Characterization of the lamellar rearrangement induced by cross-linking treatment in keratoconic corneal samples imaged by SHG microscopy

Keratoconus is an eye disorder that features a reduced stiffness of the cornea and its consequent pathological deformation. Cross-Linking (CXL) treatment has proven useful in hindering the progression of keratoconus, offering a minimally-invasive alternative to corneal surgical transplantation. In this study, the biomechanical characteristics of a human keratoconic cornea were clinically examined in vivo soon before keratoplasty, and the morphological alterations of the collagen scaffold in the same cornea were examined ex vivo by means of Second-Harmonic Generation (SHG) microscopy. A healthy cornea and a CXL-treated keratoconus were compared. In particular, the lamellar organization in the three corneal samples was characterized in different stromal layers by detecting both forward-and backwardscattered SHG signal and then considering the forward/backward (F/B) ratio as parameter. The F/B ratio was used to characterize the morphological organization of collagen lamellae within different stromal layers, finding an increased disorder at the level of Bowman's membrane, opposed to a more regular organization within deeper stromal layers in all the examined samples. The organization of collagen lamellae in CXL-treated keratoconic samples was similar to that one found in healthy corneas, demonstrating that the CXL is able to rearrange the collagen scaffold and partially recover the properties of a healthy condition. The obtained results are in agreement with previous results obtained in studies aimed at monitoring the organization of fibrillar collagen using F/B SHG ratio. In conclusion, the proposed method might be useful for both diagnosing keratoconus as well as for monitoring the effects of the CXL treatment.