人巨细胞病毒先天性感染大鼠海马空间学习记忆功能受损

Impaired spatial learning and memory skill in Sprauge-Dawley rat model of congenital human cytomegalovirus infection

目的 探讨先天性感染人巨细胞病毒（human cytomegalovirus,HCMV）的子代大鼠空间学习记忆情况及其影响机制. 方法 40只12周龄无特定病原体级Sprague-Dawley大鼠按雌雄3∶1合笼.用随机数字表法将受精大鼠分为研究组和对照组,每组15只.研究组妊娠3d腹腔接种组织半数感染量为1×10^-6的病毒悬液0.5 ml;对照组同时腹腔注射人胚肺成纤维细胞上清液0.5 ml.接种后l周检测2组孕鼠外周血HCMV特异性抗体IgM和IgG.于临产前,取研究组和对照组孕鼠各3只行剖宫产,用随机数字表法在2组剖宫产所获仔鼠中各取5只,取双侧海马组织进行病毒分离.仔鼠饲养至28日龄时,从各组随机选择6只进行神经行为学检测（包括Morris水迷宫学习后的定位航行试验和空间探索试验）.取完成神经行为学检测的大鼠脑组织,HE染色切片,光镜下观察;取仔鼠海马组织块透射电镜下观察、摄片.Morris水迷宫空间学习记忆测试数据采用广义混合线性模型,并用Origin 8.0绘制结果图.采用秩和检验、独立样本t检验和x2检验进行统计学分析. 结果 （1）研究组15只雌鼠中,11只成功分娩;对照组15只均成功分娩.研究组母鼠的产仔量低于对照组[（5.5±2.4）只与（8.7±3.1）只,t=2.366,P=0.033）,仔鼠生后1周内死亡率高于对照组[26.1％（18/69）与4.9％（6/122）,x2=191.020,P=0.000].研究组7只次海马病毒分离阳性,对照组均为阴性.（2）研究组第1-4天逃避潜伏期均长于对照组,差异有统计学意义（广义混合线性模型检验,F=499.473,P=0.000）.研究组第1-4天的总路程长于对照组（广义混合线性模型检验,F=440.167,P=0.000）.空间探索能力方面,研究组穿越平台次数少于对照组[（4.2l±1.44）次与（7.50±1.72）次,t=7.182,P=0.003],目标象限游泳时间占总时间比例少于对照组[（26±4）％与（47±5）％,t=15.487,P=0.000].（3）光镜观察可见研究组大鼠脑组织存在不同程度皮层液化坏死,空泡变性较多,海马结构紊乱;电镜下可见研究组大鼠脑组织存在典型的陈旧性髓鞘损伤及崩解. 结论 HCMV先天性感染仔鼠出现较明显的空间学习记忆功能下降,其病理机制主要与皮层和海马结构损伤有关.

Objective To investigate the influence of congenital human cytomegalovirus （HCMV） infection on spatial learning and memory skill in rats and its mechanism.Methods Forty specific pathogen free Sprague-Dawley rats （12 weeks old,male and female mated by 1：3） were selected and randomly divided into the experimental group and control group,fifteen in each group.Fertilized female rats in the experimental group were injected with 0.5 ml HCMV （1 × 10^-6 of the 50% tissue culture infective dose/rat） by intraperitoneal inoculation on the third gestational day.At the same time,a 0.5 ml suspension of human embryonic lung fibroblast cells was inoculated intraperitoneally into fertilized female rats in the control group.HCMV-specific IgM and IgG in maternal peripheral blood of the two groups were detected on one week after inoculation.Both side of hippocampus of ten neonatal rats （five in each group）,born by cesarean section,were collected for virus isolation.Spatial learning and memory skill in 4-6 week offspring in the two groups were assessed using the Morris Water Maze （MWM） test,and results were presented by Origin 8.0.Pathological damage in offspring was determined by microscopy and electron microscopy.Independent samples t-test,Chi square and rank sum tests were used for statistical analysis.Results （1） Compared with the control group,litter size and mortality of neonatal rats within one week after birth in the experimental group were significantly higher [litter size：（8.7± 3.1） vs （5.5±2.4）,t=2.366,P=0.033; mortality of neonatal rats：4.9% （6/122） vs 26.1% （18/69）,x2=191.020,P=0.000.In the MWM test,escape latency in 30-day old offspring in the experimental group was significantly increased compared with the control group during 4 days of training （F=499.473,P=0.000）.The total distance in the experimental group was significantly increased compared with the control group （F=440.167,P=0.000）.In addition,the numbers of platform crossing （4.21 ± 1.44） and swimming time in the target quadrant [（26±4）%] in the congenital infection group were decreased compared with the control group [（7.50± 1.72） and （47 ± 5）%,t=-7.182 and 15.487,P=0.003 and 0.000].In the congenital infection group,liquefaction necrosis of the cerebral cortex （light microscopy） and myelin disintegration in the hippocampus （electron microscopy） were observed.Conclusions Injury of the cortex and hippocampus in offspring with congenital HCMV infection is involved in the pathological mechanism of decreased spatial learning and memory skill.