Glutathione in rabbit corneal endothelia: the effects of selected perfusion fluids

Although the ameliorating effect of glutathione on corneal deturgescence is known, its chemical mechanism is not understood. An endeavor toward the latter was made by perfusing freshly excised rabbit corneas with selected perfusion fluids, measuring corneal thickness, and assaying the endothelial cells for reduced and oxidized glutathione after 2 and 5 hr of perfusion. Ringer's solution, containing either lactate or bicarbonate, caused significant decreases in both forms of glutathione after perfusion. The corneas increased in thickness considerably during these periods. When 5 mM glucose was added to bicarbonate-Ringer's solution, the corneas swelled about half as much as before. However, glutathione levels were as depressed as with simple Ringer's fluid. Adenosine (0.5 mM) in the presence of glucose (bicarbonate-Ringer's) caused a further swelling decrease so that the corneas were maintained at near normal thickness. The levels of glutathione were 84% of control values compared to 35% to 45% for Ringer's solutions (+/- glucose). The addition of glutathione to glucose (bicarbonate-Ringer's) caused intracellular glutathione levels to be higher than control values while allowing minimal tissue swelling. Glutathione in combination with adenosine, glucose, and bicarbonate produced the highest intracellular glutathione levels and a slight corneal deswelling. After oxidation of intracellular glutathione with t-butyl hydroperoxide in glucose (bicarbonate-Ringer's), endothelial cells were destroyed within 1 hr. The oxidant, however, may have had a direct effect upon the endothelial cell membranes.     

Intraocular irrigating solutions: a clinical study of BSS plus and dextrose bicarbonate lactated Ringer's solution

Intraocular irrigating solutions of varying compositions and costs are available for cataract surgery. We studied preoperative and two months postoperative extracapsular cataract extraction and intraocular lens implant corneal endothelial cell size in two groups. One group had received an intraoperative irrigating solution of lactated Ringer's with dextrose and bicarbonate. The other irrigating solution was BSS Plus which differs chiefly in the presence of glutathione. A nonstatistically significant trend in favor of BSS Plus was observed.     

Comparison of preservative-free bupivacaine vs. lidocaine for intracameral anesthesia: a randomized clinical trial and in vitro analysis

PURPOSE: To determine whether intracameral bupivacaine hydrochloride 0.5% is as effective as lidocaine hydrochloride 1.0% in controlling discomfort of patients during phacoemulsification and posterior chamber intraocular lens implantation. In rabbits, corneal endothelial cell function, ultrastructure, and viability were evaluated after in vitro perfusion of bupivacaine 0.5%. METHODS: In a double-masked, controlled trial, 48 eyes of 48 patients with uncomplicated age-related cataract were randomly assigned to receive bupivacaine 0.5% or lidocaine 1.0% intracamerally before phacoemulsification with a posterior chamber intraocular lens. Outcome measures such as pain, visual acuity, amount of sedation, length of surgery, pupil size, intraocular pressure, corneal clarity, and anterior chamber reaction were compared. In laboratory studies, paired rabbit corneas were evaluated by endothelial cell perfusion with either bupivacaine 0.5%, bupivacaine 0.5% and glutathione bicarbonate Ringer solution in a 1:1 ratio or bupivacaine 0.5% buffered to a pH of 7.0. The paired control corneas were perfused with glutathione bicarbonate Ringer solution and rates of corneal swelling were determined. Cell ultrastructure and viability were also evaluated. RESULTS: In the randomized trial, there was no significant difference in the pain patients had during surgery or in the early or late postoperative period. No statistically significant difference was seen between the two groups in terms of pupil size, intraocular pressure, corneal edema, anterior chamber reaction, or visual acuity immediately after the operation or on postoperative day 1. Paired rabbit corneas perfused with bupivacaine 0.5% and bupivacaine 0.5% buffered to a pH of 7.0 swelled significantly (P<.001, P = .009, respectively), and had corneal endothelial cell damage. Dilution of the bupivacaine 1:1 with glutathione bicarbonate Ringer solution prevented corneal edema and damage to the corneal endothelium. Endothelial cell viability was also decreased after perfusion of bupivacaine 0.5% (P<.001). CONCLUSIONS: Clinically, bupivacaine 0.5% is as effective as lidocaine 1.0% for anesthesia during phacoemulsification and posterior chamber intraocular lens implantation. However, in vitro perfusion of bupivacaine 0.5% damaged the corneal endothelium of rabbits except when the drug was diluted 1:1 with glutathione bicarbonate Ringer solution. Surgeons who use 0.2 to 0.5 ml of intracameral bupivacaine 0.5% should be aware of its potential to cause endothelial cell damage because of its lipid solubility. The bupivacaine 0.5% should be diluted at least 1:1 with balanced salt solution before intracameral injection, followed immediately by phacoemulsification. The surgeon should ensure that the bupivacaine 0.5% is nonpreserved and packaged in single-use vials or flip-top containers.     

