海马小胶质细胞活化介导慢性应激诱导缺血性脑卒中大鼠抑郁样行为的研究

Activation of Hippocampal Microglia Mediates Chronic Stress-induced Depression-like Behavior in Rats with Ischemic Stroke

目的:探索海马小胶质细胞活化及相关促炎性细胞因子在慢性应激诱导缺血性脑卒中大鼠抑郁样行为中的作用。方法:将健康雄性SD大鼠75只,采用线栓法构建右侧大脑中动脉闭塞(MCAO)模型,缺血时间70 min。造模术后1周,根据SPSS随机数字生成器分布至卒中组和“卒中+应激”组。根据时程,前者分为卒中1周组、卒中2周组及卒中4周组,后者分为“卒中2周+应激1周”组(简称“应激1周组”)和“卒中4周+应激3周”组(简称“应激3周组”),每组15只。应激组采用“慢性不可预见的温和应激+孤养”法进行诱导刺激。于各时间点评估5组大鼠体质量、旷场测试(OFT)、新奇抑制摄食测试(NSFT)及蔗糖偏爱指数(SPI),采用TTC染色检测残存脑体积比,运用Western blot检测患侧海马Iba-1、IL-1β及IL-18表达,采用免疫荧光染色检测患侧海马CA1区Iba-1荧光强度。结果:MCAO术后2周时,与卒中2周组比较,应激1周组大鼠体质量下降,OFT直立运动次数和穿格子数减少(P均<0.05);MCAO术后4周时,与卒中4周组比较,应激3周组大鼠体质量下降,SPI下降,OFT直立运动次数和穿格子数减少,以及NSFT潜伏期延长和摄食量减少(P均<0.05)。TTC染色显示,5组患侧胼胝体及纹状体不同程度萎缩,但残存脑体积比差异无统计学意义(P>0.05)。Western blot显示,与卒中4周组或应激1周组比较,应激3周组大鼠患侧海马Iba-1、IL-1β及IL-18表达均显著增加(P均<0.05);卒中组3个不同时间点患侧海马Iba-1、IL-1β及IL-18表达差异均有统计学意义(P均<0.05)。免疫荧光显示,与卒中4周组或应激1周组比较,应激3周组患侧海马CA1区Iba-1荧光强度均显著增加(P均<0.05);卒中组3个不同时间点患侧海马CA1区Iba-1荧光强度差异均有统计学意义(P均<0.05)。结论:慢性应激诱导缺血性卒中大鼠抑郁样行为不仅与患侧海马小胶质细胞活化及相关促炎性细胞因子释放有关,还可能呈时间相关性增强;卒中后患侧海马炎症反应可能是一个持续过程。

Objective:To explore the role of hippocampal microglia activation and related proinflammatory factors in depression-like behavior induced by chronic stress in rats with ischemic stroke.Methods:A total of seventy-five healthy male SpragueDawley rats were used to establish a right middle cerebral artery occlusion(MCAO)model with 70 min ischemia.One week after MCAO,rats were assigned to stroke and"stroke+stress"groups according to the SPSS random number generator.And according to time course,the former was divided into 1-week stroke,2-week stroke and 4-week stroke groups,while the latter was divided into"2-week stroke+1-week stress"(referred to as"1-week stress")and"4-week stroke+3-week stress"(as"3-week stress")groups,with 15 rats in each group."Chronic unpredictable mild stress plus isolation"was used in the stress group.The body weight,open field test(OFT),novelty-suppressed feeding test(NSFT)and sugar preference index(SPI)of rats were evaluated at each time point.Residual brain volume ratio was detected by TTC staining.The expression of Iba-1,IL-1βand IL-18 in the affected hippocampus was detected by Western blot.The fluorescencs intensity of Iba-1 in the CA1 region of the affected hippocampus was detected by immunofluorescence staining.Results:Two weeks after MCAO,compared with the 2-week stroke group,the body weight and counts of rearing and grid crossing of OFT decreased in the 1-week stress group(all P<0.05).Four weeks after MCAO,compared with the4-week stroke group,decreases in the body weight,SPI and counts of rearing and grid crossing of OFT,and extended latency and reduced food intake of NSFT were observed in the 3-week stress group(all P<0.05).TTC staining showed different degrees of atrophy of corpus callosum and striatum in each group,but the ratio of residual brain volume was not statistically different(P>0.05).Western blot showed that,compared with the 4-week stroke group or 1-week stress group,the expression of Iba-1,IL-1βand IL-18 in the affected hippocampus was significantly higher in the 3-week stress group(all P<0.05).The expression of Iba-1,IL-1βor IL-18 in affected hippocampus of the stroke group was significantly different at 3 different time points(all P<0.05).Immunofluorescence staining showed that,compared with the 4-week stroke group or 1-week stress group,the fluorescence intensity of Iba-1 in the CA1region of affected hippocampal was significantly higher in the 3-week stress group(all P<0.05).The fluorescence intensity of Iba-1in the CA1 region of the stroke group was significantly different at 3 different time points(all P<0.05).Conclusion:Chronic stressinduced depression-like behavior in ischemic stroke rats is not only related to the activation of affected hippocampal microglia and the release of related proinflammatory factors,but also may be time-dependent.Inflammatory response in the affected hippocampus after stroke may be a continuous process.