This work was supported by the Labex MemoLife and by the ARSEP (Fondation pour l&#8217;aide &#224; la recherche sur la scl&#233;rose en plaques)

1. Solutions and Materials &#160;
<ol>
	<li>Prepare isolation vessel solutions: B1, add 1.5 ml of HEPES 1M to 150 ml of HBSS; B2, add 3.6 g of Dextran to 20 ml of B1; B3, add 1 g of BSA to 100 ml of B1.</li>
	<li>Modify the filter holder by cutting the bottom off the upper screwing part.</li>
	<li>Prepare immunostaining solutions: Fixation solution, 4% Paraformaldehyde in PBS pH 7.4; Permeabilization/blocking solution, dilute goat serum to 5% and Triton X100 to 0.25% in PBS pH 7.4. 
	Note: Fixation depends on the type of primary antibodies used.</li>
</ol>
2. Dissection
Note: Sterile conditions are not required, unless vessels are used for cell culture purposes.
<ol>
	<li>Prepare a 150 ml beaker with 20 ml of B1. Keep on ice and cover with parafilm to avoid air contamination.</li>
	<li>Deeply anesthetize the mouse under a hood with a small paper towel soaked in 1 ml of pure Isoflurane that is added into the cage. Anesthesia is verified by a lack of reaction to a toe pinch. Kill the mouse by cervical dislocation. 
	NOTE: These steps are accomplished in compliance with national and institutional regulations.</li>
	<li>Optional: Perform intracardiac perfusion with 20 ml of PBS 1x&#160;to eliminate the blood content24.</li>
	<li>Section the skin with a scalpel from the neck to the nose and pull it away. Remove all contaminating hairs with PBS 1x.</li>
	<li>To open the skull, first insert scissors anteriorly to the olfactory bulb, and open the scissors to rupture the skull in two parts.</li>
	<li>Carefully remove the brain using a brain spatula. Dissect out the choroid plexus from the lateral ventricles as they would contaminate the blood vessel preparation38. 
	NOTE: Optional: The final preparation will contain parenchymal and meninges vessels. If not desired, meninges can be peeled off following the procedure described by&#160;38.</li>
	<li>Transfer the brain into the beaker containing B1 solution on ice. Up to 8 brains can be treated together.</li>
</ol>
3. Brain Tissue Homogenization
<ol>
	<li>Using two scalpels, manually and vigorously beat the brain in the B1 solution subsequently obtaining small pieces of about 2 mm.</li>
	<li>Homogenize the preparation with an automatized Dounce homogenizer, performing 20 strokes at 400 rpm. Ensure that the glass tube is maintained in ice and that the upper part of the douncer is in solution when moving up and down, so as to prevent the formation of air pockets. If several samples are prepared, wash the douncer with ionized water between each homogenization.</li>
</ol>
4. Vessel Purification
<ol>
	<li>Transfer the homogenate into a 50 ml plastic tube and proceed to the centrifugation at 2,000 g for 10 min at 4 &#176;C. A large white interface (mostly myelin) will form on the top of the vessel pellet (red if no perfusion was performed) .</li>
	<li>Discard the supernatant. The vessel pellet and the white interface will remain attached together. Add 20 ml of ice-cold B2 solution and shake the tube manually and vigorously for 1 min.</li>
	<li>Proceed to the second centrifugation at 4,400 g for 15 min at 4 &#176;C. The myelin will now form a dense white layer at the surface of the supernatant.</li>
	<li>Carefully detach the myelin layer from the tube walls by holding the tube and slowly rotating it to allow the supernatant to pass along the walls. Discard myelin with the supernatant. The pellet containing the vessels remains attached at the bottom of the tube.</li>
	<li>Blot the inside wall of the tube with an absorbent paper wrapped around a 5 ml plastic pipette and remove all residual fluids, avoid touching the vessel pellet. Keep the tube upside-down on an absorbent paper to drain any remaining liquid.</li>
