EphA4/EphrinB2 signaling mediates pericyte-induced transient glia limitans formation as a secondary protective barrier after subarachnoid hemorrhage in mice

Background: Most patients with subarachnoid hemorrhage (SAH) do not exhibit brain parenchymal injury upon imaging but present significant blood-brain barrier (BBB) disruption and secondary neurological deficits. The aim of this study was to investigate whether stressed astrocytes act as a secondary barrier to exert a protective effect after SAH and to investigate the mechanism of glial limitan formation.Methods: A total of 204 adult male C57BL/6 mice and an endovascular perforation SAH model were employed. The spatiotemporal characteristics of glial limitan formation after SAH were determined by immunofluorescence staining and transmission electron microscopy. The molecular mechanisms by which pericytes regulate glia limitans formation were analyzed using polymerase chain reaction, Western blotting, immunofluorescence staining and ELISA in a pericyte-astrocyte contact coculture system. The findings were validated ex vivo and in vivo using lentiviruses and inhibitors. Finally, pericytes were targeted to regulate glial limitan formation, and the effect of the glia limitans on secondary brain injury after SAH was evaluated by flow cytometry and analysis of neurological function.Results: Stress-induced glial limitan formation occurred 1 day after SAH and markedly subsided 3 days after ictus. Pericytes regulated astrocyte glia limitan formation via EphA4/EphrinB2 signaling, inhibited inflammatory cell infiltration and altered neurological function.Conclusions: Astrocyte-derived glia limitans serve as a secondary protective barrier following BBB disruption after SAH in mice, and pericytes can regulate glial limitan formation and alter neurological function via EphA4/ EphrinB2 signaling. Strategies for maintaining this secondary protective barrier may be novel treatment ap-proaches for alleviating early brain injury after SAH.