Abstract
Purpose :
Oxidative stress (OS) plays an important role in dry eye disease (DED) and other corneal injuries, including irradiation, chemical burns, and exposure to vesicating agents. Current methods utilize monolayer cell cultures or animals that result in poor data extrapolation, low throughput and high cost. Physiologically relevant, human-based in vitro models for ocular research are needed.
Methods :
This study evaluated the utility of an in vitro reconstructed 3D tissue model (EpiCorneal) to study molecular mechanisms of OS corneal damage. The 3D tissues are comprised of normal human corneal epithelial cells that are cultured at the air-liquid interface. They develop a tight barrier (TEER 1000±250 Ω*cm2) and express tight junctions, mucins, and key corneal detoxification enzymes similar to in vivo human cornea. OS was generated by exposure to DED conditions (elevated temperatures and low humidity for 24-72h), irradiation (UVB, 60/120mJ), hydrogen peroxide (HP, 20/50mM), and nitrogen mustard (NM, 7.8mM for 10/30min). Barrier function, tissue viability, reactive oxygen species (ROS) accumulation, lipid oxidation, cytokine release, histology, and gene expression were evaluated 2/24h post-exposure.
Results :
The most pronounced effects were as follows. DED: tissue thinning, barrier impairment, decline in mucin expression, and increased lipid oxidation. Topical application of lubricant eye drops improved tissue morphology, barrier, and lipid oxidation. UVB: intracellular ROS accumulation. HP: barrier impairment and decreased tissue viability, morphology deterioration 24h post-incubation of 50mM-treated tissues. NM: barrier impairment, decreased tissue viability, lipid oxidation, IL-8 release, and tissue morphology deterioration 24h post-incubation. Utilizing a PCR gene array, we compared effects of corneal damage on the expression of 84 OS responsive genes (antioxidants, ROS and superoxide metabolism, oxygen transport and others), and found specific molecular footprints for each mechanism of OS corneal damage.
Conclusions :
In summary, the in vitro 3D corneal tissues structurally and functionally reproduced key features of in vivo cornea and unique toxicity and molecular responses for various types of OS ocular damage. This model is anticipated to be a useful tool to study molecular mechanisms of DED and corneal damage and to evaluate new corneal drug formulations.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.