Temporal and spatial analysis of stromal cell and extracellular matrix patterning following lamellar keratectomy

Extracellular matrix (ECM) supplies both physical and chemical signals to keratocytes which can impact their differentiation to fibroblasts and/or myofibroblasts. It also provides a substrate through which they migrate during wound repair. We have previously shown that following transcorneal freeze injury (FI), migrating corneal fibroblasts align parallel to the stromal lamellae during wound repopulation. In this study, we compare cell and ECM patterning both within and on top of the stroma at different time points following lamellar keratectomy (LK) in the rabbit. Twelve rabbits received LK in one eye. Rabbits were monitored using in vivo confocal microscopy at 3, 7, 21 and 60 days after injury. A subset of animals was sacrificed at each time point to further investigate cell and matrix patterning. Tissue was fixed and labeled in situ with Alexa Fluor 488 phalloidin (for F-actin), and imaged using multiphoton fluorescence and second harmonic generation (SHG) imaging (for collagen). Immediately following LK, cell death occurred in the corneal stroma directly beneath the injury. At 7 and 21 days after LK, analysis of fluorescence (F-actin) and SHG results (collagen) indicated that fibroblasts were co-aligned with the collagen lamellae within this region. In contrast, stromal cells accumulating on top of the stromal wound bed were randomly arranged, contained more prominent stress fibers, and expressed alpha smooth muscle actin (α-SMA) and fibronectin. At 60 days, cells and matrix in this region had become co-aligned into lamellar-like structures; cells were elongated but did not express stress fibers. Corneal haze measured using in vivo confocal microscopy peaked at 21 days after LK, and was significantly reduced by 60 days. Cell morphology and patterning observed in vivo was similar to that observed in situ. Our results suggest that the topography and alignment of the collagen lamellae direct fibroblast patterning during repopulation of the native stroma after LK injury in the rabbit. In contrast, stromal cells accumulating on top of the stromal wound bed initially align randomly and produce a fibrotic ECM. Remarkably, over time, these cells appear to remodel the ECM to produce a lamellar structure that is similar to the native corneal stroma.