Neurodegeneration and neuroprotection in experimental models of retinal diseases

Retinal diseases like diabetic retinopathy (DR) and glaucoma are major causes of vision loss and blindness worldwide.DR has been classically defined as a vascular disease. Nevertheless, in the last decade, different studies highlighted that neuronal death and glial activation occur before any vascular changes. In particular, hyperglycaemia triggers metabolic pathways that lead to the onset of oxidative stress (OS). OS, in turn, enhances retinal metabolic dysfunction and leads to neurodegeneration. Moreover, OS induces the upregulation of vascular endothelial growth factor (VEGF).In DR, VEGF performs a double role. Indeed, in the early phases of the disease, it is released to protect retinal neurons, but, with the progression of DR, it undergoes an uncontrolled upregulation responsible for retinal pathological vascular changes and neoangiogenesis. The molecular mechanisms involved in this VEGF upregulation are not well known. I hypothesised that the OS-induced VEGF upregulation is due to an autocrine loop in which VEGF, mainly released by Müller cells, directly induces its own expression. The results confirmed that OS may induce VEGF expression through the activation of nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor involved in the antioxidant response. VEGF, in turn, would promote its own expression and release through an autocrine loop in which the growth factor would bind to its receptor VEGFR2 and activate hypoxia-inducible factor 1, the main transcription factor involved in VEGF expression, thereby favouring the upregulation of the growth factor and then the progression of DR. Similar to DR, OS plays a key role also in glaucoma, even though with different mechanisms. Indeed, an increase in intraocular pressure would lead to OS, glial reactivity, retinal ganglion cell death, and functional deficits. Therefore, it appears that, although DR and glaucoma manifest different pathophysiological features, they are characterised by the same pathological trigger, that is OS. Considering the OS related mechanisms of neuroinflammation and neurodegeneration, counteracting and preventing retinal OS could be a good strategy for treating both DR and glaucoma. Different therapeutic approaches have been directed at modulating the retinal prooxidant/antioxidant equilibrium using nutraceuticals as natural antioxidant compounds. In particular, I evaluated the antioxidant and neuroprotective effects of Lisosan G (LG), a fermented powder obtained from whole grains and composed by different metabolites(including gallic acid, 4-hydroxybenzoic acid, quercetin, and nicotinamide), using an in vivo model of glaucoma represented by DBA/2J mice. In this context, the antioxidant, anti-inflammatory and neuroprotective effects of LG were highlighted. Moreover, a pharmacokinetic analysis demonstrated that the main LG metabolites can reach the retina in a relatively short time. These data, on the one hand, confirm and expand the notion of LG as a powerful natural compound with high therapeutic potential, while on the other provide important information for further studies aimed at improving the bioavailability of the active components of LG. Within the same research line, I also investigated the antioxidant efficacy of a nature inspired hybrid (NIH1) using an ex vivo model of retinal OS. NIH1 is a hybrid derived from curcumin and diallyl sulphide, which exerts its antioxidant effect by activating Nrf2. The hybridization strategy was aimed at increasing curcumin bioavailability and enhancing its antioxidant properties. Indeed, in previous studies, NIH1 emerged for its ability to activate Nrf2 to a higher extent than curcumin alone in in vitro models. At that point, it was essential to gather information about NIH1 efficacy in models that maintain the retinal complexity. For this reason, I tested the effect of NIH1 in organotypic retinal explants. In particular, I evaluated the impact of the hybrid on the activation of antioxidant enzyme expression, neuroprotection, and glial reactivity in response to OS. The results confirmed the antioxidant properties of NIH1 and provided a rationale for further in vivo studies. Taken together, the findings of this thesis contribute to characterize a mechanism involved in VEGF upregulation in response to OS and novel therapeutic strategies for early treatment of glaucoma and DR.