The isolation and molecular characterization of cerebral microvessels.

TitleThe isolation and molecular characterization of cerebral microvessels.
Publication TypeJournal Article
Year of Publication2019
AuthorsLee Y-K, Uchida H, Smith H, Ito A, Sanchez T
JournalNat Protoc
Date Published2019 11
KeywordsAnimals, Astrocytes, Blood-Brain Barrier, Brain, Brain Chemistry, DNA, Endothelial Cells, Mice, Mice, Inbred C57BL, Mice, Transgenic, Microvessels, Pericytes, RNA

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.

Alternate JournalNat Protoc
PubMed ID31586162
Grant ListR01 HL094465 / HL / NHLBI NIH HHS / United States
Related Faculty: 
Teresa Sanchez, Ph.D.

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