The Effects Of Running Exercise And Fluoxetine On The Capillaries Of Medial Prefrontal Cortex In Depression Model Mice And Related Mechanism

Part one Effects of Running Exercise and Fluoxetine on Depression-like Behavior in the CUS MiceObjective: Fluoxetine is a commonly used antidepressant drug in clinical practice.However,clinical studies and laboratory animal studies have found limitations in the antidepressant efficacy of fluoxetine.Running exercise can effectively improve the symptoms of depression,but it is unknown what is the difference in the effects of running exercise and the classic antidepressant fluoxetine on depression-like behavior.Therefore,we compared the effects of running exercise and fluoxetine on depressive behaviors in CUS model mice.Methods: 150 male C57 mice aged 4-5 weeks were selected.After adaptive feeding for 2 weeks,139 mice that met the experimental inclusion criteria were completely randomly divided into Control groups(n = 20)and CUS model group(n = 119).CUS intervention was performed on the mice in the CUS model group,and the sucrose preference test(SPT)was conducted every weekend.After 4 weeks,the mice in the CUS model group were divided into stress resilient group(n = 62)and stress-susceptible group(n = 42)according to the results of SPT.The pressure-sensitive group was randomly divided into a CUS model / standard control group(CUS/STD group,n = 14),a CUS model / running exercise group(CUS/RUN group,n = 14),and a CUS model / fluoxetine treatment group(CUS/FLX group,n = 14).Mice in the CUS/RUN group were given a forced running exercise intervention for 4 consecutive weeks,5 days a week(16: 00-18: 00 every day)for 20 minutes a day.On the first three days of the first week,the mice were running at a speed of 5 m/min,and then the next two days at a speed of 8 m/min.For the remaining 3 weeks,the speed was maintained at 10 m/min.At the same time as running exercise intervention,mice in the CUS/FLX group were given fluoxetine(10 mg/kg/d)intraperitoneally,and mice in the Control group,CUS/STD group and CUS/RUN group were given the same dose of sterile saline intraperitoneal injection.During the entire animal experiment,in addition to behavioral tests,the mice in the Control group were fed normally,5 mice per cage.The remaining three groups of mice were given CUS intervention,1 mouse per cage,for the whole course.SPT was performed on mice at the same time every weekend to evaluate the state of anhedonia,and the body mass of mice was recorded at the end of SPT.After 4 weeks of running exercise and fluoxetine,mice in each group were subjected to open field tests(evaluation of mobility and anxiety-like behavior in mice),forced swimming test and tail suspension test(evaluation desperate behavior in mice).Results: 1.Body weight: During the baseline adjustment phase of the sugar water test,there was no significant difference between the body weight of the Control group mice and the body weight of the CUS model group mice(p = 0.316).At the end of the 4th week of CUS intervention,the body weight of the CUS model group mice was significantly lower than that of the Control group mice(p < 0.001).From the beginning of running exercise and fluoxetine treatment to the end of that,the body weight of in CUS/STD group mice,the CUS/RUN group mice and the CUS/FLX group mice was significantly lower than that of the Control group mice(p < 0.001),but there was no significant difference in body weight among the CUS/STD group mice,the CUS/RUN group mice and the CUS/FLX group mice.2.Sucrose preference test: In this study,during the baseline adjustment phase of the sucrose preference test,there was no significant difference in the percentage of sucrose preference between the Control group mice and the CUS model group mice(p = 0.462).At the end of 4th week of CUS intervention,the percentage of sucrose preference of the CUS model group mice was significantly lower than that of the Control group mice(p < 0.001).By the end of the 3rd week after running exercise or fluoxetine treatment,the percentage of sucrose preference of the CUS/STD group mice was significantly lower than that of the Control group mice(p < 0.05),and the percentage of sucrose preference of the CUS/RUN group mice was significantly higher than that of the CUS/STD group mice(p < 0.05).There was no significant difference between the percentage of sucrose preference of the CUS/FLX group mice and that of the CUS/STD group mice(p = 0.212).By the end of the 12 th week,the percentage of sucrose preference of the CUS/STD group mice,the CUS/RUN group mice and the CUS/FLX group mice was significantly higher than that of the CUS/STD group(p < 0.05).3.Forced swimming test: In this study,by the end of 12 th week,the forced swimming immobility time of the Control group mice and the CUS/RUN mice was significantly lower than that of