【作者】 甄军丽；

【导师】 王维平；

【作者基本信息】 河北医科大学 ， 神经病学， 2014， 博士

【摘要】 癫痫是一种常见的神经系统综合征。国内流行病学调查结果显示，我国目前约有900多万癫痫患者，其中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，主要特点是逐渐进展的原发性癫痫反复发作（spontaneous recurrent epileptic seizure，SRS）和海马硬化，伴有明显的药物抵抗性。临床调查表明，超过一半的癫痫患者具有不同程度的认知功能障碍，有的因癫痫发作频繁不能坚持服药，有的因长期服用抗癫痫药物（药物本身可致不同程度的认知功能障碍）所致，这无疑给个人、家庭及社会带来了沉重的负担。匹鲁卡品（pilocarpine，PILO）致痫大鼠的行为学、海马神经元损伤的病理形态学及神经电生理学等改变均与人类的TLE相似，因此该模型是目前研究癫痫持续状态（status epilepticus，SE）和TLE的经典模型之一。SE的致病机制较为复杂，目前尚未完全阐明，主要包括：苔藓纤维芽生假说；Ca2+内流引发的细胞毒性；谷氨酸和γ-氨基丁酸及其受体结构和功能异常；氧化应激损伤及凋亡等。氧化应激是指体内活性氧簇（reactive oxygen species，ROS）产生增多或体内的抗氧化系统出现障碍，体内氧自由基代谢就会失衡，蓄积过多的ROS易导致组织受损。ROS通过直接或间接氧化损伤DNA、诱发基因突变，导致蛋白质和脂质过氧化，激活非选择性钙通道，调节细胞凋亡相关基因，诱导海马神经元发生凋亡。另外，SE时常伴严重的缺氧，易致ROS的增加，而ROS也可直接或间接堆积于线粒体，损伤线粒体膜，降低膜电位，造成线粒体的结构和功能异常，使线粒体内的细胞色素C（Cytochrome c）释放入细胞质，引发caspase级联效应，诱导细胞凋亡。因此，对如何减少氧化应激ROS的产生、保护线粒体并抑制细胞凋亡具有重要的研究意义，探索难治性癫痫的替代治疗或辅助治疗措施便成为当务之急。目前已发现多种预处理方法可以诱导组织细胞对缺氧产生耐受，包括缺血、高压氧、高温、低温、睡眠剥夺等，这些方法由于本身存在明显的副作用，在临床实际应用方面受到限制。众所周知，低氧容易改变机体的生理功能，严重的低氧往往引起的是一种损伤的病理改变，而最近研究发现适度的低氧预处理对机体则起到一定的保护效应。研究已证实，低氧预处理可通过抑制肿瘤坏死因子α、上调促红细胞生成素、血管内皮生长因子及Bcl-2抗凋亡蛋白等在海马CA1和CA3区的表达，改善脑缺血缺氧后脑组织的损伤及其导致的认知功能障碍。越来越多的研究报道，慢性间歇性低压低氧（chronic intermittent hypobarichypoxia，CIHH）对机体多种组织、器官均有明显的保护作用，例如增强机体对缺血缺氧的耐受性，对抗缺血/再灌注所致的心脏功能损伤、抑制心肌超微结构受损及钙离子超载、保持钙稳态，并能够有效抗心律失常，保护神经、肝脏、肾脏等组织器官；而且CIHH还具有降压、抗氧化应激、抗凋亡、增强免疫功能等作用。在缺血性脑血管病方面的研究中，CIHH通过上调大鼠脑内的谷氨酸转运体-1（glutamate transporter-1，GLT-1）诱导其产生缺氧耐受性。而CIHH预处理是否对癫痫也具有一定的脑保护作用？目前尚未见报道。有鉴于此，本实验通过制备氯化锂-匹鲁卡品诱导的SE大鼠模型，观察CIHH对致痫大鼠的行为学、脑组织形态学及分子生物学等方面的影响，评价CIHH预处理的保护作用并进一步探讨其可能的作用机制，为此主要致力于以下四个部分的研究：第一部分CIHH预处理对匹鲁卡品致痫大鼠的脑保护效应目的：建立匹鲁卡品（pilocarpine，PILO）诱导的SE大鼠模型，研究CIHH预处理对PILO致痫大鼠的脑损伤是否具有保护作用，分别从行为学、形态学及分子生物学等方面进行评价，并初步探讨其作用机制。方法：（实验一）将成年健康雄性Sprague-Dawley（SD）大鼠随机分为6组：正常对照组（CON组）和PILO模型组（SE组），根据SE发作后不同的时间点分为SE3h、SE12h、SE24h、SE72h和SE7d五个亚组，每组各10只。应用Nissl染色法和流式细胞仪动态观察SE后不同时间点大鼠的海马神经元凋亡情况，检测该模型制备是否成功。（实验二）根据目前文献报道：SE后脑组织损伤程度最严重的时间多为发作后72h，我们选取SE72h进行研究，将动物随机分为4组：CON组（n=7），SE组（n=13），CIHH+SE组（n=13），CIHH组（n=7）。利用脑电图观察大鼠的痫性发作潜伏期和癫痫波发放的频率，并记录死亡率；对脑组织一部分行Nissl染色计数海马组织中存活的神经元；另一部分应用流式细胞仪定量分析海马神经元的凋亡情况。结果：（实验一）150只大鼠中，41只被诱发SE，成功率为82％，实验过程中共死亡9只（包括诱发SE成功后的7只），最终可纳入实验的大鼠共34只。CON组无大鼠死亡，10只均可纳入实验。2Nissl染色：CON组大鼠海马CA1区和CA3区神经元结构完整，无神经元丢失；SE3h组和SE12h组大鼠海马CA1区和CA3区结构相对完整，未见到明显的神经元丢失；SE24h组可见大鼠海马CA1区和CA3区神经元部分丢失；SE72h组神经元丢失明显，且CA1区更严重。而SE7d组海马CA1区神经元的丢失较SE72h组有所减少。与CON组相比，SE3h组、SE12h组大鼠海马CA1区和CA3区神经元数量几乎无减少（P>0.05）；SE24h组、SE72h组和SE7d组大鼠海马CA1区和CA3区的神经元数量均明显减少（P<0.05）。与SE72h组相比，其它组大鼠海马CA1区和CA3区神经元数量均明显增加（P<0.05）。3细胞凋亡率：与CON组相比，SE3h组、SE12h组的海马细胞凋亡率均无明显增加（P>0.05），而SE24h组、SE72h组和SE7d的细胞凋亡率有不同程度的增加（P<0.05）。与SE72h组相比，SE24h组和SE7d组海马细胞的凋亡率均有所下降（P<0.05），但后两者间无明显的统计学差异（P>0.05）。与Nissl染色及以往的文献报道结果一致，证明本实验模型制备是成功的、可行的。(实验二)1SE组、CIHH+SE组大鼠的死亡率分别为23.1%和0%。与SE组相比，CIHH+SE组的潜伏期延长（33.9±2.3min vs.24.5±1.7min，P<0.05）；癫痫波发放的频率更少（100.5±2.9HZ vs.118.3±4.0HZ，P<0.05）。CON组和CIHH预处理组中大鼠均无SE发作及死亡。2Nissl染色：CON组大鼠的海马CA1区和CA3区神经元结构完整，几乎无神经元丢失。与CON组相比，SE组大鼠神经元丢失明显；CIHH+SE组神经元结构部分完整，尼氏小体数量较SE组显著增加。CIHH组大鼠的海马区神经元结构相对完整，偶见神经元丢失。海马神经元存活数：与CON组相比，SE组大鼠海马CA1区和CA3区神经元数量均明显减少（P<0.05）；经CIHH预处理后明显增加了神经元的存活数量（P<0.05）。单纯行CIHH预处理组存活的神经元数量虽然少于CON组，但两者间无明显的统计学差异（P>0.05）。3细胞凋亡率：与CON组相比，SE组和CIHH+SE组的海马细胞凋亡率均明显增加（P<0.05）；单纯行CIHH组细胞凋亡率虽然稍高于CON组，但两者无明显的统计学差异（P>0.05）。