| Part one The effects of running exercise and Fluxotine treatment on behaviors of the CUS-induced depression model miceObjective : Over the past 40 years,selective serotonin reuptake inhibitor(SSRI)systems have established meaningful links between the clinical phenomena of mood disorders and the pharmacological treatments of mood disorders currently employed to treat them,but 15% to 30% of cases are unresponsive to multiple interventions or even become more worse.Running exercise has been proven to be able to prevent or relieve depression,however,the therapeutic effects of exercise on depression are still controversial.Our previous study found that running exercise could reverse the depressive-like behavior in CUS rats.However,it is unknown whether exercise reverses depression-like behavior more quickly than fluoxetine treatment does.Methods:A total of 143 male C57BL/6J mice were used in the present study.The mice(10–12 weeks old at the beginning of the CUS exposure)were housed in groups of 5 per cage under a 12-h light/dark cycle(lights on at 07:00 a.m.)at constant temperature(22 ± 1°C)and humidity with free access to food and water.The animals were allowed 1 week to habituate to the housing conditions before any experiments were initiated.All animals were age-and weight-matched(the mice weighed 10-15 g)at the beginning of the experiments.After 4 weeks of CUS stimulation,the CUS mice(n = 42)in the model group were ultimately used as the CUS mice.These mice were randomly divided into a CUS standard group(n = 14)and a CUS running group(n = 14)and a CUS fluoxetine group(n = 14).The control group(n = 14)mice did not receive any treatments but were handled every day.The mice in the CUS running group were placed on a treadmill to run regularly for four weeks(10 min per day,5 days per week).During the first two weeks,the running speed of 5 m/min was gradually increased to 10 m/min.For the rest of the experiment,the running speed was maintained at 10 m/min,and exercise protocols were performed as previously described.The mice in the CUS fluoxetine group were intraperitoneally injected with fluoxetine once daily from day 28 to day 56 of the period of CUS(a total of 28 days).The effects of stress on the hedonic state of the mice were assessed weekly using the SPT and body weight measurements.At the end of 8 weeks,the effects of stress on the hedonic and anxiety state of the mice were assessed by FST,TST and OFT.This study was approved by the Animal Care and Research Committee of Chongqing Medical University,P.R.China.Results:1.After 4 weeks of CUS interventions,the body weight of the CUS group was significantly lower than that of the control group(p < 0.01).After 4 weeks of running exercise and fluoxetine treatment,the body weight of the CUS + RN group and the CUS + FLX group was significantly lower than that of the control group(p < 0.01,p < 0.01).2.After 4 weeks of CUS interventions,the sucrose preference of the CUS group was significantly lower than that of the control group(p < 0.01).After 3 weeks of running exercise and fluoxetine treatment,the sucrose preference of mice in the CUS group was still significantly lower than that of the control group(p < 0.01).Meanwhile,the sucrose preference of mice in the CUS + RN group was significantly higher than that of the CUS group(p < 0.01).However,there was no significant difference in the sucrose preference between the CUS group and the CUS + FLX group(p > 0.05).Until the 4 weeks of running exercise and fluoxetine treatment,the sucrose preference of the mice in the CUS + RN group and the CUS + FLX group were both significantly higher than that of the CUS group(p < 0.01,p < 0.01).3.At the 4 weeks of running exercise and fluoxetine treatment,the immobility time of FST(Forced Swimming Test)in the CUS group was longer than that in the control group(p < 0.01).Meanwhile,the immobility time of FST in the CUS + RN group was significantly shorter than that of the CUS group(p < 0.05).However,there was no significant difference in the immobility time of FST between the CUS group and the CUS + FLX group(p > 0.05).4.At the 4 weeks of running exercise and fluoxetine treatment,the immobility time of