Corneal collagen cross linkingdevelopment of new in vivo methods for the mechanical characterization and assessment as treatment of acanthamoeba keratitis

Abstract

Corneal collagen crosslinking is based on the photopolymerization of the corneal collagen induced by the combined action of the riboflavin and ultraviolet-A light of 370 nm. Although it is an evolving technique, among their multiple effects, the knowledge of new aspects related to the increase of the corneal stiffness and the antimicrobial spectrum provided by this therapy, has been considered as the main motivation of this Doctoral Thesis. Based on this fact, two main goals have been proposed: the development of new methods to characterize the mechanical properties of the cornea in vivo, and the assessment of new effective topical therapies, in combination with corneal collagen crosslinking, as treatment protocol of Acanthamoeba keratitis. This Thesis is organized in 4 chapters including an introduction about the objectives, different biomechanical and clinical aspects, as well as the main conclusions and original contributions derived from this period. Regarding the mechanical characterization of the corneal tissue, three methods were used: uniaxial, inflation and indentation tests. In a first step, a new alternative during the ex vivo uniaxial tensile tests was established to prevent the modification of its mechanical properties. Afterwards, a new ex vivo experimental and in silico numerical protocol was developed to characterize the corneal tissue parameters, considering two mechanical behaviors: the response to physiological loads (intraocular pressure induced by inflation), and the response to non-physiological loading due to the indentation. Finally, a mechano-morphological experimental protocol was designed to characterize the mechanical behavior of the cornea after performing corneal collagen crosslinking on animal models. Different aspects related to the Acanthamoeba keratitis were also addressed. Firstly, a rabbit model of Acanthamoeba keratitis was developed through contact lenses infected with trophozoites and cysts, as the best method to reproduce the natural course of this disease in humans. In a second step, different pharmacological agents and its combinations, including the adjuvant treatment with riboflavin and ultraviolet A, were tested in vitro against Acanthamoeba spp. through qualitative and quantitative assays. Finally, the in vivo response to the most promising amoebicidal treatment in vitro was evaluated in the previously developed animal model.