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.
Ischemic stroke affects millions of individuals worldwide and a high prevalence of survivors experience cognitive deficits. At present, the underlying mechanisms that drive post-stroke cognitive decline are not well understood. Microglia play a critical role in the post-stroke inflammatory response, but experimental studies show that an accumulation of chronically activated microglia can be harmful and associates with cognitive impairment. This study aimed to assess the effect of acute post-stroke minocycline treatment, a tetracycline derivative that readily crosses the blood-brain barrier and has been shown to inhibit microglia activation, on chronic microglia and astrocyte expression within both the infarct and remote white matter regions, as well as determine its effect on various domains of cognitive function post-stroke. Nine-month-old male rats received an injection of endothelin-1 into the right dorsal striatum to induce a transient focal ischemic stroke, and then were treated with minocycline or saline for 4 days post-stroke. Rats were tested using a series of lever-pressing tasks and the Morris water maze to assess striatal-based learning, cognitive flexibility, and spatial learning and reference memory. We found that minocycline-treated rats had smaller stroke-induced infarcts, less microglia activation in the infarct area and less microglia activation in remote white matter regions compared to saline-treated rats at 28 days post-stroke. The behavioural testing results differed according to the cognitive domain; whereas minocycline-treated rats trended towards improved striatal-based learning in a lever-pressing task, but cognitive flexibility was unaffected during the subsequent set-shifting task. Furthermore, minocycline treatment unexpectedly impaired spatial learning, yet it did not alter reference memory. Collectively, we show that post-stroke minocycline treatment can reduce chronic microglia activation even in remote brain regions, with domain-specific effects on cognitive function.
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