Using immunohistochemistry to investigate surface protein localization in corneal epithelium injury response
OA Version
Citation
Abstract
The cornea acts to both refract light entering the eye and as a protective outer coating for the underlying globe. The wound healing response of the corneal epithelium is not entirely well understood, and inefficient or incomplete healing is a common cause of visual impairment. Wound healing can be mitigated by several factors including age and wound type. Motility of corneal epithelial cells in response to a wound is known to be coordinated by calcium signaling events, which are initiated by the binding of ATP released by damaged cells to P2X7, a purinergic ion channel. This thesis attempts to examine the role of P2X7 expression in the face of different wound modalities at different time points in the wound response. Intact globes from aged mice were wounded, allowed to heal for a set period of time, and then fixed and frozen in preparation for cryosectioning. The obtained tissue sections were then stained for P2X7 via immunohistochemistry and imaged via fluorescent confocal microscopy. Qualitative and quantitative analysis of the resultant expression data showed that the endothelial cells in the central region of wounded corneas 5 minutes after wounding lacked P2X7 expression, regardless of wound type. This suggests that there is apical-to-basal transmission of signaling, as the wounds inflicted did not penetrate the basement membrane underlying the epithelium, yet the endothelial cells below that membrane exhibit changes in protein expression. Stratified data also showed a statistically significant difference in P2X7 expression in the limbal region of scratch wounded and alkali burned globes with 120 minutes heal time, although the true significance of this difference is limited by small sample size. These results suggest that these experiments should be repeated to gain more statistical confidence in the differences found and should also be conducted in younger eyes to allow for comparison across age groups.
Description
2024
License
Attribution 4.0 International