| Depression has become a major health concern, affecting more than11%people over the world, resulting in enormous personal and economic costs and even, over10%cases, suicide. Although great improvements on antidepressant treatment have been achieved, almost30%patients with major depression have no reaction to medicine.Compared with the medicine administration, acupuncture, electroacupuncture (EA) especially, is known for the profound therapeutical value while few side effects. In China, many patients have accepted EA help in clinical practice and increasing studies have demonstrated the antidepressant effects of EA. Been laid great focus as EA has, the neurological mechanisms of its role in the reversion of depression remain complicated. Few revelations had been generally acknowledged so far. A promising mechanism was repeatedly mentioned, that EA was beneficial to neural stem cell (NSC) survival and proliferation in the process of depression treatment. However, sufficient evidences and clear mechanisms of the antidepressive-like effects, activating the neural stem/progenitor cells in the differentiation and amplification level, for example, still need further basic studies.Previous investigations have demonstrated that hippocampal neurogenesis plays a role in the depression. Exposure to stress is known to induce decreased neurogenesis in hippocampus. The dentate gyms (DG) of the adult hippocampus contains large amounts of undifferentiated progenitor cells, which contribute to hippocampal neurogenesis. It has been proposed that two major subclasses of stem/progenitor cells are quiescent neural progenitor (QNP) and amplifying neural progenitor (ANP). Both subclasses are Nestin and SOX-2staining positive. One subclass (QNP) has an elaborate tree of processes in the granule cell layer and express GFAP (Nestin+/GFAP+or SOX-2+/GFAP+), the other (ANP) does not (Nestin+/GFAP-or SOX-2+/GFAP-). Studies have shown that QNPs and ANPs are different in response of external stimulus or treatments. Then, in the etiology and EA therapy of the depressive disorder, which type of NSCs is mainly actived is still poorly understood.Based on this, we looked into the antidepressant-like activity of EA using a chronic unpredictable stress model (CUS, one of the fully validated animal models of depression). And in particular, we attempted to ascertain whether EA increased the proliferation of stem cells in DG of CUS rats. Meanwhile, we explored the proliferation of QNP and ANP cell subclasses respectively for the purpose of making clear the main target (or which neural stem cell type/types) EA anti-depression effects aimed.Furthermore, very few known basic experimental studies exist, regarding on the probable pathways in the process of EA treatment of depression and stem cell proliferation. In the light of some growth/neurotrophic factors, like nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF), known to have survival and division effects on neuronal cells, studies had indicated EA raised levels of these growth/neurotrophic factors. As the extracellular signal-regulated kinase (ERK, including ERK1and ERK2) has been widely demonstrated to mediate the processes of these factors’expression, it is extremely possible that EAmay improve the depressive syndrome and activates NSCs through pathways including ERK.In line with this, we men investigated the phosphorylated extracellular signal-regulated kinase (p-ERK) of the hippocampal stem cells.In addition, to identify whether EA stimulation resulted in a microenvironment conducive to neural stem cells (NSCs) proliferation in DG, we investigated the effects of microdialysates from the DG of EA-treated CUS rats (EA microdialysates) on the in vitro growing stem cells. It was observed that the growth of neurospheres and p-ERK level in the NSCs were up-regulated by EA microdialysates.The results are as follows: 1. Antidepressant-like effect of EA on CUS model ratsSD rats (male,220g) were randomly divided into four groups:normal group (Normal), CUS model group (Model), CUS model+sham EA treatment group (Sham EA), CUS model+EA treatment group (EA).In CUS procedure, rats are exposed over a long period to a variety of relatively severe stressors in random order. It causes neurobehavioral disturbances in rodents and mimics symptoms of depression. During our CUS procedures,7types of stresses in all were carried out. The order of stresses was set as random. Any of the stresses was carried out every1or2days, in which manner all19stresses were arranged in a period of30days. The Model, Sham EA and EA groups received this stress while the Normal group was left undisturbed as the Non-stressed control.EA or sham EA was administered every other day for14days (8times), starting on day15. EA stimulation was performed at a pair of acupoints, which were Du-20(Bai-Hui) and GB-34(Yang-Ling-Quan)(For the EA group:30min,2\100Hz,0.3mA). The Sham EA group was administered in the same protocol (in the same real acupoints) but power off; the Model group was just bounded and hung for30min.Two weeks after CUS, the CUS rats exhibited a significant decrease in physical state score (PSS). The immobility time of stressed rats significantly increased and their climbing time decreased in the forced swimming test, and these durations were similar between2groups before the stress. Anxiety behaviors of rats were measured by entries and time spent in the open arms in EPM. Analyses of ratio of open/total arm entries (OER) and open/total time ratio (OTR) revealed no difference between Normal and Model groups at the baseline level. After2weeks of CUS, stressed rats showed a significant lower preference of the open arms (or significant increase in anxiety levels), which suggests that rats exposed to CUS exhibited anxiety behaviors. Before CUS, the rearing numbers of all groups were at the same level in the open field test (OFT). After17days, stressed rats displayed fewer rearings but similar crossing distance, compared with the normal ones.Since day15, EA and Sham EA were administered to the CUS rats for14days in total8times. PSS of three CUS-exposed groups (Model, Sham EA and EA) had not shown any significant difference after2,3and4weeks of CUS period. Received one week of acupuncture administration, EA and Sham EA groups showed very significantly decreased immobility time of the FST compared with Model group. After2weeks of treatment (4weeks of the CUS