CORNEAL E N D O T H E L I U M UNDER VARIOUS STORAGE CONDITIONS RAGNIT GEERAETS, PH.D., WILLIAM D . M C L E S T E R , M . D . , A N D F R A N C E S D . M C M U L L A N , B.A. Bichmond,
Short-term storage of corneal graft material has been used more frequently since 1 9 7 4 when M c C a r e y and Kaufman developed a modified tissue culture medium, the M-K medium, which consists of a mixture of T C - 1 9 9 medium, 5 % dextran, and 100 units/ml of a streptomycin and penicillin mixture. T h e osmolarity is 2 9 0 m O s m and the p H is 7.4. T h e idea of short-term storage, however, had been mentioned by M a g i t o t as early as 1 9 1 2 . T h e purpose of short-term storage in a suitable medium is twofold. First, surgery can b e scheduled more conveniently, particularly if the recipient lives far from where the surgery is to b e performed. Secondly, experimental w o r k has shown that short-term storage of the donor cornea in a suitable medium causes antigenicity loss by diffusion o f such antigens into the storage medium. 1
2
3 - 1 2
In 1 8 7 3 , L e b e r first described the importance o f the corneal endothelium and the role it plays in corneal clarity. Quantitative examinations of corneal endothelium regeneration in vivo were reported by H o n e g g e r in 1 9 6 2 who showed the interrelationship of corneal swelling to corneal endothelial defects. T h e role of corneal hydration as a balance between the imbibition pressure of the stromal mucopolysaccharides and the metabolically active transport system in the endothelium was reemphasized by M i s h i m a and K u d o . H e n c e , the importance of the state of the corneal endotheli1 3
14
15
From the Department of Ophthalmology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia. This study was supported in part by the Old Dominion Eye Bank and Research, Inc., Richmond, Virginia. Reprint requests to Ragnit Geeraets, Ph.D., Box 262/MCV, Richmond, VA 23298.
Virginia
um and its viability in successful penetrating corneal transplantation has long been realized. Therefore, short-term storage is useful only if the storage medium is nontoxic and maintains endothelial viability. In most situations these properties had to b e maintained for only a few days to meet the two criteria o f short-term storage. Recently Friedland and F o r s t e r stated that this goal could b e achieved by keeping the donor graft material in M-K medium at 4°C for several days as well as by storing the corneas in a moist c h a m b e r in the refrigerator. However, storage o f the whole eye under similar conditions had a negative effect on the endothelium that was caused by a toxic reaction o f autolysis of the intraocular tissue on the endothelium. A similar observation had already been made by S t o c k e r . Van Horn and Schultz stored cat corneal endothelia with a scleral rim in a refrigerator for up to 120 hours. T h e corneal endothelial cells stored in the moist c h a m b e r remained viable longer than corneas stored in the serum. Whether these differences are caused by t e c h n i q u e or different animal models is not clear. 16
5
17
M A T E R I A L AND M E T H O D S
T o determine the effects o f storage on the corneal endothelium, we stored three rabbit donor corneas each in serum, in M-K medium, and in a moist c h a m b e r at 4°C, and observed the results with a scanning electron microscope. W e did not use other culture media b e c a u s e M c C a r e y and Kaufman found them inferior to the M-K medium. W e incubated an equal number of control corneas in a tissue culture incubator at 37°C with 5 % C 0 and moist air in
206
1
2
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M-K medium, serum, and minimum essential medium ( M E M ) with 1 0 % fetal calf serum and 100 units/ml of a penicillin and streptomycin mixture. T h i s part o f the study was conducted to explore the viability and integrity of the endothelium under incubation conditions that would eliminate temperature reversal at the time of transplantation which is required for graft material under refrigeration. W e examined specimens from these rabbit corneas after various periods o f storage. Three rabbit eyes were used for each storage condition and time. T h e corneas were excised with a 2-mm scleral rim after the eyes had been enucleated. Corneal tissue was placed immediately in the storage media or moist chamber. On days 1 , 2 , 3, 4 , 6, and 9 we removed the specimens from the media and processed them concurrently with a freshly obtained cornea, used as a control. T h e y were fixed in glutaraldehyde for 2V2 hours, dissected in quarters, dehydrated in a graded series of acetone, and critical-point-dried with fluorocarbon C F - C F (Freon 116). T h e endothelial surface was always face up. T h e specimens were then mounted, coated with gold-palladium, and examined with a scanning electron microscope. 3
3
Fig. 1 (Geeraets, McLester, and McMullan). Normal corneal endothelium has a distinct outline with microvilli and occasional cilia. The surface appears smooth without separation of individual cells (SEM, x 2,000).
