Role of extracellular vesicles mRNA in neuroinflammation in a mouse model of stroke

Abstract

Ischemic stroke is a leading cause of death in the world (Internet reference 1: WHO, 2020) and understanding its pathophysiology is crucial to the establishment of new therapeutical options to improve the outcome after a stroke event. Various studies have investigated EVs in the pathophysiological landscape of stroke. However, so far, only one previous study from our laboratory has focused on mRNAs being transported by EVs and their effect on different cells in the ischemic penumbra. We hypothesized that the mRNA population in sEVs changes following tMCAO. To assess this, we set up a new isolation protocol for isolating sEVs from whole brain tissue, consisting on homogenization, differential centrifugation and filtration to separate sEVs from cells, debris, and larger EVs. This was followed by a bottom-up iodixanol-based DGUC, the current gold standard for isolating EVs (Zhang et al., 2023) that further purified the sEVs based on their density. Next, we characterized our isolated sEVs by western blot, NTA and EM according with the MISEV2018 (Théry et al., 2018). Then, we analyzed the differential expression of 757 different mRNAs associated with neuroinflammation with the NanoString nCounter® technology at 24 h and 7 d after tMCAO. Of note, the NTA assay which analyzes the size and quantity of EVs showed no significant difference at both time points between tMCAO and shams. At both time points, several mRNAs were significantly upregulated. From all analyzed mRNAs, Spp1-mRNA was revealed as a potential candidate for further analysis, mainly because of its high fold change of approximately 100 times increase in tMCAO compared to sham sEVs. The nSolver cell type profiling and open-source transcriptome atlases indicated that microglia and macrophages were the main source of the differentially expressed mRNAs. Furthermore, multiple pathways could be ascribed to the upregulated mRNAs. Although the functionality of the mRNAs and their translation to proteins could not be assessed in this thesis, it provides a solid starting point for further investigations of the role on mRNA in EVs in stroke.