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 potentially harmful xenobiotics cannot accumulate. Indeed, brain microvascular endothelial cells are not fenestrated and exhibit limited pinocytosis, which ensures a lack of non-selective permeability². Additionally, brain microvessel endothelial cells express tight junction and adherens junction proteins that act to form a physical "seal" between adjacent endothelial cells and greatly restrict paracellular diffusion of blood-borne substances into brain parenchyma. Indeed, selective permeability of endogenous and exogenous substances requires functional expression of uptake and efflux transporters³. Overall, tight junctions, adherens junctions, and transporters work in concert to maintain the unique barrier properties of the BBB.

The BBB is a dynamic barrier that responds to physiological, pathophysiological, and pharmacological stimuli. For example, hypoxia/reoxygenation stress has been shown to modulate expression of critical tight junction proteins (i.e., occludin, zonulae occluden-1 (ZO-1)), which is associated with increased paracellular permeability to vascular markers such as sucrose^4,5,6. Similar observations have been made at the BBB in the setting of traumatic brain injury⁷ and peripheral inflammatory pain^8,9. These same diseases can also modulate transport mechanisms at the BBB^{10,11,12,13,14}. Indeed, hypoxia/reoxygenation injury enhances functional expression of organic anion transporting polypeptide 1a4 (Oatp1a4) at the BBB, which can lead to significant increases in the blood-to-brain transport of specific Oatp transport substrates such as taurocholate and atorvastatin¹³. BBB properties can also be altered by pharmacotherapy itself, a mechanism that can form a basis for both profound changes in the drug effectiveness in the brain and for drug-drug interactions. For example, acetaminophen targets nuclear receptor signaling mechanisms in the brain microvascular endothelial cells, increases functional expression of the critical efflux transporter P-glycoprotein (P-gp), and modifies time-dependent analgesia conferred by morphine, an opioid analgesic drug and established P-gp transport substrate¹⁵. A thorough understanding of BBB changes, that can be induced by diseases or by drugs, also requires identification and characterization of specific regulatory mechanisms that control these modifications. Indeed, discrete signaling pathways have been identified in brain microvascular endothelial cells that control the molecular expression of tight junction proteins^16,17 and transporters^15,18,19. Taken together, these observations indicate that complex molecular pathways are involved in the regulation of BBB tight junctions and transporters in both health and disease.

A significant challenge in the study of the BBB is the absolute requirement of a simple and effective method for isolation of microvessels from brain tissue derived from experimental animals and subsequent preparation of membrane samples. These samples must be prepared so that they are both enriched in brain microvascular endothelial cells and limited in presence of other cell types. Over the past several years, multiple methodologies for isolation of microvasculature from rodent brain have been reported in the scientific literature^13,20,21,22. This article describes a simple, robust, and reproducible method for isolation of microvessels from rat brain and for preparation of endothelial membrane-enriched samples that can be used for the analysis of protein expression. An advantage of this microvessel isolation protocol is the ability to obtain sample preparations of high quality and with sufficient protein yield from an individual experimental animal. This enables the consideration of inter-animal variability in protein expression. Such an advance in this protocol has greatly improved the robustness of BBB studies because over-estimation (or under-estimation) of the true magnitude of protein changes at the BBB can now be avoided. Additionally, the inclusion of multiple centrifugation steps with dextran enables improved enrichment of microvessels in experimental samples while facilitating removal of unwanted cellular constituents such as neurons.

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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 stirrer. Once the solution has been thoroughly mixed, adjust the pH to 7.4 using 1.0 M HCl.
2. Prepare 26% dextran (MW 75,000) solution (w/v) prior to anesthetizing animals. Prepare the dextran solution as described in the following steps.
   NOTE: It is critically important to prepare this solution ahead of time because dextran can take approximately 1.5-2 h to dissolve in aqueous buffer.
   1. Weigh out the powdered dextran (26 g per 100 mL buffer) into a clean beaker.
   2. Measure out the appropriate volume of BMB and dispense into a separate, clean beaker.
   3. Slowly pour BMB into the beaker containing powdered dextran. Manually mix the powdered dextran during pouring of BMB using a glass stir rod.
   4. Adjust the pH to 7.4 with 1.0 M HCl immediately prior to use.
      NOTE: A typical isolation of brain microvessels from 12 individual Sprague-Dawley rats (male or female; 3 months of age; 200-250 g each) requires 200 mL of dextran solution (i.e., 52 g dextran in 200 mL of BMB).

2. Extraction of Brain Tissue from Sprague-Dawley Rats

1. Following the desired experimental treatment, anesthetize Sprague-Dawley rats using ketamine (50 mg/kg; 2.5 mL/kg i.p.) and xylazine (10 mg/mL; 2.5 mL/kg I,p.). Dilute ketamine and xylazine in 0.9% saline to final concentrations of 20 mg/mL and 4.0 mg/mL respectively. Euthanize animals by decapitation using a sharpened guillotine in accordance with IACUC guidelines. Use the same procedure for both male and female Sprague-Dawley rats.
2. Resect the skin from the rat skull by making a single transverse cut using surgical scissors.
3. Using rongeurs, carefully remove the skull plate and expose the brain.
4. Remove the brain using a spatula. Detach the cerebrum and place the isolated brain tissue into a 50 mL conical tube containing 5 mL of BMB. Add 1.0 μL of protease inhibitor cocktail per 1.0 mL of BMB buffer immediately prior to use.