与SE组相比，CIHH+SE组的细胞凋亡率明显减低（P<0.05）。结论：PILO诱导的SE大鼠模型能较好的动态观察脑损伤后神经元凋亡情况。SE发作后72h时，海马组织CA1区损伤最为严重；CIHH预处理可能通过延长SE大鼠的脑电图痫性放电潜伏期、降低痫性波发放频率来抑制癫痫发作，降低海马神经元的凋亡率。第二部分不同的低氧预处理对匹鲁卡品致痫大鼠的海马损伤作用目的：已有研究表明，慢性间歇性常压低氧（chronic intermittentnormobaric hypoxia，CINH）对大鼠的脑组织损伤发挥着一定的保护作用。本研究分别从PILO诱导的SE大鼠的SRS、胞浆内钙离子浓度及细胞凋亡率等方面比较两种不同的预处理方式（CIHH和CINH）对大鼠海马损伤的保护作用。方法：将成年雄性SD大鼠随机分为4组：正常对照组（CON组，n=18），PILO模型组（SE组，n=34），CIHH预处理+SE组（CIHH-Pre组，n=24）和CINH预处理+SE组（CINH-Pre组，n=24）。将每组大鼠分为两部分：一部分应用视频脑电图(V-EEG)观察大鼠SE后自发性癫痫反复发作（SRS）情况（包括SRS的频率、发作级别及持续时间）；另一部分于SE72h时分别应用Nissl和FJB染色法观察大鼠海马的神经元凋亡和变性情况；流式细胞仪检测海马胞浆内钙离子浓度（[Ca2+]i）和细胞凋亡率。结果：1PILO诱导的SE大鼠死亡率为47.1%，而CIHH-Pre组和CINH-Pre组SE大鼠的死亡率分别为16.7%和20.8%。V-EEG记录SRS的情况：CIHH-Pre组和CINH-Pre组出现SRS的频率、严重程度和持续时间均较SE组明显降低（P<0.05）。与CINH-Pre组相比，CIHH-Pre组出现SRS的频率更少（P<0.05），发作级别更低（P<0.05）。CON组大鼠无SRS发作和死亡。2与CON组相比，SE组海马胞浆内[Ca2+]i明显升高（2.16±0.06vs.1.19±0.03，P<0.05）。与SE组相比，CIHH-Pre组大鼠海马胞浆内[Ca2+]i（1.51±0.03）和CINH-Pre组（1.74±0.03）均明显的降低（P<0.05）；且CIHH-Pre组海马胞浆内[Ca2+]i低于CINH-Pre组（P<0.05）。3与CON组相比，SE组大鼠海马的神经元丢失明显；而CIHH-Pre组和CINH-Pre组神经元丢失的程度较SE组减轻。SE组大鼠海马CA1区和CA3区的存活神经元数量均较CON组明显减少（P<0.05）。与SE组相比，CIHH-Pre组和CINH-Pre组均明显地增加了大鼠海马CA1区和CA3区的神经元数量（P<0.05）；而CINH-Pre组CA1区的存活神经元数量仍低于CIHH-Pre组（55.00±1.07vs.74.83±1.40，P<0.05）。4CON组未见明显的FJB阳性细胞。与CON组相比，PILO诱导的SE组海马CA1区和CA3区可见呈黄绿色荧光的FJB阳性细胞明显增多。CIHH-Pre组和CINH-Pre组大鼠海马CA1区和CA3区均明显减少了FJB阳性细胞（P<0.05）,且CIHH-Pre组CA1区的FJB阳性细胞数少于CINH-Pre组（P<0.05）。5PILO诱导的SE组大鼠海马细胞早期凋亡率（Q4）和总的凋亡率（Q2+Q4）均明显高于CON组（P<0.05）。与SE组相比，CIHH-Pre组和CINH-Pre组均明显地降低了海马细胞的凋亡率（P<0.05），且CIHH-Pre组的凋亡率低于CINH-Pre组（P<0.05）。结论：PILO诱导的SE大鼠容易出现SRS行为，使海马组织细胞内[Ca2+]i升高，导致神经元损伤。CIHH和CINH预处理均可通过有效地抑制SE大鼠的SRS行为和胞浆内钙离子超载来营救损伤的海马神经元，且CIHH预处理措施可能更有效。第三部分CIHH对匹鲁卡品致痫大鼠的海马抗氧化防御体系的影响目的：研究CIHH预处理是否对癫痫发作产生的氧化应激及线粒体损伤具有一定的保护作用，并探讨可能的作用机制。方法：将成年雄性SD大鼠随机分为3组：正常对照组（CON组），PILO模型组（SE组）和CIHH预处理+SE组（CIHH+SE组），每组各20只。于SE发作后72h时，首先制作大鼠海马组织的匀浆液，根据试剂盒说明分别检测MDA含量、总SOD、SOD1、SOD2及GSH的活力；然后应用流式细胞仪检测海马组织细胞内ROS、线粒体膜电位（mitochondrial transmembranepotential，△Ψm）的水平；Western Blot检测胞浆中Cytochrome c、Caspase3、Bcl-2、Bax蛋白的表达；并结合透射电镜观察线粒体的超微结构。结果：1与CON组相比，SE组大鼠海马内MDA的水平明显升高（P<0.05）；总的SOD和SOD2的活力均明显降低（P<0.05），GSH的含量也明显降低（P<0.05），而CIHH+SE组可不同程度地逆转上述指标的异常改变（P<0.05）。SODl在各实验组间则无明显差异（P>0.05）。2与SE组相比， CIHH+SE组能明显降低海马细胞内ROS的水平（P<0.05），明显升高△Ψ的水平（P<0.05）。3与CON组相比，SE组大鼠海马组织胞浆内Cytochrome c，Caspase3，Bax，及Bax/Bcl-2蛋白的水平均明显升高（P<0.05），而Bcl-2蛋白水平显著降低（P<0.05）。CIHH预处理后均可不同程度地逆转上述蛋白的表达水平（P<0.05）。4与CON组相比，SE组中线粒体的超微结构发生明显改变，表现为广泛的空泡化、严重的嵴断裂伴膜破坏；而CIHH+SE组可部分改善线粒体的结构变化。结论：SE发作后线粒体可能是自由基攻击的靶点。CIHH预处理可通过增强大鼠海马组织的抗氧化应激能力，改善线粒体的结构和功能来发挥神经元的保护作用。第四部分CIHH对匹鲁卡品致痫大鼠认知功能的改善作用和机制探讨目的：研究CIHH预处理对SE大鼠的认知功能障碍、海马组织中突触的超微结构和突触相关蛋白的表达有何影响，进一步探讨相关的作用机制。方法：将成年雄性SD大鼠随机分为3组：正常对照组（CON组，n=10）和PILO模型组（SE组，n=20）和CIHH预处理+SE组（CIHH+SE组，n=20）。根据文献报道及我们前期的实验结果表明：SE发作后多于14-42d出现SRS行为，由此我们选取SE发作后42d时，进行Morris水迷宫行认知功能检测；结束后分别应用透射电镜观察海马CA1区突触的超微结构；WesternBlot法检测胞浆中PSD-95和Kalirin-7蛋白的表达。结果：由于大鼠经SE发作后部分出现死亡，本部分实验中SE发作后42d时，各组大鼠的存活情况：CON组10只，PILO诱导的SE组9只，CIHH+SE组14只。1水迷宫检测：随着训练时间的延长，从第3d开始，各组大鼠寻找平台的潜伏期均逐渐缩短（P<0.05）。CIHH预处理组较SE组的潜伏期明显缩短（P<0.05）。在空间探索试验中，与CON组相比，SE组大鼠的穿台次数明显减低（5.33±0.58vs.11.8±0.76，P<0.05），在目标象限停留时间也明显缩短（52.78±2.00s vs.86.80±1.67s，P<0.05）；而CIHH+SE组大鼠的穿台次数（7.86±0.44）较SE组有所增加（P<0.05），在目标象限停留时间（67.36±2.06s）也明显延长（P<0.05）。