period), Sham EA and EA groups had kept a low immobility time, which was similar to that of the Normal group, and significantly less than the Model group. Correspondingly, EA group exhibited highly significant reverse of the climbing-time decrease. Interestingly, Sham EA also showed some anti-depressant effect especially on the immobility time, in despite of less reversal latitude than the EA group. Analysis of OER and OTR data in the EPM test revealed that EA administration made a significant reverse of their reductions caused by CUS exposure. When repeatedly administrated, CUS rats in the Sham EA group could also recover to a certain extent despite that the anti-depression effect was not such steady and marked. In the OFT, EA, as well as Sham EA, had significantly improved the rearing number in the initial period of treatment. Compared with Normal and Model rats, acupuncture groups had shown a promoted exploratory behavior. Acupuncture could excite the rearing behavior and EA had a steadier effect than Sham EA.2. BrdU cell number of NSCs was increased by EA administrationFor labeling dividing stem cells during the S phase of mitosis, rats were administered5-bromo-2-deoxyuridine (BrdU) one day before perfusion. The brains were separated and cut into sections. Immunohistochemistry results suggested:BrdU and SOX-2double positive cells were mainly located in the subgranular zone (SGZ); after2-4weeks of CUS the stem cells in stressed rats had a lower proliferation level; Both EA and Sham EA raised the positive BrdU stem cells (BrdU positive&SOX-2positive) in the DG zone. These two groups had the similar level of dividing cells as the Normal group, indicating that EA or Sham EA had the effect of activating stem/progenitor cells in CUS model. 3. QNPs and ANPs in hippocampus after EA administrationReceived CUS for4weeks, ANPs (GFAP negative and SOX-2positive) in the model group had been significantly lessened, compared with the normal group. QNPs number of the stressed rats kept a similar level to those in the normal group.After8treatments, QNPs (GFAP and SOX-2double positive) were unaffected by Sham EA but amplified to a significant level in the EA group. The decrease of ANPs was reversed by8treatments of EA and Sham EA. In addition, the total stem cell number in the EA group had highly significant differences compared with the Model group. It was attributed to that the the EA group had not only significantly higher ANPs but also QNPs level. While, Sham EA (similar to "acupuncture") only raised the number of ANPs but QNPs. Together, these results indicate that acupuncture was a contributory method for ANP generation while electric current had some QNPs-beneficial effects. EA exhibited its antidepressant role in two aspects, which are acupuncture-point penetration and electric current, and acted on both subtypes of NSCs.4. Activation of p-ERK involved in the stem cell by EA stimulationConsistent with the results of the promoting effect on stem cell proliferation, we further found a similar relation between EA and the activation of p-ERK1/2in stem cells. The number of p-ERK/Nestin double positive cells in DG was greater in the EA group than the Model group. These results show that the cascade of MAPK/ERK pathway may be involved in the stem cell activation process in the EA-treated CUS rats.5. EA Microdialysates up-regulated the sphere growth and p-ERK level of NSCsFor hippocampal microdialysates, microdialysis sampling was performed after five times of treatment. The optimal perfusion flow-rate was set at2.5μl/min and sampling time for each rat was2.5h (about300～350μl microdialysates obtained).To detect the microenvironmental effect of EA microdialysates, NSCs were first purified from the hippocampus of the postnatal SD rats. Nine days later, they were identified under the optical microscope and detected of Nestin under the inverted fluorescence microscope. A typical appearance of spheres formed after9days of purification.The purified NSCs were further cultured in different groups of medium (PBS group:70%NSC medium+30%PBS; Model group:70%NSC medium+30%microdialysates from CUS model rats; EA group:70%NSC medium+30%microdialysates from EA treated CUS rats). The microdialysates of all groups had been aseptically filtrated before application in cell culture. NSCs were well mixed slightly and mildly by pipette, inoculated into the96-orifice plate (5.23×103cells/well) with120μl medium. After3days100μl medium per orifice was replenished and then after2days70μl was supplied further. The cell viability and growth of NSC spheres were investigated48h after the second supplementation of the culture medium (7days of the different medium culture).The%cell viability of the EA group was similar to the Model group after7days of culture in the EA medium. Percentage cell viability was determined by CCK-8method. In addition, NSC spheres, which were greater than100μm in diameter, kept in EA medium were larger in number than those in Model medium. The number of big spheres (≥100μm) indicated the sphere-formation tendency and the level of maintaining the NSCs properties. The above results suggested that there would be some NSC-survival factors in the extracellular fluid of the DG zone in the EA stimulated animals.Next, we analyzed the p-ERK level of NSCs administrated by different mediums. The p-ERK positive stem cells were larger in number in the EA-microdialysates group. Therefore, the factors in the microenvironment of hippocampal DG zone in EA-administered rats could up-regulate ERK signaling pathway in NSCs. This is consistent with the results of BrdU/SOX-2double staining and may be in explanation of it, as well as EA’s role in the reversion of depression. The present study suggested:(1) EA has potent antidepressant-like effect on CUS induced depression model rats.(2) EA has proliferation promoting effects on NSCs in the hippocampal DG of CUS rats.(3) EA takes the antidepressant-like effect when it protects both QNPs and ANPs, and the effect of the electric current is more related to the QNP activity.(4) The effect of EA on hippocampal NSCs might involve the activation of ERK signaling pathways.(5) EA microdialysates from the hippocampus of EA treated rats promotes the NSCs survival and keeps their stem cell properties. The therapeutic action of EA on the depression model rats might be associated with the induced neurotrophic extracellular microenvironment. |
