Central nervous system in cerebral malaria: 'Innocent bystander' or active participant in the induction of immunopathology?

Cerebral malaria (CM) is a major life-threatening complication of Plasmodium falciparum infection in humans, responsible for up to 2 million deaths annually. The mechanisms underlying the fatal cerebral complications are still not fully understood. Many theories exist on the aetiology of human CM. The sequestration hypo-thesis suggests that adherence of parasitized erythrocytes to the cerebral vasculature leads to obstruction of the microcirculation, anoxia or metabolic disturbances affecting brain function, resulting in coma. This mechanism alone seems insufficient to explain all the known features of CM. In this review we focus on another major school of thought, that CM is the result of an over-vigorous immune response originally evolved for the protection of the host. Evidence in support of this second hypothesis comes from studies in murine malaria models in which T cells, monocytes, adhesion molecules and cytokines, have been implicated in the development of the cerebral complications. Recent studies of human CM also indicate a role for the immune system in the neurological complications. However, it is likely that multiple mechanisms are involved in the induction of cerebral complications and both the presence of parasitized erythrocytes in the central nervous system (CNS) and immunopathological processes contribute to the pathogenesis of CM. Most studies examining immunopathological responses in CM have focused on reactions occurring primarily in the systemic circulation. However, these also do not fully account for the development of cerebral complications in CM. In this review we summarize results from human and mouse studies that demonstrate morphological and functional changes in the resident glial cells of the CNS. The degree of immune activation and degeneration of glial cells was shown to reflect the extent of neurological complications in murine cerebral malaria. From these results we highlight the need to consider the potentially important contribution within the CNS of glia and their secreted products, such as cytokines, in the development of human CM.     

The crossroads of neuroinflammation in infectious diseases: endothelial cells and astrocytes

Homeostasis implies constant operational defence mechanisms, against both external and internal threats. Infectious agents are prominent among such threats. During infection, the host elicits the release of a vast array of molecules and numerous cell-cell interactions are triggered. These pleiomorphic mediators and cellular effects are of prime importance in the defence of the host, both in the systemic circulation and at sites of tissue injury, for example, the blood-brain barrier (BBB). Here, we focus on the interactions between the endothelium, astrocytes, and the molecules they release. Our review addresses these interactions during infectious neurological diseases of various origins, especially cerebral malaria (CM). Two novel elements of the interplay between endothelium and astrocytes, microparticles and the kynurenine pathway, will also be discussed.     

Protection of blood retinal barrier and systemic vasculature by insulin-like growth factor binding protein-3

Previously, we showed that insulin growth factor (IGF)-1 binding protein-3 (IGFBP-3), independent of IGF-1, reduces pathological angiogenesis in a mouse model of the oxygen-induced retinopathy (OIR). The current study evaluates novel endothelium-dependent functions of IGFBP-3 including blood retinal barrier (BRB) integrity and vasorelaxation. To evaluate vascular barrier function, either plasmid expressing IGFBP-3 under the regulation of an endothelial-specific promoter or a control plasmid was injected into the vitreous humor of mouse pups (P1) and compared to the non-injected eyes of the same pups undergoing standard OIR protocol. Prior to sacrifice, the mice were given an injection of horseradish peroxidase (HRP). IGFBP-3 plasmid-injected eyes displayed near-normal vessel morphology and enhanced vascular barrier function. Further, in vitro IGFBP-3 protects retinal endothelial cells from VEGF-induced loss of junctional integrity by antagonizing the dissociation of the junctional complexes. To assess the vasodilatory effects of IGFBP-3, rat posterior cerebral arteries were examined in vitro. Intraluminal IGFBP-3 decreased both pressure- and serotonin-induced constrictions by stimulating nitric oxide (NO) release that were blocked by L-NAME or scavenger receptor-B1 neutralizing antibody (SRB1-Ab). Both wild-type and IGF-1-nonbinding mutant IGFBP-3 (IGFBP-3NB) stimulated eNOS activity/NO release to a similar extent in human microvascular endothelial cells (HMVECs). NO release was neither associated with an increase in intracellular calcium nor decreased by Ca(2+)/calmodulin-dependent protein kinase II (CamKII) blockade; however, dephosphorylation of eNOS-Thr(495) was observed. Phosphatidylinositol 3-kinase (PI3K) activity and Akt-Ser(473) phosphorylation were both increased by IGFBP-3 and selectively blocked by the SRB1-Ab or PI3K blocker LY294002. In conclusion, IGFBP-3 mediates protective effects on BRB integrity and mediates robust NO release to stimulate vasorelaxation via activation of SRB1. This response is IGF-1- and calcium-independent, but requires PI3K/Akt activation, suggesting that IGFBP-3 has novel protective effects on retinal and systemic vasculature and may be a therapeutic candidate for ocular complications such as diabetic retinopathy.     

