Assessment of stress fiber orientation during healing of radial keratotomy wounds using confocal microscopy

Radial keratotomy (RK) is a refractive surgical procedure in which partial thickness incisions are made in the cornea in order to alter its shape. Previous studies suggest that RK wounds undergo changes in wound gape in response to the ingrowth of myofibroblasts which mediate subsequent wound contraction and may modulate changes in corneal curvature seen after RK. A recent quantitative analysis of f-actin organization in full-thickness incisional wounds in the rabbit demonstrated that microfilament bundles (stress fibers) present in myofibroblasts align parallel to the long axis of the wound during wound contraction. To investigate whether the same pattern of alignment occurs after RK, a similar analysis of f-actin organization was undertaken using the cat RK model. Radial keratotomy wounds were studies from 10 to 28 days after surgery using en block staining with fluorescein isothiocyanate (FITC) phalloidin, and three-dimensional (3-D) datasets (z- series of en face optical sections) were collected using laser confocal microscopy at various regions within the wound. In addition, conventional en face sections were double-labeled using combinations of phalloidin and antibodies to fibronectin and α5β1 integrin. Myofibroblast ingrowth started in the bottom of the wound and progressed anteriorly. At 10 to 14 days f- actin was predominantly distributed in long, thick bundles (stress fibers) within the wound. These fibers appeared to be randomly oriented anteriorly, but became progressively more aligned with the long axis of the wound posteriorly. At 21 days, the stress fibers were predominantly oriented parallel to the long axis of the wound at all levels. F-actin, fibronectin and integrin were coaligned at both the 14 and 21 day time points. Since the majority of wound closure occurs between 14 and 28 days after surgery, we conclude that parallel alignment of the actin filament-fibronectin-integrin assembly in the cat RK model is associated with wound contraction.