Corneal Uriel
Storage
at Room
Sachs, MD; Kenneth Goldman, MD; John
\s=b\tShort-term based mainly on moist chamber and McCareyKaufman medium (M-Kium) preservation.nvolve a controlled 4C temperature for storage. Warming the cornea to room temperature, however, drastically affects the endothelial viability. On enzymatic staining and histological Mmedium-stored study, the corneas had more normal endothelium than did "moist chamber" eyes when storage was prolonged for seven days at room temperature. In human corneas that were kept at 4Cfor 24 hours and then exposed to a temperature of 25 C, destruction of organelles had occurred by six hours and was increased by 12 hours. M-Kdium Corneas that were kept in had relatively intact endothelium after four days, but cell disruption and vacuolation present by the seventh day. The M-Kdium, therefore, affords protection to tissue warmed to room temperature, where metabolic activity is resumed.
(Arch
O96:1075-1077,
Temperature
Valenti; E. Kaufman, MD
dium)
are
kept
at 4 C to
prevent
of the endothe¬
possible degeneration lium that occurs at higher tempera¬ tures. At 4 C
storage temperature,
"moist chamber" eyes can be used for up to 48 hours, and "M-K medium" corneas are suitable for keratoplasty for up to seven days.-' At temperatures higher than 4 C, the cornea increases its metabolic activity, and its survival is related to the availability of nutrients and the release of metabolic wastes.
We present the effect of
temperature (25 C) storage
structure of rabbit and human
room
on
the
corneas
stored in moist chambers and in M-K medium. MATERIALS AND METHODS Adult New Zealand albino rabbits (2 to 3 kg) were killed by carbon dioxide inhala¬ tion, and the eyes were immediately enucleated. Neosporin drops were flooded over the eyes for two minutes. Twelve
intact globes were placed in jars that contained 5 ml of normal saline solution for
moisturizing.
Six of these globes were refrigerated at 4 C, while the remaining six globes were left at room temperature (25 C). Twelve additional corneas with a 2- to 3-mm scierai rim that was carefully dissected from the rest of the globe were placed in vials that contained 20 ml of M-K medium with 4 mg of gentamicin sulfate added. Six vials were stored at 4 C, and the remaining six were stored at 25 C. The closed vials were opened after seven days to study the endo¬ thelial changes. All the corneas were dissected; half of each cornea was immedi¬ ately stained with nitroblue tetrazolium (NBT) as described by Robbins et al.3 The other half was placed in 0.25% glutaralde¬ hyde in Milloning buffer at 4 C for 24 hours. A small, 2-mm wide central portion of this half of the cornea was then postfixed in 1% osmium tetroxide in Milloning buffer and embedded in exoxy resin. Thin, 0.1-µ sections were examined by transmis¬ sion electron microscopy. The remainder of the cornea was prepared for scanning elee-
Human
donor corneas, preserved in moist chambers and in McCarey-Kaufman medium' (M-K mefor publication Sept 30, 1977. From the Department of Ophthalmology, College of Medicine, University of Florida, Gainesville. Dr Sachs is now with the Soroca Medical Center, Beer-Sheva, Goldman is with the Department of Ophthalmology, Montefiore Hospital, Bronx, NY; Dr Kaufman is with the Department of Ophthalmology, Louisianae University School of Medicine, New Orleans. Reprint requests to Department of Ophthalmology,ana State University School of Medicine, 136 choman St, New Orleans, LA 70112 (Dr Kaufman).
Accepted
showing, left, few separated irregular cells and large of bare Descemet's membrane after seven days' moist chamber storage at 25 C, and right, regular mosaic pattern with prominent cell membranes after seven days at 25 C, McCarey-Kaufman medium storage.
