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Anti-vascular endothelial growth factor drugs (anti-VEGF) are used in the treatment of retinal conditions including neovascular age-related macular degeneration (nAMD), and macular oedema associated with diabetic retinopathy or vein occlusions. Despite the fact that treatment often extends over many years, requiring repeated injections, little is known about the long-term effects on the healthy retina also exposed to the treatment. The aim of this research was to investigate the relationship between cumulative dose of anti-VEGF received and parameters of extramacular function and retinal layer thicknesses in people undergoing treatment for nAMD. Chapter 2 presents a systematic review of the literature conducted to evaluate current evidence regarding the effect of anti-VEGF drugs on peripheral retinal function and structure of animals and humans. The review found that the majority of evidence was not supportive of a detrimental effect of treatment on retinal function or structure. Crucially, however, there were significant limitations in the evidence base available to date. Of particular note was the dearth of studies which evaluated the effect of multiple injections over a prolonged period of time in animals or humans (for example, only 22.5% of the assessed studies followed up the subjects/participants for longer than 9 months after the first anti-VEGF treatment). A few studies with longer follow up times did report a small effect of treatment on retinal function in humans, but sample sizes were small. It was concluded that there is a need for research evaluating the cumulative effect of multiple retreatments of anti-VEGF drugs on retinal structure and function in humans. Chapters 3 and 4 report on aspects of electroretinogram (ERG) protocol development. In Chapter 3 the basic protocol for the PhD research was piloted, and the repeatability assessed. The final protocol consisted of a Transient Cone ERG (ISCEV standard; white 3 cd.s.m-2 stimulus, 4 ms duration, 2 Hz, over a white background of 30 cd.m-2), a flicker ERG (41 Hz, red stimulus peak wavelength 635 nm, 12 ms duration, 41 Hz, mean luminance 30 cd.m-2), and a photopic negative response (PhNR) recorded to a series of stimulus intensities (0.11-3.35 cd.s.m-2 red stimulus, peak wavelength 635 nm, 4 ms duration, 2 Hz, blue LED background, peak wavelength 465 nm, 158.8 scotopic cd.m-2). In Chapter 4, the repeatability of a desktop ERG recording system (Espion™, Diagnosys) was compared to that of a handheld system which uses skin electrodes instead of conjunctival electrodes (RETeval®, LKC). Reliability was found to be similar between the two. Amplitudes, but not times to peak, were substantially different in the hand held device. An electrode placement study aimed to determine the impact of the position of the skin sensor strip when recording ERGs using the handheld RETeval® device. Placement was important with respect to amplitudes - skin surface electrodes recorded lower amplitude values when placed further away from the manufacturer recommended position (2mm below the lower lid margin). However, time to peak was relatively unaffected by electrode position. Chapter 5 presents a cross-sectional study which aimed to investigate the relationship between number of anti-VEGF injections received by people with nAMD and parameters of the full-field ERG and optical coherence tomography (OCT) layer thicknesses (adjacent to the macula). Thirty-two participants were recruited from St Thomas' Hospital. There was a tendency across all stimulus intensities for PhNR amplitude to reduce with increasing number of injections, a relationship that was statistically significant prior to Bonferroni correction at three intensities. Increasing number of injections was also associated with a reduced thickness of the nasal outer segment layer. Increasing time since first injection was significantly associated with reduced PhNR 0.11 cd.s.m-2 amplitude, and increased Transient Cone ERG b-wave time to peak, as well as reduced nasal nerve fibre layer thickness, photoreceptor outer segment layer thickness and total retinal thickness, and reduced temporal ganglion cell complex thickness, and outer segment thickness. A second study was conducted at a different test site using the RETeval® hand held device, with the aim of determining whether these ERG findings were consistent in a second set of participants (n = 19). There was a trend across all data towards reducing amplitudes and increasing implicit times with increasing number of injections. However, this was only statistically significant for the time to peak of the a-wave in the PhNR ERG protocol. The signal to noise ratio was small, however, and the lower than desired sample size means that this study was not powered to detect weak correlations. To conclude, the cross-sectional analysis of participants undergoing anti-VEGF therapy for nAMD highlighted a possible relationship between number of injections and certain parameters of the full-field ERG and retinal layer thicknesses measured adjacent to the macula. Of particular note was the relationship between PhNR amplitude and number of injections, and the apparent thinning of the photoreceptor outer segments with increasing number of injections and increasing time since first injection. However, the effect size was small. A number of potential confounding variables have been identified, including the effect of the disease process itself on the outcome measures assessed. Furthermore, interpretation of these findings should be made with caution given the low sample size, which limited the ability to detect small and medium effect sizes. The results of this study suggest that it is unlikely that long term anti-VEGF treatment causes a large negative effect on retinal structure or function which may be reassuring to practitioners and patients.

Classic literature on spatial summation suggests that the physiological basis for Ricco's area lies in retinal ganglion cell [RGC] receptive fields. However, the finding of an enlarged Ricco's area in glaucoma challenges this notion, as histological studies have found that RGC dendritic trees shrink before death in glaucoma, which should correspond to receptive field size shrinkage, rather than enlargement. Evidence has suggested a cortical contribution to determining the size of Ricco's area, rather than a solely retinal basis. Pan & Swanson (2006) found that perimetric spatial summation could only be accounted for when considering cortical pooling by multiple spatial mechanisms. If an enlargement of cortical receptive fields is found in glaucoma, and if this is related to Ricco's area measurements, this might partly explain the basis for the increase in spatial summation in the condition. The current thesis therefore aims to investigate how receptive field sizes at the retina and cortex contribute to Ricco's area. Pattern Electroretinography and functional MRI population receptive field [pRF] mapping were utilised to test for differences in retinal and cortical receptive field size respectively, in a sample of glaucoma patients and age-similar controls. While there was no apparent enlargement of pRFs in glaucoma compared to controls, patients did demonstrate a significantly steeper relationship between pRF size and eccentricity in V1d. However, evidence for a cortical contribution to Ricco's area was not found in these data, despite replicating an enlargement of Ricco's area. In addition, though glaucoma patients demonstrated evidence for larger retinal receptive field sizes, this was not significantly associated with Ricco's area. Overall, these results do not support the initial hypothesis of both retinal and cortical contributions to Ricco's area. However, future work with more specific cortical modelling is outlined for fully characterising the relationship between cortical receptive field sizes and Ricco's area.

OPA1 is the key regulator for the mitochondrial fusion and fission balance and also plays an important role in the pathological process of ADOA, causing visual dysfunction and even systemic disabilities. Retinal ganglion cells (RGCs) are vulnerable to energy disturbances, preferentially affected in most ADOA patients. The precise role of mitochondrial dynamics in retinal ganglion cell health and progression of cell death remains unclear. Thus, in this thesis it is hypothesized that alterations in mitochondrial dynamics during the early asymptomatic stages of ADOA precede the neurodegeneration and consequent RGC death. Utilising immunofluorescence, time-lapse imaging and measurement of oxygen consumption rates, it was found that Opa1 deficiency leads to significant fragmentation of mitochondrial morphology, activation of mitochondrial motility and impaired respiratory function in RGCs from the B6; C3-Opa1Q285STOP mouse model. The increased motility of mitochondria is hypothesized to be a spontaneous compensatory response which promotes fusion activity and facilitates energy production. This result highlights the significant alterations in the intricate interplay between mitochondrial morphology, motility, and energy production in RGCs with Opa1 deficiency long before the onset of clinical symptoms of the pathology. In order to explore and gain a better understanding of mitochondrial morphology and motility in RGCs under different conditions, hypertensive stress and drug administration were applied to RGCs in vitro. In the in vitro hypertension model, it was found that hypertensive stress caused shortened length of mitochondrial structures accompanied with increased mitochondrial motility. By applying nicotinamide directly into the RGC culture medium in vitro with different concentrations for 24 hours, it was found that nicotinamide increased the percentage of motile mitochondria in neurites of RGCs as well as the velocities of mitochondrial movement.

The growth of computer aided drawing systems for blind people (CADB) has long been recognised and has increased in interest within the assistive technology research area. The representation of pictorial data by blind and visually impaired (BVI) people has recently gathered momentum with research and development; however, a survey of published literature on CADB reveals that only marginal research has been focused on the use of a formal approach for on screen spatial orientation, creation and reuse of graphics artefacts. To realise the full potential of CADB, such systems should possess attributes of usability, spatial navigation and shape creation features without which blind users drawing activities are less likely to be achieved. As a result of this, usable, effective and self-reliant CADB have arisen from new assistive Technology (AT) research. This thesis contributes a novel, abstract, formal approach that facilitates BVI users to navigate on the screen, create computer graphics/diagrams using 2D shapes and user-defined images. Moreover, the research addresses the specific issues involved with user language by formulating specific rules that make BVI user interaction with the drawing effective and easier. The formal approach proposed here is descriptive and it is specified at a level of abstraction above the concrete level of system technologies. The proposed approach is unique in problem modelling and syntheses of an abstract computer-based graphics/drawings using a formal set of user interaction commands. This technology has been applied to enable blind users to independently construct drawings to satisfy their specific needs without recourse to a specific technology and without the intervention of support workers. The specification aims to be the foundation for a system scope, investigation guidelines and user-initiated command-driven interaction. Such an approach will allow system designers and developers to proceed with greater conceptual clarity than it is possible with current technologies that is built on concrete system-driven prototypes. In addition to the scope of the research the proposed model has been verified by various types of blind users who have independently constructed drawings to satisfy their specific needs without the intervention of support workers. The effectiveness and usability of the proposed approach has been compared against conventional non-command driven drawing systems by different types of blind users. The results confirm that the abstract formal approach proposed here using command-driven means in the context of CADB enables greater comprehension by BVI users. The innovation can be used for both educational and training purposes. The research, thereby sustaining the claim that the abstract formal approach taken allows for the greater comprehension of the command-driven means in the context of CADB, and how the specification aid the design of such a system.