Effects of irrigation solutions on corneal endothelial function.

Rabbit corneas were perfused in vitro with an irrigation solution for 90 minutes. This was followed by 6 hours of perfusion with tissue culture medium TC199 during which endothelial function was assessed by monitoring rates of swelling during a period of perfusion in the absence of bicarbonate ions, and subsequent rates of thinning when bicarbonate ions were restored to the perfusate. Corneal thickness (measured with an ultrasonic pachymeter) immediately following excision was 401 microns (SD 19, n = 23). During the 90 minute perfusion at 35 degrees C, corneas exposed to balanced salt solution (BSS), Hartmann's solution or 0.9% NaCl (all initially at room temperature) swelled, respectively, at 14 (SD 2.3, n = 4), 11 (SD 2.6, n = 4), and 70 (SD 4.3, n = 4) microns/h. Cold Hartmann's solution (initially at 4 degrees C) caused corneas to swell at 9 (SD 2.3, n = 4) microns/h. On the other hand, corneas perfused with BSS Plus thinned at 9 (SD 3.4, n = 4) microns/h and TC199 with Earle's salts had little effect on thickness. Rates of swelling and thinning during the following assessment perfusion showed no apparent effects of prior exposure to any of the irrigation solutions on the barrier properties or pump function of the endothelium. Despite this, the increased thickness of corneas exposed initially to BSS, cold Hartmann's solution, or 0.9% NaCl was not fully reversed, even by the end of the 6 hour assessment perfusion. In contrast, the swelling observed in corneas exposed to Hartmann's solution at room temperature was reversed and these corneas had returned to their normal thickness by the end of the assessment period. All corneas, even those exposed to 0.9% NaCl, had an intact endothelial mosaic with no evidence of damage or cell loss, although morphological differences in cell shape and the appearance of cell borders were evident compared with freshly isolated cornea.     

Effect of intraocular irrigating solutions on intracellular membrane potentials and swelling rate of isolated human and rabbit cornea.

Isolated human and rabbit corneas were incubated in glutathione bicarbonate Ringer solution (GBR), balanced salt solution (BSS), or 0.9% NaCl solution. The swelling rate of human corneas was 25.5 micron/hr in GBR and significantly increased to 32.7 in BSS and 66.1 in NaCl. The epithelial intracellular potential of human cornea was constant at about 60 mV up to 5 hr of incubation in GBR and decreased continuously to 40 mV in BSS and NaCl. Endothelial cell potentials were stable for up to 3 hr of incubation in GBR or BSS and decreased from a control value of about 18 mV to 10 mV 2 hr after bathing in NaCl. Qualitatively similar data were obtained in the isolated rabbit cornea. The results demonstrate the advantage of GBR as an intraocular irrigating solution.     

