Background
Infection with
Plasmodium falciparum remains prevalent in many areas of the world and is associated with severe disease and mortality, particularly in children living in sub-Saharan Africa [
1]. Cerebral malaria (CM) is a severe disease syndrome with mortality rates ranging from 15 to 25 % in research settings [
2]. In addition, almost a third of paediatric CM survivors develop long-term neurological complications [
2,
3]. Severe brain swelling seen on neuroimaging has been reported in paediatric CM. CM associated brain swelling is associated with poor outcomes in Kenyan children and is a significant predictor of mortality in Malawian children [
4‐
6]. Recently, CM associated brain swelling determined by magnetic resonance imaging (MRI) found that swelling in survivors was readily reversible and that mortality was not associated with peripheral parasitaemia [
5]. These observations have provided new insights into CM related morbidity and mortality and now can potentially inform development of adjuvant therapies to reverse or prevent brain swelling.
The mechanism of CM brain swelling is unknown and likely involves several factors including parasite mediated venous obstruction, increased permeability of the blood brain barrier (BBB), cytotoxic oedema or increased blood flow volume [
5,
6]. Prior evidence of alterations in BBB permeability includes the observation of fibrinogen leakage into the brain [
7]. Moreover, a reduction in endothelial cell tight junction proteins, which maintain the integrity of the BBB, has been reported, providing further support for increases in BBB permeability during CM [
8‐
10]. A variety of systemic factors can lead to transient increase of BBB permeability in other diseases. These include the metabolic derangements associated with diabetic ketoacidosis and elevated concentrations of oxidized phospholipids [
11,
12]. Systemic metabolic abnormalities are common in CM, which is often associated with a hyperlactataemia, hypoglycaemia and evidence of marked inflammation [
13‐
16]. Therefore, metabolites measured in a cohort of Malawian children with CM were correlated to brain volume, to examine their role as potential mediators of brain swelling.
Arachidonic acid, other phospholipase A
2 (PLA
2) lipid metabolites and plasma PLA
2 enzymatic activity were associated with brain swelling. Expression of the PLA
2 enzyme is upregulated via the nuclear factor-kappa B (NFκB) pathway, which in turn is regulated by pro-inflammatory cytokines, such as tumour necrosis factor alpha (TNFα) [
17]. An association of TNFα and other cytokines with brain swelling was found, suggesting that brain swelling is associated with a high inflammatory state. These data provide new biochemical insights into mechanisms of brain swelling in paediatric CM. Further experiments are needed to determine if these associated molecules induce increased brain swelling in the setting of CM.
Discussion
To identify potential mediators of brain swelling associated with paediatric CM, plasma metabolite levels of Malawian children with retinopathy positive CM were correlated to brain volume. Associations were identified between PLA2 metabolites, including arachidonic acid, with brain volume. Plasma PLA2 enzymatic activity also correlated to brain volume, suggesting an upregulation of the PLA2 pathway in children with high brain swelling. TNFα can upregulate PLA2 and this study reports that, in conjunction with other inflammatory cytokines, TNFα is correlated with brain volume. l-urobilin, a haem degradation product and mannitol were also associated with brain volume. Collectively, these results suggest a higher inflammatory state in children with increased brain volume. Further studies on how these molecules may be involved in BBB disruption, and why some children sustain a higher inflammatory state are now warranted.
Brain swelling has recently been shown to be the strongest predictor of mortality in paediatric CM, with an adjusted odds ratio of 7.5 (95 % CI 2.1–26.9) for severe brain swelling among patients who died compared to those who survived [
5]. Brain swelling induces increased intracranial pressure that may lead to brain-stem compromise and respiratory arrest. The mechanism of increased brain volume is unknown. Multiple mechanisms of increased brain volume may exist in clinical conditions such as CM [
30], including processes that impact BBB function [
5,
7,
10]. This study set out to identify circulating biochemical mediators that may contribute to BBB dysfunction. [
7,
10].