在可视平台试验中，各组间大鼠的逃避潜伏期和游泳速度均无明显的统计学差异（P>0.05）。2突触间隙宽度:与CON组相比（20.27±0.45nm），SE组和CIHH+SE组大鼠海马的突触间隙均显著增宽（P<0.05）。虽然CIHH+SE组的突触间隙较SE缩窄，但两者无明显的统计学差异（23.13±0.89nm vs.25.20±0.78nm，P>0.05）。突触后致密物厚度：与CON组相比（30.27±0.69nm），SE组和CIHH+SE组大鼠海马的突触后致密物厚度均显著降低（P<0.05）；而CIHH+SE组较SE组明显增加（27.87±0.77nm vs.22.67±0.76nm，P<0.05）。3PSD-95蛋白：与CON组相比（0.72±0.02），SE组和CIHH+SE组中大鼠海马的胞浆内PSD-95蛋白的表达水平均显著降低（P<0.05）；而CIHH+SE组中表达的水平明显高于SE组（0.40±0.02vs.0.21±0.02，P<0.05）。Kalirin-7蛋白：与CON组相比(0.68±0.02)，SE组和CIHH+SE组中大鼠海马胞浆内Kalirin-7蛋白水平均显著降低（P<0.05）；而CIHH+SE组的表达水平高于SE组（0.34±0.02vs.0.26±0.02，P<0.05）。结论： PILO诱导的SE大鼠长期反复发作可出现明显的认知功能障碍和海马突触结构的损伤。CIHH预处理可有效改善受损的突触超微结构并上调突触相关蛋白的表达，这可能是CIHH改善癫痫发作后认知功能障碍的机制之一。更多还原

【Abstract】 Epilepsy is one of the most common neurological disorders, afflictingmore than9million individuals in China according to domesticepidemiological investigations. Among patients with epilepsy, about25%-40%suffers from intractable epilepsy resistant to currently available antiepilepticdrugs (AEDs). Many such cases are of temporal lobe epilepsy (TLE),characterized by spontaneous recurrent seizures (SRS) and progressivehippocampal sclerosis. Clinical investigations have shown that over half ofepilepsy patients suffer from cognitive impairments due to neurologicaldamage associated with frequent seizures and (or) the side-effects oflong-term AED use. This cognitive dysfunction imposes an even heavierburden on epileptic individuals, their families, and society.The rat pilocarpine (PILO)-induced seizure model exhibits many of theclinical, behavioral, and neuropathological hallmarks of TLE and statusepilepticus (SE), and so is advantageous for preclinical testing of noveltreatment strategies.The pathogenesis of TLE is complex and may involve a combination ofrecurrent mossy fiber sprouting, excitotoxicity induced by neuronal calciuminflux, abnormal glutamate and γ-aminobutyric acid (GABA) receptorstructure and function, and oxidative stress with ensuing apoptosis. Oxidativestress occurs when production of reactive oxygen species (ROS) exceeds thecapacity of endogenous cellular antioxidant defense systems, resulting inmitochondrial dysfunction and destruction of macromolecules such asmembrane lipids. Status epilepticus is often associated with severe brainhypoxia. Reperfusion of hypoxic tissue is associated with ROS accumulationin mitochondria, which can damage mitochondrial membranes, decreasing the mitochondrial transmembrane potential, inducing mitochondrial cytochrome crelease into the neuronal cytoplasm, and activating the caspase cascadeleading to apoptosis. Reducing ROS production during SE to protectmitochondria and inhibit neuronal apoptosis is a possible replacement oradjuvant therapy for intractable epilepsy.A variety of pretreatment or preconditioning methods can induce hypoxicand oxidative stress tolerance, including mild transient ischemia, hyperbaricoxygen, hyperthermia, hypothermia, sleep deprivation. However, clinicalapplication of many of these methods is limited by potential side effects.Severe hypoxia often leads to pathological changes, but recent studies havethat shown moderate transient hypoxia is a physiologically