Corneal endothelial junctions and the effect of ouabain

Paired rabbit corneas were perfused in vitro for endothelial permeability (Pac) determination with glutathione bicarbonate Ringer's solution (GBR) and GBR plus ouabain (10(-4) M). Results indicated no difference in Pac between the two groups (3.39 vs 3.67, respectively) despite significantly greater stromal swelling in the group perfused with ouabain. Freeze-fracture microscopy of similarly perfused corneas revealed intact tight junctional complexes in both groups, although the tight junctional complex of perfused corneas appeared less organized than that of freshly enucleated, nonperfused controls. Gap junctions were abundant as observed in freeze-fracture replicas of GBR-perfused endothelium, and appeared to be decreased or absent in ouabain-perfused endothelium. These results indicate that corneal endothelial tight junctions are unaffected by perfusion with ouabain, whereas gap junctions appear to be lost. The permeability and freeze-fracture data reaffirms the importance of tight junctions as permeability barriers and indicates that gap junctions are not of primary importance for maintenance or control of the corneal endothelial barrier.     

Comparison of intraocular irrigating solutions used for pars plana vitrectomy and prevention of endothelial cell loss

We performed a prospective study of corneal endothelial cell loss following pars plana vitrectomy. Two different intraocular irrigating solutions were used, and the anterior capsule of the lens was removed when performing lensectomy. The central corneal endothelium was photographed with a specular microscope before and at three months after vitrectomy. Endothelial cell loss was calculated using a computer-assisted digitizer. In the 16 aphakic eyes, lactated Ringer's solution caused a significantly greater cell loss (31.1%) with resultant corneal edema when compared to BSS Plus (6.9%). All of the 17 phakic eyes showed minimal cell loss (1.0%) as well as corneal changes regardless of the kind of irrigating solution used. The results suggest that the absence of the lens is a salient factor in corneal endothelial damage during vitrectomy, and that the composition of the intraocular solution used is closely related to this endothelial damage.     

Loss of stromal glycosaminoglycans during corneal edema

This study tried to determine if glycosaminoglycans (GAGs) are released from the rabbit stroma during corneal edema. The GAGs of rabbit corneas were labeled in situ using anterior-chamber injections of 35S-sulfate and 3H-glucosamine. Labeled corneal pairs were excised and the endothelium perfused in vitro in the specular microscope. Edema was induced in one cornea by perfusion with a calcium-free balanced salt solution; the control cornea was perfused with glutathione bicarbonate Ringer's (GBR). Corneal thickness was measured every 15 minutes during the 3-hour perfusion period, and perfusate fractions were collected from each cornea and analyzed for the presence of GAGs. Edematous corneas swelled from 438 +/- 14.8 microns to 688 +/- 10.6 microns compared with control corneas (427 +/- 4.7 microns to 454 +/- 7.2 microns). Total 3H-glucosamine (4.00 +/- 0.68%) and 35S-sulfate (10.36 +/- 0.92%) released from the edematous corneas during perfusion exceeded that lost by control corneas (1.92 +/- 0.18% for 3H-glucosamine; 3.23 +/- 0.52% for 35S-sulfate). Enzymatic digestion studies showed the presence of keratan sulfate in the edematous perfusates. The results suggest that increased loss of radiolabeled components from edematous corneas represent a loss of stromal GAGs and possibly GAG fragments. Therefore, corneal edema involves loss of GAGs and water uptake.     

Effects of sodium lactate on isolated rabbit corneas

Corneal stromal lactate accumulation may result from epithelial hypoxia and contact lens wear, but the possible corneal toxicity of lactate has not been reported. Isolated superfused whole rabbit corneas were examined for thickness changes during exposure to neutral sodium lactate (NaL) or excess sodium chloride (NaCl) in Krebs-bicarbonate Ringer's solution for a 3-hr period. Placed in the tears side bath, 5 mM NaL significantly thinned corneas (swelling rates of 1 +/- 1 micron/hr in Ringer's controls vs -11 +/- 1 micron/hr in lactate-treated corneas; mean +/- SD). Excesses of 5 mM NaCl had essentially identical effects (0 +/- 1 micron/hr in controls vs -13 +/- 3 micron/hr in experimentals). When placed on the aqueous side of normal-thickness corneas, neither 20 mM NaL nor 20 mM excess NaCl affected corneal thickness, but both solutions stimulated endothelium-mediated deswelling in preswollen deepithelialized corneas. When "loaded" into the stroma of deepithelialized corneas, Ringer containing 20 mM lactate caused more swelling than Ringer's alone (491 +/- 18 microns in controls vs 558 +/- 20 microns in loaded corneas; mean +/- SEM). A similar swelling occurred when 20 mM excess NaCl was loaded into the stroma (483 +/- 15 vs 565 +/- 20 microns in controls and loaded corneas, respectively), due to fluid uptake into the hypertonic stroma across the endothelium from the aqueous side (Ringer's) bath. Corneas both loaded and superfused with either NaL or excess NaCl swelled and subsequently deswelled similar to controls swollen and superfused in Ringer's.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intraocular irrigating solutions: a comparison of Hartmann's lactated Ringer's solution,BSS and BSS Plus

