July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Transient Receptor Potential (TRP)-receptors protect the healthy murine cornea against colonization by Pseudomonas aeruginosa and environmental bacteria
Author Affiliations & Notes
  • Stephanie Wan
    Vision Science, UC Berkeley, Berkeley, California, United States
  • Sophia Ma
    Optometry, UC Berkeley, California, United States
  • Matteo Metruccio
    Optometry, UC Berkeley, California, United States
  • Diana Bautista
    Molecular and Cell Biology, UC Berkeley, California, United States
  • David J Evans
    College of Pharmacy, Touro University, California, United States
    Optometry, UC Berkeley, California, United States
  • Suzanne M J Fleiszig
    Optometry, UC Berkeley, California, United States
  • Footnotes
    Commercial Relationships   Stephanie Wan, None; Sophia Ma, None; Matteo Metruccio, None; Diana Bautista, None; David Evans, None; Suzanne Fleiszig, None
  • Footnotes
    Support  NIH Grant EY011221
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 5184. doi:
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      Stephanie Wan, Sophia Ma, Matteo Metruccio, Diana Bautista, David J Evans, Suzanne M J Fleiszig; Transient Receptor Potential (TRP)-receptors protect the healthy murine cornea against colonization by Pseudomonas aeruginosa and environmental bacteria. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5184.

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

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Purpose : The healthy murine cornea contrasts with the conjunctiva by lacking a viable bacterial microbiome. It also rapidly clears inoculated pathogens, e.g. P. aeruginosa or S. aureus. Here we tested the hypothesis that TRP (Transient Receptor Potential) ion channels, located on corneal sensory nerve endings and some corneal cells, play a role in maintaining the “amicrobiomic” state of the mouse cornea.

Methods : C57BL/6 mice or gene-knockout mice in TRPA1 and TRPV1 (TRPA1V1dKO) were anesthetized. Corneas were inoculated with ~5 x 108 CFU P. aeruginosa PAO1 every hour. Some corneas were inoculated without manipulation, others superficially-injured by Kimwipe™ blotting. After 4 h, mice were sacrificed, and eyes fixed and imaged by confocal microscopy. FISH using a universal bacterial 16S rRNA gene probe [Alexa488]-GCTGCCTCCCGTAGGAGT-[Alexa488] (Eurofins Genomics) was used to detect viable environmental bacteria. Immunohistochemistry was used to detect corneal CD45+ cells. ImageJ was used for image quantification, and Student’s t-Test for statistical analysis.

Results : Inoculated P. aeruginosa colonized TRPA1V1 dKO corneas more efficiently than wild-type (28.4 ± 5.9 versus 13.0 ± 3.5 bacteria/field, respectively, P < 0.05). A larger difference occurred after superficial-injury (103.1 ± 25.2 bacteria/field for TRPA1V1 dKO mice versus 33.8 ± 6.5 bacteria/field for wild-type, P < 0.05). Without prior inoculation, FISH showed significantly more environmental bacteria colonizing TRPA1V1 dKO corneas versus wild-type (8.0 ± 2.0 versus 1.9 ± 0.6 bacteria/field, P < 0.05). A small, but statistically significant reduction in CD45+ cells was observed in TRPA1V1 dKO corneas versus wild-type after P. aeruginosa challenge (40.8 ± 3.8 versus 57.2 ± 4.2 CD45+ cells/field, respectively, P < 0.05).

Conclusions : TRPA1 and/or TRPV1 nociceptors contribute to maintaining the absence of a viable bacterial microbiome on the mouse cornea during health and after superficial-injury. Whether the reduction in CD45+ cell numbers contributes to bacterial colonization of TRPA1V1 dKO corneas, or represents a compromised response to those microbes, remains to be determined. Establishing relative roles of TRPV1 versus TRPA1, and location of required receptors (sensory nerves and/or ocular surface cells) also requires further investigation.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.


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