Experimental Cerebral Malaria In Mice Research Articles

CD8+ T cell infiltration and proliferation in the brainstem during experimental cerebral malaria.

Cerebral malaria (CM) is a lethal neuroinflammatory disease caused by Plasmodium infection. Immune cells and brain parenchyma cells contribute to the pathogenesis of CM. However, a systematic examination of the changes that occur in the brain parenchyma region during CM at the single-cell resolution is still poorly studied. To explore cell composition and CD8+ T cell infiltration, single-cell RNA sequencing (scRNA-seq) was performed on the brainstems of healthy and experimental cerebral malaria (ECM) mice. Then CD8+ T cell infiltration was confirmed by flow cytometry and immunofluorescence assays. Subsequently, the characteristics of the brain-infiltrated CD8+ T cells were analyzed. Finally, the interactions between parenchyma cells and brain-infiltrated CD8+ T cells were studied with an astrocytes-CD8+ T cell cocultured model. The brainstem is the most severely damaged site during ECM. ScRNA-seq revealed a large number of CD8+ T cells infiltrating into the brainstem in ECM mice. Brain-infiltrated CD8+ T cells were highly activated according to scRNA-seq, immunofluorescence, and flow cytometry assays. Further analysis found a subset of ki-67+ CD8+ T cells that have a higher transcriptional level of genes related to T cell function, activation, and proliferation, suggesting that they were exposed to specific antigens presented by brain parenchyma cells. Brain-infiltrated CD8+ T cells were the only prominent source of IFN-γ in this single-cell analysis. Astrocytes, which have a high interferon response, act as cross-presenting cells to recruit and re-activate brain-infiltrated CD8+ T cells. We also found that brain-infiltrated CD8+ T cells were highly expressed immune checkpoint molecule PD-1, while parenchyma cells showed up-regulation of PD-L1 after infection. These findings reveal a novel interaction between brain-infiltrated CD8+ T cells and parenchyma cells in the ECM brainstem, suggesting that the PD-1/PD-L1 signal pathway is a promising adjunctive therapeutic strategy for ECM targeting over-activated CD8+ T cells.

Lymphotoxin-α Orchestrate Hypoxia and Immune factors to Induce Experimental Cerebral Malaria: Inhibition Mitigates Pathogenesis, Neurodegeneration, and Increase Survival.

Knockdown studies have shown lymphotoxin-α (Lt-α) as a critical molecule for Experimental cerebral malaria (ECM) pathogenesis. We investigated the role of lymphotoxin-α in regulating active caspase-3 and calpain1. T cell infiltration into the brains, and subsequent neuronal cell death are the essential features of Plasmodium berghei ANKA(PbA)-induced ECM. Our results showed increased Lt-α levels during ECM. Treatment of naïve mice with serum from ECM mice and exogenous Lt-α was lethal. We inhibited Lt-α in vivo during PbA infection by injecting the mice with anti-Lt-α antibody. Inhibition of Lt-α mitigated neuronal cell death and increased mice's survival until 30-day post-infection (p.i.) compared to only 15days survival of PbA control mice.

Ferroptosis participates in neuron damage in experimental cerebral malaria and is partially induced by activated CD8+ T cells

Cerebral malaria is the most serious complication of malaria infection, with 26% of surviving children having neurological sequelae, which may be caused by neuron damage, but the mechanism is not clear. Ferroptosis has been reported to play an important role in neuron damage in several nervous system diseases. However, the occurrence of ferroptosis in experimental cerebral malaria (ECM) pathogenesis is still unknown. In this study, we firstly detected increased levels of malondialdehyde (MDA) and iron, which are indicators of ferroptosis, in the cerebrum of ECM mice. Some important regulators of ferroptosis, including upregulated expression of transferrin receptor 1 (TfR1) and acyl-CoA synthetase long-chain family member 4 (ACSL4), and downregulation of glutathione peroxidase 4 (GPX4) levels, were also confirmed in ECM mice. Consistently, neuron damage, which was detected in the cerebrum of ECM mice, was positively correlated with reduced GPX4 expression and furtherly rescued by administration of the ferroptosis inhibitor ferrostatin-1 (Fer-1). In addition, primary neurons were damaged by activated CD8+ T cells, an effect that was also partially rescued by Fer-1 on amyloid precursor protein expression and mitochondrial membrane potential levels in vitro. Activated CD8+ T cells were also shown to infiltrate the cerebrum of ECM mice and upregulate TfR1 expression in primary neurons, which may be an important event for inducing ferroptosis in ECM. Altogether, we show that ferroptosis contributes to neuron damage in ECM pathogenesis, and activated CD8+ T cells may be important inducers of neuronal ferroptosis. Hence, targeting ferroptosis may be a promising adjuvant therapeutic strategy for neurological sequelae in patients with cerebral malaria.

