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

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% O(2); 2 × 10(6) or 2 × 10(7) 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.