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癫痫持续状态亚低温神经保护作用机制的研究
【作者】 王艺；
【作者基本信息】 复旦大学 ， 儿科学， 2009， 博士
【摘要】 第一部分亚低温对匹罗卡品癫痫持续状态幼鼠海马损伤的保护作用研究目的:探讨亚低温在癫痫持续状态时的神经保护作用,比较不同亚低温干预时间(2小时、4小时、8小时)的保护作用。方法:采用21日龄的wistar大鼠经腹腔注射匹罗卡品制作惊厥持续状态的模型。随机分成NS对照、SE30分钟单纯安定治疗、SE30分钟亚低温联合安定治疗2小时、SE30分钟亚低温联合安定治疗4小时、SE30分钟亚低温联合安定治疗8小时、SE60分钟单用安定治疗、SE60分钟亚低温联合安定治疗8小时组,共7组。干预后72小时取材,经HE染色和Nissl染色观察大鼠海马组织形态学变化和损伤情况;MAP2免疫组织化学染色评价大鼠海马神经元细胞骨架蛋白的影响;通过免疫组织化学方法检测caspase-3,并进行caspase-3和NeuN免疫荧光双标记、TUNEL原位凋亡检测大鼠海马神经元的凋亡情况。结果:SE30分钟、SE60分钟后大鼠海马神经元出现不同程度的病理损伤,HE染色和Nissl染色发现海马CA1和CA3区均出现明显的神经元间隙增宽、胞浆溶解、核固缩、核溶解,Nissl颗粒减少等现象,坏死细胞计数百分比和NS组相比明显增高,差异存在统计学意义(P＜0.05)。亚低温联合安定治疗2小时即显著减少海马CA3区神经元细胞的坏死,与安定单一治疗组相比,差异具有统计学意义(P＜0.05)。亚低温联合治疗8小时的海马神经元形态和坏死细胞计算百分比NS对照组最为接近。MAP2免疫组化检测发现,SE30分钟、SE60分钟后大鼠海马神经元细胞骨架结构损伤明显,和NS组相比差异具有显著的统计学意义(P＜0.05)。和单一应用安定治疗组相比,亚低温联合治疗2小时即可显著升高海马CA3区神经元MAP2的表达水平,差异具有统计学意义(P＜0.05)。CA1区MAP2表达水平显著升高发生在亚低温干预8小时后,差异具有统计学意义(P＜0.05)。而SE60分钟后给予亚低温联合干预2小时,MAP2表达水平的升高无统计学意义。Caspase-3免疫组织化学以及TUNEL原位检测发现,SE30分钟、SE60分钟后大鼠海马神经元细胞发生明显的凋亡,和NS组相比差异具有显著的统计学意义(P＜0.05)。亚低温联合治疗8小时后,CA1和CA3区caspase-3蛋白表达明显下降,差异具有统计学意义(P＜0.05)。而TUNEL检测发现,亚低温联合干预2小时后CA1和CA3区凋亡细胞明显减少,和未干预组相比,差异具有统计学意义(P＜0.05)。caspase3和NeuN免疫荧光双标结果发现,亚低温8小时后,双标神经元数量明显减少。结论:1.癫痫持续状态导致大鼠海马神经元细胞的坏死、凋亡以及神经元细胞骨架塌陷。2.亚低温干预能减少海马神经元的坏死、凋亡和细胞骨架蛋白的丢失,具有神经损伤保护作用。3.和亚低温持续治疗2小时、4小时相比,持续治疗8小时具有更好的神经保护作用。第二部分亚低温对匹罗卡品癫痫持续状态幼鼠海马损伤的神经保护机制的研究目的:通过研究离子型和代谢型谷氨酸受体核酸以及蛋白表达变化,探讨亚低温以及不同亚低温干预时间对癫痫持续状态幼鼠海马神经元损伤的保护机制。方法:采用21日龄的wistar大鼠共60只,经腹腔注射匹罗卡品制作癫痫持续状态的模型。随机分成5组,具体分组同第一部分,每组12只。干预后72小时,每组随机挑选6只幼鼠,灌流取脑海马切片,采用免疫组化方法检测各组幼鼠海马各区GLUR-1和GLUR-2蛋白表达水平变化;每组其余6只幼鼠活体分离出双侧海马组织,分别采用实时荧光定量PCR法和Western-blotting免疫印迹技术检测各组幼鼠海马GLUR-1、GLUR-2和mGLUR1-αmRNA和蛋白表达水平。结果:免疫组化发现,SE30分钟后,海马CA1和CA3区的GLUR-1蛋白表达明显升高而GLUR-2蛋白表达明显降低,与NS组相比,差异存在统计学意义(P＜0.05)。亚低温干预后可以降低海马CA1和CA3区GLUR-1蛋白表达而增加GLUR-2蛋白表达,亚低温干预8小时后,两者的蛋白表达与和未给予亚低温干预组相比差异具有统计学意义(P＜0.05)。Western-blotting发现海马GLUR-1和GLUR-2蛋白表达变化与免疫组化结果相似,亚低温干预4和8小时后,两者的蛋白表达与和未给予亚低温干预组相比差异具有统计学意义(P＜0.05)。SE30分钟后,海马mGLUR1-α蛋白表达有升高,亚低温联合安定治疗后可以降低海马mGLUR1-α蛋白表达,亚低温干预8小时后,mGLUR1-α蛋白表达与和未给予亚低温干预组相比差异具有统计学意义(P＜0.05)。RT-PCR结果发现,SE30分钟后,海马GLUR-1和mGLUR1-αmRNA表达增加,而GLUR-2mRNA表达下降,亚低温联合安定治疗后GLUR-1和mGLUR1-αmRNA表达下降,GLUR-2mRNA表达有升高,亚低温干预8小时后GLUR-1和mGLUR1-αmRNA表达和未给予亚低温干预组相比有统计学差异(P＜0.05),但GLUR-2mRNA表达和未给予亚低温干预组相比无统计学差异。结论:GLUR-1、GLUR-2和mGLUR1-α共同参与癫痫持续状态后神经元损伤的形成,亚低温通过下调GLUR-1mRNA和蛋白表达,上调GLUR-2蛋白表达,下调mGLUR1-αmRNA和蛋白表达,减少神经元损伤。
【Abstract】 PART ONE:The Neuroprotective Effect of General Mild Hypothermia on nippocampus in Pilocarpine-induced Status Epilepticus inYoung RatsObjective:To investigate the ncuroprotection of mild hypothermia on hippocampal neurons in young rats with pilocarpine induced status cpilepticus(SE).The different duration of mild hypothcrmia(2h,4h,8h) were used to the rats after SE for 30mins and 60 mins.Methods:42 Male Wistar rats aged 21-days