【作者】 王莲；

【导师】 黄远桂； 陈良为；

【作者基本信息】 第四军医大学 ， 神经病学， 2009， 博士

【摘要】 颞叶癫癎是一种严重的神经系统疾病,其长期的病理变化很多仍不清楚,这也是颞叶癫癎患者临床治疗的严重障碍。癫癎发生过程通常包括三个阶段:损伤→潜伏期阶段(致癎期)→自发性癫癎发作(症状性癫癎)。虽然癫癎发生和反复发作的机制在颞叶癫癎患者中很难了解,但是在动物的颞叶癫癎模型中获得了越来越多的资料。在啮齿类动物中通过匹罗卡品注射诱发癫癎持续状态(status epilepticus, SE)已广泛用于慢性癫癎的实验研究。在大鼠或小鼠中系统应用匹罗卡品诱导的中枢神经系统神经元丢失与颞叶癫癎患者的情况相似。颞叶癫癎模型中有两种主要的神经元死亡机制。一种机制是急性死亡,与水肿或缺血有关。另一种机制是迟发性死亡,可能由细胞内浓度短暂性异常升高的游离钙介导,这也许会过度激活各种在正常情况下参与突触可塑性的酶系统如钙蛋白酶和蛋白激酶,caspases介导的程序性细胞死亡(programmed cell death, PCD)可能在迟发性损伤中起作用。神经元丢失是致癎期间环路重组的主要因素之一,因此损伤因素如SE发作诱导的细胞受损程度和时程仍是非常需要阐明的重要问题,因为这也许是慢性癫癎中发生长期病理变化和反复癫癎发作的关键性神经基础。细胞凋亡是慢性癫癎发生中神经元丢失的原因之一。凋亡是机体在一定的生理或病理条件下通过启动多种机制如死亡信号受体、线粒体功能障碍、caspase酶激活及DNA破坏而进行的程序性死亡过程。进一步阐明凋亡在慢性癫癎发生过程所起的作用仍然非常重要。研究目的:本研究旨在阐明颞叶癫癎中可能发生的长期脑损伤特征及损伤的相关机制。研究方法:我们在颞叶癫癎的小鼠匹罗卡品模型中通过Fluoro-Jade C (FJC)染色方法探测了变性神经元的区域分布和时程变化,还结合免疫荧光技术阐明了受累神经元的类型及涉及的损伤机制。FJC是一种新发展的荧光染料,对正发生变性的神经元(包括胞体、树突、轴突和轴突末梢)有高度的亲和力,特异性标记中枢神经系统正发生变性的神经元。研究结果:(1)在本研究中,我们用FJC染色方法结合标记神经元核特异性蛋白(neuronal nuclear specific protein, NeuN)的免疫荧光技术揭示匹罗卡品诱发的SE导致了从嗅球到中脑的许多脑区发生了大量的神经元细胞变性。(2)而且,由FJC揭示的神经元变性(包括急性和迟发性神经元损伤)是时间依赖性的。FJC阳性神经元在SE后4h出现,在12h-3d时达到高峰,然后又逐渐下降,7-14d时在有些区域甚至恢复到基线水平或消失。(3)双标记资料揭示FJC与Hoechst33342双标,且大多数FJC阳性神经元也表达凋亡信号因子如cytochrome C、caspase-9和活化的caspase-3,提示这些FJC阳性神经元正经历凋亡过程且可能尚处于早期阶段,也可能提示caspases介导的程序性细胞死亡在迟发性损伤中起重要作用。(4)更有趣的是,大部分(88%)FJC阳性神经元显示GABA(gamma-aminobutyric acid)能性质,因为它们也对谷氨酸脱羧酶-67(glutamic acid decarboxylase-67, GAD-67)显示免疫反应性,这也许提示在小鼠的匹罗卡品模型中GABA能神经系统的抑制功能受到严重损害。结论:本研究首次在小鼠匹罗卡品癫癎模型中应用FJC染色技术探测神经元死亡的区域分布、时程变化及相关机制。结合以前的研究综合考虑,在小鼠匹罗卡品颞叶癫癎模型中通过FJC标记变性的GABA能神经元的时程变化情况及所揭示的神经元死亡机制不仅有利于更好地理解颞叶癫癎中所发生的中枢神经系统长期病理变化和自发反复发作的癫癎机制,而且从治疗干预的可能性角度考虑,有助于探测治疗干预的时间窗和致癎期间的神经保护措施以预防或减轻癫癎发作。更多还原

