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Background/aims: Glial fibrillary acidic protein (GFAP) is an established indicator of retinal stress; its expression in retinal astrocytes and Muller cells has been demonstrated to be modulated by cytokines and [...]

Objective: To examine the morphological features of macular photoreceptors in histologically normal retina from normal donor eyes and eyes with age-related macular degeneration (AMD)., Methods: The macular region was excised from 18 donor eyes (aged 22-96 years) and cryosectioned. Sections were stained with hematoxylin-eosin or double immunolabeled using opsin antibodies or synaptic markers., Results: Three of 8 retinas studied in detail had AMD lesions; the remainder were histologically normal. Immunoreactivity to cone opsin was abnormal in parts of all retinas (3.5%-95.0% of each sample) and was associated with swelling of and altered immunoreactivity in the cone distal axon. In non-AMD retinas, the anomalies were mainly in nonfoveal macular locations. The nature of the anomalies was identical in non-AMD retinas and in parts of AMD retinas adjacent to overt degeneration., Conclusion: Redistribution of opsin and anomalies in the distal cone axon are common in the aging human macula and may indicate susceptibility to AMD., Clinical Relevance: The findings are consistent with tests of cone function in aging and early AMD, which suggests that integrated cone functions--including contrast sensitivity, color matching, and short wavelength-sensitive cone sensitivity--are the most reliable prognostic indicators of progression in AMD.

The central retina in primates is adapted for high acuity vision. The most significant adaptations to neural retina in this respect are: 1. The very high density of cone photoreceptors on the visual axis; 2. The dominance of Midget pathways arising from these cones and 3. The diminishment of retinal blood supply in the macula, and its absence on the visual axis. Restricted blood supply to the part of the retina that has the highest density of neural elements is paradoxical. Inhibition of vascular growth and proliferation is evident during foetal life and results in metabolic stress in ganglion cells and Muller cells, which is resolved during formation of the foveal depression. In this review we argue that at the macula stressed retinal neurons adapt during development to a limited blood supply from the choriocapillaris, which supplies little in excess of metabolic demand of the neural retina under normal conditions. We argue also that while adaptation of the choriocapillaris underlying the foveal region may initially augment the local supply of oxygen and nutrients by diffusion, in the long term these adaptations make the region more vulnerable to age-related changes, including the accumulation of insoluble material in Bruch's membrane and beneath the retinal pigment epithelium. These changes eventually impact on delivery of oxygen and nutrients to the RPE and outer neural retina because of reduced flow in the choriocapillaris and the increasing barriers to effective diffusion. Both the inflammatory response and the sequelae of oxidative stress are predictable outcomes in this scenario.

Background/aims: Microglia are the primary antigen presenting cells in the central nervous system and the retina, and can harbour viral antigens that may damage neural tissue via the release of neurotoxins. All cells bearing CD4 molecules and co-receptors (members of the chemokine receptor and Fcgamma receptor families) are potential targets for the human immunodeficiency virus (HIV). In this study, retinal microglia (in vitro and in situ) were investigated for the expression of candidate HIV-1 binding receptors., Methods: Cultured human retinal microglia and frozen sections of human retinas were used. Immunohistochemistry was used to investigate expression of cell surface receptors necessary for HIV-1 infection: CD4, CC chemokine receptor 5 (CCR5), and Fcgamma receptors., Results: Human retinal microglia expressed detectable levels of CD4, CD16, CD64, and CCR5 in vitro and Fcgamma receptor I (CD64) in situ., Conclusions: Human retinal microglia express several candidate receptors required for viral binding and as such may be a potential reservoir for HIV-1 infection.

Purpose: The therapeutic potential of TA, an anti-inflammatory glucocorticoid, for the treatment of exudative retinopathy has been examined in several independent clinical studies. The modulation of permeability and adhesion molecule expression of an epithelial cell line has been described in vitro, with the use of cytokines and triamcinolone acetonide (TA). In the current study, the influence of proinflammatory cytokines and TA on permeability and adhesion molecule expression in human choroidal endothelial cells (CECs) was investigated., Methods: Human CEC isolates treated with IFNgamma, TNFalpha, and TA were evaluated by flow cytometry and immunocytochemistry for expression of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and major histocompatibility complex (MHC)-I and -II. The effects of IFNgamma, TNFalpha, and TA on paracellular permeability of CEC monolayers were assessed in transendothelial cell resistance (TER) assays., Results: Both IFNgamma and TNFalpha significantly upregulated expression of ICAM1 and MHC-I on CECs. Expression of VCAM1 was induced after stimulation with both IFNgamma and TNFalpha, whereas expression of MHC-II was induced only by stimulation with IFNgamma. Cytokine-induced expression of ICAM1, MHC-I, and MHC-II antigen by CECs was significantly downregulated by TA. IFNgamma stimulation also increased permeability of CEC monolayers, whereas subsequent TA treatment decreased permeability of CEC monolayers., Conclusions: Human CEC isolates provide a useful in vitro model to study choroidal neovascular membrane characteristics and their potential response to pro- and anti-inflammatory agents. In addition, the results indicate that TA has the capacity to reduce adhesion molecule expression and permeability of choroidal vessels in vitro, confirming its potential as a therapeutic agent for treatment of exudative macular degeneration.

Glial fibrillary acidic protein (GFAP) is an established marker of retinal glia and has been shown to be modulated by several cytokines and retinal pathology. The influence of a number of factors, including post-mortem delay, storage duration and retinal pathology, on the distribution and morphology of macroglia and GFAP antigenicity was examined in human retina. The effects of these parameters on GFAP expression were estimated using immunohistochemistry, confocal microscopy and image analysis. Changes in expression of antigenicity were analysed in human retinal cryosections at three levels: constitutive,aberrant and total. The results indicated that short-term and long-term storage duration had no significant effect on GFAP immunoreactivity at all three levels of expression (P > 0.2).However, a significant increase in GFAP immunoreactivity and distribution at all three levels of expression was associated with prolonged post-mortem delay (> 30 h) (P < 0.05). This study highlights the importance of rigorous matching of post-mortem delay between control specimens in histological studies of human retinae. The study further demonstrates the utility of Eye Bank retinae fixed and stored in 2% paraformaldehyde, provided that appropriate controls are applied.

