The Posterior Corneal Surface in Fuchs' Dystrophy Scanning Electron Microscope Study

The posterior corneal surface in Fuchs' dystrophy Scanning electron microscope study

Frank M. Polack

This study comprises 12 corneal buttons removed at the time of keratoplasty from patients with bullous keratopathy due to Fuchs' dystrophy. All except one were aphakic. Three had vitreous contact to the endothelium. One patient had several intraocular procedures. Specimens were processed for scanning electron microscopy and dried in a freeze-drying or critical-point machine. Portions of these specimens were processed for transmission electron microscopy or paraffin sectioning. Specimens can be separated in three groups based on our findings: (A) corneas with abnormal or absent endothelium; posterior surface with many fibroblast-like cells; long filaments over Descemet's membrane; and few and small warts. (B) Endothelial cells were present for the most part. Cells were of abnormal size and shape with fibroblastic cells present over or between them; no warts or filament were seen. (C) Absent or abnormal endothelial cells, with no fibroblastic cells; Descemet's layer was covered with a profuse number of warts of oval or rounded shape and of different sizes. It is believed that the fibrils seen in specimens of Group A with vitreous contact may correspond to vitreous strands. The fibroblastic cells present in many aphakic corneas could have originated by metaplasia of endothelial cells or from macrophages of uveal origin. Vitreous contact may have occurred before or after these changes in Descemet's membrane.

Key words: corneal endothelium, corneal edema, corneal dystrophy, bullous keratopathy, scanning electron microscopy, Fuchs' dystrophy, vitreous contact.

B'ullous keratopathy is one of the mani- ing due to stromal swelling and epithelial festations of chronic edema and is charac- bullae. The most common causes of bullous terized by various degrees of corneal cloud- keratopathy requiring corneal transplanta- tion are Fuchs' dystrophy and corneal edema following cataract surgery. This last From the Department of Ophthalmology, College condition may be associated with endo- of Medicine, J. Hillis Miller Health Center, thelial dystrophy or may be the result of Gainesville, Fla. operative complications or vitreous adhe- Supported in part by Grants EY-00446, EY-00033, sion to the endothelium. It is known that EY-00266, and EY-00415 from the National Eye excrescences or warts in Descemet's mem- Institute. brane (cornea guttata) precede the de- Submitted for publication March 21, 1974. velopment of corneal edema, and bullous Reprint requests: Dr. Frank M. Polack, Depart- ment of Ophthalmology, College of Medicine, keratopathy frequently occurs in eyes with Box 733, J. Hillis Miller Health Center, Gaines- cornea guttata after uncomplicated intra- ville, Fla. 32610. ocular surgery. 913

