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ObjectivesTo evaluate the hydration and the levels of free and total sodium in human corneal stromata preserved for up to 21 days in a dextran and chondroitin sulfate–based corneal storage medium (Optisol-GS, Chiron IntraOptics, Irvine, Calif) and to evaluate the effect of epithelial removal on stromal sodium and hydration parameters.MethodsStromal hydration was evaluated thermogravimetrically. A sodium-specific electrode and an atomic absorption spectrophotometer were used to determine the amounts of free and of total stromal sodium, respectively, of preserved human corneas. In 50% of the corneas, the epithelium was removed prior to placement in the storage medium. After 3, 7, 14, or 21 days at 4°C, corneas were removed from the storage medium and sodium measurements were taken.ResultsIn corneas with an intact epithelium, the stromal hydration as well as the stromal free sodium and total sodium levels were relatively constant up to 21 days of storage in the preservation medium. In the absence of the epithelium, the water and sodium contents of the stroma increased significantly during storage.ConclusionThe presence of an intact epithelium is required for maintaining the hydration and sodium levels within the corneal stroma during storage. Removal of the epithelium prior to storage results in increased sodium values and hydration, which may affect postkeratoplasty deturgescence.THE GOAL of corneal transplantation and eye banking is to provide physiologically functional corneas to individuals whose corneas have decompensated or are otherwise no longer transparent. Prior to transplantation, however, the explanted corneas must be stored. A dextran and chondroitin sulfate–based preservative (Optisol-GS, Chiron IntraOptics, Irvine, Calif) is currently the storage medium of choice (see Table 1for composition of Optisol-GS). It has been reported that this formulation of storage medium maintains the endothelial barrier functionand endothelial cell viabilityup to 21 days, while epithelial viability is significantly decreased by day 10 of storage.The stromal ionic composition, however, which directly determines the osmotic forces within the stroma, to our knowledge, has not previously been examined.Table 1. Constituents of Optisol-GS Corneal Storage Medium*See table graphicIn biological systems, the total concentration of an ion in solution comprises 2 components, free and bound, the ratio of which is dependent upon the complexity and macromolecular content of the tissue. The biological activity of an ion is determined solely by the free ionic component and not by the total concentration. While a bound ion is electrochemically inert, a free ion participates in electrochemical reactions and generates osmotic forces that result in water movement across a semipermeable membrane.Within the corneal stroma, the most prevalent cation is sodium, some of which is bound to the proteoglycans (PGs) within the stromal matrix.The distinction between free sodium (also known as sodium activity) and bound sodium is important in examining the deturgescence properties of the cornea. For example, we have previously demonstrated that in the adult New Zealand white rabbit, the concentration of sodium is greater within the stroma than in the aqueous humor. However, 33 mEq/L of sodium are bound and therefore are unable to generate an osmotic force. As a result, the aqueous sodium activity (142.9 mEq/L) exceeds the stromal sodium activity (134.4 mEq/L), generating an osmotic force of 98.5 mm Hg, which is large enough to account for stromal deturgescence (Figure 1).Figure 1.Illustration of the in vivo transendothelial gradient for sodium (Na+) for a rabbit cornea. A, The free sodium (sodium activity) of the aqueous humor is greater than that of the stroma. This gradient in free sodium generates an osmotic force of 98.5 mm Hg in response to which water moves out of the stroma, accounting for stromal deturgescence. B, Detail of boxed area in A showing that the total concentration of sodium within the stroma is divided into 2 components, free and bound. The net negative charge of the stromal proteoglycans attracts ionically extracellular cations, rendering some sodium molecules electrochemically inert. These bound ions are unable to participate in electrochemical reactions. The remainder of the sodium is free and able to generate osmotic forces that are proportional to the difference in activity across a semipermeable membrane, as shown in A.It is unknown if the storage of corneas in a dextran and chondroitin sulfate–based medium and/or the storage conditions alter the sodium balance within the stroma in any way. This issue has not been addressed previously, to our knowledge, and is the focus of this study. We