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>

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 brain is a very active field of research3. Many efforts have been made to develop valuable models and techniques allowing cellular, molecular and pharmacological investigations of the BBB, and in particular, protocols for isolating vessels from the rodent brain. First attempts simply collected microvessels from mechanically dissociated cerebral tissue using variable pore-sized meshes to discard contaminating brain parenchymal cells13,29,30,31. Density gradient centrifugation was later introduced associated to filtration of brain vessels on nylon meshes or on glass beads columns 32,33,15. In these procedures, glass bead versus nylon mesh filtration appeared to improve yields and high- versus low-speed centrifugation, microvessel purity, although high-speed centrifugation induces also shear stress which affects gene expression 15,32. An additional step of enzymatic digestion was also introduced in some protocols following the dissociation step 34 with the aim of detaching adherent neurons and astrocytes to increase microvessel purity 35. However, an excessive pre-digestion could also disrupt microcapillary integrity 36,37. More recently, immuno-laser capture microdissection (immuno-LCM) has been shown to enable the retrieval of vessels or even of distinct cell populations in vessels, to examine changes in BBB gene expression, although the amount of material obtained by this technique is very limited 23.
Here, we present a procedure for a mechanical isolation of brain vessels initially elaborated by Yousif et al14. It offers the possibility to obtain pure brain vessels, and to separate them according to their diameter. This protocol does not pretend to allow the purification of the entire brain vascular compartment, and we did not compare its efficacy with the previous published protocols. However, the absence of gradient, specific columns and high-speed centrifugation steps makes the overall procedure very easy and inexpensive. Importantly, we strongly recommend to remove the choroid plexus as described by 38 before homogenization of the brain, as we experienced it as a possible source of contamination. Other important recommendations are: 1) the use of &#8220;low binding&#8221; plastic material (in particular pipette tips) since vessels adhere naturally to untreated plastics. Omitting this point would result in the loss of most vessels; 2) the careful equilibration of nylon filters prior to filtration (step 5.2); 3) The resuspension of the pellet in as much buffer as possible before filtration to enhance the purity of the sample; 3) If necessary, blood cells and proteins trapped in purified vessels can be washed out by intracardiac perfusion with PBS prior to brain dissection; 4) The yield of vessels purified might be improved by repeating the separation from the myelin in dextran two or three times (step 4.2)39,40. For immunostaining, we strongly recommend: 1) to perform a nuclear labeling in order to distinguish vessels from contaminating hairs and dusts for which antibodies have great non-specific affinity; 2) to avoid collecting the vessels by centrifugation before each medium change as it may result in the formation of an inextricable ball of material.
Purification of intact brain vessels present great advantages to study the molecular characteristics of BBB. Although specific or enriched endothelial and pericyte genes and pathways have been identified by transcriptomic and proteomic studies on individual cell types18,19, it is well known that dissociation and cell sorting techniques lead to the loss of cell polarity and morphology as well as interconnection between cell types, which strongly impact their molecular profiles. In this context, keeping the entire vascular compartment intact as described here, with the exception of astrocytes, might represent a great advantage. Moreover, compared to cell sorting, purification of brain vessels does not require the use of specific transgenic reporter mouse strains or lengthy immunostaining-based procedures. Another advantage to the use of brain vessels is the possibility of adapting the purification protocol to other species as already shown for murine14, bovine13 as well as in humans 41,42. Finally, a further development of the present technique would be the in vitro culture of brain vessels fragments. Co-culture of purified vessels with primary astrocytes neurons and microglial cells might help to reassemble the neurovascular unit in an accurate way than complex 3-dimensional multicellular culture systems where cells are first isolated and purified before being reassembled 43. Thus purification of intact brain vessels might be of great help for the development of any type of in vitro assays to study the BBB.

