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(p<0.05)。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(p<0.05)。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(p<0.05)；4.5h后处理组TLR4阳性细胞数量较4.5h缺血组减少(p<0.05)。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(p<0.05)。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(p<0.05)；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(p<0.05)。
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。
Objective To study the effects of ischemic postconditioning prolong time window and investigate the possible mechanism.
Method There are three parts in this experiment. The first part:middle cerebral artery occlusion model was stablished and ischemic postconditioning was implemented in the beginning of reperfusion. Adult male SD rats were randomly divided into ischemia/reperfusion groups and ischemia/reperfusion with ischemic postconditioning groups at2h,3h,4h,4.5h,6h ischemic time points. The effects of ischemic postconditioning were evaluated by neurological score, infarct volume, brain edema and found out the best time window. To select one time point that neurological score and infarct volume is no difference compared to2h, then continuing the following experiments. The second part:the pathological changes of brain tissue in rats were observed by HE staining; the expression of TLR2and TLR4in ischemic cortex in ischemia/reperfusion groups and ischemia/reperfusion with ischemic postconditioning groups at2h and4.5h ischemic time points were measured through immunohistochemistry. The third part:the expression of TLR2, TLR4, IRAK4, IL-1β and apoptosis in ischemic cortex in ischemia/reperfusion groups and ischemia/reperfusion with ischemic postconditioning groups at2h and4.5h ischemic time points were measured through flow cytometry, qRT-PCR and Western-Blotting.
Result Neurological score of ischemic postconditioning groups at2h,3h,4h ischemic time points were improved by the same time points ischemic groups and4.5h,6h postconditioning groups in reperfusion24h and48h(p<0.05). Infarct volume and brain edema of ischemic postconditioning groups at2h,3h,4h ischemic time points were reduced by the same time points ischemic groups and4.5h,6h postconditioning groups in reperfusion48h(p<0.05). The number of TLR2and TLR4positive cells in ischemic cortex at2h postconditioning group during reperfusion48h were reduced by2h ischemic group and4.5h postconditioning group (p<0.05); the number of TLR4positive cells was reduced at4.5h postconditioning group by4.5h ischemic group. The apoptosis in ischemic cortex at2h postconditioning group during reperfusion48h were reduced by2h ischemic group and4.5h postconditioning group (p<0.05). The expression of TLR2, TLR4, IL-1βmRNA and protein as well as IRAK4mRNA in ischemic cortex at2h postconditioning group during reperfusion48h were decreased by2h ischemic group (p<0.05); the expression of TLR2, IL-1βmRNA and protein as well as TLR4protein decreased by4.5h postconditioning group (p<0.05)
Conclusion Ischemic postconditioning significantly attenuated the cerebral ischemia/reperfusion injury by neurological score, infarct volume, brain edema improved. The best time window is within4.5h. Ischemic postconditioning significantly attenuated apoptosis and inflammation caused by cerebral ischemia/reperfusion injury. The expression of TLR2, TLR4, IRAK4, IL-1βand apoptosis in ischemic cortex at2h postconditioning group significantly were inhibited.
引文
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