Retinal Pigment Epithelial Abnormalities in Fundus Flavimmaculatus

Retinal Pigment Epithelial Abnormalities in Fundus Aavimaculatus

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A Ught and Electron Microscopic Study RALPH C. EAGLE, JR., MD, * ALFRED CLUCIER, MO,t VITALIANO B. BERNARDINO, JR., MD:j: MYRON Y ANOFF, MD*

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Abstract: Light, fluorescent, and scanning and transmission electron microscopic examinations of two postmortem eyes from a 24-year-old man with well-documented fundus flavimaculatus with atrophic macular degeneration revealed striking abnormalities in the retinal pigment epithelium (RPE). Beginning near the equator, scanning electron microscopy demonstrated a progressively marked heterogeneity in the size of the RPE cells. Surrounded by a fairly regular mosaic of relatively normal appearing cells, enormously enlarged hypomelanotic cells measuring up to 80 microns in diameter occurred in irregular aggregates that became more prevalent posteriorly. Diffusely and intensely PAS-positive, the RPE was packed with a granular substance with ultrastructural, autofluorescent, and histochemical properties consistent with an abnormal form of lipofuscin. The greatest concentration of lipopigment was noted posteriorly. Stains for acid mucopolysaccharide were only mildly positive. The clinical and fluorescein angiographic manifestations of fundus flavimaculatus are consistent with accumulation of a lipofuscin-like substance in the RPE. The massive amounts of lipopigment encountered in this young individual suggest that disordered lipopigment metabolism may playa major role in the pathogenesis of this retinal pigment epithelial disorder. [Key words: fundus flavimaculatus, histopathology, hereditary macular degeneration, lipofuscin, retinal pigment epithelium, scanning electron microscopy, Stargardt's disease, transmission electron microscopy.] Ophthalmology 87:1189-1200,1980

We report the histopathologic and electron microscopic findings in two postmortem eyes from a 24-year-old white man with welldocumented fundus flavimaculatus and Stargardt's maculopathy. While largely confirming

the previous findings of Klien and Krill,I-2 our observations demonstrate that the presence of small amounts of acid mucopolysaccharide within the retinal pigment epithelium is greatly overshadowed by the massive intracellular ac-

From the Laboratory of Ophthalmic Pathology, Department of Ophthalmology, University of Pennsylvania School of Medicine: Philadelphia, and the Scheie Eye Institute: Philadelphia; The Retina Service, Wills Eye Hospital ,t Ph iladelphia ; and the Ophthalmic Pathology Laboratory, Wills Eye Hospital,:!: Philadelphia.

1980, and at the Wills Eye Hospital Clinical Conference, April 1980.

Presented in part at the Association for Research in Vision and Ophthalmology Meeting, Orlando, May

0161-6420/80/1200/1189/$01.10

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Supported in part by the Gretel and Eugene Ormandy Teaching and Research Fund , Scheie Eye Institute. Reprint requests to Ralph C. Eagle , Jr. , MD, Ophthalmic Pathology Laboratory, Scheie Eye Institute, 51 North 39th Street, Philadelphia, PA 19104

© American Academy of Ophthalmology

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cumulation of an abnormal lipofuscin-like substance. The massive accumulation and. distribution of this substance within the retinal pigment epithelium correlate well wit~ th~ clinical manifestations and course of thls dlsease and is consistent with current concepts of retinal pigment epithelial physiology and lipopigment biology.

