【作者】 王佩；

【导师】 王维平；

【作者基本信息】 河北医科大学 ， 神经病学， 2008， 博士

【摘要】 癫痫是由于脑部兴奋性过高的某些神经元突然异常放电引起的脑功能异常,是一种常见的慢性临床综合征。临床发现30%～40%的癫痫患者除癫痫发作症状外,常伴有记忆力减退、注意力分散等认知功能受损的表现,严重影响了患者的生活质量。癫痫与认知功能损害的关系已被人们广泛重视,并已成为现代癫痫病学研究的热点之一,但癫痫后认知功能损害的机制还不清楚。目前认为,突触是神经元之间结构和功能的接触点,是神经信息传递的关键部位,突触结构和功能的完整对于保证神经元获得信息并顺利进行信息传递、加工和贮存是非常重要的。突触可塑性是指突触在一定条件下通过改变形态而调整功能的能力,在神经系统的发育、成熟及学习记忆等生理过程中起重要作用。突触可塑性变化主要表现在突触囊泡数密度(synaptic vesicles density, SVD)、突触间隙的宽度(the width of synaptic cleft, WSC)及突触后膜致密物(postsynaptic density, PSD)的厚度等方面的变化。突触可塑性和海马的长时程增强(long-term potentiation,LTP)密切相关,LTP反映突触水平的信息存储过程,是学习记忆的分子基础。突触素(synaptophysin, SYN, P38)作为突触囊泡的特异性标志蛋白,其密度和分布可间接反映体内突触的数量和分布情况,与突触重建及认知过程密切相关。突触后致密物95(postsynaptic density 95, PSD-95)串集N-甲基-D-门冬氨酸(N-methy1 -D-aspartate, NMDA)受体,而此受体是诱发LTP的必要条件。LTP的形成是突触前后机制共同作用的结果。P38反映了突触前膜递质的释放,PSD-95反映了突触后膜受体的有效性。钙作为神经信号传递的重要信使,参与了学习记忆的神经机制,神经细胞内钙离子浓度(intracellular concentration of calcium, [Ca2+]i)的微小变化就可以引起一系列生理乃至病理性改变。Ca2+/钙调蛋白依赖性蛋白激酶IIα(calcium/calmodulin-dependent protein kinase IIα, CaMK IIα)是海马内含量最高的一种蛋白,也是PSD的主要成分,其自动磷酸化与LTP的维持(<3小时)密切相关,更长时间的LTP的维持需要一系列基因的表达和新蛋白质的合成,而环磷腺苷反应单元结合蛋白(cAMP response element binding protein, CREB)是细胞内的转录转导因子,调节基因的转录和蛋白质的合成,是短时记忆向长时记忆转化的关键。癫痫作为高频刺激(high frequency stimulation, HFS),可以引起谷氨酸大量释放,钙离子内流,诱导兴奋性氨基酸毒性作用及钙超载,频繁HPS是否引起学习记忆能力下降,钙信号如何沿Ca2+-CaMK II-CREB传递,同时,调节谷氨酸释放的突触前膜囊泡(P38)及固定谷氨酸受体的突触后膜成分PSD-95如何变化,突触超微结构发生了什么改变,所有这些我们并不清楚,而这些可能为研究癫痫后认知功能障碍的机制找到一些新的突破点。尼莫地平(nimodipine, NMD)是选择性作用于脑血管和神经元的双氢吡啶类钙通道阻滞剂,除具有扩张脑血管,增加脑供血等作用外,还可以阻断钙通道,减轻钙超载,保护神经元。近年的研究表明,尼莫地平可以改善多种原因导致的学习记忆障碍,但该药对癫痫后认知功能障碍的实验研究尚未见相关报道。有鉴于此,本文通过对突触可塑性及钙信号转导的研究,探讨癫痫后认知功能障碍的可能发生机制,并观察尼莫地平的脑保护作用。第一部分慢性癫痫大鼠模型的制备、行为学测定及尼莫地平的影响目的:建立戊四氮点燃慢性癫痫大鼠模型,观察其学习记忆能力的变化及尼莫地平的影响。方法:选用健康雄性SD大鼠,经两次Y-迷宫筛选合格后随机分四组。NC(normal control)组大鼠(35只)每日腹腔注射生理盐水3.5 ml/kg,共44 d。PTZ组(40只)每日腹腔注射1% PTZ 35 mg/kg,连续44 d,自给药第31天起,每日PTZ注射前15 min腹腔注射生理盐水3.5 ml/kg,共14 d。NMD1组(40只)及NMD2组(40只)每日腹腔注射1% PTZ 35 mg/kg,连续44 d,自给药第31天起,每日PTZ注射前15 min分别腹腔注射NMD 1.25 mg/kg、2.5 mg/kg ,共14 d。造模成功后描记脑电图(electroencephalogram, EEG)变化,并采用Morris水迷宫和Y-迷宫实验,进行大鼠学习和记忆成绩的测试。结果:大鼠在注射PTZ后第6～10天起开始出现凝视、点头、反复洗脸动作、面部及头部抽搐等,以后发作症状逐日加重,于给药第18～24天出现全身肌阵挛、双前肢抬起,最终发生全身强直-阵挛性发作,达到RacineⅣ～Ⅴ级的点燃标准,以后每天均有Ⅳ～Ⅴ级发作。第30天时PTZ组大鼠35只完全点燃,NMD1组34只完全点燃,NMD2组35只完全点燃。第31～44天,PTZ组大鼠每天仍有Ⅳ～Ⅴ级癫痫发作,NMD1和NMD2组大鼠癫痫发作程度减轻。NC组大鼠自始至终无癫痫发作表现。EEG表现:NC组大鼠EEG以α、β波为主,背景正常,无异常放电现象。PTZ组大鼠EEG表现为在基础波背景下,阵发性出现大量高幅尖波、棘波、棘慢复合波,持续1～2 s。NMD1组和NMD2组大鼠EEG表现为痫性放电受到抑制,在基础波背景下可见散在少量棘波或尖波,波幅降低。Morris水迷宫实验中定位航行实验结果:每日逃避潜伏期取其平均值进行比较。第1天,四组大鼠的逃避潜伏期比较无统计学意义(P>0.05);第2天,与NC组的逃避潜伏期(15.14±4.69)s比较,PTZ组逃避潜伏期(27.36±6.13)s明显延长( P<0.05);NMD1组和NMD2组的逃避潜伏期(21.66±5.21)s及(19.76±5.81)s较PTZ组缩短( P<0.05),但与NC组比较明显延长(P<0.05);NMD1组和NMD2组的逃避潜伏期比较无统计学意义。第3天,PTZ组大鼠的逃避潜伏期(7.71±1.30)s与NC组(4.46±1.11)s比较有显著性差异(P<0.01);NMD1组和NMD2组的逃避潜伏期(5.94±1.19)s及(5.69±1.03)s仍短于PTZ组( P<0.05),但长于NC组( P<0.05);NMD1组和NMD2组的逃避潜伏期比较无统计学意义(P>0.05)。空间探索试验结果:PTZ组大鼠120 s内穿越平台区域的次数(5.73±2.14)明显低于NC组(9.25±2.18)( P<0.01);NMD1、NMD2组大鼠120 s内穿越平台区域的次数(8.58±2.12)及(7.51±2.05)与PTZ组比较明显增多(P<0.01),但与NC组比较无统计学意义(P>0.05);NMD1组和NMD2组大鼠120 s内穿越平台区域的次数二者比较无统计学意义(P>0.05)。在平台象限的游泳时间四组比较无显著性差异(P>0.05)。Y迷宫检测结果:PTZ组大鼠的错误反应次数(error number, EN)(12.75±2.50)与NC组(6.25±3.10)比较明显增多(P<0.05);NMD1组和NMD2组的错误反应次数(8.75±2.22)和(8.00±3.16)与PTZ组比较又明显减少(P<0.05),但NMD1组和NMD2组与NC组比较无显著性差异(P>0.05);NMD1组和NMD2组比较也无显著性差异(P>0.05)。全天总反应时间四组比较无统计学意义(P>0.05)。结论:本实验建立的戊四氮点燃癫痫大鼠模型类似人类慢性癫痫发作,且具备学习和记忆能力缺损,是研究癫痫后认知功能障碍比较理想的动物模型,尼莫地平可以部分改善慢性癫痫大鼠的学习记忆功能缺损。第二部分慢性癫痫大鼠海马突触后致密物95与突触素表达的变化及尼莫地平的影响目的:观察戊四氮点燃慢性癫痫大鼠海马P38及PSD-95表达的变化及尼莫地平的影响,探讨P38及PSD-95在癫痫后认知功能障碍中的作用与意义。方法:每组大鼠各取8只,10%水合氯醛麻醉,4%多聚甲醛灌注固定,取含海马脑段,石蜡包埋,冠状切片,采用SP法行PSD-95及P38免疫组化染色,测定免疫反应产物吸光度。每组大鼠各取8只,迅速分离海马,Trizol提取总RNA,应用反转录-聚合酶链反应(RT-PCR)方法检测定大鼠海马PSD-95 mRNA表达的变化。