[Localisation of bevacizumab in the monkey retina after an intravitreal injection of Avastin]. / Lokalisation von Bevacizumab in der Netzhaut von Affen nach einer intravitrealen Bevacizumab-Injektion.

BACKGROUND: The aim was to investigate to what extent the vascular endothelial growth factor (VEGF) antibody bevacizumab is able to penetrate the retina in primates after an intravitreal injection of Avastin. MATERIAL AND METHODS: Monkeys (Macaca fascicularis) were injected intravitreally with radioactively labelled and free Avastin. The animals were sacrificed 1, 4, 7, or 14 days after the injection, and the eyes were examined histologically and immunohistochemically. Blood samples were also taken on several days. RESULTS: In the fundoscopic images, no pathologic changes could be found during the experiment. Using immunohistochemistry, bevacizumab was found in the choroid and the inner layers of the retina 1 day after the injection. Bevacizumab penetrated more quickly in the fovea than in the rest of the retina. It was also encountered in the photoreceptors and blood vessels. When (125)I-labelled Avastin was used, radioactivity could be determined in the blood serum 1 day after the injection. CONCLUSION: The results show that the bevacizumab molecule can penetrate the retina after intravitreal injection of Avastin. However, there is an active uptake in the retinal cells.

[Chances and risks of anti-VEGF therapy]. / Chancen und Risiken der Anti-VEGF-Therapie.

Vascular endothelial growth factor (VEGF) plays a pivotal role in angiogenesis. Through regulation of haemodynamics, haematopoesis and the immune system, endocrinology and reparative processes, inhibition of VEGF can cause multiple adverse events. Previous data suggest that--even after intravitreal injection--systemic exposure might occur, thus bearing the risk of manifestation of side effects. Experience with intravenous administration of the antibody bevacizumab (Avastin) pointed to the potential consequences of a pan-VEGF blockade. The change of haemodynamic parameters implies a potential influence on the patient's morbidity. Studies already conducted during the approval process do not provide sufficient statistical power when evaluating whether systemic events significantly differ between the treatment and control groups. Retinal perfusion showed an altered vascular tone (change in vessel diameter) following anti-VEGF treatment. Physiological fenestration of the choroicapillaris is significantly reduced. Possible effects on the local oxygen supply in ischaemic tissue have to be considered. In contrast to destructive treatment modalities (laser, cryo), VEGF inhibitors promise the prompt and efficient response of retinal neovascularisation and the preservation of a better function (visual fields). The maturation of growing vessels (pericytes) and the secondary formation of membranes are limiting factors with regard to the time-point at which anti-VEGF therapy is most effective. A diligent use of the available drugs has to take into account which types of exudative retinopathy are showing no or only very limited response to the treatment.

Immunohistochemical localisation of intravitreally injected bevacizumab at the posterior pole of the primate eye: implication for the treatment of retinal vein occlusion.

AIM: To locate bevacizumab in the posterior pole within 1-14 days after intravitreal injection in the primate eye. METHODS: Four Cynomolgus monkeys received an intravitreal injection of 1.25 mg of bevacizumab. The eyes were enucleated on days 1, 4, 7 and 14 for immunohistochemistry using donkey anti-human Cy3-IgG. Control eyes remained untreated. RESULTS: In the optic nerve, immunoreactivity for bevacizumab was most prominent on day 1 after injection and diminished rapidly. In the blood vessels of the nerve fibre layer, the staining was intense in the walls and weak in the lumen from day 1 to 4, and was only localised in the lumen thereafter. In the macula, an accumulation of bevacizumab was observed 1 day after injection in the nerve fibre layer, the ganglion cell layer and in the photoreceptors at the level of the outer nuclear layer in the fovea centralis. CONCLUSION: Bevacizumab penetrates quickly into the macula, the retinal veins and the optic nerve after intravitreal injection in the primate eye, and accumulates preferentially and specifically on the vessel walls and inside the photoreceptors localised in the fovea centralis 1 day after injection. Our finding supports the clinically observed rapid effect in the treatment of retinal vein occlusion and macular oedema.

Immunohistochemical localisation of intravitreally injected bevacizumab in the anterior chamber angle, iris and ciliary body of the primate eye.

