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The blood-retinal barrier (BRB) plays an important role in the homeostatic regulation of the microenvironment in the retina. It consists of inner and outer components, the inner BRB (iBRB) being formed by the tight junctions between neighbouring retinal capillary endothelial cells and the outer barrier (oBRB) by tight junctions between retinal pigment epithelial cells. Astrocytes, Müller cells and pericytes contribute to the proper functioning of the iBRB. In many clinically important conditions including diabetic retinopathy, ischaemic central retinal vein occlusion, and some respiratory diseases, retinal hypoxia results in a breakdown of the iBRB. Disruption of the iBRB associated with increased vascular permeability, results in vasogenic oedema and tissue damage, with consequent adverse effects upon vision. Factors such as enhanced production of vascular endothelial growth factor (VEGF), NO, oxidative stress and inflammation underlie the increased permeability of the iBRB and inhibition of these factors is beneficial. Experimental studies in our laboratory have shown melatonin to be a protective agent for the iBRB in hypoxic conditions. Although oBRB breakdown can occur in conditions such as accelerated hypertension and the toxaemia of pregnancy, both of which are associated with choroidal ischaemia and in age-related macular degeneration (ARMD), and is a feature of exudative (serous) retinal detachment, our studies have shown that the oBRB remains intact in hypoxic/ischaemic conditions. Clinically, anti-VEGF therapy has been shown to improve vision in diabetic maculopathy and in neovascular ARMD. The visual benefit in both conditions appears to arise from the restoration of BRB integrity with a reduction of retinal oedema. [ABSTRACT FROM AUTHOR]

Microglia, the resident immune cells of the brain and retina, are constantly engaged in the surveillance of their surrounding neural tissue. During embryonic development they infiltrate the retinal tissues and participate in the phagocytosis of redundant neurons. The contribution of microglia in maintaining the purposeful and functional histo-architecture of the adult retina is indispensable. Within the retinal microenvironment, robust microglial activation is elicited by subtle changes caused by extrinsic and intrinsic factors. When there is a disturbance in the cell-cell communication between microglia and other retinal cells, for example in retinal injury, the activated microglia can manifest actions that can be detrimental. This is evidenced by activated microglia secreting inflammatory mediators that can further aggravate the retinal injury. Microglial activation as a harbinger of a variety of retinal diseases is well documented by many studies. In addition, a change in the microglial phenotype which may be associated with aging, may predispose the retina to age-related diseases. In light of the above, the focus of this review is to highlight the role played by microglia in the healthy and diseased retina, based on findings of our own work and from that of others., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Glutamate that accumulates in injured brain tissue has been shown to hinder the neuroprotection rendered by insulin-like growth factor-1 (IGF-1). However, its role in attenuating the neuroprotective effect of IGF-1 in the hypoxic retina is unknown and the current study was aimed at elucidating this. One-day-old Wistar rats were exposed to hypoxia for 2 h and the retinas were studied at 3 h to 14 days after exposure. Following hypoxia, the concentrations of glutamate and IGF-1 were significantly increased over control values in the immature retina and in cultured retinal ganglion cells (RGCs). In addition to IGF-1, the relative expression of insulin receptor substrate-1 (IRS1) phosphorylated at the tyrosine residue (p-IRS1tyr), phosphorylated protein kinase B (p-AKT) and phosphorylated protein kinase A (p-PKA), which are involved in IGF-1 signalling, was also studied in hypoxic retinas and in cultured RGCs. Glutamate-mediated inhibition of the IGF-1 pathway in hypoxic RGCs was evident with a reduced expression of p-IRS1tyr and p-AKT and an increased expression of p-PKA. However, the addition of exogenous IGF-1 reversed this. Glutamate enables the phosphorylation of IRS1 at the serine residue (p-IRS1ser) through a PKA-dependent pathway. The increased expression of p-IRS1ser and its increased association with IGF-1 receptors in hypoxic RGCs suggested a possible interference by glutamate with the IGF-1 pathway. Moreover, there was increased caspase-3/7 activity in hypoxic RGCs. These results suggest that glutamate, by blocking IGF-1-mediated neuroprotection, could cause the apoptosis of RGCs in the hypoxic neonatal retina.