Pipecolic Acid, a Putative Mediator of the Encephalopathy of Cerebral Malaria and the Experimental Model of Cerebral Malaria.

We explored a metabolic etiology of cerebral malaria (CM) coma. Plasma metabolites were compared between Malawian children with CM and mild Plasmodium falciparum malaria. A candidate molecule was further studied in animal models of malaria. Clinically abnormal concentrations of pipecolic acid (PA) were present in CM plasma, and nearly normal in mild malaria samples. PA is renally cleared and the elevated PA blood levels were associated with renal insufficiency, which was present only in CM subjects. Prior studies demonstrate that PA has neuromodulatory effects and is generated by malaria parasites. PA brain levels in Plasmodium berghei ANKA-infected animals in the experimental cerebral malaria (ECM) model inversely correlated with normal behavior and correlated with blood-brain barrier (BBB) permeability. Mice infected with malaria species that do not induce neurological abnormalities or manifest BBB permeability had elevated plasma PA levels similar to ECM plasma at 7 days postinfection; however, they had low PA levels in the brain compared to ECM mice brains at 7 days postinfection. Our model suggests that malaria-generated PA induces coma in CM and in ECM. The role of BBB permeability and the mechanisms of PA neuromodulation in CM will require additional investigation.

Mast cells-derived exosomes worsen the development of experimental cerebral malaria

Cerebral malaria (CM) is the most severe neurological complication caused by Plasmodium falciparum infection. The accumulating evidence demonstrated that mast cells (MCs) and its mediators played a critical role in mediating malaria severity. Earlier studies identified that exosomes were emerging as key mediators of intercellular communication and can be released from several kinds of MCs. However, the potential functions and pathological mechanisms of MCs-derived exosomes (MCs-Exo) impacting on CM pathogenesis remain largely unknown. Herein, we utilized an experimental CM (ECM) model (C57BL/6 mice infected with P. berghei ANKA strain), and then intravenously (i.v.) injected MCs-Exo into P. berghei ANKA-infected mice to unfold this mechanism and investigate the effect of MCs-Exo on ECM pathogenies. We also used an in vitro model by investigating the pathogenesis development of brain microvascular endothelial cells line (bEnd.3 cells) co-cultured with P. berghei ANKA blood-stage soluble antigen (PbAg) after MCs-Exo treatment. The higher numbers of MCs and levels of MCs degranulation were observed in skin, cervical lymph node, and brain of ECM mice than those of the uninfected mice. Exosomes were successfully isolated from culture supernatants of mouse MCs line (P815 cells) and characterized by spherical vesicles with the diameter of 30-150 nm, and expression of typical exosomal markers (e.g., CD9, CD63, and CD81). The i.v. injection of MCs-Exo dramatically elevated incidence of ECM in the P. berghei ANKA-infected mice, exacerbated liver and brain histopathological damage, promoted Th1 cytokine response, aggravated brain vascular endothelial activation and blood brain barrier breakdown in ECM mice. In addition, the treatment of MCs-Exo led to the decrease of cells viability and mRNA levels of Ang-1, ZO-1, and Claudin-5, but increase of mRNA levels of Ang-2, CCL2, CXCL1, and CXCL9 in bEnd.3 cells co-cultured with PbAg in vitro. Taken together, our data indicated that MCs-Exo could worsen pathogenesis of ECM in mice.

