Abstract
Inflammation modulates brain injury after perinatal arterial stroke. We previously demonstrated that microglial cells play an important role in protecting neonatal brain from acute stroke by phagocytosing dying neurons, attenuating cytokine accumulation and by protecting neurovascular integrity. Microglia may modulate injury in neonatal stroke via crosstalk between cells via other mechanisms, such as by releasing extracellular vesicles from microglia (MEV), including exosomes (MExo) and microvesicles (MMV). Aim: Elucidate the mechanisms of MEV communication with brain cells in injured neonatal brain and role of these vesicles in protection. Methods: Transient middle cerebral artery occlusion (tMCAO) was performed in postnatal day 9 (P9). Microglial cells were isolated by CD11b-conjugated beads from ischemic and contralateral cortex 24h after reperfusion and plated at same density for 96h. MEV were isolated by multi-step centrifugation (MMV) and ultra-centrifugation (MExo) and labeled with MiniClaret dye. Uptake of MEV from contralateral/ injured cortex by Iba1 + -cells from contralateral and injured regions was determined 10, 30 & 120min. Images co-stained with flotillin-1, which is strongly expressed in MMV but not in MExo, were analyzed using Volocity ® . Results: The uptake of MEV from injured cortex by microglia from injured cortex was significantly higher than uptake of contralateral-MEV by microglia from uninjured cortex (5-fold at 10min; p<0.0001) regardless of time. Uptake of MEV from injured regions by microglia from contralateral cortex and contralateral-MEV by microglia from injured cortex were low. While the number of ipsilateral and contralateral flotillin-1 + -MMV was similar, uptake of flotillin-1 - -MExo and ratio of ipsilateral MExo/MEV were significantly increase. Summary: Our data demonstrate selective enhancement of microglial communication with MEV from activated microglia after acute neonatal stroke as well as distinct MEV-subtype-dependent mechanisms of communication in injured brain. This mechanism could provide a better understanding of the role of microglia on the severity of neonatal stroke. Support: AHA17IRG33430004, RO1NS44025, RO1HL139685
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