Based On The Alteration Of ATP Level In PFC Of Maternal Separation Model To Explor The Mechanism Of Antidepression In Electro-acupuncture Treatment On The Acupoints Of Baihui And Yingtang

OBJECTIVEAt present,acupuncture at Baihui and Yitang are widely used in the clinical of acupuncture and moxibustion to treat depression disorser,but the mechanism of antidepressant effect of acupuncture is still unclear.In this study,the essence of traditional Chinese acupuncture and moxibustion theory combined with the modernized detection means includes animal behavior tests,enzyme-linked immunosorption assay and neurotransmitter detection under awake condition were applied to explore the specific mechanism of electroacupuncture's anti-depression effect in the depression-like model rats caused by maternal separation(MS).From the external phenotypes to the endophenotypes of depression,in view of the possible pathogenesis of depression,we tested a variety of neurotransmitters,corticosterone(CORT)and adenosine triphosphate(ATP)levels in the key brain region of depression disorder in the prefrontal cortex(PFC)of MS depression-like rats to found the possible pivotal neurobiological mechanisms for the effectiveness of electro-acupuncture'Baihui'and 'Yitang' in MS caused depression.Our results are not only beneficial to further understand the pathogenesis of MS caused depression,but also providing theoretical foundation for acupuncture's operation in clinic for treating depression related mental diseases,besides presenting new ideas and targets for the treatment of emotional diseases such as depression.Experiment 1 Behavioral characteristics of maternal separation model of depression in adolescence and adult periodMethod1.Animal modeling and grouping methods:the Wistar rats in the gestation period were fed in separate cages and divided into healthy control group and modeling group.Set up the first day is PNDO when offspring rats were born.In model group,the mother mice were transferred out from PND1-PND21 at 8:30-13:30,and the offspring rats lost mothers' care for 4 hours per day and sustained total 21 days.In healthy control group,mother rats and offspring rats lived in a cage without disturbance.All the mother rats in the two groups were transferred in PND22 when weaned and remained offspring rats to separated into male and female cages.The male rats were fed in one cage and remained five,the female rats were eliminated.? The PND22-PND50 offspring rats were divided into young control group y-Cont(male offspring rats without maternal separation intervention)and young model group y-MS(male off spring with maternal separation intervention).?PND60 was the begin of the adult stage of the Wistar rats.Divide the offspring at PND60 into two groups includes control group Cont(male offspring rats without maternal separation intervention)and model group MS(male offspring with maternal separation intervention).2.Evaluation Indicators:week 1 of weaning:Scurose Preference Test(SPT),week 2 of weaning:Open Field Test(OFT),week 3 of weaning:Elevated Plus-maze Test(EPT),week 4 of weaning:Forced Swimming Test(FST),week 1 of being an adult:Scurose Preference Test(SPT),week 2 of being an adult:Open Field Test(OFT),week 3 of being an adult:Elevated Plus-maze Test(EPT),week 4 of being an adult:Forced Swimming Test(FST).3.Statistical methods:data were expressed by mean +standard error(x±SEM).SPSS 22.0 and Graphpad prism 7 were used for statistical analysis and mapped.Independent sample T-test analysis was used to compare the two groups,P<0.05 showed significant difference.Result1.In the week 1 of weaning,there was no significant difference in the preference of sucrose between y-MS group and y-Cont group(P>0.05).2.In the week 2 of weaning,compared with y-Cont group,y-MS group rats had higher total and corners distances,results had significant difference of(P<0.05).In the central area,y-MS group rats had significantly less residence time than Cont group rats,and the speed of crossing the central area was significantly faster than y-Cont group rats,and the number of crossings increased significantly.3.In the week 3 of weaning,compared with y-Cont group,the time and distance of open arm in y-MS group were significantly reduced(P<0.05),while the time and distance of closed arm in y-MS group were significantly increased(P<0.001),and there was no significant difference in the time in central area.4.In the week 4 of weaning,the immobility time of rats in y-MS group was significantly longer than y-Cont group(P<0.001).5.In the week 1 of being an adult,there had significant decreasing in the consumption of sucrose in MS group when compared with Cont group{P<0.001).6.In the week 2 of being an adult,there were no difference in the total distance,center distance,time in center and average speed in center area of OFT when compared with Cont group(P>0.05).7.In the week 3 of being an adult,there were no difference in the time and distance of open arms or the time and distance of close arms when compared with Cont group(P>0.05).8.In the week 4 of being an adult,the immobility time of rats in MS group was significantly longer than Cont group(P<0.01).Conclusion1)In the week 1 of weaning,y-MS rats did not show depression-like