with little change up to four days of storage ( F i g . 3). After days 6 and 9, various cells had irregular surfaces and shapes. T h e s e changes were more pronounced after nine days o f storage ( F i g . 4 ) . SERUM—After 2 4 hours the cells appeared essentially the same as those stored in M-K medium. S i n c e more microvilli were seen, these specimens more closely resembled the control corneas. T h e cell borders were more pro-
RESULTS
T h e control specimens in all experimental groups had essentially the same appearance ( F i g . 1). T h e cells were smooth with microvilli and an occasional cilium. T h e cells were the same size and were outlined by their membranes without cell separation. Storage under refrigeration (4°C)—M-K M E D I U M — A f t e r 2 4 hours o f storage we noted no significant difference in the appearance of the cells as compared with that o f the control cells although the cell outlines were more marked indicating that a mild shrinkage or dehydration o f the cells may have taken place ( F i g . 2 ) . This endothelial appearance persisted
Fig. 2 (Geeraets, McLester, and McMullan). Corneal endothelium stored 24 hours in M-K medium at 4°C. The cell outlines are more marked but the surface is smooth without separation of cells (SEM, x 2,000).
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Fig. 3 (Geeraets, McLester, and McMullan). Corneal endothelium stored four days in M-K medium at 4°C shows some irregular cell configuration and occasional cells that have some degenerative changes. The cell boundaries are still intact (SEM, x 2,000). n o u n c e d ' a n d there was more cell shrinkage ( F i g . 5 ) . T h e cell layer was intact for the first four days of storage ( F i g . 6 ) . Similar to the cells stored in M-K medium, the cell surface b e c a m e more irregular after six and nine days of storage with no gaps between individual cells that formed an intact layer over D e s c e m e t 's membrane ( F i g . 7 ) . MOIST CHAMBER—After 2 4 hours of storage the endothelium appeared similar
Fig. 4 (Geeraets, McLester, and McMullan). Corneal endothelium stored nine days in M-K medium at 4°C appears smooth, although the cell surfaces are somewhat more irregular in size and shape and the cell outlines are more distinct than those in the control cells (SEM, x2,000).
FEBRUARY, 1977
Fig. 5 (Geeraets, McLester, and McMullan). Corneal endothelium stored 24 hours in serum at 4°C. The cell outlines are more distinct than those in the control cornea probably because of some dehydration of the cells. There are many microvilli and occasional cilia present and the cells form a continuous sheet without separation (SEM, x2,000). to the specimens stored in the other two media ( F i g . 8 ) . After 4 8 hours some specimens had occasional degenerated cells in each viewing field ( F i g . 9 ) . T h u s , an early onset o f endothelial damage existed, although most of the endothelium appeared normal. T h e cell degeneration b e c a m e more pronounced and widespread after three days of storage ( F i g . 10). Storage under incubation (37°C)—M-K MEDIUM—The endothelial cells stored in this medium at this temperature kept their
Fig. 6 (Geeraets, McLester, and McMullan). Corneal endothelium stored four days in serum at 4°C has intact smooth cell surfaces with many microvilli and few differences as compared with control specimens. Microvilli are present (SEM, x 2,000).
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Fig. 7 (Geeraets, McLester, and McMullan). Corneal endothelium stored nine days in serum at 4°C shows differences in cell size and irregular surfaces although the continuity of the cell membrane is uninterrupted (SEM, x2,000).
normal shape and appeared viable for the first four days (Fig. 11). All corneal specimens kept their normal thickness. After storage day 6 slight cellular changes were observed with irregular surfaces, more shrinkage, and fewer microvilli than on the days before. After storage day 9 many cells showed severe damage, destruction, and cell dehiscence throughout all specimens (Fig. 12). However, some areas had normal cell configuration and a smooth surface.
