Purification of Mouse Brain Vessels

Summary

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.

Abstract

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’s disease, Alzheimer’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.

Introduction

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' 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 BBB^5,6. 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 flow^11,12.

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'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 procedures^18,19. 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 described^20,21. Finally, compared to laser capture microdissection^22,23 the present protocol is inexpensive, easy to perform and rapidly adaptable in to any laboratory.

Protocol

1. Solutions and Materials  

1. 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.
2. Modify the filter holder by cutting the bottom off the upper screwing part.
3. 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.

2. Dissection

Note: Sterile conditions are not required, unless vessels are used for cell culture purposes.

1. Prepare a 150 ml beaker with 20 ml of B1. Keep on ice and cover with parafilm to avoid air contamination.
2. 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.
3. Optional: Perform intracardiac perfusion with 20 ml of PBS 1x to eliminate the blood content²⁴.
4. Section the skin with a scalpel from the neck to the nose and pull it away. Remove all contaminating hairs with PBS 1x.
5. To open the skull, first insert scissors anteriorly to the olfactory bulb, and open the scissors to rupture the skull in two parts.
6. 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 ³⁸.
7. Transfer the brain into the beaker containing B1 solution on ice. Up to 8 brains can be treated together.

3. Brain Tissue Homogenization

1. Using two scalpels, manually and vigorously beat the brain in the B1 solution subsequently obtaining small pieces of about 2 mm.
2. 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.

4. Vessel Purification

1. Transfer the homogenate into a 50 ml plastic tube and proceed to the centrifugation at 2,000 g for 10 min at 4 °C. A large white interface (mostly myelin) will form on the top of the vessel pellet (red if no perfusion was performed) .
2. 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.
3. Proceed to the second centrifugation at 4,400 g for 15 min at 4 °C. The myelin will now form a dense white layer at the surface of the supernatant.
4. 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.
5. 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.
6. 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.

5. Filtration

1. Prepare a beaker on ice with 30 ml of ice-cold B3 solution. Cover with parafilm to avoid air contamination.
2. Place a 20 µm-mesh filter on a modified filter holder on the top of a becker flask and equilibrate by applying 10 ml of ice-cold B3 solution.
3. Pour the vessel preparation on the filter and rinse the vessels with 40 ml of ice-cold B3 solution.
4. 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.
5. Pour the beaker content in a 50 ml plastic tube and centrifuge at 2,000 g for 5 min at 4 °C.
6. Note: Alternatively, the brain vessels’ suspension from step 4.6 can be filtered onto a 100 µm-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 µm-mesh filter as above.
7. 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 °C.

6. Fixation, Permeabilization and Blocking

1. 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.
2. 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.
3. Remove most of the PBS and add 200 µl of fixative solution. Suspend the vessels by shaking gently and incubate for 20 min at RT.
4. Pipette out the fixative solution and replace it with 200 µl of 1x PBS (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 PBS, ensuring the vessels are not touched.
5. After the last wash, replace 1x PBS by the permeabilization/blocking solution and incubate 1 hr at RT, resuspending the vessels by shaking gently from time to time.

7. Immunostaining

1. 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 °C O/N.
2. After 3 washes in PBS at RT, incubate the vessels with the mix of secondary antibodies diluted in PBS for 2 hr at RT.
3. 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.
4. After 3 washes in PBS, resuspend the vessels in 50 µl of PBS.

8. Mounting and Observation

1. 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 μl.
2. Transfer the vessels to a glass slide using the siliconized glass capillary and carefully remove the liquid with a piece of absorbent paper.
3. Apply a single drop of mounting medium onto the vessels and hold a coverslip at 45º 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 at RT with protection from light. Vessels can be observed under a fluorescent confocal microscope.

Results

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...

Discussion

The blood–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...

Materials

List of materials used in this article

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