肠道菌群在氯胺酮减轻小鼠术后认知功能障碍中的作用:与膈下迷走神经的关系

Role of gut microbiota in amelioration of postoperative cognitive dysfunction in mice by ketamine: association with subdiaphragmatic vagal nerve

目的评价肠道菌群在氯胺酮减轻小鼠术后认知功能障碍(POCD)中的作用及其与膈下迷走神经的关系。方法 SPF级健康雄性C57BL/6J小鼠144只, 18月龄, 体质量32~35 g, 采用随机数字表法分为8组(n=18):假手术组(Sham组)、手术组(S组)、氯胺酮+手术组(SK组)、氯胺酮+手术+膈下迷走神经切除组(SK+SDV组)、伪无菌小鼠+生理盐水组(GM+V组)、伪无菌小鼠接受S组小鼠粪菌移植组(GM+S组)、伪无菌小鼠接受SK组小鼠粪菌移植组(GM+SK组)和伪无菌小鼠+膈下迷走神经切断+接受SK组小鼠粪菌移植组(GM+SDV+SK组)。吸入2.0%异氟烷麻醉下行胫骨离断髓内固定手术。SK组于缝皮开始时腹腔注射氯胺酮50 mg/kg。术后24 h时收集S组和SK组小鼠的新鲜粪便, 制备成粪菌滤液。SK+SDV组于氯胺酮给药前、GM+SDV+SK组于粪菌移植前切断膈下迷走神经。伪无菌小鼠模型:于粪菌移植或膈下迷走神经神经切断前2周将广谱抗生素混合液加入小鼠饮用水中, 每2 d更换1次。GM+SK组、GM+SDV+SK组经抗生素干预14 d后, 每天固定时间灌胃给予相应小鼠粪菌滤液200 μl, GM+V组给予生理盐水溶剂, 连续14 d。胫骨离断髓内固定术后或粪菌移植完成后24 h, 收集小鼠粪便, 行16S rRNA基因高通量测序, 采用ELISA法检测海马IL-6、IL-17、TNF-α、IL-4和IL-10的含量, 采用Morris水迷宫实验评价小鼠学习和记忆能力。结果与Sham组比较, S组术后肠道菌群Simpson多样性指数、Shannon指数、Ace指数降低, 海马IL-6、IL-17和TNF-α含量增加, 海马IL-4和IL-10含量降低, 训练阶段逃避潜伏期、测试阶段逃避潜伏期和游泳距离延长, 目标象限停留时间缩短(P<0.05);与S组比较, SK组术后肠道菌群Simpson多样性指数、Shannon指数、Ace指数升高, 海马IL-6、IL-17和TNF-α含量降低, 海马IL-4和IL-10含量增加, 训练阶段逃避潜伏期、测试阶段逃避潜伏期和游泳距离缩短, 目标象限停留时间延长(P<0.05);与SK组比较, SK+SDV组术后肠道菌群Simpson多样性指数、Shannon指数、Ace指数降低, 海马IL-6、IL-17和TNF-α含量增加, 海马IL-4和IL-10含量降低, 训练阶段逃避潜伏期、测试阶段逃避潜伏期和游泳距离延长, 目标象限停留时间缩短(P<0.05)。与GM+V组比较, GM+S组海马IL-6、IL-17和TNF-α含量增加, IL-4和IL-10含量降低, 训练阶段逃避潜伏期、测试阶段逃避潜伏期和游泳距离延长, 目标象限停留时间缩短(P<0.05);与GM+S组比较, GM+SK组海马IL-6、IL-17和TNF-α含量降低, 海马IL-4和IL-10含量增加, 训练阶段逃避潜伏期、测试阶段逃避潜伏期和游泳距离缩短, 目标象限停留时间延长(P<0.05);与GM+SK组比较, GM+SDV+SK组海马IL-6、IL-17和TNF-α含量增加, 海马IL-4和IL-10含量降低, 训练阶段逃避潜伏期、测试阶段逃避潜伏期和游泳距离延长, 目标象限停留时间缩短(P<0.05)。结论氯胺酮可改善肠道菌群紊乱, 减轻小鼠POCD, 其机制可能与膈下迷走神经传导有关。