	<li>Suspend the pellet in 1 ml of ice-cold B3 solution by pipetting up and down with low-binding tips , keeping the tube on ice, then add another 5 ml of B3 solution. Make sure that vessels are dispersed as much as possible and do not form aggregates.</li>
</ol>
5. Filtration
<ol>
	<li>Prepare a beaker on ice with 30 ml of ice-cold B3 solution. Cover with parafilm to avoid air contamination.</li>
	<li>Place a 20 &#181;m-mesh filter on a modified filter holder on the top of a becker&#160;flask and equilibrate by applying 10 ml of ice-cold B3 solution.</li>
	<li>Pour the vessel preparation on the filter and rinse the vessels with 40 ml of ice-cold B3 solution.</li>
	<li>Recover the filter using clean forceps and immediately immerse it in the beaker containing the B3 solution. Detach the vessels from the filter by shaking it gently.</li>
	<li>Pour the beaker content in a 50 ml plastic tube and centrifuge at 2,000 g for 5 min at 4 &#176;C.</li>
	<li>Note: Alternatively, the brain vessels&#8217; suspension from step 4.6 can be filtered onto a 100&#160;&#181;m-mesh filter. In this case, larger vessels are preferentially retained on the filter while the flow-through contains microvessels (mainly capillaries), which are then filtered on a 20 &#181;m-mesh filter as above.</li>
	<li>Resuspend the pellet of microvessels in 1 ml of ice-cold B3 solution and transfer it by pipetting it into a 1.5 ml Eppendorf tube. Centrifuge at 2,000 g for 5 min at 4 &#176;C.</li>
</ol>
6. Fixation, Permeabilization and Blocking
<ol>
	<li>Transfer vessels by pipetting into 0.2 ml PCR tubes containing PBS using a 1.0 ml low binding tip. Be careful not touching the vessels as they may adhere to the outside part of the tip. Perform all the following steps under a binocular microscope.</li>
	<li>For each of the following medium changes, leave the tubes on ice until the vessels have reached the bottom, and pipette out most of the liquids using long and thing gel-loading pipet tips.</li>
	<li>Remove most of the PBS and add 200 &#181;l of fixative solution. Suspend the vessels by shaking gently and incubate for 20 min at RT.</li>
	<li>Pipette out the fixative solution and replace it with 200 &#181;l of 1x&#160;PBS&#160;(first wash), incubate for 5 min at RT and repeat this step 3 times. Each time, verify that vessels have correctly sunk to the bottom of the tubes and pipette out 1x&#160;PBS, ensuring the vessels are not touched.</li>
	<li>After the last wash, replace 1x&#160;PBS by the permeabilization/blocking solution and incubate 1 hr at RT, resuspending the vessels by shaking gently from time to time.</li>
</ol>
7. Immunostaining
<ol>
	<li>Replace the permeabilization/blocking solution with the mix of primary antibodies (see references of the antibodies used here and dilutions in the Materials template table) diluted in the permeabilization/blocking solution, and incubate at 4 &#176;C O/N.</li>
	<li>After 3 washes in PBS at RT, incubate the vessels with the mix of secondary antibodies diluted in PBS for 2 hr at RT.</li>
	<li>Note: we strongly recommend performing nuclear staining with Hoechst (1:2,000) or DAPI (1:2,000) in order to distinguish vessels from possible contaminating hairs or dusts under the microscope.</li>
	<li>After 3 washes in PBS, resuspend the vessels in 50 &#181;l of PBS.</li>
</ol>
8. Mounting and Observation
<ol>
	<li>Prepare a tip with a siliconized glass capillary: cut a P200 pipette tip using a scalpel and adjust the capillary inside. The approximate volume of the capillary is 40 &#956;l.</li>
	<li>Transfer the vessels to a glass slide using the siliconized glass capillary and carefully remove the liquid with a piece of absorbent paper.</li>
	<li>Apply a single drop of mounting medium onto the vessels and hold a coverslip at 45&#186; allowing the drop to spread along the edge of the slip. Let go off the coverslip and allow medium to spread slowly. Let it dry O/N&#160;at RT with protection from light. Vessels can be observed under a fluorescent confocal microscope.</li>
</ol>

