Corneal Fibroblast Migration Patterns During Intrastromal Wound Healing Correlate With ECM Structure and Alignment

To assess keratocyte backscattering, alignment, morphology, and connectivity in vivo following a full-thickness corneal injury using the Heidelberg Retina Tomograph Rostock Cornea Module (HRT-RCM), and to correlate these findings with en bloc three-dimensional (3-D) confocal fluorescence and second harmonic generation (SHG) imaging.

Rabbit corneas were scanned in vivo both before and 3, 7, 14, and 28 days after transcorneal freeze injury (FI), which damages all corneal cell layers. Corneal tissue was also fixed and labeled for f-actin and nuclei en bloc, and imaged using 3-D confocal fluorescence microscopy and SHG imaging.

Using the modified HRT-RCM, full-thickness scans of all cell layers were consistently obtained. Following FI, stromal cells repopulating the damaged tissue assumed an elongated fibroblastic morphology, and a significant increase in cellular light scattering was measured. This stromal haze gradually decreased as wound healing progressed. Parallel, interconnected streams of aligned corneal fibroblasts were observed both in vivo (from HRT-RCM reflection images) and ex vivo (from f-actin and nuclear labeling) during wound healing, particularly in the posterior cornea. Second harmonic generation imaging demonstrated that these cells were aligned parallel to the collagen lamellae.

The modified HRT-RCM allows in vivo measurements of sublayer thickness, assessment of cell morphology, alignment and connectivity, and estimation of stromal backscatter during wound healing. In this study, these in vivo observations led to the novel finding that the pattern of corneal fibroblast alignment is highly correlated with lamellar organization, suggesting contact guidance of intrastromal migration that may facilitate more rapid wound repopulation.