缺血后处理对大鼠颈动脉狭窄解除引起的脑水肿和血脑屏障破坏的影响

发布时间：2018-03-18 10:04

本文选题：缺血后处理　切入点：血脑屏障　出处：《吉林大学》2015年博士论文　论文类型：学位论文

【摘要】：脑血管病是一种严重危害人类健康的疾病，，已成为全球人类死亡的三大原因之一。其中因颈动脉狭窄性疾病引发的卒中占所有卒中的1/4~1/2。在美国几乎1/3的卒中来源于颈动脉狭窄，而在我国来源于颅外颈动脉狭窄的缺血性脑卒中的年发病率为120~180/10万，年病死率更是可达80~120/10万。目前颈动脉狭窄的治疗方法有药物治疗、手术治疗等。但就现在来看，重度狭窄颈动脉早期的血管重建能够有效的减少卒中的复发率，降低死亡率。虽然血管重建能解决以上问题，但血管重建之后的并发症例如神经认知功能障碍、严重的脑水肿、持续头痛、颅内出血，甚至死亡严重威胁患者生存状态。因此寻找有效的方法解决血管重建之后的并发症已成为一个亟待解决的问题。血脑屏障由胶质细胞、周细胞、血管内皮细胞和细胞外间隙组成，是一个介于血液和脑组织之间的复杂的系统，也是神经系统特有的结构。在血脑屏障结构和功能完整的情况下，血脑屏障可阻挡某些大分子物质通过并对血液和脑组织之间的物质交换进行严格的筛选。而在血脑屏障功能和结构遭到破坏时，血脑屏障通透性增高，脑水肿形成。血脑屏障正常通透性主要靠血管内皮细胞之间的紧密连接（Tight junction,TJ）来维持。而紧密连接主要有紧密连接蛋白ZO，occludin，跨膜蛋白，claudins和连接黏附分子（Juntional adhesion molecule，JAM）构成。其中occludin和claudin-5是形成紧密连接的关键蛋白。它们的表达和分布变化可导致血脑屏障的完整性发生变化。除此之外与血脑屏障的完整性密切相关的还有基质金属蛋白酶家族。其中MMP-2、MMP-9在基质金属蛋白酶家族中占有重要地位。它们主要作用是水解细胞基质和基膜的主要成分Ⅳ型胶原。已有研究证明MMP-2、MMP-9与血脑屏障结构和功能的变化有密切的关系，尤其MMP-9能够通过降解claudin-5和occludin等来调节血脑屏障的通透性。因此我们重点研究MMP-9、claudin-5和occludin与血脑屏障功能、脑水肿之间的关系，并进一步明确蛋白之间的相互作用。目前，在多种器官中，缺血后处理能够减轻缺血/再灌注带来的损伤。其方法是再灌注的早期快速的、间歇性的阻断血流进而改变血流的动力学水平。虽然以往的实验中我们已经证明缺血后处理能够阻止早期解除颈动脉狭窄所导致的神经元延迟性死亡，但缺血后处理对颈动脉狭窄解除后的大脑水肿和血脑屏障破坏的影响及其机制尚不清楚。实验目的：在大鼠重度颈动脉狭窄模型中探讨缺血后处理对颈动脉狭窄解除引起的脑水肿和血脑屏障破坏的影响及其作用机制。实验方法: 通过两侧颈动脉与不锈钢微管绑定建立Wistar大鼠低灌注模型。缺血后处理步骤包括30秒缺血/30秒再灌注，循环三次。通过测量脑组织含水量对脑水肿的程度进行评估，并且通过检测伊文思蓝(EB)和荧光素钠（NaF）的在脑组织的含量来评估血脑屏障通透性的变化。分别用明胶酶谱分析和原位酶谱分析检测MMP-9的活性和定位。用免疫组织化学观察紧密连接蛋白claudin-5和occludin的分布。实验结果：（1）血脑屏障通透性研究显示颈动脉狭窄解除组与假手术组和狭窄组相比，脑组织含水量增加，EB、NaF在大脑中的浓度显著提高，但这种变化被缺血后处理减弱。（2）明胶酶谱和荧光原位明胶酶谱研究显示MMP-9主要分布在皮层并且颈动脉狭窄的解除能导致MMP-9活性的升高，但这种活性的升高能被缺血后处理显著抑制。（3）免疫组织化学揭示了缺血后处理提高了claudin-5和occlaudin的连续分布。（4）ELISA检测显示由于颈动脉狭窄解除导致MMP-9的表达上调，进行缺血后处理后以上变化减弱，但是颈动脉狭窄解除后claudin-5和occlaudin的表达下调，进行缺血后处理后这两种蛋白的表达上调。实验结论：缺血后处理是一种有效防止脑水肿和改善血脑屏障通透性的方法，并且能够在重度颈动脉狭窄的解除过程中应用。
[Abstract]:Cerebrovascular disease is a serious hazard to human health disease, has become one of the three major causes of global human death. The stroke caused by carotid artery stenosis disease stroke stroke accounted for all sources of 1/4~1/2. in the United States almost 1/3 in carotid artery stenosis and ischemic stroke in our country from the extracranial carotid artery narrow the annual incidence rate of 120~180/10 million, the annual mortality rate is up to 80~120/10 million. The treatment of carotid artery stenosis with drug therapy, surgical treatment and so on. But now, severe stenosis of carotid artery early revascularization can reduce the effective stroke recurrence rate, reduce mortality. Although vascular reconstruction to solve the above problems, but after the reconstruction of vascular complications such as neurocognitive dysfunction, severe brain edema, persistent headache, intracranial hemorrhage, and even death serious threat to patient survival. Because of this search Finding effective methods to solve the complications after vascular reconstruction has become an urgent problem.
The blood brain barrier by glial cells, pericytes, endothelial cells and extracellular space, is a complex system between blood and brain tissue, is unique to the nervous system structure. In the structure and function of blood-brain barrier integrity under the condition of the blood brain barrier can block some macromolecules through and on between blood and brain tissue material exchange strict screening. The blood brain barrier and the destruction of the structure, increase the permeability of blood brain barrier and brain edema formation. The normal blood brain barrier permeability mainly depends on the vascular endothelial cell tight junctions (Tight junction, TJ) to maintain and are closely connected closely. Connected proteins ZO, occludin, a transmembrane protein, claudins and junctional adhesion molecules (Juntional adhesion, molecule, JAM). Occludin and claudin-5 is closely connected with the