结果:PSD-95免疫组化图像分析结果显示:与NC组大鼠海马CA1区PSD-95免疫反应产物吸光度(0.7108±0.0584)比较,PTZ组大鼠海马CA1区PSD-95免疫反应产物吸光度(0.6525±0.0648)明显减少(P<0.05);与PTZ组比较,NMD1组海马CA1区PSD-95免疫反应产物吸光度(0.6921±0.0620)和NMD2组海马CA1区PSD-95免疫反应产物吸光度(0.7033±0.0659)均明显增加(P<0.05);NMD1组、NMD2组及NC组PSD-95免疫反应产物吸光度无明显差异(P>0.05)。P38免疫组化图像分析结果显示:与NC组大鼠海马CA1区P38免疫反应产物吸光度(0.6033±0.0444)比较,PTZ组大鼠P38免疫反应产物吸光度(0.4475±0.0505)明显减少(P<0.05);与PTZ组比较,NMD1组和NMD2组P38免疫反应产物吸光度(0.5819±0.0367)和(0.5723±0.0528)均明显增加(P<0.05)。NMD1、NMD2组及NC组P38免疫反应产物吸光度三组比较无明显统计学差异(P>0.05)。各组大鼠海马PSD-95 mRNA的表达水平以PSD-95 mRNA/β-actin mRNA表示。结果显示:与NC组PSD-95 mRNA表达水平(0.607±0.051)比较,PTZ组PSD-95 mRNA表达水平(0.325±0.030)明显降低(P<0.05);与PTZ组比较,NMD1和NMD2组PSD-95 mRNA表达水平(0.512±0.035)及(0.575±0.041)明显增高(P<0.05);NMD1、NMD2组及NC组PSD-95 mRNA表达水平三组比较无明显统计学差异(P>0.05)。结论:PTZ点燃癫痫大鼠海马组织P38、PSD-95及PSD-95 mRNA表达水平均降低,并且与大鼠学习和记忆成绩下降相一致,提示P38及PSD-95可能参与了癫痫后认知功能障碍的发病机制;尼莫地平可以提高癫痫大鼠海马组织P38蛋白、PSD-95蛋白及mRNA表达水平,进而促进癫痫大鼠学习和记忆能力改善。第三部分慢性癫痫大鼠海马神经元和突触超微结构的变化及尼莫地平的影响目的:观察戊四氮点燃慢性癫痫大鼠海马CA1区神经元形态结构和突触超微结构(SVD、PSD、WSC)的变化及尼莫地平的干预作用,探讨突触可塑性与癫痫后认知功能障碍的关系。方法:每组大鼠各取3只,10%水合氯醛麻醉,4 %多聚甲醛(含2.5 %戊二醛)灌注固定,利用透射电子显微镜观察各组大鼠海马CA1区神经元形态结构和突触超微结构(SVD、PSD、WSC)的变化。结果:NC组海马神经元形态完整,结构清晰,细胞器丰富,有髓神经髓鞘完整。PTZ组海马神经元减少,细胞核肿胀,核膜模糊不清,核内异染色质减少分散,胞核疏松、透明,出现核固缩现象。细胞器明显减少,线粒体空泡化或均质化。粗面内质网板层离散,核糖体脱颗粒。神经毡水肿变形,有些轴突、树突扩张、融合。可见髓鞘层裂。NMD1组及NMD2组海马神经元和神经毡水肿减轻,核膜尚完整,核形态基本正常,染色质均匀分布,胞质中细胞器尚丰富,形态基本正常。NC组海马CA1区神经毡内Gray I型突触丰富,突触前、后膜清晰,囊泡密集,PSD较厚,均匀致密。PTZ组突触数量减少,突触前、后膜模糊不清,突触间隙增大,PSD变薄,不均匀,突触囊泡减少。NMD1组和NMD2组突触数量较多,突触前后膜结构较清晰,PSD尚均匀,突触囊泡数量较多。PTZ组大鼠海马CA1区PSD为(31.37±1.94)nm,与NC组(49.86±3.38)nm比较有显著性差异(P<0.05);与PTZ组比较,NMD1组大鼠海马CA1区PSD(45.69±2.70)nm和NMD2组(48.20±2.15)nm明显增高(P<0.05);NMD1、NMD2组及NC组大鼠海马CA1区PSD三组比较无明显统计学差异(P>0.05)。与NC组大鼠海马CA1区WSC(14.62±1.19)nm比较,PTZ组WSC(22.34±1.84)nm显著增大(P<0.05);与PTZ组比较,NMD1组大鼠海马CA1区WSC(16.37±1.72)nm和NMD2组WSC(15.02±1.63)nm明显减小(P<0.05);NMD1组、NMD2组及NC组大鼠海马CA1区WSC三组比较无明显统计学差异(P>0.05)。与NC组大鼠海马CA1区SVD(3362.61±102.08)个/um2比较,PTZ组SVD(1078.36±111.03)个/um2明显减少(P<0.05);与PTZ组比较,NMD1组大鼠海马CA1区SVD(2932.01±142.95)个/um2和NMD2组SVD(3155.50±137.49)个/um2明显增高(P<0.05);NMD1、NMD2组及NC组三组大鼠海马CA1区SVD比较无明显统计学差异(P>0.05)。结论:癫痫大鼠海马CA1区存在神经元与突触超微结构异常,尼莫地平可以改善神经元及突触超微结构的病理学变化,这些与癫痫大鼠学习和记忆能力的变化相平行,说明突触可塑性与癫痫后认知功能障碍之间存在密切关系。第四部分慢性癫痫大鼠海马[Ca2+]i和钙/钙调蛋白依赖性蛋白激酶IIα的变化及尼莫地平的影响目的:观察戊四氮点燃慢性癫痫大鼠海马[Ca2+]i及CaMK IIα, P-CaMK IIα蛋白、CaMK IIαmRNA表达的变化及尼莫地平的干预作用,探讨钙信号转导与癫痫后认知功能障碍的关系。方法:每组大鼠各取6只,10%水合氯醛麻醉,迅速取海马,制备海马组织混悬液,负载Fluo-3/AM,采用流式细胞分析技术测定各组大鼠海马细胞[Ca2+]i变化; RT-PCR方法检测CaMK IIαmRNA表达的变化。每组大鼠各取8只,10%水合氯醛麻醉,迅速取海马,提取海马总蛋白及膜蛋白,Western blot方法检测海马CaMK IIα, P-CaMK IIα蛋白表达的变化。结果:与NC组[Ca2+]i(1.26±0.21)比较,PTZ组海马[Ca2+]i(1.94±0.33)明显升高(P<0.05);与PTZ组[Ca2+]i比较,NMD1组[Ca2+]i(1.35±0.28)和NMD2组[Ca2+]i(1.32±0.26)明显降低(P<0.05);NMD1、NMD2组和NC组海马[Ca2+]i之间无明显差异(P>0.05)。各组大鼠海马CaMK IIα蛋白表达水平以CaMK IIα/β-actin表示。结果显示:与NC组CaMK IIα表达水平(1.030±0.133)比较,PTZ组CaMK IIα表达水平(0.700±0.061)显著降低(P<0.05);与PTZ组比较,NMD1组和NMD2组CaMK IIα表达水平(1.000±0.130)、(0.981±0.071)显著升高(P<0.05);NC组和NMD1、NMD2组大鼠海马CaMK IIα表达水平之间无明显差异(P>0.05)。各组大鼠海马P-CaMK IIα蛋白表达水平以P-CaMK IIα/β-actin表示。结果显示:与NC组P-CaMK IIα表达水平(0.693±0.035)比较,PTZ组P-CaMK IIα表达水平(0.250±0.036)显著降低(P<0.05);与PTZ组比较,NMD1组和NMD2组P-CaMK IIα表达水平(0.679±0.058)、(0.665±0.043)显著升高(P<0.05);NC组、NMD1和NMD2组P-CaMK IIα表达水平无显著性差异(P>0.05)。大鼠海马CaMK IIαmRNA表达水平以CaMK IIαmRNA/β-actin mRNA表示。结果显示,与NC组CaMK IIαmRNA表达水平(1.217±0.074)比较,PTZ组CaMK IIαmRNA表达水平(0.758±0.050)明显降低(P<0.05);与PTZ组比较,NMD1组和NMD2组CaMK IIαmRNA表达水平(1.149±0.083)、(1.300±0.071)明显增高(P<0.05);NMD1、NMD2组及NC组大鼠CaMK IIαmRNA表达水平无明显统计学差异(P>0.05)。结论:戊四氮点燃慢性癫痫大鼠海马内存在钙超载及P-CaMK IIα,CaMK IIα蛋白及CaMK IIαmRNA表达水平降低,导致钙信号转导异常,可能参与了癫痫后认知功能障碍的发病机制;尼莫地平可以调节海马[Ca2+]i水平,增强P-CaMK IIα,CaMK IIα蛋白及CaMK IIαmRNA表达水平,与它提高癫痫大鼠学习记忆成绩相一致,提示尼莫地平具有改善认知功能的药理学作用。