Produção Nacional
Revisado por pares
Murine Cerebral Malaria Is Associated with a Vasospasm-Like Microcirculatory Dysfunction, and Survival upon Rescue Treatment Is Markedly Increased by Nimodipine
2010; Elsevier BV; Volume: 176; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2010.090691
ISSN1525-2191
AutoresPedro Cabrales, Graziela Maria Zaniní, Diana Meays, John A. Frangos, Leonardo J. M. Carvalho,
Tópico(s)Mosquito-borne diseases and control
ResumoBrain hemodynamics in cerebral malaria (CM) is poorly understood, with apparently conflicting data showing microcirculatory hypoperfusion and normal or even increased blood flow in large arteries. Using intravital microscopy to assess the pial microvasculature through a closed cranial window in the murine model of CM by Plasmodium berghei ANKA, we show that murine CM is associated with marked decreases (mean: 60%) of pial arteriolar blood flow attributable to vasoconstriction and decreased blood velocity. Leukocyte sequestration further decreased perfusion by narrowing luminal diameters in the affected vessels and blocking capillaries. Remarkably, vascular collapse at various degrees was observed in 44% of mice with CM, which also presented more severe vasoconstriction. Coadministration of artemether and nimodipine, a calcium channel blocker used to treat postsubarachnoid hemorrhage vasospasm, to mice presenting CM markedly increased survival compared with artemether plus vehicle only. Administration of nimodipine induced vasodilation and increased pial blood flow. We conclude that vasoconstriction and vascular collapse play a role in murine CM pathogenesis and nimodipine holds potential as adjunctive therapy for CM. Brain hemodynamics in cerebral malaria (CM) is poorly understood, with apparently conflicting data showing microcirculatory hypoperfusion and normal or even increased blood flow in large arteries. Using intravital microscopy to assess the pial microvasculature through a closed cranial window in the murine model of CM by Plasmodium berghei ANKA, we show that murine CM is associated with marked decreases (mean: 60%) of pial arteriolar blood flow attributable to vasoconstriction and decreased blood velocity. Leukocyte sequestration further decreased perfusion by narrowing luminal diameters in the affected vessels and blocking capillaries. Remarkably, vascular collapse at various degrees was observed in 44% of mice with CM, which also presented more severe vasoconstriction. Coadministration of artemether and nimodipine, a calcium channel blocker used to treat postsubarachnoid hemorrhage vasospasm, to mice presenting CM markedly increased survival compared with artemether plus vehicle only. Administration of nimodipine induced vasodilation and increased pial blood flow. We conclude that vasoconstriction and vascular collapse play a role in murine CM pathogenesis and nimodipine holds potential as adjunctive therapy for CM. Cerebral malaria (CM) caused by Plasmodium falciparum claims the lives of nearly 1 million children every year.1Rowe AK Rowe SY Snow RW Korenromp EL Schellenberg JR Stein C Nahlen BL Bryce J Black RE Steketee RW The burden of malaria mortality among African children in the year 2000.Int J Epidemiol. 2006; 35: 691-704Crossref PubMed Scopus (209) Google Scholar Despite antimalarial treatment, 10% to 20% of patients die, and one in every four survivors develops neurological sequelae,2McIntosh HM Olliaro P Artemisinin derivatives for treating severe malaria.Cochrane Database Syst Rev. 2000; 2: CD000527PubMed Google Scholar, 3John CC Bangirana P Byarugaba J Opoka RO Idro R Jurek AM Wu B Boivin MJ Cerebral malaria in children is associated with long-term cognitive impairment.Pediatrics. 2008; 122: e92-e99Crossref PubMed Scopus (215) Google Scholar therefore adjunctive therapies are urgently needed. A number of clinical trials addressing potential adjunctive therapies for CM showed no proven benefits and some interventions were even deleterious,4Idro R Jenkins NE Newton CR Pathogenesis, clinical features, and neurological outcome of cerebral malaria.Lancet Neurol. 