3. Brain Processing

1. Transfer the brain tissue from the 50 mL conical tube to a clean petri dish.
2. Using forceps, gently roll the brain on 12.5 cm diameter filter paper to remove outer meninges, which are loosely adhered to the cerebral cortex. Gently press the brain tissue against filter paper and roll the tissue again. Turn the filter paper frequently during this step.
3. Separate the choroid plexus from the cerebral hemispheres using forceps.
   NOTE: The choroid plexus appears as a clear membranous tissue localized to the surface of the cerebral ventricles.
4. Gently flatten the brain tissue and remove remaining meninges and olfactory bulbs using forceps.
5. Place the cortical brain tissue into a chilled glass mortar. Add 5.0 mL of BMB containing protease inhibitor cocktail to the mortar.
6. Using an overhead power homogenizer, homogenize the brain tissue using 15 up and down strokes at 3,700 rpm. Perform homogenization using a 10 mL mortar and pestle tissue grinder. Between homogenization of each individual sample, clean the pestle using 70% ethanol.
   NOTE: Homogenization strokes should be consistent in terms of pace and magnitude.
7. Pour the homogenate into labeled centrifuge tubes.

4. Centrifugation Steps

1. Add 8.0 mL of 26% dextran solution to each labeled centrifuge tube containing brain homogenate.
2. Invert the tube twice and then thoroughly vortex the sample. Conduct the vortexing of each sample using multiple angles to ensure a thorough mixing of brain homogenate solution with 26% dextran solution.
3. Centrifuge samples at 5,000 x g for 15 min at 4 °C.
   NOTE: For some analyses, it is useful to compare brain microvessels with brain parenchyma. Should the assessment of protein expression in brain parenchyma be required, the supernatant from this centrifugation step (i.e., brain parenchymal fraction) can be collected and stored at -80 °C for future use.
4. Remove the supernatant using a vacuum aspirator and glass Pasteur pipette.
   NOTE: Caution must be taken to not disturb the pellet. Otherwise, quantity of the brain microvessels collected will be reduced, which will significantly decrease the protein yield from this procedure.
5. Resuspend the pellet in 5.0 mL of BMB containing protease inhibitor cocktail (i.e., 1.0 μL protease inhibitor cocktail per 1.0 mL of BMB buffer). Vortex the pellet to ensure thorough mixing.
6. Add 8.0 mL of 26% dextran to each centrifuge tube and vortex as described in steps 4.1-4.2 of this protocol.
7. Centrifuge samples at 5,000 x g for 15 min at 4 °C.
8. Using a vacuum flask and glass pipette, aspirate supernatant and ensure that pellet containing brain microvessel is not disrupted.
   NOTE: Excess material that has adhered to the tube wall does not contain microvessels and should be carefully cleaned and removed.
9. Repeat steps 4.5 through 4.8 an additional two times.
10. Once 4 dextran centrifugation steps have been completed, add 5.0 mL of BMB to each pellet and vortex to resuspend the sample.
    NOTE: Following completion of step 4.10, whole microvessels can be collected for analysis of protein localization. This can be accomplished by taking a 50 μL aliquot of re-suspended microvessel pellet and smearing on a glass microscope slide. Microvessels are then heat-fixed at 95 °C for 10 min on a heating block followed by fixation in ice-cold ethanol for an additional 10 min. Slides can then be stored at 4 °C until required for imaging studies.

5. Ultracentrifugation to Prepare Samples of Total Brain Microvascular Membranes

1. Transfer samples to the chilled glass homogenizer.
2. Using an overhead power homogenizer, homogenize brain tissue using 8-10 up and down strokes at 3,000 rpm. Homogenization is conducted using a 10 mL mortar and pestle tissue grinder. Clean the pestle using 70% ethanol following homogenization of each individual sample.
3. Transfer samples to clean ultracentrifuge tubes. Number and weigh each individual tube. Balance and pair the tubes prior to loading into the ultracentrifuge rotor.
   NOTE: All weighing and pairing of ultracentrifuge tubes must be conducted with the caps on to ensure accurate measurement of tube weights.
4. Centrifuge samples at 150,000 x g for 1 h at 4 °C.
5. Using a vacuum flask and glass Pasteur pipette, aspirate supernatant using care not to disrupt the capillary membrane pellet.
6. Based on the pellet size, add an appropriate volume of storage buffer, which is comprised of deionized water and BMB in a 1:1 (v/v) ratio. Typically, pellets are resuspended in 400-500 μL of storage buffer. Add protease inhibitor cocktail to the storage buffer in a ratio of 1.0 μL per 1.0 mL of storage buffer.
7. Vortex samples to resuspend pellet in storage buffer.
8. Using a glass Pasteur pipette, transfer capillary membrane samples to a labeled 1.5 mL microcentrifuge tube.
9. Pass sample through a needle syringe 5x to ensure the sample is thoroughly mixed and that no cellular aggregates exist.
10. Store samples at -80 °C until required for analysis.
    NOTE: Protein content of each microvessel membrane sample is measured using the Bradford method.

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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....

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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...

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Materials

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