Purchase this article with an account.
Vickery E Trinkaus-Randall, Krisandra Kneer, Celeste Rich; A Pre-Type II diabetic mouse model confers a change in P2X7 protein trafficking that occurs with corneal injury. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1391.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The P2X7 purinergic receptor is an ion channel expressed by epithelium, intra-epithelial and stromal nerves of the cornea. In injury P2X7 expression becomes concentrated at the wound margin, and decreases away from the wound suggesting that P2X7 localization is important in corneal wound healing. We hypothesized that in pathologic corneal wound healing such as that occurs in diabetes there is a change in P2X7 localization and protein trafficking that alters the ability to properly sense the environment.
C57Bl6 mice were fed a high-fat diet to induce a pre-Type 2 diabetes model (DiO). Epithelial debridement wounds were performed on DiO mice corneas after 8, 15 and 22 weeks of enhanced diet, and compared to WT age matched controls. Corneas were examined until wound closure was achieved. Real-time PCR and immunohistochemical analyses for P2X7, GM130 (a cis-Golgi marker), and beta3-tubulin were performed and imaged using Zeiss 700 and 880 LSM with AIRYScan confocal microscopy. Brefeldin A treatment was used to verify cis-Golgi localization of P2X7.
Corneas were examined at 2, 6 and 12 hours after injury. Corneas from 15 and 22 week DiO mice exhibited a 2-fold increase in the number of cells distal from the wound edge that expressed P2X7 compared to the 8 week DiO and WT mice. There was also a significant loss in P2X7 positive intraepithelial nerves. High resolution images resolved the localization of P2X7 to punctate staining around the nuclei. After 15 or more weeks, the corneas of DiO mice had significantly greater amounts of punctae P2X7 positive staining. P2X7 was localized to the cis-Golgi. After 15 weeks, there was significantly greater cis-Golgi punctae and P2X7 at the wound margin that extended back from the leading edge suggesting that protein trafficking is altered in the DiO mice corneas and that there may be a lack of secretion. Additional experiments verified localization.
Changes in protein localization and potential trafficking of P2X7 in DiO corneas may reveal a major change in wound healing that cannot be detected in large scale assays. The enhanced expression and localization of P2X7 at the wound margin in DiO corneas suggest a potential for corneal epithelial cells to sense dietary changes that may attribute to improper secretion and trafficking of P2X7 to the membrane to promote healthy wound healing.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
This PDF is available to Subscribers Only