Retinoblastoma (Rb) is the most common intraocular tumor of childhood. In this study we examined primary Rb specimens and Rb cell lines for the expression of immunoglobulin superfamily (IgSF) antigens: MHC class I and II (MHC-I and MHC-II), neural cell adhesion molecule (NCAM), intercellular adhesion molecule-1 (ICAM-1), and Thy-1, which play an important role in immune system and tumor cell interactions. MHC-I and-II, ICAM-1 (CD54), NCAM (CD56), and Thy-1 (CDw90) immunoreactivity was studied in eight primary Rb biopsy specimens using immunohistochemistry, three using immunoelectron microscopy, and six Rb cell lines using flow cytometry (FCM). Parenchymal and vascular-associated cells, phenotypically similar to retinal microglia, strongly expressed MHC-II immunoreactivity and were distributed throughout primary Rb specimens. However, MHC-II expression on Rb cell lines was similar to nonspecific control levels. Tumor cells in primary Rb specimens displayed high NCAM, moderate Thy-1, and low MHC-I and ICAM-1 immunolabeling. Tumor vasculature expressed low to moderate MHC-I and ICAM-1 immunoreactivity and moderate Thy-1 immunoreactivity. NCAM was not detected on the vasculature of primary Rb specimens. Rb cell lines displayed variable expression of Thy-1, ICAM-1, and MHC-I. NCAM was highly expressed on five of six Rb cell lines. The high levels of constitutive NCAM immunoreactivity on Rb tumor cells confirm the neuroectodermal origins of this tumor. Additionally, the variable expression of Thy-1 may suggest separate neural lineages or differences in the maturational status ofsome Rb tumors. The presence of a population of infiltrating MHC-II-positive cells in primary Rb tumors has implications for immunomodulation of Rb growth.

Animal models, in vitro assays and pilot clinical studies suggest that intravitreal triamcinolone acetonide may be useful in the treatment of age-related macular degeneration. The present case study reports the effect of intravitreal triamcinolone acetonide injection on a subretinal neovascular lesion, microglial morphology and quantitative expression of MHC-II antigens. Triamcinolone acetonide significantly decreased MHC-II expression consistent with immunocytochemical observations which revealed condensed microglial morphology. The modulation of subretinal oedema and microglial morphology correlates with in vitro observations suggesting that downregulation of inflammatory markers and endothelial cell permeability are significant features of the mode of action of triamcinolone acetonide.

Aetiological and immunological aspects of AMD, a leading cause of blindness in Western countries, have been reviewed. Developmental studies suggest that anatomical features unique to the fovea result in a critical relationship between metabolic demand and blood supply at the macula, which is maintained throughout life. Recent studies show a sufficient degree of consistency in the link between smoking and both dry and wet AMD to regard it as causative. Dry AMD is considered to be the natural endstage of the disease; epidemiological and morphological studies point to choroidal vascular atrophy as the causative event and it is suggested that signals associated with acute vascular compromise lead to the development of subretinal neovascularisation. The relationship between sub-pigment epithelial deposits, including basal laminar deposit, and the pathogenesis of AMD is examined. Much of the literature is consistent with a choroidal origin for the constituents of drusen. The blood-retinal barrier preserves the physiological environment of the neural retina and limits inflammatory responses. The factors, including cytokines, adhesion molecules and the presence of resident immunocompetent cells (microglia), which determine the immune status of the retina are considered. Historical descriptions of the involvement of inflammatory cells are provided, evidence implicating inflammation in the pathogenesis of AMD involving macrophages, giant cells and microglia has been derived from observations of human and animal subretinal neovascular lesions. The role of humoral factors such as anti-retinal autoantibodies and acute phase proteins together with clinical observations has been surveyed. Taken together these data demonstrate the involvement of both cellular and humoral immunity in the pathogenesis of AMD. It remains to be determined to what degree the influence of immunity is causative or contributory in both wet and dry AMD, however, the use of anti-inflammatory agents to ameliorate the condition further indicates the existence of an inflammatory component.

Whilst animal studies and a pilot clinical trial suggest that intravitreal triamcinolone acetonide (TA) may be useful in the treatment of age-related macular degeneration (AMD), its mode of action remains to be fully elucidated. The present study has investigated the capacity of TA to modulate the expression of adhesion molecules and permeability using a human epithelial cell line (ECV304) as a model of the outer blood-retinal barrier (BRB). The influence of TA on the expression of ICAM-1 and MHC-I was studied on resting and phorbol myristate acetate (PMA)- or interferon-gamma (IFN-gamma)- and/or tumour necrosis factor-alpha (TNF-alpha)-activated cells using flow cytometry and immunocytochemistry. Additionally, ECV304 cells were grown to confluence in uncoated Transwell chambers; transepithelial resistance (TER) across resting and PMA-activated cells was monitored. TA significantly decreased the paracellular permeability of ECV304 cells and down-regulated ICAM-1 expression, consistent with immunocytochemical observations. PMA-induced permeability changes were dose-dependent and TA decreased permeability of both resting and PMA-activated monolayers. MHC-I expression by ECV304 cells however, was not significantly affected by TA treatment. The modulation of TER and ICAM-1 expression in vitro correlate with clinical observations, suggesting re-establishment of the BRB and down-regulation of inflammatory markers are the principal effects of intravitreal TA in vivo. The results further indicate that TA has the potential to influence cellular permeability, including the barrier function of the retinal pigment epithelium (RPE) in AMD-affected retinae.

