【作者】 陈晓刚；

【导师】 李文玲；

【作者基本信息】 河北医科大学 ， 外科学， 2008， 硕士

【摘要】 目的:癫痫是由多种病因引起的慢性脑功能障碍综合征,是大脑神经元异常放电引起的脑功能紊乱,可以表现为运动、感觉、意识、精神等多方面的功能障碍,给病人及家属造成重大的经济和社会负担。目前癫痫治疗除了传统的内科药物、外科手术治疗外,伽玛刀放射治疗因不开颅,对脑组织损伤小而被认为是安全、有效的治疗方法。如何给予合适的放射剂量,使大脑皮层I-II层细胞凋亡,减少胶质神经瘢痕的形成,而又不损伤其余四层结构,是伽玛刀治疗功能区癫痫研究的重点。基于上述原因考虑,我们建立功能区慢性氢氧化铝癫痫模型,希望在此模型的基础上,为以后更好的研究功能区外科手术及伽玛刀放射治疗剂量选择提供服务。在众多可引发癫痫样放电的慢性单纯部分性发作动物模型中,金属沉积模型是最佳模型,氢氧化铝是此类模型的代表。在动物的选择上,我们选择猫为实验动物,因为猫行动灵活,功能区皮层结构与人类相似。通过对猫功能区慢性局限性癫痫模型的制作方法的实验性研究,探讨猫功能区慢性癫痫模型的癫痫发作及不同时间点脑电图变化情况,观察致癫灶病理及超微结构改变,超氧化物歧化酶(Superoxide dismutase,SOD)和丙二醛(Malondialdehyde, MDA)的变化情况,为进一步研究癫痫的机制奠定一定的理论基础。方法:雄性健康家猫20只,体重2500-3500g,随机分为两组,氢氧化铝组和生理盐水组,各组动物用3%戊巴比妥钠麻醉后开颅在左侧大脑额叶运动区注入8%氢氧化铝乳剂50ul或生理盐水50ul。术后每天观察猫的行为学变化,每天观察3-5小时不等,即生活习惯、进食、有无抽搐发作及发作持续时间;术后2周、4周、6周、8周、12周、16周及20周用MS4000U电生理监测仪描记实验动物致痫区皮层脑电图观察并记录脑电图的变化;20周后取致痫区脑组织行HE染色及透射电镜观察病理变化情况,同时取致痫区脑组织通过分光光度计测量SOD、MDA的变化情况。结果: 1形为学结果:氢氧化铝组及生理盐水组于术后三天内进食较少,精神萎靡,三天后恢复正常进食,精神好转,氢氧化铝组中6只猫于术后11-14周的不同时间点发生临床癫痫发作,表现为单纯部分发作,发作时间持续10余秒至五分钟左右不等。生理盐水组连续观察20周内未观测到临床癫痫发作情况。2脑电图结果:氢氧化铝组于术后2-6周未见异常放电,8周开始出现棘波放电,放电频率为4.7±1.3次/分。12周可见棘波放电次数明显增多,并呈丛集性棘波或棘慢波异常放电,放电频率为37.2±8.4次/分。16周时放电次数较前有所减少,放电频率为9.2±2.4次/分。联续观测到20周时仍有棘波或棘慢波异常放电,放电频率为8.6±2.4次/分。生理盐水组术前术后一直无异常棘波放电。3 HE染色病理结果:氢氧化铝组致痫区皮层神经元数量减少,神经元细胞出现不同程度变性、坏死,灰质内胶质细胞增生和坏死的神经元细胞被胶质细胞包围吞噬,毛细血管扩张、充血。4超微病理结果:氢氧化铝组致痫区皮层神经元细胞质高度水肿,细胞器数量减少,散在线粒体的大部分嵴的部分膜融合或消失,粗面内质网扩张,可见明显脱颗粒现象,部分核膜内外层融合、模糊不清或消失缺损,吞饮小泡数量减少,毛细血管的一侧明显水肿,有髓神经髓鞘明显增生,厚薄不均,板层结构有分层现象,大部分融合,模糊不清,微丝微管数量减少。5 SOD、MDA含量:氢氧化铝组SOD含量为147.68±24.29U/mgprot ,生理盐水组SOD含量为175.01±25.29U/mgprot ,氢氧化铝组MDA含量为0.5082±0.1616umol/gprot ,生理盐水组MDA含量为0.2896±0.0528 umol/gprot,氢氧化铝组SOD含量较生理盐水组明显减少,氢氧化铝组MDA含量较生理盐水组明显增多。结论:1氢氧化铝致猫癫痫模型出现单纯部分性癫痫发作,皮层脑电图棘波持续放电以及病理形态学的异常改变,表明氢氧化铝致猫慢性局限性癫痫模型制作成功。2氢氧化铝组癫痫模型棘波放电始于术后第8周左右,在12周左右出现高峰,持续20周仍有棘波放电。12周左右可能是棘波放电的高峰时期。3氢氧化铝组致痫灶的病理机制为神经元变性、坏死,皮层胶质细胞增生,毛细血管扩张、充血等。这种病理形态学改变可能是导致大脑皮层棘波放电的结构基础。4氢氧化铝组致痫区脑组织MDA含量明显升高,而SOD含量明显降低,说明致痫区脑组织氧化和抗氧化系低于统平衡失调,产生过量的氧自由基,进一部损伤脑组织,而清除氧自由基的SOD由于过量消耗而减少,导致神经组织、细胞损伤、变性、坏死,神经元异常放电,进一步加剧癫痫的发展。更多还原

【Abstract】 Objective: Epilepsy is cerebral chronic functional disturbance syndrome by multifold etiopathogenisis, is abnormal discharage of neuron. Epilepsy show multifold functional disturbance as movement, aesthema, consciousness, mind, etc. The result induce to heavy burden for family and society. Now epileptic therapy apart from traditional medicine and operation also Gamma Knife radiosurgery. Because of Gamma Knife radiosurgery needn’t open skull, it is regarded as not only safe available method but also minor impairment. The investigative emphasis is that how much therapeutic dose is suitable, that not only apoptosis happen only the I-II layer of cerebral cortex without other four layer but also lesser gliocyte cicatricle emerge. On account of above-mentioned reason ,we establish the model of chronic and partial epilepsy induced by aluminum hydroxide in animal motor cortex, to serve both surgical operation in eloquent cortex and the choose of therapeutic dose in Gamma Knife radiosurgery. Among several animal models of chronic and partial epilepsy , Metal animal models