the CUS/STD group mice(p < 0.05),and there was no significant difference between the forced swimming immobility time of the CUS/FLX group mice and that of the CUS/STD group mice(p = 0.056).4.Tail suspension test: In this study,by the end of 4th week after running exercise or fluoxetine treatment,the tail suspension immobility time of the CUS/RUN group mice and the CUS/FLX group mice was significantly lower than that of the CUS/STD group mice(p < 0.05).5.Open field test: In this study,by the end of 4th week after running exercise or fluoxetine treatment,there was no significant difference in the open field scores among the Control group mice,the CUS/STD group mice,the CUS/RUN group mice and the CUS/FLX group mice(p = 0.459).Conclusions: 1.3 weeks of running exercise could reverse the state of anhedonia caused by CUS intervention,but fluoxetine treatment needed 4 weeks to reverse the state of anhedonia caused by CUS intervention.2.The 4 weeks of running exercise was able to reverse the desperation of the mice caused by CUS intervention,but the 4 weeks of fluoxetine treatment was not effective for the CUS-induced desperation of the mice.Part two Study on the Effects of Running Exercise and Fluoxetine on the Volume and Capillaries of Medial Prefrontal Cortex in CUS Model MiceObjective: The medial prefrontal cortex(m PFC)is one of the important brain regions involved in emotion generation and regulating stress behavior.However,it is unknown whether chronic unpredictable stress(CUS)affects the volume of m PFC in mice.It is also unknown whether running exercise and fluoxetine affect the volume of m PFC in CUS mice.There may be vascular lesions in the m PFC of depression,but there is no accurate quantitative evidence.Whether 4 weeks of running exercise and fluoxetine treatment can affect the capillaries in m PFC is currently unknown.Therefore,in this part,the effects of running exercise and fluoxetine on the volume of m PFC in CUS model mice and the effects of running exercise and fluoxetine treatment on the density,the total length,the total surface area,and the total volume of capillaries in m PFC of CUS model mice were quantitatively investigated to provide a certain experimental basis for searching for new interventions of depression.Methods: After the behavioral test was completed,5 mice were randomly selected from each group.After intraperitoneal anesthesia,4% paraformaldehyde was perfused through the heart to fix.One mouse hemisphere was randomly selected from each mouse,dehydrated by gradient concentration of sucrose solution,embedded in OCT,quick-frozen,and then 60 μm thick continuous sections were cut along the coronal plane.Every fourth section was sampled from the sections containing m PFC in a systematic-random manner,and 4 sets of consecutive equidistant sections containing m PFC structures were obtained in the end.One sets of equidistant sections were randomly selected and stained with toluidine blue staining patch.The m PFC boundary was delineated under the stereoscopic optical microscope according to the cytoarchitectionics,and the total volume of m PFC in each mouse was investigated according to the Cavalieri principle.Then,the contralateral hemisphere of the mouse brain hemisphere selected to investigate the total volume of m PFC was selected and embedded in a mouse brain mold filled with 6% agar and cut into 1 mm thick continuous coronal slices.Tissue blocks of approximately 1 mm3 were cut from m PFC and 5 blocks containing m PFC were sampled per mice,and after dehydration for four days,the samples were embedded in agar with the caudal surface facing down.The embedded samples were treated with the Isector technique to obtain isotropic and uniform random(IUR)sections.Then,one 4-μm-thick section was sliced from each block along the direction parallel to the IUR surface.Then,immunohistochemical staining of CD31(marker of vascular endothelial cells)was performed on the IUR sections,with the length density,the total length,the surface area density,the total surface area,the volume density,the total volume and the diameter of capillaries in m PFC of each group mice were investigated using the stereological methods.Results: 1.Stereological measurement results of total volume of m PFC: the total volume of m PFC of the CUS/STD group mice was significantly lower than that of the Control group mice(p < 0.05),while the total volume of m PFC of the CUS/RUN group mice was significantly bigger than that of the CUS/STD group mice(p < 0.001),but there was no significant difference between the m PFC volume of the CUS/FLX group mice and that of the CUS/STD group mice(p = 0.052).2.Stereological results of various capillary parameters in m PFC of mice: The length density and total length,the total surface area,the volume density and total volume of m PFC capillaries in the CUS/STD group