Name: a simple and reproducible method to prepare membrane samples from freshly isolated rat brain microvessels
Uploaded: 2018-05-07T15:00:00
Description: Watch this Scientific Journal Video about A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels at JoVE.com

This work was supported by grants from the National Institutes of Health (R01-NS084941) and the Arizona Biomedical Research Commission (ADHS16-162406) to PTR. WA has received past support from a pre-doctoral appointment to a National Institutes of Health Training Grant (T32-HL007249).

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
<ol>
	<li>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-(hydroxymet...

<ol>
	<li>Rolfe, D. F., Brown, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+energy+utilization+and+molecular+origin+of+standard+metabolic+rate+in+mammals.">Cellular energy utilization and molecular origin of standard metabolic rate in mammals.</a> Physiol Rev. 77 (3), 731-758 (1997).</li><li>Brzica, H., Abdullahi, W., Ibbotson, K., Ronaldson, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Transporters+in+Central+Nervous+System+Drug+Delivery+and+Blood-Brain+Barrier+Protection:+Relevance+to+Treatment+of+Stroke.">Role of Transporters in Central Nervous System Drug Delivery and Blood-Brain Barrier Protection: Relevance to Treatment of Stroke.</a> J Cent Nerv Syst Dis. 9, 1179573517693802 (2017).</li><li>Ronaldson, P. T., Davis, T. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+increases+blood-brain+barrier+permeability+and+induces+alterations+in+occludin+during+hypoxia-reoxygenation.">Oxidative stress increases blood-brain barrier permeability and induces alterations in occludin during hypoxia-reoxygenation.</a> J Cereb Blood Flow Metab. 30 (9), 1625-1636 (2010).</li><li>Lucke-Wold, B. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bryostatin-1+Restores+Blood+Brain+Barrier+Integrity+following+Blast-Induced+Traumatic+Brain+Injury.">Bryostatin-1 Restores Blood Brain Barrier Integrity following Blast-Induced Traumatic Brain Injury.</a> Mol Neurobiol. 52 (3), 1119-1134 (2015).</li><li>Campos, C. R., Ocheltree, S. M., Hom, S., Egleton, R. D., Davis, T. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia/reoxygenation+stress+signals+an+increase+in+organic+anion+transporting+polypeptide+1a4+(Oatp1a4)+at+the+blood-brain+barrier:+relevance+to+CNS+drug+delivery.">Hypoxia/reoxygenation stress signals an increase in organic anion transporting polypeptide 1a4 (Oatp1a4) at the blood-brain barrier: relevance to CNS drug delivery.</a> J Cereb Blood Flow Metab. 34 (4), 699-707 (2014).</li><li>Tome, M. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetaminophen+modulates+P-glycoprotein+functional+expression+at+the+blood-brain+barrier+by+a+constitutive+androstane+receptor-dependent+mechanism.">Acetaminophen modulates P-glycoprotein functional expression at the blood-brain barrier by a constitutive androstane receptor-dependent mechanism.</a> Mol Pharmacol. 84 (5), 774-786 (2013).</li><li>Artus, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Wnt/planar+cell+polarity+signaling+pathway+contributes+to+the+integrity+of+tight+junctions+in+brain+endothelial+cells.">The Wnt/planar cell polarity signaling pathway contributes to the integrity of tight junctions in brain endothelial cells.</a> J Cereb Blood Flow Metab. 34 (3), 433-440 (2014).</li><li>Yu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+exposure+to+ethanol+downregulates+tight+junction+proteins+through+the+protein+kinase+Calpha+signaling+pathway+in+human+cerebral+microvascular+endothelial+cells.">Long-term exposure to ethanol downregulates tight junction proteins through the protein kinase Calpha signaling pathway in human cerebral microvascular endothelial cells.</a> Exp Ther Med. 14 (5), 4789-4796 (2017).</li><li>Abdullahi, W., Brzica, H., Ibbotson, K., Davis, T. P., Ronaldson, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+morphogenetic+protein-9+increases+the+functional+expression+of+organic+anion+transporting+polypeptide+1a4+at+the+blood-brain+barrier+via+the+activin+receptor-like+kinase-1+receptor.">Bone morphogenetic protein-9 increases the functional expression of organic anion transporting polypeptide 1a4 at the blood-brain barrier via the activin receptor-like kinase-1 receptor.</a> J Cereb Blood Flow Metab. 37 (7), 2340-2345 (2017).</li><li>Mesev, E. V., Miller, D. S., Cannon, R. 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M., Decleves, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+drug+transporters+at+the+blood-brain+barrier+using+an+optimized+isolated+rat+brain+microvessel+strategy.">Expression of drug transporters at the blood-brain barrier using an optimized isolated rat brain microvessel strategy.</a> Brain Res. 1134 (1), 1-11 (2007).</li><li>McCaffrey, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+junctions+contain+oligomeric+protein+assembly+critical+for+maintaining+blood-brain+barrier+integrity+in+vivo.">Tight junctions contain oligomeric protein assembly critical for maintaining blood-brain barrier integrity in vivo.</a> J Neurochem. 103 (6), 2540-2555 (2007).</li><li>Brzica, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+liver+and+kidney+expression+of+sulfate+anion+transporter+sat-1+in+rats+exhibits+male-dominant+gender+differences.">The liver and kidney expression of sulfate anion transporter sat-1 in rats exhibits male-dominant gender differences.</a> Pflugers Arch. 457 (6), 1381-1392 (2009).</li><li>Ronaldson, P. 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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 literature13,20,21,22,24. Although the microves...