TST(Tail Suspension Test)in the CUS group was longer than that in the control group(p < 0.01).Meanwhile,the immobility time of TST in the CUS + RN group and CUS + FLX group were both significantly shorter than that of the CUS group(p < 0.05,p < 0.01).5.After 4 weeks of running exercise or fluoxetine treatment,there was no significant difference in the total score of the OFT(Open Field Test)among the control group,the CUS group,the CUS + RN and the CUS + FLX group.Conclusions:1.3 weeks of running exercise could reverse depressive-like behavior in CUS mice,whereas 3 weeks of fluoxetine treatment failed to reverse depressive-like behavior in CUS mice.Although 4 weeks of running exercise and fluoxetine treatment could increase the sucrose preference and decrese the immbolity time of TST of the CUS mice,only running exercise could decrease the immbolity time of FST in CUS mice,which suggested that running exercise is faster and prior to fluoxetine in the improvement of the depressive-like behavior of the CUS-induced depression model mice.2.8 weeks of CUS did not induce anxiety-like behavior in mice.Part two The effects of running exercise and fluoxetine treatment on the differentiation and maturation of oligodendrocytes and myelination in hippocampus of CUS depression model miceObjective:Running exercise has been shown to prior to fluoxetine in the improvement of the improve depressive-like behaviors of the depressed mice,but the cellular for the different effects is unclear.In our previous study,we found that exercise could reverse the decrease in the number of CNPase+ oligodendrocytes in the hippocampus of the CUS-induced depressed rats.However,fluoxetine had no effects on the hippocampus and its component,myelinated fibers,in the CUS rat model of depression.The current study mainly investigated whether this rapid effect of exercise occured through promoting oligodendrocyte differentiation and/or myelination in the hippocampus of CUS depression model mice,which might provide the scientific evidence for the antidepressant effect of running exercise.Methods:After being done the behavior tests,five mice from each group were randomly selected and perfused transcardially with 4% paraformaldehyde.The brains were removed and split into two hemispheres by a midsagittal section.The right or left hemisphere from each mouse was sampled at random,and the sampled hemisphere was gradiently dehydrated with sucrose solution and coronally sectioned into 50 μm thick sections on a cryostat microtome.From the sections containing hippocampus,every fifth section was sampled in a systematic-random manner,and five groups of sampled sections were acquired.Two groups of sampled sections were randomly selected for immunohistochemical staining of PDGFRa+(maker of oligodendrocyte precursor cells)and CC1+(maker of mature oligodendrocytes),and the two kinds of cells(CC1+ and PDGFRa+)in the hippocampus were counted with the stereological method.The immunofluorescence staining was performed for double-labeled Olig2/ PDGFRa+(maker of proliferation oligodendrocytes),triple-labeled CC1+/Brd U+/Olig2+(maker of new matured oligodendrocytes)and MBP(maker of myelin forming cells and myelin sheath),and the number of Olig2+/PDGFRa+,Brd U+/Olig2+ and CC1+/Brd U+/Olig2+ cells per unit area in the CA1,CA3 and DG regions of each group mice were photographed and counted using confocal microscopy.The intensity of MBP was analyzed with the NIS-element AR analysis system.Then,5 mice were randomly selected from the first part,the animals were anesthetized with 1% pentobarbital sodium(0.4 ml/100 g)and perfusion fixed with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate-buffered saline.The hemispheres were embedded in 6% agar and coronally sectioned into 1-mm-thick slabs,starting randomly at the rostral pole.One block was sampled from every slab and thus five tissue blocks per hemisphere were obtained.The g-ratio of myelinated nerve fibers in the CA1 of the hippocampus was measured with transmission electron microscopy.Results: 1.The stereological results of CC1+ cells in the CA1,CA3 and DG subfields among the four groups: In the CA1 subfield,a significant decrease of