实验性蛛网膜下腔出血对大鼠行为和海马结构的影响

发布时间：2018-05-10 16:52

本文选题：蛛网膜下腔出血 + 脑脊液　；参考：《河北医科大学》2017年博士论文

【摘要】：蛛网膜下腔出血(Subarachnoid hemorrhage,SAH),是一种临床常见的危重病症,泛指脑底部或脑表面的病变血管破裂,血液直接流入蛛网膜下腔引起的一种临床综合征。根据出血部位和出血量的不同,治疗效果差异很大。SAH常见的并发症有,再出血、脑血管痉挛和急慢性脑积水(hydrocephalus)等。有些SAH的幸存者,可出现进行性脑室增大、脑皮质萎缩和认知功能障碍甚至变得痴呆。阿尔兹海默病(Alzheimer's disease)简称AD,又称早老性或老年性痴呆,是临床上最为常见的痴呆类型。相继的研究发现,它是一种以进行性学习、记忆和认知功能障碍为主要临床表现的神经退行性疾病;目前对其发病机制仍不十分清楚,也无明确有效的防治方法。我们好奇,是否这种SAH后遗症的发生,与AD的发生和发展存在相似的病理机制?众所周知,正常的脑脊液(Cerebrospinal fluid,CSF)循环,对于脑组织代谢产物,尤其是脂质、蛋白等大分子物质的转运,维持中枢神经系统内环境的相对稳定,发挥着难以替代的作用。所以有理由推测,是否由于不同原因引起的慢性CSF循环障碍或脑积水,导致了脑组织代谢产物转运异常,诱发了脑组织出现病理性变化和功能受损?本研究模拟蛛网膜下腔出血,建立慢性CSF循环障碍的大鼠模型,观察和检测实验动物的行为变化、海马皮质神经细胞内线粒体跨膜电位、以及海马皮质、侧脑室室管膜和脉络丛的细微和超微结构变化,检测海马皮质内相关蛋白表达的变化等。本实验研究分为以下三部分。第一部分实验性蛛网膜下腔出血大鼠模型的建立和对其行为影响的观察目的:本部分研究实验性蛛网膜下腔出血(SAH)继发的脑损伤,对大鼠认知功能产生的伤害性影响,为实验性SAH对海马皮质、侧脑室室管膜以及脉络丛损伤机制的研究,提供行为学证据。方法:选取成年SD大鼠60只,平均体重为300.40±44.50 g,雌雄不拘;将60只大鼠随机分为试验组和对照组,各组30只。试验组大鼠用10%的水合氯醛进行腹腔注射麻醉后,向脑枕大池(或称小脑延髓池)内注入自体动脉血0.30 ml,制作模拟SAH的实验动物模型;对照组大鼠只是向枕大池内注射等量生理盐水,其余操作步骤同试验组。同时,对试验组和对照组大鼠(各10只),术后进行常规喂养,并于术后2个月、4个月、6个月时,进行一般行为学统计分析、神经功能整体评分和行为学研究三个方面的测试,统计和分析相关数据。结果:与对照组相比,试验组大鼠术后出现精神萎靡不振、嗜睡、饮食减少,肢体活动力减低等神经功能缺损的表现,P0.05,差异具有统计学意义;并且随着饲养时间的延长,这种现象更加明显,P0.05,差异具有统计学意义;在Morris水迷宫试验的测试中,与对照组相比,试验组大鼠的空间学习能力以及记忆能力明显下降,P0.05,差异具有统计学意义;并且随着饲养月份的延长,试验组大鼠的学习、记忆、认知功能和活动能力逐渐减退,P0.05,差异具有统计学意义。第二部分实验性蛛网膜下腔出血诱发大鼠海马皮质和侧脑室室管膜及脉络丛的形态学变化目的:本部分研究主要通过光镜、电镜和流式细胞术,观察实验性蛛网膜下腔出血(SAH),诱发大鼠海马皮质、侧脑室室管膜及脉络丛的形态学变化,海马皮质神经细胞内线粒体跨膜电位(MTP)的变化,为实验性SAH对大鼠海马皮质、侧脑室室管膜及脉络丛的损伤机制,提供实验形态学证据。方法:选取成年SD大鼠90只,平均体重为300.20±27.50 g,雌雄不拘;将90只大鼠随机分为试验组和对照组,每组各45只。试验组大鼠用10%的水合氯醛进行腹腔注射麻醉后,向脑枕大池内注入其自体动脉血液0.30 ml,制作模拟SAH的实验动物模型;对照组大鼠只是向枕大池内注射等量生理盐水,其余操作步骤同试验组。试验组和对照组大鼠(各取10只)术后进行常规喂养,并于术后2个月、4个月、6个月时,通过主动脉灌注固定取材,进行光镜、扫描和透射电镜样品的制备;同时每组随机取5只大鼠,通过深度麻醉下断头取脑的方法剥离双侧海马,应用流式细胞术检测海马皮质神经细胞内MTP;通过荧光显微镜拍照,观察海马皮质神经细胞的凋亡情况;比较试验组与对照组大鼠海马皮质、侧脑室室管膜及脉络丛,在光镜、扫描和透射电镜下的细微和超微结构差异,以及两组大鼠海马皮质神经细胞内MTP的差异,揭示实验性SAH对大鼠相关脑组织结构的影响,为行为变化提供形态学依据。结果:试验组:光镜观察显示,随着术后饲养月份的增加,大鼠侧脑室逐渐增大,室管膜细胞由立方形变为多形性甚至扁平形;海马皮质表面的室管膜结构出现细微改变,在海马槽和多形细胞层,可观察到小血管周围间隙明显增宽,锥体细胞层明显变薄,细胞数量减少,细胞排列紊乱;并且,饲养时间越长,这种现象越明显。扫描电镜观察显示,在脉络丛上皮细胞表面,出现大量火山口样凹陷,其绒毛萎缩、扭曲甚至脱落;在海马皮质被覆的室管膜细胞表面,其纤毛和微绒毛明显减少。透射电镜观察显示,脉络丛上皮细胞可见大量的吞饮泡,胞体和胞核呈现不规则状;海马皮质锥体细胞核内的异染色质明显增多,细胞内出现膨大的线粒体、神经原纤维缠结的团块,在神经毡内的淀粉样脂蛋白沉积等现象;在术后饲养到6个月时,这种现象更为明显;表现为海马室管膜细胞表面的微绒毛减少更明显,大量的淀粉样脂蛋白沉积在血管周围。JC-1流式细胞术检测发现,海马皮质神经细胞内MTP去极化现象严重,与对照组相比,P0.05,差异具有统计学意义,提示神经细胞内线粒体发生不同程度的病理变化,细胞凋亡现象明显;并且随着饲养时间的延长,这种现象逐渐加重,P0.05,差异具有统计学意义。然而在对照组:术后2个月、4个月甚至6个月,大鼠的海马皮质、侧脑室室管膜及脉络丛,以及海马皮质神经细胞内MTP,均未观察到明显的病理变化。第三部分实验性蛛网膜下腔出血大鼠海马皮质内炎性和自噬相关蛋白表达的变化目的:以上述两部分的研究为基础,通过免疫组织化学法、Western blot以及TUNEL检测法,进一步探讨实验性蛛网膜下腔出血(SAH),对大鼠与学习、记忆相关的重要脑区—海马皮质内炎性和自噬相关蛋白表达的影响,揭示实验性SAH与AD之间的关联性。方法:选取成年SD大鼠90只,平均体重为289.40±45.50 g,雌雄不拘;随机将大鼠分为试验组和对照组,每组各45只。试验组大鼠用10%的水合氯醛进行腹腔注射麻醉后,向枕大池内注入自体动脉血0.30 ml,制作模拟SAH的实验动物模型;对照组大鼠只是向枕大池内注射等量生理盐水,其余操作步骤同试验组。同时,对试验组和对照组大鼠进行常规喂养,并于术后2个月、4个月、6个月时,随机每组取10只大鼠,经主动脉灌注固定取材,石蜡切片,通过免疫组织化学染色法,观察Aβ,IL-1β,IBA-1在海马皮质的表达情况;采用TUNEL检测法,观察大鼠海马皮质锥体细胞的凋亡情况。同时随机每组取5只大鼠,在深度麻醉下快速断头取脑,剥离大鼠双侧海马皮质结构,通过Western blot法,检测炎性相关蛋白IL-1β、自噬相关蛋白(Atg-5,Beclin-l和LC-3),在大鼠海马皮质的表达;记录并分析相关数据。结果:1 Western blot结果分析:与对照组相比,试验组大鼠海马皮质炎性相关蛋白IL-1β、自噬相关蛋白(Atg-5,Beclin-l和LC-3)的表达明显增多,P0.05,差异具有统计学意义;并且随着术后饲养时间的延长表达呈现上升趋势,P0.05,差异具有统计学意义。2免疫组织化学结果显示:与对照组相比,试验组大鼠在实验性SAH术后,海马皮质内Aβ、IL-1β、IBA-1的表达明显增多,并且随着术后饲养时间的延长呈现上升趋势。3 TUNEL检测结果表明:在实验性SAH术后,与对照组相比,试验组大鼠的海马皮质内锥体细胞凋亡现象增多,P0.05,差异具有统计学意义,并且随着术后饲养时间的延长而逐渐加重,术后饲养6个月时达高峰,P0.05,差异具有统计学意义。结论:1采用枕大池单次注射微量自体动脉血的方法,可以成功复制模拟SAH的实验大鼠模型,其病理变化与行为表现相一致,具有较为可靠的基础和临床研究参考意义。2实验性SAH可导致试验组大鼠的学习、记忆以及活动能力明显减退,并且随着术后饲养时间的延长,病情逐渐加重;提示实验性SAH可能对大鼠侧脑室室管膜、脉络丛、海马皮质等结构,造成了不同程度的病理性损伤。3实验性SAH可诱发大鼠海马皮质、侧脑室室管膜以及脉络丛的细微和超微结构变化,诱发海马皮质神经细胞内MTP去极化,出现神经细胞凋亡或坏死增多现象。4实验性SAH可诱发大鼠海马皮质炎性因子表达增高、细胞自噬作用增强,淀粉样脂蛋白沉积,以及海马皮质锥体细胞凋亡等现象,并且随着术后饲养时间的延长,愈发严重。