Purpose: To determine whether MT1-MMP and MMP-2 are expressed in normal and keratoconic corneas, and to investigate the ability of MT1-MMP, expressed on cultured keratocytes after stimulation with concanavalin A, to activate pro-gelatinase A (pro-MMP 2)., Methods: Specimens of keratoconus corneas (n = 20), removed at corneal transplantation, were obtained from pathology archives, sections were cut, and were stained with an antibody to MT1-MMP, using peroxidase immunohistochemistry. Eye banked corneas served as controls (n = 14). Normal human keratocyte cultures were initiated from eye bank corneas, and after stimulation with con A, MMPs in the media were examined using gelatin zymography and immunoblotting, and MT1-MMP expression was analysed by flow cytometry and immunoblotting., Results: All corneas showed some expression of MT1-MMP and MMP-2, although the degree of staining varied greatly. The MMPs were present in the epithelium, endothelium and stroma. Expression of MT1-MMP, but not MMP-2, in the epithelium and stroma, was significantly elevated in keratoconus, compared to normal corneas. In vitro, keratocytes stimulated with con A expressed MT1-MMP and produced active MMP-2, detected by zymography. These responses to con A were concentration-dependent and MT1-MMP expression and MMP-2 activation correlated significantly (p = 0.0003) In addition, MMP inhibitors abolished MMP-2 activation, providing further evidence that MT1-MMP activated MMP-2., Conclusion: The observation that MT1-MMP expression may be up-regulated in keratoconus corneas, taken together with the demonstration that human corneal cells can express this enzyme, which in turn can activate latent MMP-2, provide evidence for a possible role for MT1-MMP in the pathogenesis of keratoconus.

The present study had investigated the roles of apoptosis and necrosis in the regression of the human fetal hyaloid vasculature. Normal human fetal hyaloid specimens (n = 67) ranging from 10 to 20 weeks' gestation were studied. Specimens were either immunolabeled with anti-von Willebrand factor and major histocompatibility complex class I antibodies or investigated using the terminal-deoxyribonucleotidyl transferase-mediated dUTP-biotin DNA nick-end labeling technique. A fluorescent DNA-binding dye acridine orange/ethidium bromide mixture was also applied to unfixed flat mounts of hyaloid vasculature and some specimens were processed for transmission electron microscopy. Vascular regression including cell loss in the connecting vessels, stretching and thinning of the vasa hyaloidea propria, tunica vasculosa lentis and the pupillary membrane was clearly evident after 13 weeks' gestation. Cresyl violet staining revealed condensed cells and pyknotic bodies throughout the hyaloid system; cell death occurred either in single cells or along small capillary segments associated with vascular regression. Acridine orange/ethidium bromide staining showed DNA condensation at early and late stages of cell death. Similarly, DNA nick-end labeling was positive in endothelial cells, pericytes and vessel and non-vessel associated hyalocytes. The observation of hyalocytes juxtaposed to cytolysed endothelial cells may indicate a role for these cells in vascular regression. Features of apoptosis were more evident during early vascular regression whilst necrosis was increasingly evident at later stages., (Copyright 2000 Academic Press.)

Wholemounts of human fetal retinas were labeled with antibodies to Ki67 or proliferating cell nuclear antigen, to map the distribution of proliferating cells in the developing primary vasculature and neural retina. Double labeling was used to determine the relative proportions of endothelial cells (CD34), astrocytes (glial fibrillary acidic protein - GFAP) and microglia (major histocompatability complex class II) associated with the developing vessels. The differentiated region of neural retina (cold spot) was 3.5 mm(2)at 15 weeks gestation (WG), centred on the incipient fovea, and increased in size with age to 80.5 mm(2)by 23-24 WG. Ki67 immunoreactive cells were distributed throughout the developing vasculature at all ages. The mean density of dividing cells in the neural retina increased with gestational age from 146 mm(-2)at 15 WG, to 624 mm(-2)at 23-24 WG. By 20 WG proliferation in the vasculature overlapped the margins of the cold spot, which was almost completely vascularized by 23-24 WG, except for a narrow strip on the horizontal meridian, which included the incipient fovea. Counts of CD34/Ki67 immunoreactive cells indicated that 15-52% of proliferations in the developing vasculature at 18 WG are endothelial cells. In contrast, in the fellow retina 65-85% cells were Ki67/GFAP immunoreactive, indicating proliferation of astrocytes in situ. No dividing microglia were observed. The findings suggest that large numbers of proliferating astrocytes accompany the developing vessels as they migrate across the primate retina., (Copyright 1999 Academic Press.)

We have investigated the expression of leucocyte markers, phenotypic characteristics and cellular relationships of the normal human fetal hyaloid vasculature using immunohistochemistry, light and electron microscopy. Antibodies against von Willebrand Factor, alpha-smooth muscle actin, glial fibrillary acidic protein, vimentin, major histocompatibility complex classes-I and -II, CD45 (leucocyte-common antigen) and calcitonin gene-related peptide were used to identify the cellular constituents of the hyaloid vasculature in whole mounts. Additional morphological features were described at the ultrastructural level. Endothelial cells throughout the hyaloid system were immunoreactive to von Willebrand Factor and major histocompatibility complex class-I antibodies. Pericytes were immunoreactive to alpha-smooth muscle actin antibody; labeled cells were distributed along large branches of the hyaloid artery, vasa hyaloidea propria, tunica vasculosa lentis and pupillary membrane but no immunoreactivity was detected on small connecting capillaries. Vessel and non-vessel-associated hyalocytes on the hyaloid artery, vasa hyaloidea propria, tunica vasculosa lentis, pupillary membrane and vitreous were immunoreactive to major histocompatibility complex classes-I and -II, CD45 and calcitonin gene-related peptide antibodies. Anti-glial fibrillary acidic protein reactivity was detected on Bergmeister's papilla but not on the hyaloid artery. Cells immunoreactive for vimentin were present throughout the hyaloid vasculature including small connecting capillaries. Ultrastructural observations of the hyaloid vasculature revealed junctional complexes, including zonulae adherens, macula adherens and possible zonulae occludens, between adjacent endothelial cells. Fenestrae were not observed in the gestational ages included in the present study. The use of whole mounts in conjunction with specific antisera has provided novel immunohistochemical definitions of the structure and cellular constituents of the human hyaloid. The results indicate that hyalocytes are a heterogeneous population of leucocyte-lineage cells., (Copyright 1999 Academic Press.)

