【作者】 林幽町；

【导师】 迟兆富；

【作者基本信息】 山东大学 ， 神经病学， 2010， 博士

【摘要】 癫痫是神经系统常见疾病,给社会、家庭及患者个人都带来了沉重的负担。我国目前约有900万癫痫患者,其中有25-40%为难治性癫痫。颞叶癫痫(temporal lobe epilepsy, TLE)是最常见的难治性癫痫类型,其发病机制目前尚未完全清楚,药物及手术治疗常难以获得明显疗效。氯化锂-匹鲁卡品致痫大鼠行为学、神经电生理学和海马神经元损伤的病理学改变均与人类颞叶癫痫相似,因此该模型是目前研究癫痫持续状态(status epilepticus, SE)和TLE的常用模型之一我们前期通过建立慢性癫痫模型发现,癫痫发作后氧化呼吸链功能受损与线粒体DNA(mitochondrial DNA, mtDNA)编码亚基mRNA及蛋白合成缺陷有关。有研究表明癫痫发作产生大量自由基堆积于线粒体,与核DNA(nuclear DNA, nDNA)相比,mtDNA更易受损,故推测癫痫发作后mtDNA可能存在严重的氧化损伤从而导致影响其编码功能、影响线粒体呼吸链功能。癫痫发作能造成线粒体呼吸链活性下降、超微结构受损已被广泛认可,线粒体功能的异常是神经元损伤的重要机制之一,因此对线粒体损伤及代偿机制的研究具有重要现实意义。正常的生理代谢活动下,线粒体可产生一定量的自由基,后者可损伤线粒体基因组,但机体存在健全的抗氧化机制,以维持遗传信息的稳定性。第一道防线为自由基清除系统,如SOD、GSH等,负责尽可能地清除自由基；第二道防线为DNA修复系统,负责修复各种原因导致的基因组的损伤。癫痫发作后,这两大代偿系统的反应性可能决定神经元及线粒体的损伤程度。为此,本课题建立氯化锂-匹鲁卡品致痫大鼠模型,探讨癫痫发作后海马神经元损伤的病理学及分子生物学特征；检测癫痫模型中海马mtDNA的氧化损伤；检测两道抗氧化防线的反应性,以此深入研究癫痫的发病机制。本研究分四个部第一部分氯化锂-匹鲁卡品大鼠急慢性癫痫模型的建立及脑电图、海马神经元损伤的病理学观察目的建立氯化锂-匹鲁卡品诱发的大鼠急、慢性癫痫模型,观察大鼠的行为学、电生理学及海马组织病理学改变。方法成年雄性Wistar大鼠腹腔注射氯化锂-匹鲁卡品诱发癫痫持续状态(status epilepticus, SE),观察大鼠行为学变化。SE持续60 min后腹腔注射地西泮终止发作。于致痫后3h对大鼠进行EEG描记；多聚甲醛灌注取脑,石蜡包埋切片,Nissl染色观察大鼠海马神经元的损伤；其后每天观察记录大鼠有无自发性痫性发作(spontaneous recurrent seizures, SRS)。结果1.根据Racine发作评分标准,达到Ⅳ-Ⅴ级发作的大鼠被认为是诱发SE成功,表现为双侧前肢的阵挛、抽搐及全面性强直-阵挛发作,双侧后肢强直伴身体直立、躯干背曲强直、跌倒。诱发SE的成功率为83.1%,潜伏期38.5±18.7min,72h内死亡率为23.2%。平均静止期为12.7±6.2天。有74%的大鼠观察到每周1-3次Ⅰ级到Ⅴ级的不同形式的自发性发作,表现基本同急性期,但持续时间较短暂,每次很少超过1min。2.癫痫发作后3 h(急性期)大鼠皮层和海马区的EEG均可记录到丛集性棘波放电。3. Nissl染色显示癫痫大鼠海马的CA1和CA3区神经元丢失,排列疏松,轮廓模糊,界限不清,可见部分神经元胞体皱缩,核固缩,胞浆深染,胞浆内尼氏小体减少。结论根据氯化锂-匹鲁卡品腹腔注射后大鼠的行为学和EEG改变,说明氯化锂-匹鲁卡品可致大鼠急、慢性癫痫发作,氯化锂-匹鲁卡品致痫后可引起大鼠海马神经元损伤。第二部分氯化锂-匹鲁卡品致痫大鼠海马线粒体超微结构改变和线粒体基因组损伤的研究目的观察氯化锂-匹鲁卡品致痫后急、慢性癫痫大鼠海马线粒体超微结构变化,研究线粒体DNA(mitochondrial DNA, mtDNA)拷贝量的改变,检测海马mtDNA氧化碱基水平,进一步探讨癫痫致海马线粒体损伤的分子生物学机制。方法成年雄性Wistar大鼠随机分为急性对照组、急性癫痫组(SE后25h)、慢性对照组、慢性癫痫组(SE后60d)。在相应的时间点断头取脑,电镜观察线粒体超微结构的变化。分离海马,提取基因组DNA。RQ-PCR法分别检测三段不同区域mtDNA与nDNA的比例,从而反应mtDNA拷贝水平；比较三段不同区域拷贝量以排除mtDNA大片段缺失对实验结果的影响。然后,以Fpg (formamidopyrimidine DNA glycosylase)孵育切除氧化损伤碱基。由于氧化碱基切除后该处形成缺口,阻止PCR通过此处扩增,RQ-PCR法检测Fpg孵育后与孵育前mtDNA的比例,可以反映完好碱基的比例；比较三段不同区域完好碱基的百分比可排除可能的氧化损伤热点对结果的影响。结果1.急性期与慢性期癫痫大鼠海马神经元的线粒体均呈现不同程度的损伤,表现为间隙肿胀,线粒体基质消失,结构破坏,重者线粒体明显空泡化。2.与急性对照组相比,急性癫痫组大鼠海马mtDNA拷贝量无明显变化(p>0.05),三段不同区域的mtDNA片段的拷贝量无统计学差异(p>0.05)。3.Fpg孵育后,急性癫痫组与急性对照组mtDNA拷贝量无明显变化(p>0.05),孵育后三段不同区域的mtDNA片段拷贝量无统计学差异(p>0.05)。4.与慢性对照相比,慢性癫痫大鼠海马mtDNA拷贝量明显降低(p<0.05),三段不同区域的mtDNA片段的拷贝量无统计学差异(p>0.05)。5.Fpg孵育后,癫痫组比对照组mtDNA拷贝量下降更为明显(p<0.05),孵育后三段不同区域的mtDNA片段拷贝量无统计学差异(p>0.05)。结论氯化锂-匹鲁卡品所致的SE模型中,mtDNA拷贝量无明显变化,未检测出明显增加的氧化位点。氯化锂-匹鲁卡品所致的慢性癫痫模型中,mtDNA拷贝量明显下降,且线粒体基因组内存在大量的氧化碱基,目前尚未发现明显的易损区域。本研究阐明了反复痫性发作可致海马线粒体基因组损伤,氧化应激可能是损伤的重要病理机制。第三部分氯化锂-匹鲁卡品致痫大鼠海马抗氧化防御体系反应性的探讨目的检测匹鲁卡品致痫大鼠模型中海马组织脂质过氧化损伤,评估自由基清除剂超氧化物歧化酶(Superoxide dismutase, SOD)的水平及海马氧化还原状态,从而进一步探讨线粒体氧化损伤的机制。方法成年雄性Wistar大鼠随机分为急性对照组、SE后3h、SE后25h、慢性对照组、慢性癫痫组(SE后60d)。