Background: We evaluated the effects of Hartmann's lactated Ringer's (HLR) solution, balanced salt solution (BSS) and BSS Plus on human corneal endothelium. Methods: Paired human corneas were mounted in the in vitro specular microscope for endothelial perfusion with HLR, BSS or BSS Plus for 15, 30, 60 and 120 min. Reversal experiments with BSS Plus after initial HLR perfusion were performed. At the end of the perfusions, electron microscopy, F-actin staining of the endothelial cytoskeleton and endothelial permeability measurements were carried out. Results: Longterm perfusion (120 min) with HLR resulted in a significantly higher swelling rate than in the paired controls perfused with BSS Plus. Short-term exposure to HLR for 15, 30 and 60 min after initial BSS Plus perfusion increased the swelling rates significantly. The increased corneal swelling after HLR perfusion for 60 min was reversed by BSS Plus perfusion. Ultrastructural changes in HLR-perfused corneas included endothelial cell edema, cytoplasmic vacuolation and mitochondrial swelling. F-actin staining showed overall cytoskeletal disorganization after perfusion with HLR. Corneal endothelial permeability was higher for BSS Plus-perfused corneas than with HLR solution. Conclusion: The results suggest that the clinically observed corneal clouding during irrigation with HLR is due to endothelial cell edema and decreased endothelial pump function. However, this increased corneal swelling is reversible by perfusion with BSS Plus.     

Quantitative evaluation of the effects of a bicarbonate and glucose-free balanced salt solution on rabbit corneal endothelium in vitro.

Whole corneas from 2- to 2.5-kg albino rabbits were perfused in vitro at 35 to 36 degrees C with a commercial balanced salt solution (BSS) that lacks bicarbonate and glucose. The corneas swelled at an average rate of 45 +/- 11 microns/h. Evaluation of the corneal endothelium by scanning electron microscopy revealed no obvious cytotoxic effect beyond the occasional small area affected by air bubble damage. Morphometric analysis of the central corneal endothelium revealed no significant differences between the endothelial mosaic of perfused corneas, when compared to freshly fixed corneas, in terms of percentage 6-sided cells (pleomorphism) and variance in cell areas (polymegethism). In this example, therefore, a corneal swelling induced by a bicarbonate and glucose-free BSS does not appear to be due to either obvious endothelial cytotoxic effects or significant morphological alteration of the endothelial mosaic.     

Effect of oxidized glutathione on the barrier function of the corneal endothelium

Effects of glutathione on the corneal endothelium were reexamined. Four kinds of solutions were made: oxidized glutathione (GSSG) was added to a basic solution which does not contain glutathione (GSSG-0) at a concentration of 0.03 mM, 0.3 mM or 3 mM to make GSSG-0.03, GSSG-0.3 or GSSG-3, respectively. Paired rabbit corneas were perfused separately, and the endothelial permeability (Pac) to carboxyfluorescein was determined. Between the paired corneas perfused with GSSG-0 and GSSG-0 or GSSG-0 and GSSG-0.03, there was no significant difference in the Pac. A significant difference in this factor was seen between the paired corneas perfused with GSSG-0 and GSSG-0.3 or GSSG-0 and GSSG-3 (P less than 0.01). The ratio of GSSG-0 to GSSG-0.3 for Pac, 1.18 +/- 0.16, and that of GSSG-0 to GSSG-3, 1.14 +/- 0.07, were significantly greater than the left-right ratio for Pac obtained when the paired corneas were perfused with GSSG-0, 1.01 +/- 0.10 (mean +/- SD, n = 8) (P less than 0.025). The corneal swelling rate (micron/hr) was 7.9 +/- 4.9 for the corneas perfused with GSSG-0 and 8.4 +/- 5.4 (mean +/- SD, n = 6) for those perfused with GSSG-0.3; difference was not significant. Addition of GSSG at a concentration of 0.3 mM or more to the irrigating solution was further beneficial to the corneal endothelial barrier function and a solution containing GSSG may be safer for patients with vulnerable corneas.     

