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The simultaneous acquisition of electroencephalography and functional magnetic resonance imaging (EEG-fMRI) is a multimodal technique extensively applied for mapping the human brain. However, the quality of EEG data obtained within the MRI environment is strongly affected by subject motion due to the induction of voltages in addition to artefacts caused by the scanning gradients and the heartbeat. This has limited its application in populations such as paediatric patients or to study epileptic seizure onset. Recent work has used a Moiré-phase grating and a MR-compatible camera to prospectively update image acquisition and improve fMRI quality (prospective motion correction: PMC). In this study, we use this technology to retrospectively reduce the spurious voltages induced by motion in the EEG data acquired inside the MRI scanner, with and without fMRI acquisitions. This was achieved by modelling induced voltages from the tracking system motion parameters; position and angles, their first derivative (velocities) and the velocity squared. This model was used to remove the voltages related to the detected motion via a linear regression. Since EEG quality during fMRI relies on a temporally stable gradient artefact (GA) template (calculated from averaging EEG epochs matched to scan volume or slice acquisition), this was evaluated in sessions both with and without motion contamination, and with and without PMC. We demonstrate that our approach is capable of significantly reducing motion-related artefact with a magnitude of up to 10mm of translation, 6° of rotation and velocities of 50mm/s, while preserving physiological information. We also demonstrate that the EEG-GA variance is not increased by the gradient direction changes associated with PMC. Provided a scan slice-based GA template is used (rather than a scan volume GA template) we demonstrate that EEG variance during motion can be supressed towards levels found when subjects are still. In summary, we show that PMC can be used to dramatically improve EEG quality during large amplitude movements, while benefiting from previously reported improvements in fMRI quality, and does not affect EEG data quality in the absence of large amplitude movements., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

We here describe the novel finding that brain endothelial cells in vitro can stimulate the growth of Plasmodium falciparum through the production of low molecular weight growth factors. By using a conditioned medium approach, we show that the brain endothelial cells continued to release these factors over time. If this mirrors the in vivo situation, these growth factors potentially would provide an advantage, in terms of enhanced growth, for sequestered parasitised red blood cells in the brain microvasculature. We observed this phenomenon with brain endothelial cells from several sources as well as a second P. falciparum strain. The characteristics of the growth factors included: <3 kDa molecular weight, heat stable, and in part chloroform soluble. Future efforts should be directed at identifying these growth factors, since blocking their production or actions might be of benefit for reducing parasite load and, hence, malaria pathology., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Previous studies have shown that IFN-gamma is essential for the pathogenesis of cerebral malaria (CM) induced by Plasmodium berghei ANKA (PbA) in mice. However, the exact role of IFN-gamma in the pathway (s) leading to CM has not yet been described. Here, we used 129P2Sv/ev mice which develop CM between 7 and 14 days post-infection with PbA. In this strain, both CD4(+) and CD8(+) T cells were involved in the effector phase of CM. When 129P2Sv/ev mice deficient in the IFN-gamma receptor alpha chain (IFN-gammaR1) were infected with PbA, CM did not occur. Migration of leucocytes to the brain at the time of CM was observed in wild type (WT) but not in deficient mice. However, in the latter, there was an accumulation of T cells in the lungs. Analysis of chemokines and their receptors in WT and in deficient mice revealed a complex, organ-specific pattern of expression. Up-regulation of RANTES/CCL5, IP-10/CCL3 and CCR2 was associated with leucocyte migration to the brain and increased expression of MCP-1/CCL2, IP-10/CCL3 and CCR5 with leucocyte migration to the lung. This shows that IFN-gamma controls trafficking of pathogenic T cells in the brain, thus providing an explanation for the organ-specific pathology induced by PbA infection.

Recent findings that levels of brain lactate and alanine were elevated in murine cerebral malaria led us to investigate the effect of dichloroacetate (DCA; 60 mg/kg), an activator of pyruvate dehydrogenase, on the levels of brain metabolites, and on the survival of mice infected with Plasmodium berghei ANKA which normally causes lethal cerebral malaria. DCA significantly reduced brain lactate and alanine levels when administered to infected mice, had no effect on the TCA cycle-related metabolites glutamate, GABA and aspartate and was associated with increased brain glutamine levels: 40% of mice thus treated survived the normally lethal infection.

An immunohistochemical method was developed, using a polyclonal antibody, to detect the enzyme indoleamine 2,3-dioxygenase (IDO) in normal and malaria-infected tissue. Plasmodium berghei ANKA, a cerebral malaria (CM) model, and P. berghei K173, a non-cerebral malaria (NCM) model, were used. It was found that vascular endothelial cells were the primary site of IDO expression in both models of malaria infection and that this response was systemic, with the vascular endothelium of brain, heart, lung, spleen and uterus all staining positive. These results suggest that IDO is part of a systemic host response to parasite infection. Although high levels of IDO production alone may not cause pathology, it is possible that when its production is combined with other features of CM, such as breakdown of the blood-brain barrier (BBB), metabolites of the kynurenine pathway may be able to influence the otherwise tightly regulated, immunologically privileged site of the CNS and cause some of the symptoms and pathology observed.

Cerebral expression of c-fos protein was studied by immunocytochemistry in murine cerebral malaria (CM) and malaria without cerebral involvement (non-CM). c-fos expression, low in the brains of uninfected mice, increased in frequency, intensity, and distribution during the course of fatal CM (e.g., a 70-fold increase on day 7 after inoculation). These changes paralleled the timing and degree of the neurologic complications and histopathologic changes. Only a slight increase in c-fos expression was detectable in non-CM mice on day 7 after inoculation. Dexamethasone treatment (days 0 and 1 after inoculation) of the CM mice largely prevented the increased cerebral c-fos expression, histopathologic changes, cerebral complications, and death. Increased c-fos expression may indicate the specific neuronal pathways activated by the immunopathologic process of fatal murine CM and could be associated with the behavioral changes and neurologic complications in this model.