209
Fig. 9 (Geeraets, McLester, and McMullan). Corneal endothelium stored 48 hours in a moist chamber at 4°C. The individual cells are not as uniform in size and configuration and there is an occasional degenerated cell although the layer is intact (SEM, x 2,000).
anee at storage day 4 was similar to that described for the M-K medium. However, some small processes crossed the membranes from one cell to another ( F i g . 13). T h e s e structures resembled the border bars described by Svedbergh and B i l l in normal corneal endothelial cells in man and monkeys. T h e i r significance is not known and in our specimens may be artifactual. After storage day 6 , the entire cornea was slightly swollen. T h e endo1 8
M E M M I X T U R E — T h e cellular appear-
Fig. 8 (Geeraets, McLester, and McMullan). Corneal endothelium stored 24 hours in a moist chamber at 4°C. The surface appears somewhat irregular although there is no separation of cells (SEM, X2.000).
Fig. 10 (Geeraets, McLester, and McMullan). Corneal endothelium stored 72 hours in a moist chamber at 4°C. The individual cell surfaces are more irregular and do not appear as smooth as those stored for shorter periods although cell integrity still persists (SEM, x2,000).
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Fig. 11 (Geeraets, McLester, and McMullan). Corneal endothelium after four days in M-K medium at 37°C shows normal configuration and cell outlines with smooth surfaces. The individual cells show microvilli and individual cilia and are similar to the control cells (SEM, x2,000). thelial cells had b e c o m e more rounded and
seemed
Descemet's days
after
to
have
membrane
detached
from
( F i g . 14). Nine
storage all endothelial
cells
were detached and formed clumps o f debris (Fig. 15). T h e corneas had swollen to about twice the normal thickness. S E R U M — A s anticipated, early swelling of the cornea and damage o f the endothelial cells with complete destruction was
Fig. 12 (Geeraets, McLester, and McMullan). Corneal endothelium after nine days in M-K medium at 37°C. Although most of the cell surface is smooth and the outline is intact, several areas in the specimen have degenerated cells and cell separation (SEM, X2.000).
FEBRUARY, 1977
Fig. 13 (Geeraets, McLester, and McMullan). Corneal endothelium after four days in MEM mixture at 37°C. The surface is wavy but intact, and all cells have microvilli and individual cilia. Fine processes seem to bridge the cell membranes, an observation not made in the other specimens (SEM, x2,000). noted two days after storage under these temperature conditions ( F i g . 16). DISCUSSION Our observations cannot b e extrapolated to stored human donor material since several variables exist: specific characteristics o f the species, the time lapse b e tween the donor's death, enucleation o f the eye, the time the cornea had
been
stored in the storage medium, and finally
Fig. 14 (Geeraets, McLester, and McMullan). Corneal endothelium after six days of storage in the MEM mixture at 37°C. The individual cells are swollen and are detached from each other. A complete layer covers Descemet's membrane (SEM, x2,000).
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chamber at 4°C, however, was not as effective as previously d e s c r i b e d . These differences may have been caused by different storage techniques or by processing the specimens for further scanning electron microscopic screening. Storing donor corneas under incubation at 37°C showed best results in M-K medium. However, corneal endothelium stored in the M E M mixture showed little change when compared with that stored in M-K medium. At days 6 and 9, some cellular changes were noted in both but the cell destruction was more severe in the M E M mixture. Graft material stored in serum under incubation did not survive the first 2 4 hours. With regard to endothelial cell integrity, we obtained best results when the tissue was stored in M-K medium, and storage under refrigeration leads to better results than storage in an incubator at 37°C. 16
Fig. 15 (Geeraets, McLester, and McMullan). Corneal endothelium after nine days of storage in MEM mixture at 37°C. The cells have degenerated and clumped in individual groups over Descemet's membrane (SEM, x2,000).
the age o f the donor. In our study, a more ideal situation existed since all corneal tissue was stored in the various media immediately after enucleation o f the eye thus preventing possible damage to the endothelium by autolysis, age, or the other variables. Nevertheless, storage of the donor cornea at 4°C either in M-K medium or host serum for up to four days seems effective in maintaining endothelial integrity and possibly reducing corneal antigenic properties. ' Storage in a moist 3
7
Fig. 16 (Geeraets, McLester, and McMullan). Corneal endothelium after 48 hours of storage in serum at 37°C. Individual cells are destroyed and rounded with some of the cells connected by bands. Cell debris covers Descemet's membrane (SEM, x2,000).
