【作者】 何选丽；

【导师】 晏勇；

【作者基本信息】 重庆医科大学 ， 神经病学， 2007， 硕士

【摘要】 目的:用卡莫司汀(BCNU)诱导大鼠皮质发育障碍(DCDs)动物模型,探索BCNU构建DCDs动物模型的最佳剂量和方法,为研究DCDs及其致痫机制提供新的动物模型。方法:1.BCNU诱导大鼠皮质发育障碍模型的建立及评估:采用BCNU不同剂量(以BCNU 5~20mg/kg梯度剂量)给妊娠17天的SD大鼠作腹腔注射,制作子代SD大鼠DCDs模型。按药物不同注射剂量分为模型①组(BCNU 5mg/kg)、模型②组(BCNU 10mg/kg)、模型③组(BCNU 15mg/kg)和模型④组(BCNU 20mg/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日常行为,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)、P21、P56和P84时间点测量各组仔鼠体重(反映仔鼠生长发育情况)。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利用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组织观察其大体形态、测脑湿重;作常规病理检查,观察各组仔鼠大脑皮质和海马结构,以确定是否存在DCDs以及DCDs的病理类型。通过以上各项指标判定BCNU建立的DCDs模型是否成功及成功建模的最佳药物剂量和方法学的可靠性。2.相关神经递质检测:用免疫组织化学方法检测神经肽Y(NPY)和钙视网膜蛋白(Calretinin)阳性神经元在最佳剂量模型组仔鼠大脑中的分布,以初步探索该模型的致痫机制。结果:1.各模型组及对照组仔鼠的存活率: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.仔鼠一般行为学观察:模型①组仔鼠各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,出现活动量减少、反应迟钝、智能障碍等表现。各模型组没有观察到明显的自发性癫痫发作。3.仔鼠生长发育情况: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差异消失;与对照组相比模型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.水迷宫实验:模型①组仔鼠水中逃避潜伏期及跨过原平台次数和对照组无统计学意义(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天无统计学差别,跨过原平台次数与对照组无差异(P>0.05);训练期间模型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.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.大脑组织形态、脑湿重及病理学检查比较:模型①组仔鼠大脑组织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组仔鼠与对照组相比脑组织重量减轻、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、神经细胞缺失发育不良,皮质及海马神经元异位等,类似人类皮质发育不良和神经元结节状异位。7.神经递质检测:免疫组织化学结果显示模型③组仔鼠大脑皮质和海马神经肽Y神经元明显减少(P<0.05);钙视网膜蛋白神经元在模型③组仔鼠大脑皮质和海马异常表达,且神经元发育不良。结论:1.本实验给予孕17d SD大鼠腹腔注射BCNU 15mg/kg可以制作出理想的仔鼠DCDs模型,而腹腔注射BCNU5mg/kg、10mg/kg不能成功建立DCDs模型,20 mg/kg的剂量过大。2.本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,又具有典型的DCDs病理改变,很好地体现了人类DCDs的行为学和病理学特征。3.本模型仔鼠大脑中神经肽Y和钙视网膜蛋白神经元的异常为DCDs易致痫性发作的机制提供了理论依据。4.本模型仔鼠DCDs能模拟人类大脑皮质发育不良及神经元异位,与国外孕15d腹腔注射BCNU 20mg/kg模型相比仔鼠存活率更高,模型的重复性更好,且操作简单,是一种新的、实用的DCDs动物模型。更多还原

【Abstract】 Objective: To investigate the mechanism underlying disorders of cortical development (DCDs), We developed an animal model of DCDs induced by Carmustine and explored the optimal dose and methodology of established animal model of DCDs.Methods:1.The establishment and assessment of rat model of DCDs induced by Carmustine: Pregnant Sprague-Dawley rats were given intraperitoneal injection of carmustine with different doses on embryonic day 17. The rats in model one were given injection of carmustine with 5mg/kg. The rats in model two were given injection of carmustine with 10mg/kg. The rats in model three were given injection of carmustine with 15mg/kg. The rats in model four were given injection of carmustine with 20mg/kg. BCNU exposure in utero produces histologic alterations suggestive of DCDs in rat offspring. The control group were untreated.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 body weight reflecting developmental condition of rat offspring were measured at P0, P7, 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 measureing the wet weight of brains and observing their appearance, we examined the changes of brain pathology in cerebral cortex and hippocampus to confirm the typies of DCDs at P21 and P84.we assessed that we could or not establish the animal model of DCDs induced by Carmustine successfully by above aspects.we also judged the best drug dose of successful model and evaluated the credibility of methodology in the animal model of DCDs.2.Detection of related neurotransmitters: To explore the seizure mechanism underlying DCDs, immunohistochemistry method was used to detect the neuronal distribution of neuropeptide Y and Calretinin in brains of rat offspring of optimal dose model.Results:1.The survival rate of rat offspring in models group and control group: The 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.The all rat offspring in model four were death at P0.2.The results of general ethology observation: The observation results of rat offspring in model one were similar with those of control group. No abnormity existed in model two at the process of growth and development. The rat offspring in model three were poor than control group on birth, followed by less activities, dullness and disturbance of intelligence in daily life. No obviously seizure was observed in all model groups.3.Condition of growth and development in rat offspring: The rat offspring in control group and model one were found to have no difference in body weight (P>0.05). The mean weight of rat offspring in model two was reduced slightly at P0 (P<0.05), but the difference of weight was disappear when they were adult. The rat offspring in model three indicated an overall reduction in mean weight at every time point, the phenomenon was significantly with age (P<0.05).4.Morris-water-maze experiment: The rat offspring in model one did not showed 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 at first day and second day, but the difference was disappear at 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 experiment: There 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 as 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 brains: The disparation of cerebral appearance and brain weight were unexist 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 displaied in model three, the difference was increased with age. The incidence rate of DCDs was 100%. Typical pathological features included that model rats showed a thin cortical plate and disorder of layer structure, nerve cells in cerebral cortex were absence and dysplasia, distinct clusters of neuronal elements that represent heterotopias emerged in cerebral cortex and hippocampus. These morphologic features of DCDs found in this model were shared with cortical dysplasia and neuronal heterotopias in humans.7.Detection of neurotransmitters: The results demonstrated that neuronal numbers of neuropeptide Y were decreased obviously in cerebral cortex and hippocampus of rat offspring in model three (P<0.05). The neuronal architecture and expression of calretinin were abnormal in rat offspring in model three.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 to pregnant rats can not establish successfully the model of DCDs in offspring.2.Rat offspring in the model show poor growth and development, abnormal behaviors, disorders of study and memory, lower threshold of epileptic seizure and have typically pathologic changes of DCDs, these commendably reveal the features of ethology and pathology in humans.3.The neuronic abnormity of neuropeptide Y and calretinin in the brain of model rat offspring provides theoretical clew for the mechanism of epileptic seizure induced by DCDs.4.DCDs of rat offspring in the model can simulate cortical dysplasia, 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. The model has better repeatability and being a new, pragmatic animal model of DCDs.更多还原