| PART ONE THE EFFECTS OF MARESIN1 ON THE BEHAVIORS OF CUS-INDUCED DEPRESSION MODEL MICEObjective: Maresin1,as a newly discovered lipid mediator,has been confirmed to have a powerful anti-inflammatory effect,reducing neuronal and synaptic plasticity injury caused by inflammation.However,whether this drug has an antidepressant effect is not clear.This study aimed to explore the therapeutic effect of Maresin1 on depressive behaviors in the CUS-induced depression mice and provide some theoretical basis for finding new antidepressant drugs.Methods: 4-6 weeks old male C57BL/6J mice were selected and sucrose preference test(SPT)was performed after 1 week of adaptive feeding.Mice without sucrose preference were excluded.The left mice were randomly divided into the Control group(n = 40)and the CUS group(n = 127).During the experiment,the mice in the Control mice were normally housed with 5 mice/cage and mice in the CUS group were given single-cage and solitary rearing.After five weeks of CUS intervention,fifty depressed mice were selected based on the results of behavior tests and randomly divided into the CUS/PBS group(n = 25),CUS/Ma R1 group(n= 25).Mice in the CUS/Ma R1 group received intraperitoneal Maresin15ug/kg/day,alternate daily administration for 28 days.Mice in the CUS/PBS group received equivalent doses of PBS at the same time.The body weight and the SPT were measured on every weekend.After the Maresin1 intervention,the tail suspension test(TST)and open field test(OFT)were performed to evaluatec the depression-like and anxiety-like behaviors of the mice and the antidepressant effects of the Maresin1 were measured using the behavior results.Results:1.The weight measurement results showed that during the period of CUS-induced depression,the weight growth of mice in the CUS group was significantly slower than that in the Control group(P < 0.001).During intraperitoneal administration of Maresin1,the body weight in the CUS/Ma R1 and CUS/PBS group were significantly less than that in the Control(P < 0.001),while there was no significance difference in the CU S/Ma R1 and CUS/PBS mice(P = 0.257).2.The results of SPT showed that the percentage of sucrose preference in the CUS depressive model group was significantly lower than that in the Control group(P < 0.01).After four weeks Maresin1 treatment,there were significant differences in the percentage of sucrose preference among the three groups(F = 3.003,P = 0.038).The percentage of sucrose preference in the CUS/PBS group were significantly lower than that in the control group(P=0.007)and the CUS/Ma R1(P =0.032).There was no significant difference between the CUS/Ma R1 group and control group(P=0.75).3.The results of TST showed that compared to the Control group,the immobility time of mice in the CUS group was significantly increased,and the corresponding struggle time in the TST was significantly reduced(P =0.028).The immobility time of TST in the CUS/Ma R1 group showed a tendency to decrease compared with CUS/PBS group,but without significant difference(P = 0.231).The mobility time in the TST in the CUS/PBS group was significantly longer than that in the Control group(P< 0.05).4.The results of OFT showed that that both the CUS/PBS group(P<0.001)and CUS/Ma R1(P=0.001)group had significantly reduced movement distance in the central area compared with the Control group.The total movement distance(P= 0.04)and peripheral movement distance(P= 0.024)in CUS/Ma R1 group were significantly higher than those in CUS/PBS group.But,the total movement distance in the two group was significantly lower than in the Control group(P <0.05).There was no statistical difference in the staying time in the central area between the CUS/Ma R1 and CUS/PBS group(P = 0.453).Conclusions:1.The CUS intervention caused slower weight growth,decreased sucrose preference,and increased immobility time in the TST.CUS inervention successfully induced depression-like behavior.2.Four weeks Maresin1 treatment partially improved the depression-like behavior,but had insufficient advantages in slowing weight growth and anxiety-like behavior.PART TWO DYNAMICALLY MONITORING THE ROLE OF GLIAL CELLS IN THE CUS-INDUCED DEPRESSION DEVELOPMENT PROCESS BASED ON[18F] DPA-714 PETObjective: Microglial activation is one of the important pathophysiological changes in depression.TSPO is widely considered as a marker of microglial activation.This study aimed to monitor neuroinflammation and the dynamic changes of the glia cells during CUS-induced depression through dynamic scanning of [18F]DPA-714 PET and evaluating of microglia,astrocytes,and the TSPO expressed on the double glia cells,in order to clarify the clinical transformation value of [18F]DPA-714 PET.Methods: Experimental animals were identical with Part 1.Six mice randomly selected from the CUS group were given [18F]DPA-714 PET scan at multiple time points(before CUS intervention,after 2 or 4 or 5weeks of CUS intervention).The PET reconstruction scheme was used for the image reconstruction.