L’objectif de cette thèse est la validation fonctionnelle d’implants rétiniens pour la restauration fonctionnelle de la vision chez des patients non voyants suite à la perte de leurs photorécepteurs. Ce travail a été réalisé sur modèle animal et a évalué expérimentalement de nouveaux protocoles de stimulation. Tout d’abord nous avons utilisé la technique de spectroscopie d’impédance pour simuler mathématiquement l’interface tissu-implantafin de caractériser la présence d’un espace entre le tissu et l’implant. La seconde partie compare par imagerie optique (IO) les caractéristiques de la réponse corticale évoquée par stimulation visuelle ou électrique de la rétine par prothèse sous rétinienne. Nous avons retrouvé que la taille de l’activation par l’implant rétinien est beaucoup plus grande que son correspondant visuel. Dans une troisième partie, est réalisée une évaluation in vitro de la performance des stimulations sur rétine isolée pour définir comment les cellules ganglionnaires réagissent à différents modes de stimulations. Ce travail a permis d’établir la courbe des réponses en fonction de l’intensité des stimulations électriques. Enfin, la thèse décrit un modèle animal de dégénérescence rétinienne qui présente des désorganisations de la rétine externe. Une analyse en IO a été réalisée sur ce modèle afin d’évaluer la réponse corticale aux stimuli visuels et électriques. Ce travail de thèse, par des approches physiques et physiologiques complémentaires, apporte un certain nombre de réponses qui devraient permettre d’améliorer l’utilisation de futures prothèses rétiniennes par une adaptation physique des matrices d’électrodes ou des patrons de stimulations utilisées / The aim of this thesis is the functional validation of retinal implants used for vision restoration in blind patients due to the loss of photoreceptors. This work was designed to develop an animal model to experimentally validate prototypes of new implants and new stimulation protocols pattern. Firstly we used the technique of impedance spectroscopy to simulate mathematically the tissue/implant interface. These data confirm the importance of reducing the space between the stimulating electrodes and retinal tissue, as well as the importance of physical characteristics of the electrical stimulus used. In a second approach, we have compared responses of visual cortical neuronal population using optical imaging (OI), evoked either by visual or electric retinal stimulation through subretinal prosthesis. This approach has demonstrated that the stimulation of an electrode induces cortical activation that the size of the cortical response to the retinal implant stimulation is much larger than its corresponding visual stimulus. In the third part, I performed in vitro experiment to measure the performance of stimulation at the level of ganglion cells of isolated retina. We have quantified the response curve as a function of the intensity of the electrical stimulation. Finally, the thesis describes a new animal model of outter retinal degeneration. OI was also performed on this model to assess the response to the visual and retinal prosthesis stimulations. This thesis, through complementary physical and physiological approaches, provides a number of responses that can potentially improve the use of retinal prostheses through specification of their design or patterns of stimulation.

Indiana University-Purdue University Indianapolis (IUPUI) / Choroidal neovascularization (CNV) is the vision-threatening characteristic of wet age-related macular degeneration (AMD), a major cause of blindness affecting almost 2 million elderly Americans. The current approved treatments target the dominant angiogenic mediator, vascular endothelial growth factor (VEGF). However, repeated injections of anti-VEGF drugs can cause ocular and systemic side effects, and about 30% of wet AMD patients are non-responsive. There is thus an unmet need to develop VEGF-independent antiangiogenic molecules to complement or combine with existing medications. I studied SH-11037, a novel homoisoflavonoid with potent and selective antiangiogenic activity against human retinal endothelial cells. Intravitreal SH- 11037 dose-dependently suppressed angiogenesis in the laser-induced CNV (LCNV) mouse model. These effects were prominent as early as 7 days post-laser treatment as measured by a novel ellipsoid quantification method of optical coherence tomography images in vivo. A supratherapeutic dose of 100 μM SH- 11037 was not associated with signs of murine ocular toxicity, and did not interfere with pre-existing retinal vasculature or retinal function. SH-11037 synergized with anti-VEGF therapy in vitro and in vivo, suggesting a VEGFindependent mechanism. By photoaffinity pulldown, I identified soluble epoxide hydrolase (sEH) as an SH-11037-binding target. sEH is a key enzyme in ω-3 and ω-6 fatty acid metabolism. sEH levels were dramatically upregulated in retinal sections from L-CNV mice and a specific sEH inhibitor, t-AUCB, significantly suppressed L-CNV lesion volume. Additionally, SH-11037 inhibited sEH enzymatic activity in vitro and in vivo in L-CNV mice. Given the role of sEH in the metabolism of docosahexaenoic acids (DHA), inhibition of sEH using small molecules like SH-11037 would enhance ocular DHA levels, with beneficial antiangiogenic and anti-inflammatory effects. SH-11037 is thus a novel sEH inhibitor, which could make it an alternative or additive therapy to existing anti- VEGF drugs for treatment of neovascular diseases in the eye and other tissues.

Indiana University-Purdue University Indianapolis (IUPUI) / Human induced pluripotent stem (iPS) cells have the unique ability to differentiate into 200 or so somatic cell types that make up the adult human being. The use of human iPS cells to study development and disease is a highly exciting and interdependent field that holds great promise in understanding and elucidating mechanisms behind cellular differentiation with future applications in drug screening and cell replacement studies for complex and currently incurable cellular degenerative disorders. The recent advent of iPS cell technology allows for the generation of patient-specific cell lines that enable us to model the progression of a disease phenotype in a human in vitro model. Differentiation of iPS cells toward the affected cell type provides an unlimited source of diseased cells for examination, and to further study the developmental progression of the disease in vitro, also called the “disease-in-a-dish” model. In this study, efforts were undertaken to recapitulate the differentiation of distinct retinal cell affected in two highly prevalent retinal diseases, Usher syndrome and glaucoma. Using a line of Type III Usher Syndrome patient derived iPS cells efforts were undertaken to develop such an approach as an effective in vitro model for studies of Usher Syndrome, the most commonly inherited disorder affecting both vision and hearing. Using existing lines of iPS cells, studies were also aimed at differentiation and characterization of the more complex retinal cell types, retinal ganglion cells (RGCs) and astrocytes, the cell types affected in glaucoma, a severe neurodegenerative disease of the retina leading to eventual irreversible blindness. Using a previously described protocol, the iPS cells were directed to differentiate toward a retinal fate through a step-wise process that proceeds through all of the major stages of neuroretinal development. The differentiation process was monitored for a period of 70 days for the differentiation of retinal cell types and 150 days for astrocyte development. The different stages of differentiation and the individually derived somatic cell types were characterized by the expression of developmentally associated transcription factors specific to each cell type. Further approaches were undertaken to characterize the morphological differences between RGCs and other neuroretinal cell types derived in the process. The results of this study successfully demonstrated that Usher syndrome patient derived iPS cells differentiated to the affected photoreceptors of Usher syndrome along with other mature retinal cell types, chronologically analogous to the development of the cell types in a mature human retina. This study also established a robust method for the in vitro derivation of RGCs and astrocytes from human iPS cells and provided novel methodologies and evidence to characterize these individual somatic cell types. Overall, this study provides a unique insight into the application of human pluripotent stem cell biology by establishing a novel platform for future studies of in vitro disease modeling of the retinal degenerative diseases: Usher syndrome and glaucoma. In downstream applications of this study, the disease relevant cell types derived from human iPS cells can be used as tools to further study disease progression, drug screening and cell replacement strategies.