weighing 60-65g were used in this study. Animals were given pilocarpine 380mg/Kg through intraperitoneal injection to induce status epilepticus.Rats with induced-sustaining status epilepticus for 30 minutes were divided into seven groups,including:SE 30 minutes treated with NS,with diazepam 10mg/Kg,diazepam with combination of mild hypothermia for 2 hours,4 hours and 8 hours,status epilepticus for 60 minutes were treated with diazepam 10mg/Kg or diazepam with combination of mild hypothermia for 2 hours.The control group received equal volume injections of saline.72 hours after SE,the hippocampal pathological changes have been observed through HE staining Nissl staining,In order to evaluated the changes of microtubule-associated protein 2(MAP2) and caspase 3 in the rat hippocampus,immunohistochemistry was performed according to previously established methods.We also did terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling(TUNEL) to observe the PCD of hippocampus neurons.Results:The hippocampal neuronal damage were found in rats after 30 min and 60 min SE.The neurons showed pyknotic nuclei and shnmken plasma in cyton.Nissl granula decreased in CA1 and CA3 regions of hippocampus.The percentage of neuron necrosis of hippocampus were significantly more than that in control group(P＜0.05).2 hours after therapeutic alliance with diazepam and mild hypothermia, necrotic neurons in CA3 region of hippocamous were significantly reduced compared with the group treated with diazepam alone(P＜0.05).8 hours after treatment,the neuron damage in hippocampus and the percentage of necrotic cells were similar to those of the NS group.Compared with the group of diazepam treatment,the expression of MAP-2 protein were enhanced in the hippocampus with mild hypothermia with 2h,4h or 8h duration(P＜0.05).The staining intensity and immunoreactivity increased 8 h after the mild hypothermia treatment.There was no significant difference in MAP2 immunoreactivity between the 60 mins SE rats and with 2h hypothermia treatment.The expression of caspase-3 increased in the hippocampus of rats with 30 mins and 60 mins SE.Significant reductions in caspase-3 immunoreactivity were seen after the intervention of mild hypothermia.TUNEL staining showed DNA fragmentation positivity in the SE with diazepam but decreasing significant in the groups with mild hypothermia.Double stained neurons with caspase3 and NeuN indicated that the positive apoptosis neurons decreased significantly after 8 hours mild hypothermia treatment..Conclusion:1.Status epilepticus can induces brain damage such as neurons necrosis,apoptosis and the collapse of neuron cystoskeleton.2.Mild hypothermia can protect hippocampal neurons from necrosis,apoptosis and lossing of MAP-2.3.Compared with 2 and 4 hours of persistent mild