【作者】 黄华；

【导师】 晏勇；

【作者基本信息】 重庆医科大学 ， 神经病学， 2009， 博士

【摘要】 目的:探讨用卡莫司汀(1-3-bis-chloroethyl-nitrosourea,BCNU)建立皮质发育障碍(disorder of cortical developments,DCDs)模型的最佳剂量和方法;探索DCDs模型鼠海马神经元的凋亡机制及与之相关的致痫机制;评价神经示踪剂生物素葡聚糖胺(biotin dextran amine,BDA)对大鼠皮质和海马神经元形态学示踪的应用价值,为DCDs的异常神经网络研究打下基础;观察托吡酯治疗皮质发育障碍所致难治性癫痫(intractable epilepsy,IE)的疗效。方法:1 BCNU诱导大鼠皮质发育障碍模型的建立采用不同剂量的BCNU (以BCNU 5～20mg/kg梯度剂量)给妊娠17天的Sprague-Dawley (SD)大鼠作腹腔注射,建立子代SD大鼠DCDs模型。按BCNU的不同注射剂量分为模型①组(BCNU 5 mg/kg)、模型②组(BCNU 10 mg/kg)、模型③组(BCNU 15 mg/kg)和模型④组(BCNU 20 mg/kg),同时设立对照组。对各模型组仔鼠进行以下观察:①统计各组仔鼠的存活率;观察各组仔鼠出生时一般状态及出生后行为学,包括意识状态、生活能力、有无癫痫发作等。②分别于出生后当天(P0)、出生后7天(P7)、14天( P14)、21天( P21)、56天(P56)和84天(P84)时间点测量各组仔鼠体重。③采用Morris水迷宫实验测试各组仔鼠学习和空间记忆能力。④红藻氨酸诱导各组仔鼠癫痫发作,比较其痫性发作阈值和死亡率。⑤于P21、P84两时间点取各组仔鼠大脑观察其大体形态、测脑湿重;常规病理检查,观察各组仔鼠大脑皮质和海马结构,确定是否存在DCDs以及DCDs的病理类型。2. DCDs模型大鼠海马神经元bcl-2、bax基因的表达用BCNU诱导建立SD大鼠皮质发育障碍动物模型,在仔鼠出生后P0、P15、P30、P45和P60天时,模型组和对照组均用TUNEL检测海马区的凋亡细胞数目,免疫组化和RT-PCR方法检测bcl-2和bax基因在mRNA和蛋白水平表达的变化,并用图像分析系统进行结果分析。3.生物素葡聚糖胺(BDA)对皮质神经元的示踪采用成年的SD大鼠,用牙科钻开颅形成左右对称的4个3mm直径的圆形骨窗,用微量注射器将10%BDA缓慢注射入运动感觉皮质区和海马区,注射总量4ul。存活14d后,取出脑组织在低温冰箱中保存,冰冻切片后,采用自由漂片法行BDA染色,观察BDA标记神经元的形态。4.托吡酯治疗DCDs致难治性癫痫的疗效观察收集经MRI和临床确诊的100例DCDs致IE患者,给予托吡酯(100mg.d-1～200mg.d-1 )单药治疗,与基线发作频率比较,探讨新一代抗癫痫药物托吡酯对DCDs所致难治性癫痫的疗效。结果:1 BCNU诱导大鼠皮质发育障碍模型评估1)仔鼠的存活率对照组与模型①、②、③和④各组仔鼠的存活率分别为100%、100%、93.1%、83.3%和0%,模型④组仔鼠出生当日全部死亡。2)仔鼠的行为学观察模型①组仔鼠的各项观察指标与对照组无差别;模型②组仔鼠出生后日常行为与对照组无明显差异;模型③组仔鼠出生时一般状态较正常对照组差,出现活反应迟钝、活动减少、智能障碍等表现。各模型组没有观察到自发性癫痫发作。3)仔鼠的生长发育情况模型①组仔鼠生长发育情况与对照组无差别(P>0.05);模型②组出生时体重轻度降低(P<0.05),但随着鼠龄的增长两组间没有差异;模型③组仔鼠各时间点平均体重明显降低,随鼠龄的增长两组间差别越显著(P<0.05)。4)仔鼠的水迷宫实验与对照组相比,模型①组仔鼠水中逃避潜伏期及跨过原平台次数无统计学意义(P>0.05);模型②组仔鼠第1、2天水中逃避潜伏期比对照组仔鼠延长(P<0.05),第3、4天无统计学差异,跨过原平台次数与对照组无差异(P>0.05);模型③组仔鼠水中逃避潜伏期始终较对照组延长,第5天撤除平台后,跨过原平台所在位置的次数明显少于对照组(P<0.05)。5)仔鼠的红藻氨酸诱发癫痫发作实验模型①组仔鼠癫痫发作的潜伏期和持续时间及死亡率与对照组比较无差别(P>0.05);模型②组仔鼠癫痫发作的潜伏期缩短(P<0.05),但癫痫发作持续时间及死亡率与对照组比较无统计学差异(P>0.05);模型③组仔鼠痫性发作的潜伏期缩短,癫痫持续状态时间较对照组延长,且死亡率明显高于对照组(P<0.05)。6)仔鼠的大脑组织形态、脑湿重及病理学比较模型①组仔鼠大脑形态和脑湿重与对照组相比无差异,无DCDs病理改变;模型②组仔鼠大脑外形及脑湿重与对照组无明显差别(P>0.05),DCDs发生率为41.67%;模型③组仔鼠脑组织重量减轻、形态异常,随着鼠龄的增长差别越明显,DCDs发生率为100%。典型病理改变包括:大脑皮质厚度变薄、层状结构紊乱、神经细胞缺失或者发育不良,皮质及海马神经元异位等,类似于人类皮质发育不良和神经元结节状异位。2 DCDs模型大鼠海马神经元bcl-2、bax表达TUNEL、免疫组化和RT-PCR检测结果显示:与相应时间点的对照组比较,P0时模型组海马区神经元凋亡和bax、bcl-2在mRNA和蛋白水平表达无明显变化;而在P15、P30、P45、P60时模型组神经元凋亡细胞数逐渐增加,有显著差异(P<0.05),模型组海马神经元bax、bcl-2在mRNA和蛋白水平表达以及Bax/Bcl-2比值均较对照组增高,具有显著统计学意义(p<0.05)。3生物素葡聚糖胺(BDA)对皮质神经元的示踪在大脑皮质层和海马区注射BDA后,皮质、海马神经元吸收良好,可以清楚看到神经元的胞体、树突及轴突。4托吡酯对DCDs致IE的疗效观察100例中37例完全停止发作,33例发作频率减少≥75%,20例发作频率减少≥50%,托吡酯治疗DCDs致IE的总有效率90%,无明显不良反应。结论:1.给予孕17d SD大鼠腹腔注射BCNU 15mg/kg可以制作出理想的DCDs动物(大鼠)模型,而腹腔注射BCNU5mg/kg、10mg/kg和20 mg/kg不能成功建立DCDs模型;DCDs模型仔鼠生长发育差、行为学异常、学习记忆障碍、癫痫发作阈值低,具有典型的DCDs病理改变,很好地模拟人类DCDs的行为学和病理学特征;本模型仔鼠DCDs能作为人类大脑皮质发育不良及神经元异位的动物模型,与国外孕15d腹腔注射BCU N20mg/kg模型相比仔鼠存活率更高,操作简单,是一种新的、实用的DCDs动物模型。2.皮质发育障碍模型鼠海马区神经元在P0时凋亡和正常鼠没有差异,而在P15、P30、P45、P60时存在显著的凋亡,与相应时间点的对照组比较有显著的差异;凋亡基因bax、bcl-2在mRNA和蛋白水平表达以及bax/bcl-2在P0时没有差异,而在P15、P30、P45、P60时,与相应时间点的对照组比较均增高,有显著的差异,说明bax、bcl-2参与了其凋亡过程,皮质发育障碍模型鼠海马神经元凋亡可能与皮质发育障碍所致癫痫和难治性癫痫等临床症状密切相关。3. BDA能较好的显示皮质和海马神经元的胞体、轴突和树突,并具有生物性稳定好、转运距离远等优点,实验方法简单,为大鼠皮质和海马神经元的形态学研究和本课题组对皮质发育障碍的与癫痫和难治性癫痫相关的异常神经网络研究提供了可靠的技术平台。4.新一代抗癫痫药物托吡酯单药治疗DCDs所致IE的控制率、显效率和有效率好,无严重的副作用,可以作为DCDs所致IE的首选药物。更多还原

