See related article on page 1306 The American Journal of Pathology, Vol. 176, No. 3, March 2010 Copyright © American Society for Investigative Pathology DOI: 10.2353/ajpath.2010.091090 Commentary Cerebral Malaria A Vasculopathy Mahalia S. Desruisseaux,*† Fabiana S. Machado,‡ Louis M. Weiss,*† Herbert B. Tanowitz,*† and Linnie M. Golightly§ From the Department of Pathology * Division of Parasitology, and the Department of Medicine,† Division of Infectious Disease, Albert Einstein College of Medicine, Bronx, New York; the Department of Biochemistry and Immunology,‡ Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Brazil; and the Department of Medicine § Division of Infectious Disease, Weill Cornell Medical College, New York, New York Cerebral malaria (CM), an important disease entity in the developing world, remains the deadliest complication of infection with P. falciparum. According to a recent World Health Organization report, half of the world’s population remains at risk of acquiring malaria. The majority of the burden of disease occurs in Sub-Saharan Africa, where children under the age of five account for more than 80% of malaria-related deaths. CM is associated with an encephalitic syndrome that includes ataxia, seizures, hemiplegia, and eventually coma and death. These symptoms of severe malaria can progress very precipitously within hours from mild to severe. Indeed, even with successful antiparasitic treatment, residual neurological damage is a common finding in CM. It is estimated that more than 10% to 20% of the children who survive an episode of CM develop long-term cognitive deficits, which can include memory impairment, learning and language impairments, visuospatial and motor deficits, and psychiatric disorders. The etiology of CM is not entirely understood because clinical studies in humans are not always feasible, and autopsy studies have only given us a limited view of this disease syndrome. In recent years, the increasing availability of computerized axial tomography (CT) and magnetic resonance imaging (MR) scanning has increased our understanding of the pathophysiology of this entity. Multiple mechanisms have been proposed to govern CM pathogenesis, including an intense upregulation of the inflammatory response, hypoxia, hypoglycemia, monocytic and/or red blood cell sequestration, and microvas- cular dysfunction leading to ischemia. The etiology of CM is likely multifactorial, and these hypotheses are not mutually exclusive. The mouse model of CM using C57BL/6 mice infected with the ANKA strain of P. berghei as described by Cabrales et al1 in the current issue of this Journal recapitulates many of the features of human CM. Although there has been some discussion as to whether this particular mouse model is a reliable model of human CM, it is now generally acknowledged to be a relevant and practical small animal model for CM. The pathological features of both human CM and the murine model described here and by others include microhemorrhages and vascular occlusion. However, the nature of the vascular occlusion in murine CM differs from that observed in human CM in that the former displays no red blood cell adherence and/or occlusion. Importantly, cognitive dysfunction has been observed in this animal model.2 Recently, a number of studies implicate a disruption in the integrity of the cerebral vasculature as an important contributing factor in the pathogenesis of CM. Both human and experimental CM studies are associated with a reduction in cerebral blood flow (CBF), which may be an important factor in the progression to CM. Single photon emission computed tomography (SPECT) in human CM demonstrated marked cerebral hypoperfusion associated with a significant decrease in oxygen saturation and neurological deficits corresponding to the areas of hypoSupported in part from grants IDSA ERF/NFID Colin L. Powell Minority Postdoctoral Fellowship in Tropical Disease Research, sponsored by GlaxoSmithKline; NIH Training Grant in Mechanisms of Cardiovascular Diseases (T32 HL-07675 to M.S.D.); Dominick P. Purpura Department of Neuroscience (neuroscience fellowship to M.S.D.) and Department of Psychiatry and Behavioral Sciences (to M.S.D.), Albert Einstein College of Medicine; Burroughs-Wellcome Funds Career Awards for Medical Scientists (to M.S.D.); Dana Foundation Program in Brain and Immunoimaging (to H.B.T.); and the CNPq and FAPEMIG Brazil (to F.S.M.) and National Institutes of Health grants NS054243 (to L.M.G.) and AI-076248 (to H.B.T.). Accepted for publication November 25, 2009. Address reprint requests to Herbert