[Abstract]:Subarachnoid hemorrhage (SAH) is a common and critically ill disease. It refers to a clinical syndrome caused by the rupture of the vessels at the base of the brain or the surface of the brain and the blood flow directly into the subarachnoid cavity. According to the site of bleeding and the amount of bleeding, there are many common complications in the treatment of.SAH. Blood, cerebral vasospasm, and acute and chronic hydrocephalus (hydrocephalus). Some of the survivors of SAH have progressive ventricular enlargement, cortical atrophy and cognitive impairment or even dementia. Alzheimer's disease (Alzheimer's disease), referred to as premature or senile dementia, is the most common type of dementia in clinical. It has been found that it is a neurodegenerative disease with progressive learning, memory and cognitive impairment as the main clinical manifestation. The pathogenesis is still not very clear, and there is no clear and effective method of prevention and control. We wonder if this SAH sequela is similar to the occurrence and development of AD. It is known that the normal Cerebrospinal fluid (CSF) cycle is difficult to substitute for the transport of brain tissue metabolites, especially lipid, protein and other macromolecular substances, to maintain the relative stability of the central nervous system. Therefore, there is reason to speculate on the chronic CSF circulation disorder or brain caused by different causes. Water accumulation has caused abnormal transport of brain tissue metabolites and induced pathological changes and functional impairment of brain tissue. This study simulated subarachnoid hemorrhage, established a rat model of chronic CSF circulation disorder, observed and detected behavioral changes in experimental animals, mitochondrial transmembrane potential in the hippocampus, and the cortex and side of the hippocampus. Changes in the ultrastructure of the ventricular ependyma and choroid plexus and the changes in the expression of related proteins in the cortex of the hippocampus were detected. The study was divided into three parts. The first part of the experiment was to establish and observe the effects of the experimental subarachnoid hemorrhage model in rats: this part studies experimental subarachnoid hemorrhage (SAH). The effects of secondary brain injury on the cognitive function of rats, and the behavioral evidence for the study of the damage mechanism of the hippocampal cortex, the lateral ventricular ependyma and choroid plexus in the experimental SAH. Methods: 60 adult SD rats were selected, the average weight was 300.40 + 44.50 g, and the female male was not restricted. The rats were randomly divided into the experimental group and the control group. After intraperitoneal injection of 10% chloral chloral, the rats in the experimental group were injected with 0.30 ml of autologous arterial blood into the big pool of the brain occipital (or cerebellopontine medullary pool) to make an experimental animal model for simulating SAH. The control group was only injected with the same amount of normal saline in the large