| ObjectiveEA treatment has been prevalently applied in treating anxiety and acupoints GV24 and GV29 were frequently used.However,the mechanism underlying EA in treating anxiety remains unclear.To investigate the mechanism of EA in treating anxiety,we observed the effect of EA on GV24 and GV29 to CUMS mice and analyzed the possible thearapeutic targets by pharmachological tools and NOX2 deletion.Methods 1.Animal grouping(1)Oberving the beneficial effect of EA treatment on GV24 and GV29 by mice subjected to CUMS.A total number of 48 C57/BL6 male adult mice(weight 21.43±1.49g)were randomly divided into 4 groups including control group(n=12),EA group(n=12),CUMS group(n=12),CUMS+EA group(n=12).In the first 3 weeks,mice from control and EA group were not dealt with any stress.CUMS and CUMS+EA group were subjected to CUMS.After CUMS procedure was finished,mice from EA and CUMS+EA group were treated with 1 week of EA therapy on acupoint Baihui(GV20)and Yintang(GV29).(2)The confirmation of the above thearaputic targets using apocynin and transgenetic mice.In the first stage of this part,A total number of 48 C57/BL6 male adult mice(weight 22.05±0.93g)were randomly divided into 4 groups including control group(n=12),apocynin group(n=12),CUMS group(n=12),CUMS+apocynin group(n=12).In the first 3 weeks,CUMS and CUMS+apocynin group were subjected to CUMS.Meanwhile,mice from apocynin and CUMS+apocynin group were treated with apocynin administration.In the second stage of this part,a total number of 32 male adult transgenic mice(weighted 22.19±0.92,16 NOX2-deficient mice and 16 littermate control mice)were used in this study All mice were male and 7 weeks old were randomly divided into 4 group including WT control group(n=8),NOX2K0 group(n=8),WT CUMS group(n=8),NOX2KO+CUMS group(n=8).In the first 3 weeks,WT GUMS and NOX2K0+CUMS group were subjected to CUMS.2.The anxiety model of CUMSBriefly,after one week of habituation,mice were subjected to CUMS stimuli.During the process,animals were exposed to various types of stress including restraining in a small tube,forced warm water bath,water/food deprivation,paired housing in wet sawdust,and reversed day/night cycle,etc.All mice were group-housed during this period.3.Interventions(1)EA treatmentMice were anesthetized with 5%isoflurane and put on a heating pad which het to 32?.Acupuncture needles were inserted into acupoints GV20 and GV29 and EA treatment was performed by an EA apparatus.(1mA,2Hz).(2)Apocynin administrationAll mice were group-housed during CUMS process except,Half of the control and CUMS mice received apocynin(5 mg/kg/day,dissolved in drinking water)during the CUMS process.Based on our measurements,the daily water intake for each mouse.4.Behavioral test and estimation(1)Test of anxiety-like behaviors? Open field test(OFT):OFT was.performed as we previously described[33].Briefly,mice were placed in a random order into a 45 x 45 x 45 cm open-field arena which located in a sound proof box.Mice were initially placed in the box through the entrance and then allowed to move freely in the arena for 30 minutes.The locomotion of mice was collected as videos with a digital camera,then the total distance travelled(m)and amount of time they spent in the center area(s)in 10 minutes were recorded by the video analysis system.? Elevated plus maze test(EPM):Briefly,the EPM apparatus consisted of two open arms(30 × 5 cm),two enclosed arms(30 × 5 × 15 cm),and a central platform(5 × 5 cm)located at the intersection of the four arms.At the start of the test,each mouse was placed in the central platform facing one of the two opened arms and allowed to explore the maze for 5 minutes.Time spent in the open arms was measured with the video analysis system.? Light/dark test:The LDB apparatus had two chambers:one dark chamber and one brightly illuminated chamber.The apparatus was enclosed in a sound-attenuating box to mask noise.The time that mice spent in each division was recorded for 5 minutes and mice were allowed move freely between the two chambers.