基于神经血管单元稳态探讨巨刺法改善大脑中动脉闭塞大鼠运动功能机制的研究

关键词：卒中  巨刺  运动功能  神经血管单元  阳明经穴

Keywords:stroke  opposing needling  motor function  neurovascular unit  Yangming meridian points

摘要：

      摘要 目的：基于神经血管单元稳态探讨巨刺法改善大脑中动脉闭塞模型（MCAO）大鼠运动功能的机制。 方法：将48只雄性SD大鼠随机分为假手术组（IS组）、模型组（IC组）、巨刺组（ON组）、非穴组（FON组）（各12只）。其中IC组、ON组及FON组大鼠制备MCAO模型，成模后ON组接受电针健侧“曲池”和“足三里”，FON组取穴位置的旁开5mm处电针干预7天，IS组和IC组不进行电针干预，仅进行模拟抓取。使用改良神经功能缺损评分（mNSS）评测各组大鼠神经功能；应用CatWalk步态分析对比各组大鼠运动功能；应用小动物磁共振成像系统行弥散张量成像（diffusion tensor imaging，DTI）扫描，使用DTI衍生参数测量运动皮质、纹状体神经传导束各向异性分数比值（ratio fractional anisotropy，rFA）；应用TTC染色观察脑梗死体积，应用免疫荧光法检测脑缺血侧运动皮质神经元核蛋白（neuronal nuclei，NeuN）的表达变化；应用免疫组织化学法检测血小板-内皮细胞黏附分子（platelet endothelial cell adhesion molecule-1 ，PECAM-1/CD31）、胶质纤维酸性蛋白（glial fibrillary acidic protein，GFAP）、钙结合蛋白（ionized calcium binding adapter molecule 1，Iba1）表达变化；应用Western Blot法检测闭锁连接蛋白-1（zonula occludens 1，ZO-1）及闭合蛋白（Occludin）的表达变化。 结果：干预7天后，与IC组相比，ON组大鼠神经功能评分、mNSS评分降低（P<0.05）；与IC组、FON组相比，ON组的平均运动速度加快、足印最大接触面积增加（P<0.05）；与IC组、FON组相比，ON组的梗死体积减少、rFA值相对增加（P<0.05）；与IC组、FON相比，ON组的NeuN阳性细胞表达明显升高（P<0.05）；与IC组、FON组相比，ON组的CD31表达显著升高、GFAP和Iba1的表达显著降低（P<0.05）；与IC组、FON组相比，ON组的ZO-1、Occludin蛋白表达均显著升高（P<0.05）。CatWalk步态参数、免疫荧光、免疫组化与Western Blot检测结果改善有一致性。 结论：巨刺法改善MCAO大鼠运动功能，其机制可能与修复神经血管单元稳态有关。

Abstract:

      Abstract Objective： The aim of present study was to explore the mechanism of the giant puncture in improving the motor function of the middle cerebral artery occlusion model（MCAO） rats based on the integrity of neurovascular unit. Method： Totally 48 male Sprague-Dawley（SD） rats were randomly divided into sham operation group（IS group），model group （IC group），giant spur group （ON group），non-acupoint group （FON group） （12 rats in each group）. MCAO models were produced for rats in the IC group， ON group， and FON group. After the modeling， the ON group received electroacupuncture at the contralateral side "Quchi" and "Zusanli"， and the FON group received electroacupuncture at a 5mm side of the acupoint selection position. After 7 days of intervention，the IS group and IC group did not perform electroacupuncture intervention， only simulated grabbing. The modified neurological deficit score（mNSS） was used to detect the neurological function of each group of rats； CatWalk gait analysis was used to compare the motor function of each group； small animal magnetic resonance imaging system was used to perform diffusion tensor imaging（DTI） scan and DTI-derived parameters measured the ratio fractional anisotropy（rFA） of motor cortex and striatum nerve conduction bundles；TTC staining was used to observe cerebral infarction volume， immunofluorescence method was used to detect neuronal nuclei in the motor cortex on the cerebral ischemic side ， NeuN expression changes； immunohistochemical method was used to detect platelet endothelial cell adhesion molecule-1（PECAM-1/CD31）， glial fibrillary acidic protein（GFAP），calcium Binding protein （ionized calcium binding adapter molecule 1， Iba1） expression changes；Western Blot method was used to detect the expression changes of zonula occludens 1，ZO-1 and Occludin. Result： After 7 days of intervention， compared with the IC group，the neurological function score and mNSS score of the ON group decreased （P<0.05）； compared with the IC group and the FON group， the ON group had faster average movement speed and maximum foot print contact area increased （P<0.05）； compared with the IC group and FON group， the infarct volume of the ON group was reduced， and the rFA value relatively increased （P<0.05）； compared with the IC group and FON， the expression of NeuN positive cells in the ON group obviously increased （P<0.05）； compared with the IC group and FON group， the CD31 expression in the ON group significantly increased， and the expression of GFAP and Iba1 significantly was reduced （P<0.05）； compared with the IC group and the FON group， the ON group the expression of ZO-1 and Occludin protein significantly increased （P<0.05）. The improvements of catwalk gait parameters， immunofluorescence， immunohistochemistry and Western Blot detection results had consistent results. Conclusion： The opposing needling improves the motor function of MCAO rats， and its mechanism may be related to the repair of neurovascular unit homeostasis.