难治性颞叶癫痫微环路中路由器细胞的组成解析和鉴定

The great concern in the field of epilepsy have been got since the findings that a few number of hub cells can trigger and regulate the abnormal synchronization of neural network activity. However, there is no evidence from the human brain. In this study, the postoperative specimens of patients with refractory temporal lobe epilepsy were used and the hippocampal dentate granular cells and moss cells were used as candidate hub cells. CLARITY, virus tracing and high resolution imaging and recording of synchronization of calcium signal were used to determine the cell type, number and distribution with the largest connection and output; analysis of the composition, topology and spatial distribution characteristics of the reciprocating and collateral loops were used at single cell resolution level; analysis of stimulation or suppression of candidate hub cells induced and regulated the abnormalities of synchronization of activity of circuit were performed. The differences between the above indexes were compared between the pathological changes of epilepsy and the prognosis of epileptic foci. The aim of this study is to identify and analyze the structural basis of the hub cells in the hippocampus circuit and the underlying mechanism of abnormal network synchronization activities and to reveal the relationship between the changes of the hub cells and the preoperative assessment of epileptic foci and the prognosis after surgery. The discovery and confirmation of the compositing and location of the hub cells can provide a basis for the accurate surgery of refractory epilepsy and development of new method of interfering abnormal circuit activity in epilepsy.

少数路由器细胞即可引起和调控神经网络的异常同步化活动的发现，引起了癫痫领域的极大关注，至今尚无来自人脑的证据。本课题以难治性颞叶癫痫患者术后标本为对象，以海马齿状回颗粒细胞和苔藓细胞为候选路由器细胞，采用透明化，病毒追踪，高分辨成像技术结合同步化钙离子信号等技术，确定具有最大联结和输出端的细胞类型，数目比例和分布特征；单细胞分辨率水平解析往返型和侧枝型环路的组成成份，拓扑结构和空间分布特征；分析刺激或抑制候选路由器细胞诱发和调控环路异常同步化的影响和机制；比较轻重度癫痫病理改变间上述指标间的差异，分析其与致痫灶预测和术后预后的关系。目标是鉴定和解析海马环路中路由器细胞的结构基础和诱发/调控异常网络同步化活动的机制，揭示路由器细胞及其微环路改变与术前致痫灶评估和术后预后之间的关系。发现和确认路由器细胞的组成和位置，可为实现难治性癫痫的精准手术，提出新型干预异常环路活动的方法提供依据

癫痫是一类严重影响病人身心健康和生活质量的慢性重大疾病，而颞叶皮层又与癫痫的发生发展有重要关系，大脑皮质分为六层，其中第一层的层间星形胶质细胞是灵长类动物所特有的胶质细胞。在本研究中，一方面在体外培养术后标本，逆行和顺行病毒追踪技术结合显微光学切片断层成像技术，解析颞叶海马星形细胞间的神经环路的高清晰度结构基础；电生理技术记录神经环路的功能联结特征和异常同步化活动；分析难治性TLE患者临床表现特征、磁共振功能和结构像、脑白质弥散张量（DTI）、皮层脑电图等与离体海马神经环路中路由器细胞及其微环路的网络特征之间的关系。另一方面以神经胶质纤维酸性蛋白（GFAP）为星型胶质细胞的标记物，采用组织透明、免疫组化和单细胞重构的方法观察了成人颞叶新皮层I层的层间GFAP阳性细胞的分布和三维形态特征以及癫痫时1层GFAP细胞的形态表征、数目和支配域的变化。在成人颞叶皮质第一层，从水平方向又分为两层，即邻近软脑膜的浅层和深层。其中包括两种亚型的GFAP阳性层间星形胶质细胞（ILA），分别称为软膜 ILA和软膜下ILA。第I层中的软膜下ILA星形胶质细胞具有明显的细胞域结构特征。其三维重构显示：第I层GFAP星形胶质细胞结构域具有位置依赖的结构特征。与V-VI层的深层的胶质细胞域相比， 第I层的细胞域分支程度明显减少。 细胞域体积的体视学分析显示，与第V-VI层的星形胶质细胞相比，第I层的星形胶质细胞域占据的体积明显较小。令人兴奋的是，本研究首次报道：癫痫患者颞叶皮质的第一层的层间星形胶质细胞的数量增加，其细胞域体积显著增大提示在癫痫发病过程中星形胶质细胞发生结构重塑。上述发现首次揭示了成人颞叶皮质第一层的层间胶质细胞域的结构特征及其在癫痫发病中可能的功能意义。