is the best and aluminum Hydroxide is most suitable metal. About the choice of animal, we choose cats as experimental animal, because cats are active, cats’motor cortexs are developed and similar with human being. After establish and study the model of chronic and partial epilepsy induced by aluminum hydroxide in cat’s motor cortex. The first we observe epileptic seizure and record the change of Electroencephalogram(EEG) at different time point; The second the tissue of epileptogenic focus are observed by the light microscope and eletron microscope; The third the contents of Superoxide dismutase(SOD) and Malondialdehyde(MDA) in the motor cortex are measured, then we can establish some theoretical foundation for researching the pathogenesy of epilepsyMethods: Twenty healthy male domestic cats, weigh 2500~3500 grams, were randomly divided into the aluminum hydroxide -induced group and normal saline control group, cats were anesthetized using 3% Nembutal, then 50 microliter of sterile 8% aluminum hydroxide or normal sodium were injected into the sigmoid gyrus after opened their cranium. We observed their behavioral changes after the procedure everyday, we had continuous observed three to five hours in the everyday of twenty weeks, including living habit, eating and epileptic seizure. Using MS4000U Biological Signals Quantitative Recording And Analyzing System recorded and analysed dynamic changes of EEG in the epileptogenic focus of the models of animal at two week, four week, six week, eight week, twelve week, sixteen week, twenty week after the operation. The epileptogenic focus were cutted and observed by the light microscope and eletron microscope after twenty weeks, at the same time the content of SOD and MDA in the motor cortex were measured by spectrophotometer.Results: 1 Ethology: The appetite of the two groups decreased significantly atter the operation and the spirit was dispirited. they have recovered normal eating and the spirit was improved after three days, six cats was found epileptic seizure in the aluminum hydroxide -induced group from 11 to 14 weeks, they showed simple partial seizures, the time of seizures lasted from ten second to five minutes. The epileptic seizure hadn’t been observed in normal saline control group .2 Results of EEG: Spike wave wasn’t monitored in the aluminum hydroxide -induced group from two weeks to six weeks after aluminum Hydroxide was injected. Epileptiform spike activity was first detected at the eight weeks after Aluminum hydroxide was injected, frequency of spike wave is 4.7±1.3 per minute. Severe Spike wave was detected at the twelve weeks, frequency of spike wave is 37.2±8.4 per minute. Spike wave was detected at the sixteen and twenty weeks, frequency of spike wave is 9.2±2.4 per minute and 8.6±2.4 per minute. Spike wave wasn’t detected in the normal saline control group from the beginning to the end.3 Results of HE stain: The epileptogenic focus of the aluminum hydroxide -induced group showed that the quantity of neurons was decreaded, the neurons showed degeneration