importantmechanism for maintaining or enhancing endogenous cytoprotection.Hypoxic preconditioning can inhibit tumor necrosis factor alphaexpression while raising erythropoietin, vascular endothelial growth factor,and anti-apoptotic Bcl-2expression in hippocampal CA1and CA3subfields.In vivo, hypoxic preconditioning can prevent hippocampal damage fromischemia/hypoxia and prevent associated cognitive dysfunction. Accumulatingevidence indicates that chronic intermittent hypobaric hypoxia (CIHH)enhances cardiac tolerance against subsequent ischemia/hypoxia, reducesheart damaged due to ischemia/reperfusion, inhibits ultrastructural damageand calcium overload in cardiac myocytes, maintains myocyte calciumhomeostasis, and prevents arrhythmia. In addition, CIHH has been shown toprotect nerve, liver, kidney, and other organs against subsequent severehypoxic insults. Furthermore, CIHH lowers blood pressure, reduces oxidativestress and apoptosis, and enhances immune function. In a rat model ofischemic cerebrovascular disease, CIHH enhanced ischemic resistance byupregulating brain expression of glutamate transporter-1(GLT-1). However,whether CIHH preconditioning may protect against SE or not, there was noreported. In this study, we examined the effects of CIHH preconditioning onbehavioristics of rat lithium-pilocarpine model of TLE, hippocampal neuronsurvival (morphology), and molecular biology in order to further explore the underling mechanisms.PartⅠ Neuroprotective effect of CIHH preconditioning on pilocarpine-induced seizures in ratsObjective: To establish a pilocarpine (PILO)-induced SE rat model andevaluate the efficacy of CIHH preconditioning for preventing SE-associateddeath of hippocampal neurons and preserving hippocampal function.Methods:(Experiment1) Healthy adult male Sprague-Dawley rats wererandomly divided into six groups of10: a normal control (CON group) and5PILO groups (SE groups) sacrificed3h,12h,24h,72h, and7days post-SE(SE3h, SE12h, SE24h, SE72h and SE7d groups, respectively). Nissl stainingand flow cytometry were used to assess morphological damage and apoptosisof hippocampal neurons post-SE.(Experiment2) According to previousreports,72h post-SE is the peak time of SE-associated brain damage, so thistime point was selected to examine the neuroprotective effects of CIHHpreconditioning. Animals were randomly divided into four groups: CON (n=7),SE (n=13), CIHH+SE (n=13), CIHH (n=7). Rat mortality from0-72h post-SEwas determined and electroencephalograms (EEGs) were recorded to measurethe frequency, severity, and duration of epileptiform discharges. Hippocampaltissues were then isolated to assess morphological damage and neuronalsurvival by Nissl staining and neuronal apoptosis rate by flow cytometery.Results:(Experiment1)1Status epilepticus was induced by PILO in41of50rats (success rate,82%). Seven SE group rats died during the procedure. The remaining34PILO-treated rats were analyzed and compared to the10(surviving) CONgroup rats.2Nissl staining: In the CON group, there was almost no visible neuronalloss in CA1and CA3subfields. Similarly, SE3h and SE12h groups showed noobvious neuronal loss and neuronal