Abstract
Purpose :
Following photorefractive keratectomy (PRK) in the rabbit, the corneal stroma proceeds through specific stages of healing which can be isolated both temporally and spatially using in vivo confocal microscopy. The purpose of this study is to characterize the gene expression patterns associated with different phases of wound healing.
Methods :
A 9.0 diopter PRK was performed on the left eye of three rabbits. Both eyes were imaged at days 7, 21 and 60 after PRK with in vivo confocal microscopy through focusing (CMTF). One rabbit was sacrificed at each timepoint, and corneas were processed for single cell RNA sequencing (scRNA-seq) using the 10X Genomics platform and sequenced on an Illumina machine. Data analysis—clustering and differential expression—was performed using Seurat.
Results :
At day 7, elongated stromal fibroblasts were evident in the anterior stroma by CMTF. At day 21, a reflective fibrotic layer formed on top of the stroma and haze was at its peak. Between days 21 and 60, stromal thickness gradually returned towards pre-op values, suggesting tissue regeneration. Analysis of scRNA-seq data with unbiased clustering consistently yielded multiple clusters expressing keratocyte markers at each time point. At day 7, a suspected fibroblast cluster exhibited increased expression of genes associated with cell-ECM adhesion, cell migration and cell-cell junctions, as well as markers of impending fibrosis. At day 21, there was increased expression of genes consistent with myofibroblast differentiation, such as α-smooth muscle actin (ACTA2), components of the contractile machinery [e.g. β-tropomyosin (TPM2), transgelin (TAGLN)], cell-ECM adhesion molecules [e.g. integrins, leucine rich repeat containing 15 (LRRC15)] and fibrotic ECM components [e.g. fibronectin (FN1), collagen type III]. At day 60, there was reduced expression of myofibroblast markers as compared to day 21. In addition, an activated keratocyte cluster was identified with upregulation of basement membrane proteins (collagen types IV, VII), higher expression of normal stromal collagens, and biglycan (BGN).
Conclusions :
Initial scRNA-seq analysis of corneas after PRK shows temporally dependent expression of genes associated with fibroblasts (day 7), myofibroblasts (day 21), and a potential regenerative subpopulation of keratocytes (day 60). Further analysis may reveal novel genes and signaling pathways regulating specific stages of corneal wound healing.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.