| Objective:To study the effects of neonatal stress on the development of the glymphatic system and its later effects on α-syn aggregation in the brain and PD progression.Methods:1.Construction of stress model:C57BL/6 mice were subjected to neonatal maternal deprivation(NMD)to model the early life stress.Neoborn mice were isolated from their mother 3 hours per day,for 14 consecutive days(from the 2nd day to the 15th day of life).2.ELISA:After the completion of NMD,the plasma corticosterone of mice was detected by ELISA.3.Behavioral experiments:① At 16 days old,body weight of mice was measured,and tail suspension experiment,forced swimming experiment,and open field experiment were conducted.② PD-like symptoms in NMD and CON mice were measured at 8 weeks of age(after 4 weeks of AAV-A53T injection),including tail suspension test,forced swimming test,open field test,rod rotation test,and olfactory sensitivity test.4.Western blot was used to detect the changes of AQP4 and GFAP protein levels in the olfactory bulb,prefrontal cortex,hippocampus,striatum,and substantia nigra of 16 day old mice(just after NMD)α-syn,phosphorylated α-syn and tyrosine hydroxylase(TH)protein levels in the olfactory bulb and substantia nigra of mice at 8 weeks of age(after 4 weeks of AAV-A53T injection)were also detected by western blot.5.RT-qPCR was used to detect the changes in transcription levels of AQP4 and related anchor protein including laminin and agrin,as well as inflammatory factors including interleukin 10(IL-10)and tumor necrosis factor(TNF α)in 16 days old mice.6.Immunofluorescent staining was used to observe the distribution of AQP4 in the olfactory bulb,prefrontal cortex,hippocampus,striatum,and substantia nigra of 16 days old mice,as well as the changes in the number of astrocytes(GFAP staining positive)cells.Moreover,the expression of α-syn in the olfactory bulb,prefrontal cortex,hippocampus,striatum and substantia nigra of mice at 8 weeks of age(after 4 weeks of AAV-A53T virus injection),and the changes in the number of dopaminergic neurons(TH staining positive)cells were similarly detected.Results:1.Establishing a method to judge whether NMD mode is successful:Compared with the CON group,the NMD-treated mice exhibited significantly increased body weight at 16 days of age(P<0.01).Whereas,there was no significant change in cortisol levels.In the tail suspension test,the immobility time of NMD-treated mice significantly increased compared with the CON group.In the forced swimming test,there was no significant change in the immobility time of NMD mice in water compared with the CON group(P>0.05).In the open field experiment,the total movement distance of mice in the NMD group was significantly reduced compared with the CON group(P<0.001).However,there was no significant difference in the time spent in the central grid between the two groups(P>0.05).2.Effects of NMD exposure on the development of the glymphatic system:Compared with the CON group,the polarized distribution of AQP4 in the olfactory bulb,prefrontal cortex,striatum,and substantia nigra of NMD group mice was significantly reduced(P<0.05),but there was no significant difference in the polarity distribution in the hippocampus(P>0.05).Compared with CON mice,the transcription levels of AQP4,Laminin,Agrin and expression levels of AQP4 protein and GFAP protein in the prefrontal cortex,hippocampus,and striatum of 16 days old NMD mice remained unchanged(P>0.05);The expression level of AQP4 in substantia nigra of NMD-treated mice decreased significantly(P<0.05),the transcription levels of AQP4 remained unchanged(P>0.05),but the transcription levels of Laminin decreased significantly(P<0.05),while there was no significant change in GFAP expression levels between the two groups;The expression levels of AQP4 and GFAP protein in the olfactory bulb of NMD-treated mice exhibited no difference compared with CON group.However,the transcription level of AQP4 and the transcription levels of Laminin and Agrin in the olfactory bulb of NMD-treated mice were also significantly decreased(P<0.05)compared with CON group;Compared with the CON group,the transcription level of TNFα in the olfactory bulb,prefrontal cortex,and hippocampus of NMD-treated mice showed no difference(P>0.05),but the transcription levels of TNFα significantly increased in the striatum,and substantia nigra(P<0.05).Compared with CON group,the transcription level of IL-10 significantly increased in the olfactory bulb of NMD-treated mice(P<0.001),while there was no significant difference in the prefrontal cortex,hippocampus,striatum,and substantia nigra between the two groups(P>0.05).Compared with CON group,the number of astrocytes in the olfactory bulb,prefrontal cortex,hippocampus,striatum,and substantia nigra of NMD mice showed no significantly difference(P>0.05).3.Effect of NMD on α-syn aggregation and PD-like pathology later in life:At 60 min following the injection of fluorescently labeled α-syn into the brain,the content of α-syn in the substantia nigra of NMD-treated mice was significantly increased compared with the CON group(P<0.05).At 4 weeks after the injection of AAV-A53T the content of α-syn in the striatum of NMD group was increased and in the substantia nigra of NMD group was decreased(P<0.05),but the expression level of phosphorylated α-syn in the substantia nigra of NMD-treated mice was increased compared with the CON group(P<0.05);We also found that the expression level of TH in the substantia nigra of NMD group was decreased compared with the CON group(P<0.05),and there was no significant difference in the expression level of TH in the olfactory bulb and striatum between the two groups.However,immunostaining showed the number of TH positive neurons in the olfactory bulb(at the edge of the olfactory glomerulus)of NMD group significantly decreased compared with CON group(P<0.05).Behavioral tests showed that in the tail suspension test and forced swimming test,the immobility time of NMD-treated mice significantly increased compared with the CON group(P<0.05);In the open field experiment,there was no significant change in the total movement distance between the two groups(P>0.05),and in the time spent in the central grid,there was no significant change between the two groups(P>0.05).In the rotarod test experiment,there was no significant change in the latency to fall from the rod in the NMD group compared with the CON group(P>0.05).In the olfactory assessment experiment,the olfactory sensitivity of the NMD mice was significantly decreased compared with the CON group(P<0.001).Conclusion:Neonatal stress leads to abnormal distribution of AQP4 in the olfactory bulb and other brain regions,and disturbs the development of the glymphatic system.The damaged glymphatic system further leads to α-syn accumulation in the brain and aggravates PD pathology in later life. |
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