morphology resembled that of the CONgroup. Neuronal loss was apparent in the CA1and CA3regions of the SE24hgroup. In the SE72h group, neuron loss appeared even greater, especially in CA1. Neuronal loss in the CA1of the SE7d group was clearly lower than inthe SE72h group. Cell counting revealed that the numbers of survivingneurons in the SE3h and SE12h groups were not significantly different fromthe CON group (P>0.05), while significant reductions were observed in theSE24h, SE72h, and SE7d groups compared to the CON group (P<0.05).Neuronal survival was significantly higher in all other SE groups compared tothe SE72h group (P<0.05).3Flow cytometry: Compared to the CON group, neuronal apoptosis rateswere significantly higher in the SE24h, SE72h, and SE7d groups (P<0.05), butnot in the SE3h and SE12h groups (P>0.05). Apoptosis rates in SE24h andSE7d groups were not significantly different (P>0.05) but were bothsignificantly lower compared than in the SE72h group (P<0.05). Thus, PILOinduced neuronal death in the hippocampus, peaking at72h post-SE.(Experiment2)1Mortality was23.1%in the SE group, while no deaths were observed inthe CIHH+SE group. Compared to the SE group, the latency to reach SE waslonger (P<0.05) and the frequency of EEG-defined epileptic discharges lower(P<0.05) in the CIHH+SE group. There was no mortality or seizures in theCON and CIHH groups.2Nissl staining: Neurons in CA1and CA3subfields of the CON groupwere clearly stained and there was almost no neuronal loss, while neuronal losswas clearly visible in the SE group. Neuronal cell death was partially reversedin the CIHH+SE group and fewer neurons showed morphological signs ofdamage in both CA1and CA3subfields compared to the SE group. There wasvery slight neuronal loss in the CIHH group. Cell counting revealed asignificant decrease in neuronal survival in the SE group compared to the CONgroup (P<0.05), while the number of surviving neurons was significantlyhigher in the CIHH+SE group compared to the SE group (P<0.05). Althoughthe number of surviving hippocampal neurons was lower in the CIHH groupthan the CON group, there was no significant statistical difference (P>0.05).3Flow cytometry: The numbers of apoptotic neurons were significantly higher in the SE and SE+CIHH groups compared to the CON group (P<0.05).However, apoptotic rate was significantly lower in the CIHH+SE groupcompared to the SE group (P<0.05), indicating partial rescue from apoptosisby CIHH preconditioning. The rate of neuronal apoptosis was slightly higher inthe CIHH group compared to the CON group, but the difference was notstatistically significant (P>0.05).Conclusion: The PILO-induced SE rat model allows for dynamicobservation of progressive hippocampal neuronal injury and apoptosis. At72hafter SE, damage was more severe in the CA1. Preconditioning with CIHHprolonged SE latency and reduced seizure frequency, PILO-induced mortality,morphological signs of hippocampal neuronal damage, and neuronalapoptosis.PartⅡ Effects of different hypoxic preconditioning protocols on thehippocampal damage of status epileptic ratsObjective: Chronic intermittent normobaric hypoxia (CINH）has