also examined stromal hydration. In addition, we evaluated the importance of the corneal epithelium as a diffusion barrier for its ability to maintain the stromal sodium environment during preservation.MATERIALS AND METHODSPROCUREMENT OF CORNEASHuman corneal tissue, unsuitable for transplantation because of systemic disease or age criteria, was provided by the Texas Lions Eye Bank (Houston). The mean ± SD donor age was 57.5 ± 20.0 years and death-to-enucleation time was 6.1 ± 3.6 hours. Corneas provided by the eye bank in a dextran and chondroitin sulfate–based corneal preservation medium were stored with an intact epithelium (5 corneas), while eyes supplied to us as whole globes had the epithelium scraped off prior to storage of the cornea and scleral rim in the storage medium (5 corneas). Corneas were maintained at 4°C for 3, 7, 14, or 21 days. Unpaired corneas were used in this analysis.MEASUREMENT OF STROMAL HYDRATIONThe stromal water content was determined thermogravimetrically as described previously.Briefly, the endothelium and epithelium were removed, and the denuded stroma was lightly blotted. Stromal punches (7.5 mm) were weighed, dried, and reweighed. The wet and dry weights of the stromal button were used to calculate stromal hydration values. These measurements were performed after measuring the free sodium within the stroma.MEASUREMENT OF SODIUM ACTIVITYAt 3, 7, 14, or 21 days, the corneas were removed from the refrigerator (4°C) and warmed to room temperature. They were mounted in a device that bathed the endothelial surface with a balanced salt solution (BSS Plus, Alcon Laboratories, Fort Worth, Tex). Sodium activity in the stroma of the central cornea was measured with a sodium-specific microelectrode (NAS 11-18 glass, Microelectrodes Inc, Londonderry, NH) as previously described.Briefly, the electrode was calibrated in sodium chloride standards (10-1000 mmol/L) containing 5 mmol/L potassium chloride, and inserted into a stromal tract made by a 22-gauge needle. A millivolt reading was recorded on a benchtop pH meter (pH/ISE Benchtop Meter, Model 340, Corning Inc, Corning, NY) and the corresponding activity was calculated using the Eisenman-Nicolsky equation: E=Eo±Slog (ax±Kyay), where Eindicates change in electrical response of the electrode; Eo, standing potential present in the electrode; S, slope of electrode response; ax, activity of ion, x; Ky, selectivity coefficient for interfering ion, y; and ay, activity of interfering ion, y. Sodium activity values were corrected by a factor empirically determined by adjusting the sodium activity of a plasma sample to equal the sodium concentration of that same sample.MEASUREMENT OF SODIUM CONCENTRATION AND BOUND VALUESSodium concentration was determined by extracting ions from dried stromata into 0.1-normal nitric acid and reading the eluant on an atomic absorption spectrophotometer (Model 2280, Perkin-Elmer Corp, Norwalk, Conn).Bound sodium was calculated as the difference between sodium concentration and sodium activity.Stromal hydration and sodium measurements were not obtained from fresh human corneas as baseline data with which to compare preserved corneal values because stromal hydration and sodium values change rapidly within hours after death. Our human corneas were obtained no less than 5 to 6 hours post mortem, precluding accurate measurements that would reflect in vivo values.DATA ANALYSISStromal sodium values have been normalized to the stromal water content because of the large increases in hydration noted in corneas stored in the preservation medium without the epithelium (Figure 2). Data obtained with the sodium-specific electrode and atomic absorption spectrophotometer are listed in Table 2for comparison.Figure 2.Stromal hydration of human corneas stored in a dextran and chondroitin sulfate–based preservation medium (Optisol-GS, Chiron IntraOptics, Irvine, Calif) at 4°C. Percent values are expressed as mean ± SEM.Table 2. Sodium Levels in Corneas With and Without Epithelium Stored in a Dextran and Chondroitin Sulfate−Based Corneal Preservation Medium*See table graphicCorneas used in this study were not paired. Data were analyzed using analysis of variance with 2 factors: presence of the epithelium and storage time. Statistically, a significant interaction is present "if a change in one of the factors produces a response at one level of the other factor different from that produced at other levels of this factor."In our study, if the interaction between the presence of the epithelium and storage time was significant, the effect of storage time was analyzed separately, comparing values from corneas stored with vs without the epithelium. If the interaction was not significant, only the main effects were studied. The multiple comparison technique