were significantly less than those in the Control group(all p < 0.001).The length density and total length,the total surface area,the volume density and total volume of m PFC capillaries in the CUS/RUN mice were significantly greater than those in CUS/STD group mice(all p < 0.001),The length density and total length,the surface area density and total surface area,the volume density and total volume of m PFC capillaries in the CUS/FLX group mice were not significantly different from those in the CUS/STD group mice(p > 0.05).The length density and total length,the surface area density and total surface area,the volume density and total volume of m PFC capillaries in the CUS/RUN group mice were significantly greater than those in the CUS/FLX group mice(p < 0.001).The average diameter of m PFC capillaries in Control group mice and that in CUS/RUN group mice were significantly less than that in CUS/STD group mice(both p < 0.05).The average capillary diameter in m PFC of the CUS/FLX group mice was not significantly different from that of the CUS/STD group mice(p = 0.587).There was no significant difference between the average capillary diameter in m PFC of the CUS/RUN group and that of the CUS/FLX group(p = 0.065).3.Correlation analysis results: the correlation coefficient between the m PFC volume of mice and their preference for source preference was 0.554 at 3rd week(p = 0.014)and was 0.422 at 4th week after running and FLX treatment(p = 0.072).The correlation coefficient of the m PFC volume of mice and their tail suspension immobile time was-0.480(p < 0.05).The correlation coefficient between the m PFC volume of mice and their body mass was 0.299(p > 0.05).The correlation coefficients between the volume of m PFC in mice and the length density,the total length,the surface area density,the total surface area,the volume density,the total volume and average diameter of capillaries in m PFC were 0.693(p <0.001),0.694(p <0.001),0.524(p <0.05),0.587(p <0.05),0.557(p <0.05),0.551(p <0.05)and-0.598(p <0.05).Conclusions: 1.Stereological results showed that CUS intervention could cause m PFC volume reduction in mice.2.The 4 weeks of running exercise could effectively improve the m PFC volume atrophy in mice induced by CUS,but the 4 weeks of fluoxetine treatment had no significant effect on it.3.CUS could cause the decreases in length density and total length,the total surface area,the volume density and total volume of the m PFC capillaries in mice,and cause the increase in average capillary diameter.4.Four weeks of running exercise could effectively improve the parameters of m PFC capillaries in CUS mice but the four weeks of fluoxetine treatment has no effective effects on them.5.The 4-week running exercise was more effective than the 4-week fluoxetine treatment in the effects on the parameters of the m PFC capillaries.6.The volume of m PFC in mice was positively correlated with the state of anhedonia,and negatively correlated with desperate behavior.The volume of m PFC in mice was positively correlated with the length density,the total length,the surface area density,the total surface area,the volume density and the total volume of capillaries in m PFC.And the volume of m PFC in mice was negatively correlated with the average diameter of capillaries in mice m PFC.Part three Running Exercise/PPARδ Promotes the Proliferation and Migration of Cerebral Cortical Vascular Endothelial Cells of Mouse through Activating e NOSObjective: Four weeks of running exercise can effectively improve the parameters of m PFC capillaries in CUS mice,but what might be the mechanism is unknown.PPARδ agonist GW501516(GW1516)is an excellent running exercise simulation agent,which can effectively simulate the various effects of running exercise,including the function of protecting vascular endothelial cells,but whether PPARδ can affect the proliferation and migration of mouse cortical endothelial cell strain(Bend.3)and their molecular mechanisms are not yet known.Therefore,in this part,the level of PPARδ and endogenous nitric oxide synthase(e NOS)in mice m PFC were detected with ELISA.Then,the effect and mechanism of PPARδ on the proliferation and migration of Bend.3 cells cultured in vitro were studied in order to provide a direction for studying the molecular mechanism for the fact that running exercise protects the capillaries in the m PFC of CUS mice.Methods: Six mice from each group in the part one were randomly selected and killed.The m PFC tissue was quickly separated on ice.One side of the m PFC tissue was randomly selected to detect the protein expression levels of e NOS and PPARδ in the m PFC of the mice with ELISA kit.Mouse cerebral cortical vascular endothelial cell strains(Bend.3)were cultured in vitro,and they were treated with GW1516(PPARδ agonist),GSK0660(PPARδ antagonist),AVE3085(e NOS agonist),and