This study was carried out to investigate the roles of tight junction (TJ) proteins and other factors in the increased permeability of the blood retinal barrier (BRB) affecting the immature neonatal retina following a hypoxic insult. The expression of endothelial TJ proteins such as claudin-5, occludin and zonula occludens-1 (ZO-1) and endothelial cell specific molecule-1 (ESM-1), and associated structural changes in the blood vessels were analyzed in the retinas of 1-day-old Wistar rats subjected to hypoxia for 2 h and subsequently sacrificed at different time points ranging from 3 h to 14 d. The mRNA and protein expression of claudin-5, occludin & ZO-1 was found to be reduced in the hypoxic retina, although, at the ultrastructural level, the TJ between the endothelial cells and retinal pigment epithelial cells appeared to be intact. Following the hypoxic insult vascular endothelial cells frequently showed presence of cytoplasmic vacuoles, vacuolated mitochondria and multivesicular aggregations projecting into the lumen of the capillaries. The expression of ESM-1 in the immature retinas was found to be increased following hypoxic exposure. The structural and molecular changes in the hypoxic neonatal retinas were consistent with a hypoxia induced impairment of the BRB. Hypoxia reduced the expression of TJ proteins in the neonatal retina, but the role of increased ESM-1 expression in this process warrants further investigation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Purpose: Hypoxic insult to the developing retina results in apoptosis of retinal ganglion cells (RGCs) through production of inflammatory mediators, nitric oxide (NO), and free radicals. The present study was aimed at elucidating the pathway through which hypoxia results in overproduction of NO in the immature retina, and its role in causing apoptosis of RGCs., Methods: Wistar rats (1 day old) were exposed to hypoxia and their retinas were studied at 3 hours to 14 days after exposure. The protein expression of nuclear factor-κB (NF-κB) and neuronal nitric oxide synthase (nNOS) in the retina and primary cultures of RGCs was analyzed using Western blotting and double-immunofluorescence, whereas the concentration of NO was determined calorimetrically. In cultured RGCs, hypoxia-induced apoptosis was evaluated by caspase-3 immunolabeling., Results: Following hypoxic exposure, NF-κB-mediated expression of nNOS, which was localized to the RGCs, and subsequent NO production was significantly increased in the developing retina. In primary cultures of RGCs subjected to hypoxia, the upregulation of nNOS and NO was significantly suppressed when treated with 7-nitroindazole (7-NINA), an nNOS inhibitor or BAY, an NF-κB inhibitor. Hypoxia-induced apoptosis of RGCs, which was evident with caspase-3 labeling, also was suppressed when these cells were treated with 7-NINA or BAY., Conclusions: Our results suggest that in RGCs, hypoxic induction of nNOS is mediated by NF-κB and the resulting increased release of NO by RGCs causes their apoptosis through caspase-3 activation. It is speculated that targeting nNOS could be a potential neuroprotective strategy against hypoxia-induced RGCs death in the developing retina., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: To determine whether progressive ametropia can be induced in chicks and reversed by manipulation of the chromaticity of ambient light., Methods: One-day-old chicks were raised in red light (90% red, 10% yellow-green) or in blue light (85% blue, 15% green) with a 12 hour on/off cycle for 14 to 42 days. Refraction was determined by streak retinoscopy, and by automated infrared photoretinoscopy and ocular biometry by A-scan ultrasonography., Results: Red light induced progressive myopia (mean refraction ± SD at 28 days, -2.83 ± 0.25 diopters [D]). Progressive hyperopia was induced by blue light (mean refraction at 28 days, +4.55 ± 0.21 D). The difference in refraction between the groups was highly significant at P < 0.001. Induced myopia or hyperopia was axial as confirmed by ultrasound biometry. Myopia induced by 21 days of red light (-2.21 ± 0.21 D) was reversed to hyperopia (+2.50 ± 0.29 D) by subsequent 21 days of blue light. Hyperopia induced by 21 days of blue light (+4.21 ± 0.19 D) was reversed to myopia (-1.23 ± 0.12 D) by 21 days of red light., Conclusions: Rearing chicks in red light caused progressive myopia, while rearing in blue light caused progressive hyperopia. Light-induced myopia or hyperopia in chicks can be reversed to hyperopia or myopia, respectively, by an alteration in the chromaticity of ambient light. Manipulation of chromaticity may be applicable to the management of human childhood myopia.

Hypoxia-induced glutamate accumulation in neural tissues results in damage to neurons through excitotoxic mechanisms via activation of glutamate receptors (GluRs). Here we examine whether hypoxia in the developing retina would cause activation of the ionotropic α-amino-3-hydroxy-5-methylisoxazole-4-propioate (AMPA) GluRs and increase in Ca(2+) influx into retinal ganglion cells (RGCs) that might ultimately lead to their death. Neonatal Wistar rats were subjected to hypoxia for 2h and then sacrificed at various time points after the exposure together with normal age matched control rats. Primary cultures of RGCs were also prepared and subjected to hypoxia. Expression of AMPA glutamate receptor (GluR) 1-4 was examined in the retina. Additionally, expression of GluRs, intracellular Ca(2+) influx, reactive oxygen species (ROS) generation and cell death were investigated in cultured RGCs. GluR1-4 mRNA and protein expression showed a significant increase (P < 0.01) over control values after the hypoxic exposure both in vivo and in vitro. Cells expressing GluR1-4 in the retina were identified as RGCs by double immunofluorescence labeling with Thy1.1. Increased intracellular Ca(2+) in cultured RGCs following hypoxic exposure was reduced (P < 0.01) by 10 μM AMPA antagonist 6, 7-dinitroquinoxaline-2,3-dione (DNQX). Our results suggest that following a hypoxic insult, an increased amount of glutamate accumulates in the neonatal retina. This would then activate AMPA receptors which may damage RGCs through increased Ca(2+) accumulation and ROS generation. The involvement of AMPA receptors in damaging the RGCs is evidenced by suppression of intracellular Ca(2+) influx by DNQX which also decreased ROS generation and cell death by 50%., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The purpose of this study was to determine whether melatonin treatment would mitigate retinal ganglion cell (RGC) death in the developing retina following a hypoxic insult. Lipid peroxidation (LPO), glutathione (GSH), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) concentrations, expression of vascular endothelial growth factor receptors, Flt-1 and Flk-1, release of cytochrome c from mitochondria, and caspase-3 expression were examined in the retinas of 1-day-old rats at 3 hr to 14 days after a hypoxic exposure. The mRNA and protein expression of Flt-1 and Flk-1 and the tissue concentration of LPO, TNF-α, and IL-1β were upregulated significantly after the hypoxic exposure, whereas the content of GSH was decreased significantly. RGC cultures also showed increased LPO and decreased GSH levels after hypoxic exposure but these effects were reversed in cells treated with melatonin. TNF-α and IL-1β expression was specifically located on microglial cells, whereas Flt-1 and Flk-1 was limited to RGCs as confirmed by double immunofluorescence labeling. Cultures of hypoxic microglial cells treated with melatonin showed a significant reduction in the release of these cytokines as compared to untreated hypoxic cells. Hypoxia induced increase in the cytosolic cytochrome c and caspase-3 in RGCs was attenuated with melatonin treatment. The results suggest that, in hypoxic injuries, melatonin is neuroprotective to RGCs in the developing retina through its antioxidative, anti-inflammatory, and anti-apoptotic effects. Melatonin suppressed Flt-1 and Flk-1 expression in retinal blood vessels, which may result in reduced retinal vascular permeability and it also preserved mitochondrial function as shown by a reduction in cytochrome c leakage into the cytosol. The results may have therapeutic implications for the management of retinopathy of prematurity., (© 2012 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.)