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<li>Warren, M. S., Zerangue, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+gene+expression+profiles+of+ABC+transporters+in+brain+microvessel+endothelial+cells+and+brain+in+five+species+including+human%5BTitle%5D)+AND+%22Pharmacological+Research%22%5BJournal%5D)">Comparative gene expression profiles of ABC transporters in brain microvessel endothelial cells and brain in five species including human.</a> Pharmacological Research. 59 (6), 404-413 (2009).</li>
<li>Geier, E. G., Chen, E. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Profiling+Solute+Carrier+Transporters+in+the+Human+Blood-Brain+Barrier%5BTitle%5D)+AND+%22Clinical+Pharmacology+and+Therapeutics%22%5BJournal%5D)">Profiling Solute Carrier Transporters in the Human Blood-Brain Barrier.</a> Clinical Pharmacology and Therapeutics. 94 (6), 636-639 (2013).</li>
<li>Seetharaman, S., Barrand, M. A., Maskell, L., Scheper, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multidrug+Resistance-Related+Transport+Proteins+in+Isolated+Human+Brain+Microvessels+and+in+Cells+Cultured+from+These+Isolates%5BTitle%5D)+AND+%22Journal+of+Neurochemistry%22%5BJournal%5D)">Multidrug Resistance-Related Transport Proteins in Isolated Human Brain Microvessels and in Cells Cultured from These Isolates.</a> Journal of Neurochemistry. 70 (3), 1151-1159 (1998).</li>
<li>Urich, E., Patsch, C., Aigner, S., Graf, M., Iacone, R., Freskgard, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multicellular+self-assembled+spheroidal+model+of+the+blood+brain+barrier%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Multicellular self-assembled spheroidal model of the blood brain barrier.</a> Sci Rep. 3, 1500(2013).</li>
</ol>

The authors have nothing to disclose.

The blood&#8211;brain barrier regulates the passage of physiological substances in and out of the CNS and protects it against potentially harmful substances present in the blood. It is involved in several CNS pathologies, including neurodegenerative diseases2 and brain tumors28. The extremely low permeability of the BBB hampers also the passage of therapeutic agents targeting neural cells and the development of methods intending to reversibly open the BBB with no deleterious consequences for the bra...

