September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Molecular Mechanism of Ocular Surface Damage: Applications to Dry Eye and Wound Healing Models on In Vitro Reconstructed Human Corneal Tissues
Author Affiliations & Notes
  • Yulia Kaluzhny
    MatTek Corporation, Ashland, Massachusetts, United States
  • Miriam W Kinuthia
    MatTek Corporation, Ashland, Massachusetts, United States
  • Alena Plotkin
    MatTek Corporation, Ashland, Massachusetts, United States
  • Patrick Hayden
    MatTek Corporation, Ashland, Massachusetts, United States
  • Mitchell Klausner
    MatTek Corporation, Ashland, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Yulia Kaluzhny, MatTek (E); Miriam Kinuthia, MatTek (E); Alena Plotkin, MatTek (E); Patrick Hayden, MatTek (E); Mitchell Klausner, MatTek (E)
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 4380. doi:
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      Yulia Kaluzhny, Miriam W Kinuthia, Alena Plotkin, Patrick Hayden, Mitchell Klausner; Molecular Mechanism of Ocular Surface Damage: Applications to Dry Eye and Wound Healing Models on In Vitro Reconstructed Human Corneal Tissues. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4380.

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

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Abstract

Purpose : Current methods used to investigate mechanisms of corneal wound healing (CWH) and pathogenesis of dry eye disease (DED) utilize monolayer cell cultures or animals. However these methods present numerous disadvantages and there is a need for more physiologically relevant, human-based in vitro models for CWH and DED research.

Methods : This study utilized EpiCorneal tissue model comprised of normal human corneal epithelial cells that are cultured at the air-liquid interface. Corneal wounds were introduced by abrasion or chemicals (1N NaOH). Wounded tissues were cultured in the presence or absence of human corneal keratocytes (HCK) or EGFR inhibitor (erlotinib, 10 μM). A DED model was generated by placing EpiCorneal tissues under desiccating stress conditions (40% RH, 40°C, and 5% CO2) that stimulate morphological, cellular, and molecular changes relevant to dry eye.

Results : CWH was analyzed by transepithelial electrical resistance (TEER), histology, confocal microscopy, and gene expression. TEER recovered to 933.7/502.4 Ω*cm2 in the presence/or absence of HCK in 4 days post-wounded cultures. mRNA expression was analyzed using a 96-gene wound healing microarray. 13 genes (including collagen, integrin, chemokine, and protein kinase families) were up-regulated in the EpiCorneal tissues 24h post-abrasion in the absence of HCK and 16 genes (including WNT, FGF, small GTPases, chemokine, and integrin families) were up-regulated in the presence of HCK, but not in control cultures. DED was analyzed by TEER, histology, tissue viability, mucins and tight junction (TJ) protein expression. Dramatic reduction in tissue thickness was observed after 48h in DSC that coincided with decreased expression of mucins, increased TEER and atypical expression of TJ proteins. Topical application (25 μl/tissue) of lubricant gel drops (GenTeal, Alcon) improved tissue morphology and barrier function.

Conclusions : The results demonstrate that the in vitro organotypic human corneal tissue structurally and functionally reproduces CWH and DED. The model will avoid species extrapolation, be more cost effective and more reproducible than animal methods, and will facilitate drug discovery by allowing screening and optimization of active pharmaceuticals prior to clinical studies.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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