于相应时间点分别断头取脑,分离海马,检测丙二醛(malonaldehyde, MDA)、SOD、谷胱甘肽(glutathione,GSH)。结果1.MDA在SE发作后3h、25h及60d均有显著升高(p<0.05)。2.GSH在SE发作后3h无明显变化(p>0.05),25h及60d组明显下降(p<0.05)。3.总SOD含量在SE发作后3h无明显变化(p>0.05),在25h及60d组则呈明显下降(p<0.05)；SOD1在各实验组间无明显差异(p>0.05)；SOD2在SE发作后3h无明显变化(p>0.05),在25h及60d组则呈明显下降(p<0.05)结论癫痫发作后,癫痫组存在脂质过氧化损伤；内源性自由基清除剂在各实验组呈现下降的趋势。这证实了癫痫发作后海马氧化应激水平的增高,以及自由基生成与清除的平衡被打破。此外,SOD1不变而SOD2下降提示癫痫发作后线粒体可能是自由基攻击的中心。第四部分氯化锂-匹鲁卡品致痫大鼠海马mtDNA修复机制的研究目的评估匹鲁卡品致痫后大鼠海马中的线粒体碱基切除修复通路(mitochondrial base excision repair, mtBER)各个关键酶在基因和蛋白水平表达的改变,从而进一步探讨线粒体基因组损伤的机制。方法成年雄性Wistar大鼠随机分为急性对照组、SE后3h、9h、25h组、慢性对照组、慢性癫痫组(SE后60d)。于相应时间点分别断头取脑,分离海马,RQ-PCR法检测关键酶1mRNA表达；取新鲜海马提取线粒体,Western blot法检测蛋白的表达。结果1. RQ-PCR显示OGG1和polγmRNA在癫痫发作后3h、9h和25h表达明显降低(p<0.05)；而各个时间点APE1 mRNA的表达无明显变化(p>0.05)2. Western blot示急性期线粒体内OGG1和polγ的表达在3h、9h和25h表达明显降低(p<0.05)；线粒体内APE1的表达在3h、9h降低(p<0.05),25h恢复至正常水平(p>0.05)；而急性期各组APE1在海马组织水平的表达未见明显差异(p>0.05)。3.慢性癫痫组polγmRNA表达较对照组增高(p<0.05),APE1的mRNA未见明显改变(p>0.05)。4.慢性癫痫组线粒体内polγ蛋白的表达较对照组增加(p<0.05), APE1的表达则明显下降(p<0.05),但APE1在海马组织水平的表达与对照组无统计学差异(p>0.05)。5.与polγ的改变相似,慢性癫痫组Tfam的mRNA和蛋白水平均显著增高(p<0.05)。结论在SE急性期线粒体内mtBER通路的关键酶整体下调,而慢性期mtBER通路中剪切酶下降、合成酶升高。mtBER通路的异常可能进一步加剧线粒体基因组的损伤。与mtDNA复制、转录相关的Tfam和polγ在慢性期呈现反馈性增高,推测可能对维持一定的mtDNA水平起重要作用。更多还原

【Abstract】 Epilepsy is one of the most common neurological disorders affecting about 9 million patients in china. About 25-40% of patients suffer with intractable epilepsy. Temporal lobe epilepsy (TLE) presents the most prevalent refractory epilepsy and its pathogenesy still remains obscure. It is difficult to gain significant curative effects through administration of antiepileptic drugs (AEDs) and operating. The alteration of behavior, electroencephalogram (EEG) and the hippocampal neuronal injury in pilocarpine-induced seizures in rats is similar to that in TLE patients, so pilocarpine-induced seizures have been one of the most frequently used models to research SE and TLE.Mitochondrial dysfunction has been implicated as one of the main mechanisms underlying neuronal injury. Our previous study revealed that seizure-induced mitochondrial DNA (mtDNA) damage might be one of the factors contributing to mitochondrial dysfunction. Compared with nuclear DNA (nDNA), mtDNA is more vulnerable. It has been proved that recurrent seizures can result in the accumulation of free radicals in mitochondrion. It is plausible that elevated oxidative stress may be the cause of mtDNA damage and mitochondrial dysfunction.mtDNA is close to the source of reactive oxygen species (ROS) and sensitive to oxidative damage, but there are endogenous antioxidant systems to protect mtDNA. Free radical scavengers, the primary antioxidant system, have been