The Role Of Gut Microbiota In Depressive Behaviors Induced By Autoimmune Prostatitis And The Neural Mechanism Of Its Effects On Emotion And Memory

ObjectiveChronic prostatitis / chronic pelvic pain syndrome(CP/CPPS)is a common urogenital disorder.The typically clinical manifestations of CP/CPPS are longstanding pelvic pain with or without voiding disturbances.In addition to these symptoms,patients with CP/CPPS often suffer from neuropsychiatric symptoms such as depression,anxiety,and memory decline in clinic.Multiple clinical studies have also indicated a correlation between CP/CPPS and these mental disorders.However,the underlying mechanism behind them is still unclear.In recent years,increasing evidence has shown that abnormal gut microbiota composition and glial activation are closely related to the mood alteration,and the structural changes in synaptic plasticity play an important role in learning and memory.Therefore,we sought to establish a mouse model of experimental autoimmune prostatitis(EAP)and explore the role of gut microbiota in EAP-induced depression.Also,we investigated the neural mechanism of EAP on emotional disorders and learning-memory decline in mice.Methods Male non-obese diabetic mice were immunized by subcutaneous injection of prostate antigen and adjuvant twice.Behavioral tests consisting of open field test(OFT),sucrose preference test(SPT),elevated plus maze(EPM),forced swimming test(FST),and tail suspension test(TST)were used to assess emotional performances of mice.Mice were also tested for learning and memory abilities by Morris water maze(MWM)and novel object recognition test(NORT).Then,fecal samples were collected,and 16 S ribosomal RNA sequencing was performed to detect differences in gut microbiota composition between control and EAP group.Subsequently,fecal bacteria from the control and EAP mice were transplanted into antibiotics?induced pseudo?germ?free mice to investigate the effects on host behaviors and composition of gut bacteria.Also,the morphology and function of microglia and astrocytes were detected by immunofluorescence,Western blotting,and transmission electron microscopy.Proinflammatory mediators(TNF-?,IL-1?,etc.)along with SLC6A4(a serotonin transporter)and IDO(a rate-limiting enzyme of serotonin synthesis)were quantified with reverse transcription-polymerase chain reaction(RT? PCR),and serum serotonin concentrations were measured by enzyme-linked immunosorbent assay(ELISA).Proton magnetic resonance spectroscopy(1H-MRS)was performed to measure hippocampal glutamate levels.In addition,dendritic complexity and spine densities were measured by using the Golgi-Cox procedure.Transmission electron microscopy was used to observe the synaptic morphology.Activation of microglia and its association with synapses were also investigated by immunofluorescence staining.Results1.(1)Establishment of EAP model:(1)Body weight: There was no significant difference in body weights between the control and EAP group.(2)HE staining:There was no obvious prostatic inflammatory in the control group;by contrast,the prostate in the EAP group showed stromal lymphocytic infiltrations.(3)Pathological score for prostatitis: Compared with the control group,EAP mice displayed a higher mean inflammation score(P < 0.001).(4)Measurement of pelvic pain: EAP showed an increase in the percentage of frequency in response changes to pelvic stimulus.(2)Emotion-related behavioral tests:(1)OFT: Center distance(P < 0.05),center time(P< 0.05),and number of rearings(P < 0.05)in EAP group were significantly reduced compared with those in control group.(2)SPT: EAP mice indicated an obvious decrement in the percentage of sucrose preference(P < 0.05).(3)EPM: EAP mice showed a marked decrement in the open arm time(P < 0.05),percentages of open arm time(P < 0.05),and open arm entries(P < 0.01).(4)FST and TST: EAP group showed a notable elevation in immobility time(P < 0.05).(3)Comparisons of gut microbiota composition in control group and EAP group:(1)?-diversity: EAP mice significantly increased the Chao 1(P < 0.05),Shannon index(P < 0.01),Simpson index(P < 0.01)and PD whole tree index(P < 0.05).(2)?-diversity: NMDS and PCA analysis showed significant differences in ?-diversity between control and EAP mice.(3)The relative abundance of several gut microbiota at six levels(phylum,class,order,family,genus,and species)exhibited obvious differences between the two groups.