Ophthalmology (Eye & Ear Hospital) - Theses
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ItemRole of microRNAs in retinal neovascularization( 2018)MicroRNAs (miRNAs) are master gene regulators that have been widely implicated in many biological processes as well as pathogenesis of many diseases in the eye. The identification of miRNAs that are altered in human diseases as well as in animal models has led to new avenues to develop novel therapeutic targets for ocular disorders. The broad focus of studies described in this thesis is the identification of miRNAs that have altered expression during neovascularization of the retina using a rat model of oxygen-induced retinopathy (OIR). miRNA-Seq studies carried out in rats, mice and humans show that retinal miRNAs in rats and mice are highly homologous with humans. In exploring the altered miRNAs in retinal neovascularization, we found four miRNAs of interest (miR-143, miR-126, miR-150 and miR-145) that were down-regulated in the retina preceding neovascularization in OIR rats. Further investigation suggested that transforming growth factor-beta activated kinase 1 (TAK1), a target gene of miRNA-143, was highly up-regulated in the retina of OIR rats. Selectively pharmacological inhibition of TAK1 activity by 5Z-7-Oxozeaenol led to significant reduction of angiogenic capability of human endothelial cells, as measured by cell proliferation, cell migration and tube formation. Moreover, 5Z-7-Oxozeaenol greatly suppressed vascular spouting of mice aortic explants. In vivo intravitreal administration of 5Z-7-Oxozeaenol profoundly attenuated the retinal neovascularization in OIR rats. Previous studies have reported the down-regulation of miR-126 and miR-150 in OIR mice, and the restoration of these altered miRNAs led to significant suppression of retinal neovascularization in vivo. Here we focus on miR-143 rather than miR-126 and miR-150 to explore its therapeutic potential and underlying mechanisms in retinal neovascularization. Intravitreal administration of a synthetic miR-143 mimic into the eyes of OIR rats suppressed retinal neovascularization by ~50%. Further RNA-Seq identified that a gene cluster mediated by Fos, an inflammatory and stress gene, plays a crucial role in the suppression of retinal neovascularization in OIR rats by the restoration of miR-143. Plasmid vectors of reporter genes and corresponding elements of altered miRNAs such as miR-143, miR-126 and miR-150 were devised. Co-transfecting the devised plasmids with corresponding mimic of the altered miRNAs in HEK293A cells inhibited reporter gene expression. In the context of gene therapy for retinal neovascularization, the results indicate that therapeutic gene expression could be regulated in accordance with the disease activity through altered miRNAs.
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ItemSecond eye at risk: predicting the development of advanced age-related macular degeneration( 2017)Hypothesis 1: In people who develop a choroidal neovascular membrane (CNVM) secondary to Age-Related Macular Degeneration (AMD), presenting visual acuity varies. Eyes with worse visual acuities at presentation have worse visual acuity outcomes compared to eyes that present with better visual acuities despite treatment. Aim 1: To determine and compare the visual acuity of first and second eyes diagnosed with CNVM secondary to AMD at presentation and through two years of follow-up to determine the long-term visual consequences of poor presenting visual acuity. Methods 1: Retrospective review of clinical notes of patients presenting with newly diagnosed CNVM secondary to AMD in their first eye from the Royal Victorian Eye and Ear Hospital from January 2012 to April 2014. Visual acuity was recorded up to two years. Visual acuity in the second eye developing CNVM was recorded at diagnosis and for a further two years from the time of diagnosis. Results 1: First eyes presented with a visual acuity of 6/52 and second eyes presented with a visual acuity of 6/21. Although this did not reach statistical significance, first eyes tended to have worse visual acuity during the 2 years of follow up. This suggests that there is room to identify and begin treatment earlier once CNVM has developed. Hypothesis 2: Reduced microperimetric macular sensitivity, or a decrease in microperimetric macular sensitivity over time, is associated with an increased risk of developing CNVM in an eye with large drusen whose fellow eye has CNVM secondary to AMD. Aim 2: To determine if reduced macular function, or a decrease in macular function over time, as determined by microperimetry, can predict the development of CNVM in eyes with large drusen whose fellow eye has CNVM secondary to AMD. Methods 2: Participants with unilateral CNVM secondary to AMD in one eye and large drusen in the