Purpose: To determine the effects on the electroretinogram (ERG) of retinal capillary closure induced in the pig by embolization with microspheres., Methods: Fourteen Yorkshine Landrace pigs of 25- to 45-kg body weight were used. With a customized cannula introduced into the external carotid artery, 10-μm diameter microspheres were delivered to the origin of the vessel that supplies blood to the eye in the pig. Fundus fluorescein angiography and electroretinography were performed between days 7 and 28 post injection. The ERG responses of embolized eyes were compared with those of the contralateral nonembolized eyes., Results: The amplitudes of the scotopic b-wave (P = 0.002), the maximal b-wave (P < 0.010), the photopic a-wave (P < 0.001) and b-wave (P < 0.001), and the scotopic oscillatory potentials (OPs) (P = 0.025) and photopic OPs (P = 0.036) were significantly reduced in embolized eyes. The reduction of these ERG amplitudes was significantly correlated with the number of microspheres in the retina. There was no significant difference in the combined rod-cone bright flash (maximal) ERG a-wave amplitude between eyes with and without microspheres. Implicit times, however, were similar in embolized and control eyes., Conclusions: In eyes embolized with microspheres, the amplitudes of most ERG components were significantly reduced without alteration of their implicit times. The magnitude of ERG amplitude reduction correlated with the number of microspheres in the retina.

It is well established that hypoxia causes excess accumulation of glutamate in developing neural tissues. This study aimed to elucidate the mechanism by which glutamate can cause retinal ganglion cell (RGC) death through the N-methyl-D-aspartate (NMDA) receptors (NR) in the developing retina. One-day-old Wistar rats were exposed to hypoxia for 2 hours and then killed at different time points. Normal age-matched rats were used as controls. NR1, NR2A-D, and NR3A messenger RNA and protein expression showed significant increases over control values, notably at early time points (3 hours to 7 days) after the hypoxic exposure, and immunoexpression of NR1, NR2A-D and NR3A on retinal ganglion cells (RGCs) was enhanced in hypoxic rats and this was confirmed in cultured hypoxic RGCs. Ca(2+) influx in cultured RGCs was increased after hypoxic exposure, and the intracellular Ca(2+) concentration was suppressed by MK-801. Mitochondrial permeability transition pore opening, mitochondrial/cytosolic cytochrome c, and cytosolic caspase-3 expression levels were significantly increased in the hypoxic RGCs. These increases were reversed by MK-801, suggesting that the NMDA receptor subunits in the retina respond rapidly to the hypoxia-induced glutamate overload that leads to the cascade of events that result in RGC death.

Hypoxic injury, including that resulting in the retinopathy of prematurity, may induce retinal ganglion cell (RGC) death in the neonatal retina. We hypothesized that this may be mediated by excess production of tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) by microglia. One-day-old Wistar rats were subjected to hypoxia for 2 h and the expression of TNF-α and IL-1β and their receptors was determined in the retina. The mRNA and protein expression of TNF-α, IL-1β, TNF-receptor 1 (TNF-R(1)), and IL-1 receptor 1 (IL-1R(1)) and the tissue concentration of TNF-α and IL-1β were up-regulated significantly after the hypoxic exposure. TNF-α and IL-1β immunoreactivity was localized in microglial cells, whereas that of TNF-R(1) and IL-1R(1) was restricted to RGCs, as confirmed by double immunofluorescence labelling. Along with this, increased expression of monocyte chemoattractant protein-1 and its receptor CCR2 was detected in the microglia. Primary cultured microglia subjected to hypoxia showed enhanced release of TNF-α and IL-1β. Primary cultured retinal ganglion cells (RGCs) treated with conditioned medium derived from hypoxic microglia showed enhanced apoptosis, which was significantly reduced when the cells were treated with microglia conditioned medium neutralized with TNF-α/IL-1β antibody. Our results suggest that activated microglial cells in hypoxic neonatal retina produce increased amounts of TNF-α and IL-1β that could induce RGC death., (Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Purpose: To investigate the feasibility of creating an animal model of selective retinal capillary closure to mimic the capillary closure that occurs in diabetic retinopathy., Methods: Fluorescent microspheres of 10- or 15-μm diameter were delivered to one eye of anesthetized pigs via a customized cannula advanced through the carotid arterial system to the origin of the external ophthalmic artery that supplies blood to the eye in this species. After preliminary trials in 10 pigs, embolization was performed in one eye of 34 animals that were allowed to survive for 7, 14, or 28 days. Embolized eyes were assessed by fluorescein angiography, electroretinography (ERG), and, after enucleation, light (LM) and electron (EM) microscopy., Results: The microspheres were detectable in the retina immediately after embolization, were restricted to the nerve fiber layer of the retina, and remained thereafter within the retina for periods up to 28 days. They effectively occluded embolized capillaries and some precapillary arterioles. No systemic or cerebral adverse effects were noted, thus allowing survival and subsequent follow-up. Embolization caused a reduction in the b-wave amplitude and the oscillatory potentials of the rod-cone bright-flash ERG but did not affect the amplitude of the a-wave. Embolization induced extracellular and intracellular edema confined to the inner and mid retina, and as a result the retinas of embolized eyes were thicker than those of fellow, nonembolized eyes. The outer retina and RPE were unaffected., Conclusions: This survival model of retinal embolization with microspheres should be useful in the study of the retinal effects of the capillary closure that may occur in diabetic eyes.

