Plasma derived extracellular vesicle biomarkers of microglia activation in an experimental stroke model

Chronic microglia activation post-stroke is associated with worse neurological and cognitive outcomes. However, measurement of microglia activation in vivo is currently limited. Plasma derived extracellular vesicles (EVs) are cell-specific indicators that may allow for non-invasive measurement of microglia phenotype. The aim of this study was to identify activation-state specific microglia EVs (MEVs) in vitro followed by validation in an experimental stroke model. Following pro-inflammatory activation, MEVs contain the microglia protein TMEM119 alongside increased expression of the Toll-like receptor 4 co-receptor CD14. Immunoprecipitation followed by fluorescent nanoparticle tracking analysis (ONI Nanoimager) was used to confirm the isolation of TMEM119(+)/CD14(+) EVs from rat plasma. Electron microscopy confirmed that TMEM119 and CD14 localize to the MEV membrane. To model ischemia, plasma was collected from 3-month wildtype Fischer344 rats prior to, 7 and 28 days after endothelin-1 or saline injection into the dorsal right striatum. Fluorescently labelled MEVs were directly measured in the plasma using nanoflow cytometry (Apogee A60 Microplus). We report a significant increase in circulating TMEM119(+)/CD14(+) EVs 28-days post-stroke in comparison to baseline levels and saline-injected rats, which correlated weakly with stroke volume. TMEM119(+)/MHC-II+ EVs were also increased post-stroke in comparison to baseline and saline-injected animals. This study is the first to describe an EV biomarker of activated microglia detected directly in plasma following stroke and represents a future tool for the measurement of microglia activity in vivo.