Proliferation and Metaplasia of Intravitreal Retinal Pigment Epithelium Cell Autotransplants

PROLIFERATION AND METAPLASIA O F INTRAVITREAL R E T I N A L P I G M E N T E P I T H E L I U M CELL AUTOTRANSPLANTS M A S K S. MANDELCORN, M.D.,

ROBERT MACHEMER, M.D.,

AND STANLEY B. HERSCH,

EDWARD FINEBERG,

M.D.

M.D.

Miami, Florida anesthetized with 0.05 ml of phencyclidine (Sernylan) intramuscularly and 5 mg of pentobarbital (Nembutal) intraperitoneally and given a retrobulbar injection of 0.3 ml of xylocaine 1% (Lidocaines). The donor eye was enucleated and the sclera sewn to a metal ring around the equa­ tor. The anterior segment including lens and vitreous was removed. Under the operating microscope we gently detached the retina by running a stream of normal saline between retina and R P E through a 21-gauge needle. With care, it was possible to completely de­ tach the retina from the pigment epithelium without leaving pigment on the under surface of the retina and without touching either retina or RPE. The detached retina was cut off at the optic disk and discarded. To separate R P E cells from each other and from Bruch's membrane 1 ml of trypsin 0.25% prepared from lyophylized trypsin was injected into the posterior segment and left for 30 minutes at 37°C.2 The trypsin was then inactivated by Dulbecco's medium MATERIALS AND METHODS containing calcium and magnesium ions. By Owl monkeys (Aotus trivirgatus) were applying gentle suction through a blunt 14gauge needle R P E cells were aspirated and From the Bascom Palmer Eye Institute, Depart­ ment of Ophthalmology, University of Miami collected in Dulbecco's medium in a cen­ School of Medicine; and the Veterans Administra­ trifuge tube. To concentrate the cells and to tion Hospital, Miami, Florida. This study was sup­ ensure complete inactivation of trypsin, this ported in part by Public Health Service research grant EY-00841; in part by the Veterans Adminis­ cell suspension was centrifuged at 1,400 rpm tration Hospital, Miami; in part by funds from Re­ and washed three times in Dulbecco's me­ search to Prevent Blindness, Inc., New York; and dium. The sediment containing R P E cells in part by the Florida Lions Eye Bank, Inc., Miami, Florida, Dr. Mandelcorn is a fellow of the Medical was aspirated into a tuberculin syringe Research Council of Canada. Dr. Fineberg is the through an 18-gauge needle for injection into recipient of a Fight-for-Sight grant. the recipient eye. Presented in part at the Association for Research The harvested R P E cells from one eye in Vision and Ophthalmology Annual Spring Meet­ ing, Sarasota, Florida, May 3-7, 1973, and the were injected into the normal fellow eye of Ninth Meeting of the Club Jules Gonin, LaBaule, the same owl monkey through the pars plana France, May 12-18, 1974. Reprint requests to Robert Machemer, M.D., P.O. 2 mm behind the corneoscleral limbus under Box 520875, Biscayne Annex, Miami, FL 33152. a conjunctival flap and aimed at the posterior After experimental retinal detachment in the owl monkey, pigmented cells appear in the vitreous cavity and along the internal limiting membrane of the retina. In some cases these pigmented cells form membranes that wrinkle the inner retinal layers and produce fixed retinal folds.1 Since in many ways they resemble those of retinal pigment epithelium it occurred to us that these mem­ brane-producing cells might be of retinal pig­ ment epithelial ( R P E ) origin. We designed an experiment to demonstrate that R P E cells are not inhibited by vitreous from proliferat­ ing and migrating and that they are inher­ ently capable of producing membranes, to­ gether with changes in their cell morphology. Therefore, R P E cells were isolated from one eye and transplanted into the vitreous cavity of the intact fellow eye of the experi­ mental animal to grow in a natural environ­ ment in which they find themselves during retinal detachment; namely, in the vitreous cavity.

