| Purpose:Depression is a current mental and psychological disorder that seriously theatens the physical and mental health of human beings.Neuroplasticity acts as an important pathological mechanism of depression and substantially provides sets of important targets for antidepressant treatment,among which acupuncture is potential one.Our previous studies have realed that,acupuncture shows a good effect on depressive disorders in clinical practice.In this experiment,based on the clarification of the electroacupuncture on improving behaviors of chronic unpredictable mild stress combined with social isolation depression model,the mechanism of electroacupuncture treatment on synaptic plasticity(structural plasticity and functional plasticity)of depression was further explored.Methods:Eighteen adult Sprague-Dawley rats,120g-170g,were randomly divided into blank group,model group and intervention group,with 6 rats in each group.The blank group was all kept in one cage,and the model group and the intervention group were kept in a single cage/rat,that is,the isolated culture mode.The blank group received 35 days of normal feeding conditions,freely ingested water and food,undergoing normal circadian rhythm.During 35 days of common feeding,the model group received chronic unpredictable mild stimulating stress which including food,drinking water deprivation,450 angled cage,horizontal cage,5 ? ice water stimulation,37 ? warm water stimulation,tail clamping,Combined cages,Day and night inversion,Foreign objects in food and Restriction,with 12 kinds of stimulation in total,2 kinds are randomly given every day.In addition to the same manipulation as the model group,the intervention group received electroacupuncture treatment for 3 weeks,21 days in total,from the 15th day to the 35th day,20 minutes/day,5 days/week.(1)Observations:1,Behavioral testing:?Sucrose preference test(SPT).The preference ratio of the rat to the sucrose solution was observed on the day before the experiment(Day 0),Day 7,Day 14,Day 21,Day 28 and Day 35;?Open field test(OFT).The number of horizontal crossing and vertical rearing was counted on the 36th day of the experiment;?Morris water maze(MWM).Platform latency,platform shuttle times and platform' s quadrant time ratios were observed and calculated on days 37 to 41 of the experiment;?Passive avoidance test(PAT).The latency of rats from the bright box into the dark box was measured repeatedly on the 40th and 41st days of the experiment.2,Changes of synaptic ultrastructure in hippocampus and prefrontal cortex:Transmission electron microscopy was used to observe synaptic ultrastructural characteristics of regions of hippocampal CA1 and prefrontal cortex:synaptic numerical density(Nv),post-synaptic density(PSD)and Synaptic cleft.3,Expression of synaptic plasticity-related proteins in hippocampus and prefrontal cortex:Western Blot immunoblotting was used to observe the expression levels of synaptic plasticity-related proteins:Synaptophysin,PSD-95,BDNF,AKT and GSK-3 p in the CA1 and prefrontal cortex.(2)Statistics:Verify that the measurement data is in accordance with the normal distribution and the homogeneity test of variance.Those who meet the above conditions are analyzed by one-way ANOVA.The LSD method is used for comparison between groups.For those who do not meet the normal distribution and homogeneity of variance,Kruskal-Wallis H rank sum test is used;repeated measures ANOVA is used for repeated measures.Adjustment of the statistical results according to the correction coefficient if the spherical tester is not satisfied.The above method applied a significance of P<0.05.Results:1.Ethology?Sucrose preference test.Repeated measurements showed that the sucrose preference values of the blank group and the electroacupuncture group fluctuated up and down within the normal range,and the values of the model group gradually decreased with the experiment.Statistical analysis indicated that there was no difference in the baseline values of the three groups at the beginning of the measurement(P>0.05),which means that groups were comparable;the difference over time between three groups was significant(P<0.05),and there was no difference between the blank group and the acupuncture group(P>0.05),but the two were significantly larger than the model group(P<0.05,P<0.05).?Open field test.It was observed that the spontaneous activity of the blank group and the electroacupuncture group was more than that of the model group.Statistical analysis showed that there was no difference on horizontal movement and vertical movement between the blank group and the electroacupuncture group(P>0.05),but the two groups were significantly higher than the model group(P<0.05,P<0.05).?Passive avoidance test.There was no statistical difference in the latency of the three groups during training(P>0.05).After electrical stimulation,the test latency of the three groups was significantly different(P<0.05):the blank group and the electroacupuncture group were larger than the model group(P<0.05),and there was no difference between the former two(P>0.05).?Morris water maze.In the latency to the platform,the number of shuttles across the platform area and the lime ratio of target quadrant,it was found that the blank group and the electroacupuncture group were larger than the model group,but the difference was not statistically significant(P>0.05).2.Synaptic ultrastructure:According to transmission electron microscopy,the synaptic ultrastructure of the blank group,the model group and the electroacupuncture group have different characteristics:compared with the blank group and the electroacupuncture group,the synaptic vesicle density of presynaptic components in hippocampal CA1 region and the prefrontal cortex of model group decreased,edge of active zone in presynaptic bouton and post-synaptic density blur;statistical analysis suggested:?synaptic number density(Nv)in hippocampal CA1 area and prefrontal cortex was significantly smaller than that of blank group and electroacupuncture group(P<0.05,P<0.05).?Width of post-synaptic density(PSD)in two brain regions of the model group was also smaller than that of the two groups(P<0.05,P<0.05).?The Nv number in the hippocampal CA1 area of the electroacupuncture group was larger than that of the blank group(P<0.05).There was no difference in the Nv number and PSD between the two groups in the hippocampal CA1 area and the prefrontal cortex(P>0.05).3.Synaptic plasticity-related protein expression:WB immunoblotting revealed that the expression patterns of synaptophysin,PSD-95,BDNF,AKT and GSK-3 ? in three groups were similar in both hippocampal CA1 area and prefrontal cortex:?The levels of Synaptophysin,PSD-95,BDNF and AKT in model group were significantly lower than those of the blank group and the electroacupuncture group(P<0.05,P<0.05).? Conversely,the level of GSK-3? in model group was significantly higher than that of other two groups(P<0.05,P<0.05).?There was no statistical difference in the expression levels of each protein between the blank group and the electroacupuncture group(P>0.05).Conclusions1.Electroacupuncture effectively improves the abnormal behavior of depre ssion rats.2.The regulation of synaptic structural and functional plasticity maybe the key mechanism for electroacupuncture treatment of depression. |
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