Sodium butyrate (SB) is a potent biological modifier that can induce diverse effects including growth inhibition, differentiation, or apoptosis of many cell types including retinoblastoma (Rb), and modulation of genes such as c-fos and p53. In this study we assessed the effects of SB on cell growth and expression of p53, critical for cell cycle control, and Bcl-2, an inhibitor of apoptosis, in two human Rb cell lines (Y79 and WERI-Rb1). Attachment cultures were treated with 1 mM SB for up to 5 days and immunocytochemistry was used to examine for the expression of neural cell adhesion molecule (NCAM), p53, and Bcl-2. Suspension cultures of both cell lines were also treated with 1 and 4 mM SB, and at selected times cell extracts were prepared and the expression of p53 and Bcl-2 proteins determined by Western blot analysis. Treatment with 1 mM SB of both cell lines for 5 days inhibited growth and induced morphological changes including extension of neurite-like processes. Up to 12 h after 1 mM SB treatment, p53 and Bcl-2 expressions were similar to control levels, then gradually decreased to very low levels at 5 days. SB (4 mM) also inhibited growth associated with cell death, which was apparent at 24 h posttreatment. Expressions of p53 and Bcl-2 were decreased below control levels at 4 h, and by 24 h only very low levels of protein were detected. SB-induced modulation of p53 and Bcl-2 expression may have implications for controlling Rb growth, particularly in combination with chemotherapy drugs, which are increasingly used in the treatment of Rb.

Purpose: To evaluate the safety and efficacy of intravitreal triamcinolone after 18 months of follow up in patients with age-related macular degeneration and subfoveal or juxtafoveal choroidal neovascularization considered unsuitable for laser photocoagulation., Methods: Thirty eyes of 28 patients, referred from general eye clinics as well as the private clinic of one of the authors to a hospital-based retinal out-patient clinic, were treated with an intravitreal injection of triamcinolone (4 mg). The primary outcome measure was the proportion of eyes with loss of six or more lines on a Bailey-Lovie Chart. The incidence of adverse events associated with treatment was also observed., Results: Of the 20 eyes with initial visual acuity (VA) of 6/60 or better, the vision was maintained (+/-1 Bailey-Lovie lines) in 11 eyes (55%), while six eyes (30%) suffered severe visual loss (six or more lines). The VA improved by five to six lines in three of 10 eyes with initial vision of 3/60 or worse. Three of four eyes receiving a second injection suffered either progressive cataract or elevated intra-ocular pressure (IOP) requiring cataract surgery and/or filtering surgery. One of 26 eyes (3%) receiving a single injection showed progression of cataract and elevation of IOP within 6 weeks of treatment and required anti-glaucoma medication for 6 weeks. Progression of nuclear sclerosis 8-12 months after treatment was observed in six of 26 eyes (23%) receiving a single injection., Conclusions: The results of the present study suggest that a single intravitreal injection of 4 mg triamcinolone is reasonably well tolerated by the human eye. The rate of development of severe visual loss was less than reported for historical controls. Because the results are preliminary and uncontrolled, the treatment should not be used routinely until its benefit to patients is established by a prospective, randomized controlled study.

Chemotherapy alone has largely been unsuccessful in controlling retinoblastoma growth, and has traditionally been limited in use as an alternative to irradiation for the treatment of retinoblastoma. Recently, clinical studies combining chemotherapy with local therapies, including radiotherapy, laser therapy or cryotherapy and in some cases, cyclosporine A, have been effective in treating retinoblastoma. Differentiating agents may also be combined with chemotherapy to enhance the action of cytotoxic drugs on tumor cell growth, although this approach has not been fully investigated in retinoblastoma. In this study, we evaluated the cytotoxic response of human retinoblastoma cell lines (Y79 and WERI-Rb1) to two chemotherapy agents commonly used in treating retinoblastoma, vincristine (VCR) and cisplatin (CDDP). Retinoblastoma cells have been shown to be sensitive to the differentiating agent sodium butyrate, and cell lines were also treated with a combination of VCR or CDDP with sodium butyrate, and the effects on retinoblastoma viability assessed. Both VCR and CDDP induced dose-dependent death of Y79 and WERI-Rb1 cells, accompanied by nuclear and cytoplasmic condensation and DNA laddering, features characteristic of apoptosis. Inhibitors of macromolecular synthesis, cycloheximide and actinomycin-D, significantly reduced VCR- and CDDP-induced apoptosis, although putative endonuclease inhibitors zinc sulphate and aurintricarboxylic acid had no apparent effect. Treatment with 0.5 mM or 1 mM sodium butyrate combined with VCR or CDDP significantly increased induction of apoptosis by these agents. This augmentation of chemotherapy-induced apoptosis may have implications for retinoblastoma therapy.

We have investigated the relationships of the cellular constituents of the retinal vasculature--astrocytes, microglia and pericytes--to the differentiating endothelium in human fetal retina. The vascular endothelium was stained using NADPH-diaphorase histochemistry in 12 human fetal retinae ranging in gestational age from 15-22 weeks. Specimens were double labeled using antibodies against glial fibrillary acid protein, alpha smooth muscle actin, or major histocompatibility complex class II antigens to label astrocytes, contractile cells and microglia, respectively. In addition, specimens of 12, 14, 16 and 20 weeks gestation were hybridized in situ for VEGF expression. In retinal wholemounts the vascularized area comprised four lobes that converged on the optic disc. The vascular network was more dense in the temporal lobes than in the nasal lobes, and different growth patterns were evident. Astrocytes were distributed in two layers--one associated with the optic axons and a deeper layer associated with the developing vessels. In retinae younger than 20 weeks, astrocytes in the deep layer were only loosely associated with the developing vessels and extended as far as 150 microns ahead of the most peripheral vessels. A closer register between retinal vessels and the distribution of astrocytes was evident in the nasal region of retinas older than 20 weeks. In situ hybridization demonstrated expression of VEGF mRNA in the vascular layer, superficial to the ganglion cell layer, at the margins of the vascularized zone. Differences were evident in the density of astrocyte coverage of developing vessels and in the extent of VEGF expression in different regions of the retina: the relationship of these differences to differentiation gradients in the neural retina is discussed. Intensely immunoreactive microglia were observed in the vascular layer, associated with the vascular endothelium as far as the most peripheral loops, but not beyond. Alpha smooth muscle actin-containing cells covered the proximal parts of large arteries, but not corresponding veins; they were absent from arterial side-arm branches, as well as the newly formed and small diameter vessels in the age range studies. The results suggest that microglia, contractile cells and astrocytes have distinct temporo-spatial relationships to the differentiating vascular endothelium in human retinas and that VEGF expression at the vascular front, presumably by astrocytes, is associated with the spread of the retinal vasculature, as described in other species.

