Stroke imposes a long-term neurological disability with limited effective treatments available for neuronal recovery. Transplantation of neural stem cells (NSCs) is reported to improve functional outcomes in the animal models of brain ischemia. However, the use of cell therapy is accompanied by adverse effects, so research is growing to use cell-free extracts such as extracellular vesicles (EVs) for targeting brain diseases. In the current study, male Wistar albino rats (20 months old) were subjected to middle cerebral artery occlusion (MCAO). Then, EVs (30 μg) were injected at 2 hours after stroke onset via an intracerebroventricular (ICV) route. Measurements were done at day 7 post-MCAO. EVs administration reduced lesion volume and steadily improved spontaneous locomotor activity.EVs administration also reduced microgliosis (ionized calcium-binding adaptor molecule 1 (Iba1) + cells) and apoptotic (terminal-deoxynucleotidyl transferase mediated nick end labelling [TUNEL]) positive cells and increased neuronal survival (neuronal nuclear (NeuN) + cells) in the ischemic boundary zone (IBZ). However, it had no effect on neurogenesis within the sub-ventricular zone (SVZ) but decreased cellular migration toward the IBZ (doublecortin (DCX) + cells). The results of this study showed neuroprotective and restorative mechanisms of NSC-EVs administration, which may offer new avenues for therapeutic intervention of brain ischemia. Significance of the study: Based on our results, EVs administration can effectively reduce microglial density and neuronal apoptosis, thereby steadily improves functional recovery after MCAO. These findings provide the beneficial effect of NSC-EVs as a new biological treatment for stroke. K E Y W O R D S extracellular vesicle (EV), ischemic boundary zone (IBZ), middle cerebral artery occlusion (MCAO), neural stem cell (NSC), neurogenesis

Background: Ischemic stroke, as a health problem caused by the reduced blood supply to the brain, can lead to the neuronal death. The number of reliable therapies for stroke is limited. MSCs exhibit therapeutic achievement. A major limitation of MSC application in cell therapy is the short survival span. MSCs affect target tissues through the secretion of many paracrine agents including EVs. This study aimed to investigate the effect of HUCPVCs-derived EVs on apoptosis, functional recovery, and neuroprotection. Methods: Ischemia was induced by MCAO in male Wistar rats. Animals were classified into sham, MCAO, MCAO + HUCPVC, and MCAO + EV groups. Treatments began at two hours after ischemia. Expressions of apoptotic-related proteins (BAX/BCl-2 and caspase-3 and -9), the amount of TUNEL-positive cells, neuronal density (MAP2), and dead neurons (Nissl staining) were assessed on day seven post MCAO. Results: Administration of EVs improved the sensorimotor function ( p < 0.001) and reduced the apoptotic rate of Bax/Bcl-2 ratio ( p < 0.001), as well as caspases and TUNEL-positive cells ( p < 0.001) in comparison to the MCAO group. EV treatment also reduced the number of dead neurons and increased the number of MAP2 + cells in the IBZ ( p < 0.001), as compared to the MCAO group. Conclusion: Our findings showed that HUCPVCs-derived EVs are more effective than their mother’s cells in improving neural function, possibly via the regulation of apoptosis in the ischemic rats. The strategy of cell-free extracts is, thus, helpful in removing the predicaments surrounding cell therapy in targeting brain diseases.