and necrosis. Glial cells were hyperplastic in the motor cortex, and you could find neurons was phagocytized by glial cells. Micrangium was dilated and congestive.4 Ultrastructure of nerve cells: The epileptogenic focus of the aluminum hydroxide -induced group showed that cytoplasm of neurons were apparent swelling, the quantity of organelles were decreasing, a part of membrane of the bulk of the sporadic mitochondrias’cristae were coalescesed or disappeared, rough endoplasmic reticulum(RER) were expanded and you could observe apparent degranulation phenomenon, a part of nuclear membrane were coalescesed, unclear or disappear. the quantities of pinocytosis vesicle were decreased, on one side of micrangiums were swollen, neural myelin sheath were hyperplastic apparently, and the thickness was inhomogeneous, the layers structure had the phenomenon of delamination, the quantities of microfilament and microtubule were decreased.5 Contents of SOD, MDA: Contents of SOD was 147.68±24.29U/mgprot on the aluminum hydroxide -induced group. Contents of SOD was 175.01±25.29U/mgprot on the normal saline control group. Contents of MDA was 0.5082±0.1616umol/gprot on the aluminum hydroxide -induced group. Contents of MDA was 0.2896±0.0528umol/gprot on the normal saline control group. Contents of SOD was lower apparently on the Aluminum Hydroxide -induced group than the normal saline control group. Contents of MDA was more apparently on the aluminum hydroxide -induced group than the normal saline control group.Conclusions: 1 The models of the aluminum hydroxide -induced group showed simple partial seizures, continued spike wave and changes of pathology, the results indicated the models of chronic and partial epilepsy induced by Aluminum hydroxide in cat’s motor cortex was successful.2 Epileptiform spike activity was first detected at the eight weeks after aluminum hydroxide was injected, severe Spike wave was detected at the twelve weeks, spike wave still was detected at the twenty weeks. Maybe Spike wave were peak time at the twelve weeks.3 The pathological mechanism of epileptogenic focus on the Aluminum hydroxide -induced group was that neurons were degeneration and necrosis, glial cells were hyperplastic in the motor cortex, micrangium was dilated and congestive etc. We thinked about the pathological changes of epileptogenic focus perhaps were structural basic of epileptic seizure and spike wave.4 Contents of SOD was lower apparently on the Aluminum Hydroxide -induced group than the normal saline control group. Contents of MDA was more apparently on the Aluminum Hydroxide -induced group than the normal saline control group. The result showed the system of oxidation and antioxidation were disequilibrium on the tissue of epileptogenic focus, producted excess oxygen free radical, and strengthened to injure normal cerebral cortex. The SOD as a biocatalyst for oxygen free radical was decreased by excess consume, the result was that cells of nerve were injured, then denaturation and necrosis, further promoted the develop of epilepsy.更多还原