confirmed to be a protective pathway against seizure-induced oxidative damage. Once DNA damage has been generated, it is the role of repair systems to prevent its accumulation. But little is known about contribution of mtDNA repair pathway. Compared with various nuclear DNA repair pathways, only the base excision repair (BER) pathway has been shown to function in mitochondria and thus is critical in the defense of mitochondrial oxidative stress.In this study, we detected the response of antioxidant defense system and mitochondrial base excision repair (mtBER) pathway to verify the underlying mechanisms of mtDNA impairment and extensively study the molecular biological mechanisms of TLE.PARTⅠA study of behavior, electroencephalogram and pathobiology in lithium-pilocarpine induced seizures in ratsObjectiveTo investigate the alteration of behavior, electrophysiology and pathology in lithium-pilocarpine-induced seizures in rats, and to explore the neuronal injury of hippocampus.MethodsAdult male Wistar rats were given lithium-pilocarpine intraperitoneally to induce SE. The change of behavior in rats was observed. Seizures were allowed to last for 60 min and then were terminated by administration of diazepam. Rats were monitored by video recordings to assure development of seizures, record EEG at 3h, study the pathological changes with Nissl at 72h and 2 months after SE induced by pilocarpine.Results 1.83.1% of the rats were induced to develop SE after administration of lithium and pilocarpine (according to Racine, the rats showing stageⅣ-Ⅴconvulsive seizures were considered to develop SE successfully). The time from pilocarpine injection to the first onset of stageⅣSE was 38.5±18.7min, and the death rate within 72h was 23.2%. The latency phase is about 12.7±6.2 days.74% showed seizures at least once a week.2. EEG in cortex and hippocampus of rats showed accumulated spike waves at 3 h after SE.3. Nissl staining showed the neuronal damage in hippocampal CA1 and CA3 regions at 72 h and 2 months after pilocarpine-induced seizures. The surviving neurons showed round and palely stained nuclei, meanwhile, the dead neurons in hippocampus showed pyknotic nuclei and shrunken plasma body.ConclusionsLithium-pilocarpine could induce acute seizures (SE) according to the alteration of behavior and EEG. Seizures induced by lithium-pilocarpine caused hippocampal neuronal damage.PARTⅡAlteration of mitochondrial ultrastructure and mtDNA damage in hippocampi of temporal lobe epilepsy modelObjectiveTo detect the alteration of the mtDNA copy number and the level of mtDNA impairment of hippocampi in rats with seizure, and to explore the molecular mechanisms of mitochondrial dysfunction in