Whole blood transfusion improves vascular integrity and increases survival in artemether-treated experimental cerebral malaria

Pathological features observed in both human and experimental cerebral malaria (ECM) are endothelial dysfunction and changes in blood components. Blood transfusion has been routinely used in patients with severe malarial anemia and can also benefit comatose and acidotic malaria patients. In the present study Plasmodium berghei-infected mice were transfused intraperitoneally with 200 μL of whole blood along with 20 mg/kg of artemether. ECM mice showed severe thrombocytopenia and decreases in hematocrit. Artemether treatment markedly aggravated anemia within 24 h. Whole blood administration significantly prevented further drop in hematocrit and partially restored the platelet count. Increased levels of plasma angiopoietin-2 (Ang-2) remained high 24 h after artemether treatment but returned to normal levels 24 h after blood transfusion, indicating reversal to quiescence. Ang-1 was depleted in ECM mice and levels were not restored by any treatment. Blood transfusion prevented the aggravation of the breakdown of blood brain barrier after artemether treatment and decreased spleen congestion without affecting splenic lymphocyte populations. Critically, blood transfusion resulted in markedly improved survival of mice with ECM (75.9% compared to 50.9% receiving artemether only). These findings indicate that whole blood transfusion can be an effective adjuvant therapy for cerebral malaria.

BCG Provides Short-Term Protection from Experimental Cerebral Malaria in Mice.

Clinical and experimental evidence suggests that the tuberculosis vaccine BCG offers protection against unrelated pathogens including the malaria parasite. Cerebral malaria (CM) is the most severe complication associated with Plasmodium falciparum infection in humans and is responsible for most of the fatalities attributed to malaria. We investigated whether BCG protected C57BL/6 mice from P. berghei ANKA (PbA)-induced experimental CM (ECM). The majority of PbA-infected mice that were immunized with BCG showed prolonged survival without developing clinical symptoms of ECM. However, this protective effect waned over time and was associated with the recovery of viable BCG from liver and spleen. Intriguingly, BCG-mediated protection from ECM was not associated with a reduction in parasite burden, indicating that BCG immunization did not improve anti-parasite effector mechanisms. Instead, we found a significant reduction in pro-inflammatory mediators and CD8+ T cells in brains of BCG-vaccinated mice. Together these data suggest that brain recruitment of immune cells involved in the pathogenesis of ECM decreased after BCG vaccination. Understanding the mechanisms underlying the protective effects of BCG on PbA-induced ECM can provide a rationale for developing effective adjunctive therapies to reduce the risk of death and brain damage in CM.

Testing the impact of a single nucleotide polymorphism in a Plasmodium berghei ApiAP2 transcription factor on experimental cerebral malaria in mice

Cerebral malaria (CM) is the deadliest form of severe Plasmodium infections. Currently, we have limited understanding of the mechanisms by which Plasmodium parasites induce CM. The mouse model of CM, experimental CM (ECM), induced by infection with the rodent parasite, Plasmodium berghei ANKA (PbANKA) has been extensively used to study the pathophysiology of CM. Recent genomic analyses revealed that the coding regions of PbANKA and the closely related Plasmodium berghei NK65 (PbNK65), that does not cause ECM, differ in only 21 single nucleotide polymorphysims (SNPs). Thus, the SNP-containing genes might contribute to the pathogenesis of ECM. Although the majority of these SNPs are located in genes of unknown function, one SNP is located in the DNA binding site of a member of the Plasmodium ApiAP2 transcription factor family, that we recently showed functions as a virulence factor alternating the host’s immune response to the parasite. Here, we investigated the impact of this SNP on the development of ECM. Our results using CRISPR-Cas9 engineered parasites indicate that despite its immune modulatory function, the SNP is neither necessary nor sufficient to induce ECM and thus cannot account for parasite strain-specific differences in ECM phenotypes.

Fenozyme Protects the Integrity of the Blood-Brain Barrier against Experimental Cerebral Malaria.