This thesis hoped to inform the practice of future individual myopia management. All myopia risk factors across global ethnic regions must be considered, instead of relying on the most widely used averaged parameters, towards the development of growth model tools. There could be possible crucial cut points of near phoria development at specified age ranges, earlier and later in life, suggesting this myopia risk factor should be measured alongside other primary outcome parameters important for treatment efficacy. Other notable human lifespan findings included: emmetropic and female patients attended eye examinations more frequently; females exhibited higher levels of near phoria and myopia; myopes were more esophoric than emmetropes, progressive myopes were more esophoric than both myopes and emmetropes, and were less likely to increase in exophoria with age. The presumed design optimisation, regarding daily CE-marked optical myopia control strategies, was based on the possible mechanism behind myopic retinal defocus (blur) and accommodative lag in myopia development and progression. Contact lens designs could have an inherent characteristic for their treatment effect in the temporal retina at 30° and J0 astigmatic component. Multifocal contact lenses for myopia control significantly impacted glare, but did not affect contrast sensivitiy differently than standard lenses, and would offer equally acceptable treatment compliance and qualifty of life expectations. Specialty instrumentation for measuring primary outcomes (refraction and axial length) should be used interchangeably for myopia control studies. This was confirmed between the gold standard biometers, IOLMaster 700 and IOLMaster 500, for the key parameters of axial length, anterior chamber depth and corneal topography, where discrepancies in white-to-white corneal diameter values, following MiSight and NaturalVue contact lens wear, were minimal and clinically irrelevant. Further novel discoveries proved myopia control contact lenses were viable non-invasive sampling vehicles for human dopamine detection. Thus, the thesis probed the viability of novelty applications of such "labelled" and/or gold standard medical devices and instrumentations towards treating individual myopic patients and highlighted that appropriate global myopia management and standardisation remain poor.

It is clear from many sources that the prevalence of myopia is increasing at an alarming rate. Although the pathogenic mechanisms behind the development of myopia remain unclear, various factors have been associated with myopic progression, including genetics, accommodative spasm, prolonged near work, race, gender, educational level, and the amount of time spent outdoors in sunlight. Because of the personal and socio-economic burdens associated with this refractive condition, the key factors in myopia progression continue to be keenly sought. Recent human and animal research suggests that the extent of myopia progression may be dependent on the extent of relative peripheral hyperopia, which in turn is dependent on the type of optical correction (glasses versus contact lenses) worn by an individual. This theory has been termed the hyperopic defocus theory, and the principal goal of this thesis was to assess this current (and popular) theory of myopia development. The methodology employed was a combination of retrospective data analyses and experimental measures of central and peripheral refractive status. Participants selected for inclusion in the study were all myopic, aged 6-24 years, and wore either contact lenses or spectacles. From theoretical arguments and experimental evidence, three hypotheses were made. First, that contact lens wearers will show less myopic progression than spectacle lens wearers. Second, that higher degrees of foveal myopia (myopic progression) arise in eyes with greater amounts of relative peripheral hyperopia. And third, that the dependence of the degree of foveal myopia on relative peripheral refraction is influenced by the type of optical correction worn (contact lenses versus spectacle lenses). In assessing the degree of myopic progression from early childhood through to late adolescence, taking into account the influence of possible covariates (i.e. initial age and initial degree of myopia), the results indicated: (a) optical correction does not have a significant influence on myopic progression; (b) there exists a significant negative correlation between foveal myopic refraction and the degree of relative peripheral hyperopia; and (c) the degree of foveal myopia on relative peripheral refraction is not influenced by the type of optical correction worn. From these results, the first and third hypotheses cannot be supported. These non-confirmatory results of the hyperopic defocus theory, however, are balanced by the supportive finding that higher degrees of foveal myopia arise in eyes with greater amounts of relative peripheral hyperopia (i.e., Hypothesis 2). The latter has important implications for the profession of optometry because, if the theory has genuine merit, it would enable ophthalmic practitioners to not only correct myopia but also minimise the continued development of myopia in children and young adults through various treatment options, including peripheral defocus contact lenses and orthokeratology.

Introduction: Strabismus surgery undertaken for psychosocial reasons in adults aims to improve eye alignment and health related quality of life (HRQoL). Additionally, patients can gain a larger visual field, unexpected binocular single vision (BSV), and improved binocular summation, eye movements and task performance from surgery. Despite these improvements, NHS funding for strabismus surgery, without expected visual benefit, has been withdrawn in some areas of England due to concern that not enough patient benefit from surgery is proven. Methods: A mixed methods feasibility study was undertaken to investigate the outcomes of adult strabismus surgery undertaken specifically for psychosocial reasons. In the qualitative phase semi-structured interviews were conducted postoperatively and the findings informed the quantitative phase design. The quantitative phase prospectively recruited surgery and control group participants to undergo standard clinical measurements and additional study measurements. Results: In the qualitative interviews participants (n=13) reported a range of improvements in their vision, task performance, physical symptoms and confidence and emotions postoperatively. Compared to the control group (n=15), the surgery group (n=12) had postoperative quantitative improvements in binocular summation at 100% contrast, coarse stereotest (CST) performance, the time to perform a touchscreen spatial localisation (TSL) task and the time to perform the clinical kinematic assessment tool (CKAT) aiming task. Improvements were also reported in vision, task performance, physical symptoms, confidence and emotions, and health related quality of life (HRQoL) using patient reported outcome measures (PROMs). Most measures were unchanged and some worsening of task performance (bead threading and grooved pegboard) was measured postoperatively. Conclusion: Strabismus surgery undertaken for psychosocial reasons can lead to objective improvements in vision and task performance and subjective improvements in vision, task performance, physical symptoms and confidence and emotions. These improvements were in addition to the typically expected outcomes of improved eye alignment and improved HRQoL.

Aims: To examine how binocular functions change with increasing age as measured by stereoacuity, motor fusion, ocular movements, near point of convergence (NPC), and ocular alignment. Methods: A preliminary questionnaire survey to establish the professionals' views on whether age affects distance stereoacuity, fusion and NPC; and what the expected value of stereoacuity was for two age groups using Titmus, TNO and Frisby stereotests. A prospective single-centre cohort study was performed on 77 normal participants aged 10 - 79 years measuring ocular alignment, ocular motility, NPC, motor fusion, and stereoacuity (with Titmus, TNO, Frisby, and Frisby-Davies distance stereotests). Results: The preliminary study results confirmed there was a gap in knowledge regarding any association between age and fusion, and between age and stereoacuity. From the cohort study, all stereotests showed a statistically significant decline in stereoacuity with increasing age (p < 0.05). As age increases NPC declines, this was a statistically significant change (p < 0.05); one year increase in age yielded a 0.032 cm decline in NPC. Age-related changes in positive distance fusion were found- as age increases distance positive fusion declines, which was a statistically significant change (p < 0.05). Age-related changes in positive near fusion, negative near fusion, negative distance fusion, vertical near fusion, vertical distance fusion, ocular alignment, and ocular motility were not found. Conclusions: Overall, stereoacuity was affected by age, but this study challenges the view that other aspects of a binocular vision examination are affected by increasing age. The normative data will provide a baseline from which to compare outcomes in clinical situations.

Despite the known visual and pathological implications of hyperopia, there has been inertia to address the modulation of refractive error in these individuals. Imposing relative peripheral hyperopic defocus using centre-near multifocal contact lenses accelerates axial growth in isohyperopic children. Axial growth and refractive error did not change during the 6 months prior to intervention in the intervention or control group. Axial growth across the 2-year period of intervention was 0.17 mm in the intervention group versus 0.06 mm in the control group. Refractive error change across the same period was -0.26 D in the intervention group and +0.01 D in the control group. Axial growth and refractive error during the final 6 months without intervention did not change in either group. The overall difference in axial growth between groups was significant whereas the change in refractive error was not. Imposing relative peripheral hyperopic defocus using centre-near multifocal contact lenses does not accelerate axial growth nor reduce refractive error in anisohyperopic children. In this paired eye study, axial growth and refractive error did not change during the 6 months prior to intervention in either eye. Axial growth across the 2-year period of intervention was 0.11 mm in the intervention eye versus 0.15 mm in the control eye. Refractive error change across the same period was -0.23 D in the intervention eye and -0.27 D in the fellow eye. Axial growth and refractive error during the final 6 months without intervention did not change in either group. The overall change in axial growth was greater in the control eye than the intervention eye, whereas the reduction in refractive error was comparable. Axial length measures are comparable and repeatable under pre- and post-cycloplegic conditions. Refractive error measures are comparable and repeatable at discrete time intervals after the instillation of a cycloplegic agent.

