Optisol corneal storage medium.

We compared the safety and efficacy of Optisol (Chiron Ophthalmics, Irvine, California), a new corneal storage medium, with McCarey-Kaufman and Dexsol corneal storage media (Chiron Ophthalmics, Irvine, California) and K-Sol corneal storage medium (Cilco, Huntington, West Virginia). Optisol contains dextran, 2.5% chondroitin sulfate, vitamins, and precursors of adenosine triphosphate (adenosine, inosine, and adenine). In in vitro studies, rabbit and human corneas stored in Optisol were thinner after 12 to 14 days at 4 C than tissue stored in other media. Thymidine uptake showed increased mitotic activity in human corneal endothelial cells cultured in Optisol, compared to Dexsol. Specular microscopic fields showed larger are-as of visibly intact endothelial cells and ultrastructural examination disclosed fewer structural changes in endothelial cells stored in Optisol, compared to tissue stored in Dexsol. In vivo, no clinical signs of epithelial toxicity or histologic evidence of intraocular inflammation were observed in rabbit eyes in which Optisol drops were instilled four times a day for 14 days. Finally, 51 patients undergoing penetrating keratoplasty with tissue stored in Optisol for one to six days (mean, 3.6 days) were enrolled in an uncontrolled, open-label clinical study. The percentage of clear grafts (93%, 41 of 44 patients examined at three months; and 98%, 42 of 43 patients examined at six months) and endothelial cell loss (5.0% and 11.5% at three and six months, respectively) were comparable to data from previous studies that used tissue stored in other short-term and intermediate-term media. The results suggest that Optisol storage preserves corneal endothelial cells for up to two weeks at 4 C, thereby permitting flexibility in the use of donor tissue for corneal transplantation, and that Optisol storage yields thinner tissue, which may allow for more accurate evaluation and more effective surgical manipulation.

P-31 NMR analysis of phospholipids from cultured human corneal epithelial, fibroblast and endothelial cells.

Corneal epithelial, fibroblast and endothelial cells, cultured from human donors, were analyzed to determine their characteristic phospholipid profiles by 31P NMR. Tissue phospholipid profiles from epithelial, fibroblast and endothelial cell cultures were evaluated to differentiate the individual cell types and to identify resonances that typically appear in high-resolution phospholipid profiles of whole corneas. Phosphatidylcholine, phosphatidylethanolamine plasmalogen, an uncharacterized phospholipid at 0.13 delta, phosphatidylinositol, phosphatidylserine and sphingomyelin were determined to be, in decreasing order of concentration, the major phospholipids detected in these three cultured corneal cell types. Indices of phospholipid metabolism representing total plasmalogen content, total choline-containing lipids and the total choline-containing lipids less those synthesized through the plasmalogen pathway were found to differentiate the three cell types. Minor phospholipids cardiolipin, lysophosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine (LPC) and LPC plasmalogen not usually reported in studies of corneal phospholipids using other techniques, were useful in discriminating between cell types. Phospholipid profiles of the whole cornea provide important information concerning the biochemistry and pathology of the tissue, however, phospholipid analysis of individual components of the cornea, such as the epithelial, fibroblast and endothelial cells, makes it possible to understand the contribution of specific cellular constituents to the spectral information obtained from the whole cornea.

Expression of two molecular forms of the complement decay-accelerating factor in the eye and lacrimal gland.