Fig
1 .—Rabbit corneal endothelium
areas
Downloaded From: http://archopht.jamanetwork.com/ by a University of Iowa User on 06/13/2015
RESULTS Rabbits Moist Chamber
Storage.-At
4
C,
than Vs of the endothelial cells appeared viable with a positive indi¬ rect NBT stain after one week of storage. A smooth and regular mosaic pattern was observed by scanning electron microscopy. Ultrastructurally, a mild degree of decreased cyto¬ plasmic density was present; mito¬ chondria and nuclei were normal. At 25 C, all moist chamber-stored corneas were opaque, and the NBT revealed few positively stained cells after a week of storage. On scanning electron microscopical examination, large areas of bare Descemet's mem¬ brane were found, and a very small number of irregularly shaped endo¬ thelial cells were scattered throughout (Fig 1, left). In thin sections, we were unable to find cells on the bare Desce¬ met's membrane. more
Fig 2.—Human eyes stored in moist chambers at 4 C for 24 hours. Left, After removal from 4 C and after warming for six hours at 25 C, normal intercellular spaces, nucleus and apical junction, and swollen mitochondria (arrows) are seen. Right, After removal from 4 C and after warming for 12 hours at 25 C, swollen cytoplasm, and mitochondria without their fine structure (arrow) are seen.
M-K Media
Fig 3.—Human corneas stored in McCarey-Kaufman medium at 25 C. Left, After four days, raised central area (arrows) has developed in some cells. Right, After seven days, cells
are
swollen and few
pits (arrow)
are
present.
Fig 4.—Human corneas stored in McCarey-Kaufman medium at 25 C. Left, After four days, smooth posterior membrane, dense cytoplasm, and distinct organelle structures are seen. Right, After seven days, thick, swollen cell with decrease in anterior cytoplasm density and few vacuoles is seen. (This is cornea in Fig 3, right.) tron
microscopy by the critical point drying
process.' Glucose concentration in the M-K media was assayed by the glucose oxidase-peroxidase method at the beginning and the end of the preservation period. At the same intervals, pH was measured with a pH
ionalyzer. Human eyes were obtained from the North Florida Lions Eye Bank, Gainesville, and used in the study if the time between death and enucleation was less than six hours. Donor ages ranged from 9 to 82
years. Eight whole eyes were kept in moist chambers at 4 C until 24 hours had elapsed from the time of death; these eyes were then stored at room temperature (25 C) for six to 12 hours. Eight additional corneas were separated with a 2- to 3-mm scierai rim and were kept at 25 C for four or seven days in M-K medium. The human corneas were bissected, and half of each cornea was prepared for scanning, and the other half was prepared for transmission electron microscopy in the method described for rabbit eves.
Downloaded From: http://archopht.jamanetwork.com/ by a University of Iowa User on 06/13/2015
4 C, a to NBT stain indi¬
Storage.-At
positive reaction cated viability in
than Vs of the scanning elec¬ examination, the
more
cells after one week. On tron
microscopical
endothelium had a flat, smooth, poste¬ rior surface; some cells had a raised central area. By transmission electron microscopical examination, the endo¬ plasmic reticulum, chromatin, and cell junctions were normal; a number of small vacuoles were present. At 25 C, the M-K medium-stored corneas appeared generally normal and were the same after NBT stain of the endothelium as those stored at 4 C. The endothelial layer, as seen by scan¬ ning, was intact with few pits and occasional cells with a prominent central area (Fig 1, right); the same ultrastructural changes that were seen at 4 C were found. The presence of the media, therefore, protected the endothelium that had been warmed to room
temperature. Humans
Moist Chamber Storage.-After six hours at 25 C, slightly less than Vs of the cells yielded a positive NBT stain. On examination by scanning microscopy, approximately 20% of the cells had a disrupted posterior mem¬ brane. On thin section, areas of decreased cytoplasmic density were
present anteriorly, and mitochondria showed signs of swelling (Fig 2, left). After 12 hours at 25 C, the cytoplasm was diffusely edematous; the mito¬
chondria
were
completely disorga¬
nized, losing both shape and structure (Fig 2, right). By scanning micros¬
patches of endothelial lysis were Room temperature, therefore, severely damaged the endothelium of copy, seen.
