Intermittent Hypoxia Preconditioning Protects Against Ischemic Brain Injury in Mice via a PF4-Dependent Paracrine Mechanism

BACKGROUND: Intermittent hypoxia (IH) preconditioning enhances brain resilience, thereby protecting against subsequent ischemic injury, yet its precise mechanisms remain elusive. We tested the novel hypothesis that peripheral paracrine mechanisms mediate IH-induced neuroprotection. METHODS: , 5-minute intervals, 10 cycles/d) was applied to generate IH-derived plasma (IHP). To test the hypothesis, IHP or normoxic plasma (100 μL/injection) was intravenously administered every 3 days (6 doses total) before distal middle cerebral artery occlusion. For therapeutic evaluation, plasma was administered daily for 3 days after distal middle cerebral artery occlusion or 60-minute transient middle cerebral artery occlusion, followed by additional doses every 3 days for 6 doses in long-term transient middle cerebral artery occlusion studies. Infarct volume and neurological deficits were primary outcomes. Candidate circulating mediators were identified via proteomics and validated by antibody-mediated depletion and recombinant protein supplementation. Blood-brain barrier integrity was further examined. RESULTS: Systemic IHP administration protected against acute brain injury after distal middle cerebral artery occlusion, reducing infarct volume and improving sensorimotor performance. Poststroke administration of IHP conferred acute and sustained neuroprotection in transient middle cerebral artery occlusion, but not distal middle cerebral artery occlusion, improving sensorimotor and cognitive recovery and reducing brain atrophy up to 4 weeks after stroke. Proteomic profiling identified 120 IH-upregulated plasma proteins, notably PF4 (platelet factor 4), a cytokine with potent neuroprotective properties. PF4 immunodepletion abolished IHP-induced neuroprotection, whereas recombinant PF4 replicated these benefits across sex and age. Furthermore, PF4 treatment protected against blood-brain barrier disruption after transient middle cerebral artery occlusion, attenuating IgG extravasation and loss of endothelial expression of zonula occludens-1. CONCLUSIONS: These findings identify PF4 as a key paracrine mediator of IH-induced neuroprotection and support the therapeutic potential of both IHP and PF4-based interventions for ischemic stroke.