Multimodal imaging in age-related macular degeneration

Age-related macular degeneration (AMD) is a leading cause of blindness and affects approximately one in seven Australians aged 50 years and above. Currently, this complex condition is not easily and uniformly assessed. The signs of AMD differ between eyes and also occur in other macular disorders. This hinders accurate diagnosis and classification, which is fundamental to optimal patient care. Ocular imaging and visual function assessment have the potential to minimise the devastating consequences of disease through early detection. However, multiple devices are now commercially available and the impact of these technologies in clinical practice may not be straightforward. For instance, their usefulness may depend on accessibility and the operator’s knowledge and clinical skills. The impact on patient management, as well as alternative models of eye-care delivery, requires clarification. This thesis aims to explore the current and potential utility of imaging technologies (optical coherence tomography, infrared imaging, monochromatic retinal photography and fundus autofluorescence) in the assessment and management of AMD and other diseases of retinal pigment epithelium dysfunction. The findings show that optometrists self-describe high levels of practice competency and make ready use of imaging in everyday practice. However, they also unwittingly demonstrated low awareness of the evidence base in AMD. Furthermore, when their interpretation of images was tested using a series of case vignettes, their diagnostic accuracy as a group improved by only five per cent (from 61 per cent to 66 per cent); their tendency to refer increased by four per cent. These factors might be improved through education. A series of open-access, chair-side reference charts were consequently devised to help optometrists use imaging technologies more effectively in clinical practice. The additive contribution of multimodal structural and functional testing was particularly emphasised. Finally, a novel model of intermediate-tier eye-care in Australia was shown to substantially reduce the number of false positive cases or cases without a specific diagnosis. Interestingly, this model was acclaimed by reviewers as “scoring highly for originality and of international relevance”. Most excitingly, the thesis concludes with future directions regarding collaborative care and multimodal imaging, where detection of disease might be facilitated via a computational approach

Lysolipids are prominent in subretinal drusenoid deposits, a high-risk phenotype in age-related macular degeneration

IntroductionAge related macular degeneration (AMD) causes legal blindness worldwide, with few therapeutic targets in early disease and no treatments for 80% of cases. Extracellular deposits, including drusen and subretinal drusenoid deposits (SDD; also called reticular pseudodrusen), disrupt cone and rod photoreceptor functions and strongly confer risk for advanced disease. Due to the differential cholesterol composition of drusen and SDD, lipid transfer and cycling between photoreceptors and support cells are candidate dysregulated pathways leading to deposit formation. The current study explores this hypothesis through a comprehensive lipid compositional analysis of SDD.MethodsHistology and transmission electron microscopy were used to characterize the morphology of SDD. Highly sensitive tools of imaging mass spectrometry (IMS) and nano liquid chromatography tandem mass spectrometry (nLC-MS/MS) in positive and negative ion modes were used to spatially map and identify SDD lipids, respectively. An interpretable supervised machine learning approach was utilized to compare the lipid composition of SDD to regions of uninvolved retina across 1873 IMS features and to automatically discern candidate markers for SDD. Immunohistochemistry (IHC) was used to localize secretory phospholipase A2 group 5 (PLA2G5). ResultsAmong the 1873 detected features in IMS data, three lipid classes, including lysophosphatidylcholine (LysoPC), lysophosphatidylethanolamine (LysoPE) and lysophosphatidic acid (LysoPA) were observed nearly exclusively in SDD while presumed precursors, including phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidic acid (PA) lipids were detected in SDD and adjacent photoreceptor outer segments. Molecular signals specific to SDD were found in central retina and elsewhere. IHC results indicated abundant PLA2G5 in photoreceptors and retinal pigment epithelium (RPE). DiscussionThe abundance of lysolipids in SDD implicates lipid remodeling or degradation in deposit formation, consistent with ultrastructural evidence of electron dense lipid-containing structures distinct from photoreceptor outer segment disks and immunolocalization of secretory PLA2G5 in photoreceptors and RPE. Further studies are required to understand the role of lipid signals observed in and around SDD