Positive correlations between brain volume and several lipids that are PLA
2 metabolites were identified. The fatty acids released by PLA
2, such as arachidonic acid, can be sources of energy, signaling, and of relevance to this study, potent mediators of inflammation. Specifically, arachidonic acid, the precursor of the eicosanoid pathway, can increase the permeability of human brain microvasculature endothelial cell monolayers via prostaglandin E
2 activation of EP
3 and EP
4 receptors [
31]. Lysophospholipids are also generated by PLA
2 and are important in cell signaling and membrane biology. PLA
2 enzymatic activity has been associated with neurological and inflammatory conditions and inhibitors of the PLA
2 enzyme are being studied to reduce pathologic inflammation [
32‐
36]. Fatty acids and lysophospholipids can be generated by enzymes other than PLA
2, therefore it was confirmed in this study that plasma PLA
2 activity was also correlated with brain swelling [
37,
38]. These data extend a prior observation where high PLA
2 plasma activity was associated with severe malaria and death in Malawian children [
39]. Why some children have higher PLA
2 enzyme activity is unknown. PLA
2 activity is tightly regulated by host responses including TNFα and reactive oxygen species (ROS), both of which can be elevated during severe malaria [
15,
40,
41]. Thus the PLA
2 pathway and its metabolites may be acting directly on brain microvasculature endothelial cells or indirectly through their effects on cell signaling or energy metabolism.
l-urobilin, a degradation product of haem also correlated with brain volume. Malaria induces erythrocyte lysis and subsequent release of haemoglobin into the circulation. This results in increase of bloodstream ROS levels, which can alter BBB permeability [
41‐
44]. Lysed erythrocytes can increase BBB permeability and result in brain oedema, typically occurring twenty-four hours after haemolysis [
45‐
47]. Direct measurement of ROS and other haemoglobin metabolites are needed to explore the role of erythrocyte lysis in CM associated brain swelling.
Plasma mannitol was also found to have a positive correlation with brain swelling. Exogenous mannitol has been associated with increased brain oedema or brain weight in animal models of vasogenic oedema and brain infarct [
50,
51]. Mannitol is used therapeutically for the treatment of increased intracranial pressure following brain injury [
48,
49]. Mannitol adjunctive therapy has been studied in CM and was found to either have no significant effect on clinical outcomes in paediatric CM in Uganda [
52] or to potentially have adverse outcomes by prolonging coma resolution time in adults with CM [
53]. Further studies to investigate the role of endogenous and exogenous mannitol in CM paediatric brain swelling are necessary.
Inflammatory cytokines were also associated with brain volume and these may directly alter BBB permeability. CCL2 and IL-8 have been previously associated with alterations in tight junction proteins including occludin, ZO-1 and claudin-5 resulting in increased brain endothelial cell permeability [
54,
55]. In addition, TNFα has been shown to increase retinal endothelial cell permeability through protein kinase C zeta (PKCζ) and the NFκB pathway by reducing the expression and distribution of claudin-5 and ZO-1 [
56]. Interestingly, inhibition of TNFα by soluble TNF p55 receptor attenuates status epilepticus-induced oedema in a rat model, which could be relevant in CM, as seizures are highly prevalent [
3,
57]. Furthermore, TNFα can regulate the transcription of PLA
2 proteins via the NFκB pathway, providing a link between inflammatory cytokines and the PLA
2 pathway [
17]. The mechanism, where inflammatory cytokines, increased PLA
2 activity and its lipid metabolites converge to disrupt the BBB, in the setting of parasites sequestration to brain endothelial cells in CM, requires further investigation. If the PLA
2 pathway is found to play a direct role in BBB dysfunction, PLA
2 inhibitors could be evaluated as potential adjunctive therapy.
The higher inflammation and brain volume do not correlate with peripheral parasitaemia or total parasite body load represented by parasite HRP2 levels [
58]. The variability in inflammatory responses during infection could be attributed to differences in disease duration, host or parasite genetic polymorphism, prior malaria exposures, uric acid levels or other host or parasite factors [
59‐
65]. Further studies examining how host and/or parasite parameters are mediating inflammation and upregulation of the PLA
2 pathway in the setting of increased brain volume could inform therapeutic intervention.
A limitation to the design of this study is that the plasma metabolome was examined after onset of brain swelling. For this reason, it is difficult to know if the metabolic profile is a cause of BBB dysfunction or the result of changes secondary to increased brain volume. A longitudinal study design with repeated metabolic sampling over time in correlation to brain swelling status would provide a more powerful approach to identify potential mediators of BBB permeability. Additionally, there were no metabolomics data from patients with brain volume scores 1, 2 and 8 due to the low prevalence of those brain volume scores. To develop biomarkers of brain swelling for regions without MRI, further plasma analyses that include all of the brain volume groups are warranted. Given the variances observed, larger sample sizes would permit corrections for multiple comparisons.