Ginical-Ulrrastructural Study of a Retinal Dystrophy

Ann H. Bunr-Milam,* Robert E. Kalina,* ond Roberta A. Pagon*j-

An ultrastructural and cytochemical study was performed on the retina and retinal pigment epithelium of an eye surgically enucleated for choroidal melanoma from an otherwise healthy 31-year-old man. The patient and his identical twin show a retinal dystrophy that, based on clinical appearance, visual fields, and electrophysiology, is most likely autosomal recessive retinitis pigmentosa. Rod and cone photoreceptors were reduced in numbers and outer segments were virtually absent in the region corresponding to the patient's poorest vision. In the region from approximately 20° to 60° (best field of vision), the outer segments of rods and cones were shortened and disorganized. The retinal pigment epithelium showed reactive changes in areas of most severe photoreceptor pathology, including reduplication, loss of melanin, increased melanolysosomes, and migration of individual cells into the retina. The acid phosphatase reactivity of both the retinal pigment epithelium and photoreceptor cells appeared normal, as were the photoreceptor cilia and inner layers of the retina. This study thus provides improved ultrastructural documentation of a relatively early case of retinitis pigmentosa that may provide a foundation for further functional studies aimed at elucidation of this enigmatic retinal dystrophy. Invest Ophthalmol Vis Sci 24:458-469, 1983

Recent ultrastructural studies of retinitis pigmen- Materials and Methods tosa (RP) have been performed upon postmortem specimens of either moderately advanced sex-linked The propositus, a 31-year-old white man of French RP1 or advanced, presumed autosomal dominant and English origin had noted delayed dark adaptation RP.2'3 Although the previous studies have provided for at least 9 years. At age 29 a choroidal tumor was new ultrastructural information on poorly under- found in the superior temporal quadrant of the left eye during a periodic eye examination. Over the next stood retinal dystrophies, each was compromised by 2 two years the mass increased markedly in size. postmortem autolytic changes or previous prolonged exposure of the patient donor to cytotoxic drugs for Our first examination of July 25, 1979 showed vi- treatment of malignant disease.1>3 This report presents sual acuity of the right eye 20/20 and the left eye 20/ the ultrastructure and cytochemistry of the retina and 100, with correction of moderate myopic astigma- retinal pigment epithelium of an eye surgically enu- tism. The refraction was: right eye, —3.50+ 1.25 X 142; left eye, -4.50 + 2.75 X 30. The eyes appeared cleated for choroidal melanoma from an otherwise normal except for the fundi, which both were lightly healthy 31-year-old man who, together with his iden- pigmented and showed midperipheral bone corpus- tical twin brother, shows a retinal dystrophy. Based cular retinal pigment alterations (Fig. 1). The periph- on the clinical appearance, visual fields and electro- eral macula in the area of the temporal vascular ar- physiology, the most likely diagnosis in our patient cades in each eye showed many faint, cream-colored and his identical twin is autosomal recessive RP. deep retinal dots that were approximately 50-100 Aim in diameter. The optic discs appeared normal, and the retinal vessels appeared normal or very From the Departments of Ophthalmology* and Pediatrics,t slightly narrowed. University of Washington School of Medicine, Seattle, Wash- The left eye showed a moderately pigmented cho- ington. roidal tumor in the temporal periphery. The mass Supported by NIH Research Grants Nos. EY-01311 and EY- was highly elevated and was more than 10 mm in 01730, a fellowship from Fight For Sight, Inc. (RAP), and grants from Research to Prevent Blindness, Inc. and the Lions Sight Con- diameter. The inferior fundus was occupied by a ret- servation Foundation of Washington and Northern Idaho. Some inal detachment with subretinal fluid that shifted su- facilities were supported in part by Public Health Service Grant periorly to reach the fovea and the inferior margin HD02274. of the mass when the patient assumed the recumbent Submitted for publication March 25, 1982. position. Reprint requests: Ann H. Bunt-Milam, PhD, Department of Ophthalmology RJ-10, University of Washington, Seattle, WA Goldmann perimetry (Fig. 2) showed a dense ring 98195. scotoma in the right eye. There was advanced visual

