Abstract
Purpose :
When a cornea is injured, the release of ATP from damaged cells activates the ionotrophic purinergic receptor P2X7, which localizes to the wound edge within two hours of injury. This correlates with the presence of prominent calcium signaling events that have a high probability of communication between clusters of adjacent cells. Our goal is to determine if calcium signaling differs between the corneal epithelium and the corneal-limbal interface, and analyze whether calcium signaling correlates with cellular motility after injury.
Methods :
A 3-D printed holder was used to stabilize the intact globe for imaging cell-cell communication and motility after injury. Globes were incubated in Fluo-4AM and CellMask DeepRed, and imaging performed over time using the Zeiss 880 Confocal Microscope. Images were collected after injury on the corneal epithelium and corneal-limbal regions of male C57Bl6 mice. Image J and MATLAB were used for analysis of clustering between cells, the probability of communication between adjacent cells, and to identify the presence and abundance of conductor cells
Results :
Calcium signaling was detected at both the central corneal epithelium and at the corneal-limbal interface. Mobilization was detected between layers of basal and wing cells in the central cornea and was absent in apical cells. In the corneal-limbal region, we again did not detect communication in the apical cells and detected a strong correlation between sensory nerves and basal cells. Only the basal cells migrated into the wound bed to close the wound. Using a unique machine learning approach we identified a subpopulation of cells that signal at a significantly greater frequency and duration than others. We hypothesize that the presence of this functionally distinct cellular population is decreased with age.
Conclusions :
Injury induces P2X7-driven calcium signaling events in both the corneal and the corneal-limbal regions. The calcium signaling events propagate between different layers of epithelial cells in both central and peripheral corneal regions, and between epithelial cells and nerves in peripheral regions. High-signaling conductor cells were found throughout the cornea after injury, with the greatest number of conductor cells found adjacent to the wound. These results demonstrate that cell-cell communication driven by a functional subpopulation of conductor cells occurs throughout the corneal region.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.