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Because the corneal epithelium invariably encounters the full concentration of the preservative that is contained in multi-dose topical ophthalmic preparations, we investigated the cytotoxicity of several of these agents by using a sensitive model of human corneal epithelial cells in vitro. Primary cultures of epithelial cells were prepared from freshly enucleated globes. At confluence, all experimental cultures received a single dose of preservative at the concentration present in marketed formulations. The serum in the culture medium simulated the possible neutralizing effect of proteins present in the tear film in vivo. The cells were observed continuously by phase-contrast microscopy and time-lapse videomicrography for 24 hrs. Benzalkonium chloride at a concentration of 0.01% and chlorobutanol at 0.5% caused immediate cell retraction, as well as cessation of normal cytokinesis, cell movement, and mitotic activity; the epithelial cells degenerated within 2 hrs and 8 hrs, respectively. Cultures treated with chlorobutanol developed conspicuous blebs on the cell surface after 3 to 5 hrs of exposure. Thimerosal (0.001%) caused cell retraction, cessation of mitotic activity, and total cell destruction within 9 hrs. Sorbic acid (0.1% and 0.2%) greatly reduced cell movement and suppressed mitotic activity, but no cell death occurred. At concentrations of 50 ppm and 30 ppm, H2O2 instantaneously caused a marked retraction of the cells, followed by cessation of cytokinesis, cell movement, and mitosis. Retraction and death of the epithelial cells occurred within 12-24 hrs after exposure to 1 ppm H2O2 in serum-free medium. Polyquaternium ammonium chloride (0.001%) and polyaminopropyl biguanide (0.00005%) had no discernible effects on cytokinetic movement or on the mitotic activity of the epithelial cells. We relate our findings in vitro to those reported in vivo and discuss the mechanism of cytotoxicity of the various preservatives.

Several of the known growth factors either have an effect on corneal tissues or can be isolated from them. Both epidermal growth factor (EGF) and fibroblast growth factor (FGF) stimulate the proliferation of corneal epithelium, keratocytes, and endothelium. Compared to FGF, EGF is more potent in stimulating the growth of endothelial cells; it also increases the tensile strength of corneal stromal wounds. The corneal epithelium produces an angiogenic growth factor as well as a neuronotrophic growth factor. Insulin-like growth factor stimulates the growth of keratocytes and enhances the effect of EGF on the corneal endothelium. Mesodermal growth factor stimulates the proliferation of keratocytes and increases the rate of healing of damaged corneal endothelial cells. It is evident that growth factors have many potential clinical applications especially in accelerating corneal wound repair after surgery, chemical burns, or ulcers, and in increasing the numbers of corneal endothelial cells in aging and diseased corneas, as well as in donor corneas to be used for transplantation. Several parameters require evaluation, e.g. the dose, concentration, combination and formulation, exposure time, receptor affinity, and tissue interdependence of the growth factor(s), as well as the variability in patient response and severity of disease and/or injury. Investigations are also needed for the development of effective delivery systems and for determining whether growth factors are specific and safe in humans. Growth factors are emerging as a new generation of ophthalmic pharmaceuticals, and they will soon be integrated into the advancing practice of ophthalmic surgery and medicine.

We investigated the cytotoxicity of hydrogen peroxide by exposing primary confluent cultures of human corneal epithelium to a single dose of this agent at concentrations ranging from 30 ppm to 100 ppm in the tissue culture medium. Our criteria for cytotoxicity included alterations in cytokinesis and movement of the cells, changes in cell morphology, and mitotic index, and cell degeneration and death. At a concentration as low as 30 ppm, hydrogen peroxide caused rapid cell retraction as well as cessation of cytokinesis and mitotic activity; formation of membranous vesicles preceded cell death, which occurred by seven to eight hours after exposure. At a concentration of 50 ppm, normal cell activity ceased almost instantaneously; numerous surface vesicles formed by 1.5 hours of exposure, and the cells died within four to five hours. Higher concentrations (70 to 100 ppm) of hydrogen peroxide caused cell death within a few minutes. Because neutralization of hydrogen peroxide and patient compliance with the manufacturer's instructions are critical in the proper use of peroxide-based disinfection systems, users should be aware of possible short- and long-term iatrogenic toxicity of residual peroxide on the cornea.

