Abstract TP20: Microglial-Derived Extracellular Vesicles Protect From Neonatal Arterial Focal Stroke

Abstract

Background: The developmental stage of the brain at the stroke onset plays a key role in the pathophysiology of injury, with neuroinflammation as a major injury modulator (Rayasam et al 2022; Hagberg et al. 2015). We reported that in the neonatal brain microglia secrete extracellular vesicles, exosomes and microvesicels, under physiological conditions and that microglial activation after transient MCA occlusion (tMCAO) in postnatal day 9 (P9) mice alters “cargo” of microglial-derived extracellular vesicles (MEV) and increases MEV uptake by microglia in vitro (Lecuyer et al., Neurobiol Dis. 2021). Here, we examined whether MEV administration protects the neonatal brain from stroke. Methods: P9 C57BL6 mice were subjected to a 3h tMCAO, microglia isolated from naïve mice and from injured (Inj) and contralateral (Contra) regions 24h after reperfusion using CD11b-conjugated beads, and cultured for 4 days in10% exosome-free FBS. MEV were purified from culture medium by multistep centrifugation [x300 g (Phase1, p1), x1,200 g (p2), x12,000 g (p3), and x100,000 g (p4)], concentration determined by Nanosight, MEV labelled with Claret-FR. MEV uptake by microglia from Inj and Contra regions was examined in vitro. 0.5 or 3.0 x 10 8 MEV were injected retro-orbital 2h after reperfusion. Behaviour outcomes (24h) and injury (at 4 days) were determined. Results: Post-vivo, uptake of p4C or p4I was increased by microglia from Inj, but not from Contra regions and reduced cell size and morphology within 30 min. Administration of p4-MEV from naïve (p4N-MEV), Contra (p4C-MEV) or Inj (p4I-MEV) regions significantly improved performance on Open Field test and reduced injury, whereas p3N-MEV did not affect inury. MEV increased survival (100%) compared to non-treated (77%) and vehicle-treated (83%) mice. We are defining p4 and p3 identity (exosomes Vs. microvesicles). Summary: p4-MEV but not p3-MEVprotect from neonatal stroke. Funding: R01 NS44025, R01 HL139685, R21 NS098514