缺血后处理延长脑缺血再灌注治疗时间窗及机制研究

发布时间：2018-05-23 21:49

本文选题：脑缺血/再灌注损伤 + 缺血后处理　；参考：《昆明医科大学》2014年博士论文

【摘要】：目的：采用线栓法建立更符合人类常见缺血性卒中类型的大鼠大脑中动脉栓塞(MCAO)模型,于再灌注即刻给予缺血后处理。观察缺血后处理对大鼠脑缺血/再灌注损伤的保护作用,并寻找其有效治疗时间窗。选择一个梗死面积和神经功能评分与缺血2h再灌注组无明显差异的最远时间点给予缺血后处理,探讨缺血后适应对炎症反应及细胞凋亡的影响。方法：本研究分为三部份进行。第一部份,缺血后处理对脑缺血/再灌注损伤的保护作用的研究。建立稳定的大鼠MCAO模型,于再灌注即刻给予缺血后处理。对单纯脑缺血/再灌注组和脑缺血/再灌注合并缺血后处理组的大鼠分别实施2h、3h、4h、4.5h、6h的脑缺血处理,观察缺血后处理对不同组大鼠脑缺血/再灌注48h后神经功能评分、脑梗死体积和脑水肿程度的影响,寻找有效的治疗时间窗。根据第一部分实验结果选择一个梗死面积和神经功能评分与缺血2h再灌注组无明显差异的最远时间点,进行第二、三部分实验。第二部分,HE染色观察大鼠脑组织的病理形态改变；采用免疫组化半定量测定脑缺血/再灌注组及脑缺血/再灌注合并缺血后处理组在2个时间点缺血侧皮层内TLR2和TLR4的表达变化。第三部分,采用流式细胞术、荧光定量PCR、Western-Blotting检测脑缺血/再灌注组及脑缺血/再灌注合并缺血后处理组在2个时间点缺血侧皮层内TLR2和TLR4及其下游信号通路分子IRAK4和炎性细胞因子IL-1β蛋白和mRNA的表达及细胞凋亡情况。结果：2h、3h、4h后处理组大鼠脑缺血/再灌注24h和48h的神经功能评分较相同缺血时间点缺血组和后处理4.5h和6h组改善(p0.05)。2h、3h、4h后处理组大鼠脑缺血/再灌注48h的脑梗塞体积和相对脑水肿程度较相同缺血时间点缺血组和后处理4.5h和6h组明显减轻(p0.05)。2h后处理组大鼠脑缺血/再灌注48h缺血侧皮层内TLR2和TLR4阳性细胞数量较2h缺血组和4.5h后处理组减少(p0.05)；4.5h后处理组TLR4阳性细胞数量较4.5h缺血组减少(p0.05)。2h后处理组大鼠脑缺血/再灌注48h缺血侧皮层内细胞凋亡比例较2h缺血组和4.5h后处理组明显减少(p0.05)。2h后处理组大鼠脑缺血/再灌注48h缺血侧皮层内TLR2、TLR4、IL-1βmRNA和蛋白及IRAK4mRNA表达量较2h缺血组降低(p0.05)；2h缺血时间点后处理组TLR2、IL-1βmRNA和蛋白及TLR4蛋白表达量较4.5h后处理组降低(p0.05)。结论：缺血后处理能减轻急性脑缺血/再灌注损伤,表现为神经功能缺损的改善、脑梗塞体积和相对脑水肿程度的减轻,其保护作用的有效时间窗为脑缺血4.5h以内。缺血后处理能有效改善大鼠脑缺血/再灌注损伤所致的细胞凋亡和炎症反应,表现为2h后处理组缺血侧皮层内TLR2和TLR4及其下游信号通路分子IRAK4和炎性细胞因子IL-1β的表达及细胞凋亡受到明显抑制。
[Abstract]:Aim: to establish a rat model of middle cerebral artery embolization (MCAO) which is more suitable to the common ischemic stroke type in human. To observe the protective effect of post-ischemic treatment on cerebral ischemia / reperfusion injury in rats and to find an effective time window for its treatment. A farthest time point with no significant difference between the infarct size and neurological function score and the ischemia 2 h reperfusion group was selected to study the effects of ischemic adaptation on inflammatory response and apoptosis. Methods: the study was divided into three parts. In the first part, the protective effect of post-ischemic treatment on cerebral ischemia / reperfusion injury was studied. A stable rat model of MCAO was established and treated immediately after reperfusion. The rats in the pure cerebral ischemia / reperfusion group and the cerebral ischemia / reperfusion combined with ischemic post-treatment group were treated with cerebral ischemia for 2 h, 3 h, 4 h and 4.5 h, respectively, and the neurological function scores were observed after 48 hours of cerebral ischemia / reperfusion in different groups. The effect of cerebral infarction volume and degree of cerebral edema to find an effective time window for treatment. According to the results of the first part of the experiment, a farthest time point with no significant difference between the infarct size and neurological function score and the ischemia 2h reperfusion group was selected, and the second and third parts of the experiment were carried out. The second