Cerebral malaria is a lethal complication of malaria infection characterized by central nervous system dysfunction and is often not effectively treated by antimalarial combination therapies. It has been shown that the sequestration of the parasite-infected red blood cells that interact with cerebral vessel endothelial cells and the damage of the blood-brain barrier (BBB) play critical roles in the pathogenesis. In this study, we developed a ferritin nanozyme (Fenozyme) composed of recombinant human ferritin (HFn) protein shells that specifically target BBB endothelial cells (BBB ECs) and the inner Fe3O4 nanozyme core that exhibits reactive oxygen species-scavenging catalase-like activity. In the experimental cerebral malaria (ECM) mouse model, administration of the Fenozyme, but not HFn, markedly ameliorated the damage of BBB induced by the parasite and improved the survival rate of infected mice significantly. Further investigations found that Fenozyme, as well as HFn, was able to polarize the macrophages in the liver to the M1 phenotype and promote the elimination of malaria in the blood. Thus, the catalase-like activity of the Fenozyme is required for its therapeutic effect in the mouse model. Moreover, the Fenozyme significantly alleviated the brain inflammation and memory impairment in ECM mice that had been treated with artemether, indicating that combining Fenozyme with an antimalarial drug is a novel strategy for the treatment of cerebral malaria.

Erg mediates downregulation of claudin-5 in the brain endothelium of a murine experimental model of cerebral malaria.

Cerebral malaria (CM) is a severe complication with brain vascular hyperpermeability. Claudin-5 is the major component of tight junctions. To investigate the expression of claudin-5 in CM, we established a murine experimental cerebral malaria (ECM) model and an invitro model by treating murine brain endothelial cells (bEnd3) with plasma from ECM mice. Expression of claudin-5 and the ETS transcription factor Erg was reduced in the brain endothelium of ECM mice. In bEnd3 cells exposed to ECM plasma, decreased expression of claudin-5 and Erg, and increased permeability were observed. Silencing of Erg significantly reduced Cldn5 expression. ChIP assays indicated that Erg binds to the -813 ETS motif of the murine Cldn5 gene promoter, and the binding is decreased by treatment with ECM plasma.

Potential role of arteether on N-methyl-D-aspartate (NMDA) receptor expression in experimental cerebral malaria mice and extension of their survival.

Cerebral malaria (CM) is the severe neurological complication causing acute non-traumatic encephalopathy in tropical countries. The mechanisms underlying the fatal cerebral complications are still not fully understood. Glutamate, a major excitatory neurotransmitter in the central nervous system of the mammalian brain, plays a key role in the development of neuronal cells, motor function, synaptic plasticity, learning and memory processes under normal physiological conditions. The subtypes of ionotropic glutamate receptor are N-methyl-D-aspartate receptors (NMDARs) which are involved in cellular mechanisms of learning and memory, synaptic plasticity and also mediate excitotoxic neuronal injury. In the present study, we found that glutamate level in synaptosomes, as well as expression of NMDAR, was elevated during the extreme condition of CM in C57BL6 mice. Arteether at 50 mg kg-1 × 1, 25 mg kg-1 × 2, days decreased the NMDAR expression and increased the overall survival of the experimental CM mice.

