Dissertação de Mestrado Investigação Biomédica 2024 Faculdade de Ciências Médicas, Universidade NOVA de Lisboa

Age-related Macular Degeneration (AMD) is a multifactorial and progressive retinal disease that affects millions of people worldwide and has become the leading cause of visual impairment in developed countries. This disease can be defined as a disruption of the retina’s normal homeostatic mechanisms, where ageing coupled with chronic inflammation, increased lipid and lipoprotein deposition, lysosomal dysfunction, and oxidative stress contributes to AMD features. The primary cause of AMD pathology appears to be the retinal pigmented epithelium (RPE) thinning, autofluorescence accumulation (lipofuscin), and depigmentation, leading to atrophy together with the accumulation of drusen between the RPE and the choroid. The main regulator of NRF2 is KEAP1, which, under homeostatic conditions, facilitates its proteasomal degradation. However, under conditions of stress, KEAP1 becomes oxidized, preventing NRF2 degradation. This allows newly synthesized NRF2 to translocate into the nucleus, where it activates genes involved in antioxidant defense, detoxification, and antiinflammatory responses, as well as those promoting proteasome function and autophagy. This study aimed to characterize and validate an inducible AMD mice model and select optimal time points. Alongside analyzing cellular and morphological changes to establish a screening process to test NRF2 activator compounds on this model. To accomplish the aim, we have characterized and validated a NaIO3 inducible model that recapitulates some AMD features in vivo. A single dose of NaIO3 intraperitoneal injection was given at day 0, followed by retinal thickness (RT) evaluated at day 3, 7, and 14 by Optical Coherence Tomography (OCT). Blue-Light Fundus Autofluorescence (BAF) scan was done at the same time point to look for lipofuscin-like granules. For a 25mg/kg dose of NaIO3, we observed a lipofuscin-like phenotype by day 3 and a subsequent increase of this phenotype over day 7 and 14. RT decreases rapidly from day 3 to day 7 and is followed by a slowed pace decrease until day 14. Retinal sections were evaluated at these time points for histological analysis and immunofluorescence assays for detection of cell death (TUNEL staining) and exploring microglia infiltration (Iba1 staining). The highest number of TUNEL positive signals were found at day 3, where Iba1 intensity was found significantly higher than the control and reached maximum values at day 7. After extensive analysis, we found the most significant time points in day 3 and 7 to be the most significant time points for evaluating the efficacy of drug compounds. We then tested an FDA-approved drug dimethyl fumarate (DMF) to explore the potential antioxidative effect by activating the Nrf2 pathway. Unfortunately, at the concentration tested DMF did not show significant cellular and morphological improvement as compared to the NaIO₃-treated mice, although more optimization is needed. Despite this, the study provided valuable insights into the molecular and cellular markers of inducible AMD mice models and well-tuned workflow plans which will be fruitful for screening other compounds