Multi-Dimensional Imaging of Cell and Extracellular Matrix Interactions During Corneal Wound Healing

Corneal haze is a leading cause of blindness, and may occur after injury, surgery, or chemical burns. The cornea's thickest layer, the stroma, accounts for two-thirds of the eye's refractive power, and contains highly aligned collagen fibrils needed for transparency. Stromal cells, or keratocytes, help maintain the extracellular matrix (ECM). After injury or surgery, keratocytes within the wound undergo apoptosis, while keratocytes in the wound margin transform and activate into fibroblasts and myofibroblasts, leading to contraction, deposition of fibrotic ECM, and haze. Thus, spatial and temporal investigations of cell/ECM interactions are needed for further insight into this healing process. In this research, freeze-injury (FI), lamellar keratectomy (LK), and photorefractive keratectomy (PRK) in vivo rabbit models are used, and cell/ECM interactions are tracked using an in vivo confocal microscope as well as second harmonic generation (SHG) and multiphoton imaging. These in vivo models revealed that in the short term, intrastromal cells migrated in parallel to the collagen lamellae, while cells over the stromal wound bed transformed to myofibroblasts and were randomly arranged. Stromal haze initially increased after injury, but gradually decreased over time. Long term, collagen became more organized, more quiescent cells replaced myofibroblasts, and normal stromal thickness regenerated (after keratectomy) below the ablation surface. Punctate F-actin labeling was detected along cells that co-aligned with native and regenerated collagen, suggesting critical involvement of cell/ECM interactions in stromal remodeling and regeneration. Collagen lamellae appeared to provide a template for fibroblast patterning during both intrastromal migration and stromal regrowth. Overall, cells appear to regenerate and remodel the ECM to produce a lamellar structure similar to the native stroma. These results provide novel insights into the mechanisms of cell/ECM interactions involved in various stages of wound healing in the cornea, and may lead to potential therapies that prevent or reverse fibrosis in the clinic.