Oxidation causes melanin fluorescence.

PURPOSE: The goal of this study is the characterization of the strong yellow fluorescence of oxidized melanin in the retinal pigment epithelium (RPE) and the choroid. METHODS: Naturally occurring melanin in the human retina and choroid was oxidized by exposing fixed and plastic-embedded sections of a human eye to light and hydrogen peroxide. Synthetic melanin was also oxidized in vitro by exposure to light and hydrogen peroxide. The fluorescence of oxidized melanin was examined by absorption spectroscopy, fluorescence spectroscopy, and fluorescence microscopy. RESULTS: Naturally occurring melanin oxidized in situ exhibited a lipofuscin-like yellow fluorescence. Oxidation of melanin in vitro degraded the melanin polymer, resulting in a fluorescent solution. Fluorescence spectroscopy gave an excitation maximum at approximately 470 nm and an emission maximum at approximately 540 nm for both natural and synthetic melanin. Increasing the time of exposure to light or hydrogen peroxide increased melanin fluorescence. CONCLUSIONS: The results indicate that the strong yellow fluorescence of melanin in the RPE and choroid in situ is a property of oxidized melanin and is not due to contamination of the melanin by proteinaceous or lipid materials. The data presented allow a reinterpretation of the results obtained from fluorescence investigations of melanin-containing tissue and suggest a link between melanin degradation and lipofuscin formation.

Subretinal injection of rod outer segments leads to an increase in the number of early-stage melanosomes in retinal pigment epithelial cells.

Our study was performed to test the hypothesis that subretinally injected protein can induce melanogenesis in the retinal pigment epithelium (RPE). Rod outer segments (ROS) were isolated from cattle eyes and injected into the subretinal space of Long Evans rats. Five days after surgery, the injected eyes were investigated by electron microscopy. The number of early-stage melanosomes and small melanin granules was compared in injected and noninjected eyes. It was found that the injected ROS were phagocytized by the RPE cells, and the number of early-stage melanosomes in the RPE was significantly increased in injected eyes compared to eyes without injection. The ROS-containing endosomes fused with melanolysosomes in which melanogenesis took place. The increased number of early-stage melanosomes indicates new formation of melanin.

Transplantation of iris pigment epithelium into the choroid slows down the degeneration of photoreceptors in the RCS rat.

BACKGROUND: Trophic factors [e.g. basic fibroblast growth factor (bFGF)] released by transplanted retinal pigment epithelial (RPE) cells are able to slow down the hereditary degeneration of the retina in the Royal College of Surgeons rat in sites distant from the site of transplantation where rod outer segment (ROS) phagocytic activity is not reconstituted by the transplants. METHODS: To investigate whether iris pigmented epithelial (IPE) cells are also able to generate this rescue by trophic factors, we transplanted IPE cells from Long-Evans rats into the choroid and subretinal space of 17 young RCS rats. The eyes were enucleated after 6 months and prepared for light microscopy. Six age-matched RCS rats served as controls. Light microscope sections from the whole choroid, healthy choriocapillaris, transplanted cells and the maximum thickness of the choroid, and outer nuclear layer parameters were analyzed by computer-assisted morphometry. RESULTS: In transplanted animals photoreceptor cells were rescued from degeneration although the majority of the transplanted IPE cells were located in the choroid. In the non-transplanted group photoreceptors were absent. CONCLUSIONS: Transplantation of IPE cells slows down degeneration of the photoreceptors in the RCS rat. This photoreceptor-sparing effect by the IPE cells was observed even when the transplants were predominantly located within the choroid. The beneficial effect observed may be related to trophic factors possibly secreted by the transplanted IPE cells.

Comparative study of ROS degradation by IPE and RPE cells in vitro.

BACKGROUND: The aim of this study was to compare the degradation of rod outer segments (ROS) in porcine iris pigment epithelial cells (IPE) and retinal pigment epithelial (RPE) cells by measuring the increase of lipofuscin-like fluorescence. METHODS: We measured the development of autofluorescence of lipofuscin-like material in living cells over a period of 4 weeks using an image-analyzing system comprising a light microscope, a filter set with an appropriate wavelength for the detection of lipofuscin-like autofluorescence and a silicon-intensified target camera connected to a computer. The lipofuscin-like fluorescence was quantified as the mean gray value of pixels over a defined area in the cell. In addition, ultrastructural examination of the cells was performed using transmission electron microscopy. RESULTS: We found that while both cell types had increased autofluorescence over time, the increase of lipofuscin-like fluorescence was significantly higher in IPE cells than in RPE cells. The ultrastructure of both cell types was similar and no accumulation of lipofuscin-like granules was observed. CONCLUSION: These findings suggest that although IPE cells are able to phagocytize ROS, their ability to degrade them may be lower than in RPE cells. The increase of lipofuscin-like fluorescence is not due to the accumulation of lipofuscin-like granules.

