Abstract
Purpose :
Our goal is to use cell signaling profiles to characterize changes in the wound response in in vitro and ex vivo corneal models in health and disease. We hypothesize that the periodicity of calcium signaling between cells is altered with age and disease, while the presence of clusters of signaling cells near the wound edge enhances the response.
Methods :
Calcium mobilization and wound healing experiments were performed using live cell imaging. Studies were performed on intact eyes from male control (C57Bl6 and BALBC) mice and male NONcNZO10/LtJ diabetic mice and along with those on human primary cultures, human primary diabetic cultures, and human corneal limbal epithelial cultures. Globes and cells were preincubated with Fluo4-AM. To image the globes, they were stabilized using a 3D printed holder. Images of the central corneal epithelium were collected for 1 to 2 hrs after injury. Images were analyzed using MATLAB and ImageJ.
Results :
Differences in calcium signaling events were observed during the wound healing process between healthy and diabetic models. Calcium signaling events were present at a distinct defined periodicity in both tissue and culture models of healthy models. Tissue cell signaling events within apical cell layers were negligible compared to those in basal cell layers. Tissue signaling events were defined by distinct signaling events with an observed periodicity of 20 min with signaling frequency and consistent initiation across five independent runs. Culture models elicited signaling events at a faster frequency and periodicity of every 5 min but with a more variable initiation post-wounding across ten independent runs. Diabetic tissue and cells displayed altered signaling events compared to heathy controls.
Conclusions :
The results indicate that disease states can be identified by diminished calcium cellular communication after injury within corneal epithelium as well as the nature and timing of cellular migration. With cellular activity periodicity observed in both ex vivo and culture, we are developing a computational program to model the activity. Future studies will focus on the changes in would healing response within aged disease states and other conditions where healing is altered.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.