We analysed the tissue plasminogen activator (TPA) content of the corneal epithelium, endothelium, and stroma and of the lens, as well as the aqueous and vitreous humors, in dog, calf, and monkey eyes. A quantitative estimation of the TPA activity in the corneal tissues by the [125I]fibrin-coated well assay showed similar levels of activity in the corneal epithelium, stroma, and endothelium. However, some differences were observed among the three mammalian species analysed. The values, expressed in urokinase (UK) units of activity per mg protein, ranged from 0.8 +/- 0.22 to 1.03 +/- 0.23 for the epithelium, 0.47 +/- 0.13 to 0.98 +/- 0.2 for the stroma, and 0.48 +/- 0.11 to 0.93 +/- 0.22 for the endothelium. The lens, the vitreous and the aqueous humor yielded low to negligible values. However, by using the enzyme-linked immunosorbent assay (ELISA) method, we detected an appreciable amount of TPA in the lens (9.49 +/- 1.5 ng TPA per ml lens), corneal epithelium (1.43 +/- 0.29 ng TPA per mg protein) and vitreous humor of the dog (5.71 +/- 0.61 ng TPA per ml vitreous humor) and of the calf (5.7 +/- 0.44 ng TPA per ml vitreous humor). Such discrepancies may reflect species-specific variation in the TPA content of the tissues and possibly the limitations of the techniques utilized, because of the problem related to cross-reactivity between the TPA of a given species and the antibody used in the assays.(ABSTRACT TRUNCATED AT 250 WORDS)

We investigated the cytotoxicity of hydrogen peroxide by exposing primary cell cultures of human corneal epithelium to a single dose of this agent at concentrations ranging from 30 to 100 ppm. Hydrogen peroxide, at a concentration as low as 30 ppm, caused cell retraction as well as cessation of cell movement and mitotic activity. Formation of membranous vesicles preceded cell death that occurred by 7 to 8 hours after exposure to 30 ppm. With a concentration of 50 ppm, normal cell activity ceased almost instantaneously. Numerous surface vesicles formed by 1.5 hours of exposure, and the cells died by 4 to 5 hours. Higher concentrations of hydrogen peroxide (70 to 100 ppm) caused cell death within a few minutes. Because neutralization of hydrogen peroxide and patient compliance are critical in the proper use of hydrogen peroxide-based contact lens disinfection systems, users will be well served if the long-term effects of residual peroxide on the cornea are subjected to continued study.

A wide-field contact specular microscope was used to examine and to photograph the corneal epithelium in 35 eyes with chronic corneal edema. Seven of the patients subsequently underwent corneal transplantation allowing for correlation of the in vivo findings with the light and electron microscopic appearance. The early findings on specular microscopy included loss of the clear superficial epithelial cell outlines with easy dislodgement of these cells and a fine pattern of edema outlining basal cells. Individual cellular edema and fluid-filled cysts occurring at various epithelial layers and subepithelially appeared with worsening swelling. Subepithelial connective tissue and anamalous basement membrane were present at later stages.

Corneal mucus plaques adhered to the anterior corneal surface in 17 of 67 advanced cases of keratoconjunctivitis sicca. The plaques were translucent to opaque and varied in size and shape, from multiple isolated islands to bizarre patterns involving more than half the corneal surface. Ultrastructurally, they consisted of mucus mixed with desquamated degenerating epithelial cells and proteinaceous and lipoidal material. The condition may be symptomatic but can be controlled and prevented in most cases by topical ocular application of 10% acetylcysteine.

By exposing primary cultures of human corneal epithelial cells to a single dose of either benzalkonium chloride or chlorobutanol, evaluated the cytotoxicity of these two preservatives which are commonly used in artificial tear solutions and other topical ophthalmic medications. Control and experimental cultures were analyzed by continuous time-lapse videomicrographic recordings as well as by sequential phase-contrast microscopy. Both benzalkonium chloride at a concentration of 0.01% and chlorobutanol at 0.5% caused immediate cessation of normal cytokinesis and mitotic activity, and epithelial cells degenerated within two hours and eight hours, respectively.

The clincopathological features and pathogenesis of secondary mosaic degeneration of the cornea (anterior crocodile shagreen of Vogt) are described. The structural basis for the normal anterior corneal mosaic pattern seems to lie in the particular arrangement of many prominent collagen lamellae of the anterior stroma that thake an oblique course to gain insertion into Bowman's layer. Since, at normal intraocular pressure, Bowman's layer is under tension, when viewed from the anterior surface the cornea appears smooth. By releasing the tension, however, a reproducible polygonal ridge pattern becomes manifest. It is suggested that a prolonged phthisical state of the eye is one condition wherein the mosaic pattern may become permanent and that, as a secondary event, this is followed by irregular calcification of Bowman's layer which particularly involves the ridges projecting into the epithelium. Biomicroscopically these ridges corresponded to the branching reticular arrangement of the mosaic opacities.

Corneal changes in passive Arthus reaction are essentially the same as that reported to occur in active Arthus reaction and consist of accumulation of polymorphonuclear leucocytes around the deposits of immune complexes, release of lysosomes and fragmentation and dissolution of collagen fibrils. In addition, degenerative changes were found in the corneal endothelium and Descemet's membrane.