[18F]DPA-714 standardized uptake value(SUV)in the whole brain and brain area were calculated using PMOD image analysis software and mouse MRI module.The changes in the [18F]DPA-714 SUV were compared during the development of CUS-induced depression.Eight mice were randomly selected from the Control group and CUS model group for euthanasia after 2 or 5 weeks CUS intervention,respectively.Three mice were randomly selected for paraffin sections,double-labeled Immunofluorescence for TSPO/Iba-1,TSPO/GFAP,TUNEL-labeld microglia.The numbers of Iba-1+cells,GFAP+cells,TSPO+/Iba-1+cells,TSPO+/GFAP+cells and TUNEL-labeld microglia were caculated by the software,and microglial morphology was analyzed by Image J software.The expressions of IL-1β、IL-18、IL-4 and NLRP3 in the hippocampus were measured by fluorescence quantitative PCR in the five left mice from each group.Results:1.After 2 weeks of CUS intervention,[18F]DPA-714 SUVs in the whole brain and several brain regions were increased significantly(P<0.05),but [18F]DPA-714 SUV in the left amygdala decreased,contrary to the changes of the remaining brain regions.After 4 weeks of CUS intervention,[18F]DPA-714 SUV in the whole brain and each brain region decreased significantly,no significantly difference from baseline(P > 0.05).After 5 weeks of CUS intervention,[18F]DPA-714 SUV in the whole brain and multiple brain regions was further decreased significantly,lower than the baseline(P <0.05).At the end of 5 weeks CUS intervention,the top brain regions with the largest changes in [18F]DPA-714 SUV were the brainstem,midbrain gray matter,amygdala,superior and inferior colliculus,cerebellum,and hippocampus.2.The results of TSPO/IBA-1 immunofluorescence staining in the hippocampus during CUS induced depression: The numbers of Iba-1+microglial cells in the hippocampus(P Control,CUS-2W = 0.008,P Control,CUS-5W< 0.001,P CUS-2W,CUS-5W < 0.001)and CA1 subregion(P Control,CUS-2W <0.001,P Control,CUS-5W < 0.001,P CUS-2W,CUS-5W < 0.001)were found to be significantly lower in mice subjected to 2 and 5 weeks of CUS than in control mice.This decrease appeared to be time-dependent.The number of microglial cells in the CA2/CA3 subregion was also significantly decreased after 5 weeks of CUS(P Control,CUS-5W = 0.032,P CUS-2W,CUS-5W = 0.006).However,only the number of microglial cells in the DG was significantly different between the two CUS subgroups.The numbers of TSPO+ cells in the whole hippocampus(P Control,CUS-2W < 0.001,P Control,CUS-5W < 0.001),CA1 subregion(P Control,CUS-2W =0.002,P Control,CUS-5W =0.002)and DG subregion(P Control,CUS-2W < 0.001,P Control,CUS-5W < 0.001)were significantly decreased after 2 and 5 weeks of CUS.The numbers of Iba-1+/TSPO+ cells in the hippocampus(P Control,CUS-2W < 0.001,P Control,CUS-5W< 0.001,P CUS-2W,CUS-5W < 0.001)and the CA1 subregion were significantly lower in mice subjected to 2 and 5 weeks of CUS than in Control mice.This decrease also appeared to be time-dependent.Further,there was a significant decrease in the number of TSPO+/Iba-1+ cells in CA2/CA3(P = 0.013)and DG(P = 0.003)subregions after 5 weeks of CUS intervention.3.Microglial morphology was altered in the hippocampus following CUS,with smaller microglial soma(P Control,CUS-2W = 0.025,P Control,CUS-5W= 0.001),shorter process length(P Control,CUS-2W = 0.039,P Control,CUS-5W =0.011)and fewer process branches.The numbers of TUNEL-stained microglia markedly increased after 2 weeks(not after 5 weeks)CUS intervention(P = 0.001).4.Changes of hippocampal neuroinflammation in mice during CUS induced depression: After CUS intervention,RNA expressions of IL-1β(P Control,CUS-2W= 0.028,P Control,CUS-5W = 0.013)and IL-18(P Control,CUS-2W =0.002,P Control,CUS-5W < 0.001)were decreased.RNA expressions of the anti-inflammatory cytokine IL-4 was increased significantly after 2 weeks of CUS intervention,but decreased with the prolonged of stress and presented a downward trend(P Control,CUS-2W < 0.001,P Control,CUS-5W =0.185).The expression of NOD-like receptor thermal protein domain associated protein 