Protein Kinase C-Theta Is Required for Development of Experimental Cerebral Malaria
2011; Elsevier BV; Volume: 178; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2010.11.008
ISSN1525-2191
AutoresMathilde Fauconnier, Marie-Laure Bourigault, Sandra Même, Frédéric Szeremeta, Jennifer Palomo, Adeline Danneels, Sabine Charron, Lizette Fick, Muazzam Jacobs, Jean‐Claude Belœil, Bernhard Ryffel, Valérie Quesniaux,
Tópico(s)Calpain Protease Function and Regulation
ResumoCerebral malaria is the most severe neurologic complication in children and young adults infected with Plasmodium falciparum. T-cell activation is required for development of Plasmodium berghei ANKA (PbA)–induced experimental cerebral malaria (CM). To characterize the T-cell activation pathway involved, the role of protein kinase C-theta (PKC-θ) in experimental CM development was examined. PKC-θ–deficient mice are resistant to CM development. In the absence of PKC-θ, no neurologic sign of CM developed after blood stage PbA infection. Resistance of PKC-θ–deficient mice correlated with unaltered cerebral microcirculation and absence of ischemia, as documented by magnetic resonance imaging and magnetic resonance angiography, whereas wild-type mice developed distinct microvascular pathology. Recruitment and activation of CD8+ T cells, and ICAM-1 and CD69 expression were reduced in the brain of resistant mice; however, the pulmonary inflammation and edema associated with PbA infection were still present in the absence of functional PKC-θ. Resistant PKC-θ–deficient mice developed high parasitemia, and died at 3 weeks with severe anemia. Therefore, PKC-θ signaling is crucial for recruitment of CD8+ T cells and development of brain microvascular pathology resulting in fatal experimental CM, and may represent a novel therapeutic target of CM. Cerebral malaria is the most severe neurologic complication in children and young adults infected with Plasmodium falciparum. T-cell activation is required for development of Plasmodium berghei ANKA (PbA)–induced experimental cerebral malaria (CM). To characterize the T-cell activation pathway involved, the role of protein kinase C-theta (PKC-θ) in experimental CM development was examined. PKC-θ–deficient mice are resistant to CM development. In the absence of PKC-θ, no neurologic sign of CM developed after blood stage PbA infection. Resistance of PKC-θ–deficient mice correlated with unaltered cerebral microcirculation and absence of ischemia, as documented by magnetic resonance imaging and magnetic resonance angiography, whereas wild-type mice developed distinct microvascular pathology. Recruitment and activation of CD8+ T cells, and ICAM-1 and CD69 expression were reduced in the brain of resistant mice; however, the pulmonary inflammation and edema associated with PbA infection were still present in the absence of functional PKC-θ. Resistant PKC-θ–deficient mice developed high parasitemia, and died at 3 weeks with severe anemia. Therefore, PKC-θ signaling is crucial for recruitment of CD8+ T cells and development of brain microvascular pathology resulting in fatal experimental CM, and may represent a novel therapeutic target of CM. Malaria is 1 of the 5 most important infectious diseases worldwide, and cerebral malaria (CM) is a frequent cause of death in children and young adults infected with Plasmodium falciparum.1Miller L.H. Good M.F. Milon G. Malaria pathogenesis.Science. 1994; 264: 1878-1883Crossref PubMed Scopus (477) Google Scholar Many aspects of the pathophysiology of CM are still poorly understood. Investigations in human beings and mice characterized the sequestration of erythrocytes (parasitized or not), platelets, and leukocytes in cerebral blood vessels,2Idro R. Jenkins N.E. Newton C.R. Pathogenesis, clinical features, and neurological outcome of cerebral malaria.Lancet Neurol. 2005; 4: 827-840Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar, 3Hunt N.H. Grau G.E. 