Comparison of K-Sol and M-K medium for cornea storage: results of penetrating keratoplasty in rabbits.

Five pairs of rabbit corneas were stored for two weeks at 4 degrees C, one of each pair in K-Sol medium, and one in McCarey-/Kaufman (M-K) medium. After transplantation all penetrating keratoplasty grafts became clear and thin. Endothelial cell loss was significantly less in the K-Sol stored corneas. Another five pairs of corneas were stored for two weeks in K-Sol or three days in M-K medium. After penetrating keratoplasty there were no significant differences in clarity, thickness, or endothelial cell loss. The results indicate that K-Sol provides satisfactory medium-term corneal storage compared with short-term storage in M-K medium at refrigerator temperatures.     

Histological study of corneas preserved in two new media.

A new corneal preserving medium (K-Sol), developed by Kaufman and others, contains purified chondroitin sulphate, TC 199, HEPES buffer, and gentamicin. Another new medium (JM) containing bicarbonate-free glucose-phosphate Ringer solution and dextran 70 has been developed in Japan. New Zealand white rabbit corneas with scleral rims were stored in each medium at 4 degrees C for one or two weeks. The condition of the endothelium was evaluated histologically. Corneas preserved in both media were in good condition at the end of one week. Corneas preserved in K-Sol for two weeks showed fewer endothelial changes than similar tissue stored in JM for two weeks. Corneal swelling was also less in corneas preserved in K-Sol, than in corneas preserved in JM.     

Lysosomal enzyme levels in corneal storage media. K-Sol versus McCarey-Kaufman

Evidence indicates that lysosomal enzymes can carry out corneal autolysis during corneal storage and that they are damaging to the corneal endothelium. The authors investigated the release of lysosomal enzymes into two corneal storage media (K-Sol and McCarey-Kaufman [M-K]) by paired human donor corneas during 4 degrees C storage. The authors also studied the interaction of these media with lysosomal enzymes from human cornea. K-Sol and M-K stimulated (P less than 0.01) both beta-glucuronidase and alpha-galactosidase about equally. beta-N-Acetyl-glucosaminidase, a major catabolic enzyme of the cornea, was inhibited by the chondroitin sulfate in K-Sol by over 90% (P less than 0.01). Corneas stored in M-K released more lysosomal enzymes than corneas stored in K-Sol. At 4 days, the values approached significance (P less than 0.06) and by day 10 significantly higher values were found in the M-K media (P less than 0.01). Both storage methods showed a linear release. Individual corneas were found to vary in their release rates. Whether corneas that release more enzyme will show higher endothelial cell loss or produce less successful penetrating keratoplasty grafts deserves further study.     

Corneal function after storage in commercial eye bank media

Rabbit corneas were stored in commercially prepared media (K-Sol, M-K, CSM, or Dexsol) for 3, 6, 9, or 12 days. Corneas stored in each medium showed decreased post-storage deturgescence with increased storage time. This effect was most pronounced in corneas stored in K-Sol, followed by those in M-K, CSM, and Dexsol. Corneas stored in K-Sol also exhibited the fastest swelling rate when cultured in the presence of 20 microM ouabain, followed by those in M-K, Dexsol, and CSM. An estimate of the active ion transport capacity of each experimental group was made by determining the area between each group's corneal thickness profiles in the presence and absence of ouabain. Corneas stored in M-K, K-Sol, or CSM retained approximately 70% of control activity after 3 days storage and 65% after 6 days storage. Corneas stored in Dexsol had 92% of control activity after 3 days of storage, 78% after 6 days, and 44% after 12 days.     

Rabbit corneal endothelial physiologic and morphologic characteristics following storage in MK medium and K-Sol

Rabbit corneas were stored for 3, 7, 10 or 14 days in MK medium or K-Sol. Corneas were thinner immediately following removal from K-Sol than following removal from MK medium at all time periods studied. Following 3 days of storage, corneas stored in both solutions showed similar temperature reversal characteristics. Corneas stored for 7 and 10 days in MK medium also temperature reversed, whereas those stored in K-Sol did not. Corneas stored for 14 days in both solutions swelled following mounting in the specular microscope. Endothelial cell morphology was similar following storage in MK medium and K-Sol at all time periods studied. It is postulated that a persistant osmotic gradient is present across the endothelium following storage in K-Sol. This osmotic gradient occurs because of retention of chondroitin sulphate in the corneal stroma thereby preventing early temperature reversal.     

