缺血再灌注脊髓运动神经元放电的信息编码规律及其致瘫机制

The treatment outcome both spinal cord injury(SCI)'s remedy and rehabilitative care of paralegia are not up to ideal effectiveness currently. It is the fundamental reasons that we still don't know the relevant occurred mechanism of paraplegia, especially the neuroinformatics mechanism of SCI causing paraplegia. Since it is very difficult to record and collect the neuronal discharges of spinal cord at the process of making spinal cord injury in vivo, additionally, and the traditional analysis of frequency coding is also very limitative,which usually cannot reveal many effective information of neuronal activity, and also restrict the relevant research progress and become the bottleneck of the research field of neuroinformatics of SCI. In recent years，we continously investigate those problem and successfully reproduce a new model of spinal cord ischemia and reperfusion, which can be completely carry out the real-time recording of the same injured neuron's spike during spinal cord ischemia and reperfusion in vivo, moreover the temporal coding analysis and other techniques are also successfully applied lately, etc. These progresses make the neuroinformatics researchs become possible. This project will be planning to use the above-mentioned new model created by the blocking and releasing of the secondary lumbar artery's blood to cause local ischemia and reperfusion of the segemantal lumber spinal cord (L4), the multi-barrel glass microelectrode was adopted to record the unit discharges of spinal cord motor neuron (MN) during pro-ischemia，ischemia and reperfuson，and observe MN's response to the electrical stimulation of cerebral motor cortex and red nucleus and the tibial nerve, etc.，then investigating effects of Glu / GABA receptor agonist and antagonist gived by the ionic microelectrophoresis on the MN's discharge; and at the same time the regulatory role of feedback inhibition loop of Renshaw cell are also examined with the immunohistochemistry and electrophysiological techniques；combination of the in vitro research with perfusion culture model of thick slices of spinal cord following hypoxia plus low glucose analog ischemia, in depth investigating the response of MN's discharge and Renshaw cell' regulation, combined with the corresponding receptor expression and functional analysis, to reveal the law of MN's information coding in the injured spinal cord including the neural coding features, functions and recurrent regulatory responses by the linear and nonlinear analysis, so as to clarify the neuroinformatics mechanism of paraplegia's occurrence following SCI，to provide a new treatment strategy for the therapy of SCI patient and rehabilitation management of paraplegia.

目前脊髓损伤(SCI)救治和截瘫康复治疗效果并不理想,根本原因与其发生的神经信息学机制尚不清楚有关。既往由于在体脊髓神经元在损伤过程中的放电信息采集困难，加之传统频率编码分析的局限性,制约该领域研究的进展。近年我们探索出脊髓损伤过程中神经元放电实时记录的新模型,加之时间编码分析等新技术的成功应用使之成为可能。本项目拟用阻断兔腰动脉致L4腰髓缺血再灌注损伤的上述模型,多管玻璃微电极实时记录缺血及再灌注脊髓运动神经元（MN）的放电及其对运动皮层和红核及胫神经等电刺激的反应；观察离子微电泳给予Glu/GABA及受体激动剂、拮抗剂对MN放电的影响；用免疫组化和电生理学技术研究润绍细胞回返抑制环路的调控；以缺氧+缺糖模拟缺血的脊髓厚切片灌流培养模型作体外验证,结合相关受体表达及功能分析,用线性和非线性分析揭示损伤脊髓MN的信息编码规律,阐明SCI致瘫的神经信息学机制，为SCI和截瘫治疗提供新的策略。

本课题中采用家兔脊髓缺血再灌注模型，通过神经病理、免疫荧光染色、ELISA、Western-blot和神经电生理学等技术方法，首先观察了缺血再灌注损伤后脊髓不同组织和细胞对损伤易感性的差异及损伤病理学特点；然后从电生理功能监测的角度观察缺血及再灌注过程中神经元放电活动的变化；最后通过ELISA和Western-blot方法观察损伤过程中神经递质及其受体以及胞内钙离子的变化，揭示脊髓缺血再灌注损伤过程中神经元损伤特点及其致瘫的分子机制。结果观察到：(1) 脊髓缺血少于30min，再灌注损伤可逆；大于30min损伤不可逆，后肢多表现为肌张力增高、牵张反射亢进、呈强直状态。提示：脊髓缺血再灌注损伤 “窗口期”为缺血30min，所导致的后肢功能障碍主要表现为痉挛性瘫痪。(2) 缺血再灌注导致的脊髓损伤，灰质病理损害较白质明显；运动神经元和中间神经元均有明显的变性坏死，但中间神经元死亡数量相对更多。表明：脊髓不同部位、不同类型神经元对缺血再灌注损伤存在易感性差异，这可能是缺血再灌注损伤致痉挛性瘫痪的组织病理学基础。（3）利用时频分析对脊髓缺血及再灌注时脊髓神经元放电进行分析，可见脊髓缺血损伤早期神经元兴奋性增加、随着缺血时间延长其兴奋性逐渐降低，整合功能失调。中间神经元（闰绍细胞）对缺血再灌注损伤更为敏感，最终使运动神经元处于“去抑制”状态，由此导致的运动神经元高兴奋性反应，是造成脊髓缺血再灌注损伤致痉挛性瘫痪的重要神经电生理机制(神经信息学机制)。（4）脊髓缺血再灌注损伤后，Glu释放增加、GABA和Gly释放减少，是造成神经元兴奋毒性损伤的重要原因，其中Gly及其受体主要在损伤早期而GABA在损伤中后期发挥作用，在此基础上引起的神经元钙离子内流增加、CalpainⅠ激活、细胞骨架蛋白68-KDNFP降解，可能是导致神经元损伤、死亡的重要分子机制。上述研究成果对深入阐明脊髓继发性损伤及其致瘫机制提供重要的实验依据，进而为此防治提供新的理论指导和依据。