BSS and BSS-plus: effect on corneal endothelial ionic and non-ionic fluxes

Rabbit corneas were mounted in water jacketed chambers and the endothelial surface perfused with either BSS (Balanced Salt Solution) or BSS-Plus for 3 hr. Unidirectional and net fluxes of sodium were similar in both groups of corneas. Bicarbonate fluxes in BSS-Plus were statistically similar to those in Krebs-Ringer solution. Bicarbonate fluxes could not be determined with BSS because the solution does not contain bicarbonate. In addition, there was no statistically significant alteration of inulin or dextran permeability when comparing perfusion with BSS and BSS-Plus. From this study it appears that BSS and BSS-Plus are comparable in their ability to maintain corneal endothelial physiologic function during in-vitro perfusion. This is in contrast to previous work which showed that BSS-Plus induced less endothelial morphologic change than BSS. It is concluded that morphologic alterations may be more sensitive parameters of endothelial stress than are fluxes and permeabilities.     

Effect of indocyanine green intraocular stain on human and rabbit corneal endothelial structure and viability. An in vitro study

PURPOSE: To evaluate the direct effect of intraocular indocyanine green (ICG) on endothelial cell function, ultrastructure, and viability in human and rabbit corneas. SETTING: A laboratory evaluation study. METHODS: Paired human and rabbit corneas were mounted in an in vitro specular microscope for endothelial cell perfusion. One corneal endothelium was perfused with 25 mg ICG dissolved in 0.5 mL aqueous solvent in 4.5 mL balanced salt solution (BSS(R)) for 3 minutes followed by washout with a control solution. The percentage of ICG exposed to the corneal endothelium was 0.5%. The paired cornea was perfused with the same solution without ICG, followed by the washout. The corneas were fixed for scanning and transmission electron microscopy (TEM). In another group, the endothelial viability was determined using a live cell/dead cell assay. RESULTS: In rabbit corneas, the mean corneal swelling rate was 12.9 microm/h +/- 1.2 (SEM) in the ICG corneas and 2.8 +/- 1.9 microm/h in the controls. Scanning electron microscopy and TEM revealed a normal endothelial cell mosaic. The control electron micrographs were similar. In human corneas, the mean swelling rate was 19.1 +/- 2.8 microm/h in the ICG corneas and 19.2 +/- 2.6 microm/h in the controls. Scanning electron microscopy and TEM revealed intact junctions with slight cellular vacuolization, similar to that in the controls. In the live cell/dead cell subgroup, the mean damage was 17.3% +/- 1.7% in the ICG-exposed corneas and 22.0% +/- 8.9% in the controls. CONCLUSIONS: Three-minute exposure to ICG in BSS had no adverse effect on corneal endothelial function, ultrastructure, or viability in human and rabbit corneas. This study provides a safety profile for the corneal endothelium when ICG is used as an intraocular tissue stain in ophthalmic surgery.     

