Lena Ivert

Photoreceptor allografts in a feline model of retinal degeneration.

Abstract · Background: Photoreceptor transplants provide a potential means to restore function in a degenerate retina and/or rescue degenerating host photoreceptors by trophic influences. We have examined photoreceptor allografts in the Abyssinian cat model of hereditary photoreceptor degeneration to determine the viability and influence of such transplants on the host retina. · Methods: Small pieces of 3- to 5-day-old normal kitten retina containing undifferentiated photoreceptors were injected into the subretinal space of adult Abyssinian cats at an early stage of retinal degeneration using standard vitreo-retinal surgical techniques. The retinas were examined by ophthalmoscopy and fundus photography, then by light and electron microscopy at different times after surgery. · Results: Such allografts survive for at least 6 months after surgery. The photoreceptors develop outer segments, invariably in rosettes. The transplants gradually integrate with the host retina but detach the host photoreceptor layer from the retinal pigment epithelium (RPE), which tends to reduce the number of host photoreceptors over the transplant. There is no slowing of the photoreceptor degeneration in neighboring non-detached retina. Inflammation or rejection was not detected. · Conclusion: Undifferentiated, neonatal photoreceptor allografts survive and develop outer segments in the subretinal space of the Abyssinian mutant feline retina. The allografts gradually integrate with the host neural retina without inducing rejection. In the vicinity of the transplant there is increased loss of host photoreceptors, considered to be due to their detachment from the RPE layer. There is no evidence of any rescue of host photoreceptors elsewhere in this mutant retina.

A novel melano-lysosome in the retinal epithelium of rhesus monkeys.

The large phagocytic load that confronts the retinal pigment epithelium (RPE) is thought to play a possible role in the pathogenesis of age related macular degeneration (AMD) that afflicts both humans and monkeys. Our knowledge of how RPE degrades phagosomes and other intra-cellular material by lysosomal action is still rudimentary. In this paper we examine organelles that play a role in this process, melanosome, lysosomes and phagosomes, in the RPE of young and old rhesus monkeys in order to better understand lysosomal autophagy and heterophagy in the RPE and its possible role in AMD. We used electron microscopy to detect and describe the characteristics of melanosomes and lysosome-like organelles in the macular RPE of rhesus monkeys (Macaca mulatta) that were 1, 6, 24, 24, 26 and 35 years of age. The measurements include the number, shape and size of these organelles located in the basal, middle and apical regions of RPE cells. Phaagosomes were also examined but not counted or measured for size or shape because of their rarity. Melanosomes were homogeneously dark with a circular or elliptical shape and decreased in number with age. Smaller melanosomes were more common at the basal side of the RPE. Among the small melanosomes, we found an organelle that was losing melanin in varying degrees; in some cases was nearly devoid of melanin. Because of the melanin loss, we considered this organelle to be a unique type of autophagic melano-lysosome, which we called a Type 1 lysosome. We found another organelle, more canonically lysosomal, which we called a Type 2 lysosome. This organelle was composed of a light matrix containing melanosomes in various stages of degradation. Type 2 lysosomes without melanosomes were rare. Type 2 lysosomes increased while Type 1 decreased in number with age. Phagosomes were rare in both young and old monkeys. They made close contact with Type 2 lysosomes which we considered responsible for their degradation. Melanosomes are being lost from monkey RPE with age. Much of this loss is carried out by two types of lysosomes. One, not defined as unique before, appears to be autophagic in digesting its own melanin; it has been called a Type 1 lysosome. The other, a more canonical lysosome, is both heterophagic in digesting phagosomes and autophagic in digesting local melanosomes; it has been called a Type 2 lysosome. Type 1 lysosomes decrease while type 2 lysosomes increase with age. The loss of melanin is considered to be detrimental to the RPE since it reduces melanins protective action against light toxicity and oxidative stress. Phagosomes appear to be degraded by membrane contacts with Type 2 lysosomes. The loss of melanin and the buildup of Type 2 lysosomes occur at an earlier age in monkeys than humans implying that a greater vulnerability to senescence accelerates the rate of AMD in monkeys.

Age-related changes in the basement membrane of the retinal pigment epithelium of Rpe65 −/− and wild-type mice

PurposeTo investigate unusual changes in the basal surface of the retinal pigment epithelium (RPE) cell layer in aging Rpe65 −/− and wild-type mice.MethodsThe retinas of Rpe65 −/− and wild-type mice of different ages—6 weeks and 3, 6, 12–13 and 16 months—were examined by electron microscopy.ResultsThere was an age-related increase in the width of the basement membrane of both Rpe65 −/− and wild-type mice which was associated with loss of basal infoldings of the plasma membrane of the RPE cells and protrusions of basement membrane material deep into the cytoplasm of these cells. These changes were evident at 6 months of age in RPE65 −/− mice and became extensive at 1 year of age. Similar changes occurred in wild-type mice but were less extensive and were only evident after 1 year of age.ConclusionsThere is an age-dependent abnormality that develops at the basal surface of murine RPE cells, which resembles some of the changes observed in human age-related macular degeneration. These changes occur earlier in life and are more extensive in Rpe65 mutant mice.