SUMMARY
Three rabbit corneas each were stored in McCarey-Kaufman (M-K) medium, rabbit serum, and in a moist chamber at 4°C refrigeration for various lengths of observation. T h e endothelial cells appeared normal—under all conditions for the first 2 4 hours as compared with control corneas processed concurrently with each experimental group. After 4 8 hours of storage the specimens in the moist chamber showed isolated endothelial cell damage. T h e endothelia in M-K medium or rabbit serum appeared viable up to six days without significant differences although those stored in rabbit serum showed a better preserva ti on ai m i cm vi 11 i on individual endothelial cells. Under all conditions a mild shrinkage of the cells seemed to have taken place as indicated by the more pronounced cell boundaries. We incubated an equal number of control rabbit corneas at 37°C with 5 % C 0 and moist air in M-K medium, serum, and 2
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minimal essential medium ( M E M ) with 1 0 % fetal calf serum and 100 units/ml of a penicillin and streptomycin mixture. In serum, the endothelia showed rapid destruction with swelling of the entire cornea. T h o s e stored in M-K medium maintained a normal endothelial covering of the cornea up to six days. At nine days of storage, marked cellular changes were observed with dehiscence of the cellular layer. When stored in the M E M mixture, the endothelial cells showed a normal layer without obvious cell damage when compared with those stored in M-K medium up to four days. However, after six and nine days o f storage, cellular destruction was greater in these specimens than in those stored in M-K medium _In_addition, there was considerable swelling of the whole ¿ornea under this storage condition.
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5. : Storage of donor corneas in the recipient's serum prior to grafting. Pacific Med. Surg. 76:31, 1968. 6. Geeraets, W. J . , Chan, G., and Guerry, D.: Corneal antigenicity. Arch. Ophthalmol. 64:413, 1960. 7. Geeraets, W. J . , Hoskins, D., and Guerry, D.: Some immunological aspects in keratoplasty. Acta Ophthalmologica 43:173, 1965. 8. Geeraets, W. J., Lederman, I. R., Woo, H., and Guerry, D., Ill: In vivo corneal graft reaction after short-term storage: An experimental study. Am. J . Ophthalmol. 60:28, 1965. 9. Tsutsui, J . , and Watanabe, H.: Heterograft of fish cornea into rabbit eyes. Am. J . Ophthalmol. 48:363, 1959. 10. Castroviejo, R.: Keratoplasty, microscopic study of the corneal grafts. Trans. Am. Ophthalmol. Soc. 35:355, 1937. 11. Sykes, J., and Girard, L.: Heterologous corneal transplants in rabbits. Am. J. Ophthalmol. 48:259, 1959. 12. Kuwahara, Y.: Studies on heterotransplantation of cornea. Am. J. Ophthalmol. 53:911, 1962. 13. Leber, Th.: Studien über den Flussigkeitswechsel im Auge. Albrecht von Graefe's Arch. Klin. Ophthalmol. 19:87, 1873. 14. Honegger, H.: Quantitative Untersuchungen über die Hornhautendothelregeneration in vivo. Albrecht von Graefe's Arch. Klin. Ophthalmol. 165: 31, 1962. REFERENCES 15. Mishima, S., and Kudo, T.: In vitro incuba1. McCarey, B. E . , and Kaufman, H. E . : Im- tion of rabbit corneas. Invest. Ophthalmol. 6:329, 1967. proved corneal storage. Invest. Ophthalmol. 13:165, 16. Friedland, B. R., and Forster, R. K.: Compari1974. son of corneal storage in McCarey-Kaufman medi2. Magitot, A.: Transplantation of the human um, moist chamber, or standard eye bank condicornea previously preserved in an aseptic fluid. tions. Invest. Ophthalmol. 15:143, 1976. J.A.M.A. 59:18, 1912. 17. Van Horn, D. L., and Schultz, R. D.: Compari3. Stocker, F . W., Levenson, D., and Georgiade, son of serum vs eye bank storage of cat corneas. N.: Medium-term preservation of corneal tissue for Arch. Ophthalmol. 92:142, 1974. grafting. Arch. Ophthalmol. 70:554, 1963. 18. Svedbergh, B., and Bill, A.: Scanning electron 4. Stocker, F. W.: Preservation of donor cornea in microscopic studies of the corneal endothelium in autologous serum prior to penetrating grafts. Am. J. man and monkeys. Acta Ophthalmol. 50:321, 1972. Ophthalmol. 60:21, 1965.
