Abstract
Purpose :
The corneal epithelium acts to protect the eye and has a unique wound healing mechanism that allows for rapid wound healing and maintains eye function. Our goal is to examine the cellular calcium signaling activity in diabetic in vitro and ex vivo models. We hypothesize that cellular activity following wounding becomes decreased and altered.
Methods :
We investigated changes in the cellular calcium signaling activity during the wound response of diabetic tissue using live cell imaging in both ex vivo and in vitro models. Experiments were performed on control (BALBC), diabetic (NONcNZO10/LtJ) intact globes and cell culture models (control (Human Corneal Limbal Epithelium), human primary corneal epithelium control and human primary epithelium diabetic, and mouse primary corneal epithelium control and diabetic). To image, cells and globes were preincubated with the calcium stain Fluo-4, AM. Images were collected for up to 2 hours after the induced injury using the Zeiss Axiovert LSM 880 and were analyzed using FIJI/ImageJ, stabilized utilizing Non-Rigid Motion Correction (NoRMCorre) algorithms, and imported into MATLAB.
Results :
The wound response in diabetic cells was significantly decreased and displayed altered patterns of cellular activation and cell-cell communication were compared to non-diabetic controls. Diabetic cell culture models display decreased cellular propagation and an altered spatiotemporal pattern of activity and intensity profile following injury. Analysis of calcium signaling activity in the corneal epithelium of intact globes revealed prominent signaling in wing and basal cells and minimal signaling in apical cells. Signal propagation was detected between basal and wing layers, however its activity was altered and decreased in diabetic models with a significant decrease in wing cell activity.
Conclusions :
These results indicated that a potential source of poor wound healing outcomes in diabetes could be caused by diminished cell-cell communication. Our newly developed analytical tools allow us to detect changes in the pattern of propagation of calcium between cell layers and we speculate that these can be used to determine expected healing outcomes. 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 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.