Abstract
Purpose :
The P2X7 purinergic receptor is localized in the corneal epithelium with a distinct apico-basal polarity. Upon injury, P2X7 expression becomes diffuse at the wound margin and decreases away from the wound. Inhibitors of the receptor not only attenuate migration but alter the cytoskeletal rearrangements that are part of wound healing. DIO mice (pre-diabetic model for Type II diabetes) exhibit altered levels of the receptor. Our goal was to examine the corneal nerves in the DIO and control mice and ask if alterations are associated with compromised cell migration and wound repair.
Methods :
Experiments were performed using in vivo, ex vivo organ culture and in vitro models. DIO and WT mice were sacrificed at 15 and 7.5 weeks after initiation of respective diets. Corneas were removed and either fixed or maintained in organ culture after epithelial debridement to monitor wound closure. A human corneal epithelial cell line was used to examine cytoskeletal rearrangements after injury.
Results :
Corneas were stained for Tuj1 and P2X7. Nine tiles of the corneas were imaged separately from the apical surface through the stromal nerves and stitched into a composite image. Compression of z-stacks revealed a striking lack of nerves in the DIO mice. In the control epithelium the nerves were concentrated in the basal region and only 20% of the nerves were detected in the most apical region. In the DIO corneas there was a 75% reduction in basal nerves and a 30% reduction in apical nerves. The decrease may explain the lack of the prototypical whorl detected in control. Analysis of the central stroma revealed a 2.3 fold greater number of Tuj1 positive nerves in the DIO that exhibited different morphologies. P2X7 was detected along the corneal nerves. While P2X7 mRNA is elevated there was no detectable difference in localization of P2X7 in the unwounded epithelium. After injury in the DIO corneas the change in P2X7 localization detected previously was absent. Additional experiments demonstrated that inhibition of P2X7 decreased turnover of focal adhesion clusters and altered organization of the cytoskeleton at the leading edge.
Conclusions :
The high fat, high calorie diet of DIO mice results in a lack of corneal sensory nerves and a lack of change in localization of P2X7 at the wound margin, indicating the importance of P2X7 in regulating proper early signaling events after corneal epithelial injury.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.