epilepsy.MethodsAdult male Wistar rats were divided randomly into acute control,25h, chronic control,60d. Mitochondrial ultrastruture damage was evaluated by electron microscope. DNA was isolated from fresh hippocampi. The ratio of mtDNA to nDNA was determined by quantitative real-time PCR to evaluate the mtDNA number. We designed three pairs of primers specific to deferent regions of mtDNA in case of the interference of mtDNA depletion. Fpg was used to specifically remove oxidized bases and mtDNA damage can be determined from the ratio of intact PCR products in cleaved versus uncleaved DNA using quantitative PCR.Results1. Mitochondrial ultrastructure was damaged and it varied from mild to profoundly severe in the hippocampus during experimental epilepsy. Mild damage was characterized by early swelling as manifested by separation of cristae. In the more severe cases, mitochondrial swelling was accompanied by clearing of matrix density and disruption of membrane integrity. The most severe damage of mitochondria was vacuolar plus rupture of inner and outer mitochondrial membranes.2. Compared with acute control group,25h group showed constant mtDNA copy number(p>0.05), and the frequencies of DNA damage in different regions we chose showed no difference (P>0.05).3. After incubation with fpg, still no difference were found between control and 25h group(p>0.05), and no statistical differences were detected in the quantification of the three mtDNA regions(p>0.05).4. The ratio of mtDNA to nDNA decreased about 1/4 in group of 60d compared with control (P<0.05). No statistical differences were detected in the quantification of the three mtDNA regions(p>0.05).5. After incubation with fpg, the percentage of intact mtDNA decreased significantly in group of 60d (p<0.05). The frequencies of DNA damage in different regions we chose showed no difference (P>0.05).ConclusionsReal-time PCR revealed a decreased copy number of mtDNA accompanied with increased amount of oxidized bases in hippocampi of rats with recurrent seizures. The results suggested that recurrent seizures lead to severe mtDNA damage in hippocampi, including oxidative alteration and strand breaks.PARTⅢThe involvement of antioxidant defense system in hippocampi of rats with seizuresObjectiveTo detect the lipid peroxidation and the response of endogenous antioxidants, and to evaluate the redox state in hippocampi of rats with seizures.MethodsAdult male Wistar rats were randomly divided into 5 groups for treatment:control, 3h, and 25h after the onset of SE, chronic control, and chronic seizures (60d). The contents of MDA, SOD1, SOD2 and GSH were measured as the methods described by the commercial assay kits.Results1. MDA was dramatically increased at 3h,25h, and 60d after the onset of SE (p<0.05).2. Compared with control the content of GSH showed no difference at 3h, and decreased at 25h after the onset of SE(p<0.05). Recurrent seizures resulted in a 1/3 decrease in GSH (p<0.05).3. Total SOD and SOD1 activity were constant at 3h and reduced at 25h and 60d. No significant differences were observed in SOD1 activity (p>0.05).ConclusionsSeizures induced lipid peroxidation in hippocampi; the endogenous antioxidants appeared to decrease in experimental groups. The results revealed elevated oxidative stress in hippocampi after seizures, and the balance between free radical producing and scavenging was broken. Besides, the reduction of SOD2 but not SOD1 demonstrated a higher risk of oxidative damage in mitochondria..PARTⅣThe involvement of mitochondrial base excision repair pathway in hippocampi of rats with seizuresObjectiveTo assess the response of enzymes of mitochondrial base excision repair (mtBER) pathway at both mRNA and protein levels, so as to explore the underlying mechanisms of mtDNA damage in epilepsy.MethodsAdult male Wistar rats were randomly divided into 6 groups for treatment:control, 3,9, and 25h after the onset of SE, chronic control, and chronic seizures (60d). Q-PCR and western blot were used to determine the level of mtBER pathway enzymes.Results1. Real-time PCR analysis demonstrated lower expression of both OGG1 and poly in experimental group at 3,9, and 25h after the onset of SE (p<0.05), with no significant difference between experimental and control rats in APE1 level (p>0.05).2. The protein levels of poly and OGG1 were significantly reduced at 3,9, and 25 h after the onset of SE (p<0.05). However, APE1 decreased at 3 and 9h (p<0.05) and then returned to the control level at 25h (p>0.05). The protein level of APE1 in the hippocampi was constant in these group (p>0.05).3. The protein level of APE1 was significantly reduced in rats with epilepsy (p<0.05). Meanwhile DNA polymeraseγ(polγ) was increased compared with the control group (p<0.05).4. The mRNA levels of poly were also raised (p<0.05), which were compatible with the results of western blot. Interestingly APE1 showed no significant changes in mRNA level and hippocampal protein level (p>0,05;t-test).5. Along with the increase of polγ, Tfam was elevated in mRNA and protein level (p<0.05; t-test).ConclusionsmtBER enzymes failed to respond to SE induced by pilocarpine in rat hippocampi, and the mtBER pathway showed unbalanced expression, which may influence the repair of oxidative damage in mtDNA generated by seizures. mtDNA replication might serve as a potential compensatory mechanism for mtDNA damage.更多还原