Immunocytochemical identification of neural elements in the central nervous systems of a snail,some insects,a fish,and a mammal with an antiserum to the molluscan cardio-excitatory tetrapeptide FMRF-amide

Summary The choriocapillaris is the fenestrated capillary network that supplies a large portion of the nutrients required by the retinal pigment epithelium, photoreceptor cells, and other cells of the outer neural retina. The permeability of these capillaries was investigated in the rat by the use of ferritin (mol. wt. approx. 480,000; mol. diam. 110Å) as a tracer. Ninety minutes after intravascular ferritin administration, a high concentration of tracer particles was distributed uniformly in the capillary lumina but few particles were present in Bruch's membrane, the multilayered basement membrane that separates the choriocapillary endothelium from the retinal pigment epithelium. The bulk of the tracer remained in the capillary lumina with a definite blockage seen at fenestral, channel, and vesicle diaphragms. These results indicate that the rat choriocapillary endothelium, unlike the fenestrated endothelia lining other capillary beds, constitutes an important barrier to the passage of ferritin and presumably of circulating native molecules of similar size.Supported by NIH grants EY 01889 and EY 07034 from the National Eye Institute and a grant-in-aid from Fight for Sight, Inc., of New York City     

Permeability of regenerating and atrophic choriocapillaris in the rabbit

When rabbits receive intravenous injections of sodium iodate, large expanses of the retinal pigment epithelium are destroyed. The adjacent capillary bed, the choriocapillaris, atrophies in response to the loss of the pigment epithelium and then regenerates. This provides a model of the permeability of regenerating and atrophic choriocapillaris, which we studied using intravenously injected horseradish peroxidase and catalase. Regenerating capillaries were permeable to peroxidase but not catalase. The permeability to peroxidase was probably due to endothelial fenestrations, since catalase (which is larger than peroxidase and does not penetrate endothelial fenestrae) was retarded at interendothelial junctional complexes, indicating that they were intact. Atrophic choriocapillaries were impermeable to catalase but displayed a heterogeneous permeability to peroxidase. This was correlated with the presence or absence of fenestrae; capillary profiles lacking fenestrae retained peroxidase in their lumina, whereas if fenestrae were present the tracer penetrated into the pericapillary space. The observations indicate that: (1) the permeability of the regenerating choriocapillaris is qualitatively similar to the mature choriocapillaris, and (2) the atrophic choriocapillaris undergoes changes in permeability that are primarily correlated with the loss of endothelial fenestrae. The observations provide a functional correlate - change in permeability - for structural changes in choriocapillaris endothelium (thickening, loss of fenestrae) in response to destruction of the retinal pigment epithelium, which has been postulated to exert a trophic effect on these capillaries.     

Choriocapillaris atrophy after experimental destruction of the retinal pigment epithelium in the rat. A study in thin sections and vascular casts

Rats that receive intravenous injections of sodium iodate develop a retinopathy characterized by the partial loss of the retinal pigment epithelium (RPE). In thin sections examined by transmission electron microscopy the choriocapillaris atrophied adjacent to areas of RPE destruction. The endothelial cells thickened and lost their fenestrae and the lumen of the capillary was reduced. At sites where the RPE remained normal in appearance the choriocapillaris did not atrophy. Scanning electron microscopy of vascular casts of the choriocapillaris showed the coexistence of atrophic and normal choriocapillaris throughout the retina, presumably adjacent to sites where the RPE was destroyed or spared, respectively. Our observations support the concept that the RPE exerts some control over the structure and function of the choriocapillaris.     

Fine structure of the retinal pigment epithelium of the mallard duck (Anas platyrhynchos)

As part of a comparative morphological study, the fine structure of the retinal pigment epithelium (RPE), the choriocapillaris and Bruch's membrane (complexus basalis) has been investigated by light and electron microscopy in the mallard (Anas platyrhynchos). In this species the RPE consists of a single layer of cuboidal cells which display numerous very deep basal (scleral) infoldings and extensive apical (vitreal) processes which enclose photoreceptor outer segments. The RPE cells are joined laterally by prominent basally-located tight junctions. Internally smooth endoplasmic reticulum is the most abundant cell organelle with only small amounts of rough endoplasmic reticulum present. Polysomes are abundant as are basally-located mitochondria which often displayed a ring-shaped profile. The cell nucleus is large and vesicular. Melanosomes are plentiful only within the apical processes of the RPE cells in the light-adapted state. Myeloid bodies are large and numerous and very often have ribosomes on their outer surface. Bruch's membrane (complexus basalis) shows a pentalaminate structure but with only a poorly represented central elastic lamina. Profiles of the choriocapillaris are relatively small and the endothelium of these capillaries while extremely thin facing the retinal epithelium is but minimally fenestrated.     

