Abstract
Purpose :
Injury to the corneal epithelium releases ATP to the local environment, initiating calcium signaling events, which are believed to play a role in coordinating cell motility into the wound bed. Previous work has identified clusters of cells that exhibit increased calcium signaling and that inhibition of P2X7, an ATP-activated calcium channel, limits motility. Furthermore in murine eyes from older mice there is decreased calcium signaling and motility in response to a wound. Our goal is to utilize live cell imaging to correlate levels of calcium signaling with motility into the wound bed as well as to use immunohistochemistry to compare localization of P2X7 in younger and older eyes.
Methods :
To examine cell motility, cultured Human Corneal Limbal Epithelial (HCLE) cells were stained with SiR-Actin and vinculin probes and scratch wounded with a 25G needle. Live cell imaging was performed using a Zeiss LSM 880 confocal microscope. To examine P2X7 localization 87 week-old ex vivo murine globes were injured with a 25G needle and allowed to heal at 37°C and 5% CO2 for 0, 5, or 120 minutes. Globes were cryosectioned and stained with an antibody targeted to the extracellular domain of P2X7. Slides were counterstained with DAPI and rhodamine-phalloidin and Z-stack tilescans of the entire cornea were imaged using either the Zeiss LSM 700 or 880 confocal microscopes.
Results :
The immunohistochemistry experiments show localization of P2X7 expression along membranes of basal cells, with overall decreased expression when compared to younger eyes. The live cell imaging experiments identified clusters of cells that exhibit higher levels of calcium signaling than the surrounding cells with lower P2X7. In addition, the surrounding cells exhibit the highest levels of motility into the wound bed.
Conclusions :
The live cell imaging experiments support the hypothesis that clusters of high signaling cells act to induce and coordinate motility of their surrounding cells into the wound bed, yet do not exhibit high levels of motility themselves. The immunohistochemistry experiments support the hypothesis that decreased levels of calcium signaling in wing cells in older eyes is correlated with decreased expression of P2X7, whose inhibition is known to attenuate cell motility. In the future, organ culture experiments will be performed to examine the extent of wound healing.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.