Purpose: To investigate antigenic and morphologic features of microglial and vascular elements in the neural retina associated with age-related macular degeneration (ARMD) compared with those features in age-matched normal and young adult retinas., Methods: Adult eyes (n = 97) were classified histopathologically into normal and ARMD-associated groups. Peroxidase imunohistochemical examination of retinal flatmounts was used to visualize major histocompatibility complex class II (MHC-II) immunoreactivity; the intensity and distribution of labeling were quantified by image analysis. In histochemical investigation, reduced nicotinamide-adenine dinucleotide phosphate diaphorase and glial fibrillary acidic protein or MHC-II double labeling were used to detect vascular changes in some preparations., Results: An increase in the proportion of the retina (percentage of total area) expressing MHC-II immunoreactivity was observed in age-matched retinas compared with that seen in normal retinas. A significant increase (P < 0.05) in the percentage of area immunoreactive for MHC-II was observed, primarily on vascular elements, in retinas with incipient ARMD compared with the area affected in the age-matched group. Increased MHC-II immunoreactivity on vessels in the normal-aged group observed with confocal microscopy was associated with irregularities in the organization of astrocytes. Hypertrophy of retinal microglia was also apparent, although the intensity of microglial MHC-II immunoreactivity was not significantly different between groups., Conclusions: The results indicate that an increase in MHC-II immunoreactivity on retinal vascular elements is associated with normal aging. A further increase in MHC-II immunoreactivity on vascular elements and morphologic changes in microglia was associated with incipient ARMD. Immunologic responses in neural retinal microglia and vascular elements appear to be related to early pathogenetic changes in retinal pigment epithelium pigmentation and drusen formation.

Suspension and attachment cultures of Y79 human retinoblastoma cells were treated with all-trans retinoic acid (RA) for up to 10 days to assess its effect on growth and cell-surface expression of immunoglobulin superfamily antigens MHC class I and class II, ICAM-1, NCAM and Thy1. RA up to 10 microM induced growth inhibition, and marked morphological differentiation with extension of prominent processes resembling neurites was seen in attachment cultures. However, above 10 microM RA produced extensive cell death. We also observed increased cell-surface expression of MHC class I, ICAM-1, NCAM and Thy1 on Y79 cells treated with 10 microM over 10 days; constitutive MHC class II expression was not apparent, nor did RA treatment appear to induce Y79 cells to express MHC class immunoreactivity. The up-modulation of cell-adhesion molecules (NCAM, ICAM-1 and Thy1) and immune recognition molecules (NCAM, ICAM-1 and MHC class I), associated with reduced growth and tumour cell differentiation, suggests that RA may have a potential role in regulating the growth and development of retinoblastoma tumours.

Retinoblastoma (Rb), derived from retinal neuroepithelial progenitor cells, is the most common intraocular malignancy of childhood. This study examined 10 human Rb biopsy specimens with light and electron microscopy for histopathological features not previously described in detail, including cell death, leukocytic infiltration, and the tumor vasculature. Rb is a solid well-vascularized tumor with regions of viable tumor cells surrounding vessels, interspersed with zones of necrosis; apoptotic cells were seen in all specimens. Mononuclear phagocyte series (MPS) cells and lymphocytes often colocalized, adjacent to tumor vessels, and MPS cells frequently invested the perivascular space. Lymphocytes were rarely seen within areas of viable tumor. Tumor vessels at early stages of formation resembled normal developing retinal vessels. While junctions were often seen between endothelial calls, disruption of these junctions and endothelial fenestrae was sometimes evident. Müller cells and astrocytes extended processes around tumor cells and blood vessels, and contributed to the formation of the vascular glia limitans, which in some mature vessels was disrupted and discontinuous. Overall, this study provides further morphological details of cell death within Rb, particularly apoptotic involution, and describes the presence of a vascular-associated leukocytic infiltration in Rb. Evidence of compromise of the normal blood-retinal barrier (BRB) within the Rb tumor vessels is presented.

Microglia of the adult human retina are a heterogeneous population of cells, some having characteristics of dendritic antigen presenting cells (DC) and others resembling macrophages, or MPS cells. Studies of the development of microglial distributions in human retina suggest that cells bearing macrophage markers are ontogenetically distinct from microglia that do not. Quantitative studies indicate that macrophage antigen immunoreactive microglia are a subpopulation CD45- and MHC-immunoreactive microglia. While CD45 and MHC-I and -II immunoreactive microglia are seen in the retina prior to the arrival of the vasculature, significant numbers of macrophage-positive microglia only arrive along with the vascular precursors, at about 14 to 15 weeks of gestation. Microglia appear to enter the retina from the ciliary margin prior to vascularization but from both the optic disc and ciliary margin, postvascularization. Macrophage antigen positive microglia enter the retina mainly via the optic nerve head. It is argued that macrophage-antigen positive microglia become established in the retina as vessel associated (perivascular and paravascular) microglia and that the MHC-positive, but macrophage-antigen negative microglia (representing DC), become established as the parenchymal, ramified microglia of adult retina.

The development of microglial topography in wholemounts of human retina has been examined in the age range 10-25 weeks gestation (WG) using histochemistry and immunohistochemistry for CD45 and major histocompatibility complex class II antigens. Microglia were present in three planes corresponding to the developing nerve fibre layer/ganglion cell layer, the inner plexiform layer and the outer plexiform layer. Distribution patterns of cells through the retinal thickness and across the retinal surface area varied with gestational age. Microglia were elongated in superficial retina, large and ramified in the middle plane, and small, rounded and less ramified in deep retina. Intensely labeled, rounded profiles seen at the pars caeca of the ciliary processes, the retinal margin and at the optic disc may represent precursors of some retinal microglia. At 10 WG, the highest densities of microglia were present in middle and deep retina in the far periphery and at the retinal margin, with few superficial microglia evident centrally at the optic disc. At 14 WG, high densities of microglia were apparent superficially at the optic disc; microglia of middle and deep retina were distributed at more central locations although continuing to concentrate in the retinal periphery. Microglia appear to migrate into the developing human retina from two mains sources, the retinal margin and the optic disc, most likely originating from the blood vessels of the ciliary body and iris, and the retinal vasculature, respectively. The data suggest that the development of microglial topography occurs in two phases, an early phase occurring prior to vascularization, and a late phase associated with the development of the retinal vasculature.