Pericyte response during choriocapillaris atrophy in the rabbit

The rabbit and rat choriocapillaris atrophies in response to experimental destruction of the retinal pigment epithelium by intravenous injection of sodium iodate. This provides a convenient model of capillary atrophy. We have observed that pericytes are spared during this process; the atrophy is due to loss of endothelium only. Extensive examination of thin sections obtained 1 day to 11 weeks after administration of iodate showed that pericytes retained their normal relationship to the remnant capillary basement membrane left behind as the endothelial tube atrophied. This was most conspicuously manifested in their retention of processes longitudinally disposed along the sleeves of remnant basement membrane. The processes retained bundles of actin filaments that had dense regions along them and inserted into subplasmalemmal densities at basement membrane attachment sites, i.e. they had the characteristics of stress fibers. The pericytes did not phagocytose the debris of endothelial necrosis, in spite of their known phagocytic abilities. Necrotic endothelial cells were eliminated by sloughing into the capillary lumen. The observations support the idea that the function of pericytes in the choriocapillaris, the major source of nutrition for the retinal photoreceptors, resides in their contractility, and that pericytes do not remove necrotic endothelium during capillary atrophy.     

Restriction of exogenous transthyretin (prealbumin) by the endothelium of the rat choriocapillaris

The endothelium of the choriocapillaris has been shown to restrict molecules with Einstein-Stokes radii greater than or equal to 3.2 nm which correspond to minimal molecular weights of approximately 64,000-68,000 daltons. The present study was undertaken to determine if the endothelium restricts exogenous transthyretin (prealbumin), a 55,000-dalton carrier of retinol-binding protein. Rats were injected with human 125I-transthyretin which was allowed to circulate for 15 and 30 min. Chromatographic analysis demonstrated that the human transthyretin did not bind to rat blood proteins. Eye tissue from injected rats was prepared for light and ultrastructural autoradiographic analysis. Autoradiographic grains were confined to areas over the lumen of the choriocapillaris with few present on Bruch's membrane, the basement membrane common to the endothelium of the choriocapillaris and the retinal pigment epithelium. These findings demonstrate that the choriocapillaris can restrict transthyretin and suggest a possible role of its endothelium in the metabolism of retinol-carrier molecules.     

The Polarity of the Retinal Pigment Epithelium

The diversity of epithelia in the body permits a multitude of organ-specific functions. One of the foremost examples of this is the retinal pigment epithelium. Located between the photoreceptors of the retina and their principal blood supply, the choriocapillaris, the retinal pigment epithelium is critical for the survival and function of retinal photoreceptors. To serve this purpose, the retinal pigment epithelium cell has adapted the classic Golgi-to-cell-surface targeting pathways first described in such prototypic epithelial cell models as the Madin-Darby canine kidney cell, to arrive at a unique distribution of membrane and secreted proteins. More recent data suggest that the retinal pigment epithelium also takes advantage of its inherent asymmetry to augment the classical pathways of Golgi-to-cell-surface traffic. As retinal pigment epithelium transplants and gene therapy represent potential cures for retinal degenerative diseases, understanding the basis of the unique polarity properties of retinal pigment epithelium cells will be a critical issue for the development of future therapies.     