Fatal murine cerebral malaria (FMCM) is an immunopathological process. The depletion of CD4+ T cells, or the administration of antioxidants or antibodies against certain cytokines, protect the mice against cerebral complications. We previously have shown that astrocytes, microglia, and monocytes play a role in the development of FMCM, suggesting that an active immune response occurs locally within the central nervous system. We now have investigated the functional involvement of glia and monocytes in FMCM by assessing 1) the production, 2) the temporal appearance, and 3) the cellular source of cytokine mRNA and protein in the brain. Brain sections from uninfected and FMCM mice were analyzed for the presence of cytokine mRNA and protein by in situ hybridization and immunohistochemistry. Tumor necrosis factor (TNF)-alpha mRNA and protein were associated with microglia and astrocytes, monocytes, and the cerebral vascular endothelium in FMCM mice but not uninfected animals. TNF-alpha mRNA was first detected several days before the animals showed cerebral symptoms and died. Interleukin (IL)-1 beta mRNA was found in the brains of both uninfected and FMCM mice. However, IL-1 beta protein was associated only with monocytes, the meningeal vascular endothelium, and neurons in the fronto-parietal cortex in the FMCM brains. No IL-4 or IL-6 mRNAs were detected in either group. These results provide the strongest evidence to date that cytokines, in particular TNF-alpha, produced locally in the central nervous system play a role in the pathogenesis of FMCM.

CBA/T6 mice inoculated with Plasmodium berghei ANKA strain (PbA) exhibited cerebral symptoms and died from cerebral malaria 6-8 days p.i. whereas DBA/2J mice developed (around days 6-9) a non-fatal cerebral malaria, with milder cerebral symptoms, and died between days 15 and 22 from other malaria-related complications. When inoculated with P. berghei K173 (Pb) these mouse strains did not develop cerebral malaria. These mouse/parasite strain combinations were used, in conjunction with the retinal whole-mount technique, to elucidate factors critical in the pathology of murine cerebral malaria. CBA/T6 mice infected with PbA (PbA-CBA mice) demonstrated mild changes in vascular permeability as early as days 2-3, prior to the appearance on day 5 of cerebral symptoms, whereas mice with non-cerebral malaria did not show any vascular permeability changes until the very late stage of the disease (days 14-22). In the PbA infections, progressive deterioration of endothelial barrier properties, demonstrated by Evans' Blue leakage both generally and from specific focal areas, as well as a developing monocytosis and adherence of mononuclear cells to the endothelium of the retinal vessels continued until death (in CBA/T6 mice) or resolution (in DBA/2J mice). Adherent monocytes, particularly in PbA-CBA mice, were associated with reduced Hoechst staining of individual endothelial cells and a banking up proximally of both parasitized and non-parasitized blood cells in the small blood vessels, often with accompanying focal leakage of Evans' Blue from the retinal vessels. The occurrence and severity of these early changes in the microcirculation correlated with the subsequent development of cerebral symptoms. Monocyte margination appeared to be the most significant factor associated with the development of cerebral symptoms.

CBA/T6 and Balb/c mice inoculated with Plasmodium berghei ANKA strain (PbA) died from cerebral malaria 6-8 days post-inoculation. DBA/2J mice similarly inoculated developed a non-fatal cerebral malaria, with mild temporary cerebral symptoms, and died between days 15 and 22 from other malaria-related complications. When inoculated with P. berghei K173 (Pb) these mouse strains did not develop a cerebral malaria but died between days 15 and 22 from other malaria-related complications. These mouse strain/parasite strain combinations allow for detailed examination of factors critical in the pathology of murine cerebral malaria. Monastral Blue, a colloid dye, when injected intravascularly between days 0 and 2 into PbA-inoculated CBA (PbA-CBA) or Balb/c (PbA-Balb/c) mice prevented death from cerebral malaria. There was no evidence of increased vascular permeability at this stage. When Monastral Blue was injected between days 5 and 8, there was increased vascular permeability in the kidney, liver, lung, spleen and brain of PbA-CBA and PbA-Balb/c mice. Injection of Monastral Blue into these animals at this time also precipitated cerebral symptoms and death, but not in Pb-infected mice. Endothelial and mononuclear cells phagocytosed, and were coated with, the Monastral Blue particles when the dye was injected between days 5 and 8 into PbA-CBA and PbA-Balb/c mice. Control, uninfected mice did not demonstrate either of these features. Pb-infected mice only demonstrated coated mononuclear cells. Mononuclear cell attachment to the endothelium, increased vascular permeability and increased association of Monastral Blue particles with monocytes and endothelial cells were correlated with cerebral symptoms and death. Monastral Blue is thus a useful agent for studying the roles of mononuclear cells and endothelium in murine cerebral malaria.

Cerebral malaria in A/J and CBA/H mice infected with Plasmodium berghei ANKA is accompanied by mononuclear cell infiltration, haemorrhage and cerebral endothelial cell damage. This damage is presumably one of the causes of the breakdown of the blood-brain barrier which was detected by measuring the movement of the dye Evans blue and radioisotope labelled albumin and erythrocytes. The density of brain tissue, measured by a Percoll gradient technique, was significantly reduced in mice exhibiting cerebral symptoms, suggesting the occurrence of cerebral oedema.