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The present study addressed the role of protein kinase C (PKC) in the pathogenesis of CM in PbA infection. PKC isozymes are important in cellular activation, differentiation, adhesion, motility, and survival. Among the PKC family, PKC-δ and PKC-θ are expressed in lymphocytes, and PKC-θ is also expressed in natural killer cells.11Vyas Y.M. Maniar H. Dupont B. Cutting edge: differential segregation of the SRC homology 2-containing protein tyrosine phosphatase-1 within the early NK cell immune synapse distinguishes noncytolytic from cytolytic interactions.J Immunol. 2002; 168: 3150-3154PubMed Google Scholar PKC-θ is a critical regulator of T-cell receptor signaling and T-cell activation,12Chaudhary D. Kasaian M. PKC-theta: a potential therapeutic target for T-cell-mediated diseases.Curr Opin Invest Drugs. 2006; 7: 432-437PubMed Google Scholar, 13Marsland B.J. Soos T.J. Spath G. Littman D.R. Kopf M. Protein kinase C theta is critical for the development of in vivo T helper (Th) 2 cell but not Th1 cell responses.J Exp Med. 2004; 200: 181-189Crossref PubMed Scopus (186) Google Scholar and has been proposed as a therapeutic target for T-cell–mediated diseases.12Chaudhary D. Kasaian M. PKC-theta: a potential therapeutic target for T-cell-mediated diseases.Curr Opin Invest Drugs. 2006; 7: 432-437PubMed Google Scholar However, the role of PKC-θ in T-cell activation is complex, and PKC-θ is not equally involved in all T-cell responses. PKC-θ regulates Th2 helper cells that mediate immune response against helminth infection.12Chaudhary D. Kasaian M. PKC-theta: a potential therapeutic target for T-cell-mediated diseases.Curr Opin Invest Drugs. 2006; 7: 432-437PubMed Google Scholar, 13Marsland B.J. Soos T.J. Spath G. Littman D.R. Kopf M. Protein kinase C theta is critical for the development of in vivo T helper (Th) 2 cell but not Th1 cell responses.J Exp Med. 2004; 200: 181-189Crossref PubMed Scopus (186) Google Scholar It is involved in interferon I and II signaling in human T cells.14Srivastava K.K. Batra S. Sassano A. Li Y. Majchrzak B. Kiyokawa H. Altman A. Fish E.N. Platanias L.C. Engagement of protein kinase C-theta in interferon signaling in T-cells.J Biol Chem. 2004; 279: 29911-29920Crossref PubMed Scopus (51) Google Scholar PKC-θ is also important for T-cell survival and differentiation into functional cytotoxic T cells; however, it may be dispensable, compensated by other innate signals, for CD8+ T-cell proliferation and development.15Marsland B.J. Nembrini C. Schmitz N. Abel B. Krautwald S. Bachmann M.F. Kopf M. Innate signals compensate for the absence of PKC-(theta) during in vivo CD8(+) T cell effector and memory responses.Proc Natl Acad Sci USA. 2005; 102: 14374-14379Crossref PubMed Scopus (64) Google Scholar PKC-θ is not essential for mounting normal protective Th1 immune responses to clear some virus infections.16Giannoni F. Lyon A.B. Wareing M.D. Dias P.B. Sarawar S.R. Protein kinase C theta is not essential for T-cell–mediated clearance of murine gammaherpesvirus 68.J Virol. 2005; 79: 6808-6813Crossref PubMed Scopus (40) Google Scholar Further, TLR9 ligand CpG was recently shown to directly act on T-cell proliferation and survival, bypassing the PKC-θ pathway.17Marsland B.J. Nembrini C. Grun K. Reissmann R. Kurrer M. Leipner C. Kopf M. TLR ligands act directly upon T cells to restore proliferation in the absence of protein kinase C-theta signaling and promote autoimmune myocarditis.J Immunol. 