| BackgroundDepression is a recurrent mental illness characterized by changes in emotional,autonomic and cognitive symptoms.It is a major global mental health challenge,with negative impacts on individuals and families,as well as a huge financial strain on society.The global economic costs associated with depression are projected to double by 2030.The pathogenesis of depression is complex,and the current therapies for depression have some problems,such as long effective time,multi-drug combination therapy and obvious side effects.About one-third to one-half of depressed patients do not respond to multiple antidepressants,and many more receive only a partial response.Therefore,there is an urgent need to find new antidepressant treatments.Vagus Nerve Stimulation(VNS),a type of physical therapy,was approved by the U.S.Food and Drug Administration for the treatment of treatment-resistant depression.Clinical studies have confirmed its effectiveness in treating depression.However,due to the need for surgical implantation and adverse reactions such as cough,hoarseness and gastrointestinal discomfort,it has limitations in clinical application.Transcutaneous auricular vagus nerve stimulation(taVNS)is a new treatment based on the auricular acupoint theory of traditional Chinese medicine combined with VNS.TaVNS works by acting on the auricular point,the heart,and the liver in the auricular concha,which is also the distribution area of the auricular branch of vagus nerve,the only superficial branch of the vagus nerve.A body of evidence showed that taVNS could effectively improve depressive symptoms without significant adverse reactions reported.Compared with traditional VNS,taVNS has the advantages of safety and noninvasive,which is a new treatment for depression.The cholinergic system,including neurotransmitters acetylcholine(ACh),acetylcholinesterase(AChE),choline acetyl transferase(ChAT),etc..It plays an important role in mediating physiologic function,related to emotion,perception,memory and so on.The hyper-cholinergic tone,characterized by abnormal cholinergic factors such as increased ACh level,decreased AChE activity and increased ChAT expression is closely related to the onset and development of depression.In the animal model of depression,central cholinergic signaling was altered,with the most obvious changes in brain regions such as the hippocampus and medial septum nucleus,and the hippocampal hyper-cholinergic tone promoted anxiety and depression-like behaviors.It is noteworthy that cholinergic drugs can induce memory impairment,which may be related to their pro-inflammatory effects.Intraperitoneal injection of the classic anticholinergic drug scopolamine induced memory impairment in rats,accompanied by an increase in hippocampal tumor necrosis factor-α(TNF-α)level.This limits the use of cholinergic drugs in clinical antidepressant therapy.Previous studies have shown that taVNS exerts a central antidepressant effect by modulating the hippocampal cholinergic component α7 nicotinic acetylcholine receptor(α7nAChR),but it is unknown whether taVNS exerts a central antidepressant effect by modulating the activity of the hippocampal cholinergic system.Therefore,the present study was designed to investigate the mediate effects of taVNS on the hippocampal cholinergic system activity in mice with chronic unpredictable mild stress(CUMS)by measuring the hippocampal AChE and ChAT expression in the medial septum nucleus,the hippocampal major cholinergic input nucleus.And measuring the hippocampal TNF-α,interleukin 6(IL-6)and interleukin 1β(IL-1β)expression and morphology of microglia to investigate the mediation of hippocampal neuroinflammatory response in chronic stress mice by taVNS while modulating the activity of hippocampal cholinergic system.Objectives(1)To observe the effect of taVNS on depression-like behavior in chronic stress mice.(2)To compare the effects of taVNS on hippocampal ChAT and AChE expression in mice,and to observe the effect of taVNS on the hippocampal cholinergic system activity in chronic stress mice.