Proper function of the central nervous system (CNS) requires a highly regulated extracellular environment, and its metabolic demands are huge compared to other organs1. The CNS is also extremely sensitive to a wide range of chemicals, generally harmless to peripheral organs but to it, neurotoxic. To ensure correct functioning, most of the CNS&#39; vasculature forms an endothelial barrier; the blood-brain barrier (BBB), which controls the flow of molecules and ions as well as the passage of immune cells between the blood and the brain, thereby maintaining proper homeostasis2, but also limiting the entry of therapeutic drugs, thus hampering treatments of neurological disorders3. At the cellular level, the BBB is mainly sustained by extensive tight junctions between endothelial cells, polarized expression of efflux transporters and a very low transcytosis rate4. Properties and functions of the BBB are mostly induced by neighboring cells4. In particular, pericytes play an important role in inducing and maintaining the BBB5,6. Being contractile cells, pericytes also regulate blood flow7 as do the smooth muscle cells surrounding large vessels. Finally, astrocytes, the major glial cells of the brain, send large processes named endfeet around most of the brain vasculature8 and modulate BBB integrity and immune quiescence9, the transfer of metabolites to neurons10, and induce the tight coupling between neuronal activity and blood flow11,12.
The ability to study the molecular and cellular properties of the cerebrovascular system is crucial to characterize better its contribution to brain physiology and physiopathology. To tackle this question, strategies to isolate the brain&#39;s cerebrovascular system have been developed, which allow for the preparation of intact brain vessel fragments. Cerebral vessel purification was initially described using bovine brains13 and improved and adapted to other species, in particular rodents14. In this last study, the use of filters of varying size was introduced to separate brain vessels in to fractions enriched in vessels of different diameters. Interestingly, in such preparations, endothelial cells kept their metabolic properties15, transporter functionality16 and polarization17. Here, we describe in detail this protocol and further demonstrate that isolated vessels retain most of their in situ structures. Endothelial cells remain linked by tight junctions and surrounded by a continuous basal lamina. Pericytes and smooth muscle cells remain attached to the endothelial layer, as well as perivascular astrocyte membranes. However, astrocytes, microglial cells, neurons and oligodendrocytes are eliminated. Lastly, we describe a procedure to perform immunostaining on isolated brain vessels.
Until now most of the molecular and cellular studies concerning the cerebrovascular system have been performed on purified brain vessel cells dissociated by cell-sorting using cell specific reporter mouse strains or immunostaining-based procedures18,19. Although these techniques allows for the isolation of almost pure cerebrovascular cell populations, isolated cells completely lose their in situ morphology and interactions, which in turn, greatly affects their molecular and cellular properties. The protocol described here, allowing for the isolation of whole cerebrovascular fragments with no need of specific antibodies or transgenic mouse stains, offers a good alternative as the overall structure of isolated cerebral vessels is conserved, thus, lessening repercussions on their molecular properties. Isolated vessels might then be used for studying gene activity, protein synthesis and regulation at the BBB as recently described20,21. Finally, compared to laser capture microdissection22,23&#160;the present protocol is inexpensive, easy to perform and rapidly adaptable in to any laboratory.