beenproved playing an important role in protecting the damaged brain of rats. Inthis study, we compared the effects of different hypoxic preconditioningprotocols (CIHH and CINH) on spontaneous recurrent seizures (SRS),intracellular free calcium concentration ([Ca2+]i), and apoptosis rate followingPILO-induced status epilepticus (SE).Methods: Adult male Sprague-Dawley rats were randomly divided intofour groups: normal control group (CON group, n=18), PILO group (SE group,n=34), CIHH+SE group (CIHH-Pre, n=24) and CINH+SE group (CINH-Pre,n=24). The rats in each group were divided into two parts: a part of that wascompared by video monitoring of behavioral seizure activity (frequency, rank,and duration); another part of that were used to examine changes in themorphology of hippocampal pyramidal neurons by menas of Nissl stainingand Fluoro-Jade B (FJB) staining, and further to detect the quantification of[Ca2+]i and cell apoptosis by means of flow cytometry at SE72hour.Results:1Mortality and SRS of rats induced by PILO: In the SE group, mortality rate was47.1%, while in the CIHH-Pre group and CINH-Pre group, mortalityrate was16.7%and20.8%, respectively.SRS by the V-EEG: There was a significant decrease in the number,mean severity, and duration of seizures in both CIHH-treated andCINH-treated SE rats compared to those treated by PILO (P<0.05). However,the CIHH-Pre protocol induced a greater reduction in both seizure frequencyand severity compared to the CINH-Pre group (P<0.05). There was no SRSseizures and animal died in the CON group.2Comparation of the [Ca2+]i: A substantial increase was measured in theSE group compared to the CON group (P<0.05), while both CIHH-Pre andCINH-Pre significantly reduced the post-SE [Ca2+]i elevation compared to theSE group (P<0.05). Hippocampal [Ca2+]i was lower in the CIHH-Pre groupthan that of the CINH-Pre group (P<0.05).3Nissl staining: There was a marked loss of neurons in CA1and CA3subfields of SE group compared to the CON group. Compared to the SE group,there was a significantly reduced neuronal loss in the CIHH-Pre andCINH-Pre groups.The number of surviving neurons in both subfields of the hippocampus inSE group obviously decrease compared to the CON group (P<0.05).Compared to the SE group, the number of neurons was significantly increasein the CIHH-Pre and CINH-Pre groups (P<0.05), while the average CA1pyramidal neuron density was lower in the CINH-Pre group than in theCIHH-Pre group (P<0.05).4FJB staining: No FJB-positive cell was observed in the CON group.Many FJB-positive cells with kelly fluorescent were observed in the CA1andCA3subfields in the SE group. CIHH-Pre and CINH-Pre groups showed asignificant reduction in the number of FJB-positive cells in the CA1and CA3subfiels (P<0.05). The average numbers of FJB-positive cells in the CA1subfield was lower in the CIHH-Pre group than in the CINH-Pre group(P<0.05).5Apoptosis rate of the hippocampus: Both the number of cells in early apoptosis (Q4) and total apoptotic cells (Q2+Q4) were significantly higher inthe SE group than in the CON group (P<0.05). Both the CIHH-Pre group andthe CINH-Pre group exhibited markedly reduced