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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.

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.

To analyze the elemental composition and topology of the extracellular compartments of the compound eye, the eyes of blowflies Calliphora vicina were rapidly frozen and ultrathin cryosections were freeze dried. Three zones of an ommatidium, peripheral cytosol of visual cells, rhabdomeres, and ommatidial cavities were analyzed by X-ray microprobe analysis. The ommatidial cavity was found to contain sodium and potassium in proportion similar to that in the blowfly hemolymph. Potassium-to-sodium ratio in a cytosol was typical for a cytosol. The rhabdomeres displayed an electrolyte content intermediate between the above compartments. Three topologically connected extracellular compartments were characterized by the experiments with tracers, monastral blue and lanthanum: (1) common intercellular space of ommatidia including peripheral clefts between the visual cells, both tracers entered this compartment; (2) the ommatidial cavity, which is not accessible for monastral blue, however, as revealed by our X-ray microanalysis, it was reachable for lanthanum; (3) rhabdomeric loops, which were accessible for lanthanum entering either via the cavity or from the common intercellular clefts. The above characteristics of the ionic content and topology of ommatidial compartments might suggest higher sodium and lower potassium content in the microvilli as compared with the cytosol. The rhabdomeric and “cavital” plasma membranes are assumed to be permeable for these ions so that a voltage of only 25–30 mV, negative inside, is probably formed across them, much lower than the known resting potential −60 mV across the peripheral plasma membrane of a visual cell.

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.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.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.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.

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.

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.

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.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.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.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.

Localization of light-induced lipid peroxides in the rat retina at an ultrastructural level as benzidine-reactive substances.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.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.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.

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.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.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.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.

The aim of this study was to investigate the possible rescue effect of subretinal iris pigment epithelial (IPE) cell transplantation in Royal College of Surgeons (RCS) rats by light and electron microscopic histology.IPE cells were harvested from 20- to 26-day-old Long-Evans rats and were directly trans planted transsclerally into the subretinal space of 32 16- to 20-day-old RCS rats using a 32-gauge Hamilton syringe. Specimens of transplanted eyes were embedded for electron microscopy after 8 weeks. Specimens from the iris and retinal pigment epithelium (RPE) of Long-Evans rats and RPE from RCS rats without surgical treatment were also embedded. Sham surgery was also performed in 8 eyes.The IPE cells transplanted into the subretinal space were localized between host RPE and retina, had round cell shapes without polar organization, and contained phagosomes resulting from rod outer segment (ROS) uptake. The underlying host RPE cells were heavily pigmented. RPE cells from RCS rats revealed fragmentation of endoplasmic reticulum, which distinguishes them ultrastructurally from pigment epithelial cells of Long-Evans rats. Ultrastructural alterations were observed in the cytoplasm of transplanted cells. Melanin granules in the IPE cells were found in large vacuoles, which also contained phagosomes originating from ROS uptake. In 13 eyes, 1 to 4 rows and 5 to 8 rows of saved photoreceptors were detected facing transplanted IPE cells in 6 (46%) and 4 (31%) eyes, respectively, 2 months after surgery. However, in 10 (53%) and 7 (37%) of 19 eyes, 1 to 4 rows and 5 to 8 rows, respectively, were also found at sites without IPE cells in the plane of section. ROS directed toward transplanted IPE cells were seen in one case, but these rods were shortened and disorganized. At most sites between transplanted cells and inner segments of photoreceptors, outer segments and cellular debris were absent. In eyes without transplanted cells no photoreceptor cells were alive at the age of 2 months. After sham surgery 6 (75%) eyes had 1 to 4 rows and 2 (25%) 5 to 8 rows of photoreceptors.Transplanted IPE cells can take up and degrade ROS in vivo in RCS rats. Uptake of ROS alters the morphology of pigment granules in transplanted IPE cells. Pigmentation is an uncertain marker for identifying transplanted pigment cells. IPE transplants are not as good as RPE transplants in rescuing photoreceptors. However, there is a significant difference between transplanted eyes and nontreated eyes. The rescue effect of IPE cells was not significantly different from that of sham surgery.

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.(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.(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.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.

