星形胶质细胞在卵巢摘除结合注射D-半乳糖大鼠复合AD模型中的病理改变与功能障碍

【作者】 花向东；

【导师】 丁炯； 肖明； 韩群颖；

【作者基本信息】 南京医科大学 ， 人体解剖学， 2008， 硕士

【摘要】 目的:1.通过OVX结合注射D-gal建立复合因素AD动物模型,阐明雌激素剥夺和氧化应激在AD发生发展中的协同作用;2.研究与探索星形胶质细胞参与此AD模型的发病过程及机制。方法:1.3月龄雌性SD大鼠,随机分为对照组(C)、OVX结合注射D-gal模型组(O+D)、单纯OVX组(O)、单纯注射D-gal组(D)和17-β雌二醇替代治疗组(O+D+E),6周后进行行为学测定、基底前脑胆碱能神经元ChAT免疫组化染色、海马区Aβ免疫组化染色、海马及皮质电镜超微结构观察和脂褐素与突触的定量分析来验证此模型。2.应用此模型(O+D),以2周和6周为观察时间点,通过海马区GFAP的免疫组化染色和western-blotting检测、GSH的生化检测、AChE组织化学染色与光密度分析及AChE活性测定、突触和血-脑屏障周围超微结构观察以及HRP通透性实验来研究星形胶质细胞在此模型中的病理改变与功能障碍。结果:1.与C组相比,O+D组、O+D+E组、D组、O组均出现不同程度的行为学和病理学损害,以O+D组损害最为明显,具体表现为:(1)开场实验中,穿越格数明显减少;Y型迷宫达到学会标准的测试次数增多;跳台实验中,错误次数增加、潜伏期缩短。(2)在MS、NDB区域的ChAT免疫阳性胆碱能神经元明显减少,染色变浅,突起变短。(3)在海马的齿状回、CA1和下托观察到了其他组没有出现的细胞内Aβ沉积。(4)超微结构损害:细胞核变形扭曲;核内染色质聚集成不规则的高电子密度的块状;核膜破裂、不完整;胞浆内含有大量脂褐素、次级溶酶体;线粒体出现肿胀扭曲、变形,嵴断裂、空泡样变;细胞质内出现类似NFT改变;大量变性的突触结构。脂褐素数量明显增加,突触数量明显减少。2.(1)与C组和2w O+D组相比,6w O+D组大鼠在水迷宫行为学实验中,到达平台的潜伏期明显延长;穿越平台次数和在目标象限时间明显减少,而C组与2wO+D组大鼠之间没有统计学差异。(2)与C组和2w O+D组大鼠相比,6w O+D组大鼠在海马区出现了明显的胆碱能纤维终末的密度下降与AChE活性降低,而C组与2w O+D组大鼠之间没有统计学差异。(3)与C组相比,2w与6w O+D组大鼠海马区GFAP水平明显增加,但两组间没有统计学差异。(4)与C组和2w O+D组相比,6w O+D组出现了明显的GSH水平的降低。而2w O+D组则观察到了GSH水平的轻度升高,但与C组相比并没有明显的差异。(5)在2w和6wO+D组大鼠海马区出现了广泛的星形胶质细胞活化。与C组静息态星形胶质细胞细长的突起相比,两组O+D活化的星形胶质细胞胞体肥大,突起增粗深染,呈高度分支状;此外,许多6w O+D组GFAP阳性的星形胶质细胞出现了胞体类似崩解的退行性改变。(6)电镜结果显示6w O+D组大鼠海马内星形胶质细胞出现了糖原颗粒和胶质纤维丝的聚集或缺失、线粒体的肿胀或分布异常为特征的退行性改变。尤其在海马突触密集的区域,水样变性的星形胶质细胞突起高度肿胀,包绕着变性的突触结构。此外,在许多2w和6w O+D组大鼠海马脑毛细血管周围包绕着许多肿胀的星形胶质细胞终足。尤其在6w O+D组大鼠毛细血管内皮细胞出现扭曲变形和空泡化。上述病理改变并没有在C组出现。(7)6w O+D组大鼠的海马及大脑皮质的一些区域出现了血管周围区域HRP的泄漏。而在C组HRP反应产物则分布在血管内,没有明显的外泄。结论:1.雌激素剥夺和氧化应激在AD发生发展中的协同作用。2.OVX结合注射D-gal可以作为一个较好模拟AD病症的动物模型。3.星形胶质细胞在此模型病变的发生发展中起到了重要作用。更多还原