performence of anhedonia.2)In the week 2 of weaning,y-MS rats showed behavioral tendency of hyperactivity or mania.3)In the week 3 of weaning,y-MS rats showed significantly anxiety-like performence.4)In the week 4 of weaning,y-MS rats showed performance of despair and depression.5)Stable depression-like behaviors including anhedonia and despairing behaviors without anxiety-like changes were observed in adult MS rats.Experiment 2 Effectiveness of Electroacupuncture on Baihui and Yitang in MS modeled depressive ratsMethod1.Animal Modeling and Grouping:modeling method is the same as Experiment 1.In PND60,male offspring Wistar rats were randomly divided into MS group(model control group),MS+EA group(model+electro-acupuncture group)and MS+Sham-EA group(model+pseudo-electro-acupuncture group).In order to reduce the differences caused by different cages of rats,each group of rats came from different offspring of four mother rats.The male offspring rats who did not participate in modeling were Cont group(control group).2.Interventions:the therapy of experimental rats began in PND61.?Electroacupuncture Intervention Group:in the electro-acupuncture intervention group,Baihui(GV20)and Yintang(GV29)were selected for acupuncture treatment with reference to‘Experimental Acupuncture Science'.The experimental rats were anesthetized with isoflurane to therapy by Electroacupuncture.The anesthesia was induced by 4%isoflurane and oxygen mixture,and the rats were anaesthetized after about 5 minutes.After anesthesia,the rats were fixed on the Ryward anesthetic mask and maintained mild anesthesia with 2%isoflurane.Then disinfected the skin of the acupoint with 75%alcohol,used Huatuo acupuncture needle(0.25*13 mm),pierced the Baihui and Yintang points about 5 mm,and then connected to the Master8 electrostimulator.The parameters of stimulation intensity:current was 2 mA,frequency was 2 HZ,and electro-acupuncture time was 15 minutes.Electro-acupuncture was started at 8:30 a.m.Took it every other day for 21 days.?Sham Electro-Acupuncture Intervention Group:the rats in the MS+Sham-EA electro-acupuncture group were anesthetized with the same method as those in the electro-acupuncture group.After skin disinfection,the tip of the acupuncture needle was gently touched on the skin,but the needle was not penetrated.The model control group and blank control group received the same amount of isoflurane oxygen mixed gas anesthesia intervention as the electro-acupuncture group and the sham electro-acupuncture group,and the anesthesia method was the same as the electro-acupuncture group.Intervention was conducted every other day for 21 days.3.Evaluation Indicators:behavioral tests were performed in all groups on the first day after the end of treatment:Open Field Test(OFT),Elevated Plus-maze Test(EPT),Forced Swimming Test(FST).4.Statistical methods:data were expressed by mean+standard error(x±SEM).SPSS 22.0 and Graphpad prism 7 were used for statistical analysis and mapped.One-way ANOVA-LSD test was used to compare the data among groups,and K-S test was used when the variance was uneven.P<0.05 was significant difference.Result1.The results of OFT showed that there was no significant difference in total distance and central area distance between groups(P>0.05).The results of EPT showed that there was no significant difference in the time spent in closed arms or open arms between groups(P>0.05).2.SPT results showed that compared with Cont group,the preference of sucrose in MS group was significantly lower(P<0.05),while the preference of sucrose in MS+EA group was significantly higher than that in MS group(P<0.05)and with no difference compared with Cont group(P>0.05),and there was no significant difference between MS+Sham-EA group and MS group(P>0.05).3.FST results showed that the immobility time of rats in MS group was significantly longer than that in Cont group(P<0.001).Compared with MS group,the immobility time of MS+EA group decreased significantly(P<0.001)though did not improve to the same behavior as control group(P<0.05).And there was no significant difference between MS group and MS+Sham-EA group(P>0.05).Conclusion1)model of maternal separation for 4 hours a day in early life did not produce anxiety-like lesions in adulthood.Anxiety-like test of electro-acupuncture on adult MS model did not produce any effect,nor did sham electro-acupuncture;2)Electro-acupuncture at Baihui and Yin tang acupoints can improve depression-like anhedonia and despair behavior in adult rats caused by maternal separation.Experiment 3 The moderating effect of Electroacupuncture on Baihui and Yintang in PFC neurotransmitters and CORT levels of depressive MS rats Method1.Animal Modeling,Grouping and Intervention:the modeling method is the same as experiment 1.with MS+Sham-EA group was cancelled,other groups and intervention methods were the same as experiment 2.2.Evaluation Indicators:?Using ultra high liquid mass spectrometry(HPLC-MS/MS)method teated the concentration of monoamines neurotransmitters and Ach in the tissues supernatant of PFC region.?Utilizing the HPLC-MS/MS tested the concentration of CORT in the tissues supernatant of PFC region.? Using microdialy conbined with HPLC-MS/MS method tested the microthe concentration