2007; 178: 3466-3473PubMed Google Scholar Therefore, the implication of PKC-θ in the T-cell pathway during development of experimental CM remains to be established. In the present study, PKC-θ–deficient mice were used to identify PKC-θ as an obligatory pathway for development of CM in PbA infection. PKC-θ–deficient mice were protected from CM development in blood stage infection with PbA, and magnetic resonance imaging (MRI) and magnetic resonance angiography (MRA) confirmed the lack of ischemia and microvascular pathology and brain morphologic changes in these mice. Further, it was demonstrated that experimental CM protection is associated with reduced T-cell recruitment and activation in the brain, whereas lung edema is unaffected in the absence of functional PKC-θ. Mice deficient in PKC-θ18Sun Z. Arendt C.W. Ellmeier W. Schaeffer E.M. Sunshine M.J. Gandhi L. Annes J. Petrzilka D. Kupfer A. Schwartzberg P.L. Littman D.R. PKC-theta is required for TCR-induced NF-kappaB activation in mature but not immature T lymphocytes.Nature. 2000; 404: 402-407Crossref PubMed Scopus (789) Google Scholar were obtained from Manfred Kopf (Molecular Biomedicine, ETH Zurich, Switzerland) and bred in our animal facility at the Transgenose Institute (CNRS, Orleans, France). Mice were of C57BL/6J genetic background (back-crossed at least 15 times), and wild-type C57BL/6J mice were used as controls. For experiments, adult (aged 8 to 10 weeks) mice were kept in filtered-cages in a P2 animal facility. All animal experiments complied with ethical and animal experiments regulations of the French government. Recombinant PbA that constitutively expresses green fluorescent protein at a high level throughout the complete life cycle, from a transgene controlled by the strong promoter from a P berghei elongation factor-1α gene, was obtained from Dr. Andrew P. Waters (Glasgow Biomedical Research Center, University of Glasgow, Glasgow, United Kingdom).19Franke-Fayard B. Trueman H. Ramesar J. Mendoza J. van der Keur M. van der Linden R. Sinden R.E. Waters A.P. Janse C.J. A Plasmodium berghei reference line that constitutively expresses GFP at a high level throughout the complete life cycle.Mol Biochem Parasitol. 2004; 137: 23-33Crossref PubMed Scopus (389) Google Scholar Mice were infected via intraperitoneal injection of 105 parasitized erythrocytes as described previously.20Rudin W. Eugster H.P. Bordmann G. Bonato J. Muller M. Yamage M. Ryffel B. Resistance to cerebral malaria in tumor necrosis factor-alpha/beta–deficient mice is associated with a reduction of intercellular adhesion molecule-1 up-regulation and T helper type 1 response.Am J Pathol. 1997; 150: 257-266PubMed Google Scholar Mice were observed daily, grip strength was evaluated during daily body weight measurement, and clinical neurologic signs of CM culminating in ataxia, paralysis, and coma were assessed. Parasitemia was assessed in 2 μL of blood collected from the tail vein at various time points after infection with EGFP-PbA. Blood was diluted in 3 ml of PBS containing 0.5% bovine serum albumin, and fluorescent cells were analyzed using a flow cytometer (FACScan LSR; Becton Dickinson, Grenoble, France)21Togbe D. de Sousa P.L. Fauconnier M. Boissay V. Fick L. Scheu S. Pfeffer K. Menard R. Grau G.E. Doan B.T. Beloeil J.C. Renia L. Hansen A.M. Ball H.J. Hunt N.H. Ryffel B. Quesniaux V.F. Both functional LTbeta receptor and TNF receptor 2 are required for the development of experimental cerebral malaria.PLoS ONE. 2008; 3: e2608Crossref PubMed Scopus (40) Google Scholar using FlowJo software (TreeAge Software, Inc, Williamstown, Massachusetts). Blood was drawn with the mice under anesthesia with isofluorane (CSP, Fontenay sous Bois, France) into tubes containing EDTA (Vacutainer; Becton Dickinson) as indicated, and hematologic parameters were determined using a 5-part-differential hematology analyzer (MS 9.5; Melet Schloesing Laboratoires, Osny, France). Mice were injected intravenously with 0.2 ml of 325 mg/ml Evans blue solution (Sigma-Aldrich, St. Louis, MO) on day 7, shortly before the death of the wild-type mice. One hour later, mice were sacrificed, and the coloration of the brain was assessed. Brain extravasation was evaluated using Evans blue in formamide (Sigma-Aldrich) measured by absorbance at 610 nm of the tissue extracts as an indicator of increased capillary permeability.20Rudin W. Eugster H.P. Bordmann G. Bonato J. Muller M. Yamage M. Ryffel B. Resistance to cerebral malaria in tumor necrosis factor-alpha/beta–deficient mice is associated with a reduction of intercellular adhesion molecule-1 up-regulation and T helper type 1 response.Am J Pathol. 