Changes Of Adult Hippocampal Neurogenesis During Treatment Of Electroacupuncture With Depression Model Rats

Changes of adult hippocampal neurogenesis during treatment of electroacupuncture with depression model ratsDepression is a severe illness with a lifetime prevalence of between 10 and 20% according to large epidemiological studies.Suicide is a major risk in depression,with about 15%of depressed patients committing suicide.Conventional treatments like psychotherapy and medication alleviate the blues in a whopping 50%to 70%of patients who complete the regimen.In addition,about 1/3 patients have poor compliance to their antidepressant treatments and stop taking medication is common in clinical treatment.It is crucial to look for the alternative treatments with balanced effect between the antidepressant effects and side effects.Acupuncture has long been used as a treatment for somatic and mental disorders in Traditional Chinese Medicine (TCM).This approach generates few side effects,and is considered a new,alternative method of medicine in Western countries.During the past 50 years,electric stimulation has been applied to acupuncture needles,which is called electroacupuncture(EA) and thus appears to be more effective for the treatment of depression.Many clinical data indicate that complementary,alterative therapies are effective in treating depression patients and a growing number of people with depression are choosing to be treated in this manner,notably with acupuncture. However there is little study regarding basic experimental studies on the effect of acupuncture treatment with depression.The dentate gyrus(DG) of the adult hippocampus contains undifferentiated,rapidly proliferating progenitor cells.Approximately,70-80%of the newly formed cells differentiate into granule neurons,which ultimately fully integrate into the hippocampal network.Hippocampal neurogenesis can be influenced by several environmental factors and stimuli,among which stress play a key role in the regulation.Adult neurogenesis is decreased by many different types of stressors, including social stress,acute and chronic restraint stress,footshock stress and chronic mild stress.In contrast to the effects of stress,antidepressant treatment increases adult neurogenesis.Administration of one of the several different classes of antidepressants, including 5-HT or NE selective reuptake inhibitors,increases neurogenesis in adult hippocampus.Interestingly,studies indicated that acupuncture increased cell proliferation in DG after transient global ischemia in gerbils and in DG of streptozotocin-induced diabetic rats.These observations raised the question of whether EA can influence the cell proliferation in the DG of stress induced depression model rats.Therefore,to investigate to role of hippocampal neurogenesis in depression and antidepressant-like treatment with EA is one of the main targets of the present study.In addition,a very recent study showed the notable changes of astroglial structural plasticity in response to stress and antidepressant treatment,which support the notion that glial changes may contribute to the pathophysiology of affective disorders as well as to the cellular actions of antidepressants.These observations raised the question of whether EA can influence astrocytes in the hippocampus during EA treatment with depression model rats.Brain-derived neurotrophic factor(BDNF) is one member of the neurotrophin family of growth factors that is widely expressed throughout the mammalian brain including the hippocampus.Chronic antidepressant administration regulates expression of BDNF in the hippocampus,which supports the hypothesis that this neurotrophin may contribute to the treatment of depression.Glial cell line-derived neurotrophic factor (GDNF) was originally purified from a rat glioma cell-line supernatant as atrophic factor for embryonic midbrain dopamine neurons,and was later found to have potent survival-promoting effects on various types of neurons.GDNF is thought to bind preferentially to GFRα-1(the GDNF family receptor) and GDNF fails to exert its biological action in the absence of GFRα-1.Recent studies showed that expression of GDNF is altered in humans with mood disorders.Furthermore,acute and chronic electroconvulsive seizure(ECS) treatment significantly increases expression of GDNF receptors in the DG of rodents.However,the expression of BDNF,GDNF and their receptors TrkB,GFRα-1 in behavioral depression model rats has been unknown. Previous study suggested that GDNF might be involved in the therapeutic effect of EA on Parkinson's disease.It remains unclear whether BDNF or GDNF is involved in antidepressant-like effect of EA.Therefore,to investigate the change of BDNF, GDNF and their receptors in depression and antidepressant-like effect of EA is the another main target of the present study.Recent study showed that administration of BDNF directly into specific brain regions mimicked antidepressant effects in animal models of depressive-like behavior. However,there are difficulties for systemic delivery of BDNF or GDNF on depression patients.Thus the present study was also aimed to investigate the effect of BDNF or GDNF on the proliferation and differentiation of neural stem cells using the culture of hippocampal stem cells in newbom rats in vitro.Using the chronic unpredictable stress induced depression model rats,which is a valid model of depression,as it satisfies the criteria of correlation,isomorphism,and homology,the present study was aimed to:(1) observe the antidepressant-like effect of EA on depression model rats;(2) examine the change of adult hippocampal neurogenesis during depression and EA treatment using BrdU incorporation and BrdU immunohistochemistry;(3) examine the expression of GFAP during depression and EA treatment using immunohistochemistry,Western Blot and RT-PCR;(4) examine the