Metabolomics and metabolites in ischemic stroke.

Stroke is a major reason for disability and the second highest cause of death in the world. When a patient is admitted to a hospital, it is necessary to identify the type of stroke, and the likelihood for development of a recurrent stroke, vascular dementia, and depression. These factors could be determined using different biomarkers. Metabolomics is a very promising strategy for identification of biomarkers. The advantage of metabolomics, in contrast to other analytical techniques, resides in providing low molecular weight metabolite profiles, rather than individual molecule profiles. Technically, this approach is based on mass spectrometry and nuclear magnetic resonance. Furthermore, variations in metabolite concentrations during brain ischemia could alter the principal neuronal functions. Different markers associated with ischemic stroke in the brain have been identified including those contributing to risk, acute onset, and severity of this pathology. In the brain, experimental studies using the ischemia/reperfusion model (IRI) have shown an impaired energy and amino acid metabolism and confirmed their principal roles. Literature data provide a good basis for identifying markers of ischemic stroke and hemorrhagic stroke and understanding metabolic mechanisms of these diseases. This opens an avenue for the successful use of identified markers along with metabolomics technologies to develop fast and reliable diagnostic tools for ischemic and hemorrhagic stroke.

CM-Dil Staining and SEC of Plasma as an Approach to Increase Sensitivity of Extracellular Nanovesicles Quantification by Bead-Assisted Flow Cytometry.

The quantification of the specific disease-associated populations of circulating extracellular membrane nanovesicles (ENVs) has opened up new opportunities for liquid biopsy in cancer and other chronic diseases. However, the sensitivity of such methods is mediated by an optimal combination of the isolation and labeling approaches, and is not yet sufficient for routine clinical application. The presented study aimed to develop, characterize, and explore a new approach to non-specific ENV staining, followed by size-exclusive chromatography (SEC), which allows us to increase the sensitivity of bead-assisted flow cytometry. Plasma from healthy donors was purified from large components, stained with lipophilic CM-Dil dye, and fractionated by means of SEC. The obtained fractions were analyzed in terms of particle size and concentration using NTA, as well as vesicular markers and plasma protein content via dot-blotting. We characterized the process of CM-Dil-stained plasma fractionation in detail and indicated the fractions with optimal characteristics. Finally, we explored the sensitivity of on-bead flow cytometry for the analysis of specific populations of plasma ENVs and demonstrated the advantages and limitations of the proposed technique.