moist chamber-stored human eyes. M-K Media Storage.-The endothe¬ lium on the fourth day of storage at 25 C had a regular pattern, and occasional cells had a central elevation (Fig 3, left). Transmission electron micros¬ copy showed relatively normal cells, with dense nuclei and cytoplasm; minimal chromatin vacuolation and disorganized mitochondria were pres¬ ent (Fig 4, left). After seven days, the picture remained relatively normal. A few clefts appeared near the posterior surface, and areas of decreased cyto¬ plasmic density were found (Fig 4, right). Scanning electron microscopy revealed less distinct cellular borders and many pits in edematous-looking cells (Fig 3, right), with some degener¬ ated cells on linear folds (which are thought to be artifactual). The pH of the media in the vials that were left at 25 C varied from 6.10 to 6.80 and was lower than in media refrigerated at 4 C, where the pH range was 6.80 to 7.06. At 4 C, the glucose concentration remained stable, whereas at 25 C, the glucose level fell from 160 mg/100 ml to 110 mg/100 ml. COMMENT
Storage
in moist chambers and M-K
medium is
usually performed at 4 C to
prolong the
survival of the cornea. We have found that at room temperature (25 C), rabbit corneas that have been stored in M-K medium for seven days still retain an intact endothelial layer. Eyes stored in moist chambers had almost total endothelial degeneration, with only a few abnormal endothelial cells remaining. As in other reports,'1 rabbit eyes stored at 4 C by either method had a continuous endothelial layer, but corneas without medium, when warmed to room temperature, were
destroyed.
Shorter periods of storage were used for human eyes because endothe¬ lium in them degenerates more rap¬ idly.3 Moist chamber-stored eyes that were removed from the cold began to show degenerative vacuolization, and disruption of organelles was present. The second six-hour period was more deleterious than the first one. These eyes had been initially cooled to 4 C, and very likely, metabolic activity took place only after one or two hours when the temperature of the eye isolated in a glass-air jacket ap¬ proached 25 C. Similarly, substantial storage time before warming would almost certainly increase this dam¬ age. In contrast, corneas kept in M-K medium at 25 C fared much better. At four days, minimal endothelial dam¬ age was present; by seven days,
although small
areas
of
lysis
were
present, cellular organelles
were still of the fact that recognizable. spite M-K medium was not designed for organ culture, our results for the first
In
Downloaded From: http://archopht.jamanetwork.com/ by a University of Iowa User on 06/13/2015
four days compare well with those of Lindstrom et al," who used a modified Eagle minimal essential medium with 10% calf serum added. In M-K medium at 25 C, corneas had some metabolic activity, as evidenced by the decrease in glucose concentration. For normal eye banking procedures, especially when temperature control during shipping is uncertain, the use of M-K medium for preservation of donor corneas is a safer method than moist chamber storage. Where the donor tissue is warmed and metabolic activity is thus resumed, moist cham¬ ber tissue can be seriously damaged. This investigation was supported in part by grants EY-00446 and EY-00266 from the Nation¬ al
Eye
Institute.
Nonproprietary Name and Trademark of Drug Gentamicin
sulfate—Garamycin. References
1. McCarey BE, Kaufman HE: Improved corneal storage. Invest Ophthalmol 13:165-173, 1974. 2. Bigar F, Kaufman HE, McCarey B, et al: Improved corneal storage for penetrating keratoplasties in man. Am J Ophthalmol 79:115-120,
1975. 3. Robbins JE, Capella JA, Kaufman HE: A study of endothelium in keratoplasty and corneal preservation. Arch Ophthalmol 73:242-247, 1965. 4. Polack FM: Scanning electron microscopy of corneal graft rejection. Invest Ophthalmol 11:1\x=req-\
14, 1972.
5. McCarey BE, Sakimoto T, Bigar F: Ultrastructure of M-K and refrigerated moist chamber-stored corneas. Invest Ophthalmol 13:859-863, 1974. 6. Lindstrom RL, Doughman DJ, Van Horn DL, et al: Organ culture corneal storage at ambient room temperature. Arch Ophthalmol 95:869-878, 1977.
