CNS Hypoxia Is More Pronounced in Murine Cerebral than Noncerebral Malaria and Is Reversed by Erythropoietin
2011; Elsevier BV; Volume: 179; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2011.06.027
ISSN1525-2191
AutoresCasper Hempel, Valéry Combes, Nicholas H. Hunt, Jørgen A. L. Kurtzhals, Georges E. Grau,
Tópico(s)Hemoglobinopathies and Related Disorders
ResumoCerebral malaria (CM) is associated with high mortality and risk of sequelae, and development of adjunct therapies is hampered by limited knowledge of its pathogenesis. To assess the role of cerebral hypoxia, we used two experimental models of CM, Plasmodium berghei ANKA in CBA and C57BL/6 mice, and two models of malaria without neurologic signs, P. berghei K173 in CBA mice and P. berghei ANKA in BALB/c mice. Hypoxia was demonstrated in brain sections using intravenous pimonidazole and staining with hypoxia-inducible factor-1α–specific antibody. Cytopathic hypoxia was studied using poly (ADP-ribose) polymerase-1 (PARP-1) gene knockout mice. The effect of erythropoietin, an oxygen-sensitive cytokine that mediates protection against CM, on cerebral hypoxia was studied in C57BL/6 mice. Numerous hypoxic foci of neurons and glial cells were observed in mice with CM. Substantially fewer and smaller foci were observed in mice without CM, and hypoxia seemed to be confined to neuronal cell somas. PARP-1–deficient mice were not protected against CM, which argues against a role for cytopathic hypoxia. Erythropoietin therapy reversed the development of CM and substantially reduced the degree of neural hypoxia. These findings demonstrate cerebral hypoxia in malaria, strongly associated with cerebral dysfunction and a possible target for adjunctive therapy. Cerebral malaria (CM) is associated with high mortality and risk of sequelae, and development of adjunct therapies is hampered by limited knowledge of its pathogenesis. To assess the role of cerebral hypoxia, we used two experimental models of CM, Plasmodium berghei ANKA in CBA and C57BL/6 mice, and two models of malaria without neurologic signs, P. berghei K173 in CBA mice and P. berghei ANKA in BALB/c mice. Hypoxia was demonstrated in brain sections using intravenous pimonidazole and staining with hypoxia-inducible factor-1α–specific antibody. Cytopathic hypoxia was studied using poly (ADP-ribose) polymerase-1 (PARP-1) gene knockout mice. The effect of erythropoietin, an oxygen-sensitive cytokine that mediates protection against CM, on cerebral hypoxia was studied in C57BL/6 mice. Numerous hypoxic foci of neurons and glial cells were observed in mice with CM. Substantially fewer and smaller foci were observed in mice without CM, and hypoxia seemed to be confined to neuronal cell somas. PARP-1–deficient mice were not protected against CM, which argues against a role for cytopathic hypoxia. Erythropoietin therapy reversed the development of CM and substantially reduced the degree of neural hypoxia. These findings demonstrate cerebral hypoxia in malaria, strongly associated with cerebral dysfunction and a possible target for adjunctive therapy. 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We assessed the extent of hypoxia and subsequent HIF-1α response in CM and non-CM using several murine models and neuroprotective treatment. Furthermore, the possible role of cytopathic hypoxia was tested as a driving force for CM progression in PARP-1 gene knockout (PARP-1−/−) mice. Female, 7-week-old, CBA mice (Animal Resources Centre, Canning Vale, Western Australia) were housed under standard conditions with ad libitum access to pellet food and water. After 1 week of acclimatization, mice were divided into three groups of seven mice each and were injected i.p. with either isotonic saline solution (noninfected control mice), 2 × 106 P. berghei K173 (PbK)–infected erythrocytes (non-CM) or 106 PbA-infected erythrocytes (CM), as previously described.38Grau G.E. Piguet P.F. Engers H.D. Louis J.A. Vassalli P. Lambert P.H. L3T4+ T lymphocytes play a major role in the pathogenesis of murine cerebral malaria.J Immunol. 