电引导治疗颞叶癫痫的引导电流与疗效的量效关系及其神经保护机制研究

There are about 1/3 patients with epilepsy with continuous seizure dispite of the using of new anti-epilepsy drugs, surgical treatment and neuro-modulation therapy. We have performed the current conduction treatment in rats with temporal lobe epilepsy (TLE), and reached promising outcomes in seizure control and neuroprotection. However, our previous study was lack of theraputic quantitative indicators, such as conductive current intensity, and the action mechanism of neuroprotection is still unknown. Basing on the previous research and preliminary experiments, we propose that there should be a dose-response relationship between conductive current intensity and therapeutic outcomes of current conduction, and that interleukin-1β (IL-1β), high-mobility group box-1(HMGB-1), glumate, and calcium pathway may play key roles in the neuroprotection of current conduction treatment. In this study, we design the one-way conduction electrode and a micro-amperemeter circuit which is connected to the conduction electrode to test real-time current intensity. The one-way conduction electrodes were divided into high-current, low-current and zero-current electrodes according to their electrical resistances. The changes of seizure frequency and epileptiform discharges on EEG were recorded in either rat model with TLE or clinical patients with TLE before and after current conduction treatment to analyze the relationship of current intensity and theraputic outcomes. The current conduction rat model with kainate-induce TLE, built in our previous research, and some techniques, including real-time quantitative PCR, Elisa, patch clamp whole-cell recording technique and microdialysis, will be employed in this study to study the effects of IL-1β, HMGB-1, glumate, and calcium pathway in neruoprotection during different current intensity level in conduction therapy. We want to confirm that there is a dose-response relationship between conduction current intensity and therapeutic effect in rats and patients with TLE who underwent current conduction treatment, and that IL-1β, HMGB-1, glumate, and calcium pathway may play important roles in neuroprotection of current conduction therapy for rats with TLE from experiences in multilevel，including gene expression, protein content, cell ion channel, tissue pathology, seizure attack and memory. The research is very important to help lay a foundation for current conduction therapy in TLE into clinical research as soon as possible.

针对当前近1/3癫痫患者治疗困难的现状，申请人利用海马电引导治疗方法，减少颞叶癫痫大鼠癫痫发作、降低细胞凋亡率，但存在缺乏电流量等量化指标及神经保护机制不明确等问题。本项目基于前期研究和预实验，研制单向引导的新型引导电极，并提出电引导治疗颞叶癫痫中引导电流与癫痫控制和神经保护存在量效关系及其神经保护机制与白细胞介素1β（IL-1β）、高迁移率族box-1、谷氨酸、钙离子等通路相关的假设。利用电阻差异制作高、低和零电流三种引导电极，通过将自制微电流计与引导电极串联，适时检测电引导治疗颞叶癫痫大鼠和颞叶癫痫患者中电流量，分析其与癫痫发作、癫痫放电变化的量效关系；采用实时定量PCR、Elisa、膜片钳等，分析电引导治疗对颞叶癫痫大鼠海马IL-1β、谷氨酸等相关通路的影响，探索其神经保护机制。从基因、蛋白、细胞、组织、大体等多层次进行实验研究，证明所提假设。推动海马电引导治疗颞叶癫痫进入临床研究。

针对当前近1/3癫痫患者治疗困难的现状，前期申请人利用海马电引导治疗方法，减少颞叶癫痫大鼠癫痫发作、降低细胞凋亡率，但存在缺乏电流量等量化指标及神经保护机制不明确等问题。本项目基于前期研究和预实验，通过可控引导电极的持续改进和设计，研制单向引导的新型引导电极，改进了近零电压的交流引导电路相关，3项专利已经获得授权，另外两项发明专利仍在申请过程中。基于海人藻酸海马局部注射颞叶癫痫大鼠电引导模型，根据引导不同可以分为模型对照组、低电阻引导阻和高电阻引导组进行的电引导相关量疗关系及其机制研究，结果显示低引导阻在癫痫放电和临床癫痫发作频率均显著低于对照阻和高电阻引导组，而高引导阻在癫痫发作总量和扩散的癫痫发作数量低于对照组，相应低电组引导组局部Glu/GABA显著低于模型对照组和高电阻引导组，而在一定时间内高引导组低于对照组。同时，利用实时定量PCR检测IL-1β/IL-1R1、HMGB-1/TLR-4和NMDA受体mRNA 表达水平，提示IL-1R1和NMDA参与了电引导的抗痫作用，而NMDA与量疗关系更为密切。Western Blotting测得IL-1β/IL-1R1、HMGB-1/TLR-4和NMDA受体蛋白含量，显示HMGB1、TLR4、IL-1β、IL-R1、NMDA均参与了电引导的抗痫作用相关，而TLR4、NMDA和IL-R1与量疗关系更为密切。另外从免疫组化和免疫荧光检测中也显示IL-1β/IL-1R1、HMGB-1/TLR-4表达在低引导组更低。水迷宫空间探索试验中低电阻引导组站台穿越次数显著高于模型对照组和高电阻引导组，提示其认知保护可能。本研究还利用进行颅内电极检查的16例颞叶癫痫患者进行了电引导治疗的临床前研究，结果显示引导后海马、杏仁核和颞叶皮层的棘波明显减少，而海马、杏仁核的涟波和海马的快速涟波，以及杏仁核和中颞皮层的癫痫指数明显下降。本研究首次证实了电引导治疗存在量效关系，在颞叶癫痫患者中也同样有效果，此外也证实除了Glu-NMDA/GABA通路，IL-1β/IL-1R1和HMGB-1/TLR-4也在电引导的抗痫机制中有重要作用，同时，电引导可以产生一定的认知保护作用。为电引导治疗的应用起到了明显的推动作用。