CASE REPORT

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A 21-year-old white man was evaluated by one of the authors (ACL) in December 1975. During the preceding two Years, the patient had noted a slight decrease in central vision and some difficulty in night vision. The visual acuity was 20/25 bilaterally with a minor myopic correction. Results of the ocular examination were normal except for the fundi. Ophthalmoscopic examination revealed variably sized, irregular, linear to pisciform, yellowish-white opacities. in the deep retinal layers throughout the postenor pole (Fig O. In the fovea, an annular zone of retinal pigment epithelial atrophy was present. The optic disc, retinal vessels, and peripheral fundus appeared normal. Intraven~us fl~ores­ cein angiography revealed a generahzed dlffuse hypofluorescence of the choroidal pattern (Fig 2). In the fovea, a ring of hyperfluorescence corresponded to the zone of pigment epithelial atrophy. The yellowish-white flecks were generally hypofluorescent. The clinical diagnosis w~s fundus flavimaculatus with Stargardt's atrophlc macular degeneration. The patient was in good general health. Past history showed no abnormality except a previous renal stone. One of his five siblings, an older sister, has fundus flavimaculatus with severe Stargardt' s maculopathy. Her best corrected visual acuity on examination in April 1980 at the age of 26 years was 20/300 (right eye), 20/200 (left eye). There was no additio~al family history of blindness or nonrefr~c!lve ocular disease and no history of consangUlmty. Early in November 1978, the patient was examined for cosmetic contact lenses at the Pennsylvania College of Optometry. Although approximately 8-degree pericentral scotomata were present on visual field examination, the visual acuity was 20/30 bilaterally. On Thanksgiving Day 1978, a motor vehicle .accide~t sustained by the patient resulted m multlpIe traumatic injuries to the head, thorax, and abdomen including transection of the brain stem and an avulsion laceration ofihe aorta, resulting in death. At his family's request, his eyes were forwarded to the Eye Bank of Philadelphia. After removal of the corneas for transplantation purposes, the globes were fixed in neutral buffered formaldehyde.

MATERIALS AND METHODS Representative portions of each previously opened, normal sized globe were processe~ for light (including serial sections), ultraviOlet fluorescent, and scanning and transmission electron microscopy. Paraffin-embedded sections (7-microns) were stained with hematoxylin and eosin; periodic acid-Schiff (PAS) before and after incubation with diastase, hyaluronidase and neuraminidase; alcian blue (pH 2.5) and modified Mowry's colloidal iron stains for acid mucopolysaccharide before and after ovine hyaluronidase; the Fontana stain for melanin· the Oil Red-O and Kinyoun' stech" niques for lipofuscin; an~ the Prus~ian blue stain for iron. Deparaffimzed unstamed sections as well as saline-suspended flat mounts of retin~l pigment epithelium and choroid, were examined by ultraviolet fluorescent microscopy using a Zeiss universal photomicroscope with high intensity mercury or halogen light sour~es and either a dark-field condenser and ZelSS BG-12 excitor and #47, 50, GG-14 or OG-53 barrier filters, or with vertical illuminator III RS for epi-excitation (FITC excitor barrier filter and reflector combination). In addition, 8micron paraffin sections were prepared from paraffin blocks of nonocular tissue prepared during the general autopsy. Sections were stained with hematoxylin and eosin, and PAS . Deparaffinized sections were examine~ with the fluorescent microscope. Representative samples of wet tissue from peripheral and posterior portions of the retinal pigment epithelium were taken from the calottes and processed for scanning and transmission electron microscopy. Specimens for scanning were prepared. by peeling the retina off the surface of the retmal pigment epithelium, dehydrat.ing i~ gr~d~d acetones and critical point drymg wlth hqUld CO 2 • After sputter coating with gold, the mounted specimens were examined with the Phillips SEM 500 scanning electron microscope. The formaldehyde-fixed tissue was processed for transmission electron microscopy by transferring initially to it glutaraldehyde-formaldehyde solution, post-fixing with os~ium tetroxide, and embedding in epon. One-mlcron orientation sections were stained with toluidine blue. Thin sections stained with lead citrate and uranyl acetate were examined with the Hitachi HU-12 transmission electron microscope.

RESULTS Macroscopically, the only abnormality noted was an irregular depigmentation of the retinal pigment epithelium posteriorly and scattered

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Fig 1. Top left. characteristic yellow-white flecks and annular zone of retinal pigment epithelial atrophy in the fovea are seen in fundus photograph taken three years prior to the patient's death. Fig 2. Top right. on fluorescein angiography, visibility of the choroidal circulation is largely abolished (" sign of choroidal silence"). Ring of hyperfluorescence corresponds to foveal pigment epithelial atrophy. Fig 3. Middle, peripheral retinal pigment epithelium shows prominent line of apicalized pigment. Cells are uniform in size. Outer segments of artifactitiously disrupted retina remain adherent to pigment epithelium (hematoxylin and eosin, original magnification x 400). Fig 4. Bottom, cells of posterior retinal pigment epithelium vary markedly in diameter and melanin content. Retina is artifactitiously disrupted (hematoxylin and eosin, original magnification x400).

