Anatomy and Neuroscience - Theses

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    Using induced pluripotent stem cell-derived retinal pigment epithelium cells to characterise phenotypic differences associated with reticular pseudodrusen in age-related macular degeneration
    Hall, Jenna ( 2024-02)
    Age-Related Macular Degeneration (AMD) is one of the leading causes of severe vision loss in individuals aged over fifty in Western populations. A hallmark of AMD pathogenesis is the accumulation of lipid and protein deposits, termed drusen, in the macula. Past studies have established that retinal pigment epithelium (RPE) dysfunction alone initiates drusen-like deposit formation, with disease lines exhibiting greater volume and number with respect to deposit formation in vitro. Despite this, automated quantification methods for these deposits were lacking in the literature. While conventional drusen on the basal side of the RPE is a hallmark of AMD pathogenesis, the recognition of reticular pseudodrusen (RPD) on the apical side indicates a distinct AMD phenotype. This thesis focuses on the modelling of RPD using human induced pluripotent stem cells (hiPSCs) as part of this extensive investigation into molecular distinctions between conventional AMD and RPD. Initial identity confirmation of hiPSC-derived RPE cells involved rigorous characterisation and functional assays to establish baseline phenotypic presentations. Subsequent investigations delved into pathological AMD signatures, initially confirming the appearance of hallmark drusen deposits in culture. Recognising the time-intensive and manual nature of existing quantification methods, significant efforts were made to develop and publish two semi-automated quantification tools, fostering standardised approaches in the scientific community. These tools facilitated stressor experiments with N-Retinylidene-N-Retinylethanolamine (A2E), revealing its potential exacerbation of AMD phenotypes in vitro. A large-scale proteomics study aimed to identify differentially expressed proteins between AMD and RPD samples uncovered distinctions in extracellular matrix proteins, the spliceosome complex, and cellular homeostasis, including mitochondrial health. Validation of mitochondrial hits involved assays comparing baseline oxidative states. Utilising the developed pipelines for quantifying drusen-like deposits at baseline and post-stressor treatment, and investigating baseline oxidative stress in RPE cultures derived from patient-specific iPSCs revealed differences between AMD cohorts with and without RPD. This study underscores the utility of patient-specific iPSCs for in vitro modelling of AMD pathogenesis, elucidating variations in phenotypic presentations within stratified AMD disease cohorts. The data suggests discernible differences in disease profiles concerning mitochondrial dysfunction and drusen accumulation between AMD samples with or without RPD, emphasising the potential of iPSCs in unravelling the complexities of AMD.
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    Nanosecond Laser Therapy for the Management of Age-related Macular Degeneration
    Findlay, Quan ( 2021)
    Age-related macular degeneration (AMD) is a chronic, progressive disease affecting the central retina and remains the leading cause of irreversible vision loss in developed nations. AMD is a multifactorial disease encompassing a complex interplay between ageing, environmental and genetic risk factors which ultimately lead to chronic inflammation and death of photoreceptors. While the pathophysiology of AMD is still not fully understood, recent advances have highlighted the involvement of the innate immune system and RPE dysfunction to be fundamental to the development and progression of this debilitating condition. As such, therapies that can influence these two systems have the potential to slow progression of disease. Despite advances in the treatment of neovascular AMD, there remains no effective treatment for non-neovascular AMD and importantly, interventions that address the progression from early to sight-threatening late AMD remain limited. The progression to late stages is usually slow, providing a large window of opportunity to intervene. Novel laser treatments, specifically short-duration pulsed lasers such as the subthreshold nanosecond laser (SNL), offer a potential therapeutic option. The fundamental aims of this study were to investigate SNL-induced cellular response in the RPE that may have potential benefits for addressing pathologic changes that occur in the RPE/BM interface in AMD, and to determine whether changes to the RPE are associated alterations to systemic innate immunity. SNL treatment, delivered in a single session, to the posterior eye in wildtype mice was confirmed using immunohistochemistry to selectively target the RPE without causing damage to the overlying retina. The laser-ablated RPE monolayer was healed within 3 days post-treatment with evidence indicative of newly proliferated daughter cells. Importantly, this proliferative response was also observed in the non-laser treated fellow eye. RNA sequencing and pathway analysis of laser-treated RPE identified biologically-relevant pathways including the promotion of cell survival and cell proliferation, and the inhibition of inflammation and apoptosis. Together, the fellow eye effect and RNA sequencing results suggest that a SNL-induced systemic change involving innate immunity may likely be involved. Systemic change in leukocyte phagocytosis function following SNL treatment was examined for 12-month old wildtype and in the mouse model of AMD (P2X7-null mice). Real-time flow cytometry showed a laser-induced reduction in total phagocytosis capacity of yellow-green fluorescent beads 3 months following SNL treatment. Subsequent ultrastructural studies in the P2X7-null mice showed a significant thinning of the pathologically thickened Bruch’s Membrane (a hallmark feature in AMD). The role of SNL in altering the systemic response, including phagocytosis and cytokine changes, was investigated in a cohort of patients with intermediate AMD. Similar to the rodent results, a substantial reduction in peripheral monocyte phagocytosis was also observed for all subsets of monocyte populations occurring from 3 months following SNL treatment. This reduction was sustained for at least 12 months post-treatment without any significant recovery to pre-laser levels. Cytometric bead array demonstrated significant changes to cytokine-profile with an acute elevation of 9 to 13 cytokines involved in inflammation (of a total of 13 cytokines) at the 2 weeks to 3 month post-treatment time points using 200 SNL spots. At the 6 month time point, a significant increase in interleukin-6 remains in the laser-treated participants compared to pre-laser levels. In summary, these findings indicate that SNL can induce a rejuvenating response by promoting RPE proliferation in both the treated eye and the non-treated eye while also reducing the thickness of BM in the eye that received SNL treatment. This study also provides evidence that SNL therapy has the potential to modify systemic immune responses, including phagocytosis function and cytokine-profile. Taken together, our results indicate a potential role for SNL treatment of AMD to slow progression of disease.