<table><tbody><tr><td>Tissue Grinder Size C</td><td>Thomas scientific</td><td>3431E25</td><td></td></tr><tr><td>centrifuge 5415&amp;nbsp;R</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>centrifuge 5810 R</td><td>Eppendorf</td><td>5811000320</td><td></td></tr><tr><td>High-performance, Modular Stereomicroscope</td><td>Leica</td><td>MZ6</td><td></td></tr><tr><td>Compact System Provides High Quality Leica LED1000</td><td>Leica</td><td>LED1000</td><td></td></tr><tr><td>low binding tips (P1000)</td><td>Sorenson BioScience</td><td>14200T</td><td></td></tr><tr><td>Swinnex 47 mm filter holder PP 8/Pk</td><td>Millipore</td><td>&lt;a href=&quot;http://www.millipore.com/catalogue/itemdetail.do?id=SX0004700&quot;&gt;SX0004700&lt;/a&gt;</td><td></td></tr><tr><td>Nylon net filter disc Hydrophilic 20 &amp;micro;m 47 mm 100/Pk</td><td>Millipore</td><td>&lt;a href=&quot;http://www.millipore.com/catalogue/itemdetail.do?id=NY2004700&quot;&gt;NY2004700&lt;/a&gt;</td><td></td></tr><tr><td>Nylon net filter disc Hydrophilic 100 &amp;micro;m 47 mm 100/Pk</td><td>Millipore</td><td>&lt;a href=&quot;http://www.millipore.com/catalogue/itemdetail.do?id=NY1H04700&quot;&gt;NY1H04700&lt;/a&gt;</td><td></td></tr><tr><td>Standard Wall Borosilicate Tubing</td><td>Sutter Instrument</td><td>B150-86-7.5</td><td></td></tr><tr><td>Microscope Slides</td><td>Thermo Scientific</td><td>1014356290F</td><td></td></tr><tr><td>Cover Slips, Thickness 1</td><td>Thermo Scientific</td><td>P10143263NR1</td><td></td></tr><tr><td>0.2 ml Thin-walled tubes and domed cap</td><td>Thermo Scientific</td><td>AB-0266</td><td></td></tr><tr><td>&amp;nbsp;PARAFILM M (roll size 4 in. &amp;times; 125 ft)</td><td>Sigma</td><td>P7793-1EA</td><td></td></tr><tr><td>HBSS, no calcium, no magnesium, no phenol red</td><td>Life technology</td><td>&lt;a href=&quot;https://www.lifetechnologies.com/order/catalog/product/14175129&quot;&gt;14175-129&lt;/a&gt;</td><td></td></tr><tr><td>HEPES (1M)</td><td>Life technology</td><td>15630056</td><td></td></tr><tr><td>Dextran from Leuconostoc spp. Mr ~70,000</td><td>Sigma</td><td>31390</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A2153</td><td></td></tr><tr><td>10x PBS</td><td>Euromedex</td><td>ET330</td><td></td></tr><tr><td>16% Formaldehyde (w/v), Methanol-free&amp;nbsp;</td><td>Thermo Scientific</td><td>28908</td><td></td></tr><tr><td>Triton&lt;sup&gt; &lt;/sup&gt; X-100</td><td>Sigma</td><td>X100</td><td></td></tr><tr><td>bisBenzimide H 33342 trihydrochloride (Hoechst)</td><td>Sigma</td><td>14533</td><td></td></tr><tr><td>Mounting medium Fluoromount-G</td><td>Southern Biotech</td><td>0100-01</td><td></td></tr><tr><td>Isolectin GS-IB4 From Griffonia simplicifolia, Alexa Fluor 488 Conjugate; Dilution 1/100</td><td>Life technology</td><td>I21411</td><td></td></tr><tr><td>Agrin (rabbit) ; dilution 1/400</td><td>kindly provided by Dr Markus A Ruegg</td><td></td><td></td></tr><tr><td>Anti ZO-1 (mouse, clone 1A12)</td><td>Life technology</td><td>33-9100</td><td>dilution 1:500</td></tr><tr><td>Anti Smooth Muscle Actin (mouse, clone 1A4)</td><td>Sigma</td><td>A2547</td><td>&amp;nbsp;dilution 1:500</td></tr><tr><td>Anti GFAP (mouse, clone GA5)</td><td>Sigma</td><td>G3893</td><td>&amp;nbsp;dilution 1:500</td></tr><tr><td>Anti AQP4 (rabbit)</td><td>Sigma</td><td>A5971</td><td>&amp;nbsp;dilution 1:500</td></tr><tr><td>Anti Cx43 (mouse, Clone&amp;nbsp; 2)</td><td>BD Biosciences</td><td>610061</td><td>&amp;nbsp;dilution 1:500</td></tr><tr><td>Anti Olig2 (rabbit)</td><td>Millipore</td><td>AB9610</td><td>&amp;nbsp;dilution 1:200</td></tr><tr><td>Anti NF-M (mouse)</td><td>provided by Dr Beat M. Riederer, University of Lausanne, Switzerland.</td><td>&amp;nbsp;dilution 1:10</td><td></td></tr><tr><td>Anti Iba1 (rabbit)</td><td>Wako</td><td>019-19741</td><td>&amp;nbsp;dilution 1:400</td></tr><tr><td>Alexa Fluor 488 Goat Anti-Mouse IgG (H+L) Antibody</td><td>Life technology</td><td>A11029</td><td>&amp;nbsp;dilution 1:2,000</td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td><td>Life technology</td><td>A11034</td><td>&amp;nbsp;dilution 1:2,000</td></tr><tr><td>Alexa Fluor 555 Goat Anti-Mouse IgG (H+L) Antibody</td><td>Life technology</td><td>A21424</td><td>&amp;nbsp;dilution 1:2,000</td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 555 conjugate</td><td>Life technology</td><td>A21429</td><td>&amp;nbsp;dilution 1:2,000</td></tr></tbody></table>

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.

Here, we describe a protocol allowing for the mechanical isolation of brain vessels14. Figure 1 summarizes the main steps of this technique. The architecture of brain vessels is complex and includes several cell types, i.e., endothelial cells sealed by tight junctions and surrounded by pericytes, smooth muscle cells, and astrocyte foot processes9. Thus, following isolation of brain vessels, we aimed to characterize the structure of purified vessels by immunostaining as desc...

