Experimental design

All animal procedures were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University, Shanghai, China and reported according to the ARRIVE guidelines. Eighty adult (aged approximately 16 weeks) male ICR mice (Jiesijie, Shanghai, China) weighing 25-28 g were used and randomly divided into the sham, phosphate-buffered saline (PBS), M2-sEV, and miR-124 knockdown (miR-124-kd) groups (12 mice in the PBS group and 15 each in the sham, M2-sEV, and miR-124-kd groups) by an investigator blinded to the experimental design. M2 BV2 cell miR-124 was knocked down using the miR-124-kd lentivirus (OBiO, Shanghai, China; virus titer, 109 TU/mL) or lentiviral vector alone, and the exosomes isolated from vector alone cells were considered as the control of miR-124-kd. Mice were housed with free access to food and water under a 12 h light-dark cycle (light on at 8:00 and off at 20:00). The PBS, M2-sEV, and miR-124-kd mice underwent 90 min of tMCAO. Animals in the M2-sEV and miR-124-kd groups were treated daily with sEVs from M2 BV2 cells through tail intravenous injections (4 µg/kg) ²¹ from day 1 to day 7 after tMCAO. Mice in the PBS group were treated with equal volumes of PBS in the same manner. Aldh1l1- CreERT2:Ai14 (gift from Korea Advanced Institute of Science and Technology) transgenic mice were used to trace reactive astrocyte transdifferentiation. Bromodeoxyuridine (BrdU) (Sigma-Aldrich, B2531, San Louis, CA) was intraperitoneally injected into all mice (50 mg/kg per day) beginning on day 7 after ischemia ¹⁹.