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Table I. List of cases with their trophy with abnormal Descemet's layer preoperative clinical diagnosis. Three without warts is another manifestation of groups (A, B, and C) are outlined for Fuchs' dystrophy not usually recognized.10 descriptive purposes (see text). The The presence of isolated warts in this mani- presence of warts in Descemet's membrane festation of Fuchs' dystrophy, or warts (guttata) as seen in cross-sections of the buried in fibrillar basement membrane-like cornea is indicated in each case material"1 indicates that they are different manifestations of the same disease. Vitreous Case adhesions to the dystrophic endothelium No. Clinical diagnosis Age Guttata are likely to occur in these cases; however, A. 1. Bilateral Fuchs' dys- 70 + trophy, aphakic similar histologic alterations are found in 2. Bilateral bullous ker- 65 + the endothelium after chronic vitreous ad- atopathy, aphakic hesion. The sequence of events must then (vitreous adhesion) be evaluated together with the clinical his- 3. Bullous keratopathy 70 + with vitreous touch tory and examination of the other eye. 4. Bilateral bullous kera- 61 The purpose of this study is to demon- topathy, aphakic (vitreous adhesion) strate the alterations in the posterior cor- 5. Bilateral bullous kera- 67 + neal surface of corneas with bullous keratopathy, aphakic topathy of varying degrees due to Fuchs' 6. Bilateral bullous kera- 76 + topathy, aphakic dystrophy, some of them with vitreous ad- B. 7. Monocular aphakic 64 hesion, as seen with the (SEM). bullous keratopathy 8. Monocular bullous 70 Materials and methods keratopathy, apha- This study was done on twelve corneal buttons KlC (7 to 7.5 mm.) removed at the time of kerato- 9. Bilateral bullous ker- 67 atopathy, aphakic plasty by myself or Dr. H. E. Kaufman. Eleven C. 10. Bullous keratopathy, 74 +++ patients had various degrees of bilateral aphakic aphakic bullous keratopathy, except for two patients who 11. Bilateral bullous ker- 62 +++ had a normal contralateral cornea to slit lamp atopathy, aphakic examination. One patient had cornea guttata with 12. Bilateral bullous kera- 61 +++ moderate edema and was phakic in both eyes topathy, phakic (Table I). As soon as the corneal discs were removed from the patient they were immediately placed in cold Since the original ultramicroscopic study 4 per cent glutaraldehyde where they remained of "cornea guttata" by Feeney and Garron for a period of time from eight to 72 hours. A in 19611 and by Jakus in 1962,2 this entity small segment of the corneal disc was removed has been the subject of several reports here for light or transmission electron microscopy and and abroad.30 Except for two publica- the remaining tissue was processed for SEM. This 78 tissue was rinsed in distilled water, postfixed in tions, the surface morphology of cornea 1 per cent osmium tetroxide for 30 minutes, and guttata in Fuchs' dystrophy as seen with again rinsed in distilled water and run through a the scanning electron microscope (SEM) graded series of ethyl alcohols. Once in 100 per has not been well studied. cent alcohol it was dehydrated in freeze-drying or in a critical-point machine. Thereafter, the speci- Cornea guttata and Fuchs' dystrophy are men was mounted on an aluminum stub and terms interchangeably used by clinicians coated with gold palladium. Specimens were ex- when corneal edema develops. Warts in amined in a scanning electron microscope (Stereo- Descemet's layer are one of the manifesta- scan, Cambridge, England) at 10 or 20 KV. Pic- tions of the dystrophic endothelium which tures were recorded on Polaroid P/N film. may go on for years before it fails to main- Paraffin specimens were processed in a standard way and stained with hematoxylin and eosin and tain the cornea deturgesced, or before the 9 periodic acid-Schiff (PAS). Transmission electron dystrophy decompensates. Endothelial dys- microphotographs were taken with a Zeiss 9-S2

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Fig. 1. SEM of the endothelial surface of a cornea with bullous keratopathy showing an abnormal cell surface and multiple fibroblast-like cells randomly oriented. (SEM, x200.) Fig. 2. Microphotograph of an area of the previous picture at higher magnification. Abnormal endothelial cells (E) are seen together with fibroblastic cells. The endothelial cells are broad and elongated with serrated borders and filamentous prolongations over Descemet's membrane (D). This layer is covered by fibrinous material. At times these endothelial cells seem to take the shape of fibroblastic cells (arrow). {SEM, xl,000.) Fig. 3. Another area showing what appears to be transitional figures of flat endothelial cells to fibroblastic elements. Debris in the form of amorphous clumps are seen between cells of Des- cemet's (D) membrane. (SEM, xl,000.) Fig. 4. High-power microphotograph of an endothelial cell (E) with niicrovilli and cytoplasmic filaments which make contact with fibroblastic cells. Descemet's membrane (D) is covered by fine filamentous material. (SEM, x5,000.) Fig. 5. Microphotograph shows the surface of Descemet's membrane (D) with nodular excrescences (arrow) and absence of endothelial cells. Filamentous deposits are seen spreading over the surface, at times forming clumps. (SEM, xl,000.) Fig. 6. When these long filaments are examined at higher magnification, they appear like a fine brush laid over the rough-surfaced Descemet's membrane. (SEM, x2,000.)