第五部分慢性癫痫大鼠海马CREB表达水平的变化及尼莫地平的影响目的:观察戊四氮点燃慢性癫痫大鼠海马P-CREB蛋白及CREB mRNA表达的变化及尼莫地平的干预作用,探讨核转录因子CREB表达与癫痫后认知功能障碍的关系。方法:Western blot方法检测海马P-CREB(Ser133)蛋白表达的变化, RT-PCR方法检测CREB mRNA表达的变化。结果:各组大鼠海马P-CREB(Ser133)表达水平以P-CREB/β-actin表示。结果显示:与NC组P-CREB表达水平(0.643±0.054)比较,PTZ组大鼠海马P-CREB表达水平(0.275±0.041)显著降低(P<0.05);与PTZ组比较,NMD1组和NMD2组P-CREB表达水平(0.602±0.045)、(0.621±0.071)显著升高(P<0.05);NC组及NMD1、NMD2组大鼠海马P-CREB蛋白表达水平比较无显著性差异(P>0.05)。各组大鼠海马CREB mRNA表达水平以CREB mRNA/β-actin mRNA表示。结果显示,与NC组CREB mRNA表达水平(0.605±0.071)比较,PTZ组CREB mRNA表达水平(0.319±0.037)明显降低(P<0.05);与PTZ组比较,NMD1组和NMD2组CREB mRNA表达水平(0.560±0.058)、(0.582±0.083)明显增高(P<0.05);NMD1、NMD2组及NC组大鼠海马CREB mRNA表达水平之间无明显统计学差异(P>0.05)。结论:慢性癫痫大鼠海马CREB蛋白磷酸化水平及转录水平下调,尼莫地平可以提高CREB蛋白及转录水平,这与其学习和记忆成绩变化相一致,提示核转录因子CREB可能参与了癫痫后认知功能障碍的发病机制。更多还原

【Abstract】 Epilepsy, which is essentially an abnormality of cerebral function resulting from the discharges of some cerebral neuron in over high excitement state, has been a common chronic clinical syndrome. Besides the common symptoms of epilepsy, about 30%~40% of the epileptic patients have been found to have deleterious cognitive consequences, which may affect the patients’life greatly, such as the declination of memory, the scattering of attention, etc. Despite the fact that the relationship between epilepsy and the cognitive deficiency has drawn much attention and become one of the focuses of modern epilepsy research, the mechanics of damaged cognition after epilepsy is not known yet.Synapse has been well recognized as the key of transmitting neural information for its being touch points among neurons. It is therefore that whether synapse is intact or not determines the quality of information transmitting, processing and storage. The synaptic plasticity refers to the ability of synapse to change shape and adjust functions under some certain conditions, which plays a crucial role in the whole developing process of neural system, study ability and memory. The synaptic plasticity, which is closely related with long-term potentiation (LTP) of hippocampus, is measured by the changes of the number of synaptic vesicles density (SVD), the width of synaptic cleft (WSC) and the postsynaptic density (PSD).The density and distribution of synaptophysin (SYN, P38), a typical proteinous marker of synaptic vesicles, is not only an indirect reflection of the quantity and distribution of synapse but also closely related to the reconstruction of synapse and cognitive process. Postsynaptic density 95(PSD-95) is an essential factor of inducing LTP for its role in clustering N-methy1-D-aspartate (NMDA). As the co-functioning results of the presynaptic and postsynaptic mechanics, the formation of LTP requires the increase of both the release of presynaptic transmitter and the efficiency of postsynaptic receptor. P38 reflects the release of presynaptic transmitter and PSD-95 reflects the efficiency of postsynaptic receptor.Calcium, an important messager of neural information, plays an important part in learning ability and memory. Any change of the intracellular concentration of Calcium ([Ca2+]i) will lead to a series of biological and even pathological changes. Calcium/calmodulin-dependent protein kinase IIα(CaMK IIα) is the most protein inside hippocampus and a main element of PSD simultaneously. There is a close relationship between autophosphorylation of CaMK IIαand the maintenance of LTP within 3 hours. Moreover the maintenance of LTP more than 3 hours requires the expression of a series of genes and the synthesis of new proteins. And cAMP response element binding protein (CREB), the intracellular transcription factor, which