南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (7): 1194-1203.doi: 10.12122/j.issn.1673-4254.2023.07.16

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改良的四血管间断阻塞法制作大鼠全脑缺血再灌注损伤模型

孙 伟，陈 平，唐小杭，谷颖敏，田雪松   

  1. 上海中医药大学创新中药研究院，科技实验中心，上海 201203
  • 出版日期:2023-07-20 发布日期:2023-07-20

An improved 4-vessel intermittent occlusion method for establishing rat models of global cerebral ischemia-reperfusion injury

SUN Wei, CHEN Ping, TANG Xiaohang, GU Yingmin, TIAN Xuesong   

  1. Innovation Research Institute of Traditional Chinese Medicine, Experiment Center for Science and Technology, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
  • Online:2023-07-20 Published:2023-07-20

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摘要： 目的 改良Pulsinelli与Brierley建立的经典四血管阻塞（4VO）模型。方法 80只雄性SD大鼠。其中32只随机分为：假手术组、I4VO-Con10 组、I4VO- Int10 组和I4VO- Int15 组。假手术组暴露双侧椎动脉与颈总动脉但不夹闭；I4VO-Con10 为持续缺血组，夹闭双侧椎动脉与颈总动脉10 min，再灌注24 h；I4VO- Int10 组和I4VO- Int15 组为间断缺血组，I4VO- Int10 组进行5 min缺血、5 min再灌注及5 min缺血，然后再灌注24 h；I4VO- Int15 组缺血5 min、再进行2次5 min/5 min的再灌注/缺血循环，随即再灌注24 h。激光多普勒监测局部脑血流量，观察大鼠生存情况，HE染色观察海马组织病理改变，以此确定最优改良方法；48只大鼠按照最优改良方法（I4VO-Int15）建立I4VO模型，随机分为假手术组和5个I4VO模型组。5个I4VO模型组根据再灌注时间点（1、3、7、14、28 d）分为I4VO-D1、I4VO-D3、I4VO-D7、I4VO-D14和I4VO-D28组。记录各组大鼠体质量与生存状况，HE染色观察海马、视网膜与视束形态学改变；Y迷宫实验、明暗箱实验评估I4VO-D28组大鼠认知功能与视功能。结果 I4VO-Int15是最优改良方法；I4VO-D1组、I4VO-D3组、I4VO-D7组、I4VO-D14组和I4VO-D28组体质量较对应时间点假手术组的体质量明显降低，存活率无显著性差异，海马神经元损伤、丢失进行性加重；I4VO-D28组大鼠出现认知障碍；I4VO-D28组大鼠视网膜、视束未见明显缺血性损伤。结论 改良的I4VO模型能够成功复制大鼠全脑缺血再灌注损伤的主要病理过程；改良的I4VO模型未引起视功能损伤。

关键词: 四血管阻塞；脑缺血再灌注损伤；动物模型；大鼠

Abstract: Objective To improve the classical 4-vessel occlusion (4VO) model established by Pulsinelli and Brierley. Methods Thirty-two male SD rats were randomized into sham operation group, I4VO-Con10 group, I4VO-Int10 group and I4VO-Int15 group. The sham surgery group underwent exposure of the bilateral vertebral arteries and carotid arteries without occlusion to block blood flow. The I4VO- Con10 group experienced continuous ischemia by occluding the bilateral vertebral arteries and carotid arteries for 10 minutes followed by reperfusion for 24 hours. The I4VO-Int10 and I4VO-Int15 groups were subjected to intermittent ischemia. The I4VO- Int10 group underwent 5 minutes of ischemia, followed by 5 minutes of reperfusion and another 5 minutes of ischemia, and then reperfusion for 24 hours. The I4VO- Int15 group experienced 5 minutes of ischemia followed by two cycles of 5 minutes of reperfusion and 5 minutes of ischemia, and then reperfusion for 24 hours. The regional cerebral blood flow (rCBF) was monitored with laser Doppler scanning, and survival of the rats was observed. HE staining was used to observe hippocampal pathologies to determine the optimal method for modeling. Another 48 rats were randomized into 6 groups, including a sham operation group and 5 model groups established using the optimal method. The 5 I4VO model groups were further divided based on the reperfusion time points (1, 3, 7, 14, and 28 days) into I4VO-D1, I4VO-D3, I4VO-D7, I4VO- D14, and I4VO- D28 groups. Body weight changes and survival of the rats were recorded. HE staining was used to observe morphological changes in the hippocampal, retinal and optic tract tissues. The Y-maze test and light/dark box test were used to evaluate cognitive and visual functions of the rats in I4VO-D28 group. Results Occlusion for 5 min for 3 times at the interval of 5 min was the optimal method for 4VO modeling. In the latter 48 rats, the body weight was significantly lower than that of the sham-operated rats at 1, 3, 7, 14 and 28 days after modeling without significant difference in survival rate among the groups. The rats with intermittent vessel occlusion exhibited progressive deterioration of hippocampal neuronal injury and neuronal loss. Cognitive impairment was observed in the rats in I4VO-D28 group, but no obvious ischemic injury of the retina or the optic tract was detected. Conclusion The improved 4VO model can successfully mimic the main pathological processes of global cerebral ischemia-reperfusion injury without causing visual impairment in rats.

Key words: 4-vessel occlusion; global cerebral ischemia-reperfusion injury; animal model; rats