An Ex Vivo Model to Study Early Changes in Keratoconus

Abstract

Purpose: To develop an ex vivo model for keratoconus that simulates eye rubbing, a hallmark characteristic of patients with this disease.

Methods: Uniaxial stretch (3% strain, 6 h) was applied to human stromal caps placed in stretch chambers, followed by hematoxylin and eosin staining and evaluation of Bowman layer (BL) breaks in stretched corneas compared with unstretched controls. The impact of mechanical strain on corneal epithelium was also assessed by applying similar cyclical strain to mouse eyes, and changes in basal epithelium morphology were examined using F-actin immunostaining. In addition, Wnt10a and Col12a1 mRNA levels were analyzed in full-thickness epithelium.

Results: Human corneas subjected to mechanical strain showed a significantly higher number of BL breaks compared with unstretched controls (2.0 ± 1 vs. 0.4 ± 0.53, P = 0.003). In mouse corneas, mechanical strain increased the basal epithelial cell width by 15% (7.16 ± 1.67 vs. 8.25 ± 1.82 μm, P < 0.001), while reducing the apical cell height by 8% (7.88 ± 1.93 vs. 7.30 ± 1.72, P = 0.025). Wnt10a and Col12a1 mRNA levels were reduced in stretched mouse epithelium compared with unstretched controls (67%, P = 0.0312; 65%, P = 0.0312, respectively).

Conclusions: Eye rubbing is a well-known risk factor for the development and progression of keratoconus. By simulating eye rubbing using our ex vivo cyclical mechanical strain model reproduces changes that occur in early keratoconus. Specifically, this model recapitulates at least 3 elements of the keratoconus phenotype (BL breaks, enlargement of the basal epithelium, and reduction in transcript levels of Wnt10a and Col12a1 ), which makes it a valuable tool for the development of targeted drug therapies for progressive keratoconus.

Keywords: corneal biomechanics; corneal epithelium; ex vivo model; keratoconus.