| BackgroundHCMV is the virus most frequently transmitted to a developing fetus. HCMV infection is more widespread in developing countries and in communities with lower socioeconomic status and represents the most significant viral cause of birth defects in industrialized countries. Now, congenital cytomegalovirus infection is a common cause of mental retradation and difficulty of learning.In order to study the mechanism of HCMV congenital infection induced learning and memory dysfunction, we have successfully built a series of mouse and rats model for HMCV congenital infection in central nervous system since the beginning of2002. In this research, a Sprague Dawley (SD) model of HCMV congential infection was selected, neurobehavioral development in offspring were assessmented by Morris Water Maze, open field behavioral testand etc.Then, the characteristics of synaptic plasticity in the DG and CA1region of offspring’s hippocampus were detected by field potential recording method. Final, the level of metabolic calcium disorder, CaMK Ⅱ and NMDA receptor subunit expression in the hippocampus neurons of the model were detected by immunohistochemistry and protein electrophoresis technology.Part oneAssessment the Change of Offspring’s Neurobehavioral Development in a SD Rat Model of HCMV Congential Infection in Central Nervous SystemObjectiveIn order to research the influence on neurobehavioral development of rats by Human cytomegalovirus congential infetion.Method40SPF SD rats (12weeks old, male and female mated by1:3) were selected and randomly divided into experiment group and control group. Fertilized female rats in experiment group were injected with0.5ml HCMV (10-6TCID50in/rat) by intraperitoneal inoculation on4gestation days. At the same times,0.5ml suspension of HELF cell were inoculated through intraperitoneal on the fertilized female rats of control group. To verify the reliabilty of rat’s model, pathological change of brain, virus particles, HCMV pp65specific antigen and major immediate early antigen (MIE) of HCMV were detected. Then, neurobehaviorial developments of4-6week offspring in two groups were assessmented by Morris Water Maze (MWM), inclined plane, activities and open field test and so on.Result(1) Compared with control group, litter size, mortality of neonatal rats in one week and positive rate of virus isolation on the hippocampus in the experiment group were significantly higher than that in control group (P<0.05). In congenital group, pathological damage of virus infection, virus particles and HCMV-specific DNA were successfully detected in the offspring of rats, characteristic enlargements of cells with intranuclear inclusions were found in the brain of offspring.(2) Morris water maze testIn Morris water maze test, the time of escape latency for30-day old offspring in the congenital groups were significantly increased than in the control group during4days training (F=499.473, P<0.05). The total distance in experiment group were significantly increased compared with the control group (F=440.167, p<0.01). At same time, the numbers of cross paltform (4.21±1.44) and the swimming time in the target quadrant (26±4%) of congenital infection group were decreased than that in control group (7.50±1.72, P<0.05;47±5%, P<0.05)A series of above results show that function of spatial learning and memory of offsprings in the experiment group was damaged by HCMVcongential infection.Swimming speed of30-day old offspring in the congenital groups was significantly decreased than in the control group during the4days training (F=7.211, p=0.008<0.05).(3) Motor function test and othersCompared to control group, the offspring in the experiment group showed bad performance in the suspension test, inclined plane test, involuntary movement and spontaneous movement test (P<0.05),which demonstrated that HCMV congential infection may damage the movement function of offspring in experiment group.At same time, the socle of resistance to capture for offspring in the experiment group was3.56±0.92, which significantly decreased than that in the control group (5.28±0.83)(Z=-4.248, P<0.001). In the open field test, numbers of stand up and lattice span in the congenital infection group (15.44±0.86,80.56±3.16) were signficantly decreased than in the control gruop (30.28±3.44,84.94±3.70)(t=9.684, P<0.05; t=1.815, P=0.129), which demonstrated that the level of emotional and behavioral development of rats in the experiment group was delayed by HCMV congenital infection.Conclusion(1) Pathological damage of virus infection, virus particles and HCMV-specific DNA were successfully detected in the offspring, a characteristic enlargement of cells with intranuclear inclusions were found in the brain and livers.(2) A SD rat’s model of Human cytomegalovirus congential infection in centre nervous system has been build.(3) Human cytomegalovirus congenital infection may impairs spatial learning and memory in rats, which accompany with damaged the abilty of movement and level of emotional and behavioral development. Part twoEffects of Congenital HCMV Infection on Synaptic Plasticity (LTP/DP) in Dentate Gyrus (DG) and CA1of Rat HippocampusObjectiveSynaptic plasticity in hippocampal DG and CA1area is an important model of spatial learning and memory. This study investigate whether the congenital HCMV infection affect the induction and maintenance of LTP/DP in Dentate Gyrus (DG) and CA1of HCMV congenital infection rat’s hippocampus by in vivo field potential recording method.Method40SPF SD rats (12weeks old, male and female mated by1:3) were selected and randomly divided into experiment group and control group. Fertilized female rats in experiment group were injected with0.5ml HCMV (10-6TCID50in/rat) by intraperitoneal inoculation on4gestation days. At the same times,0.5ml suspension of HELF cell were inoculated through intraperitoneal on the fertilized female rats of