Lipofuscin In Retinal Pigment Epithelium Research Articles

The photochemistry of human retinal lipofuscin as studied by EPR.

Fluorescent material generated in the human retina accumulates within lipofuscin (HLF) granules of the retinal pigment epithelium (RPE) during aging. We have been investigating the possible light-induced contribution of these fluorophores to various diseases including age-related macular degeneration. Our studies have shown that some of the fluorescent components of HLF are products of the reaction of retinaldehyde with ethanolamine and that synthetic mixtures of this reaction can serve as a useful model for photophysical studies. Previous research by us has demonstrated that irradiation of either natural or synthetic lipofuscin resulted in the formation of a triplet state and possibly a free radical. Here EPR studies were performed to verify the formation of that radical. The UV irradiation of either synthetic or natural human retinal lipofuscin extracts in oxygen-free methanol led to the formation of a 5,5-dimethylpyrroline-N-oxide (DMPO) spin-trapped carbon-centered radical resulting from either hydrogen atom or electron abstraction from solvent molecules. In the presence of oxygen superoxide was formed, which was observed as a DMPO adduct. It is concluded that certain components of the chloroform-soluble fluorophores of human RPE lipofuscin granules and the fluorescent reaction products of retinaldehyde and ethanolamine are photophysically similar but not the same. Electron or hydrogen abstraction from a substrate by these fluorophores in vivo and the resulting radical products may contribute to the age-related decline of RPE function and blue light damage in the retina.

Drusen-like Deposits in the Outer Retina of Japanese Quail

Histological examination of the outer retina of Japanese quail ( Coturnix coturnix Japonica) ranging in age from 4-48 months revealed the presence of drusen-like deposits which closely resemble those described in primate and human retinas. At both light and electron microscopic levels, these deposits were characterized by randomly distributed, granular and heterogenous materials. Larger deposits with pleomorphic inclusions, often globular in shape, occurred more frequently in older quail, particularly in males. An increase in the incidence and size of drusenoid deposits occurred with increasing age, with a greater rate of accumulation in males than in females. Previously, we have shown that retinal pigment epithelial lipofuscin increases more rapidly with age in female quail and that cumulative light damage is associated with increased lipofuscin in females but not in males. In the present study, no increase in drusen-like deposits was observed in single light-damaged or double light-damaged retinas of either sex. Thus, lipofuscin and the frequency and size of drusenoid deposits do not appear to be directly linked, although both increase with age in both sexes.

Experimental Light Damage Increases Lipofuscin in the Retinal Pigment Epithelium of Japanese Quail (Coturnix coturnix Japonica)

Lipofuscin accumulation in the retinal pigment epithelium (RPE) of Japanese quail was investigated in normal and light-damaged animals at 4 months and 12 months of age following a single, 18-hr light-damaging exposure (3000-3200 lx) evaluated 6 weeks postexposure. Quail in a second experimental group light-damaged at 4 months of age were exposed to a second, identical light-damaging exposure at 12 months of age. RPE lipofuscin was quantitatively assessed in three ways: (1) total lipofuscin, expressed as a percentage of the basal RPE cell area, (2) mean number of lipofuscin granules per RPE cell, and (3) mean area of lipofuscin granules.At 12 months of age, control female RPE cells showed substantially more lipofuscin than did males. Animals that received a single damaging light-exposure showed no significant differences in lipofuscin at either age when compared with controls, and none were observed between males and females. However, in the double light-exposure condition, both total lipofuscin and the number of lipofuscin granules were significantly higher in females compared to controls and also, when compared with animals in the single light-exposure condition. Mean lipofuscin granule size decreased in females in the double light-exposure condition when compared with control values.Histopathological evaluation of photoreceptor outer segments and outer nuclear layer indicated that double-exposed, 12-month-old females showed the most severe effects, including a substantial decrease (70%) in rod photoreceptor densities. However, only a small change was observed in cone densities for either sex (12-15%) in this condition. Overall, a strong, negative correlation (r = -0·78) was obtained between total lipofuscin and average rod density across age, sex, control and experimental conditions. A somewhat greater correlation (r = -0·82) was obtained for females, alone. These results suggest that rods, when severely damaged by light exposure, contribute to increased RPE lipofuscin.Age, sex and light-exposure history appear to be critical variables that can influence both the amount of lipofuscin present in RPE cells as well as the relative vulnerability of rod and cone photoreceptors to cumulative light-exposure damage. These results have implications for the development of a variety of age-related changes and diseases of the outer retina, including age-related macular degeneration.