M-K medium and postmortem cytologic damage.

Freshly enucleated rabbit eyes were refrigerated at +4 degrees C under standard eye bank conditions for 2, 6, 9, and 21 days. One group of corneas with a scleral rim were excised and placed in M-K medium, stored for 18, 24, or 48 hr at +4 degrees C; they were then removed, and endothelial cell viability was evaluated with nitroblue tetrazolium. The cells were examined by light microscopy and scanning and transmission electron microscopy. A second group of corneas were similarly obtained and then used as donor corneas from 6 mm transplants. Each recipient rabbit was evaluated daily by slit-lamp biomicroscopy and corneal pachometry. Fourteen days postoperatively, the rabbits were sacrificed, the eyes enucleated, and the excised corneas were evaluated in a fashion similar to those of group 1. M-K medium storage protected the morphology and functional integrity of the rabbit corneal endothelium up to 48 hr beyond moist chamber storage for 2, 6, and 9 days. However, M-K medium appeared to have no such effect on corneas that had been moist chamber--stored for 21 days. These results suggest that some human corneas with a prolonged time from death to moist chamber storage may be utilized for corneal transplantation after further storage in M-K medium.     

Corneal storage in MK medium and K-Sol. Effect on ionic and non-ionic fluxes

Rabbit corneas were stored at 4 degrees C for 3, 7 or 14 days in either modified MK medium or K-Sol. Corneal endothelial permeability to inulin following storage in modified MK was significantly less at each time examined than that found in corneas stored for either 3, 7 or 14 days in K-Sol. Inulin permeability after storage in K-Sol was increased at all times relative to unstored control corneal tissue, but only at 7 and 14 days in MK medium. Dextran permeability was similar following 3 days of storage in either solution, but dextran permeability following storage in modified MK was significantly less than the values found in corneas stored for 7 and 14 days in K-Sol. Dextran permeability was not significantly increased relative to control, at any storage time in MK medium but was increased at 7 and 14 days in K-Sol. Inulin and dextran permeabilities after storage in MK medium were maintained more closely to values found in fresh tissue than corneas stored in K-Sol. Net endothelial sodium fluxes following storage in modified MK medium were markedly less than those found in corneas stored for 3, 7 and 14 days in K-Sol. Net sodium fluxes are maintained better in K-Sol than in MK medium relative to control values. Net bicarbonate fluxes following storage in modified MK medium were significantly less than the 3-day values in K-Sol, but similar to the values after 7 and 14 days of K-Sol storage.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of UW solution and its components on corneal thickness during and after storage.

The ability of rabbit corneas to undergo energy-dependent deturgescence was examined after the corneas were stored at 4 degrees C in UW solution, M-K media, or selected modifications of these media. All corneas slowly increased in thickness during storage, despite the presence of colloidal osmotic agents. Corneas stored for 2.5 days in M-K became slightly thinner when cultured over a 24-hour period. Corneas stored in UW swelled quickly in culture and became too opaque to measure within three hours. Corneas stored in UW with 1.8 mM CaCl2 swelled transiently, then maintained their thickness and exhibited deturgescence in the latter stages of the culture period. Deturgescence of corneas stored for 7 days in M-K was only slightly worse than those stored for 2.5 days. Corneas stored for 7 days in UW, however, became opaque almost immediately in culture and those stored in calcium-supplemented UW became opaque within 4.5 hours. Replacement of the dextran in M-K with hydroxyethyl starch produced a slower rate of corneal swelling during storage and a substantially better corneal deturgescence profile during culture. Use of high concentrations of potassium ion in the M-K formulation had no significant effect on post-storage deturgescence. Replacement of glucose in M-K with the impermeable sugar, raffinose had a slight deleterious effect on corneal deturgescence in subsequent culture. Use of the impermeable anions gluconate or lactobionate to replace chloride ion caused profound corneal swelling during culture, compared with those stored in M-K. These experiments show that UW solution is inferior to M-K at preserving post-storage corneal function.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human corneal storage in modified McCarey-Kaufman and K-Sol media: effect on endothelial Na+/K+ ATPase pump site density and permeability.