Electron Microscopy of the Tapetum Lucidum of the Cat

The fine structure of the tapetum of the cat eye has been investigated by electron microscopy. The tapetum is made up of modified choroidal cells, seen as polygonal plates grouped around penetrating blood vessels which terminate in the anastomosing capillary network of the choriocapillaris. The tapetal cells are rectangular in cross-section, set in regular brick-like rows, and attain a depth of some thirty-five cell layers in the central region. This number is gradually reduced peripherally, and is replaced at the margin of the tapetum by normal choroidal tissue. The individual cells are packed with long slender rods 0.1 µ by 4 to 5 µ. The rods are packed in groups and with their long axes oriented roughly parallel to the plane of the retinal surface. Each cell contains several such groups. Cells at the periphery or in the outer layers of the tapetum are frequently seen to contain both tapetal rods and melanin granules, the latter typical of the choroidal melanocytes. Also melanocyte granules may have intermediate shapes. These observations plus the similar density of the two inclusions lead to the belief that the tapetal rods may be melanin derivatives. A fibrous connective tissue layer lies between the tapetum and the retina. The subretinal capillary network, the choriocapillaris, rests on this layer and is covered by the basement membrane of the retinal epithelium. The cytoplasm of the retinal epithelium exhibits marked absorptive modifications where it comes in contact with the vessels of the choriocapillaris. This fibrous layer and the basement membrane of the retinal epithelium apparently comprise the structural elements of Bruch's membrane.     

Retinal epithelial fine structure in the grey seal (Halichoerus grypus)

The morphology of the retinal epithelium and the closely associated choriocapillaris and Bruch's membrane (complexus basalis) has been investigated in the eye of the grey seal (Halichoerus grypus) by electron microscopy. The retinal epithelium is composed of a single layer of cuboidal cells joined laterally by apically-located junctional complexes. Basally (sclerally) these cells display numerous infoldings while apically (vitreally) abundant processes enclose rod outer segments. Internally the large vesicular nucleus is centrally located. Smooth endoplasmic reticulum, mitochondria and lysosome-like bodies are abundant. Rough endoplasmic reticulum and polysomes while present are not plentiful. Phagosomes of outer-segment discs are only occasionally noted in the light-adapted state. As the entire fundus is overlain with a choroidally located tapetum cellulosum, only at the extreme periphery is an occasional melanosome present in these epithelial cells. The choriocapillaris endothelium is highly fenestrated and the profiles of these capillaries are deeply indented into the epithelial layer. Bruch's membrane (complexus basalis) is reduced to a trilaminate structure rather than the typical pentalaminate membrane seen in most mammalian species and when associated with capillary profiles is further reduced to a single thick basal lamina.     

Demonstration of microfibrils in Bruch's membrane of the eye

The cationic dyes ruthenium red and alcian blue were used to visualize a population of microfibrils in Bruch's membrane, a compound basement membrane located in the uveal tract of the eye between the retinal pigment epithelium and choriocapillaris. Microfibrils were tubular structures, 10-12 nm in diameter, that showed a characteristic beaded pattern. The majority of microfibrils appeared as a dense mantle around the layer of amorphous elastin. Microfibrils and collagen fibers were also present as a loosely organized meshwork in the collagenous zone of the membrane. Microfibrils were also seen along the basal surface of the retinal pigment epithelium where they appeared to insert into the substance of the basal lamina. Ruthenium red staining of microfibrils was not abolished by prior exposure of tissue to several kinds of degradative enzymes. The findings suggest that the elastic properties of Bruch's membrane may depend on both the elastin and microfibrillar components.     

Retinal epithelial fine structure in the vervet monkey (Cercopithecus aethiops)

The morphology of the retinal pigment epithelium (RPE) and closely associated Bruch's membrane (complexus basalis) and choriocapillaris have been investigated by electron microscopy in the vervet monkey (Cercopithecus aethiops). The RPE is composed of a single layer of cuboidal cells joined laterally by apically-located junctional complexes. Basally (sclerally) these cells display numerous infoldings while apically (vitreally) abundant processes enclose and interdigitate with rod outer segments. Internally the large vesicular nucleus is centrally located and smooth endoplasmic reticulum, mitochondria and lysosome-like bodies, are plentiful. Rough endoplasmic reticulum, polysomes and melanosomes while present are not abundant. Phagosomes of outer segment discs are noted in various stages of uptake and degradation. The choriocapillaris is highly fenestrated over large areas. Bruch's membrane shows the typical pentalaminate structure noted in other mammalian species without a tapetum lucidum.     