AIMS: To locate bevacizumab in the tissues related to neovascularisation in the anterior segment within 1-14 days after intravitreal injection in the primate eye. METHODS: Four cynomolgus monkeys received an intravitreal injection of 1.25 mg bevacizumab. Control eyes remained untreated. The eyes were enucleated on day 1, 4 and 14 for immunohistochemistry, using donkey anti-human Cy3-IgG. RESULTS: Immunoreactivity for bevacizumab was found in the blood vessels walls of the iris, anterior chamber angle and ciliary body. In the iris and chamber angle, immunoreactivity was most prominent on day 1 after injection and diminished until day 14. In the ciliary body, staining was most intense on day 4 and remained prominent until day 14. Immunoreactivity was also present in certain vessel lumens, especially in the ciliary body and the iris on day 4 and 14. CONCLUSION: Bevacizumab penetrates quickly into the iris, anterior chamber angle and ciliary body after intravitreal injection in the primate eye and accumulates particularly in blood-vessel walls. The highest concentration of bevacizumab in these tissues is present on day 1-4, the iris and anterior chamber angle being penetrated slightly earlier than the ciliary body. Our findings support the clinically observed rapid effect in the treatment of iris neovascularisation.

Transplantation of bone marrow-derived mesenchymal stem cells rescue photoreceptor cells in the dystrophic retina of the rhodopsin knockout mouse.

BACKGROUND: Retinitis pigmentosa belongs to a large group of degenerative diseases of the retina with a hereditary background. It involves loss of retinal photoreceptor cells and consequently peripheral vision. At present there are no satisfactory therapeutic options for this disease. Just recently the use of mesenchymal stem cells has been discussed as one therapeutical option for retinal degeneration, as they have been shown to differentiate into various cell types, including photoreceptor cells. In this article we wanted to investigate the potency of mesenchymal stem cells to induce rescue effects in an animal model for retinitis pigmentosa, the rhodopsin knockout mouse. METHODS: For the experiments, three experimental groups of 10 animals each were formed. The first group consisted of untreated rhodopsin knockout (rho(-/-)) animals used as controls. The second group consisted of rho(-/-) mice that had received an injection of mouse mesenchymal stem cells, which were transduced using an adenoviral vector containing the sequence for the green fluorescent protein (GFP) prior to transplantation. In the third sham group, animals received an injection of medium only. Thirty-five days after transplantation, GFP-expressing cells were detected in whole-mount preparations of the retinas as well as in cryostat sections. For the detection of rescue effects, semi-thin sections of eyes derived from all experimental groups were produced. Furthermore, rescue effects were also analysed ultrastructurally in ultrathin sections. RESULTS: Histological analysis revealed that after transplantation, cells morphologically integrated not only into the retinal pigment epithelium but also into layers of the neuroretina displaying neuronal and glial morphologies. Furthermore, significant rescue effects, as demonstrated by the occurrence of preserved photoreceptor cells, were detected. CONCLUSIONS: Our data indicate that mesenchymal stem cells can prolong photoreceptor survival in the rhodopsin knockout mouse, also providing evidence of a therapeutical benefit in retinitis pigmentosa.

Quantitative chemical analysis of ocular melanosomes in the TEM.

Melanosomes in retinal tissues of a human, monkey and rat were analyzed by EDX in the TEM. Samples were prepared by ultramicrotomy at different thicknesses. The material was mounted on Al grids and samples were analyzed in a Zeiss 912 TEM equipped with an Omega filter and EDX detector with ultrathin window. Melanosomes consist of C and O as main components, mole fractions are about 90 and 3-10 at.%, respectively, and small mole fraction ratios, between 2 and 0.1 at.%, of Na, Mg, K, Si, P, S, Cl, Ca. All elements were measured quantitatively by standardless EDX with high precision. Mole fractions of transition metals Fe, Cu and Zn were also measured. For Fe a mole fraction ratio of less than 0.1at.% was found and gives the melanin its paramagnetic properties. Its mole fraction is however close to or below the minimum detectable mass fraction of the used equipment. Only in the human eye and only in the retinal pigment epitelium (rpe) the mole fractions of Zn (0.1 at.% or 5000 microg/g) and Cu were clearly beyond the minimum detectable mass fraction. In the rat and monkey eye the mole fraction of Zn was at or below the minimum detectable mass fraction and could not be measured quantitatively. The obtained results yielded the chemical composition of the melanosomes in the choroidal tissue and the retinal pigment epitelium (rpe) of the three different species. The results of the chemical analysis are discussed by mole fraction correlation diagrams. Similarities and differences between the different species are outlined. Correlation behavior was found to hold over species, e.g. the Ca-O correlation. It indicates that Ca is bound to oxygen rich sites in the melanin. These are the first quantitative analyses of melanosomes by EDX reported so far. The quantitative chemical analysis should open a deeper understanding of the metabolic processes in the eye that are of central importance for the understanding of a large number of eye-related diseases. The chemical analysis also allows a correlation with structural changes observed at the various regions of the eye.

Iris pigment epithelial cells: a possible cell source for the future treatment of neurodegenerative diseases.

