睡眠剥夺对盲肠结扎穿孔术脓毒症小鼠海马神经元和小胶质细胞的影响

Effects of sleep deprivation on hippocampal neurons and microglia in mice with cecal ligation and puncture-induced sepsis

目的探讨睡眠剥夺对盲肠结扎穿孔术脓毒症小鼠海马神经元和小胶质细胞的影响。方法24只8周龄C57BL/6小鼠购自河南省实验动物中心,随机分为3组(n=8):正常对照组(control)、盲肠结扎穿孔术组(CLP)、盲肠结扎穿孔术+睡眠剥夺组(CLP+SD),分别给予无处理,盲肠结扎穿孔术和盲肠结扎穿孔术后睡眠剥夺48 h处理。术后48 h小鼠心脏灌流处死全脑剥离,提取小鼠海马进行免疫荧光染色及共聚焦观察,并记录神经元Fluoro Jade B和离子钙接头分子1(Iba1)阳性细胞数。组间比较采用单因素方差分析(ANOVA),两两比较采用独立样本t检验。结果Control组、CLP组和CLP+SD组的海马齿状回Iba1阳性细胞数分别为(36.75±8.71)、(78.50±11.94)、(147.38±43.40)个,同control组比较,CLP组和CLP+SD组Iba1阳性细胞数均显著升高(F=41.75、110.60,P<0.05),且CLP+SD组的Iba1阳性细胞数比CLP组显著增加(F=68.88,P<0.05)。Control组、CLP组和CLP+SD组的海马CA区神经元Fluoro Jade B阳性细胞数分别为(106.75±19.62)、(167.25±16.18)、(211.13±54.40)个,同control组比较,CLP组和CLP+SD组海马神经元Fluoro Jade B平均阳性细胞数均显著升高(F=60.50、104.40,P<0.05)且CLP+SD组的Fluoro Jade B阳性细胞数比CLP组显著增加(F=43.88,P<0.05)。结论睡眠剥夺联合盲肠结扎穿孔术可导致小鼠术后48 h海马小胶质细胞激活和退变神经元数量增加。

Objective To investigate the effect of sleep deprivation on hippocampal neurons and microglia in septic mice with cecal ligation and puncture.Methods Totally,248-week-old C57BL/6 mice were randomly divided into three groups(n=8 each):normal control(control)group,cecal ligation and puncture(CLP)group,cecal ligation and puncture+sleep deprivation(CLP+SD)group,which were given no treatment,cecal ligation and puncture,and cecal ligation and puncture with sleep deprivation for 48 h,respectively.Then the mice were sacrificed by heart perfusion and the whole brain was stripped 48 h after operation.The hippocampus of the mice was extracted for immunofluorescence staining and confocal observation,and the number of Fluoro Jade B and Iba1 positive neurons was recorded.One-way analysis of variance(ANOVA)was used for comparison between groups,and independent sample t-test was used for pairwise comparison.Results The number of Iba1 positive cells in hippocampal dentate gyrus of control group,CLP group and CLP+SD group was 36.75±8.71,78.50±11.94 and 147.38±43.40,respectively.Compared with control group,the number of Iba1 positive cells in CLP group and CLP+SD group was significantly increased(F=41.75,P<0.05).The number of Iba1 positive cells in CLP+SD group was significantly greater than that in CLP group(F=68.88,P<0.05).The number of Fluoro Jade B positive cells in hippocampal CA area of control group,CLP group and CLP+SD group was 106.75±19.62,167.25±16.18 and 211.13±54.40,respectively.Compared with control group,the average number of Fluoro Jade B positive cells in hippocampal CA area of CLP group and CLP+SD group was significantly increased(F=60.50,P<0.05).The number of Fluoro Jade B positive cells in CLP+SD group was significantly greater than that in CLP group(F=43.88,P<0.05).Conclusion Sleep deprivation combined with cecal ligation and puncture leads to an increase in the number of degenerative neurons and microglial activation in the hippocampus of mice 48 h after surgery.