Ophthalmology (Eye & Ear Hospital) - Theses
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ItemNew approaches to the photopic negative response of the electroretinogram in glaucoma( 2022)Background: Glaucoma is an optic neuropathy characterised by the progressive loss of retinal ganglion cells (RGCs) and is a leading cause of blindness worldwide. Prior to irreversible cell death, RGCs exhibit functional changes that if detectable, could aid in predicting the disease trajectory and help better guide the management of this condition. The photopic negative response (PhNR) of the electroretinogram (ERG) shows potential as such a functional test, but its utility has been limited due to problems with repeatability and interpretation. Purpose: To explore novel techniques to improve the repeatability of the electroretinogram in glaucoma, describe new feature extraction techniques, and to develop models that predict glaucoma severity from the ERG. Methods: Normal controls and individuals with glaucoma were recruited for these studies. ERGs were collected with red flashes on a blue background or with a white-on-white stimulus. Various novel denoising techniques were evaluated on the normal control group to improve measurement repeatability, and complexity measures were used as new features to better discriminate between those with and without glaucoma. Novel predictive models informed by novel features extracted with time-frequency techniques were trained evaluated and compared to models using various amplitude markers from the ERG. Results: The denoising technique termed complete ensemble empirical mode decomposition with adaptive noise (CEEMDAN) was shown to be effective and efficient in removing baseline drift and wander of the ERG, thus significantly improving the repeatability of the recordings. Eyes with and without glaucoma showed significant differences based on novel complexity measures of the ERG, beyond the standard PhNR measure. New models informed by time-frequency features significantly added to the predictive performance for glaucoma severity when used alongside conventional amplitude markers of the ERG. Conclusion: This body of work developed a new method of denoising of the ERG, which also could have wider application in electrophysiology. New features based on complexity showed potential for providing important additional measures of RGC dysfunction useful for disease discrimination. Time frequency features extracted from the ERG also showed that there is substantial information in the ERG beyond what is captured by standard amplitude markers that could aid in better detecting early RGC dysfunction.
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ItemOptimising the clinical utility of the photopic negative response to probe changes in glaucoma( 2019)Glaucoma is one of the leading causes of irreversible blindness and early detection and treatment are key to preventing or slowing the progression of vision loss. An objective, non-invasive method to detect early functional changes in glaucoma would be invaluable, and a potential surrogate marker of retinal ganglion cell health may lie in the photopic negative response (PhNR) of the electroretinogram (ERG). Purpose: To develop and validate a reliable testing protocol for the PhNR recorded using a portable ERG device (RETeval®) that can be easily implemented in clinical practice. Methods: Normal controls and glaucoma participants were recruited for the study. Responses were elicited using a red stimulus on a steady blue background with the RETeval®. A comparison of baseline detrending methods as well as electrode performance was evaluated between the groups. The PhNR stimulus-response function was characterised, and the correlation between PhNR parameters and current measures of retinal structure and function were assessed. The sensitivity of the PhNR to detect glaucomatous changes was evaluated in glaucoma participants receiving treatment for intraocular pressure lowering. The PhNR was also used to follow glaucoma participants over 12-months to assess its relative ability to detect disease progression. Results: A method of baseline detrending was developed which improved the test-retest repeatability of the PhNR without compromising its clinical utility in glaucoma. Sensor strip electrodes provided a viable alternative to conventional Dawson, Trick and Litzkow (DTL) electrodes, although had inferior signal-to-noise performance that may reduce the sensitivity to detect subtle changes in disease. Measuring the PhNR across a range of stimuli to determine the maximum response provided reliable data and may be more appropriate than a single stimulus strength. However, the maximum response showed only weak correlation with standard automated perimetry and retinal nerve fibre layer thickness in glaucoma. With the optimised protocol, short-term changes in the PhNR were detected after intraocular pressure lowering in a subset of participants. Disease progression was also suggested by monitoring the PhNR over 12-months in a pilot study. Conclusion: This body of work developed a standardised, reliable method for PhNR recording with a portable ERG device which can complement current tests in monitoring glaucoma progression and evaluating treatment response in the clinic.
