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。
Low-dose ketamine attenuates microcirculatory deficits after traumatic brain injury in micevia microglial NF-κB/iNOS pathway
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.
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