2005; 4: 827-840Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar stressing the need for a better understanding of CM pathogenesis to develop effective therapies. An unresolved issue of CM pathogenesis regards the role of brain hemodynamic perturbations and ischemia. Sequestration of parasitized red blood cells (pRBCs) containing mature forms of the parasite in the brain microvasculature is a characteristic postmortem finding in human CM cases5MacPherson GG Warrell MJ White NJ Looareesuwan S Warrell DA Human cerebral malaria. A quantitative ultrastructural analysis of parasitized erythrocyte sequestration.Am J Pathol. 1985; 119: 385-401PubMed Google Scholar and together with rosetting6Fernandez V Wahlgren M Rosetting and autoagglutination in Plasmodium falciparum.Chem Immunol. 2002; 80: 163-187Crossref PubMed Google Scholar and reduced RBC deformability7Dondorp AM Pongponratn E White NJ Reduced microcirculatory flow in severe falciparum malaria: pathophysiology and electron-microscopic pathology.Acta Trop. 2004; 89: 309-317Crossref PubMed Scopus (158) Google Scholar may result in the obstruction of blood flow potentially leading to ischemia and hypoxia. In vivo studies of the microcirculation in human CM support this mechanism, with direct observation of retinal microvasculature showing impaired perfusion, retinal whitening, vascular occlusion, and ischemia.8Beare NA Harding SP Taylor TE Lewallen S Molyneux ME Perfusion abnormalities in children with cerebral malaria and malarial retinopathy.J Infect Dis. 2009; 199: 263-271Crossref PubMed Scopus (133) Google Scholar Accordingly, microvascular obstruction observed in the rectal mucosa of CM patients was proportional to the severity of the disease.9Dondorp AM Ince C Charunwatthana P Hanson J van Kuijen A Faiz MA Rahman MR Hasan M Bin Yunus E Ghose A Ruangveerayut R Limmathurotsakul D Mathura K White NJ Day NP Direct in vivo assessment of microcirculatory dysfunction in severe falciparum malaria.J Infect Dis. 2008; 197: 79-84Crossref PubMed Scopus (187) Google Scholar In addition, hypovolemia, shock and intracranial hypertension, commonly associated with poor outcomes in CM,4Idro R Jenkins NE Newton CR Pathogenesis, clinical features, and neurological outcome of cerebral malaria.Lancet Neurol. 2005; 4: 827-840Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar reduce tissue perfusion, and tissue hypoxia is one of the likely explanations for the acidosis frequently observed in severe malaria.7Dondorp AM Pongponratn E White NJ Reduced microcirculatory flow in severe falciparum malaria: pathophysiology and electron-microscopic pathology.Acta Trop. 2004; 89: 309-317Crossref PubMed Scopus (158) Google Scholar, 10Maitland K Newton CR Acidosis of severe falciparum malaria: heading for a shock?.Trends Parasitol. 2005; 21: 11-16Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar Ischemic damage has also been shown in children with CM and was associated with severe neurological sequelae.11Newton CR Peshu N Kendall B Kirkham FJ Sowunmi A Waruiru C Mwangi I Murphy SA Marsh K Brain swelling and ischaemia in Kenyans with cerebral malaria.Arch Dis Child. 1994; 70: 281-287Crossref PubMed Scopus (133) Google Scholar On the other hand, transcranial Doppler sonography studies showed normal or even increased cerebral blood flow (CBF) velocities12Newton CR Marsh K Peshu N Kirkham FJ Perturbations of cerebral hemodynamics in Kenyans with cerebral malaria.Pediatr Neurol. 1996; 1: 41-49Abstract Full Text PDF Scopus (73) Google Scholar, 13Warrell DA White NJ Veall N Looareesuwan S Chanthavanich P Phillips RE 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, 14Clavier N Rahimy C Falanga P Ayivi B Payen D No evidence for cerebral hypoperfusion during cerebral malaria.Crit Care Med. 1999; 27: 628-632Crossref PubMed Scopus (30) Google Scholar, 15Kampfl A Pfausler B Haring HP Denchev D Donnemiller E Schmutzhard E Impaired microcirculation and tissue oxygenation in human cerebral malaria: a single photon emission computed tomography and near-infrared spectroscopy study.Am J Trop Med Hyg. 1997; 56: 585-587PubMed Google Scholar in large arteries during CM, which associated with microcirculatory obstruction has been suggested to increase cerebral blood volume leading to intracranial hypertension.16Newton CR Kirkham FJ Winstanley PA Pasvol G Peshu N Warrell DA Marsh K Intracranial pressure in African children with cerebral malaria.Lancet. 