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.

Fibrinogen primes the microglial NLRP3 inflammasome and propagates pro-inflammatory signaling via extracellular vesicles: Implications for blood-brain barrier dysfunction.

The brain's response to acute injury is characterized by increased permeability of the blood-brain barrier (BBB) and pro-inflammatory microglia signaling, both of which have been linked to poor cognitive outcomes and neurological disease. The damaged BBB has increased leakiness, allowing serum proteins like fibrinogen into the brain, which interacts with local cells in a deleterious manner. At the same time, in response to injury, microglia demonstrate increased NLRP3 inflammasome activity and heightened release of pro-inflammatory cytokines. The relationship between increased fibrinogen uptake and microglial inflammasome signaling in the injured brain has not been well described. In this work, we investigate fibrinogen mediated NLRP3 inflammasome priming of BV-2 cells and primary adult microglia and propose a role for extracellular vesicles (EVs) as propagators of this interaction. Following exposure to fibrinogen microglia significantly upregulate transcription of IL-1ß, IL-6, NLRP3 and other pro-inflammatory cytokines which was sustained by repeated fibrinogen exposure. Inhibition of fibrinogen mediated NLRP3 signaling was achieved at the transcriptional and assembly level using cannabidiol (CBD) and the NLRP3 inhibitor MCC950, respectively. EVs released following NLRP3 priming carry IL-1ß, IL-18 mRNA and fibrinogen, propagate inflammatory signaling and can be detected in the circulation following BBB disruption in a preclinical stroke model. In conclusion, the interplay between fibrinogen extravasation, microglial NLRP3 signaling, and EV release can perpetuate chronic pro-inflammatory signaling and represents a novel method of inflammatory propagation.

Ganglioside Detection from Formalin-Fixed Human Brain Tissue Utilizing MALDI Imaging Mass Spectrometry.

Matrix assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) is used to perform mass spectrometric analysis directly on biological samples providing visual and anatomical spatial information on molecules within tissues. A current obscuration of MALDI-IMS is that it is largely performed on fresh frozen tissue, whereas clinical tissue samples stored long-term are fixed in formalin, and the fixation process is thought to cause signal suppression for lipid molecules. Studies have shown that fresh frozen tissue sections applied with an ammonium formate (AF) wash prior to matrix application in the MALDI-IMS procedure display an increase in observed signal intensity and sensitivity for lipid molecules detected within the brain while maintaining the spatial distribution of molecules throughout the tissue. In this work, we investigate the viability of formalin-fixed tissue imaging in a clinical setting by comparing MALDI data of fresh frozen and postfixed rat brain samples, along with postfixed human brain samples washed with AF to assess the capabilities of ganglioside analysis in MALDI imaging of formalin-fixed tissue. Results herein demonstrate that MALDI-IMS spectra for gangliosides, including GM1, were significantly enhanced in fresh frozen rat brain, formalin-fixed rat brain, and formalin-fixed human brain samples through the use of an AF wash. Improvements in MALDI-IMS image quality were demonstrated, and the spatial distribution of molecules was retained. Results indicate that this method will allow for the analysis of gangliosides from formalin-fixed clinical samples, which can open additional avenues for neurodegenerative disease research.