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 border="0">
	<tbody>
		<tr>
			<td>Tissue Grinder Size C</td>
			<td>Thomas scientific</td>
			<td>3431E25</td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge 5415&#160;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 47mm filter holder PP 8/Pk</td>
			<td>Millipore</td>
			<td><a href="http://www.millipore.com/catalogue/itemdetail.do?id=SX0004700">SX0004700</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon net filter disc Hydrophilic 20&#181;m 47mm 100/Pk</td>
			<td>Millipore</td>
			<td><a href="http://www.millipore.com/catalogue/itemdetail.do?id=NY2004700">NY2004700</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon net filter disc Hydrophilic 100&#181;m 47mm 100/Pk</td>
			<td>Millipore</td>
			<td><a href="http://www.millipore.com/catalogue/itemdetail.do?id=NY1H04700">NY1H04700</a></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>&#160;PARAFILM&#174; M (roll size 4 in. &#215; 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><a href="https://www.lifetechnologies.com/order/catalog/product/14175129">14175-129</a></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>PBS 10X</td>
			<td>Euromedex</td>
			<td>ET330</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Formaldehyde (w/v), Methanol-free&#160;</td>
			<td>Thermo Scientific</td>
			<td>28908</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton&#8482; 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&#174; 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 colspan="2">kindly provided by Dr Markus A Ruegg</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>&#160;dilution 1:500</td>
		</tr>
		<tr>
			<td>Anti GFAP (mouse, clone GA5)</td>
			<td>Sigma</td>
			<td>G3893</td>
			<td>&#160;dilution 1:500</td>
		</tr>
		<tr>
			<td>Anti AQP4 (rabbit)</td>
			<td>Sigma</td>
			<td>A5971</td>
			<td>&#160;dilution 1:500</td>
		</tr>
		<tr>
			<td>Anti Cx43 (mouse, Clone&#160; 2)</td>
			<td>BD Biosciences</td>
			<td>610061</td>
			<td>&#160;dilution 1:500</td>
		</tr>
		<tr>
			<td>Anti Olig2 (rabbit)</td>
			<td>Millipore</td>
			<td>AB9610</td>
			<td>&#160;dilution 1:200</td>
		</tr>
		<tr>
			<td>Anti NF-M (mouse)</td>
			<td colspan="2">provided by Dr Beat M. Riederer, University of Lausanne, Switzerland.</td>
			<td>&#160;dilution 1:10</td>
		</tr>
		<tr>
			<td>Anti Iba1 (rabbit)</td>
			<td>Wako</td>
			<td>019-19741</td>
			<td>&#160;dilution 1:400</td>
		</tr>
		<tr>
			<td>Alexa Fluor&#174; 488 Goat Anti-Mouse IgG (H+L) Antibody</td>
			<td>Life technology</td>
			<td>A11029</td>
			<td>&#160;dilution 1:2000</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor&#174; 488 conjugate</td>
			<td>Life technology</td>
			<td>A11034</td>
			<td>&#160;dilution 1:2000</td>
		</tr>
		<tr>
			<td>Alexa Fluor&#174; 555 Goat Anti-Mouse IgG (H+L) Antibody</td>
			<td>Life technology</td>
			<td>A21424</td>
			<td>&#160;dilution 1:2000</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor&#174; 555 conjugate</td>
			<td>Life technology</td>
			<td>A21429</td>
			<td>&#160;dilution 1:2000</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 described in the second part of our protocol. Agrin, a component of the endothelial basal lamina was continuously and homogeneously labeled at the endothelial surface (Figure 2A). Zona occludens-1 (ZO-1), one of the components of endothelial tight junctions was immunostained with no apparent discontinuity, suggesting that endothelial tight junctions were preserved (Figure 2B). The pericyte endothelial coverage, labeled by NG2, appeared almost continuous as previously described25 (Figure 2C). Finally, smooth muscle actin labeling revealed the presence of a dense smooth muscle cell layer at the surfaces of large purified vessels (Figure 2D). These results showed that the overall in situ brain vessel structure was conserved in the isolated vessel fragments.
Astrocytes have been shown to send large processes, or &#39;endfeet&#39; to the surface of the vessels, sheathing almost entirely the cerebrovascular system8. Their tight interaction with vessels and their role in maintaining and regulating various cerebrovascular functions, designated them as a crucial part of the neurovascular unit9. We further analyzed the astrocyte vascular coverage of isolated brain vessels. Immunolabelling of the astrocytic intermediate filament Gfap, present normally in the whole astrocyte (Figure 3A), was only partially present on purified vessels showing some short fibers at the endothelial surface (Figure 3B-C). In contrast, Connexin 43, a gap junction protein highly enriched in perivascular astroglial membranes26, was highly detected at the surface of purified large vessels (Figure 3D) and capillaries (Figure 3E). Similarly, immunolabelling of Aquaporin-4 (Aqp4), a member of the water channel family greatly expressed at the level of astrocyte membranes facing the vessels 27,26, outlining the walls of isolated vessels (Figure 3F-G). Although astrocytes were not co-purified with vessels, their perivascular endfeet membranes remained attached during the mechanical procedure of brain vessel isolation.
Finally, we examined the possible contamination of our preparations by other neural cell types. Immunolabelling of neuronal intermediate filaments (NFM) (Hornung et al, 1999) revealed the presence of few remaining fibers attached to the vessels, indicating a possible co-purification of a few neuronal terminals (Figure 4A-B). Microglia, labeled by Iba1 (Figure 4C-D) and oligodendrocytes, labeled by Olig2 (Figure 4E-F) were not detected in isolated vessels. Thus, neurons, microglia and oligodendrocytes were not co-purified with the brain vessels.
Together, these results indicate that the described protocol allows the purification of structurally preserved brain vessel fragments, upon which perivascular astrocytic membranes remain attached.
<img alt="Figure 1" src="/files/ftp_upload/53208/53208fig1.jpg" /> 
Figure 1.&#160;Summary Scheme of the Brain Vessel Purification Protocol. The principal steps are presented. This sketch indicates the three possible vessel fractions that can be obtained depending on the filters used (20 or 100 &#181;m-mesh). S1: microvessels and large vessels, S2: large vessels and S3: microvessels. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53208/53208fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53208/53208fig2.jpg" /> 
Figure 2. Isolated Vessels Retain Most of Their In Situ Structure. Confocal image projections of immunostained isolated mouse brain vessels. (A) Basal lamina immunolabelled for Agrin (red). (B) Endothelial cell tight junctions immunolabelled for Zonula Occludens (ZO-1) (red). (C) Pericytes immunolabelled for NG2 (red). (D) Smooth muscle cells immunolabelled for Smooth Muscle Actin (SMA) (red). Nuclei are stained with Hoechst (blue). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53208/53208fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53208/53208fig3.jpg" /> 
Figure 3. Astrocyte Endfeet Membranes Remain Attached to the Purified Vessels. Confocal image projections. (A) Image of a brain section showing a cortical vessel immunostained for endothelial Pecam (white), astrocytic Gfap (red) and Connexin 43 (Cx43) (green). (B, C) Gfap immunolabelling (green) at the surface of an isolated capillary labeled by Isolectin b4 (white) (B) or of a large vessel labeled for Smooth Muscle Actin (SMA) (red) (C). (C) is a Re-print with the permission from20 (D, E) Cx43 immunolabelling (green) at the surface of an isolated capillary labeled by Isolectin b4 (white) (D) or of a large vessel labeled for Smooth Muscle Actin (SMA) (red) (E). (F, G) Aquaporin 4 (Aqp4) immunolabelling (green) at the surface of an isolated capillary labeled by Isolectin b4 (white) (F) or of a large vessel labeled for Smooth Muscle Actin (SMA) (red) (G). Nuclei are stained with Hoechst (blue). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53208/53208fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53208/53208fig4.jpg" /> 
Figure 4: Neurons, Microglia and Oligodendrocytes are Not Co-purified with Brain Vessels. Confocal image projections. (A, B) Neurofilament (NFM) immunostaining on a 20 &#181;m-thick hippocampal slice (red) (A) and purified brain vessels (B). (C, B) Microglial Iba1 immunostaining on a 20 &#181;m-thick cortical slice (red) (C) and purified brain vessels (D). (E, F) Oligodendrocyte Olig2 immunostaining on a 20 &#181;m-thick cortical slice (red) (E) and purified brain vessels (F). Nuclei are stained by Hoechst (white), endothelium by Isolectine b4 (Ib4) (green). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53208/53208fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