Storage
at Room
Sachs, MD; Kenneth Goldman, MD; John
\s=b\tShort-term based mainly on moist chamber and McCareyKaufman medium (M-Kium) preservation.nvolve a controlled 4C temperature for storage. Warming the cornea to room temperature, however, drastically affects the endothelial viability. On enzymatic staining and histological Mmedium-stored study, the corneas had more normal endothelium than did "moist chamber" eyes when storage was prolonged for seven days at room temperature. In human corneas that were kept at 4Cfor 24 hours and then exposed to a temperature of 25 C, destruction of organelles had occurred by six hours and was increased by 12 hours. M-Kdium Corneas that were kept in had relatively intact endothelium after four days, but cell disruption and vacuolation present by the seventh day. The M-Kdium, therefore, affords protection to tissue warmed to room temperature, where metabolic activity is resumed.
(Arch
O96:1075-1077,
Temperature
Valenti; E. Kaufman, MD
dium)
are
kept
at 4 C to
prevent
of the endothe¬
possible degeneration lium that occurs at higher tempera¬ tures. At 4 C
storage temperature,
"moist chamber" eyes can be used for up to 48 hours, and "M-K medium" corneas are suitable for keratoplasty for up to seven days.-' At temperatures higher than 4 C, the cornea increases its metabolic activity, and its survival is related to the availability of nutrients and the release of metabolic wastes.
We present the effect of
temperature (25 C) storage
structure of rabbit and human
room
on
the
corneas
stored in moist chambers and in M-K medium. MATERIALS AND METHODS Adult New Zealand albino rabbits (2 to 3 kg) were killed by carbon dioxide inhala¬ tion, and the eyes were immediately enucleated. Neosporin drops were flooded over the eyes for two minutes. Twelve
intact globes were placed in jars that contained 5 ml of normal saline solution for
moisturizing.
Six of these globes were refrigerated at 4 C, while the remaining six globes were left at room temperature (25 C). Twelve additional corneas with a 2- to 3-mm scierai rim that was carefully dissected from the rest of the globe were placed in vials that contained 20 ml of M-K medium with 4 mg of gentamicin sulfate added. Six vials were stored at 4 C, and the remaining six were stored at 25 C. The closed vials were opened after seven days to study the endo¬ thelial changes. All the corneas were dissected; half of each cornea was immedi¬ ately stained with nitroblue tetrazolium (NBT) as described by Robbins et al.3 The other half was placed in 0.25% glutaralde¬ hyde in Milloning buffer at 4 C for 24 hours. A small, 2-mm wide central portion of this half of the cornea was then postfixed in 1% osmium tetroxide in Milloning buffer and embedded in exoxy resin. Thin, 0.1-µ sections were examined by transmis¬ sion electron microscopy. The remainder of the cornea was prepared for scanning elee-
Human
donor corneas, preserved in moist chambers and in McCarey-Kaufman medium' (M-K mefor publication Sept 30, 1977. From the Department of Ophthalmology, College of Medicine, University of Florida, Gainesville. Dr Sachs is now with the Soroca Medical Center, Beer-Sheva, Goldman is with the Department of Ophthalmology, Montefiore Hospital, Bronx, NY; Dr Kaufman is with the Department of Ophthalmology, Louisianae University School of Medicine, New Orleans. Reprint requests to Department of Ophthalmology,ana State University School of Medicine, 136 choman St, New Orleans, LA 70112 (Dr Kaufman).
Accepted
showing, left, few separated irregular cells and large of bare Descemet's membrane after seven days' moist chamber storage at 25 C, and right, regular mosaic pattern with prominent cell membranes after seven days at 25 C, McCarey-Kaufman medium storage.