Endothelial permeability (Pac) to carboxyfluorescein and Na+/K+ ATPase pump site density were determined in human corneas following storage for 4 or 7 days at 4 degrees C in either modified McCarey-Kaufman (mMK) or K-Sol media. Following 4 days of storage, Pac values for mMK- and K-Sol-preserved corneas were not significantly different from those of their prestorage mates. After 7 days of storage, however, corneas stored in K-Sol media showed a significant increase in Pac compared to their prestorage mates, whereas the mMK-stored corneas showed no change in Pac. Na+/K+ ATPase pump site density determined using [3H]ouabain was similar to a control group in the K-Sol-stored tissue but higher in the mMK-stored tissue following 7 days of storage. These studies suggest that mMK medium maintains endothelial barrier function and Na+/K+ ATPase pump site density at least as well as K-Sol medium through 7 days of corneal storage.     

Endothelial cell damage in human and rabbit corneas stored in K-Sol without antioxidants.

Human and rabbit corneas were stored at 4 degrees C in K-Sol with and without antioxidants (ascorbic acid, reduced glutathione, alpha-tocopherol, and retinol acetate) for two to three weeks. All the corneas were then examined visually and by scanning electron microscopy. They appeared clear and slightly oedematous. Scanning electron micrographs were used to grade corneal endothelial cell morphology in a masked manner in terms of cell shape, cell borders, cell swelling, and apical holes. Corneas stored in K-Sol without antioxidants showed changes in cell shape, cell borders, and apical holes. Human corneas showed more morphological changes than rabbit corneas. The results suggest that antioxidants in K-Sol have an important role in the preservation of endothelial cell morphology.     

Hypothermic preservation of corneas in a hyperkalaemic solution (CPTES): II. Extended storage in the presence of chondroitin sulphate.

Periods of preservation for donor corneas, even for short times, are necessary to facilitate optimum conditions in penetrating keratoplasty. However, current techniques for corneal storage at low temperatures may not provide optimal conditions for maintaining tissue integrity. In particular, the ionic composition of the storage medium has received little attention since it has been assumed throughout that the normal complement of ions in tissue culture media will also be suitable for preservation at reduced temperatures. This study extends our previous investigations on the merits of using CPTES (corneal-potassium-TES), a potassium-rich balanced salt solution containing an impermeant anionic pH buffer (TES), as a storage solution specifically designed to prevent the loss of intracellular potassium and minimise endothelial cell swelling during the time that the normal regulatory processes are switched off. The effect of adding the natural polymer chondroitin sulphate (CS) as a colloid osmotic agent to the hyperkalaemic storage medium is now examined. Corneas stored in CPTES containing 2.5% chondroitin sulphate retained a very high level of structural and functional integrity after three, five, and seven days storage at 0 degrees C; furthermore, stromal swelling was restricted to only 21%. All corneas stored in CPTES + 2.5% CS showed active endothelial function by thinning efficiently at rates that were greater than those previously reported for rabbit corneas stored for similar lengths of time in either M-K medium or K-sol. The zwitterionic buffers TES and HEPES were interchangeable in the hyperkalaemic solution and were non-toxic to corneal endothelium at a concentration of 100 mM. These compounds offer excellent pH buffering in bicarbonate-free medium.     

Morphologic changes of K-Sol preserved human corneas

Supplementation of tissue culture medium with chondroitin sulfate has been shown to enhance donor corneal preservation. We assessed the efficacy of one of these chondroitin-supplemented media (K-Sol) in comparison with McCarey-Kaufman (MK) medium in maintaining corneal cellular morphology. Thirty-six human corneas, obtained within 8.6 h after death, were placed into K-Sol medium for up to 20 days preservation, and five paired control corneas were placed into MK medium for up to 6 days preservation. Specular photomicrographs were obtained every second to third day for a predetermined storage interval, then studied morphologically in a masked protocol by light microscopy, transmission electron microscopy, and scanning electron microscopy. Endothelial cell loss by specular microscopy averaged 5.8% after 1 week (6 to 8 days) and 7.4% after 13 days in K-Sol medium. Epithelial changes were erratic throughout the 20 day K-Sol preservation period. However, substantial keratocyte changes occurred after 10 days, and endothelial morphology uniformly deteriorated after 17 days. The morphologic data suggest that human corneas may be able to be preserved in K-Sol medium at 4 degrees C for up to 10 days but should be cautiously used thereafter.     

