The Effect Of Chronic Stress On The AQP4 Mediated Glymphatic System In The Brain

Objective: To investigate the effect of chronic stress on glymphatic system mediated by aquaporin 4(AQP4)in the brain and its molecular mechanism.Methods: 1.Stress protocol and drug administration: Chronic mild unpredictable stress model(CUMS,6 weeks)was established.Stress response was evaluated by measuring the body weight,plasma corticosterone concentration,open field test(OFT),forced swimming test(FST)and tail suspension test(TST).Mice were randomly divided into four group: Control,CUMS,dexamethasone(DEX)and mifepristone(CUMS+MFP).Mice of DEX group were intraperitoneally injected with DEX 5 mg/kg per day,mice of CUMS+MFP group were orally administrated MFP 35 mg/kg per day besides stress exposure.2.Fluorescence imaging was used to determine the glymphatic function: Fluorescent tracer was infused into the subarachnoid CSF of the anesthetized mice via cistern magna puncture.At 30 or 90 min after the start of tracer infusion,the brain was sliced on a vibratome and prepared for observation.Tracer distribution within the brain was visualized by a fluorescence microscope.The coverage area of fluorescent tracers in the brain was calculated and analyzed by Image J.Tracer distribution at 30 min and 90 min respectively represented the glymphatic influx and glymphatic efflux.3.Evaluating the transcription and expression of AQP4 and GFAP in mice brain: Western blot,immunofluorescence and Q-PCR were used to determine the protein expression,transcription and localization of AQP4 in the cerebral cortex;Q-PCR was used to determine the transcriptional level of AQP4 related anchoring protein in cerebral cortex.Western blot was used to determine the expression of GFAP in cerebral cortex.Results: 1.The approach to determine the glymphatic function was established by using fluorescence imaging: After injection,two CSF tracers with different molecularweight rapidly moved into the brain parenchyma along the perivascular space or across the pia mater.FITC-d3 moved faster and wider than OA-45.At 30 min post-injection,the amount of tracer penetration reached the peak.At 90 min,tracers were mostly cleared from the brain and gathered in the cervical lymph nodes.Tracer distribution differed cross different brain regions and mainly accumulated in the lateral cortex,ventral cortex,hypothalamus and vertical limb of the diagonal band(VDB).2.The increase of body weight of CUMS,DEX and CUMS+MFP group was significantly slowed down compared with Control(p<0.01).There was no significant difference between the CUMS+MFP and CUMS(p>0.05);The concentration of plasma corticosterone in CUMS group was significantly higher than Control(p<0.05).However,there was no significant difference between DEX and CUMS+MFP group;Compared with Control,the movement distance and immobility time in the central grids of CUMS and DEX treated mice in OFT was significantly reduced(p<0.05).The immobility time of CUMS and DEX treated mice in FST and TST was significantly increased(p<0.001).Compared with the CUMS,the movement distance of CUMS+MFP group was significantly increased(p<0.001)and the immobility time was significantly decreased(p<0.001);Immunofluorescence results showed that the expression of A?1-40 in the cerebral cortex of CUMS treated mice was significantly increased(p<0.05),while MFP largely reduced the accumulation of A?1-40 caused by CUMS(p<0.05).3.At 30 min after tracers injection,the influx of both OA-45 and FITC-d3 into the brain of CUMS and DEX treated mice significantly decreased compare with Control(p<0.01).Moreover,the change was more remarkable in the Lateral and Ventral cortex(p<0.001).At 90 min,the amount of the residual tracers in the brain of CUMS and DEX treated mice was significantly increased both in the prefrontal(Bregma +2.5),anterior(Bregma +1.1)and posterior(Bregma-1.5)brain(p<0.05).Compared with the CUMS group,the amount of OA-45 and FITC-d3 influx into the brain significantly increased at30 min in CUMS+MFP,and more remarkable in the dorsal and lateral cortex(p<0.001).While,the amount of the residual tracers in the brain at 90 min significantly decreased in the prefrontal(Bregma +2.5),anterior(Bregma +1.1)and posterior(Bregma-1.5)brain(p<0.05).4.Compared with Control,the protein expression and transcriptional level of AQP4 in the cerebral cortex of CUMS and DEX treated mice were significantly decreased(p<0.001,p<0.05 respectively).The expression of GFAP was also significantly decreased(p<0.01).Compared with CUMS,the expression of AQP4 and GFAP in the cerebral cortex of CUMS+MFP treated mice was significantly increased(p<0.05).Moreover,AQP4 in the cortex of CUMS and DEX mice lost its polarity around the large and small vessels,and the change was more significant at the prefrontal(Bregma +2.5)and anterior(Bregma +1.1)brain(p<0.05).MFP treatment partly reversed the polarity distribution of AQP4(p<0.01).What's more,AQP4-anchoring molecules such as agrin and laminin were significantly decreased in the cerebral cortex of CUMS and DEX treated mice at transcriptional level(p<0.05),while MFP reversed their transcription reduction caused by CUMS(p<0.01).Conclusion: Chronic stress could damage the glymphatic function through reducing the expression and polarized localization of AQP4 in mice brain,in which the activation of glucocorticoid receptor is involved.