Purpose: To assess the feasibility of using two-photon microscopy to study the pattern of diffusion through the sclera of a tracer (tazarotenic acid [TA])., Methods: Polyvinyl alcohol films containing 1% tazarotenic acid (PVA-TA) were applied to the equatorial sclera of isolated perfused bovine eyes. Two-photon microscopy (TPM) was used to determine the lateral spread and depth of penetration of TA in the sclera over time. Protein and collagen binding were determined, and calibration standards were prepared by TPM imaging at 10 μm depth in scleral samples that had been immersed for 24 hours in solutions of TA of 0.7, 7.0, or 70 μg/mL., Results: TA was weakly bound to collagen and sclera (<55%) but strongly bound to plasma protein (95%). In perfused eyes, 50 minutes after PVA-TA application, peak fluorescence in the sclera was detected at a 210-μm depth. By 85 minutes after application of the PVA-TA film, fluorescence had disappeared from surface layers of the sclera and was at maximum at 250 to 290 μm. Penetration of the tracer followed the track of scleral collagen bundles rather than that of the proteoglycan ground substance between collagen bundles., Conclusions: TPM can image in real time the progressive diffusion of TA from its source in a PVA-TA film applied to the equatorial sclera of the isolated perfused bovine eye and follow its subsequent penetration deeper into the sclera. The data suggest that lateral spread and deeper penetration of the test compound occurred along the course of scleral collagen bundles. Imaging was possible to a depth of 340 μm, the average thickness of the human equatorial sclera.

Purpose: To investigate the possible roles of retinal photoreceptors in macular oedema and retinal angiogenesis with particular reference to the mode of action of laser therapy., Methods: (i) Studies in rats made hypoxic for 2 h by administering an oxygen/nitrogen mixture of reduced oxygen content, and growth factors determined by RT-PCR, western blotting, and immunohistochemistry. Assessment of blood-retinal barrier integrity using fluorescent and electron-dense tracers. (ii) Studies in pigs with one retina made hypoxic by selective embolisation of the retinal capillary circulation with fluorescent microspheres. (iii) Assessment of laser therapy in selected cases of retinal neovascularisation indicating a role for photoreceptors., Results: In the hypoxic retina, angiogenic and vascular permeability factors such as vascular endothelial growth factor (VEGF), nitric oxide synthases (NOSs), and insulin-like growth factor-1 are upregulated in retinal astrocytes and Müller cells but are also present in large amount in the photoreceptors. Hypoxia-inducible factor-1 (HIF-1) is upregulated in retinal glial cells but not in the photoreceptors, suggesting that growth factors in the photoreceptors may not have been generated there. The tracer dye, rhodium isothiocyanate, leaking from an abnormally permeable inner blood-retinal barrier in the hypoxic retina accumulates in the photoreceptors., Conclusions: The results indicate that laser treatment of macular oedema or retinal neovascularisation may obtain its effect not only by improving oxygen availability in the inner retina, but also by reducing the load of angiogenic/permeability factors that accumulate in the photoreceptors in hypoxic/ischaemic conditions.

Purpose: This study was undertaken to examine the effects of an acute hypoxic exposure on the retinal cells and production of vascular factors such as vascular endothelial growth factor (VEGF) and nitric oxide (NO), which may affect vascular permeability in the developing retina., Methods: Retinas of 1-day-old rats were examined at 3 hours to 14 days after hypoxic exposure. The mRNA and protein expression of hypoxia-inducible factor-1alpha (HIF-1alpha), VEGF, endothelial nitric oxide synthase (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS) were determined by real-time RT-PCR, Western blot analysis, and immunohistochemistry. Electron microscopy was used to examine the structural alterations in retinal cells, and rhodamine isothiocyanate (RhIC) or horseradish peroxidase (HRP) was administered intraperitoneally or intravenously to determine vascular permeability., Results: The mRNA and protein expression of HIF-1alpha, VEGF, eNOS, nNOS, and iNOS, along with VEGF concentration and NO production, were increased in response to hypoxia. Swollen Müller cell processes, apoptotic and necrotic cells in the inner nuclear layer, and changes in ganglion cells such as swollen and disrupted mitochondria were observed in hypoxic animals. Increased leakage of RhIC and HRP from retinal and hyaloid vessels was seen after hypoxic exposure., Conclusions: The authors suggest that increased VEGF and NO production in hypoxia resulted in increased vascular permeability, leading to changes in Müller cells and degeneration of neural cells. Melatonin administration reduced VEGF and NO production, diminished leakage of RhIC and HRP, and promoted cell proliferation, suggesting this as a potential therapeutic agent in reducing hypoxia-associated damage in the developing retina.

Retinal hypoxia is the potentially blinding mechanism underlying a number of sight-threatening disorders including central retinal artery occlusion, ischemic central retinal vein thrombosis, complications of diabetic eye disease and some types of glaucoma. Hypoxia is implicated in loss of retinal ganglion cells (RGCs) occurring in such conditions. RGC death occurs by apoptosis or necrosis. Hypoxia-ischemia induces the expression of hypoxia inducible factor-1alpha and its target genes such as vascular endothelial growth factor (VEGF) and nitric oxide synthase (NOS). Increased production of VEGF results in disruption of the blood retinal barrier leading to retinal edema. Enhanced expression of NOS results in increased production of nitric oxide which may be toxic to the cells resulting in their death. Excess glutamate release in hypoxic-ischemic conditions causes excitotoxic damage to the RGCs through activation of ionotropic and metabotropic glutamate receptors. Activation of glutamate receptors is thought to initiate damage in the retina by a cascade of biochemical effects such as neuronal NOS activation and increase in intracellular Ca(2+) which has been described as a major contributing factor to RGC loss. Excess production of proinflammatory cytokines also mediates cell damage. Besides the above, free-radicals generated in hypoxic-ischemic conditions result in RGC loss because of an imbalance between antioxidant- and oxidant-generating systems. Although many advances have been made in understanding the mediators and mechanisms of injury, strategies to improve the damage are lacking. Measures to prevent neuronal injury have to be developed.