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Fig 5. Posterior retinal pigment epithelium . Stain for melanin demonstrates greater concentration of pigment in smaller cells seen on the right. (Fontana, original magnification

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pigment clumping, best appreciated after peeling off portions of the semi-opaque fixed retina. A high-powered dissecting microscope revealed markedly enlarged pigment epithelial cells within the zones of hypopigmentation. By light microscopy, the retinal pigment epithelium was totally absent in the region of the fovea. Bruch's membrane appeared normal, but the choriocapillaris was atrophic. Overlying the zone of retinal pigment epithelial atrophy, the photo receptors and outer nuclear layer of the retina could not be identified. The normal outer layers were replaced by a layer of cells that contained abundant eosinophilic cytoplasm and enlarged plump moderately vesicular nuclei, presumably glial cells. Scattered foci of pigmented macrophages were noted within the glial scar. Peripheral to the macular scar, the choriocapillaris, choroid, and retina appeared normal. Although retinal pigment epithelial abnormalities were present throughout .the eye , comparison of the posterior and peripheral retinal pigment epithelium revealed striking differences. The cells of the peripheral retinal pigment epithelium were mildly enlarged and fairly uniform in size. The most striking feature was a marked apical condensation of the neuroepithe~ . lial melanin granules (Fig 3). The melanin granules were confined to approximately the apical fifth of the pigment epithelial cells, forming a prominent scalloped pigment line. Posteriorly, there was a marked variability in the diameter of the retinal pigment epithelial cells (Fig 4). Relatively normal pigment epithelial cells occurred adjacent to aggregates of markedly enlarged cells. The line of apicalized pigment was not as prominent posteriorly as . peripherally. The Fontana stain for melanin confirmed that the apicalized granules were melanin. Although the melanin content of the posterior retinal pigment epithelium was generally diminished, the degree of hypopigmentation was variable. Aggregates of nearly

amelanotic enlarged cells were seen adjacent to cells that contained pigment (Fig 5). In general, smaller cells contained more melanin than larger cells. Posteriorly, scattered large cells with histochemical staining characteristics identical to the retinal pigment epithelium were noted in the subretinal space (Figs 4, 7). On the basis of size, some of these cells appeared to represent desquamating pigment epithelial cells rather than macrophages. Posteriorly, the cytoplasm of the retinal pigment epithelium appeared somewhat smudged and glassy with an increased eosinophilia. Large intracellular vacuoles filled with eosinophilic, PAS-positive granules were found occasionally within the pigment epithelium. No drusen of Bruch ' s membrane were present. The most striking finding histochemically was the marked positivity of the retinal pigment epithelium when stained with the PAS technique. Both peripheral and posterior retinal pigment epithelium were intensely PASpositive (Figs 6, 7). Digestion with diastase, neuraminidase, and hyaluronidase failed to alter this positivity. Both Kinyoun's and the Oil Red-O stains for lipofuscin were positive. Stains for acid mucopolysaccharide were minimally positive in the retinal pigment epithelium and moderately positive in the choroid (Fig 8). Treatment with hyaluronidase extinguished the positivity, suggesting that the retinal pigment epithelium and the choroid contained hyaluronic acid (Fig 9). The Prussian blue stain for iron was negative. Ultraviolet fluorescent microscopy of deparaffinized unstained sections (7-microns) using transillumination revealed an intense persistent autofluorescence of the entire retinal pigment epithelium. Large amounts of granular autofluorescent pigment were noted in the retinal pigment epithelium when pigment epithelial flat mounts were examined by epi-illumination. Peripherally, the retinal pigment epithelial mosaic was fairly uniform (Fig 10). The auto-

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Fig 6. Top, the peripheral retinal pigment epithelium is intensely PAS-positive (PAS, original magnification x 400). Fig 7. Center, intense PAS-positive reaction characterizes posterior retinal pigment epithelium. Desquamating cells in subretinal space have identical histochemical properties (PAS, original magnification x 400). Fig 8. Bottom, colloidal iron stain reveals minimal to moderate amounts of acid mucopolysaccharide in pigment epithelium and choroid. Adherent photoreceptor matrix is strongly positive. (AMP, original magnification x 400).

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Fig 9 . Tup . incubation with hyaluronidase extinguishes positive reaction in RPE and choroid suggesting that acid mucopolysaccharide is hyaluronic acid . Hyaluronidase resistant photoreceptor matrix AMP serves as built-in control (AMP after hyaluronidase , original magnification x 400). Fig 10. Center , UV fluorescent microscopy of peripheral retinal pigment epithelial flat mount reveals large amount of autofluorescent pigment which is obscured partially by melanin. The epitheli a l mosaic is fairly uniform (UV fluorescent micro scopy , epi-excitation , original magnification x 390) . Fig 11. Bottom , marked variation in cell diameter and enormously enlarged cells are seen in flat mount of posterior retinal pigment epithelium. Large quantities of autofluorescent pigment are present. Melanin content is sparse (UV fluorescent microscopy , epi-excitation, original magnification x 390).

