Gas6/Axl signaling promotes hematoma resolution and motivates protective microglial responses after intracerebral hemorrhage in mice

Abstract

Background: Intracerebral hemorrhage (ICH) stands out as the most fatal subtype of stroke, currently devoid of effective therapy. Recent research underscores the significance of Axl and its ligand growth arrest-specific 6 (Gas6) in normal brain function and a spectrum of neurological disorders, including ICH. This study is designed to delve into the role of Gas6/Axl signaling in facilitating hematoma clearance and neuroinflammation resolution following ICH.

Methods: Adult male C57BL/6 mice were randomly assigned to sham and ICH groups. ICH was induced by intrastriatal injection of autologous arterial blood. Recombinant mouse Gas6 (rmGas6) was administered intracerebroventricularly 30 min after ICH. Virus-induced knockdown of Axl or R428 (a selective inhibitor of Axl) treatment was administrated before ICH induction to investigate the protective mechanisms. Molecular changes were assessed using western blot, enzyme-linked immunosorbent assay and immunohistochemistry. Coronal brain slices, brain water content and neurobehavioral tests were employed to evaluate histological and neurofunctional outcomes, respectively. Primary glia cultures and erythrophagocytosis assays were applied for mechanistic studies.

Results: The expression of Axl increased at 12 h after ICH, peaking on day 3. Gas6 expression did not remarkably changed until day 3 post-ICH. Early administration of rmGas6 following ICH significantly reduced hematoma volume, mitigated brain edema, and restored neurological function. Both Axl-knockdown and Axl inhibitor treatment abolished the neuroprotection of exogenous Gas6 in ICH. In vitro studies demonstrated that microglia exhibited higher capacity for phagocytosing eryptotic erythrocytes compared to normal erythrocytes, a process reversed by blocking the externalized phosphatidylserine on eryptotic erythrocytes. The erythrophagocytosis by microglia was Axl-mediated and Gas6-dependent. Augmentation of Gas6/Axl signaling attenuated neuroinflammation and drove microglia towards pro-resolving phenotype.

Conclusions: This study demonstrated the beneficial effects of recombinant Gas6 on hematoma resolution, alleviation of neuroinflammation, and neurofunctional recovery in an animal model of ICH. These effects were primarily mediated by the phagocytotic role of Axl expressed on microglia.

Keywords: Axl; Gas6; Hematoma; Intracerebral hemorrhage; Microglia; Neuroinflammation.