【Abstract】 Objective:The dissertation is to investigate the optimal dose and methods of the established animal model of disorder of cortical developments (DCDs) induced by Carmustine, to explore the mechanism of apoptosis in hippocampus of the rats with DCDs and correlated apoptosis, to estimate the application effects of BDA as a nerve tracer in rat cortical and hippocampus neuron for the study of abnormal neural network of DCDs, and to study the clinical effective of topiramate for intractable epilepsy by DCDs.Methods:1 The establishment of rat model of DCDs induced by CarmustinePregnant Sprague-Dawley rats were given intraperitoneal injection of carmustine with different doses at embryonic day 17. The rats in model one were given injection of carmustine with 5mg/kg, the rats in model two 10mg/kg, the rats in model three 15mg/kg, and the rats in model four 20mg/kg. The histologic alterations are suggestive of DCDs in rat offspring with the control group treated shamly.The following aspects were observed in all groups. We calculated the survival rate of rat offspring in each group and observed their general state on birth and the postnatal daily behaviors including consciousness, viability and epileptic seizure. The weight reflecting developmental condition of rat offspring were measured at P0,P7,P14,P21,P56 and P84. We adopted Morris-water-maze experiment to test the study and memory capacity of rat offspring in each group and used Kainic acid inducing seizure to compare their seizure threshold and mortality induced by seizure. After measuring the wet weight of brains and observing their appearance, we examined the changes of brain pathology in cerebral cortex and hippocampus to confirm the types of DCDs at P21 and P84. We assessed whether we could establish the animal model of DCDs induced by Carmustine successfully by above aspects or not. We also judged the best drug dose of successful model and evaluated the credibility of methodology in the animal model of DCDs.2 The expression of bax and bcl-2 in hippocampus of the rats with disorder of cortical developmentsRats with DCDs were established by BUCN. AT P0, P15, 30, P45, and P60, the number of apoptosis cell in hippocampus were detected by TUNE,the protein expression of Bax and Bcl-2 were detected by immunohistochemistry method, the mRNA of bax and bcl-2 were detected by RT-PCR method in the BCNU-exposed groups and the control groups at the same age, then whose outcomes was analyzed by computer micrograph analysis system.3. Application of BDA as a nerve tracer in rat cortical neuronAdult SD rats were used in this experiment and 10%BDA was injected slowly into the bilateral sensory and motor cortex and hippocampus with a mini syringe through four round-shaped bone windows with 3mm diameter, formed by a dental dril1, with the total volume 4u1.After survival for 14 days, brains were removed and stored in a freezer. Sections were made in a cryostat,and stained by the free-floating method for BDA before the morphous of these neurons were observed.4 Clinical effects of Topiramate for the Intractable Epilepsy by Disorders of Cortical DevelopmentA group of 100 patients with intractable epilepsy by DCDs were diagnosed by MRI and physician of neurology, and treated with topiramate(100mg.d-1～200mg.d-1). We observed their change of seizures frequency compared with the basic-line before treatment.Results:1. The assessment of rat model of DCDs induced by Carmustine1) The survival rate of rat