We have investigated the ontogeny of MHC class I, class II, CD45, and macrophage antigens in whole mounts of normal human fetal retina at 10-25 weeks gestation (WG) using monoclonal antibodies and immunogold histochemistry. MHC class I antigens were expressed on retinal vascular endothelial cells and provided a useful marker of vessel organization from 14-25 WG. Microglial cells expressed immunoreactivity to MHC class I, class II, and CD45 antigens from 10 WG (pre-vascularization) and macrophage S22 (Mac S22) antigen from 14 WG (post-vascularization), although none of the antigens tested were detected on neuronal or macroglial elements. Microglia expressing MHC, CD45, and macrophage antigens occurred in both ramified and rounded forms with no close correlation being observed between morphology and antigenicity. The numbers of immunoreactive cells labeled with each of the four markers increased steadily throughout gestation in all specimens studied. Equivalent numbers of microglia expressed MHC class I, class II, and CD45 antigens in retinae at similar gestational ages; however, our data indicate that microglia expressing Mac S22 antigen comprise approximately 40% or less of the population of MHC and CD45-immunoreactive cells during development. Topographical analyses suggest that MHC class I, class II, and CD45-positive microglia enter the retina from both the peripheral retinal margin and the optic disc from at least 10 WG; Mac S22-positive cells appear in association with the development of the retinal vasculature and enter the retina via the optic disc after 14 WG.

Purpose: To study the efficacy of the anti-inflammatory agent triamcinolone (Kenacort A-40) in patients with exudative age-related macular degeneration and subfoveal and juxtafoveal choroidal new vessels, considered unsuitable for laser photocoagulation., Method: Thirty eyes of 28 patients were treated with intravitreal injection of triamcinolone. The subsequent visual acuity (VA) of treated eyes was compared with published VA outcomes of untreated eyes. Patients were classified into three types according to their responses to treatment., Results: Within two weeks of receiving treatment, exudation decreased and vision improved in the majority of Types I and II patients (87%), the trend continuing in longer term follow-up. The overall VA outcome for treated eyes was significantly better than published VA data for untreated exudative macular lesions., Conclusions: The preliminary results are encouraging and no serious side effects of a single injection of triamcinolone have been detected in patients followed for up to 18 months. The treatment should, however, continue to be regarded as unproven and only administered in the context of a prospective, case-controlled clinical trial.

The immunoreactivity, morphology and relationship to the glia limitans of microglia were investigated in flatmounts and sections of normal human retina, using immunogold histochemistry, electron microscopy (EM), and antibodies directed against CD45, major histocompatability complex class I (MHC-I), MHC-II, and human macrophage antigens. Immunoreactivity was evident for all antibodies tested, including MHC-I, which labeled both microglia and retinal vascular endothelium. Most consistent labeling was obtained using antibodies to CD45, MHC-II, and anti-human macrophage (S22) antigen. Immunoreactive cells were seen in the perivascular space (perivascular cells), where they were closely adherent to the vessel profile, and in the retinal parenchyma (microglia). Some parenchymal microglia were also vessel associated and by EM were seen to be closely related to the glia limitans (paravascular microglia). Paravascular microglia were shown by optical densitometry, to express higher levels of MHC antigens than neighboring, non-vessel associated, parenchymal microglia. In addition, paravascular microglia were macrophage (S22) antigen positive, while other parenchymal microglia did not express macrophage antigens. Quantitative data indicate that similar populations of microglia are immunoreactive to CD45, MHC-I, and MHC-II, while relatively few microglia (approximately 10%) are immunoreactive for human macrophage (S22) antigens, supporting previous suggestions that microglia are a heterogeneous population.

Purpose: To investigate the distribution and phenotype of major histocompatibility complex (MHC) class II-positive dendritic cells and macrophages in normal human fetal cornea in the age range 10 to 25 weeks gestation., Methods: Peroxidase and gold immunohistochemistry were used to visualize MHC class II and macrophage antigen (S22) immunoreactive cells. Cell distributions were analyzed quantitatively, and topographic maps were produced., Results: Immunoreactive cells, concentrated centrally, were present at 10 weeks gestation in the corneal epithelium and stroma. Average densities increased steadily up to 25 weeks gestation. Two morphologic forms of MHC class II and S22 immunoreactive cells were observed--large, dendritiform cells and small, rounded cells with short processes. Electron microscopy revealed that most MHC class II-positive cells were morphologically consistent with previous ultrastructural descriptions of corneal Langerhans cells. Immunoreactive cells were more numerous in immunogold-labeled specimens than in peroxidase-labeled specimens of similar ages. However, quantitative analysis of both techniques revealed that S22-positive cells comprised 30% to 50% of MHC class II-positive cells., Conclusions: This study provides a detailed description of heterogeneous populations of MHC class II and S22 immunoreactive cells in the human fetal cornea. In contrast to the adult cornea, which is typically devoid of MHC class II-positive cells, immunoreactive cells in the fetal cornea are concentrated centrally and increase in density up to at least 25 weeks gestation. These results indicate that reduction in Langerhans cell numbers to adult levels must occur after 25 weeks gestation. The presence of dendritic cells and macrophages in the fetal cornea has important implications for the understanding of corneal immunology.

The mode of cell death induced in the Y-79 human retinoblastoma cell line by sodium butyrate (SB), a short-chain fatty acid with potent inhibitory effects on the growth of many transformed cell lines, was investigated by fluorescence and transmission electron microscopy, agarose gel electrophoresis, and metabolic studies. While SB (< 1 mM) resulted in marked morphological differentiation, higher concentrations (1-4 mM) induced predominantly apoptotic involution in Y-79 in a concentration-dependent fashion after a latent period of 24 h. Dying cells displayed the characteristic morphology of apoptosis accompanied by DNA laddering with agarose gel electrophoresis. Extensive cell necrosis was apparent with 0.5 M SB. Induction of apoptosis and DNA laddering by SB was reduced by putative inhibitors of RNA and protein synthesis, but not putative endonuclease inhibitors. These results are important for understanding the mode of action of sodium butyrate as a potential cancer chemotherapeutic agent.

