急性睡眠片段化对老年大鼠认知功能及海马Homer1a表达的影响

Effect of acute sleep fragmentation on cognitive function and hippocampal Homer1a expression in aged rats

目的:探讨急性睡眠片段化(sleep fragmentation,SF)对老年大鼠认知功能的影响及海马Homer1a和突触可塑性的关系。方法:取108只22~24月龄SPF级雄性SD大鼠,按照随机数字表法分为对照组(Control组)、非睡眠片段化组(NSF组)和睡眠片段化组(SF组),每组36只大鼠。采用睡眠剥夺杆法建立睡眠片段化模型。采用Morris水迷宫实验、新物体识别实验评估大鼠学习记忆功能;采用Western blot法检测海马组织Homer1a蛋白表达水平,免疫组化染色观察CA1区Homer1a表达分布;采用Golgi染色观察海马CA1区树突棘密度,离体电生理膜片钳实验检测海马CA3-CA1场兴奋性突触后电位(field excitatory postsynaptic potential,fEPSP)斜率变化。采用SPSS 22.0和GraphPad Prism 9.3软件分析数据和制图,多组间比较采用单因素方差分析,进一步两两比较采用Tukey-Kramer检验。结果:(1)在行为学实验中,3组大鼠穿越原平台次数、目标象限停留时间、1 h和24 h新物体识别指数均差异有统计学意义(F=13.63,11.34,21.26,16.22,均P<0.01)。SF组大鼠穿越原平台次数[(2.00±1.27)次]低于Control组[(5.67±2.16)次]和NSF组[(6.50±2.35)次](均P<0.05),SF组的目标象限停留时间[(9.02±4.84)s]短于Control组[(24.73±7.37)s]和NSF组[(27.81±8.37)s](均P<0.05)。SF组在1 h和24 h两个时间点的新物体识别指数均低于Control组和NSF组(均P<0.05)。(2)在Western blot检测中,SF组大鼠海马Homer1a蛋白表达(0.91±0.13)高于Control组(0.70±0.05)和NSF组(0.74±0.04)(均P<0.05)。(3)在免疫组化染色中,SF组大鼠海马CA1区Homer1a蛋白的光密度值高于Control组和NSF组(P<0.05)。(4)在Golgi染色中,SF组大鼠海马CA1区树突棘密度低于Control组和NSF组(P<0.05)。(5)离体电生理实验显示:SF组海马CA3-CA1区fEPSP斜率均低于Control组和NSF组(均P<0.05)。结论:老年大鼠急性SF干预会引起认知功能损害,这可能与海马Homer1a过表达从而抑制海马突触可塑性相关。

Objective To explore the effects of acute sleep fragmentation(SF)on cognitive function and the relationship between hippocampal Homer1a and synaptic plasticity in aged rats.Methods One hundred and eight SPF grade male SD rats aged 22 to 24 months were divided into three groups according to random number table:control group(Control group),non-sleep fragmentation group(NSF group)and sleep fragmentation group(SF group),with 36 rats in each group.A sleep fragmentation model was established by sleep deprivation rod method.Morris water maze and novel object recognition tests were used to evaluate the learning and memory function of rats.Homer1a expression in hippocampus was detected by Western blot,and its distribution in CA1 area of hippocampus was observed by immunohistochemical staining.Golgi staining was used to observe the density of dendritic spines in CA1 area of hippocampus,and in vitro electrophysiological patch clamp test was used to detect the slope of field excitatory postsynaptic potential(fEPSP)from CA3 to CA1 in hippocampus.SPSS 22.0 and GraphPad Prism 9.3 softwares were used for data statistical analysis and mapping.One-way ANOVA was used for comparison among groups,and Tukey-Kramer test was used for further pairwise comparison.Results(1)In the behavioral tests,there were statistical differences in the times of crossing the original platform,the target quadrant residence time and the new object recognition index at 1 h and 24 h among the three groups(F=13.63,11.34,21.26,16.22,all P<0.01).The times of crossing the original platform in SF group((2.00±1.27)times)was lower than that of Control group((5.67±2.16)times)and NSF group((6.50±2.35)times)(both P<0.05).The target quadrant residence time in SF group((9.02±4.84)s)was shorter than that in Control group((24.73±7.37)s)and NSF group((27.81±8.37)s)(both P<0.05).The new object recognition index at 1 h and 24 h in SF group were lower than those in Control group and NSF group(all P<0.05).(2)In Western blot assay,the expression of Homer1a protein in hippocampus of SF group(0.91±0.13)was higher than that of Control group(0.70±0.05)and NSF group(0.74±0.04)(both P<0.05).(3)In immunohistochemical staining,the optical density value of the Homer1a protein in CA1 area of hippocampus in the SF group was higher than that in the Control group and NSF group(both P<0.05).(4)In Golgi staining,the density of dendritic spines in CA1 area of hippocampus in SF group was lower than that in Control group and NSF group(both P<0.05).(5)In vitro electrophysiological test showed that the slope of fEPSP in CA3-CA1 area of hippocampus in SF group were lower than that in Control group and NSF group(both P<0.05).Conclusion Acute SF intervention in aged rats can cause cognitive impairment,which may be associated with the inhibition of hippocampal synaptic plasticity induced by hippocampal Homer1a overexpression.