1986; 137: 2348-2354PubMed Google Scholar, 39Mitchell A.J. Hansen A.M. 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All experiments complied with Australian, Danish, and European guidelines for animal research and were approved by the respective national or state boards for animal studies. For detection of hypoxia at a comparable time point, all mice were euthanized in an experiment when susceptible mice exhibited clinical signs of CM. All PbA-infected CBA7Warrell D.A. White N.J. Veall N. Looareesuwan S. Chanthavanich P. Phillips R.E. Karbwang J. Pongpaew P. Krishna S. Cerebral anaerobic glycolysis and reduced cerebral oxygen transport in human cerebral malaria.Lancet. 1988; 2: 534-538Abstract PubMed Scopus (115) Google Scholar and C57BL/65Maude R.J. Beare N.A. Fluorescein angiography findings strengthen the theoretical basis for trialing neuroprotective agents in cerebral malaria.Trends Parasitol. 2009; 25: 350-351Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar mice demonstrated signs of CM at days 7 and 8 after infection, respectively, and most of these mice had entered the terminal phase of murine CM. Signs of CM included ruffled fur, loss of coordination, fitting, ataxia, coma, and body temperature lower than 32°C. Body temperature lower than 32°C was considered a proxy for a terminal outcome of the infection, as previously described.35Wiese L. Hempel C. Penkowa M. Kirkby N. Kurtzhals J.A. Recombinant human erythropoietin increases survival and reduces neuronal apoptosis in a murine model of cerebral malaria.Malar J. 2008; 7: 3Crossref PubMed Scopus (64) Google Scholar, 44Curfs J.H. Schetters T.P. Hermsen C.C. Jerusalem C.R. van Zon A.A. Eling W.M. Immunological aspects of cerebral lesions in murine malaria.Clin Exp Immunol. 1989; 75: 136-140PubMed Google Scholar On the day of euthanasia, mice were first briefly anesthetized using isoflurane (Baxter Healthcare Corp., Deerfield, IL). Packed cell volume was measured in PbK-infected mice after high-speed centrifugation of blood collected in capillary tubes. During anesthesia, mice were injected i.v. retro-orbitally with 80 mg/kg pimonidazole HCl (Hypoxyprobe-1 kit; HPI, Inc., Burlington, MA) and 15 mg/kg Hoechst 33342 (Catalog No. H3570; Invitrogen Corp., Carlsbad, CA) diluted in PBS (total volume, 300 μL), the latter to validate the success of the i.v. injection. Mice were allowed to recover, and the solution was left circulating for 30 minutes before euthanasia via cervical dislocation under deep isoflurane anesthesia. The brain was removed quickly, split sagittally, and immersion-fixed in formalin for 24 hours at room temperature before transfer to 70% (v/v) ethanol. Tissue was paraffin-embedded automatically using a Histokinette (Shandon, Inc., Pittsburgh, PA) and cut into 5-μm thin sagittal sections and 30-μm thick sections for Z-stacks. Sections were cleaned of paraffin and rehydrated according to standard procedures. Heat-induced epitope retrieval was performed by boiling sections in citrate buffer (pH 6) in a microwave oven. Endogenous peroxidase activity was quenched via incubation in 0.5% (w/v) H2O2 (diluted from 30% H2O2; Sigma-Aldrich Corp., St. Louis, MO) dissolved in Tris-buffered saline solution with 0.5% (v/v) Tween-20 (Merck KGaA, Darmstadt, Germany). Nonspecific binding was blocked using serum-free protein block (Catalog No. X0909; Dako A/S, Glostrup, Denmark). Primary antibodies used included mouse anti-pimonidazole (50× dilution; HPI, Inc.) and mouse anti–HIF-1α (600× dilution; Catalog No. ab1; Abcam, Inc., Cambridge, MA). Primary antibodies were diluted in 10% (v/v) goat serum (In Vitro A/S, Fredensborg, Denmark) and incubated overnight at 4°C. Primary antibodies were detected using a biotinylated goat anti-mouse secondary antibody (200× dilution; Catalog No. B8774; Sigma-Aldrich Corp.). Biotinylated antibody was labeled using an avidin-biotin-peroxidase complex according to the manufacturer's recommendations (Vectastain ABC kit; Catalog No. PK4000; Vector Laboratories, Inc., Burlingame, CA) and was visualized using 3,3-diaminobenzidine tetrahydrochloride tablets (Kem-En-Tec Diagnostics A/S, Taastrup, Denmark) dissolved in Tris-buffered saline solution–0.5% Tween 20 with 0.015% H2O2 (Sigma-Aldrich Corp.). Sections were counterstained using Mayer's hematoxylin (VWR International ApS, Herlev, Denmark) before mounting. Chromogenically stained samples were visualized using an Imager.Z1 microscope fitted with an AxioCam MRc5 Camera (Carl Zeiss MicroImaging GmbH, Göttingen, Germany). To assess the co-localization of pimonidazole reactivity with a specific cell type, anti-pimonidazole was co-incubated overnight with rabbit anti-glial fibrillary acidic protein (250× dilution; Catalog No. Z334; Dako A/S) for co-localization with astroglia. The primary antibodies were detected using goat anti-mouse IgG–Alexa 568 (1000× dilution; Catalog No. A11031; Invitrogen Corp.) and goat anti-rabbit IgG–Alexa 488 (1000× dilution; Catalog No. A11034; Invitrogen Corp.). For neuronal co-localization, the anti-pimonidazole was first incubated alone overnight, detected using goat anti-mouse IgG–Alexa, and incubated for 40 minutes at room temperature with mouse anti-neuronal nuclei–Alexa 488 (100× dilution; Catalog No. MAB377X; Chemicon, Milipore Corp., Billerica, MA). For labeling of vessels, a fluorescein isothiocyanate–conjugated tomato lectin (100× dilution; Catalog No. FL-1171; Vector Laboratories, Inc.) was incubated simultaneously with the primary antibody. Nuclei were labeled using DAPI (20,000× dilution; Catalog No. D1306; Invitrogen Corp.). Low-magnification fluorescence microscopy was performed using an Olympus IX-71 equipped with an F-view CCD camera (Olympus Corp., Tokyo, Japan) illuminated with a mercury burner. Confocal immunofluorescence microscopy was performed using a Nikon TE 2000E Eclipse with 60× numerical aperture 1.4 Apoplan oil immersion objective lens (Nikon Instruments, Inc., Melville, NY), with gain adjusted for each laser (408 nm, 450/35; 488 nm, 515/30; and 543 nm, 605/75). Optical sectioning was performed in 600-nm increments. Standard negative control staining, without any primary antibody, was performed simultaneously for each primary antibody. All slides were randomized, blinded, and assessed using digital image analysis by one individual (C.H.). The degree of hypoxia was assessed by thresholding the staining intensity for pimonidazole-labeled areas in various parts of the brain including the olfactory lobe, cortex, corpus callosum, hippocampus, thalamus, hypothalamus, cerebellum, midbrain, pons, and medulla. Photographs were taken at identical settings using an RGB filter at 200× magnification with 2 × 2 mosaic function to increase the area sampled (area per micrograph, 1.456 mm2). If the region did not fill the entire frame (eg, when tissue boundaries and ventricles were included), these areas were cropped using ImageJ software (version 1.43I; National Institutes of Health, Bethesda, MD). The segmentation plug-in (ImageJ) was used to perform color-based thresholding on the brownish diaminobenzidine precipitation. Thresholding of the images was performed by sampling tissue with positive staining repeatedly in various areas and sections. From these randomly chosen areas, it was possible to set hue (stop), saturation (pass), and brightness (pass), which convincingly differentiated intensely stained tissue from unstained tissue and artifacts. The filtered image was converted to eight-bit gray scale and thresholded in a manner similar to that previously described.45Jankovic B. Aquino-Parsons C. Raleigh J.A. Stanbridge E.J. Durand R.E. Banath J.P. MacPhail S.H. Olive P.L. Comparison between pimonidazole binding, oxygen electrode measurements, and expression of endogenous hypoxia markers in cancer of the uterine cervix.Cytometry B Clin Cytom. 2006; 70: 45-55Crossref PubMed Scopus (76) Google Scholar For presentation purposes, the thresholded areas have been normalized to the mean area of noninfected mice. A systematic uniform random sampling principle was used for assessment of HIF-1α–positive cells.46Larsen J.O. Stereology of nerve c
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