was employed to study the changes among the storage times. P<.05 was considered statistically significant. SAS statistics software (SAS, Cary, NC) was used.RESULTSHYDRATIONA significant interaction was evident between presence of the epithelium and storage time (Figure 2, P<.001). Stromal hydration was unchanged in corneas stored with the epithelium for up to 21 days in the dextran and chondroitin sulfate–based medium (P=.133). However, there were significant differences in hydration in corneas stored without an epithelium (P<.001), with the mean percent water content increasing significantly from 3 to 14 days of storage, after which time it did not increase further.FREE SODIUM (ACTIVITY)A significant interaction between presence of the epithelium and storage time was found (Figure 3, A; P<.001) for free sodium. No significant differences in free sodium were found during storage in human corneas stored with the epithelium (P=.10). In corneas stored without an epithelium, however, there were significant differences in free sodium among the days in storage (P<.001). The mean free sodium significantly increased from day 3 to day 14, then it decreased at day 21 to the level of day 7.Figure 3.A, Free sodium (Na+) values, B, total sodium values, and C, bound sodium values for human stromata stored in a dextran and chondroitin sulfate–based preservation medium (Optisol-GS, Chiron IntraOptics, Irvine, Calif) at 4°C. Milliequivalents per liter values are expressed as mean ± SEM.TOTAL SODIUM (CONCENTRATION)There was a significant interaction effect between presence of the epithelium and storage time (Figure 3, B; P<.001) for total sodium. In corneas stored with the epithelium, there were no significant differences in the mean total sodium among storage times (P=.36). In corneas stored without the epithelium, there were significant differences among the storage times (P<.001). In those corneas, the mean total sodium increased, although insignificantly, from day 3 to day 7, followed by a significant increase at day 14, after which time the total sodium stabilized.BOUND SODIUMWhen the stromal bound sodium was analyzed, the interaction effect between presence of the epithelium and storage time was insignificant (Figure 3, C; P>.05). However, the corneas stored with the epithelium had significantly lower bound sodium values (P<.001). In corneas stored both with and without the epithelium, there was a decrease in bound sodium from day 3 to day 14 of storage followed by a significant increase at day 21 (P<.001).COMMENTData from this study suggest that in the presence of the epithelium the amount of free sodium within the stroma is constant in corneas stored in the preservation medium for up to 21 days. In contrast, corneas stored without the epithelium have a progressive increase in free sodium until 14 days of storage, after which time the level decreases (Figure 3, A). The total sodium values follow a similar trend: in the presence of the epithelium, the total amount of sodium (bound + free) is constant until 21 days of storage, and it progressively increases in those corneas in which the epithelium was removed (Figure 3, B). While it is unknown if an elevated stromal sodium activity level will affect the ability of stored human corneas to regain transparency, our previous findings suggest that it does not favor stromal deturgescence.The relationship among corneal endothelial function, stromal composition (both ionic and PG), hydration, and transparency is complicated. In vivo, the endothelium pumps sodium and other ions into the anterior chamber, and it also serves as a semipermeable membrane between the corneal stroma and the aqueous humor. The combination of these endothelial functions produces an osmotic force in the direction of the anterior chamber. The stromal osmotic pressure, in turn, affects the hydration and thickness, and ultimately the transparency, of the cornea.While it has been reported that there is no reduction in collagen fibril interweaving or lamellar adhesion,Slack et aldocumented a small progressive loss of stromal PGs during corneal storage in various preservation media, with the loss being greater in the absence of both the endothelium and epithelium. In addition, previous reportshave shown that colloids (dextran and chondroitin sulfate), added to preservation media to limit the increased corneal hydration that occurs with storage time and reduced temperatures, are taken up by tissues and can cause rebound stromal swelling after surgery. The aforementioned observations prompted us to examine the ionic environment of the stroma and to determine if changes in sodium occur during storage in a dextran and chondroitin sulfate–based storage medium. To our knowledge, no previous study has examined the ionic composition of