Influence of dietary vitamin a on autofluorescence of leupeptin-induced inclusions in the retinal pigment epithelium

Substantial evidence indicates that the major precursors for retinal pigment epithelial (RPE) lipofuscin are molecular components of the photoreceptor outer segments. Previous experiments have demonstrated that the retinoids required for transduction by the photoreceptors promote RPE lipofuscin fluorophore formation. Animals deficient in these retinoids accumulate lipofuscin in the RPE at greatly reduced rates. The protein components of the photoreceptor outer segments also appear to be involved in the formation of RPE lipofuscin fluorophores. When degradation of phagocytosed photoreceptor outer segment proteins by the RPE is blocked, this tissue rapidly becomes engorged with phagosome-derived inclusions that have fluorescence properties similar to lipofuscin. Experiments were conducted to determine whether the development of lipofuscin-like fluorescence in these inclusions was dependent on the availability of retinoids. Rats were fed diets containing vitamin A either in the form of retinyl palmitate (+A), which can be metabolically converted into the retinoids involved in vision, or retinoic acid (-A), which does not support visual function. After the retinas of the -A animals had been depleted of retinoids involved in vision, animals from both groups were given intraocular injections of the protease inhibitor leupeptin. Two days later, the amounts of lipofuscin-like autofluorescence from the RPEs of rats in each group were determined. Leupeptin treatment produced an increase in this fluorescence in RPEs of the +A animals, but not in the RPEs of the rats fed the -A diet, despite the fact that phagosome-like inclusions accumulated in the RPE in both dietary groups. This finding suggests that retinoids involved in the visual process are probably directly involved in RPE lipofuscin fluorophore formation.

Fluorophores of the human retinal pigment epithelium: Separation and spectral characterization

Ten fluorescent fractions orginating from the chloroform extracts of retinal pigment epithelial (RPE) cells of human donor eyes (ages 52–98 yr) have been separated and characterized by UV-vis absorbance and corrected fluorescence spectroscopy. The semipurified fluorophores fall into four categories based upon their spectral properties: green-emitting fluorophores, a golden yellow-emitting fluorophore, yellow-green-emitting fluorophores and orange-red-emitting fluorophores. All share common absorbance peaks around 280- and 330 nm, and the orange-red-emitting fluorophores also exhibit a strong absorbance peak at 420 nm. No significant visible-emitting fluorophores were detected in the methanol-water phase of these extracts. While these fluorophores are abundant in extracts from adult-derived RPE, most of the fluorophores occur in much lower amounts in RPE extracts from human donors under 10 yr of age. Eyes from child human donors also have much less RPE lipofuscin than those from adult donors, suggesting that most of the fluorophores are lipofuscin derived. This interpretation is supported by the previous finding that all of the fluorophores from whole RPE are also present in extracts of purified lipofuscin granules. Characterization of the chromatographic and spectral properties of the chloroform-soluble fluorescent components from the human RPE provides an important tool for determining the mechanism of RPE lipofuscin fluorophore formation. The absorbance properties defined here are of significance to investigations into the photobiology of the RPE and to those using laser therapy in treatment of age-related retinal diseases.

Lipofuscin autofluorescence: Evidence for vitamin A involvement in the retina

A lipofuscin-like autofluorescence develops in the degenerating photoreceptor cells of the RCS rat, a strain with inherited retinal dystrophy. In animals with normal retinas, age-related lipofuscin accumulation in the eye is restricted to the retinal pigment epithelium (RPE). Previous investigations have established that RPE lipofuscin accumulation in the normal rat retina can be reduced by dietary vitamin A deficiency. In order to determine whether the photoreceptor-derived fluorescence in the RCS rat retina is related to RPE lipofuscin fluorescence, the influence of dietary vitamin A on the fluorophore content of the RCS rat retina was studied. Vitamin A deficiency substantially reduced the autofluorescence associated with degenerating photoreceptor cells of the RCS rat retina. A specific vitamin A-dependent fluorophore was isolated from these retinas using thin-layer chromatography (TLC). The mobility of this fluorophore on TLC differs from that of the major age-dependent fluorophore isolated from the RPE of normal rats. Thus, if the vitamin A-dependent fluorophores of the photoreceptors and RPE are related, it appears that the fluorophore generated in the photoreceptor cells must undergo chemical modification once it has been taken up by the RPE. The fact that both the RPE- and photoreceptor-associated fluorophores are vitamin A-dependent suggests that such a relationship between them is likely. These experiments indicate that the RPE is somewhat different from other lipofuscin-accumulating tissues in that a major precursor of RPE lipofuscin fluorophores originates in another cell type and enters the RPE via phagocytosis.

