大鼠心脏骤停心肺复苏后脑Occludin蛋白表达及其对血脑屏障的影响

Expression of Occludin protein and its influence on blood brain barrier after cardiopulmonary resuscitation in sudden cardiac arrest rats

目的 探讨大鼠心脏骤停心肺复苏后脑Occludin蛋白表达及其对血脑屏障的影响.方法 将200只Sprague-Dawley雄性大鼠根据随机数字表法分为对照组和复苏组,各100只.对照组未进行窒息及心肺复苏,仅进行麻醉和动静脉及气管插管;复苏组制作心脏骤停模型,常规心肺复苏.对照组50只大鼠术后稳定10min后和复苏组50只大鼠自主呼吸恢复（ROSC）后于2、6、12、24、48 h（每个时点10只）断头处死取脑组织,采用免疫组织化学染色方法检测脑组织Occludin蛋白的表达;对照组余50只大鼠术后稳定10 min后和复苏组余50只大鼠ROSC后于0、4、10、22、46 h（每个时点10只）经大鼠尾静脉按3 mg/kg标准注射20 g/L伊文氏蓝,在相应时点2h后断头处死取脑组织,用荧光分光光度计计算血脑屏障对伊文氏蓝的通透性.结果 对照组术后各时点Occludin蛋白表达阳性细胞数和伊文氏蓝含量差异均无统计学意义（均P＞0.05）.复苏组在ROSC后2、6、12、24、48 h Occludin蛋白表达阳性细胞数先降低后升高,各时点均明显低于对照组[（68.2±1.0）个/高倍视野比（86.8±1.3）个/高倍视野、（42.1±1.6）个/高倍视野比（85.1±2.7）个/高倍视野、（18.1 ±1.1）个/高倍视野比（84.9±2.8）个/高倍视野、（37.8±0.7）个/高倍视野比（82.1 ±3.1）个/高倍视野、（61.2±1.0）个/高倍视野比（83.8±2.5）个/高倍视野],伊文氏蓝含量先升高后降低,各时点均明显高于对照组[（0.496±0.042） μg/g比（0.245±0.012） μg/g、（0.704±0.021） μg/g比（0.236 ±0.016） μg/g、（0.898±0.047） μg/g比（0.239±0.011）μg/g、（0.874±0.035）μg/g比（0.251±0.019） μg/g、（0.692±0.036）μg/g比（0.232±0.043）μg/g],差异均有统计学意义（均P＜0.05）.复苏组大鼠脑Occludin蛋白表达阳性细胞数与伊文氏蓝含量呈负相关（r=-0.862,P＜0.01）.结论 大鼠心脏骤停心肺复苏后脑Occludin蛋白表达降低,紧密连接结构受损,导致血脑屏障通透性增加.

Objective To investigate the expression of Occludin protein after cardiopulmonary resuscitation （CPR） in sudden cardiac arrest rats and its effect on blood brain barrier.Methods Totally 200 Sprague-Dawley male rats were randomly divided into control group （n =100） and CPR group （n =100）.Only arteriovenous and endotracheal intubations were given in control group,without choking and CPR;sudden cardiac arrest model was established,then CPR was performed in CPR group.Fifty rats were sacrificed with decollation to collect the brain tissue 2,6,12,24 and 48 h after postoperative stable phase （10 min after operation） in control group and 2,6,12,24 and 48 h after return of spontaneous circulation （ROSC） in CPR group （n =10 at each time points）;the Occludin expression in the brain tissue was measured by immunohistochemistry.Fifty rats were injected with 3 mg/kg Evans blue via caudal veins,then were sacrificed with decollation 2 h later to collect the brain tissue;the brain barrier permeability rates of Evans blue were measured by fluorescence spectrophotometer.Results The cell number of positive Occludin protein and Evans blue content in brain tissue were not significantly different between groups （P 〉 0.05）.Along with time,the cell number of positive Occludin protein in brain tissue firstly decreased then increased in CPR group,being significantly lower than that in control group at all the time points [（68.2± 1.0）/high power field vs （86.8 ± 1.3）/high power field,（42.1 ± 1.6）/high power field vs （85.1 ±2.7）/high power field,（18.1 ± 1.1）/high power field vs （84.9 ±2.8）/high power field,（37.8 ± 0.7）/high power field vs （82.1 ± 3.1）/high power field,（61.2 ± 1.0）/high power field vs （83.8 ± 2.5）/high power field] （P 〈 0.05）;along with time,the contents of Evans blue in brain tissue firstly increased then decreased in CPR group,being significantly higher than that in control group at all time points [（0.496 ± 0.042） μg/gvs （0.245 ±0.012） μg/g,（0.704±0.021） μg/g vs （0.236±0.016） μg/g,（0.898 ± 0.047） μg/g vs （0.239±0.011） μg/g,（0.874±0.035） μg/gvs （0.251 ±0.019） μg/g,（0.692± 0.036） μg/g vs （0.232 ±0.043） μg/g] （P 〈0.05）.The cell number of positive Occludin proteins in CPR group was negatively correlated with Evans blue content （r =-0.862,P 〈 0.01）.Conclusion Occludin protein expression in brain is decreased and the close connecting structure is damaged after CPR in rats,leading to increased permeability of blood-brain barrier.