异氟醚后处理对大鼠脑缺血再灌注时血管生成的影响及Shh信号通路在其中的作用

Effects of isoflurane postconditioning on angiogenesis during cerebral ischemia-reperfusion in rats and the role of Shh signaling pathway

目的评价异氟醚后处理对大鼠脑缺血再灌注时血管生成的影响及Shh信号通路在其中的作用.方法清洁级健康雄性SD大鼠32只,6~8周龄,体重220~280g,采用随机数字表法分为4组(n=8):假手术组(Sham组)、缺血再灌注组(I∕R组)、异氟醚后处理组(ISO组)和异氟醚后处理+Shh信号通路特异性抑制剂环巴胺组(ISO+CYC组).采用线栓法制备大鼠脑缺血再灌注损伤模型,缺血1.5h,再灌注24h.再灌注24h时,行神经功能缺陷评分,然后处死大鼠取脑组织,TTC染色法测量脑梗死体积,尼氏染色法观察病理学变化,Westernblot法测定脑皮质神经胶质瘤相关癌基因同源物(Gli1)、血管内皮生长因子(VEGF)和抗人原始造血细胞抗原(CD34)的表达水平.结果与Sham组比较,I∕R组和ISO组神经功能缺陷评分和脑梗死体积升高,脑皮质Gli1、VEGF和CD34的表达上调(P<0.05);与I∕R组比较,ISO组神经功能缺陷评分和脑梗死体积降低,脑皮质Gli1、VEGF和CD34的表达上调(P<0.05),脑组织病理学损伤减轻,ISO+CYC组上述指标差异无统计学意义(P>0.05);与ISO组比较,ISO+CYC组神经功能缺陷评分和脑梗死体积升高,脑皮质Gli1、VEGF和CD34的表达下调(P<0.05).结论异氟醚后处理减轻大鼠脑缺血再灌注损伤的机制与激活Shh信号通路,促进血管生成有关.

Objective To evaluate the effects of isoflurane postconditioning on angiogenesis during cerebral ischemia-reperfusion(I/R)in rats and the role of Shh signaling pathway.Methods Thirty-two clean-grade healthy male Sprague-Dawley rats,aged 6-8 weeks,weighing 220-280 g,were divided into 4 groups(n=8 each)by a random number table method:sham operation group(Sham group),I/R group,isoflurane postconditioning group(ISO group),and isoflurane postconditioning plus Shh signaling pathway specific inhibitor cyclopamine group(ISO+CYC group).Cerebral ischemia was produced by inserting a 3-0 nylon thread with a rounded tip into the internal jugular vein.The nylon thread was threaded cranially until resistance was met.Occlusion was maintained for 1.5 h followed by 24 h reperfusion.Neurological deficit was scored at 24 h of reperfusion.Rats were then sacrificed,and brains were removed for determination of cerebral infarct volume(by TTC)and expression of glioma-associated oncogene homolog 1(Gli1),vascular endothelial growth factor(VEGF)and transmembrane phosphoglycoprotein protein(CD34)in cerebral cortex(by Western blot)and for examination of the pathological changes(by Nissl staining).Results Compared with Sham group,the neurological deficit score and cerebral infarct volume were significantly increased,and the expression of Gli1,VEGF and CD34 in the cerebral cortex was up-regulated in I/R and ISO groups(P<0.05).Compared with I/R group,the neurological deficit score and cerebral infarct volume were significantly decreased,and the expression of Gli1,VEGF and CD34 in the cerebral cortex was up-regulated(P<0.05),and the pathological changes of brain tissues were significantly attenuated in ISO group,and no significant change was found in the parameters mentioned above in ISO+CYC group(P>0.05).Compared with ISO group,the neurological deficit score and cerebral infarct volume were significantly increased,and the expression of Gli1,VEGF and CD34 in the cerebral cortex was down-regulated in ISO+CYC group(P<0.05).Conclusion The mechanism by which isoflurane post-conditioning attenuates cerebral I/R injury is related to activating Shh signaling pathway and promoting angiogenesis in rats.