Nanyang Technological University

Research

JoVE Journal

Neuroscience

23.3K 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

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), including gene therapy applications. AAV vectors are non-replicating, able to infect both dividing and non-dividing cells&#160;and provide long-term transgene expression. Importantly, some serotypes, such as the newly described PHP.B, can cross the blood-brain-barrier (BBB) in animal models, following systemic delivery. AAV vectors can be efficiently produced in the laboratory. However, robust and reproducible protocols are required to obtain AAV vectors with sufficient purity levels and titer values high enough for in vivo applications. This protocol describes an efficient and reproducible strategy for AAV vector production, based on an iodixanol gradient purification strategy. The iodixanol purification method is suitable for obtaining batches of high-titer AAV vectors of high purity, when compared to other purification methods. Furthermore, the protocol is generally faster than other methods currently described. In addition, a quantitative polymerase chain reaction (qPCR)-based strategy is described for a fast and accurate determination of the vector titer, as well as a silver staining method to determine the purity of the vector batch. Finally, representative results of gene delivery to the CNS, following systemic administration of AAV-PHP.B, are presented. Such results should be possible in all labs using the protocols described in this article.

Here, we describe an efficient and reproducible strategy to produce, titer, and quality-control batches of adeno-associated virus vectors. It allows the user to obtain a vector preparation with high-titer&#160;(&#8805;1 x 1013 vector genomes/mL) and a high purity, ready for in vitro or in vivo use.

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), ...

