An Overview of Cerebral Malaria: Lessons Learnt from Observations in Humans and Experimental Models

Malaria has been a significant threat to human health
throughout history. As an effective vaccine against malaria is....


Introduction
Malaria has been a significant threat to human health throughout history. As an effective vaccine against malaria is not available yet vector control measures like the use of insecticidetreated mosquito nets and indoor residual spraying has received significant attention to prevent and reduce malaria transmission. Five different species of protozoan parasites belonging to the genus Plasmodium are capable of infecting humans and causing malaria [2]. Out of these, two major species such as P. vivax and P. falciparum, are responsible for most of the morbidity and mortality in humans.
The signs and symptoms that are seen in individuals who are infected and develop clinical signs of illness, varies widely between children and adults [3]. Malaria manifests itself by inducing the following types of conditions in humans -it may be accompanied by electrolyte and metabolic imbalances, respiratory failure, severe anemia, jaundice, pulmonary edema or a severe neuropathological condition termed as Cerebral Malaria (CM) [4]. The World Health when it is not attributable to other causes [5]. According to hospital admission records, 1-2 % of all P. falciparum infections result in CM, having a fatality rate of 10-20% [6]. Furthermore, at least 10 to 20% of those who survive and recover from CM suffer significant post-disease neurological sequelae for several years after cure [7].
In the regions of high malaria transmission, the age of the affected individuals and the degree of exposure to the parasite causing the infection are key determinants to the susceptibility to severe malaria. Developing complete immunity from malaria has not yet been possible for humans due to the complexity of the parasite's life cycle; however acquisition of clinical immunity, which confers protection from life-threatening illnesses are achievable. But the acquisition of clinical immunity requires numerous exposure to the parasite during an individual's life span through infective mosquito bites [8]. Most children, who get repeatedly exposed to malaria in areas of high transmission, acquire some form of resistance against the severe forms of the disease, provided they manage to survive the first few years of their lives after getting infected [8]. In areas of low transmission however, Severe Malaria (SM) occurs equivalently in all age groups, and is slightly more common in adults. This is because clinical immunity to malaria does not occur at all or takes a very long time to acquire and may persist for a short period. Although adults generally have a fairly good resistance against developing SM, infants are at an increased risk of developing malignant malaria, especially during their first years of life. However, the older children who've had atleast a single prior exposure to P. falciparum infection, are disproportionately at risk of developing CM symptoms [9]. The epidemiology of CM is suggestive of the fact that the predisposition to disease manifestation or its prevention is ultimately determined not only by the virulence of the invading parasite; but is also attributable to the host's own immune response which contributes to the degree of disease pathogenesis and outcome [10][11][12].