Cerebral Vascular Function in a Murine Model of Experimental Cerebral Malaria

Cerebral Malaria (CM) is among the most severe complications of malarial infection, with a mortality rate of 15–20%. Many survivors of CM develop lasting neurological sequalae, cognitive and behavioral impairments, or epilepsy. However, the etiology of CM is poorly defined, and treatment strategies are limited.Studies in the murine model of experimental cerebral malaria (ECM) have demonstrated cerebral blood flow (CBF) impairments that are associated with decreased nitric oxide (NO) bioavailability, blood brain barrier disruption, endothelial activation, impaired pial vessel reactivity, and cerebral ischemia. While these complications have been observed in vivo, cerebral artery function has not specifically been evaluated in ECM. Therefore, the aim of this study was to interrogate vasoreactivity and NO bioavailability in cerebral arteries (CAs) isolated from ECM mice, with the hypothesis that arterial vasodilatory function would be impaired by ECM.ECM was induced by Plasmodium berghei ANKA (PbA) infection in 10–12 week old C57Bl/6J mice and confirmed by murine coma score. On day 6 post‐infection, CBF was measured by laser speckle imaging. Cerebral arteries (CAs) were dissected from the brain into a modified Krebs buffer. Cerebral vasoreactivity was assessed by ex vivo pressure myography in middle cerebral arteries (MCA) obtained from control (n=4) and ECM (n=7) mice. NO production from dissected vessels was measured in an ex vivo Daf‐2 fluorescence assay (n=6, n=6).As expected, ECM mice had a significant, 25% reduction in CBF. However, ex vivo MCAs from ECM mice maintained robust endothelium‐dependent vasodilation to methacholine (MCh), which was inhibited by L‐NAME. ECM MCAs also exhibited normal dose responses to serotonin‐induced vasoconstriction and nitroprusside‐induced vasodilation, suggesting normal vasoreactivity of ECM cerebral arteries. Similarly, the results of the ex vivo Daf‐2 assay showed that the MCh‐induced NO production from ECM arteries was comparable to controls. To test whether ECM plasma would induce vascular dysfunction in ex vivo CAs, we incubated CAs obtained from ECM (n=2) and control (n=6) mice in plasma collected from ECM or control animals. Surprisingly, we found that plasma from ECM mice increased NO production of both ECM and control CAs.These unexpected results indicate that NO bioavailability and vasoreactivity are preserved in cerebral vessels in ECM, and that circulating factors in the plasma during PbA infection actively stimulate NO production. Our next focus will be on reconciling decreased CBF with preserved vascular function, as well as identifying the NO‐stimulating mediator(s) present within ECM plasma.Support or Funding InformationResearch and funding support from NIAID Division of Intramural Research.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Reversal of cerebrovascular constriction in experimental cerebral malaria by L-arginine

Vascular dysfunction associated with low nitric oxide (NO) biavailability and low plasma L-arginine levels is observed in both human and experimental cerebral malaria (ECM). In ECM, cerebrovascular constriction results in decreased pial blood flow and hypoxia, and administration of NO donors reverses constriction and increases survival. Supplementation of L-arginine, the substrate for NO synthesis by NO synthases, has been considered as a strategy to improve vascular health and act as adjunctive therapy in human severe malaria. We investigated the effect of L-arginine supplementation on pial vascular tonus of mice with ECM after direct superfusion on the brain surface or systemic delivery. Pial arteriolar diameters of Plasmodium berghei-infected mice with implanted cranial windows were measured using intravital microscopy methods, before and after L-arginine administration. Systemic delivery of L-arginine was performed intravenously, at 10, 50, 100 and 200 mg/kg, as bolus injection or slowly through osmotic pumps, combined or not with artesunate. Direct superfusion of L-arginine (10−7M, 10−5M and 10−3M) on the brain surface of mice with ECM resulted in immediate, consistent and dose-dependent dilation of pial arterioles. ECM mice showed marked cerebrovascular constriction that progressively worsened over a 24 h-period after subcutaneous saline bolus administration. L-arginine administration prevented the worsening in pial constriction at all the doses tested, and at 50 mg/kg and 100 mg/kg it induced temporary reversal of vasoconstriction. Slow, continuous delivery of L-arginine by osmotic pumps, or combined bolus administration of artesunate with L-arginine, also prevented worsening of pial constriction and resulted in improved survival of mice with ECM. L-arginine ameliorates pial vasoconstriction in mice with ECM.

Human Cerebral Malaria and Experimental Cerebral Malaria in Mice: Relevance and Applicability

Human Cerebral Malaria and Experimental Cerebral Malaria in Mice: Relevance and Applicability

Angiotensin II Moderately Decreases Plasmodium Infection and Experimental Cerebral Malaria in Mice.

