Experimental Retinal Detachment in the Owl Monkey

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2. Kümmel, R. : Zur Anatomie der frischen Netzhautablösung. Arch. f. Augenheilk. 314:100, 1936. 3. Kronfeld, P. C. : Histologie appearance of recent retinal tears. Arch. Ophth. 13:779, 1935. 4. Hervouet, F. : L'anatomie pathologique du décollement de la rétine. Bibl. Ophth. 3:1, 1965. 5. Birch-Hirschfeld, A . and Inouye, T. : Experi-

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mentelle und histologische Untersuchungen über Netzhautabhebung. Arch. f. Ophth. 70:486, 1909. 6. Machemer, R. and Norton, E. W . D. : Experimental retinal detachment in the owl monkey. I. Methods of production and clinical picture, Am. J. Ophth. 66:388,1968. 7. Hogan, M. J. and Zimmerman, L. E. : Ophthalmic Pathology. Philadelphia, Saunders, ed. 2, 1962.

RETINAL DETACHMENT

IN T H E O W L

MONKEY

I I I . ELECTRON MICROSCOPY OF RETINA AND PIGMENT EPITHELIUM ARNOLD J. KROLL, M . D . AND ROBERT MACHEMER, M . D . * Miami, Florida

It has been shown in the preceding paper that experimental retinal detachment in the owl monkey produced characteristic histologic changes. In the present paper, we describe the electron microscopic characteristics of the retina and pigment epithelium in retinal detachment. 1

MATERIALS AND METHODS In a series of 14 owl monkeys (Aotus trivirgatus), retinas were experimentally detached as previously described. Eyes were enucleated, without regard to conditions of light- or dark-adaptation, at intervals ranging from one hour to 14 weeks. Control eyes were subjected to intravitreous hyaluronidase injection alone, or injection plus vitreous aspiration and reinjection. Areas of attached retina in eyes with partial detachment also served as controls. 2

From the Bascom Palmer Eye Institute, Department of Ophthalmology University of Miami School of Medicine. This investigation was supported in part by U S P H S Research Grants No. NB05918 and NB06841 from the National Institute of Neurological Diseases and Blindness, by the National Council to Combat Blindness, Inc. Grantin-Aid Special Fellowship No. F-211; by funds from Research to Prevent Blindness, Inc. and by the Florida Lions Eye Bank, Inc. Read in part before the 17th annual session of the New Orleans Academy of Ophthalmology, Symposium on Retina and Retinal Surgery, New Orleans, February 15, 1968, and before the Club Jules Gonin, Cambridge, England, April 2, 1968. •Visiting research fellow University Eye Clinic, Göttingen, Germany.

After enculeation, each globe was opened near the ora serrata. A portion of midperipheral retina and pigment epithelium remote from the retinal breaks was excised with a sharp razor blade. The specimens were then fixed in 2 % phosphate buffered osmium tetroxide, dehydrated in a graded series of ethanol, embedded in epon, and sectioned and stained for both phase-contrast and electron microscopy as described elsewhere. 3

OBSERVATIONS The detailed observations are recorded as legends which accompany the figures. In essence, experimental retinal detachment produced the following changes: The horizontally layered discs in the photoreceptor outer segments underwent marked degeneration. This included loss of horizontal orientation, fragmentation, and eventual atrophy. Macrophages appeared and phagocytized fragments of outer segments. The pigment epithelial lamellar inclusion bodies " disappeared. The apical surface of the pigment epithelial cells became convexly protuberant, and the apical processes increased in number, and became thickened. Melanin granules withdrew from the apical processes. Cystoid extracellular spaces of varying sizes appeared in the middle and inner retina. These spaces consisted of enlargement of extracellular spaces. They were bordered by the cytoplasmic membranes of contiguous retinal 4

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Fig. 1 (Kroll and Machemer). Control eye. Low-power survey of region of photoreceptor outer segments (os) and pigment epithelium ( p ) . Note close morphologic relationship of outer segments to apical processes (p) of pigment epithelial cells, n, nucleus of pigment epithelial cell. (Mark is equivalent to lu;x6,000.)