Ultrastructural localization of light-induced lipid peroxides in the rat retina.

PURPOSE: Localization of light-induced lipid peroxides in the rat retina at an ultrastructural level as benzidine-reactive substances. METHODS: Long-Evans rats with nondilated pupils were exposed to intense light of 6000 lux for 12 or 24 hours. Control animals were kept under physiological light conditions. Rats with dilated pupils were exposed to a light intensity of 50 lux or 150,000 lux for 1 hour. For ultrastructural localization the enucleated eyes were fixed in a 0.1-M cacodylate buffer (pH 7.4) containing 2% glutaraldehyde for 2 hours. Pieces of the superior part of the central eyecup were incubated overnight with tetramethylbenzidine (TMB; pH 3.0) at 4 degrees C, postfixed with 1.5% OSO4, and embedded for electron microscopy. RESULTS: In animals exposed to intense light, electron-dense structures appeared exclusively throughout the rod outer segments after an irradiation of 6000 lux for 24 hours or 150,000 lux for 1 hour and were absent in animals with nondilated pupils kept at physiological light conditions. Dilation of the pupils leads to the appearance of electron-dense structures after just 1 hour of 50 lux, whereas rats with nondilated pupils withstand even a 12-hour irradiation with 6000 lux. No electron-dense structures were found when no TMB was used in incubation. CONCLUSIONS: The appearance of electron-dense structures in the rod outer segments depends on the incubation with TMB and intensive light exposure of the rat. Dilation of the pupils lowers the threshold for the emergence of electron-dense structures significantly. This strongly supports the view that light-induced lipid peroxides in the rat retina are localized at an ultrastructural level as benzidine-reactive substances. This protocol presents a tool for the generation and ultrastructural localization of lipid peroxides in rat retinas.

Tracing of benzidine-reactive substances in ROS, RPE and choroid after light-induced peroxidation.

BACKGROUND: A new method for the ultrastructural localization of lipid peroxides as benzidine-reactive substances (BRS) was recently developed in our laboratory. The aim of the present study was to localize BRS in the eye after intense light exposure. The light protocol was chosen to either hamper disc shedding or to induce a shedding peak. METHODS: Long-Evans rats were either kept under constant irradiation to enhance lipid peroxidation or under physiological light conditions. The light-induced peroxidation was carried out either by constant irradiation for 24 h or by constant irradiation for 20 h followed by a dark period of 4 h. The eye cups were fixed by glutaraldehyde, incubated with or without tetramethylbenzidine and embedded for electron microscopy. RESULTS: After constant irradiation for 24 h smooth nonlamellar BRS appear exclusively intracellularly over the complete rod outer segments (ROS). After the initiation of disc shedding smooth BRS are localized in the extracellular space of the ROS and extracellularly in the basal labyrinth of the retinal pigment epithelium (RPE). Fine-lamellar BRS emerge in vacuoles of the RPE, in the basal labyrinth and in the lumen of choroidal capillaries. CONCLUSION: Light conditions that trigger the disc shedding possibly activate a mechanism to extrude peroxidative damaged material over the complete ROS into the extracellular space to diminish peroxidative damage inside the ROS. Indigestible residual material from the ROS degradation in the phagosomes consists of membranous lipids associated with peroxidative damaged proteins. The residual material seems to be transported through Bruch's membrane into the choriocapillaris.

Ultrastructural localization of lipid peroxides as benzidine-reactive substances in the albino mouse eye.

BACKGROUND: Lipid peroxidation is considered to be a prominent feature of retinal degeneration and has also been proposed to be involved in the pathogenesis of age-related macular degeneration. Melanin protects against lipid peroxidation and takes part in the detoxification of lipid peroxides (LP). LP can be ultrastructurally detected as benzidine-reactive substances (BRS) using tetramethylbenzidine (TMB). Albino mice lack melanin. In the present study, LP were localized as BRS in the eyes of albino and pigmented mice. METHODS: Eye cups of an albino mouse lineage and of wild-type mice were fixed with 2% glutaraldehyde, incubated with 0.5 mg/ml TMB and embedded for electron microscopy. RESULTS: BRS were detected in the eyes of albino mice, but no reaction product was seen in pigmented eyes. BRS located in the retinal pigment epithelium (RPE) and in the choroid of the albino mouse; no BRS were found in intact rod outer segments (ROS). CONCLUSION: The lack of melanin in albino mice is associated with a higher level of lipid peroxidation in RPE and choroid. Melanin seems to protect against LP in RPE and choroid. A lack of melanin is not associated with lipid peroxidation in intact ROS. The present investigation demonstrates the significance of melanin in protection against LP in RPE and choroid.