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fluorescent pigment was partially obscured, however, by an uneven distribution of melanin. Posteriorly, markedly enlarged retinal pigment epithelial cells were present, and the concentration of melanin was greatly diminished (Fig 11). Although autofluorescent, the enlarged cells were largely devoid of melanin. The average diameter, as well as the proportion of large retinal pigment epithelial cells, increased with progression toward the posterior pole. By scanning electron microscopy (SEM), the peripheral retinal epithelial mosaic appeared fairly uniform. Only occasional cells with a mildly increased diameter were noted. As one progressed posteriorly, however, the epithelial mosaic became more irregular. Posterior to the equator, focal aggregates of enormously enlarged pigment epithelial cells were seen within the mosaic of relatively normal-sized cells (Fig 12). These aggregates of large cells were irregular in shape with a generally angular rather than circular configuration. As one moved posteriorly, the proportion of large cells increased dramatically. It was estimated that adjacent to the optic nerve at least 50% of the surface of Bruch's membrane was covered by large cells. Peripherally, the retinal pigment epithelial cells averaged approximately 14 microns in diameter, while posteriorly, cells measuring up

to 83 microns in diameter were noted. On the basis of cell counts performed on scanning micrographs taken at identical magnifications, the density of cells posteriorly was only approximately 25% of that seen in the periphery. In several instances, suboptimal fixation, as well as fortuitous artifact, permitted examination of superficial cellular contents (Fig 13). Large ovoid granules consistent with neuroepithelial melanin were present on the apical surface of smaller cells. Such granules were seen infrequently on the surface of the relatively smooth large cells. These latter cells contained smaller spheroidal bodies consistent with lipofuscin-like material. Transmission electron microscopy demonstrated a massive accumulation of secondary lysosomal material in the cytoplasm of the retinal pigment epithelial cells (Figs 14, 15). Although heterogeneity and lesser density distinguished most of this material from the normal lipofuscin of aging human eyes, a majority of electron microscopists consulted thought the substance was an abnormal form of lipofuscin. The melanin granules and other organelles of the peripheral retinal pigment epithelium were displaced to the periphery of the cells by the massive amount of lipofuscin-like material (Fig 14). Posteriorly, the accumulation of abnormal

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Fig 13. Small intact pigment epithelial cell (top left) and large artifactitiously ruptured cell (top right) are examined under higher magnification below. Numerous oval neuroepithelial melanin granules stud apical surface of small cell (bottom left). Cytoplasm of large ruptured cell contains numerous spheroidal bodies. Only rare melanin granules are seen (bottom right); (top lefi and right, SEM, original magnification x 1,250; bottom left and right, SEM, original magnification x 5,000).

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lipofuscin was greater than peripherally (Fig 15). Here, only scattered melanin granules were present and the large quantities of abnormal pigment made it difficult to discern cellular detail. Scattered phagosomes containing degenerating photoreceptor outer segments were present, documenting the phagocytic capacity of the retinal pigment epithelium. The foveal scar was not examined. Elsewhere, Bruch's membrane and the choriocapillaris appeared normal. Light and fluorescent microscopy revealed substantial amounts of PAS-positive and autofluorescent pigment in the patient's cardiac muscle, renal tubules, and central nervous system. The amount of pigment was not, however, qualitatively greater than that found in several age-matched controls.

DISCUSSION The term, fundus flavimaculatus, was ongInally applied by Franceschetti to a group of pa-

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tients who had a heredomacular degeneration that was characterized by ill-defined, yellow or yellow-white, oval, linear or pisciform spots located in the deep retinallayers. 3 .4 Now thought to be part of Stargardt's disease,5-7 fundus flavimaculatus commonly presents as an insidious bilateral loss of vision secondary to progressive atrophic macular degeneration that starts in the fovea and spreads centrifugally. The evanescent yellow spots characteristically form a garland that surrounds the enlarging zone of retinal pigment epithelial and choriocapillary atrophy. As the disease progresses, new flecks form peripherally as older posterior flecks resorb. Fluorescein angiography in early cases characteristically shows a generalized hypofluorescence and obscuration of the underlying choroidal pattern. 8- 9 This appearance which has been termed the "sign of choroidal silence"8 is thought to result from an increased opacity of the retinal pigment epithelium. Fresh flecks are initially hypofluorescent but may progress to hyperfluorescence with partial or complete resorption. l ,6

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Fig 14. Cytoplasm of peripheral retinal pigment epithelial cell contains massive accumulation of abnormal lipofuscin which has displaced melanin granules and other organelles. Bruch's membrane and choriocapillaris appear normal (TEM, original magnification x5,350).

