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

Abstract

Corneal scars originate from keratocyte-derived fibroblasts and myofibroblasts that are ultimately cleared through apoptosis or revert to keratocytes. A mouse model expressing a keratocyte lineage-specific reporter KeraRT/tetO-Cre/mTmG (I-Kera mTmG) was interrogated to elucidate cell phenotype dynamics during scar maturation. This mouse model expresses tdTomato (red) in all keratocan-negative cells while eGFP (green) is expressed only by keratocytes. A 1mm full-thickness keratotomy was created in adult I-KeramTmG mice. The presence or absence of keratocytes was examined at 3-, 6- and 10-months post injury. At 3- and 6-months post-injury, 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 post-injury, green cells can be seen throughout the scar, but most cells were red. Proliferation of stromal cells after injury was studied by EdU labeling and Ki-67 staining and both assays showed proliferation only during the first 2 weeks after injury. Second harmonic generation (SHG) microscopy showed thickened and irregularly arranged collagen fibers in scars suggesting that neither extracellular matrix organization nor cell phenotype had changed significantly 10-months post injury. In vivo experiments suggest that in old corneal scars, a non-keratocyte phenotype persists in an abnormal matrix with unique characteristics that probably prevent regression of fibroblasts and myofibroblasts to keratocytes or invasion of surrounding keratocytes.