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vitreous near the disk. The scleral opening was immediately closed with a preplaced 5-0 nylon mattress suture. This was done in 20 monkeys. The harvested cells were studied under the microscope for possible contam­ ination with other cells. To study whether migration and prolifera­ tion of R P E cells are affected by removal of vitreous,3 a second experiment was designed in which the vitreous of the recipient eye was first removed as completely as possible by aspirating the animal's relatively liquid vitre­ ous and replacing it with air. Ten to 14 days later when all the air had definitely been reabsorbed and replaced by clear fluid we transplanted R P E cells from the animal's normal fellow eye into the vitrectomized eye. This was done in eight monkeys. In two control groups, each consisting of six monkeys, 0.15 ml of Dulbecco's medium or cardiac blood was injected into the vitre­ ous cavity. All eyes were observed daily with binoc­ ular indirect ophthalmoscopy. Eyes were enucleated two days, three days, one, two, four, and nine weeks after autotransplantation (Table). Two hours before enucleation 50 (xCi (0.05 ml) of thymidine-methyl H-3 (specific activity, 5.0 curie/Mmol) was in­ jected into the vitreous cavity. The enucleated eye was fixed immediately in 6% buffered glutaraldehyde. While in fixative, the globe TABLE RECORD OF NUMBER OF EYES IN EACH EXPERIMENT

Vitreous Normal Vitreous Humor Duration Humor, Removed. Buffer Blood RPE Auto­ RPE Auto­ transplant transplant 2 days 3 days 1 wk 2wk 4 wk 9 wk

2 2 4 4 4 4

2 2 2 2

2 2 1 1

2 2 2

Total

20

8

6

6

AUGUST, 1975

was bisected behind the equator and left for two hours during which photographs of the R P E cell autotransplant were taken through a dissecting microscope. The R P E cell autotransplant was dissected, postfixed in phosphate-buffered osmium tetroxide 2%, dehydrated in alcohol, and em­ bedded in Epon. Sections 1.5 u, thick were stained with paraphenylenediamine and ex­ amined under phase contrast microscopy. Thin sections, 0.05 (i, stained with uranyl acetate and lead citrate were examined by electron microscopy (JEM-7). For autoradiographic evaluation sections were coated with Kodak nuclear track emulsion NTB-2, exposed for five days and developed in Kodak D19 developer, and then examined under phase contrast. RESULTS

Gross findings—Immediately after injec­ tion the R P E cell autotransplant dispersed inferiorly in tiny clumps into the cortical vitreous overlying the pars plana and ora serrata and remained dispersed throughout the first four weeks (Fig. 1). Between four and nine weeks after injec­ tion the tiny pigmented clumps became thicker and developed processes which seemed to anchor them to the surrounding vitreous (Fig. 2 ) . They also formed long lightly pigmented strands (Fig. 3). Microscopic findings—Freshly harvested R P E cells consisted microscopically not only of individual round pigmented cells but also of sheets of hexagonal cells. There were also some free pigment granules. Contamination by other cell types was not detected. During the first four weeks after trans­ plantation but less frequently thereafter, round heavily pigmented cells containing rodshaped and round pigment granules typical of the retinal pigment epithelium were found predominantly in small clumps but also free in the vitreous (Fig. 4). By the fourth week some round pigmented cells were found lying on or enclosed within newly formed intra-

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Fig. 1 (Mandelcorn and associates). Photomacrograph of RPE autotransplant dispersed in vitreous base (one week after injection).

Fig. 2 (Mandelcorn and associates). Photomacrograph of RPE autotransplant four weeks after injec­ tion. Note the large irregularly pigmented cluster with lightly pigmented processes.

