Neuroscience
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A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels
Chapters
Summary May 7th, 2018
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.
Transcript
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.
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