Abstract WP376: Small extracellular vehicles derived from cerebral endothelial cells promote white matter remodeling and improve functional recovery following cerebral ischemia/reperfusion in aged rats

Background: Ischemic strokes due to large vessel occlusion (LVO) predominantly affect the elderly and often result in high mortality and long-term disability. Although endovascular thrombectomy (EVT) has revolutionized LVO treatment, nearly half of patients fail to achieve functional independence despite successful reperfusion. Using a model of ischemia/reperfusion in aged rats, we tested the hypothesis that small extracellular vesicles (sEVs) derived from healthy cerebral endothelial cells (CEC-sEVs) enhance functional recovery. Methods: Aged male rats (18–20 months old) were subjected to 3h transient middle cerebral artery occlusion (MCAO) by a filament. CEC-sEVs were isolated from CEC cultures of healthy young-adult rats. Upon reperfusion (at 3h), the rats were randomly treated with CEC-sEVs (1x10 11 particles/injection) or saline administered via the internal carotid artery followed by a second dose given intravenously 24h after MCAO (n=10/group). Neurological outcomes were assessed weekly for 4 weeks with an array of behavioral tests. Results: The CEC-sEV treatment significantly reduced infarct volume by 23% and promoted neurological function recovery measured by modified neurological severity scores, foot-fault tests, and adhesive removal tests from weeks 2 to 4 post-stroke compared to the saline treated rats. Immunohistochemistry (IHC) revealed that CEC-sEVs substantially increased myelin basic proteins by 32% which is associated with significantly reduced oxidative damage in the NG2-positive oligodendrocyte progenitor cells (OPCs), as demonstrated by decreased 8-OHdG immunoreactivity (15±5% vs 26±8% of NG2-positive cells in saline) in the peri-infarct regions. Additionally, CEC-sEVs significantly increased neurofilament heavy chain positive axons by 26%. RNAseq and bioinformatics analyses showed that CEC-sEV cargo was enriched with miR-34a, -146a, -183, -202, and -450, which potentially target genes involved in cellular oxidative stress regulation. Conclusions: The CEC-sEVs treatment initiated at reperfusion substantially improved functional recovery in aged rats with 3h transient MCAO. Our IHC and CEC-sEV cargo miRNA data suggest that CEC-sEVs promote OPC differentiation into myelinating oligodendrocytes (OLs), likely by miRNA-mediated attenuation of oxidative stress, and thereby facilitating white matter remodeling after stroke. Our findings support the development of CEC-sEVs as a potential adjuvant therapy to optimize stroke outcome after EVT.