May 2005
Volume 46, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2005
VIP Promotes Survival of Cornea Endothelium Under Severe Oxidative Stress ex vivo
Author Affiliations & Notes
  • R.M. Dodson
    Ophthalmology, University of Maryland School of Medicine, Baltimore, MD
  • S.–W.M. Koh
    Ophthalmology, University of Maryland School of Medicine, Baltimore, MD
  • Footnotes
    Commercial Relationships  R.M. Dodson, None; S.M. Koh, None.
  • Footnotes
    Support  NIH RO1EY11607, RPB, Inc
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 4520. doi:
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      R.M. Dodson, S.–W.M. Koh; VIP Promotes Survival of Cornea Endothelium Under Severe Oxidative Stress ex vivo . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4520.

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

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Abstract

Abstract: : Purpose. Previously, we have demonstrated that vasoactive intestinal peptide (VIP), an autocrine of corneal endothelial (CE) cells, which is also detected in the aqueous humor, promotes the survival of CE cells under the acute killing effects of H2O2 ex vivo. Here, we demonstrate that this effect of VIP is long–term and also decreases the inflammatory potential of dying CE cells. Methods: Bovine corneoscleral explants were conditioned in Eagle's MEM/ 20 mM HEPES (1h, 37oC), transferred and incubated in medium containing VIP (0, 10–14–10–6 M, 15 min , 37oC), and then treated with 1.4 mM H2O2/PBS (17 h, 37 o C). CE cells were then treated with one of the following protocols: To reveal viability: cornea cups were treated with reagents in Viability/Cytotoxicity kit (Molecular Probes) to show the red nuclei of the dead CE cells on a carpet of green (live) CE cells in flat–mounted corneas under a fluorescence microscope. To reveal fragmented genomic DNA: CE cells were lysed in cornea cup and DNA was isolated (BioRad kit) for agarose (1.8%) gel electrophoresis, and examined with ethidium bromide stain under UV light. For CE cell attachment assay: Cornea cups were fixed in formalin/PBS and stained with Toluidine Blue, followed by lysis of CE cells in SDS and measurement of their absorbance at 600 nm in a spectrophotometer. To reveal apoptotic and necrotic CE cells: CE cells were detached from the corneas by collagenase/BSA/EDTA and labeled with annexin–V–fluorescein and propidium iodide (Molecular Probes), respectively, and examined under a fluorescence microscope. Results: VIP–promoted increase in CE cell viability persisted 17 h after the VIP/ H2O2 treatment. The number of CE cells remaining attached to the corneas increased in a VIP concentration–dependent manner, as demonstrated by DNA quantification and cell attachment assay. Numerous CE cells in control corneas displayed necrotic cell death as the predominant mode of death. VIP–treated corneas demonstrated a dramatic increase in viable attached CE cells and in these corneas apoptosis, as demonstrated by DNA laddering, was observed in both detached and viable attached CE cells. Apoptotic and necrotic CE cells labeled with annexin–V–fluorescein and propidium iodide, respectively, confirmed that necrotic CE cell death was the prominent mode of cell death in control corneas, whereas apoptosis was prominent in VIP treated corneas. Conclusions: Necrotic cell death causes damage to the neighbors of dying cells, due to leakage of cellular contents, and is associated with initiation of an inflammatory response. Thus, VIP protects CE cells from dying and attenuates the inflammatory potential of dying CE cells.

Keywords: cell death/apoptosis • cornea: endothelium • neuroprotection 
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