stored human corneas. Because of the important role ions play in corneal deturgescence owing to the osmotic forces they generate,it is essential that the stromal ionic composition be unaltered during storage.We propose the following models to explain the measured sodium values obtained in this study. With an intact epithelium and endothelium (Figure 4, A), the passive diffusion of all molecules across the cell layers is limited during corneal storage in a dextran and chondroitin sulfate–based preservation medium. Dextran and chondroitin sulfate increase the colloidal osmotic pressure of the storage medium and limit movement of water into the stroma by maintaining a high osmotic pressure. The presence of both limiting cell layers may also serve to minimize both stromal PG loss (Figure 4, A), as previously suggested,and colloidal uptake from the medium. Our bound sodium data suggest that the PG loss in the presence of the epithelium may be greater than colloidal uptake. This may explain the large progressive drop (≈60%, Figure 3, C) in bound sodium observed between days 3 and 14 in corneas with an epithelium. While the in vivo total sodium content of human corneas is unknown, using rabbit data as a referencethe observed loss in bound sodium predicts a reduction of approximately 14% in total sodium. Indeed, the total sodium at day 14 was not significantly reduced compared with day 3; however, there is a reduction of approximately 10%, which is consistent with the earlier observation. Therefore a statistically significant reduction in bound sodium does not correspond to a statistically significant reduction in total sodium. These data from this study indicate that the absolute amount of free and total sodium ions remains unaltered within the stroma of the human cornea during and up to 21 days of storage in a chondroitin sulfate–based storage medium. We postulate that an intact epithelium prevents stromal losses of sodium by acting as a barrier to diffusion.Figure 4.Illustrative interpretation of the fluxes of sodium (Na+), water, and colloids across the corneal barriers during storage in a dextran and chondroitin sulfate–based preservation medium (Optisol-GS, Chiron IntraOptics, Irvine, Calif). A, The epithelium serves as a barrier to limit the diffusion of dextran, chondroitin sulfate, and sodium into the corneal stroma. These same molecules can then osmotically keep water from diffusing into the stroma. Stromal proteoglycan loss is also minimized. By limiting the diffusion of these substances, the number of free and total sodium molecules within the stroma remains constant. There is minimal movement of larger molecules across the endothelial surface. B, Without the epithelium, dextran, chondroitin sulfate, sodium, and water diffuse into the stroma because the epithelial barrier is no longer present. Stromal proteoglycan loss is increased. A combination of all these factors leads to an increase in the number of total, free, and bound sodium molecules within the stroma by diffusion from the storage medium. There is minimal diffusion of larger molecules across the endothelial surface.Without the epithelium during storage in the preservation medium, there is no barrier for the movement of molecules or ions into or out of the stroma across the anterior surface of the cornea. Dextran, chondroitin sulfate, and sodium will diffuse down their respective gradients (see Table 1for sodium values of the storage medium) uninhibited into the stroma (Figure 4, B). Water will also flow down its concentration gradient into the stroma and greatly increase stromal hydration, as noted in Figure 2. Consistent with this model, the absolute amounts of stromal free sodium (Figure 3, A) and total sodium (Figure 3, B) increased progressively in the absence of the epithelium. The stroma was able to gain sodium from the storage medium.The behavior of bound sodium was more complex. It remained stable during the first week of storage, then decreased by approximately 25% at day 14. At day 21, the amount of bound sodium increased significantly in the absence of the epithelium (P<.001) compared to both days 3 (+22%) and 14 (+40%). Slack et alstudied the effect of corneal epithelial removal on stromal PG losses during a 2-week period. They demonstrated that the loss in stromal PGs is several times greater following removal of the epithelium and greatest at day 14 of storage. We propose that the changes in bound sodium may be explained by a biphasic variation in the stromal concentrations of PGs and colloids. At day 14, the loss of PGs is greater than the uptake of colloids, resulting in a transient reduction in bound sodium. Stromal colloidal uptake has been shown to account for postkeratoplasty stromal swelling.Therefore, the uptake of dextran and chondroitin