The Improvement Effects Of Acupuncture On Synaptic Reorganization In Rats With Cerebral Ischemia

Cerebrovascular disease has ranked the second of the leading cause of death for human. One third of the people who survive this disease will be disabled.It is important to search for effective prevention and treatment on ischemic cerebrovascular disease.The synaptic is one of the key positions in the brain plasticity on the treated and recovered mechanism of ischemic brain damage.We try to explore the underlying mechanism of acupuncture on the improvement of synaptic reorganization in rats with cerebral ischemia.1.Literature researchWe found that stroke has various etiological factors,complicated pathogenesis,sudden onset and different clinical manifestations through researched on the etiology and pathogenesis theory of stroke from ancient to modern literatures.Lots of experimental studies have showed the underlying mechanism of acupuncture in the treatment of cerebral ischemia.It was reviewed of the research progress on astrocyte and synaptic plasticity in modern medicine,and found that the theory of brain plasticity laid a theoretical foundation for the treatment and recovery of stroke,and astrocyte connect closely with synapses,so reactive astrocyte has played an important role in restoration of brain injured and participation in the regulation to synaptic plasticity.Moreover,acupuncture is one of effective stimulating methods to central nervous system,which has played an important role in functional reorganization of cerebra.2.Experiments research PurposeTo observe the effects of electroacupuncture(EA) on structure parameters of synapse and reactive changes of astrocyte in the marginal zone of focal cerebral ischemia in rats at different time courses so as to further explores its underlying mechanism in the treatment of cerebral ischemia.MethodsA total of 90 male Wistar rats were divided randomly into sham group,model group and EA group.According to different drawing materials time there was 1 hour,1 day,3days, 7days,and 21days time point after cerebral ischemia in each group,with 6 cases in each time point.Heat-coagulation-induced occlusion of the middle cerebral artery was performed to establish the model of focal cerebral ischemia in model group and EA group, but the middle cerebral arteries were exposed without occlusion in sham group.EA was immediately applied to Baihui(GV20) and Dazhui(GV14) for 30min in EA group.The treatment was given once daily.The sham and model groups did not receive acupuncture. Main outcome measures are following:(1) Neurological deficit scores and pathomorphology changes dyed by the way of HE were observed in every group.(2) Synaptic number density(Nv),the thickness of postsynaptic density(PSD),the width of synaptic cleft,and the curvature of synaptic interface were observed by using transmission electronic microscope in the marginal zone of focal cerebral ischemia in rats at different time points.(3) In the marginal zone of focal cerebral ischemia in rats at different time points,the ultrastructure changes of astrocytes were observed by using transmission electronic microscope,and glial fibrillary acidic protein(GFAP) expression of astrocytes were detected by using immunohistochemical method and laser confocal scanning microscope (LCSM).(4) In the marginal zone of focal cerebral ischemia in rats at different time points, excitatory amino acid transporters-1(EAAT 1 ) and excitatory amino acid transporters-2(EAAT2) expression of astrocytes were assayed with immunohistochemical method in different groups.(5) In the marginal zone of focal cerebral ischemia in rats at different time points, connexin 43(Cx43) expressions were measured by using immunohistochemical method.(6) In the marginal zone of focal cerebral ischemia in rats at different time points, [Ca²⁺]i of astrocytes were detected by using LCSM.(7) Canonical correlation analysis was taken between structure parameters of synapse x1- x4(Nv,PSD,the width of synaptic cleft,and the curvature of synaptic interface) and parameters of astrocyte y1- y4(CX43,EAAT2,GFAP,[Ca²⁺]i) in the same time and group by PEMS3.1.Results(1) There had significant differences in the neurological deficit scores of model group and sham group at all stages,which showed that the models were made successfully.There had no obvious difference between EA and model group at 1 day.Neurological deficit symptoms were aggravated in model group at 3days,and compared with that in model group,neurological deficit scores of EA group significantly decreased(P＜0.01).At 7days and 21days,neurological deficit scores of EA group obviously lower than those in model group(P＜0.01,P＜0.05).The results of HE staining showed that neuronal necrosis appeared.The models were made successfully according to signs of cerebral ischemia such as neural cells arranged in disorder,reticular formation,cell nuclear pyknosis and anachromasis,cytoplasm degeneration appeared,especially at 3days the neuron degeneration and necrosis most obviously.With the prolongation of ischemia,the lesion reduced.But in comparison with that in model group,smaller liquefaction lesion,cell outline and nucleolus could be seen clearly in EA group.