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Christian Hammer, Johannes Menzel-Severing, Corinna Petsch, Naresh Polisetti, Bjoern Bachmann, Jörg Klenke, Katrin Skerl, Christian Wüllner, Christof Donitzky, Friedrich Kruse; Wound healing in rabbit corneas after flapless refractive lenticule extraction with a novel 345nm ultraviolet femtosecond laser. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3889.
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© ARVO (1962-2015); The Authors (2016-present)
To preclinically characterize corneal wound healing in New Zealand White (NZW) rabbits after flapless refractive lenticule extraction (FLE) with a novel 345nm ultraviolet femtosecond laser (UV-FSL) developed by Alcon-WaveLight.
A total of 20 NZW rabbits was included in this study. One eye received FLE while the contralateral eye served as untreated control. The lenticules (-5dpt) had a diameter of 5.5mm and were cut with 80nJ and a spot separation of 4µmx4µm and were removed via two extraction canals. Four animals were sacrificed after 48 hours, 1 week, 2 weeks, 4 weeks and 3 months, respectively. Lenticular beds and extraction canals were prepared for standard histology and fluorescence microscopy. To investigate cell death rate, proliferation, myofibroblastic transdifferentiation of keratocytes and inflammation, TUNEL assay (Roche) as well as immunostaining for Ki67, αSMA and CD11b was performed, respectively, on sagittal sections of kryoembedded specimens and subjected to qualitative and quantitative analysis.
Standard histological analysis revealed a zone of keratocyte depletion of approximately 50µm thickness at the extraction site. Epithelial and endothelial cells appeared unharmed. At the sites of epithelial incision, a callus of epithelial cells formed. At 48h, TUNEL-assay analysis showed pronounced staining of keratocytes near the extraction site (159.9±18.4cells/mm), which steadily decreased to 74.9±19.8cells/mm at 1 week and 5.7±4.8 cells/mm at 2 weeks. At 4 weeks, no TUNEL-positive keratocytes were detected anymore. Marked Ki67 staining of keratocytes was evident at 48h (10.0±3.8 cells/mm), which had decreased conspicuously at one week (5.2±1.7 cells/mm) and 2 weeks (0.4±0.5cells/mm). At 4 weeks, no Ki67 staining was found anymore. The corneal stroma was free of αSMA- and CD11b-positive cells at all times. Only in close proximity to the epithelial callus, some isolated keratocytes were αSMA-positive and very few CD11b-positive cells were observed.
Preclinical in-vivo assessment of corneal wound healing after application of the novel 345nm UV-FSL showed promising results after FLE in NZW rabbits. Together with the benign development of cell death rate and keratocyte proliferation, the absence of inflammation and transdifferentiated keratocytes advocates an initiation of the clinical phase.
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