基于脑白质重塑机制的rTMS干预脑梗死后执行功能障碍的状态依赖性研究

Executive dysfunction is the most difficult part of rehabilitation after stroke. In clinical work we found that rTMS has potential to improve executive function. Our experimental study showed that rTMS could improve performance of learning and memory of rats with cerebral infarction. However, the underlying mechanism remains unknown. Our previous study found that executive dysfunction was associated with white matter changes in subjects with infarction. Nogo-A/NgR1/RhoA pathway mediated white matter plasticity. Recent studies indicated that the effect of rTMS was influenced by white matter structure and was state-dependent. The hypotheses is that rTMS plays a facilliated- or interferent-effect on executive function by modulating white matter connectivity among executive metwork. On the basis of established tMCAO model, we will observe the different effects of rTMS combined with executive function training at different interval on executive functional recovery. Immunofluorescence and BDA tracing will be carried out to detect axonal sprouting and growth of neural fibers in frontal-parietal, cortical-subcortical area, where the executive network locates.Molecular biology method will be used to investigate the expression of Nogo-A pathway and endogenous antagonist of NgR1. The aim of this study is to demonstrate the state-dependent characteristics of rTMS intervention on executive function. The results will provide evidence of rTMS in modulating white matter plasticity The study will help find new targets and strategies for executive function rehabilitation after cerebral infarction.

执行功能障碍是脑卒中康复的难点，我们在临床中发现rTMS具有改善执行功能的潜能，亦证实rTMS改善脑梗死大鼠的学习记忆功能，其机制未明。前期研究表明脑梗死后执行功能障碍与脑白质改变有关，脑白质重塑受Nogo-A/NgR1/RhoA通路的调控。最近发现rTMS的作用受脑白质结构的影响且存在状态依赖性。据此假设，rTMS通过调控执行功能脑区间白质连接发挥易化或干扰执行功能训练的作用。本研究在已建立的tMCAO模型基础上，观察rTMS与执行功能训练不同时序性结合对执行功能影响的差异性，通过免疫荧光、BDA神经纤维示踪等方法检测额-顶叶、皮质-纹状体等执行功能网络结构的轴突出芽和神经纤维生长，采用分子生物学方法检测Nogo-A通路和NgR1内源性拮抗剂的表达，旨在阐明rTMS干预执行功能的状态依赖性特点，获得rTMS调控脑白质重塑的证据，为脑梗死后执行功能障碍的康复提供新的靶点和策略。

为探讨rTMS是否通过调控执行功能脑区间白质连接发挥易化或干扰认知功能训练的作用，以及阐明rTMS干预认知功能的状态依赖性特点，本研究在已建立的tMCAO模型基础上，采用改良大鼠神经功能缺损评分和水迷宫测试评估大鼠的神经及认知功能，结果表明，20Hz rTMS刺激较10Hz rTMS刺激更好地改善脑梗死恢复期大鼠的神经功能及认知功能；与rTMS刺激后1h训练组、rTMS刺激后4h训练组相比，rTMS后即刻认知训练对改善脑梗死大鼠的认知功能具有更佳的效果，表明rTMS对脑梗死大鼠认知功能的改善具有状态依赖性；固蓝染色结果表明rTMS可减轻脑梗死后白质的损害。通过免疫荧光观察脑梗死周围Nogo-A通路和NgR1内源性拮抗剂的表达，结果表明rTMS减少Nogo-A下游信号通路NgR1和RhoA的表达，增加NgR1内源性拮抗剂LGI1的表达。通过基因测序实验及数据分析发现，脑梗死大鼠和假手术大鼠相比，脑梗死后认知功能障碍的生物标志物可能为脑梗死后上调的基因Il15，Acta2，Snrpg，Cxcl12，Pth1r和Moxd1和下调的基因Calb2，Cbln4，Rxfp3，Spock3，Kcnk9和Adarb2；rTMS改善脑梗死大鼠的认知功能的机制可能与调控海马突触可塑性及调控Calb2、Zic1、Kcnk9和Grin3a等功能基因有关；rTMS后即刻认知训练组与20Hz rTMS组相比，差异基因主要富集于吞噬相关通路，且Calb2, Rsph1, Ccdc114, Acta2, Ttll9, Dnah1, Dlx2, Dlx1, Ccdc40和Ccdc113可能为调控的关键基因。本研究表明rTMS对脑梗死后认知功能的恢复存在状态性，且与调控白质损害有关，从基因调控的角度探讨了rTMS改善认知功能的新机制，为rTMS的临床应用提供了科学依据。