Retinal pigment epithelial fine structure in the bobtail goanna (Tiliqua rugosa)

The retinal pigment epithelium (RPE), the choriocapillaris and Bruch's membrane (complexus basalis) have been studied by light and electron microscopy in the bobtail goanna (Tiliqua rugosa) an Australian diurnal lizard. The RPE consists of a single layer of cuboidal cells which display very deep and tortuous basal (choroidal) infoldings as well as numerous apical (vitreal) processes which interdigitate with the photoreceptor cells. The lateral cell borders are relatively smooth and joined by basally located tight junctions. Internally smooth endoplasmic reticulum is abundant while rough endoplasmic reticulum is not. The RPE cell nucleus is large and vesicular and basally located in the light-adapted state. Polysomes, mitochondria and myeloid bodies are present and widely distributed. Melanosomes are plentiful in the apical region of the epithelial cells in light-adaptation. Bruch's membrane is pentalaminate with the basal lamina of the choriocapillaris being exceptionally thick. The choriocapillaris is a single layer of large-caliber capillaries with thin but only moderately fenestrated endothelium. Numerous dense granules are always present within these endothelial cells.     

Identification of a synergistic interaction between endothelial cells and retinal pigment epithelium

The retinal pigment epithelium located between the neurosensory retina and the choroidal vasculature is critical for the function and maintenance of both the photoreceptors and underlying capillary endothelium. While the trophic role of retinal pigment epithelium on choroidal endothelial cells is well recognized, the existence of a reciprocal regulatory function of endothelial cells on retinal pigment epithelium cells remained to be fully characterized. Using a physiological long‐term co‐culture system, we determined the effect of retinal pigment epithelium‐endothelial cell heterotypic interactions on cell survival, behaviour and matrix deposition. Human retinal pigment epithelium and endothelial cells were cultured on opposite sides of polyester transwells for up to 4 weeks in low serum conditions. Cell viability was quantified using a trypan blue assay. Cellular morphology was evaluated by H&E staining, S.E.M. and immunohistochemistry. Retinal pigment epithelium phagocytic function was examined using a fluorescent bead assay. Gene expression analysis was performed on both retinal pigment epithelium and endothelial cells by quantitative PCR. Quantification of extracellular matrix deposition was performed on decellularized transwells stained for collagen IV, fibronectin and fibrillin. Our results showed that presence of endothelial cells significantly improves retinal pigment epithelium maturation and function as indicated by the induction of visual cycle‐associated genes, accumulation of a Bruch's membrane‐like matrix and increase in retinal pigment epithelium phagocytic activity. Co‐culture conditions led to increased expression of anti‐angiogenic growth factors and receptors in both retinal pigment epithelium and endothelial cells compared to monoculture. Tube‐formation assays confirmed that co‐culture with retinal pigment epithelium significantly decreased the angiogenic phenotype of endothelial cells. These findings provide evidence of critical interdependent interactions between retinal pigment epithelium and endothelial cell involved in the maintenance of retinal homeostasis.     

The fine structure of the adepidermal reticulum in the basal membrane of the skin of the newt,Triturus

The fine structure of the tapetum of the cat eye has been investigated by electron microscopy. The tapetum is made up of modified choroidal cells, seen as polygonal plates grouped around penetrating blood vessels which terminate in the anastomosing capillary network of the choriocapillaris. The tapetal cells are rectangular in cross-section, set in regular brick-like rows, and attain a depth of some thirty-five cell layers in the central region. This number is gradually reduced peripherally, and is replaced at the margin of the tapetum by normal choroidal tissue. The individual cells are packed with long slender rods 0.1 micro by 4 to 5 micro. The rods are packed in groups and with their long axes oriented roughly parallel to the plane of the retinal surface. Each cell contains several such groups. Cells at the periphery or in the outer layers of the tapetum are frequently seen to contain both tapetal rods and melanin granules, the latter typical of the choroidal melanocytes. Also melanocyte granules may have intermediate shapes. These observations plus the similar density of the two inclusions lead to the belief that the tapetal rods may be melanin derivatives. A fibrous connective tissue layer lies between the tapetum and the retina. The subretinal capillary network, the choriocapillaris, rests on this layer and is covered by the basement membrane of the retinal epithelium. The cytoplasm of the retinal epithelium exhibits marked absorptive modifications where it comes in contact with the vessels of the choriocapillaris. This fibrous layer and the basement membrane of the retinal epithelium apparently comprise the structural elements of Bruch's membrane.     