(2)Test of depressive-like behaviors? Sucrose preference test:Briefly,mice were presented with two drinking bottles:one contained 1%sucrose and the other contained water.On day-2,mice were individually housed and habituated to 1%(w/v)sucrose for 24 hours.On day-1,mice were deprived of water with free access to food for 16 hours.On the test day,mice were housed singly and given access to two pre-weighed bottles,one containing water and the other containing 2%(w/v)sucrose.These two bottles were weighed again after 24 hours of consumption.sucrose The preference for sucrose over water was measured by sucrose/sucrose+water ×100%?? Forced swim test:In brief,mice were placed into a transparent plastic cylinder(10-cm diameter,30-cm height)which was filled with 20-cm-deep water(24 ±2?).The forced swimming lasted 6 minutes,and the immobility time,which defined as mice were absence of all motions except those required to keep their head above water,in the last 4 min were manually recorded.5.El ectrophysi ologyAfter the behavioral tests,the mice were anesthetized with 100 mg/kg intraperitoneal injection of 1%pentobarbital sodium.After perfusion,brain slices were taken immediately and the thickness of the brain slices was 300?m.The brain slices were incubated for 30 min(34?)in water bath and incubated for 1 h(23?26?)at room temperature.The circulating perfusion system was filled with 50 ml of incubation solution and mixed with 95%oxygen and 5%carbon dioxide.The heater was set at 32?.After finding the target brain area under low magnification,switch the high-power microscope to find the cells.The glass electrode is given a positive pressure of 0.1 ml before the liquid is introduced.When the cells are sealed,the cells are removed and the cells are clamped at-70 mV and given a certain negtive pressure.After the cells were stabilized,the membrane was broken,and the clamp voltage was set to-60 mV and+10 mV on the same cell to record sEPSC and sIPSC,respectively.Each recording time is 2min.6.Test of oxidative stress levelROS was assessed by dihydroethidium(DHE)imaging and the levels of lipid peroxides.In brief,DHE(Sigma)was injected intraperitoneally(2 mg/kg)1 h before the mice were anesthetized.Then the brain was taken and sliced into 10 ?m thick sections with a cryostat microtome.The DHE fluorescence in brain sections was observed using a confocal microscope.The intensity of the fluorescence was quantified on 4-6 sections per mouse using NIH ImageJ software and the averaged intensity of fluorescence was expressed as a percentage(mean of control group was set as 100%).To determine the level of lipid peroxides,the concentration of malondialdehyde(MDA),a byproduct of lipid peroxides,was measured in homogenates of isolated ventral hippocampus.The MDA test kit was purchased from Nanjing Jian-cheng Bioengineering Institute(Nanjing,China).According to the instructions of the MDA test Kit,tissues were processed with corresponding reagents.Then,the samples were heated at 95?for 40 min and centrifuged at 4000 rpm from additional 10 min at 4?.The OD values were measured using an ultra-violet spectrophotometer at 532 nm.7.ImmunofluorescenceMice were deeply anesthetized with 1%sodium pentobarbital and transcardially perfused with normal saline and 4%paraformaldehyde(in PBS;PH=7.4).Then,brains were immediately harvested and post fixed in 4%paraformaldehyde for 24 h and separately dehydrated in 15%and 30%sucrose at 4?.Brains were embedded in optimum cutting compound and cut into 40 ?m thick sections.The sections were blocked with 3%BSA(containing 0.25%Triton X-100 and 10%goat serum)for 1 h at room temperature.The sections were then exposed to monoclonal mouse antibody against NOX2(1:100,BD)and rabbit antibody against Iba-1(1:200,Abcam)as primary antibody and incubated at room temperature for 14 h.We then exposed the sections to Abcam-488 goat anti-mouse(1:1000)and Abcam-594 goat anti-rabbit(1:2000)secondary antibody,incubated for 1 h at 37?.Then,4',6'-Diamidino-2-phenylindole(DAPI)counterstaining was performed to label cell nuclei.The fluorescent images were taken using a confocal microscope.The total number of NOX2+microglia in vCAl(4-6 sections per mouse)were manually counted for each double-immunolabeled z-stack.8.Real-time quantitative PCRIn brief,total RNA from the ventral hippocampus was isolated using E.Z.N.A.