七氟烷麻醉后大鼠觉醒机制:光遗传学和化学遗传学技术调控中缝背核食欲素能神经元投射

Arousal mechanism after sevoflurane anesthesia in rats:orexinergic modulation in dorsal raphe nucleus by optogenetic and chemogenetic techniques

目的采用光遗传学和化学遗传学技术调控中缝背核食欲素能神经元投射的方法,探讨七氟烷麻醉后大鼠觉醒的机制。方法健康雄性Hcrt-Cre转基因大鼠45只,10~12周龄,体质量220~250 g,采用随机数字表法分为6组:光遗传兴奋组(CHR2组)、光遗传抑制组(eNpHR组)和光遗传对照组(O-CON组),每组5只;化学遗传兴奋组(hm3Dq组)、化学遗传抑制组(hm4Di组)和化学遗传对照组(C-CON组),每组10只。分别采用光遗传学和化学遗传学技术调控策略进行处理。在大鼠病毒注射3周后,采用2.7%七氟烷与纯氧1.5 L/min对大鼠进行麻醉诱导并维持稳定麻醉状态,全程脑电监测,记录光刺激前2 min与刺激时2 min大鼠脑电爆发性抑制率(%BSR)。结合光遗传及化学遗传策略,观察兴奋或抑制食欲素能神经元投射至中缝背核的神经末梢是否可引起七氟烷麻醉后大鼠翻正反射行为的改变。结果与C-CON组比较,hm3Dq组大鼠七氟烷麻醉后翻正反射恢复(RORR)时间缩短,hm4Di组和eNpHR组RORR时间延长(P<0.05);与O-CON组或光刺激前2 min%BSR比较,CHR2组光刺激期间%BSR降低(P<0.05),eNpHR组光刺激期间%BSR差异无统计学意义(P>0.05);与O-CON组比较,CHR2组大鼠七氟烷麻醉后RORR时间缩短(P<0.05)。结论大鼠中脑中缝背核的下丘脑外侧区食欲素能神经末梢发挥主动全麻后促觉醒效应。

Objective To investigate the arousal mechanism after sevoflurane anesthesia using orexinergic modulation in dorsal raphe nucleus(DRN)by optogenetic and chemogenetic techniques in rats.Methods Forty-five healthy male Hcrt-Cre rats,aged 10-12 weeks,weighing 220-250 g,were divided into 6 groups by the random number table method:optical-excitatory group(CHR2 group,n=5),optical-inhibitory group(eNpHR group,n=5),optical-control group(O-CON group,n=5);chemogenetic-excitatory group(hm3Dq group,n=10),chemogenetic-inhibitory group(hm4Di group,n=10)and chemogenetic-control group(C-CON group,n=10).The optogenetic or chemogenetic techniques were used in each group.Three weeks after injecting the rat virus,anesthesia was induced and maintained with 2.7%sevoflurane anesthesia in 1.5 L/min O_(2),and the EEG data were continuously recorded throughout the process.The burst suppression ratio(%BSR)was recorded at 2 min before and of laser stimulation.Combining optogenetic and chemogenetic strategies,it was investigated that whether activation of orexinergic projection to DRN could modulate anesthetic behaviors during sevoflurane anesthesia.Results Compared with C-CON group,the recovery of righting reflex(RORR)time was significantly shortened after sevoflurane anesthesia in hm3Dq group(P<0.05),and the RORR time was significantly prolonged after sevoflurane anesthesia in hm4Di group and eNpHR group(P<0.05).Compared with O-CON group or the baseline at 2 min before light stimulation,the%BSR was significantly decreased during 473nm laser stimulation in CHR2 group(P<0.05),and no statistically significant change was found in the%BSR during 473nm laser stimulation in eNpHR group(P>0.05).Compared with O-CON group,the RORR time was significantly shortened after sevoflurane anesthesia in CHR2 group(P<0.05).Conclusions Lateral hypothalamic areaorexin-DRN neural circuit plays a key role in promoting arousal from general anesthesia in rats.