血管内皮细胞凋亡在兔脑血管痉挛模型的初步研究

The apoptosis of cerebral vascular endothelial cells: a preliminary study in rabbit models with cerebral vasospasm

目的采用兔脑血管痉挛模型研究脑血管内皮细胞(vascular endothelial cell,VEC)凋亡的表达及时间窗,初步探讨VEC凋亡在脑血管痉挛中的作用。方法 35只健康清洁级新西兰大白兔经血管内穿刺产生蛛网膜下腔出血(SAH),制作脑血管痉挛模型,按处死时间将实验动物分为12 h、1、2、3、7 d共5组,每组7只。每组又分为SAH亚组5只及对照亚组2只(即导丝仅进入但未刺破颈内动脉)。动物处死后,选取后交通动脉及基底动脉上段血管制成石蜡切片,采用三磷酸脱氧尿嘧啶缺口末端标记法(TUNEL法)观察VEC凋亡情况并计数。每组取动物2只,对照动物2只,共12只右颈内动脉行电镜检查,了解血管内皮病理改变及VEC凋亡情况。结果 TUNEL法显示,TUNEL阳性细胞出现在SAH 1 d后,在2～7 d高表达,3 d组TUNEL阳性细胞数量最多(凋亡率为39%)。电镜显示,SAH亚组VEC出现空泡化、胞质浓缩及弹力膜扭曲等改变,1 d组出现VEC核固缩改变,2、3 d组VEC核固缩现象出现概率较高,7 d组VEC未见核固缩改变。结论研究证实VEC凋亡峰值略早于迟发性痉挛高峰期,表明内皮细胞凋亡在脑血管痉挛中起重要作用。

Objective To investigate the expression and time window of endothelial cells apoptosis in rabbit models with cerebral vasospasm, and to discuss the action of vascular endothelial cell （VEC） apoptosis on cerebral vasospasm. Methods By endovascular puncturing method subarachnoid hemorrhage （SAH） was created in 35 heahhy New Zealand rabbits, which were used to establish cerebral vasospasm models. According to the scheduled sacrifice time （12 hours, one, 2, 3 and 7 days）, the rabbits were randomly divided into five groups with 7 rabbits in each group. Every group were further subdivided into SAH subgroup （n = 5） and control subgroup （n = 2）. In control subgroup, the guide-wire was inserted in the internal carotid artery but did not break the artery. After the rabbits were sacrificed, the posterior communicating artery and tile upper segment of basilar artery were collected and sent for pathologic examination. The TUNEL stain technique was used to observe the apoptosis of endothelial ceils in sectioned slices. Every two rabbits from each SAH subgroup and two rabbits of control subgroup （12 rabbits in total） were collected. The right carotid arteries of these 12 rabbits were examined with electron microscopy to observe the pathological changes and the apoptosis of vascular endothelial cells. Results TUNEL immunohistochemical study showed that TUNEL positive staining cells appeared one day after SAIl, and the expression reached its peak in 2 - 7 days. Themaximum number of TUNEL positive staining cells was found in the rabbits of 3- day SAH subgroup, and the apoptosis rate was 39%. Electron microscopy examination demonstrated vacuolization, cytoplasm condensation, torsion of elastic membrane, etc. in VEC of SAHsubgroups. Nuclear condensation of VEC was found in the rabbits of one- day SAH subgroup, the nuclear condensation of VEC was more frequently seen in the rabbits of 2-day and 3- day SAH subgroups, while no nuclear condensation of VEC was found in the rabbits of 7- day SAH subgroup. Conclusion This study proves that the peak value of VEC apoptosis appears slightly earlier than that of the delayed cerebral vasospasm in the rabbit models of cerebral vasospasm produced by endovascular puncturing method, indieating that the apoptosis of endothelial cells plays an important role in the pathogenesis of vasospasm.