Background: There is growing evidence that HMG-CoA reductase inhibitors, otherwise known as the statin family, can exert pleiotropic effects in many areas. Of these, potential neuroprotective effects have gained significant attention. It is well established that microglia, the brain's resident phagocytes, play a pivotal role in the pathogenesis of neuroinflammation and neurodegeneration. This process is thought to be, in part, due to the presence of chronically activated microglia. In this study, we investigated the potential neuroprotective properties of HMG-CoA reductase using an in vitro model of inflammatory cell activation and in vivo models of posterior uveitis and multiple sclerosis. The focus of this study was to investigate the effect of simvastatin on the microglial cell and its activation products. Emphasis was placed on the production of reactive species released by this cell type and the subsequent damage these cause to biological macromolecules. Methods: The microglial cell line BV2 were treated with simvastatin (1μM; 2 to 120 h), in vitro before being activated for 48 hours with a pro-inflammatory mix of LPS, TNFa and IFNg. Supernatants were taken and nitric oxide levels measured using the Griess assay. The animal model of posterior uveitis, experimental autoimmune uveoretinitis (EAU), was established in wild type C57BL/6 mice through subcutaneous injection of IRBP1-20. Mice were treated orally with simvastatin at 50, 75 or 100 mg/kg. Fundus images were taken before and after treatment administration for evaluation of clinical ocular pathology. Retinal flat mounts were prepared from simvastatin treated mice to assess cellular infiltrates. Experimental autoimmune encephalomyelitis (EAE), an in vivo model of MS, was induced by rMOG subcutaneous immunisation. The effect of simvastatin treatment was assessed clinically and by immunohistochemical analysis of tissue sections to determine cellular infiltrates and levels of oxidative damage to biological macromolecules, consistent with those assessed in EAU. Results: The levels of nitric oxide produced by microglial cells were significantly reduced when exposed to a pre-incubation of simvastatin for 3 48 hours, compared to cells receiving the pro-inflammatory mix alone. In EAU, fundoscopic analysis revealed that high-dose simvastatin halts clinical disease progression in IRBP1-20 induced posterior uveitis. Retinal flat mounts prepared from these cohorts showed a significant decrease in the expression of the innate immune cell surface receptor CD11b. Additionally, histological examination of eye sections displayed a significant reduction in lipid peroxidation as revealed by the marker 4-Hydroxynonenal (4HNE), nitrosylated proteins, as measured by 3-nitrotyrosine and oxidised DNA/RNA as determined by 8-OHdG. In line with this study, results from our EAE model demonstrated an important role for microglial cell number in disease, whilst also providing evidence of simvastatin decreasing oxidative damage to macromolecules in areas of extensive pathology. Discussion: These data provide evidence to support the notion that microglial cell activation may contribute to the pathogenesis of neuroinflammatory disease and that statins may attenuate damage through their ability to inhibit the production of reactive species. Further to this, we provide therapeutic, chemical and physical evidence that simvastatin can provide protection against 1) nitric oxide production in an inflammatory environment 2) clinical disease attenuation in EAU and EAE and 3) reduction in peroxynitrite levels in vivo. Collectively, these data provide evidence that statins may attenuate microglial cell activation by ways of inhibiting the production of reactive species. Thus, the evidence presented in this thesis points to the importance and potential use of simvastatin therapy as a neuroprotective therapeutic agent.

The WHO estimates 10 million people in the World are blinded by corneal disease. For many conditions, transplantation of a donor cornea may restore vision. However, there is a global shortage of suitable tissue and a high risk of rejection. The potential of stem cell therapies and tissue engineering approaches to address this significant unmet clinical need was investigated. Limbal epithelial stem cells (LESC) maintain the epithelium, the outermost layer of the cornea, and can be successfully transplanted to restore vision. However, when scarring occurs, transplantation of corneal stroma is required. Human corneal stromal stem cells (CSSC) are involved in stroma maintenance and have previously been shown to restore transparency in cloudy mouse corneas without rejection. This study investigated the development of a surgeon friendly tissue equivalent (TE) for the therapeutic delivery of CSSC and LESCs. For the first time, human corneal rims were rendered transparent for imaging under the iDISCO protocol. CSSC were successfully isolated and characterised with mesenchymal stem cell (MSC) properties confirmed. RAFT-TE, a potential artificial ocular surface, has been extensively investigated by our group using research grade collagen (First Link; not suitable for clinical use). In this thesis, a comparative study was performed to show that Koken collagen (Good Manufacturing Practice compliant) is a suitable replacement for research grade collagen as it did not compromise RAFT-TE properties. Next, co-culture conditions for LESC and CSSC in RAFT-TE were optimised. First, the idea of co-delivering CSSC together with LESCS to the surface of RAFT-TE as a mixed population was trialled. This resulted in unexpected epithelial cell peeling. To overcome this challenge, CSSC were successfully cultured for the first time inside Koken RAFT-TE. CSSC formed cell clusters, remodelled the matrix, and migrated to the surface of the TE. It was also shown that they can be induced to differentiate towards the keratocyte lineage inside the TE. This work highlights the importance of considering clinical manufacturing standards early in the process of development. Overall, it provides valuable insights to develop personalised autologous therapies and off the shelf allogeneic strategies for restoring vision in patients with corneal blindness.

Diabetic Retinopathy (DR) is a leading cause of visual impairment but its pathophysiology is not well understood. Moderate/severe non-proliferative DR (NPDR) is characterised by the presence of three features: deep haemorrhages (DH), venous beading (VB) and intraretinal microvascular abnormalities (IRMA). They are grouped together as risk factors for progression to sight threatening DR. It remains unclear whether these individual features have similar pathophysiologies, and whether they respond equally to anti-VEGF, a new therapy for NPDR. Optomap images of 504 NPDR eyes were examined to evaluate the distribution and prevalence of these three features. DNA samples from 199 patients with NPDR and 397 diabetic patients with no DR were collected. The genotype of specific candidate genes were evaluated in patients with DR, VB or IRMA vs no DR. Optical coherence tomography angiography (OCTA) images of 30 patients were examined for focal ischemia adjacent to VB and IRMA. The responses of these three features to anti-VEGF treatment were also re-examined in the images from the CLARITY trial. DH were present in most cases of NPDR. VB and IRMA did not always co-exist in the same eye and when they do, were often in different locations. VEGF, TGFb-1 and ARHGAP22 polymorphisms (ischaemia-related genes) were more common in patients with DR and IRMA, but not VB. Areas of focal ischaemia were more frequently adjacent to IRMA than to VB. DH and IRMA responded to anti-VEGF therapy but VB did not. These findings suggest that VB and IRMA do not share the same pathophysiology, and that IRMA are more likely to be ischaemic driven. Nonetheless, some IRMA may not be driven by ischaemia as they have no adjacent ischaemia on OCTA, do not carry the specific genotype, and do not respond to anti-VEGF. Furthermore, patients with VB may not benefit from anti-VEGF therapy.

Aims To investigate the clinical efficacy of primary selective laser trabeculoplasty (SLT) as initial therapy in newly-diagnosed treatment-naïve open-angle-glaucoma (OAG)/ ocular hypertension (OHT) patients. To also investigate patient-reported outcome measures related to health-related quality of life (HRQL) between primary SLT and topical medication. Methods Pre-specified and post-hoc analyses performed using data derived from the Laser in Glaucoma and Ocular Hypertension ('LiGHT') Trial, a multi-centre randomised-controlled trial. Results 718 patients (1235 eyes) were randomised: 356 patients (613 eyes) were allocated to SLT (Laser-1st pathway) and 362 patients (622 eyes) to medical treatment (Medicine-1st pathway). Early absolute IOP-lowering following primary SLT was no different between OHT and OAG eyes (adjusted mean difference = -0.05mmHg; 95% confidence interval (CI) -0.6 to 0.5mmHg; p=0.85). No difference was noted in early absolute IOP-lowering between topical medication and primary SLT (adjusted mean difference = -0.1mmHg; 95% CI, -0.6 to 0.4mmHg; p=0.67). At 36-months, 536 eyes (87.7% of 611 eyes) of 314 patients (88.5% of 355 patients) were available for analysis in Laser-1st pathway. 74.6% of eyes (400 eyes) treated with primary SLT achieved drop-free "disease-control" at 36-months; 58.2% (312 eyes) following single SLT. 6 eyes of 6 patients experienced immediate post-laser IOP spike with 1 eye requiring treatment. 115 eyes of 90 patients received repeat SLT during the first 18 months of the trial. Repeat treatment maintained drop-free IOP control in 67% of these eyes for a subsequent 18 months, with no clinically-relevant adverse events. At 36-months, there was no significant difference in all HRQL measures between the treatment arms, including EQ-5D-5L (adjusted mean difference = 0.01; 95% CI, -0.01 to 0.03; p=0.23). Conclusions This work supports primary SLT to be a safe and clinically effective alternative to topical treatment that could be offered as a first-line IOP lowering treatment to patients with OAG or OHT.

