IN-VIVO CONFOCAL MICROSCOPIC STUDIES OF ENDOTHELIAL WOUND-HEALING IN RABBIT CORNEA

Corneal endothelial wound healing in living rabbit eyes after mechanic al scrape (MS) and transcorneal freeze (TCF) injury was studied using tandem scanning confocal microscopy (TSCM). MS injury was created on t he central corneal endothelium with an olive tip cannula; TCF injury w as created using a 3-mm-diameter stainless steel probe cooled with liq uid nitrogen. In vivo observation of wound healing using TSCM was corr elated with scanning electron microscopy (SEM) for fixed tissues. At 6 h after MS, migrating endothelial cells at the leading edge showed la mellipodial processes on in vivo TSCM and SEM. After 24 h, the denuded area was almost fully resurfaced by migrating endothelial cells showi ng wide spaces between nuclei by TSCM. After 28 days, resurfaced endot helial cells showed normal hexagonal mosaic appearance with enlarged c ells by TSCM and SEM. TCF injury produced fibroblastic changes in the endothelial cells with elongation and spreading by 24 h after injury. After 3 days, the wounded area was resurfaced with two cell types: (a) migrating endothelial cells at the peripheral area, which appeared po lygonal in shape with wide intracellular spaces and (b) fibroblast-lik e cells at the center of the wound, which formed a retrocorneal fibrou s membrane (RCFM). The RCFM was posteriorly covered with normal endoth elium after 28-60 days. TSCM of the stroma demonstrated spindle-shaped , activated keratocytes migrating into the wounded stroma at 3-14 days . In conclusion, TSCM allows viewing of dynamic four-dimensional morph ologic changes (x, y, z, and time) during in vivo cellular repair of c orneal wound healing after either MS or TCF injury.