As the response of the adult retina to hypoxia is likely to differ from that already established in the neonatal animal, this study was undertaken to examine the expression patterns of insulin-like growth factor-I (IGF-I) and -II (IGF-II), angiopoietin-2 (Ang-2), and pigment epithelium-derived growth factor (PEDF) in normal and hypoxic retinas of adult rats. In the latter, the retinas were examined from 3 hr to 14 days after hypoxic exposure. The mRNA and protein expression of IGF-I, IGF-II, Ang-2, and PEDF in the retina was determined by real-time RT-PCR, Western blotting, and immunohistochemistry. The results showed up-regulated expression of IGF-I, IGF-II, and Ang-2 mRNA and protein in response to hypoxia, whereas PEDF expression was drastically reduced, suggesting that increased expression of IGF-I and IGF-II may be involved not only in neovascularization but also in neuroprotection in hypoxic conditions. The up-regulation of Ang-2, a proangiogenic factor, and the down-regulation of PEDF, an antiangiogenic factor, is indicative of an imbalance between pro- and antiangiogenic factors in the hypoxic retina that may favor neovascularization. This was supported by the increased density of rat endothelial cell antigen-1 (RECA-1) protein quantification and RECA-1-stained blood vessels in the inner retina., ((c) 2007 Wiley-Liss, Inc.)

Objective: To investigate the multifocal electroretinogram (mfERG) in myopic children in relation to the rate of myopia progression., Design: Observational study., Participants: Eighty-one school children with myopia., Methods: Cycloplegic refraction, ocular biometry, and mfERG recordings were performed in myopic children aged 9 to 11 years in 2002. The refraction and ocular biometry assessments were repeated 2 years later in 2004. The 2-year myopia progression rate was calculated for a randomly selected eye of each individual. The mfERG parameters recorded at the initial visit in 2002 were compared with subsequent progression rates., Main Outcome Measures: First-order kernel mfERG responses., Results: Of the 81 eyes, 12 eyes had a high progression rate (defined as a progression rate of >1 diopter [D]/2 years), 44 eyes had a moderate progression rate (progression rate of >0.25 D but < or =1 D/2 years), and 25 eyes showed no progression or a low progression rate (progression rate of < or =0.25 D/2 years). The P1 amplitude of the mfERG in the high progression group was significantly smaller than that in the moderate (P = 0.023) and non/low-progression groups (P = 0.030) but only within the central 5 degrees (ring 1). None of the other mfERG parameters of the central ring were significantly different among the groups. The mfERG parameters of the outer rings were similar in all groups., Conclusions: Decreased foveal function as determined by the mfERG is associated with a high rate of myopia progression. Electrophysiologic examination of central retinal function may predict the progression and severity of myopia in school children.

Purpose: The present study was undertaken to examine the involvement of nitric oxide (NO) and excitotoxicity in the development of hypoxia-induced retinopathy in adult rats., Methods: Retinas of adult rats were examined at 3 hours to 14 days after hypoxia. The mRNA and protein expression of endothelial, neuronal, and inducible nitric oxide synthase (eNOS, nNOS, and iNOS, respectively), hypoxia-inducible factor-1alpha (HIF-1alpha), vascular endothelial growth factor (VEGF), N-methyl-D-aspartate receptor subunit 1 (NMDAR1), and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid glutamate (AMPA GluR2 and GluR3) receptors in the retina was determined by real-time RT-PCR, Western blot analysis, and immunohistochemistry. The response of retinal microglial cells to hypoxia was also studied by immunohistochemistry., Results: Hemorrhages were observed in the retina after hypoxia. Upregulated mRNA and protein expression of HIF-1alpha, NMDAR1, GluR2, GluR3, VEGF, eNOS, nNOS, and iNOS in the retina was observed in response to hypoxia. Complement type 3 (CR3) receptors and major histocompatibility complex (MHC) class I and II antigen expression on the microglial cells was increased after exposure to hypoxia., Conclusions: The findings of this study indicate that NO and excitotoxicity may produce damage to retina in response to hypoxia. Increased expressions of eNOS and VEGF in response to hypoxia are indicative of vasodilatation and increased permeability of retinal blood vessels. Increased phagocytosis by retinal microglial cells evidenced by increased expression of CR3 receptors may occur for the removal of hemorrhagic debris. Upregulation of MHC antigens indicates the readiness of these cells to participate in an immune response.

Aims: This study reports the metastatic death rate after trans-scleral local resection of uveal melanoma and identifies relevant risk factors., Methods: Local resection was performed in 332 patients (mean age 51 years), with follow up ranging to 20.9 years (median for living patients 33 months). The tumours had a mean largest basal diameter of 13.1 mm and mean thickness of 7.5 mm, with 135 containing epithelioid cells. Risk factors were identified by Cox analysis and metastatic rates demonstrated using Kaplan-Meier curves., Results: There were 52 deaths from metastatic melanoma. The significant risk factors were (i) age more than 60 years at treatment (p = 0.001), (ii) mixed/epithelioid tumours (p = 0.003), (iii) superior location of mixed/epithelioid tumours (over and above (ii)) (p = 0.001), (iv) largest basal tumour diameter of 16 mm or more (p < 0.001), (v) lack of adjunctive radiotherapy (p = 0.031), (vi) secondary enucleation for bulky residual/recurrent tumour (p = 0.002), and (vii) secondary enucleation for small residual/recurrent tumour extraocularly (p = 0.019). Metastatic death was not significantly associated with (i) incomplete tumour excision (p = 0.163), and (ii) small residual/recurrent tumour treated by enucleation (p = 0.855)., Conclusions: Survival diminished from 92% at 15 years if less than two risk factors were present to less than 30% in 3.5 years if more than three risk factors were present.