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electron microscope after the specimen had been fibrin-like fibrillar material. Fig. 4 shows, cut into small pieces and postfixed in 1 per cent at higher magnification, the surface of a osmium tetroxide with phosphate buffer for 90 minutes. After dehydration through a graded series fibroblast-like cell next to a flattened cell of alcohols, specimens were finally embedded in to which it is in contact by means of cyto- epon. plasmic processes. Fig. 5 and 6 show the corneal endothelial surface of an eye with Results severe postcataract bullous keratopathy of Based on our histologic findings, speci- long standing (Case 2). Cross-sections mens can be divided into three main showed a Descemet's layer of 15 to 22 groups (Table I): microns in thickness with occasional small A. Corneas with abnormal or absent en- guttata and absence of endothelium. In dothelium. Fibroblast-like cells present in SEM specimens, endothelial cells could small or profuse number over abnormal not be identified; Descemet's membrane Descemet's membrane. Occasional small was covered by amorphous material of warts. Thick fibril deposition over Desce- rough texture as in the previous case and met's membrane and warts. Thick or lami- there were a few small warts. Long fila- nated Descemet's mambrane with warts in ments were deposited on its surface and cross-sections. Thickness, 12 to 16 microns. over the warts (Fig. 5). In some areas B. Endothelial cells present for the most they were clumped together, whereas in part. Cells of abnormal size and shape. other areas the filaments spread out like Fibroblast-like cells present over or be- brushes over large areas (Fig. 6). Similar tween endothelial. cells. No warts or long findings were observed in three other cases. filaments. In cross-sections, Descemet's The one illustrated in Figs. 7 through 11 membrane appears thick with overlying fi- showed occasional abnormal cells over brous membrane. Wavy or laminated at Descemet's membrane which had few warts times. Thickness, 18 to 28 microns. of small size. Fibrin-like deposits were C. Endothelium absent in many areas. not observed but there were large numbers Multiple warts of round or oval shape and of filaments randomly distributed over the of different sizes. Descemet's membrane of corneal surface (Figs. 7 and 8). Vitreous irregular thickness with numerous warts. adhesion to the posterior cornea was pres- Thickness varied from 12 to 18 microns. ent at the time of surgery and vitreous fibers were found in some areas of PAS- Group A. Comprises six cases of ad- stained cross-sections of the cornea (Fig. vanced bullous keratopathy, three of them 9). It is possible, therefore, that these fila- with vitreous contact to the endothelium. ments and those seen in other cases may Case 1 showed a profuse number of fibro- correspond to vitreous fibers or strands. blast-like cells scattered and randomly oriented over the endothelial surface. Normal Group B. Figs. 12 and 13 correspond endothelial cells, however, could not be to an eye with bullous keratopathy follow- identified. Survey of the graft posterior sur- ing surgery for congenital cataracts (Case face showed that the cellular elements pres- 7) 20 years previously and multiple intra- ent were flat and elongated with serrated ocular procedures. Cross-sections examined borders or long lateral prolongations which with the transmission electron microscope interconnected with each other (Fig. 2). showed a thick (24 to 28 micron) Desce- They formed monolayers, although occa- met's layer in which Descemet's membrane sionally more than one layer of cells were material alternated with fibrillary and fi- seen, some of them apparently transform- brous tissue. Fibroblast-like cells were pres- ing to spindle-like cells (Fig. 2, arrow). ent in the membrane and over and be- Large areas were devoid of cells as shown tween endothelial cells (Fig. 12). SEM in Fig. 3 (D); but the surface of Desce- microphotographs showed areas of absent met's membrane was covered by a layer of or abnormal endothelium. Endothelial cells

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Fig. 7. Several guttata formations from another case of aphakic bullous keratopathy. Descemet's membrane surface is devoid of endothelial cells and criss-crossed by many long filamentary structures. (SEM, xl,000.) Fig. 8. These filaments follow the contour of Descemet's surface and the guttata excrescence. (SEM, x2,000.) Fig. 9. Light microscope microphotograph of a portion of the same cornea showing vitreous filaments adherent to the surface of the abnormal Descemet's layer. (PAS, xl.OOO.) Fig. 10. Medium-power view of the surface of Descemet's membrane showing randomly oriented fibrillary structures of varying diameters. (SEM, x2,000,) Fig. 11, High-power view of a portion of the previous photograph showing two filaments over Descemet's layer, Much finer filaments cover this layer and extend over the large ones. (SEM, xlO,000.) of irregular shape intertwined with fibro- thelial mosaic (Fig. 14) with variations in blast-Kke cells. At times these were partial- size and shape frequently found in this ly visible under the endothelial layer (Fig. age group. Guttata excrescences were not 13). Similar findings were observed in an- seen but there were rows of fibroblast-like other case with bilateral corneal disease. cells on their surface. These cells were Case 9 differed somewhat from the pre- similar to those previously described, ex- vious case in which the corneal edema cept that these had a cellophane-like' ex- had appeared only two years after un- pansion on one or both ends to which complicated cataract extraction and it was other cells attached. In contrast to previous of moderate degree. Low-power micro- specimens, groups of these cells seemed photographs showed a complete endo- to be oriented in the same direction. Endo-