Complement is present in ocular fluids, but the molecular mechanism(s) restricting its activation to exogenous targets and not to autologous ocular cells are currently unknown. To clarify how this control is achieved, monoclonal antibody (mAb)-based techniques were used to examine the eye, the lacrimal gland, and ocular fluids for the decay-accelerating factor (DAF), a membrane regulatory protein which protects blood cells from autologous complement activation on their surfaces. Immunohistochemical staining of tissue sections revealed DAF antigen on corneal and conjunctival epithelia, corneal endothelium, trabecular meshwork, and retina, as well as on lacrimal gland acinar cells and in adjacent lumens. By flow cytometry, cultures of conjunctival epithelium exhibited the highest DAF levels and levels on corneal epithelium greater than corneal endothelium greater than conjunctival fibroblasts. Biosynthetic labeling of corneal endothelium yielded de novo DAF protein with an apparent molecular weight (Mr) of 75 kD, approximating that of blood cell DAF protein, and digestions of conjunctival epithelium with phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme which cleaves glycoinositolphospholipid membrane anchors, released approximately 70% of the ocular surface DAF protein similar to leukocyte surface DAF protein. Quantitations of DAF by radioimmunometric assay employing mAbs against two DAF epitopes revealed 325 ng/ml (n = 12), 4.8 ng/ml (n = 10), and 22.0 ng/ml (n = 8) of soluble DAF antigen in tears, aqueous humor, and vitreous humor, respectively. Western blot analyses of the tear DAF antigen revealed two DAF forms, one with an apparent Mr of 72 kD resembling membrane DAF forms in other sites, and a second with an apparent Mr of 100 kD, which is previously undescribed. Since DAF activity is essential physiologically in protecting blood cells from autologous complement attack, the identification of DAF on the ocular surface, intraocularly, in the lacrimal gland, and in tears suggests that DAF-mediated control of complement activation is also required in these locations.

An in vitro and clinical comparison of corneal storage with chondroitin sulfate corneal storage medium with and without dextran.

The safety and efficacy of 1% dextran in Chondroitin Sulfate Corneal Storage Medium (CSM) in reducing corneal swelling after 4 degrees C storage was assessed in a corneal endothelial cell culture system. No difference was found in 3H-thymidine incorporation by cells incubated in either CSM-dextran medium or CSM medium alone. Subsequently, 21 pairs of corneas, stored in either CSM or CSM-dextran from 30 to 112 hours, were transplanted into 42 eyes of 42 patients, paired by diagnostic group and procedure. All CSM grafts and 19 of 21 CSM-dextran grafts were clear at 4 months with no primary donor failures in either group. Intraoperative corneal thickness was significantly greater in the CSM group (0.82 +/- 0.07 mm) than the CSM-dextran group (0.76 +/- 0.06 mm); however, the two groups did not differ thereafter. No differences in all endothelial morphometric parameters were noted between the two groups pre- and postoperatively. Average endothelial cell loss by 4 months was 13.0 +/- 16.4% for the CSM group and 16.4 +/- 15.5% for the CSM-dextran group. The addition of dextran to CSM medium results in significant intraoperative corneal thinning without adversely affecting endothelial DNA synthesis in vitro and endothelial survival in vivo.

Preclinical evaluation of a new donor corneal trephination system.

A new corneal trephination system developed by the University of Minnesota, Department of Ophthalmology and Vision Care/3M features a corneal holder that orients and stabilizes the donor cornea during storage, transportation, and trephination. A gravity corneal punch, and disposable trephine and cutting block minimize corneal button irregularities that in other devices may contribute to postoperative astigmatism. This new system was compared with the Iowa Press by evaluating trephinated corneas on the basis of profile projectors. Buttons trephined from 10 pairs of human corneas stored in (CSM) preservation solution at 4 degrees C were evaluated by measuring diameters and edge angles and correlating them with the trephine size used. In contrast with the Iowa Press, this new system produces corneal buttons that are consistently circular, parallel-edged, and identical in size to the trephine used.

Viscoelastic agents.

Viscoelastic materials possess a unique set of properties that result from their chemical structure. These properties enable them to protect the corneal endothelium and epithelium from mechanical trauma and to maintain an intraocular space, such as the anterior or vitreous chambers, even in the face of an open incision. Hence viscoelastic materials have been successfully applied to many areas of ophthalmic surgery, most notably anterior segment surgery, with few complications. There are currently three commercially available viscoelastic preparations, and several new preparations are in various stages of development.

pH of organ-culture-stored corneas.

Changes in intracorneal and storage-medium pH values of organ-culture-stored cat corneas were monitored over a 4-week period. The intracorneal pH was determined using the phosphorus-31 magnetic resonance spectroscopy (31P MRS) chemical shift of inorganic orthophosphate in conjunction with a standard pH titration curve. We incubated 32 adult cat corneas using two similar standard organ-culture methods, one with chondroitin sulfate (method 1) and the other without (method 2). Time-course data at 0, 1, 3 and 4 weeks of storage were used to calculate the rate of pH change. The intracorneal pH was not changed significantly for either organ-culture method; however, the storage-medium pH rate of change declined significantly for both methods (method 1, 0.15 pH units/week; method 2, 0.12 pH units/week). The difference between intracorneal and storage-medium pH values over time increased at a rate of 0.12 and 0.11 pH units/week for method 1 and method 2, respectively. The declining storage-medium pH in conjunction with the maintenance of intracorneal pH contributes to an increased metabolic demand on the cornea.