mediates the genetic transcription and the proteinous synthesis, plays a key role in the transition from short term memory to long term memory.Epilepsy, one of the high frequency stimulations (HFS), will cause the release of glutamic acid (Glu) and the inward flow of Calcium ion, which will induce the poisonous effect of excitatory amino-acid (EAA) and the overloading of Ca2+. And we have not found the truths, which may help find some new breakthroughs in the research on the mechanics of damaged cognition after epilepsy, such as whether the HPS will possibly lead to the declination of learning ability and memory, how the Calcium message transmits along Ca2+-CaMK II-CREB, how P38 in charge of the Glu release and PSD-95 in charge of clustering the Glu receptor change, and what the change of synaptic ultrastructure is, etc.Nimodipine (NMD) is an inhibitor of L-type voltage-dependent Ca2+ channels (VDCCs). Recent researches have showed that NMD could help improve the impairments of learning ability and memory resulted from various diseases. Still not published is the experimental research on NMD’s effect on damaged learning ability and memory after epilepsy. Based on the above mentioned facts, the dissertation intends to explore the possible mechanics of the damaged cognition after epilepsy through the research of synaptic plasticity and Calcium message transcription and observe the positive effects of NMD upon epilepsy and damaged cognition after epilepsy.Part I Establishment of chronic epileptic rat model, evaluation of behavioral features and the effect of NMDObjective: To establish a chronic epileptic rat model kindled by PTZ and observe the changes of learning ability and memory of epileptic rats and the effect of NMD.Methods: Adult male Sprague–Dawley (SD) rats were selected through Y-Maze tests and then divided into 4 groups randomly. The 35 rats in the normal control group (NC) were injected saline intraperitoneally (i.p) by 3.5 ml/kg for consecutive 44 days. The 40 rats in PTZ group received intraperitoneal injection of 1% pentylenetetrazol (PTZ) by 35 mg/kg for consecutive 44 days. From the 31st day on, each rat was injected saline intraperitoneally by 3.5 ml/kg 15 minutes before PTZ in the rest 14 days. Rats in NMD1 group and in NMD2 group (40 rats each group) were injected intraperitoneally 1% PTZ by 35mg/kg for consecutive 44 days. From the 31st day on, each rat was injected NMD intraperitoneally by 1.25 mg/kg and by 2.5 mg/kg respectively 15 minutes before PTZ in the rest 14 days. Then those rats were recorded with electroencephalogram (EEG) and tested with Morris Water Maze (MWM) and Y-Morris for the measurement of the rats’learning ability and memory.Results: Beginning from day 6 on, rats in epileptic and NMD groups had seizures induced by repetitive PTZ, which were characterized by head shaking, squealing and crawling. Moreover, wild running, loss of righting reflex and generalized tonic–clonic convulsions were seen on day 18~day 24, which indicated reaching the kindling standard. On day 30, the numbers of the rats being kindled in PTZ group, NMD1 group and NMD2 group were 35, 34, and 35 respectively. The Racine’s stage greatly reduced after NMD injection in the NMD groups. No seizures were found in the normal group. EEG changes: The EEG records of the rats in NC group were dominant withαwaves andβwaves and the background was normal without any discharges. The rats in PTZ group were recorded in EEG as sharps, spike-slow waves and most polyspike discharge, with high potential on a relatively normal background. The EEG records of the rats in NMD1 group