MEP structure was less,the staining became lighter,and the structure was not clear.Acetylcholinesterase staining(cuprous ferricyanide staining) showed that the MEP morphology of the poisoned rats became smaller and distributed sparsely in a dose-dependent manner.Compared with the control group,the content of acetylcholinesterase in the low,medium and high dose groups was significantly reduced(P<0.01).IHC and WB detection showed that,compared with the control group,the expression of AchE and CGRP in the low,medium and high dose groups decreased with the increase of dose(P<0.01).Conclusion:1.ACR damages the normal structure and function of spinal anterior horn motor neurons,which may be related to the decrease of AChE,CGRP and GAP43 content caused by ACR,thus resulting in impaired growth and regeneration of spinal anterior horn motor neurons,axonal degeneration,and impaired synaptic structure and signaling impairment.2.ACR exposure damaged the normal structure and function of the sciatic nerve in rats,which may be related to the axonal degeneration caused by ACR exposure,the decreased expression of MBP and MAG protein,and the nerve demyelinating caused by ACR exposure.3.ACR poisoning in rats resulted in decreased wet weight of gastrocnastus muscle and changes in MEP structure,which may be because ACR led to axonal degeneration,resulting in axoplasmic transport dysfunction,and then the content of AChE,CGRP and other functional proteins in neuromuscular junction decreased.4.ACR may reduce the ability of protein synthesis and transport by damaging the cell body and axon of the anterior horn motor neurons of the spinal cord,demyelinating the sciatic nerve and weakening the signal transmission of nerve endings,eventually leading to MEP degeneration and muscle atrophy leading to skeletal muscle weakness.