Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

Summary

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

Abstract

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans ^1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages ³ and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.

Unlike other infections, i.e neutrotopic viruses^4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.

This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Protocol

1. Infection of Mice With P. berghei-ANKA

1. Defrost an aliquot of cryopreserved P. berghei ANKA pRBC.
2. Restrain a cerebral malaria-resistant BALB/c donor mouse (8-12 weeks-old) using the two-handed restraint technique. Inject mouse with 100-200 μl of pRBC using a 1 ml insulin syringe (28G needle). Routinely, 1-2 donor mice are injected.
3. On days 4-5 post-infection (p.i) remove donor mouse from cage and place it on a workstation or a disposable working pad.
4. Gently restrain mouse by the end of the tail and using small scissors cut the tail tip (approximately 1 mm). Alternatively, a small tail puncture using a 25 G needle could be performed.
5. Place a drop of blood from the mouse's tail vein near the end of a frosted-end microscope slide.
6. Place a second slide (spreader) on a 45° angle and back it into the drop of blood. Once the blood spreads along the edge, push the spreader-slide evenly across the microscope slide to make the blood smear. Allow the smear to air dry.
7. Fix blood smears in 100% methanol for 30 sec and then stain slides in freshly prepared Giemsa for 10 min.
8. Rinse blood smear with running water for 1 min, allow to air-dry and examine slide under the microscope (100x, oil immersion). Enumerate pRBC within 1,000 erythrocytes and calculate percent parasitemia. Donor mice should reach parasitemia levels between 2.5-5% before they can be bled for infection of experimental animals.
9. Collect blood from the donor mouse by retro-orbital bleeding using a heparinized micro-hematocrit capillary tube. All experiments are performed in compliance with The Walter & Eliza Hall Institute Animal Ethics Committee requirements. Depending on local Animal Ethic Committee requirements other bleeding procedures might be used. Dissolve required amount of blood in RPMI medium at a final concentration of 1X10⁶ pRBC/0.2 ml. Consider that a normal mouse hematocrit is ~6x10⁹ red blood cells/1 ml.
10. Infect experimental C57BL/6 mice (8-12 weeks old) by injecting intraperitoneally (i.p) 1x10⁶ pRBC/0.2 ml.
11. To monitor parasitemia levels of infected mice follow steps 1.3-1.8. Parasitemia should be determined every 2-3 days starting on day 2 or 3 p.i.
12. Onset of severe malaria in C57BL/6 mice might manifest as hunched appearance, ruffed fur and low activity. Some mice may recover at this stage, but progression to loss of self-righting reflex indicates irreversible disease and animals must be euthanized.

2. Intracardial Perfusion of Mice and Brain Extraction

1. Assemble a manual gravity perfusion system by securing a 500 ml reservoir to a column holder attached to the top of a 60 cm column stand. Connect plastic tubing to the bottom end of the reservoir and secure a tubing clamp to control buffer flow. Attach tubing to a 23 G needle. Fill up the reservoir with PBS (kept at 22 °C), open clamp and allow buffer to run through the tubing to remove all air bubbles.
2. Euthanize P. berghei ANKA-infected mouse by CO₂ inhalation. Confirm death by the absence of pedal, orbital and respiratory responses.
3. Pin euthanized mouse by the hind and front paws dorsally on a styrofoam dissection board contained in a plastic tray. Depending on local Animal Ethic Committee requirements anesthesia could be administered prior commencement of intracardial pefusion.
4. Wipe ventral side with 70% ethanol.
5. Use large scissors and forceps to open skin along the midline to expose the thoracic cavity. Fold and pin skin to the sides.
6. Hold the sternum with fine forceps and cut the diaphragm and along both sides of sternum severing the ribs. Take care not to damage any large blood vessels.
7. Pin the rib cage by the sternum loosely next to the head.
8. Hold ventricles with fine forceps and carefully incise right atrium with fine scissors.
9. Insert 23G needle attached to gravity perfusion system into left ventricle towards the ascending aorta while PBS is running. Insert only 0.5 cm of the needle tip.
10. Perfuse mouse for 5 min or until effluent is clear.
11. Unpin mouse and lay it on its abdomen. Wipe head with 70% ethanol.
12. Using large scissors, make a cut just above the cervical spinal cord area.
13. Use fine scissors to make a median caudal-rostral cut starting at the base of the skull and peel skin away to expose the skull.
14. Holding the head, place the blades of a small scissors into each orbital cavity and make a cut between the orbits.
15. Make a longitudinal cut along the sagittal suture and carefully peel the skull on each brain hemisphere outward.
16. Using a spatula, gently lift the brain and place it into a 10 ml centrifuge tube containing RPMI medium.

