小鼠大脑中动脉栓塞模型的改良及评价

小鼠大脑中动脉栓塞模型的改良及评价

【摘要】目的：对小鼠大脑中动脉栓塞（MCAO）模型进行改良并进行评价。方法：C57BL/6J小鼠50只，随机分为假手术组10只，MCAO组40只；改良线栓法制作小鼠MCAO模型；术后24 h行神经功能评分，TTC染色测脑梗死体积；术后30 d，水迷宫测试小鼠远期学习和记忆能力；术后90 d，头部MRI观察梗死病灶及脑组织变化。结果：造模成功率为67.5％；MCAO组小鼠神经功能评分为（2.36±0.21）分，相对梗死体积为（25.36±3.65）％；MCAO组小鼠逃避潜伏期明显长于假手术组，穿越原平台位置次数明显少于假手术组（P<0.05）；小鼠头部MRI T2加权像可见缺血侧皮质和/或皮质下梗死病灶。结论：线栓法是简单、可靠的制作小鼠MCAO模型的方法。

【关键词】小鼠；大脑中动脉栓塞；动物模型

Establish and Optimize Middle Cerebral Artery Occlusion Model in Mice

TANG Ying-xin▲, LIU Min-zhen, DING Feng-fei, ZHANG Qiang, WANG Wei, P AN Deng-ji.▲Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China 【Abstract】Objective: To establish and optimize middle cerebral artery occlusion model in mice. Methods: Fifty C57BL/6J mice were randomly divided into sham group (n=10) and MCAO group (n=40). The mice in MCAO group were used to establish the MCAO model by intraluminal occlusion using monofilament.

Twenty-four hours after operation, the nervous function was evaluated and the infarct volume was calculated by TTC staining. Thirty days after operation, Morris water maze test was carried out to investigate spatial learning and memory of mice. Ninety days after operation, head MRI scan was conducted. Results: In MCAO group, the successful rate of execution was 67.5%. The neurological deficit score was (2.36±0.21) and the relative infarct volume was (25.36±3.65)% in MCAO group.Mice in sham group performaced better in water maze task than those in MCAO group.

T2-weighted MRI imagines showed infarction clearly. Conclusion: Intraluminal occlusion of MCA using monofilament is a kind of simple and reliable method to establish mouse MCAO model.

【key words】mice; middle cerebral artery occlusion; animal model

大脑中动脉栓塞（middle cerebral artery occlusion, MCAO）模型是目前使用最为广泛的、研究局灶性脑缺血再灌注损伤的理想模型。线栓法[1]是制作MCAO 模型常用方法，大鼠是最常用的模型动物。随着基因技术的发展，越来越多的研究选择转基因或基因敲除小鼠为实验动物，但关于构建小鼠MCAO模型的研究不多[2,3]。本研究对小鼠MCAO模型进行一定的改良并进行相关评价。

1 材料与方法

1.1 材料

1.1.1 实验动物雄性C57BL/6J小鼠50只，体质量20～25 g，随机分为假手术组10只，MCAO组40只，由华中科技大学同济医学院实验动物中心提供。

1.1.2 主要试剂与材料2,3,5-氯化三苯基四氮唑（triphenyltetrazolium chloride, TTC）（购于Sigma公司）；1-FR型动物手术显微镜（购于Zeiss公司）；三洋牌渔线（直径0.128 mm）（购于三洋渔具公司）；硅橡胶（PROVIL NOVO）（购于Heraeus Kulzer公司）；水合氯醛（购于北京化学试剂公司）；7.0T 磁共振仪（购于Bruker BioSpin公司）。

1.2 方法

1.2.1 线栓制备将渔线切割为2.5 cm长度/段，将硅胶均匀裹于鱼线表面，直径≤0.2 mm，手术显微镜下仔细将线头修整圆润，避免毛刺，线栓即制作完毕。

1.2.2 小鼠MCAO模型建立MCAO模型制备参照Connolly等[4]和Hata等[5]

的方法，根据小鼠体型及血管直径的不同而进行改良。10％水合氯醛腹腔麻醉（0.35 mL/100g）小鼠，仰卧于可以加热的护垫上；在手术显微镜下，行颈部正中切口，分离皮下组织，暴露右侧颈动脉鞘，小心将颈动脉和迷走神经分离，其间注意避免牵拉损伤迷走神经；细心游离颈总动脉至分叉处，结扎颈外动脉和颈总动脉，用血管夹夹闭颈内动脉；用显微眼科剪自颈总动脉近心端剪开，将线栓沿颈总动脉插入颈内动脉约10 mm左右，稍遇阻力时停止，以阻塞同侧的大脑中动脉；于颈总动脉切口上方用丝线固定线栓。彻底止血，逐层缝合肌肉和皮肤；将动物置于饲养笼中，注意保温，待其苏醒。缺血1 h后将线栓取出。假手术组采用同样的手术操作但不插入线栓。手术操作过程中维持肛温在（37.0±0.5）℃。