sulfate from the storage medium may exceed the PG loss at day 21, leading to a net increase in stromal negative charges and, in turn, to an increase in bound sodium. These phenomena may also be favored by qualitative changes in PG synthesis by keratocytes, as shown by van der Want et al.In summary, the results of this study suggest that the stromal sodium balance and hydration are maintained in intact human corneas stored in a dextran and chondroitin sulfate–based medium. Removal of the epithelium prior to storage results in increased sodium values and hydration. It has been documented that the epithelium undergoes a significant breakdown by 10 days of storage in this medium; our data indicate, however, that even though the epithelium may be partly compromised it still retains enough of its barrier function to maintain the stromal sodium environment, and perhaps also the stromal macromolecular composition. Based on these data, every effort should be made to store corneas with an intact epithelial layer to minimize changes in stromal ion composition and hydration, which may optimize postkeratoplasty deturgescence.KKimHFEdelhauserGPHolleyDHGeroskiMLynnGEWalshOptisol stored corneal endothelial permeability.Am J Ophthalmol.1994;117:385-393.TLMeansDHGeroskiAHadleyMJLynnHFEdelhauserViability of human corneal endothelium following Optisol-GS storage.Arch Ophthalmol.1995;113:805-809.TLMeansDHGeroskiNL'HernaultHEGrossniklausTKimHFEdelhauserThe corneal epithelium after Optisol-GS storage.Cornea.1996;15:599-605.KGreenMHFriedmanPotassium and calcium binding in corneal stroma and the effect on sodium binding.Am J Physiol.1971;221:363-367.KGreenBHastingsMHFriedmanSodium ion binding in isolated corneal stroma.Am J Physiol.1971;220:520-525.MMStiemkeRJRomanMLPalmerHFEdelhauserSodium activity in the aqueous humor and corneal stroma of the rabbit.Exp Eye Res.1992;55:425-433.MMStiemkeHFEdelhauserDHGeroskiThe developing corneal endothelium: correlation of morphology, hydration and Na/K ATPase pump site density.Curr Eye Res.1991;10:145-156.DCCowellDMBrowningSClarkeDKilshawJRandellRSingerSodium and potassium ion selective electrodes: a review of theory and calibration.Med Lab Sci.1985;42:252-261.AHJMaasOSiggaard-AndersenHFWeisbergWGZijlstraIon-selective electrodes for sodium and potassium: a new problem of what is measured and what should be reported.Clin Chem.1985;31:482-485.WWDanielBiostatistics: A Foundation for Analysis in the Health Sciences.4th ed. New York, NY: John Wiley & Sons Inc; 1987:307-311.SDiksteinDMMauriceThe metabolic basis of the fluid pump in the cornea.J Physiol.1972;221:29-41.DMMauriceThe location of the fluid pump in the cornea.J Physiol.1972;221:43-54.SHodsonThe regulation of corneal hydration by a salt pump requiring the presence of sodium and bicarbonate ions.J Physiol.1974;236:271-302.MVRileyAnion-sensitive ATPase in rabbit corneal endothelium and its relation to corneal hydration.Exp Eye Res.1977;25:483-494.BCBarronMKSmolekBEMcCareyA biomechanical evaluation of rabbit corneas in M-K and Dexsol.Ophthalmic Surg.1992;23:733-737.JWSlackTAKangasHFEdelhauserDHGeroskiMLMcDermottComparison of corneal preservation media for corneal hydration and stromal proteoglycan loss.Cornea.1992;11:204-210.CWBreslinHEKaufmanYMCentifantoDextran flux in M-K medium-stored human corneas.Invest Ophthalmol Vis Sci.1977;16:752-756.DSHullKGreenKBowmanDextran uptake into, and loss from, corneas stored in intermediate-term preservative.Invest Ophthalmol.1976;15:663-666.JHLassWMBourneDCMuschA randomized, prospective, double-masked clinical trail of Optisol vs DexSol corneal storage media.Arch Ophthalmol.1992;110:1404-1408.HJLvan der WantEPelsYSchuchardBOlesenSSperlingElectron microscopy of cultured human corneas: osmotic hydration and the use of a dextran fraction (Dextran T 500) in organ culture.Arch Ophthalmol.1983;101:1920-926.Accepted for publication January 9, 1998.This study was supported in part by grants from the Texas Lions Eye Bank (Houston) and the Knights Templar Eye Foundation, Springfield, Ill; and by an unrestricted grant from Research to Prevent Blindness Inc, New York, NY. Dr Edelhauser is a Research to Prevent Blindness Senior Scholar.We thank Alessandro Iannaccone, MD, for his critical review of the manuscript; Alice Chuang, PhD, for her assistance with statistics; and Mark Udden, PhD, for the use of his atomic absorption spectrophotometer.Part of this study was completed while Dr Jablonski-Stiemke was at Baylor College of Medicine, Houston, Tex.Reprints: Monica M. Jablonski, PhD, The University of Tennessee–Memphis, Department of Ophthalmology, 956 Court Ave, Memphis, TN 38163 (e-mail: email@example.com).
JAMA Ophthalmology – American Medical Association
Published: May 1, 1998
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