The distributions of the hydrolases acid and alkaline phosphatase (AP and ALP), N-acetyl-beta-D-glucosaminidase (NAG), beta-glucuronidase (beta-Gluc), beta-galactosidase (beta-Gal), non-specific esterase (UE), dipeptidylpeptidases II and IV (DPPII and DPPIV), aminopeptidases M and A (APM and APA), and gamma-glutamyltransferase (GGT) were investigated in the human, pig and Lewis rat normal anterior segment by histochemical methods. The distribution of the above hydrolases, particularly that of proteases, varied between ocular tissues and between the three species. Lysosomal hydrolases together with GGT and ALP were consistently active in the corneal epithelium, stroma and endothelium in all three species; the corneal distribution and activity of beta-Gal, APM, APA and DPPIV, however, displayed interspecies variation. The angular tissues showed similarities for most hydrolases with the exceptions of beta-Gal, UE, APM, APA and DPPIV. In all eyes examined strong ciliary epithelial activity for AP, beta-Gal, UE, GGT and ALP was observed in the pars plicata; only the pig eye also displayed strong DPPIV activity in this area. Regional differences in hydrolase distribution in the iris were observed in all species. A post-mortem freezing delay of longer than 24 h resulted in a decrease in hydrolase activity.

This study describes the expression of MHC class II antigens in retinal flat mounts from normal donors and patients with Alzheimer's disease (AD). We confirm previous observations of MHC class II immunoreactivity on microglia in normal retinae, while observing insignificant levels of reactivity on endothelial cells (EC). A significantly increased level of MHC class II expression was detected in AD retinae. This increased immunoreactivity was found to occur in the absence of lymphocytic infiltrates, suggesting that the pathogenesis of AD in the retina may be distinct from that reported to occur in some regions of the brain. MHC class II expression, measured using computerized optical densitometry, appeared to be increased principally as a result of induced MHC class II immunoreactivity on EC. Ramified microglia and perivascular macrophages, although hypertrophied, appeared to show unchanged levels of MHC class II expression. These findings are consistent with earlier suggestions that both aberrant MHC class II expression and suppressor activity of resident macrophages may restrict immune responses.

Purpose: Intermediate filament expression by retinal macroglia was studied in normal eyes, normal eyes of older subjects, and eyes of subjects with age-related macular degeneration (AMD), classified on the basis of histopathologic assessment of the retinal pigment epithelium (RPE), choroid, and donor age., Methods: Adult human retinae (N = 43) were divided into three groups: normal (< 50 years of age, with normal RPE and choroid); normal eyes of older subjects (> 50 years with normal RPE and choroid); and eyes of subjects with AMD (> 50 years with histopathologic findings indicative of AMD), on the basis of histopathologic assessment of the RPE/choroid and donor age. Intermediate filament expression by retinal macroglia was studied in cryostat sections and in retinal flatmounts using avidin-biotin-peroxidase immunolabeling of antibodies to glial fibrillary acidic protein (GFAP) and vimentin. RESULTS. Analyses of immunohistochemically labeled retinal sections revealed similar distributions of vimentin reactivity in retinae of each group. Distributions of GFAP in normal and normal aged retinae were similar, but sections of AMD-affected retinae showed evidence of GFAP expression by Müller cells. In flatmounts, vimentin distribution was similar in the three groups, but GFAP labeling revealed hypertrophic astrocytes, which were absent from normal retinae, in 17% of aged retinae and 55% of AMD-affected retinae. Deeply penetrating, GFAP-positive processes were observed in 17%, 27%, and 55% of normal, normal aged, and AMD-affected retinae, respectively., Conclusions: Variation in GFAP and vimentin expression in retinal macroglia is affected by increasing age, and a distinctive variation of intermediate filament expression in retinal macroglia is associated with the pathogenesis of AMD.

The distributions of the lysosomal enzymes [acid phosphatase (AP), N-acetyl-beta-D-glucosaminidase (NAG), beta-glucuronidase (beta-Gluc), beta-galactosidase (beta-Gal), dipeptidylpeptidase II (DPP II)] and of the membrane-bound proteases [aminopeptidase M (APM), aminopeptidase A (APA), gamma-glutamyltransferase (GGT), dipeptidylpeptidase IV (DPP IV)] were investigated in the normal human adult and foetal anterior segment by histochemical methods. The distribution of these hydrolases varied between ocular tissues. The most active enzymes in the adult corneal epithelium and endothelium were AP, beta-Gluc, NAG, beta-Gal and GGT; in the keratocytes, APM, APA, beta-Gluc and GGT predominated. The adult trabecular meshwork cells were stained by AP, beta-Gluc, NAG, APM, GGT, DPP II and DPP IV. The enzymes AP, beta-Gluc, APM and APA, however, displayed greater activity in the endothelium of Schlemm's canal. The adult ciliary epithelium stained strongly for all lysosomal hydrolases; GGT was the most active protease here. Differences in enzyme activity were noted in some tissues when foetal and adult anterior segments were compared. There appeared to be a decrease in the activity of some enzymes with age and post-mortem delay greater than 24 h. The function(s) of each enzyme and their possible roles in the respective tissues are discussed.

Neural tissue has been considered to be immunologically privileged and major histocompatibility complex (MHC) class II antigens not expressed in normal human brain grey matter and retina. In the present study we compare phenotypic characteristics of human retinal microglia and dendritic Langerhans cells, including their morphologies and distribution, MHC class II and CD45 antigen expression and nucleotidase reactivity. Levels of class II expression were measured using optical densitometry in combination with standard immunohistochemical techniques applied to retinal flatmounts. The results indicate that ramified retinal microglia have features in common with dendritic antigen presenting cells of cornea and conjunctivum, including the constitutive expression of MHC class II antigens.