expression of key proteins. The distribution and changes to the integrity of the blood-brain barrier changes. And matrix metalloproteinase family integrity besides and blood brain barrier are closely related. The MMP-2, MMP-9 plays an important role in the matrix metalloproteinase family. Their main function is the main component of type IV collagen hydrolysis of cell matrix and basement membrane. The research proved that MMP-2, there is a close relationship between MMP-9 and changes of blood brain barrier structure and function, especially MMP-9 through degradation of claudin-5 and occludin to regulate the permeability of the blood-brain barrier. So we focus on the study of MMP-9, claudin-5 and occludin and blood brain barrier function, the relationship between brain edema, and further clarify the interaction between proteins role.
At present, in a variety of organs, postprocessing can reduce the damage of ischemia and ischemia / reperfusion brings. The method is fast in the early period of reperfusion, intermittent occlusion of blood flow and change the blood flow dynamics. Although previous experiments we have shown that ischemic postprocessing can prevent early termination of carotid artery stenosis caused by delayed neuron death, but ischemic postprocessing effects on carotid artery stenosis after relieving brain edema and blood-brain barrier damage and its mechanism is not clear.
Objective:
The effects and mechanisms of ischemic postconditioning on cerebral edema and blood-brain barrier damage induced by carotid artery stenosis were studied in a rat model of severe carotid stenosis.
Experimental methods:
Through both sides of the carotid artery and the stainless steel tube binding model of Wistar rat ischemia reperfusion model. Low postprocessing steps include 30 seconds /30 seconds after ischemia reperfusion, three cycles. By measuring the brain water content on the degree of cerebral edema were evaluated, and detected by Evans blue (EB) and fluorescein sodium (NaF) in the brain the content of organization to assess the changes of blood-brain barrier permeability respectively. The spectrum analysis of the activity and location of detection of MMP-9 by gelatin zymography and in situ enzyme distribution. By immunohistochemical observation of tight junction protein claudin-5 and occludin.
Experimental results:
(1) blood brain barrier permeability study showed that compared with sham operation group and stenosis group, the water content of brain tissue increased, and the concentration of EB and NaF increased significantly in the carotid artery stenosis relieving group, but this change was weakened by ischemic postconditioning.
(2) gelatin zymography and fluorescence in situ gelatinase analysis showed that MMP-9 was mainly distributed in the cortex, and the release of carotid stenosis could lead to the increase of MMP-9 activity. However, the increase of this activity could be significantly inhibited by ischemic postconditioning.
(3) immuno histochemistry revealed that post ischemic treatment enhanced the continuous distribution of claudin-5 and occlaudin.
(4) ELISA detection showed that the expression of MMP-9 was upregulated due to the release of carotid artery stenosis, and the above changes decreased after ischemic postconditioning. However, the expression of claudin-5 and occlaudin was down regulated after carotid artery stenosis, and the expression of these two proteins increased after ischemia.
Experimental conclusions:
Ischemic postconditioning is an effective method to prevent brain edema and improve the permeability of the blood brain barrier, and can be used in the process of severe carotid stenosis.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R743.31

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