A Core of Keratocan-Negative Cells Survives in Old Corneal Scars

Corneal scars originate from keratocyte-derived fibroblasts and myofibroblasts that are ultimately cleared through apoptosis or revert to keratocytes. A mouse model expressing the keratocyte lineage-specific reporter KeraRT/tetO-Cre/mTmG (I-KeramTmG) was used to elucidate cell phenotype dynamics during scar maturation. In this model, tdTomato (red) is expressed in all keratocan-negative cells, while enhanced green fluorescent protein (green) is expressed only by keratocytes. A 1-mm full-thickness keratotomy was generated in adult I-KeramTmG mice. The presence of keratocytes was determined at 3, 6, and 10 months after injury. At 3 and 6 months, few green cells were visualized at the scar borders, while few or no green cells were seen in the central (core) scar. At 10 months, a few green cells and a majority of red cells were observed throughout the scar. Proliferation of stromal cells after injury was studied by 5-ethynyl-2'-deoxyuridine labeling and Ki-67 staining. Both assays showed proliferation only during the first 2 weeks after injury. Second harmonic generation microscopy showed thickened and irregularly arranged collagen fibers in scars, suggesting that neither extracellular matrix organization nor cell phenotype had changed significantly at 10 months after injury. Findings from in vivo experiments suggest that in old corneal scars, a nonkeratocyte phenotype persists in an abnormal matrix with unique characteristics that probably prevent the regression of fibroblasts and myofibroblasts to keratocytes or invasion of surrounding keratocytes.