Atomic Force Microscopic Observation of Trehalose-treated and Dried Corneal Epithelial Surface

To search for the mechanism of the effect of trehalose on the ocular surface applied as eye drops, we observed trehalose-treated and dried corneal epithelial surfaces of porcine eyes with atomic force microscopy. Full-thickness corneal pieces, 5 × 5 mm in size, were cut out from enucleated porcine eyes. Three kinds of disaccharides, trehalose, maltose, and sucrose, dissolved in saline at 100 mM concentration, and saline as a control, were applied to the epithelial surface of the full-thickness corneal pieces for 1 min. Excess fluid was then absorbed by cotton tips at the epithelial edge of the pieces, and the corneal pieces were dried naturally at room temperature and in room humidity for 72 h. The dried corneal epithelial surfaces were observed first with a dissecting microscope and then fixed on the observation platform for atomic force microscopy. With dissecting microscopy, trehalosetreated corneal epithelial surfaces appeared smoother than saline-, maltose-, or sucrose-treated surfaces. With atomic force microscopy in a tapping mode, saline-, maltose-, and sucrose-treated corneal epithelial surfaces had a larger number of pits in greater sizes than trehalose-treated surfaces. The number of pits measuring 0.14 µm or larger was significantly less in trehalose-treated corneal epithelial surfaces than in saline-treated surfaces (p = 0.0326, analysis of variance [ANOVA], p < 0.05, Tukey-Kramer test). No significant difference was found in the number of pits between maltose- or sucrose-treated surfaces and saline-treated surfaces. Mean roughness, an indicator for surface roughness, was significantly lower in trehalose-treated surfaces than in sucrose-treated surfaces (p = 0.0181, ANOVA, p < 0.05, Tukey-Kramer test). Trehalose-treated corneal epithelial surfaces are smoother than saline-, maltose-, or sucrose-treated surfaces. One effect of trehalose on the ocular surface is to keep the corneal epithelial surface smooth in drying condition.