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Fig. 12. Microphotograph showing a fibroblastic cell (Fib.) over Descemet's membrane. This cell is partially covered by endotheiial cells (En) (arrow). (SEM, x5,000.)x Fig. 13. Transmission electron microphotograph of a section of the same cornea. It shows a thick Descemet's layer (Des.) and a cross-section of a fibroblastic cell which has characteristics of a macrophage. Arrow shows rest of endotheiial cell (En), (x 12,000.)

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Fig. 15. Microphotograph showing the extreme of Fig. 14. Specimen obtained at the time of kerato- one fibroblastic cell which lays over the cell nu- plasty shows an intact endothelial layer covered clei. A long endothelial cell filament is seen over by multiple fibroblastic cells, most of them ori- the nucleus. (SEM, x5,000.) ented in one direction and arranged in chains. Other areas did not show such cells. (SEM, x500.) met's membrane under the ruptured cyto- plasm of an endothelial cell. In many areas thelial cell size varied between 10 and these cells are totally absent revealing the and 30 microns. Nuclei were of similar surface structure of Descemet's membrane size and cell borders had tight junctions. warts (Fig. 18). These warts vary in size Most of the cells had microvilli and one from 5 to 25 microns or more. Their shapes or two long villous filaments (Fig. 15). vary from round to oval, often two warts Frequently, these were located over the are joined by a solid bridge. The top was nuclei and showed a cytoplasmic thick- usually flat but some warts had a slightly ening at its base where the filament also dimpled or umbilicated surface. Most of became thicker. This structure was often the times they had steep borders, occa- in contact with or partially wrapped around sionally they showed sloping sides, but the fibroblast-like cell. PAS-stained sections warts with undermined sides, as would showed a thickened Descemet's membrane be expected in collar-button warts, could with lamination as well as flat endothelial not be found. Descemet's membrane sur- cells. Cross-sections of fibroblastic cells face had a fibrillar surface which, in a were found frequently. Transmission elec- whorl-like fashion, circled the warts. They tron microscopy also showed a thick lami- probably corresponded to fine collagen nated Descemet's layer with thin endo- fibrils (long spacing) found around the thelial cells over fi brill ary material, but no warts in transmission electron microscopy fibroblastic cells could be found in these specimens. Fig. 19 corresponds to Case 12 specimens. which was phakic. It shows several warts Group C. Figs. 16 through 18 correspond with a flatter surface surrounded by fi- to Case 10 and demonstrate the appear- brillar material. ance of a cornea with numerous guttate formations which can be easily visualized Comment in some areas due to the absence of en- Fuchs' corneal dystrophy is characterized dothelial cells. Absence of endothelial cells by primary changes in the corneal endo- is, in part, artifactual as determined by the thelium and in Descemet's membrane. It presence of cell residues; in other areas it has been shown that endothelial cells ac- seems to be due to actual cell degenera- quire the morphology and the function of tion. The artifact shown in Fig. 17 fa- fibroblasts5 while they produce basement cilitates the observation of a wart in Desce- membrane-like material characteristic of