Endothelial cell survival on transplanted human corneas preserved at 4 C in 2.5% chondroitin sulfate for one to 13 days

A comparison of transplantation of 37 corneas preserved in 2.5% chondroitin sulfate (K-Sol) at 4 C for one to 13 days (mean, 6.8 days) with those of 37 corneas preserved in McCarey-Kaufman medium at 40 C for one to 81 hours (mean, 39 hours) yielded the following results. The mean endothelial cell loss noted two months after keratoplasty was 6% in each group. The grafts preserved in K-Sol were significantly thinner two months postoperatively. Within the K-Sol group, there was a significant positive correlation between preservation time and both epithelialization time and corneal thickness three weeks after keratoplasty. There was also a significant positive correlation between preservation time and endothelial cell loss in both groups, with the highest cell losses (greater than or equal to 30%) all occurring after two days of storage in the McCarey-Kaufman group and after ten days of storage in the K-Sol group. These results appear to justify the use of chondroitin-sulfate-based preservation media to extend donor storage time; they also indicate that donor corneas should be used as early as feasible.     

Comparison of corneal preservation media for corneal hydration and stromal proteoglycan loss.

Changes in the composition of stromal proteoglycans (PGs) have been previously demonstrated in corneal edema, wound healing, and disease. To examine if PGs are lost during corneal preservation, rabbit corneal PGs were radiolabeled in situ with 35S-sulfate and 3H-glucosamine, excised and stored in either modified McCarey-Kaufman medium (MMK), K-Sol (Coopervision-Cilco, Bellevue, WA, U.S.A.), corneal storage medium (CSM), or Dexsol (Chiron Ophthalmics, Irvine, CA, U.S.A.) for up to 14 days. The percentage of total radio-label lost was significantly greater from de-epithelialized corneas (p less than 0.05) and from corneas stored in CSM (p less than 0.05) or K-Sol (p less than 0.05). Corneas stored in CSM for 4 and 7 days were significantly more hydrated than corneas stored in MMK, K-Sol, or Dexsol. After 14 days of storage, all corneas were hydrated above control values with the Dexsol-stored cornea showing the least hydration. Results suggest that loss of stromal PGs during corneal storage is reduced with epithelial integrity and with preservation media containing dextran.     

Corneal endothelium under various storage conditions.

Three rabbit corneas each were stored in McCarey-Kaufman (M-K) medium, rabbit serum, and in a moist chamber at 4 degrees C refrigeration for various lengths of observation. The endothelial cells appeared normal under all conditions for the first 24 hours as compared with control corneas processed concurrently with each experimental group. After 48 hours of storage the specimens in the moist chamber showed isolated endothelial cell damage. The 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 preservation of microvilli 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 degrees C with 5% CO2 and moist air in M-K medium, serum, and minimal essential medium (MEM) with 10% 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. Those 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 MEM 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 of 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 cornea under this storage condition.     

Preoperative metabolic analysis of donor corneas using magnetic resonance spectroscopy

Successful corneal transplantation was accomplished following metabolic phosphorus magnetic resonance analysis. Four cat corneas were analyzed using phosphorus-31 magnetic resonance following storage in modified McCarey-Kaufman (M-K) medium for 24 h. Corneas were re-stored in M-K medium and transplanted 24 h after MR analysis. Four control corneas (contralateral eye, no magnetic resonance analysis performed) were also transplanted following storage in M-K medium under identical conditions. Successful corneal transplantation was accomplished with minimal ATP tissue levels. Corneas stored for 48 h maintained a pH of 7.3. The phosphorus-31 spectral modulus, which is the ratio of the high-energy phosphates to the low-energy phosphates, was calculated using the spectral integral (range, 0.49-0.77). No difference in endothelial cell density or morphology was detected between corneas following magnetic resonance analysis and control corneas when evaluated by specular microscopy.     