Concentration of astrocytic filaments at the retinal optic nerve junction is coincident with the absence of intra-retinal myelination: comparative and developmental evidence

The structure of the lamina cribrosa (LC) and astrocytic density were examined in various species with and without intra-retinal myelination. Sections of optic nerve from various species were stained with Milligan's trichrome or antibodies to glial fibrillary acidic protein, myelin basic protein (MBP) and antibody O4. Marmoset, flying fox, cat, and sheep, which lack intraretinal myelination, were shown to possess a well-developed LC as well as a marked concentration of astrocytic filaments distal to the LC. Rat and mouse, which lack intraretinal myelination, lacked a well-developed LC but exhibited a marked concentration of astrocytic filaments in this region. Rabbit and chicken, which exhibit intraretinal myelination, lacked both a well-developed LC and a concentration of astrocytes at the retinal optic nerve junction (ROJ). A marked concentration of astrocytes at the ROJ of human fetuses was also apparent at 13 weeks of gestation, prior to myelination of the optic nerve; in contrast, the LC was not fully developed even at birth. This concentration of astrocytes was located distal to O4 and MBP immunoreactivity in human optic nerve, and coincided with the site of initial myelination of ganglion cell axons in marmoset and rat. Myelination proceeded from the chiasm towards the retinal end of the human optic nerve. Moreover, the outer limit of oligodendrocyte precursor cells (OPC) migration into the rabbit retina was restricted by the outer limit of astrocyte spread. These observations indicate that a concentration of astrocytic filaments at the ROJ is coincident with the absence of intraretinal myelination. Differential expression of tenascin-C by astrocytes at the ROJ appears to contribute to the molecular barrier to OPC migration (see Bartsch et al., 1994), while expression of the homedomain protein Vax 1 by glial cells at the optic nerve head appears to inhibit migration of retinal pigment epithelial cells into the optic nerve (see Bertuzzi et al., 1999). These observations combined with our present comparative and developmental data lead us to suggest that the astrocytes at the ROJ serve to regulate cellular traffic into and out of the retina.     

Age related changes in NAD+ metabolism oxidative stress and Sirt1 activity in wistar rats

The cofactor nicotinamide adenine dinucleotide (NAD+) has emerged as a key regulator of metabolism, stress resistance and longevity. Apart from its role as an important redox carrier, NAD+ also serves as the sole substrate for NAD-dependent enzymes, including poly(ADP-ribose) polymerase (PARP), an important DNA nick sensor, and NAD-dependent histone deacetylases, Sirtuins which play an important role in a wide variety of processes, including senescence, apoptosis, differentiation, and aging. We examined the effect of aging on intracellular NAD+ metabolism in the whole heart, lung, liver and kidney of female wistar rats. Our results are the first to show a significant decline in intracellular NAD+ levels and NAD:NADH ratio in all organs by middle age (i.e.12 months) compared to young (i.e. 3 month old) rats. These changes in [NAD(H)] occurred in parallel with an increase in lipid peroxidation and protein carbonyls (o- and m- tyrosine) formation and decline in total antioxidant capacity in these organs. An age dependent increase in DNA damage (phosphorylated H2AX) was also observed in these same organs. Decreased Sirt1 activity and increased acetylated p53 were observed in organ tissues in parallel with the drop in NAD+ and moderate over-expression of Sirt1 protein. Reduced mitochondrial activity of complex I-IV was also observed in aging animals, impacting both redox status and ATP production. The strong positive correlation observed between DNA damage associated NAD+ depletion and Sirt1 activity suggests that adequate NAD+ concentrations may be an important longevity assurance factor.     

Changes in kynurenine pathway metabolism in the brain, liver and kidney of aged female Wistar rats

The kynurenine pathway of tryptophan catabolism plays an important role in several biological systems affected by aging. We quantified tryptophan and its metabolites kynurenine (KYN), kynurenine acid (KYNA), picolinic acid (PIC) and quinolinic acid (QUIN), and activity of the kynurenine pathway enzymes indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO) and quinolinic acid phosphoribosyltransferase (QPRTase), in the brain, liver and kidney of young, middle-aged and old female Wistar rats. Tryptophan levels and TDO activity decreased in all tissues with age. In contrast, brain IDO activity increased with age, while liver and kidney IDO activity decreased with age. The levels of KYN, KYNA, QUIN and PIC in brain all increased with age, while the levels of KYN in the liver and kidney showed a tendency to decrease. The levels of KYNA in the liver did not change, but the levels of KYNA in the kidney increased. The levels of PIC and QUIN increased significantly in the liver but showed a tendency to decrease in the kidney. QPRTase activity in both brain and liver decreased with age but was elevated in the kidney in middle-aged (12-month-old) rats. These age-associated changes in tryptophan metabolism have the potential to impact upon major biological processes, including lymphocyte function, pyridine (NAD(P)(H)) synthesis and N-methyl-d-aspartate (NMDA)-mediated synaptic transmission, and may therefore contribute to several degenerative changes of the elderly.     

Aging-related changes in astrocytes in the rat retina: imbalance between cell proliferation and cell death reduces astrocyte availability

The aim of this study was to investigate changes in astrocyte density, morphology, proliferation and apoptosis occurring in the central nervous system during physiological aging. Astrocytes in retinal whole-mount preparations from Wistar rats aged 3 (young adult) to 25 months (aged) were investigated qualitatively and quantitatively following immunofluorohistochemistry. Glial fibrillary acidic protein (GFAP), S100 and Pax2 were used to identify astrocytes, and blood vessels were localized using Griffonia simplicifoli a isolectin B4. Cell proliferation was assessed by bromodeoxyuridine incorporation and cell death by TUNEL-labelling and immunolocalization of the apoptosis markers active caspase 3 and endonuclease G. The density and total number of parenchymal astrocytes in the retina increased between 3 and 9 months of age but decreased markedly between 9 and 12 months. Proliferation of astrocytes was detected at 3 months but virtually ceased beyond that age, whereas the proportion of astrocytes that were TUNEL positive and relative expression of active caspase 3 and endonuclease G increased progressively with aging. In addition, in aged retinas astrocytes exhibited gliosis-like morphology and loss of Pax2 reactivity. A small population of Pax2⁺/GFAP⁻ cells was detected in both young adult and aged retinas. The reduction in the availability of astrocytes in aged retinas and other aging-related changes reported here may have a significant impact on the ability of astrocytes to maintain homeostasis and support neuronal function in old age.     

Perforin mediated apoptosis of cerebral microvascular endothelial cells during experimental cerebral malaria

Cerebral malaria is a serious complication of Plasmodium falciparum infection. We have investigated the role of perforin in the pathogenesis of cerebral malaria in a murine model (Plasmodium berghei ANKA (PbA) infection). C57BL/6 mice demonstrated the typical neuropathological symptoms of experimental cerebral malaria infection from day 5p.i. and became moribund on day 6p.i. This pathology was not seen in PbA-infected, perforin-deficient (pfp-/-) mice. From days 5-6p.i. onwards there was a significant increase in mRNA for granzyme B and CD8, but not CD4, in brain tissue from PbA-infected C57BL/6 and pfp-/- mouse brains. Perforin mRNA was strongly increased in the brains of PbA-infected C57BL/6 mice on day 6p.i. Immunohistochemistry revealed increased perforin staining and elevated numbers of CD8(+) cells within the cerebral microvessels in PbA-infected C57BL/6 at days 5 and 6p.i. compared with uninfected animals. At day 6p.i., there were TUNEL-positive cells and activated caspase-3 positive cells of endothelial morphology in the CNS of PbA-infected C57BL/6 mice. The TUNEL-positive cells were greatly reduced in pfp-/- mice. These results suggest that CD8(+)T lymphocytes induce apoptosis of endothelial cells via a perforin-dependent process, contributing to the fatal pathogenic process in murine cerebral malaria.     

Immunolocalization of occludin and claudin-1 to tight junctions in intact CNS vessels of mammalian retina

The distributions of occludin and claudin-1, two tight junction–associated integral membrane proteins were investigated by immunohistochemical analysis of whole-mount preparations of the blood vessels in the myelinated streak of the rabbit retina. Light microscopy revealed that occludin and claudin-1 immunoreactivities were abundant along the interface of adjacent endothelial cells of all blood vessels. Electron microscopy revealed that both proteins were distributed in a regular pattern (at regular intervals of approximately 80 nm) along the length of tight junctions, probably in the regions of tight junction strands. No other structures or cell types expressed either of these two proteins in the myelinated streak. Whereas occludin immunoreactivity was concentrated only at the tight junction interface, claudin-1 immunoreactivity also extended into the cytoplasm of the endothelial cells, suggesting a different structural role for claudin-1 than for occludin at tight junctions. Retinal pigment epithelial cells expressed occludin around their entire circumference, consistent with the function of these cells as a barrier separating the retina from the leaky vessels of the choroid. Also consistent with the association of occludin expression with vessels that exhibit functional tight junctions, this protein was expressed at only a low level in, and showed an irregular distribution along, the vessels of the choroid, a vascular bed that lacks blood-barrier properties. Further, the distribution of occludin was examined during formation and remodelling of the rat retinal vasculature. Occludin expression was evident at the leading edge of vessel formation and was found on all vessels in both the inner and outer vascular plexus. Numerous vascular segments at the early stage of vascular formation and regression lost occludin expression. The biological significance of this transient loss of occludin expression in terms of barrier function remains to be elucidated.     

Differentiation and migration of astrocyte precursor cells and astrocytes in human fetal retina: relevance to optic nerve coloboma.

The presence of astrocyte precursor cells (APCs) and time course and topography of astrocyte differentiation during development were investigated by triple-label immunohistochemistry with intact fetal and adult human retinas. Throughout retinal development and adulthood, expression of Pax2 was restricted to cells of the astrocytic lineage. Three distinct stages of astrocytic differentiation were identified during development: i) Pax2+/vimentin+/GFAP- APCs; ii) Pax2+/vimentin+/GFAP+ immature perinatal astrocytes; and iii) Pax2+/vimentin-/GFAP+ mature perinatal astrocytes. In adult, cells with the antigenic phenotype of mature perinatal astrocytes were restricted to a region surrounding the optic nerve head (ONH), whereas cells at a fourth stage of differentiation, adult astrocytes (Pax2-/vimentin-/GFAP+), were apparent throughout the vascularized retina. APC appearance was centered around the ONH and preceded the appearance of perinatal astrocytes. A cluster of Pax2+ somas was also present in a small region surrounding the ONH at the ventricular surface of the developing retina, which suggests the existence of two distinct sites of astrocytic differentiation. The coincidence in the location of APCs and perinatal astrocytes at the ventricular zone with that of optic nerve colobomas, together with the association of Pax2 gene mutations with this condition, suggests that coloboma formation may result from impaired astrocyte differentiation during development.