Fine structure of the retinal epithelium and tapetum lucidum of the ranch mink Mustela vison

The morphology of the retinal pigment epithelium (RPE), Bruch's membrane (complexus basalis), choriocapillaris and tapetum lucidum has been studied in the eye of the ranch mink (Mustela vison) by light and electron microscopy. The RPE is composed of a single layer of cells joined laterally by apically located junctional complexes. Basally (sclerally) these cells display numerous infoldings whereas apically (vitreally) two types of processes are associated with rod and cone outer segments. Smooth endoplasmic reticulum and mitochondria are abundant in these cells whereas rough endoplasmic reticulum and polysomes, although present, are not plentiful. An occasional wandering phagocyte is noted at the RPE-photoreceptor interface. In the posterosuperior part of the fundus, a degenerative tapetum lucidum is present. The presence of only a few layers of tapetal cells containing but little reflective material and the haphazard arrangement of this material makes it very unlikely that this area functions as an effective tapetum lucidum. The RPE over the aberrant tapetum, however, shows the morphology that is seen when a functioning tapetum cellulosum is present, namely the absence of melanosomes and an indented choriocapillaris. Bruch's membrane in non-tapetal areas is pentalaminate but, over the tapetum and where it is associated with capillary profiles, it is reduced to a single, thickened basal lamina. The choriocapillary endothelium is highly fenestrated and in nontapetal areas these capillaries are not indented into the epithelial layer.     

Remodelling of the retinal pigment epithelium in response to intraepithelial capillaries: evidence that capillaries influence the polarity of epithelium

Summary Light- and urethane-induced retinopathies in rats are characterized by loss of photoreceptors. Retinal capillaries subsequently become incorporated into the normally avascular retinal pigment epithelium. These models provided an opportunity to study the response of epithelial cells to closely apposed capillaries, in order to determine if capillaries contribute to the polar organization of epithelial cells. Pigment epithelial cells reorganized their lateral plasma membrane where the latter faced intraepithelial capillaries. This normally flat, undifferentiated membrane developed attachment sites, folds and intracytoplasmic tubules, and exhibited endocytosis and putative basal lamina secretion. These structural and functional specializations are normally restricted to the basal plasma membrane — the normal vascular front of the cell facing the dense meshwork of capillaries constituting the choriocapillaris. We conclude that RPE cells, and perhaps epithelia in general, polarize in response to an adjacent capillary bed.     

Ultrastructural study of the retinal pigment epithelium during metamorphosis in the sea lamprey (Petromyzon marinus L.)

Summary The sequence of morphological changes in the retinal pigment epithelium during the metamorphic period of the sea lamprey Petromyzon marinus L. has been investigated using electron microscopy. At early metamorphic stages (stages I and II), photoreceptors are present in a small zone of the retina. During these stages, the lateral surface of the epithelial cells shows zonulae occludentes and adhaerentes. The degree of cell differentiation varies throughout the retinal pigment epithelium. Cells covering the differentiated photoreceptors in the central retina have phagosomes, whereas pigment granules appear only in the retinal pigment epithelium dorsal to the optic nerve head. Most epithelial cells have myeloid bodies; their morphology is more complex around the optic nerve head. At stage III, when photoreceptors develop over the whole retina, the distribution of cytoplasmic organelles is almost homogeneous in the retinal pigment epithelium. Subsequently, the basal plasma membrane of the epithelial cells becomes progressively folded and their apical processes enlarged. In addition, extensive gap junctions develop between retinal pigment cells. In late metamorphic stages, noticeable growth of myeloid bodies occurs and consequently the retinal pigment epithelium resembles that of the adult. This study also describes, for the first time, the presence of wandering phagocytes in the retinal pigment epithelium of lampreys; their role in melanosome degradation is discussed.     

Ocular tissue interactions during the development of human fetal neuroretina grafted into nude mice