other eye (study eye, group 1) were recruited prospectively from the Royal Victorian Eye and Ear Hospital, East Melbourne, Victoria Australia between February and May 2014. All participants underwent microperimetry testing at baseline and 12 months study eye developed CNVM or GA. Results 2: Reduced microperimetric sensitivity is associated with the development of CNVM or GA collectively (P=0.045). All eyes that developed CNVM or GA had a mean macular microperimetric sensitivity less than 25dB at baseline. This confirms our hypothesis, and provides further evidence that macular function, as tested by microperimetry, may be a useful biomarker for predicting disease progression. Hypothesis 3: Microstructural changes, such as drusen, EZ disruption, HF, RPD and nGA, are independently associated with decreased retinal function, which partially explain the reduced microperimetric retinal sensitivity seen in eyes that progress to late AMD from Aim 2. Aim 3: To determine if macular structure-function relationships explain the reduced microperimetric macular sensitivity in fellow eyes with large drusen that ultimately go on to develop late AMD of people with unilateral CNVM secondary to AMD in their other eye. Methods 3: Group 1 from Aim 2 was used in Aim 3. Controls with bilateral intermediate AMD in both eyes (group 2) were recruited as a comparison group. All participants from groups 1 and 2 underwent multimodal imaging, including spectral domain optical coherence tomography, fundus autoflourescence, and fundus infrared reflectance. Results 3: Mean microperimetric macular sensitivity is lower in cases compared to controls (P<0.001) at baseline and is independently associated with the development of CNVM or GA collectively (P=0.039), and GA alone (P=0.005) when taking into account microstructural changes. Conclusion: People with CNVM secondary to AMD are presenting with poor vision in an Australian population, which limits their long-term visual outcome despite treatment. Within our study population, all eyes developing late AMD had a baseline mean microperimetric macular sensitivity less than 25dB. Furthermore, microperimetric macular sensitivity is associated with the development of late AMD whose fellow eye already has established CNVM independent of microstructural changes. This provides substantial evidence that microperimetric macular sensitivity, in conjunction with the quantification of structural changes, may prove to be a useful biomarker that could potentially allow for greater risk stratification of those likely to develop late AMD. As such, macular microperimetry has the potential to become a useful clinical tool with practical implications in terms of monitoring.
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ItemBlind feline model for retinal prosthesis( 2015)Hereditary photoreceptor degenerations such as retinitis pigmentosa are a common and incurable cause of blindness in the western world. The development of visual prostheses that use electronic stimulation of the brain or retina to generate a visual percept is a novel field aimed at the restoration of functional vision in patients with photoreceptor degenerations. It is not well understood how changes in the visual pathway as a result of photoreceptor degeneration might impact the efficacy of an implanted visual prosthesis. In order to examine this relationship it is necessary to test the functioning of visual prostheses in a large eyed blind animal model. The aim of this study was to develop and characterise a blind feline model of photoreceptor degeneration using intravitreal injection of adenosine triphosphate (ATP), and to examine the effects of a chronic model of blindness on the efficacy of suprachoroidal electronic stimulation. In order to develop a blind model of photoreceptor degeneration we performed unilateral intravitreal injections of ATP in normal sighted cats. We assessed animals over a 12 week period post injection using electroretinography (ERG), optical coherence tomography (OCT) and retinal histology to determine a dose response and time course of retinal degeneration. Intravitreal injection of ATP lead to a rapid loss of rod photoreceptor function and a gradual loss of cone photoreceptor function within 12 weeks post ATP injection. The outer retina showed a progressive reduction in thickness over the 12 week assessment period, with the inner retina remaining intact. Our data shows that unilateral, intravitreal ATP injection can be used as a rapid, safe and effective model of photoreceptor degeneration in the feline. We examined the effect of ATP-induced retinal degeneration on the efficacy of a suprachoroidal prosthesis. At the conclusion of the previous experiment, four ATP-injected animals were anaesthetised and implanted with a suprachoroidal stimulating array in each