offspring in models group and control groupThe survival rates of rat offspring in control group , model one, model two, model three and model four were 100%, 100%, 93.1%, 83.3%and 0% respectively. All the rat offsprings in model four died at P0.2) The results of general ethology observationThe observation results of rat offspring in model one were similar with those of control group. No abnormality existed in model two at the process of growth and development. The rat offspring in model three were poorer than control group on birth, followed by fewer activities, dullness and disturbance of intelligence in daily life. No obvious seizure was observed in all models group.3) Condition of growth and development in rat offspringThe rat offsprings in control group and model one were found to have no difference in weight (P>0.05). The mean weight of rat offspring in model two reduced slightly at P0(P<0.05), but the difference of weight disappeared when they became adult. The rat offspring in model three indicated an overall reduction in mean weight at every time point, so the phenomenon was significantly with age(P<0.05).4) Morris-water-maze experimentThe rat offspring in model one did not show any disparity with control group on the escape latency and times of crossing platform (P>0.05.The escape latency of rat offspring in model two was longer than that of control group on first day and second day, but the difference disappeared on third day and fourth day. The times of crossing platform of rat offspring in model two was equal with that of control group(P>0.05. In the training period escape latency of rat offspring in model three was always longer than that of control group(P<0.05). After removing platform on fifth day, the times of crossing platform of rat offspring in model three was not so many as that of control group(P<0.05).5) Kainic acid inducing seizure experimentThere was no difference between model one and control group on latent period and time duration of Kainic acid inducing seizure and mortality induced by seizure of rat offspring(P>0.05). The latent period of Kainic acid inducing seizure was shortened in model two(P<0.05), but the time of epileptic state and the mortality induced by seizure of rat offspring in model two were the same as that of control group(P>0.05). The rat offspring in model three had a significantly lower seizure threshold and longer time of epileptic state than those of control group, the mortality induced by seizure of rat offspring was obviously higher than that of control group(P<0.05).6) Comparison of cerebral appearance and brain weight and pathological examination of brainThe disparation of cerebral appearance and brain weight didn’t exist between model one and control group. There was no pathologic change in model one. Rat offspring in model two have no significant change in weight and construction of brain, the incidence rate of DCDs was 41.67%. Reduced weight and abnormal construction of brain were displayed in model three, the difference increased with age. The incidence rate of DCDs reached 100%. Typical pathological features included that model rats showed a thin cortical plate and disorder of layer structure, nerve cells in cerebral cortex became thinner and dysphasia, and distinct clusters of neuronal elements that represented heterotopias emerged