Clinical Attributes of the Pathology Observed in CM Patients
Post-mortem examination of human brain have helped to uncover the extent and type of brain pathology that occurs due to CM. leading to death. The prevalance of haemorrhages and swelling in the white matter of the subcortical rim and corpus callosum as well as presence of petechial and ring haemorrhages in cerebral and cerebellar cortices have been some of the most commonly reported findings from such examinations [13].
Histopathological analyses of brain of CM patients after death has revealed the presence of parasitized erythrocytes clogging the cerebral capillaries [13]. The pathologic features of CM also include the presence of monocytes and macrophages within cerebral vessels together with the sequestration of pigmented macrophages and parasitized erythrocytes confined to cerebral blood vessels [14]. Due to the lack of detailed comparative histopathological analysis, it is extremely difficult to determine the variations in CM pathology between children and non-immune adults, but given the number of differences in the symptoms of pediatric and adult CM it is plausible that there may be some age-related variations in the cerebral pathology observed in these groups [15]. Although parasite sequestration,haemorrhages and inflammation are some of the common findings in histological analyses of the brains of the majority of CM patients, it is quite probable that CM is not a homogenous syndrome. In fact, three distinct and different patterns of histopathological changes have been observed in African children.
Firstly, there is the 'classical' pattern with parasite sequestration, perivascular haemorrhages and immune cell infiltration observed within brain micro-vessels due to CM. Next, there are incidences where parasite sequestration has been observed within the brain, but without any of the other aforementioned abnormalities. And lastly, numerous cases have been observed where individuals with high peripheral parasitemia develop a syndrome that is clinically defined as CM but where there has been no evidence of parasite sequestration within the brain [16,17] but these studies are severely restricted by ethical constraints and the availability of the expensive specialized equipment in malariaendemic areas [20][21][22]. In order to observe and understand the immunological pathways that are primarily elicited to control the infection together with the manipulations brought about at the level of the invading pathogen for its own survival and persistence, it is imperative to implement direct intervention studies and move beyond purely descriptive conjectures, which is an extremely difficult undertaking. Examination of the peripheral blood often provides very limited information about the immunological and parasitological environment in the brain as the peripheral blood parasitaemia does not accurately reflect total parasite biomass.
Total parasite biomass is a stronger correlate of severe malarial disease than the peripheral parasitaemia [23]. The situation is made further complicated as the patients usually present themselves to the hospitals only when the disease has reached a very advanced stage. It is clear that other approaches -in combination with human studies -are required to fully understand the pathogenesis of CM. Much of our understanding of mammalian physiology has come from studies of animals and the extent of the conservation of basic immunological and neuropathological processes between laboratory rodents and humans has become more apparent [24,25]. Primate models of CM, including P. knowlesi and P. coatneyi infections in Rhesus monkeys and P. falciparum infection in squirrel monkeys [26,27], have allowed the investigation of some aspects of CM, but these models are prohibitively expensive and are done with smaller number of animals due to ethical reasons.
neuropathological syndromes have been shown to develop in certain strains of inbred mice infected with various strains of Plasmodium berghei [28,29]. There has been and continues to be significant disagreement within the malaria research community as to whether the murine models share sufficient similarities with human cerebral malaria to make them relevant or useful. However, experimental models have proven invaluable for understanding the pathogenesis of numerous autoimmune and infectious diseases of humans and many vaccines and immuno-therapies currently in use were initially developed and tested in experimental models [25]. Therefore from that aspect, the use of relevant experimental animal models has significantly aided in the study of CM and are indispensable tools for gaining valuable insight and improving our understanding of the disease pathogenesis.

Plasmodium Yoelii XL and Plasmodium berghei K173
Although more extensively studied as a model of hyperparasitaemia and failure of parasite control, PyXL has been shown to sequester within the brain microvasculature and produce a cerebral syndrome in mice comparable with human cerebral malaria. [30][31][32]; however, the hyper-parasitaemia associated with this infection (rapidly ascending peripheral parasitaemia that can reach 80-100%) is not typical of human CM cases and this model is not widely used to study CM. In a few studies, P. berghei K173 has been found to induce CM-like symptoms, but the dose-  [37][38][39]. ECM is also associated with the significant accumulation of platelets within the brain vasculature [40]: platelets have been shown to directly promote endothelial cell damage during infection. Cognitive dysfunction in mice during P. berghei ANKA infection, as shown by impaired memory, is directly correlated with haemorrhage and inflammation, including microglial activation [41]. Indeed, the accumulation of monocytes and macrophages, and activation of brain resident mononuclear cells, including astrocytes and microglial cells is believed to be a key feature of ECM. As in humans, genetic and environmental factors determine the susceptibility of mice to ECM. For example, the resistance of F1 intercrossed BALB/c (resistant) and C57BL/6 (susceptible) mice to the development of ECM is determined by age, and environmental exposure, with young mice (8-10 weeks) susceptible to ECM and older mice (16-20 weeks) resistant to the development of cerebral pathology [41].

The Involvement of Blood Brain Barrier in CM Pathology
Several studies on patients in Africa and Asia have shown that the disruption of the Blood Brain Barrier (BBB) in CM leads to severe neurological complications including

2.
Seizures resulting from electrolyte imbalance, and

All of these factors ultimately result in Central Nervous
System (CNS) dysfunction and death. The BBB is a semipermeable membrane that separates the peripheral blood from the brain parenchyma. The BBB comprises a monolayer of Endothelial Cells (ECs) joined together by tight junctions and the underlying basal lamina. The integrity of the BBB is further supported by pericytes and the astrocyte end-feet [46]. The BBB, together with microglia and neurons, forms the neurovascular unit. Apart from serving as an anatomical barrier, the BBB plays a crucial role in the homeostasis of the CNS by facilitating the transport of nutrients such as glucose and amino acids from the blood to the CNS and removal of metabolic waste products from the CNS into the blood by means of specific transport channels [46]. Up to now, the precise underlying mechanisms leading to the disruption of BBB integrity during CM have remained unclear. Several mutually nonexclusive events have been associated with BBB breakage in human and murine studies, namely

