The cellular mechanism of corneal
wound contraction after
radial keratotomy (RK) was studied in a feline eye model. A total of 10 cat eyes were evaluated at various times from 0-30 days after surgery. Changes in the distribution of intracellular filamentous actin, nonmuscle
myosin,
alpha-actinin, surface membrane alpha 5 beta 1
integrin, and extracellular
fibronectin were studied using immunofluorescence and
laser confocal and electron microscopy. From day 3-7, staining for
fibronectin increased along the
wound margin. By day 7, keratocytes adjacent to the
wound margin showed increased
f-actin staining with intense staining for
fibronectin compared with normal keratocytes.
Myosin and alpha 5 beta 1
integrin expression was very weak at this time;
alpha-actinin was not found. By day 14, fibroblasts within the
wound formed
f-actin microfilament bundles (stress fibers) which colocalized with
fibronectin. Wound-healing fibroblasts also stained positively for alpha 5 beta 1
integrin,
myosin, and
alpha-actinin (the latter two were colocalized). The presence of
myosin and
alpha-actinin in the
wound fibroblasts and the re-organization of
f-actin into stress fibers by day 14 correlated with the development of
wound contraction. A comparison of the cellular distribution of actin,
myosin, and
alpha-actinin with alpha 5 beta 1
integrin 14 days after injury suggested that
integrin was localized along stress fiber bundles during
wound contraction. The data from this study suggest that modulation of
wound gape during healing of RK
wounds may involve transformation of the corneal keratocyte to a myofibroblast-like cell and the subsequent formation of intracellular stress fibers composed of
f-actin, nonmuscle
myosin, and
alpha-actinin. Based on the colocalization of
fibronectin filaments and
f-actin filaments and the unique distribution of alpha 5 beta 1
integrin, these findings support the hypothesis that the tension within the
wound is generated by the formation of intracellular stress fibers and the interactions between stress fibers and the extracellular matrix, mediated by specific membrane receptor molecules.