Abstract
Purpose :
Corneal opacity caused by fibrotic scar tissue accumulates in response to corneal inflammatory response and remains long-term in the stroma, often correctable only by surgery. Numerous recent studies have investigated innovative approaches to reversal of corneal scarring using non-surgical means, however, existing mouse models limit these studies due to the optical properties the mouse cornea which exhibits little opacity in response to fibrotic matrix accumulation. The current study investigates the hypothesis that corneal scaring can be modeled using a transgenic mouse in which enhanced green fluorescent protein (EGFP) is expressed by a collagen 3 promoter.
Methods :
The transgenic line Tg(Col3a1-EGFP) DJ124Gsat (Col3-EGFP) was obtained from MGI via Mouse Biology Program at UC Davis. The animals were bred and used as heterozygotes, used experimentally at 6-8 weeks. Debridement and alkali wounding of corneas were carried out using published protocols under ARVO guidelines and approved IACUC protocols. Corneal fluorescence in vivo was examined using Olympus dissecting microscope. Ex vivo, corneas were examined using standard fluorescence microscopy, histology, microplate fluorimeter, ELISA for EGFP and by qPCR. Image analysis used Nikon NIS Elements software. Statistical significance was determined by t-test and ANOVA.
Results :
Col3-EGFP mice are healthy, vigorous, and breed with large litters showing no morphological or behavioral abnormalities. After physical or chemical wounding, corneal opacity was observed at 3 days, but corneal EGFP expression was not observed until 7 days, peaking at 10-14 days and still detected at 28 days. EGFP could be readily detected by in vivo imaging. Ex vivo, EGFP was observed in stromal cells peripheral to the wound and could be quantified by counting individual cells, or by ELISA for GFP in corneal extracts. qPCR showed EGFP mRNA to be upregulated synchronously with mRNA for Col3a1 and smooth muscle actin (Acta2).
Conclusions :
Mouse corneal opacity in response to wounding does not provide an accurate measure of stromal fibrosis, a prime component of long-lasting human corneal scars. The Col3-EGFP mouse provides a new tool to detect corneal scarring in real-time and will be useful in modeling the ability of stem cells and other innovative treatments in preventing and reversing corneal blindness.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.