A system for long-term corneal perfusion

Seventy-two human corneas were maintained in a perfusion system at 37 degrees C and 18 mm Hg intracameral pressure for 1 to 3 weeks. Corneal thickness, which was initially greater than normal because the enucleated eyes were kept at 4 degrees C before excision of the corneas, decreased slowly during the period of incubation. Endothelial removal or perfusion with ouabain (10(-4) M) induced irreversible stromal swelling. Cooling to 4 degrees C for 8 hr during perfusion caused stromal swelling that disappeared after rewarming to 37 degrees C; less stromal swelling occurred with cooling after 3 weeks of perfusion than after 3 days. No enlargement of central endothelial cells was noted in most corneas by serial specular microscopy. Electron microscopy demonstrated reversal of postmortem changes and maintenance of normal intracellular ultrastructure for 3 weeks. This system for long-term corneal perfusion will allow controlled studies of the effects of new methods of corneal preservation and other perturbations upon the corneal endothelium in situ.     

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)     

High-resolution MAS 1H NMR spectroscopic analysis of rabbit cornea after treatment with dexamethasone and exposure to UV-B radiation

Metabolic changes in rabbit cornea after combined long-term steroid treatment and UV-B exposure were investigated. Corneas were exposed to UV-B radiation (2.05 J/cm2) after 36 days topical pretreatment with either 0.1% dexamethasone or saline. Twenty-four hours after UV-B exposure, corneas were excised and aqueous humour aspirated. Intact corneal tissues were analyzed by magic angle spinning proton NMR spectroscopy and pattern recognition methods. UV-B decreased corneal ascorbate (63% reduction), taurine (62%), and choline (63%), whereas glucose was elevated. Dexamethasone pretreatment further depleted corneal taurine and ascorbate, decreased aqueous ascorbate (85%), and accumulated glucose in cornea and aqueous humour. The results reflect antioxidative mechanisms and osmoregulation.     

Contact lens-induced edema in vitro--amelioration by lactate dehydrogenase inhibitors

Isolated rabbit corneas bathed in Krebs-bicarbonate Ringer solution were observed for thickness changes after a 90 minute equilibration period. Control corneas swelled an average of 0.5 micron/hr, and placement of a polymethylmechacrylate (PMMA) contact lens on the epithelial surface caused the corneas to swell 24.5 microns/hr, an effect similar to 0.5 mM epithelial cyanide exposure. The pronounced swelling induced by PMMA lens placement was much less however, in the epithelial presence of 3.2 mM sodium oxalate (3.22 microns/hr) or 3.2 mM sodium oxamate (5.38 microns/hr). An equiosmotic excess of 4.8 mM NaCl was least active (15.89 microns/hr). On normal isolated corneas (without contact lenses), the Ringer containing an excess of 4.8 mM NaCl significantly deswelled the corneas (-13.44 microns/hr), which contrasted with oxalate and oxamate containing Ringer solutions (1.17 and 1.33 micron/hr respectively). The present study supports the notion that contact lens-induced edema results from stromal lactate accumulation, and suggests a potential alternative to osmotic therapy for its amelioration. These LDH inhibitors, in the concentrations used, have no acute osmotic or toxic effect on normal corneas in vitro.     

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.     

Comparative toxicity of intraocular irrigating solutions on the corneal endothelium.

Isolated rabbit and human corneas were perfused in vitro with Plasma-lyte 148 solution and with a glutathione bicarbonate Ringer's solution. The corneal swelling rate and ultrastructure were compared to corneas perfused with three commonly used intraocular irrigating solutions. Corneas perfused with Plasma-lyte 148 swelled at a rate of 47 mu/gr and the endothelial cells separated from each other and showed extensive damage after three hours of perfusion. By comparison, corneas perfused with 0.9% NaCl increased in thickness by 98 mu/hr, lactated Ringer's by 39 mu/hr, balanced salt solution by 24 mu/hr, and glutathione bicarbonate Ringer's solution by 4 mu/hr. These results indicate that endothelial cell damage and increased corneal thickness observed during perfusion was related to the incomplete composition of 0.9% NaCl, Plasma-lyte 148, and lactated Ringer's and that endothelial cell damage can be prevented if the intraocular irrigating solution contains concentrations of inorganic and organic constituents that are similar to those in aqueous humor.     

