BACKGROUND: Hematoma clearance has been a proposed therapeutic strategy for hemorrhagic stroke. This study investigated the impact of CX3CR1 (CX3C chemokine receptor 1) activation mediated by r-FKN (recombinant fractalkine) on hematoma resolution, neuroinflammation, and the underlying mechanisms involving AMPK (AMP-activated protein kinase)/PPARγ (peroxisome proliferator-activated receptor gamma) pathway after experimental germinal matrix hemorrhage (GMH). METHODS: A total of 313 postnatal day 7 Sprague Dawley rat pups were used. GMH was induced using bacterial collagenase by a stereotactically guided infusion. r-FKN was administered intranasally at 1, 25, and 49 hours after GMH for short-term neurological evaluation. Long-term neurobehavioral tests (water maze, rotarod, and foot-fault test) were performed 24 to 28 days after GMH with the treatment of r-FKN once daily for 7 days. To elucidate the underlying mechanism, CX3CR1 CRISPR, or selective CX3CR1 inhibitor AZD8797, was administered intracerebroventricularly 24 hours preinduction of GMH. Selective inhibition of AMPK/PPARγ signaling in microglia via intracerebroventricularly delivery of liposome-encapsulated specific AMPK (Lipo-Dorsomorphin), PPARγ (Lipo-GW9662) inhibitor. Western blot, Immunofluorescence staining, Nissl staining, Hemoglobin assay, and ELISA assay were performed. RESULTS: The brain expression of FKN and CX3CR1 were elevated after GMH. FKN was expressed on both neurons and microglia, whereas CX3CR1 was mainly expressed on microglia after GMH. Intranasal administration of r-FKN improved the short- and long-term neurobehavioral deficits and promoted M2 microglia polarization, thereby attenuating neuroinflammation and enhancing hematoma clearance, which was accompanied by an increased ratio of p-AMPK (phosphorylation of AMPK)/AMPK, Nrf2 (nuclear factor erythroid 2–related factor 2), PPARγ, CD36 (cluster of differentiation 36), CD163 (hemoglobin scavenger receptor), CD206 (the mannose receptor), and IL (interleukin)-10 expression, and decreased CD68 (cluster of differentiation 68), IL-1β, and TNF (tumor necrosis factor) α expression. The administration of CX3CR1 CRISPR or CX3CR1 inhibitor (AZD8797) abolished the protective effect of FKN. Furthermore, selective inhibition of microglial AMPK/PPARγ signaling abrogated the anti-inflammation effects of r-FKN after GMH. CONCLUSIONS: CX3CR1 activation by r-FKN promoted hematoma resolution, attenuated neuroinflammation, and neurological deficits partially through the AMPK/PPARγ signaling pathway, which promoted M1/M2 microglial polarization. Activating CX3CR1 by r-FKN may provide a promising therapeutic approach for treating patients with GMH.

Subarachnoid hemorrhage (SAH) is a devastating stroke, in which acidosis is one of detrimental complications. The extracellular pH reduction can activate G protein-coupled receptor 4 (GPR4) in the brain. Yet, the extent to which proton-activated GPR4 contributes to the early brain injury (EBI) post-SAH remains largely unexplored. Ferroptosis, iron-dependent programmed cell death, has recently been shown to contribute to EBI. We aimed to investigate the effects of GPR4 inhibition on neurological deficits and neuronal ferroptosis after SAH in rats. A total 253 Sprague Dawley (SD) male rats (weighing 275-330g) were utilized in this study. SAH was induced by endovascular perforation. NE-52-QQ57 (NE), a selective antagonist of GPR4 was administered intraperitoneally 1-hour post-SAH. To explore the mechanisms, RhoA activator U-46619 and YAP activator PY-60 were delivered intracerebroventricularly. Short- and long-term neurobehavior, SAH grading, western blot assay, ELISA assay, immunofluorescence staining, and transmission electron microscopy was performed post-SAH. Following SAH, there was an upregulation of GPR4 expression in neurons. GPR4 inhibition by NE improved both short-term and long-term neurological outcomes post-SAH. NE also reduced neuronal ferroptosis, as evidenced by decreased lipid peroxidation products 4HNE and MDA levels in brain tissues, and reduced mitochondrial shrinkage, increased mitochondria crista and decreased membrane density. The application of either U-46619 or PY-60 partially offset the neuroprotective effects of NE on neuronal ferroptosis in SAH rats. This study demonstrated that acid-sensing receptor GPR4 contributed to neuronal ferroptosis after SAH via RhoA/YAP pathway, and NE may be a potential therapeutic strategy to attenuate GPR4 mediated neuronal ferroptosis and EBI after SAH.

Background and Purpose: Ferroptosis is a distinct form of cell death characterized by iron‐dependent lipid peroxidation and plays a crucial role in the early brain injury (EBI) following subarachnoid hemorrhage (SAH). As a newly discovered endogenous ligand for the C‐KIT receptor tyrosine kinase, meteorin‐like protein (Metrnl) exerts regulatory functions in oxidative stress and protects against various diseases. However, the specific role of the Metrnl/C‐KIT axis in neuronal ferroptosis during EBI following SAH remains to be elucidated. Methods: Sprague Dawley rats were used to establish the SAH model through endovascular perforation. r‐Metrnl was administered intranasally 1 h after SAH. Metrnl shRNA, C‐KIT inhibitor ISCK03, AMPK inhibitor dorsomorphin, and Nrf2 inhibitor ML385 were administered intracerebroventricularly or intraperitoneally before r‐Metrnl treatment to explore the underlying mechanisms. Neurobehavioral assessments, immunofluorescence, western blot, ELISA, Fluoro‐Jade C staining, transmission electron microscopy, and Nissl staining were conducted to evaluate the effects. Additionally, primary neuron culture with hemoglobin (Hb) stimulation was used for in vitro studies. Results: Phosphorylated C‐KIT and endogenous Metrnl levels were upregulated after SAH. Knockdown of Metrnl aggravated neurobehavioral deficits and neuronal ferroptosis, whereas r‐Metrnl treatment showed a protective effect. Mechanistically, r‐Metrnl significantly increased the protein levels of SLC7A11, GPX4, FTH, FSP1, and GSH, whereas it decreased the levels of ACSL4, 4HNE, and MDA in the ipsilateral hemisphere 24 h after SAH. Also, r‐Metrnl reduced mitochondrial shrinkage, increased mitochondrial crista, and decreased membrane density. However, the beneficial effects of r‐Metrnl were partially reversed by ISCK03, dorsomorphin, or ML385 treatment both in vivo and in vitro. Conclusions: Our study demonstrated that r‐Metrnl reduced neuronal ferroptosis and improved neurological outcomes after SAH by modulating the C‐KIT/AMPK/Nrf2 signaling pathway.