PKA信号通路在脑缺血后突触可塑性中的作用及针刺干预研究
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摘要
在当今社会,脑血管疾病是目前严重危害人类健康的主要疾病之一,其中缺血性脑血管病疾病占60%～80%,由于神经元能量储备极少,对缺血缺氧十分敏感,缺血性神经元死亡是脑血管病致残和致死的重要因素,因此,探讨缺血性神经元损伤的机制及其防治具有重大的理论和现实意义。关于其机理研究方面近几年脑可塑性尤其突触可塑性成为了研究重点,对针刺促进脑血管病的恢复机制研究提供了很多依据。PKA系统可以作用于突触可塑性,增加递质释放持续增多以及调节突触结构的变化,参与“慢突触传递”,因此本课题通过观察电针对脑缺血大鼠及其注射H89后突触结构可塑性及相关物质的变化来探讨PKA信号通路在脑缺血中的可能机制及针刺对其的影响。本文从以下几个方面进行了研究。
1.文献研究
本部分就古代中医学关于中风的命名,缺血中风的病因病机,中风与五脏六腑的关系以及针灸治疗中风的古代记述方面进行了论述,从而对中风的古文献研究背景有了深入的认识。近年来在脑血管病的研究中发现卒中后恢复的不同阶段,大脑功能和组织结构可以发生一定的自我修复和重建能力,其中突触可塑性是大脑可塑性的主要表现形式,本文就近几年缺血后突触可塑性以及针刺干预作用的研究进行了综述,为进一步研究针刺治疗脑缺血的机制奠定了理论基础。
2.实验研究
目的:本研究观察了脑缺血及注射H89后不同时间段的学习记忆能力、突触超微结构的变化,生长相关蛋白GAP-43、突触素P38表达的动态变化,并探讨针刺督脉“百会”“大椎”干预其变化的时效关系为临床针刺治疗缺血性脑血管疾病提供坚实的理论基础。
方法:
(1)本实验选用250只Wistar大鼠分为假手术组、缺血组、缺血+电针组、缺血+H89组、缺血+H89+电针组共五组,各组再按距离手术结束时间的不同再分为1h组、3h组、6h组、12h组、2周组5个时间段小组进行观察。动物模型延用以往的电凝闭大脑中动脉法,造成局灶性脑缺血模型。其中假手术组只作手术暴露大鼠大脑中动脉,不予凝闭;缺血组采用电凝闭大脑中动脉法复制局灶性脑缺血模型:缺血+H89组造模后侧脑室给予H89药物注射;缺血+电针组、缺血+H89+电针组造模并药物注射后立即给予电针治疗,选取督脉经穴百会(GV20)、大椎(GV14)两穴进行针刺,针刺后接电针30min。
(2)Y迷宫学习记忆功能的观察:应用Y迷宫观察各组大鼠空间辨别的学习记忆能力,于手术或治疗后第3h、6h、12h和2周进行试验,数值越高代表分辨学习的能力越差。
(3)突触的超微结构:12h、2周后,随机取10小组大鼠每组各3只左心室灌注取材,参数的观察采用JEM-1200EX透射电镜,底片放大1.5万倍印相,进行电镜观察缺血区大脑皮层突触界面参数PSD、间隙宽度和界面曲率的变化规律及电针对其的影响。
(4)生长相关蛋白GAP-43的表达变化:150只大鼠于术后1h、3h、6h、12h和2周每个时间段分别左心室灌注取材后,采用免疫组化方法检测生长相关蛋白GAP-43的表达并观察其变化。
(5)突触素P38表达变化:150只大鼠于术后1h、3h、6h、12h、2周组5个时间段分别左心室灌注取材后,采用免疫组化的方法检测突触素P38的表达并观察其变化。
结果:
(1)Y迷宫学习记忆功能
缺血组学习成绩从3h开始始终差于假手术组,脑缺血后6h至2周,大鼠的学习记忆能力逐渐有所提高;缺血+针刺组大鼠Y迷宫测试成绩始终优于缺血组,且至2周学习成绩与假手术组无明显差异(P＞0.05),缺血+H89组的学习成绩较缺血组为差;缺血+H89+针刺组优于缺血+H89组。
(2)突触界面参数
脑缺血后,大鼠的PSD厚度下降,注射H89后下降更明显,与假手术组比较均有显著差异(P＜0.001);电针治疗后12h、2周,PSD厚度改善明显,缺血+H89+针刺组明显高于缺血+H89组,并且高于缺血组。
12h、2周脑缺血后间隙宽度变窄,在注射H89后,突触间隙宽度变小最明显,经电针治疗后,缺血组及注射H89后的大鼠间隙宽度均增加,电针可以促进突触间隙恢复正常。
脑缺血损伤12h、2周,突触界面曲率减小,减少了神经元的接触面,大大降低了神经信息传递的有效性,注射H89后,突触界面曲率下降更明显,电针治疗后界面曲率增加明显,缺血+H89+针刺组明显高于缺血+H89组,并且也高于缺血组。
(3)生长相关蛋白GAP-43的表达