Because earlier studies indicate that the choroid close to the ora serrata may have unique anatomic features such as wandering cells, blood vessels in Bruch's membrane, and accumulated pigment in the retinal pigment epithelium (RPE), the morphology of the normal human eye at the ora serrata region was investigated.Specimens from the ora serrata region of two normal human eyes (male donors, 48 and 52 years old) were investigated by light and electron microscope. Specimens from all quadrants were studied in one eye.The elastic layer of Bruch's membrane extended as far as 15 microm into the peripheral choroid; capillaries were included between the elastin layer and the RPE. Nasally, from the anterior end to 2 mm posterior of the ora serrata, the RPE cells contained more melanin than did those in the adjacent posterior region. Melanin granules in the RPE cells close to the ora either formed large clusters or appeared unusually small because of fragmentation. A unique, fine lamellar, membranous material with a fingerprint-like structure was found between the basal folds of the RPE. This material is also found within the extracellular matrix of the choroid and in association with red blood cells.The morphology of Bruch's membrane is varied near the ora serrata because capillaries and wandering cells are present in its outer collagenous layer. Unique, fine lamellar, fingerprint-like structures are extruded from the RPE and are removed from the eye together with red blood cells. Capillaries within the inner collagenous region of Bruch's membrane at the ora serrata may not necessarily represent a pathologic response but may be a normal characteristic of thick regions of Bruch's membrane.

In this study, melanophore cytodifferentiation in the fins of xanthic goldfish that had been exposed to osmotic stress for 18 days was investigated. It was found that multi-vesicular bodies (MVB) are not the only type of premelanosome. Granules having a homogeneous matrix also function as premelanosomes. The presence of acid phosphatase reaction product inside the melanin granules indicated that these organelles in this animal were also related to lysosomes. DOPA-oxidase of tyrosinase, the key enzyme in melanogenesis, was surprisingly not only detected in melanocytes but also in the Golgi stacks of dermal cells. Due to the mechanisms of premelanosome formation it is evident that cytoplasmic material also serves as substrate for melanogenesis. EDX microanalysis was performed to measure the ionic composition of the melanin granules. After aldehyde fixation the newly-formed melanin granules did not contain Na, but had accumulated Ca.

This paper is a review about the ultrastructural data dealing with the origin of the melanin granules in retinal pigment epithelial cells, in melanocytes, in the ink gland of cuttle fish, in Kupffer cells of the liver, in neuronal tissues, in cultured pigment cells. The role and structure of lysosomes in melanogenesis are discussed in a separate chapter. The early steps of melanogenesis are ultrastructurally very heterogeneous, even in the same cell types. With respect to this heterogeneity and the considerably different views on melanosome origin in the literature, the author hypothesizes that pigment cells may use protein matrices originated from different cellular pathways. 1) They may either produce a specific protein matrix and be converted into melanin in the classical way, or 2) alternatively, a matrix resulting from lysosomal protein degradation or endocytotic pathways may be used and converted into melanin, as found in fibroblasts transfected with the tyrosinase gen or in Kupffer cells. The very heterogeneous ultrastructure of the polymerizing melanin may be influenced by the amount and sterical availability of tyrosine residues in the protein moieties and the activity of tyrosinase.

The eggshells of young developmental stages in the uterus are rather thin and homogenous. In the brezel stage of Brugia malayi they are 35 nm thick and 20 nm in Litomosoides carinii. In young developmental stages up to brezel stages the eggshells bind the lectins WGA, DBA and PNA labelled with colloidal gold. This shows that GlcNAc, GalNAc and Gal residues are present at the surface of the sheath. In intrauterine microfilariae of B. malayi the original sheath is reduced to a thickness of 7 nm. It is reinforced by secretions from a specialized area of the epithelium of the uterus which do not appear as a homogeneous layer but look like a string of pearls. This layer may be called the "uterine layer". It has a thickness of 40-80 nm. In the microfilaria of L. carinii, the thickness of the original sheath is reduced to 2-3 nm and the uterine layer has a thickness of 7 nm. The uterine layer does not react with any of the lectins, which shows that the surface lacks N-acetylglucosamine, N-acetylgalactosamine and galactose residues. The uterine layer appears to be an ancestral (plesiomorphic) feature which is present in free-living nematodes and the highly specialized bloodforms of filariae. The uterine layer seems to protect and disguise the original sheath against the immune reactions of the host.