【作者】 李艺；

【导师】 肖波； Carolyn R. Houser；

【作者基本信息】 中南大学 ， 神经病学， 2010， 博士

【摘要】 第一章：匹罗卡品致痫小鼠自发性癫痫发作模型的建立及癫痫后海马新生神经细胞增生的研究目的：探讨匹罗卡品致痫小鼠自发性癫痫发作模型的特点,观察癫痫发作后海马结构齿状回颗粒细胞下层中新生神经细胞增生的变化。方法：1.6-8周成年雄性C57BL/6小鼠进行随机分配为实验组和对照组,建立匹罗卡品致痫后自发性癫痫发作小鼠模型。对实验组小鼠进行连续50天的行为学监测,观察其自发性痫性发作的频率、时程和发作规律(n=8)；并在50天后行海马结构尼氏染色观察其病理学改变。2.将造模成功的小鼠在癫痫持续状态(SE)后1周,2周,4周,8周,12周和正常对照组(各组n=3)的脑组织进行Doublecortin (DCX)的免疫组化染色观察新生神经细胞的分布；同时应用western blot方法检测各组小鼠海马组织中DCX的表达变化(各组n=3)。结果：1.对模型小鼠连续50天的行为学监测显示：匹罗卡品诱发癫痫持续状态后87.5%的模型小鼠存在自发性癫痫发作。模型小鼠在3-14天不等的潜伏期后开始出现自发性痫性发作；其发作时程约10-40秒,平均每周出现2.18±0.45次,并多在昼夜交替时出现。其次,模型小鼠的自发性痫性发作具有簇发性。2.匹罗卡品致痫后有自发性癫痫发作的小鼠模型海马结构尼氏染色显示门区神经元显著减少的现象恒定出现,同时可见CA1、CA3区锥体细胞排列紊乱,CA1区神经元丢失；此外一部分的模型小鼠表现为齿状回颗粒细胞减少、排列紊乱,或颗粒细胞层弥散化。3.自发性癫痫发作小鼠模型中各时间点DCX的免疫组化和Western Blot检测发现DCX+细胞和DCX蛋白的表达在模型小鼠中的显著增多。这种现象在SE后1周即可见到,在SE后4周最为明显；在SE后8周开始下降,但仍与正常对照组无明显差异；至SE后12周,DCX+细胞显著减少,其蛋白表达亦下降。在最初时增生的DCX+细胞呈簇状分布,随时间推移出现迁移分散；DCX+细胞逐渐增多的同时伴有DCX+细胞树突的分支增加。结论：匹罗卡品致痫后自发性癫痫发作小鼠模型具备与颞叶癫痫相似的行为学特点和病理学改变,并且存在有持续的新生神经元增生。第二章：匹罗卡品致痫小鼠自发性癫痫发作相关的ERK通路激活及其对新生神经细胞影响的研究目的：研究匹罗卡品致痫小鼠自发性癫痫发作模型海马结构中ERK通路激活的改变,并探索性研究自发性癫痫发作后ERK通路的活化影响新生神经细胞增生的可能机制。方法：1.将SE后2-7周具有自发性痫性发作的小鼠进行行为学观察,在正常对照组(n=14),自发性癫痫发作后2min (n=5),3min (n=3),6min (n=2),30min (n=3)(在此之前12 h无其它痫性发作)和发作静止期(n=3)进行脑组织ERK通路活化(pERK)的检测,并比较了自发性癫痫发作后pERK与Fos, GABAAR 6亚基的表达差异。2.对自发性癫痫发作后2min的模型小鼠海马结构进行pERK与BLBP,GFAP, S100β,Nestin, Pax6, NeuroD, PSA-NCAM, DCX, Tuj-1, Prox1,NeuN, Ki67, Mcm2的免疫荧光,检测表达pERK的细胞类型(各组n=5)。3.半定量分析正常组和自发性癫痫发作后颗粒细胞下层细胞增殖指标Mcm2标记的细胞密度的变化,并通过检测BLBP与Mcm2的共表达(n=5)观察自发性癫痫发作后海马结构中多能神经干细胞的增殖潜能改变。4.利用pERK/BLBP/Mcm2免疫荧光三标技术检测自发性癫痫发作后颗粒细胞下层ERK通路活化与多能神经干细胞增殖的关系(n=3)。结果：1.在正常对照组中,少数pERK阳性的细胞散在于颗粒细胞层中,在发作静止期,其表达显著下降。而在自发性癫痫发作后ERK通路被迅速激活：在自发性癫痫发作后2min,pERK在颗粒细胞下层中的表达显著增加,随后扩散到整个颗粒细胞下层；在自发性癫痫发作后6min达高峰,30 min后回到基础水平。在与Fos, GABAARδ亚基的表达比较发现Fos表达较pERK表达延迟,并且出现Fos和pERK表达上调的区域表现为GABAARδ亚基表达的下调。2.在模型小鼠自发性癫痫发作后2min,颗粒细胞下层中有77.62±4.89%的pERK+细胞表达多能神经干细胞标记物BLBP,77.68±5.50%的pERK+细胞表达GFAP,21.67±7.12%的pERK+细胞表达具有神经元潜能的神经干细胞指标NeuroD,少于5%的pERK+细胞表达PSA-NCAM。pERK+细胞不表达胶质细胞标记物S100p,亦不表达神经元前体细胞标记物DCX, Tuj-1, Prox1,NeuN。3.在模型小鼠自发性癫痫发作后2min,Mcm2标记的细胞密度显著高于正常对照组；同时其BLBP+Mcm2+细胞在BLBP+细胞中的比率显著增加。4.在模型小鼠自发性癫痫发作后2min,所有BLBP+Mcm2+细胞亦同时表达pERK。结论：1.pERK作为ERK通路活化的指标,在匹罗卡品致痫后自发性癫痫发作小鼠模型中随着其癫痫活动状态的不同呈现相应的改变,是衡量其神经元活动敏感的指标之一。2.在匹罗卡品致痫后自发性癫痫发作小鼠模型中,其多能神经干细胞的增殖潜能显著增加。3.在匹罗卡品致痫小鼠模型自发性癫痫发作后,ERK通路首先在多能神经干细胞和早期具有神经元潜能的干细胞中激活,并可能促进其增殖潜能。4.自发性癫痫发作可能通过影响新生神经元形成过程中相关环节从而促进癫痫后新生神经元的增生。更多还原

【Abstract】 Part one:A mouse model of temporal lobe epilepsy characterized with spontaneous seizures and its influence on adult neurogenesisObjective:To investigate a useful pilocarpine model of temporal lobe epilepsy (TLE) characterized by recurrent spontaneous seizures, and to explore its influence on the adult neurogenesis of hippocampal formation.Methods:1.Young adult (6-8 week of age) C57BL/6 male mice were used in the study. Sustained seizures were induced in experimental animals by the administration of pilocarpine. After recovery from status epilepticus (SE), the behaviors of pilocarpine-treated mice were continuously monitored for 50 days (n=8).Then the mice were stained with cresyl violet for the historical study.2.DCX, a neural progenitor cell marker, was detected by immunohistochemistry and