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ItemUsing induced pluripotent stem cells to model primary open-angle glaucoma( 2019)Glaucoma is a group of optic neuropathies that may be characterized by gradual degeneration of retinal ganglion cells (RGCs) and their axons leading to irreversible vision loss [1, 2]. Glaucoma is the second leading cause of blindness worldwide [3-5] and it is estimated that the number of people affected by the disease will reach 80 million by 2020, while more than 11 millions will be bilaterally blind [6]. In this project I focus on primary open-angle glaucoma (POAG), as it accounts for the majority of glaucoma cases worldwide. So far, multiple risk factors for glaucoma have been identified; however, the exact mechanism causing RGC loss in patients remains elusive. Furthermore, examination of RGCs affected in POAG is difficult pre-mortem due to their anatomical location. To overcome this problem, somatic cells can be reprogrammed into patient-specific induced pluripotent stem cells (iPSCs), which can be then differentiated into cell type of interest, i.e. RGCs. This PhD project consisted of several steps. First, I assessed the feasibility of transferring the iPSC culture into the automated platform. Using automation was essential to generate large number of samples required for analysis. The transition to automation was successful, as evidenced by maintenance of iPSC morphology, expression of pluripotency markers and ability to differentiate into derivatives of three germ layers. I also demonstrated that incorporating automation into human (h) iPSC culture allows standardization of maintenance and passaging procedures reducing inter-sample variability and human error. I subsequently used the platform to generate over 300 hiPSC lines for POAG modelling. In parallel, I optimized RGC differentiation protocol to obtain sufficient number of cells for their examination with single cell RNA sequencing (scRNA-seq). Next, iPSC-derived RGCs were subjected to scRNA-seq to gain in-depth information about transcriptomic differences between healthy controls and POAG patients. Understanding mechanisms underlying RGC function, maintenance of homeostasis and those conferring susceptibility to POAG is crucial to discover new therapeutic targets and commence the process of drug discovery.
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ItemGeneration of induced pluripotent stem cells for patient-specific modelling of glaucoma in vitro( 2016)Glaucoma is a group of optic neurodegenerative diseases of the retinal ganglion cells (RGCs) leading to reduced vision loss (Quigley, 1999). Despite glaucoma being prevalent in the developed world (Quigley and Broman, 2006), the aetiology of glaucoma remains unknown. Several known risk factors exist including: age, increased intraocular pressure, genetics and oxidative stress (Almasieh et al., 2012, Agarwal et al., 2009). As glaucoma is insidious, and coupled with the anatomical location of the RGCs, pre-mortem studies of its pathophysiology in vivo remains difficult. Induced pluripotent stem cell (iPSC) technology has revolutionized the medical research field enabling a generation of in vitro cellular models. Human induced pluripotent stem cells (hiPSCs) can therefore be used for interrogation of disease pathophysiology, such as for glaucoma. Addressing the aims of this project, this dissertation has two main parts: optimization of an RGC differentiation protocol and disease modelling. Part A describes the testing and optimization of differentiation protocols for generating RGCs from a commercially available human embryonic stem cell (hESC) line, showing that these methods can be applied to hiPSCs. During this process, an adherent retinal differentiation protocol was optimized to include an RGC enrichment step by magnetic activated cell sorting (MACS) utilizing an retina-specific RGC surface marker, Thy1.1. Following enrichment, hESC-derived RGCs were comprehensively characterized, including by: gene and protein expression; electrophysiology; and mitochondrial transport assays, a feature that is underwhelming performed in other RGC protocols. Further, RNA sequencing (RNAseq) analysis revealed that the hESC-derived RGCs exhibit a similar gene expression profile to publicly available human ganglion cell layer (GCL) data confirming the enrichment of the hESC-differentiated population of retinal cells. Part B describes the generation of iPSC lines reprogrammed from the fibroblasts of individuals harbouring genetic risk variants associated with primary open angle glaucoma (POAG), the most common form of glaucoma. To elucidate a potential molecular mechanism for POAG, RNAseq was performed upon iPSC-derived RGCs harbouring different genetic variants. This data indicated that significant misexpression of genes involved in extracellular matrix organization, collagen synthesis and integrin signalling, may be the cause for differences between POAG affected and unaffected individual iPSC lines. These findings support the mechanical theory for glaucoma (Morgan, 2000). To investigate a disease-associated phenotype, apoptotic death of the iPSC-derived RGCs in response to an oxidative stress insult was analysed by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). The results indicated a statistically insignificant (p=0.0503) trend towards increased susceptibility of the POAG affected iPSC-derived RGCs. Instead, these findings may indicate a subtle effect of oxidative stress that may be suitable for the slow, progressive development of POAG in vivo. As POAG is an age-related disease, increased levels of oxidative stress occurring during the normal ageing process may expose individuals with underlying genetic predispositions. Therefore, individuals predisposed towards POAG may develop RGCs with a reduced threshold for ROS and other by-products of oxidative stress. In summary, the data presented within this PhD describes an in vitro disease model for POAG with the use of patient-specific iPSCs that has advanced our understanding of potential molecular mechanisms for its development in vivo.
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ItemCharacterising retinal ganglion cell response to injury( 2016)Glaucoma is a group of progressive optic neuropathies that have in common the slow progressive degeneration and eventual death of retinal ganglion cells (RGCs). There is evidence, however, from clinical and laboratory work, to suggest that early dysfunction in glaucomatous RGCs may be reversible. This thesis explores characteristics of healthy, injured and recovered RGCs using a model of acute intraocular pressure injury in mice. Patch clamping was used to interrogate single cell electrophysiology. RGCs exhibited reversibly impaired excitability following injury. A study into differences manifest with age suggested that older mice undergo a slower recovery process following injury. This is the first evidence that single RGCs can undergo a period of reduced function with recovery at a later stage and provides insights into possible mechanisms for reversible ganglion cell dysfunction in early glaucoma.