乳酸林格氏液和复方电解质灌注液在白内障超声乳化术中的应用比较

目的：比较白内障超声乳化手术中复方电解质液和乳酸林格氏液对角膜功能的影响,为临床选择适宜的灌注液提供科学依据。

方法：收集老年性白内障患者随机分为以乳酸林格氏液为前房灌注的对照组和以复方电解质液为前房灌注液的试验组。术式为白内障超声乳化手术摘除伴人工晶状体植入术。分别在术前、术后各随访点测量角膜内皮细胞密度、中央角膜厚度、视力、角膜水肿、六角形细胞比例和内皮细胞变异系数等。

结果：患者60例60眼顺利完成术后随访,试验组30例30眼,对照组30例30眼。两组患者术后1、3d复方电解质液组角膜内皮细胞密度大于乳酸林格氏液组角膜内皮细胞密度,两组间差异有统计学意义(P=0.030、0.046)。术后1、14d,乳酸林格氏液组角膜内皮细胞变异系数与复方电解质液组比较,两者差异有统计学意义(P=0.025、0.014)。复方电解质液组患者术后第1d视力优于乳酸林格氏液组,差异有统计学意义(P=0.040)。

结论：在老年性白内障超声乳化手术中,复方电解质灌注液有较好的组织相容性,可以更好地维持角膜内皮细胞结构的稳定,减轻角膜内皮细胞的损伤; 复方电解质灌注液较乳酸林格氏液更适用于老年性白内障超声乳化手术。     

Evaluation of the effects of saline versus bicarbonate-containing mixed salts solutions on rabbit corneal epithelium in vitro

Rabbit corneas were incubated, over 4 h in vitro, with the corneal epithelial surface exposed to various solutions to assess their utility as incubation solutions for physiological, pharmaceutical or toxicological studies. The corneal endothelium was perfused with a 35 mm bicarbonate–mixed salts solution equilibrated at 36°C. Corneal thickness, corneal hydration or epithelial cell appearance (as assessed by scanning electron microscopy) were found to be similar to in vivo if a 35 mm bicarbonate, mixed salts solution (equilibrated with 5% CO₂–air) was used for the epithelium. Some swelling (14 μmh^?1), increased hydration and minor cell exfoliation were seen if this 35 mm bicarbonate solution was equilibrated with 5% CO₂–95% O₂ (hyperoxia). Solutions with only 5 mm bicarbonate (0.5% CO₂–air) produced rapid swelling, large increases in hydration and marked cellular damage. Slightly hypertonic (310 mOsm kg^?1) solutions containing 5 mm bicarbonate caused some swelling at 15 μm h^?1, small increases in hydration and some cell damage but the swelling and cellular damage were further reduced by making the solution slightly more hypertonic (325 mOsm kg^?1) by addition of NaCl and KCl. Saline (NaCl 0.9% or 0.97%) or phosphate-buffered saline (PBS) (300 mOsm kg^?1) produced swelling at 21–28 μm h^?1, 30% increases in hydration and almost total destruction of the superficial cell layers. These studies confirm in vivo experiments that saline (and also buffered saline solutions) are rather toxic to the corneal epithelium and thus should not be used as epithelial incubation solutions. Even when using mixed salts solutions and even with bicarbonate present, small differences in composition can have marked effects on corneal thickness, hydration or cell appearance. Hyperoxic solutions appear to be mildly cytotoxic compared with normoxic solutions.     

NaCl osmotic perturbation can modulate hydration control in rabbit cornea

The corneal endothelium transports solute from the stroma to the aqueous humor, maintaining corneal hydration. Currently, little is known about how this active transport system is controlled. The purpose of this study is to investigate in greater detail the corneal response to small NaCl osmotic perturbations using a more refined automatic thickness measurement system in a search for response signatures of transport control. Adult New Zealand White rabbit corneas were debrided of their epithelium, excised and mounted in perfusion chambers. The endothelium, thus isolated, was bathed in isotonic Glutathione Bicarbonate Ringer's (GBR) solution and the bare anterior stroma was covered with silicone oil. Following stabilization in isotonic GBR, the endothelial perfusate was altered by +/-15 mOsm or+/-45 mOsm for 1 hr and 45 min by addition or removal of NaCl and returned (reversal) to GBR for 1 hr and 45 min. An enhanced, automatic scanning specular microscope monitored stromal thickness. The effective membrane transport coefficients were determined from the stromal thickness vs. time curves using an established numerical model of corneal hydration dynamics.It was found that the small (+/-15 mOsm) NaCl perturbations of the rabbit corneal endothelium resulted in a rapid trans-endothelial stromal volume control response that was not reversible after return to GBR. Long after the expected dissipation of the induced transients, this thickness 'controlling' response ultimately resulted in a sustained net thinning of 14 microm following the hypotonic perturbation and reversal, and a net swelling of 16 microm following the hypertonic perturbation and reversal. Model calculations indicated that the change induced by the perturbation could be explained by an immediate and persistent reduction of the passive endothelial NaCl permeability by 26% for the -15 mOsm perturbation compared to the +15 mOsm perturbation. This change persisted even after return to GBR. In contrast, the larger (+/-45 mOsm) perturbations did not elicit a similar response consistently.Our data suggest that trans-endothelial fluid transport can be rapidly modulated to control stromal hydration in response to small NaCl osmotic stresses in a way that cushions the shock and reduces the change in corneal thickness. Moreover, this behavior is not reversible in the short term, and may assist the regulation of corneal hydration homeostatically.     

