【作者】 陈伟；

【导师】 胡向阳；

【作者基本信息】 安徽医科大学 ， 病理学与病理生理学， 2009， 硕士

【摘要】 目的:利用立体定位技术在大鼠海马CA3中心区域微量注射海人酸(kainate acid,KA)建立大鼠颞叶癫痫动物模型,利用此种动物模型做慢性实验,观察其发作表现、脑电生理改变和致痫病理学依据,比较大鼠癫痫与人类癫痫的相似性;并以此动物模型模拟人类复杂部分性发作,探讨癫痫对睡眠的影响,为临床上寻找对癫痫相关睡眠障碍诊断和治疗提供基础资料。方法:1.动物分组将24只雄性Wistar大鼠随机分为两组:正常生理盐水假手术组(NS组)12只;KA致痫组(KA组)12只。2.模型建立利用立体定向仪在大鼠海马CA3区(AP:- 4.0 mm,ML:- 4.4 mm,DV:3.8 mm)缓慢注射2.5μl KA (0.04μg/μl)(约10 min注射完毕),留针3 min,缝合头皮,按照Racine分级法对发作进行分级,发作达4或5级的大鼠用于进一步的慢性实验。对照组注射等体积的生理盐水。3.动物体重测量:在大鼠模型建立后第1天/3天/5天/1周/ 2周/4周和8周,分别用电子计数天平称量大鼠体重,并做好记录。4.动物行为学观察:在KA注射两周后,将大鼠放置于有视频摄像头监控的实验室内饲养,每天于9:00～17:00视频监控,每周5天(8小时/天, 5天/周)。评价大鼠的行为及痫样发作情况。5.皮层电极和颈肌电极安装:动物行为学观察2月后,将大鼠用水合氯醛(350 mg/kg)腹腔麻醉后,4个铜质电极穿透颅骨接触硬脑膜(左右各2个),用于记录皮层脑电(EEG)活动。在双侧颈肌内插入银丝电极用于记录肌电(EMG)活动。皮层与颈肌电极分别连接一微型底座,均以牙科水泥固定。6.多导睡眠图( PSG )的描记:通过脑电图机同步记录脑电和肌电活动。为了尽可能避免大鼠24 h睡眠-觉醒周期的影响,每次描记均从9: 00开始,连续记录3～4 h。7.睡眠分期:采用25 s为一分段时间,将睡眠-觉醒周期分为:①觉醒期(W):以额-顶叶引导出低幅快波脑电和明显的肌电活动为特征;②慢波睡眠( SWS):以睡眠梭形波和高幅慢波为特征,肌电活动明显减少,其中δ波少于50%属浅慢波睡眠( SWS1 ) ,超过50%属深慢波睡眠( SWS2 );③异相睡眠( PS):以低幅快波为特征,除偶尔有肌肉抽动外,无明显肌电活动。8.形态学分析脑电记录完毕后,对大鼠进行内灌注取脑,选出有典型海马结构的脑组织脱水后利用冰冻切片机切片,连续切片4张,片厚20μm,分成2组,每组各2张。一组做Nissl染色,以评价各组实验大鼠海马各区神经元损伤情况;另一组做Timm染色,以评价各组实验大鼠苔状纤维发芽程度。9.统计学分析统计分析采用SPSS 13.0统计分析软件。统计数据用均数±标准误表示。结果:1.大鼠体重变化:术后癫痫组大鼠体重明显下降,3天后癫痫组和对照组体重无统计学差异。2.行为学结果:NS组(n = 12)未见一只实验动物有自发发作;KA组(n = 12)大鼠在麻醉清醒后均出现急性发作期,发作大约在KA注射24 h后减弱并消失。但在4周后又逐渐出现自发性发作(n = 10)。3.造模8周后PSG描记显示,与NS组相比,KA组大鼠深慢波睡眠时间和异相睡眠时间减少,癫痫大鼠睡眠时痫样放电比率比清醒时升高。4.形态学结果:4.1. Nissl染色结果显示与NS组大鼠(n = 6)相比,KA组大鼠海马CA1区、CA3区以及齿状回的门区均有大量的神经元丢失(n = 6)。4.2. Timm染色结果显示NS组大鼠海马的黑色颗粒全部位于齿状回门区和CA3区辐射层(n = 6);KA组大鼠的海马除了在DG门区和CA3区锥体细胞辐射层能观察到Timm阳性颗粒外,在颗粒细胞内分子层和CA3区锥体细胞分子层可观察到大量Timm阳性颗粒(n = 6)。结论:1. KA诱导的癫痫大鼠出现类似人类颞叶癫痫发病行为学改变及神经电生理改变。2. KA诱导的癫痫大鼠出现类似人类颞叶癫痫的海马神经元丢失和苔状纤维发芽的病理学特征,可作为人类颞叶癫痫研究的可靠动物模型。3.癫痫发作能引起癫痫大鼠睡眠结构的改变,主要表现为觉醒次数增加,深慢波睡眠减少,异相睡眠减少。更多还原