1991; 337: 573-576Abstract PubMed Scopus (177) Google Scholar Alternatively, collateral flow has been proposed as a mechanism to reconcile the findings of normal or increased CBF velocities and impaired perfusion,17White NJ Cerebral perfusion in cerebral malaria.Crit Care Med. 1999; 27: 478-479Crossref PubMed Scopus (7) Google Scholar an interpretation supported by findings of hyperdynamic flow in capillaries adjacent to obstructed vessels.9Dondorp AM Ince C Charunwatthana P Hanson J van Kuijen A Faiz MA Rahman MR Hasan M Bin Yunus E Ghose A Ruangveerayut R Limmathurotsakul D Mathura K White NJ Day NP Direct in vivo assessment of microcirculatory dysfunction in severe falciparum malaria.J Infect Dis. 2008; 197: 79-84Crossref PubMed Scopus (187) Google Scholar Interventions that improve cerebral perfusion have been proposed to be beneficial in CM.8Beare NA Harding SP Taylor TE Lewallen S Molyneux ME Perfusion abnormalities in children with cerebral malaria and malarial retinopathy.J Infect Dis. 2009; 199: 263-271Crossref PubMed Scopus (133) Google Scholar, 18Maitland K Pamba A English M Peshu N Marsh K Newton C Levin M Randomized trial of volume expansion with albumin or saline in children with severe malaria: preliminary evidence of albumin benefit.Clin Infect Dis. 2005; 40: 538-545Crossref PubMed Scopus (140) Google Scholar The murine model of CM by Plasmodium berghei ANKA (PbA) shares many features with the human pathology,19Hunt NH Grau GE Cytokines: accelerators and brakes in the pathogenesis of cerebral malaria.Trends Immunol. 2003; 9: 491-499Abstract Full Text Full Text PDF Scopus (376) Google Scholar including the presence of multiple brain microhemorrhages and vascular obstruction, although the nature of the sequestered cell (leukocytes) differs. In murine CM, magnetic resonance imaging (MRI) and spectroscopy studies showed the presence of brain edema, decreased CBF, and ischemia.20Penet MF Viola A Confort-Gouny S Le Fur Y Duhamel G Kober F Ibarrola D Izquierdo M Coltel N Gharib B Grau GE Cozzone PJ Imaging experimental cerebral malaria in vivo: significant role of ischemic brain edema.J Neurosci. 2005; 25: 7352-7358Crossref PubMed Scopus (136) Google Scholar, 21Sanni LA Rae C Maitland A Stocker R Hunt NH Is ischemia involved in the pathogenesis of murine cerebral malaria?.Am J Pathol. 2001; 159: 1105-1112Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar Lack of resolution in MRI, however, precludes detailed studies of the microcirculation, which is a major target and player in CM pathogenesis. A few studies have addressed the in vivo microcirculatory changes in murine models of severe malaria,22Kaul DK Nagel RL Llena JF Shear HL Cerebral malaria in mice: demonstration of cytoadherence of infected red blood cells and microrheologic correlates.Am J Trop Med Hyg. 1994; 50: 512-521PubMed Google Scholar, 23Kaul DK Liu XD Nagel RL Shear HL Microvascular hemodynamics and in vivo evidence for the role of intercellular adhesion molecule-1 in the sequestration of infected red blood cells in a mouse model of lethal malaria.Am J Trop Med Hyg. 1998; 58: 240-247Crossref PubMed Scopus (40) Google Scholar, 24Martini J Gramaglia I Intaglietta M van der Heyde HC Impairment of functional capillary density but not oxygen delivery in the hamster window chamber during severe experimental malaria.Am J Pathol. 2007; 170: 505-517Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar however in sites other than the brain (cremaster muscle or skin). In the present work, we used for the first time brain intravital microscopy to follow the dynamic changes in the pial microcirculation during the course of PbA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow, and eventually vascular collapse, events similar to postsubarachnoid hemorrhage (SAH) vasospasm.25Keyrouz SG Diringer MN Prevention and therapy of vasospasm in subarachnoid hemorrhage.Crit Care. 2007; 11: 220-229Crossref PubMed Scopus (156) Google Scholar We also show that nimodipine, a calcium channel blocker used to treat post-SAH vasospasm,25Keyrouz SG Diringer MN Prevention and therapy of vasospasm in