Neodymium:YAG laser interaction with Alcon IOGEL hydrogel intraocular lenses: an in vitro toxicity assay.

The objective of this study was to determine the potential toxicity generated by the interaction of the Nd:YAG laser and Alcon IOGEL intraocular hydrogel lens material. The IOGEL lens is composed of poly 2-hydroxyethylmethacrylate, containing 38% water, previously shown to be highly biocompatible in a wide range of tissue culture and implantation experiments. In this study, intraocular lenses (IOLs) immersed in serum-free cell culture medium were purposely exposed to exaggerated doses of laser energy to cause extensive damage. An IOLAB polymethylmethacrylate (PMMA) lens served as a control lens material. The resultant solutions were assayed for cytotoxicity in a bioassay system using fourth passage human corneal endothelial cells. No cytotoxicity was seen in the bioassay for the IOGEL hydrogel IOLs or the PMMA control IOL at any laser range/dosage tested over a 72-hour incubation period. Hydrogel lenses exhibited decreasing yellowing with decreasing energy levels, and no lens discoloration was apparent at the lowest level of irradiation, 5 mJ/50 laser bursts; the PMMA control lens exhibited moderate yellowing at 15 mJ/50 bursts. Lens marking was moderate for all IOGEL IOLs; the PMMA lens marking was severe at the power level tested.

Minnesota system corneal preservation.

The clinical and laboratory results with a modified Minnesota system of organ culture corneal preservation are presented. A refinement of our preservation technique using a closed system, as well as the addition of chondroitin sulphate to the medium is presented. Laboratory results show preservation of corneal endothelial integrity for at least 21 days with maintenance of normal corneal thickness. In addition, a 10-day quarantine system reduces the risk of donor contamination and secondary endophthalmitis. Preliminary results of the 34 degrees C and 4 degrees C closed Minnesota corneal preservation system using chondroitin sulphate show that it is safe and efficacious and allows intermediate to long-term maintenance of sterile thin tissue prior to corneal transplantation.

Neodymium:YAG laser interaction with intraocular lenses: an in vitro toxicity assay.

Use of the Nd:YAG laser is an effective technique to open an opacified posterior lens capsule. However, in the presence of a posterior chamber intraocular lens (IOL), precise focusing of the laser on the capsule is required to avoid pitting the lens optic. The question has been raised whether toxic products may result from laser damage to the IOL. We addressed this issue in the present study by exposing primary human corneal endothelial cell and human corneal organ cultures to solutions produced by purposefully hitting IOLs immersed in cell growth medium with a Nd:YAG laser. The lenses studied were lathe-cut polymethylmethacrylate (PMMA), injection-molded non-UV PMMA, injection-molded UV PMMA, and cast-molded UV PMMA. Samples of each material were irradiated in a holder containing 1 ml of cell culture medium using the following conditions: 5, 10, and 50 laser bursts at 10 millijoules (mJ), and 50 laser bursts at 5 mJ. The solutions were applied to the endothelial cell cultures (all materials) and to the corneal organ cultures (injection-molded non-UV lenses only). There was no toxicity in either assay for any of the materials studied.

Glutathione metabolism in lenses of dogs and rabbits: activities of five enzymes.

Several biochemical parameters were examined in clear dog and rabbit lenses as functions of age, and in posterior subcapsular cataracts in the Alaskan malamute. Tabulated data include soluble protein, reduced sulfhydryl content of soluble protein, reduced glutathione, water, and activity of five enzymes of glutathione metabolism. The enzymes include the glutathione biosynthesis system consisting of gamma-glutamylcysteine synthetase and glutathione synthetase, as well as glutathione peroxidase, glutathione reductase and glutathione-S-transferase. Each enzyme, acting last in a sequential reaction of either two or three reactions, was in excess activity over the preceding enzyme(s) in every case but one. In the exception, the ratio of glutathione reductase to glutathione peroxidase activity was about 1:600 and 1:155 in the dog and rabbit lens, respectively.