Abstract
Purpose :
To determine the localization and presence of pannexin1 in diabetic unwounded and wounded models and correlate with mechanical properties of the cornea. Previously, investigators proposed that pannexin1 plays a role in cytoskeletal integrity along with passage of ATP through channels and subsequent activation of purinoreceptors. Our ultimate goal is to determine if the presence and localization of ion channels correlates with corneal integrity, calcium mobilization and cell migration.
Methods :
Epithelial debridement wounds were performed on corneas from C57BL/6J wildtype and the polygenic Type 2 diabetic mouse (NONcNZO10/LtJ; NO). Immunohistochemical analyses were performed and tile images were captured using confocal microscopy. Corneas were incubated in the presence or absence of inhibitors to examine changes at the leading edge. Stiffness of epithelium and stroma were measured using nanoindentation.
Results :
Pannexin 1 was localized along apical epithelial cell borders in wildtype and NO unwounded corneal epithelium. The epithelium was intact.Two hours after injury to WT corneas, pannexin1 became intense but was diffuse immediately at the edge. After 20 h, the intensity decreased by half with pannexin1 present evenly throughout. In male and female NO epithelium, the intensity of Pannexin1 was 75% less at 2h and negligible 20 h after injury. There was no detectable difference between males and females. To examine the role of pannexin, control corneas were incubated in the presence of probenocid or 10panx or control peptide and stained for F-actin and cells at the wound edge did not display typical lamellipodial extensions and wound repair was impaired. There was less fibronectin along the stromal nerves and the wound edge. In addition, the diabetic models displayed significantly softer epithelium and the slope of increased stiffness from anterior to posterior stroma was more gradual.
Conclusions :
The localization and polarity of the ion channel pannexin1 is lost in diabetic corneal epithelium compared to wildtype suggesting that the channel function is altered with less subsequent activation of P2X7. We hypothesize that cell-cell communication will be diminished in the NO mice as shown using calcium mobilization and smaller actin patches at the wound edge will ultimately provide less strength for migration.
This is a 2020 ARVO Annual Meeting abstract.