control group. Field excitatory postsynaptic potentials (EPSPs) were recorded in the hippocampal slices of offspring (50-65days) to study alterations of LTP/DP in area dentate gyrus (DG) and CA1of the hippocampus after congenital infection.ResultsThe input/output (I/O) curves of the EPSP slope PS amplitude in area DG in experiment group were significantly depressed when compared to control group (P<0.05). LTP of the EPSP slope and PS amplitude in area DG of the hippocampus was137.90±7.84%(EPSP) and240.11±34.20%(PS) in control rats and123.28±7.20%(EPSP) and136.84±12.37%(PS) in experiment rats (EPSP:F=22.749, P<0.05; PS:F=6.439, P<0.05, one-wayANOVA with Tukey test). DP of the EPSP slope and PS amplitudewas88±5%(EPSP) and72±5%(PS) in control rats and91±2%(EPSP) and91±5%(PS) in congenitally infected rats (EPSP:F=4.463, P=0.052; PS: F=130.947, P<0.05).LTP of the EPSP slope and PS amplitude in area CA1of the hippocampus were151.17±9%(EPSP) and235±10%(PS) in control rats and124.63±6.71%(EPSP) and168±7%(PS) in experiment rats (EPSP:F=96.953, P<0.05; PS:F=F=127.624, P<0.05). DP of the EPSP slope and PS amplitudewas88±9%(EPSP) and82±9%%(PS) in control rats and98±2%(EPSP)and83±7%(PS) in congenitally infected rats (EPSP:F=7.064, P<0.05P=0.052; PS:F=0.079, P>0.05).ConclusionOur results show that Congenital HCMV infection could significantly decrease I/O function, PPR and LTP in experiment group, which may indicate that congenital cytomegalovirus infection could severely inhibit synaptic plasticity of adult rats.Our pathological results show that the structural of hippocampus in experiment rats were disorder, granular cells damaged and reduced, a large number of vacuolation cell were seen. The structural disorder and damage in the hippocampus are directly damage the I/O function and PPR in DG. Part ThreeLevel of NMDA Receptor Subunit, Ca2+/calmodulin-dependent Protein Kinase Ⅱ and Ca2+in Rat Hippocampus Induced by HCMV Congenital Infection:a Mechanism for Learning ImpairmentObjectiveThe N-methyl-D-aspartate (NMDA) subtype of glutamate receptors plays an important physiological role in synaptic plasticity and cognitive functions by mediating an influx of Ca2+ions through channels gated by these receptors. Here we investigated the effects of HCMV congenital infection on the expression of NMDA receptors, Ca2+/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) and level of Ca2+in the hippocampal neurons of neonatal Sprague Dawley rat.Method and ResultsExpression level of NMDA receptor subunits (NMDA-R1,NMDA-R2A, NMDA-R2B) of DG areas in experiment rats were0.181±0.06(GluN1),0.174±0.094(GluN2A) and0.123±0.013(GluN2B), which were significantly decreased than that in the control rats0.288±0.027(GluN1),0.235±0.018(GluN2A) and0.184±0.023(GluN2B)(GluN1:t=8.409, P<0.01; GluN2:t=8.683, P<0.01; GluN2B:t=6.523, P<0.01) by image analysis of immunohistochemistry.Expression level of NMDA receptor subunits (NMDA-R1, NMDA-R2A, NMDA-R2B) of CA1areas in experiment rats were0.223±0.020(GluN1),0.215+0.024(GluN2A) and0.17±0.020(GluN2B), which were significantly decreased than that in the control rats0.316±0.038(GluN1),0.273±0.024(GluN2A) and0.280±0.023(GluN2B)(GluNl:t=6.124, P<0.01; GluN2:t=4.826, P<0.01; GluN2B:t=10.132, P<0.01) by image analysis of immunohistochemistry. Expression level of NMDA receptor subunits (NMDA-R1,NMDA-R2A, NMDA-R2B) of hippocampus in experiment rats were0.463±0.042(GluN1),0.156±0.020(GluN2A) and0.177±0.019(GluN2B), which were significantly decreased than that in the control rats0.686±0.037(GluN1),0.248±0.020(GluN2A) and0.233±0.033(GluN2B)(GluN1:t=11.171, P<0.01; GluN2:t=9.120, P<0.01; GluN2B:t=4.118, P<0.01) by West Blot.The level of CaMK II in congenital infected group (0.2184±0.020, OD value)were significantly increased than (0.3323±0.0252, OD value) in the control group (t=8.924, p<0.01) by image analysis of immunohistochemistry.In the HCMV-infected group, the [Ca2+] in the hippocampus neurons (237.97±25.83) were higher than the controls (126.85±11.29)(r=11.125, p<0.05) by Fura-2fluorescence method.ConclusionThe function of learning and memory impaired by HCMV congenital infection in SD rat model. This research complemented prior studies and indicated that disrupting the calcium balance and decreasing expression of GluN1, GluN2A, GluNR2B and CaMKII in the neurons of the hippocampus of rats may result in impairment in LTP. Functional studies investigating the role of HCMV congenital infection damaged NMDA receptor subunits in the development of hippocampus are also needed. In this research, a SD rat’s model of Human cytomegalovirus congential infection in centre nervous system has been built. For SD rats with HCMV congential infection, function of spatial learning and memory, abilty of movement and development of emotion and behavior were significantly decreased than control rats by neurobehavioral assessment. The induction and maintenance of LTP/DP in CA1and DG area of hippcampus were affected by congenital HCMV infection. Compared to control group, the expression levle of NMDA receptors and Ca2+/calmodulin-dependent protein kinase II (CaMKII) in the hippocampal neurons of neonatal rat in congenital HCMV infection group were decreased. At same times, the [Ca2+] in the hippocampus neurons are higher than in the controls. These results demonstrate that congenital HCMV infection could reduce the range of synaptic plasticity in the Sprague-Dawley rats, which may trigger the dysfunction of learning and memory through disrupting the calcium balance.Abbreviations:HCMV, human cytomegalovirus; LTP, Long-term potentiation; DG,dentate gyrus; I/O, Input/output function; PPR, Paired-pulse reaction; CPE, cytopathic effect; SD, Sprague-Dawley; NMDA, N-methyl-D-aspartate; HF, Inhuman fibroblast; CaMK II, Ca2+/calmodulin-dependent protein kinase II; GluN, subunit of N-methyl-D-aspartate receptor. |
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