Melanolipofuscin Granules Research Articles

Aceruloplasminemia

To examine the retinal histopathologic manifestation of aceruloplasminemia, an autosomal recessive disease caused by mutation of the ferroxidase ceruloplasmin, resulting in tissue iron overload. The morphologic features of the human aceruloplasminemic retina were studied with light and electron microscopy. Retinal iron accumulation was assessed with Perls Prussian blue staining, immunohistochemistry, and secondary ion mass spectrometry. Light and electron microscopic analysis revealed several ocular pathologic findings that resembled age-related macular degeneration, including retinal pigment epithelium (RPE) depigmentation, atrophy and hypertrophy, nodular and diffuse drusen, and lipofuscin and melanolipofuscin granules. Complement deposition was detected in drusen. The RPE cells and neural retina had increased levels of iron. Two major types of RPE cells were observed: melanosome rich and melanosome poor. Melanosome-rich cells had increased levels of iron and melanolipofuscin. The melanolipofuscin granules were observed in large aggregates, where some of the melanosomes were degrading. Melanosome-poor cells lacked melanosomes, melanolipofuscin, and lipofuscin but contained electron-dense aggregates high in iron, phosphorus, and sulfur. The findings in the aceruloplasminemic retina resemble some of those found in age-related macular degeneration. Also, they suggest that melanosomes in the RPE can be degraded via iron-mediated reactive oxygen species production. Mechanisms underlying the pathologic mechanisms found in aceruloplasminemia also may be important in age-related macular degeneration.

Chemical composition of melanosomes, lipofuscin and melanolipofuscin granules of human RPE tissues

Energy-filtered analytical transmission electron microscopy was used to image the ultrastructure and determine quantitatively the chemical composition of pigment granules of the choroid and retinal pigment epithelium of two healthy human donors, aged 68 and 85 years. The electron microscopy preparation procedure did not affect the autofluorescence of melanolipofuscin and lipofuscin granules, since staining was omitted during sample preparation. Oval melanosomes, melanolipofuscin and lipofuscin granules were observed, having sizes of about 1.5 μm × 0.5 μm, and were analyzed using energy-dispersive X-ray microanalysis and electron energy loss spectroscopy. Up to now, these pigments could only be identified by scattering contrast in bright field images, with melanosomes having dark contrast and lipofuscin being much brighter. High-precision energy-dispersive X-ray microanalysis of pigment granules (>15,000 integrated counts in the oxygen K α peak) yielded minimum detectable mole fractions of about 0.02 at% for copper and zinc. For the first time, quantitative analytical electron microscopy yielded the chemical composition of the different pigments without prior isolation from the tissue. This is important to better understand physical and chemical properties of the pigments and their metabolism and turnover. The composition of melanosomes and lipofuscin can clearly be distinguished by the applied methods. Melanosomes were the pigments with largest oxygen (about 5 at%) and nitrogen (about 10 at%) mole fractions. The S/N ratio determination demonstrated a high pheomelanin content of the melanosomes. Lipofuscin had a significantly smaller oxygen mole fraction (about 4 at%) and nitrogen was found to be only slightly above the limit of detection (0.4 at%). For comparison, the cytoplasm contained oxygen and nitrogen mole fractions of 3 at% and 0.8 at%. Bright field images showed melanolipofuscin granules having a core-shell structure with a dark inner and a bright outer fraction. The dark fraction had a chemical composition close to the melanosomes and the composition of the bright fraction could be distinguished from that of lipofuscin due to a significantly increased nitrogen mole fraction in the melanolipofuscin granule. For all pigments observed the oxygen mole fraction yielded a positive correlation with the calcium mole fraction as previously established for melanosomes. Only lipofuscin contained measurable phosphorus mole fractions, which also correlated positively with oxygen. In lipofuscin, mole fractions of nitrogen were significantly smaller than in melanosomes and only indicated a small fraction of proteins. In contrast, the phosphorus mole fraction was significantly larger indicating the presence of significant amounts of phospholipids. Copper and zinc mole fractions were larger than 0.1 at% in the melanosomes, but were below the detection limit in the lipofuscin granules. Compared to melanosomes of monkeys and rats analyzed beforehand, human retinal pigment epithelium melanosomes contained the highest amount of zinc, which even exceeded the calcium mole fraction. Trace elements like zinc are of great importance for metabolism and anti-oxidative mechanisms and also play a role in the progression of age related macular degeneration. They can now be investigated by quantitative analytical electron microscopy.

Retinal pigment epithelium pigment granules stimulate the photo-oxidation of unsaturated fatty acids

The cellular pigments of the retinal pigment epithelium (RPE) have been shown to catalyze free radical activity, especially when illuminated with visible or ultraviolet light. This activity is sufficient to cause photooxidation of several major cellular components. The present investigation determined the relative ability of melanin, lipofuscin, and melanolipofuscin granules isolated from human and bovine eyes to oxidize polyunsaturated fatty acids, specifically linoleic and docosahexaenoic acids. The dark reactivity as well as the light-stimulated reactions were determined. The production of hydroperoxide derivatives of the linoleic and docosahexaenoic acids were determined by NADPH oxidation coupled to the activity of glutathione peroxidase, and also by production of thiobarbituric acid reactive substances. All RPE pigment granules stimulated fatty acid oxidation when irradiated with short wavelength (<550 nm) visible light, with the melanosomes exhibiting the greatest light-induced activity. Only lipofuscin granules, however, caused peroxidation of fatty acids in the dark. These findings provide additional support for the role of RPE pigments in “blue light toxicity” as well as indicating that accumulation of lipofuscin may contribute to increased photooxidation in the aging RPE.