Angiotensin II, a peptide hormone that regulates blood pressure, has been proposed as a protective factor against cerebral malaria based on a genetic analysis. In vitro studies have documented an inhibitory effect of angiotensin II on Plasmodium growth, while studies using chemical inhibitors of angiotensin II in mice showed protection against experimental cerebral malaria but not major effects on parasite growth. To determine whether the level of angiotensin II affects Plasmodium growth and/or disease outcome in malaria, elevated levels of angiotensin II were induced in mice by intradermal implantation of osmotic mini-pumps providing constant release of this hormone. Mice were then infected with P. berghei and monitored for parasitemia and incidence of cerebral malaria. Mice infused with angiotensin II showed decreased parasitemia seven days after infection. The development of experimental cerebral malaria was delayed and a moderate increase in survival was observed in mice with elevated angiotensin II, as confirmed by decreased number of cerebral hemorrhages compared to controls. The results presented here show for the first time the effect of elevated levels of angiotensin II in an in vivo model of malaria. The decreased pathogenesis observed in mice complements a previous human genetic study, reinforcing the hypothesis of a beneficial effect of angiotensin II in malaria.

The HFE genotype and a formulated diet controlling for iron status attenuate experimental cerebral malaria in mice

Plasmodium falciparum infects approximately 500million individuals each year. A small but significant number of infections lead to complications such as cerebral malaria. Cerebral malaria is associated with myelin damage and neurological deficits in survivors, and iron status is thought to impact the outcome of infection. We evaluated whether a mouse model of experimental cerebral malaria with Plasmodium berghei ANKA strain was altered by dietary iron deficiency or genetic iron overload (H67D HFE). We found that H67D mice had increased survival over H67H (wild type) mice. Moreover, a specifically designed formulation diet increased survival regardless of whether the diet was iron deficient or iron adequate. To determine potential mechanisms underlying demyelination in experimental cerebral malaria, we measured Semaphorin4A (Sema4A) protein levels in the brain because we found it is cytotoxic to oligodendrocytes. Sema4A was increased in wild type mice that developed experimental cerebral malaria while consuming standard rodent chow, consistent with a decrease in myelin basic protein, an indicator of myelin integrity. The brains of iron deficient and H67D mice had lower levels of Sema4A. Myelin basic protein was decreased in brains of mice fed the iron deficient diet as has been previously reported. We also examined erythropoietin, which is under consideration for treatment of cerebral malaria, and IL-6, which is known to increase during infection. We found that plasma erythropoietin was elevated and IL-6 was low in H67D mice and in the mice fed the formulation diets. These data reveal a paradigm-shifting concept that maintaining iron status may not increase the mortality associated with malaria and provide a dietary strategy for further examination. Moreover, the data provide clues for exploring the mechanism to limit the co-morbidity associated with experimental cerebral malaria that appears to include decreased Sema4A in brain as well as elevated erythropoietin and lower IL-6 in plasma.

Vitamin D inhibits the occurrence of experimental cerebral malaria in mice by suppressing the host inflammatory response.

In animal models of experimental cerebral malaria (ECM), neuropathology is associated with an overwhelming inflammatory response and sequestration of leukocytes and parasite-infected RBCs in the brain. In this study, we explored the effect of vitamin D (VD; cholecalciferol) treatment on host immunity and outcome of ECM in C57BL/6 mice during Plasmodium berghei ANKA (PbA) infection. We observed that oral administration of VD both before and after PbA infection completely protected mice from ECM. VD administration significantly dampened the inducible systemic inflammatory responses with reduced circulating cytokines IFN-γ and TNF and decreased expression of these cytokines by the spleen cells. Meanwhile, VD also resulted in decreased expression of the chemokines CXCL9 and CXCL10 and cytoadhesion molecules (ICAM-1, VCAM-1, and CD36) in the brain, leading to reduced accumulation of pathogenic T cells in the brain and ultimately substantial improvement of the blood-brain barriers of PbA-infected mice. In addition, VD inhibited the differentiation, activation, and maturation of splenic dendritic cells. Meanwhile, regulatory T cells and IL-10 expression levels were upregulated upon VD treatment. These data collectively demonstrated the suppressive function of VD on host inflammatory responses, which provides significant survival benefits in the murine ECM model.

Targeting the olfactory bulb during experimental cerebral malaria

Malaria is responsible for over 500 million clinical cases and over 500000 deaths annually. Fatalities arise from a range of overlapping syndromes, such as cerebral malaria, whose pathogenesis is still incompletely understood. In a new study, Coban and colleagues provide new clues on the involvement of the olfactory bulb during experimental cerebral malaria in mice that open the way to testable hypotheses and potentially earlier intervention in humans.