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field loss in the left eye due to the retinal detachment but some inferior field was preserved. Electroretin- ography4 of the right eye (Fig. 3) showed prolonged latency and decreased amplitude of the b-waves to single light flashes in both the dark-adapted and light- adapted state. In the dark-adapted state, there was no detectable response to a single blue flash; a single white flash had a b-wave latency of 48.4 msec (predicted 47.7 msec) and a b-wave amplitude of 75.0 nV (predicted 586.4 /*V). A flickering white light at 30 Hz gave a b-wave latency of 34.8 msec (predicted 29.2 msec) and a b-wave amplitude of 36.6 fiV (predicted 84.6 ixV). A single white flash in the light- adapted state gave a b-wave latency of 30.8 msec (predicted 28.8 msec) and a b-wave amplitude of 29.4 pV (predicted 122.4 MV). The patient and his twin brother were identical for Fig. 1. The retinal periphery of the propositus showed generally both HLA A and B antigens, 16 blood group antigens, light choroidal pigmentation and a bone spicule disturbance of the and 19 red blood cell antigens. The patient's twin retinal pigment epithelium (arrow). brother had noted delayed dark adaptation for 9 years, and a diagnosis of retinal dystrophy had been made at another institution. Our examination showed in phosphate buffer, tissue chopped at 40 nm and visual acuity of 20/20 in each eye with correction of reacted for the cytochemical demonstration of acid myopic astigmatism. The refraction was: right eye, phosphatase.5 Control specimens were processed -4.25 + 1.75 X 155; left eye, -6.25 + 3.50 X 30. The identically with omission of the B-glycerophosphate ocular fundi were similar to that of the propositus substrate. Following osmication, the specimens were with the exception of the tumor and retinal detach- processed for electron microscopy as above. ment. Visual fields and electroretinographic findings were indistinguishable from those of the right eye of Results the propositus. The patient's father was 70 years old and his Gross Examination mother, 65 years old. Neither had evidence of retinal The globe appeared normal with the exception of dystrophy by history, fundus examination, or visual a moderately pigmented mass (15 mm in diameter field testing. His parents were unavailable for further X 11 mm high) arising from the choroid in the su- evaluation. The patient's two brothers and three sis- perior temporal quadrant and a retina with fine, bone ters had no visual problems, nor did the seven sons corpuscular pigment that was detached inferiorly and of his sisters. Two maternal uncles had no history of temporally to the level of the fovea and inferior mar- visual difficulty. The patient's only child was an 18- gin of the mass. month-old boy who was not examined. The patient's twin had no children. Microscopic Examination Because of the increasing size of the tumor in a visually compromised eye, enucleation of the left eye Histologically the choroidal tumor was comprised of the propositus was performed on August 16, 1979. of spindle B cells with prominent nucleoli; there was The cornea was slit, the vitreous chamber was in- no scleral extension. The fovea was detached and jected through the pars plana with 2% paraformal- showed the typical ultrastructure of retinal detach- dehyde-2% glutaraldehyde in 0.18 M phosphate buffer, ment,6 including loss of photoreceptor outer segments and the globe was placed immediately into the same and migration of pigment and lipid-laden macro- fixative. Both attached and detached regions of the phages into the fluid-filledsubretina l space. The pho- retina were processed by routine procedures for elec- toreceptor layer was reduced to a single layer of cone tron microscopy. Semi-thin (1 /mi) sections were inner segments, most of which appeared normal, al- stained with Richardson's mixture; thin sections though a few had swollen mitochondria. The cone (1000 A) were stained with uranyl acetate and lead pedicles were atrophic with few synaptic vesicles and citrate and examined with an AEI 801 or JEOL 100S ribbons. The inner layers of the detached retina ap- electron microscope. Following overnight, fixation, peared normal but contained occasional pigment- some samples were washed overnight in 10% sucrose laden macrophages in the inner nuclear layer. The

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ILIW Ull m' Fig. 2. Goldmann perim- IJW 0 Mi M« ~ etry showed a dense ring r •_ "'* Ul * 1 scotoma in the right eye Ul • « *jm l¥ (upper) and advanced su- V •• perior field loss in the left eye (lower) due to inferior retina) detachment. Note preservation of some inferior fieldi n the left eye, corresponding to the regions of the retina studied by electron microscopy.

optic nerve head and choroid appeared normal, and were much shorter (2.0 ixm long) (Fig. 4) than corie no preretinal membrane7 was present. outer segments (approximately 12 ^m) found in this In the attached region of the retina extending out region of normal retinae (Bunt-Milam, unpublished). to approximately 20°, which corresponded to a region In scattered foci the retinal pigment epithelium (RPE) of severe loss of visual field, rod and cone inner seg- was reduplicated, usually with depigmentation of the ments were present in reduced numbers, but outer sclerad layer, and a pigmented RPE cell and/or mac- segments were, for the most part, absent. Occasional rophage appeared to be invading the retina. cone cells had very disorganized outer segments that In the region of the retina from approximately 20°

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A C

Fig. 3. Electroretinogra- phy showed prolonged im- plicit times and decreased amplitudes for the right eye. 50;JV 5QjuV All values are abnormal at a confidence level of at least 98% for our laboratory based I upon means and values pre- 100 200 msec 50 100 150msec dicted from age-matched normal subjects. A, Blue B flash, dark-adapted (normal, 189.9 MV). B, White flash, dark-adapted (normal, 586.4 fiV). C, 30 Hz flicker in dark- ness (normal, 84.6 fiV). D, White flash, light-adapted IOOJJV 50JJV (normal, 122.4 MV).