Influence of early photoreceptor degeneration on lipofuscin in the retinal pigment epithelium

Experiments were conducted to evaluate the role played by photoreceptor cells in the accumulation of age pigment, or lipofuscin, in the retinal pigment epithelium (RPE). The age-related accumulation of RPE lipofuscin was compared between rats with hereditary photoreceptor degeneration (RDY) and congenic rats with normal retinas. In the RDY animals, the age-related increase in RPE lipofuscin content was substantially less than in normal controls. This suggests that the photoreceptor cells play a significant role in RPE lipofuscin deposition, although they may not be the sole contributors to RPE lipofuscin formation. Evidence that outer-segment components may be converted into lipofuscin fluorophores was provided by the discovery that in young RDY rats, fragments of outer segments from degenerating photoreceptor cells had fluorescence properties similar to those of RPE lipofuscin. Chloroform-methanol extraction of retina-RPE tissue from young normal and dystrophic rats, and analysis of the chloroform fractions by thin-layer chromatography, revealed three distinct fluorescent components associated with the lipofuscin-like fluorescence of the outer-segment fragments in the RDY rats.

Relationship between dietary retinol and lipofuscin in the retinal pigment epithelium

A variety of evidence suggests that autoxidation of cellular components probably plays a significant role in the age-related accumulation of lipofuscin, or age-pigment, in the mammalian retinal pigment epithelium (RPE). Among the likely candidates for conversion into RPE lipofuscin fluorophores via autoxidative mechanisms are vitamin A compounds, which are present in the retina and RPE in high concentrations. Vitamin E, an important lipid antioxidant, is likely to inhibit vitamin A autoxidation. Experiments were conducted to evaluate the significance of vitamin A autoxidation in the deposition of lipofuscin in the RPE. Albino rats were fed diets either supplemented with or lacking vitamin E. Each of these two groups of animals was further subdivided into three groups which were fed different levels of vitamin A palmitate: none, 14.0 μmol/kg diet, and 80.5 μmol/kg diet. After 26 weeks, the animals were killed and the RPE lipofuscin contents were determined by both fluorescence measurements and quantitative ultrastructural morphometry. Vitamin A palmitate deficiency led to significant reductions in RPE lipofuscin deposition, relative to the amounts of this pigment present in the groups receiving vitamin A palmitate in their diets. The relative magnitude of the vitamin A effect was greater in the vitamin E-supplemented groups than in the groups fed the diets deficient in vitamin E. This finding suggests that vitamin E interacts with vitamin A ester metabolites in vivo in a more complex manner than simply acting as an antioxidant protectant. Rats fed the diets containing the higher level of vitamin A palmitate failed to display elevated RPE lipofuscin contents relative to those in the rats fed 14.0 μmol of vitamin A palmitate/kg diet. Failure of high vitamin A intake to enhance RPE lipofuscin deposition may have been due to the fact that intake of vitamin A above normal levels did not lead to an elevation in vitamin A content of the retinal tissue. Establishing an effect of vitamin A deficiency on RPE lipofuscin deposition and characterization of the interactions between vitamins E and A are inmportant steps toward defining precisely the molecular and cellular mechanisms underlying age-pigment accumulation in the RPE.

Lipofuscin in retinal pigment epithelium of rhesus monkey: lack of diminution with centrophenoxine treatment

An experiment was performed to test the ability of Centrophenoxine to reduce the amount of lipofuscin (age pigment) in the retinal pigment epithelium (RPE) of aged rhesus monkeys. Centrophenoxine is reputed to have this action in neurons of lower mammals. Quantitative electron microscopic analysis was performed on sections from the perifovea of ten rhesus monkeys, all approximately 20 years of age. Four of the animals received 80 mg/kg Centrophenoxine (IM injection) daily for 12 weeks. No significant difference between the treated and control groups could be demonstrated statistically (Mann-Whitney U-test) either in the fraction of RPE cell cytoplasm occupied by lipofuscin granules or in the average size of the granules.