【作者】 姚远；

【导师】 吴立文； 许琪；

【作者基本信息】 北京协和医学院 ， 临床医学， 2012， 博士

【摘要】 癫痫是脑部神经元超同步化放电所导致的一组异质性疾病。内侧颞叶癫痫(mesial temporal lobe epilepsy, MTLE)是最常见的癫痫综合征之一,也是最常见的成年人药物难治性癫痫。海马硬化(hippocampal sclerosis, HS)是MTLE常见的病理表现,以海马区星形胶质细胞增生、神经元丢失、颗粒细胞散布和苔状纤维发芽为特征,但目前海马硬化的形成过程及MTLE发病的分子机制仍不清楚。近年来,研究发现Notchl信号通路对许多海马硬化中出现的或与之类似的病理表现有促进功能；在包括癫痫持续状态在内的损伤后神经再生过程中发挥作用,并能在神经元兴奋性增加时表达上调；在动物模型中发现,Notchl信号关键分子NICD1表达量在MTLE形成过程中上调,且MTLE患者手术切除的硬化海马中NICD1表达水平也存在上调。因此,选择Notchl信号通路研究其在海马硬化和MTLE发生中的作用,对理解疾病的发生过程具有重要意义。目的探讨Notchl信号通路的表达上调与HS-MTLE的形成是否有关；Notchl信号通路的激活是否推动了慢性期的自发癫痫发作。方法1.在C57BL/6小鼠右侧海马内植入电极-加药装置,同侧海马内注射Jaggedl激活Notchl信号通路(对照组BSA)或DAPT(对照组DMSO)抑制该通路后,海马内注射7ng海人酸构建HS-MTLE急性期模型,通过录像脑电图(video-electroencephalogram, vEEG)监测观察激活或抑制Notchl信号通路对急性期发作时间、次数和起始时间的影响。另一组小鼠右侧海马内注射200ng海人酸构建慢性期模型,右侧海马内植入电极-加药装置,通过vEEG监测确定稳定的发作基线后,右侧海马内注射Jaggedl,观察激活Notchl信号通路对慢性期发作时间的影响；洗脱期过后右侧海马内注射DAPT+Jaggedl,观察取消激活后慢性期的发作时间。2.以免疫荧光方法检测200ng海人酸模型5w时海马Notchl分子的分布。结果1.正常小鼠EEG表现为5-7Hz θ波；慢性期及急性期海人酸模型均出现高幅尖波、阵发性海马放电,及扩散至大脑皮层的发作；此外,急性期模型还出现阵发性棘慢波或多棘慢波综合、逐渐进展的高幅棘波及癫痫持续状态。2.慢性期注射Jaggedl后发作时间显著减少(n=5,p=0.00014),统计方法为重复ANOVA法继之Dunnet检验。注射DAPT+Jaggedl后,发作时间无明显减少(n=2)。3.急性期注射Jagged1后,发作时间(p=0.0048)、发作次数(p=0.019)较对照组显著增加(n=6或10)；注射DAPT后,发作时间(p=0.009)和发作次数(p=0.0111)较对照组显著减少(n=7)。统计方法为Mann-Whitney检验。4.200ng海人酸模型小鼠海马中,与对照组相比5w时主要分布在齿状回中,且表达量明显上调。结论1.成功构建了HS-MTLE的海马内电极-加药模型。2. Notchl信号通路在急性期模型中促进癫痫放电和发作,而在慢性期模型中抑制自发性癫痫发作。证明Notchl分子在HS-MTLE的形成和进展中存在不同作用。3. Notchl分子在慢性期海马组织中主要分布于DG区,且表达明显上调。更多还原

【Abstract】 Epilepsy is a type of heterogeneous disease caused by hyper synchronized discharges generated by excited neurons. Mesial temporal lobe epilepsy (MTLE) is one of the most common epilepsy syndromes and the most common intractable epilepsy in adults. Hippocampal sclerosis (HS) is frequently seen in MTLE, characterized by proliferation of astrocytes, loss of neurons, dispersion of granule cells, and mossy fiber sprouting in hippocampus. However, the molecular mechanism of hippocampal sclerosis and pathogenesis of MTLE is still unclear. During the past decade, Notchl signaling pathway has been found responsible for pathological phenomenon presented in or similar to those in hippocampal sclerosis. Meanwhile, researches indicate that Notchl signaling pathway takes part in neurogenesis following injuries including status epilepticus, and can be activated in response to neuronal activity. Moreover, researchers find that the critical component of Notchl signaling pathway, NICD1, is increased during epileptogenesis in the animal model of HS-MTLE, and there were similar findings in the sclerotic hippocampi of MTLE patients underwent surgery. Thus, it is important to uncover the roll of Notchl signaling pathway in the pathogenesis of hippocampal sclerosis and MTLE.ObjectiveTo discover whether the activation of Notchl signaling pathway is related to the development of HS-MTLE or not, and if it promotes spontaneous seizures during the chronic phase of epilepsy.Methods1. Implant a depth electrode and drug-adding device in the right hippocampus of each of the experiment C57BL/6mouse. Inject Jaggedl to activate Notchl pathway(BSA control) or DAPT to inhibit the pathway(DMSO control) via the device before injecting in the right hippocampus(r. i. h.)7ng kainic acid to induce the acute model of HS-MTLE. Monitoring video-electroencephalogram (vEEG), and calculate the respective seizure parameters after activation or inhibition of the pathway. Induce another group of mice with200ng kainic acid (r. i. h) to build chronic model of HS-MTLE and implant them with the same device. After confirming stable baselines of time in seizure by vEEG monitoring, they were injected with Jaggedl (r. i. h.) to determine its effect on the time of seizure during a certain period of time. Inject DAPT+Jaggedl (r. i. h) on the same mice after a washout period, and calculating the time of seizure after de-activating the pathway.2. Identify the distribution of Notchl by immunofluorescence in the hippocampus in the chronic phase (5w) of200ng kainic acid mouse model.Results1. Normal mouse EEG shows5-7Hz θ background. Both acute and chronic model show high voltage sharp waves, paroxysmal hippocampal discharge, and discharges propagating to cerebral cortex. In addition, acute model shows paroxysmal spike-and-wave or poly spike-and-wave, high voltage spikes buildup and status epilepticus.2. In chronic model, time in seizure was significantly reduced after Jaggedl injection (n=5, p=0.00014versus baseline by repeated measures ANOVA followed by Dunnet’s test), but without much change after DAPT+Jaggedl injection (n=2).3. In acute model, time in ictal activity (p=0.0048) and number of ictal activity (p=0.0109) were significantly increased after Jaggedl injection versus control by Mann-Whitney’s test (n=6or10). Time in ictal activity (p=0.009) and number of ictal activity (p=0.0111) were significantly reduced versus control by the same test (n=7).4. Excessive activation of Notchl is seen in the hippocampus of5w mouse model, mainly located in the dentate gyrus.Conclusion1. The model of intrahippocampal electrode and drug adding device was successfully built.2. Notchl signaling pathway promotes epileptiform discharges and seizures in acute model of HS-MTLE, but suppresses spontaneous seizures in chronic model, indicating its different effects on the development and progression of HS-MTLE. 3. Notchl is activated within dentate gyrus in chronic phases of HS-MTLE model.更多还原