假手术组GAP-43的表达较低,缺血组3h、6h、12h、2周GAP-43的表达均较假手术组增强,于12h升高最明显;缺血+针刺组GAP-43于五个时间段表达大大高于缺血组;缺血+H89组给予PKA通路抑制剂H89后,3小时开始GAP-43表达较缺血组减少;缺血+H89+针刺组5个时间段GAP-43表达均较缺血+H89组明显增加,但较缺血+针刺组表达为少。
(4)突触素P38的表达:
缺血+针刺组的表达各时段均高于缺血组。注射H89后,突触密度功能的下降从3小时开始尤为突出,缺血+H89组P38的表达明显低于其它四组,且注射H89后大鼠自我修复能力差,从3小时开始缺血+H89组表达低于缺血组,缺血+针刺组表达高于缺血组,缺血+H89+针刺组的表达明显高于缺血+H89组。
结论:
(1)通过对3h、6h、12h和2周Y迷宫测试的观察说明脑缺血早期可以导致学习记忆能力的下降,缺血大鼠存在自我修复能力,电针可以从脑缺血急性期开始改善脑缺血大鼠的学习记忆能力:且提示PKA抑制剂H89可能作用于海马区从而影响大鼠学习记忆能力,PKA通路在脑缺血后学习记忆的恢复中起重要的作用,针刺可以逆转H89的抑制作用,作用于PKA通路从而促进学习能力的恢复。
(2)通过对12h、2周突触结构指标PSD、间隙宽度、界面曲率的变化的观察,发现脑缺血后突触超微结构发生损害,运用PKA抑制剂H89后突触超微结构损害进一步加重;电针可以促进脑缺血后突触超微结构的修复,并逆转运用H89后突触超微结构损害的加重。
(3)通过观察大鼠脑缺血后1h、3h、6h、12h、2周5个时间段GAP-43表达的变化,发现脑缺血急性期GAP-43的表达增强,针刺可以明显增高GAP-43的表达,促进轴突再生及突触重塑;PKA信号通路系统参与了脑缺血后早期神经元突触的再生,电针可以作用于PKA信号通路从而促进轴突的再生和修复。
(4)通过观察大鼠脑缺血后P38在1h、3h、6h、12h、2周5个时间段表达的变化,说明脑缺血急性期突触素的表达下降,针刺治疗可以促进突触素的表达:PKA信号通路系统参与了突触的修复,电针可以作用于PKA信号通路从而促进脑缺血后早期神经元突触囊泡功能密度的恢复。
通过以上实验结论,我们认为针刺可以促进脑缺血急性期突触可塑性,PKA信号通路的抑制是脑缺血后突触结构功能进一步损害的原因之一;作用于PKA信号通路促进突触的再生和修复是电针促进神经功能恢复的机理之一。
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 research
These 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 research
Purpose
This 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.
1.文献研究
本部分就古代中医学关于中风的命名,缺血中风的病因病机,中风与五脏六腑的关系以及针灸治疗中风的古代记述方面进行了论述,从而对中风的古文献研究背景有了深入的认识。近年来在脑血管病的研究中发现卒中后恢复的不同阶段,大脑功能和组织结构可以发生一定的自我修复和重建能力,其中突触可塑性是大脑可塑性的主要表现形式,本文就近几年缺血后突触可塑性以及针刺干预作用的研究进行了综述,为进一步研究针刺治疗脑缺血的机制奠定了理论基础。
2.实验研究
目的:本研究观察了脑缺血及注射H89后不同时间段的学习记忆能力、突触超微结构的变化,生长相关蛋白GAP-43、突触素P38表达的动态变化,并探讨针刺督脉“百会”“大椎”干预其变化的时效关系为临床针刺治疗缺血性脑血管疾病提供坚实的理论基础。
方法:
(1)本实验选用250只Wistar大鼠分为假手术组、缺血组、缺血+电针组、缺血+H89组、缺血+H89+电针组共五组,各组再按距离手术结束时间的不同再分为1h组、3h组、6h组、12h组、2周组5个时间段小组进行观察。动物模型延用以往的电凝闭大脑中动脉法,造成局灶性脑缺血模型。其中假手术组只作手术暴露大鼠大脑中动脉,不予凝闭;缺血组采用电凝闭大脑中动脉法复制局灶性脑缺血模型:缺血+H89组造模后侧脑室给予H89药物注射;缺血+电针组、缺血+H89+电针组造模并药物注射后立即给予电针治疗,选取督脉经穴百会(GV20)、大椎(GV14)两穴进行针刺,针刺后接电针30min。
(2)Y迷宫学习记忆功能的观察:应用Y迷宫观察各组大鼠空间辨别的学习记忆能力,于手术或治疗后第3h、6h、12h和2周进行试验,数值越高代表分辨学习的能力越差。
(3)突触的超微结构:12h、2周后,随机取10小组大鼠每组各3只左心室灌注取材,参数的观察采用JEM-1200EX透射电镜,底片放大1.5万倍印相,进行电镜观察缺血区大脑皮层突触界面参数PSD、间隙宽度和界面曲率的变化规律及电针对其的影响。