eye. Cortical thresholds to stimulation were obtained with multi-unit recordings from the visual cortex. Suprachoroidal electrode thresholds were then correlated to histological markers of neural and glial thickness. ATP-induced retinal degeneration increased the charge threshold necessary to produce a cortical response to suprachoroidal stimulation. Higher thresholds correlated spatially to areas with increased gliotic activity within the retina. Our data suggests that neural and glial changes resulting from ATP induced photoreceptor degeneration influences the efficacy of electronic stimulation using a suprachoroidal prosthesis. Finally, we characterised the extent of neural and glial remodelling present within the retina 6 to 12 weeks following intravitreal ATP injection, using immunohistochemical markers for cell death, neural remodelling and gliosis. Ongoing photoreceptor cell death was present at 6 and 12 weeks after ATP injection. Retinal remodelling events including cell migration and aberrant neurite growth were present throughout retina at 12 weeks after ATP injection. Ganglion cell axons in the optic nerve appeared to remain intact. A thick glial scar containing no neural tissue was observed entombing the outer retina, which may help to explain the correlation between electrode threshold and glial thickness we had observed previously. Our data show that the ATP injected feline model of retinal degeneration undergoes secondary retinal degeneration and remodeling events similar to those commonly seen in other models of retinal degeneration. The primary outcome of this study was the development and characterisation of a feline model of photoreceptor degeneration. This new model has potential as a tool for the future development and optimisation of visual prostheses. We found that pathological gliosis as a result of ATP induced photoreceptor degeneration appeared to influence the efficacy of suprachoroidal electronic stimulation. This research can be used to further inform the optimisation of device placement and stimulation strategy for visual prostheses.
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ItemAmyloid precursor protein-mediated neuroprotection and processing in the aged retina( 2013)The world’s population is ageing and this means an increase in diseases in which age is a risk factor including Alzheimer’s disease (AD), Parkinson’s disease (PD), age-related macular degeneration (AMD) and glaucoma. Classic pathological hallmarks of AD include intracellular neurofibrillary tangles (NFTs), rich in phosphorylated tau protein, and extracellular amyloid plaques containing amyloid beta (Aβ). This is accompanied by neuronal loss and decline in cognitive function. Neuronal loss is also a feature of glaucoma with the selective death of retinal ganglion cells (RGCs) and there is RGC loss observed in AD patients. There is also evidence of increased incidence of glaucoma in AD patients compared to control populations. Aβ is produced as part of amyloidogenic cleavage of the amyloid precursor protein (APP); a large membrane bound protein with a putative receptor-type function. An alternative cleavage pathway, termed non-amyloidogenic, prevents Aβ formation and instead produces the neuroprotective and neurotrophic molecule soluble APPα (sAPPα). The majority of APP cleavage is via the non-amyloidogenic pathway but conditions such as oxidative stress drive increased cleavage through the amyloidogenic pathway. This reduces the pool of protective sAPPα available and may render neurons, including ageing neurons more susceptible to injury. Soluble APPα has demonstrated protection against a wide range of insults including the mitochondrial complex I inhibitor rotenone and this protection is linked to activation of the PI3K/Akt pathway. The work described in this thesis aimed to further elucidate mechanisms of APP-driven neuroprotection against mitochondrial insult, and whether this may represent a drug target in the retina. To examine the mechanism by which sAPPα acts we used rotenone toxicity in human neuronal cells. Cells overexpressing wild-type APP695 (APPwt) displayed increased survival after exposure to rotenone and a second complex I inhibitor MPP+. APPwt overexpression lead to activation of Akt in rotenone treated cells. Akt is a serine/threonine kinase central to survival and apoptosis pathways. We found that APPwt overexpressing cells also showed inactivation of pro-apoptotic Gsk3β downstream of Akt while a second downstream target Bad was not affected by APP. Control cells did show Bad inactivation suggesting that sAPPα-mediated protection overcomes the need for this aspect of the PI3K pathway to be utilised. Intravitreal delivery of rotenone in rodents induces an optic neuropathy specifically targeting the inner retina. We found that sAPPα treatment