Short-term storage of corneal graft material has been used more frequently since 1 9 7 4 when M c C a r e y and Kaufman developed a modified tissue culture medium, the M-K medium, which consists of a mixture of T C - 1 9 9 medium, 5 % dextran, and 100 units/ml of a streptomycin and penicillin mixture. T h e osmolarity is 2 9 0 m O s m and the p H is 7.4. T h e idea of short-term storage, however, had been mentioned by M a g i t o t as early as 1 9 1 2 . T h e purpose of short-term storage in a suitable medium is twofold. First, surgery can b e scheduled more conveniently, particularly if the recipient lives far from where the surgery is to b e performed. Secondly, experimental w o r k has shown that short-term storage of the donor cornea in a suitable medium causes antigenicity loss by diffusion o f such antigens into the storage medium. 1
2
3 - 1 2
In 1 8 7 3 , L e b e r first described the importance o f the corneal endothelium and the role it plays in corneal clarity. Quantitative examinations of corneal endothelium regeneration in vivo were reported by H o n e g g e r in 1 9 6 2 who showed the interrelationship of corneal swelling to corneal endothelial defects. T h e role of corneal hydration as a balance between the imbibition pressure of the stromal mucopolysaccharides and the metabolically active transport system in the endothelium was reemphasized by M i s h i m a and K u d o . H e n c e , the importance of the state of the corneal endotheli1 3
14
15
From the Department of Ophthalmology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia. This study was supported in part by the Old Dominion Eye Bank and Research, Inc., Richmond, Virginia. Reprint requests to Ragnit Geeraets, Ph.D., Box 262/MCV, Richmond, VA 23298.
Virginia
um and its viability in successful penetrating corneal transplantation has long been realized. Therefore, short-term storage is useful only if the storage medium is nontoxic and maintains endothelial viability. In most situations these properties had to b e maintained for only a few days to meet the two criteria o f short-term storage. Recently Friedland and F o r s t e r stated that this goal could b e achieved by keeping the donor graft material in M-K medium at 4°C for several days as well as by storing the corneas in a moist c h a m b e r in the refrigerator. However, storage o f the whole eye under similar conditions had a negative effect on the endothelium that was caused by a toxic reaction o f autolysis of the intraocular tissue on the endothelium. A similar observation had already been made by S t o c k e r . Van Horn and Schultz stored cat corneal endothelia with a scleral rim in a refrigerator for up to 120 hours. T h e corneal endothelial cells stored in the moist c h a m b e r remained viable longer than corneas stored in the serum. Whether these differences are caused by t e c h n i q u e or different animal models is not clear. 16
5
17
M A T E R I A L AND M E T H O D S
T o determine the effects o f storage on the corneal endothelium, we stored three rabbit donor corneas each in serum, in M-K medium, and in a moist c h a m b e r at 4°C, and observed the results with a scanning electron microscope. W e did not use other culture media b e c a u s e M c C a r e y and Kaufman found them inferior to the M-K medium. W e incubated an equal number of control corneas in a tissue culture incubator at 37°C with 5 % C 0 and moist air in
206
1
2
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M-K medium, serum, and minimum essential medium ( M E M ) with 1 0 % fetal calf serum and 100 units/ml of a penicillin and streptomycin mixture. T h i s part o f the study was conducted to explore the viability and integrity of the endothelium under incubation conditions that would eliminate temperature reversal at the time of transplantation which is required for graft material under refrigeration. W e examined specimens from these rabbit corneas after various periods o f storage. Three rabbit eyes were used for each storage condition and time. T h e corneas were excised with a 2-mm scleral rim after the eyes had been enucleated. Corneal tissue was placed immediately in the storage media or moist chamber. On days 1 , 2 , 3, 4 , 6, and 9 we removed the specimens from the media and processed them concurrently with a freshly obtained cornea, used as a control. T h e y were fixed in glutaraldehyde for 2V2 hours, dissected in quarters, dehydrated in a graded series of acetone, and critical-point-dried with fluorocarbon C F - C F (Freon 116). T h e endothelial surface was always face up. T h e specimens were then mounted, coated with gold-palladium, and examined with a scanning electron microscope. 3
3
Fig. 1 (Geeraets, McLester, and McMullan). Normal corneal endothelium has a distinct outline with microvilli and occasional cilia. The surface appears smooth without separation of individual cells (SEM, x 2,000).