and NMD2 group were demonstrated the repressed discharges. Place navigation results in MWM test: Comparisons were made among the mean escape latencies on each day. On day 1, comparisons of the four groups were of no difference in significance (P>0.05). On day 2, the mean escape latency of rats in PTZ group (27.36±6.13) s was much longer than that in NC group (15.14±4.69) s (P<0.05). Moreover, the mean escape latencies of the rats in both NMD1 group (21.66±5.21) s and NMD2 group (19.76±5.81) s were shorter than that of the rats in PTZ group (P<0.05), but much longer than that in NC group (P<0.05). The comparison of mean escape latency in NMD1 group and NMD2 group had no distinct difference (P>0.05). On day 3, the mean escape latency of the rats in PTZ group (7.71±1.30) s was obvious different from that in NC group (4.46±1.11) s (P<0.01). The mean escape latencies of the rats in both NMD1 group (5.94±1.19) s and NMD2 group (5.69±1.03) s were shorter than that in PTZ group (P<0.05), but longer than that of those in NC group (P<0.05). The comparison of mean escape latencies of the rats in NMD1 group and NMD2 group had no statistic difference (P>0.05). Spatial probe test: The number of crossing times through the platform quadrant within 120 s in PTZ group (5.73±2.14) decreased obviously compared with that in NC group (9.25±2.18) ( P<0.01). And the numbers of crossing times through the platform quadrant within 120 s in both NMD1 group (8.58±2.12) and NMD2 group (7.51±2.05) increased significantly compared with that in PTZ group (P<0.01), but were of no statistic significance compared with that in NC group(P>0.05). And the comparison of the numbers in NMD1 group and NMD2 group was not statistically significant (P>0.05). There was no significant difference of the swimming time spent in the platform quadrant of the rats in four groups (P>0.05). Y-maze results: The error number (EN) of the rats in PTZ group (12.75±2.50) increased obviously compared with that in NC group (6.25±3.10) (P<0.05). The ENs in both NMD1 (8.75±2.22) and NMD2 group (8.00±3.16) significantly decreased compared with that in PTZ group (P<0.05), but was of no obvious difference from that in NC group (P>0.05). There was no obvious difference between NMD1 group and NMD2 group (P>0.05). Comparisons of the total reaction time (TRT) of the rats in four groups were of no significant difference (P>0.05).Conclusion: The epileptic model kindled by PTZ in the experiment did have impairments in learning ability and memory. This model is the perfect animal model which is feasible to study the damaged cognition after epilepsy. NMD could partly improve the damaged cognitive function of epileptic rats.Part II Changes of postsynaptic density 95(PSD-95) and synaptophysin (P38) expression levels in hippocampus of rats kindled by PTZ and the effect of NMDObjective: To observe the changes of P38 protein, PSD-95 protein and PSD-95 mRNA in hippocampus of rats kindled by PTZ and the effect of NMD and to explore the role of PSD-95 and P38 in damaged cognition after epilepsy.Methods: 8 rats in each group were anesthetized by 10% chloral hydrate and perfused with 4% paraformaldehyde solution. The brain tissue containing hippocampus was cut and embedded with paraffin. The changes of P38 and PSD-95 were tested with immunohistochemical SP methods and the absorbency of immunohistochemical products was also detected. Another 8 rats in each group were anesthetized by 10% chloral hydrate and the hippocampi were quickly separated. Total RNA was extracted strictly by Trizol. The expression of PSD-95 mRNA was measured by RT-PCR.Results: The