A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels

Summary

Here, a method for isolation of rat brain microvessels and for the preparation of membrane samples is described. This protocol has the clear advantage of producing enriched microvessel samples with acceptable protein yield from individual animals. Samples can then be used for robust protein analyses at the brain microvascular endothelium.

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.

Introduction

The blood-brain barrier (BBB) exists at the interface between the central nervous system (CNS) and the systemic circulation and plays an essential role in the maintenance of brain homeostasis. Specifically, the BBB functions to precisely control solute concentrations in brain extracellular fluid and to efficiently supply those nutrients that are required by brain tissue to fulfill the considerable metabolic demands of the CNS¹. These roles imply that the BBB, which exists primarily at the level of the microvascular endothelial cell, must possess discrete mechanisms that enable some substances to access brain parenchyma while ensuring that poten....

Protocol

All procedures outlined below have been approved by an Institutional Animal Care and Use Committee (IACUC) and conform to National Institutes of Health (NIH) and Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines. The procedural flow for the protocol is depicted in Figure 1.

1. Set-up for the Procedure

1. Prepare the brain microvessel buffer (BMB). Start by weighing 54.66 g D-mannitol, 1.90 g EGTA, and 1.46 g 2-amino-2-(hydroxymethyl)-1,3-propanediol (i.e., Tris) base into a clean beaker. Add 1.0 L of deionized water. Mix the components of the BMB buffer using a magnetic stirre....

Results

The experimental flow for isolation of rat brain microvessels and for the preparation of microvessel membrane samples is shown in Figure 1. Using the procedure presented here, successful isolation of intact microvessels from rat brain is demonstrated (Figure 2A). These vessels were obtained following completion of centrifugation with dextran and immediately prior to commencing ultracentrifugation to prepare membrane samples (i.e........

Discussion

In this article, a simple and effective method of preparing membrane protein samples from microvessels freshly isolated from rat brain tissue is described. Several approaches for isolation of rat brain microvessels and/or generation of membrane preparations from isolated microvasculature have been reported in the literature^{13,20,21,22,24}. Although the microves.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Protease Inhibitor Cocktail	Sigma-Aldrich	#P8340	Component of brain microvessel buffer
D-mannitol	Sigma-Aldrich	#M4125	Component of brain microvessel buffer
EGTA	Sigma-Aldrich	#E3889	Component of brain microvessel buffer
Trizma Base	Sigma-Aldrich	#T1503	Component of brain microvessel buffer
Dextran (MW 75,000)	Spectrum Chemical Mftg Corp	#DE125	Dextran used in centrifugation steps to separate microvessels from brain parenchyma
Zetamine	MWI Animal Health	#501072	General anesthetic
Xylazine	Western Medical Supply	#5530	General anesthetic
0.9% saline solution	Western Medical Supply	N/A	General anesthetic diluent
Filter Paper (12.5 cm diameter)	VWR	#28320-100	Used for removal of meninges from brain tissue
Centrifuge Tubes	Sarstedt	#60.540.386	Disposable tubes used for dextran centrifugation steps
Pierce™ Coomassie Plus (Bradford) Assay	ThermoFisher Scientific	#23236	Measurement of protein concentration in membrane preparations
Wheaton Overhead Power Homogenizer	DWK Life Sciences	#903475	Required for homogenization of samples
10.0ml glass mortar and pestle tissue grinder	DWK Life Sciences	#358039	Required for homogenization of samples
Hydrochloric Acid	Sigma-Aldrich	#H1758	Required for pH adjustment of buffers
Bovine Serum Albumin	ThermoFisher Scientific	#23210	Protein standard for Bradford Assay
Standard Forceps	Fine Science Tools	#91100-12	Used for dissection of brain tissue
Friedman-Pearson Rongeurs	Fine Science Tools	#16020-14	Used for opening skull to isolate brain
50 ml conical centrifuge tubes	ThermoFisher Scientific	#352070	Used for collection of brain tissue following isolation
Glass Pasteur Pipets	ThermoFisher Scientific	#13-678-20C	Used for aspiration of cellular debris following dextran spins
Ethanol, anhydrous	Sigma-Aldrich	#459836	Used for cleaning tissue grinder; diluted to 70% with distilled water
Ultracentrifuge tubes	Beckman-Coulter	#41121703	Used for ultracentrifugation of samples