Age-related macular degeneration (AMD) is the leading cause of visual impairment in the developed world among people over 50 years of age. Although AMD is clinically characterised by the presence of drusen, subretinal drusenoid deposits (SDD) have also been recognized as a distinct morphological feature that confers increased risk of developing advanced AMD. To date, there has been a lack of validated biomarkers that can capture early changes in visual function that strongly correlate to the anatomical alterations which also include SDD phenotype. This thesis aimed to explore functional and structural markers to differentiate between healthy eyes (n=11) and intermediate AMD (iAMD) with SDD (n=11) and without SDD (n=17) and non-foveal atrophic AMD (n=11). Firstly, I assessed scotopic thresholds using a novel dark-adapted chromatic (DAC) perimeter, in healthy aging and in varying AMD disease. Individuals with SDD had depressed retinal sensitivity centrally, particularly inferiorly and nasally. Functionally, eyes with SDD were comparable to eyes with non-foveal atrophy, but structurally differed in outer nuclear layer (ONL) and total retinal volumes and thicknesses. Importantly, only rod-mediated tests were able to distinguish iAMD with and without SDD. Another aim of this thesis was to explore the efficacy of 670nm light on aging and AMD. Although an improvement in scotopic thresholds was observed in healthy aged eyes (n=4) compared to younger eyes (n=5), a pilot study conducted in 40 participants over the age 55 years (12 control, 28 with intermediate AMD) refuted any clinical benefit. In conclusion, this thesis supports the need to re-classify the AMD severity scale by incorporating eyes with SDD as a separate group. This phenotype should be sub-analysed in clinical trials evaluating potential prophylactic agents to delay the progression. Scotopic sensitivity offers diagnostic value, but rod intercept time offers both prognostic and diagnostic value as candidate biomarkers.

The ability of humans to perceive polarized light was first documented by Haidinger in 1844, who discussed the entoptic phenomenon of what was later to be known as Haidinger's brushes. Until recently, Haidinger's brushes were believed to constitute the full extent of human polarization sensitivity. It is now known that the human visual system is capable of detecting visual stimuli modulated solely by light polarization (Misson et al., 2015, Temple et al., 2015). Misson and Anderson (2017) developed the technique of polarization pattern perception (PPP) and showed that human polarization sensitivity was significantly more acute and quantifiable than previously thought. Furthermore, like its related phenomenon of Haidinger's brush, they showed that PPP is confined to the macula, and matches the spectral characteristics and distribution of the macular pigments. The known protective functions of macular pigments, and the association of its deficiency with susceptibility to macular degeneration, make a measure of PPP potentially useful as a clinical screening tool for at-risk individuals and for the early detection of macular disease. Normative polarization pattern perception values had not yet been established in humans, and the repeatability of the technique was yet to be explored. The effect of age and variations in corneal and macular characteristics on PPP are also currently unknown. The principal aim of this research project is to quantify normative monocular sensitivity values for PPP in healthy individuals and address these gaps in the field. Grating stimuli were displayed in polarization-only contrast on a delaminated LCD screen. This technique was shown to give rapid, inexpensive, quantifiable data, which, with some development, could be used to assess and monitor macular function and screen at risk individuals. PPP values across a range of ages are presented and discussed, with reference to each participant's corneal and macular characteristics. The monocular polarization pattern sensitivity for healthy participants aged 19-59 years was 5.17, which equates to an average ability to discriminate stimuli differing by 8 degrees. This provided evidence in support of the human ability to perceive polarized light to a much higher degree than previously expected, and was similar to data from previously published pilot studies developing the technique (Misson et al., 2019, Misson and Anderson, 2017). There was no significant change in human PPP across the age range 19-59 years. Test-retest measures showed a positive correlation, but the overall repeatability of the technique would need improvement if it were to become a useful clinical measurement. A significant positive correlation between MPOD and PPP was found, which together with the lack of influence from variations in other ocular characteristics (age, refraction, central foveal thickness, central corneal thickness, corneal retardance, corneal birefringence and ocular dominance) make a measure of PPP potentially highly beneficial for macular assessment.

Age-related macular degeneration (AMD) is a sight-threatening disease caused by degeneration of the macula. Late disease presents as neovascular AMD (NV-AMD) or geographic atrophy (GA). Advances in angiogenesis research have led to the development of treatments for patients with NV-AMD. However, there are no clinically available treatments for GA. Placental growth factor (PlGF) is a seemingly dispensable member of the VEGF family, in normal physiology. However, it has been shown to propagate retinal pathology in animal models. Reports also suggest that PlGF can induce epithelial cell dysfunction and its inhibition has shown efficacy in a mouse model of GA. In this thesis, it was hypothesised that PlGF can induce dysfunction of retinal pigment epithelium (RPE), and its inhibition can attenuate pathology in a sodium iodate (NaIO3) model of GA. In chapter 2 PlGF and its receptors were detected in human macula in late AMD and could be detected in cultured RPE. PlGF treatment of RPE increased permeability via ERK1/2 signalling and altered ZO-1 localisation. In chapter 3 the anti-PlGF antibody THR-317 inhibited the effects of PlGF on RPE, however could not protect against hypoxia or oxidative stress. In a co-culture model, THR-317 improved barrier resistance without altering the choroidal phenotype of endothelial cells. In chapter 4 PlGF inhibition provided some protection against RPE degeneration in the NaIO3 model. However, it did not protect against secondary complications and losses in visual function. In conclusion, while PlGF and its receptors may be present in the human macula and can regulate RPE permeability, targeting PlGF is unlikely to be protective during GA. However, this should not detract from the value of PlGF as a target during outer retinal neovascular disease. The dispensable role for PlGF in maintenance of choroidal phenotype suggests it is a potentially safer target than the vasotrophic factor VEGF-A.

The current system of retinal screening is manual; It requires repetitive examination of a large number of retinal images by professional optometrists who try to identify the presence of abnormalities. As a result of the manual and repetitive nature of such examination, there is a possibility for error in diagnosis, in particular in the case when the progression of disease is slight. As the sight is an extremely important sense, any tools which can improve the probability of detecting disease could be considered beneficial. Moreover, the early detection of ophthalmic anomalies can prevent the impairment or loss of vision. The study reported in this Thesis investigates computer vision and image processing techniques to analyse retinal images automatically, in particular for diabetic retinopathy disease which causes blindness. This analysis aims to automate registration to detect differences between a pair of images taken at different times. These differences could be the result of disease progression or, occasionally, simply the presence of artefacts. The resulting methods from this study, will be therefore used to build a software tool to aid the diagnosis process undertaken by ophthalmologists. The research also presents a number of algorithms for the enhancement and visualisation of information present within the retinal images, which under normal situations would be invisible to the viewer; For instance, in the case of slight disease progression or in the case of similar levels of contrast between images, making it difficult for the human eye to see or to distinguish any variations. This study also presents a number of developed methods for computer analysis of retinal images. These methods include a colour distance measurement algorithm, detection of bifurcations and their cross points in retina, image registration, and change detection. The overall analysis in this study can be classified to four stages: image enhancement, landmarks detection, registration, and change detection. The study has showed that the methods developed can achieve automatic, efficient, accurate, and robust implementation.

Aims: To investigate the association between subjectively and objectively measured environmental and lifestyle factors on eye growth. Rationale: Emmetropisation is the process of visual regulation of eye growth towards an optimal refraction. Disruptions in emmetropisation have been thought to lead to the development of myopia which has increased in prevalence worldwide. It is a condition which brings significant socio-economic burden and sight-threatening complications. This has led to a significant interest in furthering our understanding of the influential factors driving eye growth, which is the focus of this thesis. Methods: Two age cohorts were recruited, 226 aged 7-12 years and 87 aged 18-25 years. 55.3% (n=173) were followed up longitudinally after 12 months and 18.5% (n=58) after 24 months. Time spent outdoors was measured by both subjective and objective methods, including questionnaires, a wrist-worn actigraphy device and a surrogate biomarker, Conjunctival UV autofluorescence (CUVAF). Other lifestyle factors were assessed via questionnaires. Results: Significant differences in objectively measured light exposure were found between seasons and day of the week. UK children were found to spend more time outdoors on weekdays than weekends. This study has shown for the first time a lack of CUVAF in UK children and a low prevalence of CUVAF in UK young adults. This suggests that CUVAF may not be a suitable surrogate measure of time outdoors in the UK. A normative dataset of sleep patterns of UK children is presented and has shown emerging evidence that sleep/wake cycles are altered in myopes. Urbanisation, BMI and birth weight were found to be significantly associated with eye growth, however all other factors were found not be to significant. Conclusions: The role of illuminance and eye growth is a prominent area of current research and this study has provided valuable data on environmental risk factors in the UK.

In light of new data regarding pathology of multiple sclerosis (MS), more research is needed into the vascular aspects of the disease. Demyelination caused by inflammation is historically thought of as the main cause of disability in the disease. Recent studies, however, have suggested that MS is in fact a spectrum of overlapping phenotypes consisting of inflammation, oxidative damage and hypoperfusion. The microvasculature plays an important role in all of these pathogenic processes and its dysfunction may therefore be of crucial importance to the development and progression of the disease. This thesis focuses on investigating the microvasculature of the retina as a surrogate for the brain by assessing the vascular structure, blood flow dynamics and oxygen transfer of the retinal blood vessels in secondary progressive multiple sclerosis (SPMS). Studying the retinal microvasculature using a multimodal imaging approach has allowed us to develop a more detailed understanding of blood flow in MS and to identify new imaging markers for trials into neuroprotective drugs in MS. The work done in this thesis demonstrated; i) a higher rate of retinal microvascular abnormalities in MS which progresses with disease severity, ii) evidence of retinal vascular remodelling in SPMS and iii) changes in blood velocity and flow in the retina in SPMS. These observations pave the way for future investigations into the mechanisms of vascular alterations and vascular dysfunction in MS, and provide a set of imaging markers to further explore other cerebrovascular diseases through the retina.