subarachnoid hemorrhage.Crit Care. 2007; 11: 220-229Crossref PubMed Scopus (156) Google Scholar, 26Dorhout Mees SM Rinkel GJ Feigin VL Algra A van den Bergh WM Vermeulen M van Gijn J Calcium antagonists for aneurysmal subarachnoid haemorrhage.Cochrane Database Syst Rev. 2007; 18: CD000277Google Scholar markedly increased survival when given off-label to mice with CM as adjunctive therapy to artemether. All protocols were approved by the La Jolla Bioengineering Institutional Animal Care and Use Committee. Eight- to 10-week-old C57Bl/6 (Jackson Laboratories, Bar Harbor, ME) were inoculated intraperitoneally (IP) with 1 × 106 PbA parasites expressing the green fluorescent protein (PbA-GFP, a donation from the Malaria Research and Reference Reagent Resource Center – MR4, Manassas, VA; deposited by C.J. Janse and A.P. Waters; MR4 number: MRA-865). Parasitemia, body weight, and rectal temperature were checked daily from day 4. A motor behavior assessment modified from the SHIRPA protocol was used to determine the clinical status of the animals.27Lackner P Beer R Heussler V Goebel G Rudzki D Helbok R Tannich E Schmutzhard E Behavioural and histopathological alterations in mice with cerebral malaria.Neuropathol Appl Neurobiol. 2006; 32: 177-188Crossref PubMed Scopus (97) Google Scholar Five tests were performed: transfer arousal, locomotor activity, tail elevation, wire maneuver, and righting reflex. For each test, mice received an individual score, and the sum of scores was used to create a composite score (scale 0 to 23, where 0 represents complete impairment in all individual tests—usually comatose animals—and 23 represents maximum performance). CM was defined as the presentation of one or more of the following clinical signs of neurological involvement: ataxia, limb paralysis, poor righting reflex, seizures, roll-over, coma. We used the closed cranial window model as described.28Mostany R Portera-Cailliau C A craniotomy surgery procedure for chronic brain imaging.J Vis Exp. 2008; 15: 680Google Scholar Briefly, mice were anesthetized with ketamine- xylazine and were administered dexamethasone (0.2 mg/kg), carprofen (5 mg/kg), and ampicillin (6 mg/kg) subcutaneously, to prevent swelling of the brain, inflammatory response, and infection. After shaving the head and cleansing with ethanol 70% and betadine, the mouse was placed on a stereotaxic frame and the head immobilized using ear bars. The scalp was removed with sterilized surgical instruments, lidocaine-epinephrine was applied on the periosteum, which was then retracted exposing the skull. A 3- to 4-mm-diameter skull opening was made in the left parietal bone using a surgical drill. Under a drop of saline, the craniotomy was lifted away from the skull with very thin tip forceps and gelfoam previously soaked in saline applied to the dura mater to stop any eventual small bleeding. The exposed area was covered with a 5-mm glass coverslip secured with cyanocrylate-based glue and dental acrylic. Carprofen and ampicillin were given daily for three to five days after recovery from surgery. Mice presenting signs of pain or discomfort were euthanized with 100 mg/kg of euthasol IP. Two to three weeks after surgery, mice were lightly anesthetized with isoflurane (4% for induction, 1% to 2% for maintenance) and held on a stereotaxic frame. A panoramic picture of the vessels under the window was taken, and then mice were transferred to an intravital microscope stage (customized Leica-McBain, San Diego, CA). Body temperature was maintained using a heating pad. Using water-immersion objectives (×20), blood vessel images were captured (COHU 4815, San Diego, CA) and recorded on video-tape. An image shear device (Image Shear, Vista Electronics, San Diego, CA) was used to measure baseline vessel diameters (D), and RBC velocities (V) were measured off line by cross correlation (Photo Diode/Velocity Tracker Model 102B, Vista Electronics, San Diego, CA). Measurements of 6 to 10 pial venules (diameter range: 22 to 80 μm, velocity range: 2 to 4 mm/s) and 2 to 6 pial arterioles (diameter range: 18 to 70 μm, velocity range: 3 to 6 mm/s) were performed in each animal, and blood flow (Q) in each individual vessel was calculated using the