absorbency of PSD-95 immunohistochemical products in the hippocampal CA1 area of the rats in PTZ group (0.6525±0.0648) decreased distinctly compared with that in NC group (0.7108±0.0584) (P<0.05). Compared with the absorbency of PSD-95 products in PTZ group, those in NMD1 group (0.6921±0.0620) and NMD2 group (0.7033±0.0659) increased obviously (P<0.05). There was no distinct difference among NMD1 group, NMD2 group and NC group (P>0.05). The absorbency of P38 immunohistochemical products in the hippocampal CA1 area of the rats in PTZ group (0.4475±0.0505) reduced distinctly compared with that in NC group (0.6033±0.0444) (P<0.05). Compared with the absorbency in PTZ group, those in NMD1 group (0.5819±0.0367) and NMD2 group (0.5723±0.0528) increased significantly (P<0.05). The difference of the absorbencies among NMD1 group, NMD2 group and NC group was not distinct (P>0.05). The expression levels of PSD-95 mRNA were determined by calculating the density ratio of PSD-95 mRNA/β-actin mRNA. The expression level in PTZ group (0.325±0.030) reduced obviously compared with that in NC group (0.607±0.051) (P<0.05). Compared with that in PTZ group, the PSD-95 mRNA expression levels in both NMD1 group (0.512±0.035) and NMD2 group (0.575±0.041) increased significantly (P<0.05). The expression levels in NMD1 group, NMD2 group and NC group were of no statistic significance (P>0.05).Conclusion: There were reductions in expression levels of P38, PSD-95 and PSD-95 mRNA in the hippocampus of the PTZ-kindled rats in accordance with the decline of the rats’learning ability and memory, which suggests that both P38 and PSD-95 might participate in the mechanism of damaged cognition after epilepsy. NMD could improve the expression levels of P38, PSD-95 and PSD-95 mRNA and the damaged cognitive function of epileptic rats.Part III Changes of neuronal and synaptic ultrastructure in hippocampus of rats kindled by PTZ and the effect of NMDObjective: To observe the changes of neuronal ultrastructure and synaptic ultrastructure (SVD, PSD and WSC) in hippocampal CA1 area of rats kindled by PTZ and the effect of NMD and to explore the relationship between synaptic plasticity and damaged cognition after epilepsy.Methods: 3 rats in each group were anesthetized by 10% chloral hydrate and perfused with 4% paraformaldehyde solution (containing 2.5% glutaraldehyde). The changes of neuronal ultrastructure and synaptic ultrastructure (SVD, PSD and WSC) in hippocampal CA1 area of rats in each group were observed through the electron microscopy.Results: In hippocampal CA1 area of the rats in NC group, there were intact neurons, distinct structure, uniform chromatin, abundant apparatus, and integrate myelin. In PTZ group, however, there were condensed nucleus, edematous neuron, swollen perikaryon with vacuole, reduced mitochondria and Golgi apparatus and polyribosome; there was also Myelin-splitting nerve fiber. In NMD1 and NMD2 group, the neuron and neuropile were less swollen, karyotheca were normally intact, chromatin were uniform, the apparatus were rich and basically normal, and the myelin was complete and integrate. In NC group, Gray I type synapses in hippocampal CA1 area were abundant with distinct pre-and-post synaptic membranes and rich synaptic vesicles. The synaptic cleft was clear and the PSD was thick and uniform. In PTZ group, there were reduced synapses with indistinct pre-and-post synaptic membranes, increased WSC, thinner PSD, and decreased SVD. In both NMD1 group and NMD2 group, there were more synapses than that in PTZ group. The synapses had relatively distinct pre-and-post synaptic membranes, and