Department of Rehabilitation, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, China.
Department of Neurology, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, China.
Exp Neurol. 2024 Dec;382:114964. doi: 10.1016/j.expneurol.2024.114964. Epub 2024 Sep 15.
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.
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.
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.
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.
脑出血（ICH）是最致命的中风亚型，目前尚无有效的治疗方法。最近的研究强调了 Axl 及其配体生长停滞特异性 6（Gas6）在正常大脑功能和一系列神经疾病中的重要性，包括 ICH。本研究旨在深入研究 Gas6/Axl 信号在促进脑出血后血肿清除和神经炎症消退中的作用。
成年雄性 C57BL/6 小鼠随机分为假手术和 ICH 组。ICH 通过纹状体注射自体动脉血诱导。ICH 后 30 分钟，通过侧脑室给予重组小鼠 Gas6（rmGas6）。在 ICH 诱导前给予病毒诱导的 Axl 敲低或 R428（Axl 的选择性抑制剂）治疗，以研究保护机制。使用 Western blot、酶联免疫吸附试验和免疫组织化学评估分子变化。冠状脑切片、脑水含量和神经行为测试分别用于评估组织学和神经功能结果。原代神经胶质细胞培养和红细胞吞噬实验用于进行机制研究。
ICH 后 12 小时 Axl 表达增加，第 3 天达到峰值。Gas6 表达直到 ICH 后第 3 天才明显增加。ICH 后早期给予 rmGas6 可显著减少血肿体积，减轻脑水肿，并恢复神经功能。Axl 敲低和 Axl 抑制剂治疗均消除了外源性 Gas6 在 ICH 中的神经保护作用。体外研究表明，与正常红细胞相比，小胶质细胞吞噬凋亡红细胞的能力更强，而阻断凋亡红细胞上的外翻磷脂酰丝氨酸可逆转这一过程。小胶质细胞的红细胞吞噬作用是 Axl 介导的，依赖于 Gas6。Gas6/Axl 信号的增强减轻了神经炎症，并促使小胶质细胞向促修复表型转化。
本研究在 ICH 动物模型中证明了重组 Gas6 对血肿溶解、减轻神经炎症和神经功能恢复的有益作用。这些作用主要是通过小胶质细胞上表达的 Axl 的吞噬作用介导的。
