异氟醚全身麻醉对腰椎间盘突出症大鼠海马区小胶质细胞活化及炎症的影响

Effect of general anesthesia with isoflurane on hippocampal microglia activation and inflammation in rats with lumbar disc herniation

目的探讨异氟醚全身麻醉对腰椎间盘突出症(LDH)大鼠海马区小胶质细胞活化及炎症的影响。方法将40只大鼠随机分为对照组10只、模型组30只。模型组大鼠在异氟醚全身麻醉下行自体髓核移植术以建立LDH模型,对照组不进行麻醉和手术操作。建模后1 d,采用旷场实验及条件恐惧实验观察大鼠的行为学表现;检测对照组(实验后2 d)和模型组(建模后2、4、8 d)海马CA1区小胶质细胞数量,小胶质细胞中含NLR家族Pyrin域蛋白3(NLRP3)活化情况,海马组织中的白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)水平,以及NLRP3、凋亡相关斑点样蛋白(ASC)及含半胱氨酸的天冬氨酸蛋白水解酶1(Caspase1)蛋白表达情况。结果模型组环境诱发僵直时间占比较对照组降低(P<0.05),但两组大鼠中心区域活动时间、声音诱发僵直时间占比差异均无统计学意义(均P>0.05)。模型组建模后2、4、8 d小胶质细胞数量均多于对照组,且建模后2、4、8 d小胶质细胞数量依次减少(均P<0.05)。对照组海马组织细胞质内活化NLRP3较少,模型组建模后2 d海马组织细胞质内NLRP3大量激活,但随着时间的延长,细胞质内活化NLRP3逐渐减少。模型组建模后2 d海马组织IL-1β、TNF-α水平及建模后4 d TNF-α水平均高于对照组,且建模后4、8 d海马组织IL-1β、TNF-α水平均低于建模后2 d(均P<0.05)。模型组建模后2、4、8 d海马组织NLRP3、ASC及Caspase1蛋白相对表达量均高于对照组,且建模后2、4、8 d大鼠海马组织NLRP3、ASC及Caspase1蛋白相对表达量依次降低(均P<0.05)。结论异氟醚全身麻醉可能通过促进LDH大鼠海马区小胶质细胞胞质中NLRP3的活化以上调IL-1β、TNF-α表达,从而导致中枢神经系统炎症反应的发生。

Objective To investigate the effect of general anesthesia with isoflurane on hippocampal microglia activation and inflammation in rats with lumbar disc herniation(LDH). Methods Forty rats were randomly divided into control group(n=10) and model group(n=30). Rats in the model group underwent autologous nucleus pulposus transplantation under general anesthesia with isoflurane to establish LDH models, whereas the control group did not undergo anesthesia or surgical operation. One day after the modeling, rats′ behavioral manifestations were observed by open field test and fear conditioning test. Multiple indexes were detected in the control group two days after the experiment as well as in the model group two, four and eight days after the modeling, including the number of microglia in the hippocampal CA1 region, the activation of NLR family Pyrin domain containing 3(NLRP3) in microglia, the levels of interleukin(IL)-1β and tumor necrosis factor α(TNF-α) in hippocampal tissues, as well as the protein expression of NLRP3, apoptotic speckle-like protein containing a CARD(ASC), and cysteinyl aspartate specific proteinase 1(Caspase1).Results The model group had a decreased proportion of environment-induced stiffness time than the control group(P<0.05), but there was no statistically significant difference in the proportion of central region activity time or sound-induced stiffness time between the two groups(all P>0.05). The model group yielded more microglia than the control group two, four and eight days after the modeling, and the number of microglia in the model group two, four and eight days after the modeling declined successively(all P<0.05). The control group exhibited decreased activated cytoplasmic NLRP3 in hippocampal tissues, while the model group saw a dramatic increase in the number of activated cytoplasmic NLRP3 in hippocampal tissues two days after the modeling, but the activated cytoplasmic NLRP3 declined gradually over time. The model group produced higher hippocampal IL-1β and TNF-α levels two days after the modeling as well as a higher TNF-α level four days after the modeling in comparison with the control group, the hippocampal IL-1β and TNF-α levels four and eight days after the modeling were lower than the levels two days after the modeling(all P<0.05). The relative hippocampal expression of NLRP3, ASC, and Caspase1 proteins two, four and eight days after the modeling in the model group was higher than that in the control group, which declined successively(all P<0.05). Conclusion General anesthesia with isoflurane may up-regulate the expression of IL-1β and TNF-α via promoting the activation of cytoplasmic NLRP3 in hippocampal microglia among LDH rats, thus leading to the occurrence of inflammatory responses in the central nervous system.