(?)Total RNA Kit I.Total RNA was reversed transcribed with the PrimeScriptTM RT reagent Kit to generate cDNA.Gene expression was determined by quantitative PCR with SYBR Green Dye Gene Expression Assays,which was performed on an ABI7500 system.The primers were synthesized by Shanghai Sangon Biological Engineering Technology Company(Shanghai,China).The cycle threshold,determined as the initial increase in fluorescence above background,was determined for each sample.?-actin was used as internal control for normalization.9.StatisticsData were analyzed using SPSS 19.0.All results were expressed as mean± SD.The main effects of factors were analyzed using two-way ANOVA and the differences between each groups were analyzed using LSD post hoc tests.For all tests,a probability of P<0.05 was considered statistically significant.Results1.Oberving the beneficial effect of EA treatment on GV24 and GV29 by mice subjected to CUMS.In the OFT and EPM,we found that,for the total distance travelled in the open field,there is no significant difference between each group(P>0.05).However,the time spent in the center field and the time spent in the open arms were significantly decreased after CUMS exposure(P<0.05).After EA treatment,CUMS-decreased time in the center field and open arms were ameliorated(P<0.05).In addition,no significant difference was observed between non-CUMS mice with and without EA treatment(P>0.05).2.Oberving the effect of EA treatment on GV24 and GV29 to the vCAl pyramidal cells of mice subjected to CUMS.After CUMS exposure,vCA1 pyramidal neurons exhibited significantly increased frequency and charge of sEPSC(P<0.05),and EA treatment significantly remitted this increasement(P<0.05).Neither CUMS nor EA treatment led to any significant changes in sEPSC amplitude of all groups of mice(P>0.05).We also found that CUMS and EA showed no effect on either frequency,amplitude or chage of sIPSC(P>0.05).CUMS also increased the sEPSC/sIPSC charge transfer ratio(P<0.05),which significantly remitted by EA treatment(P<0.05).In the second part of the study,results showed that apocynin administration and NOX2 deletion both remitted CUMS-caused anxiety-like behaviors in the OFT,EPM and LDB.And neither apocynin administration or NOX2 deletion exert any influence in these behaviors of mice without CUMS stimuli.3.Oberving the effect of EA treatment on GV24 and GV29 to the vCA1 oxidative stress induced by CUMS.Control group vs CUMS group,CUMS increased the DHE intensity and MDA content in vCA1(P<0.01).CUMS group vs CUMS+EA group,EA treatment decreased the DHE intensity and MDA content in vCA1(P<0.01).Control group vs Control+EA group,EA treatment caused no significant change of DHE intensity and MDA content in vCA1 of mice without CUMS exposure(P<0.01).4.Oberving the effect of EA treatment on GV24 and GV29 to the level of vCA1 NOX2 epression of mice subjected to CUMS.Control group vs CUMS group,CUMS significantly increased the number of NOX2-positive microglia and microglia in vCA1 of mice(P<0.01).CUMS group vs CUMS+EA group,EA treatment significantly decreased the number of NOX2-positive microglia and microglia in vCA1 of mice with CUMS stimuli(P<0.01).Control group vs Control+EA group,EA treatment led no significantly change in the number of NOX2-positive microglia and microglia in vCA1 of mice without exposure to CUMS(P<0.01).5.The confirmation of the above thearaputic targets using apocynin and transgenetic mice.(1)Anxiety and depressive behaviorsControl group vs CUMS group,CUMS significantly decreased the time spent in the center area of open field and in the open arms of elevated plus maze(P<0.01).CUMS group vs CUMS+EA group,EA treatment significantly ameliorated these changes led by CUMS exposure(P<0.01).Control group vs Control+EA group,EA treatment led no significantly change in the behaviors which non-CUMS mice exhibited during behavior test.