Retinal ischemia is a key feature in sight threatening eye diseases such as retinopathy of prematurity (ROP) or diabetic retinopathy (DR). Whilst the longterm consequences of ROP and DR are well described, our understanding of the early pathobiological events is much less clear. In particular cellular changes during the early stages of these disease are poorly studied so far. Most of our current insights about the pathobiological events are derived from animal models, with little confirmation in humans. The aim of this thesis is therefore to fill this gap by carefully characterizing vascular features and cellular damage in post-mortem tissue from patients with early stages of ROP and DR. To better understand the early cellular events in ROP, post-mortem eyes from postnatal, premature infants were collected. Different vascular phenotypes could be distinguished in whole mount retinal vasculature stains. Differences in branching profiles and capillary free zone morphology implicated different levels of oxygen exposure in the infants studied. Furthermore, characterizing a hyperplastic ridge, distal to the edge of the growing vascular plexus, revealed a correlation between the retinal astrocyte marker PAX2 and increased expression of vascular endothelial growth factor (VEGF). This suggests that retinal astrocytes make an important, but so far overlooked, contribution to the pathology in ROP. To better understand early stages of DR, eyes from diabetic donors without diagnosed DR were collected. Whole mount imaging revealed an indistinguishable retinal vasculature phenotype compared to controls, confirming the absence of DR. However, detailed quantification of vessel profiles on retinal cross sections demonstrated a 5-fold increase in acellular (and presumed non-perfused) capillaries (7-fold in the deeper plexuses) in retinas from diabetics without DR. Interestingly, localized capillary dropout of individual capillaries in the deeper plexuses did not correlated with a reduction of cells in the vicinity of the non-perfused capillaries. Instead, there was a panretinal loss of cells in the inner nuclear layer (INL) in diabetic retina, suggesting an ischemia independent mechanism for INL cell loss in diabetic retina.

Inherited retinal dystrophies (IRDs) are a large group of genetic diseases that lead to retinal degeneration and represent a major cause of vision impairment or blindness. The high genetic heterogeneity of IRDs hinders a broad application of gene-specific therapies. There is an unmet need for therapies that can target common pathological mechanisms, regardless of the genetic cause. MicroRNAs (miRNAs) are important players in retinal biology and my group has demonstrated that miR-204 has a pathogenic role in human IRDs. Due to their pleiotropic actions, miRNAs represent promising therapeutic tools. On this basis, I hypothesized that the modulation of miR-204 levels, as well as other miRNAs, could represent a valid approach to tackle retinal degeneration. The first part of my thesis elucidates the potential of miR-204 administration as a therapeutic approach for IRDs. I found that administration of miR-204 by an adeno-associated viral (AAV) vector at patient-relevant stages of disease progression led to a long-term preservation of retinal function in a mouse model for a dominant form of Retinitis Pigmentosa (RP). Interestingly, transcriptome analysis revealed that miR-204 effect is mediated by dampening pathological processes shared by different IRDs (e.g. innate immune response). The second part of my work was focused on identifying additional miRNAs that can exert a protective action in IRDs in a gene-independent manner. Using a High Content Screening (HCS) approach I tested 560 miRNAs in a model of oxidative stress-induced retinal degeneration, the light-damage in cone-like cells (661W). As a result, I found that miR-429 significantly preserved 661W viability during photo-stress. Further analysis revealed that miR-429 overexpression increases the activated form of the pro-survival AMP-activated protein kinase (AMPK), recently demonstrated to be protective during retinal degeneration. Overall, the results of this thesis indicate that modulation of miRNAs can be a promising approach to develop mutation-independent treatments for IRDs.

Worldwide, the burden of diabetes mellitus (DM) and its complications are becoming a major public health concern, particularly in low-middle income countries (LMICs), where 80% of people with DM (PwDM) reside. The main sight-threatening complication of DM is diabetic retinopathy (DR). Early detection through diabetic eye screening (DESPs) is fundamental to reduce the risk of avoidable DR-related blindness or visual impairment. Since the introduction of systematic DESPs in high-income countries such as the UK, DR is no longer the leading cause of blindness among working age adults. The main aim of this thesis is to enable capacity building for DESPs in LMICs, focusing on two low-resource settings, Vietnam and Bangladesh. Identifying gaps in national level DR policies and planning in LMICs is crucial to inform and enact DR policies. A mixed methods approach (quantitative and qualitative analyses) was used to evaluate the feasibility of DESPs in Bangladesh and Vietnam, and enabling capacity building in both settings was key. Gaps in DR policy processes and DESP implementation in LMICs exist, especially in low-income countries. To build capacity for DESPs in Vietnam nurses were trained to perform DR screening and grading; however, their diagnostic test accuracy was low, demanding further exploration and evaluation. The think-aloud study assessed the decision-making processes of novice and expert DR graders to improve DR training protocols in Vietnam. Compared to novices, expert graders were more likely to systematically grader fundus images and use all available grading tools, such as the red-free filter to view more discrete signs of DR. Furthermore, high costs and distance to hospitals were two main barriers to DR screening and treatment uptake in Bangladesh and Vietnam; therefore, intervention strategies to improve uptake are necessary. To ensure DESPs are sustainable, fully integrated and universally accessible, careful planning, development and evaluation are required.

Dominant Optic Atrophy (DOA) is the most common inherited optic neuropathy in the UK, characterised by the preferential loss of retinal ganglion cells (RGCs) and progressive blindness. 60-70 % of DOA patients harbour mutations in the OPA1 gene, encoding a mitochondrial protein that regulates mitochondrial morphology, bioenergetics and mitochondrial DNA (mtDNA) quality. Currently, DOA has no therapeutic options and the mechanisms driving RGC degeneration are poorly understood. In this study, a biobank of induced pluripotent stem cells iPSCs (iPSCs) encompassing the clinical and genetic DOA spectrum was created using patient-derived OPA1 mutant fibroblast cell lines, and CRISPR/Cas9 gene editing to generate isogenic cell lines. RGC differentiation was optimized and characterised in 2D and 3D in vitro methods, demonstrating expression of RGC-associated genes including BRN3B and ISL1. OPA1 mutant iPSCs showed no differentiation deficit compared to wild-type control cell lines, exhibiting comparable expression of all relevant markers. 2D-RGCs demonstrated enrichment of neuronal associated markers, including ELAVL3 and TAU, when compared to 3D retinal organoids. Phenotypic analysis demonstrated significant deficits in respiration, ATP production and increased mtDNA mutation in fibroblasts, iPSCs and 2D-RGCs compared to isogenic controls. Characterisation of mitochondrial stress through induction of stress associated gene expression demonstrated significant levels of upregulation in iPSCs, however, 3D- and 2D-RGCs exhibited fewer upregulated genes indicating that mitochondrial stress may be a cell type specific response. Importantly, correction of patient-derived iPSCs restored mitochondrial homeostasis, demonstrating that restoration of WT OPA1 expression is able to mitigate mutant associated phenotypes. Thus, an OPA1 mutant iPSC biobank has been established encompassing the clinical disease spectrum, enabling effective in vitro modelling to establish RGC specific disease mechanisms. OPA1 mutant RGCs demonstrate significant reductions in mitochondrial homeostasis, including reduced bioenergetic output and mtDNA quality. This work provides a platform for further investigation of OPA1-mediated disease mechanisms and therapeutic design.

The overarching aim of this thesis is to develop a novel glaucoma drainage device that meets the '10-10-10' goal for glaucoma treatment: specifically, a surgical intervention that can reduce the intraocular pressure to 10mmHg, can be implanted in 10 minutes and lasts for 10 years. The thesis encompasses elements from the research and design phase of device development to the preclinical and clinical testing of the device. The doctoral proposal is divided into four chapters. The first describes the role of minimally invasive surgery in the management of glaucoma; the second elucidates the physiochemical factors that impact the outflow facility of microtubes used in minimally invasive glaucoma surgery; the third presents a novel way to titrate aqueous outflow from a novel glaucoma microstent; finally, the fourth determines the bioburden, extractables and leachables of a novel glaucoma device, along with its implications for in vivo biocompatibility.

Microglia are a tissue-resident immune cell of the central nervous system known to possess functions involved in immune surveillance and tissue homeostasis. Transcriptomics has characterised microglia and enabled discovery of microglial heterogeneity in addition to core microglial transcriptional programmes. However, investigation of microglia during severe inflammatory contexts has been challenging because no markers reliably discriminate them from the monocyte populations that ingress during inflammation. Nonetheless, candidate markers have been identified; these show promise in specific microglial identification yet remain to be widely validated. Within the literature, there are conflicting reports on how microglia regulate or promote inflammation depending on the tissue insult. However, it is well-recognised that the homeostatic state of microglia is altered yet it remains unknown if this state is restored post-resolution. Furthermore, understanding the plasticity of microglial responses to both acute and persistent inflammation within the eye will help to determine the extent to which different pathways are perturbed. The purpose of this thesis was to investigate the transcriptional changes that occur in retinal microglia in response to inflammation and whether the homeostatic threshold remains perturbed after acute and/or chronic inflammation. The data presented herein demonstrates how an ultra-low input mRNA-Seq approach was optimised and validated to permit transcriptomic assessment of low numbers of cells isolated from individual retinas. The Cx3cr1CreER:R26-tdTomato mouse line was then validated as microglial-specific during inflammation. mRNA-Seq was utilised to profile the temporal kinetics of the microglial transcriptome in the acute endotoxin-induced uveitis (EIU) model. Restoration of the microglial homeostatic state was confirmed, and key marker changes were orthogonally validated. Furthermore, C5AR1 was validated as a marker for differentiating microglial subsets during inflammation. The next steps have begun to examine microglial behaviour in experimental autoimmune uveitis (EAU), a model of chronic inflammation, and new approaches are being optimised to better understand tissue heterogeneity.