equation: Q = V × π(D/2)2. The next day mice were inoculated IP with 1 × 106 PbA-GFP pRBC. The intravital microscopy procedure was repeated daily from day 4 of infection until the mice died or were euthanized. Noninfected control mice were submitted to the same procedures. To enhance imaging of the vascular network, including poorly perfused vessels, in two experiments animals with clinical signs of CM (n = 8) and controls (n = 3) were infused i.v. with albumin-FITC (1 mg/kg; Molecular Probes, Irvine, CA). Adherent and rolling leukocytes were visualized by anti-CD45-TxR antibodies (CalTag, Carlsbad, CA), also infused i.v. Green fluorescence (518 nm) emitted by albumin-FITC and GFP (PbA-GFP pRBC) was captured using ALPHA Vivid: XF100-2 (Omega Optical, Brattleboro, VT), and anti-CD45-TxR fluorescence (615 nm) was exited and captured with a Vivid Standard: XF42. To evaluate the effect of nimodipine on pial blood flow, PbA-infected mice with clinical CM and noninfected controls were imaged, vessel diameter and RBC velocities were measured, and then they were injected with artemether plus nimodipine at 4 mg/kg (as described below) and measurements were repeated at 30, 60, and 120 minutes. PbA-infected mice presenting poor righting reflex, hypothermia, and/or other clinical signs of neurological involvement such as ataxia, limb paralysis, seizures, and/or roll-over were treated with artemether (Artesiane, Dafra Pharma, Belgium, a kind gift of Dr. Alberto Moreno, Emory University, Atlanta, GA) given IP at 50 mg/kg, in combination with nimodipine (Sigma, St Louis, MO) or vehicle. Nimodipine was dissolved in ethanol (EMD, NJ), dispersed with polyethyleneglycol 400 (PEG, Sigma), and then saline was added (1:1:8 v/v) and mixed thoroughly. This solution was administered IP in three different doses: 1.3 mg/kg, 4 mg/kg, and 12 mg/kg. Artemether was given daily for five days, and nimodipine or vehicle were given at 0, 12, 24, and 36 hours. Parasitemia, motor behavior, and rectal temperature were checked at each time point and daily afterward. After treatment, parasitemia was checked by microscopical examination of Giemsa-stained blood smears to differentiate viable from dead parasites. Statistical analyses were performed using the Student t test with Mann–Whitney correction when comparing two groups, analysis of variance with Kruskall–Wallis post hoc analysis when comparing more than two groups, and survival curves were compared with a nonparametric log-rank test, using the Graphpad Prism software (GraphPad Software Inc., La Jolla, CA). A P value <0.05 was considered significant. Reported data are the mean ± SEM unless otherwise indicated. Mice with an implanted cranial window and infected with PbA presented an overall CM incidence of 83% (19/23 mice, four separate experiments), deaths occurring on days 5 to 8 (Figure 1A) with parasitemias between 10% and 30% (Figure 1B). Infected mice developed hypothermia, more intense in CM mice (Figure 1C). Pial vascular hemodynamics was sequentially studied in the 23 PbA-infected and 10 noninfected control mice. Three of the 19 PbA-infected mice that developed CM died before the measurements could be performed at the time of presentation of clinical signs of CM. Therefore, complete hemodynamics data are available for 16 mice with CM, with a total of 56 arterioles and 114 venules analyzed. In the four PbA-infected mice that did not develop CM, 8 arterioles and 38 venules were analyzed, and in the 10 control mice a total of 33 arterioles and 83 venules were analyzed. Marked decreases in arteriolar and venular blood flows were observed during infection, particularly in mice presenting clinical signs of CM (Figure 2, A and B; see also Supplemental Videos S1, S2, and S3 at http://ajp.amjpathol.org). In mice presenting clinical CM, the mean decrease in arteriolar blood flow was 60% of baseline, with one (6%) mouse showing preserved blood flow, 8/16 (50%) CM mice a decrease in blood flow between 40% and 60%, and 7/16 (44%) CM mice more than 75% decrease (Figure 2C). This last group includes three mice with vascular network collapse (see below), two of them with no visible patent vessels and whose blood flow was considered to be zero in the