Western blot method at 1 week,2 weeks,4 weeks,8weeks,12 weeks after SE, as well as in control animals (n=3, respectively).Results:1.Fifty-day behavior study showed that 87.5% of pilocarpine-treated mice developed spontaneous seizures.They usually experienced a latent period from 3-14 days after SE before they begin to have spontaneous seizures.The duration ranged from 10-40 seconds. Spontaneous seizures occurred 2.18±0.45 per week on average, usually during the light on/off period and appeared in clusters.2.The Nissl staining of the pilocarpine treated mice consistently showed hilus cells loss, and usually accompanied by cell loss in CA1 area and disorganization of CA1,CA3 pyramidal cell layers.In addition, some animals also showed cell loss in dentate granule cell layer or granule cell layer dispersion.3.The Western bolt results revealed that the level of DCX began to increase at 1 week after SE in the hippocampus formation, and downregulated to basal level at 8 weeks after SE. The immunohistochemistry of DCX showed that the DCX-labeled cells were located in the subgranular zone (SGZ).After SE, they were originally appeared in clusters, and then immigrated to dentate inner granule cell layer and occasionally to hilus or outer layer of dente gurus.Conclusions:The mouse model of TLE with spontaneous seizures shares most of the characteristics of TLE patients, meanwhile it showed consistently increased neurogenesis which could also be observed in TLE patients.It offered a useful tool to help investigate the ongoing epilepsy-related processes associated with spontaneous seizures. Part two:Activation of ERK pathway by spontaneous seizures and its potential effects on neurogenesis in a mouse model of TLEObjective:To evaluate the dynamic changes of ERK pathway in the mouse model of epilepsy with spontaneous seizures,and to explore the influence of ERK pathway activated by spontaneous seizures on neurogenesis.Methods:1.Pilocarpine-treated mice from 2 to 7 weeks following SE were monitored for spontaneous seizures, and a total of 17 pilocarpine-treated animals were included in the pERK study. The mice were purfused at different intervals following a spontaneous seizures, including 2min (n=6),3min (n=3),6min (n=2),30min (n=3)following seizure detection. Animals that demonstrated additional spontaneous seizures during the previous 12-24h before perfusion were excluded. For comparison, age-matched epileptic mice (n=3)following SE that had not experienced a behavioral seizure in the last 24h prior to perfusion were included. Age-matched control animals were included in the study and were perfused at the same time as the pilocarpine-treated animals.In addition, for comparison of pERK labeling, the immunohistochemistry of Fos and GABAARδsubunit were also studied at the onset of a spontaneous seizure (2 min).2.In the pilocarpine-treated animals which experienced a spontaneous seizure at 2 min before perfusion, several neural progenitor cell (NPC) markers were used for double immunofluorescence labeling with pERK in the hippocampal formation:(1)radial glia-like NPC markers, including GFAP, BLBP, Nestin and Pax6; (2) a marker of intermediate stage NPCs