Retinal imaging allows assessment of ocular health, and is important for the detection and monitoring of retinal diseases. One such device for imaging the retina is the ultra-wide field of view scanning laser ophthalmoscope (UWFoV-SLO), manufactured by Optos plc, which is capable of imaging up to 200 degrees of the retina in a single scan. This thesis details work in two areas, both relating to the image processing of UWFoV-SLO images. The first is the investigation of automated image quality assessment algorithms for UWFoV-SLO images. A novel image quality metric (referred to as the GVC metric) was developed, which is based on a textural measure of image patches that contain retinal vasculature. This metric is demonstrated to correlate with the assessment of image quality as graded by experts. The GVC metric was applied to images captured at a UWFoV-SLO manufacturing facility, and timeseries analysis of this data discovered a number of potential system modifications that could improve the quality of manufactured devices. These included the correction of a software bug that was impairing image quality, and to install all manufactured devices with a detector that captured higher quality images. These observations were further confirmed with forced-choice preference testing with expert assessors of image quality. The second investigation concerns the automated localisation of two retinal landmarks (the optic disc and fovea) in UWFoV-SLO images with convolutional neural networks. On a test set of 1485 images, optic disc localisation accuracy of 96.70% less than 1 optic disc radius from the ground truth was achieved, and 93.27% of fovea predictions were less than 1 optic disc radius from the ground truth. It is also shown that the laterality of the image (whether it is of the left or right eye) can be reliably inferred from the landmark coordinates. Finally, three methods to automatically detect unreliable landmark predictions are presented. These are based on prior knowledge of the spatial distribution of landmarks --- two methods employ Gaussian mixture models, and the third applies a threshold to the Euclidean distance between the predicted landmark coordinates.

The endothelial cells that line retinal blood vessels play vital roles in angiogenesis and microvascular barrier formation and are supported by retinal mural cells. When these cells become dysfunctional during diabetes, they can lead to sight threatening complications such as diabetic retinopathy (DR) and diabetic macular oedema (DMO). Gene expression changes that underlie endothelial dysfunction in diabetes have been studied by PCR and bulk RNA sequencing methods. These methods give the impression of uniform expression across all cells within a sample; however, great variability has been found in gene expression between individual cells even within a relatively homogeneous population. To uncover this cell-to-cell variation a new technology known as single cell RNA sequencing (scRNA-seq) has been developed in recent years, which identifies gene expression within single cells. Therefore, the aim of my PhD project was to use scRNA-seq to better understand the biology of the retinal endothelium and retinal vasculature and how this becomes dysfunctional within a diabetic milieu. A single cell RNA-sequencing workflow was established to uncover novel insights into retinal vascular biology including the expression of novel alternative splice variants, endothelial heterogeneity, patterns of active transcription factor expression and development of a more complete model of the inner blood retinal barrier (iBRB). Key changes in gene expression triggered by high glucose (HG) were related to the upregulation of inflammatory and proliferation-related genes with many genes not previously linked to these processes within hyperglycaemia. We found that during experimental diabetes, retinal endothelial cells adopt an angiogenic phenotype even during very early diabetes in the absence of overt hypoxia and a neovascular response. Retinal mural cells were found to switch to a more migratory phenotype which is in agreement with recent publications that have shown that pericyte migration is a key event in DR progression. In addition, numerous novel high glucose and diabetes specific gene expression and TF changes were found which warrant further investigation. Current therapeutics for DR mainly focus on the end stages of the disease process and can be ineffective in many patients with some adverse side effects. Through this study, we have identified many novel potential therapeutic targets for the early-stage treatment of diabetic retinopathy that could potentially slow or prevent the development of the disease. This paves the way for numerous studies to further investigate these novel findings to develop more effective therapeutics against DR and DMO.

Introduction: There is increasing evidence that vitreomacular adhesion (VMA) has a detrimental effect on the clinical outcome of certain retinal diseases, including neovascular age-related macular degeneration (nAMD). Conversely, the presence of vitreomacular separation or posterior vitreous detachment may be protective. This thesis focuses on two of the potential mechanisms by which the vitreous may influence retinal disease, namely vitreous oxygenation (pO2) and VMA. Methods: Vitreous pO2 was investigated using a MRI technique that is able to measure vitreous pO2 in vivo. VMA was investigated via retrospective and prospective studies using ocular coherence tomography scans that were correlated to visual acuity (VA), central macular thickness (CMT) and the number of intravitreal treatments. Results: Vitreous pO2 in non-vitrectomised eyes pooled from two studies was 12.5±7.1 mmHg when measured with MRI. Following vitrectomy, vitreous pO2 increased from 13.2±5.8 to 34.5±8.0 mmHg in non-ischaemic eyes. In cases of ischaemic central retinal vein occlusion, vitreous pO2 was 50.8±24.3 mmHg. No evidence was found showing that VMA adversely affects VA, CMT or the number of intravitreal injections required, in the clinical treatment of nAMD. Conxluaion: MRI is able to measure vitreous pO2 in vitrectomised and nonvitrectomised healthy eyes with reasonable accuracy. The finding of high vitreous pO2 in eyes with ischaemic CRVO is unexpected and warrants further investigation. Unlike some previous published series, our results do not support the hypothesis that VMA negatively influences nAMD outcomes.

Keratoconus is a corneal disease characterized by the affected tissue adopting a conical shape, leading to loss of vision and compromised structural integrity. The disease is manageable in its early stage with lenses or glasses and can be treated in its later stages with surgery or crosslinking. The underlying cause of the disease is still poorly understood, a model would aid the exploration of this disease. In this study, primary stromal cells harvested from healthy and keratoconic corneas using a variety of extraction methods were cultured in compressed collagen gels and serum-free medium containing retinoic acid to simulate the cornea's natural environment. The survival of stromal cells and their gene expression was assayed, paying special attention to genes related to differentiation and ECM maintenance. Among healthy cells, differences were identified between limbal and central population while central keratoconic cells more closely resembled healthy limbal cells. Use of retinoic acid as a medium supplement allowed for the culture of cells in serum-free conditions and caused gene expression that resembled in vivo behaviour. The final addition of ECM in the form of curved, compressed collagen gels to this culture system caused the stromal cells to radically change their behaviour and reaction to retinoic acid. Overall, this study identified a stromal tissue model as well as the multiple considerations in the construction of such a model. The use of this model in keratoconus research may lead to breakthroughs in attempts to better understand the disease.

The majority of previous research investigating the impact of low vision on the completion of activities of daily living (ADLs) have examined visual impairment as a whole. The aim of this thesis was to provide a comprehensive overview of ADLs to determine what the most difficult areas are for people with Retinitis Pigmentosa (RP), a particular type of visual impairment. This research was achieved through both self-report questionnaire and objective analysis of human movement. 681 participants (570 with RP) were examined throughout this research. Identified through self-report, at the objective level, the most difficult ADLs amongst those with RP was mobility. In particular, at the goal level, this was identified as mobility outdoors (experimental chapter 1). Further, at the task level, orientation and walking around safely without bumping into things and tripping over or stepping off something were identified as most difficult (experimental chapter 2). Those who support people with RP perceived most of the ADLs significantly more difficult to complete (for those with RP), with greatest difference in perceptions between two groups being practical tasks. When assessing balance through measuring postural control (experimental chapter 3), those with RP showed similar postural control to those with normal vision when standing on a firm surface, regardless of the vision condition (eyes open or eyes closed). However, when standing on a foam surface with eyes open, the reduction in postural control among people with RP, compared to those with normal vision, highlighted the added importance of the somatosensory information to maintaining standing balance for those with RP. However, it was only apparent when the somatosensory system was disturbed. The examination of gait among people with RP (experimental chapter 4) demonstrated that those who used a mobility cane adopted a cautious walking behaviour in both level walking and obstacle crossing tasks. Such cautious behaviour was not evident for people with RP who did not use a cane, or for the normally sighted individuals. This thesis is the first to provide a comprehensive overview of self-report difficulties among those with RP. Findings also demonstrate the importance of maintaining adequate foot (somatosensory) and eye (vision) health for those with RP to regulate balance control. The additional mobility training for those with RP who use a cane is necessary for their walking gait. Furthermore, the support from the carers should reflect the needs of those with RP, which helps them with their independence in completing ADLs rather than overprotecting them.