Behavior of retinal epithelium to bleb detachment versus retinectomy

PurposeTo compare the behavior of the retinal pigment epithelium to a bleb detachment versus removal of a segment of neural retina.MethodsA bleb detachment was performed on 14 adult pigmented rabbits. In seven rabbits, the neural retina was removed within the bleb detachment. The rabbits were followed for months after surgery by scanning laser ophthalmoscopy and post mortem histology using light and electron microscopy.ResultsBleb detachment produces a transformation of the retinal epithelial layer that results in migration, enlargement of cell size and accumulation of large amounts of lysosomal material, resembling that found in Chediak-Higashi syndrome. Retinectomy causes migration of the epithelial cells, development of multiple layers with vacuoles but no accumulation of lysosomal material.ConclusionThe neural retina appears to exert a strong influence on the behavior of the retinal epithelial layer. Brief separation of the neural retina from the epithelium provokes a rapid transformation of this cell layer leading to migration of the cells and apparent faulty digestion of phagosomes, causing an enormous buildup of lysosomal debris. Removal of the neural retina also provokes retinal epithelial cell migration but no buildup of lysosomal debris occurs, presumably due to the absence of photoreceptor outer segments and consequently phagosomes. This migratory tendency, which appears to alter lysosomal degradation, could lead to apoptosis of these epithelial cells.

Alteration in choroidal blood flow produced by local pressure

BackgroundTo investigate how transient pressure applied to the retinal pigment epithelium (RPE) layer and choroid affects choroidal blood flow in rabbits.MethodsTwelve rabbits underwent vitrectomy and local retinectomy. In nine of the rabbits a glass rod was used to exert brief pressure on the RPE layer and choroid. Three of the rabbits had no pressure indentation and were considered to be controls. The choroidal circulation was studied by indocyanine green (ICG) angiography. The retina and choroid were studied by postmortem histology.ResultsPressure on the RPE layer and choroid caused nonfluorescence in segments of retinal arteries and veins and reduced fluorescence in adjacent choroidal capillaries, producing a black region at the pressure site in the angiograms. The size of this region decreased during the angiogram, often accompanied by the appearance of fine channels considered to be flow through the partially blocked vessels; the obstructed ends of the vessels became increasingly hyperfluorescent. These changes lasted for about 24 h before the choroidal circulation recovered. Histology showed evidence of thrombotic-like material in choroidal arteries and veins at the areas of absent perfusion. After local retinectomies, there was no evidence of thrombosis in control eyes where no pressure had been applied.ConclusionBrief pressure on the RPE and choroid causes immediate reduction in flow through choroidal vessels, which appears to be due to local thrombosis in small segments of these vessels that resolves slowly. This may reflect a tendency for thrombi to form rapidly in choroidal vessels; it may also depend on neural reflexes causing vasoconstriction. The long time course of recovery could result in retinal ischemia and may underlie the pathophysiology of other pressure insults to the choroid.

The Ultrastructure, Spatial Distribution, and Osmium Tetroxide Binding of Lipofuscin and Melanosomes in Aging Monkey Retinal Epithelium

ABSTRACT Purpose: To examine the ultrastructure of lipofuscin bodies and melanosomes in retinal epithelium of elderly rhesus monkeys and determines changes in their number and morphology as a function of retinal eccentricity. Methods: Electron microscopy was used to describe and quantify two major organelles in elderly monkey retinal epithelium, lipofuscin bodies and melanosomes, at different retinal loci extending from the macula to the peri-macula, equator, periphery and ora serrata. Osmium tetroxide was used to distinguish lipofuscin bodies from melanosomes. Results: Lipofuscin bodies and melanosomes diminished in number with advanced age but there was an inverse relationship between these two organelles. Lipofuscin bodies were more numerous in the macula and melanosomes more numerous in the peripheral retina. Three types of lipofuscin bodies were identified: 1) smaller and tending to locate in the middle third of the epithelial cell, 2) larger, less common, and located more basally, and 3) extremely rare, melano-lipofuscin, containing a melanosome. When osmicated, all lipofuscin bodies contained electron dense materials. When osmium tetroxide was not used for fixation, the first two types of lipofuscin bodies lost their electron densities while the third type retained its electron density due to the melanosome it contained. Conclusion: As previously reported for human retina, lipofuscin is most abundant in the macular and peri-macular epithelium and least abundant in the periphery, whereas melanosomes show the opposite relationship. This distribution pattern could contribute to the macula’s greater vulnerability to photo-toxicity. Three types of lipofuscin bodies are found in aging monkey retinal epithelium. All types contain electron dense material, but the most prominent two types lose their densities in the absence of osmium tetroxide during fixation. Most of the electron densities in lipofuscin bodies must contain a material that binds strongly to osmium tetroxide such as polyunsaturated fatty acids.