Fig
1 .—Rabbit corneal endothelium
areas
Downloaded From: http://archopht.jamanetwork.com/ by a University of Iowa User on 06/13/2015
RESULTS Rabbits Moist Chamber
Storage.-At
4
C,
than Vs of the endothelial cells appeared viable with a positive indi¬ rect NBT stain after one week of storage. A smooth and regular mosaic pattern was observed by scanning electron microscopy. Ultrastructurally, a mild degree of decreased cyto¬ plasmic density was present; mito¬ chondria and nuclei were normal. At 25 C, all moist chamber-stored corneas were opaque, and the NBT revealed few positively stained cells after a week of storage. On scanning electron microscopical examination, large areas of bare Descemet's mem¬ brane were found, and a very small number of irregularly shaped endo¬ thelial cells were scattered throughout (Fig 1, left). In thin sections, we were unable to find cells on the bare Desce¬ met's membrane. more
Fig 2.—Human eyes stored in moist chambers at 4 C for 24 hours. Left, After removal from 4 C and after warming for six hours at 25 C, normal intercellular spaces, nucleus and apical junction, and swollen mitochondria (arrows) are seen. Right, After removal from 4 C and after warming for 12 hours at 25 C, swollen cytoplasm, and mitochondria without their fine structure (arrow) are seen.
M-K Media
Fig 3.—Human corneas stored in McCarey-Kaufman medium at 25 C. Left, After four days, raised central area (arrows) has developed in some cells. Right, After seven days, cells
are
swollen and few
pits (arrow)
are
present.
Fig 4.—Human corneas stored in McCarey-Kaufman medium at 25 C. Left, After four days, smooth posterior membrane, dense cytoplasm, and distinct organelle structures are seen. Right, After seven days, thick, swollen cell with decrease in anterior cytoplasm density and few vacuoles is seen. (This is cornea in Fig 3, right.) tron
microscopy by the critical point drying
process.' Glucose concentration in the M-K media was assayed by the glucose oxidase-peroxidase method at the beginning and the end of the preservation period. At the same intervals, pH was measured with a pH
ionalyzer. Human eyes were obtained from the North Florida Lions Eye Bank, Gainesville, and used in the study if the time between death and enucleation was less than six hours. Donor ages ranged from 9 to 82
years. Eight whole eyes were kept in moist chambers at 4 C until 24 hours had elapsed from the time of death; these eyes were then stored at room temperature (25 C) for six to 12 hours. Eight additional corneas were separated with a 2- to 3-mm scierai rim and were kept at 25 C for four or seven days in M-K medium. The human corneas were bissected, and half of each cornea was prepared for scanning, and the other half was prepared for transmission electron microscopy in the method described for rabbit eves.
Downloaded From: http://archopht.jamanetwork.com/ by a University of Iowa User on 06/13/2015
4 C, a to NBT stain indi¬
Storage.-At
positive reaction cated viability in
than Vs of the scanning elec¬ examination, the
more
cells after one week. On tron
microscopical
endothelium had a flat, smooth, poste¬ rior surface; some cells had a raised central area. By transmission electron microscopical examination, the endo¬ plasmic reticulum, chromatin, and cell junctions were normal; a number of small vacuoles were present. At 25 C, the M-K medium-stored corneas appeared generally normal and were the same after NBT stain of the endothelium as those stored at 4 C. The endothelial layer, as seen by scan¬ ning, was intact with few pits and occasional cells with a prominent central area (Fig 1, right); the same ultrastructural changes that were seen at 4 C were found. The presence of the media, therefore, protected the endothelium that had been warmed to room
temperature. Humans
Moist Chamber Storage.-After six hours at 25 C, slightly less than Vs of the cells yielded a positive NBT stain. On examination by scanning microscopy, approximately 20% of the cells had a disrupted posterior mem¬ brane. On thin section, areas of decreased cytoplasmic density were
present anteriorly, and mitochondria showed signs of swelling (Fig 2, left). After 12 hours at 25 C, the cytoplasm was diffusely edematous; the mito¬
chondria
were
completely disorga¬
nized, losing both shape and structure (Fig 2, right). By scanning micros¬
patches of endothelial lysis were Room temperature, therefore, severely damaged the endothelium of copy, seen.