1997; 150: 257-266PubMed Google Scholar Similar mean (SD)baseline levels were observed in noninfected wild-type and PKC-θ−/− mice: 0.50 (0.21) μg/ml and 0.51 (0.26) μg/ml, respectively. Mice were euthanized and perfused with intracardiac sterile PBS/EDTA, 0.002 mmol/L, to remove circulating red blood cells and leukocytes from the brain and lung. The tissues were then fixed with a second perfusion of PBS/paraformaldehyde 4% (Sigma-Aldrich). The brains and lungs from PbA-infected mice were fixed in 3.6% PBS/formaldehyde for 72 hours as described previously.20Rudin W. Eugster H.P. Bordmann G. Bonato J. Muller M. Yamage M. Ryffel B. Resistance to cerebral malaria in tumor necrosis factor-alpha/beta–deficient mice is associated with a reduction of intercellular adhesion molecule-1 up-regulation and T helper type 1 response.Am J Pathol. 1997; 150: 257-266PubMed Google Scholar Longitudinal sections of 3 μm were stained with H&E. Brain microvascular disease with endothelial damage, accumulation of mononuclear cells, and extravasated erythrocytes in the Virchov-Robin space was assessed semiquantitatively from a whole brain section, typically between −5.64 and −6.84 from the bregma. Erythrocyte accumulation in alveoli and thickening of alveolar septae of the lung were quantified using a semiquantitative score with increasing severity of changes (0 to 5) by 2 independent observers including a trained pathologist (B.R.). Brain microvascular obstruction was evaluated by scoring the number of vessels containing sequestered blood cells on multiple fields corresponding to whole brain sections (0% microvascular obstruction = 0, 1% to 19% microvascular obstruction = 1, 20% to 39% microvascular obstruction = 2, 40% to 59% microvascular obstruction = 3, 60% to 79% microvascular obstruction = 4, and 80% microvascular obstruction = 5). Lung sections were scored on 10 fields per section for erythrocyte accumulation in alveoli (less than 1 cell per alveoli = 0, 1 cell per alveoli = 1, 2 to 5 cells per alveoli = 2, 5 to 10 cells per alveoli = 3, 10 to 30 cells per alveoli = 4, and 30 cells pre alveoli = 5); alveolar size (all normal alveoli = 0, mix of normal and medium-sized alveoli = 1, medium-sized alveoli only = 2, mix of medium-sized and small alveoli = 3, all small alveoli = 4, and full obstruction = 5 (not observed); and thickening of alveolar septae due to inflammatory cells and edema was scored with increasing severity from 0 to 5 as an estimate of interstitial inflammation. Macrophages and T-cell populations were analyzed in situ using immunohistologic staining. Euthanized mice were perfused as described above, and brains and lungs were harvested, imbedded in OCT media (Tissue-Tek; Sakura Finetek Japan Co, Ltd, Tokyo, Japan), and frozen rapidly. Longitudinal cryosections of 10 μm were fixed in acetone, washed in PBS/Tween 0.05% and PBS, and incubated for 30 minutes with wild-type mouse serum at 2% in PBS. All incubations were carried out in a wet chamber. Brain and lung sections were then incubated overnight at 4°C with biotinylated rat antibodies to CD3e (clone 145-2C11), CD8α (clone 53-6.7), CD4 (clone RM4-5), CD54 (clone 3E2,), CD11b (clone M1/70), or IA/IE (clone 2G9) (all from BD Pharmingen, Inc, San Diego, CA) as indicated. After washing, sections were incubated for 30 minutes with an avidin-biotin peroxydase complex (ABC; Vector Laboratories, Inc, Burlingame, CA), followed by the peroxydase substrate (diaminobenzidine; Vector Laboratories, Inc). Finally, sections were washed in PBS, mounted using Eukitt medium (O. Kindler GmbH & Co, Freiburg, Germany), and examined microscopically. MRI experiments were performed as described previously21Togbe D. de Sousa P.L. Fauconnier M. Boissay