Actin filament organization during endothelial wound healing in the rabbit cornea: comparison between transcorneal freeze and mechanical scrape injuries

Abstract

Purpose: To compare and contrast the in vivo mechanism of wound healing after mechanical scrape and transcorneal freeze (TCF) injury in a rabbit eye model by examining changes in the cytoskeletal organization of contractile, filamentous actin (f-actin) microfilaments as relates to differences in cell migration or translocation during endothelial repair.

Methods: Endothelial wound healing after mechanical scrape and transcorneal freeze injury was studied in rabbit eyes using laser scanning confocal microscopy (LSCM). Central corneal mechanical scrape injury was made using an olive tip cannula, and TCF injury was made using a 3-mm diameter stainless steel probe cooled with liquid nitrogen. Cytoskeletal changes in f-actin stained with phalloidin-FITC were observed during wound healing using LSCM.

Results: At 6 hours after mechanical scrape, the leading edge of the migrating sheet showed a decrease in the intensity of phalloidin-FITC staining, suggesting a decrease in cortical f-actin. Migrating endothelial cells in vivo did not appear to develop stress fibers after mechanical scrape, which is consistent with an in vitro cell spreading mechanism of endothelial wound healing. By 24 hours, the denuded area was almost fully resurfaced by migrating endothelial cells. On the other hand, TCF injury produced fibroblastic changes in the endothelial cells with extension and elongation of spindle-shaped endothelial cells at the leading edge by 24 hours after injury. Fibroblastic endothelial cells developed prominent actin stress-fibers, which is consistent with an in vitro cell migration mechanism of endothelial wound healing. Three days after TCF, the wounded area was resurfaced with two cell types: rough, fibroblast-like cells forming a retrocorneal fibrous membrane having prominent f-actin bundles or stress fibers with few cell-cell junctions, and smooth, polygonal-shaped endothelial cells having tight cell junctions with a cortical distribution of f-actin. After 28 days the retrocorneal fibrous membrane was posteriorly covered with normal endothelium.

Conclusions: These data support the hypothesis that endothelial wound healing involves two separate, injury-dependent, mechanisms--cell spreading and cell migration.