Aims: The aims of this study were to report local tumour control after trans-scleral local resection of uveal melanoma and to identify risk factors for (i) clinical residual tumour recognised immediately after surgery, and (ii) delayed tumour recurrence from subclinical microscopic deposits., Methods: The sample included 310 patients, treated by choroidectomy (188), cyclochoroidectomy (87), or iridocyclectomy (35), with follow up ranging from 42 days to 20.9 years (median 36 months), a mean basal largest tumour diameter of 13.2 mm, and a mean tumour thickness of 7.4 mm., Results: There were 24 patients with residual tumour. Forward stepwise logistic regression indicated that posterior extension to within 1 disc diameter of the optic disc or fovea was the sole best indicator of the risk of residual disease (p < 0.001). After excluding these cases, 286 patients were studied for the development of delayed local recurrence, which occurred in 57 cases. Forward stepwise multivariate analysis showed the statistically significant predictors for recurrent tumour to be epithelioid cellularity (p = 0.002), posterior tumour extension to < 1 disc diameter of disc of fovea (p = 0.002), large tumour diameter > or = 16 mm (p = 0.019) and lack of adjunctive plaque radiotherapy (p = 0.018)., Conclusions: The recurrence rate at 4 years varied from 6% if no risk factors were present to 57% if there were more than two risk factors.

Mortality after enucleation for intraocular melanoma is 50% in 10 to 15 years. Death rates after conservative management by local resection, proton beam irradiation, or plaque radiotherapy appear to be similar to post-enucleation mortality in retrospective studies. Risk factors for metastatic death include tumor size, location, cell type, and age of patient and are common to all forms of management including enucleation. The eye and vision can be retained in most cases treated conservatively but choice of conservative management is dependent on patient and tumor characteristics. Any adverse effect on survival posed by conservative management is likely to be slight. Randomized prospective trials that would be needed to establish whether this is true in the case of local resection or proton beam irradiation would be difficult to mount and are unlikely to be conducted.

Local resection of choroidal melanomas is not widely performed so that the indications for this operation have not previously been defined statistically. Univariate and multivariate Cox regression analyses were used to identify the factors influencing visual acuity after 163 completed local resections for choroidal melanoma in patients with a preoperative visual acuity of counting fingers or better. The variables included in the analyses were patient age and sex; eye laterality and preoperative visual acuity; location of anterior and posterior tumour margins; tumour location (coronal and sagittal); tumour diameter, thickness, and cell type; ocular decompression by vitrectomy; and adequacy of surgical clearance. The surgical resections were performed using a lamellar scleral flap for eye closure, hypotensive anaesthesia for haemostasis, and, in the later years, ocular decompression by pars plana vitrectomy to improve access. The patients (94 men, 69 women) had a mean age of 50 years. The tumours had a mean diameter of 13.3 mm and a mean thickness of 7.4 mm, with 38 tumours extending to within 1 disc diameter (DD) of the optic disc, fovea or both (that is, 'posterior tumour extension'). Cox multivariate analysis showed that the most significant preoperative factors for predicting retention of good vision (6/12 or better) were nasal tumour location (p = 0.002) and distance of more than 1 DD between the tumour and the optic disc or fovea (p = 0.010). The most significant predictive risk factor for severe visual loss (hand movements or worse) was posterior tumour extension to within 1 DD of the optic disc and/or fovea (p = 0.009). One year post-operatively, all 28 patients with nasal tumours not extending to within 1 DD of the optic disc or fovea retained the eye with 57% having vision of 6/12 or better and 93% having vision of counting fingers or better. In 68 patients with temporal tumours, 90% retained the eye at 1 year with preservation of vision of counting fingers or better in 82% of 56 eyes without posterior tumours extension and in 50% of 12 eyes with posterior tumour extension. In patients with choroidal melanoma, conservation of the eye and vision can be achieved by local resection, especially if the tumour is located nasally and does not extend close to the disc or fovea.

The histopathological features of uveitis and retinal vasculitis in acute experimental allergic encephalomyelitis (EAE) were investigated using light and electron microscopy. Lewis rats were immunized by spinal cord homogenate, complete Freund's adjuvant and Bordetella pertussis. The eyes of rats with EAE exhibited vasculitis in the iris, trabeculitis and endothelial abnormalities in the retinal vessels; vasculitis was observed in the optic nerve and brain. Endothelial cells in the vessels in the iris, retina, optic nerve and central nervous system were noted to be elevated (high endothelial-like venules, or HELV). Inflammatory cells in the vascular lumen were attached to the surface of endothelial cells in abnormal areas in the iris. By comparison with the findings in the iris and retina, there were no significant changes in the vessels of the ciliary body and choroid. The ultrastructural features indicated that anterior uveitis in acute EAE resulted from vasculitis in the iris due to changes of the endothelial cells and was not due to a reaction against the myelinated nerves or any other particular components of the iris. In addition, our results suggested that vasculitis in the iris was consequent upon specialized changes of the endothelial cells similar to HELV which were responsible for the transcellular emigration of lymphocytes in other inflammatory diseases or in experimental models. HELV change plays an important role in the perivascular inflammatory process in the iris, retina, optic nerve and central nervous system in EAE and possibly in multiple sclerosis.

