兔单侧椎动脉结扎致颈髓急性缺血改变的研究

Study on acute ischemia of cervical medulla caused by ligation of unilateral vertebral artery in rabbits

中文关键词:  椎动脉损伤  椎动脉结扎  中枢缺血  动物实验  日本大耳白兔

英文关键词:Vertebral artery injury  Vertebral artery ligation  Ischemia  Zoopery  Japanese white rabbit

中文摘要:

  【摘要】 目的：通过兔与人解剖学异同的结扎方法损伤椎动脉，观测急性期颈髓组织缺血变化，了解单侧椎动脉损伤对颈髓的影响。方法：选取30只日本大耳白兔（育龄126~140d；雌性17只，雄性13只），随机分为实验组和对照组各15只，实验组行右侧椎动脉第一段结扎，对照组行右侧椎动脉第一段分离作为对照。每组分别于术后2h、6h、24h各处死5只，通过免疫组织化学染色、Western Blotting和qRT-PCR方法观测BCL2相关X蛋白（BCL2-associated X protein，BAX）、B细胞淋巴瘤-2（B-cell lymphoma 2，BCL2）、含半胱氨酸的天冬氨酸蛋白酶3（cysteine-aspartic protease 3，CASP3）、FBJ三氏小鼠骨肉瘤病毒致癌基因同源物（Finkel-Biskis-Jinkins murine osteosarcoma viral oncogene homolog，FOS）等指标及其mRNA表达。结果：免疫组织化学染色和Western Blotting检测结果显示，结扎组兔颈髓组织中的BAX、BCL2、CASP3和FOS蛋白表达所有时间点均高于对照组，其中BAX（免疫组织化学染色：39975.00±1007.00；Western Blotting检测：3.81±0.04）和BCL2（免疫组织化学染色：49865.00±1783.00；Western Blotting检测：5.26±0.07）在术后24h时表达最多，CASP3在术后6h时最多（免疫组织化学染色：82218.00±1256.00；Western Blotting检测：3.46±0.08），FOS在术后2h时最多（免疫组织化学染色：23840.00±1584.00；Western Blotting检测：2.46±0.04），和对照组相比差异具有统计学意义（P<0.05）；结扎组上述蛋白的表达在不同观察节点也不相同，两两比较时差异具有统计学意义（P<0.05），其中，BAX和BCL2随时间增加而增大，CASP3随时间增加先增大后减小，FOS随时间增加而减小。qRT-PCR检测结果显示，结扎组兔颈髓组织中的BAX、BCL2、CASP3和FOS表达均高于对照组，BAX（14.48±1.16）和BCL2（10.35±1.67）在术后24h时表达最多，CASP3在术后6h时最多（30.37±2.27），FOS在术后2h时最多（15.65±1.21），和对照组相比差异具有统计学意义（P<0.05）；结扎组上述mRNA的表达在不同观察节点也不相同，两两比较时差异具有统计学意义（P<0.05），其中，BAX和BCL2随时间增大，CASP3随时间增加先增大后减小，FOS随时间增加逐渐减小。结论：结扎兔右侧椎动脉第一段会造成兔颈髓轻度急性缺血损伤，对CASP3的检测提示术后6h时损伤最严重，该缺血损伤可以很快恢复，在24h时已基本恢复至术前状态。

英文摘要:

  【Abstract】 Objectives: To ligate the vertebral artery based on the comparative anatomy between rabbit and human, and to investigate the changes in cervical spinal cord ischemia in acute phase in order to study the effect of vertebral artery injury made on cervical spinal cord. Methods: The rabbits were randomly divided into experimental group and control group equally. After ligating the first part of right vertebral artery in rabbits in grouping, ex vivo evaluation was carried out at 2 hours after ligation, 6 hours after ligation and 24 hours after ligation. The ex vivo observation indexes were immunohistochemistry, Western Blotting and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) focus on BCL2-associated X protein (BAX), B-cell lymphoma 2(BCL2), cysteine-aspartic protease 3(CASP3) and Finkel-Biskis-Jinkins murine osteosarcoma viral oncogene homolog (FOS). Results: According to the results of immunohistochemically staining and Western Blotting test on the cervical spinal cord of rabbits in ligation groups, protein BAX, BCL2, CASP3 and FOS expressed significantly highly than those in the control group(P<0.05). The maximums of BAX (immunohistochemically staining 39975.00±1007.00; Western Blotting test 3.81±0.04) and BCL2 (immunohistochemically staining 49865.00±1783.00; Western Blotting test 5.26±0.07) were at 24 hours, the maximum of CASP3(immunohistochemically staining 82218.00±1256.00; Western Blotting test 3.46±0.08) was at 6 hours and the maximum of FOS (immunohistochemically staining 23840.00±1584.00; Western Blotting test 2.46±0.04) was at 2 hours after surgery. And the levels of these proteins were different from each other at three time points(P<0.05). BAX and BCL2 increased with time, CASP3 increased at the beginning and later on decreased, FOS decreased with time. qRT-PCR test showed that the mRNA levels of BAX, BCL2, CASP3 and FOS in ligation groups were higher than those in the control group and the differences were statistically significant(P<0.05). The maximums of BAX(14.48±1.16) and BCL2(10.35±1.67) were at 24 hours, the maximum of CASP3(30.37±2.27) was at 6 hours and the maximum of FOS(15.65±1.21) was at 2 hours after surgery. The levels of these mRNAs were different from each other at three time points(P<0.05). BAX and BCL2 increased with time, CASP3 increased at the beginning and later on decreased, FOS decreased with time. Conclusions: The cervical spinal cord experienced a process of obtaining mild acute ischemic injury at the beginning and then quickly recovering along with time according to immunohistochemically staining results, Western Blotting results and qRT-PCR results. The most serious damaged time point was at 6 hours after ligation and the cervical spinal cord returned to baseline by 24 hours after ligation.