The Mechanism Of NBP Effectively Reduce Amyloid-β Deposition In AD Mice’s Brain Via The Glymphatic Clearance

Background:Alzheimer’s disease(AD)is a severe age-related neurodegenerative disease characterized by abnormal deposition of Aβ plaques,which affects millions of people world wide.So far no effective treatment was found for this most common form of dementia.Several Aβ clearance system exert Aβ efflux from the parenchyma.Aβclearance impairment reduced the waste elimination from the brain and contributed to the Aβ deposits in brain parenchyma.Glymphatic system is important in removing parenchymal Aβ and is disrupted in Alzheimer’s disease.Then the impaired glymphatic function could be a target for Aβ clearance in the AD.The drug L-3-N-butylphthalide(NBP)has been demonstrated effective in cerebral vascular diseases through multiple pharmacological effects.It has been reported NBP could reduce the Aβ plaques and improve the cognitive behavior in the AD rodent models.But the underlying mechanisms how NBP decreases Aβ deposit remains not fully understood.Objective:In the present study,we purposed to explore the influence of NBP on the glymphatic system and the mechanism of its protective effect on AD by scavenging Aβ.Methods:(1)Firstly,24 C57BL/6 mice were divided into NBP group and vehicle group.After the corresponding drug treatment,mice were injected with CSF tracer Alexa Fluor 647-conjugated ovalbumin(OVA-647,0.5 mg/ml in aCSF)in cerebellomedullary cistern or cerebral cortex,and brain tissue sections were taken to observe the clearance of fluorescent markers.(2)48 C57BL/6 mice were divided into four groups according to the feeding drugs and the intervention drugs of intraventricular injection,including NBP+aCSF group,vehicle+aCSF group,NBP+propranolol group and vehicle+propranolol group.After treatment with corresponding drugs,cerebral vessels were visualized after mice intravenously injected with FITC-2000kd(1%in saline)and FITC labeled Aβ-40 was injected into hippocampus.The cerebrovascular pulsatility were detected in vivo by two-photon microscope,and the brain tissue sections were taken to observe the clearance of fluorescent labeled Aβ-40.(3)28 APP/PS1 transgenic AD model mice and 28 wild-type(WT)mice(total of 56 mice)were divided into four groups:(APP/PS1+NBP)group,(APP/PS1+vehicle)group,(WT+NBP)group,(WT+vehicle)group.NBP or vehicle treated for 3 months.Morris water maze test was used to evaluate the cognitive behavior of mice.Immunofluorescence staining was used to quantitatively detect the amyloid burden,Aβplaques number and the AQP4 polarity in brain parenchyma.The levels of soluble/insoluble Aβ-40 and Aβ-42 in cortex and hippocampus were measured by enzyme-linked immunosorbent assay(ELISA).Results:(1)NBP increase CSF influx and clearance in glymphatic system:Thirty minutes after CSF tracer OVA-647 was injected into cisternal magma,OVA-647 was more rapidly infiltrated into the NBP treated brains,compared with vehicle group.After injecting ova-647 into the cerebral cortex,the remained dye reduced remarkably in the brain of NBP treated animals when compared to vehicle group,while fluorescent intensity was dramatically higher in the dCLNs of NBP treatment than that of vehicle treatment,indicating that the interstitial solute efflux from the parenchyma and drainage into dCLNs was markedly increased after NBP treatment.(2)NBP increase cerebral vascular pulsatility in brain:the cerebral pulsation of arteries was dramatically increased after NBP treatment.No significantly alteration in the cerebral pulsation of veins was found in the brain of NBP treated mice.(3)The beneficial effect of NBP on perivascular drainage was inhibited by propranolol:the vascular pulsatility of arteries was significantly reduced after propranolol treatment.NBP could significantly decrease the fluorescence intensity of Aβ40-FITC and promote the clearance of Aβ40.However,in the NBP groups,the reduced fluorescent intensity of Aβ40-FITC was reversed by propranolol treatment,indicating that the NBP effect was dependent on the cerebral pulsation.(4)Prolonged NBP treatment improves cognitive behavior in APP/PS1 mice:shorter escape latency and higher percentage of time exist in target quadrant were presented in NBP treated APP/PS1 mice when compared to vehicle group.(5)Prolonged NBP treatment increases the perivascular AQP4 expression and reduces Aβ deposition in the parenchyma of APP/PS1 mice:After 3 months of NBP treatment,the reduced AQP4 polarization was remarkably reversed in APP/PS1 mice.Quantification analysis indicated that the amyloid burden and Aβ plaques number were significantly reduced by NBP treatment.Moreover,NBP could dramatically decrease Aβ40 and Aβ42 levels in cortex and hippocampus from APP/PS1 mice.Conclusions:NBP can enhance the cerebral pulsation of arteries,thus improving the efficiency of the glymphatic system and accelerating the clearance of various metabolites in the brain.NBP can also increase the perivascular polarity of AQP4 in APP/PS1 mice,which promote glymphatic clearance and reduce the Aβ deposition in parenchyma of APP/PS1 mice,and eventually improve the impared cognitive and behavioral abilities in AD.