The Mechanism Of PKA Signaling Pathway In Synaptic Plasticity After Cerebral Ischemia And Intervention Studies Of Acupuncture

In today's society,cerebral vascular disease is a serious hazard to human health, including 60%～80%of ischemic cerebrovascular diseases.As a result of less energy reserves of neurons,and sensitive to hypoxia-ischemia,neuronal death due to ischemia is an important factor to disability and mortality of cerebrovascular disease,therefore,to explore mechanism of prevention and control of ischemic neuronal damage has great theoretical and practical significance.In recent years research on its mechanism of brain plasticity in particular synaptic plasticity has become the focus,providing lots of research basement to mechanism of acupuncture which promote the restoration of cerebrovascular disease.PKA system can act on the synaptic plasticity,increased neurotransmitter release, as well as provide a sustained changes in synaptic structure,participation in " slow synaptic transmission",so this research by observing the plasticity of synaptic structure and related material after electro-acupuncture to cerebral ischemia and with the injection of H89 aim to explore the changes in PKA signaling pathway in cerebral ischemia and the possible mechanisms of the influence of acupuncture.The following aspects were studied in this article.1.Literature researchThese parts discuss the nominating of stroke in ancient Chinese medicine.The etiology and pathogenesis of stroke,the relationship between stroke and five zang/six fu as well as the ancient records relating to Acupuncture treatment were also discussed which can guide us to deep understanding to ancient background of stroke.In recent years the study to cerebralvascular disease found that the function and organizational structure of brain can self-recover and reconstruct at different stages,in which synaptic plasticity is the main form. In this article the synaptic plasticity as well as effects of acupuncture are reviewed in order to lay a theoretical basis to further research on cerebral ischemia mechanism with the treatment by acupuncture.2.Experiments researchPurposeThis study observed dynamic changes of learning and memory capacity,synaptic ultrastructure,expression of growth associated protein GAP-43,synaptophysin P38 after cerebral ischemia and injection of H89 at different periods,as well as the influence of Acupuncture on above by using Baihui(GV20) and Dazhui(GV14) acupoints.Methods(1) 250 Wistar rats were divided into sham-operated group,ischemic group, ischemia+electro-acupuncture(EA) group,ischemic+H89 group,ischemia+H89+EA group, according to the different time after the operation each group further divided into 1h group,3h group,6h group,12h group,2w group.Heat-coagulation-induced occlusion of the middle cerebral artery(MCAO) was performed to establish the model of focal cerebral ischemia.The middle cerebral arteries were exposed without operation in sham-operated group,ischemia model +H89 group given intracerebroventricular H89 drug injection after the operation of MCAO;ischemia+EA group,ischemia+H89+EA group given the electro-acupuncture treatment 30min immediately after operation,EA was delivered to Du Meridian Baihui(GV20) and Dazhui(GV14).(2) Learning and memory function test with Y-maze:the application of Y-maze to observe the spatial learning and memory ability,in the surgery or treatment after 3h,6h, 12h and 2 weeks,higher results on behalf of the poorer ability to learn.(3) Ultrastructure of synapses:3 rats of each group in 10 groups were chosen at random in 12hour and 2weeks.The rats were fixed by intracardial perfusing.The brains were exposed and the samples were taken from ischemic area of cerebral cortex.Electron micrographs of synapses were observed by transmission electron microscope JEM-1200EX. The film prints were enlarged 15,000 times.Synaptic Number Density(Nv),Surface Density(Sv),the thickness of postsynaptic density(PSD),the width of synaptic cleft,the length and curvature of synaptic interface were measured to observe the changes and the effects of EA.(4) The expression of growth associated protein GAP-43:150 rats were intracardially perfused at 1,3,6 and 12hours as well as 2 weeks.Growth associated protein GAP-43 was detected with immunohistochemical method.(5) The expression of synaptophysin P38:150 rats were intracardially perfused at 1,3, 6 and 12hours as well as 2 weeks.Synaptophysin P38 was detected with immunohistochemical method.Results (1) Learning and memory function test with Y-maze:Ischemia group is always worse than the sham-operated group after 3hours.The capacity of learning and memory in rats increased gradually from 6hours to 2weeks.Y maze test of ischemia+EA group is always better than the ischemic group,and there had no significant difference(P＞0.05) with the sham-operated group at 2weeks.Ischemia+H89 group performance was worse than the ischemia group.Ischemia+H89+acupuncture group was better than the ischemia+H89 group.