?-syn In CSF Enters The Brain Parenchyma Via Glymphatic Influx

Purposes:To investigate the movement of ?-synuclein(?-syn)in CSF into the brain via the glymphatic influx and the potential influence on brain function.Methods:1.Fluorescence imaging on the glymphatic influx:Fluorescently conjugated cadaverine(A488-ca)was infused into the subarachnoid CSF of the anesthetized mice via cistern magna puncture.At 10 min and 30 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 ImageJ.After 10 min of injection,some brain sections were immunostained in order to observe tracer distribution and cellular uptake,Brain slices were visualized by a confocal laser microscope.2.The entry of ?-syn in CSF into the brain via the glymphatic influx:fluorescently conjugated A53T ?-syn(SYN-488)and no fluorescently conjugated A53T ?-syn protein(Hm-SYN)were separately infused into the subarachnoid CSF of the anesthetized mice via cistern magna puncture.At 30 min after the start of tracer infusion,mice brains were sliced and prepared for fluorescence imaging.Some brain sections were immunostained in order to observe tracer distribution and cellular uptake,Brain slices were visualized by a confocal laser microscope.Brain sections harvested from A53T transgenic mice with various ages were used for immunostaining,in order to observe whether ?-syn and its aggregates are present in the paravascular space and the brain tissue.3.The effects of ?-syn in CSF on glymphatic function:SYN was injected into cistern magna of C57BL/6 mice for 40 min,then CSF tracers(FITC-d3,OA-45)was intracisternally infused.At 30 min after infusion with FITC-d3,brains were sliced and prepared for fluorescence imaging.A53T transgenic mice aged 8-12 months were injected with fluorescent tracer(OA-45,FITC-d3)via cistern magna puncture.At 30 min and 90 min after the start of tracer infusion,brains were sliced 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 ImageJ.Tracer distribution at 30 min and 90 min respectively represented the glymphatic influx and glymphatic efflux respectively.In order to evaluate glymphatic system function.Results:1.Fluorescence imaging on the glymphatic influx:A488-ca moved into the brain parenchyma rapidly,and the influx was time and region dependent.A488-ca entered the mice brain more readily and spread more widely than another commonly used CSF tracer fluorescently conjugated ovalbumin(OA-45).Furthermore,A488-ca could enter the brain parenchyma either along the paravascular space or across the pial surface.More importantly,relative to OA-45 largely remained in the extracellular space,A488-ca exhibited obvious cellular uptake by astrocytes surrounding the blood vessels and neurons in the cerebral cortex.The glymphatic influx especially the transcellular pathway was faithfully traced by intracisternal infusion with fluorescently conjugated cadaverine.2.The entry of ?-syn in CSF into the brain via the glymphatic influx:After intracisternal injection with SYN-488 and Hm-SYN separately,?-syn in CSF was found to enter the brain parenchyma along the paravascular space,and then was absorbed by some neurons located in the cerebral cortex and aggregated in the substantia nigra pars compacta.A53T transgenic mice aged 2-4 months displayed almost no ?-syn aggregates formed in their brain.Some ?-syn aggregates could be observed in the paravascular space and some neurons in the brain of 8-12 months old mice,while a large number of aggregates was found in the brain of 16-18 months old mice.3.The effects of ?-syn in CSF on glymphatic function:At 30 min after tracers injection,the influx of FITC-d3 and OA-45 into the brain of in the SYN treated mice was significantly decreased compare with control(P<0.05).At 30 min after tracers injection,the influx of both OA-45 and FITC-d3 into the brain of A53T mice significantly decreased compare with WT mice(P<0.05).At 90 min after tracers injection,the residual volume of OA-45 and FITC-d3 significantly increased in the brain tissue of A53T mice compared with WT group(P<0.05).Conclusion:?-syn in the CSF could enter the brain parenchyma via the paravascular space,and then reduce the glymphatic function.