cistern, the rest of the operation were the same as the experimental group. At the same time, the experimental group and the test group were used in the control group. Rats in the control group (10 rats each) were fed with regular feeding after operation, and 2 months, 4 months and 6 months after the operation, the general behavioral statistical analysis, the overall neurological score and the behavioral study were tested in three aspects, and the related data were analyzed. Results: compared with the control group, the rats in the experimental group were depressed, drowsiness, drinking. P0.05, the difference has statistical significance, and with the prolongation of feeding time, the difference has statistical significance. In the test of the Morris water maze test, the spatial learning ability and memory ability of the rats in the test group were compared with the control group. The difference was statistically significant in P0.05, and with the prolongation of the feeding month, the learning, memory, cognitive function and activity ability of rats in the experimental group gradually decreased, and the difference was statistically significant. The second part of experimental subarachnoid hemorrhage induced the morphology of the cortex and lateral ventricular ependyma and choroid plexus in the hippocampus of rats. Objective: To observe the morphological changes of the hippocampal cortex, the ependyma and choroid plexus of the rats, the changes in the mitochondrial transmembrane potential (MTP) in the hippocampal neurons of the hippocampus, and the experimental SAH to the hippocampus and the lateral ventricle of the rat, mainly through the observation of the experimental subarachnoid hemorrhage (SAH) by light microscopy, electron microscopy and flow cytometry. The damage mechanism of ependyma and choroid plexus was provided with experimental morphological evidence. Methods: 90 adult SD rats, with an average weight of 300.20 + 27.50 g, were randomly divided into experimental and control groups, which were randomly divided into experimental and control groups, with 45 rats in each group. The rats in the experimental group were injected with 10% chloral chloral anesthesia and injected into the large cerebral cisterns. The blood of the body artery was 0.30 ml, and the experimental animal model of the simulated SAH was made. The rats in the control group were only injected with the same amount of saline in the large cistern, the rest of the operation were the same as the experimental group. The experimental group and the control group (each taken from each group) were given routine feeding after the operation, and the material was fixed by the aorta for 2 months, 4 months and 6 months after the operation. The samples of microscope, scanning and transmission electron microscope were prepared. At the same time, 5 rats in each group were randomly taken from each group. The bilateral hippocampus was stripped by the method of head extraction under deep anesthesia. Flow cytometry was used to detect the MTP in the hippocampal neurons. The apoptosis of the hippocampus was observed by the fluorescence microscope, and the experimental group and the control group were compared. Hippocampal cortex, lateral ventricle ependyma and choroid plexus, microscopic and ultrastructural differences under light microscopy, scanning and transmission electron microscopy, and the difference