part was to observe the histopathological changes of rat brain by HE staining. The expression of TLR2 and TLR4 in ischemic cortex of cerebral ischemia / reperfusion group and cerebral ischemia / reperfusion combined with ischemia group were measured by semi-quantitative immunohistochemical method at two time points. The third part, flow cytometry, Fluorescence quantitative PCR Western-blotting for the detection of TLR2 and TLR4 and their downstream signaling molecules IRAK4, IL-1 尾 protein and mRNA in cerebral ischemia / reperfusion group and cerebral ischemia / reperfusion combined with ischemic post-treatment group at two time points Expression and apoptosis. Results compared with ischemia group at the same ischemic time point and 4.5 h and 6 h post-treatment group, the neurological function scores of rats in the brain ischemia / reperfusion 24 h and 48 h after treatment with 1: 2 h + 3 h + 4 h improved the infarct volume and phase of cerebral ischemia / reperfusion 48h in the rats treated for 4 h after brain ischemia / reperfusion for 4 h. The amount of TLR2 and TLR4 positive cells in cerebral ischemic cortex of rats treated with cerebral ischemia / reperfusion for 48 h after cerebral ischemia and reperfusion was significantly reduced compared with that of ischemia group at 2 h and 4. 5 h and 6 h after treatment, compared with that of 2 h ischemia group and 4. 5 h post-treatment group. The number of TLR4 positive cells in the treatment group was significantly lower than that in the 4.5 h ischemia group compared with that in the 4.5 h ischemia group. The percentage of apoptosis in the ischemic cortex of the rats in the 48 h cerebral ischemia / reperfusion group was significantly lower than that in the 2 h ischemia group and the 4.5 h post-treatment group compared with the 4. 05 h ischemia group. The expression of IL-1 尾 mRNA, protein and IRAK4mRNA in the cerebral cortex of the cerebral ischemia / reperfusion 48 h group was lower than that in the 2h ischemia group. The expression of TLR2TLR4 尾 mRNA and protein and TLR4 protein in the post-treatment group was significantly lower than that in the control group (P 0.05). Conclusion: the acute cerebral ischemia / reperfusion injury can be alleviated after ischemic treatment, which is characterized by the improvement of neural function defect, the reduction of cerebral infarction volume and the relative degree of cerebral edema. The effective time window of its protective effect is within 4.5 hours of cerebral ischemia. The apoptosis and inflammatory response induced by cerebral ischemia / reperfusion injury were effectively improved after ischemic treatment in rats. The expression of TLR2, TLR4 and its downstream signaling pathway, IRAK4, IL-1 尾, and apoptosis were significantly inhibited in the ischemic cortex of 2 h post-treatment group.
【学位授予单位】：昆明医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743.3

【相似文献】