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Carmen Martinez-Garcia, Francisco J. Avila, Lucía Ibares-Frías, Raquel Palacios, Patricia Gallego-Muñoz, Roberto Cantalapiedra, Juan M Bueno, Tecnicas opticas de diagnostico; Cross-linking Wound Healing In Animal Models: A Complementary Study Using Two-photon Microscopy And Histological Analysis. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1629. doi: https://doi.org/.
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<br /> To analyze and compare the wound healing after cross-linking (CXL) treatment in two animal models (hens and rabbits) using second harmonic generation (SHG) microscopy imaging and histological analysis.
Monolateral CXL treatment was performed on 10 rabbits and 10 hens. The central 7-mm of the corneal epithelium was removed and 0.125% riboflavin+20% dextran solution was instilled during 30 minutes, then every 5 min during of UVA radiation. The eye was illuminated with UV-light (370 nm, 3 w/cm2) during 30 minutes. Animals were euthanized after 30 days of CXL treatment, the eyes enucleated and the corneas excised. The unstained ex-vivo corneas were imaged using a custom two-photon microscope (Bueno et al., 2010), which allows optical sectioning. Later, a histological analysis was done under bright-field microscopy after hematoxylin-eosin staining. Treated and contralateral corneas were compared using both techniques
SHG images showed sets of collagen bundles running parallel to each other with some orthogonal interweaving. One month after the CXL treatment, central and posterior stromal organization seemed not to be affected by the treatment. However, for the anterior stroma the effects of CXL differed between hens and rabbits. In the former, the collagen distribution recovered its usual arrangement and did not differ from control corneas. In rabbits, this part of the stroma presented changes that remain one month after CXL treatment. Collagen bundles lost their regular orientation and appeared less delineated and less interwoven after CXL compared to the control samples. Histology from hens showed corneas depleted of cells in the anterior stroma and light hipercellularity in the medium stroma. In rabbits, treated corneas were similar to control.
The wound healing after CXL was different in both animal models. SHG imaging and histological analyses provided complementary information on corneal structures: collagen organization and cell distribution respectively. The former revealed that the regular stromal arrangement was recovered faster in hens than in rabbits. The later showed that the cellular repopulation was slower in chickens than in rabbits.
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