Retinal pigment epithelium transplantation in retinal diseases

Age-related macular degeneration (AMD) and inherited macular diseases (IMD) are retinal disorders that can cause blindness through atrophy of the retinal pigment epithelium (RPE) or choroidal neovascularisation (CNV). RPE transplantation in severe forms of neovascular AMD has been performed with promising short-term outcomes. However, this approach has not been evaluated in atrophic types of AMD or IMD. Furthermore, the long-term outcomes of photoreceptors cell function rescue by RPE reconstruction in neovascular AMD is unknown. Current surgical techniques are complex with associated high complication rates. Therefore, other treatment approaches to reconstruct the RPE are required. This thesis aims to examine whether long-term photoreceptor cell function rescue can be achieved through RPE reconstruction by investigating the outcomes of autologous RPE transplantation or full macular translocation in AMD and IMD. A further aim is to determine the feasibility of a new approach to reconstruct the RPE using human embryonic stem cell (hESC). A prospective study of autologous RPE-choroid grafts in 9 patients with atrophic macular disease secondary to AMD or IMD demonstrated that submacular RPE graft can support retinal function and fixation. However, there was a high surgical and post-operative complication rates and the overall visual acuity and reading ability declined. Long-term follow-up demonstrated that the graft can maintain retinal function for over 2 years in some patients. A retrospective review of long-term outcomes following autologous RPE-choroid grafts and full macular translocation in 12 and 40 patients with neovascular AMD, respectively, showed that rescue of retinal function beyond 2 years is possible. A visual acuity of 6/12 was achieved and maintained for over 2 years in 8% and 15% of patients who had patch graft and translocation, respectively. However, overall visual acuity outcomes were limited by delayed post-operative complications such as recurrent CNV and cystoid macular oedema. A prospective porcine experiment showed that subretinal implant of hESC derived-RPE was feasible and human donor cell can survive in vivo for up to 6 weeks. However, there was significant loss of the hESC-RPE which may have occurred intra-operatively or during the first 2 weeks post-operatively. Macrophages were noted at the site of the graft suggesting some inflammatory and immunological responses to the human cells, polyester substrate or surgical trauma. The work in this thesis has provided the proof of principle that reconstruction of the RPE can maintain retinal function in atrophic and neovascular macular diseases over the long-term. A novel approach using hESC-RPE on an artificial substrate may be a more feasible and safer alternative to current clinical techniques of RPE reconstruction

Assessing neurodegeneration of the retina and brain with ultra-widefield retinal imaging

The eye is embryologically, physiologically and anatomically linked to the brain. Emerging evidence suggests that neurodegenerative diseases, such as Alzheimer’s disease (AD), manifest in the retina. Retinal imaging is a quick, non-invasive method to view the retina and its microvasculature. Features such as blood vessel calibre, tortuosity and complexity of the vascular structure (measured through fractal analysis) are thought to reflect microvascular health and have been found to associate with clinical signs of hypertension, diabetes, cardiovascular disease and cognitive decline. Small deposits of acellular debris called drusen in the peripheral retina have also been linked with AD where histological studies show they can contain amyloid beta, a hallmark of AD. Age-related macular degeneration (AMD) is a neurodegenerative disorder of the retina and a leading cause of irreversible vision loss in the ageing population. Increasing number and size of drusen is a characteristic of AMD disease progression. Ultra-widefield (UWF) retinal imaging with a scanning laser ophthalmoscope captures up to 80% of the retina in a single acquisition allowing a larger area of the retina to be assessed for signs of neurodegeneration than is possible with a conventional fundus camera, particularly the periphery. Quantification of changes to the microvasculature and drusen load could be used to derive early biomarkers of diseases that have vascular and neurodegenerative components such as AD and other forms of dementia.Manually grading drusen in UWF images is a difficult, subjective and a time-consuming process because the area imaged is large (around 700mm2) and drusen appear as small spots ( 0.8 and < 0.9), achieving AUC 0.55-0.59, 0.78-0.82 and 0.82-0.85 in the central, perimacular and peripheral zones, respectively. Measurements of the retinal vasculature appearing in UWF images of cognitively healthy (CH) individuals and patients diagnosed with mild cognitive impairment (MCI) and AD were obtained using a previously established pipeline. Following data cleaning, vascular measures were compared using multivariate generalised estimation equations (GEE), which accounts for the correlation between eyes of individuals with correction for confounders (e.g. age). The vascular measures were repeated for a subset of images and analysed using GEE to assess the repeatability of the results. When comparing AD with CH, the analysis showed a statistically significant difference between measurements of arterioles in the inferonasal quadrant, but fractal analysis produced inconsistent results due to differences in the area sampled in which the fractal dimension was calculated.When looking at drusen load, there was a higher abundance of drusen in the inferonasal region of the peripheral retina in the CH and AD compared to the MCI group. Using GEE analysis, there was evidence of a significant difference in drusen count when comparing MCI to CH (p = 0.02) and MCI to AD (p = 0.03), but no evidence of a difference when comparing AD to CH. However, given the low sensitivity of the system (partly the result of only moderate agreement between human observers), there will be a large proportion of drusen that are not detected giving an under estimation of the true amount of drusen present in an image. Overcoming this limitation will involve training the system using larger datasets and annotations from additional observers to create a more consistent reference standard. Further validation could then be performed in the future to determine if these promising pilot results persist, leading to candidate retinal biomarkers of AD