with neuronal lineage potential, NeuroD; (3) immature neuronal markers, including DCX, Tuj-1;(4) an early granule cell-specific marker, Prox1;(5)a general neuronal marker, NeuN; (6) A marker of mature astrocytes,S100β.In addition, proliferation markers, Ki67 and Mcm2 were also used to determine if pERK labeled cells were in the proliferative cycle.3.To evaluate the difference of Mcm2 labeled cells and the BLBP-expressing NPCs that were in the proliferateive cycle in the SGZ between pilocarpine-treated animals at the onset of a spontaneous seizure and control animals, the density of Mcm2 labeled cells and the percentage of double-labeled cells that expressed BLBP and Mcm2 were calculated (n=5).4.Triple immunofluorescece of pERK, BLBP and Mcm2 were conducted in the hippocampal formation of pilocarpine-treated animals at the onset of a spontaneous seizure, and the percentages of single-, double-and triple-labeled cells were calculated (n=3).Results:1.In the control animals, the pERK-labeled cells were scattered in the dentate gyrus.In the pilocarpine-treated animals, when the mice had not experience spontaneous seizure in the last 24h before perfusion, the pERK labeling was obviously decreased. At the onset of a spontaneous seizure, the pERK labeling dramatically increased, it first appeared in the cells located in the SGZ at 2 min after a spontaneous seizures, and quickly spread to the whole granule cell layer, then went back to basic level within 30min. For comparison of pERK labeling with Fos and GABAARδsubunit, The Fos labeling were relatively delayed compared to pERK staining although they shared the similar changes; The area with pERK upregulation showed downregulation of GABAARδsubunit expression.2.In the animals that just experienced a spontaneous seizure at 2 min before perfusion,77.62±4.89% of pERK labeled cells colocalized with BLBP,77.68±5.50% of pERK labeled cells colocalized with GFAP, 21.67±7.12% of pERK labeled cells colocalized with NeuroD, and less than 5% of pERK labeled cells meanwhile expressed PSA-NCAM. Virtually no expression of DCX, Tuj-1, Prox1 or NeuN was found in pERK labeled cells.3.In the animals that just experienced a spontaneous seizure at 2 min before perfusion, the density of Mcm2 labeled cells were increased compared to that in the control animals. Similarly, the potential of BLBP labeled cells that were proliferating was also increased.4.In the animals that just experienced a spontaneous seizure at 2 min before perfusion, virtually all BLBP and Mcm2 double-labeled cells meanwhile showed ERK activation.Conclusions:1.ERK pathway activation showed dynamic changes following spontaneous seizures, which suggests it could be a sensitive marker considering the spontaneous seizure activity.2.In the pilocarpine treated animals with spontaneous seizures, the proliferative potential of neural stem cells that located in the SGZ were increased.3.At the onset of a spontaneous seizure, ERK pathway activation was first appeared in the neural stem cells and the NPCs with neuronal potential.4.Spontaneous seizure could exert its influence on the proliferation of NPCs and may contribute to the epilepsy-related neurogenesis.更多还原