To determine the roles of different ocular tissues in the development of the human fetal neuroretina, a study ethically and technically impossible in human subjects, human embryonic and fetal retinas were heterotopically implanted into nude mice. Ninety-five eyeballs were obtained from legally aborted 6- to 7-week-old embryos or 8- to 10-week-old fetuses. Ten isolated neuroretinas with vitreous but without pigment epithelium, 20 half-eyeballs and 70 intact eyeballs, of which 12 had a thick layer of periocular tissue, were microsurgically grafted. Five intact eyeballs were used for reference. Over a period of 1-245 days, all of the grafts were removed for light and electron microscopy observations. All of the isolated neuroretinas had disappeared by the second day after transplantation. Grafts of the posterior section of the eyeball contained only some clusters of pigment epithelium, occasionally covered with undifferentiated neuroretinal cells. Grafts of the retrolental section of the eyeball contained small areas of dysplasic neuroretina with folds and rosettes. Grafts of the 70 intact eyeballs were successful, but only 26 showed normal histological organization of the choriocapillaris, the retinal pigment epithelium and the neuroretina in the posterior part of the posterior chamber. Photoreceptor differentiation was evident in these retinas after approximately 80 days of transplantation and was complete after 166 days. Their anterior part was always dysplasic, with occasional ciliary differentiation. Twenty-three grafted eyeballs had a dysplasic neuroretina with folds, rosettes and necrotized areas. Twenty-one were atrophic, 12 of which were the eyeballs grafted with periocular tissue. These results demonstrate the role of the fetal mesenchyme and pigment epithelium in the rapid revascularization, and subsequent survival and tissue organization, of the neuroretina. The stratified development of the neuroretina required a thin mesenchymal environment for revascularization of the graft by human vasculogenesis or neoangiogenesis and a normal retinal pigment epithelium for normal neuroretinal differentiation. When these conditions were not satisfied, the neuroretina disappeared or was dysplasic, partly necrotized or atrophic. This model might prove useful for a number of therapeutic or clinical studies.     

Labelling of regenerating retinal pigment epithelium by colloidal iron oxide and ferritin conjugated to wheat germ agglutinin

Summary In order to determine if there are biochemical changes in plasma-membrane oligosaccharides of regenerating retinal pigment epithelium, the binding of colloidal iron oxide at low pH and ferritin-conjugated wheat germ agglutinin — probes of sialic acid and N-acetylglucosamine on the cell surface — was examined electron-microscopically. An animal model of retinal pigment epithelium regeneration — rabbits with sodium iodate induced retinopathy — was used. In this model, large expanses of regenerating pigment epithelium are present for comparison with zones of spared pgiment epithelium in the same animals. In thin sections examined by transmission electron microscopy, ferritin-conjugated wheat germ agglutinin appeared to bind more intensely to the exposed plasma membrane of regenerating retinal pigment epithelium than to spared pigment epithelium, or that of normal rabbits. Morphometry verified this. Colloidal iron oxide intensely labelled the plasma membranes of regenerating, spared, and normal pigment epithelium, and was visibly reduced after exposure of tissue to neuraminidase. The observations indicate that the plasma membrane of regenerating retinal pigment epithelium bears sialic acid and N-acetylglucosamine residues as in normal retinal pigment epithelium. However, the amount of plasma membrane bearing exposed N-acetylglucosamine increases during regeneration.     

Oxidized low density lipoprotein-induced senescence of retinal pigment epithelial cells is followed by outer blood-retinal barrier dysfunction

Age-related macular degeneration is the most common cause of vision loss in the elderly, which starts from aging processes of retinal pigment epithelial cells. Among variable risk factors in occurrence and progression of age-related macular degeneration, oxidized low density lipoprotein could be causally involved in pathobiological changes of RPE cells. Herein we showed that oxidized low density lipoprotein-induced senescence of retinal pigment epithelial cells is followed by outer blood-retinal barrier dysfunction. Under sub-lethal concentration, oxidized low density lipoprotein could promote advanced senescence of retinal pigment epithelial cells. Interestingly expression of CRALBP and RPE 65, indicators of retinal pigment epithelial cell differentiation, was decreased by oxidized low density lipoprotein. In addition, oxidized low density lipoprotein induced reactive oxygen species production and up-regulated inflammatory factors such as tumor necrosis factor-α and vascular endothelial growth factor, when β-catenin, a critical mediator of the canonical Wnt pathway, was also elevated. Oxidized low density lipoprotein increased paracellular permeability of retinal pigment epithelial cells, when zonula occludens-1 at intercellular junctions markedly decreased as well. Furthermore, in retinal pigment epithelial cells and choriocapillaris of human apolipoprotein E2 transgenic mouse eye, increased vascular endothelial growth factor and decreased zonula occludens-1 expression was observed. Therefore, our results suggest that oxidized low density lipoprotein could promote senescence of retinal pigment epithelial cells which leads to induce outer blood-retinal barrier dysfunction as an early pathogenesis of age-related macular degeneration.