Assessment of f-actin organization and apical-basal polarity during in vivo cat endothelial wound healing

Abstract

Purpose: To assess the relationships between cytoskeletal changes and apical-basal polarity during healing of mechanical scrape injuries in the cat corneal endothelium.

Methods: Ten cats (20 eyes) were used in this study. One mechanical scrape injury was created in the corneal endothelium of each eye using a blunt olive tip cannula. Tandem scanning confocal microscopy (TSCM) was performed at sequential time points after injury for in vivo assessment of cell morphology and wound healing rates. In two eyes, scanning electron microscopy was performed to allow verification of TSCM observations. Ten eyes were collected between 6 and 48 hours after wounding for in situ labeling of f-actin, ZO-1, or both.

Results: Cat endothelial cell morphology observed using in vivo microscopy was identical to that shown using scanning electron microscopy. During healing, endothelial cells always remained attached to the endothelial sheet, although some showed extensions of lamellipodia into the open wound area. The in situ localization of f-actin also correlated with the TSCM in vivo wound morphology. Quantitative analysis showed that there was a decrease in the intensity of phalloidin-fluorescein isothiocyanate staining at the leading edge of the wound, suggesting a decrease in f-actin; a significant correlation was found between the relative intensity of f-actin staining and the distance from the wound margin (R = 0.98, P < 0.01). At 24 and 48 hours after injury, both ZO-1 and f-actin maintained an apical localization within cells immediately adjacent to the leading edge, despite the considerable distance of movement and dramatic decrease in the intensity of f-actin staining.

Conclusions: Overall, these data demonstrate that after scrape injury in the cat, endothelial cells exhibit a pattern of healing in which total intracellular f-actin is reduced, but normal cell connectivity and apical-basal polarity are maintained throughout.