雷帕霉素对X-射线诱导的大脑皮质发育畸形大鼠老年期认知功能及行为学的影响

发布时间：2018-03-03 06:00

本文选题：大脑皮质发育畸形　切入点：雷帕霉素　出处：《西南医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:探讨雷帕霉素(RAP)对X-射线宫内照射诱导大脑皮质发育畸形(MCD)模型大鼠老年期认知损害及行为学的影响,为进一步研究MCD的发病机制及早期干预、治疗打下基础。方法:(1)将6只健康妊娠17天的SD(E17)大鼠随机分配为3组,即正常组、二甲基亚砜(DMSO)注射的175cGy X-射线照射组(DMSO组)、RAP治疗的175cGy射线照射组(RAP组)各2只,各孕鼠所生鼠一代(F1)饲养至老年期(满18月龄)后作为实验对象;(2)行为学观察:采用视频监控自然光线下进行连续24小时录像,对治疗前、治疗中、及治疗后期间监控,持续3月,抽取其中30天,每天抽取10小时,人工观察分析大鼠活动、饮水、摄食、昼夜节律及是否有自发性癫痫发作等;(3)水迷宫实验:通过Morris水迷宫(MWM)测定大鼠药物治疗前后的定位航行及空间探索实验,比较大鼠空间学习记忆能力的变化。(4)实验动物于实验终止后或死亡后取脑组织,观察大鼠脑大体标本,进行巨检标本采图,对脑组织分别进行冰冻切片,行Nissl染色分析。结果:(1)行为学观察:药物治疗前、治疗中及治疗后三月期间,老年大鼠活动性较青年均减少,正常组无自发性癫痫发作,MCD大鼠较正常组夜间活动多(洗脸、舔毛、搔痒等动作较多,追逐、打架行为频繁)。随着鼠龄的增加,活动逐渐减少,行动变缓等情况。治疗前,DMSO组、RAP组≤2级(Racine分级)的癫痫发作次数较正常组多,药物治疗后RAP组癫痫发作次数减,整个实验过程中未见典型的Racine分级三级及其以上的自发性癫痫发作。(2)MWM药物治疗前定位航行实验:3组大鼠随着训练天数的增加,逃避潜伏期逐渐缩短,DMSO组、RAP素组逃避潜伏期较正常组延长,差异具有统计学意义(p0.05),DMSO组与RAP组比较差异无统计学意义;药物治疗后:DMSO组、RAP组逃避潜伏期仍较正常组延长,差异有统计学意义(p0.05)。DMSO组与RAP组相比,逃避潜伏期无明显差异,药物治疗后各组逃避潜伏期与药物治疗前比较差异无统计学意义。空间探索测定:药物治疗前DMSO组、RAP组穿越平台次数、目标象限时间比明显少于正常组(p0.05),DMSO组与RAP组比较无差异;药物治疗后RAP组、DMSO组目标象限时间比、穿越平台次数较正常组减少(p0.05),RAP组与DMSO组比较无差异。正常组、DMSO组、RAP组治疗后与治疗前比较无差异。(3)大体标本及病理学分析:DMSO组、RAP组脑体积较正常组减小,以大脑半球缩小明显,四叠体暴露。冠状切面观察:胼胝体可见缺失,皮质变薄;切片过程中,3组均可见脑组织多发白色钙化点,主要分布于白质。Nissl染色:DMSO组、RAP组大鼠与正常组比较六层皮质变薄,皮质结构紊乱,纵向、横向皮质层次分界模糊,细胞排列紊乱、稀疏,皮质内及皮质下组织可见大小不等的神经元异位结节。RAP组、DMSO组可见海马结构异常,连续性中断,CA 1、CA 2区可见数量、大小不等的异位结节。海马结构可见神经元分散现象。根据FCD分级:RAP组、DMSO组均可见有皮层异位结节,胼胝体缺失、海马异常,FCD分型为FCDⅠ-Ⅱ型。结论:(1)X-射线宫内照射E17大鼠能较好的模拟人类MCD,具有脑皮质结构紊乱、室管膜下结节、海马结构异常、认知损害及行为学改变等特点。(2)X-射线诱导的MCD大鼠老年期具有出现认知能力功能早,认知损害严重的特点。(3)RAP对MCD模型大鼠老年期具有保护作用,改善行为学表现及低级别的癫痫发作,但不能改善认知损害及逆转病理学损害。
[Abstract]:Objective: To investigate the effect of rapamycin (RAP) on X- ray irradiation induced intrauterine cortical malformation (MCD) effect of senile rat model of cognitive impairment and behavior, for further study of pathogenesis of MCD and early intervention, to lay the foundation for treatment. Methods: (1) 6 healthy pregnant 17 days SD (E17) rats were randomly divided into 3 groups, namely the normal group, two dimethyl sulfoxide (DMSO) 175cGy X- ray injection group (group DMSO), 175cGy ray irradiation group RAP treatment (group RAP) 2 rats each, the pregnant rats born generation rats (F1) fed to the old age (18 month old) as the experimental object; (2) behavioral observation: the use of video surveillance under natural light for 24 hours of video, before treatment, treatment, after treatment and during continuous monitoring, March, from which 30 days, every day from 10 hours, were analyzed by artificial observation activities, drinking water, food intake the circadian rhythm, and whether there is spontaneous epilepsy Seizure; (3) water maze test: the Morris water maze (MWM) determination of positioning navigation and space of rats before and after drug treatment experiment, changes of spatial learning and memory abilities of rats. (4) experimental animal experiment after the termination or after the death of brain tissue, observing specimen of rat brain giant, biopsy specimens of brain tissue were collected, frozen section, Nissl stain analysis. Results: (1) the behavior observation: drug therapy before March, during and after the treatment period, aged rats compared to young activity decreased, the normal group without spontaneous seizures, compared with normal MCD rats group activities at night (wash his hair, more, itch