Toll样受体4在机械通气致小鼠海马小胶质细胞活化中的作用

Role of Toil-like receptor 4 in mechanical ventilation-induced activation of microglias in hippocampi of mice

目的 评价Toll样受体4（TLR4）在机械通气致小鼠海马小胶质细胞活化中的作用。方法 健康雄性TLR4基因敲除型C57BL/10ScNJNju小鼠和野生型C57BL/6小鼠各42只，10～12周龄，体重20～25 g，采用随机数字表法，将2组小鼠各随机分为2个亚组（n＝21）：自主呼吸亚组（SB亚组）和机械通气亚组（MV亚组）。SB亚组放置在异氟醚麻醉箱内自主呼吸6 h，MV亚组行气管插管术后异氟醚麻醉维持下机械通气6 h。于机械通气后1、3 d时随机取12只小鼠，进行恐惧条件化实验评价认知功能，记录僵直时间比率。于机械通气后1 d随机处死3只小鼠，取全脑组织，采用免疫荧光双标法观察海马小胶质细胞形态，计数CD11b阳性细胞，计数TLR4与CD11b的共表达（TLR4/CD11b）阳性细胞。于机械通气后1 d随机处死6只小鼠，取海马组织，采用ELISA法检测海马TNF-的含量。结果 与野生型组的SB亚组比较，野生型组的MV亚组机械通气后1、3 d时僵直时间比率降低，海马CD11b阳性细胞数增加，TLR4/CD11b阳性细胞数增加，TNF-α含量增加（P〈0.05）；基因敲除型组的MV亚组海马CD11b阳性细胞数增加，TNF-α含量增加（P〈0.05）。与基因敲除型组的SB亚组比较，基因敲除型组的MV亚组机械通气后1、3 d时僵直时间比率降低，海马CD11b阳性细胞数增加，TNF-α含量增加（P〈0.05）。与野生型组的MV亚组比较，基因敲除型组的MV亚组机械通气后1、3 d时僵直时间比率升高，海马CD11b阳性细胞数减少，TNF-α含量降低（P〈0.05）。结论 机械通气致小鼠海马胶质细胞活化的机制部分与TLR4有关。

Objective To evaluate the role of Toll-like receptor 4（TLR4）in mechanical ventilation-induced activation of microglias in the hippocampi of mice.Methods Forty-two healthy male TLR4 gene knocked out C57BL/10ScNJNju mice and 42 wild type C57BL/6 mice, aged 10-12 weeks, weighing 20-25 g, were used in the study.The mice of either group were divided into 2 subgroups（n=21 each）using a random number table： spontaneous breathing subgroup（subgroup SB）and mechanical ventilation subgroup（subgroup MV）. In subgroup SB, the mice were exposed to isoflurane for 6 h in an anesthesia chamber.After tracheal intubation, the mice were mechanically ventilated for 6 h when anesthesia was maintained with isoflurane in subgroup MV.Twelve mice were selected from each subgroup at 1 and 3 days after ventilation, fear conditioning test was performed to assess the cognitive function, and the rate of freezing time was recorded.Three mice in each subgroup were sacrificed at 1 day after ventilation, and the brains were removed to observe the morphological changes of microglias（by double immunofluorescent staining）and to count CD11b positive cells and co-expression of TLR4 with CD11b（TLR4/CD11b）positive cells in hippocampal microglias.Six mice in each subgroup were sacrificed at 1 day after ventilation, and the hippocampi were isolated for determination of the content of tumor necrosis factor-alpha（TNF-α）in hippocampal tissues by enzyme-linked immunosorbent assay.Results Compared with subgroup SB of wild type group, the rate of freezing time was significantly decreased at 1 and 3 days after ventilation, and the number of CD11b positive cells and TLR4/CD11b positive cells were increased, and the content of TNF-α was increased in subgroup MV of wild type group（P〈0.05）, and the number of CD11b positive cells and content of TNF-α were significantly increased in subgroup MV of gene knockout group（P〈0.05）. Compared with subgroup SB of gene knockout group, the rate of freezing time was significantly decreased at 1 and 3 days after ventilation, and the number of CD11b positive cells and content of TNF-α were increased in subgroup MV of gene knockout group（P〈0.05）. Compared with subgroup MV of wild type group, the rate of freezing time was significantly increased at 1 and 3 days after ventilation, and the number of CD11b positive cells and content of TNF-α were decreased in subgroup MV of gene knockout group（P〈0.05）.Conclusion The mechanism by which mechanical ventilation induces activation of microglias in hippocampi is partially related to TLR4 in mice.