海马Aβ_(42)沉积诱发中性粒细胞聚集在七氟烷麻醉导致老龄大鼠血脑屏障损伤中的作用

Role of hippocampalβ-amyloid 42 deposition-induced accumulation of neutrophils in blood-brain barrier damage caused by sevoflurane anesthesia in aged rats

目的评价海马β淀粉样蛋白42(Aβ_(42))沉积诱发中性粒细胞聚集在七氟烷麻醉导致老龄大鼠血脑屏障损伤中的作用。方法取鞘内置管成功的健康雄性Wistar大鼠72只,18~20月龄,体重600~650 g,采用随机数字表法分为4组(n=18):对照组(C组)、γ-分泌酶抑制剂DAPT组(D组)、七氟烷麻醉组(S组)和DAPT+七氟烷麻醉组(DS组)。C组和S组鞘内注射二甲基亚砜10μl,30 min后C组吸入60%氧气2 h,S组吸入3.6%七氟烷+60%氧气2 h,同时行胫骨骨折手术。D组和DS组鞘内注射100μmol/L DAPT 10μl,30 min后D组吸入60%氧气2 h,DS组吸入3.6%七氟烷+60%氧气2 h,同时行胫骨骨折手术。分组处理结束后12 h时行条件恐惧实验,计算僵直时间比率。行为学测试结束后处死大鼠,取海马组织,采用Western blot法测定Aβ_(42)、Occludin和基质金属蛋白酶-9(MMP-9)的表达水平,采用免疫组化法计数中性粒细胞,采用伊文思蓝(EB)染色法测定EB含量。结果与C组比较,S组和DS组僵直时间比率下降,海马Aβ_(42)和MMP-9表达上调,Occludin表达下调,中性粒细胞计数和EB含量增加(P<0.05),D组上述指标差异无统计学意义(P>0.05);与S组比较,DS僵直时间比率升高,海马Aβ_(42)表达和MMP-9表达下调,Occludin表达上调,中性粒细胞计数和EB含量降低(P<0.05)。结论七氟烷麻醉导致老龄大鼠术后认知功能障碍的机制与海马Aβ_(42)沉积诱发中性粒细胞聚集,造成血脑屏障损伤有关。

Objective To evaluate the role of hippocampalβ-amyloid 42(Aβ_(42))deposition-induced accumulation of neutrophils in blood-brain barrier damage caused by sevoflurane anesthesia in aged rats.Methods Seventy-two healthy male Wistar rats in which IT catheters were successfully planted,aged 18-20 months,weighing 600-650 g,were divided into 4 groups(n=18 each)using a random number table method:control group(group C),γ-secretase inhibitor DAPT group(group D),sevoflurane anesthesia group(group S)and DAPT plus sevoflurane anesthesia group(group DS).Dimethyl sulfoxide 10μl was intrathecally injected in group C and group S,and 30 min later group C inhaled 60%oxygen for 2 h,and group S inhaled 3.6%sevoflurane and 60%oxygen for 2 h and tibial fracture surgery was performed at the same time.DAPT 10μl was intrathecally injected in group D and group DS,and 30 min later group D inhaled 60%oxygen for 2 h,and group DS inhaled 3.6%sevoflurane and 60%oxygen for 2 h and tibial fracture surgery was performed at the same time.The fear conditioning test was performed at 12 h after the end of treatment in each group,and the ratio of time spent freezing was calculated.The rats were sacrificed after the end of behavioral test,and hippocampal tissues were removed for determination of the expression of Aβ_(42),occludin and matrix metalloproteinase-9(MMP-9)(by Western blot),neutrophil count(by immuno-histochemistry),and content of Evans blue(EB)(by EB staining).Results Compared with group C,the ratio of time spent freezing was significantly decreased,the expression of Aβ_(42) and MMP-9 was up-regulated,the expression of occludin was down-regulated,the neutrophil count and content of EB were increased in group S and group DS(P<0.05),and no significant change was found in the parameters mentioned above in group D(P>0.05).Compared with group S,the ratio of time spent freezing was significantly increased,the expression of Aβ_(42) and MMP-9 was down-regulated,the expression of occludin was up-regulated,the neutrophil count and content of EB were decreased in group DS(P<0.05).Conclusion The mechanism by which sevoflurane anesthesia leads to postoperative cognitive dysfunction is related to hippocampal Aβ_(42) deposition-induced accumulation of neutrophils and causing damage to blood-brain barrier in aged rats.