低剂量氯胺酮通过小胶质细胞NF-κB/iNOS通路减轻小鼠颅脑创伤后的微循环障碍

Low-dose ketamine attenuates microcirculatory deficits after traumatic brain injury in mice via microglial NF-κB/iNOS pathway

目的:探讨低剂量氯胺酮对颅脑创伤(TBI)小鼠神经炎症和微循环的影响。方法:采用随机数字表法将60只成年雄性C57BL/6小鼠分成假手术组、TBI组、假手术+氯胺酮组和TBI+氯胺酮组,每组15只;后2组小鼠采用控制性皮质撞击法(CCI)建立开放性TBI模型。假手术+氯胺酮组和TBI+氯胺酮组于造模后30 min腹腔注射氯胺酮30 mg/kg,1次/d,连续3 d。假手术组和TBI组分别于相同时间点经腹腔注射等量生理盐水。每组取6只小鼠分别于造模前、造模后即刻、造模后30 min、造模后1 d及造模后3 d利用激光散斑对比血流成像(LSCI)技术测量脑皮层血流量。另取6只小鼠于造模后第3天经心脏灌注取材行免疫组织化学染色和免疫荧光双标染色,检测小胶质细胞标志物离子钙接头蛋白抗体-1(Iba-1)及核因子(NF)-κB p65核转位情况。剩余每组3只小鼠于造模后第3天处死后制成组织原浆,通过Western blotting实验检测皮质脑组织NF-κB p65、p-NF-κB p65、p-IκB及诱导型一氧化氮合酶(iNOS)蛋白水平,通过ELISA法检测皮层脑组织肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、iNOS、氧自由基(ROS)、氮自由基(RNS)表达水平。结果:LSCI检测结果显示,造模后3 d,与TBI组比较,TBI+氯胺酮组小鼠脑局部微循环相对血流量明显增加,差异有统计学意义( P<0.05)。免疫组织化学染色结果显示,与假手术组及假手术+氯胺酮组比较,TBI组、TBI+氯胺酮组小鼠脑皮质区域内Iba-1阳性细胞数明显增加,差异有统计学意义( P<0.05);与TBI组比较,TBI+氯胺酮组小鼠Iba-1阳性细胞数明显下降,差异有统计学意义( P<0.05)。ELISA法检测结果显示,与假手术组及假手术+氯胺酮组比较,TBI组、TBI+氯胺酮组小鼠损伤皮质脑组织中炎性因子TNF-α、IL-1β和IL-6、iNOS及自由基ROS和RNS表达水平均明显升高,差异有统计学意义( P<0.05);与TBI组比较,TBI+氯胺酮组小鼠损伤皮质脑组织中炎性因子TNF-α、IL-1β、IL-6、iNOS及自由基ROS和RNS表达水平均明显下降,差异均有统计学意义( P<0.05)。免疫荧光双染结果显示,与TBI组比较,TBI+氯胺酮组小鼠NF-κB p65核转位明显受到抑制。Western blotting实验结果显示,与假手术组及假手术+氯胺酮组比较,TBI组和TBI+氯胺酮组小鼠损伤皮质脑组织中iNOS、NF-κB p65、p-NF-κB p65、p-IκB蛋白表达水平均明显升高,差异均有统计学意义( P<0.05);与TBI组比较,TBI+氯胺酮组小鼠损伤皮质脑组织中iNOS、NF-κB p65、p-NF-κB p65、p-IκB蛋白表达水平均明显降低,差异均有统计学意义( P<0.05)。 结论:低剂量氯胺酮可减轻开放性TBI后神经炎症,改善脑微循环血流,其作用机制可能与抑制小胶质细胞NF-κB/iNOS通路有关。

Objective To investigate the effect of low-dose ketamine on neuroinflammation and microcirculation in mice with traumatic brain injury(TBI).Methods Sixty adult male C57BL/6 mice,weighing 22-28 g,were randomly divided into sham-operated group,TBI group,Sham+ketamine group,and TBI+ketamine group(n=15).A controlled cortical impingement(CCI)method was used to establish TBI models in the later 2 groups.Sham+ketamine group and TBI+ketamine group were intraperitoneally injected with 30 mg/kg ketamine once daily for 3 d at 30 min after TBI;sham-operated group and TBI group were intraperitoneally injected same amount of saline at the same time points.Cerebral cortical blood flow in 6 mice from each group was measured by laser speckle contrast imaging(LSCI)before,immediately after,30 min after,1 d after and 3 d after modeling,respectively.Three d after modeling,immunohistochemical staining and immunofluorescent double label staining were used to detect the nuclear translocation of microglia markers,ionized calcin-antibody-1(Iba-1)and nuclear factor(NF)-κB p65 in damaged cortical brain tissues in 6 mice from each group.The remaining 3 mice in each group were sacrificed and tissue plasma was extracted 3 d after modeling;levels of NF-κB p65,phosphorylated(p)-NF-κB p65,p-IκB and inducible nitric oxide synthase(iNOS)in cortical brain tissues were detected by Western blotting.Expressions of tumor necrosis factor-α(TNF-α),interleukin-1-β(IL-1β)and interleukin-6(IL-6),iNOS,reactive oxygen species(ROS)and reactive nitrogen species(RNS)in cortical brain tissues were detected by ELISA.Results LSCI indicated that,3 d after modeling,relative blood flow in local cerebral microcirculation of TBI+ketamine group was significantly increased compared with that of TBI group(P<0.05).Immunohistochemical staining indicated that compared with the sham-operated group and Sham+ketamine group,the TBI group and TBI+ketamine group had significantly increased number of Iba-1 positive cells in the cerebral cortex(P<0.05);compared with the TBI group,the TBI+ketamine group had significantly decreased number of Iba-1 positive cells(P<0.05).ELISA indicated that compared with the sham-operated group and Sham+ketamine group,the TBI group and TBI+ketamine group had significantly increased expressions of TNF-α,IL-1β,IL-6,iNOS,ROS and RNS in damaged cortical brain tissues(P<0.05);compared with the TBI group,the TBI+ketamine group had significantly decreased expressions of TNF-α,IL-1β,IL-6,iNOS,ROS and RNS in damaged cortical brain tissues(P<0.05).Immunofluorescent double label staining indicated obviously inhibited NF-κB p65 nuclear translocation in TBI+ketamine group when it was compared with TBI group.Western blotting indicated that compared with the sham-operated group and Sham+ketamine group,the TBI+ketamine group had significantly increased iNOS,NF-κB p65,p-NF-κB p65 and P-IκB protein expressions in damaged cortical brain tissues(P<0.05);compared with the TBI group,the TBI+ketamine group had significantly decreased protein expressions of iNOS,NF-κB p65,p-NF-κB p65 and p-IκB in damaged cortical brain tissues(P<0.05).Conclusion Low-dose ketamine reduces neuroinflammation and improves cerebral microcirculatory blood flow after open TBI,whose mechanism may be related to inhibition of microglia NF-κB/iNOS pathway.