Alterations in the metabolic functions of trabecular meshwork (TM) cells are thought to be involved in the pathogenesis of primary open-angle glaucoma (POAG). In an investigation of this possibility, 30 trabeculectomy specimens from patients with POAG were examined histochemically for 11 lysosomal and membrane-bound enzymes. The patients ranged from 48 to 87 years in age. The degree of enzyme staining was compared with that of 15 age-matched controls obtained from an eye bank at less than 24 h after death. There was no history of eye disease in the controls. The enzymes examined were: dipeptidylpeptidases II and IV (DPPII and IV); beta-glucuronidase (beta-GLUC); acid-beta-galactosidase (s beta-GAL); N-acetyl-beta-D-glucosaminidase (NAG); nonspecific esterase (UE); acid phosphatase (SP); alkaline phosphatase (ALP); gamma-glutamyltransferase (GGT); and aminopeptidase A and M (APA and APM). Evaluation of the specimens was performed by two observers and by computer-aided optic densitometry. Results showed increased staining of SP, UE, GGT and APM in the pathological specimens as compared with the controls. SP and UE indicate phagocytic activity, APM is involved in collagen turnover and GGT participates in both drug detoxification and the breakdown of glutathione in the gamma-glutamyl cycle. Our observations show different hydrolase activities in the TM cells of human glaucomatous eyes as compared with normal values, suggesting that such metabolic differences may be related to the pathogenesis of POAG.

A number of reports have suggested that oxidative damage in the retina may contribute to the pathogenesis of Age-related macular degeneration (AMD). The present study was designed to investigate the hypothesis that serum levels of the antioxidants, Vitamin E and selenium are related to the pathogenesis of AMD. Fasting bloods were obtained from 80 patients with AMD and 86 controls. Assays for serum levels of Vitamin E, selenium, cholesterol and triglycerides were performed. Assessment of patients and controls was based upon eye examination, fundus photography and medical history. No significant difference was found in serum levels of Vitamin E between subjects and controls, however, there was a borderline association between AMD and both serum selenium levels and current smoking status. The results suggest that if oxidative damage is a factor in the pathogenesis of AMD, it is not reflected in serum levels of Vitamin E; further studies are required to clarify the possible relationship between serum selenium levels, smoking and AMD.

Monoclonal antibodies to human leucocyte antigens, including anti-CD45 and anti-CD68, have been used to describe microglia in flatmounts of normal adult human retina for the first time. Anti-CD45 (the leucocyte common antigen) intensely labeled large numbers of cells in a regular distribution across the retina; anti-CD68 and anti-macrophage antibodies labeled fewer cells with distinctive morphologies, suggesting the presence of subpopulations of microglia in the human retina expressing leucocyte antigens.

Deep stromal vascularization was observed in corneas of four cynomolgus monkeys after extended monocular hydrogel contact lens wear. Keratocytes and extravascular leukocytes, including macrophages, lymphocytes, and in one case, neutrophils, were associated with the vessels in three monkeys. Vessel regression was observed in some animals, but not when lens wear was continuous. In one animal, neutrophil infiltration of the stroma associated with gross epithelial edema was observed. Involution of neutrophils by apoptosis and phagocytosis of pyknotic cells by macrophages was noted. The present study describes the ultrastructural features of deep corneal vascularization developed during extended contact lens wear and illustrates an association between leukocytes and new vessels.

Recent studies have suggested a role for mononuclear phagocytes series (MPS) cells in neovascularisation associated with retinal pathology and experimentally induced subretinal neovascularisation. The present study is concerned with the normal development of the human retinal vasculature. Morphological details are provided of developing vascular structures including the formation of tight junctions and canalisation of angioblast cords. The relationships of astrocytes and pericytes to developing structures and the presence of a perivascular collagenous matrix are described. Ultrastructural and histochemical analyses reveal an association between MPS cells and developing vascular structures. It is suggested that MPS cells may influence angiogenesis in normal retinal development, as well as in retinal pathology.

Sera from 128 patients with age-related macular degeneration (AMD) were examined and profiles of a variety of serum constituents, including immunoglobulins, alpha and beta globulins and autoantibodies, were tabulated. A similar series of tests were carried out on 20 control sera. The results indicate a higher incidence of serum abnormalities, particularly involving alpha-2 globulin, in patients with disturbance of pigmentation of the retinal pigment epithelium (RPE). The sera were further tested for the presence of autoantibodies with specificity for retinal tissue, and five major staining patterns were observed. Many sera produced patterns of labelling on human retina identical to that observed using labelled monoclonal anti-glial fibrillary acid protein (GFAP) antibodies, which are an established marker of retinal astrocytes. Although anti-retinal autoantibodies have been reported in association with a number of ocular pathologies, the observation of anti-astrocyte autoantibodies is new. Astrocytes are involved in the maintenance of the blood-retinal barrier (BRB) and also appear to be the facultative antigen-presenting cells of neural tissue. The present results indicate that the formation of anti-astrocyte autoantibodies may be an early feature of the pathogenesis of AMD.

Alloimmune and normal mouse (C57Bl/10) spleen cells and tumour cells (P815Y) were cultured together in flat-bottomed microtitre plates. Cytolytic activity was assessed by the 51Cr release assay in combination with histological analyses. Cells were fixed and embedded in situ and substrates to reveal ATPase or, alternatively, endogenous peroxidase activities, were applied. Both lymphocytes and monocytes formed junctions with the target cells and estimates of their relative frequencies were produced. Junctions between lymphocytes and tumour cells were completely ablated by treatment with anti-brain-associated theta and complement. Junctions involving immune lymphocytes were typically associated with locally increased ATPase activity in the target cell membrane. Neither monocytes nor normal lymphocytes increased local ATPase activity or 51Cr release to any significant extent.

Apoptosis is a natural form of cell death and has features in common with aspects of cell deletion observed in the course of human retinal development. In this report, we describe the occurrence of apoptotic cells in various layers of the developing retina. Pyknotic residues were observed within phagosomes of neighbouring retinal cells. Our observations imply that most of the debris resulting from cell death is taken up by adjacent tissue cells rather than by mononuclear phagocyte series cells (macrophages) during early stages of human retinal development.

Senile macular degeneration (SMD) is a leading cause of registered blindness in the United States and other Western countries. Loss of central vision develops as a result of atrophy of the retinal pigment epithelium or subretinal neovascularisation. The histopathology of the atrophic form of SMD has not been extensively studied. This paper illustrates at the light and electron microscope level the involvement the atrophic form of SMD. Additional features including pigment clumping and detachment of the retinal pigment epithelium at the advancing edge of the lesion are illustrated. Giant cells and MPS cells are typical features of granulomatous inflammation, and results suggest that they may play a role in the pathogenesis of SMD.