(2) Synaptic ultrastructure:After cerebral ischemia,the thickness of PSD decreased and then decreased more significantly after injected with H89.There have significant differences of ischemic group compared with sham-operated(P＜0.001).After electro-acupuncture for 12hours and 2 weeks,PSD thickness improved significantly.Ischemia+H89+EA group was significantly higher than ischemia+H89 group,and higher than ischemic group.The width of synaptic cleft decreased significantly at 12hour and 2weeks in the ischemic group.The width of synaptic cleft decreased obviously after the injection of H89. After electro-acupuncture treatment,the width of synaptic cleft of ischemia+EA group and ischemica+H89+EA group increased.Electro-acupuncture can promote synaptic cleft back to normal.The curvature of synaptic interface decreased obviously at 12hours and 2weeks in the ischemic group,greatly reduced the effectiveness of information transmission with the decreasing of the contact surface of neurons.After injection of H89,the synaptic interface curvature decline more.Electro-acupuncture treatment can increase the interface curvature. Ischemia+H89+acupuncture group was significantly higher than ischemic+H89 group,and also higher than the ischemic group.(3) The expression of growth associated protein GAP-43:The expression of GAP-43 of Sham-operated group was lower than other groups.The expression of ischemic group at 3hours,6hours,12hours and 2weeks increased than those in sham-operated group with the most significant increasing at 12hours.The expression of ischemia+acupuncture group at five times is much higher than the ischemic group.After ischemia+H89 group was given the PKA pathway inhibitor H89,the expression of GAP-43 began to reduce more than the ischemia group from 3hours.GAP-43 expression of the ischemic+H89+acupuncture group at five times was significantly increased than those of in ischemic+H89 group,but less than the expression of ischemia+acupuncture group.(4) The expression of synaptophysin P38:The expression of ischemia+EA group was higher than those of the ischemia group at different periods.After injection of H89,the decline of synaptic density and function was in particular at 3hours.The expression of P38 of Ischemia+H89 group was significantly lower than those in other four groups with the poor self-repair capability.From 3 hours the expression of ischemia+H89 group was lower than the ischemic group.The expression of ischemia+EA group higher than the ischemia group and the expression of ischemia+H89+EA group were significantly higher than those of ischemia+H89 group. Conclusion(1) By 3h,6h,12h and 2 weeks of the observation of Y maze test that cerebral ischemia can lead to early decline in learning and memory and rats have self-repair capacity. Electro-acupuncture treatment in acute stage of cerebral ischemia can improve the ability of learning and memory in rats,and prompted the possible role of PKA inhibitor H89 which working in the hippocampus thus affecting the capacity of learning and memory in rats. PKA pathway acts an important role in learning and memory in the recovery of cerebral ischemia.Acupuncture can reverse the effects of H89 inhibition,act on PKA pathway so as to promote recovery of learning ability.(2) By 12h,2 weeks with the observation of the thickness of PSD,the width of synaptic cleft and the interface curvature of the synaptic structure,found that synaptic ultrastructure damaged after cerebral ischemia,after the application of PKA inhibitor H89 synaptic ultrastructure was further damaged.Electro-acupuncture could contribute to the restoration of synaptic ultrastructure,and reverse the damage alter application of H89 to synaptic ultrastructure.(3) By observing the expression of GAP-43 after ischemia lh,3h,6h,12h and 2 weeks found that expression increased in acute phase of cerebral ischemia.Acupuncture could significantly increase GAP-43 expression,and promote axonal regeneration and synaptic remodeling.PKA signaling pathway is involved in the early stage of neuron synaptic regeneration of ischemia.Electro-acupuncture may act on PKA signaling pathway to promote axon regeneration and repairment.(4) By observing the expression of P38 after ischemia 1h,3h,6h,12h and 2 weeks found that expression decreased in the acute stage,acupuncture treatment can promote the synaptophysin expression;PKA signaling pathway is involved in synaptic repairment, electro-acupuncture may act on PKA signaling pathway so as to promote the recovery of the density and function of synaptic vesicles at early stage after ischemia.From the above experimental results,we believe that acupuncture can promote synaptic plasticity in the acute phase of cerebral ischemia,the inhibition of PKA signaling pathway is one of the reasons contributing to the impairment of brain synaptic structure and function after ischemia.Acting on PKA signaling pathway to promote the regeneration and repairment of synapses is one of the mechanisms of Electro-acupuncture which final to promote the recovery of neurological function.