(3)To compare the effects of taVNS on the morphology of hippocampal microglia and the expression of TNF-α,IL-1β and IL-6 in mice,and to observe the effects of taVNS on hippocampal neuroinflammatory response in chronic stress mice.MethodsForty male SPF C57BL/6 mice with body weight of 20±2g were adaptive feeding for one week,including blank group(Control group),model group(CUMS group),transcutaneous auricular vagus nerve stimulation group(taVNS group)and transcutaneous non-auricular vagus nerve stimulation group(tnVNS group),with 10 mice in each group.Except the Control group,other groups were housed in single cages and molded by CUMS for 9 weeks.Ten chronic stressors were used,including fasting(24h),water prohibition(24h),rhythm disturbance(12h/12h),crowding(12h),wet bedding(8h),restricted freedom(6h),tail clamping(5min),no bedding(24h),tilted squirrel cage(6h),and ice water swimming(4℃,3min).Two stressors were randomly given each day,and each stressor was not repeated for three consecutive days,increasing the unpredictability of the stimulus.At the sixth week of modeling,interventions were given to mice:Control group and CUMS group without intervention.In taVNS group,HANS stimulator was used for continuous intervention for three weeks.Conductive circular magnets were fixed in auricular concha(the auricular-point "heart"and "liver").Stimulation parameters were as follows:2/1 5Hz,dilatational,1mA,30min/time/day.2%isoflurane inhalation anesthesia was used during the intervention.In tnVNS group,the circular self-made conductive magnet was fixed on the superior scapha non-invasively,and the other operations were the same as in taVNS group.The body weight and forced swim test(FST),sucrose preference test,tail suspension test(TST)and open field test(OFT)were conducted at week 0,week 3,week 6 and week 9 to evaluated the model.After the intervention,perfusion and tissue sampling will be carried out.The expression of ChAT in medial septum,AChE,TNF-α,IL-6 and IL-1βin hippocampus were detected by Western Blot.ChAT and c-Fos in medial septum,AChE in hippocampus and morphological changes of microglia were observed by immunofluorescence.Results(1)Body weight:Before modeling(week 0),there was no significant difference in body weight between groups(P>0.05);At week 3 and week 6,other groups decreased significantly(P<0.001,P<0.001,P<0.001;P<0.001,P<0.001,P<0.001)compared to the Control group.After 3 weeks(week 9)of taVNS,the body weight of the CUMS group decreased significantly compared to the Control group.(P<0.001),and the body weight of the taVNS group increased significantly compared to the CUMS group(P<0.001),the body weight was reduced in the tnVNS group compared to the taVNS group(P<0.001).(2)Sucrose preference test:Before modeling(week 0)and week 3,there was no significant difference in sucrose preference ratio between groups(P>0.05).At week 6,sucrose preference ratio for other groups decreased significantly compared to the Control group(P<0.001,P<0.05,P<0.001).After 3 weeks(week 9)of taVNS,compared to the Control group,the sucrose preference ratio decreased significantly in the CUMS group(P<0.001),the ratio in the CUMS group was significantly increased(P<0.001).Compared to the taVNS group,ratio was reduced in the tnVNS group(P<0.01).(3)FST:Before modeling(week 0)and week 3,there was no significant difference in immobility time between groups(P>0.05).At week 6,the immobility time of the other groups was longer compared to the Control group(P<0.05,P<0.01,P<0.01).After 3 weeks(week 9)of taVNS,the immobility time in the CUMS group was much longer than that in the Control group(P<0.001),and the immobility time in the taVNS group was shorter than that in the CUMS group(P<0.001).The immobility time was extended in the tnVNS group compared to the taVNS group(P<0.01).(4)TST:Before modeling(week 0)and week 3,there was no significant difference in immobility time between groups(P>0.05).At week 6,the immobility time of the other groups was longer compared to the Control group(P<0.001,P<0.001,P<0.001).After 3 weeks(week 9)of taVNS,the immobility time in the CUMS group was much longer than that in the Control group(P<0.001),and the immobility time in the taVNS group was shorter than that in the CUMS group(P<0.001).The immobility time was extended in the tnVNS group compared to the taVNS group(P<0.001).