the CC1+ cells was found in the CA1 subfield of the CUS mice compared with control group(p < 0.05),and the number of the CC1+ cells in the CUS + RN group was significantly increased compared with the CUS group in the CA1 subfield(p < 0.05).No significant difference in the maker of CC1+ cellswas detected between the CUS + FLX group and the CUS standard group in the CA1 subfield(p > 0.05).In the CA3 subfield,the number of the CC1+ cells was not significantly different between the CUS group mice and the control group mice(p > 0.05).No significant difference in the number of the CC1+ cells was detected among the CUS group,CUS + RN group and CUS + FLX group in the CA3 subfield(p > 0.05,p > 0.05).In the DG subfield,the number of the CC1+ cells was not significantly different between the CUS group mice and the control group mice(p > 0.05).No significant difference in the number of the CC1+ cells was detected among the CUS group,CUS + RN group and CUS + FLX group in the DG subfield(p > 0.05,p > 0.05).2.The stereological results of the PDGFRα+ cells in the CA1,CA3 and DG subfield among the four groups: In the CA1 subfield,the number of the PDGFRα+ cells was significantly increased in the CUS standard mice compared with the control group(p < 0.05).The number of the PDGFRα+ cells in the CUS + RN group and CUS + FLX group were significantly decreased compared with the CUS standard group in the CA1 subfield(p < 0.05).In the CA3 subfield,the number of the PDGFRα+ cells was significantly increased in the CUS standard mice compared with the control group mice in the CA3 subfield(p < 0.05),but the number of PDGFRα+ OPCs in the CUS + RN group and CUS + FLX group was not significantly different from that of the CUS standard group mice in the CA3 subfield(p > 0.05,p > 0.05).In the DG subfield,the number of the PDGFRα+ cells was significantly increased in the CUS group mice compared with the control group mice(p < 0.05).The number of the PDGFRα+ OPCs in the CUS + RN group and CUS + FLX group were significantly decreased compared with the CUS group mice in the DG subfield(p < 0.05,p < 0.05).3.The immunofluorescence results of the Brd U+/Olig2+,CC1+/Olig2+/Brd U+ cells in the CA1,CA3 and DG subfields among the four groups: In the CA1 sufield,the density of the Brd U+/Olig2+ cells of the CUS group was significantly decreased compared with that of the control group(p < 0.05),and the density of the Brd U+/Olig2+ cells of the CUS + RN group was significantly increased compared with that of the CUS group(p < 0.05),whereas no significant difference was found between the CUS group and the CUS + FLX group in the CA1 subfield(p > 0.05).The density of the CC1+/Olig2+/Brd U+ cells in the CUS group was greatly reduced compared to the control group in the CA1 subfield(p < 0.05).The density of the CC1+/Olig2+/Brd U+ cells of CUS + RN group was significantly increased compared with that of the CUS group in the CA1 subfield,whereas no significant difference was found between the CUS group and the CUS + FLX group in the CA1 subfield(p > 0.05).In the CA3 subfield,the density of the Olig2+/Brd U+ positive cells in the CUS group was significantly reduced compared to the control group(p < 0.05).The density of the Olig2+/Brd U+ cells of the CUS + RN group was significantly increased compared with that of the CUS standard group(p < 0.05),whereas no significant difference of the density of the Olig2+/Brd U+ cells was found between the CUS group and the CUS + FLX group(p > 0.05).Meanwhile,the density of the CC1+/Olig2+/Brd U+ cells in CUS standard group was significantly reduced compared to the control group in the CA3 subfield(p < 0.05).The density of the CC1+/Olig2+/Brd U+positive cells of CUS + RN group was significantly increased compared with that of the CUS group(p < 0.05),whereas no significant difference was found between the CUS group and CUS + FLX group in the CA3 subfield(p > 0.05).In the DG subfield,the density of the Brd U+/Olig2+ cells of the CUS group was significantly decreased compared with that of the control group(p < 0.05),and the density of the Brd U+/Olig2+ cells of the CUS + RN group was significantly increased compared with that of the CUS group in the DG subfield(p < 0.05),whereas no significant difference was found between the CUS group and the CUS + FLX group in the DG subfield(p > 0.05).The density of the CC1+/Olig2+/Brd U+ cells of the CUS group was significantly decreased compared with that of the control group in the DG subfield(p < 0.05).The density of the CC1+/Olig2+/Brd U+ cells of CUS + RN group was increased compared with that of the CUS group in the DG subfield(p < 0.05),whereas no significant difference was found between the CUS group and the CUS + FLX group in the DG subfield(p > 0.05).4.The immunofluorescence results of the PDGFRα+/Olig2+ cells in the CA1,CA3 and DG subfields among the four groups: In the CA1 subfield,the density of the PDGFRα+/Olig2+ cells of the CUS group was significantly increased compared with that of the control group(p < 0.05)and the density of the PDGFRα+/Olig2+ cells of the CUS + RN group and the CUS+FLX group was significantly decreased compared with that of the CUS group in CA1 subfield(p < 0.05).In the CA3 subfield,the density of the PDGFRα+/Olig2+ cells of the CUS group was not significantly different from that of the control group in CA3 subfield(p > 0.05),and the density of the PDGFRα+/Olig2+ cells of the CUS + RN group and the CUS+FLX group also was not significantly different from that of the CUS standard group in CA3 subfield(p < 0.05,p < 0.05).In the DG subfield,the density of the PDGFRα+/Olig2+ cells of CUS group was significantly increased compared with that of the control group(p < 0.05).The density of the PDGFRα+/Olig2+ cells of the CUS + RN group and the CUS+FLX group was significantly decreased compared with that of the CUS group(p < 0.05,p < 0.05).The density of the PDGFRα+/Olig2+ cells of the CUS group was signicantly increased compared with that of the control group in hilus of the DG subfield(p < 0.05).The density of the PDGFRα+/Olig2+ cells of the CUS + RN group and the CUS+FLX group was significantly decreased compared with that of the CUS group in the hius DG subfield(p < 0.05,p < 0.05),whereas the density of the PDGFRα+/Olig2+ cells in the molecular layer of the DG subfield was not significantly different among the control group,the CUS group,the CUS + RN group and the CUS+FLX group(p > 0.05).5.The immunofluorescence results of MBP in the CA1,CA3 and DG subfields among the four groups: In the CA1 subfield,a significant difference in the percentage of MBP positive myelinated fibers was found between the control group and the CUS group mice(p < 0.05),while the percentage of MBP+ myelinated fibers of the CUS + RN group was significantly increased compared with CUS group(p < 0.05),but no significant difference between CUS group and CUS + FLX group in the CA1 subfield was found(p > 0.05).In the CA3 subfield,no significant difference in the percentage of MBP+ myelinated fibers was found among the control group,the CUS group,the CUS+ RN group,the CUS+ FLX group(p > 0.05).In the DG subfield,no significant difference in the percentage of MBP+ myelinated fibers was found among the control,the CUS group,the CUS+ RN group,the CUS+ FLX group(p > 0.05).6.Ultrastructural analyses revealed a restoration of myelin thickness in the CA1 subfield among the four groups: Quantification of myelin thickness relative to axonal diameter(g-ratio)revealed that CUS resulted in thinner myelin in the CUS group compared with the control group(p < 0.05).This was reversed when the CUS mice were treated with running exercise(p < 0.05).No significant difference in g – ratio was detected between the CUS group and the CUS + FLX group(p > 0.05).Conclusions:1.The differentiation and maturation of Oligodendrocytes Precursor Cells(OPCs)in the hippocampus CA1 and DG of the CUS depression model mice were impaired,and the myelin formation ability was also decreased.2.4 weeks of running exercise might alleviate the depressivelike behaviors in the CUS-induced depressed mice through promoting the maturation of the oligodendrocytes and myelin formation in CA1 and DG subfields of the hippocampus.However,4 weeks of fluoxetine treatment did not promote the differentiation and maturation of OPCs and myelin formation in the hippocampus of depression model