Early treatment of ischemic stroke can significantly reduce disability and mortality rates. Stem cell-derived extracellular vesicles (EVs) have shown potential as therapeutics for neurological disorders. This study explored whether intranasal administration of EVs from human bone marrow mesenchymal stem cells (BM-MSCs) enhances forelimb motor function recovery in a mouse model of motor cortex stroke and investigated their mechanism of action, focusing on neuroinflammation. C57BL/6JRj mice received EV treatment of 0.1 × 10EVs per dose per day, 48 h post-stroke and twice weekly for four weeks. EV-treated mice showed significant improvement in forelimb deficits, as evaluated using a series of motor tests. Histopathological assessments revealed reduced infarct volume and decreased astrogliosis and microglial activation in EV-treated mice. EV treatment led to changes in microglial morphology in the peri-infarct area, associated with increased anti-inflammatory cytokines interleukin (IL)-10 and IL-13 and decreased pro-inflammatory cytokines IL-1β, IL-6, and tumor necrosis factor-alpha. Reduced expression of nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome and Cleaved Caspase-1 following EV treatment supports their role in dampening inflammation. In vitro experiments using oxygen-glucose deprivation confirmed that EVs attenuated the inflammatory phenotype of microglia and reduced neuronal apoptosis. EV cargo analysis revealed neuroprotective molecules, including anti-inflammatory cytokines and brain-derived neurotrophic factor (BDNF), which may contribute to their immunomodulatory properties. These findings show that EVs mitigate post-stroke brain immune response, promoting tissue healing and recovery. Our comprehensive characterization of the effects of human BM-MSC-derived EVs, encompassing functional, tissue, cellular, and molecular aspects, underscores their therapeutic potential and supports their use in stroke treatment. 9