Effect of HEPES buffer on corneal storage in MK medium

Rabbit corneas were stored for 7 days in either MK medium containing gentamicin or modified MK medium containing HEPES buffer, gentamicin and phenol red. Corneas stored for 7 days in modified MK medium were thicker than corneas stored in MK medium. Corneal endothelial permeability to inulin and dextran was similar following 7 days of storage in either solution. Transmission electron microscopy of corneal endothelial cells stored in either solution showed intact cell membranes and organelles. In vitro perfusion of rabbit corneas in the specular microscope with Krebs Ringer bicarbonate containing HEPES buffer swelled at 17 +/- 1 micron/h, whereas those perfused with Krebs Ringer bicarbonate alone swelled at 7 +/- micron/h. Perfusion with Krebs Ringer bicarbonate containing phenol red did not result in an increased corneal swelling rate. The work indicates that HEPES buffer has an adverse effect on corneal endothelial pumping function, and this results in corneal swelling during storage as well as during perfusion in the specular microscope. The adverse effect appears to be, at least in part, transient: however, the ultimate, long term effect of HEPES buffer on corneas stored prior to penetrating keratoplasty is not known and deserves continued investigation.     

Eurosol versus fetal bovine serum-containing corneal storage medium

PURPOSE: To evaluate the usability of Eurosol, a new medium-term corneal storage medium without components of bovine origin. METHODS: Ten pairs of human donor corneas were placed in tissue culture at 31 degrees C for 7, 14, 21, 28, or 35 days. One cornea of each pair was cultivated in conventional storage medium on Earls' minimum essential medium base containing 2% fetal bovine serum; the other one was stored in Eurosol. Corneas were examined with inverse light microscopy; corneal thickness was measured; and scanning electron microscopy was performed. RESULTS: No significant difference in corneal thickness and endothelial cell count was found at any time. Scanning electron microscopy showed a complete endothelial cell layer on all corneas. CONCLUSION. The findings indicate a potential clinical applicability of the tested serum-free medium-term storage medium, offering a safer alternative to conventional media containing fetal bovine serum.     

Increased endothelial cell loss after transplantation of corneas preserved by a modified organ-culture technique

Forty-seven donor corneas were preserved in McCarey-Kaufman (M-K) medium at 4 degrees C for 1 day, then in organ culture at 34 degrees C for approximately 1 month, then in M-K medium at 4 degrees C for an additional two days before transplantation. The central donor endothelium was examined by specular microscopy before and after organ culture and 2 months after keratoplasty. No significant change in central endothelial cell density occurred during organ culture. The 47 transplants were compared with 47 grafts preserved only in M-K medium at 4 degrees C for approximately 36 hours. All transplants were performed by the same surgeon over the same period, and the two groups contained similar types of surgical procedures. The organ-cultured grafts were thicker on the first post-operative day and took longer to epithelialize . Two months after keratoplasty all of the 94 grafts were clear and thin, but the mean central endothelial cell loss was 28% in the 47 organ-cultured transplants and 10% in the 47 transplants preserved only in M-K medium (P less than 0.0001). These results indicate that the endothelium of corneas preserved by organ culture at 34 degrees C and then placed in M-K medium at 4 degrees C for 2 days may be more susceptible to surgical trauma than those preserved only in M-K medium at 4 degrees C.     

Decreased endothelial cell survival after transplantation of corneas preserved by three modifications of corneal organ culture technique

We placed human donor corneas in M-K medium at 4 degrees C for 24 hours, cultured them in minimal essential medium at 34 degrees C for two to five weeks, and then either (1) placed the corneas in M-K medium at 4 degrees C for 48 hours before transplantation (Group 1, 47 eyes); (2) placed the corneas in M-K medium at 4 degrees C for 16 hours before transplantation (Group 2, 17 eyes); or (3) transplanted the corneas without postculture cooling to 4 degrees C (Group 3, 11 eyes). We compared the corneas preserved by organ culture with an equal number of corneas transplanted during the same period, but preserved only in M-K medium at 4 degrees C for one to four days. The central endothelial cell losses noted two months after keratoplasty were significantly greater in the organ-cultured corneas than in the M-K-preserved corneas in each of the three groups. The mean endothelial cell loss in the 11 organ-cultured corneas in Group 3 was significantly less than that in the 64 organ-cultured corneas in Groups 1 and 2. The corneas in Group 1 were also examined one year after keratoplasty, and the cell losses in the organ-cultured grafts remained significantly greater than those in the M-K-preserved grafts.(ABSTRACT TRUNCATED AT 250 WORDS)