Neonatal Stress Impairs The Glymphatic Function By Down-regulating The Expression Of PDGF-B

Objective: To investigate the effect of neonatal stress on the glymphatic system in brain and the underlying molecular mechanism.Methods: 1.Stress model establishment: neonatal maternal deprivation(NMD)model was used,from the second day after birth,C57BL/6 mice underwent neonatal maternal deprivation(NMD,3 hours per-day)for 14 consecutive days.2.Model evaluation: Body weight,plasma corticosterone concentration,visceral hyperalgesia(colorectal distension test),tail suspension test(TST),forced swimming test(FST)and open field test(OFT)were measured in mice aged 6 weeks.3.Evaluation of the glymphatic function: a.Fluorescence imaging approach: the fluorescent tracer was injected into the cisterna magna,at 30 min and 90 min after the start of tracer infusion,the brain was sliced,then tracer distribution within the brain was photographed by a fluorescence microscope;b.Microplate assay: at 30,90 min following intracisternal tracer infusion,the left hippocampus,cortex and right hemisphere of the brain were dissected.Using a microplate reader analyze the supernatant after centrifugation of the homogenate;c.Magnetic resonance imaging(MRI): at 20,40,60 min following intracisternal contrast agent Gd-DPTA infusion,the anesthetized mice were scanned by MRI.4.Western blot and RT-q PCR were used to detect the expression and transcription levels of AQP4,PDGF-B and PDGFRb in the hippocampus and fore cortex of mice.RTq PCR was used to determine the transcription changes of AQP4-related anchor proteins Agrin,Laminin,α-syntrophin and Dystrophin in the hippocampus and fore cortex of the mice aged 6weeks.5.Immunofluorescence was used to determine the distribution of AQP4 in the perivascular space in the hippocampus and fore cortex of the mice aged 6weeks.The expression of PDGFRb and the changes of pericytes were detected by immunofluorescence.Results: 1.There was no significant difference(P > 0.05)for the body weight between NMD group and Control(CON)group.The plasma corticosterone concentration in the NMD group was significantly higher than that in the CON group(P < 0.05).The behavioral experiments showed the immobility time of NMD group mice was significantly increased in TST and FST(P < 0.05),the movement distance and immobility time in the central area of NMD group mice in the open-field experiment was significantly reduced(P < 0.05).The NMD group mice showed visceral hyperalgesia compared with CON group(P < 0.05),at the age from 6 to 13 weeks old.2.Fluorescence imaging approach showed that,at 30 min after tracers injection,the influx of FITC-d3 into the brain of NMD group mice was significantly decreased in the brain slices(P < 0.05)when compared with CON group.While at 90 min,the amount of the residual tracers in the brain of NMD group mice was significantly increased(P < 0.05).The results of Microplate assay showed that: at 30 min after tracers injection,the fluorescence tracer contents in the left-brain hippocampus,cortex and the right hemisphere were significantly reduced in NMD group(P < 0.05).However,at 90 min,the residual amounts of fluorescence tracer in brain were significantly increased(P < 0.05).Magnetic resonance imaging(MRI)showed that at 60 min following intracisternal injection,the penetration of contrast agent in the brain from NMD mice was significantly decreased(P < 0.05)when compared with CON group.3.Compared with the CON group,the transcription and expression levels of AQP4 in the NMD group has no significant change(P > 0.05),but the polarized distribution of AQP4 in the hippocampus and fore cortex was compromised.The transcription level of Agrin and Dystrophin in the hippocampus and fore cortex of NMD group was significantly reduced(P < 0.05).4.At the age of 6 weeks,the transcription and expression level of PDGF-B in hippocampus and fore cortex of NMD group mice was significantly decreased(P < 0.05)than CON,and the expression level of PDGFRb in pericytes was significantly decreased(P < 0.05).5.At the age of 16 days(right after stress),AQP4 expression in hippocampus and fore cortex remained unchanged(P > 0.05),the transcription level of Agrin and Dystrophin in hippocampus was significantly decreased(P < 0.05),the transcription level of Agrin,Dystrophin and Laminin in fore cortex was significantly decreased(P < 0.05).The transcription and expression levels of PDGF-B in hippocampus and fore cortex were significantly decreased(P < 0.05),and the expression levels of PDGFRb were significantly deceased(P < 0.05).Conclusion: Neonatal stress could damage the glymphatic system through reducing the polarized localization of AQP4 in the hippocampus and fore cortex of mice,which was related to decreased expression of PDGF-B and dysfunction of pericytes.