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

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 mechanisms 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.

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 immunohistochemistry, 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.

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 abundance 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 (cytochalasin 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.

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.