of Glu and GABA in the cerebrospinal fluid of PFC region.3.Statistical method was the same as experiment 2.Result1.In PFC tissue supernatant,compared with Cont group,5-HT level in MS group and MS+EA group decreased significantly(P<0.001).Compared with MS group,5-HT level in MS+EA group did not change significantly(P>0.05).The changes of NE and DA level were similar to those of 5-HT.2.In PFC tissue supernatant,compared with Cont group,ACh level in MS group and MS+EA group decreased significantly(P<0.001,P<0.05).Compared with MS group,ACh level in MS+EA group has a little evelated with no biological significances(P>0.05).3.The CORT level between Cont,MS and MS+EA groups has no significant diference(P>0.05).4.In PFC cerebrospinal fluid,compared with Cont group,the content of Glu in MS group had no change while GABA had increased(P<0.05).EA could elevate the level of Glu(P<0.05)but had no effect on changing the increased GABA(P>0.05).Conclusion1)Electro-acupuncture on Baihui and Yintang may not play an antidepressant role by altering neurotransmitters in PFC tissue supernatant of MS depression-like model.2)Electro-acupuncture on Baihui and Yintang may not play an antidepressant role by altering CORT level in PFC tissue supernatant of MS depression-like model.3)Electro-acupuncture on Baihui and Yintangmay may play an antidepressant role through elevating the Glu level to inhibit the GABA level relatively in PFC cerebrospinal fluid of MS depressive model for improving the unbalance of excitatory and inhibitory neurotransmitters.Experiment 4 The relationship between electroacupuncture on Baihui and Yintang improving the depressive behavior and the ATP level in PFC of MS ratsMethod1.Modeling method is same as Experiment 12.Grouping and intervention:? MS rats were divided into three groups,the maternal separation model group(MS group),the electro-acupuncture treatment group(EA group),and the intraperitoneal injection of ATP group(ATP group).Among them,MS group,MS+EA group and blank control group(Cont group)were given intraperitoneal injection of 0.9%saline intervention,according to the amount of lml/kg.The ATP group was intervened by intraperitoneal injection of ATP solution at a certain concentration,and was also injected at a dose of 1 ml/kg.?After the intervention of acupuncture,ACSF(MS+EA+Vehicle)was injected by Stereotaxic PFC of parts of electro-acupuncture group rats,another parts of rats used for stereotaxic PFC injection of ATP antagonist suramin(MS+EA+Suramin).parts of the MS rats without electro-acupuncture intervention were used to inject ACSF(MS+Vehicle)by stereotaxic PFC and ATP agonist(MS+ATP)by stereotaxic PFC.Parts of healthy control rats were injected with ACSF(Cont+Vehicle)by stereotactic PFC,while the rest were injected with ATP antagonist suramin(Cont+Suramin)by stereotactic PFC.3.Evaluation indicators:?The expression of UCP3 gene in PFC tissues.? ATP content in cerebrospinal fluid of PFC area.?After intraperitoneal injection of ATP agonists,the consumption of sugar and water and the immobility time of forced swimming of rats in each group.?The immobility time of forced swimming and the total distance of open field after stereotaxic injection of three drugs.4.Statistical method was the same as experiment 2.Result1.Took the value of Cont group as control value 1.The expression of UCP3 in PFC of MS group was significantly higher than that of Cont group(P<0.05).Compared with MS group,the expression of UCP3 in PFC of MS+EA group was significantly lower(P<0.05).2.In cerebrospinal fluid of PFC area,the ATP content in MS group was significantly lower than that in Cont group(P<0.01),and the ATP content in MS+EA group was significantly higher than that in MS group(P<O.05).3.After intraperitoneal injection of ATP agonists for 7 days,compared with the control group,the consumption of sugar and water in MS group decreased significantly(P<0.001),and electro-acupuncture could reverse this change(P<0.01).Compared with the control group,the immobility time of MS rats increased significantly(P<0.05),which could be reversed by Electroacupuncture(P<0.05).4.Behavioral tests of forced swimming were conducted immediately after 30 minutes of intracerebral injection,and AT P agonists were injected into PFC of MS rats.It was found that the immobility time of MS rats was significantly lower than that of MS rats injected with vehicle(P<0.01).After injecting the ATP antagonist suramin into the PFC of healthy Cont rats,the immobility time was significantly higher than that of the control rats injected with vehicle(P<0.05);after injecting the ATP antagonist suramin into the PFC of EA rats,the immobility time was significantly higher than that of EA rats injected with vehicle(P<0.05).5.The rats in each group were injected with stereotactic brain injection again and were given open field test after 30 minutes' rest.There was no significant difference in the total distance of open field between the groups(P>0.05).Conclusion1)The decrease of ATP level in PFC region maybe one of the key mechanisms of MS manifestations induced depressant model.2)Electro-acupuncture at Baihui and Yin tang performer antidepressant effect in MS depression-like model through increasing ATP level in PFC region.