Cerebral tissue oxygenation impairment during experimental cerebral malaria

Ischemia and hypoxia have been implicated in cerebral malaria (CM) pathogenesis, although direct measurements of hypoxia have not been conducted. C57BL/6 mice infected with Plasmodium berghei ANKA (PbA) develop a neurological syndrome known as experimental cerebral malaria (ECM), whereas BALB/c mice are resistant to ECM. In this study, intravital microscopy methods were used to quantify hemodynamic changes, vascular/tissue oxygen (O2) tension (PO2), and perivascular pH in vivo in ECM and non-ECM models, employing a closed cranial window model. ECM mice on day 6 of infection showed marked decreases in pial blood flow, vascular (arteriolar, venular), and perivascular PO2, perivascular pH, and systemic hemoglobin levels. Changes were more dramatic in mice with late-stage ECM compared with mice with early-stage ECM. These changes led to drastic decreases in O2 delivery to the brain tissue. In addition, ECM animals required a greater PO2 gradient to extract the same amount of O2 compared with non-infected animals, as the pial tissues extract O2 from the steepest portion of the blood O2 equilibrium curve. ECM animals also showed increased leukocyte adherence in postcapillary venules, and the intensity of adhesion was inversely correlated with blood flow and O2 extraction. PbA-infected BALB/c mice displayed no neurological signs on day 6 and while they did show changes similar to those observed in C57BL/6 mice (decreased pial blood flow, vascular/tissue PO2, perivascular pH, hemoglobin levels), non-ECM animals preserved superior perfusion and oxygenation compared with ECM animals at similar anemia and parasitemia levels, resulting in better O2 delivery and O2 extraction by the brain tissue. In conclusion, direct quantitative assessment of pial hemodynamics and oxygenation in vivo revealed that ECM is associated with severe progressive brain tissue hypoxia and acidosis.

Pharmacologic inhibition of CXCL10 in combination with anti-malarial therapy eliminates mortality associated with murine model of cerebral malaria.

Despite appropriate anti-malarial treatment, cerebral malaria (CM)-associated mortalities remain as high as 30%. Thus, adjunctive therapies are urgently needed to prevent or reduce such mortalities. Overproduction of CXCL10 in a subset of CM patients has been shown to be tightly associated with fatal human CM. Mice with deleted CXCL10 gene are partially protected against experimental cerebral malaria (ECM) mortality indicating the importance of CXCL10 in the pathogenesis of CM. However, the direct effect of increased CXCL10 production on brain cells is unknown. We assessed apoptotic effects of CXCL10 on human brain microvascular endothelial cells (HBVECs) and neuroglia cells in vitro. We tested the hypothesis that reducing overexpression of CXCL10 with a synthetic drug during CM pathogenesis will increase survival and reduce mortality. We utilized atorvastatin, a widely used synthetic blood cholesterol-lowering drug that specifically targets and reduces plasma CXCL10 levels in humans, to determine the effects of atorvastatin and artemether combination therapy on murine ECM outcome. We assessed effects of atorvastatin treatment on immune determinants of severity, survival, and parasitemia in ECM mice receiving a combination therapy from onset of ECM (day 6 through 9 post-infection) and compared results with controls. The results indicate that CXCL10 induces apoptosis in HBVECs and neuroglia cells in a dose-dependent manner suggesting that increased levels of CXCL10 in CM patients may play a role in vasculopathy, neuropathogenesis, and brain injury during CM pathogenesis. Treatment of ECM in mice with atorvastatin significantly reduced systemic and brain inflammation by reducing the levels of the anti-angiogenic and apoptotic factor (CXCL10) and increasing angiogenic factor (VEGF) production. Treatment with a combination of atorvastatin and artemether improved survival (100%) when compared with artemether monotherapy (70%), p<0.05. Thus, adjunctively reducing CXCL10 levels and inflammation by atorvastatin treatment during anti-malarial therapy may represent a novel approach to treating CM patients.