(4)生长相关蛋白GAP-43的表达变化:150只大鼠于术后1h、3h、6h、12h和2周每个时间段分别左心室灌注取材后,采用免疫组化方法检测生长相关蛋白GAP-43的表达并观察其变化。
(5)突触素P38表达变化:150只大鼠于术后1h、3h、6h、12h、2周组5个时间段分别左心室灌注取材后,采用免疫组化的方法检测突触素P38的表达并观察其变化。
结果:
(1)Y迷宫学习记忆功能
缺血组学习成绩从3h开始始终差于假手术组,脑缺血后6h至2周,大鼠的学习记忆能力逐渐有所提高;缺血+针刺组大鼠Y迷宫测试成绩始终优于缺血组,且至2周学习成绩与假手术组无明显差异(P＞0.05),缺血+H89组的学习成绩较缺血组为差;缺血+H89+针刺组优于缺血+H89组。
(2)突触界面参数
脑缺血后,大鼠的PSD厚度下降,注射H89后下降更明显,与假手术组比较均有显著差异(P＜0.001);电针治疗后12h、2周,PSD厚度改善明显,缺血+H89+针刺组明显高于缺血+H89组,并且高于缺血组。
12h、2周脑缺血后间隙宽度变窄,在注射H89后,突触间隙宽度变小最明显,经电针治疗后,缺血组及注射H89后的大鼠间隙宽度均增加,电针可以促进突触间隙恢复正常。
脑缺血损伤12h、2周,突触界面曲率减小,减少了神经元的接触面,大大降低了神经信息传递的有效性,注射H89后,突触界面曲率下降更明显,电针治疗后界面曲率增加明显,缺血+H89+针刺组明显高于缺血+H89组,并且也高于缺血组。
(3)生长相关蛋白GAP-43的表达
假手术组GAP-43的表达较低,缺血组3h、6h、12h、2周GAP-43的表达均较假手术组增强,于12h升高最明显;缺血+针刺组GAP-43于五个时间段表达大大高于缺血组;缺血+H89组给予PKA通路抑制剂H89后,3小时开始GAP-43表达较缺血组减少;缺血+H89+针刺组5个时间段GAP-43表达均较缺血+H89组明显增加,但较缺血+针刺组表达为少。
(4)突触素P38的表达:
缺血+针刺组的表达各时段均高于缺血组。注射H89后,突触密度功能的下降从3小时开始尤为突出,缺血+H89组P38的表达明显低于其它四组,且注射H89后大鼠自我修复能力差,从3小时开始缺血+H89组表达低于缺血组,缺血+针刺组表达高于缺血组,缺血+H89+针刺组的表达明显高于缺血+H89组。
结论:
(1)通过对3h、6h、12h和2周Y迷宫测试的观察说明脑缺血早期可以导致学习记忆能力的下降,缺血大鼠存在自我修复能力,电针可以从脑缺血急性期开始改善脑缺血大鼠的学习记忆能力:且提示PKA抑制剂H89可能作用于海马区从而影响大鼠学习记忆能力,PKA通路在脑缺血后学习记忆的恢复中起重要的作用,针刺可以逆转H89的抑制作用,作用于PKA通路从而促进学习能力的恢复。
(2)通过对12h、2周突触结构指标PSD、间隙宽度、界面曲率的变化的观察,发现脑缺血后突触超微结构发生损害,运用PKA抑制剂H89后突触超微结构损害进一步加重;电针可以促进脑缺血后突触超微结构的修复,并逆转运用H89后突触超微结构损害的加重。
(3)通过观察大鼠脑缺血后1h、3h、6h、12h、2周5个时间段GAP-43表达的变化,发现脑缺血急性期GAP-43的表达增强,针刺可以明显增高GAP-43的表达,促进轴突再生及突触重塑;PKA信号通路系统参与了脑缺血后早期神经元突触的再生,电针可以作用于PKA信号通路从而促进轴突的再生和修复。
(4)通过观察大鼠脑缺血后P38在1h、3h、6h、12h、2周5个时间段表达的变化,说明脑缺血急性期突触素的表达下降,针刺治疗可以促进突触素的表达:PKA信号通路系统参与了突触的修复,电针可以作用于PKA信号通路从而促进脑缺血后早期神经元突触囊泡功能密度的恢复。
通过以上实验结论,我们认为针刺可以促进脑缺血急性期突触可塑性,PKA信号通路的抑制是脑缺血后突触结构功能进一步损害的原因之一;作用于PKA信号通路促进突触的再生和修复是电针促进神经功能恢复的机理之一。
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 research
These 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 research
Purpose
This 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.
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