亚低温对缺血性脑卒中小鼠血脑屏障损伤保护作用_钟超超.pdf

安 徽 医 药 Anhui Medical and Pharmaceutical Journal 2023 Feb，27（2）亚低温对缺血性脑卒中小鼠血脑屏障损伤保护作用钟超超，徐兴国，苗海航，乔峤，秦毅彬作者单位：南通大学附属医院麻醉科，江苏 南通226001通信作者：秦毅彬，男，硕士生导师，研究方向为围术期重要脏器的保护，Email：基金项目：南通市科技发展计划项目（MS12020026）摘要：目的 探讨亚低温对缺血性脑卒中小鼠血脑屏障的保护作用，寻找亚低温发挥保护作用的机制。方法 选取由南通大学实验动物中心提供的SPF级别雄性C57/BL6小鼠54只，分为假手术组（n=18）和卒中组（n=36），卒中组分为亚低温组（n=18）和常温组（n=18）。卒中组在行大脑中动脉阻塞手术后对亚低温组小鼠予以亚低温处理，在口腔温度监测下维持小鼠温度为（340.5），其他组术后均置于保温毯上正常复温。术后每日对各组神经功能进行评分，同时使用错步时间检测卒中后小鼠行为运动功能。于术后第3天处死小鼠，取梗死脑区行蛋白质印迹法（Western blotting）检测血脑屏障黏附分子紧密连接蛋白（ZO-1），紧密连接跨膜蛋白（Claudin-5）的蛋白表达以判断血脑屏障损伤的程度。结果 假手术组、亚低温组、常温组小鼠存活率分别为100%（18/18），94.44%（17/18），88.89%（16/18）。三组存活率比较，差异无统计学意义（2=53.72，P=0.122）。卒中组小鼠术后第1、2、3天神经功能评分 （2.80.2）、（2.6 0.3）、（2.40.3）分 明显高于假手术组 （0.6 0.04）、（0.5 0.03）、（0.4 0.01）分，差异有统计学意义（t=2.94，P0.001；t=2.86，P0.001；t=2.98，P0.001）；卒中组中，常温组小鼠术后第1、2、3天神经功能评分均明显高于亚低温组 （2.00.3）、（1.90.2）、（1.80.2）分，差异有统计学意义。卒中组小鼠术后第1、2、3天前后肢的平均错步比例（26.8%前、32%后；29.0%前、31.1%后；31.8%前、33.1%后）均高于假手术组（均为3%前、3%后），差异有统计学意义（2=29.05，P=0.002；2=28.76，P=0.003；2=26.42，P=0.005），且常温组小鼠术后第1、2、3天前后肢平均错步的比例高于亚低温组（16.6%前、16.9%后；18.6%前、17.9%后；20.8%前、19.9%后），差异有统计学意义（2=27.35，P=0.004；2=26.59，P=0.004；2=24.12，P=0.007）。存活的小鼠，脑卒中可以显著降低小鼠脑卒中脑组织中血脑屏障黏附分子ZO-1，Claudin-5的蛋白表达（t=2.54，P=0.001；t=2.46，P=0.003），亚低温处理组小鼠脑组织血脑屏障黏附分子ZO-1，Claudin-5的蛋白表达比常温处理组显著增高（t=2.48，P=0.003；t=1.68，P=0.043）。结论 脑卒中术后予以6 h的亚低温处理可降低卒中后小鼠神经功能评分，改善术后短期运动行为功能，减少血脑屏障的破坏。关键词：低温，人工；脑梗死；缺血性脑卒中；血脑屏障Protective effect of subhypothermia on bloodbrain barrier injury in mice with ischemic strokeZHONG Chaochao,XU Xingguo,MIAO Haihang,QIAO Qiao,QIN YibinAuthor Affiliation:Department of Anesthesiology,the Hospital Affiliated of Nantong University,Nantong,Jiangsu 226001,ChinaAbstract：Objective To investigate the protective effect of subhypothermia on bloodbrain barrier injury in mice with ischemic stroke,and to explore the mechanism of the protective effect of subhypothermia.Methods A total of 54 SPF-grade male C57/BL6 mice provided by the Experimental Animal Center of Nantong University were selected and divided into a sham-operated group(n=18)and a stroke group(n=36),and the stroke group was divided into a subhypothermia group(n=18)and a normothermic group(n=18).In the stroke group,mice in the subcritical group were treated with subcritical temperature after middle cerebral artery obstruction surgery,and the temperature of the mice was maintained at(340.5)under oral temperature monitoring,while all other groups were placed on a thermal blanket for normal rewarming after surgery.The neurological function of each group was scored daily after surgery,and the poststroke behavioral-motor function of the mice was also measured using staggered time.The mice were sacrificed on the third day after surgery,and the protein expression of ZO-1 and Claudin-5 was measured by western blotting to determine the extent of bloodbrain barrier damage.Results The survival rates of the sham group,subhypothermia group and normothermic groups of mice were 100%(18/18),94.44%(17/18),and 88.89%(16/18),respectively.There was no significant difference in the survival rate among the three groups(2=53.72,P=0.122).The neurological function scores on postoperative days 1,2,and 3 were significantly higher in the stroke group(2.80.2),(2.60.3),(2.40.3)than in the sham group(0.60.04),(0.50.03),and(0.40.01),with statistically signifi临床医学引用本文：钟超超，徐兴国，苗海航，等.亚低温对缺血性脑卒中小鼠血脑屏障损伤保护作用 J.安徽医药，2023，27（2）：337-340.DOI：10.3969/j.issn.1009-6469.2023.02.028.337安 徽 医 药 Anhui Medical and Pharmaceutical Journal 2023 Feb，27（2）cant differences(t=2.94,P0.001;t=2.86,P0.001;t=2.98,P0.001).In the stroke group,the neurological function scores on postoperative days 1,2 and 3 were significantly higher in the normothermic group than in the subhypothermia group(2.00.3),(1.90.2),and(1.80.2),and the differences were statistically significant.In the stroke group,the average proportion of hind limb missteps before the 1st,2nd,and 3rd postoperative days was higher in the mice(26.8%forelimb and 32%posterior limb;29.0%forelimb and 31.1%posterior limb;31.8%forelimb and 33.1%posterior limb)than in the sham-operated group(both forelimb and posterior limb 3%),and the differences were statistically significant(2=29.05,P=0.002;2=28.76,P=0.003;2=26.42,P=0.005).The average proportion of hind limb missteps before the 1st,2nd,and 3rd postoperative days were higher in the normothermic group than in the sham-operated group(16.6%forelimb and 16.9%posterior limb;18.6%forelimb and 17.9%posterior limb;20.8%forelimb and 19.9%posterior limb),and the difference was statistically significant(2=27.35,P=0.004;2=26.59,P=0.004;2=24.12,P=0.007).In surviving mice,stroke significantly reduced the protein expression of ZO-1 and Claudin-5 in the brain tissue of mice with stroke(t=2.54,P=0.001;t=2.46,P=0.003),and the protein expression of ZO-1 and Claudin-5 in the brain tissue of mice treated with subhypothermia was significantly higher than that in the normothermic treatment group(t=2.48,P=0.003;t=1.68,P=0.043).Conclusion Subhypothermia treatment for 6 h after s