Abstract
Purpose :
Previous studies have demonstrated that UV cross-linking (CXL) increases corneal stromal stiffness and produces alterations in extracellular matrix (ECM) microstructure. We assessed whether these changes modulate cell patterning and mechanical phenotype during stromal wound repopulation using multidimensional cellular imaging in vivo and in situ.
Methods :
A total of 20 rabbits was included in this study. All rabbits underwent a 70 μm phototherapeutic keratectomy (PTK) with an excimer laser to remove the epithelium and basement membrane. In 12 rabbits, standard UV CXL was performed in the same eye immediately after PTK. Contralateral eyes served as controls. In vivo confocal microscopy through focusing (CMTF) was used to analyze corneal epithelial and stromal thickness, as well as stromal keratocyte activation and corneal haze. CMTF scans were collected one week pre-operatively, and at 7, 21 and 90 days after the procedure. A subset of rabbits was sacrificed at each time point, and corneas were fixed, labeled in situ for F-actin, and imaged using multiphoton fluorescence microscopy and second harmonic generation imaging.
Results :
Following both PTK and PTK+CXL, an area of cell death was produced in the central corneal stroma. After PTK alone, the stroma was repopulated by keratocytes within 7 days. During repopulation, migrating cells formed thin chains that were coaligned with collagen lamellae. A similar pattern of migration was observed posterior to the cross-linked stromal tissue after PRK+CXL. In contrast, repopulation of the cross-linked anterior stromal region following PTK+CXL was significantly delayed, and was still not complete at 90 days in most rabbits. At days 7 and 21, keratocytes near the edge of the cross-linked stromal region formed clusters and expressed more stress fibers as compared to PTK alone. Interestingly, a thin layer of subepithelial fibrosis was observed on top of the native stroma 21 days after PTK alone, but this fibrotic layer did not form after PTK+CXL. Average corneal haze increased after both procedures, and peaked at day 21. By day 90 stromal haze was similar to pre-operative levels in all rabbits.
Conclusions :
We hypothesize that CXL induced changes in ECM stiffness and porosity inhibit interlamellar stromal cell migration, and lead to a disruption of normal keratocyte patterning and increased keratocyte mechanical activation during healing.
This is a 2020 ARVO Annual Meeting abstract.