June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Corneal epithelial-neuronal signaling is compromised under environmental stress
Author Affiliations & Notes
  • Vickery Trinkaus-Randall
    Ophthalmology L904, Boston University Sch of Med, Boston, MA
  • Albert Lee
    Ophthalmology L904, Boston University Sch of Med, Boston, MA
  • Kelsey Derricks
    Ophthalmology L904, Boston University Sch of Med, Boston, MA
  • Matthew Nugent
    Ophthalmology L904, Boston University Sch of Med, Boston, MA
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 5977. doi:
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      Vickery Trinkaus-Randall, Albert Lee, Kelsey Derricks, Matthew Nugent; Corneal epithelial-neuronal signaling is compromised under environmental stress. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5977.

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

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Abstract

Purpose: Damage to corneal nerves can originate from trauma including surgery, infection and chemical injury. Previously we demonstrated that injured neuronal and epithelial cells release ATP and induce a rapid response that is detected by imaging Ca2+ mobilization. Our immediate goal was to understand how neuronal-epithelial signaling was altered under environmental stress.

Methods: Cell cultures and organ cultures were used to determine cell communication. To perform the experiments we used a combination of a confocal (Carl Zeiss inverted LSM 700 and 510) and a multiphoton/confocal microscope system (Carl Zeiss upright LSM 710 NLO). Ca2+ mobilization was monitored in cell-cultures and in corneas from Thy1-GFP-rats. To analyze the response to stimuli we developed a custom MATLAB software script to analyze calcium flux movies. The script can individually identify cells, normalize each calcium signal value to the cell’s individual background, and elucidate the magnitude of response, and clusters of coordinated cells.

Results: In intact corneas from Thy1-GFP-rats, the fluorescent sensory neurons were detected passing through the basal lamina and the thin apical processes entered the basal epithelium labeled with a Ca2+ indicator dye. In cultured cells the ATP stimulated response generated by epithelial wound medium generated a rapid wave that was inhibited using Apyrase, a nucleotidase. In contrast media from injured neuronal cells elicited both the rapid wave and a second slower wave that was present in multiple cell clusters. Communication between cells was delineated with custom MATLAB cell clustering analysis. The neuronal wound media was dissected using ionotropic (N-methyl-D-aspartate (NMDA) receptor inhibitors and Apyrase where the latter abrogated the first peak but left the oscillatory glutamatergic response, while NMDA inhibitors diminished only the secondary response. Factors released by neuronal cells facilitated directed epithelial cell migration. When cells were subjected to neuronal media under environmental stress there was a decrease in Ca2+ mobilization and cell clusters. In addition there was a change in localization of an NMDA receptor (NR1).

Conclusions: These results suggest that two separate modes of Ca2+ mobilization mediate cell communication and provide insight into mechanisms that corneal nerves and epithelia signal to each other in response to injury.

Keywords: 765 wound healing • 482 cornea: epithelium • 447 cell-cell communication  
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