apoptosis rate compared tothe SE group (P<0.05). Moreover, apoptosis rate was lower in the CIHH-Pregroup than in the CINH-Pre group (P<0.05).Conclusion: SE rats induced by PILO prone to SRS leads to neuronaldamage of hippocampus by increasing the [Ca2+]i. CIHH-Pre and CINH-Precan effectively inhibit the SRS behavior and reduce the post-SE [Ca2+]i so asto rescue the hippocampal neurons. What’s more, hypobaric preconditioningmay be more effective.Part Ⅲ Effect of CIHH on the antioxidant defense system of thehippocampus in epileptic ratsObjective: To examine whether CIHH preconditioning protects againstoxidative stress and mitochondrial damage induced by SE and to explore theunderlying mechanisms.Methods: Animals were randomly divided into three groups: CON(n=20), SE (n=20), and CIHH+SE (n=20). Following these treatments,homogenates were prepared from hippocampus to measure MDAaccumulation, an index of oxidative stress, as well as the activities of theantioxidants superoxide dismutase (SOD) isoforms and glutathione (GSH)using commercial assay kits72hour post-SE. Second, levels of ROS and themitochondrial transmembrane potential (△Ψm) in the hippocampus weremeasured by flow cytometry. Third, Cytochrome c, activated Caspase3, Bcl-2,and Bax proteins in cytoplasm were measured by Western Blot. Theultrastructure of mitochondria under the three experimental conditions wasexamined by transmission electron microscopy.Results:1Compared to the CON group, the level of MDA was significantlyhigher (P<0.05), total SOD and SOD2activities lower (P<0.05), and GSHconcentration lower (P<0.05) in the SE group. These “pro-oxidant” changes inthe SE group were partially reversed in the CIHH+SE group (P<0.05). There were no significant group differences in SOD1activity (P>0.05).2Compared to the SE group, ROS was significantly lower (P<0.05) and△Ψm significantly greater (P<0.05) in the hippocampus of the CIHH+SEgroup.3Compared to the CON group, the protein expression levels ofCytochrome c, Caspase3, and Bax, as well as the Bax/Bcl-2ratio weresignificantly higher in the SE group (P<0.05), while expression of Bcl-2wassignificantly lower (P<0.05). These pro-apoptotic changes were partiallyreversed by CIHH preconditioning prior to SE (CIHH+SE group)(P<0.05).4Compared to the CON group, mitochondria from the SE groupexhibited obvious morphological abnormalities, such as vacuolization, ridgefracture, and membrane damage, effects partially reversed by CIHHpreconditioning.Conclusion: Mitochondria were damaged by SE, possibly due to freeradical generation and oxidative stress. Preconditioning by CIHH inhibitedoxidative stress and maintained mitochondrial structure and function, possiblyaccounting for the neuroprotection and inhibition of apoptosis observed in theCIHH+SE group compared to the SE group.Part Ⅳ Effect of CIHH on cognitive impairment in SE ratsObjective: To examine if CIHH can reduce cognitive impairments inSE-treated rats with spontaneous recurrent seizures (SRSs) and to determinethe underlying mechanisms.Methods: Animals were randomly divided into three groups: CON(n=10), SE (n=20), and CIHH+SE (n=20). According to results of