Congenital CMV Centrol Nervous System Infection Impaired Learning And Memory And Hippocampal Synaptic Plasticity In The Murine Model

Part1Effect of congenital MCMV centrol nervous system infection on learning and memory in miceObjective To study the effect of congenital MCMV centrol nervous system infection on the abilities of learning and memory in the mice model.Methods①MCMV Smith strain and the BALB/C mice were used to build the congenital MCMV nervous system infection model:the BALB/C mice with MCMV IgG and IgM negative were screened by ELISA and mated with the proportionl:2of male and female. The fetal mice were randomly divided into infection model group(n=7) and control group(n=7) on the day of delivery. The fetal mice in infection model group were inoculated with1.75μl of MCMV suspension (TCID50=10"5) via intracalvarium, the mice in control group were inoculated with1.75μl cell maintenance medium.②After30days, the exploration times on new object and the discrimination index (DI) were surveied via Object Recognition Test.③Survey the learning and memory abilities with Morris Water Mize test after the Object Recognition Test.Results①Object recognition test results:the exploration times on new object of infection model group and control group are14.50±5.32(s) and24.17±4.71(s), n=7, P<0.01; the discrimination index(DI) are0.37±0.09and0.54±0.15, n=7, P<0.05.②The swimming tracks, percentage of swimming distance and the average escaping latency of the first day trainning were no different between the infection group and the control group. But from the second to fifth days, the swimming tracks, percentage of swimming distance and the average escaping latency of infection group were longer than control group (P<0.05).Conclusion①The congenital MCMV nervous system infection decrease the abilities of learning and memory of mice.②The congenital MCMV nervous system infection model which was built via MCMV intracalvarium is an effective model for research on learning and memory damage caused by CMV infection. Part2Effect of congenital MCMV centrol nervous system infection on Synaptic plasticity in miceObjective To study the effect of congenital MCMV centrol nervous system infection on the Synaptic plasticity in the mice model.Methods①The congenital MCMV centrol nervous system infection model was established (same with part1).②Hippocampal brain slice preparation:5infection model group mice and5control group mice were sacrificed after ether anesthesia. The brain tissues were obtained and put in the cold artificial cerebrospinal fluid (aCSF) quickly. Removed the tissues we didn’t need, exposed the hippocampal tissue. Fixed the tissue on the stage of the slicer by502glue. Sliced the hippocampal tissue into400μm slice. Put the slice into incubation tank at least1.5hours.③The fEPSP evoked by stimulation of Scharff, LTP in60min after stimulation, PPF in hippocampal CA3-CA1area were detected via patch clamp. The accumulative plots of mEPSC amplitude and accumulative plots of mEPSC frequency in CA1pyramidal neuron were detected via patch clamp. Representative traces of AMPAR-mediated EPSCs, representative traces of NMDAR-mediated EPSCs and summary histogram for the ratio of NMDAR to AMPAR-mediated EPSC amplitudes were detected via patch clamp.Results①The fEPSPs evoked by stimulation of Scharff inputs recorded in hippoc-ampus slices from MCMV in infection model mice (n=9slices from5mice) were smaller compared with those in control mice (n=11slices from5mice).②The level of LTP60min after HFS in the CA3-CA1pathway in model group was smaller compared with that in control group, P<0.01.③Paired-pulse facilitation in the CA3-CA1pathway measured by varying the intervals (25,50,75and100ms) between pairs of stimuli before HFS stimulation were no significant differences between model group(n=9slices from5mice) and control group (n=11slices from5mice).④Input-output curves in the CA3-CA1pathway illustrating the relationship between the stimulation intensity and evoked response for fEPSP recorded in brain slices from model group(n=9slices from4mice) and control group(n=11slices from4mice) were no significantly different.⑤Accumulative plots of mEPSC amplitudes in model group(n=7slices from5mice) were smaller compared with those in control group(n=6slices from4mice). Accumulative plots of mEPSC frequencys in both group were no significant differences.⑥The NMDAR/AMPAR ratio was significantly smaller in model group mice (0.42±0.03, n=7cells from5mice) than that in control group mice (0.66±0.02, n=7cells from5mice), P<0.01. The mean amplitudes of AMPAR-mediated EPSCs of model group mice (45.8±10.0pA n=6cells from5mice) were significantly decreased compared with those in control group mice (59.9±4.3pA n=11cells from5mice), P<0.05. The mean amplitudes of NMDAR-mediated EPSCs of model group mice (27.6±2.3pA n=7cells from5mice) were significantly decreased compared with those in control group mice (53±4.8pA n=11cells from5mice), P<0.01.Conclusion①The congenital MCMV centrol nervous system infection impairs LTP in the CA3-CA1pathway with no effect on basal synaptic transmission.②The congenital MCMV centrol nervous system infection impairs LTP in the CA3-CA1pathway via a postsynaptic mechanism.③The congenital MCMV centrol nervous system infection selectively decreases NMDAR-mediated currents and AMPAR-mediated currents in the CA1. The damages of NMDAR-mediated currents are more significant. Summary:The congenital MCMV centrol nervous system infection impairs LTP in hippocampal via a postsynaptic mechanism, the decrease of NMDAR and NMDAR may be the reason. Part3Effect of congenital MCMV centrol nervous system infection on NMDA and AMPA Receptors synthesized and excretedObjective To study the effect of congenital MCMV centrol nervous system infection on NR1, NR2A, NR2B, GluRl and GluR2synthesized and excreted in hippocampal.Methods①The congenital MCMV centrol nervous system infection model was established (same with part1). The brain tissues of7mice in each group were obtained asepticly. Hippocampal tissues were collected under dissecting microscope.②The transcription and protein expression of NMDA receptors NRl, NR2A, NR2B, AMPA receptors GluRl, GluR2were detected with Real-time PCR and western blotting.Results①Compared with control group, the transcription of NMDA receptors NRl, AMPA receptors GluRl, GluR2were decreased in the infection group (P<0.05).②The NMDA receptors NR2B was significantly decreased in infection group (P<0.01).③NMDA receptors NR2A had no difference between the two groups.Conclusion①The congenital MCMV infection in centrol nervous system can decrease the transcription and protein expression of NMDA receptors NRl, NR2B, AMPA receptors GluRl, GluR2.②The CMV damaging the transcription of NMDA receptors NRl, NR2B, AMPA receptors GluR1, GluR2may be the mechanism of cognitive impairment in congenital CMV infection. Part4The effect of congenital MCMV infection on cholesterol in the murine hippocampusObjective To observe the effect of congenital MCMV centrol nervous system infection on the cholesterol in the murine hippocampus.Methods①The congenital murine cytomegalovirus centrol nervous system infection model and control were established (same with Part1).②On the3rd,15th,30th day, the cholesterol in hippocampus was analysed by High-performance liquid chromatography (HPLC) both in model and control groups.Results①The average recovery of the HPLC for murine hippocampus was99.12%. The coefficient of variation of instrument precision experiments was0.98%.②The cholesterol was increasing as days going on in both groups. In control group the concentration of3rd,15th,30th day were4.55±0.38mg/g,7.80±0.55mg/g,12.27±0.50mg/g. In infection model group the concentration of3rd,15th,30th day were3.49±0.51mg/g,5.57±0.45mg/g,8.37±0.48mg/g.③The cholesterol in infection model group was significant lower than that in the control group.Conclusion①PLC was an effective technology to detect cholesterol in the murine hippocampus.②Cholesterol played an important role during the hippocampus development. MCMV infection could decrease cholesterol in hippocampus for a long time, which could be the mechanism of permanent neurodevelopmental disability caused by congenital CMV infection.