The isolation and molecular characterization of cerebral microvessels

• Published in: Nature protocols Vol. 14; no. 11; pp. 3059 - 3081
• Main Authors: Lee, Yun-Kyoung, Uchida, Hiroki, Smith, Helen, Ito, Akira, Sanchez, Teresa
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.11.2019; Nature Publishing Group
• Subjects: 631/1647/2230; 631/1647/767/1658; 692/617/375/1370; 692/617/375/365; Analysis; Analytical Chemistry; Animals; Astrocytes - chemistry; Astrocytes - cytology; Biological Techniques; Biomedical and Life Sciences; Bisulfite; Blood-brain barrier; Blood-Brain Barrier - chemistry; Blood-Brain Barrier - cytology; Brain; Brain - blood supply; Brain cells; Brain Chemistry; Chromatin; Computational Biology/Bioinformatics; Cortex; Deoxyribonucleic acid; DNA; DNA - analysis; DNA methylation; Endothelial cells; Endothelial Cells - chemistry; Endothelial Cells - cytology; Gene expression; Head injuries; Life Sciences; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microarrays; Microvasculature; Microvessels - chemistry; Microvessels - cytology; Molecular modelling; Neurodegenerative diseases; Optimization; Organic Chemistry; Pericytes; Pericytes - chemistry; Pericytes - cytology; Phosphorylation; Post-translation; Protocol; Ribonucleic acid; RNA; RNA - analysis; Sepsis; Shearing; Stroke; Structure; Traumatic brain injury; Vascular endothelium; United States
• ISSN: 1754-2189; 1750-2799; 1750-2799
• DOI: 10.1038/s41596-019-0212-0

The study of cerebral microvessels is becoming increasingly important in a wide variety of conditions, such as stroke, sepsis, traumatic brain injury and neurodegenerative diseases. However, the molecular mechanisms underlying cerebral microvascular dysfunction in these conditions are largely unknown. 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 protocol for microvessel isolation from mouse brain cortex that yields microvessel fragments with consistent populations of discrete blood–brain barrier (BBB) components (endothelial cells, pericytes and astrocyte end feet) while retaining high RNA integrity and protein post-translational modifications (e.g., phosphorylation). We demonstrate that this protocol allows the quantification of changes in gene expression in a disease model (stroke) and the activation of signaling pathways in mice subjected to drug administration in vivo. We also describe the isolation of genomic DNA (gDNA) and bisulfite treatment for the assessment of DNA methylation, as well as the optimization of chromatin extraction and shearing from cortical microvessels. This optimized protocol and the described applications should improve the understanding of the molecular mechanisms governing cerebral microvascular dysfunction, which may help in the development of novel therapies for stroke and other neurologic conditions. Microvessels are isolated from mouse brain cortex, minimizing cell activation and yielding microvessel fragments with consistent populations of discrete blood–brain barrier components that retain RNA integrity and protein post-translational modifications.