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Fig. 3 (Mandelcorn and associates). Photomacrograph of RPE autotransplant nine weeks after injec­ tion. Note long lightly pigmented strands.

vitreal strands. By electron microscopy the free round pigmented cells had microvilli. They had neither basement membranes nor cell junctions and retained many free pig­ ment granules typical of R P E cells. A few of these granules seemed to have been phagocytized since they lay within phagosomes (Fig. 5). Some nuclei of these pigmented round cells were labeled with radioactive thymidine in the first two weeks after autotransplantation but not thereafter. By the fourth week, predominantly spin­ dle-shaped cells appeared that were much less pigmented. These cells had an oval nucleus containing a prominent nucleolus. Although some spindle-shaped cells were found alone in the vitreous, most were densely packed together forming membranes. These cells also formed thick capsules around clumps of round pigmented cells (Fig. 6). Spindleshaped cells showed marked nuclear labeling with radioactive thymidine throughout the

nine weeks of observation (Fig. 7). Electron microscopy of these long spindle-shaped cells demonstrated that they had basement membranes, cell junctions, and sometimes contained rod-shaped and round pigment granules. Fine filaments were observed in their cytoplasm. Collagen fibrils were found lying between individual spindle-shaped cells (Fig. 8 ) . By nine weeks after transplantation a fur­ ther transformation in the appearance of some of these cells was observed. Cells along the outer surface of newly formed mem­ branes lined up side by side in a single layer and acquired a brush-like border on their free side (Fig. 9). None of these cells were observed to take up radioactive thymidine. By electron microscopy these cells were cuboidal and had apical microvillous pro­ cesses below which pigment granules were found. Prominent cell junctions between neighboring cells were seen in the apical part

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Fig. 4 (Mandelcorn and associates). Free pigmented cells of RPE autotransplant one week after injection. Note round and rod-shaped pig­ ment granules.

of these cells. Underlying them was a base­ ment membrane which lined the cells' basal infoldings (Fig. 10). The eight eyes in which the vitreous had been almost completely removed and replaced by aqueous before autotransplantation ex­ hibited the same gross, histologic, autoradiographic, and electron microscopic appear­ ances after autotransplantation of R P E cells as did eyes in which vitreous had been left intact. Removal of nearly all the vitre­ ous humor had no stimulating effect on R P E cell proliferation or cytologic appearance. Controls—Injection of Dulbecco's medium free of R P E cells resulted neither in gross nor microscopic change in the vitreous cav­ ity. Injection of cardiac blood resulted in changes completely different from those ob­ served after R P E cell injection. Injected red blood cells tended to diffuse throughout the vitreous cavity in the first few weeks after injection. Later, syneresis of the vitre­ ous developed, leaving many areas where

vitreous collagen condensed to form filmy strands. Microscopically, these strands con­ sisted of blood breakdown products often within macrophages lying along vitreous fibers. No cellular membranes resembling those produced by R P E autotransplants were observed. DISCUSSION

In this experiment R P E cells were trans­ planted from one eye into the vitreous cavity of the other eye of owl monkeys. Many cells not only survived but also demonstrated ac­ tive proliferation. This intravitreal autotrans­ plant, therefore, must be regarded as an in vivo tissue culture with the advantage of a more natural environment than is possible in any in vitro tissue culture. It is probably for this reason that we observed greater vari­ ety of cellular changes in the injected R P E cells than has previously been reported in in vitro tissue cultures.2-4"6 First, there were large round pigmented

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Fig. 5 (Mandelcorn and associates). Free pigmented cell of RPE autotransplant. P indicates pigment granule; PH, phagosome (X 16,875).