uniformed PSD, and more synaptic vesicles. The PSD in hippocampal CA1 area of the rats in PTZ group (31.37±1.94) nm reduced notably compared with that in NC group (49.86±3.38) nm (P<0.05). Compared with that in PTZ group, PSD in both NMD1 group (45.69±2.70) nm and NMD2 group (48.20±2.15) nm increased notably (P<0.05). Comparisons of PSD in NMD1, NMD2 group and NC group were of no significant difference (P>0.05). The WSC in PTZ group (22.34±1.84) nm also increased obviously compared with that in NC group (14.62±1.19) (P<0.05). Compared with that in PTZ group, the WSC in both NMD1 group (16.37±1.72) nm and NMD2 group (15.02±1.63) nm reduced significantly (P<0.05). Comparisons of WSC of NMD1 group, NMD2 group and NC group were of no distinct difference (P>0.05). Compared with that in NC group (3362.61±102.08)/um2, the SVD in PTZ group (1078.36±111.03)/um2 decreased significantly (P<0.05). Compared with that in PTZ group, the SVD in hippocampal CA1 area in both NMD1 group (2932.01±142.95)/um2 and NMD2 group (3155.50±137.49)/um2 increased significantly (P<0.05). Comparisons of SVD of NMD1 group, NMD2 group and NC group had no obvious difference (P>0.05).Conclusion: There were abnormal changes in neuronal and synaptic ultrastructure in hippocampal CA1 area of the epileptic rats. NMD could ameliorate the neuronal and synaptic abnormal ultrastructure, which was consistent with the changes of learning ability and memory. There might be a close relationship between synaptic plasticity and damaged cognition after epilepsy.Part IV Changes of [Ca2+]i and calcium/calmodulin-dependent protein kinase IIα(CaMK IIα) expression levels in hippocampus of rats kindled by PTZ and the effect of NMDObjective: To observe the changes of [Ca2+]i, CaMK IIα, P-CaMK IIαprotein and CaMK IIαmRNA in hippocampus of rats kindled by PTZ and the effect of NMD and to explore the relationship between calcium signal transduction and damaged cognition after epilepsy.Methods: 6 rats in each group were sacrificed and the hippocampi were quickly separated, homogenized and the suspension was collected and incubated with Fluo-3/AM. The changes of [Ca2+]i were tested with flow cytometry; The expressions of CaMK IIαmRNA of the hippocampus in each group were tested with RT-PCR; 8 rats in each group were anesthetized by 10% chloral hydrate and the hippocampi were quickly separated. Total protein for CaMK IIαand membrane protein for P-CaMK IIαwas extracted. The expression levels of CaMK IIαand P-CaMK protein were detected by Western blot.Results: The [Ca2+]i in hippocampus of the rats in PTZ group (1.94±0.33) increased obviously compared with that in NC group (1.26±0.21) (P<0.05). Compared with that in PTZ group, the [Ca2+]i in hippocampus of the rats in both NMD1 group (1.35±0.28) and NMD2 group (1.32±0.26) reduced notably (P<0.05). The comparisons of [Ca2+]i in hippocampus among NMD1 group, NMD2 group and NC group were of no significant difference (P>0.05). The protein levels of CaMK IIαand P-CaMK IIαin hippocampus of rats in each group were determined by CaMK IIα/β-actin and P-CaMK IIα/β-actin respectively. The protein levels of CaMK IIαin hippocampus in PTZ group (0.700±0.061) reduced significantly compared with that in NC group (1.030±0.133) (P<0.05). Compared with that in PTZ group, the protein levels in hippocampus of the rats in both NMD1 group (1.000±0.130) and NMD2 group (1.000±0.130) increased significantly (P<0.05). The comparisons of the protein levels of CaMK IIαin hippocampus among NMD1 group, NMD2 group and NC group had no statistic difference (P>0.05). The protein levels of P-CaMK IIαin