Protective effects of NGF on diabetic retinopathy in vitro models

Retina is a layered structure of the eye, composed of different cellular components working together to produce a complex visual output. Because of its crucial role in visual function, pathologies such as diabetic retinopathy (DR), generally represent the main causes of blindness. Among retinal layers, under DR conditions is observed a loss of integrity of the blood brain barrier (BRB), constituted by the choroid and retinal pigmented epithelium (RPE), accompanied by Müller cells gliosis and neurodegeneration in the neural retina. Among other neurotrophins, NGF/proNGF imbalance was linked to several microvascular problems associated with diabetes, including DR. Also, it was found that the diabetic environment impairs proNGF maturation, leading to elevated proNGF expression and reduced NGF expression. Therefore, restoring the proper ratio of mature neurotrophin to proneurotrophin is a potentially effective treatment approach for treating retinal degenerative illnesses, such as DR. For this reason, the aim of this thesis was to develop in vitro platforms of DR to then investigate the use of rhNGF for the restoration of NGF/proNGF imbalance as a therapeutic strategy to prevent early signs of DR. ARPE-19 RPE cell line was used to study the alteration of barrier function induced by high glucose conditions and to built-up a 3D BRB model. The barrier function of the epithelium has been assessed by transepithelial electrical resistance (TEER) measurements and immunofluorescence staining for ZO-1. Moreover, rMC-1 Müller cell line and R28 cell line (modelling neural retina) underwent high glucose conditions to induce the injury. All the models were then treated with rhNGF to evaluate the protective effect of this important neurotrophin. Using these models, live cell assays were then- performed to evaluate effects on cell viability. TEER was used to study the effects on barrier permeability. The protein levels involved in pro-survival pathways were studied using Western Blotting analysis. Furthermore, dicarbonyl stress products, oxidative stress enzymes activity and mature BDNF levels were investigated with ELISA assays. Using Seahorse analysis were then observed: the basal respiration, proton leak and mitochondrial respiration. Collectively, all the data obtained dissected, at cellular levels, the protective effect of rhNGF in counteracting the detrimental consequences of hyperglicemia on retinal cells.