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.     

Recovery of endothelial function after vitrification of cornea at -110 degrees C

PURPOSE: To determine whether endothelial function is retained after ice-free cryopreservation of cornea by vitrification at -110 degrees C. METHODS: Rabbit corneas, mounted on support rings, were exposed to a solution containing 6.8 M propane-1,2-diol (PROH) and cooled at approximately 7 degrees C/min to -110 degrees C, which was below the glass transition temperature (T(g)) of the solution. After rewarming at approximately 12 degrees C/min and removal of the PROH, endothelial function was assessed by monitoring corneal thickness during perfusion at 34 degrees C. RESULTS: Addition and removal of 6.8 M PROH without cooling to -110 degrees C did not markedly impair endothelial function, although corneas were thicker than control samples. There was no visible crystallization of ice during cooling to -110 degrees C; but a few small, discrete sites of crystallization remote from the endothelium, were observed during warming. After removal of the PROH, corneas approximately doubled in thickness during the first 3 hours of perfusion, but they then started to thin, which suggested active control of stromal hydration by the endothelium. This was confirmed in a further set of experiments by removal of bicarbonate ions from the perfusate at this point, which resulted in further swelling at +58 +/- 2 microm/hour (SD; n = 4). Restoring bicarbonate to the perfusate halted this swelling, and the corneas then thinned at -13 +/- 2 microm/hour (n = 4). Morphologically, staining with trypan blue and alizarin red S showed an apparently intact endothelial monolayer. CONCLUSIONS: Rabbit corneal endothelium tolerated exposure to 6.8 M PROH, and endothelial function was evident after vitrification at -110 degrees C. Preliminary morphologic results with vitrified human cornea also showed retention of endothelium.     

Corneal temperature reversal after storage in Chen medium compared with Optisol GS.

PURPOSE: To compare corneal endothelial cell function by measuring corneal thickness during temperature reversal between corneas stored in two different storage media, Optisol GS and Chen Medium (CM). METHODS: Twenty paired corneas from 10 human donors were randomly assigned for storage at 4 degrees C in Optisol GS (10 corneas) or CM (10 corneas). The storage media were masked, and measurements were done in a masked fashion. After storage for 48 hours, corneal thickness was measured by ultrasonic pachymetry at 2-hour intervals for 12 hours, during which time the corneas were perfused with BSS (balanced salt solution) Plus at 37 degrees C. Scanning electron microscopy of two pairs of corneas from two donors was performed to assess ultrastructural change after 12 hours of warming. RESULTS: Corneal thickness decreased during the first 4 hours of the warming period and then increased during the 6-to 12-hour warming period. These changes in corneal thickness over time were similar for the two storage media (p = 0.212). Scanning electron microscopy showed greater amounts of endothelial cell disruption in Optisol GS-stored corneas than those stored in CM after 12 hours of warming and perfusion. CONCLUSIONS: The endothelial pump of corneas stored in CM appear to be as well-preserved as those stored in Optisol GS, although greater endothelial disruption may be present with Optisol GS by scanning electron microscopy. Further studies are required to compare the clinical effectiveness of these two media.