The authors would like to thank Miss Liana Mackiewicz for technical assistance. This work was made possible through Victorian State Government Operational Infrastructure Support and Australian Government National Health and Medical Research Council IRIISS and Project Grant 1031212.

1. Infection of Mice With P. berghei-ANKA
<ol>
 <li>Defrost an aliquot of cryopreserved P. berghei ANKA pRBC.</li>
 <li>Restrain a cerebral malaria-resistant BALB/c donor mouse (8-12 weeks-old) using the two-handed restraint technique. Inject mouse with 100-200 &mu;l of pRBC using a 1 ml insulin syringe (28G needle). Routinely, 1-2 donor mice are injected.</li>
 <li>On days 4-5 post-infection (p.i) remove donor mouse from cage and place it on a workstation or a disposable working pad.</li>
 <li>Gently...

<ol>
	<li>Brian de Souza, J., Riley, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+malaria:+the+contribution+of+studies+in+animal+models+to+our+understanding+of+immunopathogenesis.">Cerebral malaria: the contribution of studies in animal models to our understanding of immunopathogenesis.</a> Microbes and Infection. 4, 291-300 (2002).</li><li>Schofield, L., Grau, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+processes+in+malaria+pathogenesis.">Immunological processes in malaria pathogenesis.</a> Nature. 5, 722-735 (2005).</li><li>Miller, L. H., Baruch, D. I., Marsh, K., Doumbo, O. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathogenic+basis+of+malaria.">The pathogenic basis of malaria.</a> Nature. 415, 673-679 (2002).</li><li>Howe, C. L., Lafrance-Corey, R. G., Sundsbak, R. S., Lafrance, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+monocytes+damage+the+hippocampus+during+acute+picornavirus+infection+of+the+brain.">Inflammatory monocytes damage the hippocampus during acute picornavirus infection of the brain.</a> Journal of neuroinflammation. 9, 50 (2012).</li><li>LaFrance-Corey, R. G., Howe, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Brain-infiltrating+Leukocytes.">Isolation of Brain-infiltrating Leukocytes.</a> J. Vis. Exp. (52), e2747 (2011).</li><li>Lim, S. M., Koraka, P., Osterhaus, A. D., Martina, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=West+Nile+virus:+immunity+and+pathogenesis.">West Nile virus: immunity and pathogenesis.</a> Viruses. 3, 811-828 (2011).</li><li>Belnoue, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+pathogenic+role+of+brain-sequestered+alphabeta+CD8++T+cells+in+experimental+cerebral+malaria.">On the pathogenic role of brain-sequestered alphabeta CD8+ T cells in experimental cerebral malaria.</a> Journal of Immunology. 169, 6369-6375 (2002).</li><li>Campanella, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokine+receptor+CXCR3+and+its+ligands+CXCL9+and+CXCL10+are+required+for+the+development+of+murine+cerebral+malaria.">Chemokine receptor CXCR3 and its ligands CXCL9 and CXCL10 are required for the development of murine cerebral malaria.</a> Proceedings of the National Academy of Science U S A. 105, 4814-4819 (2008).</li><li>Hansen, D. S., Bernard, N. J., Nie, C. Q., Schofield, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NK+cells+stimulate+recruitment+of+CXCR3++T+cells+to+the+brain+during+Plasmodium+berghei-mediated+cerebral+malaria.">NK cells stimulate recruitment of CXCR3+ T cells to the brain during Plasmodium berghei-mediated cerebral malaria.</a> Journal of Immunology. 178, 5779-5788 (2007).</li><li>Amante, F. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+natural+regulatory+T+cells+in+the+pathogenesis+of+experimental+cerebral+malaria.">A role for natural regulatory T cells in the pathogenesis of experimental cerebral malaria.</a> The American journal of pathology. 171, 548-559 (2007).</li><li>Nie, C. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IP-10-mediated+T+cell+homing+promotes+cerebral+inflammation+over+splenic+immunity+to+malaria+infection.">IP-10-mediated T cell homing promotes cerebral inflammation over splenic immunity to malaria infection.</a> PLoS pathogens. 5, e1000369 (2009).</li><li>Franke-Fayard, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+malaria+parasite+sequestration:+CD36+is+the+major+receptor,+but+cerebral+pathology+is+unlinked+to+sequestration.">Murine malaria parasite sequestration: CD36 is the major receptor, but cerebral pathology is unlinked to sequestration.</a> Proceedings of the National Academy of Science U S A. 102, 11468-11473 (2005).</li><li>Nitcheu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perforin-dependent+brain-infiltrating+cytotoxic+CD8++T+lymphocytes+mediate+experimental+cerebral+malaria+pathogenesis.">Perforin-dependent brain-infiltrating cytotoxic CD8+ T lymphocytes mediate experimental cerebral malaria pathogenesis.</a> Journal of Immunololgy. 170, 2221-2228 (2003).</li><li>Lundie, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-stage+Plasmodium+infection+induces+CD8++T+lymphocytes+to+parasite-expressed+antigens,+largely+regulated+by+CD8alpha++dendritic+cells.">Blood-stage Plasmodium infection induces CD8+ T lymphocytes to parasite-expressed antigens, largely regulated by CD8alpha+ dendritic cells.</a> Proceeding of the National Academy of Science U S A. 105, 14509-14514 (2008).</li></ol>

No conflicts of interest declared.

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...

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

isolation and analysis of brain-sequestered leukocytes from plasmodium berghei anka-infected mice

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 viruses4-6, 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.

The results in Fig. 2 show percentages and absolute numbers of different BSL populations recovered from brains of perfused or unperfused malaria-infected and na&iuml;ve control mice. Isolated BSL were stained with PE-anti-NK1.1 and APC-anti-TCR-&beta; antibodies as indicated in the Protocol text. Consistent with previous findings 7-9, &alpha;&beta;TCR+ 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...

Watch this Scientific Journal Video about Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice at JoVE.com

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

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.

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

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

isolation-analysis-brain-sequestered-leukocytes-from-plasmodium

Research

JoVE Journal

Immunology and Infection

12.7K vistas.  The Walter and Eliza Hall Institute of Medical Research. Un método para el aislamiento de los leucocitos inflamatorios adherentes de los vasos sanguíneos cerebrales de los Plasmodium berghei ANKA de ratones infectados se describe. El método permite la cuantificación así como la caracterización fenotípica de los leucocitos aislados después de la tinción con anticuerpos fluorescentes y el posterior análisis por citometría de flujo.