脓毒症对小鼠内侧隔区-内侧前额叶皮质胆碱能投射的影响

Effects of sepsis on medial septal-medial prefrontal cortex cholinergic projection in mice

目的:观察脓毒症对小鼠内侧隔区-内侧前额叶皮质(MS-mPFC)胆碱能投射的影响。方法:选取6周龄雄性C57BL/6小鼠,于mPFC区注射逆向跨单突触狂犬病毒,通过神经元示踪,观察MS区病毒表达情况,明确mPFC的投射来源。于8周龄雄性C57BL/6小鼠侧脑室注射脂多糖(LPS)诱导急性脓毒症相关性脑病模型,使用递质探针技术,观察mPFC区乙酰胆碱(ACh)递质水平改变。进一步采用盲肠结扎穿孔术(CLP)构建小鼠脓毒症模型,利用胆碱乙酰基转移酶(ChAT)试剂盒检测Sham与CLP两组小鼠MS区ChAT活力,Western Blot方法检测两组小鼠mPFC区整体α7nAChR的表达,免疫荧光染色检测MS区胆碱能神经元坏死性凋亡。结果:侧脑室注射LPS后,小鼠mPFC区ACh递质水平明显下降。同时CLP诱导的小鼠SAE模型中,MS区ChAT活力下降,mPFC区α7nAChR表达减少。进一步免疫荧光染色结果显示,CLP小鼠MS区胆碱能神经元坏死性凋亡增加。结论:脓毒症导致MS区胆碱能神经元坏死性凋亡增加、ACh合成酶活性下降、突触末端ACh释放减少、mPFC区ACh关键受体α7nAChR表达下降等MS-mPFC胆碱能投射障碍,是导致SAE的重要机制。

Objective:To observe the influences of sepsis on medial septal-medial prefrontal cortex(MS-mPFC)cholinergic projection in mice.Methods:Retrograde transsynaptic tracing rabies virus was injected into the mPFC region of 6-week-old male C57 BL/6 mice to tag neurons.The expression of the virus in the MS region was observed through neuronal tracer to determine the projection source of mPFC.Lipopolysaccharide(LPS)was given to the lateral ventricle of 8-week-old male C57 BL/6 mice to induce acute SAE,and the changes of acetylcholine(ACh)transmitter level in mPFC region were detected by transmitter probe technology.Further,the cecal ligation and perforation(CLP)operation was implemented to construct mouse sepsis model for molecular biology test.Choline acetyltransferase(ChAT)kit was used to detect the ChAT activity in MS region of Sham and CLP groups.The expression ofα7 nAChR in mPFC region of Sham and CLP mice was quantified by Western Blot.Immunofluorescence staining was used to observe the expression of necroptosis-associated proteins of cholinergic neurons in MS region.Results:After injection of LPS into the lateral ventricle,ACh transmitter level in mPFC region of mice decreased significantly.At the same time,both the ChAT activity in MS region and the expression ofα7 nAChR in mPFC region were decreased in CLP-induced SAE model.Further immunofluorescence staining results indicated that the expression of necroptosis-associated protein of cholinergic neurons in MS region was increased.Conclusion:MS-mPFC cholinergic system was destroyed in sepsis,including the death of MS cholinergic neurons,the reduced activity of ACh synthase,the dysfunction of neural transmitter signal and the decreased expression ofα7 nAChR in mPFC,which contributes to the pathologies of SAE.