For the treatment of neurodegenerative disorders such as Parkinson's disease cell or gene therapeutical options are increasingly verified. For such approaches, neural stem cells or astrocytes are discussed as possible cell candidates. As also fetal retinal pigment epithelial cells have been successfully tested for such therapeutical options, we investigated the potential of iris pigment epithelial cells as an autologous source for future cell replacement therapies. Using the ELISA technique, we looked for the secretion of neurotrophic factors under basal and stimulated conditions by iris pigment epithelial cells (IPE) cells and compared them with the secretion of retinal pigment epithelial cells (RPE) cells. As iron plays a causative role in cell death during Parkinson's disease, the iron-binding capacity by IPE cells was investigated. Furthermore, we checked the integrative capacity of IPE cells after transplantation into the striatum of adult rats. Our data reveal that IPE cells produce and secrete a variety of neurotrophic factors which can be stimulated after treatment with cytokines. Following transplantation, the cells can be easily detected by their pigmentation, survive for at least 8 weeks and as shown by electron microscopy integrate within the host tissue. Moreover, cells can be transduced with high efficiency using a third generation adenoviral vector, making them promising vehicles to locally deliver therapeutic proteins for the treatment of neurodegenerative diseases in a combined cell and gene therapeutical approach.

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.

Subretinally transplanted embryonic stem cells rescue photoreceptor cells from degeneration in the RCS rats.

The Royal College of Surgeons (RCS) rat is an animal model for retinal degeneration such as the age-related macular degeneration. The RCS rat undergoes a progressive retinal degeneration during the early postnatal period. A potential treatment to prevent this retinal degeneration is the transplantation into the subretinal space of cells that would replace functions of the degenerating retinal pigment epithelium (RPE) cells or may form neurotrophic factors. In this study we have investigated the potential of subretinally transplanted embryonic stem cells to prevent the genetically determined photoreceptor cell degeneration in the RCS rat. Embryonic stem cells from the inner cell mass of the mouse blastocyst were allowed to differentiate to neural precursor cells in vitro and were then transplanted into the subretinal space of 20-day-old RCS rats. Transplanted and sham-operated rats were sacrificed 2 months following cell transplantation. The eyes were enucleated and photoreceptor degeneration was quantified by analyzing and determining the thickness of the outer nuclear layer by light and electron microscopy. In the eyes transplanted with embryonic cells up to 8 rows of photoreceptor cell nuclei were observed, whereas in nontreated control eyes the outer nuclear layer had degenerated completely. Transplantation of embryonic stem cells appears to delay photoreceptor cell degeneration in RCS rats.

[Characteristics and functions of melanin in retinal pigment epithelium]. / Charakteristika und Funktionen des Melanins im retinalen Pigmentepithel.

Melanin granules in the retinal pigment epithelium (RPE) have many important functions which are not yet completely understood. Melanin in the RPE protects the cell from damage caused by oxidative stress. This pigment acts as a free radical sink and diminishes cytotoxic lipid peroxidation. Thus, melanin protects against light toxicity and against cytotoxic effects caused by ocular inflammation. Many enzymes, e.g. superoxide dismutase or carboanhydrase, are only activated in the presence of zinc. Melanin can store zinc and release it when required. The absence of melanin in patients with oculocutaneous albinism is accompanied by photophobia, poor visual acuity and nystagmus. Furthermore melanin is said to protect against damaging lipofuscin accumulation in the RPE. Melanosomes are involved in the lysosomal degradation pathways and possibly take part in the degradation of rod outer segments (ROS) in the RPE.

Transplantation of autologous iris pigment epithelium after removal of choroidal neovascular membranes.

BACKGROUND: Transplantation of autologous iris pigment epithelium (IPE) into the subretinal space has been suggested as one approach for the treatment of age-related macular degeneration, as well as for other conditions in which loss of retinal pigment epithelium (RPE) occurs. Surgical removal of choroidal neovascular membranes is associated with traumatic loss of the RPE cell layer, disruption of the integrity of the photoreceptor-RPE complex, and limited visual outcome. OBJECTIVE: To examine whether IPE cells can substitute for RPE cells to be transplanted to the subretinal space of patients with either RPE degenerative disease or traumatic loss of the RPE cell layer after subretinal surgery. METHODS: Autologous IPE cells were transplanted to the subretinal space in 20 consecutive patients undergoing removal of subretinal fibrovascular membranes using pars plana vitrectomy. Autologous IPE cells were harvested by iridectomy, isolated, and transplanted directly to the subretinal spaces. Transplants were evaluated for 6 to 11 months by funduscopy, fluorescein angiography, and scanning laser ophthalmoscopic (SLO) microperimetry. RESULTS: For the entire follow-up period, no evidence of any immunologic response was observed. Revisional surgery was necessary in 3 patients because of complications (rhegmatogenous retinal detachment [n = 1]; proliferative vitreoretinopathy [n = 1]; and macular pucker [n = 1]); 1 patient did not receive IPE cells. Five of 19 phakic eyes underwent cataract surgery; in 1 case this was combined with the vitrectomy. Five patients showed improved visual acuity of 3 to 4 lines, 13 patients had stable visual acuity (+/-2 lines), and 2 patients had reduced visual acuity of 6 lines. CONCLUSIONS: In this pilot study, the transplantation of autologous IPE cells was done as an addition to conventional surgical excision of choroidal neovascular membranes. Transplanted cells were well tolerated in the subretinal space and did not adversely affect the function of the photoreceptors, since improvement or stable visual acuity was observed in 18 patients after IPE transplantation. These results suggest that autologous IPE cells may be used as a substitute for autologous RPE cells to transplant to the subretinal space to treat age-related macular degeneration.

