Abstract
Abstract: :
Purpose:It has been shown that loss of limbal epithelial stem cells is followed by conjunctivalization of the corneal surface. In this study, limbal stem cells together with the entire corneal epithelium were removed to determine: 1) which conjunctival epithelial cells participate in repair of the corneal injury, and 2) whether centripetal cell movement takes place in the conjunctivalized cornea in the absence of limbal stem cells. Methods:Ubiquitous GFP mice were used, in which only some of the ocular surface epithelial cells were strongly positive with GFP, allowing tracking of their movement. Total epithelial removal was carried out by mechanical scraping after n–heptanol treatment. At various times after the injury, a flat whole–mount of ocular surface was prepared to determine distribution of GFP positive cells in an entire area of cornea and conjunctiva. Movement of GFP positive cells in the conjunctivalized cornea was determined with a living mouse by in vivo time–lapse microscopy. Results:After total removal of corneal and limbal epithelium, conjunctival cells invaded the cornea to completely cover the denuded surface in about one week, as has been described by others. Goblet cells, normally concentrated in the fornix, were found throughout the bulbar conjunctiva and the cornea. In the uninjured cornea, GFP positive cells exhibited radial stripes from the limbus to the central cornea, whereas conjunctiva contained randomly oriented patches of GFP positive cells. Upon epithelial scrape injury and subsequent wound healing reactions, some GFP positive epithelial cells formed narrow streaks that were continuous from the fornix to the limbus to the central cornea. No such cell streaks were obvious in the palpebral conjunctiva where patches of GFP positive cells remained apparently unchanged. Time–lapse analyses revealed that epithelial cells of the conjunctivalized cornea moved centripetally at a rate similar to the centripetal movement of corneal epithelial cells in an uninjured eye. Conclusions:After a limbal injury, a signal for cell movement travels from the limbus to the fornix through an uninjured sheet of bulbar conjunctival epithelium, but apparently not to the palpebral region. Thus, epithelial cells in the fornix, as well as those in the bulbar conjunctiva, are recruited in the repair of the corneal surface, suggesting that proliferative capacity of bulbar conjunctival cells is inadequate. Centripetal cell movement in the conjunctivalized cornea suggests that limbal stem cells are not required for such movement.
Keywords: conjunctiva • cornea: epithelium • microscopy: light/fluorescence/immunohistochemistry