Abstract
Purpose :
During wound healing following surgery, disease, or trauma, corneal keratocytes often differentiate into myofibroblasts, which can lead to fibrosis and corneal haze. In contrast, following a transcorneal freeze injury (FI), keratocytes repopulate the wound without myofibroblast differentiation. The purpose of this study is to identify gene expression patterns that underlie this non-fibrotic wound healing process.
Methods :
A FI was performed on one eye of four rabbits. Both eyes were imaged in vivo before, and at day 7 and 28 after FI using in vivo confocal microscopy through focusing (CMTF) with a modified HRT-RCM system to assess corneal sublayer thickness, stromal backscatter and cell morphology. Two rabbits were sacrificed at 7 and 28 days, respectively. Corneas from both the procedure eye and the contralateral control eye were collected and 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 :
CMTF analysis showed an increase in stromal thickness and haze, as well as streams of elongated stromal fibroblasts within the wound at day 7 after FI, all of which resolved by day 28. In addition to clusters associated with other cell types, a single cluster was identified in all control eyes that expressed genes associated with corneal keratocytes, such as keratocan (KERA), lumican (LUM), and stromal collagens. At day 7 an additional cluster expressing keratocyte markers was identified after FI. This suspected migratory fibroblast cluster had increased expression of genes associated with cell junctions, cell-ECM adhesion, cell migration, and fibroblast differentiation, but not myofibroblast specific markers (e.g. α-SM actin). At day 28, both FI and control eyes had only one cluster expressing keratocyte markers.
Conclusions :
Gene expression analysis by scRNA-seq of corneas after FI shows temporally dependent fibroblast differentiation consistent with in vivo analysis on the same corneas. The gene expression patterns are also consistent with previous in situ imaging results showing interconnected streams of migrating fibroblasts 7 days after FI. Overall, this pipeline combining in vivo imaging with subsequent single cell analysis allows for the characterization of gene expression patterns associated with specific wound healing phenotypes.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.