Abstract Neonatal (perinatal) arterial ischemic stroke is as common as in the elderly and is a major cause of long-term neurological and cognitive deficits, including cerebral palsy and neurodevelopmental disabilities. Literature has emerged that the stage of brain development at the time of stroke has a major impact on the pathophysiological mechanisms of brain damage, but there are no effective treatments. Inflammation is a hallmark of perinatal brain injury and affects both early injury and brain repair and connectivity later in life. We discovered that microglia serve as endogenous protectants after acute stroke in neonatal rats and mice, including protection by removing apoptotic neuronal debris, limiting neuroinflammation and protecting blood-brain barrier integrity. We will determine whether microglial cells exert neuro- and vasoprotective effects via release of extracellular vesicles (EV). The fast-growing EV field as the fundamental way of cell-cell communication without direct cell-cell contacts in healthy and diseased organism has demonstrated heterogeneity of EV depending on the cell type releasing them, the mechanism of release and a neurodegenerative scenario. We hypothesize that microglial cells alleviate injury after neonatal stroke in part via released microglia- derived EV (MEV). In three aims, we will determine how neonatal stroke changes MEV properties and their communication with cells in brain slices and microglial cells that isolated after acute stroke (Aim 1); examine effects of disrupted lipid CD36-dependent signalling and disrupted EV release in the brain on neuroinflammation and injury in two mouse models—neonatal stroke or intracerebral IL1beta injection. We will analyse in vivo effects of halted EV release on neuronal and microglial transcriptome/proteome profiles in same injured neonatal brain (Aim 2); and determine if MEV administration early after neonatal stroke protects short-term and enhance long-term brain repair (Aim 3). To understand the mechanistic role of MEV and their therapeutic potential for neonatal stroke, we will utilize state-of-the art experimental tools, including a clinically relevant perinatal focal arterial stroke model that we invented, in conjunction with loss-of-function and gain-of function genetic and pharmacological approaches and advanced non-invasive imaging methodologies. We will manipulate EV release from neurons (CRISP editing approach), isolate and characterize individual EV and MEV fractions (super-sensitive ImageStreamX technology and MEV “cargo”), image MEV communication with resting/activated microglia and the vasculature (live imaging), and utilize novel double transgenic RiboTag mice to examine in vivo real time microglia-neuron molecular crosstalk. The proposed studies will enhance the understanding of cell signalling and communication and help achieve our long-term goal to identify novel therapeutic targets to create effective and safe therapy for neonatal stroke.