L-NAME(e NOS antagonist).Bend.3 was devided into N group(Normal control group,i.e.10% FBS DMEM high glucose medium containing 1% DMSO),A Group(10 μM AVE3085 solution),L Group(100 μM L-NAME solution),GW group(10 n M GW1516 solution),GS group(1 μM GSK0660 solution),A+GS group(10 μM AVE3085 + 1 μM GSK0660 solution)and GW+L group(10 n M GW1516 + 100 μM L-NAME),and the growth status of endothelial cells in each group was observed.A CCK-8 cell proliferation test and an endothelial cell scratch test were conducted to calculate the survival rate and the cell migration rate of Bend.3 cells after 48 hours of treatment in each group.After 48 hours of drug treatment,the cells of each group were collected,and the nitric oxide(NO)content and e NOS expression level of Bend.3 cells were detected with the ELISA kit.Results: 1.ELISA test results of the mice m PFC: The expression levels of PPARδ and e NOS in m PFC of the Control group mice and the CUS/RUN group mice were significantly higher than those of the CUS/STD group(p < 0.05).The correlation coefficient between the expression level of e NOS in m PFC of each group and the expression level of PPARδ in m PFC of each group was 0.537(p < 0.05).2.CCK-8 cell proliferation test: Bend.3 cell survival rate of the A group and the GW group were significantly higher than that of the N group(p < 0.05),and Bend.3 cell survival rate of the L group and the GS group was significantly lower than that of the N group(p < 0.05).The Bend.3 cell survival rate of the GW+L group was significantly lower than that of the GW group(p < 0.05)and was not significantly different from that of the L group.The Bend.3 cell survival rate of the A+GS group was significantly higher than that of the GS group(p < 0.05)but that was not significantly different from that of A group.3.Cell scratch test: The migration rate of Bend.3 cells in the A group and the GW group were significantly higher than that of the B group(p < 0.05),and the migration rate of Bend.3 cells in the L group and the GS group were significantly lower than that of N group(p < 0.05).The Bend.3 cells migration rate of the GW+L group was significantly lower than that of the GW group(p < 0.05)and significantly higher than that of the L group(p < 0.05).While the Bend.3 cell migration rate of the A+GS group was significantly higher than that of the GS group(p < 0.05),but that was not significantly different from that of the A group.4.NO content and e NOS expression level: the expression of e NOS in Bend.3 of the A group and the GW group were significantly higher than that of N group(p < 0.05),and the expression of e NOS in Bend.3 of the L group and the GS group were significantly lower that of the N group(p < 0.05).The expression of e NOS in Bend.3 of the GW+L group was significantly lower than that of the GW group(p < 0.05),but it was not significantly different from that of L group.The expression of e NOS in Bend.3 of the A+GS group was significantly higher than that of the GS group(p < 0.05)but it was not significantly different from that of the A group.However,there were no significant differences of the NO content in Bend.3 cells among each group.Conclusions: 1.CUS could reduce the expression levels of PPARδ and e NOS in m PFC of mice.2.4 weeks of running exercise could effectively improve the decrease of PPARδ and e NOS expression levels in m PFC mice caused by CUS.3.The expression level of e NOS in mouse m PFC was positively correlated with the expression level of PPARδ.4.PPARδ agonist GW1516 and e NOS agonist AVE3085 could effectively promote the proliferation and migration of Bend.3 cells,while PPARδ antagonist GSK0660 and e NOS antagonist L-NAME could effectively inhibit the proliferation and migration of Bend.3 cells,which indicated that PPARδ and e NOS might play key roles in the proliferation and migration of mouse cortical cells.5.The e NOS antagonist L-NAME could effectively block the positive effect of the PPARδ agonist GW1516 on the proliferation and migration of Bend.3 cells,while the PPARδ blocker GSK0660 failed to significantly block the positive effect of the proliferation and migration of the Bend.3 cells by the e NOS agonist AVE3085,which indicated that the positive effect of PPARδ on the proliferation and migration of Bend.3 cells might be depended on e NOS.6.PPARδ agonist GW1516 and e NOS agonist AVE3085 could effectively promote the expression of Bend.3 e NOS,while e NOS blocker L-NAME and PPARδ blocker GSK0660 could effectively inhibit the expression of Bend.3 e NOS.7.The e NOS blocker L-NAME could effectively block the promotion effect of the PPARδ agonist GW1516 on the expression of Bend.3 e NOS,while the PPARδ blocker GSK0660 failed to block the promotion of the Bend.3 e NOS expression by the e NOS agonist AVE3085,which indicated that e NOS might be the downstream part of PPARδ in the pathway.