特异性抑制海马CA1区GABA能神经元NLRP3表达对小鼠TBI后认知障碍的改善作用研究

Specific inhibition of NLRP3 expression in GABAergic neurons in CA1 area of the hippocampus improves cognitive dysfunction in mice after traumatic brain injury

目的探讨海马CA1区γ-氨基丁酸(GABA)能神经元NOD样受体热蛋白结构域相关蛋白3(NLRP3)基因敲除对小鼠创伤性颅脑损伤(TBI)后认知障碍的改善作用。方法将48只清洁级健康雄性NLRP3flox/flox小鼠按随机数字表法分为假手术+对照病毒组(SV组)、假手术+GABA能神经元NLRP3基因特异性敲除组(SG组)、TBI+对照病毒组(TV组)及TBI+GABA能神经元NLRP3基因特异性敲除组(TG组),每组12只。其中,TV组、TG组小鼠采用自由落体法构建TBI模型,SV组、SG组小鼠仅行头皮剪开及开骨窗等外科操作、不予打击,SG组、TG组小鼠于TBI造模前21 d于海马CA1区注射腺病毒制备GABA能神经元NLRP3基因特异性敲除模型,SV组、TV组小鼠仅于海马CA1区注射空载病毒作为对照。TBI造模后第30、31天应用新物体识别实验评价各组小鼠的认知功能,第32~36天应用Morris水迷宫实验评估各组小鼠的学习及记忆功能,第31天应用在体电生理记录小鼠在新物体识别实验中探索新物体时海马CA1区场电位。上述实验结束后,处死小鼠并取材,采用免疫荧光染色检测各组小鼠海马CA1区微管相关蛋白2(MAP2)、谷氨酸脱羧酶67(GAD67)、突触后密度蛋白95(PSD95)的荧光强度,以及焦亡相关炎性因子白细胞介素-18(IL-18)/GAD67双阳性神经元占总GAD67阳性神经元的百分比。结果与SV组、SG组比较,TV组、TG组小鼠的新物体识别指数明显下降,水迷宫实验中实验阶段的穿越平台次数明显下降、训练阶段第3、4天的逃避潜伏期明显上升,海马CA1区θ、γ振荡功率在探索新物体时明显下降,海马CA1区MAP2、GAD67、PSD95的荧光强度明显减弱,IL-18/GAD67双阳性神经元百分比明显上升,差异均有统计学意义(P<0.05)。与TV组比较,TG组小鼠的新物体识别指数明显上升,水迷宫实验中实验阶段的穿越平台次数明显上升、训练阶段第3、4天的逃避潜伏期明显下降,海马CA1区θ、γ振荡功率在探索新物体时明显上升,海马CA1区MAP2、GAD67、PSD95的荧光强度明显增强,IL-18/GAD67双阳性神经元百分比明显下降,差异均有统计学意义(P<0.05)。结论海马CA1区GABA能神经元NLRP3基因敲除能够改善小鼠TBI后的认知障碍,其机制可能与抑制GABA能神经元焦亡相关。

Objective To explore the effect of NOD-like receptor thermal protein 3(NLRP3)knockout inγ-aminobutyric acid(GABA)-ergic neurons in the hippocampal CA1 area on improving cognitive dysfunction in mice after traumatic brain injury(TBI).Methods Forty-eight healthy male NLRP3flox/flox mice weighing 25-28 g were randomly divided into 4 groups(n=12):sham-operated+control virus group(SV group),sham-operated+NLRP3 specific knockout group(SG group),TBI+control virus group(TV group),TBI+NLRP3 specific knockout group(TG group).TBI in the TV and TG groups was established by free-fall method,while surgical procedures such as scalp incision and cranial window opening without impact were given to the SV and SG groups.Adenovirus was injected into the hippocampal CA1 area of SG and TG groups 21 d before TBI to induce NLRP3 specific knockout in GABA-ergic neurons in the hippocampal CA1 area;empty virus was injected into the CA1 area of SV and TV groups.Cognitive function was evaluated using novel object recognition test 30 and 31 d after TBI,and learning and memory functions were assessed using Morris water maze test 32-36 d after TBI.Field potentials in the hippocampal CA1 area were recorded during novel object recognition 31 d after TBI.After behavioral tests,these mice were sacrificed.Immunofluorescent staining was used to detect the fluorescent intensity of microtubule-associated protein2(MAP2),glutamic acid decarboxylase 67(GAD67),and postsynaptic density protein 95(PSD95)in the hippocampal CA1 area,as well as percentage of pyroptosis-associated inflammatory factor interleukin-18(IL-18)/GAD67 double-positive neurons in total GAD67 positive neurons.Results Compared with the SV and SG groups,the TV and TG groups had decreased novel object recognition index,decreased number of platform crossings during the experimental period,increased escape latency on day 3 and day 4 of the training period in Morris water maze test,decreasedθandγoscillation power in the hippocampal CA1 area during novel object recognition,decreased fluorescent intensity of MAP2,GAD67,and PSD95 in the hippocampal CA1 area,increased percentage of IL-18/GAD67 double-positive neurons,with significant differences(P<0.05).Compared with the TV group,the TG group had increased novel object recognition index,increased number of platform crossings in Morris water maze test,decreased escape latency during the training period,increasedθandγoscillation power in the hippocampal CA1 area during novel object recognition,increased fluorescence intensity of MAP2,GAD67,and PSD95 in the hippocampal CA1 area,decreased percentage of IL-18/GAD67 double-positive neurons,with significant differences(P<0.05).Conclusion Specific inhibition of NLRP3 expression in GABA-ergic neurons in the hippocampal CA1 area can improve cognitive dysfunction in mice after TBI,whose mechanism may be related to inhibited GABA-ergic neuronal pyroptosis in the hippocampal CA1 area.