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ItemFunctional and structural changes in the visual pathway associated with ageing and glaucoma( 2013)Glaucoma is a leading cause of irreversible blindness. While early treatment is beneficial, diagnosis and monitoring are problematic: values of anatomical indicators of potential damage in the visual pathway vary with age, and values in glaucoma patients overlap substantially with the normal physiological range. A potential solution to this dilemma may exist if certain pathway structures are resistant to the disease and could provide an indication of pre-morbid anatomy. Aim: The first aim of my thesis was to identify the normal range of anatomical variation and establish the profile of normal ageing for structures in the visual pathway. The second aim was to use this normal characterisation as an age-stratified comparator for glaucoma patients to identify structures which were resistant to the disease and may provide an indication of pre-morbid anatomy. The final aim was to correlate an objective measure of cortical response by functional MRI with both subjective measures of visual function and anatomical change in glaucoma patients. Method: Normal controls (n = 83) and glaucoma patients (n = 42) with a range of disease severity were recruited for the study. The anterior visual pathway was imaged in vivo using conventional clinical techniques to obtain measures of the retinal nerve fibre layer and optic nerve head. 3T MRI was used to image the posterior visual pathway in vivo by T1, T2, diffusion imaging and functional scanning. Measurements of the retrobulbar optic nerve were taken directly from T2 scans and statistical parametric mapping software used to calculate brain volumes including visual cortices. High angular diffusion imaging was used to identify and quantify the optic radiations using spherical deconvolution and tracking software. Functional assessment was carried out by Humphrey 24-2 visual field evaluation and by functional MRI scanning with novel software developed for analysis. Results: An age stratified characterisation of the visual pathway was developed with ageing changes found for the retinal nerve fibre layer, optic nerve head parameters and the optic radiations. Visual cortex volume was only found to change in males with ageing, and the retrobulbar optic nerve did not exhibit any age-related anatomical loss. Glaucoma patients showed a gradually increasing loss of anterior anatomy and retrobulbar optic nerve diameter related to disease severity. However, optic radiations only showed a significant difference with more advanced disease, while visual cortex volumes were unaffected by disease state. Functional MRI scanning showed significantly less activation for moderate and severe glaucoma patients compared to normal controls and there was a significant correlation between functional activation and visual field mean defect scores for binocular and left eye viewing. Conclusion: Glaucomatous damage in the visual pathway is related to disease severity as assessed by functional loss, however changes in the posterior pathway lag those which occur anteriorly. This thesis presents an age-stratified characterisation of the normal visual pathway which can be used on an individual basis for glaucoma patients to assess anatomical status and amount of anatomical redundancy for each pathway structure which may give an indication of the relative risk of further progression.
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ItemTemporal processing in auditory and visual systems: evidence of wider central nervous system involvement in glaucoma( 2011)Background: Open Angle Glaucoma (OAG) is a condition that is characterised by a loss of retinal ganglion cells; which in turn inhibits visual signals, through a reduction in the levels of information being sent to the brain for processing. The degree and rate at which this inhibition occurs can vary greatly between individuals and therefore may be a consequence of innate differences in sensory nerve susceptibility to injury. In the event of an underlying neuronal susceptibility, signs of auditory processing dysfunction may be observed in individuals with OAG. Aims: The purpose of this study was to examine whether individuals with OAG display signs of central auditory processing dysfunction. Concurrent investigation of specific visual processing functions was conducted to assess if any similarities in neural processing dysfunction were evident across both visual and auditory systems within the same individuals. Methods: Experiment 1: A comprehensive range of peripheral and central auditory processing tests were conducted in 42 OAG individuals and matched controls. Central auditory function tests included evoked potential measures, tests for auditory temporal processing and speech perception. Experiment 2: Based on the findings of Experiment 1, specific auditory temporal processing tasks were performed in conjunction with hierarchical visual temporal processing tasks. Results: Experiment 1: Compared to age equivalent controls, OAG participants displayed significantly poorer amplitude modulation detection and speech perception under high levels of background noise. Amplitude modulation detection moderately correlated with speech perception and ABR performance within both groups. Collectively, signs indicated evidence of impaired auditory neural temporal processing in a significant proportion of the OAG cohort. Experiment 2: OAG Individuals showed a tendency toward poorer performance on both auditory and visual discriminatory tasks that require a high degree of temporal resolution. Specifically, poorer speech perception under competing noise as well as impaired visual speed discrimination for slow velocities were identified in a significant proportion of OAG individuals (39.13% and 47.82% respectively, were below the 90th percentile of control performance). Overall, performance on auditory processing tasks did not correlate with performance on visual processing tasks for either group. Conclusions: A subgroup of OAG individuals display evidence of central auditory neural processing dysfunction which implies that glaucoma may be associated with other non-visual sensory neural deficits. As such, the results indicate increased neuronal susceptibility in these individuals. Clinical Significance: This is the first study to explore auditory function in OAG individuals beyond assessing sound detection or the integrity of the middle and inner ear. A proportion of OAG individuals have shown signs of central auditory processing dysfunction with subsequent difficulty in perceiving speech in noisy environments. It is therefore suggested that individuals with OAG, who report hearing difficultly, be subsequently assessed to explore the impact and extent of this difficulty in relation to challenging auditory environments. This process will ensure a more comprehensive assessment is undertaken which will lead to more direct and appropriate treatment options offered. In regard to the visual findings, OAG individuals with impaired speed and motion discrimination may require interventional strategies to improve driving/pedestrian safety and navigation, an area for future research. Overall, further research to explore other specialised auditory functions in glaucoma patients would be warranted along with an investigation into how having both auditory and visual impairment impacts upon communication and quality of life.