Low-dose ultraviolet-B irradiation of donor corneal endothelium and graft survival

Donor rabbit corneal endothelium was pretreated with different doses of ultraviolet (UV-B) irradiation (302 nm) before grafting to test whether allograft survival could be favorably affected in comparison with untreated corneas grafted into the same recipients. Endothelial rejection was observed in 19 of 32 (59%) eyes that received no treatment compared with five of 32 (16%) eyes that received UV-B (P less than 0.001), and increasing doses of UV-B were associated with lower rejection rates (P less than 0.05). Although exposure of donor endothelium significantly reduced endothelial rejection at all doses tested, it resulted in primary graft failure in a substantial proportion of corneas treated at high doses. Class II (Ia) antigen staining of corneal tissue was present in conjunction with clinical evidence of rejection, and the magnitude of staining correlated with the histologic extent of inflammation. Scanning electron microscopy revealed various endothelial cell surface irregularities and membrane defects in high-dose UV-treated corneas. Endothelial cell cultures exposed in vitro to UV-B light showed a dose-dependent loss in cell viability. These data suggest that UV-B pretreatment of donor corneal endothelium prolongs graft survival but that toxic side effects must be carefully controlled.     

The pH tolerance of rabbit and human corneal endothelium.

The endotheliums of rabbit corneas were perfused in an in vitro perfusion specular microscope up to 3 hr with solutions varying in pH from 3.5 to 10.0. Corneal thickness was monitored throughout the experiment, and at appropriate times the corneas were prepared for SEM and TEM. Analysis of the corneal thickness data and interpretation of the electron micrographs reveals that outside of the pH range of 6.5 to 8.5, structural and functional alterations occur. Direct cellular damage, as well as disruption of junctional complexes, lead to a breakdown in the barrier function of the corneal endothelium. The extent of this breakdown is dependent upon both the magnitude of the pH change and the exposure time. Further experiments on banked human eyes support this finding.     

Response of the cornea to far ultraviolet light: an ultrastructural study.

Quantitative changes were measured and ultrastructural alterations were detected in rabbit corneas after exposure to a far ultraviolet irradiation (UV). Low dose exposure (105 - 8 x 105 erg/cm2) resulted one day later in a thicker epithelium and loss of the intercellular permeability barrier. In corneas exposed to higher doses (5 x 106 erg/cm2), the epithelium was considerably thinner one day later, whereas 4 days later it was of normal thickness and the endothelial cells became degenerated. The highest UV dose exposure used (a total of 20 x 106 erg/cm2 given in 4 equal daily doses) resulted in (1) swelling of the stroma and disorganization of the collagen fibers; (2) disappearance of the basement membrane; (3) degeneration of the endothelial cells; and (4) considerable reduction of epithelium thickness.     

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.     

In vivo assessment of mechanisms controlling corneal hydration

The endothelial pump and evaporation components of corneal recovery were studied in the in vivo human cornea by inducing corneal swelling with the use of hypoxia and monitoring the subsequent decrease in corneal thickness. Corneal recovery follows a nonlinear time course with the rate of recovery decreasing as the cornea thins. Following 60 micron of induced edema, recovery with the eyes open required an average of 2.5 hr to reach baseline corneal thickness, while recovery with the eyes closed took an average of 4.0 hr to reach the normal physiologic corneal swelling (17 micron). Our analysis indicates that for open eye recovery from 60 micron of swelling, the endothelial pump provides 20%, while the osmotic thinning caused by tear evaporation contributes 80% of recovery. During recovery, the rate of water evaporation from the anterior corneal surface remained relatively steady at 2.5 microliter/cm2 X hr. Comparison of measured vs calculated recovery rates during recovery with the eyes closed suggests that the endothelial pump functions at one speed and that the "pump-leak" theory of corneal hydration control is applicable for the human cornea.     

Corneal NF-kappaB activity is necessary for the retention of transparency in the cornea of UV-B-exposed transgenic reporter mice

To determine the dynamics of Nuclear Factor-kappaB (NF-kappaB) in murine corneal pathology and the role of NF-kappaB in maintaining corneal clarity after ultraviolet B radiation insult, transgenic mice containing NF-kappaB-luciferase reporter were exposed to LPS (bacterial lipopolysaccharide), TNF-alpha (Tumor Necrosis Factor-alpha) or 4 kJ m(-2) UV-B radiation. NF-kappaB decoy oligonucleotides were also administered in some of the UV-B experiments. Following various exposure times, the mice were sacrificed and whole eyes or corneal tissues were obtained. Whole eyes were examined for scattering using a point-source optical imaging technique. Tissue homogenates were examined for luciferase activity using a luminometer. TNF-alpha and LPS-injected NF-kappaB-luciferase transgenic mice demonstrated 3-10-fold increases in cornea NF-kappaB with peak activities at 4 and 6 hr post-injection, respectively. Mice exposed to 4 kJ m(-2) UV-B exhibited a 3-fold increase in NF-kappaB activity 4 hr post-exposure. The administration of NF-kappaB-decoy oligonucleotides to mice had the effect of reducing UV-B-induced NF-kappaB activity in the cornea and significantly increasing the amount of light scattering in UV-B exposed corneas 7 days post-UV-B exposure when compared to sham injected mice. These results indicate that NF-kappaB is activated in cornea in pathologies that involves increased plasma levels of LPS and TNF-alpha, as well as direct UV-B exposure, and suggest that NF-kappaB activation play an essential part in the corneal healing process.     