(2) Synaptic structure of cortex in the marginal zone of focal cerebral ischemia in rats was broke,and the number of synapse was decreased obviously.The Nv in model group were significantly lower than those in sham group at 1 hour,1day,3days,7days,and 21days post-surgery(P＜0.01,P＜0.05),especially at 1day and 3days the Nv decreased most obviously,increased gradually after 3days.The Nv in EA group were significantly lower than those in sham group at 1 hour,1day,3days and 7days(P＜0.01,P＜0.05),but the Nv in EA group were significantly higher than those in model group at 1day,3days, 7days,and 21days(P＜0.05,P＜0.01).In comparison with that in sham group at 1 hour,1day,3days,7days,and 21days post-surgery,the thickness of PSD in model group were decreased obviously(P＜0.01 ).The thickness of PSD in EA group were significantly lower than those in sham group at 1 hour, 1 day,3days and 7days(P＜0.05,P＜0.01),while which were significantly higher than those in model group at the same time point post-surgery(P＜0.05,P＜0.01).In comparison with that in sham group at 1 hour,1day,3days,7days,and 21days post-surgery,the width of synaptic cleft in model group were decreased obviously(P＜0.01, P＜0.05).The width of synaptic cleft in EA group were significantly lower than those in sham group at 1 hour,1day,3days and 7days(P＜0.01),but the width of synaptic cleft in EA group were higher than those in model group at lday,3days and 7days(P＜0.05).The curvature of synaptic interface in model group were significantly lower than those in sham group at 1day,3days,7days,and 21days post-surgery(P＜0.01).The curvature of synaptic interface in EA group were significantly lower than those in sham group at 1day and 3days(P＜0.05,P＜0.01),while there have no significant differences in EA group and in sham group at 7days and 21days(P＞0.05).Compared with that in model group,The curvature of synaptic interface in EA group increased significantly at 1day,3days,7days, and 21days(P＜0.05,P＜0.01).(3) Swollen and increasing astrocytes were observed after cerebral ischemia,while compared with that in model group,the degree of swelling of astrocytes was decreased obviously in EA group.In comparison with that in sham group at 1 hour post-surgery,there have no significant differences of the expression of average fluorescence intensity of GFAP in EA group and model group(P＞0.05),while the expression of GFAP in EA group and model group both increased significantly at 1 day,3days,7days,and 21days post-surgery(P＜0.01),and the expression of GFAP in EA group were significantly lower than those in model group(P＜0.05,P＜0.01).(4) In comparison with that in sham group,Optical Density(OD) values of EAAT1 in model group increased obviously at 1 day,3days,7days,and 21days post-surgery(P＜0.05, P＜0.01),while compared with that in sham group,the OD value of EAAT1 in EA group increased significantly at 1 hour(P＜0.05),and the change of EAAT1 expression in EA group was similar to that in model group at 1day,3days,7days,and 21days,but OD values of EAAT1 in EA group were significantly higher than those in model group(P＜0.01 ).In comparison with those in sham group,OD values of EAAT2 in model group increased obviously at 1 hour,1day,3days and 7days(P＜0.01),and the change of EAAT2 expression in EA group was similar to that in model group,however,in comparison with that in sham group,the OD values of EAAT2 in EA group still increased significantly at 21days(P＜0.01),and OD values of EAAT2 in EA group were significantly higher than those in model group(P＜0.01,P＜0.05).(5) Compared with that in sham group,the expression of CX43 in model and EA group both decreased obviously at 1day,3days and 7days post-surgery(P＜0.01),especially at 3days decreased most obviously.While compared with that in model group,the expression of CX43 in EA group increased significantly at 3days and 7days(P＜0.01,P＜0.05).The expression of CX43 in model group still lower than those in sham group at 21days(P＜0.01),but there have no significant differences of the expression of CX43 in EA group and sham group(P＞0.05).(6)In comparison with that in sham group at 1 hour,1day,3days,7days,and 21days post-surgery,Ca²⁺ average fluorescence intensity of astrocytes were increased significantly in model and EA group(P＜0.01,P＜0.05),especially at 1day and 3days the expression increased most obviously,decreased gradually after 3days.While Ca²⁺ average fluorescence intensity of astrocytes in EA group were significantly lower than those in model group at 1 hour,1day,3days and 7days(P＜0.05,P＜0.01).(7) There have closely correlation between the changes of structure parameters of synapses and CX43,EAAT2,GFAP,[Ca²⁺]i of astrocytes at 1 hour,1day,3days and 7days after cerebral ischemia.The changes of structure parameters of synapse were still closely related to the changes of CX43,EAAT2,GFAP,[Ca²⁺]i of astrocytes by EA treatment at 1 hour,1day,3days,7days,and 21days after cerebral ischemia.Conclusion(1)Neurologic impairment and pathomorphology changes of rats have the trend of self-repair after ischemic brain damage.The development and outcome of brain damage is affected by synaptic plasticity,reactive changes of astrocyte,the changes of glumatic acid transporters,the changes of intercellular gap junction and[ca²⁺]i,which indicate that brain plasticity is taken place after brain damage.(2)EA can promote the repair of synaptic structure and improve structure parameters of synapses after ischemic brain damage,which may stimulate compensatory effect of synaptic morphology plasticity in survival neurons.Therefore,we consider that EA can treat cerebral ischemia,which may be related to its effect in promoting synaptic reorganization.(3) After ischemic brain damage,EA can intervene the activation state of astrocytes, inhibit excessive hyperplasy of astrocytes,strengthen uptake ability of glutamic acid,keep stable expression of connexin in gap junctions,and reduce the reactive changes of intracellular[Ca²⁺]i of astrocytes,which may contribute to regeneration and reparation of cerebral neurons.(4)The changes of structure parameters of synapse were closely related to the changes ofCX43,EAAT2,GFAP,[Ca²⁺]i ofastrocytes in rats after ischemic brain damage.EA is helpful to synaptic reorganization,which may be related to its effect in intervening the activation state of astrocytes and promoting the beneficial interaction between astrocytes and synapses.(5)The results support that acupuncture can start the adjustment of neuron-glial network so to promote the synaptic reorganization,which may be the key mechanisms of treating cerebral ischemia.