The experiments described in this study conform to the ARVO
Statement for the Use of Animals in Ophthalmic and Vision Research and
the NIH guidelines for use of animals. Corneas were harvested from
adult Hartley guinea pigs, which were anesthetized with halothane and
killed by cervical dislocation, and day 17 chicken embyros, which were
killed by cervical dislocation. The corneal epithelia were wounded by
outlining the wound area with 3 mm corneal trephine and then scraping
the epithelium with a scalpel blade. This procedure produced
superficial scrape wounds, removing the epithelium, but leaving the
basement membrane and Bowman’s layer intact. After wounding of the
epithelium, the endothelium was removed by scraping, and the corneas
were placed in organ culture on stainless steel rafts at the
air–culture medium interface. Dulbecco’s modified Eagle’s medium
(DMEM) (Bio-Whittaker, Walkersville, MD), supplemented with 10% fetal
calf serum (Cellgro; Mediatech, Herndon, VA) and a 1% glutamine,
pen/strep cocktail (Bio-Whittaker) was used in all cultures. Both
freshly isolated corneas and 4% formaldehyde–fixed corneas were
embedded in OCT medium (Tissue Tek; Miles, Naperville, IL) and frozen
for cryosectioning. Cryosections of 6 μm were cut perpendicular to
the epithelial axis.