(5)OFT:Before modeling(week 0)and week 3,there was no significant difference in distance in central zone between groups(P>0.05).At week 6,the distance in central zone of the other groups was decreased compared to the Control group(P<0.001,P<0.001,P<0.001).After 3 weeks(week 9)of taVNS,the distance in the CUMS group was decreased than that in the Control group(P<0.001),and the distance in the taVNS group was increased than that in the CUMS group(P<0.01),and the distance in the tnVNS group was decreased than that in the taVNS group(P<0.05).Before modeling(week 0)and week 3,there was no significant difference in time in central zone between groups(P>0.05).At week 6,the time in central zone of the other groups was decreased compared to the Control group(P<0.05,P<0.05,P<0.01).After 3 weeks(week 9)of taVNS,the time in the CUMS group was decreased than that in the Control group(P<0.001),and the time in the taVNS group was increased than that in the CUMS group(P<0.001),and the time in the tnVNS group was decreased than that in the CUMS group(P<0.01).(6)Expressions of ChAT in medial septum,and AChE in hippocampus:After the experiment(week 9),the expressions of ChAT in medial septum and AChE in hippocampus of mice in each group were measured by Western Blot and immunofluorescent staining.The results showed that the ChAT expression in CUMS group was significantly higher than that in the Control group(P<0.001),and the AChE expression was significantly decreased(P<0.001),ChAT neurons are more co-labeled with c-Fos.Compared with CUMS group,ChAT expression in taVNS group was significantly decreased(P<0.001),and AChE expression was significantly increased(P<0.001),ChAT neurons are less co-labeled with c-Fos.Compared with taVNS group,AChE expression in tnVNS group was significantly decreased(P<0.001).(7)Morphology of hippocampal microglia and expression of TNF-α,IL-1β and IL-6 in hippocampus:①Western Blot:After experiment(week 9),expression of TNF-α,IL1β and IL-6 in the hippocampus of mice was detected by Western Blot.TNF-α,IL-1βand IL-6 of the CUMS group increased significantly compared to the Control group(P<0.001,P<0.001,P<0.01).Compared to the CUMS group,the expressions of TNFα,IL-1β and IL-6 in the taVNS group were significantly reduced(P<0.001,P<0.001,P<0.05).Compared to the taVNS group,the expressions of TNF-α and IL-1β in the tnVNS group were significantly increased(P<0.001,P<0.01).②Immunofluorescence:After the experiment(week 9),perfusion and sectioning were performed,and Ionized Calcium Binding Adapter Molecule 1(IBA1)was stained with immunofluorescence to observe the morphologic changes of microglia in the hippocampus.The results showed that the hippocampal microglia in the Control group had smaller cell bodies and elongated branches,showing a resting state.Compared with the Control group,the hippocampal microglia in the CUMS group increased irregularly,and the branches were short and activated.Compared with CUMS group,hippocampal microglia in taVNS group showed a resting state.Compared with taVNS group,tnVNS group showed an activated state of hippocampal microglia.Conclusions(1)After CUMS modeling,the mice showed weight loss,the sucrose preference ratio decreased,the immobility time of FST and TST increased,and the central time and central distance of OFT decreased.And the depression-like behaviors of chronic stress mice were improved after three weeks of taVNS.The antidepressant effect of tnVNS was weaken.(2)The expression of ChAT in the medial septum of CUMS group was increased,and the expression of ACNE in hippocampus was decreased,indicating that a hippocampal hyper-cholinergic tone.After three weeks of taVNS,ChAT expression was decreased and AChE expression was increased.The results showed that taVNS could inhibit the hippocampal hyper-cholinergic tone in chronic stress mice and exert the antidepressant effect.The anticholinergic hyperactivation effect of tnVNS was weaken.(3)Hippocampal microglia were activated in CUMS group,and the expressions of TNF-α,IL-1β and IL-6 were increased,indicating hippocampal neuroinflammatory response.3 weeks of taVNS inhibited the activation of microglia and decreased the expression of TNF-α,IL-1β and IL-6 in hippocampus.The results showed that taVNS could inhibit hippocampal inflammatory response in chronic stress mice and exert antidepressant effect.The anti-inflammatory effect of tnVNS was weaken.(4)In conclusion,taVNS may exert central antidepressant effect by inhibiting hippocampal hyper-cholinergic tone and hippocampal neuroinflammatory response in chronic stress mice. |
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