缺血性脑中风对小鼠神经元突触结构变化的影响

发布时间：2019-03-15 11:16

【摘要】：正常生理条件下,大脑的微循环足以维持神经元环路的完整性及活动。脑中风后脑内微循环发生障碍,血流明显下降,以往的研究表明供血的不足可以导致神经元结构遭到破坏,而及时进行血液再灌注时神经元结构可发生可逆性恢复变化。然而迄今为止这种神经元结构可逆性变化的机理和影响因素尚不清楚。本文采用YFP荧光小鼠进行双侧颈总动脉结扎手术(BCAL)制作可逆性全脑缺血模型,结合双光子活体成像技术、透射电镜技术、组织学染色技术及行为测试等方法研究了突触结构在脑中风后的变化及不同缺血时间对神经元结构可逆性变化的影响。双光子活体成像研究结果显示,短暂性脑缺血可以造成神经元损伤呈念珠状结构,此外可以导致13.2%的树突棘消失。在血液再灌注后神经元结构可以发生快速的可逆性恢复,而这种可逆性恢复并不是结构上简单的重复,它伴随着突触结构的重组,主要体现在消失树突棘的重现及新生树突棘的出现。神经元超微结构研究结果显示,在缺血再灌注过程中,细胞器、细胞质组分及突触超微结构同样经历了破坏又重建的过程。长期活体观察显示,可逆性恢复的结构可以长期存在(30 d),而树突棘更新率统计结果发现树突棘的更新率却存在长期的不断的动态变化(7-30 d)。行为测试结果显示,短暂性脑缺血对行为的影响仅限于血液再灌注后的初期(3 d以内),行为上的快速恢复可能有赖于神经元结构的快速恢复和突触结构的快速及长期的重组。此外,神经元结构可逆性恢复具有时间依赖性,随着缺血时间的延长,神经元结构发生可逆性恢复也越来越困难。缺血20 min、1 h、3 h和6 h后再灌注1 h时树突结构恢复率分别为:93%、56%、17%和0%。当缺血时间超过3 h时,神经元结构发生可逆性恢复的程度大幅度下降(缺血3 h后再灌注6 h时有36%的树突结构恢复),当缺血时间超过6 h时,神经元结构很难再进行可逆性恢复(缺血6 h后再灌注6h时仅有2%的树突结构恢复)。双光子活体观察结果显示,神经元结构发生可逆性恢复的时间窗为缺血3-6 h,这与临床上溶栓治疗中风的时间窗相一致。YFP小鼠脑切片和Golgi染色观察皮层深层部位神经元结构损伤情况,结果显示神经元的损伤模式是随着缺血时间的延长神经元的损伤由顶端蔓延至主干。当缺血小于3 h时念珠状损伤主要在树突的顶端分枝,当缺血达到6 h时念珠状损伤延伸至整个树突主干。Fluoro-Jade C染色结果显示当缺血3-6 h时,大量变性神经元出现在皮层、海马及皮层下核团部位。进一步采用透射电镜观察了不同程度脑缺血后神经元细胞核超微结构的变化情况,结果显示短暂性脑缺血时神经元细胞核内染色质发生聚集,当血液及时再灌注时染色质结块聚集现象可以恢复,而当缺血6 h时神经元细胞核染色质发生边集甚至发生凋亡。与之相应,动物行为测试结果显示,动物行为的恢复具有缺血时间依赖性。综上研究结果,脑缺血很快破坏了大脑内的神经元结构,及时进行血液再灌注后神经元结构可以发生可逆性恢复,而这种可逆性恢复依赖于神经元结构的受损伤程度。本论文的研究结果为脑中风后神经元的修复及保护提供了实验数据,对脑中风的临床治疗具有一定的参考意义。
[Abstract]:Under normal physiological conditions, the microcirculation of the brain is sufficient to maintain the integrity and activity of the neuronal loop. The present study indicated that the insufficiency of blood supply could lead to the destruction of the neuronal structure and the reversible recovery of the neuronal structure in time of reperfusion. However, the mechanism and influencing factors of the reversible change of this kind of neuronal structure to date are not clear. In this paper, the reversible complete cerebral ischemia model was made by using YFP fluorescent mouse for bilateral common carotid artery ligation (BCAL), combined with two-photon in-life imaging and transmission electron microscopy (TEM). The changes of the synaptic structure in the brain and the effects of different time on the reversible change of the structure of the neurons were studied by histological staining and behavioral test. The results of the two-photon in-vivo imaging study show that transient cerebral ischemia can cause neuronal damage to be a bead-like structure, and may result in the disappearance of 13.2% of the dendritic spines. The reversible recovery of the neuronal structure after reperfusion of the blood is not a simple repetition of the structure, which is accompanied by the recombination of the synaptic structure, which is mainly manifested in the reproduction of the disappearance of the dendritic spine and the appearance of the new dendritic spines. The results of the neuronal ultrastructure show that in the process of ischemia-reperfusion, the organelles, the cytoplasmic components and the ultrastructure of the synapse also undergo the process of destruction and reconstruction. Long-term in-life observation shows that the structure of the reversible recovery can be long-term (30d), while the statistical results