【Abstract】 Objective:1.To develop a new multiplicity AD model by long-term intraperitoneal administration of D-gal in OVX rats to further address the synergistic effects of estrogen deprivation and oxidative stress on the development and progression of AD.2.To investigate whether astrocytes are involved in the pathogenesis of this AD model.Methods:Three-month-old female Sprague-Dawley rats were randomly and equally divided into five groups:the intact control group(C),the OVX and D-gal injected group(O+D),the OVX-only group(O),the sham operated and D-gal injected group(D)and the OVX,D-gal and 17-βestradiol injected group(O+D+E).After six weeks,several behavioral tests as well as ChAT immunohistochemistry of cholinergic neuron in basal forebrain and Aβimmunohistochemistry in hippocampus, observation of electron microscope,ultrastructural analysis of lipofuscin deposition and the density of synapses have been performed to evaluate the success of this AD model.To investigate the involvement of astrocytes in the pathogenesis of this AD model,We detected the expression of GFAP by immunohistochemistry and western-blotting, levels of GSH,ultrastructural changes of the astrocytes,with special attention on astrocytes surrounding the axonal terminals and brain microvessels as well as the permeability of blood-brain barrier by intravascular injection of HRP in the hippocampus in ovariectomized rats injected with D-gal for 2 and 6 weeks respectively.Results:1.Compared with C groups,behavior and pathology of the rats was prominently impaired in the D,O,O+D and O+D+E groups, especially the O+D groups,versus with which the learning impairment was attenuated significantly in the O+D+E animals.The more serious damages of O+D groups as follows:(1)The mean number of square entries was increased in the open field testing;The training frequency required to attain the learning criterion was markedly increased in the Y-maze;The number of errors was highest and the step-down latency decreased dramatically in step-down type passive avoidance testing.(2) The decreased number of cholinergic neuron suffered atrophy,indicated by the small soma and deficiency of the processes.(3)Many Aβimmunoreactive neurons which absent in the other four groups,were easily detected in the hippocampus mainly located at the pyramidal layer of CA1,subiculum and granular cell layer of the dentate gyrus.(4) Ultrastructural changes:Nuclear degeneration with condensed chromatin aggregated in the rim of the nucleus with a disrupted and incomplete nuclear membrane.Lipofuscin accumulated in the cytoplasm some of which contained granulovacuolar bodies.NFT appeared in the cytoplasm or dendrites of neurons.Some mitochondria showed disrupted cristae and a swollen,twisted or vacuolar appearance.Synapse degeneration was frequently observed in the CA1 region of the hippocampus,the obvious increased lipofuscin and decreased synapses.2.(1)The 6w O+D rats required longer times to reach the platform than control and 2w experimental rats.The control and 2w O+D rats spent the majority of their time swimming in the quadrant where the platform was located,while the 6w O+D animals tended to spend their time in all quadrants.No statistical differences were observed between the control and 2w O+D groups.(2)6w O+D rats showed a prominent loss of cholinergic terminals and AChE activity in the hippocampus,compared to the control and 2w O+D groups.No significant differences were found between the control and 2w O+D groups.(3)2w and 6w O+D groups showed significant increase in the protein levels of GFAP,compared to the control.No statistical significances were observed between the two O+D groups.(4)The 6w O+D group exhibited significant decrease of GSH levels,compared to the control and 2w groups.A slight increase in GSH levels was observed in the 2w O+D group,but the difference was not significant,relative to the control.(5)Activated astrocytes were widespread in the all regions of the hippocampus in 2w and 6w O+D rats, relative to the controls.These activated glial cells exhibited hypertrophy, with very thick,highly ramified and intensely immunostained branches, compared to resting form with long,slender processes in the controls. Furthermore,many GFAP-positive astrocytes in the 6w O+D rats underwent degeneration,characterized by apparent breakdown of cell bodies.(6)Eelectron microscopy showed that some astrocytes were degeneration in the hippocampus of 6w O+D rats,identified by reduction or devoid of glycogen granules and glial intermediate fibers,and swollen or aberrant appearance of mitochondria.Especially,in the aggregated area of synapse,extremely swollen astrocytic process with watery appearance often engulfed the degenerated synaptic structures.Swollen perivascluar astrocytic endfeet surrounding the brain capillary were frequently observed in both 2w and 6w O+D rats.Especially,in the 6w O+D rats,irregular-shaped microvessels were lined by distorted or vacuolized endothelium.The above pathological changes were not evident in the controls.(7)Perivascular areas of HRP leakages were observed in some regions in the hippocampus or the cerebral cortex of O+D rats,the marker showed an intravascular localization in the controls.Conclusions:1.These results strongly suggest that estrogen deprivation and oxidative stress behave synergistically to enhance the development and progression of AD.2.OVX combined with D-gal injection may serve as an ideal AD rodent model capable of mimicking pathological,neurochemical and behavioral alterations in AD.3. Biochemical and pathological alterations of astrocytes may partially contribute to exacerbating neuronal deficits in course of this AD model.更多还原