七氟烷麻醉对新生期大鼠脑电波与远期行为的影响及其机制研究

The effect and possible mechanism of sevoflurane exposure on electroencephalographic seizures and long-term cognitive function in neonatal rats

目的研究七氟烷麻醉6 h对新生期大鼠脑电监测的癫痫波及远期行为的影响及机制研究。方法健康新生4~6 d SD大鼠141只(雄性66只,雌性75只)按照随机数字表法分为3大组(每组雄性22只,雌性25只)。对照组:正常笼内喂养,不接受麻醉;七氟烷组:仅接受2.1%七氟烷麻醉6 h;NKCC1阻断剂组:2.1%七氟烷麻醉前30 min接受腹腔注射1.82 mg/kg布美他尼。其中对照组在七氟烷组与NKCC1阻断剂组动物接受药物干预的同一时间接受同等剂量的二甲基亚砜(dimethyl sulfoxide,DMSO)皮下注射,以排除溶媒引起的影响。麻醉完成后进行苏醒,观察30 min后继续笼内正常母乳喂养。其中一部分新生大鼠养至9~11 d接受皮层脑电波(electroencephalogram,EEG)监测;其余大鼠养至60 d、70 d时分别进行高架十字迷宫(elevated plus maze,EPM)和前脉冲抑制(prepulse inhibition,PPI)实验。结果EEG结果中,与对照组相比较,七氟烷组雄性大鼠EEG中癫痫波出现的次数增多[(0.429±0.787)次,(1.571±0.787)次;t=2.753,P<0.01],单个癫痫波平均时长增多[(1.575±2.349)s,(6.392±3.374)s;t=3.880,P<0.01],总时长明显升高[(1.800±3.617)s,(10.957±6.028)s;t=3.929,P<0.01],差异有统计学意义;与七氟烷组相比较,NKCC1阻断剂组雄性大鼠EEG中癫痫波出现的次数减少,单个癫痫波平均时长减少,总时长明显缩短,均差异有统计学意义[(0.286±0.756)次,(0.925±1.733)s,(1.043±2.759)s;t=3.097,4.404,4.254,均P<0.01]。三组雌性大鼠之间比较,各指标均差异无统计学意义(均P>0.05)。雌雄大鼠间比较:七氟烷组雌性大鼠与雄性大鼠相比较,雌性大鼠七氟烷组的平均时长减少[(6.392±3.374)s,(2.515±2.992)s;t=3.044,P<0.01],总时长缩短[(10.957±6.028)s,(3.270±5.883)s;t=2.626,P<0.01],差异有统计学意义。行为学结果中,雄性大鼠:与对照组相比较,七氟烷组EPM实验中开放臂停留时间明显缩短(P<0.05),PPI实验中惊吓反应明显降低(P<0.05),差异有统计学意义;与七氟烷组相比较,NKCC1阻断剂组EPM实验中开放臂停留时间明显延长(P<0.05),PPI实验中惊吓反应明显增加(P<0.05),差异有统计学意义。雌性大鼠:各组变化趋势与雄性大鼠相似,但差异无统计学意义(P>0.05)。雌雄之间比较:与雄性大鼠七氟烷组相比较,雌性大鼠七氟烷组EPM实验中开放臂停留时间较长(P<0.05),差异有统计学意义。结论七氟烷麻醉6 h可以明显增加雄性新生大鼠EEG中癫痫波的产生,引发成年后雄性大鼠行为学异常,其机制可能与NKCC1相关;雄性大鼠脑神经发育更容易受到七氟烷麻醉的负面影响。

Objective To investigate the effect of sevoflurane anesthesia on electroencephalographic(EEG)seizures and long-term behavior and possible mechanism in neonatal rats.Methods A total of 141 postnatal days 4-6 Sprague-Dawley rats(66 male,75 female)were divided into 3 groups(n=47 in each group)according to random number table method:control group,sevoflurane group,and NKCC1 inhibitor group,with 22 males and 25 females in each group.Rats in the control group were fed in normal cage without anesthesia;rats in the sevoflurane group were anesthetized with 2.1%sevoflurane for 6 hours;rats in the NKCC1 blocker group received intraperitoneal injection of 1.82 mg/kg bumetanide 30 minutes before anesthesia with 2.1%sevoflurane.The rats in the control group and sevoflurane group were injected subcutaneously with the same dose of DMSO at the same time when the NKCC1 blocker group received the drug intervention,so as to eliminate the influence caused by the solvent.The rats were observed for 30 minutes after recovery from anesthesia and then continued to breastfeed normally.Some of the new born rats received EEG monitoring from 9 to 11 days after being raised;the other rats received EPM and PPI respectively at 60 and 70 days after being raised.Results The results of EEG showed that,compared with the control group,the number of epileptic waves((0.429±0.787),(1.571±0.787),t=2.753,P<0.01),the average duration of single epileptic wave((1.575±2.349),(6.392±3.374),t=3.880,P<0.01),the total duration increased significantly((1.800±3.617),(10.957±6.028),t=3.929,P<0.01)were all increased,the differences were statistically significant.Compared with sevoflurane group,the number of epileptic waves in EEG of male rats in NKCC1 blocker group decreased,the average duration of single epileptic wave decreased,and the total duration of epileptic wave shortened significantly,with statistical significance((0.286±0.756),(0.925±1.733),(1.043±2.759),t=3.097,4.404,4.254,all P<0.01).There were no significant differences in female rat among the three groups(all P>0.05).Compared with male rats,the average duration of female rats in sevoflurane group decreased((6.392±3.374),(2.515±2.992),t=3.044,P<0.01),the total duration shortened((10.957±6.028),(3.270±5.883),t=2.626,P<0.01),the difference was statistically significant.The behavioral results showed that,compared with the control group,the open arm dwell time of male rats in sevoflurane group was significantly shorter(P<0.05),and the panic response in PPI group was significantly lower(P<0.05),the difference was statistically significant.Compared with the sevoflurane group,the open arm dwell time in NKCC1 blocker group was significantly longer(P<0.05),and the panic response in PPI group was significantly increased.The difference was statistically significant(P<0.05).The change trend in female rats of each group was similar to that of male rats,but there was no significant difference(all P>0.05).Comparison between male and female rats:compared with male rats in sevoflurane group,the female rats in sevoflurane group had a longer open arm stay time in EPM experiment(P<0.05),the difference was statistically significant.Conclusion Sevoflurane anesthesia for 6 hours can significantly increase the generation of epileptic waves in EEG of male newborn rats,and cause behavioral abnormalities in adult male rats,which may be related to NKCC1.And male rats are more vulnerable to the negative effects of sevoflurane anesthesia on brain nerve development.