Identification of Surrogate Anatomic Identifiers of Disease Progression in Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is the leading cause of vision loss in patients over 50 in the developed world. The visual impairment is due to either choroidal neovascularisation (wet AMD) or geographic atrophy (GA). Drusen is the hallmark of AMD but the presence of drusen does not inform progression to wet AMD. Although the disease is mostly bilateral, the rate of progression of disease in both eyes may not be simultaneous. If one eye is affected by wet AMD, the risk of progression of the fellow eye to wet AMD increases by 10% every year. However, there are no markers that inform the time of conversion to wet AMD. For this reason, there is an unmet need to identify biomarkers that can fully predict the progression to wet AMD in order to allow early intervention before permanent damage. My thesis aimed to assess whether changes in imaging characteristics can more precisely explain conversion. I studied various cohorts including (a) normal aging eyes (b) eyes with early/ intermediate AMD and (c) fellow eyes of unilateral wet AMD to study the conversion to wet AMD. Firstly, I evaluated longitudinally volume changes in inner and outer retinal layers of 71 eyes with early/intermediate AMD using optical coherence tomography (OCT). Our results showed that inner and outer retina layer volumes may differentiate AMD eyes from healthy eyes. When comparing those who progressed to wet AMD at year 2 to those who did not, we found that baseline volume of GCIPL may differentiate between the 2 groups. As it is an inner retinal change, I hypothesized that heritability of the retinal layers may influence the rate of retinal layer changes and that may in turn help understand the changes seen in aging and AMD. I worked with the TWIN Study database, in which OCT was done in eyes of twins of different age groups and OCT data were available on 364 eyes of 184 (92 pair) twins. I evaluated whether heritability was responsible for ageing changes of the retinal layers. I found that total retinal volume and inner retinal layer volumes may be affected by genetic factors

The Impact of Fundus Autofluorescence on the Management of Age-related Macular Degeneration

Background: Fundus autofluorescence (FAF) has been described as a topographical map of fluorophores that accumulate within the retinal pigment epithelium as a result of disease. Study aims: To evaluate whether FAF offers information relevant to age-related macular degeneration over that gathered via colour fundus photography (CFP) and optical coherence tomography (OCT). Methods: Ninety-three patients were imaged via CFP, OCT and FAF and the results analysed using Orange Data Mining artificial intelligence and SPSS software. Results: Pupillary dilation makes a significant improvement to FAF image quality. Nuclear sclerotic cataract of > 1.5 on the World Health Organisation scale indicates that there is ≃85% probability that the FAF image will not be of high quality. At > 1.9 there is ≃50% probability of the image not being clinically useful as defined by a novel grading scale. Age was negatively associated with FAF comfort. There is ≥ 90% probability of an abnormal FAF result for an eye with any of the following: > 50 small, > 40 intermediate, > 20 large drusen. Age > 92 years. > 30 packet years of smoking. Any pigmentary abnormalities. ≃80% for any reticular pseudodrusen (RPD). FAF results can be predicted via CFP and OCT data using machine learning with informedness of up to 70.2% and area under the curve (AUC) of 0.903. For transfer learning to be useful within primary care, image pre-processing is likely to be required. Geographic atrophy and pigment epithelial detachments appear to be linked to a patchy FAF pattern. RPD are linked to a reticular FAF pattern. Principle component analysis indicates that drusen were responsible for the greatest percentage of variability in this study’s data (38.6%). Conclusions: Clinical impact: FAF results can be predicted from CFP/OCT via machine learning with 70.2% informedness and AUC of 0.903. Drusen number/size were the most informative variables

Visual function in aging and age-related macular degeneration including subretinal drusenoid deposits

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 Monogenic Architecture of Retinal and Neurological Diseases