Purification of the Dendritic Filopodia-rich Fraction

Dendritic filopodia are thin and long protrusions based on the actin filament, and they extend and retract as if searching for a target axon. When the dendritic filopodia establish contact with a target axon, they begin maturing into spines, leading to the formation of a synapse. Telencephalin (TLCN) is abundantly localized in dendritic filopodia and is gradually excluded from spines. Overexpression of TLCN in cultured hippocampal neurons induces dendritic filopodia formation. We showed that telencephalin strongly binds to an extracellular matrix molecule, vitronectin. Vitronectin-coated microbeads induced phagocytic cup formation on neuronal dendrites. In the phagocytic cup, TLCN, TLCN-binding proteins such as phosphorylated Ezrin/Radixin/Moesin (phospho-ERM), and F-actin are accumulated, which suggests that components of the phagocytic cup are similar to those of dendritic filopodia. Thus, we developed a method for purifying the phagocytic cup instead of dendritic filopodia. Magnetic polystyrene beads were coated with vitronectin, which is abundantly present in the culture medium of hippocampal neurons and which induces phagocytic cup formation on neuronal dendrites. After 24 h of incubation, the phagocytic cups were mildly solubilized with detergent and collected using a magnet separator. After washing the beads, the binding proteins were eluted and analyzed by silver staining and Western blotting. In the binding fraction, TLCN and actin were abundantly present. In addition, many proteins identified from the fraction were localized to the dendritic filopodia; thus, we named the binding fraction as the dendritic filopodia-rich fraction. This article describes details regarding the purification method for the dendritic filopodia-rich fraction.

In this protocol, we introduce a method for purifying the dendritic filopodia-rich fraction from the phagocytic cup-like protrusion structure on cultured hippocampal neurons by taking advantage of the specific and strong affinity between a dendritic filopodial adhesion molecule, TLCN, and an extracellular matrix molecule, vitronectin.

Dendritic filopodia are thin and long protrusions based on the actin filament, and they extend and retract as if searching for a target axon. When the ...

Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum

Most cerebellar neurons arise from two embryonic stem niches: a rhombic lip niche, which generates all the cerebellar excitatory glutamatergic neurons, and a ventricular zone niche, which generates the inhibitory GABAergic Purkinje cells, which are neurons that constitute the deep cerebellar nuclei and Bergman glia. Recently, a third stem cell niche has been described that arises as a secondary germinal zone from the ventricular zone niche. The cells of this niche are defined by the cell surface marker prominin-1 and are localized to the developing white matter of the postnatal cerebellum. This niche accounts for the late born molecular layer GABAergic interneurons along with postnatally generated cerebellar astrocytes. In addition to their developmental role, this niche is gaining translational importance in regards to its involvement in neurodegeneration and tumorigenesis. The biology of these cells has been difficult to decipher because of a lack of efficient techniques for their purification. Demonstrated here are efficient methods to purify, culture, and differentiate these postnatal cerebellar stem cells.

Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum

Demonstrated here is an efficient and cost-effective method to purify, culture, and differentiate white matter stem cells from postnatal mouse cerebellum.

Most cerebellar neurons arise from two embryonic stem niches: a rhombic lip niche, which generates all the cerebellar excitatory glutamatergic neurons, ...

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell resolution using electrophysiology, microscopy, and pharmacology. However, a major consideration in the design of in vitro experiments is the extent to which different slice preparations recapitulate naturalistic patterns of neural activity as observed in vivo. In the intact brain, the hippocampal network generates highly synchronized population activity reflective of the behavioral state of the animal, as exemplified by the sharp-wave ripple complexes (SWRs) that occur during waking consummatory states or non-REM sleep. SWRs and other forms of network activity can emerge spontaneously in isolated hippocampal slices under appropriate conditions. In order to apply the powerful brain slice toolkit to the investigation of hippocampal network activity, it is necessary to utilize an approach that optimizes tissue health and the preservation of functional connectivity within the hippocampal network. Mice are transcardially perfused with cold sucrose-based artificial cerebrospinal fluid. Horizontal slices containing the hippocampus are cut at a thickness of 450 &#956;m to preserve synaptic connectivity. Slices recover in an interface-style chamber and are transferred to a submerged chamber for recordings. The recording chamber is designed for dual surface superfusion of artificial cerebrospinal fluid at a high flow rate to improve oxygenation of the slice. This protocol yields healthy tissue suitable for the investigation of complex and spontaneous network activity in vitro.

This protocol describes the preparation of horizontal hippocampal-entorhinal cortex (HEC) slices from mice exhibiting spontaneous sharp-wave ripple activity. Slices are incubated in a simplified interface holding chamber and recordings are performed under submerged conditions with fast-flowing artificial cerebrospinal fluid to promote tissue oxygenation and the spontaneous emergence of network-level activity.

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...