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Fig. 7 (Mandelcorn and associates). Photomicrograph of autoradiographic label (circle) in nucleus of spindle-shaped cell of RPE autotransplant. Another area of same specimen as in Figure 6.

cells that, because they contained some phagosomes, probably were acting as macrophages. We believe these originated from R P E cells rather than blood or tissue macrophages since most of the melanin granules they contained appeared to be native to these cells and were not enclosed within a mem­ brane packet which is more indicative of phagocytosis. Secondly, there were long, spindleshaped cells forming membranes. Because the spindle-shaped cells resembled fibroblasts this has been called fibrous metaplasia,7-12 although by electron microscopy these cells retain epithelial characteristics such as base­ ment membranes and cell junctions. That the pigment content decreased in the later phases may be due to the fact that as these cells di­ vided; successive generations of cells re­ ceived fewer pigment granules than had been

present in the preceding generation. During the ninth week some injected cells changed from a phase in which they were spindle shaped and actively proliferating into a phase in which they were well-differenti­ ated normal pigment epithelium cells and no longer proliferating. Similar changes were observed following experimental retinal de­ tachment.1 Cellular transformations were previously observed in studies with pigment epithelial cells.6'13 When epithelial cells pro­ liferate they become less differentiated and change their appearance, although ultrastructurally they retain epithelial characteristics. Once the proliferative phase is ended they change back to their more familiar, welldifferentiated appearance. In this experiment we could not eliminate the possibility that some cells might have originated from sources other than the pig-

Fig. 6 (Mandelcorn and associates). Photomicrograph of spindle-shaped cells of RPE autotransplant four weeks after injection. These cells form a capsule around more heavily pigmented cells.

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Fig. 8 (Mandelcorn and associates). Spindle-shaped cell of RPE autotransplant. B indicates basement membrane; P, pigment granule; C, collagen; and ZO, zonula occludens (X16.87S).

Fig. 9 (Mandelcorn and associates). Photomicrograph of cells of RPE autotransplant lined up side-byside nine weeks after injection. Note brush-like border and orientation which resembles normal in-itu RPE.

Fig. 10 (Mandelcorn and associates). Cell of RPE autotransplant with apex to base orientation resem­ bling normal RPE cell. P indicates pigment granule; JC, junctional complex; B, basement membrane; M, microvilli ( x 15,000).

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ment epithelium. Therefore we paid careful attention to the possibility of contamination by cells from other sources, especially from blood. Harvested R P E cell autotransplants were carefully scrutinized and no contam­ inating cells were ever found. On the other hand, since the membrane-forming cells had cell junctions and basement membranes, they were definitely epithelial and could not have originated from connective tissue or blood elements. This conclusion is supported by the results of a separate experiment in which R P E cell autotransplants were placed in intravitreal Millipore chambers impermeable to cells so that no cells could enter or leave the chamber. Similar transformations of RPE cells and membrane production were ob­ served.14 Since cells had been injected into the vitre­ ous cavity of an eye in which the retina was still attached, there was no effect on the pro­ liferation of the cells by either the detached retina or altered RPE. Control experiments using intravitreal injections of buffer and blood did not result in changes resembling those seen after transplantation of R P E cells. Nor could the trypsin have given rise to membrane production since before injection of R P E cells trypsin had been completely in­ activated. Previous work suggested that vitreous constituents, specifically hyaluronic acid, might prevent cellular migration and prolif­ eration in the vitreous humor.3 We found, however, that R P E cells do proliferate and form membranes, both in eyes with intact vitreous humor and in eyes in which vitreous has been removed. The presence of intact vitreous humor, therefore, does not prevent migration and proliferation of R P E cells. Of great clinical interest is the obvious ability of R P E cells to form membranes while proliferating. These membranes are of considerable size and are even visible clin­ ically. In our particular experiment, cells grew over the vitreous base where the vitre­ ous structure is dense. Therefore, they did not have access to the retinal surface. But it

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can easily be imagined that these cells might also grow over the retinal surface and pro­ duce membranes. These findings, taken in the light of the histologic and electron micro­ scopic findings after experimental retinal detachment,1 suggest that cells derived from the retinal pigment epithelium may play a major role in the production of preretinal and intravitreal membranes. SUMMARY

