A simple and reproducible method to prepare membrane samples from freshly isolated rat brain microvessels

Hrvoje Brzica, Wazir Abdullahi, Bianca G. Reilly, Patrick T Ronaldson

Research output: Contribution to journalArticle

3 Scopus citations

Abstract

The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting from these stimuli can greatly modulate drug delivery to the brain and, by extension, cause considerable challenges in the treatment of central nervous system (CNS) diseases. Many BBB changes that affect pharmacotherapy, involve proteins that are localized and expressed at the level of endothelial cells. Indeed, such knowledge on BBB physiology in health and disease has sparked considerable interest in the study of these membrane proteins. From a basic science research standpoint, this implies a requirement for a simple but robust and reproducible method for isolation of microvessels from brain tissue harvested from experimental animals. In order to prepare membrane samples from freshly isolated microvessels, it is essential that sample preparations be enriched in endothelial cells but limited in the presence of other cell types of the neurovascular unit (i.e., astrocytes, microglia, neurons, pericytes). An added benefit is the ability to prepare samples from individual animals in order to capture the true variability of protein expression in an experimental population. In this manuscript, details regarding a method that is utilized for isolation of rat brain microvessels and preparation of membrane samples are provided. Microvessel enrichment, from samples derived, is achieved by using four centrifugation steps where dextran is included in the sample buffer. This protocol can easily be adapted by other laboratories for their own specific applications. Samples generated from this protocol have been shown to yield robust experimental data from protein analysis experiments that can greatly aid the understanding of BBB responses to physiological, pathophysiological, and pharmacological stimuli.

Original languageEnglish (US)
Article numbere57698
JournalJournal of Visualized Experiments
Volume2018
Issue number135
DOIs
StatePublished - May 7 2018

Keywords

  • Blood-brain barrier
  • Brain Microvessels
  • Dextran separation
  • Differential centrifugation
  • Endothelial cell
  • Issue 135
  • Membrane proteins
  • Molecular pharmacology
  • Neuroscience
  • Tight junctions
  • Transporters
  • Western blotting

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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