with little change up to four days of storage ( F i g . 3). After days 6 and 9, various cells had irregular surfaces and shapes. T h e s e changes were more pronounced after nine days o f storage ( F i g . 4 ) . SERUM—After 2 4 hours the cells appeared essentially the same as those stored in M-K medium. S i n c e more microvilli were seen, these specimens more closely resembled the control corneas. T h e cell borders were more pro-
RESULTS
T h e control specimens in all experimental groups had essentially the same appearance ( F i g . 1). T h e cells were smooth with microvilli and an occasional cilium. T h e cells were the same size and were outlined by their membranes without cell separation. Storage under refrigeration (4°C)—M-K M E D I U M — A f t e r 2 4 hours o f storage we noted no significant difference in the appearance of the cells as compared with that o f the control cells although the cell outlines were more marked indicating that a mild shrinkage or dehydration o f the cells may have taken place ( F i g . 2 ) . This endothelial appearance persisted
Fig. 2 (Geeraets, McLester, and McMullan). Corneal endothelium stored 24 hours in M-K medium at 4°C. The cell outlines are more marked but the surface is smooth without separation of cells (SEM, x 2,000).
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Fig. 3 (Geeraets, McLester, and McMullan). Corneal endothelium stored four days in M-K medium at 4°C shows some irregular cell configuration and occasional cells that have some degenerative changes. The cell boundaries are still intact (SEM, x 2,000). n o u n c e d ' a n d there was more cell shrinkage ( F i g . 5 ) . T h e cell layer was intact for the first four days of storage ( F i g . 6 ) . Similar to the cells stored in M-K medium, the cell surface b e c a m e more irregular after six and nine days of storage with no gaps between individual cells that formed an intact layer over D e s c e m e t 's membrane ( F i g . 7 ) . MOIST CHAMBER—After 2 4 hours of storage the endothelium appeared similar
Fig. 4 (Geeraets, McLester, and McMullan). Corneal endothelium stored nine days in M-K medium at 4°C appears smooth, although the cell surfaces are somewhat more irregular in size and shape and the cell outlines are more distinct than those in the control cells (SEM, x2,000).
FEBRUARY, 1977
Fig. 5 (Geeraets, McLester, and McMullan). Corneal endothelium stored 24 hours in serum at 4°C. The cell outlines are more distinct than those in the control cornea probably because of some dehydration of the cells. There are many microvilli and occasional cilia present and the cells form a continuous sheet without separation (SEM, x2,000). to the specimens stored in the other two media ( F i g . 8 ) . After 4 8 hours some specimens had occasional degenerated cells in each viewing field ( F i g . 9 ) . T h u s , an early onset o f endothelial damage existed, although most of the endothelium appeared normal. T h e cell degeneration b e c a m e more pronounced and widespread after three days of storage ( F i g . 10). Storage under incubation (37°C)—M-K MEDIUM—The endothelial cells stored in this medium at this temperature kept their
Fig. 6 (Geeraets, McLester, and McMullan). Corneal endothelium stored four days in serum at 4°C has intact smooth cell surfaces with many microvilli and few differences as compared with control specimens. Microvilli are present (SEM, x 2,000).
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Fig. 7 (Geeraets, McLester, and McMullan). Corneal endothelium stored nine days in serum at 4°C shows differences in cell size and irregular surfaces although the continuity of the cell membrane is uninterrupted (SEM, x2,000).
normal shape and appeared viable for the first four days (Fig. 11). All corneal specimens kept their normal thickness. After storage day 6 slight cellular changes were observed with irregular surfaces, more shrinkage, and fewer microvilli than on the days before. After storage day 9 many cells showed severe damage, destruction, and cell dehiscence throughout all specimens (Fig. 12). However, some areas had normal cell configuration and a smooth surface.
