持续光照建立斑马鱼失眠模型及评价

Modeling and evaluation of zebrafish insomnia by continuous illumination

目的 利用斑马鱼建立睡眠剥夺模型,为失眠的基础研究提供更多可靠的实用建模参考方案。方法 选取4月龄同一批次野生型AB系雄性斑马鱼160尾作为实验对象,随机分为对照组(CK组)和睡眠剥夺1~7 d组(SD1~SD7组)。对照组置于正常14 h/10 h明/暗交替环境,睡眠剥夺组分别利用模拟日光对其进行不同时间的持续光照达到剥夺斑马鱼睡眠的效果。造模结束后,比较各组斑马鱼运动形式、学习记忆能力、生物钟基因表达、脑部超微结构的改变。结果 运动形式结果显示:与CK组斑马鱼相比,SD1、SD2、SD3组斑马鱼静息时间均增多(P<0.05),SD3组斑马鱼大运动时间及大运动计数较CK组减少(P<0.05);学习记忆能力结果显示:CK组斑马鱼学习记忆能力较SD1、SD2、SD3组强,(P<0.05、0.01),SD1组斑马鱼学习记忆能力较SD2、SD3组斑马鱼强(均P<0.01),SD2组斑马鱼学习记忆能力较SD3组斑马鱼强(P<0.01);qRT-PCR结果显示:与CK组相比,SD3组斑马鱼脑部周期蛋白1a(Per1a)、周期蛋白2(Per2)、脑和肌肉组织芳香烃受体核转运蛋白的类似蛋白1(Bmal1)、隐色素1b(Cry1b)mRNA表达差异有统计学意义(P<0.05);电镜结果显示:SD2及SD3组斑马鱼大脑神经元出现神经元坏死现象,镜下可见细胞核塌陷,边界破裂甚至溶解,核染色质固缩,线粒体出现肿胀,甚至大量空泡。结论 持续光照3d可改变斑马鱼运动形式,使其学习记忆能力下降,改变了与睡眠相关的时钟基因表达,诱发了脑部神经元凋亡,可以用来建立斑马鱼失眠模型。

Objective To establish a sleep deprivation model using zebrafish,and to provide more reliable practical modeling schemes for basic research on insomnia.Methods A total of 160 male 4-month-old wild type AB zebrafish were randomly divided into control group(CK)and sleep deprivation group(SD1-SD7).The control group was placed in a normal 14 h/10 h alternating light/dark environment,and the sleep deprived group was subjected to different days of continuous light with simulated sunlight to achieve the effect of sleep deprivation.After modeling,the changes of movement form,learning and memory ability,biological clock gene expression and brain ultrastructure of zebrafish in each group were compared.Results The results of movement form showed that compared with CK group,the resting time of zebrafish in SD1,SD2 and SD3 groups increased(P<0.05),and the movement time and movement count of zebrafish in SD3 group decreased(P<0.05).The results of learning and memory ability showed that zebrafish in CK group had better learning and memory ability than SD1,SD2 and SD3 groups(P<0.05,0.01),and zebrafish in SD1 group had better learning and memory ability than SD2 and SD3(all P<0.01),and zebrafish in SD2 group had better learning and memory ability than SD3(P<0.01).qRT-PCR results showed that compared with CK group,the mRNA expressions of Per1a,Per2,Bmal1 and Cry1b in the brain of zebrafish in SD3 group were significantly different(P<0.05).The results of electron microscopy showed that the neurons of zebrafish brain in SD2 and SD3 groups showed neuronal necrosis.Under the microscope,there were nuclear collapse,boundary cracking and even dissolution,chromatin shrinkage,mitochondrial swelling and even a large number of vacuoles.Conclusion Light exposure for 3 days can change the movement pattern of zebrafish,reduce its learning and memory ability,change the expression of clock genes related to sleep maintenance,and induce the apoptosis of brain neurons,which can be used to establish the model of zebrafish insomnia.