3. Isolation of Brain-sequestered Leukocytes (BSL)

1. Working in a safety cabinet, place freshly harvested brain on top of a stainless steel cell strainer (40-60 mesh size) in a Petri dish containing 3-5 ml of RPMI tissue culture medium.
2. Cut brain tissue into small pieces.
3. Push small pieces of brain tissue through the cell strainer using a crystal plunger.
4. Transfer brain homogenate to a 10 ml tube and centrifuge at 250 x g for 10 min at 4 °C.
5. Dissolve pellet in 3 ml of RPMI medium, containing 0.05% Collagenase D and 2U/ml DNAse I.
6. Rotate mixture for 30 min at room temperature. Remove cell debris by pushing the mixture through a 70 μm nylon cell strainer and/or by incubating on ice for 5 min.
7. Lay brain homogenate onto a 7 ml 30% Percoll cushion and centrifuge at 400 x g (no brakes) for 20 min at room temperature.
8. Resuspend pellet in 1 ml of red blood cell lysis buffer and incubate on ice for 5 min to lyse adherent pRBC, which could not be removed from the brain by intracardial perfusion.
9. Add 9 ml of RPMI medium, wash cells and centrifuge at 250 x g for 10 min at 4 °C.
10. Resuspend BSL-containing pellet in 50-100 μl of RPMI medium and transfer cells to an Eppendorf tube.
11. Dilute a 5-10 μl aliquot of the cell sample 1:2 in Trypan blue for identification of viable cells.
12. Count viable cells under the microscope using a hemocytometer. From day 6 p.i onwards, when P. berghei ANKA-infected mice display neurological signs, 20,000-100,000 BSLs can be recovered.

4. Immunophenotyping of BSL by Multicolour Flow Cytometry

1. Centrifuge cells at 250 x g for 10 min at 4 °C, aspirate the supernatant and resuspend pellet in 50 μl of staining buffer (PBS, 1% foetal calf serum (FCS), 2 mm EDTA).
2. Add the 1 μl of purified anti-CD16/32 antibody (Fcblock).
3. Incubate cells for 10 min on ice.
4. Wash cells with 1 ml of Staining Buffer. Centrifuge cells at 250 x g for 10 min at 4 °C and aspirate the supernatant.
5. Resuspend cells in 50 μl of Staining Buffer containing pre-determined optimal dilutions of required fluorescent antibodies, i.e. anti-CD4, anti-CD8, anti-TCR and anti-NK1.1.
6. Incubate for 1 hr on ice.
7. Wash cells with 1 ml of Staining Buffer. Centrifuge cells at 250 x g for 10 min at 4 °C and aspirate the supernatant.
8. Resuspend cells in 100 μl of PBS.
9. Transfer cells to a flow cytometry plastic tube.
10. Using a flow cytometer, acquire at least 5,000-10,000 events. Appropriate unstained cell controls and fluorochrome compensation samples should be included as required.
11. Analyze results using appropriate flow cytometry software.

Results

The results in Fig. 2 show percentages and absolute numbers of different BSL populations recovered from brains of perfused or unperfused malaria-infected and naïve control mice. Isolated BSL were stained with PE-anti-NK1.1 and APC-anti-TCR-β antibodies as indicated in the Protocol text. Consistent with previous findings ^7-9, αβTCR⁺ T cells comprised a high proportion of the BSL pool in brains of perfused malaria-infected mice (day 6 p.i). This population appeared to be significa...

Discussion

The isolation and analysis of BSL is a method that allows characterization and quantification of inflammatory cells migrating to the brain in response to tissue injury or infection in experimental mouse models. The introduction of an intracardial perfusion step for removal of the blood compartment before organ extraction and subsequent cell isolation is useful to prevent contamination of inflammatory cells with non-inflammatory circulating leukocytes. This might not be an essential requirement in neurotropic infec...

Materials

Name	Company	Catalog Number	Comments
Solutions and buffers
Giemsa's azur eosin methylene blue solution	Merck Millipore	1.09204.0500	1:10 dilution in distilled water
RPMI medium			Mouse tonicity
Mouse tonicity PBS			20 mM Sodium Phosphate, 0.149 NaCl, pH 7.3
0.4%Trypan Blue	Sigma Aldrich	T-8154	1:2 dilution
Collagenase D	Worthington Biochemical
Deoxyribonuclease (DNAse) I	Sigma Aldrich	D4263-5VL	From bovine pancreas
Percoll	GE Healthcare	17-0891-01	30% solution in PBS
Ultrapure Tris	Invitrogen	15505-020
Ammonium Chloride (NH4Cl)	AnalaR	10017
Red Cell Lysis Buffer			17 mM Tris,14 nM NH4Cl, pH 7.2
FCS	Gibco	1009
EDTA disodium salt	Merck	10093.5V	0.1M, pH 7.2
Antibodies and conjugates
Anti-mouse CD16/CD32 (Fc Block), clone 2.4G2	BD Pharmingen	553142	1 μl in 50 μl staining buffer (0.5 mg/50 ml)
FITC-anti-mouse CD4, clone H129.19	BD Pharmingen	553651
PE-anti-mouse NK1.1, clone PK136	BD Pharmingen	553165
PerCPCy5.5-anti-mouse CD8, clone 53-6-7	BD Pharmingen	551162
APC-anti-mouse TCR-β, clone H57-597	BD Pharmingen	553174
PE-anti-mouse CXCR3, clone 220803	R&D Systems	FAB1685P
Biotinylated-anti-mouse CCR5, clone C34-3448	BD Pharmingen	559922
Steptavidin-PerCP-Cy5.5	BD Pharmingen	551419
Equipment and material
SuperFrost microscope slide	Lomb Menzel-Gläser
Dissection forceps, scissors	REDA Instrumente
500 ml PBS reservoir	Nalgene
Rubber tubing
23G needle	BD PrecisionGlide	302008
Cell dissociation kit containing metal sieve	Sigma Aldrich	CD-1
70 μm nylon cell strainer	BD Falcon	352350
Hemocytometer	GmbH Neubauer	717810
Flow cytometry tubes	BD Falcon	352008