a method for the isolation of neurovascular units from rat brain

A Method for the Isolation of Neurovascular Units from Rat Brain

Place the cortical brain tissue into a chilled glass mortar. Then, add 5 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 4 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.

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Nanyang Technological University

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JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB 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 (Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid), 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.</li>
	<li>Prepare a 26% dextran (MW 75,000) solution (w/v) prior to anesthetizing animals. Follow the following steps to prepare the dextran solution. 
	NOTE: It is critically important to prepare this solution ahead of time because dextran can take approximately 1.5-2 h to dissolve in an aqueous buffer.
	<ol>
		<li>Weigh out the powdered dextran (26 g per 100 mL buffer) into a clean beaker.</li>
		<li>Measure out the appropriate volume of BMB and dispense it into a separate, clean beaker.</li>
		<li>Slowly pour BMB into the beaker containing powdered dextran. During the pouring of BMB, manually mix the powdered dextran using a glass stir rod.</li>
		<li>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).</li>
	</ol>
	</li>
</ol>
2. Extraction of Brain Tissue from Sprague-Dawley Rats
<ol>
	<li>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 (Institutional Animal Care &#38; Use Committee) guidelines. Use the same procedure for both male and female Sprague-Dawley rats.</li>
	<li>Resect the skin from the rat skull by making a single transverse cut using surgical scissors.</li>
	<li>Using rongeur, carefully remove the skull plate and expose the brain.</li>
	<li>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 &#956;L of protease inhibitor cocktail per 1.0 mL of BMB buffer immediately prior to use.</li>
</ol>
3. Brain Processing
<ol>
	<li>Transfer the brain tissue from the 50 mL conical tube to a clean petri dish.</li>
	<li>Using forceps, gently roll the brain on 12.5 cm diameter filter paper to remove the 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.</li>
	<li>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.</li>
	<li>Gently flatten the brain tissue and remove the remaining meninges and olfactory bulbs using forceps.</li>
	<li>Place the cortical brain tissue into a chilled glass mortar. Add 5.0 mL of BMB containing protease inhibitor cocktail to the mortar.</li>
	<li>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.</li>
	<li>Pour the homogenate into labeled centrifuge tubes.</li>
</ol>
4. Centrifugation Steps
<ol>
	<li>Add 8.0 mL of 26% dextran solution to each labeled centrifuge tube containing brain homogenate.</li>
	<li>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.</li>
	<li>Centrifuge samples at 5,000 x g for 15 min at 4 &#176;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 &#176;C for future use.</li>
	<li>Remove the supernatant using a vacuum aspirator and a glass Pasteur pipette. 
	NOTE: Caution must be taken not to disturb the pellet. Otherwise, the quantity of the brain microvessels collected will be reduced, which will significantly decrease the protein yield from this procedure.</li>
	<li>Resuspend the pellet in 5.0 mL of BMB-containing protease inhibitor cocktail (i.e., 1.0 &#956;L protease inhibitor cocktail per 1.0 mL of BMB buffer). Vortex the pellet to ensure thorough mixing.</li>
	<li>Add 8.0 mL of 26% dextran to each centrifuge tube and vortex as described in steps 4.1-4.2 of this protocol.</li>
	<li>Centrifuge samples at 5,000 x g for 15 min at 4 &#176;C.</li>
	<li>Using a vacuum flask and glass pipette, aspirate supernatant and ensure that the pellet containing the 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.</li>
	<li>Repeat steps 4.5 through 4.8 an additional two times.</li>
	<li>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 &#956;L aliquot of re-suspended microvessel pellet and smearing it on a glass microscope slide. Microvessels are then heat-fixed at 95 &#176;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 &#176;C until required for imaging studies.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>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>ketamine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ice bucket&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Brzica, H., et al,. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57698">A simple and reproducible method to prepare membrane samples from freshly isolated rat brain microvessels.</a> J. Vis. Exp. (2018).
This video demonstrates the process of isolating brain microvessels from euthanized rats. It includes the dissection of the brain, cleaning of the tissue, and mechanical digestion, followed by repeated centrifugation to isolate the pure fraction of microvessels.

<img alt="Figure 1" src="/files/ftp_upload/22553/22553_Figure1.jpg" /> 
Figure 1: Outline of procedures and protocols to isolate rat brain microvessels and to prepare membrane samples

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines. The procedural flow for the protocol ...

Begin with a cold glass mortar containing rat cortical brain tissue, which is the brain&#39;s outermost layer.
Add a brain microvessel buffer or BMB, containing protease inhibitors.
Now, move the pestle up and down to dissociate the tissue.
This agitation releases the neurovascular units, or NVUs, comprising microvessels, pericytes, neurons, and glial cells. The protease inhibitors block the released proteases, protecting the microvessels.
Transfer this mixture to a tube.
Add a dense polysaccharide solution. Use a vortex to mix the solution with other contents uniformly.
Centrifuge. The dense polysaccharides allow sedimentation of NVUs, which are denser than tissue debris.
Discard the supernatant and resuspend the NVUs in BMB containing protease inhibitor and polysaccharide solution.
Use the vortex to mix the contents.
Repeat the centrifugation process. Discard the supernatant to remove any residual debris.
Resuspend the NVUs in BMB and store them for further analysis.

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Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

设置了一个<em&gt;体外</em大鼠血脑屏障&gt;模型（BBB）:在BBB抗渗性和受体介导的运输为中心

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

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JoVE Journal

医学

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

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Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

免疫与感染

Obtaining Human Microglia from Adult Human Brain Tissue

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining homeostasis, and during infection or injury. Several independent research groups have highlighted the central role that microglia play in autoimmune diseases, autoinflammatory syndromes and cancers. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. Primary microglia are a powerful tool to understand the immune responses in the brain, cell-cell interactions and dysregulated microglia phenotypes in disease. Primary microglia mimic in vivo microglial properties better than immortalized microglial cell lines. Human adult microglia exhibit distinct properties as compared to human fetal and rodent microglia. This protocol provides an efficient method for isolation of primary microglia from adult human brain. Studying these microglia can provide critical insights into cell-cell interactions between microglia and other resident cellular populations in the CNS including, oligodendrocytes, neurons and astrocytes. Additionally, microglia from different human brains may be cultured for characterization of unique immune responses for personalized medicine and a myriad of therapeutic applications.

This protocol is an efficient, cost effective and robust method of isolating primary microglia from live, adult, human brain tissue. Isolated primary human microglia can serve as a tool for studying cellular processes in homeostasis and disease.

A Method for the Isolation of Neurovascular Units from Rat Brain

成績單

A Method for the Isolation of Neurovascular Units from Rat Brain