CXCL14-CXCR4-mediated platelet migration across the blood-brain barrier and subsequent microglia and astrocyte activation in arterial baroreflex dysfunctionOA

Background:Arterial baroreflex(ABR)dysfunction is linked to various central nervous system disorders and is associated with platelet accumulation in the brain,contributing to neuroinflammation.However,the mecha-nisms for platelets crossing the blood-brain barrier(BBB)and the role of platelets in ABR dysfunction remain poorly understood.We hypothesize that CXCL14-CXCR4 signaling facilitates platelet migration across the BBB and promotes glial activation in the context of ABR dysfunction.Methods:ABR dysfunction was induced in Sprague Dawley rats via sinoaortic denervation(SAD).Four weeks post-surgery,12 rats were randomly divided into two groups(n=6 per group;Sham and SAD).An additional 18 were divided into three groups(n=6 per group;Sham,SAD,and SAD+clopidogrel).Platelet infiltration in the brain was assessed by immunohistochemistry.Levels of CXCL14 in the brain were evaluated using immuno-histochemistry,western blotting,and enzyme-linked immunosorbent assays.CXCR4 levels on platelets was detected by flow cytometry.We constructed an in vitro BBB model to study platelet migration across the BBB.Platelets were co-cultured with BV2 cells and C6 cells,and the levels of inflammatory factors were measured using enzyme-linked immunosorbent assays.Polarization was assessed by immunofluorescence.Results:Immunofluorescence revealed significant platelet infiltration in ABR dysfunction model rat brains.In vitro BBB models and in vivo experiments confirmed increased platelet migration in ABR-dysfunctional rats.Further analysis showed elevated levels of the chemokine CXCL14 in brain tissue and increased CXCR4 abun-dance on platelets.In vitro assays demonstrated that platelet migration across the model BBB was driven by CXCL14 and was significantly reduced by inhibitors targeting CXCL14(neutralizing antibody),CXCR4(AMD3100),G-protein signaling(pertussis toxin),PI3K signaling(LY294002),and actin polymerization(cyto-chalasin B).Moreover,CXCL14 stimulation enhanced the phosphorylation of neural Wiskott-Aldrich syndrome protein(N-WASP),a key regulator of cytoskeletal dynamics.Migrating platelets also promoted the polarization of microglia and astrocytes toward pro-inflammatory M1 and A1 phenotypes,respectively.Treatment with clopidogrel,a platelet inhibitor,suppressed platelet migration and glial activation.Conclusion:Our findings indicate that the CXCL14-CXCR4 axis mediates platelet migration across the BBB in states of ABR dysfunction via G-protein and PI3K-dependent pathways,ultimately triggering glial activation.Our study provides new insights into the mechanisms of platelet migration and neuroinflammation associated with ABR dysfunction.