Author:

Abstract:

Glaucoma is a group of neurodegenerative eye diseases that constitute a major cause of blindness and visual impairment world-wide. It is characterized by progressive degeneration of retinal ganglion cells (RGCs), the cells that convey visual information from the eye to the brain. Current treatments focus on controlling the intraocular pressure because an elevated pressure is a major risk factor for glaucoma. Unfortunately, these treatments are often insufficient to halt disease progression, resulting in an unmet need for novel therapies to protect the RGCs. Currently, considerable research efforts are dedicated to understand the pathological mechanisms of glaucoma and the preclinical development of novel neuroprotective therapies. This research is often performed using rodent models. Each of these glaucoma models captures various aspects of the disease and most of them are associated with the loss of RGCs. Therefore, reliable quantification of RGC degeneration in these animal models is a must to assess the extent of induced glaucomatous damage and potential efficacy of a new treatment. With the murine retina containing over 40 000 RGCs, manual quantification of the full retina is impractical and so computer algorithms have been developed to facilitate counting. However, most of the published algorithms are tailored to a specific RGC visualization method. Additionally, they lack accessibility, as they are often written for expensive commercial software, with the source code unavailable. In the first part of this project, an RGC counting algorithm was developed for the free-to-use software ImageJ and its source code was published online. The semi-automated script was found to provide similar results as manual counting, but without the intra- and interobserver variation associated with the manual approach. The script demonstrated correct assessment of RGC degeneration in three commonly used mouse glaucoma models. This efficient tool was then leveraged for rapid quantification of RGC survival in the second stage of this project. The second part of this PhD dissertation builds on various reports suggesting that that RGC survival is supported by the target neurons to which they project. Additionally, target-derived factors had been shown to induce signaling pathways distinct from those activated when the same molecules were provided at the RGC soma. Therefore, we sought to explore the potential of optogenetics for a controllable, prolonged induction of neuronal activity in a visual target center in the brain and thereby enhance RGC survival in a mouse glaucoma model. A novel optogenetic activation paradigm for the principal murine RGC target area, the superior colliculus (SC), was developed and demonstrated to provide consistent neuronal activation over repeated stimulations. Then, the neuroprotective potential of this optogenetic protocol was investigated in mice subjected to laser photocoagulation of the perilimbal and episcleral vessels of the eye, a commonly used glaucoma model. Our optogenetic stimulation paradigm indeed significantly reduced RGC loss in this glaucoma model by 63%, indicating that increased neuronal activity in the SC confers retinal neuroprotection. This finding highlights the retino-collicular system as a model to identify key players mediating neuroprotective retrograde signaling mechanisms. Further investigation of the molecular pathways responsible for this retrograde survival effect could lead to the identification of novel therapeutic targets for treatment of glaucoma. In conclusion, this dissertation contributes important methodological advances to the scientific community investigating optic neuropathies, with both an accessible RGC counting algorithm and an established optogenetic stimulation paradigm for the SC. These newly developed research tools enabled seeding evidence that increasing neuronal activity in visual target centers in the brain is neuroprotective for the projecting neurons and encourage further exploration of the molecular signaling pathways mediating retrograde neuroprotective communication.