Extracellular Vesicles Obtained from Hypoxic Mesenchymal Stromal Cells Induce Neurological Recovery, Anti-inflammation, and Brain Remodeling After Distal Middle Cerebral Artery Occlusion in Rats.

Abstract

Small extracellular vesicles (sEVs) obtained from mesenchymal stromal cells (MSCs) have shown considerable promise as restorative stroke treatment. In a head-to-head comparison in mice exposed to transient proximal middle cerebral artery occlusion (MCAO), sEVs obtained from MSCs cultured under hypoxic conditions particularly potently enhanced long-term brain tissue survival, microvascular integrity, and angiogenesis. These observations suggest that hypoxic preconditioning might represent the strategy of choice for harvesting MSC-sEVs for clinical stroke trials. To test the efficacy of hypoxic MSCs in a second stroke model in an additional species, we now exposed 6-8-month-old Sprague-Dawley rats to permanent distal MCAO and intravenously administered vehicle, platelet sEVs, or sEVs obtained from hypoxic MSCs (1% O₂; 2 × 10⁶ or 2 × 10⁷ cell equivalents/kg) at 24 h, 3, 7, and 14 days post-MCAO. Over 28 days, motor-coordination recovery was evaluated by rotating pole and cylinder tests. Ischemic injury, brain inflammatory responses, and peri-infarct angiogenesis were assessed by infarct volumetry and immunohistochemistry. sEVs obtained from hypoxic MSCs did not influence infarct volume in this permanent MCAO model, but promoted motor-coordination recovery over 28 days at both sEV doses. Ischemic injury was associated with brain ED1⁺ macrophage infiltrates and Iba1⁺ microglia accumulation in the peri-infarct cortex of vehicle-treated rats. Hypoxic MSC-sEVs reduced brain macrophage infiltrates and microglia accumulation in the peri-infarct cortex. In vehicle-treated rats, CD31⁺/BrdU⁺ proliferating endothelial cells were found in the peri-infarct cortex. Hypoxic MSC-sEVs increased the number of CD31⁺/BrdU⁺ proliferating endothelial cells. Our results provide evidence that hypoxic MSC-derived sEVs potently enhance neurological recovery, reduce neuroinflammation. and increase angiogenesis in rat permanent distal MCAO.