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 &#34;seal&#34; 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 sucrose4,5,6. Similar observations have been made at the BBB in the setting of traumatic brain injury7 and peripheral inflammatory pain8,9. These same diseases can also modulate transport mechanisms at the BBB10,11,12,13,14. 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 proteins16,17 and transporters15,18,19. 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 literature13,20,21,22. 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.

<table>
	<tbody>
		<tr>
			<td>Protease Inhibitor Cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>#P8340</td>
			<td>Component of brain microvessel buffer</td>
		</tr>
		<tr>
			<td>D-mannitol</td>
			<td>Sigma-Aldrich</td>
			<td>#M4125</td>
			<td>Component of brain microvessel buffer</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>#E3889</td>
			<td>Component of brain microvessel buffer</td>
		</tr>
		<tr>
			<td>Trizma Base</td>
			<td>Sigma-Aldrich</td>
			<td>#T1503</td>
			<td>Component of brain microvessel buffer</td>
		</tr>
		<tr>
			<td>Dextran (MW 75,000)</td>
			<td>Spectrum Chemical Mftg Corp</td>
			<td>#DE125</td>
			<td>Dextran used in centrifugation steps to separate microvessels from brain parenchyma</td>
		</tr>
		<tr>
			<td>Zetamine</td>
			<td>MWI Animal Health</td>
			<td>#501072</td>
			<td>General anesthetic</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Western Medical Supply</td>
			<td>#5530</td>
			<td>General anesthetic</td>
		</tr>
		<tr>
			<td>0.9% saline solution</td>
			<td>Western Medical Supply</td>
			<td>N/A</td>
			<td>General anesthetic diluent</td>
		</tr>
		<tr>
			<td>Filter Paper (12.5 cm diameter)</td>
			<td>VWR</td>
			<td>#28320-100</td>
			<td>Used for removal of meninges from brain tissue</td>
		</tr>
		<tr>
			<td>Centrifuge Tubes</td>
			<td>Sarstedt</td>
			<td>#60.540.386</td>
			<td>Disposable tubes used for dextran centrifugation steps</td>
		</tr>
		<tr>
			<td>Pierce&#8482; Coomassie Plus (Bradford) Assay</td>
			<td>ThermoFisher Scientific</td>
			<td>#23236</td>
			<td>Measurement of protein concentration in membrane preparations</td>
		</tr>
		<tr>
			<td>Wheaton Overhead Power Homogenizer</td>
			<td>DWK Life Sciences</td>
			<td>#903475</td>
			<td>Required for homogenization of samples</td>
		</tr>
		<tr>
			<td>10.0ml glass mortar and pestle tissue grinder</td>
			<td>DWK Life Sciences</td>
			<td>#358039</td>
			<td>Required for homogenization of samples</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Sigma-Aldrich</td>
			<td>#H1758</td>
			<td>Required for pH adjustment of buffers</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>ThermoFisher Scientific</td>
			<td>#23210</td>
			<td>Protein standard for Bradford Assay</td>
		</tr>
		<tr>
			<td>Standard Forceps</td>
			<td>Fine Science Tools</td>
			<td>#91100-12</td>
			<td>Used for dissection of brain tissue</td>
		</tr>
		<tr>
			<td>Friedman-Pearson Rongeurs</td>
			<td>Fine Science Tools</td>
			<td>#16020-14</td>
			<td>Used for opening skull to isolate brain</td>
		</tr>
		<tr>
			<td>50 ml conical centrifuge tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>#352070</td>
			<td>Used for collection of brain tissue following isolation</td>
		</tr>
		<tr>
			<td>Glass Pasteur Pipets</td>
			<td>ThermoFisher Scientific</td>
			<td>#13-678-20C</td>
			<td>Used for aspiration of cellular debris following dextran spins</td>
		</tr>
		<tr>
			<td>Ethanol, anhydrous</td>
			<td>Sigma-Aldrich</td>
			<td>#459836</td>
			<td>Used for cleaning tissue grinder; diluted to 70% with distilled water</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes</td>
			<td>Beckman-Coulter</td>
			<td>#41121703</td>
			<td>Used for ultracentrifugation of samples</td>
		</tr>
	</tbody>
</table>

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

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.

