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Juan M Bueno, Francisco Javier Avila, Dolores Párraga, Elvira Lorenzo, Patricia Gallego-Muñoz, M. Carmen Martínez-García; Analysis of the Corneal Collagen Organization After Chemical Burn Using Second Harmonic Generation Microscopy. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3441. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Healthy corneas present particular distributions of collagen fibers. This organization is modified with pathologies, surgery or external damage. Second harmonic generation (SHG) microscopy is a useful biomedical tool to explore collagen architecture. We propose to investigate the re-organization and natural healing of the cornea after chemical burn using SHG imaging.
A research multiphoton microscope was used to record SHG images of the ex-vivo rabbit corneas. Images were acquired at different depth locations within the corneal stroma and under different experimental conditions. Alkali burn (0,5N NaOH solution for 60 seconds) was performed on the corneal apex of the left eye of 18 New Zealand rabbits. Contra-lateral eyes were used as control. The animals were euthanized by an intracardiac injection of sodic pentobarbital after 1, 3, 5 and 6.5 months of burning. Then, the ocular globes were enucleated and the corneas excised. Before SHG imaging, the specimens were fixed in paraformaldehyde. The structure tensor was used to characterize the collagen fiber organization from SHG images. Quantitative parameters such as the degree of isotropy (DoI) and the preferential orientation (PO) of the collagen fibers (lamellae) were computed.
Control corneas showed a regular lamellar distribution with high DoI (>0.70) and a marked PO. This pattern turned into a non-organized (DoI<0.20) arrangement at 1 month of burning. SHG signal levels noticeably decreased and a PO was not present in the collagen matrix. While healing, the organization of the collagen fibers progressively increased with time, as seen at 3 and 5 months. Moreover, at 6.5 months the DOI reaches values similar to those of control eyes and a dominant direction of the fibers re-appears. Although some remaining edema is still present in some specimens, it did not affect the local organization of the collagen bundles.
SHG microscopy imaging and the structure tensor approach were used to characterize the structural changes and explore collagen healing process in the corneas after chemical burn. Herein results show that this technique is able to track the temporal regeneration of the corneal organization in an objective manner. The implementation of this tool in clinical environments would help to characterize the corneal collagen arrangement under pathologies, external damage or surgical procedures.
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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