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Fig, 16. Low-power view of cornea] endothelium in a case of advanced cornea guttata. Areas of endothelium have been artifactually detached. Arrow indicates one area of Descemet's layer devoid of endothelium. Large warts are seen all over the surface. (SEM, x200.) Fig, 17, Figure shows a partially disrupted endothelial cell (En) demonstrating a single guttata formation in Descemet's membrane (arrow), (SEM, x2,000.) Fig. 18. In areas totally devoid of endothelium several guttate excresences (W) show flat or slightly umbilicated surface. Descemet's membrane has a fine or slightly fibrillar surface which is frequently arranged around the warts. (SEM, x2,000.) Fig, 19. This microphotograph from another case shows a large wart (W) with a rather flat surface and other smaller warts. The fibrillary surface of Descemet's membrane around and blending with the warts is demonstrated. (SEM, x2,00Q.)

endothelial cells and collagen fibrils typi- to artifacts, however, care was taken not cally produced by fibroblasts. The distri- to disrupt vitreous adherent to the endo- bution of this newly produced material over thelial surface. The long fibrils present in the original Descemet's layer will originate several cases of aphakic bullous keratop- the typical warts, the thickened Descemet's athy suggest that they correspond to vitre- membrane, or the intermediate forms we ous fibers or strands. They could not be have discussed here. Endothelial cells demonstrated in transmission electron mi- eventually degenerate and leave a bare crophotographs nor have they been de- fibrillar surface, or a more compact Desce- scribed before, but they have been seen met's wart. frequently in PAS-stained sections. Absence of endothelial cells is a fre- Our SEM microphotographs support the quent finding in this condition.*15'G; 18"31 view that endothelial cells may transform The large areas without endothelium seen into fibroblasts because, in addition to in our first cases could have been due the deposition of collagen material, they

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have cytoplasmic fibrils, coated vesicles, to the almost normal appearance of the prominent cytoplasmic processes, increased endothelial surface which showed abnor- RER, and desmosomes. mal cells and fibroblasts on their surface. As mentioned before, transmission elec- These are the corneas which may appear tron microscopic studies of endothelial cells normal by slit lamp examination before in Fuchs' dystrophy suggest that these cells they decompensate following cataract sur- transform into fibroblasts.5 Our SEM studies gery and possibly be used as normal donor support this view on a morphologic basis material. Stocker10 has called attention to as seen in Figs. 1 through 4, corresponding this little recognized aspect of Fuchs' dys- to Case 1. We have seen, in others cases of trophy which has important clinical sig- Fuchs' dystrophy, what appear to be tran- nificance. Guttate formations, on the other sitional figures from flat endothelial cells hand, are early and easily recognized be- with prominent cytoplasmic processes to cause they produce a typical morphologic spindle-shaped processes with one or both change in the posterior corneal surface. ends tapered. Since these cells have been Probably, this is a more severe type of found only in aphakic eyes (only one disease, but not all patients with cornea phakic eye case was studied) it is possible guttata develop edema.0 Even though the that some fibroblastic cells are actually morphology of Descemet's membrane with fibroblasts or macrophages of uveal origin, multiple warts does not add new informa- and that the vitreous plays a major role tion to the pathogenesis of this disease, in their access to the cornea and in their it helps to understand the magnitude of proliferation or transformation from endo- the disease, particularly when the warts thelial cells to fibroblasts. Fibroblastic cells can be seen without their endothelial cover. have been seen in regenerating endothelial 11 12 H. E. Kaufman, M.D., J. Chandler, M.D., P. injuries, in retrograft membranes, and 13 Binder, M.D., and R. Abel, M.D., contributed with after chemical injuries. Once the stimulus specimens. Kenneth Faust, M.D., referred two for fibroblastic cell activity or transforma- cases included in this study. The collaboration of tion disappears, these cells may revert to T. Sakimoto, M.D., is acknowledged as well as their original endothelial aspect.11 We have the technical assistance of W. M. Chisholm, J. Valenti, and E. J. Jenkins. Comments to a portion seen such fibroblastic cells in SEM speci- of this study were made by Professor G. K. Smel- mens of retrocorneal membrane formation ser. Dr. E. Balazs gave comments about some pho- 15 10 and graft failures " and in clinical speci- tographs. mens of rejected grafts.17 Cross-sections of specimens described in REFERENCES Group B show the structure of those cells 1. Feeney, M. L., and Garron, L.K.: The Struc- which appear to be the fibroblasts seen ture of the Eye, Smelser, G. K., editor. New York, 1961, Academic Press, Inc., pp. 367-380. in SEM pictures. Their internal structure 2. Jakus, M. A.: Further observations on the seems to correspond to macrophages in fine structure of the cornea, INVEST. OPHTHAL- two cases. MOL. 1:202, 1962. We found great variations in the thick- 3. Kayes, J., and Holmberg, A.: The fine struc- ness of Descemet's membrane between ture of the cornea in Fuchs' endothelial dystrophy, INVEST. OPHTHALMOL. 3: 47, 1964. cases (16 to 28 microns) and in the same 4. McTigue, J. W.: The human cornea: a light specimen. The thicker membranes did not and electron microscope study of the normal show guttate formations in SEM specimens cornea and its alterations in various dystro- and in transmission electron microscopy phies, Trans. Am. Ophthalmol. Soc. 65: 591, sections they were small and buried in 1967. fibrous or basement membrane-like ma- 5. Iwamoto, T., and DeVoe, A.: Electron microscopic studies on Fuchs' combined dystrophy. terial. The severe degree of pathology I. Posterior portion of the cornea, INVEST. seen in cross-sections did not correspond OPHTHALMOL. 10: 9, 1971.