V. Fick L. Scheu S. Pfeffer K. Menard R. Grau G.E. Doan B.T. Beloeil J.C. Renia L. Hansen A.M. Ball H.J. Hunt N.H. Ryffel B. Quesniaux V.F. Both functional LTbeta receptor and TNF receptor 2 are required for the development of experimental cerebral malaria.PLoS ONE. 2008; 3: e2608Crossref PubMed Scopus (40) Google Scholar using a horizontal 9.4 T/20 Bruker Biospec MR system (Bruker BioSpin SA, Wissembourg, France). A 12-element linear birdcage coil (Bruker BioSpin SA) with inner diameter of 35 mm and length of 60 mm was used to achieve uniform excitation and reception. A custom-built stereotaxic head holder was used to fix the animals in the birdcage coil. The mice were anesthetized using 1.5% isoflurane in a 1:1 oxygen/nitrous oxide mixture administered using a face mask, allowing free breathing. Respiration was monitored using a balloon taped to the abdomen and connected to a pressure transducer (SA Instruments, Inc, Stony Brook, NY). Mean (SD) mouse body temperature was maintained at 37°C (0.5°C) throughout the experiment, using warm water circulation. Preparatory anatomical sagittal MRI was performed using a multisection multiecho sequence with the following parameters: rapid acquisition with relaxation enhancement factor = 8, repetition time/effective echo time = 4000/46 ms, field of view = 20 × 20 mm2, matrix = 128 × 128, 1 section 1-mm thick, number of averages = 1, and experimental time = 1 minute. Brain lesions and global changes in tissue structure were accessed via T2-weighted MRI using a multisection multiecho sequence with the following parameters: rapid acquisition with relaxation enhancement factor = 8, repetition time/effective echo time = 5000/46 ms, field of view = 17 × 17 mm2, matrix = 256 × 192 (zero-filled to 256 × 256), 37 axial sections 0.5-mm thick, number of averages = 8, and experimental time = 16 minutes. Three measures were used to estimate morphologic changes of the brain: line 1 from the pituitary gland to the aqueduct of Sylvius, the median line crossing the medial cerebellar nucleus, and the medium line stemming from the cerebellar obex. For each group, a region of interest was drawn manually in the corpus callosum, and gray-level mean was measured using Mazda software (Mazda version 3.20, copyright 1998–2002 by Piotr Szczypinski). Vascular cerebral blood flow was measured at MRA using a fast low-angle shot sequence with the following parameters: field of view = 14 × 14 mm2, matrix = 128 × 128, repetition time/echo time = 30/5 ms, 51 axial sections 0.3-mm thick, number of averages = 4, and experimental time = 13 minutes. Angiograms were produced by generating maximum intensity projections after interpolating raw data to obtain an isotropic resolution (109 μm3). Image analysis and processing were performed using the public domain software Image J. Mice were euthanized and perfused as described,10Belnoue E. Kayibanda M. Vigario A.M. Deschemin J.C. van Rooijen N. Viguier M. Snounou G. Renia L. On the pathogenic role of brain-sequestered alphabeta CD8+ T cells in experimental cerebral malaria.J Immunol. 2002; 169: 6369-6375PubMed Google Scholar on day 7, when all wild-type mice exhibited neurologic symptoms of CM. Brains were removed and homogenized gently (30 seconds at 4000 rpm) using a sterile disposable homogenization system (Dispomix; Medic Tools AG, Zug, Switzerland) in RPMI 1640 medium containing 2% fetal calf serum. Homogenates were passed through a 100-μm nylon cell strainer (Becton Dickinson France SAS, Grenoble, France), and cells were centrifuged at 400g for 10 minutes.21Togbe D. de Sousa P.L. Fauconnier M. Boissay V. Fick L. Scheu S. Pfeffer K. Menard R. Grau G.E. Doan B.T. Beloeil J.C. Renia L. Hansen A.M. Ball H.J. Hunt N.H. Ryffel B. Quesniaux V.F. Both functional LTbeta receptor and TNF receptor 2 are required for the development of experimental cerebral malaria.PLoS ONE. 2008; 3: e2608Crossref PubMed Scopus (40) Google Scholar The mononuclear cells