50 100 150 msec 50 msec

to 60°, corresponding to the patient's field of best density that was generally irregular in shape (Fig. 12). vision, rods and cones had shortened outer segments The external limiting membrane was prominent and with disorganized lamellae (Fig. 5). By light micros- contiguous to the apical surface of the RPE (Fig. 14). copy, the longest rod and cone outer segments found were 5 ^m and 2.5 /zm, respectively. Occasional phagocytosed outer segment tips were found in the RPE (Fig. 5, inset). Apical pyramid-shaped protruberances of the RPE were filled with melanin granules and enclosed individual short cone but not rod outer segments (Fig. 6). This appeared to represent an ac- centuation of the microvillus sheaths normally found around individual cone outer segments of normal length.8 The inner segments of the rods had swollen mitochondria and electron dense inclusions free in the cytoplasm with granular, heterogeneous contents (Figs. 7, 8), which were also prominent in the Miiller cell perikarya. An occasional rod inner segment (Fig. 9) appeared degenerate as evidenced by cytoplasmic densification, as well as glycogen inclusions.9 The cone inner segments contained the normal comple- ment of organelles (Fig. 10), and the connecting cilia of the rods and cones (Figs. 10, inset, 11), and the RPE (Fig. 10) appeared normal morphologically. The far periphery of the retina contained very few photoreceptor cells and showed hypertrophy of the Miiller cells (Fig. 12). The rods and cones lacked outer segments, although some inner segments appeared normal. Macrophages that had paler cytoplasm than Fig. 4. A, Light micrograph of superior retina, approximately the RPE cells and contained ingested melanin gran- 20° eccentricity. Note presence of rod and cone inner segments and occasional short cone outer segment (arrow). B, The retinal ules were found in the inner layers of the retina as pigment epithelium is reduplicated with depigmentation of some well as among the RPE cells (Fig. 13). Often a mac- of the more sclerad cells {*). Note apparent invasion of retina by rophage had a nuclear inclusion of medium electron a pigmented cell (open arrow) from the vitread layer (X630).

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Fig. 5. Electron micrograph from retina at approximately 50° eccentricity. Note short, disorganized cone outer segment but normal appearing inner segment (IS). The adjacent rod inner segment (R) contains swollen mitochondria but the retinal pigment epithelium (RPE) appears normal (XI 2,560). Inset: phagocytosed outer segment tips in the RPE (X26,400).

An occasional cell in the RPE was necrotic matrix was recognizable (Fig. 15). The photoreceptors (Fig. 14). also showed the normal distribution of acid phos- The RPE cells from all regions showed normal acid phatase in the Golgi region and occasional autopha- phosphatase activity and distribution, most promi- gosomes. The granular, electron dense inclusions in nently in the Golgi apparatus, in lysosomes, and at the rod inner segments were negative for acid phos- the periphery of melanin granules in which the linear phatase (Fig. 16). No enzyme activity was present in

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Fig. 6. Retina as in Figure 5. Electron micrograph illustrating apical pyramidal-shaped protruberances of the retinal pigment epithelium which are filled with melanin and enclose individual cone but not rod outer segments (X20,000).

the interphotoreceptor space. Control sections incu- sponses on ERG, and typical fundus appearance. The bated in substrate-free media showed no enzyme ac- mode of inheritance may be autosomal recessive. tivity. However, the advanced age of the patient's father at the time of his birth and the relatively mild clinical Discussion involvement are compatible with a new mutation for an autosomal dominant gene. Similarly, transmission The diagnosis of RP in our patient and his mono- of an X-linked recessive gene from the phenotypically zygotic twin is based on the clinical finding of pro- normal mother cannot be excluded.10 gressively abnormal dark adaptation, ring scotomata The ultrastructural findings correlate well with the on visual field testing, abnormal rod and cone re- patient's visual fields, in that in the region of loss of

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Fig. 7. The inner segments of the rods have swollen mitochondria and electron dense, granular inclusions (arrows). C, connecting cilium (x 8,610).

