基于周围神经损伤大鼠外周至脊髓感觉与运动通路研究推拿手法参数对疗效的影响及机理

As shown by research on the mechanism of early stage tuina treatment for peripheral nerve injury, tuina can: ①improve behavioral indicators in peripheral nerve damaged rats②increase neurotrophic factor expression ③protect neurons ④repair myelin sheath morphology ⑤prevent muscular atrophy. However, the relationship between tuina technique parameters and efficacy has not been elucidated; the mechanism of tuina techniques and their efficacy have not been expounded. This study uses sciatic nerve injury as the model to study peripheral nerve injury, and utilizes an analog tuina technique simulator to simulate tuina techniques while maintaining qualitative and quantitative controls. Intervention was carried out on rats injuried via clamping of their sciatic nerves, and the experimental plan was refined using orthogonal design principles. Functional and behavioral indicators were used as indicators to evaluate treatment efficacy, the effect of the research parameters on the efficacy were examined, resulting in the optimization of the practice of tuina techniques. Furthermore, light and electron microscopy was used to examine the key regions of both the sensory and motor pathways from the periphery to the spinal cord (skin- - -the location of injury- - - dorsal root ganglion- - -ending in the dorsal horn; ventral horn of the spinal cord- - -the location of injury- - -the neuromuscular junction- - -muscle) to search for the mechanism and evidence of tuina techniques in improving peripheral nerve injury morphology. Immunohistochemistry techniques were utilized to test for the expression of neuroactive substances（SP、MBP、GAP-43、CGRP；GFAP、MAG、AchE、MDNF）in these key regions. The eventual aim being to elucidate the relationship between the parameters, efficacy and mechanism of tuina techniques, so as to further reveal the scientific basis of tuina treatment of peripheral nerve injury.

通过前期研究，主要结论如下：推拿可以①改善周围神经损伤大鼠行为学指标；②促进神经营养因子表达；③保护神经元；④修复神经髓鞘；⑤防止肌肉萎缩。然而手法参数与疗效的关系尚未理清，手法产生疗效的机理尚未阐明。本课题以坐骨神经损伤为例研究周围神经损伤，采用推拿手法模拟仪定性定量模拟手法，对坐骨神经夹持损伤模型大鼠进行干预，运用正交设计精简实验方案，将行为学、功能学作为疗效评价指标，研究参数对疗效的影响，得出手法参数优化方案。并基于优化方案，从外周至脊髓感觉通路（皮肤→损伤点→背根节→脊髓背角）、外周至脊髓运动通路（脊髓腹角→损伤点→神经肌肉接头→肌肉），采用光镜、电镜寻找推拿改善周围神经损伤的形态学证据；运用免疫组化技术检测上述关键位置中相关神经活性物质SP、MBP、GAP-43、CGRP；GFAP、MAG、AchE、MDNF的表达，探寻推拿产生疗效的机理；最终理清推拿参数、疗效与机理之间的关系。

背景：本课题组前期研究得出推拿可以①改善周围神经损伤大鼠行为学指标；②促进神经营养因子表达；③保护神经元；④修复神经髓鞘；⑤防止肌肉萎缩。.研究内容：本次研究继续以坐骨神经损伤（sciatic nerve injury, SNI）为例研究周围神经损伤，采用推拿手法模拟仪进行定性定量“三法三穴”推拿干预。参数研究方面以三因素三水平正交设计精简实验方案，从行为学角度，得出促进SNI大鼠感觉及运动功能恢复的最佳刺激参数。机理方面通过免疫组织化学及分子生物学的综合评价，从感觉通路及运动通路两个角度，多层面系统分析推拿促进周围神经损伤恢复的潜在起效途径。.重要结果及关键数据：行为学研究得出：①推拿干预可以提升SNI大鼠痛觉敏感度，促进后肢肌力和精细运动功能的恢复；②轻度力量、长时间、高频率的手法可更好的促进感觉功能恢复；③中重度力量、短时间、低频率的手法可更好促进运动功能的恢复。形态学研究得出：①推拿可促进SNI大鼠坐骨神经纤维髓鞘的再生，使有髓神经数量增加；②促进再生神经与靶器官建立连接，修复受损神经的传导；③中等力量、短时间、低频率的手法可更好促进腓肠肌的恢复；④轻度力量、长时间、高频率的手法可更好促进腓肠肌肌细胞直径的恢复。组织细胞学及分子生物学研究得出：推拿可以①调节神经再生活性物质（CGRP、SP）的表达，维持微环境修复能力；②促进神经营养因子及其受体（NT-3，TrkC）的表达；③调控神经元细胞骨架蛋白（MAP-2、NF-M、MAG、ROCKⅡ、LN、MBP）的表达，维持神经元骨架的完整性；④调控轴突导向因子及其受体（Netrin1、DCC/UNC5A、Slit2、Robol）的表达，影响轴突的定向生长；⑤调控细胞凋亡因子（Bcl-2、caspase-3）的表达，发挥抗神经细胞凋亡作用。.科学意义：本实验①为临床工作者采用推拿治疗周围神经损伤类疾病时的施力轻重、操作时间长短及频率大小提供了参考依据，可以更有针对性地解决患者感觉或运动功能障碍；② 通过多层面分析推拿治疗周围神经损伤的内在机制，为推拿临床治疗此类疾病提供了理论依据。