B. Tanowitz, M.D., Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. E-mail: herbert.tanowitz@einstein.yu.edu. 1075 1076 Desruisseaux et al AJP March 2010, Vol. 176, No. 3 perfusion.3,4 These abnormalities include decreased or absent perfusion in the capillaries and in larger retinal vessels, intravascular filling defects and leakage of dye material, which is indicative of a breakdown of the blood– retinal barrier, and ischemia.5 The ischemic changes often correlate with neurological sequelae including seizures, obtundation, and coma. In the current issue of the Journal, Cabrales et al1 present substantial evidence for a role for vasoconstriction in the setting of CM and highlight the importance of vascular dysfunction in the pathogenesis of CM. Through the use of intravital microscopy, these authors obtained direct visualization of the pial microvasculature of the brain and correlated vascular dysfunction with progression of CM. Importantly, this disease progression was reversed when the vasculopathy was corrected by the calcium-channel blocker nimodipine. Previously, it was demonstrated that in the murine model of CM, a reduction in CBF at advanced stages of the disease as measured by MRI/MRA directly correlated with significant decreases in the levels of certain metabolic markers in areas of the brain that were indicative of neuronal damage.5 Specifically, a decrease in CBF was reported to be associated with a reduction in the ratio of N-acetyl aspartate (NAA) to creatine.5 NAA has been widely used as an inverse marker of neuronal loss and injury in a variety of pathologies. It is synthesized almost exclusively in neuronal mitochondria, and a decrease in NAA levels usually reflects a mixture of both neuronal loss and recent or ongoing neuronal injury/dysfunction. A reduction in cerebral perfusion has also been associated with damage in the neuron/axon compartment with CM.5 Conversely, MR spectroscopy studies of mice resistant to murine CM demonstrated no change in CBF or metabolic profile and no central nervous system lesions. These data indicate that alterations in the vasculature are an important component of CM. In the present report, Cabrales et al1 demonstrated a clear correlation with neurological deficits such as ataxia, limb paralysis, poor righting reflex, and seizures and the changes in the pial vessels. These deficits appear to be lesion-dependent, as mice with more severe neurological symptoms had a greater degree of vascular constriction and even sustained complete vascular collapse, whereas those with no signs of CM had a minimal decrease in CBF. Importantly, treatment with nimodipine together with the antimalarial agent artemether not only resulted in improved survival but also in a more rapid return to normal neurological function. The authors suggest that the reason for this observation is the partial restoration of CBF in affected mice. The vasculopathy associated with CM is likely a result of endothelial cell damage, ischemia, activation of vascular cell adhesion molecules, and an associated breakdown in the blood– brain barrier.6,7 Recently, we have focused on the role of vasoactive compounds in the setting of CM, particularly the 21-aa vasopeptide endothelin (ET-1).8 Elevated plasma levels of ET-1 and big ET-1 have been reported in patients with P. falciparum. This occurs during acute infection and persists days after treatment of malaria. The increase in ET-1 correlates with elevated levels of TNF-␣ and likely reflects damage to the endothelium. A similar observation has been reported in an experimental CM model. In this same mouse model, there was an increase in the expression of ET-1 and endothelin converting enzyme, the enzyme that is responsible for the synthesis of ET-1 from Big ET-1, as well as increased expression of the endothelin receptors (ETA and ETB). This increase in the components of the endothelin pathway was associated with a reduction in CBF and neuronal dysfunction and inflammation.8 The increase in ET-1 in the brain of mice with CM is consistent with the findings of increased plasma levels of big ET-1 in patients with acute complicated disease. Interestingly, the intraventricular injection of ET-1 has been reported to result in behavioral changes, including barrel rolling, body tilting, nystagmus, clonus, and tail extension. Most of these effects occur at doses that do not cause any changes in CBF. Low levels of nitric oxide (NO) derived from endothelial nitric oxide synthase (NOS) and neuronal NOS have also been implicated in the vasculopathy of CM, further supporting the observation that vasoconstriction is an integral part in the development of CM. Cabrales