Watch this Scientific Journal Video about Purification of Mouse Brain Vessels at JoVE.com

Purification of Mouse 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 ...

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Neuroscience

23.4K Views.  Institut National de la Santé et de la Recherche Médicale. 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. 

Video: Purification of Mouse Brain Vessels

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). Animal models are necessary for studying the etiopathology and potential therapies of cerebrovascular diseases. Imaging the vasculature in large animals is relatively easy. However, developing vessel imaging methods of murine brain disease models is desirable due to the cost and availability of genetically-modified mouse lines. Imaging the murine cerebral vascular tree is a challenge. In humans and larger animals, the gold standard for assessing the angioarchitecture at the macrovascular (conductance) level is x-ray catheter contrast-based angiography, a method not suited for small rodents.
In this article, we present a method of cerebrovascular casting that produces a durable skeleton of the entire vascular bed, including arteries, veins, and capillaries that may be analyzed using many different modalities. Complete casting of the microvessels of the mouse cerebrovasculature can be difficult; however, these challenges are addressed in this step-by-step protocol. Through intracardial perfusion of the vascular casting material, all vessels of the body are casted. The brain can then be removed and clarified using the organic solvent methyl salicylate. Three dimensional imaging of the brain blood vessels can be visualized simply and inexpensively with any conventional brightfield microscope or dissecting microscope. The casted cerebrovasculature can also be imaged and quantified using micro-computed tomography (micro-CT)1. In addition, after being imaged, the casted brain can be embedded in paraffin for histological analysis.
The benefit of this vascular casting method as compared to other techniques is its broad adaptation to various analytic tools, including brightfield microscopic analysis, CT scanning due to the radiopaque characteristic of the material, as well as histological and immunohistochemical analysis. This efficient use of tissue can save animal usage and reduce costs. We have recently demonstrated application of this method to visualize the irregular blood vessels in a mouse model of adult BAVM at a microscopic level2, and provide additional images of the malformed vessels imaged by micro-CT scan. Although this method has drawbacks and may not be ideal for all types of analyses, it is a simple, practical technique that can be easily learned and widely applied to vascular casting of blood vessels throughout the body.

In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.
Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the F&ouml;rster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The ...

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

Dolichoectasia: Vascular Dementia and Alzheimer&#39;s Disease Models

A Visual Approach for Inducing Dolichoectasia in Mice to Model Large Vessel-Mediated Cerebrovascular Dysfunction

The blood-brain (BBB) is a crucial system that regulates selective brain circulation with the periphery, as an example, allowing necessary nutrients to enter and expel excessive amino acids or toxins from the brain. To model how the BBB can be compromised in diseases like vascular dementia (VaD) or Alzheimer's disease (AD), researchers developed novel methods to model vessel dilatation. A compromised BBB in these disease states can be detrimental and result in the dysregulation of the BBB leading to untoward and pathological consequences impacting brain function. We were able to modify an existing technique that enabled us to inject directly into the Cisterna magna (CM) to induce dilatation of blood vessels using elastase, and disrupt the tight junctions (TJ) of the BBB. With this method, we were able to see various metrics of success over previous techniques, including consistent blood vessel dilatation, reduced mortality or improved recovery, and improving the fill/opacifying agent, a silicone rubber compound, delivery for labeling blood vessels for dilatation analysis. This modified minimally invasive method has had promising results, with a 19%-32% increase in sustained dilatation of large blood vessels in mice from 2 weeks to 3 months post-injection. This improvement contrasts with previous studies, which showed increased dilatation only at the 2 week mark. Additional data suggests sustained expansion even after 9.5 months. This increase was confirmed by comparing the diameter of blood vessels of the elastase and the vehicle-injected group. Overall, this technique is valuable for studying pathological disorders that affect the central nervous system (CNS) using animal models.

