缺血性腦中風(fēng)對(duì)小鼠神經(jīng)元突觸結(jié)構(gòu)變化的影響
發(fā)布時(shí)間：2019-03-15 11:16
【摘要】：正常生理?xiàng)l件下,大腦的微循環(huán)足以維持神經(jīng)元環(huán)路的完整性及活動(dòng)。腦中風(fēng)后腦內(nèi)微循環(huán)發(fā)生障礙,血流明顯下降,以往的研究表明供血的不足可以導(dǎo)致神經(jīng)元結(jié)構(gòu)遭到破壞,而及時(shí)進(jìn)行血液再灌注時(shí)神經(jīng)元結(jié)構(gòu)可發(fā)生可逆性恢復(fù)變化。然而迄今為止這種神經(jīng)元結(jié)構(gòu)可逆性變化的機(jī)理和影響因素尚不清楚。本文采用YFP熒光小鼠進(jìn)行雙側(cè)頸總動(dòng)脈結(jié)扎手術(shù)(BCAL)制作可逆性全腦缺血模型,結(jié)合雙光子活體成像技術(shù)、透射電鏡技術(shù)、組織學(xué)染色技術(shù)及行為測試等方法研究了突觸結(jié)構(gòu)在腦中風(fēng)后的變化及不同缺血時(shí)間對(duì)神經(jīng)元結(jié)構(gòu)可逆性變化的影響。雙光子活體成像研究結(jié)果顯示,短暫性腦缺血可以造成神經(jīng)元損傷呈念珠狀結(jié)構(gòu),此外可以導(dǎo)致13.2%的樹突棘消失。在血液再灌注后神經(jīng)元結(jié)構(gòu)可以發(fā)生快速的可逆性恢復(fù),而這種可逆性恢復(fù)并不是結(jié)構(gòu)上簡單的重復(fù),它伴隨著突觸結(jié)構(gòu)的重組,主要體現(xiàn)在消失樹突棘的重現(xiàn)及新生樹突棘的出現(xiàn)。神經(jīng)元超微結(jié)構(gòu)研究結(jié)果顯示,在缺血再灌注過程中,細(xì)胞器、細(xì)胞質(zhì)組分及突觸超微結(jié)構(gòu)同樣經(jīng)歷了破壞又重建的過程。長期活體觀察顯示,可逆性恢復(fù)的結(jié)構(gòu)可以長期存在(30 d),而樹突棘更新率統(tǒng)計(jì)結(jié)果發(fā)現(xiàn)樹突棘的更新率卻存在長期的不斷的動(dòng)態(tài)變化(7-30 d)。行為測試結(jié)果顯示,短暫性腦缺血對(duì)行為的影響僅限于血液再灌注后的初期(3 d以內(nèi)),行為上的快速恢復(fù)可能有賴于神經(jīng)元結(jié)構(gòu)的快速恢復(fù)和突觸結(jié)構(gòu)的快速及長期的重組。此外,神經(jīng)元結(jié)構(gòu)可逆性恢復(fù)具有時(shí)間依賴性,隨著缺血時(shí)間的延長,神經(jīng)元結(jié)構(gòu)發(fā)生可逆性恢復(fù)也越來越困難。缺血20 min、1 h、3 h和6 h后再灌注1 h時(shí)樹突結(jié)構(gòu)恢復(fù)率分別為:93%、56%、17%和0%。當(dāng)缺血時(shí)間超過3 h時(shí),神經(jīng)元結(jié)構(gòu)發(fā)生可逆性恢復(fù)的程度大幅度下降(缺血3 h后再灌注6 h時(shí)有36%的樹突結(jié)構(gòu)恢復(fù)),當(dāng)缺血時(shí)間超過6 h時(shí),神經(jīng)元結(jié)構(gòu)很難再進(jìn)行可逆性恢復(fù)(缺血6 h后再灌注6h時(shí)僅有2%的樹突結(jié)構(gòu)恢復(fù))。雙光子活體觀察結(jié)果顯示,神經(jīng)元結(jié)構(gòu)發(fā)生可逆性恢復(fù)的時(shí)間窗為缺血3-6 h,這與臨床上溶栓治療中風(fēng)的時(shí)間窗相一致。YFP小鼠腦切片和Golgi染色觀察皮層深層部位神經(jīng)元結(jié)構(gòu)損傷情況,結(jié)果顯示神經(jīng)元的損傷模式是隨著缺血時(shí)間的延長神經(jīng)元的損傷由頂端蔓延至主干。當(dāng)缺血小于3 h時(shí)念珠狀損傷主要在樹突的頂端分枝,當(dāng)缺血達(dá)到6 h時(shí)念珠狀損傷延伸至整個(gè)樹突主干。Fluoro-Jade C染色結(jié)果顯示當(dāng)缺血3-6 h時(shí),大量變性神經(jīng)元出現(xiàn)在皮層、海馬及皮層下核團(tuán)部位。進(jìn)一步采用透射電鏡觀察了不同程度腦缺血后神經(jīng)元細(xì)胞核超微結(jié)構(gòu)的變化情況,結(jié)果顯示短暫性腦缺血時(shí)神經(jīng)元細(xì)胞核內(nèi)染色質(zhì)發(fā)生聚集,當(dāng)血液及時(shí)再灌注時(shí)染色質(zhì)結(jié)塊聚集現(xiàn)象可以恢復(fù),而當(dāng)缺血6 h時(shí)神經(jīng)元細(xì)胞核染色質(zhì)發(fā)生邊集甚至發(fā)生凋亡。與之相應(yīng),動(dòng)物行為測試結(jié)果顯示,動(dòng)物行為的恢復(fù)具有缺血時(shí)間依賴性。綜上研究結(jié)果,腦缺血很快破壞了大腦內(nèi)的神經(jīng)元結(jié)構(gòu),及時(shí)進(jìn)行血液再灌注后神經(jīng)元結(jié)構(gòu)可以發(fā)生可逆性恢復(fù),而這種可逆性恢復(fù)依賴于神經(jīng)元結(jié)構(gòu)的受損傷程度。本論文的研究結(jié)果為腦中風(fēng)后神經(jīng)元的修復(fù)及保護(hù)提供了實(shí)驗(yàn)數(shù)據(jù),對(duì)腦中風(fēng)的臨床治療具有一定的參考意義。
[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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R743.3
本文編號(hào)：2440576
[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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R743.3
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