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Elvira Lorenzo-Martín, Patricia Gallego-Muñoz, Cristina Herrero-Pérez, M. Carmen Martínez-García; Molecular networks during wound healing after corneal alkali burn.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4346. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The cornea is a peculiar tissue to investigate wound healing since it must recover transparency. Although it is well known that the presence of myofibroblasts as well as the new extracellular corneal matrix (ECM) are implicated in the corneal healing process, the molecules that take part in this process and their expression as well as evolution have yet to be fully described.Since these molecules provide basic knowledge vis-à-vis developing new therapies and expanding treatment targets, we performed a multidisciplinary study to evaluate their expression combined with clinical and biological methods.
Left eyes of thirty New Zealand rabbits were burned for 60 seconds with an 8-mm filter paper soaked in 0.5N NaOH solution. Right eyes were used as controls. Corneal opacity was graded following Fantes (scale 0-4). Fifteen corneas were removed after 15 and 30 days of burning, fixed and paraffin-embedded. Corneal sections were stained with different techniques and myofibroblast differentiation was assayed with α-smooth muscle actin (α-SMA) immunofluorescence. The other fifteen corneas were excised in wound and periphery, and immersed in RNA later. mRNA expression levels were determined by real-time polymerase chain reaction using threshold cycle (Ct) relative quantification method (2-ΔΔCt) and represented as log(2-ΔΔCt). Actin beta was used as housekeeper gene and control corneas as calibrator.
α-SMA mRNA expression increased in wound and periphery, and was found to be higher at day 15 than 30. Collagen expression increased in wound area, with collagen-III being higher than collagen-I at 15 days although collagen-III was gradually replaced by collagen-I. Lumican expression increased noticeably at 15 days in both areas and was lower at 30 days. Moreover, keratocan expression increased while the healing process advanced, and was higher at 30 than at 15 days in both areas. These results were tested clinically by corneal opacity reduction following Fantes’ scale. Similarly, histological analysis and anti-α-SMA immunofluorescence revealed the new synthesis of ECM and numerous α-SMA positive-cells in the wound area.
Rabbit alkali burn offers a good model to study molecular variations during corneal wound healing and to assay new treatments based on mRNA expression. Moreover, our study allows clinical, biological and molecular results to be linked.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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