post-rotenone injury reduced apoptosis in both the ganglion cell and inner nuclear layers. Soluble APPα treatment also reduced levels of the stress response indicator protein, heme oxygenase-1 (HO-1), in a second retinal stress model of mild intraocular pressure elevation. Together these studies suggest that sAPPα has therapeutic value worthy of further exploration. Mitochondrial abnormalities, specifically at OXPHOS complexes I and IV, are well documented in AD and APP overexpression models in vitro and in vivo. This is commonly attributed to Aβ toxicity. Using neuronal cells with APPwt or with APP harbouring the Swedish mutation (APPswe), which drives increased amyloidogenic cleavage, we examined OXPHOS effects. Both cell lines display elevated Aβ compared to controls however, as expected, significantly more is produced by APPswe than APPwt cells. We found that APPwt but not APPswe cells have decreased complex IV protein levels and decreased rates of respiration. At the transcriptional level there was no alteration in these proteins nor was there any observed effect on mitochondrial biogenesis factors. We propose that this may be a novel form of protective down-regulation induced by APP or one of its cleavage products. Relatively little is known about APP levels in the human retina and even less in regards to the vitreous humour. We analysed APP and sAPPα protein levels in ageing cohorts of both tissue types from post-mortem eyes. We found decreasing amounts of APP in ageing retina with unchanged levels of sAPPα which suggests an up-regulation of non-amyloidogenic cleavage. Novel detection of sAPPα in the human vitreous show sAPPα levels were not affected by ageing. We postulate that although aged, these non-diseased eyes do not represent truly stressed tissues in which changes in APP cleavage are detectable. In vitreous from patients affected by type 1 or type 2 diabetes there are clear increases in sAPPα. Whether or not this is a protective up-regulation, possibly via increased secretion from RGCs is still unresolved.
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ItemMitochondrial dysfunction in the retina contributes to vision loss( 2012)Mitochondrial impairment in ocular disease pathogenesis has been well recognised since the first disease-linked mitochondrial DNA mutation was described for Leber’s Hereditary Optic Neuropathy in 1988. Our increased understanding of mitochondrial function and dynamics since then has suggested that mitochondrial dysfunction may be a key underlying factor contributing to retinal ganglion cell loss in a range of optic neuropathies. Mitochondrial dysfunction is traditionally attributed to heritable nuclear and mitochondrial DNA mutations but may be exacerbated by aging, oxidative stress and environmental factors. Mitochondrial impairment limits endogenous energy production and sensitises cells to stress and injury. Understanding how impairment of this key organelle can lead to loss of specific neuronal populations provides possibilities for neuroprotective intervention to prevent retinal ganglion cell degeneration. To address mechanisms of disease pathogenesis associated with vision loss we performed a detailed analysis of mitochondrial function using human cell lines from Autosomal Dominant Optic Atrophy family pedigrees bearing an identical mutation of the OPA1 gene. These patients may suffer specific loss of retinal ganglion cells, but the disease phenotype varies dramatically within family pedigrees. We compared patients with severe vision loss and patients with relatively preserved vision and found a clear defect in mitochondrial energy production and respiration in patients with poor vision. In Autosomal Dominant Optic Atrophy patients with normal vision numerous components of oxidative phosphorylation were increased. These data suggest that OPA1 deficiency impairs oxidative phosphorylation efficiency, but compensation through increases in the distal complexes of the respiratory chain may preserve mitochondrial energy production in patients who maintain normal vision. Evaluation of in vivo retinal mitochondrial function provides insight into early events occurring in optic nerve degeneration during aging and with mitochondrial impairment. We demonstrated that mitochondrial capacity decreased in the aging rodent retina, and was further decreased in the presence of a heritable mitochondrial dysfunction (xenomitochondrial mouse). These mitochondrial impairments (heritable and with aging) sensitised retinal ganglion cells to intraocular pressure elevation. Novel findings from dietary restriction demonstrated that improved mitochondrial function provided a resistance of the retinal ganglion cells to stress. Therapies that promote enhanced mitochondrial function may protect neurons against injury the detrimental effects of aging.