The ageing population will become one of the biggest issues affecting Singapore in the near future. Ophthalmic practitioners will need to be ready to deal with an increased prevalence of glaucoma, cataract, age related maculopathy and even presbyopia. As presbyopes lose their ability to accommodate at near vision, visual aids such as progressive lenses, bifocal lenses, reading glasses, monovision contact lenses and multifocal contact lenses are prescribed to help them with reading difficulties and improve their daily lives. Interestingly, an international survey in 2011 revealed zero percent soft multifocal contact lenses was prescribed in Singapore for presbyopia correction. Although there are improvements in multifocal lens design and material, no new research being conducted to investigate the presbyopic lens fitting status in Singapore. Nonetheless, recent studies have shown an increased in multifocal contact lenses prescribing trends, perhaps reflecting not just the availability of newer multifocal contact lenses, but also improvement in practitioners' confidence and knowledge in multifocal contact lenses. However, in spite of the available guides on choosing multifocal contact lenses, there is no comprehensive way to help the practitioner in selecting the best option for an individual. As such, an examination of the simplest way of predicting the most suitable multifocal lens for a patient will only enhance and add to the current evidence available. A survey was conducted to understand the Singaporean practitioners' attitude towards soft multifocal lenses and its prescribing trend. In this survey, an increase in the rate of soft multifocal contact lens fitting was observed, the perception of the unavailability of an 'ideal' multifocal contact lens, and increased chair time in fitting soft multifocal contact lenses were identified as significant barriers. However, enablers such as the increased in practitioners' motivation, confidence and proactiveness in fitting soft multifocal contact lenses were gathered. Additionally, this study aimed to compare the relative performance of three daily-replacement soft contact lenses for presbyopic correction in an optometric practice population in Singapore. The three daily-disposable multifocal contact lenses included in this study were 1-day Acuvue® Moist Brand Multifocal Contact Lenses for Presbyopia (Johnson & Johnson Vision Care, Jacksonville, FL), Clariti 1-day Mulitfocal (Cooper Vision, NY) and Dailies AquaComfort Plus Multifocal (Alcon, Fort worth, TX). In this crossover study design, 35 presbyopic participants with myopia were fitted in a random order with three different types of multifocal contact lens. After 1 month, visual performance was quantified by high contrast distance, intermediate and near visual acuity, defocus curve under photopic and mesopic conditions, reading speed, Near Activity Visual Questionnaire rating and Photographic questionnaire for Photic Phenomena. The results showed comparable levels of binocular distance, intermediate and near visual acuity achieved with the three different types of multifocal contact lens at 1-month follow up. However, a better distance acuity at distance under mesopic condition for AquaComfortPlus. In terms of subjective participant lens preference, nine participants (26%) preferred Moist multifocal, 16 participants (46%) preferred Clariti multifocal and 10 participants (28%) preferred AquaComfortPlus multifocal. However, lens preference was not related to demographic factors relating to age, gender, refractive error and the magnitude of reading addition or physiological characteristic such as pupil size. In terms of the performance of participants with their preferred lens when observing the defocus curve under mesopic condition, it emerged that there was an interaction between lens types and acuity at different levels of defocus. From this, it seems that lens preference may perhaps be driven by a change in visual experience that only manifested in low illumination conditions, suggesting it may be important to conduct objective measure such as visual acuity under mesopic condition when fitting modern-day multifocal contact lenses. It remains a hope for the future that new clinical tests or more diverse lens designs would be valuable to help the practitioner to improve the chances of first time success when fitting a multifocal contact lens for presbyopic correction.

At present optometric examination of the retinal microcirculation consists of a subjective assessment of artery size when compared to a neighbouring vein; the arterio-venous ratio (AVR). Despite it's documented limitations, the AVR still features in UK optometric clinical guidance. An objective method of recording the central retinal artery and vein equivalent sizes (CRAE and CRVE) has been used for research purposes for two decades but has yet to be validated in a clinical setting. A review of the present literature identified correlations between CRAE and CRVE and three key cardiovascular pathologies; hypertension, diabetes mellitus and stroke. A methodological study was undertaken to establish whether retinal photographs acquired in clinical practice yielded results with reproducibility comparable with those in the literature. It was shown that calibre measurements do not fluctuate significantly with the use of mydriatics, or subject to minor temporal fluctuations. When the technique was applied in clinical practice, a cross-sectional cohort (n = 271) revealed vessel correlations with systemic biomarkers in agreement with those identified in the literature review. CRAE was seen to be reduced in those with raised blood pressure, and objective AVR was reduced in those with increased cardiovascular risk (QRISK). There did not appear to be significant fluctuations in vessel measurements when the cohort was observed longitudinally across a period of 12 - 24 months, suggesting changes either too slow or too small to be detected at present; supporting theories of gradual morphological changes. When considered as part of scope of primary care, improved cardiovascular assessment through retinal vessel analysis (especially when supplemented with blood pressure and cardiovascular risk calculations) relieves pressure on GPs and identifies a significant number of previously undiagnosed and unmanaged cases of cardiovascular disease. The results build a strong case for the incorporation of objective retinal vessel analysis into routine optometric clinical guidelines.

Objective: This study examined three main objectives: 1. The validity of Rebound Tonometry (RBT) measurements in children. 2. The reliability of suboptimal RBT readings and the relationship between co-existing characteristics and these measurements. 3. The reliability of suboptimal RBT measurements in children with heritable connective tissue disease (HCTD).Design: A cross-sectional study design was used for objectives 1 and 2 and a case control study was used for objective 3.Setting: The Eye Department of Birmingham Women's and Children's Hospital (BWCH).Participants: Fifty children were recruited, including 34 with glaucoma for objectives 1 and 2 and 16 for objective 3 (8 HCTDs, 8 healthy controls).Interventions: RBT measurements were taken at the geometric centre of the cornea of one eye (RBTon) and at 3 mm temporally (RBToff), followed by Goldmann tonometry (GAT). Additional data regarding sex, age, nystagmus, strabismus, type of glaucoma, treatment, visual acuity, spectacle prescription, ethnicity, health and corneal scars were recorded from the participants' clinical notes. The same procedure was conducted on 8 children with HCTD and 8 controlsResults: Mean RBTon was significantly higher than GAT by 2.4 (SD 3.0) mmHg. A statistical difference was found between the age groups and the IOP status (p < 0.05). Mean RBToff readings were not significantly different from RBTon in children with glaucoma (p = 0.100) and this difference was not associated with co-existing characteristics (p > 0.05). Mean (RBToff - RBTon ) was not significantly different between children with HCTDs and healthy controls (p = 0.06).Conclusion: This study achieved its main objectives and found that:• RBTon measurements differ from GAT but are useful clinically.• The relationship between RBTon and GAT varies with the age of the child.• Suboptimal RBToff measurements are reliable in children with glaucoma with a range of co-existing conditions and in children with HCTDs.

The aim of this thesis was to understand the demand and uptake of private optometric community services outside the scope of traditional services having the outcome of supplying spectacles, and of locally commissioned shared care schemes. Dry eye is known to reduce quality of life, be highly prevalent, underdiagnosed and clinically significant. Dry eye is a condition with poor association and discordance between signs and symptoms, and so is poorly managed. Dry eye does not command priority in secondary care where patients sometimes present when self-help measures fail, considering it is generally not sight threatening. The global consensus on dry eye have recommended a tiered management approach highlighting advanced pharmacological care options that could be applied by optometrists with an independent prescribing qualification. Emerging technologies also show promise in advanced dry eye diagnostics and management but the investments required means that practices need to develop a strong business plan to make them commercially viable. This research was based on a single independent optometric practice and two hospitals with a relationship to the practice. Service blueprinting was applied to the dry eye service to demonstrate its usefulness in optometric service innovation. Decision tree analysis and principal component analysis were used to discriminate between people self-reporting dry eye, to predict severity, and to identify clinical tests to explain the variability between those with predominately evaporative dry eye from a sample of patients. Despite having signs of dry eye, there were no differences in visual outcomes post-lens surgery based on dry eye sign status, and there were no strong trends to link discontinuation of contact lens wear to dry eye. Dry eye is a condition that presents to the local eye casualty department that can be managed within the community along with other conditions that may have an acute or recurring presentation.

Improving reading ability is of high priority for most patients visiting optometric practice. Thus, it is expected that reading performance is one of the most important outcome measures for judging reading ability and the effectiveness of ocular interventions. Measuring near and distance visual acuity are simple and quick, but they cannot predict reading acuity, reading speed and critical print size which better reflect real-life reading performance. In contrast to distance visual acuity charts, there is not yet agreement on the best test to evaluate reading performance. There are many reading test charts available. Reading test charts should be equally reliable. However, the work described in this thesis has shown that using different reading test charts, such as Radner, MNread, Colenbrander, Bailey-Lovie and IReST, resulted in different reading performance metrics. There are no studies that have undertaken a direct comparison between all these reading tests, hence in this thesis test-retest and inter-chart reliability of the reading test charts has been compared for pre-presbyopic, presbyopic and cataract subjects. Although the reading test charts presented in this thesis are considered as standardized tests in terms of the test item, the reliability results vary and can be classified as poor, acceptable and very good. Using a reliable reading chart to evaluate the efficacy of the presbyopia treatment is a useful tool to investigate the reading performance rather than isolated letter near visual acuity. Automated measurement of the reading performance metrics by using a computer-based reading test could overcome the variation of the results between the practitioners but needs to be further calibrated. The findings of this thesis have a number of important implications for current and future ophthalmic practice.