et al1 suggest that microhemorrhages may be important in this disease process because of the alterations in NO levels, which are known to be associated with cerebral hemorrhages. Using calcium-channel blockers to ameliorate vascular spasm is not entirely novel in experimental cardiovascular disease. For example, Factor et al9 demonstrated that the administration of verapamil to Syrian cardiomyopathic hamsters ameliorated the vasospasm of the coronary microvasculature, thus resulting in improvement of the cardiomyopathic phenotype. When verapamil was administered early, but not late, in the course of experimental Trypanosoma cruzi infection, it prevented the appearance of cardiomyopathy.10 Furthermore, Tanowitz et al11 used a cremaster muscle preparation to demonstrate that the T. cruzi--associated vasospasm was prevented by the administration of verapamil, similar to the findings in CM in the current report with nimodipine.1 Nimodipine, the calcium-channel blocker used in the present study, ameliorates the vasospasm accompanying subarachnoid hemorrhage.1 Drugs of seemingly disparate classes show some efficacy in experimental CM. For example, Serghides et al12 recently reported that the peroxisome proliferatoractivated receptor-␥ (PPAR-␥) agonist rosiglitazone improved the outcome in CM in the mouse model. The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor (HMG-CoA reductase inhibitor or statin) atorvastatin has been shown to be highly efficacious when used in combination with artesunate, resulting in a significant decrease in mortality.13 Erythropoietin has also been reported as effective in ameliorating CM.14 These agents are used clinically because of their effects on cholesterol metabolism, insulin resistance, and erythropoiesis, respectively. Pertinent to the current study, however, they share with calcium-channel blockers the ability to modulate the repair of microvascular damage. Commentary 1077 AJP March 2010, Vol. 176, No. 3 Damage to the microvasculature, as elegantly demonstrated in the current study, is generally repaired either by replication of local existing endothelial cells, or by bone marrow– derived circulating endothelial progenitor cells (cEPCs), which are stimulated to migrate and incorporate into damaged sites in the microvasculature.15 Their mobilization, release, and ultimate incorporation into sites of microvascular damage are mediated by the activation of a series of molecules, including stromal cell derived growth factor 1 (SDF-1). The subsequent incorporation of cEPCs into sites of microvascular damage is essential to the maintenance of microvascular integrity. They play a critical role in diseases associated with chronic or extensive damage. Indeed, insufficient or dysfunctional cEPCs and levels of associated factors that effect cEPC mobilization and function are predictive of poor outcomes in diseases associated with microvascular damage such as in cardiovascular disease and stroke16,17. PPAR-␥ agonists, HMG-CoA reductase inhibitors (statins), erythropoietin and calcium channel blockers have all been reported to modulate the cEPC response. PPAR-␥ agonists, which promote the differentiation and mobilization of cEPCs, have been shown to promote revascularization postangioplasty and are being evaluated in the treatment of stroke and ischemia/reperfusion injuries.18 Statins increase cEPC mobilization, numbers, and functional activity, as well as delay cEPC aging and onset of senescence. These effects are independent of their ability to lower cholesterol and are believed to be responsible for the ability of statins to reduce myocardial and cerebral ischemia and cardiovascular risk.19 Erythropoietin also increases the number of cEPCs in the bone marrow as well as the peripheral circulation. It improves vascularization in murine models of ischemia and has been shown to be safe and effective in the treatment of stroke patients.20 Finally, calcium-channel blockers increase cEPC levels, promote their differentiation and migration as well as their vasodilatory effects. They have therefore been proposed as therapeutic agents to enhance microvascular repair by preserving endothelial cell integrity.21 A recent study of children with CM in Ghana suggests that the cEPC response to malaria-induced microvascular damage might also play a role in the pathogenesis of CM.22 Children with CM have lower levels of cEPCs as compared with children with uncomplicated malaria, asymptomatic parasitemic children, children with severe malarial anemia, and healthy controls. In addition, levels of SDF-1 are elevated in children with acute disease (uncomplicated