The heat shock response was examined in cultured human retinal pigment epithelium (RPE) using indirect immunofluorescence. Mild head shock (39.5-40 degrees C for 1 hr) caused no changes in cell morphology and cells continued to produce the intermediate filament proteins, cytokeratin (keratin) and vimentin. In addition, cells subjected to mild heat shock demonstrated the presence of a heat shock protein (HSP-90). After severe heat shock (45.5-46 degrees C for 1 hr) most cells showed marked morphological changes and, in addition, HSP-90 and/or stress-induced 40 kDa protein production was significantly enhanced. The expression of vimentin was relatively well preserved whereas that of keratin was markedly reduced. When the more severe grade of heat shock was preceded by mild heat shock 20-24 hr earlier, the subsequent severe heat shock resulted in less marked morphological change than in cells not preconditioned and, in addition, the expression of both vimentin and keratin was relatively well preserved. Mildly heat shocked cells appeared to gain thermal resistance supporting the theory that the concomitant synthetic capacity for HSP and normal cellular proteins contributes to thermal resistance. In doubly heat shocked cells, however, HSP-90 expression was not enhanced. The discrepancy between the expression of HSP and thermal resistance is discussed.

P1 and P2 components of visual evoked responses (VERs) to flash stimuli were compared between patients diagnosed as suffering from Senile Dementia of the Alzheimer Type (AD) and elderly controls. Additionally the P100 component of the VER to pattern reversal stimuli at high and low contrast and using large and small check patterns was compared. Significant differences between moderate AD patients and the normal elderly were found for the P2 component of the flash stimulus and for the P100 component of the high spatial frequency pattern reversal stimuli at low contrast. These findings suggest a deficit in primary visual processing and a selective deficit in secondary visual processing in moderate cases of dementia. This indicates that visual processing capacities of AD patients decline as the disease progresses in line with the continual degeneration of neuronal populations involved with vision.

Based upon the author's considerable experience of trans-scleral resection of malignant melanoma of the choroid, a technique has been developed for the biopsy of tissues of the posterior segment of the eye. Its use in the management of atypical malignancy posing diagnostic difficulty and in the investigation of selected case of acute retinal necrosis, uveitis and retinal pigment epitheliopathy is described. In 34 trans-scleral biopsies of choroid, RPE and in some cases, retina, an adverse result occurred in only one case, this it was thought being due to not including pars plana vitrectomy as part of the biopsy technique. Pars plana vitrectomy is now regarded as an integral part of this form of biopsy.

Twenty-eight patients from 25 maternal lineages with Leber's hereditary optic neuropathy (LHON) were investigated by restriction enzyme analysis for the presence or absence of the point mutation described by Wallace et al. The mutation was identified in 18 of 25 (72%) families with LHON. This provides further evidence that this mutation is present in the majority of patients with LHON. In 19 of these families with LHON, additional analysis using sequencing, oligonucleotide probing, and competitive oligonucleotide priming of PCR products was performed. In 14 cases with the site loss the point mutation was present, and five without the site loss had the wild type sequence in this region.

A sensitive radioimmunoassay has been developed which allows rapid quantitation of rod outer segment (ROS) phagocytosis by retinal pigment epithelial (RPE) explants in vitro. It involves the use of an antiopsin antiserum, in conjunction with 125I-protein A as a second antibody, and utilizes permeabilization with ethanol to distinguish between the binding and ingestion phases of phagocytosis. This procedure will be used in the future to investigate potential regulatory factors of ROS phagocytosis by retinal pigment epithelium and to evaluate animal models of retinal degeneration.

The visual acuity, the difference in sensitivity of the two eyes to light (brightness ratio), and contrast sensitivity were assessed in 28 patients with chronic open angle glaucoma and compared with those of 41 normal controls of similar ages and visual acuity. The results obtained were related to the results of Tübingen visual field analysis in patients with glaucoma. Twenty-four of the 28 glaucoma patients (86%) had a significant disparity in brightness ratio between the two eyes. This was found to match the frequency of visual field loss. Moreover, there was a significant relationship between the interocular differences in brightness sense and the difference in the degree of visual field loss between the two eyes. Of the glaucoma patients 39% had sum contrast sensitivities outside the normal range for age-matched normal controls. No significant correlation was found between the interocular difference in brightness sense and the visual acuity or the interocular difference in sum contrast sensitivity. It is concluded that, in the presence of a normal visual acuity, the brightness ratio test warrants evaluation as a potential screening test for chronic open angle glaucoma.

Previous studies have shown that bovine retinas incubated with [3H]galactose incorporated it, unmodified, into large molecules. Light and electron microscope autoradiography showed a significant proportion of the label to be in cone inner segments, and pulse-chase studies showed it was subsequently transported to the synaptic pedicles. In this report, evidence is presented to show that the galactose-labelled macromolecules are resistant to hydrolysis by proteolytic enzymes, testicular hyaluronidase, chondroitinase ABC, beta-glucosidase and beta-glucuronidase, but are readily degraded by alpha-amylase and beta-galactosidase, and to a lesser extent by beta-amylase. Treatment with alpha-amylase also leads to specific removal of radioactivity from cone inner segments and pedicles, as judged by light-microscopic autoradiography. These studies appear to indicate that the cone-specific galactose label is in glycogen or glycogen-like molecules.

The production of a 57K stress protein (StrP) after herpes simplex virus type 1 (HSV-1) infection was examined in cultured neonatal rat retinal cells. StrP expression in individual cells was identified using a monoclonal antibody, TI56. Indirect immunofluorescence of uninfected retinal cultures showed that approximately 40% of cells expressed neurofilament (NF+) and 5% expressed a low level of StrP. Following HSV infection the proportion of NF+ cells decreased while the proportion of StrP positive cells became greater and the intensity of staining increased. The number of cells labelled with a polyclonal anti-HSV antibody increased with time after infection. Retinal neurons in culture can be infected with HSV, after which StrP expression is significantly upregulated.