Effect of UV-A and UV-B irradiation on the metabolic profile of aqueous humor in rabbits analyzed by 1H NMR spectroscopy

PURPOSE: This study was conducted to investigate metabolic changes in aqueous humor from rabbit eyes exposed to either UV-A or -B radiation, by using (1)H nuclear magnetic resonance (NMR) spectroscopy and unsupervised pattern recognition METHODS: methods. Both eyes of adult albino rabbits were irradiated with UV-A (366 nm, 0.589 J/cm(2)) or UV-B (312 nm, 1.667 J/cm(2)) radiation for 8 minutes, once a day for 5 days. Three days after the last irradiation, samples of aqueous humor were aspirated, and the metabolic profiles analyzed with (1)H NMR spectroscopy. The metabolic concentrations in the exposed and control materials were statistically analyzed and compared, with multivariate methods and one-way ANOVA. RESULTS: UV-B radiation caused statistically significant alterations of betaine, glucose, ascorbate, valine, isoleucine, and formate in the rabbit aqueous humor. By using principal component analysis, the UV-B-irradiated samples were clearly separated from the UV-A-irradiated samples and the control group. No significant metabolic changes were detected in UV-A-irradiated samples. CONCLUSIONS: This study demonstrates the potential of using unsupervised pattern recognition methods to extract valuable metabolic information from complex (1)H NMR spectra. UV-B irradiation of rabbit eyes led to significant metabolic changes in the aqueous humor detected 3 days after the last exposure.     

Extended incubation times improve corneal endothelial cell transplantation success

To investigate the ability of extended incubation times to improve the success of endothelial cell transplantation, eight human donor corneas were denuded of their native endothelium, seeded twice during a 1-hr interval with a suspension of cultured infant human corneal endothelial cells, and then incubated for 144 hr under standard conditions. Subsequently the corneas were transplanted into African green monkeys using routine penetrating keratoplasty techniques. Rotational autografts and corneas devoid of endothelial cells served as controls. The seeded corneas appeared hazy at the time of surgery (mean pachymetry 48 hr postoperatively, 0.794 mm). Six corneas (75%) subsequently cleared, yielding a mean corneal thickness of 0.541 +/- 0.040 and 0.554 +/- 0.040 at 6 and 12 postoperative months, respectively. All control eyes showed advanced edema (thickness, greater than 1.0 mm) and developed extensive neovascularization. Clinically, the extended postseeding incubation corneas were observed to clear more rapidly and stabilize their thickness earlier than corneas incubated for only 24-48 hr. Scanning electron microscopy of extended postseeding incubation corneas revealed an intact monolayer of contact-inhibited cells with the hexagonal mosaic typical of corneal endothelium in vivo and improved intercellular contact compared with corneas incubated for only 24-48 hr.     

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.     

Effect of ultraviolet-B irradiation pretreatment on a second-set rejection model of orthotopic corneal grafts

PURPOSE: To investigate the effect of ultraviolet-B irradiation (UV-B) on the donor cornea in a second-set rejection model of rat orthotopic corneal transplantation. MATERIALS AND METHODS: Three patterns of fully mismatched allogeneic bilateral orthotopic corneal transplantations (DA to AO) after 10-week interval were studied: Untreated primary corneal grafts into the right eyes followed by untreated secondary grafts into the left eyes, UV-B treated primary corneal grafts into the right eyes followed by untreated secondary grafts into the left eyes, and untreated primary corneal grafts into the right eyes followed by UV-B treated secondary grafts into the left eyes. RESULTS: The rejection of untreated secondary grafts was hastened in recipients who had previously rejected untreated primary grafts (p = 0.00507). Recipients bearing long-surviving UV-B pretreated corneal grafts rejected secondary untreated corneal grafts at the first-set tempo. Recipients who had previously rejected untreated primary grafts rejected UV-B pretreated secondary grafts at the first-set tempo. CONCLUSIONS: The UV-B pretreatment to donor corneas may induce dysfunction of antigen presentation so that the recipients' immune system fails to be sensitized in second-set phenomena.     