cells. Control eyes did not show these changes. COMMENT

of vitamin A in the pigment epithelium rises. In dark adaptation the amount of retinene in the retina rises and the amount of vitamin A in the pigment epithelium falls. In the present study, electron microscopy has shown that the photoreceptor outer segments degenerate following detachment of the retina. The outer segments contain the visual pigments and thus are the photosensitive portions of the retina. In addition, this study has shown that the lamellar inclusion bodies of the pigment epithelium degenerate and disappear following retinal detachment. Inclusion bodies are heterogeneous, membrane-bound cytoplasmic 8

As long ago as 1878, Kühne showed that a thin piece of porcelain slipped between the retina and pigment epithelium would prevent bleached retina from regaining its normal color. It is now well established that the processes of light and dark adaptation involve exchanges of vitamin A between the outer segments of photoreceptors and the pigment epithelium: that is, in light adaptation the amount of retinene (vitamin A aldehyde) in the retina falls, and the amount 7

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Fig. 2 (Kroll and Machemer). Control eye. Photoreceptor outer segment in radial section consists of stacks of flattened membranous saccules ( s ) enclosed by cytoplasmic membrane Cc). (Mark is equivalent to lu.; X20,000.)

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Fig. 3 (Kroll and Machemer). Control eye. Photoreceptor outer segment in flat section consists of circular structure with scalloped edges ( s ) enclosed by cytoplasmic membrane ( c ) . Comparing this with Figure 2 permits three-dimensional construction of outer segment as similar to stack of coins enclosed in closely fitting thin bag. (Mark is equivalent to lu.; X20,000.)

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Fig. 4 (Kroll and Machemer). Retinal detachment one week. Low-power survey of region of outer segments (cf. Fig. 1 ) . Some outer segments (os) retain relatively normal configuration. Others (arrow) contain saccules which are fragmented, loosely bound, vertically arranged, and irregular. In still others the cytoplasmic membrane has ruptured and discs have become swollen and globular. Inner segments (is) are morphologically normal. (Mark is equivalent to In; X7,000.)

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Fig. 5 (Kroll and Machemer). High-power micrograph of one region of Figure 4. Abnormally arranged and fragmented saccules (s) of outer segments still retain layered appearance in some cells. In other cells the cytoplasmic membrane has ruptured. (Mark is equivalent to In ; X26,000.)

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Fig. 6 (Kroll and Machemer). Retinal detachment, four weeks. In older detachments the outer segments become attenuated, devoid of saccules, cystic, or assume bizarre forms. (Mark is equivalent to lu ; X20,000.)

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Fig. 7 (Kroll and Machemer). Retinal detachment, four weeks. Low-power survey of region of outer segments showing presence of macrophages ( M ) in this layer. Latter appear within a few days after retinal detachment. (Mark is equivalent to In ; X6,000.)

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Fig. 8 (Kroll and Machemer). High-power micrograph of macrophage seen in Figure 7. Cell is in process of phagocytizing two portions of outer segment material ( O S ) . (Mark is equivalent to In; Xl5,000.)

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Fig. 9 (Kroll and Machemer). Control eye. Low-power survey of pigment epithelium. Cells contain several kinds of cytoplasmic structures including (1) inclusion bodies (ib) which consist of membranebound stacked flattened saccules, (2) melanin granules (mg) which are dense homogeneous elliptical bodies enclosed by a single membrane and (3) lipofuscin granules (lg) which are spherical and less dense. (Mark is equivalent to In; x6,000.)

structures within the pigment epithelium which measure approximately 1.5p; in diameter and consist of stacked, flattened saccules, not dissimilar to those of photoreceptor outer segments. " Inclusion bodies may be broken or detached portions of photoreceptor outer segments engulfed by the pigment epithelial cells. It is impressive that retinal detachment produces disintegration of only two structures, the outer segments of the photoreceptors and the lamellar inclusion bodies of the pigment epithelium. There is only one other experimental condition which is known to cause outer segment degeneration and disappearance of inclusion bodies—vitamin A deficiency. Could the two conditions have anything in common? It is conceivable that the separation of the photoreceptor and pigment epithelial layers in retinal detachment interferes enough with the exchange of vitamin A, and possibly other materials between them, that a situa4

