Purpose: The purpose of this study was to correlate the temporal expression of alpha-smooth muscle specific actin (alpha-SM actin), a molecular marker for myofibroblast transformation, with corneal wound contraction.
Methods: After full-thickness, central corneal injury in rabbit eyes, the anterior width of the wound (wound gape) was measured in the same animals using in vivo confocal microscopy. In addition, animals were sacrificed at various times after injury for the determination of alpha-SM actin expression by immunofluorescent microscopy using a mouse monoclonal antibody specific for human alpha-actin. Antibody specificity was confirmed by Western blot analysis of normal and wound fibroblasts. Expression of alpha-SM actin also was related spatially to f-actin and the wound margin by co-localization with phalloidin and DTAF (5([4,6-dichlorotriazin-2yl]amino)fluorescein), a fluorescent marker bound to the wound margin.
Results: Wound contraction was most evident from days 7 to 42, when wound gape progressively decreased from 574 +/- 120 microns to 250 +/- 61 microns. Thereafter, the wound remained stable to day 84 (304 +/- 58 microns). Expression of alpha-SM actin directly correlated with wound contraction--appearing across the wound at day 7, the full thickness of the wound at day 14, and the posterior wound at day 28. alpha-SM actin was localized exclusively to phalloidin-stained, f-actin microfilament bundles or stress fibers within wound healing fibroblasts, and the disappearance of alpha-SM actin correlated with the concomitant disappearance of stress fibers at days 28 to 42. Staining of the wound margin with DTAF confirmed that the expression of alpha-SM actin was limited to fibroblasts within the wound.
Conclusions: The expression of alpha-SM actin was directly correlated to corneal wound contraction, appearing at the initiation of and disappearing at the completion of the contraction process. Furthermore, the exclusive expression of alpha-SM actin by fibroblasts present only within the wound suggests that local environmental factors unique to the wound may play an important role in myofibroblast transformation.