The release of a neutrophil chemotactic factor from UV-B irradiated rabbit corneas in vitro

Rabbit corneas were isolated, mounted on plastic rings to form a cup and the endothelium was covered with RPMI tissue culture medium. The preparation was then irradiated with 1 J. cm-2 of 300 nm light over 1 hour and then incubated for a further two hours in the dark. The supernatant fluid was assayed for chemotactic activity toward rabbit neutrophils in an in vitro Boyden chamber assay. The results indicated that medium from irradiated corneas had a chemotactic activity that was 42% of that produced by the standard chemoattractant f-met-leu-phe, (10(-9) M) while medium from unexposed corneas and exposed medium alone had less than 3% activity. An in vivo assay using sub-epidermal injection into the back of a rabbit gave qualitatively similar results, only f-met-leu-phe and the medium from irradiated corneas causing neutrophil infiltration of the tissue. A checkerboard analysis confirmed that the activity was chemotactic rather than chemokinetic. Release of a chemotactic factor following UV-B irradiation provides a mechanism for the recruitment of neutrophils, at specific localized areas of the endothelium, that is seen after discrete in vivo irradiation. The results also confirm the importance of corneal inflammatory mediators in the development of tissue damage subsequent to exposure to toxic agents.     

The ultrastructure of contact lens induced changes

The endothelium of 15 human corneas was studied with specular and electron microscopy after exposure to a thick, low water content, soft contact lens (SCL). Five control corneas (no lens wear) were studied using the same methods. SCL wear produced obvious changes in endothelial morphology in 12 of the 15 eyes. With specular microscopy, the changes consisted of an apparent increase in separation of cells and development of areas of loss of membrane reflectivity (blebs). When viewed with electron microscopy, the changes in the same corneas consisted of oedema in the nuclear area of the cells and bulging of the posterior endothelial surface, in some cases over an area of several cells. In 4 cases, the cellular oedema was marked showing both intracellular and intercellular vacuoles. It was concluded that the transient endothelial changes seen with specular microscopy following SCL lens wear were produced by alterations in the contour of the posterior endothelial surface resulting from disturbance to the endothelial environment.     

The influence of contact lens wear on the corneal response to ultraviolet radiation

One eye of each of 15 male pigmented rabbits was fitted with a gas-permeable rigid contact lens. Lenses were worn on a daily basis for an initial period of 7 days with the other eye serving as a control. After this initial period the lenses were removed and both eyes were irradiated with UV-B (305 nm +/- 9 full width half maximum) using either 0.03 J cm-2 or 0.12 J cm-2 which were approximately the epithelial and endothelial damage thresholds for this waveband respectively. The responses of the cornea were followed by microscopy and pachometry. Biomicroscopically there was a dramatic difference in response between contact lens wearing and non-wearing eyes for the higher radiant exposure, whereas little difference was observed at the lower level. For both levels of radiant exposure there was a significantly less corneal thickness increase in contact lens wearing eyes after UV-B irradiation. Recovery patterns were similar.     

Impact of the environment on the mammalian corneal epithelium

PURPOSE: To evaluate whether the content of ascorbic acid in the corneal epithelium and aqueous humor reflects seasonal fluctuations in parallel with environmental changes. METHODS: Reindeer, cattle, rabbits, and humans were examined, to cover a broad spectrum of overlapping habitats. Ascorbic acid was determined by high-performance liquid chromatography. The thickness of the corneal epithelium was measured, and the number of cells was counted in the tissue sections. RESULTS: Three groups of reindeer eyes were used, two of them collected during summer, the third group during winter. Ascorbate content did not show seasonal variation in either the corneal epithelium or the aqueous humor, whereas epithelial thickness and number of cells decreased significantly from summer to winter. In cattle, ascorbate content, thickness of the epithelium, and number of cells were lower in animals tended indoors compared with those tended outdoors, whereas ascorbate level in the aqueous humor remained similar in both cases. The rabbit showed significantly reduced ascorbate content in the corneal epithelium but not in the aqueous humor in tarsorrhaphy-treated eyes. This procedure did not change epithelial thickness, but the number of cells was slightly increased. The mean epithelial thickness in human corneas successively decreased with increasing latitude and decreasing radiation exposure from the summer season in Oslo to the midnight sun, polar night, conditions in Troms?, 10 degrees far north, although the differences did not reach statistical significance. CONCLUSIONS: Ambient radiation is needed to sustain high ascorbic acid concentration in the corneal epithelium. Corneal epithelial thickness and number of cells are prone to seasonal fluctuations regulated by ambient radiation. In contrast, ascorbate content of the aqueous humor is uninfluenced by environmental change. It is suggested that seasonal adaptation of mammalian corneal epithelium in response to variation in ambient radiation may be nature's strategy for countering radiation damage to the eye.