of the dendritic spine update rate show that the renewal rate of the dendritic spine has long-term dynamic changes (7-30d). The results of the behavior test show that the effect of transient cerebral ischemia on the behavior is limited to the initial (within 3 days) after the blood re-perfusion, and the rapid recovery of the behavior may depend on the rapid recovery of the neuronal structure and the rapid and long-term recombination of the synaptic structure. In addition, the reversible recovery of the neuronal structure has time-dependence, and the reversible recovery of the neuronal structure is becoming more and more difficult as the time of the ischemia is prolonged. The recovery rate of dendritic structure was 93%,56%,17% and 0% after ischemia for 20 min,1 h,3 h and 6 h, respectively. When the ischemia time was more than 3 h, the degree of reversible recovery of the neuronal structure decreased significantly (36% of the dendritic structure recovered at 6 h after 3 h of ischemia), and when the ischemia time was more than 6 h, The neuronal structure was difficult to perform reversible recovery (only 2% of the dendritic structures were recovered after 6 h of ischemia). The results of the two-photon in-life observation show that the time window of reversible recovery of the neuronal structure is 3-6 hours of ischemia, which is consistent with the time window for the treatment of stroke in clinic. The damage of the neurons in the deep part of the cortex was observed by the brain slices and the Golgi staining of the YFP mice. The results showed that the damage pattern of the neurons was spread from the top to the trunk with the prolongation of the ischemia time. When the ischemia is less than 3 h, the bead-like injury is mainly branched at the top of the dendrites, and the bead-like injury extends to the whole of the dendritic backbone when the ischemia reaches 6 h. The Fluorio-Jade C staining showed that a large number of degenerative neurons appeared in the cortex, hippocampus and subcortical nuclei at 3-6 hours of ischemia. The changes of the ultrastructure of the nucleus of the neurons in different degrees of cerebral ischemia were observed by transmission electron microscope. The results showed that the chromatin in the nucleus of the neurons in the nucleus of the neuron during transient ischemic attack could be recovered. In that case of the ischemia of 6 h, the chromatin of the nucleus of the neuron was even apoptosis. In contrast, animal behavior test results show that the recovery of animal behavior has an ischemic time-dependent. As a result of the study, the cerebral ischemia quickly destroyed the structure of the neurons in the brain, and a reversible recovery of the neuronal structure after the blood reperfusion was performed in a timely manner, and the reversible recovery was dependent on the degree of damage of the neuronal structure. The results of this study provide experimental data for the repair and protection of the post-stroke neurons, which is of reference to the clinical treatment of cerebral apoplexy.
【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R743.3

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