action chase, frequent fights). With the age of rats, activity decreased, action slow. Before treatment, DMSO group, RAP group is less than or equal to 2 grade (Racine grade) the number of seizures compared with the normal group, drug therapy Group RAP after seizure frequency reduction, the whole experiment process no typical Racine grade three and above spontaneous seizures. (2) MWM before drug treatment navigation test: 3 groups of rats with the increase of the number of training days, the escape latency shortened gradually, DMSO group, RAP group, the escape latency longer than normal group, the difference was statistically significant (P0.05), DMSO group and RAP group had no significant difference; after drug treatment: DMSO group, RAP group, the escape latency is still longer than normal group, the difference was statistically significant (P0.05) in.DMSO group compared with RAP group, the escape latency had no significant difference after treatment were compared to escape the incubation period and before drug treatment was not statistically significant. The determination of space exploration: before drug treatment group DMSO, RAP group the number of crossing platform, the target quadrant time ratio is lower than normal group (P0.05), there was no significant difference between DMSO group and RAP group; After treatment, RAP group, DMSO group of target quadrant time ratio, the number of crossing platform less than the normal group (P0.05), there was no significant difference between RAP group and DMSO group. The normal group, DMSO group, RAP group before and after treatment. No difference (3) specimens and pathological analysis: DMSO group, RAP brain volume group than the normal group decreased to cerebral hemisphere reduced significantly, quadrigeminal exposure. Coronal section observation: corpus callosum visible lack of cortical thinning; slice process, 3 groups were seen in brain tissue of multiple white calcification, mainly distributed in the white matter of.Nissl staining: DMSO group, RAP group and normal group rats comparison of six layers of cortical thinning, disorder, cortical structure of longitudinal, lateral cortical level fuzzy boundaries, the cells arranged in disorder, sparse, ectopic neuronal nodules of.RAP group showed the size of the organization within the cortical and subcortical hippocampus, DMSO group showed abnormal structure, continuous interruption, CA 1, CA 2 showed a large number. Small nodules ranging from ectopic hippocampal neurons. The visible dispersion phenomenon. According to FCD classification: RAP group, DMSO group showed a cortical heterotopic nodules, agenesis of the corpus callosum, hippocampus, FCD type of FCD I / II. Conclusion: (1) X- ray irradiation of intrauterine E17 rats can mimic human MCD. That is the cerebral cortex structure disorder, subependymal nodules, hippocampal structural abnormalities, characteristics of cognitive impairment and behavioral changes. (2) X- ray induced senile MCD rats with cognitive function appeared early, severe cognitive impairment characteristics. (3) RAP has a protective effect on senile MCD rats rats, improve the behavioral performance and low level of epilepsy, but can not improve the cognitive impairment and reversion of pathological damage.

【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742

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