Pigment epithelium cells autotransplanted into the vitreous cavity of owl monkey eyes, proliferated and metaplased. The metaplastic cells looked like pigmented macrophages, membrane forming fibrocyte-like cells, and frank epithelium cells. This in vivo experi­ ment demonstrated that the vitreous cavity is an adequate culture medium for cells de­ riving from the pigment epithelium and that pigment epithelium cells could be the source of intraocular proliferation seen in massive periretinal proliferation. ACKNOWLEDGMENTS

We thank Mr: Ernest Reeves for technical as­ sistance; Mr. Wayne Hinecker for his help in the animal experiments; Mrs. Marcilia Halley for technical assistance and electron microscopy; Ms. Barbara French for photography; and Mrs. Char­ lotte Rowlette for typing the manuscript. REFERENCES

1. Machemer, R., and Laqua, H.: Pigment epi­ thelium proliferation in retinal detachment (mas­ sive periretinal proliferation). Am. J. Ophthalmol. 80:1, 1975. 2. Mannagh, J., Aryo, D. V., and Irvine, A. R., Jr.: Tissue culture of human retinal pigment epi­ thelial cells. Invest. Ophthalmol. 12 :S2, 1973. 3. Balasz, E. A., Freeman, M. I., Kloeti, R., Meyer-Schwickerath, G., Regnault, F., and Sweeney, D. B.: Hyaluronic acid and replacement of vitreous and aqueous humor. Mod. Probl. Oph­ thalmol. 10:3, 1972. 4. Albert, D. M., Ts'o, M. O. M., and Raylon, A. S.: In vitro growth of pure cultures of retinal pigment epithelium. Arch. Ophthalmol. 88:63, 1972. 5. Barishak, Y. R.: In vitro behavior of the pig­ mented cells of the retina and uvea of the adult human eye. Acta Ophthalmol. 38:339, 1960. 6. Whittaker, J. R.: Loss of melanocyte phenotype in vitro by differentiated retinal pigment cells. Demonstration of mechanisms involved. Dev. Biol. IS :SS3, 1967. 7. Leber, T.: In: Graefe-Saemisch-Hess: Hand-

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buch der gesamten Augenheilkunde, 2nd ed., vol. 7. Leipzig, Engelmann, 1916, pt. 2, p: 126. 8. Manschot, W. A., and deBruijn, W. C.: Coats' disease. Definition and pathogenesis. Br. J. Oph­ thalmol. 51:145, 1967. 9. Frayer, W.: Reactivity of retinal pigment epithelium. An experimental and histopathological study. Trans. Am. Ophthalmol. Soc. 69:586, 1966. 10. Klien, B.: Diseases of the macula. Arch. Ophthalmol. 60:175, 1958. 11. Ts'o, M. O. M.: Photic maculopathy in rhesus monkey. A light and electron microscopy study. Invest. Ophthalmol. 12:17, 1973.

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12. Wallow, I. H. L., and Ts'o, M. O. M.: Pro­ liferation of the retinal pigment epithelium over malignant choroidal tumors. A light and electron microscopic study. Am. J. Ophthalmol. 73:914, 1972. 13. Rodesch, F.: Differentiation, contact inhibi­ tion, and redifferentiation. Intracellular communica­ tions in retinal pigment cells. Exp. Cell Res. 76:55, 1973. 14. Mueller-Jensen, K., Machemer, R., and Azarnia, R.: Autotransplantation of retinal pig­ ment epithelium in intravitreal diffusion chambers. Am. J. Ophthalmol. In press.

OPHTHALMIC MINIATURE

When he grew blind he would sit hour after hour in those two rooms that he had painted, looking at his works with sightless eyes, and seeing, perhaps, more than he had ever seen in his life before. Ata told me that he never complained of his fate, he never lost courage. To the end his mind remained serene and undisturbed. But he made her promise that when she had buried him—did I tell you that I dug his grave with my own hands, for none of the natives would approach the infected house, and we buried him, she and I, sewn up in three pareos joined together, under the mango-tree—he made her promise that she would set fire to the house and not leave it till it was burned to the ground and not a stick remained. W. Somerset Maugham, the Moon and Sixpence