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ItemTrabeculectomy: methods to improve safety, optimise outcomes, and assess filtering blebs( 2010)Trabeculectomy is the most commonly performed incisional glaucoma procedure worldwide. This thesis incorporates a compilation of papers with the aims of improving the surgical approach to trabeculectomy, and via improved assessment of results of surgery, optimising outcomes. In the first weeks after surgery, success of trabeculectomy hinges on the absence of complications, with the more important complications resulting from loss of control of intraocular pressure. The interactions between aqueous flow, scleral flap construction, and the placement and tension of sutures in the scleral flap can effectively control IOP, and are under the control of the surgeon. The surgical approach, independent of antimetabolite use, also affects bleb morphology. Via papers that are included in this thesis, I describe how I and coworkers have tested, modeled, and validated a ‘Safe Surgery System’ for trabeculectomy, an approach initiated by Professor Peng Khaw at Moorfields Eye Hospital, with excellent results and external validation and recognition. Later in the postoperative course, once the sclera in the trabeculectomy site has started to heal and remodel, the scleral component effectively becomes a fistula*; the functional component and the important determinant of long term success is the filtering bleb. In addition to descriptions of some minor complications of filtering blebs that had not previously been described, this thesis contains the story of the development and testing of a bleb grading system that became the Moorfields Bleb Grading System (MBGS). Although the current MBGS has flaws as described in the included publications, I believe it is still the best available tool for bleb grading since it captures important information that others ignore. My interest in the surgical and post-surgical management of blebs has led to related projects including textbook chapters regarding bleb assessment and management, and also to investigate the possibility of in-vivo imaging of blebs at tissue and cellular levels. Two papers at the end of this thesis show the potential for fluorescence-mode confocal microscopy to be applied to bleb and other wound healing research, perhaps with practical applications once suitable methods for selective fluorescent labelling become available.
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ItemRetinal associations of diabetes and vascular disease( 2009)Background: Diabetes mellitus and vascular diseases have a significant impact on the eye. Aim: To determine the prevalence, risk factors, and racial/ethnic differences of major eye conditions, particularly retinal conditions, associated with diabetes and vascular diseases. Scope: To date, the majority of studies have examined the association of retinal vascular calibre and diabetes in predominantly white Caucasian populations. Further elucidation of ethnic differences in effects of hyperglycaemia on early microvascular disease is relevant, particularly amongst Asians where diabetes is likely to see the largest increase in prevalence over the next decade. We therefore examined these findings from three Asian population-based studies, the Singapore Malay Eye Study (n=3280), Singapore Prospective Cohort Study and Singapore Cardiovascular Cohort Study 2 (n=3748). Results: The prevalence of diabetic retinopathy in the Singapore Malay Eye Study was 35%, and associated with longer duration of diabetes, poorer glycemic and blood pressure control. More importantly, 9.0% had vision-threatening retinopathy, and retinopathy was found in 6.0% of people without diabetes. Retinal vascular calibre changes were incriminated in diseases such as diabetes and hypertension, independent of traditional cardiovascular risk factors. Wider venular calibre was independently associated with early age-related macular degeneration. We also found a novel association between peripheral artery disease and glaucoma, stronger in persons with diabetes, independent of vascular risk factors, supporting the vascular theory of glaucoma. Implications: Subtle changes in retina, including retinal vascular calibre may be early markers of widespread microvascular changes in diabetes, resulting from chronic hyperglycaemia and other pathogenic processes. These results will have broad implications for understanding the impact of both microvascular and macrovascular complications of diabetes in the Asia Pacific region and targeting relevant therapeutic interventions.