3(NLRP3)was also significantly decreased in the hippocampal region after 5 weeks of CUS intervention,which is closely related to the secretion of IL-1β and IL-18.5.We observed that the number of GFAP+ cells was significantly increased in the hippocampus(P = 0.042)and in the CA2/CA3 subregion(P = 0.001)after 2 weeks of CUS.However,there was no significant difference between the control group and stressed group after 5 weeks of CUS(P = 0.072).But,the number of TSPO+/GFAP+ cells in the hippocampus was significantly increased after 5 weeks of CUS(P = 0.042),compared to the Control group.Conclusion:1.CUS caused [18F]DPA-714 uptake to first increase at 2 weeks and then decrease after 5 weeks of CUS.Therefore,the time-dependent changes in SUVs showed an “inverted V”-shaped graph.[18F]DPA-714 SUV in the hippocampal region was one of the significantly changed brain regions.2.CUS intervention resulted in a progressive decrease in microglia in the hippocampus as well as microglia expressing TSPO,with the most prominent changes in the hippocampal CA1 region.The CUS intervention caused the morphological changes in microglia,showing a reduction of microglial soma,shorter processes,and fewer branches.Increased microglial apoptosis caused by CUS intervetion partly resulted in microglial loss.3.The CUS intervention resulted in a significant decrease in RNA expression levels of IL-1β,IL-18,and NLRP3 in the hippocampus,and an increase in RNA expression level of IL-4 in the hippocampus only after 2weeks of CUS intervention.The CUS intervention may cause low levels of inflammation in the hippocampus.4.Early CUS intervention caused a significant increase in astrocytes in the hippocampus,as well as astrocytes expressing TSPO,with the most prominent hippocampal CA2/CA3 subregion changes.5.Microglial loss and early astrocytes activation led to dynamic changes in [18F]DPA-714 SUV and cytokine in the hippocampus during CUS-induced depression.PART THREE STUDY ON THE MECHANISM MECHANISM OF MARESIN1 FOR IMPROVING DEPRESSION-LIKE BEHAVIOR IN CUS DEPRESSION MODEL MICE BASED ON THE [18F] DPA-714 PETObjectives: [18F]DPA-714 PET can be sensitive and effectively evaluate the status of glial cells and neuroinflammation in the brain during CUS-induced depression.This study aims to explore the mechanism of Maresin1 for improving depression-like behavior induced by CUS intervention combined by [18F]DPA-714 PET/CT,immunofluorescence,and molecular biology to provide a theoretical basis for finding new targets for the treatment of depression.Methods: Experimental animals were identical with Part 1.Five mice randomly selected from the Maresin1 group were given [18F]DPA-714 PET scan at multiple time points(before Maresin1 treatment,after 2 or 4 weeks of Maresin1 treatment).The changes in the [18F]DPA-714 SUV were compared during the Maresin1 treatment.Eight mice were randomly selected from Control group,CUS/Ma R1 group,and CUS/PBS group for euthanasia after 2 or 4 weeks Maresin1 treatment,respectively.Three mice were randomly selected for paraffin sections,double-labeled Immunofluorescence for TSPO/Iba-1 and TSPO/GFAP.The numbers of Iba-1+microglia,GFAP+astrocytes,TSPO+/Iba-1+ cells,TSPO+/GFAP+cells were caculated by the software.The expressions of TSPO,IL-1β,IL-18,IL-4 and NLRP3 in hippocampus were analyzed by western blot and RT-PCR in the left 5 mice from each group.Results:1.Compared with that before treatment,[18F]DPA-714 SUV in the whole brains and several brain regions were increased significantly after 2and 4 weeks Maresin1 treatment,and the increase was greater 2 weeks after treatment.At the end of 4 weeks Maresin1 treatment,the top brain regions with the largest changes in [18F]DPA-714 SUV were brainstem,superior colliculus,midbrain,central gray matter,and hippocampus.2.TSPO/Iba-1 immunofluorescence staining results after Maresin1treatment: compared to the Control group,the number of Iba-1 + cells in the CUS/PBS-2W(P < 0.001)and CUS/Ma R1-2W(P = 0.007)was significantly reduced,but the number of Iba-1+ cells in the CUS/Ma R1-2W group than CUS/PBS group(P = 0.003).Furthermore,we observed the numbers of TSPO+ cells(P CUS/PBS-2W,Control < 0.001,PCUS/Ma R1-2W,Control =0.006)and TSPO+/Iba-1+ cells(P CUS/PBS-2W,Control < 0.001,PCUS/Ma R1-2W,Control= 0.001)in the CUS depression model mice were significantly lower than that in the Control group.However,the number of TSPO+ cells(P = 0.0341)and TSPO+/Iba-1+ cells(P = 0.0341)were significantly higher than those in the CUS/PBS-2W group.After four weeks Maresin1 