Fig. 5.

Softening of central corneal tissue with collagenase increases expression of limbal markers in vivo. a Schematic representation of the collagenase treatment method used to soften the central region of intact corneas in live rabbits. Clinical observation and slit-lamp examination was performed 1 and 5 days post-intervention, and compared to pre-intervention results (baseline). The central corneal epithelium was also analysed by confocal immunofluorescence (3D reconstruction) 5 days after collagenase treatment (softened), and the corresponding marker expression was quantified. Cells on softened corneas expressed (b) higher levels of ABCG2, CK15, ΔNp63, integrin-α9, and laminin-γ3 (limbal markers) and lower levels of CK3+12 and integrin-α3β1 (differentiation markers) compared to cells growing on the stiffer, untreated corneas (control). Collagen-IV and collagen-VII were also detected in collagenase-treated (softened) corneas, albeit at lower levels compared to control. c The expression of the mechanotransduction marker YAP was also significantly lower in the CK15-positive cells on softened corneas, where it mostly presented a non-nuclear (inactive) form compared to that in CK3-positive cells on untreated corneas (control). Cell nuclei were detected using DAPI. Scale bars, 50 µm. Marker expression was represented as average ± S.D. from three independent experiments (n = 3; *, **, and *** corresponds to p < 0.05, 0.01, and 0.001 after one-way ANOVA, respectively). Source data are provided as a Source Data file