were separated over a 35% Percoll gradient (Amersham Biosciences AB, Uppsala, Sweden). Brain-sequestered cells were collected, washed, and saturated with mouse serum before staining with fluorescence-labeled antibodies for 30 minutes. The cells were then phenotyped at flow cytometry using the following rat antibodies: anti-mouse CD3 conjugated to fluorescein isothiocyanate (clone 145-2C11), CD4 conjugated to peridinin-chlorophyll-protein complex (clone L3T4 RM4-5), CD8α conjugated to allophycocyanin cyanin7 (clone 53-6.7), CD69 (clone H1.2F3) conjugated to phycoerythrin, and isotype-matched control antibodies (all from BD Pharmingen Inc). For each sample, 10,000 cells from the mononuclear population were scored. Data were analyzed using a flow cytometer and FlowJo software (Tree Star Inc, Ashland, Oregon). Unless otherwise indicated, data are given as mean (SD), indicated by error bars. Statistical significance was determined using commercially available software (Prism version 4.0; GraphPad Software, Inc, San Diego, CA). Differences between multiple groups were analyzed for statistical significance via 1-way parametric analysis of variance followed by the Bonferroni posttest for analyzing large sample groups or by a nonparametric Mann-Whitney test for analysis of small groups. P < 0.05 was considered statistically significant. The role of PKC-θ was assessed by investigating the development of neurologic signs of CM, survival, and parasitemia in mice deficient in PKC-θ. After injection of PbA-infected erythrocytes, wild-type control mice developed typical neurologic symptoms of CM such as ataxia, loss of grip strength, progressive paralysis, and coma, and all died within 1 week, whereas 75% of PKC-θ–deficient mice survived longer than 10 days, and 60% of PKC-θ–deficient mice survived longer than 14 days with no sign of CM (Figure 1A). Parasitemia was analyzed at flow cytometry using parasites transfected with green fluorescent protein.19Franke-Fayard B. Trueman H. Ramesar J. Mendoza J. van der Keur M. van der Linden R. Sinden R.E. Waters A.P. Janse C.J. A Plasmodium berghei reference line that constitutively expresses GFP at a high level throughout the complete life cycle.Mol Biochem Parasitol. 2004; 137: 23-33Crossref PubMed Scopus (389) Google Scholar Although the onset of parasitemia was slightly lower in PKC-θ–deficient mice on day 4, parasitemia was similar in both groups on days 5 to 7, and PKC-θ–deficient mice failed to control parasite growth thereafter, with parasitemia increased to 25% at day 14 and reaching 58% at day 21 (Figure 1B). PKC-θ–deficient mice died after day 21 with a high parasite load (Figure 1, A and B) and severe anemia (Figure 1C) in the absence of neurologic signs. However, a retrospective analysis revealed that PKC-θ–deficient mice that died before day 15 without neurologic signs exhibited a slightly higher parasite load at day 7 compared with PKC-θ–deficient mice that survived beyond day 15 [mean (SD), 13.7 (3.8) and 9.6 (3.7%), respectively], indicating that early death in PKC-θ–deficient mice might be associated with a higher parasite load. Therefore, our data demonstrate a critical role of PKC-θ in CM development, but no drastic effect on parasite growth. Both MRI and MRA were used to explore neuropathologic disease in the brain of PbA-infected mice. These noninvasive techniques enable investigation of neurologic disease and are used in human beings to study brain changes during CM.21Togbe D. de Sousa P.L. Fauconnier M. Boissay V. Fick L. Scheu S. Pfeffer K. Menard R. Grau G.E. Doan B.T. Beloeil J.C. Renia L. Hansen A.M. Ball H.J. Hunt N.H. Ryffel B. Quesniaux V.F. Both functional LTbeta receptor and TNF receptor 2 are required for the development of experimental cerebral malaria.PLoS ONE. 2008; 3: e2608Crossref PubMed Scopus (40) Google Scholar, 22Kampfl A.W. Birbamer G.G. Pfausler B.E. Haring H.P. Schmutzhard E. Isolated pontine lesion in algid cerebral malaria: clinical features, management, and magnetic resonance imaging findings.Am J Trop Med Hygiene. 