NgR1与内源性拮抗剂LOTUS/LGI1/ADAM22调控轴突可塑性在运动训练促进脑梗死后执行功能恢复中的作用机制

Executive dysfunction is a key factor that influence rehabilitation of stroke.How to promote the recovery of executive dysfunction post stroke and what is the potential mechanism is still unknown.We have found exercise can increase the axonal plasticity and corticorubral regeneration,which was associated with the down regulation of the expression of Nogo-A/NgR1/RhoA. Recent studies indicate that both NgR1/ROCK and its endogenous antagonist LOTUS/LGI1/ADAM22 are involved in cognition.Executive functions are higher order cognitive processes.Our hypothesis is that exercise can improve executive functions after cerebral ischemia through modulation of the balance between NgR1 pathway and LOTUS/LGI1/ADAM22.In this study, transient MCAO RHRSP model will be used. The effect of exercise on executive functions will be evaluated.Immunofluorescence,BDA fiber tracing will be used to detect the axonal remodeling in cortical-thalamus-striatum and hippocampus.The expression of Nogo-A/NgR1/RhoA/ROCK and NgR1 engogenous antagonists will be demonstrated by immunofluorescence, western blot，PCR.Exogenous antagonist of NgR1, ROCK inhibitor will be delivered to explore if the effecte of exercise can be changed.In this way, the effect of exercise on executive functions and the mechanism of neural network plasticity will be demonstrated.The study will provide a new target of executive function rehabilitation post stroke.

执行功能障碍是影响脑卒中康复的关键因素，促进执行功能恢复的方法及机制尚未明确。我们已证实运动训练促进梗死灶周围轴突重塑及皮质红核束再生，与下调Nogo-A/NgR1/RhoA的表达有关。最近发现NgR1/ROCK与内源性拮抗剂LOTUS/LGI1/ADAM22均参与认知过程，而执行功能是高级的认知功能。据此假设，运动训练通过调控执行功能相关脑区NgR1与拮抗剂之间的平衡改善执行功能。本研究采用RHRSP tMCAO模型，观察运动训练对执行功能的影响，采用免疫荧光、BDA示踪检测皮质-丘脑-纹状体和海马轴突重塑的变化,采用免疫荧光、western blot、PCR观察Nogo-A/ROCK通路及NgR1内源性拮抗剂的表达，探讨外源性NgR1拮抗剂、ROCK抑制剂能否影响运动训练的作用，阐明运动训练对脑梗死后执行功能的影响及神经网络可塑性机制，为脑卒中后执行功能障碍的康复提供新的靶点。

为探讨运动训练促进脑梗死后认知功能及偏瘫肢体运动功能恢复是否与Nogo-A信号通路相关，本研究采用线栓法建立短暂性大脑中动脉闭塞的大鼠脑梗死模型，通过对造模后大鼠运动功能、认知功能及死亡率的观察，确定了最佳缺血时间；观察了跑笼训练和技巧性取食训练后脑梗死大鼠神经功能和患侧前肢抓握力的变化以及Morris水迷宫的表现；对比了采用运动训练联合Nogo-A信号通路外源性抑制剂羟基法舒地尔与单独运动训练、单独外源性抑制剂对脑梗死后运动功能及认知功能的影响；通过免疫荧光、Western blot动态地观察脑梗死周围、梗死灶对侧、海马及丘脑NgR1/RhoA/ROCK及NgR1内源性拮抗剂LGI1/LOTUS的表达。先将大鼠随机分为60min组、90min组、120min组、假手术组，观察术后7d、14d、21d、28d时的情况，结果表明缺血90min的大鼠存在运动及认知功能障碍且死亡率较低。然后将大鼠随机分为跑笼训练组、技巧性取食训练组、对照组、假手术组、跑笼+法舒地尔组、法舒地尔组、生理盐水组，观察术后7d，14d，21d、28d，42d或56d时的情况。结果表明，梗死后21d开始，训练组较对照组大鼠抓握力改善，且技巧性取食训练较跑笼训练效果好，跑笼组、跑笼+法舒地尔组较法舒地尔组效果好；梗死后56d时，技巧性取食训练较跑笼训练更好的改善认知功能，跑笼+法舒地尔组较跑笼组、法舒地尔组效果好；通过Western blot和免疫荧光检测，术后28d时训练组大鼠梗死周围皮层、梗死对侧皮层、海马及丘脑的NgR1、RhoA、ROCK表达较对照组少，LOTUS、LGI1表达较对照组多。提示运动训练能够通过抑制Nogo-A信号通路各分子的表达，促进NgR1内源性拮抗剂LGI1/LOTUS的表达改善脑梗死大鼠偏瘫肢体的运动功能及认知功能；技巧性取食训练对运动功能和认知功能恢复的促进作用优于跑笼训练。本研究阐明了运动训练改善脑梗死动物运动功能及认知功能的Nogo-A信号通路机制，为运动训练的临床应用提供新的靶点。