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....

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 significantly underrepresented.......

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

List of materials used in this article

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