Fig. 8. Higher magnification of granular inclusions in a rod inner segment (X26.630).

Fig. 9. A, Dense rod inner segment which contains glycogen (G) and dense, granular inclusions (*) (X 10,030). B, Higher magnification of glycogen (G) (X26.630).

Downloaded from iovs.arvojournals.org on 09/30/2021 Fig. 10. A cone inner segment appears normal morphologically but lacks an outer segment and abuts the retinal pigment epithelium. Note hypertrophy of Miillercell processes (X6490). Inset: Cross section of normal appearing connecting cilium of cone at higher magnification (X54,360).

Fig. 11. Longitudinal section of normal appearing rod connecting cilium and basal body. The rod outer segment membranes (OS) are disorganized (X44,950).

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Fig. 12. The far periphery of the retina contains very few photoreceptor cells (R, rod inner segment) and shows hypertrophy of the Miiller cells (M). A macrophage with pale cytoplasm, pigment granules and characteristic nuclear inclusion (*) is found in the photoreceptor layer (X7.840).

vision due to RP (from fovea to 20°), the photore- deposits also resemble those interpreted as autopha- ceptors were decreased in number and had virtually gosomes in RP retinae,1-3 but the deposits here were no outer segments. The few remaining outer segments negative for acid phosphatase, suggesting that they were small and disorganized, although all connecting are not components of the lipofuscin or lysosome- cilia examined were morphologically normal. This autophagosome system. The acid phosphatase reac- stands in contrast to the suggestion11 that RP might tivity of the RPE cells appeared normal, although the represent one expression of a generalized cilium de- number of melanolysosomes seemed higher than nor- fect affecting other organs as well as the retina. In the mally found in RPE cells at this age.12 In contrast to region of the retina from approximately 20° to 60°, some previous studies,1314 there was no evidence of corresponding to the patient's fieldo f best vision, both extracellular acid phosphatase around the degener- rods and cones had outer segments, although they ating photoreceptors; this is in agreement with the were shortened and disorganized. The rod inner seg- cytochemical findings of Essner and Gorrin15 on the ments contained abnormal, electron dense inclusions dystrophic retina of the RCS rat. 12 that are similar but not identical to lipofuscin. These Pigmented cells of two types were found invading

Fig. 13. A pale macrophage lies in the vitread layer of reduplicated retinal pigment epithelium. N, pigment epithelium nucleus; M, macrophage nucleus (X7,430).

Fig. 14. Very rarely a necrotic cell with melanin granules free in the cytoplasm was found in the retinal pigment epithelium. E, junctions of external limiting membrane (X6,770).

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14 E,

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15 B Fig. 15. A-B, Retinal pigment epithelium which has been reacted for the demonstration of acid phosphatase activity. Note reaction product in lysosomes (L) and in interior of melanin granule (M) in which linear matrix is recognizable. Some melanin granules (double arrows) are rimmed by reaction product (A, XI 5,660; B, XI6,660).

Fig. 16. Tissue processed as in Figure 14. Note positive acid phosphatase reaction in Golgi apparatus (G) of retinal pigment epithelium and absence of reaction in dense, granular inclusion (arrow) of rod inner segment. M, Muller Cell microvilli; IS, swollen mitochondria in rod inner segment (XI 5,800).

the retina. Some appeared to be typical RPE cells that matogenous in origin15 or represent RPE cells that were in the process of migrating away from the RPE have undergone metaplasia. The RPE showed other layer. Others resembled macrophages with a paler reactive changes, including islands of reduplication cytoplasm and clusters of melanin granules within and loss of melanin, particularly in the sclerad layers. phagosomes. The macrophages may be local or he- These islands of reduplication appeared to corre-