of MTP in the hippocampal neurons of two groups of rats, reveal the effects of experimental SAH on the brain tissue structure of rats, and provide morphological basis for behavioral changes. Results: experimental group: light microscope observation showed that With the increase of the month after the operation, the lateral ventricle of the rat gradually increased. The ependymal cells were formed from cubic deformation to pleomorphic or even flat shape, and the ependymal membrane structure of the hippocampal cortical surface changed slightly. In the hippocampal groove and the multiform cell layer, the space around the small vessels was obviously widened, the pyramidal cell layer became thinner and the number of cells decreased. Cells were arranged in disorder; and the longer the feeding time, the more obvious the phenomenon was. A large number of crater like sags appeared on the surface of the choroid plexus epithelial cells, and its villi atrophied, twisted and even dropped off, and its cilia and microvilli decreased significantly on the surface of the ependymal cells covered by the hippocampal cortex. Transmission electron microscopy showed that veins The epithelial cells of the collaterals showed a large number of swallowing vesicles and irregular shape of the cell and nucleus; the heterochromatin in the pyramidal nucleus of the hippocampal cortex increased obviously, the cells appeared expanded mitochondria, the neurofibrillary clumps, and the amyloid lipoprotein deposition in the felt; after 6 months of rearing, this phenomenon was more important. Obviously, the decrease of microvilli on the surface of the hippocampal ependymal cells was more obvious. A large number of amyloid lipoproteins were detected by.JC-1 flow cytometry around the vessels, and the MTP depolarization in the hippocampal neurons was serious. Compared with the control group, the difference was statistically significant, suggesting that the mitochondria in the nerve cells were different. In the control group, the hippocampus, the lateral ventricular ependyma and the choroid plexus, and the MTP in the hippocampal neurons were not observed in the control group for 2 months, 4 months or even 6 months after the operation. Obvious pathological changes. The changes in the expression of inflammatory and autophagy related proteins in the hippocampal cortex in third experimental subarachnoid hemorrhage rats: Based on the two parts of the above study, the experimental subarachnoid hemorrhage (SAH) was further explored by immunohistochemistry, Western blot and TUNEL detection. The relationship between the inflammatory and autophagy related protein expression in the hippocampal cortex was studied. Methods: the relationship between experimental SAH and AD was revealed. Methods: 90 adult SD rats were selected, the average weight was 289.40 + 45.50 g, and the male and female were not restricted. The rats were randomly divided into experimental group and control group, 45 rats in each group. The rats in the experimental group were hydrated with 10% hydration. After intraperitoneal injection of chloral anaesthesia, 0.30 ml of autologous arterial blood was injected into the large cistern to make an experimental animal model of simulated SAH. The control group was only injected with the same amount of saline in the large cistern, the rest of the operation were the same as the experimental group. At