【Abstract】 Objective:The rats were induced temporal lobe epilepsy (TLE) by kainate acid (KA) injected to the center site of CA3 region of right hippocampus with the stereotaxic technology. The following chronic experiments were carried on this model. The epileptic model effects were analysesed via three levels: behavior monitoring, intracalvarium electrocorticographic recording and histological analyses. The changes of the configuration of sleep architecture were detected in epilepsic model rats.Methods:1. Grouping. All the rats in the experiments were divided randomly into the following two groups: normal saline (NS) group and KA + vehicle solution (KA) group.2. KA-induced TLE model. Adult male Wistar rats (220-260 g and clean stage) were used in the experiments. Under chloral hydrate (350 mg/kg, i.p.) anesthesia, rats were placed on the stereotaxic apparatus and 2.5μl KA (0.04μg/μl) was slowly injected (about 10 min) to CA3 region of right hippocampus (4.0 mm posterior to bregma, 4.4 mm lateral to the midline, 3.8 mm below dura). The needle was left for 3 min and the rats’scalps were sutured. The behavioral progression of KA-induced seizures was scored according to Racine’s standard classification. Those rats that could reach at least the class 4 or 5 seizures were used in this study. The same volumes of normal saline to the same site were injected in the control group.3. Weight measure. The body weights of both groups were recorded on the 1st /3rd /5th /1st week/2nd week/ 4th week/8th week after KA injected.4. Behavior monitor. Two weeks after KA injection, the spontaneous seizures number of rats was recorded 5 days every week from 9:00 to 17:00 (8 h/d, 5 d/week) by video camera.5. Cortex electrodes and nuchal muscle electrodes install. After the behavior monitoring, under chloral hydrate (350 mg/kg, i.p.) anesthesia, four copper cortical electrodes were screwed into the skull (two on the left and two on the right) in order to record electroencephalogram (EEG). Two silver electrodes were placed under the nuchal muscles in order to record electromyogram (EMG). EEG/EMG electrodes were connected to a micropedestal socket. Dental cement was then used to affix all the leads and cannulaes to the skull.6. EEG record. The EEG/EMG signals were synchronously recorded by an EEG machine. In order to reduce the effect of sleep-wake cycle, the EEG record was started from 9:00 every day, and continuously recorded for 4～6 h.7. Sleep phases. The sleep-wake cycle was devided into four phases:①Wake (W) was identified by the presence of desynchronized-EEG and high-EMG activity;②Slow wave sleep (SWS) was identified by the presence of a high-amplitude slow-wave EEG and low-EMG activity, relative to that of W;③Paradoxical sleep (PS) was identified by the presence of regular theta activity on EEG, coupled with low-EMG activity relative to that of SWS and W.8. Morphological analysis. After the EEG recording, rats were deeply anesthetized by an overdose chloral hydrate and perfused transaorticly with the modified fixation procedure. The hippocampus was cut in 20μm thin for histological analyses. Nissl staining evaluated the degenerating neurons of CA1, CA3 and dentate gyrus. Timm staining evaluated mossy fiber reorganization in the inner molecular layer of dentate gyrus that accompanied epileptogenesis.9. Statistical analysis. The SPSS 13.0 software was performed in all statistical analyses. Values were expressed as mean±SEM. Results:1. Weight changes: The weight of epileptic rats lightened greatly after surgery versus the control rats. Three days after surgery, the weights of the two groups were no significant difference.2. Behavior results: In the NS group (n = 12), no rats appeared spontaneous seizures. In KA groups, the rats were appeared epileptic seizures when they sober up from anesthesia. The seizures disappeared spontaneously within 24 h after KA microinjection. The earliest spontaneous recurrent seizures were observed about 4 weeks after KA administration (n = 10).3. After 8 weeks, all of rats were performed with PSG. The SWS1 and PS decreased, while the wake was enhanced in the epileptic animal models compared with control group. The frequency of epileptic discharges in epileptic animal models was increased during sleep.4. Morphological results:4.1. Lots of neurons were lost in the CA1, CA3, and hilus region detected by Nissl staining in KA group (n = 6), compared with NS group (n = 6).4.2. Mossy fibers abnormally invaded the granule cell layer and the third of inner molecular layer detected by Timm staining in KA group (n = 6). However, neither neurons lost nor mossy fiber sprouting (MFS) appeared in NS group (n = 6).Conclusion:1. KA-induced epileptic rat model appears similar behavior changes with human.2. KA-induced epileptic rat model appears similar pathological characters in hippocampus neurons lost and MFS with human.3. Epileptic seizures can alter the configuration of sleep architecture. The main representative characters are that the wake was enhanced, SWS1 and PS were declined, and the frequency of epileptic discharges was increased.更多还原