209
Fig. 9 (Geeraets, McLester, and McMullan). Corneal endothelium stored 48 hours in a moist chamber at 4°C. The individual cells are not as uniform in size and configuration and there is an occasional degenerated cell although the layer is intact (SEM, x 2,000).
anee at storage day 4 was similar to that described for the M-K medium. However, some small processes crossed the membranes from one cell to another ( F i g . 13). T h e s e structures resembled the border bars described by Svedbergh and B i l l in normal corneal endothelial cells in man and monkeys. T h e i r significance is not known and in our specimens may be artifactual. After storage day 6 , the entire cornea was slightly swollen. T h e endo1 8
M E M M I X T U R E — T h e cellular appear-
Fig. 8 (Geeraets, McLester, and McMullan). Corneal endothelium stored 24 hours in a moist chamber at 4°C. The surface appears somewhat irregular although there is no separation of cells (SEM, X2.000).
Fig. 10 (Geeraets, McLester, and McMullan). Corneal endothelium stored 72 hours in a moist chamber at 4°C. The individual cell surfaces are more irregular and do not appear as smooth as those stored for shorter periods although cell integrity still persists (SEM, x2,000).
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210
Fig. 11 (Geeraets, McLester, and McMullan). Corneal endothelium after four days in M-K medium at 37°C shows normal configuration and cell outlines with smooth surfaces. The individual cells show microvilli and individual cilia and are similar to the control cells (SEM, x2,000). thelial cells had b e c o m e more rounded and
seemed
Descemet's days
after
to
have
membrane
detached
from
( F i g . 14). Nine
storage all endothelial
cells
were detached and formed clumps o f debris (Fig. 15). T h e corneas had swollen to about twice the normal thickness. S E R U M — A s anticipated, early swelling of the cornea and damage o f the endothelial cells with complete destruction was
Fig. 12 (Geeraets, McLester, and McMullan). Corneal endothelium after nine days in M-K medium at 37°C. Although most of the cell surface is smooth and the outline is intact, several areas in the specimen have degenerated cells and cell separation (SEM, X2.000).
FEBRUARY, 1977
Fig. 13 (Geeraets, McLester, and McMullan). Corneal endothelium after four days in MEM mixture at 37°C. The surface is wavy but intact, and all cells have microvilli and individual cilia. Fine processes seem to bridge the cell membranes, an observation not made in the other specimens (SEM, x2,000). noted two days after storage under these temperature conditions ( F i g . 16). DISCUSSION Our observations cannot b e extrapolated to stored human donor material since several variables exist: specific characteristics o f the species, the time lapse b e tween the donor's death, enucleation o f the eye, the time the cornea had
been
stored in the storage medium, and finally
Fig. 14 (Geeraets, McLester, and McMullan). Corneal endothelium after six days of storage in the MEM mixture at 37°C. The individual cells are swollen and are detached from each other. A complete layer covers Descemet's membrane (SEM, x2,000).
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chamber at 4°C, however, was not as effective as previously d e s c r i b e d . These differences may have been caused by different storage techniques or by processing the specimens for further scanning electron microscopic screening. Storing donor corneas under incubation at 37°C showed best results in M-K medium. However, corneal endothelium stored in the M E M mixture showed little change when compared with that stored in M-K medium. At days 6 and 9, some cellular changes were noted in both but the cell destruction was more severe in the M E M mixture. Graft material stored in serum under incubation did not survive the first 2 4 hours. With regard to endothelial cell integrity, we obtained best results when the tissue was stored in M-K medium, and storage under refrigeration leads to better results than storage in an incubator at 37°C. 16
Fig. 15 (Geeraets, McLester, and McMullan). Corneal endothelium after nine days of storage in MEM mixture at 37°C. The cells have degenerated and clumped in individual groups over Descemet's membrane (SEM, x2,000).
the age o f the donor. In our study, a more ideal situation existed since all corneal tissue was stored in the various media immediately after enucleation o f the eye thus preventing possible damage to the endothelium by autolysis, age, or the other variables. Nevertheless, storage of the donor cornea at 4°C either in M-K medium or host serum for up to four days seems effective in maintaining endothelial integrity and possibly reducing corneal antigenic properties. ' Storage in a moist 3
7
Fig. 16 (Geeraets, McLester, and McMullan). Corneal endothelium after 48 hours of storage in serum at 37°C. Individual cells are destroyed and rounded with some of the cells connected by bands. Cell debris covers Descemet's membrane (SEM, x2,000).