4.
Dysregulation of vascular ECs [52]. In addition, a growing Patients with CM show an expansion of parasites expressing PfEMP1 that bind to both ICAM-1 and EPCR [57,58]. Platelets induce the adhesion of iRBCs to one another, forming large autoagglutinates [59]. Furthermore, noninfected erythrocytes form rosettes around the iRBCs, contributing further to microvascular obstruction [59].
Since sequestration of iRBCs to the brain endothelium plays a critical role in the development of CM, prevention of sequestration and desequestration of the iRBCs are attractive therapeutic approaches, discussed in detail by Glennon, et al. [60]. Epidemiological investigations suggest that repeatedly exposed individuals eventually develop protective immunity, which may be characterized by the ability to control parasite replication by keeping parasite densities below the critical threshold for induction of hyper inflammatory immune responses to prevent pRBC sequestration and impede neurological damage [68,69].

Adjunctive Therapies For Management of CM Pathology:
The major reasons for concern for CM in humans stem from the fact that the disease can develop rather suddenly, after just initial bouts of fever that lasts only for 2 Therefore, there is an urgent need to develop adjunctive therapies that can be administered with anti-malarials that can, both halt the progression of CM and also promote healing of brain dysfunction. Based on successful case reports corticosteroids were one of the first treatments proposed as an adjunctive therapy for CM with the aim of reducing swelling and inflammation in the brain. However, dexamethasone failed to demonstrate a decrease in mortality in two clinical trials [82]. Similarly, treatment with intravenous immunoglobulin resulted in increased deleterious outcomes compared to the placebo group, including higher mortality and more neurological sequelae in children [83]. The failure of this therapy was attributed to not being able to reverse cytoadherence and sequestration. Curdlan Sulfate (CS), a sulfated 1 → 3-β-d glucan, a known inhibitor of P. falciparum in vitro, has also been tested due to its capacity to modulate the immune response to P. falciparum [84]. CS was expected to have some anticoagulant properties, and confer certain direct and nonspecific effect on cytoadhesion and rosetting. However the studies failed to demonstrate differences in mortality, possibly because of small sample sizes, but CS was safe and appeared to reduce the severity of the disease process [85]. Since high circulating levels of TNF has been attributed to the severity of CM pathology, therapy targeting Tumour Necrosis Factor (TNF) and its effects have also been explored. However, no difference in mortality was shown and moreover, there was an increased risk of neurological sequelae in the experimental group [86]. The failure of this therapeutic approach was thought to be due to antibody mediated retention of TNF in circulation, [86]. Lithium has been proposed to act as a neuroprotective agent by its ability to inhibit glycogen synthase kinase 3 (GSK3β), activate the PI3 K/Akt and MAPK signalling pathways, and by inducing the expression of brain-derived neurotrophic factors in neurons [90].
Lithium chloride administered to mice with ECM significantly increased the activation of Akt, which was associated with the prevention of adverse neurocognitive outcomes. Adjunctive treatment with lithium chloride was associated with better spatial and visual memory, and motor coordination in mice recovering from ECM [91]. Targeting endothelial activation and preventing microvascular permeability and vascular leak in CM can be another potential target for adjunctive therapy [92]. The angiopoietin (Ang)-Tie2 axis critically regulates endothelial cell function [93].
Perturbation of Ang-1, Ang-2 and soluble Tie2 concentrations are associated with disease severity and death in CM in both murine models and humans [94]. A mechanistic role for the Ang-Tie2 axis was established in ECM, where it was shown that Ang-1-deficient mice were more susceptible to ECM and adjunctive administration of a recombinant Ang-1 construct preserved BBB integrity and improved survival beyond artesunate monotherapy alone [94].

Conclusion
Malaria remains a major global health problem, associated with high morbidity and mortality. Strategies have to be developed to improve early detection and recognition of cases so that immediate treatment can be made available in order to prevent progress of the disease towards the severe condition and prevent death.