1993; 48: 818-822PubMed Google Scholar, 23Looareesuwan S. Wilairatana P. Krishna S. Kendall B. Vannaphan S. Viravan C. White N.J. Magnetic resonance imaging of the brain in patients with cerebral malaria.Clin Infect Dis. 1995; 21: 300-309Crossref PubMed Scopus (123) Google Scholar, 24Cordoliani Y.S. Sarrazin J.L. Leveque C. Goasdoue P. Sabbah P. Imaging of encephalic parasitic diseases [in French].J Neuroradiol. 1998; 25: 290-305PubMed Google Scholar, 25Patankar T.F. Karnad D.R. Shetty P.G. Desai A.P. Prasad S.R. Adult cerebral malaria: prognostic importance of imaging findings and correlation with postmortem findings.Radiology. 2002; 224: 811-816Crossref PubMed Scopus (81) Google Scholar In murine experimental CM, MRI and MRA enable semiquantitative analysis of swelling and edema, focal ischemia, morphologic changes, and vascular blood flow. This could be indicative of microvascular disease of the brain due to obstruction of small vessels with parasitized erythrocyte and leukocyte sequestration and damaging of endothelial cells.21Togbe D. de Sousa P.L. Fauconnier M. Boissay V. Fick L. Scheu S. Pfeffer K. Menard R. Grau G.E. Doan B.T. Beloeil J.C. Renia L. Hansen A.M. Ball H.J. Hunt N.H. Ryffel B. Quesniaux V.F. Both functional LTbeta receptor and TNF receptor 2 are required for the development of experimental cerebral malaria.PLoS ONE. 2008; 3: e2608Crossref PubMed Scopus (40) Google Scholar, 26Penet M.F. Viola A. Confort-Gouny S. Le Fur Y. Duhamel G. Kober F. Ibarrola D. Izquierdo M. Coltel N. Gharib B. Grau G.E. Cozzone P.J. Imaging experimental cerebral malaria in vivo: significant role of ischemic brain edema.J Neurosci. 2005; 25: 7352-7358Crossref PubMed Scopus (136) Google Scholar, 27Stoelcker B. Hehlgans T. Weigl K. Bluethmann H. Grau G.E. Mannel D.N. Requirement for tumor necrosis factor receptor 2 expression on vascular cells to induce experimental cerebral malaria.Infect Immun. 2002; 70: 5857-5859Crossref PubMed Scopus (30) Google Scholar, 28Engwerda C.R. Mynott T.L. Sawhney S. De Souza J.B. Bickle Q.D. Kaye P.M. Locally up-regulated lymphotoxin alpha, not systemic tumor necrosis factor alpha, is the principle mediator of murine cerebral malaria.J Exp Med. 2002; 195: 1371-1377Crossref PubMed Scopus (214) Google Scholar Wild-type and PKC-θ–deficient mice were examined at day 7, when sensitive mice start developing acute CM. Typical MRI and MRA images of uninfected and infected mice are shown in Figure 2,A and B, respectively. While wild-type mice exhibited distinct signs of ischemic brain damage on blood stage PbA infection, PKC-θ–deficient mice demonstrated normal MRI parameters without any signs of edema or infection, similar to findings in uninfected mice. PbA-infected wild-type mice exhibited distinct changes at MRI, observable as a bilateral hyperintense signal at the corpus callosum (Figure 2A). The gray level intensity in the corpus callosum was significantly increased after PbA infection in wild-type mice, whereas no significant variation was observed in PKC-θ–infected or PKC-θ–noninfected mice (Figure 2C). Brain edema or swelling was documented in infected wild-type mice during acute CM by measuring 3 distances in the brain (Figure 2D): (1) line 1 from the pituitary gland to the aqueduct of Sylvius, (2) median line 2 crossing the medial cerebellar nucleus, and (3) medium line 3 stemming from the cerebellar obex.26Penet M.F. Viola A. Confort-Gouny S. Le Fur Y. Duhamel G. Kober F. Ibarrola D. Izquierdo M. Coltel N. Gharib B. Grau G.E. Cozzone P.J. Imaging experimental cerebral malaria in vivo: significant role of ischemic brain edema.J Neurosci. 2005; 25: 7352-7358Crossref PubMed Scopus (136) Google Scholar Distances 1 and 3 were significantly increased and distance 2 was significantly reduced in infected wild-type mice compared with nonin
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