mice.3.The positive effects of running exercise on the oligodendrocytes in the hippocampus might be one of the important cellular mechanisms for the fact that running exercise is faster and prior to fluoxetine in the improvement of the depressive-like behavior of the CUS depressed mice.Part three Running exercise promotes differentiation and maturation of oligodendrocytes through PGC-1α pathway in the hippocampus to reverse the depressive-like behaviorObjective:The above study found that running exercise could enhance the oligodendrocyte differentiation and myelination of the hippocampus,which might contribute to reverse the depression-like behaviors in the CUSinduced depression mice.Peroxisome proliferator activated receptor coactivator 1α(PGC-1α)is an important coactivator which is closely related to running exercise and regulates the energy metabolism of the cells.PGC-1α plays an important role in mitochondrial biosynthesis,skeletal muscle fiber type conversion,glucose metabolism and lipid metabolism.Previous studies found that PGC-1α could promote the proliferation and differentiation of OPCs.This part mainly investigated that whether running exercise could promote differentiation and maturation of oligodendrocytes through PGC-1α pathway in the hippocampus of CUS depression model mice.Method: 3~4 weeks C57 BL male mice were group-housed 5 mice / cage.The animals were allowed 1 week to habituate to the housing conditions before any experiments were initiated.These mice were randomly divided into AAV-GFP group(n = 16),AAV-PGC-1α(n = 16),AAV-GFP + RN(n = 16),AAV-PGC-1α + RN(n = 17).Four groups of mice were injected with AAV-PGC-1α to null the gene of the PGC-1α in the hippocampus with stereotactic injection technique.Mice were anesthetized with 4% isoflurane and placed in a stereotaxic frame.Using a 5-ml Hamilton syringe,the AAVs were injected using the following coordinates from bregma:-2.3 mm anteroposterior,±1.8 mm lateral/medial,-2 mm dorsoventral from the skull.The experimental virus was generated to target the PGC-1α gene through the usage of a doxycycline-inducible Sh RNA.The final viral titer was 1.6*1012 vg/ml.To avoid fighting and injury after surgery,the mice were single housed in this study.4 weeks after the sugery,the mice in the four groups were tested with TST,FST and OFT.Then,the AAV-GFP + RN group and AAV-PGC-1α + RN group received treadmill running for four weeks,five days a week.The four groups of mice were intraperitoneally injected with Brd U once daily from day 28 to day 40(a total of 12 days).At the end of 8th week,the mice in the four groups were tested with TST,FST and OFT.After all the behavioral tests,the brain tissue was treated in the same ways as in part 1 and part 2,and the relevant stainings of oligodendrocytes were mentioned above.Results: 1.The frozen sections of the mice were observed under a fluorescence microscope,and the results showed that the GFP+ green fluorescence was only expressed in the hippocampus,suggesting that the injection site of the virus was located inside the hippocampus.2.After 4 weeks of surgery,the sucrose preference of the AAV-PGC-1α group was significantly lower than that of the AAV-GFP group(p < 0.05).Meanwhile,the immobility time of FST and TST of the AAV-PGC-1α group was significantly lower than that of the AAV-GFP group(p < 0.05).3.After 4 weeks of running exercise treatment,the sucrose preference of the AAVPGC-1α group was significantly lower than that of the AAV-GFP group(p < 0.05).Meanwhile,the sucrose preference of the AAV-PGC-1α + RN group was significantly lower than that of the AAV-GFP + RN group(p < 0.05).However,no significant difference was found between the AAV-PGC-1α + RN group and AAV-PGC-1α group(p > 0.05).4.After 4 weeks of running exercise treatment,the immobility time of FST of the AAV-PGC-1α group was significantly longer than that of the AAV-GFP group(p < 0.05).Meanwhile,the immobility time of FST of the AAV-PGC-1α + RN was significantly longer than that of the AAV-GFP + RN group(p < 0.05).However,no significant difference of the immobility time of FST was found between the AAV-PGC-1α + RN group and the AAV-PGC-1α group(p > 0.05).5.After 4 