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....

Watch this Scientific Journal Video about A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels at JoVE.com

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

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.

The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting ...

The overall goal of this procedure is to isolate cerebral microvessels from rat brain for biochemical studies aimed at understanding molecular characteristics of the mammalian blood-brain barrier. This method can help to answer key questions in blood-brain barrier physiology and neuropharmacology, such as regulation, localization, and expression of tight junction proteins and endogenous transporters in health and disease. The main advantage of this technique is that we can isolate a sufficient number of high-quality microvessels from an individual animal while taking into account the natural variability in protein expression between individual animals.Demonstrating this technique will be Bianca Reilly, an outstanding undergraduate student from my laboratory. After euthanizing and decapitating a Sprague-Dawley rat according to the text protocol, use surgical scissors to resect the skin from the rat skull by making a single transverse cut. Using rongeurs, carefully remove the skull plate, and expose the brain.Then, with a spatula, remove the organ. Detach the cerebrum, and place the isolated brain tissue into a 50-milliliter conical tube with five milliliters of BMB and 5.0 microliters of protease inhibitor cocktail. To process the tissue, transfer it to a clean Petri dish.Then, using forceps, gently roll the brain on a 12.5-centimeter diameter filter paper to remove the outer meninges, which are loosely adhered to the cerebral cortex. During this step, care should be taken to ensure that all meningeal tissue has been removed from the brain tissue specimen. The meninges can be visualized as a clear membranous tissue that can be easily pulled away from the brain tissue.Gently press the brain tissue against the filter paper, and roll the tissue again, turning the filter paper frequently during this step. Then, using forceps, separate the choroid plexus from the cerebral hemispheres. Then, gently flatten the brain tissue again, and use forceps to remove the olfactory bulbs adjacent to white matter.Place the cortical brain tissue into a chilled glass mortar. Then, add five milliliters of BMB with protease inhibitor cocktail. Using an overhead power homogenizer, insert a pestle into the mortar, and homogenize the brain tissue using 15 up and down strokes at 3, 700 rpm.Then, pour the homogenate into labeled centrifuge tubes. Between samples, use 70%ethanol to clean the pestle. To carry out centrifugation, add 8.0 milliliters of 26%dextran solution to each labeled centrifuge tube containing brain homogenate.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. Centrifuge the samples at 5, 000 g and four degrees Celsius for 15 minutes.Aspirate the supernatant, and resuspend the pellet in 5.0 milliliters of BMB with protease inhibitor cocktail. Then, vortex the pellet to ensure thorough mixing. Next, add 8.0 milliliters of 26%dextran to each centrifuge tube, and vortex as just demonstrated.Then, centrifuge the samples again. Using a vacuum flask and glass pipette, aspirate the supernatant, and ensure that the pellet containing the brain microvessel is not disrupted. Resuspend the pellet in BMB with protease inhibitor and then 26%dextran two more times.After the final centrifugation, add 5.0 milliliters of BMB to each pellet and vortex to resuspend the sample. Using the protocol demonstrated in this video, successful isolation of intact microvessels from rat brain was carried out. In this image, the microvessel is stained using an antibody against Oatp1a4, a transporter that is well-expressed at the plasma membrane of brain microvascular endothelial cells.As demonstrated by western blot, membrane preparations from microvessels harvested from female Sprague-Dawley rats were enriched in PECAM-1, also known as CD31, a marker protein for brain microvasculature. During optimization of this protocol, PECAM enrichment was observed in membrane samples prepared using two and four dextran centrifugation steps. As determined by the Bradford protein assay and listed in this table, membrane preparations typically yield protein concentrations ranging between 5.0 milligrams per milliliter and 10.0 milligrams per milliliter.The protein values were determined based on a standard curve that was generated using BSA as the standard. Shown here, is a western blot for the BBB transport protein Glut1. Clear single bands at 54 kilodaltons were detected for this highly expressed BBB protein in each membrane sample.This graph illustrates a higher expression of Glut1 at the BBB in male versus female rats. No difference in expression of sodium/potassium ATPase was observed between male and female animals. Once mastered, this technique can be done in two to 2 1/2 hours if it is performed properly.While attempting this procedure, it is important to remember to keep all samples on ice throughout the protocol and to take care when aspirating the supernatant from the microvessel pellet after the centrifugation steps. Following this procedure, other methods, like co-immunoprecipitation analysis, can be performed on microvessel samples to answer additional questions, like the association of different proteins within the same protein complex at the blood-brain barrier. After its development, this technique paved the way for researchers in the field of blood-brain barrier physiology and neuropharmacology to explore molecular characteristics of the blood-brain barrier in health and in rodent models of neurological diseases.After watching this video, you should have a good understanding of how to isolate high-quality microvessels from rat brain for your own studies of blood-brain barrier biology in health and disease.

Research

JoVE Journal

Neuroscience

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