expression of BDNF,GDNF and their receptors TrkB,GFRα-1 during depression and EA treatment using immunohistochemistry,Western Blot and RT-PCR;(5) observe the effect of BDNF or GDNF on the proliferation and differentiation of neural stem cells using the culture of hippocampal stem cells in newborn rats in vitro.The results are as follows:1.Antidepressant-like effect of EA or Clomipramine on depression model rats EA was performed on acupoints 'Bai-Hui'(Du 20) and 'Yang-Ling-Quan'(GB-34) once daily for 3 consecutive weeks,2 weeks post CUS procedure.Open field test, forced swimming test and physical scores were employed to evaluate the behavioral activity during the stress period or EA treatment.The results revealed that exposure to CUS resulted in a decrease of behavioral activity,whilst the session of EA treatment significantly reversed the behavioral deficit of these depression model rats.In addition, repeated administration of clomipramine(5 mg/kg i.p.) for 3 weeks,starting 2 weeks after the beginning of the stress procedure,significantly reversed the reduction of behavior measured by those behavioral tests.2.Change of adult hippocampal neurogenesis during depression and EA or Clomipramine treatmentBrdU immunohistochemistry revealed dividing cells in the DG of different group rats. The majority of BrdU-positive cells were located in the subgranular zone(SGZ) and generally occurred singly or in small clusters of three to five cells.Chronic unpredictable stress for 5 weeks resulted in a significant 49.9%decrease in the number of BrdU-positive cells relative to normal rats.Clomipramine treatment of stressed rats showed a 102.3%increase in the number of BrdU-positive cells in the DG compared with the saline group.EA treatment of stressed rats showed a 103.5% increase in the number of BrdU-positive cells in the DG compared with the sham EA group.There were no differences in the cell proliferation between the normal group and the EA group or Clomipramine group,which suggested EA or Clomipramine treatment suppressed the decrease of hippocampal cell proliferation induced by CUS.3.Change of GFAP expression during depression and EA treatment Immunohistochemistry and Western Blot were used to examine the expression of GFAP protein in hippocampus(including DG and CA3 region) of depression model rats.RT-PCR was used to examine the expression of GFAP mRNA in hippocampus of depression rats.The results showed that:(1) the expression of GFAP protein significantly decreased in hippocampus of depression model rats,which could be significantly counteracted the decrease by EA treatment;(2) the expression of GFAP mRNA significantly decreased in hippocampus of depression model rats,which could be significantly counteracted the decrease by EA treatment.4.Change of BDNF,GDNF and their receptors TrkB,GFRα-1 expression during depression and EA treatmentImmunohistochemistry and Western Blot were used to examine the expression of BDNF,GDNF and their receptors TrkB,GFRα-1 protein in hippocampus(including DG and CA3 region) of depression model rats.RT-PCR was used to examine the expression of BDNF mRNA,GDNF mRNA,TrkB mRNA and GFRα-1 mRNA in the hippocampus of depression rats.The results showed that:(1) the expression of BDNF, GDNF and their receptors TrkB,GFRα-1 protein significantly decreased in hippocampus of depression model rats and could be significantly counteracted the decrease by EA treatment;(2) the expression of BDNF mRNA,GDNF mRNA,TrkB mRNA and GFRα-1 mRNA significantly decreased in hippocampus of depression model rats and could be significantly counteracted the decrease by EA treatment.5.Effect of BDNF or GDNF on the proliferation and differentiation of hippocampal neural stem cells in vitroCulture and identification of neural stem cells from the hippocampus tissue in neonatal rats were the first step of this part study.Possible role of BDNF or GDNF in hippocampal neurogenesis was to be investigated by detecting the proliferation or differentiation of neural stem cells exposed to BDNF or GDNF.CCK-8 assay was used to detect the proliferation of neural stem cells and the absorb score were significantly increased in neural stem cells exposed to GDNF.Although the BDNF had the potential increase trend,the statistical analysis had not been different. Forty-eight hours after being cultured under the differential condition,part of neural stem cells differentiated to astrocyte(GFAP-positive) and most of them remained in undifferentiated state(Nestin-positive).When the neural stem cells exposed to the BDNF or GDNF,there are more neurites from neurospheres compared with the normal condition.This study indicated that the two neurotrophic factors have the potential effect on the differentiation of neural stem cells.The present study suggested:(1) EA has potent antidepressant-like effect on CUS induced depression model rats.(2) CUS results in a state of behavioral depression and decreases hippocampal cell proliferation and that these effects can be counteracted by EA or Clomipramine treatment,suggesting that hippocampal neurogenesis might be involved in the etiology of depression and EA or Clomipramine treatment.(3) Expression of GFAP decreased significantly in hippocampus of depression model rats and could be counteracted by EA treatment,suggesting that astrocyte might play a role in the etiology of depression and the antidepressant-like effect of EA.(4) Expression of BDNF/TrkB and GDNF/GFRα-1 decreased significantly in hippocampus in depression model rats and could be counteracted by EA treatment,suggesting that they might involve in the etiology of depression and the antidepressant-like effect of EA.(5) GDNF and BDNF respectively increased the proliferation and differentiation of hippocampal neural stem cells in vitro,suggesting that the two neurotrophic factors might be involved in the regulatory effect of hippocampal neurogenesis.