【Abstract】 Temporal lobe epilepsy presents a serious neurological disorder in human beings and its long-term pathological events largely remain an obscure and severe obstacle in clinical treatment of patients. The epileptic process usually consists of three phases: initial insult→latency period (epileptogenesis)→recurrent seizures (symptomatic epilepsy). The epileptogenesis and recurrent seizure mechanisms in humans are poorly understood, but growing evidences have been obtained from animal models of epilepsy. The rodent animal models of status epilepticus (SE) have been extensively utilized in experimental studies of chronic epilepsy by injection of pilocarpine. Systemic administration of pilocarpine can induce neuronal loss in central nervous system, which shows striking similarities to human temporal lobe epilepsy in rats or mice.There are two major mechanisms of neuronal death in models of temporal lobe epilepsy. One mechanism is acute oedemic or ischemic death of the effected neurons. The other mechanism is delayed, and is likely to be mediated by a transient but abnormal rise in intracellular free calcium concentration. This may overactivate various enzyme systems normally involved in synaptic plasticity, e.g. calpain and protein kinases. Caspase-mediated programmed cell death (PCD) can contribute to delayed damage. Neuronal loss is one of the major components of circuitry reorganisation during epileptogenesis. Thus,the injury extent and time-course of neuronal death induced by SE attack still remain crucial question to answer, which may encase key neural basis of long-term changes and recurrent seizure in chronic epilepsy. Evidences have showed that neuronal loss in the chronic epilepsy could partially result from cell apoptosis, which is a programmed physiological event but also occurred in various toxic insults and neurological diseases through multiple ways of death signaling receptors, mitochondrial dysfunction, activation of caspase enzymes, and DNA damage. It is still important to further elucidate the contribution of an apoptotic mechanism to the pathological process of chronic epilepsy.Purpose: In the present study, we are interested in elucidating long-term brain injury and related mechanisms that may occur in the temporal lobe epilepsy. Methods: The regional distribution and time-course of degenerative neurons were examined in a mouse pilocarpine model of chronic epilepsy by Fluoro-Jade C (FJC) dye that can specifically stain degenerating neurons in the central nervous system. The type of affected neurons and mechanisms involved in cellular damage were also elucidated by combining with immunofluorescence technique. FJC is a new-developed fluorescent Fluoro-Jade (FJ) dye which has high affinity for degenerating neurons including cell body, dendrites, axons and axon terminals. Results: (1) The FJC stain combined with neuronal nuclear specific protein (NeuN) immunofluorescence revealed that pilocarpine-induced SE resulted in massive degenerative death of neuronal cells in many brain regions from olfactory bulb to midbrain. (2) Moreover, cellular degeneration including acute and delayed neuronal death revealed by FJC stain was time-dependent. The FJC-positive degenerating neurons occurred at 4h, increased into peak levels at 12h–3d, and then gradually went down, even resolved to baseline or disappeared at 7d–14d after onset of SE. (3) Double-labeling data revealed that cellular co-localization of FJC and Hoechst was abundantly observed and most of FJC-positive degenerating neurons also expressed apoptosis signaling molecules such as cytochrome C, caspase-9, and activated caspase-3, indicating that these FJC-positive cells maybe were undergoing apoptotic processes and were in an early phase of apoptosis. These may also imply that caspase-mediated PCD plays an important role in the delayed damage. (4) More interestingly, a large percentage (about 88%) of FJC-positive degenerative neurons were GABAergic as indicated with their immunoreactivity to glutamic acid decarboxylase-67 (GAD-67), implying that inhibitory function of GABAergic neural system might by seriously damaged in brains subject to SE attack in this mouse pilocarpine model.Conclusion: This study has first applied FJC staining to demonstrate regional distribution, time-course, and related mechanisms of neuronal death in the mouse pilocarpine model. Taken together with previous studies, time-course and death mechanisms of degenerative GABAergic neurons in the mouse pilocarpine model revealed by FJC staining benefit further understanding of long-term brain pathological changes and recurrent seizure mechanism, and, from the point of possible pharmacological intervention, may also result in finding the most suitable time-window in therapeutic manipulation of the chronic epilepsy in human beings and appropriate neuroprotective treatment to prevent or lessen seizures during the epileptogenic phase.更多还原