Retinal damage by light in the golden hamster: an ultrastructural study in the retinal pigment epithelium and Bruch's membrane.

The mechanism of the toxicity of light on the retina remains unclear despite a large number of investigations. The purpose of this study is to identify and localize the ultrastructural changes and the site of the earliest damage after intense light exposure. Nine adult Syrian golden hamsters (Mesocricetus auratus) have been maintained under constant illumination with a high-pressure mercury lamp (HQJ R 80 W Deluxe, Osram, Berlin, light intensity 1000 lx) for 12 h, followed by an additional 3 h in the dark. Light damage is assessed by light and electron microscopy. Morphological evaluation reveals focal damage to the retinal pigment epithelial (RPE) cells in close proximity to less-affected RPE cells and normal photoreceptors. Collagen fibers in Bruch's membrane lose their parallel orientation. Occasionally, fusion of cell membranes of neighboring rod outer segments (ROS) is also observed. Continuous, 12 h exposure of hamsters to intense light results in initial focal damage to some RPE cells, such that severely damaged RPE cells are found adjacent to intact RPE cells. Only slight damage to the photoreceptors is evident, suggesting that the sequence of the pathological changes resulting from light begins with damage to the RPE cells and associated Bruch's membrane.

Cellular transport of subretinal material into choroidal and scleral blood vessels: an electron microscopic study.

BACKGROUND: The fate of indigestible material injected into the subretinal space of rats was investigated. METHODS: The non-toxic dye Monastral Blue (MB), which cannot be digested within the lysosomal compartment, was injected transsclerally into the subretinal space of Long Evans and Wistar rats. After 5 and 12 days respectively the eyes were enucleated and examined by light and electron microscopy. Cryo sections were made of eyes 5 days after MB injection for the application of immunohistochemical techniques using markers for epithelial cells (cytokeratin) and macrophages (ED 1). RESULTS: Retina, choroid and sclera were not altered in their morphology in the circumference of the MB-containing bubble generated by subretinal injection. After both 5 and 12 days no injected material was found extracellularly in the subretinal space. Especially high amounts of MB were found, in particular 5 days after injection, in lysosomes and melanosomes of RPE cells as well as in cells between choroidal melanocytes. Cells containing MB were seen in contact with choroidal and scleral blood vessels. These MB-containing cells in the choroid and in the sclera were positive for macrophage antibodies. CONCLUSION: Subretinal injection was confirmed as a suitable method for placing fluids into the subretinal space without affecting the morphology of the retina. Subretinal injected material was shown to be incorporated into lysosomes and melanosomes of RPE cells. The injected material was subsequently transported through Bruch's membrane to be finally removed from the eye via choroidal and scleral veins, the process involving macrophages.

[Ultrastructural localization of lipid peroxides in the eye. Presentation of a new method]. / Ultrastrukturelle Lokalisation von Lipidperoxiden im Auge. Vorstellung einer neuen Methode.

UNLABELLED: Lipid peroxidation is considered a prominent feature of age-related retinal degeneration. It is known that lipid peroxides can oxidize benzidine. This property was used to localize lipid peroxides ultrastructurally in the retina. METHODS: (1) Lipid peroxides were formed by incubation of linoleic acid with lipoxygenase from soybean, separated by thin layer chromatography and incubated with tetramethylbenzidine. (2) Lipid peroxides were formed by incubation of porcine retinae with soybean lipoxygenase in an oxygensaturated atmosphere. For ultrastructural localization, isolated retinae with and without enzymatically synthesized lipid peroxides were fixed with 2% glutaraldehyde, incubated with 0.5 mg/ml tetramethylbenzidine and embedded for electron microscopy. (3) Eye cups from Syrian golden hamsters were treated in the same way except for incubation with lipoxygenase. The hamsters were kept under constant illumination (1000 lux) for 12 h to enhance lipid peroxidation. RESULTS: (1) Tetramethylbenzidine was oxidized by linoleic acid peroxides. (2) In the isolated retinae of pigs lipid peroxides became visible as electron-dense structures in the rod outer segments (ROS) after treatment with lipoxygenase and were lacking in the other parts of the retina. Without treatment with lipoxygenase lipid peroxides were only infrequently seen in ROS. (3) In the eyes of light-exposed hamsters, electron-dense reaction products of lipid peroxides were particularly prominent between the basal infoldings of the RPE and within the apical parts of the ROS. CONCLUSION: Light or enzymatically induced lipid peroxides can be localized ultrastructurally due to their ability to react with tetramethylbenzidine and osmium in the absence of H2O2 to an electron-dense reaction product. Lipid peroxides seem to be removed from the RPE via Bruch's membrane and blood vessels. Disturbance of this pathway may enhance lipofuscin or drusen formation.