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Hyperfluorescence secondary to extensive retinal pigment epithelial degeneration is seen within the enlarging zone of macular degeneration. Electrophysiologic tests (electroretinograms and electro-oculograms) are usually abnormal only in the late stages of the disease when the retinal pigment epithelial, choriocapillary, and secondary retinal atrophy are widespread. 6 To our knowledge, only one eye with this disease has been previously examined histopathologically, and that eye had been traumatized. 1,2 Our light microscopic findings generally are in agreement with those reported by Klien and Krill. They noted that the entire retinal pigment epithelium was "packed with PAS-positive granules." The stain for acid mucopolysaccharide illustrated in their color Fig 15F would be considered minimally positive by most ophthalmic pathologists. Yet, they concluded that a hyaluronidase-sensitive acid mucopolysaccharide (presumably hyaluronic acid) localized in the inner-half of the retinal pigment epithelial cells was the pathologic material responsible for the characteristic fundus manifestations of this disease. Acid mucopolysaccharide (such as vitreous hyaluronic

acid and photoreceptor matrix AMP) is transparent, not yellow and does not block fluorescence. Also, the predominantly apical intraepithelial locus of the AMP is inconsistent with other histologic findings such as the apicalization of melanin granules. Klien and Krill, therefore, seem not to have appreciated the significance of the massive amounts of PAS-positive material stored in the retinal pigment ·epithelium. Using additional histochemical techniques, ultraviolet fluorescent microscopy, and transmission electron microscopy, we have demonstrated that this intense reaction is due to the massive intracellular accumulation of a lipofuscin-like substance. Lipofuscin is a yellow-brown "aging" or " wear and tear" pigment that accumulates in many organs as the body ages. The substance is especially prominent in cardiac muscle, renal tubules, certain portions of the central nervous system, and the retinal pigment epithelium. 1O- 12 A recent study has demonstrated that the lipofuscin content of the retinal pigment epithelium increases with age and topographically parallels the density of retinal rods with the greatest concentration noted posteriorly. 13 Probably derived from the peroxidation of

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Fig 15. Right. larger amounts of abnormal lipofuscin are present in the pigment epithelium posteriorly. Top left and bottom left. cytoplasm contains scattered melanin granules and phagosomes and numerous granules of abnormal lipofuscin seen under higher power below. Many abnormal lipofuscin granules have multiple dense cores surrounded by finely granular matrix . Phagosome contains degenerating outer segment (right: TEM, original magnification x 5,250; top left: TEM, original magnification x 12,600; bottom left: TEM, original magnification x 26,400).

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polyunsaturated fatty acids in cell membranes, lipofuscin is thought to represent nondegradable intracellular "waste products" resistant to further lysosomal enzymatic degradation.14 In the retinal pigment epithelium, accumulation of the pigment is thought to be related to the continual phagocytosis and digestion of photoreceptor outer segments. 12 • 15 It has been suggested that the gradual lifelong accumulation of lipofuscin within amitotic or slowly dividing tissues (such as the retinal pigment epithelium) may ultimately lead to a mechanical disruption of intracellular organization, cellular dysfunction and death. 13 Excessive amounts of lipofuscin-like material / have been demonstrated previously within the retinal pigment epithelium in several human ocular disorders. 16-20 The foveal retinal pigment epithelium in a man with dominantly inherited retinitis pigmentosa contained an increased concentration of this pigment. 16 Large quantities of an abnormal lipofuscin recently have been demonstrated in Best's vitelliform degen-

