Abstract
Abstract: :
Purpose: We have previously reported that both Smad2 and 4 were translocated into the nucleus of the basal cells in corneal epithelium after wounding. This localization was mainly confined to the epithelium migrating to cover the wound area. We have also reported that this localization disappears by one week, at which point the basement membrane appears to be continuous. Our current study examines whether there is a correlation between the presence of the basement membrane and the translocation of the Smad proteins during wound repair. Methods: Adult Sprague–Dawley rats were anesthetized and a 3mm superficial keratectomy or debridement was performed. The eyes were allowed to heal from 4 to 72 hours. Four rats for each time point had the right eye wounded, and the contralateral eye served as a control. After the appropriate time, the rats were sacrificed; the eyes were enucleated and frozen in OCT on dry ice. Six µm sections were cut and indirect immunofluorescence was performed with anti–Smad2/3 (BD Transduction Laboratories; San Diego, CA) and anti–laminin (Dako; Carpenteria, CA), a marker of basement membrane. The tissue was rehydrated, blocked and incubated for 1 hour at room temperature with anti–Smad2/3 and anti–laminin. After an hour, the tissue was rinsed, blocked and incubated with anti–mouse IgG and anti–rabbit IgG for another hour at room temperature. Tissues were then rinsed and coverslipped with Vectashield mounting media containing DAPI (Vector Labs; Burlingame, CA), a marker of nuclei. Results: In unwounded tissue, Smad 2/3 is cytoplasmic. By 4 hours after a keratectomy, Smad 2/3 nuclear localization can be seen in a few of the basal cells at the leading edge of the wound. This localization appears to be maintained and the number of basal cells stained appears to increase with time. By 48 hours post–keratectomy, it appears that all the basal cells in the epithelial tissue covering the wound area have nuclear Smad 2/3. However, in the debridement model, Smad 2/3 localization remains cytoplasmic at 4 and 8 hours. By 16 hours, a few basal cells in the migrating epithelium have nuclear Smad 2/3. Patches of nuclear Smad 2/3 in the basal epithelial cells of the wound area can be seen at all the later time points. This staining pattern is not as consistent and obvious as with a keratectomy. Conclusions: The presence of the basement membrane appears to have an effect on the amount of translocation of the Smad 2 protein during corneal epithelial wound repair.
Keywords: wound healing • cornea: epithelium • transcription factors