area under the cranial window. The observed decreases in blood flow in mice with CM were attributable to low RBC velocities and also to vasoconstriction in 11 (79%) of 14 mice (Figures 3, A and B). Three (21%) CM mice presented vasodilation instead. Interestingly, on the day before CM development, half the mice actually presented increased (6% to 25%) vessel diameters, and aneurism-like “balloon” vessel changes were observed in four mice. The outcome of the balloon lesions was not determined in three of the four affected mice because of death before next examination (two mice) and lack of record in one mouse; in the one mouse followed up, the balloon vessel evolved to vascular collapse. The four PbA-infected mice that did not develop CM presented maximal decreases of arteriolar blood flow between 14% and 37% (mean: 25%) during follow up (Figure 2, A–C). Noninfected control mice showed stable vessel diameters and a mean 10% decrease in RBC velocities on days 6 to 10 of follow-up, resulting in slight to moderate decreases in blood flow in this period (mean: 5% on day 6; 15% on day 8; 9% on day 10; Figures 2 and 3). Three of the 10 noninfected control mice were followed up to day 16, with measurements performed every other day, and showed stable blood flow during this period (data not shown).Figure 2PbA infection leads to decreased blood flow in pial vessels. Arteriolar (A) and venular (B) blood flow in PbA-infected mice with or without CM and in uninfected control mice. Results are expressed as the percentage change in relation to baseline measurements performed before infection. Flow was significantly decreased on day 6 in mice that developed CM (arteriolar: P = 0.0003; venular: P = 0.0003). Data are the mean ± SEM. C: Arteriolar blood flow in mice with CM, at the time of presentation of clinical signs (irrespective of the day of infection). Flow was significantly decreased in mice with CM (P < 0.0001). Values shown for uninfected controls mice, and infected mice without CM are the lowest recorded for each mouse on days 5 to 8. Bars indicate the mean value.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Changes in diameters and in RBC velocities in arterioles during PbA infection. A: Mice with CM showed a significant decrease in arteriolar diameters compared with uninfected controls (P = 0.0151). B: Mice with CM also showed significant decreases in RBC velocities in relation to uninfected controls (P < 0.0001). Bars indicate the mean value.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In two experiments, after vessel diameter and RBC velocities were measured in mice presenting clinical CM, albumin-FITC and anti-CD45-TxR antibodies were injected in the tail vein to enhance imaging of the vascular network. In many venules of CM mice the large number of adherent leukocytes functioned as barriers to blood flow and caused marked reductions in luminal diameters (Figure 4, A–D; see also Supplemental Video S4 at http://ajp.amjpathol.org). RBC velocities were heterogeneous, with some larger vessels presenting sluggish RBC velocities and non-perfused feeding vessels (see Supplemental Videos S2 and S3 at http://ajp.amjpathol.org). Capillaries and smaller venules were frequently nonperfused, and in some cases adherent leukocytes were found to obstruct the lumen (Figure 4, E and F; see also Supplemental Videos S5 and S6 at http://ajp.amjpathol.org). We observed real-time obstruction of capillaries occurring during intravital microscopy (see Supplemental Video S6 at http://ajp.amjpathol.org). Nonperfused capillaries eventually collapsed (Figure 4, G and H). Most vessels with obstructed flow, showing no blood cell transit, still presented albumin-FITC–derived fluorescence and the adherent leukocytes, when present, showed anti-CD45 staining, suggesting that plasma flow was not completely impaired in such vessels. Sequestered pRBC were rarely observed and, in such cases, the trapped cells were usually attached to the surface of an endothelium-adherent leukocyte (Figures 5, A and B; see also Supplemental Video S7 at http://ajp.amjpathol.org), confirming previous observations by histology.29Martins YC Smith MJ Pelajo-Machado M Werneck GL Lenzi HL Daniel-Ribeiro CT Carvalho LJ Characterization of cerebral malaria in the outbred Swiss Webster mouse infected by Plasmodium berghei ANKA.Int J Exp Pathol. 