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spond in size (~ 50-100 nm) and location to the clin- msec expected]), yellow-red light (31.39 msec [27.88 msec expected]), and blue green light (31.76 msec [23.32 msec ically observed, cream-colored deep retinal dots. No expected]). These ERG changes are not typical of the ma- other abnormal structures of the appropriate size and jority of women who are heterozygous for the X-linked distribution could be found that might correspond to retinitis pigmentosa gene as reported by Berson et al. (Am these deep dots. J Ophthalmol 87: 460, 1979) who found abnormalities of These observations of a well-preserved specimen B-wave amplitude to white light (dark-adapted) in 19 of 23 obligate heterozygotes. We continue to believe that our of RP from an otherwise healthy 31-year-old man 16 patient most likely has the autosomal recessive form of corroborate previous suggestions that the disease retinitis pigmentosa. results from a primary defect in the rod and cone photoreceptors. The outer segments in this case are References severely affected, although relatively normal inner 1. Szamier RB, Berson EL, Klein R, and Meyers S: Sex-linked segments appear to persist, with some actual loss of retinitis pigmentosa: Ultrastructure of photoreceptors and pig- photoreceptor somata. Morphologically, the RPE ment epithelium. Invest Ophthalmol Vis Sci 18:145, 1979. does not appear to be affected initially, appearing for 2. Kolb H and Gouras P: Electron microscopic observations of the most part normal in the region from 20° to 60° human retinitis pigmentosa, dominantly inherited. Invest where the photoreceptor outer segments show patho- Ophthalmol 13:487, 1974. 3. Szamier RB and Berson EL: Retinal ultrastructure in advanced logic changes. Of course, a primary metabolic defect retinitis pigmentosa. Invest Ophthalmol Vis Sci 16:947, 1977. in the RPE might not be manifest there initially but 4. Chatrian GE, Lettich E, Nelson TL, Miller RC, McKenzie RJ, rather be expressed early on as abnormal photore- and Mills RP: Computer assisted quantitative electroretinog- ceptor morphology and even photoreceptor death. raphy. I. A standardized method. Am J EEG Technol 20:57, The changes observed in the RPE in the more central 1980. 5. Etherton JE and Botham CM: Factors affecting lead capture and far peripheral retina (reduplication and migration methods for the fine localization of rat lung acid phosphatase. into the retina) may possibly be reactive in nature, Histochem J 2:507, 1970. perhaps in response to significant photoreceptor 6. Machemer R and Kroll AJ: Experimental retinal detachment death. This study thus provides improved ultrastruc- in the owl monkey. VII. Photoreceptor protein renewal in tural documentation of a relatively early case of RP normal and detached retina. Am J Ophthalmol 71:690, 1971. 7. Szamier RB: Ultrastructure of the preretinal membrane in ret- that may provide a foundation for further functional initis pigmentosa. Invest Ophthalmol Vis Sci 21:227, 1981. studies aimed at elucidation of this enigmatic retinal 8. Steinberg RH, Wood I, and Hogan MJ: Pigment epithelial dystrophy. ensheathment and phagocytosis of extrafoveal cones in human retina. Philos Trans R Soc Lond [Biol] 277:459, 1977. Key words: retinal, dystrophy, electroretinogram of retinitis 9. Kuwabara T and Cogan DG: Retinal glycogen. Arch Ophthal- pigmentosa, inherited human retinal dystrophy, retinitis mol 66:680, 1961. pigmentosa ultrastructure 10. Berson EL, Rosner B, and SimonoffE: Risk factors for genetic typing and detection in retinitis pigmentosa. Am J Ophthalmol Acknowledgments 89:763, 1980. 11. Arden GB and Fox B: Increased incidence of abnormal nasal The authors are grateful to David M. Smith, MD, for cilia in patients with retinitis pigmentosa. Nature 279:534, providing this specimen, D. F. Milam, MD, and M. J. Reeh, 1979. MD, for histopathologic consultation, R. Patterson, I. 12. Feeney L: Lipofuscin and melanin of human retinal pigment Klock, and D. Ichikawa for technical assistance, J. Foltz epithelium. Fluorescence, enzyme cytochemical, and ultra- and B. Clifton for photographic help, and J. Seng for sec- structural studies. Invest Ophthalmol Vis Sci 17:583, 1978. retarial assistance. 13. Burden EM, Yates CM, Reading HW, Bitensky L, and Chayen J: Investigation into the structural integrity of lysosomes in the Addendum normal and dystrophic rat retina. Exp Eye Res 12:159, 1971. 14. Ansell PL and Marshall J: The distribution of extra-cellular Following acceptance of this manuscript for publication, acid phosphatase in the retinas of retinitis pigmentosa rats. the patient's mother consented to be examined. Her fundi Exp Eye Res 19:273, 1974. showed no carrier signs of retinitis pigmentosa. A corneal, 15. Essner E and Gorrin G: An electron microscopic study of full-field ERG, using the same technique employed to eval- macrophages in rats with inherited retinal dystrophy. Invest uate the proband, showed no abnormalities in the dark- Ophthalmol Vis Sci 18:11, 1979. adapted state to single blue, red, or white flashes. In the 16. Mizuno K and Nishida S: Electron microscopic studies of hu- light-adapted state there was mild increase in B-wave la- man retinitis pigmentosa. Part 1. Two cases of advanced ret- tency to single flashes of white light (34.48 msec [31.05 initis pigmentosa. Am J Ophthalmol 63:791, 1967.

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