脑缺血再灌注致听力损伤的机制研究

The Mechanism of Hearing Loss after Focal Cerebral Ischemia-reperfusion Injury

目的探讨大鼠局灶性脑缺血再灌注后听力损伤的机制。方法选择健康白色雄性SD大鼠60只,随机分为假手术组和缺血再灌注组,每组30只。缺血再灌注组大鼠采用线栓法制备大脑中动脉栓塞模型,脑缺血60min,再灌注24h;假手术组只分离颈部血管,不插入线栓。造模前及造模24小时后两组分别检测听性脑干反应,行神经功能评分,然后处死动物,检测脑组织含水量和脑梗死体积,通过Evans blue的渗出情况评估血脑屏障的完整性,末端标记法观察神经细胞凋亡状况,计算凋亡指数;Western blot法检测金属蛋白酶9(MMP-9)、紧密连接蛋白5(Claudin-5)、闭锁蛋白(Occludin)、连接蛋白43(CX-43)的表达。结果与假手术组比较,缺血再灌注组ABR反应阈值明显升高,神经功能评分升高,脑组织含水量增多,脑梗死体积增大,脑神经细胞凋亡指数显著增加,MMP-9、CX-43蛋白表达明显上升,Claudin-5、Occludin蛋白表达显著下降,差异均具有统计学意义(均为P<0.05)。结论大鼠局灶性脑缺血再灌注脑损伤后其听力损伤可能与MMPs激活、细胞凋亡、血脑屏障破坏及缝隙连接损伤有关。

Objective To investigate the mechanism of hearing loss after focal cerebral ischemia-reperfusion injury in rats.Methods A total of 60 healthy male adult SD rats were included in this study and randomly divided into 2groups,ischemia-reperfusion（I/R）group and sham operated control,with 30 rats in each group.The rats in I/R group were operated for suture-occluded method to establish middle cerebral artery occlusion（MCAO）model,with ischemia for 60 mins followed by reperfusion for 24 hrs.The control group was only to be isolated cervical vessels,with no thread embolism inserted.The auditory brainstem response（ABR）was tested before operation and at24 hrs post-operation respectively.At 24 hrs post-operation,we scored neurological functions,measured the changes of water content in the brain using the dry-wet weight method,and determined the infarct volume through TTC method.We also evaluated the integrity of blood-brain barrier（BBB）by viewing the exudation of Evans blue and observed the apoptosis of neurocyte by TUNEL method to conclude apoptotic index（AI）.The expression of MMP-9,Claudin-5,Occludin and CX-43 were detected by Western blot.Results Compared with the sham group,the neurological function scores,the infarct volume and water content of the brain increased,with the elevated thresholds of ABR significantly and AI went up in I/R group.The expression levels of MMP-9and CX-43 were significantly up-regulated,but the expressions of Claudin-5and Occludin were obviously down-regulated.All of the differences above had statistical significances.Conclusion The mechanism of hearing loss after focal cerebral ischemia-reperfusion injury in rats is possibly related to MMPs activation,neurocyte apoptosis,BBB breakage and gap junction damage.