moist chamber-stored human eyes. M-K Media Storage.-The endothe¬ lium on the fourth day of storage at 25 C had a regular pattern, and occasional cells had a central elevation (Fig 3, left). Transmission electron micros¬ copy showed relatively normal cells, with dense nuclei and cytoplasm; minimal chromatin vacuolation and disorganized mitochondria were pres¬ ent (Fig 4, left). After seven days, the picture remained relatively normal. A few clefts appeared near the posterior surface, and areas of decreased cyto¬ plasmic density were found (Fig 4, right). Scanning electron microscopy revealed less distinct cellular borders and many pits in edematous-looking cells (Fig 3, right), with some degener¬ ated cells on linear folds (which are thought to be artifactual). The pH of the media in the vials that were left at 25 C varied from 6.10 to 6.80 and was lower than in media refrigerated at 4 C, where the pH range was 6.80 to 7.06. At 4 C, the glucose concentration remained stable, whereas at 25 C, the glucose level fell from 160 mg/100 ml to 110 mg/100 ml. COMMENT
Storage
in moist chambers and M-K
medium is
usually performed at 4 C to
prolong the
survival of the cornea. We have found that at room temperature (25 C), rabbit corneas that have been stored in M-K medium for seven days still retain an intact endothelial layer. Eyes stored in moist chambers had almost total endothelial degeneration, with only a few abnormal endothelial cells remaining. As in other reports,'1 rabbit eyes stored at 4 C by either method had a continuous endothelial layer, but corneas without medium, when warmed to room temperature, were
destroyed.
Shorter periods of storage were used for human eyes because endothe¬ lium in them degenerates more rap¬ idly.3 Moist chamber-stored eyes that were removed from the cold began to show degenerative vacuolization, and disruption of organelles was present. The second six-hour period was more deleterious than the first one. These eyes had been initially cooled to 4 C, and very likely, metabolic activity took place only after one or two hours when the temperature of the eye isolated in a glass-air jacket ap¬ proached 25 C. Similarly, substantial storage time before warming would almost certainly increase this dam¬ age. In contrast, corneas kept in M-K medium at 25 C fared much better. At four days, minimal endothelial dam¬ age was present; by seven days,
although small
areas
of
lysis
were
present, cellular organelles
were still of the fact that recognizable. spite M-K medium was not designed for organ culture, our results for the first
In
Downloaded From: http://archopht.jamanetwork.com/ by a University of Iowa User on 06/13/2015
four days compare well with those of Lindstrom et al," who used a modified Eagle minimal essential medium with 10% calf serum added. In M-K medium at 25 C, corneas had some metabolic activity, as evidenced by the decrease in glucose concentration. For normal eye banking procedures, especially when temperature control during shipping is uncertain, the use of M-K medium for preservation of donor corneas is a safer method than moist chamber storage. Where the donor tissue is warmed and metabolic activity is thus resumed, moist cham¬ ber tissue can be seriously damaged. This investigation was supported in part by grants EY-00446 and EY-00266 from the Nation¬ al
Eye
Institute.
Nonproprietary Name and Trademark of Drug Gentamicin
sulfate—Garamycin. References
1. McCarey BE, Kaufman HE: Improved corneal storage. Invest Ophthalmol 13:165-173, 1974. 2. Bigar F, Kaufman HE, McCarey B, et al: Improved corneal storage for penetrating keratoplasties in man. Am J Ophthalmol 79:115-120,
1975. 3. Robbins JE, Capella JA, Kaufman HE: A study of endothelium in keratoplasty and corneal preservation. Arch Ophthalmol 73:242-247, 1965. 4. Polack FM: Scanning electron microscopy of corneal graft rejection. Invest Ophthalmol 11:1\x=req-\
14, 1972.
5. McCarey BE, Sakimoto T, Bigar F: Ultrastructure of M-K and refrigerated moist chamber-stored corneas. Invest Ophthalmol 13:859-863, 1974. 6. Lindstrom RL, Doughman DJ, Van Horn DL, et al: Organ culture corneal storage at ambient room temperature. Arch Ophthalmol 95:869-878, 1977.
