Analysis of animal model of corneal wound healing

Skin and corneal wound healing (WH) are promoted by mesenchymal SCs (MSCs). MSCs favor WH via paracrine mechanisms, and this function can be mediated by the secretome including extracellular vesicles (EVs). MSC-derived EVs carry microRNAs (miRNAs) which seem to be endowed with remarkable therapeutic potential for the treatment of chronic epithelial wounds. In this study, we established a suitable animal model of corneal-injury (alkaline burn) protocol in order to assess, ex vivo and in vivo, the therapeutic effect of EVs on the resolution of wound healing with a multi-layered approach at the molecular, cellular and organismal level in preclinical settings. Histological and MRI analysis showed main alkali burn animal model features: thickening of sub-corneal connective tissue, angiogenesis and reduction of crystalline lens. In in vivo experiments, neovascularization was studied with the use of the Gd-AAZTA-MADEC contrast agent that binds to albumin and is transported through the blood circulation, demonstrating that it is suitable to study angiogenesis. In ex vivo and in vivo experiments, the effect of EV treatment, was studied through the follow up of angiogenesis, fibrosis and inflammation in order to monitor the corneal epithelium WH. Immunohistochemistry, real-time PCR and MRI analyses revealed that the application of 1 dose of EVs caused a reduction in such processes. Furthermore, MRI analysis highlighted that the positive effect of EVs-treatment was enhanced by increasing the intensity of the treatment from 1 to 2 doses per each day for 7 days. In conclusion, we demonstrated that EVs promote corneal wound healing in vivo.