hippocampus in PTZ group (0.250±0.036) reduced significantly compared with that in NC group (0.693±0.035) (P<0.05). Compared with that in PTZ group, the protein levels in hippocampus of the rats in both NMD1 group (0.679±0.058) and NMD2 group (0.665±0.043) increased significantly (P<0.05). The comparisons of the protein levels of P-CaMK IIαin hippocampus among NMD1 group, NMD2 group and NC group were of no statistic difference (P>0.05). The expression levels of CaMK IIαmRNA in hippocampus of rats in each group were determined by calculating the density ratio of CaMK IIαmRNA/β-actin mRNA. The expression levels of CaMK IIαmRNA in hippocampus in PTZ group (0.758±0.050) reduced notably compared with that in NC group (1.217±0.074) (P<0.05). Compared with that in PTZ group, the CaMK IIαmRNA levels in hippocampus of the rats in both NMD1 group (1.149±0.083) and NMD2 group (1.300±0.071) increased notably (P<0.05). The comparisons of the CaMK IIαmRNA levels in hippocampus among NMD1 group, NMD2 group and NC group had no statistic difference (P>0.05).Conclusion: The [Ca2+]i in hippocampus of the epileptic rats increased and the expression levels of P-CaMK IIαprotein,CaMK IIαprotein and CaMK IIαmRNA in hippocampus reduced, which resulted in the imbalance of calcium signal transduction pathway. These might be one part of the molecule pathogenesis of damaged cognition after epilepsy. NMD could adjust [Ca2+]i and improve the expression levels of CaMK IIαprotein and mRNA in accordance with the improvement of learning ability and memory of the epileptic rats, which demonstrates NMD’s pharmacological effects on improving cognitive functions.Part V Changes of expression levels of cAMP response element binding protein (CREB) in hippocampus of rats kindled by PTZ and the effect of NMDObjective: To observe the changes of P-CREB protein and CREB mRNA in hippocampus of rats kindled by PTZ and the effect of NMD and to explore the relationship between transcription factor CREB and damaged cognition after epilepsy.Methods: The changes of the expressions of P-CREB(Ser133) and CREB mRNA were tested with Western blot and RT-PCR methods, respectively.Results: The protein levels of P-CREB (Ser133) in hippocampus of rats in each group were determined by P-CREB/β-actin. The protein levels of P-CREB in hippocampus in PTZ group (0.275±0.041) decreased significantly compared with that in NC group (0.643±0.054) (P<0.05). Compared with that in PTZ group, the protein levels in hippocampus of the rats in both NMD1 group (0.602±0.045) and NMD2 group (0.621±0.071) increased obviously (P<0.05). The comparisons of the protein levels of P-CREB in hippocampus among NMD1 group, NMD2 group and NC group had no statistic difference (P>0.05). The expression levels of CREB mRNA in hippocampus of rats in each group were determined by calculating the density ratio of CREB mRNA/β-actin mRNA. The CREB mRNA levels in hippocampus in PTZ group (0.319±0.037) reduced notably compared with that in NC group (0.605±0.071) (P<0.05). Compared with that in PTZ group, the CREB mRNA levels in hippocampus of the rats in both NMD1 group (0.560±0.058) and NMD2 group (0.582±0.083) increased notably (P<0.05). The comparisons of the CREB mRNA levels in hippocampus among NMD1 group, NMD2 group and NC group had no statistic difference (P>0.05).Conclusion: The expression levels of both CREB protein and mRNA in hippocampus of the epileptic rats reduced and NMD could improve that, which was consistent with the changes of learning ability and memory. Transcription factor CREB might participate in the mechanism of damaged cognition after epilepsy.更多还原