An optimized method for the isolation and the molecular characterization of cerebral microvessels in vivo

The molecular characterization of cerebral microvessels in experimental disease models has been hindered by the lack of a standardized method to reproducibly isolate intact cerebral microvessels, with consistent cellular compositions, and without the use of enzymatic digestion, which causes undesirable molecular and metabolic changes. Herein, we describe an optimized method for microvessel isolation from mouse brain cortex, which yields microvessel fragments (diameter <50 m, 89.3% 3-5 m) with consistent populations of discrete blood-brain barrier components (endothelial cells, pericytes, and astrocyte end feet), retaining high RNA integrity and protein postranslational modifications (e.g. phosphorylation). We demonstrate that this method allows the quantification of changes in gene expression in a disease model (stroke) and the activation of signalling pathways in mice subjected to drug administration. We also describe the isolation of genomic DNA and bisulfite treatment for the assessment of DNA methylation, as well as the optimization of chromatin extraction and shearing from cortical microvessels. Therefore, this protocol will be of great use to improve the understanding of the molecular mechanisms governing cerebrovascular dysfunction, which may help the development of novel therapies for stroke and other neurodegenerative diseases.