malaria and CM), indicative of host attempts to mobilize cEPCs to sites of microvascular damage. These findings place CM within in the context of current paradigms for microvascular repair mechanisms and suggest that equilibrium exists between malaria-induced microvascular damage and host-mediated repair. This balance is maintained by bone marrow– derived cEPCs, which augment local microvascular repair mechanisms in the brain of P. falciparum-infected children. Acute malarial infection is associated with an increase in SDF-1 levels, leading to mobilization and homing of cEPCs to sites of microvascular damage. Thus, hosts able to maintain the balance between damage and repair do not develop CM. If there are low or insufficient numbers of cEPCs to mediate repair, the equilibrium is lost. CM develops, in part, because of breaches in the integrity of the brain microvasculature. Children able to reestablish equilibrium recover, whereas those that are unable to do so likely will die. As described in other diseases associated with microvascular damage, this equilibrium may be lost either because of the exhaustion of bone marrow progenitor cells or because of cEPC dysfunction stemming from reduced migratory capacity, survival, and/or differentiation.23 Thus, chemotherapeutic agents such as PPAR-␥ agonists, statins, erythropoietin, and calcium-channel blockers that encourage microvascular repair could be effective in the prevention and/or treatment of CM. This is consistent with the present study and highlights the role of the microvascular axis as an important target for adjuvant therapies. The demonstration that vasospasm with post-subarachnoid hemorrhage is ameliorated by nimodipine used in this study supports the notion that the host response to the microvascular damage induced by malaria is similar to that of other diseases involving microvasculature dysfunction. This is supported by current paradigms of microvascular repair, which invoke common repair mechanisms and physiological responses regardless of the initial insult to the vasculature. As suggested by the authors, nimodipine, which has been used safely and effectively in humans in the treatment of diseases associated with microvascular damage, may be useful as an adjunctive therapy in the treatment of human CM. The authors are understandably cautious regarding the need for further study of such agents and their effects in murine models of CM attributable to the heterogeneity of case presentations of CM and the multifactorial nature of the disease before clinical trials in humans. The pleomorphic effects of these drugs also require a comprehensive study of their mechanisms of action in malaria using enhanced models such as that described by Cabrales et al.1 For example, the finding that vasodilatation may precede vasoconstriction as observed in some mice with CM might indicate that calcium-channel blockers could be detrimental depending on the time of administration. Indeed in other microvasculature diseases the potential need for “cocktails” of different agents affecting the microvascular axis has been raised with the possibility of both beneficial and detrimental effects. Optimization of murine models of CM permits the direct study of the effects of these agents before use in humans and therefore is an important advance in the field. However, the apparent safety of several modulators of the response to microvascular damage in recent clinical trials in patients with malaria is encouraging.24,25 Finally, it is important to reiterate that in the future, the treatment of CM may involve some form of adjuvant therapy in addition to a potent antimalarial drug. The inclusion of adjuvants, which maintain the integrity of the vasculature, promises to be an important addition to the malaria pharmacopoeia, a point underscored by Carbrales et al.1 1078 Desruisseaux et al AJP March 2010, Vol. 176, No. 3 References 1. Cabrales P, Zanini GM, Meays D, Frangos JA, Carvalho L.J.M: Murine Cerebral malaria associated with a vasospasm-like microcirculatroy dysfunction and survival upon rescue treatment is markedly increased by nimodipine. Am J Pathol 2009, 176:1306 –1315 2. Desruisseaux MS, Gulinello M, Smith DN, Lee SC, Tsuji M, Weiss LM, Spray DC, Tanowitz HB: Cognitive dysfunction in mice infected with Plasmodium berghei strain ANKA. J Infect Dis 2008, 197:1621–1627 3. Kampfl 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, 56:585–587 4. Beare NA, Riva CE, Taylor TE, Molyneux ME, Kayira K, White VA, Lewallen S, Harding SP: Changes in optic nerve head blood flow in children with cerebral malaria and acute papilloedema. J Neurol