(4)Effects of fecal transplantation on emotional behaviors in host mice:(1)Antibiotic-treated pseudo-germ-free mice exhibited decreased central time(P <0.05)and rearings(P < 0.01)in the OFT and prolonged immobility time in FST(P <0.01)and TST(P < 0.05).(2)Fecal transplant from control mice effectively improved those behavioral abnormalities in host mice.By contrast,transplantation of fecal microbiota from EAP mice aggravated those abnormal behavioral performances.(5)Effects of fecal transplantation on gut microbiota composition in host mice:(1)Fecal transplant from control and EAP mice induced distinct changes in the ?-diversity and ?-diversity of gut microbiota in host mice.(2)Twenty-four bacteria at six levels(phylum,class,order,etc.)were significantly altered among different groups.2.(1)Morphology and functions of hippocampal glial cells:(1)Immunofluorescence:Both the number of Iba1-labeled activated microglial cells(P < 0.05)and the percentages of GFAP-stained areas for astrocytes(P < 0.05)were significantly larger in the hippocampus of EAP group.(2)Western blot: The protein levels of both Iba1(P < 0.05)and GFAP(P < 0.01)increased significantly in the hippocampus of EAP mice.(3)Transmission electron microscopy: EAP group showed more microglial activation as evidenced by increased and enlarged lysosomes in the cytoplasm.Similarly,EAP mice also showed intense activation of astrocytes characterized by increased cytosol in the cytoplasm.(2)The m RNA levels of TNF-?(P < 0.01),IL-1?(P < 0.05),IL-6(P < 0.01),IL-8(P < 0.05)were significantly higher in EAP group than that in control group.(3)Changes in neurotransmitter systems:(1)Serotonin system: EAP increased the m RNA expression level of SLC6A4(P < 0.05)and IDO(P < 0.05),accompanied by a decrement in serum concentration of serotonin(P <0.05).Furthermore,the serum concentration of serotonin in EAP mice were positively correlated with center time in the OFT(r = +0.692;P = 0.027)and open arm time in the EPM(r = +0.657;P = 0.039)and negatively correlated with immobility time in the FST(r =-0.733;P = 0.016)and TST(r =-0.755;P = 0.012).(2)Glutamatergic system: In EAP group,1H-MRS showed that the glutamate + glutamine(Glx)/ total creatine(t Cr)ratio was significantly reduced in the hippocampus(P < 0.05).3.(1)Memory-related behavioral tests:(1)MWM: During the place navigation test,EAP mice showed significant decreases in escape latency from day 4 to day 5(P <0.05).In the spatial probe test,EAP mice presented significantly less time(P < 0.01)and platform crossings(P < 0.05)in the target quadrant.(2)NORT: The recognition index in EAP mice was significantly decreased(P < 0.01).(2)Sholl analysis of hippocampal dendritic complexity: EAP resulted in a significant change in dendritic branch intersections in hippocampal neurons,especially in the shell distance 60?90?m.Furthermore,the number of dendritic branches(P < 0.05)and total dendritic length(P < 0.01)in EAP mice were reduced more apparently than that of control animals.(3)Analysis of spine density and synapse-associated proteins: EAP mice showed significantly lower spine density counted in both apical(P < 0.05)and basal(P < 0.01)dendritic segments.Also,EAP significantly reduced expressions of synaptic-related proteins SYN(P < 0.01)and PSD95(P < 0.05).(4)Stereological analysis of synaptic ultrastructure: EAP decreased the thickness of postsynaptic density(P < 0.01)and synaptic curvature(P < 0.01)and increased the width of synaptic cleft(P < 0.001).(5)Evaluation of microglial activation:(1)EAP miceshowed a significant increase in the number of partially(P < 0.05)and fully(P < 0.05)amoeboid microglia in the hippocampus.(2)In EAP group,the expression of CD11 b protein was significantly increased(P < 0.05).(6)Relationship between microglia and synapse:(1)The percentage of neurons associated with microglia was significantly larger in the EAP group(P < 0.05).(2)Microglia increased in association with synapses in the EAP group(P < 0.001).Conclusions1.Abnormal gut microbiota composition contributes to EAP-induced depressive-like behaviors in mice.2.EAP-induced emotional alteration is linked to microglial activation and its involvement in the abnormality of serotonin and glutamate neurotransmitter system.3.EAP induces cognitive declines and structural neuroplastic changes in mice,accompanied by microglial activation and microglia-synapse contacts.