Corneal endothelial wound healing in living rabbit eyes after mechanical scrape (MS) and transcorneal freeze (TCF) injury was studied using tandem scanning confocal microscopy (TSCM). MS injury was created on the central corneal endothelium with an olive tip cannula; TCF injury was created using a 3-mm-diameter stainless steel probe cooled with liquid nitrogen. In vivo observation of wound healing using TSCM was correlated with scanning electron microscopy (SEM) for fixed tissues. At 6 h after MS, migrating endothelial cells at the leading edge showed lamellipodial processes on in vivo TSCM and SEM. After 24 h, the denuded area was almost fully resurfaced by migrating endothelial cells showing wide spaces between nuclei by TSCM. After 28 days, resurfaced endothelial cells showed normal hexagonal mosaic appearance with enlarged cells 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 polygonal in shape with wide intracellular spaces and (b) fibroblast-like cells at the center of the wound, which formed a retrocorneal fibrous membrane (RCFM). The RCFM was posteriorly covered with normal endothelium 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 morphologic changes (x, y, z, and time) during in vivo cellular repair of corneal wound healing after either MS or TCF injury.
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