Retinal microglia - A key player in healthy and diseased retina.

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.

Glutamate Inhibits the Pro-Survival Effects of Insulin-Like Growth Factor-1 on Retinal Ganglion Cells in Hypoxic Neonatal Rat Retina.

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.

Vascular changes in the developing rat retina in response to hypoxia.

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.

NF-κB-mediated nitric oxide production and activation of caspase-3 cause retinal ganglion cell death in the hypoxic neonatal retina.

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.

Progressive myopia or hyperopia can be induced in chicks and reversed by manipulation of the chromaticity of ambient light.

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.

Neuroprotective effect of melatonin against hypoxia-induced retinal ganglion cell death in neonatal rats.

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-1ß (IL-1ß) 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-1ß 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-1ß 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.

Hypoxia-induced activation of N-methyl-D-aspartate receptors causes retinal ganglion cell death in the neonatal 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.

Electrophysiological findings in a porcine model of selective retinal capillary closure.

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.

Retinal ganglion cell death is induced by microglia derived pro-inflammatory cytokines in the hypoxic neonatal retina.

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-1ß (IL-1ß) by microglia. One-day-old Wistar rats were subjected to hypoxia for 2 h and the expression of TNF-α and IL-1ß and their receptors was determined in the retina. The mRNA and protein expression of TNF-α, IL-1ß, TNF-receptor 1 (TNF-R(1)), and IL-1 receptor 1 (IL-1R(1)) and the tissue concentration of TNF-α and IL-1ß were up-regulated significantly after the hypoxic exposure. TNF-α and IL-1ß 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-1ß. 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-1ß antibody. Our results suggest that activated microglial cells in hypoxic neonatal retina produce increased amounts of TNF-α and IL-1ß that could induce RGC death.

A porcine model of selective retinal capillary closure induced by embolization with fluorescent microspheres.

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.

Two-photon fluorescence excitation microscopy to assess transscleral diffusional pathways in an isolated perfused bovine eye model.

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.

Cellular and vascular changes in the retina of neonatal rats after an acute exposure to hypoxia.

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.

Hypoxia-ischemia and retinal ganglion cell damage.

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.

Features of the multifocal electroretinogram may predict the rate of myopia progression in children.

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.

Early response of neurons and glial cells to hypoxia in the retina.

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.