June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
A Long-Lasting Scar in Murine Cornea
Author Affiliations & Notes
  • Andrew Hertsenberg
    Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA
  • Sayan Basu
    Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA
  • Yiqin Du
    Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA
  • James Funderburgh
    Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 5229. doi:
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      Andrew Hertsenberg, Sayan Basu, Yiqin Du, James Funderburgh; A Long-Lasting Scar in Murine Cornea. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5229.

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

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Abstract

Purpose: Penetrating keratoplasty is a successful therapy for corneal scarring but, due do graft failure and declining donor tissue availability, new treatment methods for corneal scarring and haze are under investigation. Animal models of long-term scarring are important tools to assess these new approaches. The purpose of this study is to evaluate a penetrating murine corneal injury model for long-term changes in stromal transparency and stromal connective tissue that replicate those typical of human corneal scarring.

Methods: A wound was created by surgical removal of a 1 mm full thickness button from C57B6 mouse corneas. Corneas were imaged by optical coherence tomography (OCT) 48 hr after wounding and weekly for 2 months. Average pixel intensity for each OCT stack was measured using ImageJ. After 2 months, eyes were enucleated and fixed. Corneal collagen was examined with single harmonic generated (SHG) images by 2-photon microscopy. Corneas were digested in chondroitinase ABC and stained as whole mounts with antibodies to collagen III, biglycan, chondroitin-4-sulfate, and decorin. Birefringence was analyzed by viewing the mounted corneas through a cross polarizing filters.

Results: Two months after wounding, polarized light microscopy revealed a regular, patterned collagen birefringence in unwounded corneas, but a markedly irregular pattern in wounded area. 2-photon images confirmed the loss of collagen organization in the scar. Immunohistochemistry revealed localized concentrations of collagen III and biglycan at the site of the wound. OCT images documented corneal thickening and a distinct increase in light scatter intensity in the wound region persisting at least 2 months after wounding.

Conclusions: Previous studies showed the transient appearance of fibroblasts and myofibroblasts in penetrating murine corneal wounds, however, presence and persistence of non-transparent scar tissue has not been documented. This study demonstrated light scatter, haze, disruption in collagen organization, and accumulation of extracellular matrix components typical of human corneal scars to persist in the central cornea for at least 2 months after wounding. The mouse is an ideal animal model due to its low cost, genetic manipulability, and relative homology to humans. As such, a mouse model that closely resembles corneal scarring would expedite the search for an alternative treatment to corneal haze in humans.

Keywords: 484 cornea: stroma and keratocytes • 765 wound healing • 480 cornea: basic science  
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