Rod outer segment (ROS) phagocytosis by explanted bovine retinal pigment epithelium (RPE) was evaluated by a procedure using an indirect double radioimmunoassay which distinguished between ROS attached to the RPE cell surface and those which had been ingested. This approach has been used to investigate the effect of a variety of glycoconjugates on the phagocytic process. Inclusion of the glycosaminoglycans (GAGs) chondroitin sulphate type-A (CS-A) and type-C (CS-C), hyaluronic acid (HA) or dermatan sulphate (DS) in the incubation medium significantly inhibited the ingestion phase of ROS phagocytosis, whereas the binding phase was inhibited to a lesser extent. The interphotoreceptor matrix (IPM), containing these GAGs as part of proteoglycans, also had an inhibitory effect on phagocytosis. The free monosaccharides mannose, fucose and galactose all stimulated the ingestion of ROS by RPE cells. These findings support the suggestion that glycoconjugates may have a physiological role in the photoreceptor renewal process.

Choroidal tumours are associated with several degenerative changes in the overlying tissues, which can be called 'Tumour-Associated Retinal Pigment Epitheliopathy (TARPE)'. These changes include (i) proliferation, detachment, atrophy, and metaplasia of the retinal pigment epithelium, (ii) the accumulation of hard and soft drusen and basal laminar deposits in Bruch's membrane, (iii) disorganisation of the choriocapillaris, (iv) atrophy, cystic degeneration and detachment of the retina. The changes at the chorio-retinal interface are clinically relevant because they can exacerbate visual loss. In addition, they can be misinterpreted on ophthalmoscopy and fluorescein angiography. An amelanotic choroidal tumour may appear to be pigmented on ophthalmoscopy because of lipofuscin and melanin accumulation overlying the tumour. The hyperfluorescence associated with pigmented choroidal melanomas is more likely to be related to degenerative changes in the retinal pigment epithelium than to dye leakage from abnormal tumour vessels.

The retinal and neurological complications of abetalipoproteinaemia may be preventable by replacing vitamins A and E from an early age, but their role in adult presentations is less clear. Two adult females with abetalipoproteinaemia have received 8 and 10 years respectively of replacement therapy with vitamins A, E and linoleic acid. In Case 1, visual function improved objectively on commencing therapy but has subsequently deteriorated and her neuropathy has slowly progressed. The rate of progression of neurological impairment in Case 2 was slowed but not halted by therapy, and her severe visual disturbance was unaffected. Replacement by fat soluble vitamins has only a limited role in the management of abetalipoproteinaemia once irreversible neurological/retinal damage has occurred.

Blood rheology and haemostasis were assessed in 18 diabetics with proliferative retinopathy and in 18 diabetics without proliferative retinopathy, matched for age, sex, smoking habit and type, duration and treatment of diabetes. Proliferative retinopathy was associated with significantly higher levels of blood viscosity at high and low shear rates, which were related to higher levels of plasma viscosity and fibrinogen. Blood urea, glucose, glycosylated haemoglobin and white cell count were also significantly higher, whereas haematocrit, red cell deformability and several other haematological and biochemical variables did not differ significantly in the 2 groups. In view of these findings, and of our recent demonstration that increased blood viscosity also exists in those patients with retinal vein occlusion who develop a similar proliferative retinopathy, we suggest that hyperviscosity may contribute to retinal ischaemia and hence proliferative retinopathy.

Eighty-three patients with retinal pigment epitheliopathy have been identified from the photographic and fluorescein records of the Tennent Institute of Ophthalmology in Glasgow. Many have been brought back for follow-up assessment. Twenty-seven patients had placoid lesions, 22 geographic lesions and 15 a condition which we propose to rename the 'not ocular histoplasmosis' (NOH) syndrome. Patients with placoid lesions could be divided into typical acute multifocal placoid pigment epitheliopathy (AMPPE), where the long-term visual prognosis was poor because of recurrent disease leading to extensive areas of atrophy, and acute diffuse placoid pigment epitheliopathy which seemed to be self-limiting. No consistent evidence of viral or immunological abnormality was found in either group of patients. Geographic lesions appeared to result either from a coalescence of focal lesions or from a slow (serpiginous) spread from a single focus. Four patients with helicoid atrophy lost central vision from disciform lesions. Seven patients had pigment epithelial disorder associated with sarcoidosis. The pigment epithelial disease was unresponsive to systemic corticosteroid therapy and tended to be complicated by disciform lesions. Disciform lesions may occur in any type of pigment epitheliopathy, as may serous retinal detachment or pigment epithelial detachment. A technique for the biopsy of choroid and retinal pigment epithelium is described. This may lead to a better understanding of these disorders.

The production of stress- or heat-shock proteins (SP) which are defined by three monoclonal antibodies (TI56, TG5E and TG7A) were examined in cultured retinal glial cells with and without herpes simplex virus (HSV) infection. Indirect immunofluorescence showed that 80-90% of uninfected cells reacted with anti-glial fibrillary acidic protein (GFAP) and that 10-20% of uninfected cells were weakly labelled with anti-SP antibodies. By 6 hr after HSV infection, the proportion of GFAP labelled cells decreased to 60-70% whereas cells strongly expressing SP antigens were demonstrated. At 24 hr, GFAP+ cells were markedly reduced in number and immunolabelling with anti-SP antibodies was evident in approximately 50% of cells, directly demonstrating the accumulation of SP in cultured retinal cells after HSV infection. Double labelling with GFAP/TI56 indicated that 30% of GFAP+ cells were labelled with TI56 and 30-50% of TI56+ cells were also GFAP+, despite the abrupt loss of GFAP+ cells during HSV infection. These results indicate that SP normally expressed at low level are significantly upregulated in retinal glial cells following HSV infection.