Supplementary Material for: Intranasal Administration of Extracellular Vesicles Mitigates Apoptosis in a Mouse Model of Neonatal Hypoxic-Ischemic Brain Injury

<b><i>Introduction:</i></b> Neonatal hypoxic-ischemic brain injury (HIBI) results in significant morbidity and mortality despite current available therapies. Seeking a potential supplemental therapy for HIBI, we investigated the neuroprotective effects of extracellular vesicles derived from neural stem cells (NSC-EVs) and hypoxia-preconditioned brain cells (brain-EVs). <b><i>Methods:</i></b> HIBI was induced in postnatal day 9 mice by carotid ligation followed by hypoxia. Following injury, NSC-EVs, brain-EVs, or saline were administered intranasally. Brains were assessed for infarct size, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and caspase-3 expression. Additionally, brain-EV microRNA (miRNA) contents were analyzed by miRNA sequencing. <b><i>Results:</i></b> Both EV treated groups showed decreased infarct size (brain-EVs <i>p</i> = 0.004 and NSC-EVs <i>p</i> = 0.052), and although NSC-EV administration resulted in significantly fewer TUNEL+ cells (<i>p</i> = 0.0098), there was no change in caspase-3 expression after NSC-EV administration, suggesting a caspase-3-independent mechanism. Brain-EVs resulted in a nonsignificant decrease in TUNEL+ cells (<i>p</i> = 0.167) but significant decreases in caspase expression (cleaved <i>p</i> = 0.015 and intact <i>p</i> = 0.026). Brain-EVs consistently expressed several miRNAs, including two which have been shown to be downregulated after HIBI: miR-342-3p and miR-330-3p. <b><i>Conclusion:</i></b> Understanding the regenerative effects and contents of NSC-EVs and brain-EVs could allow for the development of targeted EV-based therapies that could reduce morbidity and mortality for neonates affected by HIBI.