ourpreliminary study, SRSs occur1442days after SE. Thus, the Morris watermaze (MWM) test of hippocampus-dependent spatial reference memory wasperformed at day42post-SE. After the MWM test, synaptic ultrastructure inthe hippocampal CA1was examined by electron microscopy and theexpression levels of synaptic proteins PSD-95and Kalirin-7were detected byWestern Blot.Results: As some rats in the SE group died, the numbers of rats examined by the MWM on day42post-SE were as follows: CON group(n=10), PILO-induced SE group (n=9), CIHH+SE group (n=14).1In the Morris water maze, all animals exhibited a progressive decline inescape latency starting on the third day of training (P<0.05). Rats in theCIHH+SE group exhibited a significantly shorter escape latency than the SEgroup (P<0.05). On the probe trial, the number of platform crossing wasmarkedly lower (P<0.05) in the SE group and these rats spent significantlyless time in the target quadrant compared to the CON and CIHH+SE group(P<0.05). Thus, SE group rats demonstrated both impaired (hippocampus-dependent) spatial learning during training and poor spatial reference memory.These deficits were partially reversed in the CIHH+SE group as indicated bysignificantly shorter escape latencies and a longer mean time in the targetquadrant compared to the SE group (P<0.05). There were no significant groupdifferences in escape latency and swimming speed in the visible platformversion of the MWM test (P>0.05), indicating that motor and sensory effectscannot account for the effects of CIHH preconditioning on cognition.2The width of synaptic cleft: The PILO-treated rats exhibitedsignificantly wider synaptic clefts compared to the CON group (P<0.05)(SE:25.20±0.78nm, CIHH+SE:23.13±0.89nm, CON:20.27±0.45nm). AlthoughCIHH reduced the synaptic cleft width, there was no statistical differencebetween SE and CIHH+SE groups (P>0.05).The thickness of the postsynaptic density (PSD): PILO-treated ratsexhibited a significant decrease in PSD compared to the CON group (P<0.05)(SE:22.67±0.76nm,CIHH+SE:27.87±0.77nm,CON:30.27±0.69nm). However,PSD thickness was significantly greater in the CIHH+SE group compared tothe SE group (P<0.05).3Expression levels of synaptic proteins: Compared to the CON group(mean density,0.72±0.02), expression levels of PSD-95were significantlylower in the SE and CIHH+SE groups (P<0.05), but expression wassignificantly higher in the CIHH+SE group compared to the SE group(0.40±0.02vs.0.21±0.02, P<0.05). Similarly, compared to the CON group (0.68±0.02), protein expression levels of kalirin-7were significantly lower inthe SE and CIHH+SE groups (P<0.05), but higher in the CIHH+SE groupthan SE group (0.34±0.02vs.0.26±0.02, P<0.05).Conclusion: PILO-induced SE rats demonstrated significant cognitiveimpairments and changes in hippocampal synaptic ultrastructure.Preconditioning by CIHH partially ameliorated hippocampus-dependentlearning and memory deficits, changes in synaptic ultrastructure, and impairedexpression levels of PSD-95and kalirin-7in the hippocampus. Rescue ofsynaptic dysfunction by CIHH preconditioning may be one of the underlyingmechanisms for improved cognition function.更多还原