Monogenic diseases, or single-gene disorders, are clinical manifestations that can be traced to genetic variation in a single gene that alters the biologically intended (wildtype) function of its protein (or mRNA) product. Although the causal gene and its function are well-understood in many monogenic diseases, this knowledge alone often does not fully encapsulate the extensive clinical spectrum of phenotypes seen in patients. This is due in part to the numerous types of pathogenic variants that can arise in a single gene, all of which can have distinct effects on disease expression. Understanding the relationship between the vast number of possible genotypes and corresponding disease phenotypes defines a gene’s monogenic disease architecture—an important but poorly understood concept that can yield informative mechanistic and clinical insight. This doctoral dissertation integrates traditional sequencing approaches with in-depth characterization of patient phenotypes to elucidate the monogenic disease architecture of three etiologically distinct disorders: retinal degeneration caused by autosomal recessive variation in ABCA4 and neurodevelopmental disease entities caused by autosomal dominant variants in CERT1 and PUM1. Genetic modifiers are identified as a significant factor in the penetrance of the major disease-causing allele of ABCA4 and several other genetic inconsistencies are resolved to create a coherent genotype-phenotype model for the disease. Insight from this model is then applied to demonstrate the effect of allele differences in disease progression and evaluation of treatment efficacy in patients. A large cohort of affected individuals with CERT1 variation is assembled to (1) validate the causal role of CERT1 in disease, (2) delineate the precise mechanism of CERT protein dysfunction in sphingolipid metabolism and (3) demonstrate therapeutic efficacy of an inhibitor compound for a newly described syndrome. Finally, the mutational spectrum of PUM1 is expanded to previously unattributed variant classes with unexpected pathophysiological consequences to patients. Not only do the findings in this dissertation advance the prospects of delivering personalized, precision medicine to patients, the overall impact underscores the importance of this integrated approach in reconciling knowledge gaps between observations at the molecular and organismal level

The development of a clinical trial protocol and functional biomarkers for age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of blindness amongst older adults in the developed world. With the predicted rise in the ageing population over the next decades, the prevalence of this debilitating disease will simply continue to increase. The only treatments currently available are for advanced neovascular AMD. The retina is already severely compromised by this stage in disease development. Therefore, there is a pressing need to evaluate potential novel interventions that aim to prevent the development of advanced disease in people with early AMD, to prevent sight loss from occurring. Furthermore, it is necessary to develop tests that are sensitive to subtle changes in visual function in order to evaluate the efficacy of these emerging treatments. There is a growing body of evidence to suggest that hypoxia contributes to the development of AMD. Hypoxia is most acute at night when the retinal photoreceptors are most metabolically active, due to the demands of the rod dark current. Increasing the light levels at night will cause the oxygen demand, and hence the hypoxia, to be substantially diminished. This leads to the hypothesis that providing low level night time light therapy to people with early AMD may slow disease progression by reducing hypoxia. In order to evaluate the potential effectiveness of such an intervention, it is necessary to select appropriate outcome measures. The inherent variability of the standard test of visual function, visual acuity, renders it inappropriate for use as a primary outcome measure in proof of concept clinical trials. Therefore, the first aim of this thesis was to evaluate the diagnostic validity and repeatability of alternative functional tests that may be used as biomarkers for early macular disease. Dark adaptation was evaluated using three stimuli, a spot of 2o radius and annuli of 7o and 12o radii, in 21 healthy adults (on two occasions) and in 11 participants with early AMD. All stimuli were found to be highly diagnostic for early AMD. The spot of 2o radius provided the best separation between groups with respect to the time constant of cone recovery (area under the ROC curve 0.91). The repeatability of chromatic and flicker thresholds were also assessed in 30 healthy adults. The coefficient of repeatability, expressed as a percentage of the mean threshold, was 17.1% for red-green chromatic thresholds, 31.1% for blue-yellow, 53.4% for 14Hz flicker thresholds, and ranged between 36.4%-53.3% for parameters of dark adaptation. A small learning effect was found for both chromatic thresholds and the 14-Hz flicker test, indicating that a control group is needed in studies of new therapeutic interventions. The second aim of this thesis was to develop a protocol for a clinical trial that seeks to determine if low level night time light therapy can prevent the progression of early AMD. The level of retinal illuminance required to suppress the rod dark current, the maximum retinal illuminance which prevents substantial suppression of melatonin secretion, and the most appropriate means of delivering the dose of retinal illumination were evaluated. The final protocol employed an organic LED illuminated light mask, worn during hours of sleep, as the mode of intervention. In conclusion, this thesis has confirmed that cone dark adaptation is a sensitive functional biomarker for AMD, and that all three functional tests have a good inter-session repeatability. These biomarkers will be validated in the prospective clinical trial of low-level light therapy to confirm their prognostic and predictive capabilities. The proposed trial will also evaluate the effectiveness of the low level night time light therapy, delivered by means of an illuminated light mask, at slowing the progression of early AMD