treatment,the number of Iba-1+ cells in the hippocampus in the CUS/Ma R1-4W group was significantly higher than that in the Control group(P < 0.001)and CUS/PBS-4W group(P < 0.001),and there was still a decrease trend in the number of Iba-1+ cells in the CUS/PBS-4W group compared with the Control group.We also observed the numbers of TSPO+ cells(P < 0.001)and TSPO+/Iba-1+ cells(P < 0.001)in the CUS/Ma R1-4W group were significantly higher than that in the Control group and CUS/PBS-4W group.But,the numbers of TSPO+ cells(P < 0.001)and TSPO+/Iba-1+ cells(P =0.01)in the CUS/PBS-4W group were significantly lower than that in the Control group.3.Compared with the Control group,the number of GFAP+ astrocytes in the CUS/PBS-2W group and CUS/Ma R1-2W group was increased,but only a significant difference between the CUS/PBS-2W group and Control group was found(P = 0.029).After 4 weeks Maresin1 intervention,the number of GFAP+ astrocytes in CUS/Ma R1-4W group was lower than in the Control group(P = 0.029)and CUS/PBS-4W group(P = 0.051),while GFAP+ astrocytes was still higher in CUS/PBS-4W group(P = 0.001).The numbers of and TSPO+/GFAP+ cells in CUS/Ma R1-2W had no significant difference compared with the Control group and CUS/PBS-2W group.However,the number of TSPO+/GFAP+ cells in the CUS/PBS-2W group was higher than in the Control group(P = 0.008).After 4 weeks Maresin1 treatment,the number of TSPO+/GFAP+ cells in the CUS/PBS-4W group was significantly lower compared with the Control group(P = 0.003)and CUS/Ma R1-4W group(P = 0.045).4.Changes of hippocampal neuroinflammation in mice after Maresin1 treatment:after 2-Week Maresin1 treatment,the RNA expression of IL-1β in the CUS/Ma R1-2W group(P = 0.032)and the CUS/PBS-2W group(P = 0.001)was still significantly lower than that in the Control group.However,compared to CUS/PBS-2W group,it was significantly higher in the CUS/Ma R1-2W group(P = 0.043).After 4-Week Maresin1 treatment,the RNA expression of IL-1β in the CUS/Ma R1-4W group(P =0.009)and the CUS/PBS-4W group(P = 0.015)was still significantly lower than that in the Control group,but there was no significant difference in the protein expression of IL-1β.Meanwhile,we found the change trend of IL-18 RNA expression after Maresin1 treatment was consistent with the change of IL-1β.In addition,the gene expression of IL-4 in the CUS/Ma R1-4W group was significantly higher than that in the CUS/PBS-4W group(P = 0.011),but having an increasing trend compared with the Control group(P = 0.085).And,the protein expression of IL-4 in CUS/Ma R1-4W group was significantly higher than that in the CUS/PBS-4W group(P < 0.001)and Control group(P = 0.013).Furthermore,the gene expression of TSPO in the CUS/Ma R1-2W and CUS/PBS-2W groups was significantly lower than that in the Control group(P < 0.001),but after 4-Week Maresin1 treatment,the gene expression of TSPO in the CUS/Ma R1-4W group was higher than that in the Control group(P = 0.017),and the protein expression of TSPO in the CUS/Ma R1-4W group was also significantly higher than that in the CUS/PBS-4W group(P < 0.001)and the Control group(P = 0.054).The secretion of IL-1β and IL-18 is closely related to NLRP3.After 2 weeks Maresin1 treatment,The gene expression of NLRP3 in CUS/Ma R1-2W group and Control group was significantly lower than that in CUS/PBS-2W group(P < 0.001).However,after 4 weeks Maresin1 treatment,there was no significant difference in the RNA expression of NLRP3 among CUS/Ma R1-4W group、CUS/PBS-4W group and Control group.Conclusions:1.Maresin1 treatment reversed the CUS intervention resulting in a reduction of [18F]DPA-714 SUV in multiple brain regions.The top brain regions with the largest change in [18F]DPA-714 SUV were: brainstem,midbrain gray matter,amygdala,superior and lower colliculus,cerebellum,and hippocampus.2.Maresin1 treatment increased the numbers of microglia,TSPO+cells,and TSPO-expressing microglia in the hippocampus with a "time-dependent" manner,which reversed the loss of microglia induced by CUS.After 4 weeks Maresin1 treatment,the number of microglia,TSPO+cells,and microglia expressing TSPO in the hippocampus were increased compared with the Control group.3.The number of astrocytes decreased in the hippocampal decreased after Maresin1 treatment4.Maresin1 promoted IL-4 as well as TSPO elevated,and treatment partially reversed the low inflammation level caused by CUS.5.Maresin1 may have a role in restoring the balance of glial cells and neuroinflammation. |
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