the same time, the experimental group and the control group were routinely fed, and 2 months, 4 months, and 6 months after the operation. At random, 10 rats in each group were taken at random. The expression of A beta, IL-1 beta and IBA-1 in the hippocampus was observed by immunohistochemical staining. The apoptosis of hippocampal pyramidal cells in the hippocampus of rats was observed by TUNEL detection. At the same time, 5 rats in each group were taken to quickly break the head under deep anesthesia. Take the brain and peel off the hippocampal cortex structure in rats and detect the expression of inflammatory related protein IL-1 beta, autophagy related protein (Atg-5, Beclin-l and LC-3) in the hippocampus cortex of rats by Western blot method, and record and analyze the related data. Results: 1 Western blot results were analyzed: compared with the control group, the inflammatory related protein IL- in the hippocampus cortex of the experimental group was compared with the control group. The expression of 1 beta, autophagy related proteins (Atg-5, Beclin-l and LC-3) was significantly increased, P0.05, and the difference was statistically significant. And with the extension of postoperative feeding time, the expression of the protein showed an upward trend, P0.05, the difference was statistically significant,.2 immunohistochemical results showed that compared with the control group, the experimental group was in the hippocampus cortex after the experimental SAH operation. The expression of A beta, IL-1 beta, and IBA-1 increased significantly, and increased with the increase of post operation feeding time.3 TUNEL results showed that after experimental SAH, the pyramidal cell apoptosis in the hippocampus of the experimental group increased, and the difference of P0.05 was statistically significant compared with the control group, and with the postoperative feeding time. The difference has statistical significance at 6 months after operation, and the difference has statistical significance at 6 months after operation. Conclusion: 1 the experimental rat model of simulated SAH can be successfully replicated by single injection of micro autologous arterial blood in the occipital big pool. The pathological changes are in accordance with the behavior expression, which has a more reliable basis and clinical reference significance.2. Experimental SAH could lead to the learning, memory, and activity of rats in the experimental group, and the condition was gradually increased with the prolongation of the feeding time. It suggested that the experimental SAH may cause the lateral ventricular ependyma, choroid plexus, and the hippocampal cortex in rats, causing different degrees of pathological damage to.3 experimental SAH can induce the rat sea. The fine and ultrastructural changes in the horse cortex, the lateral ventricular ependyma and choroid plexus, induced MTP depolarization in the hippocampal neurons, apoptosis or necrosis of the neurons,.4 experimental SAH can induce the increased expression of inflammatory factors in the hippocampus, the enhancement of autophagy, the deposition of amyloid lipoprotein, and the cortex of the hippocampus Pyramidal cell apoptosis and other phenomena, and with the extension of postoperative feeding time, more serious.

【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R743.35

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