The corneal epithelial cells are constantly replaced by the stem cells located at the limbus, the peripheral edge of the cornea, therefore known as limbal stem cells (LSCs). LSCs can be destroyed by numerous factors which results in the condition called limbal stem cell deficiency (LSCD). Ex vivo expansion of LSCs is a well-established technique used successfully to cure patients with LSCD. Therapeutic use of LSCs must be performed in compliance with good manufacturing practice (GMP) as a quality assurance system. However, traditional culture media for ex vivo expansion of LSCs contains a number of ingredients derived from animal sources which may compromise its safety profile for human transplantation. The first aim of the study was to define new GMP grade medium for cultivation and maintenance of LSCs in vitro. Formulation of new GMP compliant media resulted in equal growth to non-GMP grade media. Strick regulations for cell therapy promote centralization of culture units, therefore definition of reliable and practical transportation strategies is vitally important. The second aim of this study was to optimise the transport conditions for limbal biopsies (LBs) and cultured limbal epithelial cells (LECs). Transport of LBs at room temperature proved to be significantly superior to 4°C transport. We also showed that cultured LECs may be stored in serumfree media and transported up to 7 days at 23°C without any negative effect on cell number, viability, colony forming efficiency or gene expression profile. Due to the absence of specific LSC markers, identification and isolation of putative LSCs is a complicated task. The third and final aim of this study was to identify novel cell surface markers for LSCs. We reported herein the identification of a new cell surface marker for LSCs (CD200) as well as a cell surface marker for proliferating progenitor cells (CD109).

Glaucoma remains the leading cause of irreversible blindness in the world. Since retinal ganglion cell (RGC) axonal degeneration precedes permanent vision loss, identification of ONH parameters affected in the earliest stages of primary open angle glaucoma (POAG) is critical to ensure early diagnosis. This cross-sectional study used enhanced-depth imaging optical coherence tomography (EDI-OCT; 1040/70nm) to acquire 10° and 20° scans centred on the ONH (glaucomatous; n=128 or healthy controls; n=60). Regional measures of prelamina and LC depth and thickness, nerve fibre layer thickness at ONH border (bNFL) and peripapillary (pNFL), neuroretinal minimum rim width; (MRW) and area; (MRA) were analysed. This is the first study to quantify volumetric parameters including optic cup, prelamina and LC volume, and also Bruch's membrane opening (BMO) surface area. Furthermore, LC connective tissue alignment was probed regionally and depth-wise within the LC. Statistical modelling was performed to identify ONH parameters that best contributed to characterisation of ONHs in the earliest stages of POAG. Regional measures of prelamina depth and thickness, and LC thickness were able to differentiate between control eyes and preperimetric (PG), and early glaucoma (EG) (P<0.05). Additionally, EG LC volume was significantly less than in controls (P<0.05). Significant associations of these parameters with loss of VF sensitivity (VF Mean deviation [MD]) were identified. Border and pNFL thickness, MRW (but not MRA) significantly differed between controls and PG and EG (P<0.05); and decreased with VF MD. Lamina cribrosa connective tissue alignment altered in a region and depth specific manner between PG LC and controls, or EG LCs (P<0.05), providing an original in vivo indicator of disease. In conclusion, in vivo ONH and NFL parameters are able to discriminate between healthy ONHs and early POAG ONHs; providing a group index with potential as a novel biomarker for early diagnosis, critical to personalised clinical decision making.

Corneal blindness is the third leading cause of blindness worldwide. Current treatments require the use of donor corneas or amniotic membrane as a cell carrier. While these treatments are successful to a degree, they present downsides including accessibility of tissue and the risk of disease transmission. The use of synthetic scaffolds is a potential solution; current research has exhibited the importance of controlling mechanical properties when designing new approaches to corneal regeneration. The aim of this project is to create synthetic electrospun scaffolds with mechanically tailored stiffness to explore corneal cell behaviour in vitro, as well as to explore the inclusion of topographical cues in these membranes. Mechanically tailored membranes were fabricated using electrospinning and blending PLGA (poly(lactic-co-glycolic acid)) and PCL (Polycaprolactone) in different concentrations. Membranes were characterised in dry (storage) and wet conditions (submerged in PBS at 37°C). Characterisation of the membranes was done using Scanning Electron Microscopy (SEM) to analyse microstructure and fibre diameter. Uniaxial tensile testing was used to obtain stiffness, Ultimate Tensile Strength (UTS), and strain at UTS from the membranes. Gas chromatography was performed to measure the remnant solvents in them. Differential Scanning Calorimetry (DSC) was executed to analyse the thermal properties in the membranes. Biological testing was accomplished using rabbit and porcine limbal explants on the membranes and growing them for 2 and 3 weeks. Cell outgrowth in the membranes was analysed using different microscopy techniques (SEM, Light Sheet microscopy, and epifluorescence microscopy). Topographical cues were introduced in electrospun membranes by casting metal collectors with particular designs after the input of specialist of L. V. Prasad Eye Institute. Fibre alignment and fibre orientation were examined in the membranes by analysing SEM images and processing them with the ImageJ software with a protocol developed by us introduced in this thesis. Electrospun scaffolds with different mechanical properties were successfully manufactured blending PLGA and PCL. DCM and DMF as solvents produced a lower amount of remnant solvents in the electrospun scaffolds made of PLGA and PCL than the maximum permissible by EMA and FDA. Adding PCL showed statistical differences in the stiffness of the membrane for blends with 10% or more PCL on it, an effect that is increased when the membranes are analysed in dry and wet conditions. From all the conditions analysed, 30%PCL - 70%PLGA is able to maintain its mechanical properties in wet/warm and dry conditions which we envisage is a very important point for the material to be used in theatre. PLGA and PLGA-PCL membranes showed outgrowth from rabbit and porcine limbal explants at 2 and 3 weeks no differences were observed in the amount of cell outgrowth between them. All mechanically tailored membranes developed in this research showed good capacity as cell carriers of limbal corneal cells. 30%PCL - 70%PLGA displayed cell outgrowth in both sides of the membrane, suggesting that the cells penetrated through the membrane and populated the other side. As well, Topographical cues in the membranes were successfully introduced and characterised analysing fibre orientation and fibre alignment in different zones on them. With all this in mind, 30%PCL - 70%PLGA showed the best results in its mechanical properties and its cell outgrowth when seeded with porcine limbal explants for two weeks and we suggest moving forward the research using this electrospun membrane with longer culture times.

In order to increase spectacle independence following cataract surgery and intraocular lens (IOL) implantation; correction of spherical refractive error, astigmatic error and presbyopia should all be given careful consideration. There are many premium IOLs, including multifocal intraocular lenses (MIOLs) and toric intraocular lenses (TIOLs), available to surgeons. In order to select the appropriate IOL to meet a patient's lifestyle and expectations, clinicians must fully understand the characteristics of MIOL and TIOL designs. To date, there remain unanswered questions pertaining to MIOLs and TIOLs and by rigorous comparison of such lenses, this thesis aims to address some of the gaps in the current literature. This thesis aims to evaluate a robust protocol for investigating clinical outcomes in MIOLs that would allow for comparison between future studies. This methodology was used in a randomised control trial and a cohort study. Included in this protocol is the detailed analysis of defocus profiles. This thesis investigates polynomial curve fitting to establish the most suitable curve and curve fitting method for use in future analysis of MIOLs with detailed defocus metrics. Defocus curves can highlight the differences in optical performance in MIOLs of differing addition powers, however, to add further complexity, previous literature has highlighted that addition power can vary individual to individual based on their ocular anatomy. Thus, investigation of an easily accessible clinical method to predict the likely achieved addition power post-implantation was performed. A randomised intra-patient contralateral eye study assessed refractive outcomes and rotational stability in TIOLs. In addition, the performance of the corresponding manufacturer's calculators was evaluated in regard to refractive predictability and appropriate TIOL selection. This thesis highlights the clinical features of modern MIOL and TIOL designs, demonstrating both the benefits and challenges incurred following implantation.

Neuronal dendritic and synaptic degeneration are early markers of neurodegeneration occurring in the brain of Alzheimer's disease (AD). AD is the most common type of dementia characterised by the accumulation of extracellular amyloid plaques and neurofibrillary tangles, accompanied by neuroinflammation leading to neurodegeneration. AD diagnosis is often complex, expensive and/ or invasive, and only detect late stages of pathological changes. The retina, an extension of the central nervous system (CNS), develops AD-like pathology and may reflect ongoing pathological changes in the AD brain. Retinal ganglion cells (RGCs), the output neurons of the retina, are vulnerable to degeneration and may offer as a potential target for detecting and monitoring AD pathology. However, it is unclear whether RGC degeneration is a consistent feature in the AD retina. This thesis utilises DiOlistic labelling to investigate RGC dendritic pruning and hippocampal dendritic spine loss in the AD mouse models and assesses the impact of immune system modulation via deficiency of the complement system, elevated systemic inflammation (bacterial and viral) and dietary modulation (high fat diet and probiotics). All AD mouse models (Tg2576, 3xTg-AD and APPN-G-F) displayed excessive RGC dendritic pruning that occurred contemporaneously with dendritic spine loss in the hippocampus. In the 3xTg-AD model, deficiency in the complement system offered neuroprotection against both RGC dendritic and hippocampal synaptic pruning whilst elevated systemic inflammation (bacterial and viral) and high fat dietary modulation exacerbated neurodegeneration. Probiotic dietary supplementation was also associated with synaptic protection in the hippocampus. Overall, this thesis demonstrates that excessive RGC dendritic pruning is a common feature in the retina of AD mouse models and occurs at the time of ongoing synaptic loss in the hippocampus. These findings support the prospective use of retinal analysis to monitor AD progression and severity, providing an accessible measure that reflects pathology within the brain.