Ratones manipulados genéticamente y sistemas de imagen para una mejor comprensión de la fisiopatología de la córnea

Résumé

This doctoral thesis is presented, in part, as an evolution of the approaches used to investigate the cornea. The progress in the development of methodology can be chronologically evaluated in the successive chapters. As a main achievement, herein we present a significant improvement in the methodology compared with previous techniques currently in use. In the first chapter, we showed that the deficiency in ¿n the first impairs corneal wound healing. By using null mice in neal wound heal--/--), we observed that both basement membrane and hemidesmosomes are not recovered in corneas of ß6--/--. These mice show subepitelial blisters such as those observed in patients with bullous keratopathy. In the Chapter 2 we used a mouse deficient in thrombospondin-1 (THSB1) to study the process of corneal repair after a penetrating incision. THBS1 is crucial for both stromal repair and endothelial regeneration. The corneas of mice lacking THBS1 show a complete failure in the process of healing and tissue repair. THBS1 deficient mice developed chronic corneal edema. In this work, for the first time, we used an in vivo confocal microscope (IVCM) to evaluate the healing process in a living mouse. Using the IVCM system, we studied the inflammation of the ocular surface in an allergic conjunctivitis mouse model (chapter 3). The cell infiltration into the ocular surface was observed in vivo at real time. The novelty of this study was to observe a corneal involvement (cellular infiltration) during the inflammatory process long before it was detected with the slit lamp. In sum, our observation indicated that the number of infiltrated cells and their spatiotemporal distribution in the cornea is related to the progression of clinical symptoms of the allergic conjunctivitis. Next, we incorporated a multiphoton intravital microscope (MP-IVM) and genetically-engineered reporter expressing mice strains to study the resident immune population of cornea. With this technology we were able of non-invasive examination of the cornea in a living mouse. The myeloid-derived population in the mouse cornea was viusalized and stratified with high resolution and three-dimensional space (Chapter 4). Additionally, we generated chimeric mice by lethal irradiation of the host and subsequent transplant with bone marrow-derived cells of another mouse strain. This model allows for in vivo observation of the turnover of the myeloid population of the cornea. Moreover, we developed an experimental in vivo model to evaluate the myeloid fate mapping of the adult cornea. This model suggests myeloid population of the cornea might have an embrionary origin rather than hematopoietic as has been established before. Furthermore, we also visualized the corneal nerves in a living mouse (Chapter 5). In this model we comprehensively mapped the corneal nerves with high-resolution and three-dimensional space. Remarkably, we observed the presence of radial distributed nerve bundles in the deep part of the stroma. These nerve fibbers supply innervation from bellow to the subbasal plexus ramifying in both directions toward the center and periphery. The interface of both immune and peripheral nervous system (PNS) is shown in the chapter 6. This interaction is suggested to be critical for corneal transplant immune rejection. Herein, we proposed the cornea as a much more complex organ than it has been described before. The stochastic interaction of both ¿supersystems¿ (PNS and immune system) seems to be critical for maintaining corneal homeostasis. A chimeric mouse model was generated by lethally irradiation of a host mouse expressing neurofluorescence. This mouse was engrafted with fluorescent myeloid-derived bone marrow cells. Three months later, we intravitally observed both super-systems are physically interconnected, which demonstrates the proposed neuroimmune interface. Finally, we show the possible future applications of MP-IVM. First, we evaluated in vivo the behavior of murine adipocyte-derived stem cells (mADSCs) engrafted in different parts of the cornea. Second, we monitored the efficiency of drug delivery in the anterior chamber, trabecular meshwork and Schlemm¿s canal. Also we intravitally evaluated the efficiency of the viral infection in the endothelial cells by adenovirus. Finally, we detected the turnover of a specific CD11c dendritic cells population after earlier depletion with diphtheria toxin.