June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Basement Membrane Removal Reduces Inflammation in the Mouse Cornea and Enhances Wound Healing
Author Affiliations & Notes
  • Sonali Pal-Ghosh
    Anatomy & Regenerative Biology, George Washington University Medical Center, Washington, DC
  • Ahdeah Pajoohesh-Ganji
    Anatomy & Regenerative Biology, George Washington University Medical Center, Washington, DC
  • Gauri Tadvalkar
    Anatomy & Regenerative Biology, George Washington University Medical Center, Washington, DC
  • Daniel Saban
    Ophthalmology and Immunology, Duke University School of Medicine, Durham, NC
  • Mary Ann Stepp
    Anatomy & Regenerative Biology, George Washington University Medical Center, Washington, DC
  • Footnotes
    Commercial Relationships Sonali Pal-Ghosh, None; Ahdeah Pajoohesh-Ganji, None; Gauri Tadvalkar, None; Daniel Saban, Schepens Eye Res Inst, Mass Eye and Ear, (P), Eleven Biotherapuetics (R); Mary Ann Stepp, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 550. doi:
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      Sonali Pal-Ghosh, Ahdeah Pajoohesh-Ganji, Gauri Tadvalkar, Daniel Saban, Mary Ann Stepp; Basement Membrane Removal Reduces Inflammation in the Mouse Cornea and Enhances Wound Healing. Invest. Ophthalmol. Vis. Sci. 2013;54(15):550.

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

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Abstract

Purpose: An in vivo mouse model has been developed that reproducibly induces recurrent epithelial erosions in wild-type mice spontaneously within two weeks after a single 1.5 mm corneal debridement wound using a dulled blade. Using a rotating burr rather than a dulled blade to create a 1.5 mm wound allows mice to heal without developing erosions. These experiments were conducted to determine the cause of the difference in healing outcomes after dulled blade compared to rotating burr wounds.

Methods: A 1.5 mm area of corneal epithelium was removed from the corneal surface of adult C57BL/6 mice using either a dulled blade or rotating burr. Corneas were allowed to heal in vivo for 0, 3, 6 hr or 5 days. After sacrifice, tissues were used for immunofluorescence, QPCR, flow cytometry, and/or chemokine protein array studies. Flow experiments were repeated three times. QPCR was performed on control (n=8) and 6 hr dulled blade or rotating burr wounded (n=8) corneas. For QPCR studies, RNA isolated from epithelial cells only and whole dissected corneas were compared. All experiments were repeated at least twice.

Results: Data show that 1) erosions form after dulled blade but not after rotating burr wounds, 2) the basement membrane (BMZ) is left behind after dulled blade wounds but is removed by the rotating burr, 3) there are more monocytes (CD45+/ Ly6C hi/ Ly6G+/ CD11b hi/ F480 low/ CD11c+) and γδ T cells (CD45+/GL3 hi) recruited 6 hr after dulled blade wounds, and 4) chemokine array studies show that, at the time of wounding and 5 days later, chemokines are elevated after dulled blade compared to rotating burr wounds.

Conclusions: Despite the fact that rotating burr wounds remove the BMZ, damage more of the subbasal nerves, and induce more cytokine mRNAs within corneal epithelial cells, they heal without developing erosions. Improved healing after trauma is associated with basement membrane removal and the reduced influx of monocytes and γδT cells 6 hr after wounding. Data from chemokine arrays indicate more CCL8 and CCL12 and complement proteins (5a and Factor D) retained on the corneal stroma after dulled blade wounds that may account for the early increase in monocyte and γδT cells seen after dulled blade wounds.

Keywords: 482 cornea: epithelium • 765 wound healing • 529 flow cytometry  
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