Neuronal apoptosis, metallothionein expression and proinflammatory responses during cerebral malaria in mice
Introduction
Plasmodium falciparum malaria remains a massive burden of disease and death. The two major complications, cerebral malaria (CM) and severe anemia, are responsible for more than 1 million deaths/year mainly in children under 5 years of age in sub-Saharan Africa (Snow et al., 2005). At least a similar number of children are likely to be left with serious neurological impairment following CM (Mung'Ala-Odera et al., 2004).
Although CM has been studied extensively, many of the pathophysiological mechanisms remain unclear and treatment other than antiparasitic medication is not available. CM is an acute encephalopathy with increased levels of proinflammatory cytokines like tumor necrosis factor (TNF)-α, interferon-γ (IFN-γ) and lymphotoxin (LT) (Brown et al., 1999, Hunt and Grau, 2003). Postmortem examination of the brain reveals hemorrhages, edema and adherence of parasitized red blood cells (pRBCs) to the cerebral microvasculature. The CNS microvasculature may eventually become obstructed by the pRBC adherence, and thus ischemia and metabolic dysfunctioning are seen, which contribute to the CM pathophysiology (Medana et al., 2001a). However, it is not known how pRBC, which do not leave the vascular bed, influence parenchymal brain function to induce coma and in some cases lead to persisting brain damage. The endothelial lining is likely to play a major role in this respect.
A histopathological equivalent to the clinically observed persisting brain damage has not been identified. Neuronal necrosis is not a feature of CM, but high levels of reactive oxygen species (ROS) are generated leading to oxidative stress and neuroglial degeneration (Barnham et al., 2004, Pino et al., 2003). ROS and oxidative stress are important inducers of apoptosis, a genetically controlled, active form of cell death characterized by surface blebbing, contraction of cells and their nuclei, proteolysis and DNA digestion (Hasnain et al., 2003). It differs from necrosis in its programmed manner, complex regulatory mechanisms and distinctive morphological changes (Zhang et al., 2004).
Metallothioneins (MTs) constitute a family of small proteins (molecular weight 6–7 kDa) characterized by a high metal [Zn(II), Cu(I)] content and also by an unusual cysteine abundance. Most cells of the mammalian body possess the ability to express MTs. Within the mammalian brain, MTs occur in isoforms designated MT-I to MT-III; MT-I + II are the best characterized MT isoforms which are regulated and expressed coordinately and rapidly induced by many pathogens. During CNS pathology, levels of MT-I + II mRNA and proteins are significantly increased in reactive astrocytes and microglia/macrophages (Hidalgo et al., 2001).
MT-I + II have a wide range of protective functions in the CNS. They are antioxidant and antiapoptotic factors scavenging ROS and reducing oxidative stress and apoptosis during various CNS disorders such as epilepsy, traumatic injury, meningoencephalitis and neurodegenerative diseases (Hidalgo et al., 2001). Following traumatic brain injury, MT-I + II induce wound healing, glial scar formation and angiogenesis (Giralt et al., 2002, Penkowa et al., 1999, Penkowa et al., 2002). MT-I + II also reduce cerebral edema and infarct volume and improve functional outcome after transient focal cerebral ischemia (van Lookeren et al., 1999). Treatment with MT-II in mice with experimental autoimmune encephalomyelitis can decrease the expression of proinflammatory cytokines in the brain such as IL-6 and TNF-α as well as significantly reduce the number of apoptotic neurons (Penkowa and Hidalgo, 2001).
Experimental cerebral malaria (ECM) in mice is an animal model widely used for the study of the CM pathophysiology (de Souza and Riley, 2002). Infection with Plasmodium berghei ANKA (PbA) in specific murine strains causes neurological symptoms such as seizures and coma followed by death. The histopathological features are comparable to those of human CM, including petechial hemorrhages, edema and disruption of the blood–brain barrier (BBB). However, in contrast to humans, the mice show vascular adhesion of monocytes rather than pRBCs (de Souza and Riley, 2002, Gitau and Newton, 2005). The number of pRBCs used for inoculation affects the development of ECM in mice. A low infective dose frequently leads to ECM, whereas a high infective dose does not cause ECM but severe anemia and hyperparasitemia (Curfs et al., 1992).
In this report, we have analyzed cerebral apoptosis and MT-I + II expression in the brains of mice during the course of ECM and compared to normal controls using immunohistochemistry and histochemistry.
Section snippets
Animals
Female pathogen-free C57BL/6j mice 8–10 weeks old, weighing 18–25 g, were purchased form Møllegaard, Denmark. All animals were pathogen-free and were kept under standardized conditions at the animal facilities at the Panum Institute, University of Copenhagen, with free access to food and water ad libitum. All experiments adhered to Danish and European guidelines for animal research and were approved by the national board for animal studies. All efforts were made to minimize animal suffering and
Results
Mice killed on days 7 and 9 did not show clinical signs of ECM nor did the body temperature drop. The remaining 5 infected mice presented signs of severe ECM-like ataxia, convulsions or coma. Four of these mice were killed and perfused when showing signs of terminal illness, i.e. coma and hypothermia, and one mouse was found dead on day 11 and could not be perfused. No significant difference in the severity of neurological symptoms was observed in this group. No clinical signs of ECM were seen
Discussion
In this study, we have demonstrated apoptotic cell death and the expression of MT-I + II protein in the brains of mice with cerebral malaria. In addition, we describe a number of histopathological findings in the brains of mice with CM that parallel the human disease. As the availability of tissues from postmortem is restricted by religious and cultural objections to autopsy, only a limited number of histopathological studies in patients that died from CM have been carried out. An animal model
Acknowledgments
Thanks to Lars Hviid for critically reviewing the manuscript and to Ming Chen for valuable practical advice and help. The excellent technical assistance of Grethe Gomme, Hanne Hadberg, Pernille S. Froh and Ha Nguyen is gratefully acknowledged. These studies were supported by The Lundbeck Foundation, The Danish Medical Research Council, IMK Almene Fond, Kathrine og Vigo Skovgaards Fond, The Danish Medical Association Research Fund, Toyota Fonden, Fonden til Lægevidenskabens Fremme, Eva and Henry
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