Author Spotlight: Modeling Vascular Contributions to Alzheimer's Disease in Transgenic Mice

We demonstrate chemically inducing large blood vessel dilatation in mice as a model for investigating cerebrovascular dysfunction, which can be used for vascular dementia and Alzheimer's disease modeling. We also demonstrate visualizing the vasculature by injecting silicone rubber compound and providing clear visual guidance for measuring changes in blood vessel size.

The blood-brain (BBB) is a crucial system that regulates selective brain circulation with the periphery, as an example, allowing necessary nutrients to ...

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 ...

Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma

Cerebral blood flow is conveyed by vascular resistance arteries and downstream parenchymal arterioles. Steady-state vascular resistance to blood flow increases with decreasing diameter from arteries to arterioles that ultimately feed into capillaries. Due to their smaller size and location in the parenchyma, arterioles have been relatively understudied and with less reproducibility in findings than surface pial arteries. Regardless, arteriolar endothelial cell structure and function&#8212;integral to the physiology and etiology of chronic degenerative diseases&#8212;requires extensive investigation. In particular, emerging evidence demonstrates that compromised endothelial function precedes and exacerbates cognitive impairment and dementia.
In the parenchymal microcirculation, endothelial K+ channel function is the most robust stimulus to finely control the spread of vasodilation to promote increases in blood flow to areas of neuronal activity. This paper illustrates a refined method for freshly isolating intact and electrically coupled endothelial &#34;tubes&#34; (diameter, ~25 &#181;m) from mouse brain parenchymal arterioles. Arteriolar endothelial tubes are secured during physiological conditions (37 &#176;C, pH 7.4) to resolve experimental variables that encompass K+ channel function and their regulation, including intracellular Ca2+ dynamics, changes in membrane potential, and membrane lipid regulation. A distinct technical advantage versus arterial endothelium is the enhanced morphological resolution of cell and organelle (e.g., mitochondria) dimensions, which expands the usefulness of this technique. Healthy cerebral perfusion throughout life entails robust endothelial function in parenchymal arterioles, directly linking blood flow to the fueling of neuronal and glial activity throughout precise anatomical regions of the brain. Thus, it is expected that this method will significantly advance the general knowledge of vascular physiology and neuroscience concerning the healthy and diseased brain.

Intensive preparation of intact mouse cerebral endothelial &#34;tubes&#34; from cerebral parenchymal arterioles is illustrated for studying cerebral blood flow regulation. Further, we demonstrate the experimental strengths of this endothelial study model for fluorescence imaging and electrophysiology measurement of key cellular signaling pathways, including changes in intracellular [Ca2+] and membrane potential.

Cerebral blood flow is conveyed by vascular resistance arteries and downstream parenchymal arterioles. Steady-state vascular resistance to blood flow ...

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.

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 ...

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Cancer Research

Cancers of the Nervous System

In vivo studies

Preparing Cerebral Endothelial Tubes from Mouse Parenchymal Arterioles

Source: Hakim, M. A., et al., <a href="https://app.jove.com/v/63463">Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma.</a> J. Vis. Exp. (2022)
This video demonstrates a method for isolating the endothelial tubes from mouse cerebral parenchyma. Through a combination of enzymatic digestion and mechanical dissociation, the process yields an intact tube with endothelial cells.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1.&#160;Solutions and drugs

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Neuronal Culture Techniques

Advanced Neuronal Models

Purifying Capillaries From a Porcine Brain

Source: Nielsen, S. S. E., et al. <a href="https://app.jove.com/v/56277">Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells.</a> J. Vis. Exp. (2017).
This video demonstrates the isolation of capillaries from fresh porcine brains. The process involves removing the meninges, isolating the grey matter from the brain tissue, homogenizing and filtering the grey matter to isolate the capillaries, and finally using enzymatic digestion to purify the capillaries further.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Purification of Porcine Brain ...

Purification of Mouse Brain Vessels

Samenvatting

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