Neurosurg Psychiatry 2006, 77:1288 –1290 5. Kennan RP, Machado FS, Lee SC, Desruisseaux MS, Wittner M, Tsuji M, Tanowitz HB: Reduced cerebral blood flow and N-acetyl aspartate in a murine model of cerebral malaria. Parasitol Res 2005, 96:302–307 6. Hunt NH, Grau GE: Cytokines: accelerators and brakes in the pathogenesis of cerebral malaria. Trends Immunol 2003, 24:491– 499 7. Newton CR, Krishna S: Severe falciparum malaria in children: current understanding of pathophysiology and supportive treatment. Pharmacol Ther 1998, 79:1–53 8. Machado FS, Desruisseaux MS, Nagajyothi, Kennan RP, Hetherington HP, Wittner M, Weiss LM, Lee SC, Scherer PE, Tsuji M, Tanowitz HB: Endothelin in a murine model of cerebral malaria. Exp Biol Med (Maywood) 2006, 231:1176 –1181 9. Factor SM, Minase T, Cho S, Dominitz R, Sonnenblick EH: Microvascular spasm in the cardiomyopathic Syrian hamster: a preventable cause of focal myocardial necrosis. Circulation 1982, 66:342–354 10. De Souza AP, Tanowitz HB, Chandra M, Shtutin V, Weiss LM, Morris SA, Factor SM, Huang H, Wittner M, Shirani J, Jelicks LA: Effects of early and late verapamil administration on the development of cardiomyopathy in experimental chronic Trypanosoma cruzi (Brazil strain) infection. Parasitol Res 2004, 92:496 –501 11. Tanowitz HB, Kaul DK, Chen B, Morris SA, Factor SM, Weiss LM, Wittner M: Compromised microcirculation in acute murine Trypanosoma cruzi infection. J Parasitol 1996, 82:124 –130 12. Serghides L, Patel SN, Ayi K, Lu Z, Gowda DC, Liles WC, Kain KC: Rosiglitazone modulates the innate immune response to Plasmodium falciparum infection and improves outcome in experimental cerebral malaria. J Infect Dis 2009, 199:1536 –1545 13. Bienvenu AL, Picot S: Statins alone are ineffective in cerebral malaria but potentiate artesunate. Antimicrob Agents Chemother 2008, 52: 4203– 4204 14. Bienvenu AL, Ferrandiz J, Kaiser K, Latour C, Picot S: Artesunateerythropoietin combination for murine cerebral malaria treatment. Acta Trop 2008, 106:104 –108 15. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM: Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997, 275: 964 –967 16. Werner N, Kosiol S, Schiegl T, Ahlers P, Walenta K, Link A, Bohm M, Nickenig G: Circulating endothelial progenitor cells and cardiovascular outcomes. N Engl J Med 2005, 353:999 –1007 17. Taguchi A, Matsuyama T, Nakagomi T, Shimizu Y, Fukunaga R, Tatsumi Y, Yoshikawa H, Kikuchi-Taura A, Soma T, Moriwaki H, Nagatsuka K, Stern DM, Naritomi H: Circulating CD34-positive cells provide a marker of vascular risk associated with cognitive impairment. J Cereb Blood Flow Metab 2008, 28:445– 449 18. Aicher A, Zeiher AM, Dimmeler S: Mobilizing endothelial progenitor cells. Hypertension 2005, 45:321–325 19. Walter DH, Rittig K, Bahlmann FH, Kirchmair R, Silver M, Murayama T, Nishimura H, Losordo DW, Asahara T, Isner JM: Statin therapy accelerates reendothelialization: a novel effect involving mobilization and incorporation of bone marrow-derived endothelial progenitor cells. Circulation 2002, 105:3017–3024 20. Heeschen C, Aicher A, Lehmann R, Fichtlscherer S, Vasa M, Urbich C, Mildner-Rihm C, Martin H, Zeiher AM, Dimmeler S: Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization. Blood 2003, 102:1340 –1346 21. Sugiura T, Kondo T, Kureishi-Bando Y, Numaguchi Y, Yoshida O, Dohi Y, Kimura G, Ueda R, Rabelink TJ, Murohara T: Nifedipine improves endothelial function: role of endothelial progenitor cells. Hypertension 2008, 52:491– 498 22. Gyan B, Goka BQ, Adjei GO, Tetteh JK, Kusi KA, Aikins A, Dodoo D, Lesser ML, Sison CP, Das S, Howard ME, Milbank E, Fischer K, Rafii S, Jin D, Golightly LM: Cerebral malaria is associated with low levels of circulating endothelial progenitor cells in African children. Am J Trop Med Hyg 2009, 80:541–546 23. Schmidt-Lucke C, Rossig L, Fichtlscherer S, Vasa M, Britten M, Kamper U, Dimmeler S, Zeiher AM: Reduced number of circulating endothelial progenitor cells predicts future cardiovascular events: proof of concept for the clinical importance of endogenous vascular repair. Circulation 2005, 111:2981–2987 24. Boggild AK, Krudsood S, Patel SN, Serghides L, Tangpukdee N, Katz K, Wilairatana P, Liles WC, Looareesuwan S, Kain KC: Use of peroxisome proliferator-activated receptor gamma agonists as adjunctive treatment for Plasmodium falciparum malaria: a randomized, doubleblind, placebo-controlled trial. Clin Infect Dis 2009, 49:841– 849 25. Picot S, Bienvenu AL, Konate S, Sissoko S, Barry A, Diarra E, Bamba K, Djimde A, Doumbo OK: Safety of epoietin beta-quinine drug combination in children with cerebral malaria in Mali. Malaria J, 2009: 8:169