May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
High Glucose Attenuates Corneal Epithelial Wound Healing: Role of Oxidative Stress and PI3K Signaling
Author Affiliations & Notes
  • K. Xu
    Ophthalmology, Wayne State Univ/Kresge Eye Inst, Detroit, Michigan
  • F.-S. X. Yu
    Ophthalmology, Wayne State Univ/Kresge Eye Inst, Detroit, Michigan
  • Footnotes
    Commercial Relationships  K. Xu, None; F.X. Yu, None.
  • Footnotes
    Support  NIH/NEI R01 EY10869 & 14080, Research to Prevent Blindness
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 5039. doi:
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      K. Xu, F.-S. X. Yu; High Glucose Attenuates Corneal Epithelial Wound Healing: Role of Oxidative Stress and PI3K Signaling. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5039.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : We previously showed that corneal epithelial wound healing was impaired in porcine corneas cultured in high glucose. This study sought to investigate if oxidative stress is one of the contributing factors for delayed epithelial wound healing in high glucose condition.

Methods: : Epithelial debridement wounds in cultured porcine corneas were allowed to heal in the presence of antioxidants of N-acetylcysteine (NAC), propyl gallate (PG) and/or heparin-binding EGF-like growth factor (HB-EGF) in normal glucose (5 mM D-glucose), high glucose (25 mM D-glucose) or mannitol (5 mM D-glucose + 20 mM mannitol) media for 48 hours. Corneal epithelium was scraped off at the end of the culture and analyzed by Western blotting for detection of phosphorylation of ERK and AKT (a major substrate of phosphatidylinositol 3'-kinase, PI3K). Intracellular concentration of reduced glutathione (GSH) was quantified by Glutathione Assay Kit. To quantitate intracellular generation of reactive oxygen species (ROS), primary human corneal epithelial (HCE) cells in normal glucose or in high glucose conditions with or without NAC were treated with cell permeable fluorogenic substrate of 2,7-dichlorofluorescein (DCF), oxidation and generation of fluorescence positive cells were assessed by flow cytometry.

Results: : In corneal organ culture model, epithelial wound healing was significantly delayed in high glucose condition compared with that in normal glucose and high mannitol. While individual antioxidants and growth factors are less effective, the delayed corneal epithelial wound healing was significantly reversed by a combination of NAC and HB-EGF. Moreover, porcine corneal epithelium in high glucose exhibited lower levels of phosphorylated AKT and GSH than that in normal glucose and in high mannitol. No changes in the levels of phospho-ERK. Primary HCE cells in high glucose generated much higher levels of ROS than that in normal glucose and in mannitol. The increase of ROS in high glucose was significantly blocked by NAC.

Conclusions: : Our data suggest that high glucose causes ROS generation and impairs PI3K/AKT pathway and that oxidative stress and the impairment of PI3K/AKT pathway are two key factors contributing to the delayed epithelial wound healing in diabetic corneas.

Keywords: wound healing • diabetes • antioxidants 
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