weeks of running exercise treatment,the immobility time of TST of the AAV-PGC-1α group was significantly longer than that of the AAV-GFP group(p < 0.05).However,the immobility time of TST of the AAV-PGC-1α + RN was not significantly different from that of the AAVGFP + RN group(p > 0.05).No significantly difference of the immobility time of TST was found between the AAV-PGC-1α + RN group and the AAVPGC-1α group(p > 0.05).7.The stereological results of CC1+ cells in the CA1 subfield among the four groups: a significant decrease in the number of the CC1+ cells of the CA1 subfield in the AAV-PGC-1α group was identified compared with the AAV-GFP group(p < 0.05),and the number of the CC1+ cells in the AAV-PGC-1α + RN group was significantly increased compared with the AAV-GFP + RN group in the CA1 subfield(p < 0.05),whereas no significant difference was detected between the AAV-PGC-1α + RN group and the AAV-PGC-1α group in the CA1 subfield(p > 0.05).8.The stereological results of the CC1+ cells in the CA3 subfield among the four groups: no significant difference of CC1+ cell was detected among the AAVGFP group,the AAV-GFP + RN group,the AAV-PGC-1α group and the AAV-PGC-1α + RN group in the CA3 subfield(p > 0.05).9.The stereological results of the CC1+ cells in the DG subfield among the four groups: a significant decrease in the CC1+ cells of the CA1 subfield in the AAV-PGC-1α group was identified compared with the AAV-GFP group(p < 0.05),however,the number of the CC1+ cells in the AAV-PGC-1α + RN group was not significantly different from that of the AAV-GFP + RN group in the CA1 subfield(p > 0.05),and no significant difference was detected between the AAV-PGC-1α + RN group and the AAV-PGC-1α group in the CA1 subfield(p > 0.05).10.The immunofluorescence results of the Brd U+/Olig2+ and CC1+/Olig2+/Brd U+ cells in the CA1 subfield among the four groups: the density of the Brd U+/Olig2+ and CC1+/Olig2+/Brd U+ cells of the AAV-PGC-1α group was significantly decreased compared with that of the AAV-GFP group(p < 0.05,p < 0.05),and the density of the Brd U+/Olig2+ and CC1+/Olig2+/Brd U+ cells of the AAV-PGC-1α + RN group was significantly decreased compared with that of the AAV-GFP + RN group(p < 0.05,p < 0.05),whereas no significant difference of the density of the Brd U+/Olig2+ and CC1+/Olig2+/Brd U+ was found between the AAVPGC-1α group and the AAV-PGC-1α + RN group in the CA1 subfield(p > 0.05,p > 0.05).11.The immunofluorescence results of the Brd U+/Olig2+ and CC1+/Olig2+/Brd U+ cells in the CA3 subfield among the four groups: no significant difference of the density of the Brd U+/Olig2+ and CC1+/Olig2+/Brd U+ cells was detected among the AAV-GFP group,the AAV-GFP + RN group,the AAV-PGC-1α group and the AAV-PGC-1α + RN group in the CA3 subfield(p > 0.05,p > 0.05).12.The immunofluorescence results of the Brd U+/Olig2+ and CC1+/Olig2+/ Brd U+ cells in the DG subfield among the four groups: no significant difference of the density of the Brd U+/Olig2+ cells was detected among the AAV-GFP group,the AAV-GFP + RN group,the AAV-PGC-1α group and AAV-PGC-1α + RN group in the CA3 subfield(p > 0.05).The density of the CC1+/Olig2+/Brd U+ cells of the AAV-PGC-1α group was significantly decreased compared with that of the AAV-GFP group(p < 0.05),and the density of the CC1+/Olig2+/Brd U+ cells of the AAV-PGC-1α + RN group was significantly decreased compared with that of the AAV-GFP + RN group(p < 0.05),whereas no significant difference of the density of the CC1+/Olig2+/Brd U+ was found between the AAV-PGC-1α group and the AAV-PGC-1α + RN group in the CA1 subfield(p > 0.05).Conclusions: 1.After 4 weeks of surgery,the mice in the AAV-PGC-1α group appeared depressive-like behaviors.2.Running exercise failed to reverse the depressive-like behaviors of the mice in the AAV-PGC-1α group 3.Running exercise failed to promote the differentiation and myelination in the CA1 subfield of the hippocampus in the AAV-PGC-1α mice.4.Running exercise might reverse the depressive-like behavior by promoting the differentiation and maturation of OPC through PGC-1α pathway in the CA1 subfield of the hippocampus. |
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