in cerebral cortex and hippocampus. These morphologic features of DCDs found in this model were shared with cortical dysphasia and neuronal heterotopias in humans.2. Expression of Bax and Bcl-2 in hippocampus of the rats with DCDs at distinct time pointAt P15, P30, P45, and P60, the number of apoptosis and Bax/ bcl-2 masculine neuron, and the mRNA of bax/ bcl-2 in hippocampus in the model group are higher than that in the control group. The ratio of Bax/Bcl-2 in model group was significantly higher than those in the control groups (P<0.05), but at P0 there were not differences.3. Application of BDA as a nerve tracer in rat cortical neuronThe BDA injected into the bilateral sensory and motor cortex and hippocampus is taken by the axon and soma well,and the soma, the axon and dendrite of neuron are observed clearly with a 200×magnification.4. Clinical effects of Topiramate for the Intractable Epilepsy by Disorders of Cortical DevelopmentThere are 37 patients seizure free, 33 patients the frequency reduced more than 75%, and 20 patients more than 50% with the total effective reached 90%. All subjects didn’t have obvious side effects.Conclusions:1.The method that pregnant SD rats are given intraperitoneal injection of carmustine with 15mg/kg on embryonic day 17 can establish an ideal model of DCDs in offspring. But carmustine treatment with 5mg/kg or 10mg/kg or 20mg/kg of pregnant rats can not successfully establish the model of DCDs in offspring. Rat offsprings in the model show poor growth and development, abnormal behaviors, disorders in study and memory , lower threshold of epileptic seizure and have typically pathologic change of DCDs. They commendably reveal the features of ethology and pathology in humans. The neuronic abnormality of neuropeptide Y and calretinin in the brain of model rat offspring provide theoretical clue for the mechanism of epileptic seizure induced by DCDs. DCDs of rat offspring in the model can simulate cortical dysplasia and neuronal heterotopia that is similar in humans. The rat offspring in our model have higher survival rate than that of rat offspring in mode that carmustine treatment with 20mg/kg of pregnant rats on embryonic day 15 in abroad. Our model has better repeatability and being a new , pragmatic animal model of DCDs.2.At P15, P30, P45 and P60, Apoptosis exists notably in hippocampus of rat of disorder of cortical developments.The protein and the mRNA expression of bax and bcl-2 are higher in model group than those in the control group, but at P0, there were no differences. So we can conclude that bax and bcl-2 take part in apoptosis of hippocampus neuron, which may be associated with the clinical spectrum of DCDs, such as epilepsy and IE.3. BDA can demonstrate the soma, the axon and dendrite of neuron in cortex and hippocampus. It is biologically stable and has good availability , and can be transported for a long distance. The experiment process is simple and provides a reliable method for the morphological study of cortical and hippocampus neuron and for the study of abnormal neural network of DCDs correlated to epilepsy and IE.4.As a new antiepilepsy drug, topiramate to IE by DCDs, has a good control rate, excellence rate and effective rate, and has no severe side effect. So we can conclude that topiramate can be a choice drug for IE by DCDs.更多还原