eration. 17 A five-fold increase in fluorescent pigment has been reported in a case of senile choroidal dystrophy.13 We also have observed grossly enlarged, PAS-positive, autofluorescent cells adjacent to the foveal zone of retinal pigment epithelial atrophy in an eye obtained at autopsy from an elderly man with central areolar choroidal dystrophy.18 Although the degree of pigment epithelial involvement in this case was much less severe, the histologic picture was quite similar to that seen in fundus flavimaculatus. Extensive accumulation of lipopigment, as well as focal hypertrophy of retinal pigment epithelial cells, is also a prominent finding in central progressive retinal atrophy, a hereditary pigment epithelial dystrophy of dogs that may be a canine counterpart of fundus flavimaculatus. 19 Characteristic areas of hypofluorescence seen on fluorescein angiography in other hereditary chorioretinal or macular diseases also may result from deposits of lipofuscin or a similar material. 20 Indeed, lipofuscin or similar lipopigments may play a

greater role in determining the ophthalmoscopic or fluorescein angiographic manifestations of heredodegenerative disorders than previously recognized. We believe that the clinical findings and course, as well as the histopathologic manifestations of fundus flavimaculatus, are readily explained by the massive accumulation and characteristic distribution of an abnormal lipofuscin-like substance within the retinal pigment epithelium. The generalized choroidal hypofluorescence seen in early cases, as well as the hypofluorescence offresh flecks , are readily explained by the well-known ability of lipofuscin to absorb blue excitatory wavelengths during fluorescein angiography. The aggregates of enormously enlarged retinal pigment epithelial cells seen posteriorly have a size and configuration that approximate those of the yellowwhite flecks seen clinically. Presumably, these aggregates would appear lighter because the large cells have a decreased melanin content. The relatively greater content of apicalized melanin in the intervening smaller cells accentuates this picture. Desquamating pigment epithelial cells or macrophages in the subretinal space may contribute further to the flecked retinal appearance. Initially hypofluorescent, flecks may become hyperfluorescent as large "constipated" epithelial cells degenerate and desquamate. Probably, fluorescein is able to enter the damaged retinal pigment epithelium cells and stain them. Paralleling the distribution of normal lipofuscin within the retinal pigment epithelium, retinal pigment epithelial atrophy begins in the fovea and spreads centrifugally with time. Because the primary defect most probably resides in the retinal pigment epithelium, atrophy of the choriocapillaris and the outer retina seen within the zone of absent pigment epithelium appears to be a secondary phenomenon. The accumulation of a lipofuscin-like substance within the retinal pigment epithelium would seem to be a significant factor in the pathogenesis of fundus flavimaculatus. Theoretically , the most likely cause of the accumulation is an inability of the retinal pigment epithelium to digest properly photoreceptor outer segments. The failure to demonstrate increased quantities of autofluorescent pigment in our patient's non-ocular tissues suggests that the basic metabolic defect is confined to the retinal pigment epithelium. It is possible, however, that fundus flavimaculatus could be an otherwise unrecognized benign systemic disorder whose ocular manifestations result from an unfortunately heavy load of lipid substrate in a tissue that has a critical sensory function. Experimental data suggest that the retinal pigment epithelium may be particularly sensitive to the

accumulation of lipofuscin . 21 - 23 Increased amounts of lipofuscin have been demonstrated in the retinal pigment epithelium of antioxidant-deficient dogs,21 rats,22 and monkeys.23 The accumulation of pigment is greater in the retinal pigment epithelium than in the heart, kidney, or intestine. 22 In antioxidant-deficient animals, accumulation of autofluorescent pigment in the retinal pigment epithelium appears to correlate with a decrease in pigment epithelial melanin content. Although this experimental model may have no relevancy to human disease , * we believe that antioxidant metabolism should be studied in patients with fundus flavimaculatus.

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Tissue for transmission electron microscopy was processed by Linda Byam and Dr. Nan Pillsbury, Department of Surgical Pathology, Hospital of the University of Pennsylvania. Transmission electron microgra phs were reviewed by Drs. Ben S. Fine , Ramon L. Font , Mark O . M. Tso, Barbara W. Streeten , Kenneth R. Kenyon , and Lynette FeeneyBurns . Dolores Ventura and Nestor Menocal provided valuable technical assistance . Photographic prints and enlargements were made by James Stripling and Tim Bennet. Ultraviolet fluorescent microscopy was performed in the laboratories of Drs. John Rockey and Alan Laties. Dr. Lois Johnson performed the serum vitamin E and erythrocyte fragility determination . The manuscript was typed by Debbie Klock and Phyllis DiPietro.