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6. Pouliquen, Y., Graf, B., and Offret, G.: Etude 14. Sanchez, J., and Polack, F. M.: Effect of histologique et ultrastructurale de la cornee topical steroids on the healing of corneal dans une dystrophie endo-epitheliale de endothelium, INVEST. OPHTHALMOL. 13: 17, Fuchs' (cornea-guttata), Arch. Ophthalmol. 1974. 31: 427, 1971. 15. Polack, F. M.: Electron microscopic studies 7. Manning, W. M., and McLean, H.: Scanning of graft endothelium in corneal graft rejec- electron microscopy of human cornea, Twen- tion, Am. J. Ophthalmol. 73: 711, 1972. tieth Concilium in Ophthalmology, Mexico, 16. Polack, F. M.: Scanning electron microscopy 1970, pp. 651-656. of corneal graft rejection. I. Epithelial rejec- 8. Smelser, G. K., and Ozanics, V.: Modern tion; II. Endothelial rejection; III. The forma- views of corneal anatomy, Transactions New tion of posterior graft membranes, INVEST. Orleans Academy Ophthalmology Symposium OPHTHALMOL. 11: 1, 1972. on the Cornea, St. Louis, 1972, The C. V. 17. Polack, F. M.: Clinical and pathologic aspects Mosby Company, pp. 1-10. of the corneal graft reaction, Trans. Am. Acad. 9. Donn, A.: Cornea and sclera (annual review), Ophthalmol. 77: 418, 1973. Arch. Ophthalmol. 75: 261, 1966. 18. Chi, H. H., Teng, C. C, and Katzin, H. M.: 10. Stocker, F. W.: The endothelium of the Histopathology of corneal endothelium. A cornea and its clinical implications, ed. 2., study of 176 pathologic discs removed on Springfield, 111., 1971, Charles C Thomas, keratoplasty, Am. J. Ophthalmol. 53: 215, Publisher. 1962. 11. Inomata, H. J., Smelser, G. K., and Polack, 19. Kurz, G. H., and D'Amico, R. A.: Histo- F. M.: The fine structural changes in the pathology of corneal graft failures, Am. J. corneal endothelium during graft rejection, Ophthalmol. 66: 184, 1968. INVEST. OPHTHALMOL. 9: 263, 1970. 20. Winter, F. C: The histopathology of un- 12. Kanai, A., and Kaufman, H. E.: Electron successful penetrating keratoplasty in man, microscopic studies of corneal endothelium: in: Corneo-Plastic Surgery, Rycroft, P. V., edi- the abnormal endothelium associated with tor. New York, 1969, Pergamon Press, pp. retrocorneal membrane, Ann. Ophthalmol. 4: 557-569. 564, 1972. 21. Hales, R. H., and Spencer, R.: Unsuccessful 13. Matsuda, H., and Smelser, G. K.: Endothelial penetrating keratoplasties, Arch. Ophthalmol. cells in alkali-burned corneas, Arch. Ophthal- 70: 805, 1963. mol. 89: 402, 1973.

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