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-(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 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.
	<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 into a separate, clean beaker.</li>
		<li>Slowly pour BMB into the beaker containing powdered dextran. Manually mix the powdered dextran during pouring of BMB 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 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 rongeurs, 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 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 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&#160;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 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.</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 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.</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 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>
5. Ultracentrifugation to Prepare Samples of Total Brain Microvascular Membranes
<ol>
	<li>Transfer samples to the chilled glass homogenizer.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Centrifuge samples at 150,000 x g for 1 h at 4 &#176;C.</li>
	<li>Using a vacuum flask and glass Pasteur pipette, aspirate supernatant using care not to disrupt the capillary membrane pellet.</li>
	<li>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 &#956;L of storage buffer. Add protease inhibitor cocktail to the storage buffer in a ratio of 1.0 &#956;L per 1.0 mL of storage buffer.</li>
	<li>Vortex samples to resuspend pellet in storage buffer.</li>
	<li>Using a glass Pasteur pipette, transfer capillary membrane samples to a labeled 1.5 mL microcentrifuge tube.</li>
	<li>Pass sample through a needle syringe 5x to ensure the sample is thoroughly mixed and that no cellular aggregates exist.</li>
	<li>Store samples at -80 &#176;C until required for analysis. 
	NOTE: Protein content of each microvessel membrane sample is measured using the Bradford method.</li>
</ol>

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The authors have nothing to disclose.

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&amp;trade; 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 ...

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Research

JoVE Journal

Neuroscience

10.1K Views.  University of Arizona, Tucson. 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.

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

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.

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

Medicine

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

Preparation of Newborn Rat Brain Tissue for Ultrastructural Morphometric Analysis of Synaptic Vesicle Distribution at Nerve Terminals

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial organization of synaptic vesicles. In order to obtain high-quality electron micrographs that can yield the degree of morphological detail necessary for quantitative analysis of pre-synaptic vesicle distribution, optimal specimen preparation is critical. Chemical fixation is the first step in the process of specimen preparation, and of utmost importance to preserve fine ultrastructure. Vascular fixation with a glutaraldehyde-formaldehyde solution, followed by treatment of vibratome-sectioned specimens with osmium tetroxide, stabilizes the maximum number of molecules, especially proteins and lipids, and results in superior conservation of ultrastructure. Tissue is then processed with counterstaining, sequential dehydration and resin-embedding. En bloc staining with uranyl acetate (i.e., staining of vibratome-sectioned tissue before resin embedding) enhances endogenous contrast and stabilizes cell components against extraction during specimen processing. Contrast can be further increased by applying uranyl acetate as a post-stain on ultrathin sections. Double-staining of ultrathin sections with lead citrate after uranyl acetate treatment also improves image resolution, by intensifying electron-opacity of nucleic acid-containing structures through selective binding of lead to uranyl acetate. Transmission electron microscopy is a powerful tool for characterization of the morphological details of synaptic vesicles and quantification of their size and spatial organization in the terminal bouton. However, because it uses fixed tissue, transmission electron microscopy can only provide indirect information regarding living or evolving processes. Therefore, other techniques should be considered when the main objective is to study dynamic or functional aspects of synaptic vesicle trafficking and exocytosis.

We describe procedures for processing newborn rat brain tissue to obtain high-resolution electron micrographs for morphometric analysis of synaptic vesicle distribution at nerve terminals. The micrographs obtained with these methods can also be used to study the morphology of a number of other cellular components and their dimensional structural relationships.

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...

Biology

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...

Endothelial Cell Culture from Rat Brain Microvessels: A Procedure to Isolate and Culture Endothelial Cells from Microvessels of Rat Brain

Source: Molino, Y. et al. <a href="https://www.jove.com/v/51278/setting-up-an-vitro-model-rat-blood-brain-barrier-bbb-focus-on-bbb">Setting-up an&#160;In Vitro&#160;Model of Rat Blood-Brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport.</a> J. Vis. Exp.&#160;(2014)
This video presents the isolation of microvessels from a rat brain using a combination of density gradient centrifugation and enzymatic digestion mehods. The isolated microvessels can be cultured in a culture flask pre-coated with collagen and fibronectin matrix to obtain endothelial cell colonies.

Hodowla komórek śródbłonka z mikronaczyń mózgu szczura: procedura izolacji i hodowli komórek śródbłonka z mikronaczyń mózgu szczura

Source: Molino, Y. et al. Setting-up an&#160;In Vitro&#160;Model of Rat Blood-Brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated ...

JoVE Encyclopedia of Experiments

Cancer Research

Cancers of the Nervous System

In vitro studies

Isolation and Culture of Microglia from Rat Brains using Serum-Free Media

Source: Collins, H. Y., et al., <a href="https://app.jove.com/v/57122">Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium.</a> J. Vis. Exp. (2018)
This video demonstrates a method for isolating and culturing microglia from rat brains. It entails separating microglia from other neuronal and non-neuronal cells using plates coated with a monoclonal antibody specific to microglia.

Izolacja i hodowla mikrogleju z mózgów szczurów przy użyciu pożywek bez surowicy

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Prepare a Petri Dish for CD11b ...

Neuronal Culture Techniques

Primary Neuronal Culture

Isolating Mouse Brain Vessels: A Protocol to Isolate Intact Vessels from Murine Brain Sample

Source: Boulay, A. C. et al. <a href="https://www.jove.com/video/53208" target="_blank">Purification of Mouse Brain Vessels.</a> J. Vis. Exp. (2015)
This video demonstrates the isolation of intact vessels from a mouse&#39;s brain that constitute the blood-brain barrier (BBB) along with neurons and glial cells. The isolated vessels can be used as an in vitro model to study their role in maintaining brain homeostasis.

Izolowanie naczyń mózgowych myszy: protokół izolowania nienaruszonych naczyń z próbki mózgu myszy

Source: Boulay, A. C. et al. Purification of Mouse Brain Vessels. J. Vis. Exp. (2015)
This video demonstrates the isolation of intact vessels from a ...

In vivo studies

Obtaining a Single-Cell Suspension from Frozen Rat Brain Tissue

Source: Rubio, F. J., et. al. <a href="https://app.jove.com/v/54358">Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue.</a> J. Vis. Exp. (2016)
This video demonstrates the dissociation of frozen rat brain tissue into a single-cell suspension using enzymatic digestion and mechanical trituration. The prepared cells are fixed and permeabilized with ethanol for downstream applications like flow cytometry or gene expression analysis.

Source: Rubio, F. J., et. al. Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat ...