Biomedical subjects

T Chan-Ling

Publications and source records attributed to T Chan-Ling.

At least 19 recordsLinked to original sources

PubMed2001

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.

Adult↗

PubMed2001

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.

Animals↗

PubMed2001

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.

Animals↗

PubMed2000

Reactive changes of retinal microglia during fatal murine cerebral malaria: effects of dexamethasone and experimental permeabilization of the blood-brain barrier.

Microglial activation and redistribution toward blood vessels are some of the earliest observable events occurring within the central nervous system (CNS) during fatal murine cerebral malaria (FMCM). To investigate stimuli that might modulate microglial reactivity during FMCM we have performed two experimental manipulations and observed microglial responses in retinal whole mounts. First, to determine whether increased blood-brain barrier (BBB) permeability in the absence of the malaria parasite initiates the microglial changes, BBB function was compromised experimentally by intracarotid injection of arabinose and retinae were examined 12, 24, or 36 hours later. Second, to determine whether the immune response against the malaria parasite modulates microglial reactivity, infected mice were treated with dexamethasone before day 4 postinoculation. This treatment regime ameliorates cerebral complications without affecting parasite growth. We observed that increased BBB permeability was sufficient to elicit thickening of microglial processes and redistribution of microglia toward the vasculature, characteristic of the early stages of FMCM. However, despite the presence of plasma constituents in the CNS for up to 36 hours, microglia with amoeboid and vacuolated morphology were not observed. Dexamethasone treatment inhibited the up-regulation of alpha-D-galactose expression and reactive morphological changes in microglia during FMCM. These results suggest that disruption of the CNS milieu by entry of plasma constituents, or circulating malaria parasites in the absence of an immune response, by themselves are insufficient to induce the reactive microglial changes that are characteristic of FMCM. In addition, dexamethasone-sensitive event(s), presumably associated with immune system activation, occurring within the first few days of malaria infection are essential for the development of reactive microglia and subsequent fatal neurological complications.

Animals↗

PubMed2000

Breakdown of the blood-retinal barrier induced by activated T cells of nonneural specificity.

The cellular and microvascular responses of JC Lewis rats to an intravenous injection of activated T cells specific for ovalbumin were examined with the retinal whole mount technique. The retina was examined at various times post-injection (pi) with the use of antibodies to the alphabeta T cell receptor (TCR) or to major histocompatibility complex class II (MHC II), the monoclonal antibody ED1, and intravascular tracers. By 12 hours pi, small numbers of TCR(+), ED1(+), and MHC II(+) cells were present within the lumen of retinal vessels, and minor breakdown of the blood-retinal barrier (BRB) and microglial activation were evident. The intensity of these responses had increased by 1 day pi, when small numbers of TCR(+) cells had also undergone extravasation. By 2 to 3 days pi, the numbers of TCR(+), ED1(+), and MHC II(+) cells in the retinal parenchyma had increased, but the BRB breakdown and microglial activation had subsided. Thus, in the absence of target antigen, activated T cells induced limited and transient breakdown of the BRB, microglial activation, and the extravasation of ED1(+), MHC II(+) monocytes. In contrast, the retina of rats that received an intraocular injection of ovalbumin in addition to the intravascular injection of T cells showed massive cellular recruitment and breakdown of the BRB. These results indicate that an increase in the number of activated T cells in the circulation, such as that which occurs during viral or bacterial infection, has the potential to result in transient breakdown of the BRB and a mild local microglial response.

Animals↗

PubMed2000

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.

Aging↗

PubMed2000

Vascularization of the human fetal retina: roles of vasculogenesis and angiogenesis.

PURPOSE: To characterize the topography of and the cellular processes that underlie vascularization of the human retina. METHODS: The vasculature of human eyes obtained from fetuses ranging in age from 14 to 38 weeks of gestation (WG) was examined in Nissl-stained, whole-mount preparations and by anti-CD34 immunohistochemistry. RESULTS: The first event in retinal vascularization, apparent before 15 WG, was the migration of large numbers of spindle-shaped mesenchymal precursor cells from the optic disc. These cells proliferated and differentiated to produce cords of endothelial cells. By 15 WG, some cords were already patent and formed an immature vascular tree in the inner retinal layers that was centered on the optic disc. These processes are consistent with vessel formation by vasculogenesis. Angiogenesis then increased the vascular density of this immature plexus and extended it peripherally and temporally. Maturation of the plexus was characterized by substantial remodeling, which involved the withdrawal of endothelial cells into neighboring vascular segments. The outer plexus was formed as a result of the extension of capillary-sized buds from the existing inner vessels, a process that began around the incipient fovea between 25 and 26 WG. CONCLUSIONS: These observations suggest that the formation of primordial vessels in the central retina is mediated by vasculogenesis, whereas angiogenesis is responsible for increasing vascular density and peripheral vascularization in the inner retina. In contrast, the outer plexus and the radial peripapillary capillaries are formed by angiogenesis only. These mechanisms of retinal vascularization appear similar to those of vascularization of the central nervous system during development.

Antigens, CD34↗

PubMed1999

Contribution of O4+ oligodendrocyte precursors and astrocytes to the glial ensheathment of vessels in the rabbit myelinated streak.

The barrier properties and glial ensheathment of blood vessels in the retinal myelinated streak of adult New Zealand White rabbits were characterized at the ultrastructural level by intravascular injection of horseradish peroxidase (HRP) and immuno-electron microscopy with monoclonal antibody O4 and antibodies to glial fibrillary acidic protein (GFAP). Vessels within the myelinated streak did not leak HRP, and they exhibited tight junctions between adjacent endothelial cells. However, unlike their adult counterparts, the retinal blood vessels at postnatal day 18 exhibited substantial endocytotic activity. Both GFAP+ astrocytes and O4+ cells were evident surrounding the preretinal blood vessels of the myelinated streak. Furthermore, O4+ cells exhibited features indicative of high synthetic activity, including a large proportion of extended chromatin and prominent nucleoli within the nucleus, as well as a well-developed Golgi apparatus and numerous mitochondria in the cytoplasm. O4+ cells also exhibited variable quantities of heterochromatin, indicative of early stages of cellular differentiation. These observations are consistent with previous data showing that O4+ cells in the myelinated streak include oligodendrocyte precursor cells, pre-oligodendrocytes, and immature oligodendrocytes (Morcos Y, Chan-Ling T. Glia 21:163-182, 1997). The present data indicate that the preretinal vessels of the myelinated streak possess barrier properties typical of microvasculature in the central nervous system, and that both O4+ cells and astrocytes contribute to the glial ensheathment of these vessels. These vessels thus differ markedly from the leaky preretinal vessels associated with pathological conditions such as retinopathy of prematurity.

Animals↗

PubMed1998

Microvascular and cellular responses in the optic nerve of rats with acute experimental allergic encephalomyelitis (EAE).

The optic nerve of rats with EAE was examined at various times to determine the integrity of the blood-brain barrier (BBB) and to assess monocyte-macrophage, T cell, and microglial responses. In naive control animals, leakage of horseradish peroxidase (HRP) and the presence of cells expressing major histocompatibility complex (MHC) class II antigen were evident in the meninges of the retrobulbar optic nerve. In rats with EAE, microglia in the region of the lamina cribrosa and in the regions adjacent to the meninges became activated from day 7 to 8 postinduction (pi). HRP leakage was also evident in the region of the lamina cribrosa from day 7 to 8 pi. On day 8 pi, infiltration of inflammatory cells and Monastral blue leakage were apparent in the myelinated region of the optic nerve. The intensity of these cellular and vascular changes peaked at day 12 pi, when signs of clinical disease became manifest. Monocytes-macrophages expressing MHC class II and the ED1 antigen, together with lymphocytes expressing the alphabetaT cell receptor, constituted the major proportion of cells associated with inflammatory lesions. Thus: (i) the inherent weakness of the BBB as well as the presence of both antigen (myelin) and MHC class II+ cells in the retrobulbar optic nerve are likely susceptibility factors for the frequent involvement of this region in EAE and multiple sclerosis; and (ii) activation of microglia occurs early in the pathogenesis of experimental optic neuritis.

Animals↗

PubMed1998

Microvascular and cellular responses in the retina of rats with acute experimental allergic encephalomyelitis (EAE).

The microvascular and cellular responses in the retina during acute EAE were characterized using whole-mount preparations. The earliest detectable event was the accumulation of monocytes and T cells within veins on day 7 postinduction (pi). Mild breakdown of the blood-retinal barrier (BRB), activation of microglia and infiltration of monocytes and T cells into the retinal parenchyma were first evident on days 7 to 8 pi. Monocyte adhesion to the vessel wall and breakdown of the BRB were colocalized in the same vessel segments and occurred predominantly in veins. The marked difference in response observed in the retina versus the myelinated region of the optic nerve suggests that two types of inflammatory cascades are initiated. A mild response, characterised by very low numbers of T cells and monocytes and an absence of expression of MHC class II by resident microglia, is initiated when only small amounts of the encephalitogenic antigen are present in the perivascular space or associated with perivascular antigen-presenting cells. A full blown inflammatory reaction, as observed in the optic nerve, is initiated in the presence of substantial amounts of encephalitogenic antigen. This severe response is characterised by the infiltration of large numbers of CD4+, CD8+ T cells and ED1+ monocytes, and by abundant MHC class II expression by resident microglia as well as other cell types. Thus, MHC class II expression by resident microglia may be a possible effective amplifier mechanism if the encephalitogenic antigen is encountered in the tissue parenchyma.

Animals↗

PubMed1997

Differentiation and morphogenesis in pellet cultures of developing rat retinal cells.

We previously developed a reaggregate cell culture system (pellet cultures) in which retinal neuroepithelial cells proliferate and give rise to rod photoreceptor cells (rods) in vitro (Watanabe and Raff, 1990, Neuron 4:461-467). In the present study, we analyzed cell differentiation and morphogenesis in pellet cultures by using both cell-type-specific markers with immunofluorescence and electron microscopy. We demonstrated that, in addition to rods, the other major retinal cell types, including amacrine cells, bipolar cells, Müller cells, and ganglion cells were all present in the pellets, where most were able to develop from dividing precursor cells in vitro. The different cell types in the pellets became organized into two distinct structures: dark rosettes and pale rosettes. The cellular composition of these structures indicated that the dark rosettes correspond to the outer nuclear layer and the pale rosettes to the inner nuclear layer of the normal retina. Ultrastructural studies have indicated that the thin layer of neuronal processes surrounding the dark rosettes correspond to the outer plexiform layer, and the central region of the pale rosettes correspond to the inner plexiform layer of the normal retina. Other features of normal retinal development also occurred in the pellets, including programmed cell death and the formation of inner and outer rod cell segments and synapses. Thus, pellet cultures provide a convenient way to study different aspects of retinal development where one can control the size and the cellular composition of the initial reaggregate.

Animals↗

PubMed1997

Glial, vascular, and neuronal cytogenesis in whole-mounted cat retina.

A method was developed for detecting cytogenesis in retinal whole-mount preparations by bromodeoxyuridine (BrdU) immunohistochemistry. Because BrdU is a nonspecific marker that labels all cells in the S phase of the cell cycle, it is ideally combined with other cell-specific markers to study the cytogenesis of specific cell types. Double-label protocols to visualize mitotically active astrocytes and cells associated with the forming vasculature have been developed and applied to the retina. This approach revealed that, during normal development of the kitten retina, vascular mitogenesis occurs predominantly in the ganglion cell and nerve fiber layers, where the inner retinal plexus is formed by a process involving transformation of mesenchymal precursor cells and division of vascular endothelial cells. The peak density of vascular mitogenesis moved in a central-to-peripheral manner and was associated with the leading edge of the forming capillary plexus. A small number of dividing vascular endothelial cells was also associated with angiogenesis, the process responsible for the formation of the outer retinal plexus, vessels at the area centralis, and the radial peripapillary capillaries. Cytogenesis associated with astrocytes occurred in the ganglion cell and nerve fiber layers but was apparent predominantly at or close to the optic nerve head. Confirming earlier studies, neuronal mitogenesis was shown to occur predominantly at the ventricular zone, first at the area centralis and spreading peripherally with increasing maturity. A second region of neuronal cytogenesis, at the subventricular zone, was also apparent. Tissue hyperoxia decreased the rate of vasculogenic cell division but had no apparent effect on neurogenic or astrocytic cell division. Four distinct zones of cell generation were therefore identified within the retina, each associated with either glial, vascular, or neuronal cytogenesis. Thus, BrdU immunohistochemistry in whole-mounted retinal preparations offers a fast and reliable alternative to [3H]thymidine autoradiography for the study of the topography of cytogenesis during development.

Animals↗

PubMed1997

Compromised blood-nerve barrier, astrogliosis, and myelin disruption in optic nerves during fatal murine cerebral malaria.

We examined the optic nerve, as an analogous tissue to brain white matter, to assess possible relationships between changes in the blood-nerve barrier, axonal integrity, and astrocyte morphology in the central nervous system during fatal murine cerebral malaria (FMCM). In the FMCM model, namely, CBA mice infected with Plasmodium berghei ANKA, neurological symptoms begin around day 5 post-inoculation (p.i.) and mice become increasingly ill by day 7 p.i., at which time they lapse into coma and die. Using intravascular perfusion with horseradish peroxidase combined with light and electron microscopy, and GFAP immunohistochemistry, the optic nerves in malaria-infected mice were found to display i) breakdown of the blood-nerve barrier, detectable as early as day 3 p.i. (about 2 days before the onset of neurological symptoms) increasing to peak severity by day 7 p.i.; ii) monocytosis, vascular congestion, and monocyte adherence to the endothelium in the microvasculature during the later stages of the disease process; iii) an increased incidence of patchy axonal demyelination and degeneration, mostly associated with vascular changes and astrogliosis, beginning at day 5 p.i. and more evident by day 7 p.i.; and iv) an increased intensity of GFAP immunostaining, detectable from day 3 p.i. and peaking at day 7 p.i. These optic nerve changes were always seen in the infected individuals, though they varied in intensity. The temporal and anatomical coincidence between the compromised blood-nerve barrier, monocyte adherence to the vascular endothelium, astrocyte changes, neuronal degeneration, and demyelination in the optic nerve in FMCM suggests that these factors are mechanistically inter-related. These findings are consistent with the proposed immunopathological nature of FMCM and provide further evidence for the pivotal role of the CNS microvasculature in the disease process. This is the first investigation of involvement of the optic nerve in FMCM and the first demonstration, to our knowledge, of loss of axonal viability in this condition in any CNS tissue. The observed demyelination is consistent with reports by other workers on such changes in the brain in human cerebral malaria.

Animals↗

PubMed1997

Early activation of microglia in the pathogenesis of fatal murine cerebral malaria.

Microglia are pluripotent members of the macrophage/monocyte lineage that can respond in several ways to pathological changes in the central nervous system. To determine their role in the pathogenesis of fatal murine cerebral malaria (FMCM) we have conducted a detailed study of the changes in morphology and distribution of retinal microglia during the progression of the disease. Adult CBA/T6 mice were inoculated with Plasmodium berghei ANKA. These mice died 7 days post inoculation (p.i.) with the parasite while exhibiting cerebral symptoms, increased permeability of the blood-brain barrier, and monocyte adherence to the vascular endothelium. Mice were injected i.v. with Monastral blue 2 h prior to sacrifice to identify "activated" monocytes, and their isolated retinae were incubated with the Griffonia simplicifolia (GS) lectin or reacted for the nucleoside diphosphatase enzyme to visualize microglia and the vasculature. Changes in microglial morphology were seen within 2-3 days p.i., that is, at least 3 days prior to the onset of cerebral symptoms and 4 days before death. Morphological changes included retraction of ramified processes, soma enlargement, an increasingly amoeboid appearance, and vacuolation. There was also increased staining intensity and redistribution of "activated" microglia toward retinal vessels, but no increase in density of NDPase-positive cells. The GS lectin only labeled a small population of microglia in the uninfected adult mouse retina. However, there was a striking increase in the focal density of GS-positive microglia during the progression of the disease. Extravasation of monocytes also was observed prior to the onset of cerebral symptoms. These results provide the first evidence that microglial activation is a critical component of the pathological process during FMCM.

Acid Anhydride Hydrolases↗

PubMed1997

Identification of oligodendrocyte precursors in the myelinated streak of the adult rabbit retina in vivo.

Whole-mount techniques have been applied to the myelinated axons of the rabbit retina in order to study oligodendrocytes in the developing and adult central nervous system in vivo. Retinas from rabbits on embryonic day (E) 26 to postnatal day (P) 50 and from adults were subjected to immunohistochemistry with antibodies to glial fibrillary acidic protein (GFAP), vimentin, glycolipid O4, myelin basic protein (MBP), galactocerebroside (Gal-C), 4D6, and Griffonia simplicifolia isolectin, markers chosen for their specificity for astrocytes, cells of the oligodendrocyte lineage, Müller cells, and microglia. The first glial cells labelled within the myelinated streak (MS) were vimentin+ astrocytes. These cells were first apparent near the optic disc at E26. Their numbers and distribution increased markedly between E26 and E30, but between E29 and P3, vimentin expression was replaced by GFAP expression. Between P3 and P4, O4+, Gal-C-, MBP oligodendrocyte precursor cells, O4+, Gal-C-, MBP- pre-oligodendrocytes, and O4+, Gal-C-, MBP- immature oligodendrocytes appeared in low numbers in the region adjacent to the optic nerve head. O4-/+, Gal-C+, MBP+ mature oligodendrocytes appeared soon after at P4 to P5. With age, the outer extent of Gal-C and MBP immunoreactivity expanded, with positive cells forming a continuous sheath around nerve fibre bundles. The sequence of gliogenesis in the (MS) of the rabbit retina thus appears similar to that in the rat optic nerve. In adult rabbits, a large population of O4+ cells was distributed across the MS, the outer limit of the cells coinciding with the outer limit of retinal vessels. These O4+ cells could be classified into three stages of differentiation on the basis of expression of developmental markers and morphology: O4+, Gal-C-, MBP- oligodendrocyte precursor cells with a bipolar, unipolar, or simple morphology; O4+, Gal-C-, MBP- pre-oligodendrocytes with a complex multipolar morphology; and O4+, Gal-C+, MBP- immature oligodendrocytes with a complex multipolar morphology. The final stage of differentiation was characterized as O4-, Gal-C+, MBP+ mature oligodendrocytes with multiple parallel processes aligned along nerve fibre bundles. These results provide in vivo evidence for the existence of a substantial population of oligodendrocyte precursor cells and non-myelin-producing immature oligodendrocytes in the MS of the adult rabbit retina. This preparation makes possible in situ patch clamping and determination of other functional responses of these cell types in vivo.

Animals↗

PubMed1996

Redistribution and degeneration of retinal astrocytes in experimental murine cerebral malaria: relationship to disruption of the blood-retinal barrier.

To determine whether astrocytes play a critical role in the pathogenesis of experimental murine cerebral malaria (EMCM), we examined changes in astrocyte morphology and distribution, using retinal wholemounts, in three models: a fatal cerebral malaria (CM) model, in which mice die showing cerebral symptoms; a "resolving" model, in which mice exhibit mild cerebral symptoms, but then recover; and a non-CM model, in which cerebral symptoms are not seen. In the fatal model, retinal astrocytes lost their even distribution from day 3 post-inoculation (p.i.) with malaria parasites, progressing to gliosis (day 5 p.i.), well before the onset of cerebral symptoms on day 6-7 p.i. At the terminal stage of the disease there was a loss of astrocyte processes contacting retinal vessels, often along vessel segments containing adherent monocytes. These features occurred in a mild form in the resolving model and were absent in the non-CM models. To investigate the mechanisms underlying these astrocytic changes, we carried out two experimental manipulations. Firstly, since dexamethasone ameliorates cerebral complications in the fatal CM model, the astrocytic response was monitored after dexamethasone treatment on days 0 and 1 p.i., or days 3 and 4 p.i. Second, to determine whether increased blood-retinal barrier (BRB) permeability initiates the astrocyte changes, breakdown of the BRB was induced experimentally by intra-carotid injection of arabinose and astrocyte morphology and distribution were examined 12, 24, and 48 h later. Retinal astrocytes in both the dexamethasone- and the arabinose-treated groups showed loss of even astrocyte distribution but no loss of astrocyte ensheathment of vessels. It is concluded that: i) astrocytes are involved in the pathogenesis of EMCM, since these changes are only prominent in the fatal model and occur substantially before the onset of cerebral symptoms; ii) the initial changes in astrocyte distribution may be a consequence of the increase in BRB permeability; and iii) the immune response triggered by the malaria parasite may be responsible for the loss of astrocyte ensheathment of vessel segments.

Animals↗

PubMed1996

Roles of vascular endothelial growth factor and astrocyte degeneration in the genesis of retinopathy of prematurity.

PURPOSE: To assess the role of vascular endothelial growth factor (VEGF) in the feline model of retinopathy of prematurity (ROP). METHODS: Retinopathy of prematurity was induced in neonatal cats by raising them in an oxygen-enriched (70% to 80%) atmosphere for 4 days to suppress vessel formation and then returning them to room air for 3 to 27 days. In situ hybridization was used to detect the expression of VEGF and its high-affinity receptor, flk-1, in the retina of neonatal cats, and glial fibrillary acidic protein immunocytochemistry was used to assess astrocyte status. RESULTS: The expression of VEGF in the innermost layers of retina fell in hyperoxia and increased on return to room air. Vascular endothelial growth factor expression was transient; it was maximal where vessels were about to form, and it rapidly downregulated after vessels had formed. During the proliferative vasculopathy of ROP, VEGF expression was stronger than in the normally developing retina, and the astrocytes that normally express VEGF degenerated. After the degeneration of astrocytes, VEGF was expressed by neurones of the ganglion cell layer. flk-1 was expressed by intraretinal and preretinal vessels. Supplemental oxygen therapy reduced or eliminated the overexpression of VEGF expression, astrocyte degeneration, and formation of preretinal vessels. CONCLUSIONS: Regulation of VEGF by tissue oxygen mediates the inhibition of vessel growth during hyperoxia and the subsequent proliferative vasculopathy. Degeneration of retinal astrocytes creates conditions for the growth of preretinal vessels.

Animals↗

PubMed1996

Correlation between enhanced vascular permeability, up-regulation of cellular adhesion molecules and monocyte adhesion to the endothelium in the retina during the development of fatal murine cerebral malaria.

The relationships between increased vascular permeability to protein, monocyte adherence to the endothelium, and expression of the cell adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in the central nervous system microvasculature were studied during the progression of fatal murine cerebral malaria. CBA mice were inoculated with Plasmodium berghei ANKA, and changes in the retinal microvasculature were examined on days 3, 5, and 7 postinoculation (p.i.). Evans blue dye and horseradish peroxidase (HRP) were administered intravenously to assess vascular permeability to macromolecules macroscopically and by light and electron microscopy. ICAM-1 and VCAM-1 expression were examined by immunohistochemistry. HRP leakage into the retinal parenchyma was seen macroscopically at a low level on day 3 p.i., increasing progressively at day 5 (the earliest time at which cerebral symptoms were observed) and day 7 (the day on which animals showed severe behavioral abnormalities and died). The inner retinal vascular plexus showed a slight increase in vascular permeability to intravenous Evans blue at day 3 p.i. and congestion, monocyte adherence to the endothelium, and increased vascular permeability to both Evans blue and HRP at day 7 p.i. Electron microscopic observations were consistent with these findings and also revealed disrupted light junctions and the coating of monocytes and endothelium with HRP at day 7 p.i. Immunohistochemical staining and densitometry showed a progressive increase from day 3 to day 7 p.i. in the densities of ICAM-1 and VCAM-1 on the venular endothelium of the inner retinal vascular plexus, with the appearance of adherent ICAM-1+ monocytes at the terminal stage of the disease. None of the pathological changes associated with the inner retinal plexus were seen at any stage in the outer retinal plexus. These results suggest the following sequence of events in the inner retinal vessels, particularly the venules, during the progression of fatal murine cerebral malaria: 1) a mild increase in vascular permeability at approximately day 3 p.i., 2) a progressive increase in endothelial expression of the cell adhesion molecules ICAM-1 and VCAM-1, commencing at approximately day 3 p.i., 3) monocyte adhesion to the endothelium starting at approximately day 5 p.i., and 4) frank disruption of endothelial integrity at the terminal stage (day 7 p.i.), leading to edema and hemorrhage. Similar changes in cerebral vessels may underlie the neurological complications of the disease.

Animals↗