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Purpose: To understand the biological activity of entactin, a component of the basement membrane of the corneal epithelium, we investigated the ability of rabbit corneal epithelial cells to attach to an entactin matrix and the effect of entactin on the cells' attachment to other corneal basement proteins., Methods: Multiwell plastic plates were coated with bovine serum albumin (BSA), alone or with BSA and entactin, laminin, fibronectin or collagen type IV. Cultured rabbit corneal epithelial cells were seeded on the plates. After incubation (usually 90 min), the cells were fixed and stained with 1% crystal violet. The number of attached cells was counted under a light microscope., Results: The numbers of attached cells increased in proportion to both the incubation period and the concentration of entactin coated. Furthermore, the number of cells attached to the entactin-coated plate was greater than the number attached to the BSA-coated plate for each incubation period (30 to 120 min). Likewise, when laminin-coated plates were treated with entactin, the number of the attached cells increased in proportion to the concentration of entactin. However, entactin did not affect the cellular attachment of fibronectin or type IV collagen. Cellular attachment to entactin was partially inhibited by the cells' preincubation with the synthetic peptide (GRGDSP)., Conclusions: The present results showed that cultured corneal epithelial cells adhere to entactin and that entactin stimulated the attachment of these cells to the laminin matrix. These findings suggest that entactin plays a specific role in maintaining the normal integrity of the corneal epithelium.

We investigated the combined effects of hyaluronan and fibronectin on corneal epithelial wound closure in vivo. When fibronectin eye drops (1 mg/ml) were administered for the first 4 hours after chemical cautery and hyaluronan eye drops (1 mg/ml) were administered for following 32 hours, the mean healing rate was significantly higher than in control eyes treated with phosphate buffered saline instead of fibronectin or with initial fibronectin alone. These results demonstrated that hyaluronan and fibronectin have a synergistic effect, with fibronectin pretreatment augmenting hyaluronan-stimulated corneal epithelial wound closure.

The slowed healing rates observed by some investigators may be caused by vehicles or preservatives in the antimicrobials preparations tested. To determine whether antimicrobials directly inhibit corneal epithelial wound healing, we cultured blocks of the rabbit cornea in media containing various concentrations of antibiotics or antimicrobials (at 1, 10, or 100 micrograms/ml); after 24 hours, we measured the distance of epithelium that had migrated down the side of each block. The higher concentrations of fluoroquinolones (ofloxacin; 74 +/- 5.8% of control at 100 micrograms/ml, p < 0.05, ciprofloxacin; 4.4 +/- 1.5% of control at 100 micrograms/ml, p < 0.01, or norfloxacin; 71 +/- 7.0% at 10 mu g/ml, p < 0.01, and 1.5 +/- 0.4% of control at 100 mu g/ml, p < 0.01) and the highest concentrations of peptides (polymyxin B; 64 +/- 3.0% of control at 100 micrograms/ml, p < 0.01, or colistin; 67 +/- 5.7% of control at 100 micrograms/ml, p < 0.01) or fosfomycin (79 +/- 6.2% of control at 100 micrograms/ml, p < 0.05) had an inhibitory effect on corneal epithelial migration. Among aminoglycosides tested, sisomicin (85 +/- 10.0% of control, not significant), dibekacin (76 +/- 11.6% of control, p < 0.05) and streptomycin (77 +/- 9.4% of control, not significant) were inhibitory at 100 micrograms/ml, but tobramycin had no effect. Penicillins (aspoxicillin, sulbenicillin or ampicillin), cephalosporins (cefmenoxime or cefminox), oxytetracycline, erythromycin and chloramphenicol did not affect epithelial migration at all. These results demonstrate that some antimicrobials are inhibitory at high concentrations, but penicillins, cephalosporins, oxytetracycline, erythromycin or chloramphenicol has no inhibitory effect on corneal epithelial migration.

In order to understand the mechanisms that bring about maintenance and restoration of the integrity of corneal epithelium, we investigated independent and combined effects of transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF) on rabbit corneal epithelial cells in cell and organ culture. Specifically, we determined whether incubation with these factors influenced 1) cellular proliferation, 2) ability of cells to attach to a fibronectin matrix, and 3) the rate of epithelial migration over corneal stroma. Incubation with TGF-beta caused a dose-related decrease in the incorporation of 3H-thymidine by the epithelial cells. EGF increased 3H-thymidine incorporation, but this effect was antagonized by the addition of TGF-beta into the incubation medium. Incubation with EGF increased the numbers of cells that attached to a fibronectin matrix. TGF-beta itself did not affect the number of attached cells but, again, it antagonized the stimulatory effect of EGF. Similarly, when corneal blocks were cultured with EGF, epithelial migration increased in a dose-related manner. TGF-beta itself did not affect epithelial migration at any of the concentrations tested (0.1-10 ng/ml), but it antagonized EGF-stimulated epithelial migration. These findings suggest that the proliferation and the migration of corneal epithelial cells are regulated by different mechanisms, and that TGF-beta serves as a modulator of the effects of EGF.

Unidirectional Na+ and Cl- fluxes were determined in the isolated rabbit ciliary body under short-circuited conditions. A statistically significant net Cl- flux towards aqueous was detected in the standard bathing solution (HCO3- = 115 mM), but no net Na+ flux was demonstrated in this solution. The net Cl- flux exceeded the short-circuit current, suggesting the existence of a net flux of other ion(s). In a bathing solution containing 5 mM HCO3-, the net Cl- flux was abolished, suggesting that HCO3- in the bathing solution enhances the net Cl- flux.