Changes in the Aging Rat Retina

To characterize aging as a factor responsible for structural changes, the retinae of 47 Wistar-derived albino rats and 50 pigmented rats of the Norway and BDE (Han) strains between the ages of 1 and 36 months were examined by light and electron microscopy and analyzed for changes in cell densities. In all 3 rats strains there was an overall decline in nuclear densities of outer layer nuclei by 38-50% and inner layer nuclei by 27-33% between the ages of 1 and 27 months. Over the same age range, the ganglion cell loss was comparable to the decline in the inner nuclear layer. Neuronal cell death occurred at all ages and was more pronounced in albino rats. Moreover, in albino rats, cones were more resistant than rods to destruction by age and ambient light. As age-related ultrastructural changes in the retinal pigment epithelium (RPE) (1) a substantial accumulation of lipofuscin; (2) an apparent thickening of the basement membrane; (3) greatly enlarged and pleomorphic basal infoldings, and (4) shorter apical microvilli were encountered. Besides mature stage IV melanosomes 27-month-old BDE (Han) and 36-month-old Norway rats also revealed stage III melanosomes. Tyrosinase activity in peripheral RPE cells could be demonstrated in Norway rats up to 12 months old and BDE (Han) rats up to 24 months old. Characteristics of RPE cells in old rats of these two pigmented strains were also melanolysosomes, melanophagolysosomes and melanolipofuscin granules. Age-related changes in retinal vessels were found in the superficial and deep capillary network and consisted of a 2- to 3-fold increase in thickness of the capillary basement membrane.

Histopathologic Effects of Mitomycin C After Trabeculectomy in Human Glaucomatous Eyes With Persistent Hypotony

We evaluated the histopathologic findings in seven patients who underwent surgical revision of the filtration site after trabeculectomy with mitomycin C because of persistent hypotonous maculopathy. Light microscopic examination of subconjunctival tissue and sclera demonstrated hypocellularity of fibroblasts and disruption of the normal architecture. Tissue fragments at the margin of the bleb wall demonstrated scarring and contained multiple fibroblasts. Additionally, we investigated the histopathologic changes in an eye obtained from a patient who died one week after a trabeculectomy with mitomycin C. Transmission electron microscopy showed myelin figures, increased melanolipofuscin granules, vacuolated cytoplasm, and disrupted mitochondria of the ciliary body epithelium underlying the site of mitomycin C application. On the basis of these findings, both overfiltration because of tissue disorganization of the filtering bleb and aqueous hyposecretion because of ciliary body toxicity might be involved in the causes of persistent hypotony after mitomycin C trabeculectomy.

Autosomal-dominant Fundus Flavimaculatus: Clinicopathologic Correlation

The authors report the first clinicopathologic study of autosomaldominant fundus flavimaculatus with late-onset atrophic macular degeneration in a 62-year-old man. Results of histopathologic examination disclosed the retinal pigment epithelium (RPE) to be distended by a periodic acid-Schiff (PAS)-positive, acid mucopolysaccharide-negative material. Transmission electron microscopy showed marked accumulation of lipofuscin and melanolipofuscin granules within the RPE. The different modes of genetic transmission and ultrastructural heterogeneity suggest that fundus flavimaculatus is a clinical syndrome representing several genetically and mechanistically distinct disorders whose common endstage is a topographically similar accumulation of lipofuscin.

Ocular Clinicopathologic Correlation of Hallervorden-Spatz Syndrome with Acanthocytosis and Pigmentary Retinopathy

We studied the eyes of a 10-year-old girl with retinal degeneration, acanthocytosis, and normal betalipoprotein levels. The ophthalmoscopic pattern was characterized initially by a flecked retina and later by bone-spicule formation and "bull's-eye" annular maculopathy. On ultrastructural study, the retinal pigment epithelium varied in size and contained large, round single-membrane-bound aggregates composed of complex melanolipofuscin granules. Cells that had migrated into the outer retinal layers contained similar melanolipofuscin aggregates; these cells were identified as macrophages and correlated with the flecks and macular annulus seen on ophthalmoscopy. The cells around the retinal blood vessels contained normal melanin pigment, were identified as retinal pigment epithelial cells, and correlated with the bone spicule pigmentation found on ophthalmoscopic examination.

The organization of human retinal pigment epithelium in vitro

The in vitro growth characteristics of retinal pigment epithelium from post mortem human eyes have been described. Our studies demonstrated that only a small percentage of the RPE cells initially plated out enter the division phase 12–21 days after plating and give rise to confluent cultures. The duration of the initial lag phase and the size of the proliferative fraction are age-dependent; cultures established from younger donor eyes have a larger fraction of proliferating cells and enter the division phase earlier than those from older donor eyes. Using the melano-lipofuscin granules as cell markers, we identified two distinct cell populations in the confluent cultures. One population consisted of small, diploid, non-pigmented cells that were undergoing division; the second, larger, densely pigmented cells that appeared stationary. Electron microscopy studies confirmed that both cell populations are epithelial-like in ultrastructure and that the RPE cells in vitro retain many of their in vivo ultrastructural characteristics.