June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Corneal myofibroblasts and TGFβ inhibit nerve regeneration during wound healing post-PRK
Author Affiliations & Notes
  • Krystel R Huxlin
    University of Rochester, Rochester, New York, United States
  • Kye-Im Jeon
    University of Rochester, Rochester, New York, United States
  • Holly Butler Hindman
    University of Rochester, Rochester, New York, United States
  • Footnotes
    Commercial Relationships   Krystel Huxlin, None; Kye-Im Jeon, None; Holly Hindman, None
  • Footnotes
    Support  NIH grant R01 EY015836, Unrestricted Grant from RPB to the Flaum Eye Institute
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 130. doi:
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    • Get Citation

      Krystel R Huxlin, Kye-Im Jeon, Holly Butler Hindman; Corneal myofibroblasts and TGFβ inhibit nerve regeneration during wound healing post-PRK. Invest. Ophthalmol. Vis. Sci. 2017;58(8):130.

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

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Abstract

Purpose : Corneal injury is often followed by incomplete nerve regeneration, long-term pain and sensory dysfunction. Here, we used in vivo and in vitro approaches to test the hypothesis that corneal myofibroblasts, key players in corneal wound healing, can actively block corneal nerve regeneration after photorefractive keratectomy (PRK).

Methods : We analyzed corneas from adult domestic short hair cats: four unoperated, six 2 weeks post-PRK, and six 4 weeks post-PRK. Corneal sections were stained with antibodies against α-smooth muscle actin (αSMA), β-tubulin (Tuj1) and DAPI. We measured total Tuj1-positive nerve lengths and density in the epithelium, sub-basal layer and stroma. In vitro, we co-cultured ND7/23 cells (a peripheral sensory neuron cell line) with corneal fibroblasts (fibros) or myofibroblasts (myos). We quantified neurite extension, and the relative expression of phosphorylated collapsing response mediating protein (pCRMP)-2 in ND7/23 cells (the latter, using western blots). Finally, SB431542, a transforming growth factor-β (TGFβ) receptor inhibitor, was used to assess dependence of CRMP2 phosphorylation on signalling downstream of TGFβ activation.

Results : Two and 4 weeks after PRK, though the epithelium had fully regenerated and sub-ablation stromal nerve density was 3x greater than normal, αSMA-positive stroma and the sub-basal layer and epithelium above it were completely devoid of Tuj1-positive nerves. In vitro, ND7/23 cells had significantly fewer, shorter neurites when co-cultured with myos versus fibros. When co-cultured with myos, neurons also showed time-dependent induction of pCMRP2, which was mimicked by exposing monocultures of both ND7/23 and trigeminal neurons to TGFβ1. SB431542 blocked the increase in pCRMP2 and simultaneously increased neurite outgrowth.

Conclusions : Even 1 month post-injury, corneal nerve endings do not repopulate αSMA-positive zones or the overlying sub-basal layer and epithelium. This was consistent with the inhibitory effect of myofibroblasts and TGFβ1 on neurite growth in vitro, which was correlated with increased levels of neuronal pCRMP2. Our findings support the hypothesis that myofibroblasts and secreted TGFβ1 can inhibit nerve regeneration in injured corneas long after the epithelium has healed. We are now investigating signals upstream and downstream of pCRMP2 that mediate these inhibitory effects.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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