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REFERENCES

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1. Klien BA, Krill AE. Fundus flavimaculatus : clinical, functional and histopathologic observations. Am J Ophthalmol 1967; 64:3-23. 2. Newell FW, Krill AE, Farkas TG. Drusen and fundus flavimaculatus: clinical functional and histologic characteristics. Trans Am Acad Ophthalmol Otolaryngol 1972; 76:88 - 100. 3. Franceschetti A. Ober tapeto-retinale Degenerationen im Kindesalter . Dritter Fortbildungs-kurs der Deutschen Ophthalmologischen Gesellschaft, Hamburg 1962 . In : Sautter H, ed. Entwicklung und Fortschrift in der Augenhielkunde . Stuttgart : Enke, 1963; 107 - 20. 4. Franceschetli A, Franc;ois J. Fundus flavimaculatus. Arch Ophthalmol (Paris) 1965; 25:505-30. 5. Stargardt K. Ober familiiire progressive Degeneration in der Maculagegend des auges . Albrecht von Graefes Arch Ophthalmol 1909; 71 :534-50.

• Serum vitamin E and erythrocyte fragility determinations were performed on blood drawn from the patient's involved sister. Both tests were within normal limits (vitamin E - 1.2 units; erythrocyte fragility by malondialdehyde - 113).

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6. Fishman GA. Fundus flavimaculatus: a clinical classification. Arch Ophthalmol 1976; 94:2061-7. 7. Noble KG, Carr RE. Stargardt's disease and fundus flavimaculatus. Arch Ophthalmol 1979; 97:1281-5. 8. Bonnin P, Passot M, Triolaire-Cotten M-TH. Le signe du silence choro'idien dans les degenerescences tapeto-retiniennes posterieures. In: De Lacy JJ, ed. International Symposium on Fluorescein Angiography. The Hague: Dr. W. Junk Publishers, 1976; 461-3. 9. Anmarkrud N. Fundus fluorescein angiography in fundus flavimaculatus and Stargardt's disease. Acta Ophthalmol 1979; 57:172-82. 10. Streeten BW. The sudanophilic granules of the human retinal pigment epithelium. Arch Ophthalmol 1961;

66:391-8. 11 . Feeney L, Grieshaber JA, Hogan MJ. Studies on

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human ocular pigment. In: Rohen JW, ed. The Structure of the Eye. Stuttgart: FK Schattauer-Verlag, 1965;

535-48. 12. Feeney L. Lipofuscin and melanin of human retinal

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pigment epithelium; fluorescence, enzyme, cytochemical, and ultrastructural studies. Invest Ophthai mol 1978; 17:583-600. 13. Wing GL, Blanchard GC, Weiter JJ. The topography and age relationship of lipofuscin concentration in the retinal pigment epithelium. Invest Ophthalmol Vis Sci

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14 . Tappel AL. Lipid peroxidation and fluorescent

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1978; 17:601-7. molecular damage to membranes. In: Trump BF, Ar-

15.

16.

17.

18. 19. 20.

stila AU, eds. Pathobiology of Cell Membranes. New York: Academic Press, 1975; 145-72. Young RW, Bok O. Participation of the retinal pigment epithelium in the rod outer segment renewal process. J Cell Bioi 1969; 42:392-403. Kolb H, Gouras P. Electron microscopic observations of human retinitis pigmentosa, dominantly inherited. Invest Ophthalmol 1974; 13:487 -98. Weingeist TA, Kotbrin JL, Watzke RC. Histopathology of Best's vitelliform macular dystrophy. ARVO Abstracts. Invest Ophthalmol Vis Sci April, 1980; Suppl: 258. Eagle RC Jr. Unpublished data. Aguirre GO, Laties A. Pigment epithelial dystrophy in the dog. Exp Eye Res 1976; 23:247-56. Hsieh RC, Fine BS, Lyons JS. Patterned dystrophies of the retinal pigment epithelium. Arch Ophthalmol

1977; 95:429-35. 21. Hayes KC, Rousseau JE Jr, Hegsted OM. Plasma tocopherol concentrations and vitamin E deficiency in dogs. J Am Vet Assoc 1970; 157:64-71. 22. Katz ML, Stone WL, Oratz EA. Fluorescent pigment accumulation in retinal pigment epithelium of antioxidant-deficient rats. Invest Ophthalmol Vis Sci

1978; 17:1049-58. 23. Hayes KC. Retinal degeneration in monkeys induced by deficiencies of vitamin E or A. Invest Ophthalmol

1974; 13:499-510.