Multidrug resistance-associated proteins in glaucoma surgery.

BACKGROUND: Multidrug resistance (MDR) describes the phenomenon of cross-resistance between different cytostatic agents which are structurally and functionally dissimilar. Two recently discovered proteins, lung resistance protein (LRP) and the multidrug resistance-related protein (MRP) have been implicated in the development of MDR. Since resistance to chemotherapeutic agents is a common problem in filtration surgery, especially in cases of complicated glaucoma, we decided to investigate the presence of MRP and LRP in surgically removed Tenon specimens from glaucoma patients. METHODS: The presence of MRP and LRP in surgically removed Tenon tissue (n=15) was analyzed by immunohistochemistry. The expression by cultured Tenon fibroblasts was assessed by reverse-transcriptase polymerase chain reaction (RT-PCR) and fluorocytometry. RESULTS: LRP expression was detected in 8 of 10 Tenon specimens. Positive staining for MRP was obtained in 5 of 10 specimens. Negative controls with non-immune mouse IgG did not display any specific staining. RT-PCR and fluorocytometry revealed constitutive expression of MRP and LRP, at the RNA and protein level respectively, that was unaltered by pretreatment of the cells with mitomycin C or 5-fluorouracil. CONCLUSION: Our results demonstrate, that besides P-glycoprotein, other components of the MDR-system are present in conjunctival fibroblasts. Future developments in the use of chemotherapeutic agents in association with of filtration surgery need to take account of the presence of these counteracting mechanisms.

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.

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.

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 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.

Transplantation of autologous iris pigment epithelium to the subretinal space in rabbits.

BACKGROUND: Transplantation of autologous iris pigment epithelium (IPE) into the subretinal space has been suggested as one approach for the treatment of age-related macular degeneration. Autologous rabbit IPE cells were transplanted to the subretinal space to define the technique of transplantation and examine the survival of the transplanted cells. METHODS: Autologous IPE cells were harvested by iridectomy and transplanted directly to the subretinal space of the fellow eye in 25 rabbits, using the parsplana approach. Animals were killed over a period of 5 months, and the retinas were examined morphologically by light and electron microscopy. RESULTS: Autologous IPE cells survived and formed a polarized monolayer above the retinal pigment epithelium in the subretinal space, with apical microvilli adjacent to photoreceptors. Fragments of phagocytosed photoreceptor rod outer segments were observed in phagosomes in the cytoplasm of IPE cells. Adjacent rod outer segments remained healthy throughout the experimental period. No signs of a cell-mediated immunologic response were observed. CONCLUSIONS: Our results show that in rabbits, autologous IPE cells transplanted to the subretinal space survive and do not adversely affect the photoreceptors. These results suggest that in humans, IPE cells might provide a substitute for retinal pigment epithelium cells as autologous transplants for the treatment of age-related macular degeneration.

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.

Current understanding on the role of retinal pigment epithelium and its pigmentation.

Retinal pigment epithelium (RPE) is a monolayer of cuboidal cells that is strategically placed between the rod and cone photoreceptors and the vascular bed of the choriocapillaris. It has many important functions, such as phagocytic uptake and breakdown of the shedded photoreceptor membranes, uptake, processing, transport and release of vitamin A (retinol), setting up the ion gradients within the interphotoreceptor matrix, building up the blood-retina barrier, and providing all transport from blood to the retina and back. This short review focuses on the role of the pigment granules in RPE. Although the biology of the pigment granules has been neglected in the past, they do seem to be involved in many important functions, such as protection from oxidative stress, detoxification of peroxides, and binding of zinc and drugs, and, therefore, serve as a versatile partner of the RPE cell. Melanin plays a role in the development of the fovea and routing of optic nerves. New findings show that the melanin granules are connected to the lysosomal degradation pathway. Most of these functions are not yet understood. Deficit of melanin pigment is associated with age-related macula degeneration, the leading cause of blindness.