hypothermia treatment,8 hours of treatment have most neuroprotection effects after SE.. PART TWO:The Mechanism of Neuroprotection with Mild Hypothermia in Pilocarpine-induced Status Epilepticus in Young RatsObjective:Study on expression of mRNA and protein of GluR1,GluR2 and metabotropic glutamate receptors mGluR1 a after SE.To explore the mechanism of neuroprotection of mild hypothermia on hippocampal neurons in young rats.Methods:A total of 50 Male Wistar rats aged 21-days were randomly assigned into 5 groups:NS group,diazepam group,diazepam with 2h,4h,and 8h mild hypothermia treatment groups.Animals were given pilocarpine 380mg/Kg through intraperitoneal injection to induce status epilepticus,72 hours after SE,the changes in GluR1 and GluR2 as well as mGluR1 a protein levels were determined using Western-blotting analysis.The protein bands were quantified by densitometric analysis.Real-time PCR was used to investigate the mRNA expression of GluR1,GluR2 and mGluR_α.Results:After pilocarpine-induced status epilepticus,the GluR1 immunopositive nrurons increased obviously in CA1 and CA3 area of hippocampus,however protein expression of GluR2 obviously reduced compared with NS group(P＜0.05).After therapeutic alliance with diazepam and mild hypothermia,GluR1 protein expression in CA1 and CA3 reduced while GluR2 protein expression increased.After 8 hours of treatment,both protein expression were significant increased compared with the group treated with diazepam alone(P＜0.05).Through Western-blotting,after 4 hours and 8 hours therapeutic alliance,the expression GluR1 and GluR2 protein in hippocampal decreased significantly compared with the group treated with diazepam alone(P＜0.05).The expression of mGluR1_αprotein increased after status epilepticus and reduced by therapeutic alliance with diazepam and mild hypothermia.With 8 hours therapeutic alliance,there was a significant reduction in mGluR1_αprotein expression compared with the group treated with diazepam alone(P＜0.05).The mRNA expression of GluR1 and mGluR1_αincreased but the expression of GluR2 mRNA decreased.With diazepam and mild hypothermia treatment,the mRNA expression of GluR1 and mGluR1_αdecreased while GluR2 increased.With 8 hours therapeutic alliance,GluR1 and mGluR1_αmRNA expression decreased significantly compared with the group treated with diazepam alone(P＜0.05) There was no significant up-regulation of GluR2 mRNA in different groups.Conclusion:GluR1,GluR2 and mGluR1_αplay an important role in the brain injury induced by SE.The protein and mRNA for GluR1,GIuR2 and mGluR1_αwere different regulated by hypothermia treatment.Hypothermia attenuated neuron injury through down-regulating the expression of GluR1 as well as mGluR1_αmRNA and protein,and up-regulating the protein expression for GluR2.