(2)The level of oxidative stressControl group vs CUMS group,CUMS significantly increased the DHE intensity and MDA content in vCA1 of mice(P<0.01).CUMS group vs CUMS+apocynin group,apocynin administration significantly decreased the DHE intensity and MDA content in vCA1 of mice with CUMS stimuli(P<0.01).Control group vs Control+apocynin group,apocynin administration also significantly decreased the DHE intensity and MDA content in vCA1 of mice without exposure to CUMS(P<0.01).WT group vs WT+CUMS group,CUMS significantly increased the DHE intensity and MDA content in vCA1 of WT mice(P<0.01).WT+CUMS group vs NOX2KO+CUMS group,NOX2 deletion significantly decreased the DHE intensity and MDA content in vCA1 of mice with CUMS stimuli(P<0.01).WT group vs NOX2KO group,NOX2 deletion also significantly decreased the DHE intensity and MDA content in vCA1 of mice without exposure to CUMS(P<0.01).(3)The efficacy of excitatory transmissionCUMS exposure significantly increased the frequency and charge of sEPSC in mice without CUMS exposure(P<0.05),and the increased frequency and charge were significantly reversed by apocynin administration(P<0.05).There is no significant difference between the sEPSC amplitude of each group(P>0.05).Neither CUMS nor apocynin administration induced significant changes in sIPSC frequency,amplitude and charge(P>0.05).CUMS also increased the sEPSC/sIPSC charge transfer ratio(P<0.05),which can be significantly remitted by apocynin administration(P<0.05).(4)The level of NOX2 expressionControl group vs CUMS group,CUMS significantly increased the level of Nox2 mRNA expression in vHPC of WT mice(P<0.01).CUMS group vs CUMS+apocynin group,NOX2 deletion significantly decreased the level of Nox2 mRNA expression in vHPC of WT mice subjected to CUMS(P<0.01).Control group vs Control+apocynin group,NOX2 deletion also significantly decreased the level of Nox2 mRNA expression in vHPC of mice which not exposed to CUMS(P<0.01).Control group 'vs CUMS group,CUMS significantly increased the number of NOX2-positive microglia and microglia in vCA1 of mice(P<0.01).CUMS group vs CUMS group,apocynin administration significantly decreased the number of NOX2-positive microglia and microglia in vCA1 of mice with CUMS stimuli(P<0.01).Control group vs Control+apocynin group,apocynin administration also significantly decreased the number of NOX2-positive microglia and microglia in vCA1 of mice which not exposed to CUMS(P<0.01).WT group vs WT+CUMS group,CUMS significantly increased the level of Nox2 mRNA expression in vHPC of WT mice(P<0.01).WT+CUMS group vs NOX2K0 group,NOX2 deletion significantly decreased the level of Nox2 mRNA expression in vHPC of WT mice subjected to CUMS(P<0.01).WT group vs NOX2K0 group,NOX2 deletion also significantly decreased the level of Nox2 mRNA expression in vHPC of mice which not exposed to CUMS(P<0.01).WT group vs WT+CUMS group,CUMS significantly increased the number of NOX2-positive microglia and microglia in vCA1 of WT mice(P<0.01).WT+CUMS group vs NOX2K0 group,NOX2 deletion significantly decreased the number of NOX2-positive microglia and microglia in vCA1 of WT mice subjected to CUMS(P<0.01).WT group vs NOX2K0 group,NOX2 deletion also significantly decreased the number of NOX2-positive microglia and microglia in vCA1 of mice which not exposed to CUMS(P<0.01).Conclusion1.EA treatment on GV24 and GV29 ameliorated the anxiety behaviors induced by CUMS.2.EA treatment on GV24 and GV29 regulated the excitatory synaptic transmission and consequently adjusted the E/I balance of vCA1 pyramidal cells.3.EA treatment on GV24 and GV29 suppressed the level of NOX2-derived oxidative stress,which increased by CUMS.4.Inhibiting NOX2-derived oxidative stress by pharmacological tools or NOX2 deletion also ameliorated CUMS-induced anxiety-like behaviors. |
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