SUMMARY
Three rabbit corneas each were stored in McCarey-Kaufman (M-K) medium, rabbit serum, and in a moist chamber at 4°C refrigeration for various lengths of observation. T h e endothelial cells appeared normal—under all conditions for the first 2 4 hours as compared with control corneas processed concurrently with each experimental group. After 4 8 hours of storage the specimens in the moist chamber showed isolated endothelial cell damage. T h e endothelia in M-K medium or rabbit serum appeared viable up to six days without significant differences although those stored in rabbit serum showed a better preserva ti on ai m i cm vi 11 i on individual endothelial cells. Under all conditions a mild shrinkage of the cells seemed to have taken place as indicated by the more pronounced cell boundaries. We incubated an equal number of control rabbit corneas at 37°C with 5 % C 0 and moist air in M-K medium, serum, and 2
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minimal essential medium ( M E M ) with 1 0 % fetal calf serum and 100 units/ml of a penicillin and streptomycin mixture. In serum, the endothelia showed rapid destruction with swelling of the entire cornea. T h o s e stored in M-K medium maintained a normal endothelial covering of the cornea up to six days. At nine days of storage, marked cellular changes were observed with dehiscence of the cellular layer. When stored in the M E M mixture, the endothelial cells showed a normal layer without obvious cell damage when compared with those stored in M-K medium up to four days. However, after six and nine days o f storage, cellular destruction was greater in these specimens than in those stored in M-K medium _In_addition, there was considerable swelling of the whole ¿ornea under this storage condition.
FEBRUARY, 1977
5. : Storage of donor corneas in the recipient's serum prior to grafting. Pacific Med. Surg. 76:31, 1968. 6. Geeraets, W. J . , Chan, G., and Guerry, D.: Corneal antigenicity. Arch. Ophthalmol. 64:413, 1960. 7. Geeraets, W. J . , Hoskins, D., and Guerry, D.: Some immunological aspects in keratoplasty. Acta Ophthalmologica 43:173, 1965. 8. Geeraets, W. J., Lederman, I. R., Woo, H., and Guerry, D., Ill: In vivo corneal graft reaction after short-term storage: An experimental study. Am. J . Ophthalmol. 60:28, 1965. 9. Tsutsui, J . , and Watanabe, H.: Heterograft of fish cornea into rabbit eyes. Am. J . Ophthalmol. 48:363, 1959. 10. Castroviejo, R.: Keratoplasty, microscopic study of the corneal grafts. Trans. Am. Ophthalmol. Soc. 35:355, 1937. 11. Sykes, J., and Girard, L.: Heterologous corneal transplants in rabbits. Am. J. Ophthalmol. 48:259, 1959. 12. Kuwahara, Y.: Studies on heterotransplantation of cornea. Am. J. Ophthalmol. 53:911, 1962. 13. Leber, Th.: Studien über den Flussigkeitswechsel im Auge. Albrecht von Graefe's Arch. Klin. Ophthalmol. 19:87, 1873. 14. Honegger, H.: Quantitative Untersuchungen über die Hornhautendothelregeneration in vivo. Albrecht von Graefe's Arch. Klin. Ophthalmol. 165: 31, 1962. REFERENCES 15. Mishima, S., and Kudo, T.: In vitro incuba1. McCarey, B. E . , and Kaufman, H. E . : Im- tion of rabbit corneas. Invest. Ophthalmol. 6:329, 1967. proved corneal storage. Invest. Ophthalmol. 13:165, 16. Friedland, B. R., and Forster, R. K.: Compari1974. son of corneal storage in McCarey-Kaufman medi2. Magitot, A.: Transplantation of the human um, moist chamber, or standard eye bank condicornea previously preserved in an aseptic fluid. tions. Invest. Ophthalmol. 15:143, 1976. J.A.M.A. 59:18, 1912. 17. Van Horn, D. L., and Schultz, R. D.: Compari3. Stocker, F . W., Levenson, D., and Georgiade, son of serum vs eye bank storage of cat corneas. N.: Medium-term preservation of corneal tissue for Arch. Ophthalmol. 92:142, 1974. grafting. Arch. Ophthalmol. 70:554, 1963. 18. Svedbergh, B., and Bill, A.: Scanning electron 4. Stocker, F. W.: Preservation of donor cornea in microscopic studies of the corneal endothelium in autologous serum prior to penetrating grafts. Am. J. man and monkeys. Acta Ophthalmol. 50:321, 1972. Ophthalmol. 60:21, 1965.