2009; 90: 119-130Crossref PubMed Scopus (48) Google Scholar It is conceivable that infusion of albumin and anti-CD45 antibodies might have beneficial effects in mice with CM and therefore altered outcome, albumin by expanding blood volume and improving perfusion,18Maitland K Pamba A English M Peshu N Marsh K Newton C Levin M Randomized trial of volume expansion with albumin or saline in children with severe malaria: preliminary evidence of albumin benefit.Clin Infect Dis. 2005; 40: 538-545Crossref PubMed Scopus (140) Google Scholar, 30Cabrales P Tsai AG Ananda K Acharya SA Intaglietta M Volume resuscitation from hemorrhagic shock with albumin and hexaPEGylated human serum albumin.Resuscitation. 2008; 79: 139-146Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar and anti-CD45 antibodies by potentially mediating destruction of leukocytes and decreasing inflammation.31Wulf GG Luo KL Goodell MA Brenner MK Anti-CD45-mediated cytoreduction to facilitate allogeneic stem cell transplantation.Blood. 2003; 101: 2434-2439Crossref PubMed Scopus (36) Google Scholar However, even if present, these effects might have been minor. The amount of albumin infused (1 mg/kg, or less than 20 μg/mouse) is almost insignificant compared with the albumin concentration in the plasma of mice (50 mg/ml), and the amount of anti-CD45 antibody infused (2 to 4 μg/mouse) was also small. In line with this interpretation, in the experiments in which albumin and anti-CD45 antibody infusion was performed, all CM mice died within 24 hours as expected, similar to the experiments in which infusion was not performed. In addition, no measurements of blood flow were performed after infusion.Figure 5PbA pRBCs do not directly adhere to pial endothelial cells but may be found trapped by adherent leukocytes. A: A fluorescent PbA-GFP pRBC attached to a leukocyte (arrow). Other visible adherent leukocytes are indicated by asterisks. B: The same vessel section evidencing the adherent leukocytes, highlighted after changing the filter to detect TxR fluorescence. The arrow points to the leukocyte with the attached pRBC. See also supplemental video S7 at http://ajp.amjpathol.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A striking feature observed in 7 of 16 (44%) mice with CM (three mice died before images could be taken), and in none of the control or non-CM mice, was the collapse of large pial vessels (Figures 6, A–C and D–F) or even of a microvascular network (Figures 6, G–I). Four mice presented one or few collapsed vessels, and three mice presented vascular network collapse. In two of the three cases of vascular network collapse, this was preceded by the occurrence of hemorrhage in a major vessel (Figures 6, J–L). Noteworthy, 71% (5/7) of the mice with vascular collapse presented decreases in blood flow over 75% in relation to baseline, against 22% (2/9) in the group of CM mice without vascular collapse. Mice with vascular collapse presented also more severe vasoconstriction, with a mean 32% decrease in overall arteriolar diameter of the remaining noncollapsed vessels in relation to baseline, against a mean 19% decrease in all mice with CM (P = 0.0162). The observations of vasoconstriction and vascular collapse, which may occur in association with pial hemorrhage, are reminiscent of the vasospasm phenomenon that frequently occurs after SAH and is associated with neurological deterioration and poor prognosis.25Keyrouz SG Diringer MN Prevention and therapy of vasospasm in subarachnoid hemorrhage.Crit Care. 2007; 11: 220-229Crossref PubMed Scopus (156) Google Scholar, 32Pluta RM Hansen-Schwartz J Dreier J Vajkoczy P Macdonald RL Nishizawa S Kasuya H Wellman G Keller E Zauner A Dorsch N Clark J Ono S Kiris T Leroux P Zhang JH Cerebral vasospasm following subarachnoid hemorrhage: time for a new world of thought.Neurol Res. 2009; 31: 151-158Crossref PubMed Scopus (320) Google Scholar The standard drug for prevention and treatment of post-SAH vasospasm is nimodipine,26Dorhout Mees SM Rinkel GJ Feigin VL Algr
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