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tion mimicking a localized deficiency of the vitamin is present. This is supported perhaps, by the observation that the photoreceptors in areas of flat detachment are much better preserved than in areas of high bullous detachment. The present work also suggests that the lamellar inclusion bodies may indeed be phagocytized portions of photoreceptor outer segments. Recently, it has been reported that prolonged exposure of albino rats to light caused a temporary marked increase in the number of inclusion bodies in the pigment epithelium associated with degenerative changes in the photoreceptor outer segments. It is not likely that ischemia resulting from the separation of the photoreceptors from their blood supply in the choroid could account for the observed changes. It is difficult to postulate that ischemia would selectively damage the outer segments only and not the photoreceptor cells proper. Besides, 1

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Fig. 10 (Kroll and Machemer). Control eye. High-power micrograph showing inclusion body (ib), melanin granules ( m g ) , and lipofuscin granules (lg) in pigment epithelium. The apical processes of pigment epithelial cells (p) are long, thin and in close contact with photoreceptor outer segments. The dense elliptical melanin granules may lie in the apical processes. Note structural similarity between outer segments and inclusion body. "* The function of the latter is not known, however inclusion bodies do disappear from the pigment epithelium in experimental vitamin A deficiency." (Mark is equivalent to lu,; X20,000.) 4

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Fig. 11 (Kroll and Machemer). Pigment epithelium in eye with detached retina, one day. Two degenerate inclusion bodies (ib) are present. Apical processes (p) are thickened and no longer contain melanin granules. In older detachments, inclusion bodies completely disappear. (Mark is equivalent to l|i; X20,000.)

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Fig. 12 (Kroll and Machemer). Pigment epithelium in eye with detached retina, eight weeks. Lowpower survey (cf. fig. 9 ) . The pigment epithelium is taller. Inclusion bodies are absent, but melanin and lipofuscin granules are present. The melanin granules have withdrawn from the apical processes (p) leaving a melanin-free apical zone. The apical processes are thickened and increased in number. The pigment epithelial cell apices are convexly protuberant, n, nucleus. (Mark is equivalent to lu,; X6,000.)

it has recently been shown that retinal ischemia produces swelling of mitochondria, endoplasmic reticulum, and cell cytoplasm. None of these changes were found in the present study. The appearance of cystoid spaces in the retina after retinal detachment also occurs in the human disease. ' The cause of the retinal hydration with detachment is unknown. It is possible that one function of the pigment epithelium and choroid is to keep the retina in a normal state of dehydration. The presence of a retinal break and subretinal fluid apparently interferes with this function. The blood supply to the retina does not seem to be the determining factor in cystoid space formation. The spaces form in the inner and 11

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middle retina where the blood supply from the central retinal artery is intact, and not in the outer retina. SUMMARY Electron microscopy of a series of experimentally detached retinas in the owl monkey (Aotus trivirgatus) revealed fine structural changes in the retina and pigment epithelium. The photoreceptor outer segments degenerated, lamellar inclusion bodies disappeared from the pigment epithelium, melanin granules withdrew from the pigment epithelial apical processes, and cystoid extracellular spaces appeared in the middle and inner retinal layers. Control eyes did not show these changes.

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Fig. 13 (Kroll and Machemer). Apical region of pigment epithelium in eye with detached retina, 2 weeks (cf. Fig. 10). There is a melanin-free zone of pigment epithelial apical processes. (Mark is equivalent to l u ; X20.000.)

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Fig. 14 (Kroll and Machemer). Retinal detachment, four weeks. Low-power micrograph of cystoid space in bipolar cell layer. Note borders of space (arrows) have no lining structure. Space is therefore enlargement of the extracellular space. (Mark is equivalent to lu ; X5,000.)

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Fig. 15 (Kroll and Machemer). Detached retina, eight weeks. Border of cystoid space may be smooth. It consists of the cytoplasmic membrane (c) of adjacent retinal cells. (Mark is equivalent to In; x20,000.)

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Fig. 16 (Kroll and Machemer). Detached retina, four weeks. Border of cystoid space may be highly irregular. It nevertheless consists of cytoplasmic membrane of adjacent retinal cells. Note enlargement of extracellular spaces. (Mark is equivalent to In ; X20,000.)

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ACKNOWLEDGMENTS

The authors acknowledge the capable technical assistance of Mr. Barry Davis and Mrs. Marcilia Halley.