Objective To evaluate the role of ketamine in postoperative cognitive dysfunction(POCD)and to clarify the association with subdiaphragmatic vagus nerve in mice.Methods One hundred and forty-four SPF healthy male C57BL/6J mice,aged 18 months,weighing 32-35 g,were divided into 8 groups(n=18 each)using a random number table method:sham operation group(group C),surgery group(group S),ketamine+surgery group(group SK),ketamine+surgery+subdiaphragmatic vagotomy group(group SK+SDV),pseudo germ-free mice+normal saline group(group GM+V),pseudo germ-free mice that received fecal microbiota transplantation(FMT)from group S mice group(group GM+S),pseudo germ-free mice that received FMT from group SK mice group(group GM+SK),and pseudo germ-free mice received subdiaphragmatic vagotomy and FMT from group SK mice group(group GM+SDV+SK).Tibial fracture internal fixation was performed under anesthesia with 2.0%isoflurane.Ketamine 50 mg/kg was intraoperatively administered at the beginning of the suture in group SK.The fecal samples were collected at 24 h after surgery to prepare fecal bacteria filtrate in S and SK groups.The dorsal and ventral branches of subdiaphragmatic vagus nerve were cut prior to ketamine administration in SK+SDV group and prior to FMT in GM+SDV+SK group.Broad-spectrum antibiotics dissolved in drinking water were given ad libitum to mice for 14 consecutive days at 2 weeks before FMT or subdiaphragmatic vagotomy and replaced once every 2 days to establish the pseudo germ-free model.Pseudo germ-free mice were gavaged with normal saline or fecal suspension 200μl obtained from mice for 14 consecutive days at fixed time starting from 14 days after antibiotic intervention in GM+SK and GM+SDV+SK groups.While normal saline was given instead for 14 consecutive days in GM+V group.The fecal samples were collected after tibial fracture internal fixation or at 24 h after completion of FMT for 16S rRNA gene sequence analysis.The contents of interleukin-6(IL-6),IL-17,tumor necrosis factor-alpha(TNF-α),IL-4 and IL-10 in the hippocampus were measured by enzyme linked-immuno-sorbent assay at 24 h after surgery or FMT.The spatial memory and learning ability was assessed by the Morris water maze test at 24 h after surgery or FMT.Results Compared with group Sham,Simpson′s diversity index,Shannon index and Ace index were significantly decreased,the contents of IL-6,IL-17 and TNF-αwere increased,contents of IL-4 and IL-10 were decreased,the escape latency in the training and testing phases and swimming distance were prolonged,and the time spent in the target quadrant was shortened in group S(P<0.05).Compared with group S,Simpson′s diversity index,Shannon index and Ace index were significantly increased,the contents of IL-6,IL-17 and TNF-αwere decreased,contents of IL-4 and IL-10 were increased,the escape latency in the training and testing phases and swimming distance were shortened,and the time spent in the target quadrant was prolonged in group SK(P<0.05).Compared with group SK,Simpson′s diversity index,Shannon index and Ace index were significantly decreased,the contents of IL-6,IL-17 and TNF-αwere increased,contents of IL-4 and IL-10 were decreased,the escape latency in the training and testing phases and swimming distance were prolonged,and the time spent in the target quadrant was shortened in group SK+SDV(P<0.05).Compared with group GM+V,the contents of IL-6,IL-17 and TNF-αwere significantly increased,contents of IL-4 and IL-10 were decreased,the escape latency in the training and testing phases and swimming distance were prolonged,and the time spent in the target quadrant was shortened in group GM+S(P<0.05).Compared with group GM+S,the contents of IL-6,IL-17 and TNF-αwere significantly decreased,contents of IL-4 and IL-10 were increased,the escape latency in the training and testing phases and swimming distance were shortened,and the time spent in the target quadrant was prolonged in group GM+S(P<0.05).Compared with group GM+SK,the contents of IL-6,IL-17 and TNF-αwere significantly increased,contents of IL-4 and IL-10 were decreased,the escape latency in the training and testing phases and swimming distance were prolonged,and the time spent in the target quadrant was shortened in group GM+SDV+SK(P<0.05).Conclusions Ketamine can improve intestinal flora disorders and reduce POCD in mice,and the mechanism may be related to subdiaphragmatic vagal nerve conduction.