REFERENCES 1. Machemer, R. : Experimental retinal detachment in the owl monkey. II. Histology of retina and pigment epithelium. Am. J. Ophth. 66:396, 1968. 2. Machemer, R. : Experimental retinal detachment in the owl monkey. I. Methods of production and clinical picture. Am. J. Ophth. 66:388, 1968. 3. Kroll, A . J. : Fine structural classification of orbital rhabdomyosarcoma. Invest. Ophth. 6:531, 1967. 4. Dowling, J. E. and Gibbons, I. R. : The fine structure of the pigment epithelium in the albino rat. J. Cell Biol. 14:459, 1962. 5. Bairati, A . J. and Orzalesi, N. : The ultrastructure of the pigment epithelium and the photore-

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ceptor-pigment epithelium junction in the human retina. J. Ultrastructure Res. 9:484, 1963. 6. Cohen, A . I. : Vertebrate retinal cells and their organization. Biol. Rev. 38:427, 1963. 7. Kühne, W . : On the photochemistry of the retina and on visual purple. Edited with notes by M i chael Foster. London, Macmillan, 1878. 8. Dowling, J. E . : Chemistry of visual adaptation in the rat. Nature 188:114, 1960. 9. Dowling, J. E. and Gibbons, I. R. : The Effect of Vitamin A Deficiency on the Fine Structure of the Retina. In: Eye Structure I. Symp., Smelser, G.(ed.), New York, Academic Press, 1961, p. 85. 10. Kuwabara, T. and Gorn, R. A . : Retinal damage by visible light—an electron microscopic study. Arch. Ophth. 79:69, 1968. 11. Kroll, A . J. : Experimental central retinal artery occlusion. Arch. Ophth. 79:453, 1968. 12. Hogan, M . and Zimmerman, L. E. : Ophthalmic Pathology, A n Atlas and Textbook. Philadelphia, Saunders 1962, ed. 2, p. 560. 13. Keith, C. G. : Retinal cysts and retinoschisis. Brit. J. Ophth. 50:617, 1966.

PENICILLIN-RESISTANT

CONJUNCTIVITIS W I T H

GONOCOCCAL

AMPICILLIN

GEORGE LINK SPAETH,

M.D.

Philadelphia, Pennsylvania

Certain strains of Neisseria gonorrhoeae are now relatively resistant to penicillin, previously considered to be the antibiotic of choice in the treatment of gonococcal infections. This report of a case of gonococcal conjunctivitis suggests that ampicillin, a semisynthetic penicillin, may aid in the therapy of this serious condition. CASE REPORT A 44-year-old unmarried sales manager was admitted to the Graduate Hospital of the University of Pennsylvania with a severely painful, copiously discharging left eye. He had no previous ocular problems until two nights prior to admission when he awakened from sleep with his left eyelids crusty and stuck together. By noon the eyelids were painfully swollen and he consulted a physician in the city to which his business trip had taken him; treatment with an ointment containing neomycin, polymyxin, and prednisolone was directed at a presumed staphylococcal infection. By evening the eyelids were more swollen and the eye more painful despite the use of a topical anesthetic. The patient's From the Glaucoma Service, Wills Eye Hospital. Reprint requests to 15 Laughlin Lane (19118).

local physician added erythromycin, 250 mg orally four times a day, but the following morning the condition was still worse and he was referred to us. On examination, the left eye was swollen shut, and the eyelids were covered with thick yellow exudate and antibiotic ointment, making scrutiny of the globe difficult. The conjunctiva was chemotic and markedly hyperemic ; a few tiny fluorescein-staining areas on the cornea were present; the preauricular node was enlarged and tender. The right eye was normal. The patient admitted sexual relations, but vigorously denied any contact which he thought could have led to infection with gonorrhoea. Though there was no history of urethritis, specifically none of gonorrhoeal etiology, the patient occasionally had a white urethral discharge in the morning. He was free from any other urinary symptoms. A smear of the exudate from the left eye showed many polymorphonuclear leukocytes; gram-positive cocci, single and in pairs, were present, but gramnegative cocci were not seen. A culture was planted. The patient was admitted to the hospital the same afternoon. Physical examination was within normal limits except for temperature of 100 F and the previously described inflammation of the left eye. There was no urethral discharge, nor was there inflammation of the tip of the urethra. The prostate gland was questionably enlarged, but was not tender. Following copious irrigation, polymyxinbacitracin eye drops were administered hourly. Pen-