Background: the primary site of injury in glaucoma is likely to be at the lamina cribrosa (LC), deep within the optic nerve head (ONH). Optical coherence tomography (OCT) in glaucoma has, to date, focused on the detection of nerve fibre loss. Spectral domain OCT (SDOCT) has improved speed and axial resolution, allowing acquisition of three-dimensional ONH volumes and may capture targets deep within the ONH. This thesis explores the capabilities and potential of deep SDOCT imaging in the monkey ONH. Plan of research: an investigation was conducted into the detection of key landmarks that would be necessary for future quantification strategies. In particular, detection of the neural canal opening (NCO) was assessed and how the NCO relates to what is clinically identified as the disc margin. The next phase involved clarifying the anatomical and histological basis of ONH structures observed within SDCOT volumes, by comparison with histological sections and disc photographs. Finally, quantification strategies for novel parameters based on deep targets were developed and used to detect chronic longitudinal changes in experimental glaucoma and acute changes following IOP manipulation. Results: SDOCT reliably detects the NCO, which can be used as an anchoring structure for reference planes. Usually the NCO equates to the disc margin but disc margin architecture can be complex and highly variable. SDOCT captures the prelaminar tissue and anterior LC surface. Prelaminar thinning and posterior LC displacement were both detected longitudinally in experimental glaucoma. Prelaminar thinning was observed with acute IOP elevation; posterior LC movement was rare. Significance: deep ONH structures, including the LC, are realistic targets for clinical imaging. These imaging targets may be useful in the detection of glaucoma progression and in the verification of ex-vivo models of ONH biomechanical behaviour.

Mutations in the E3 ubiquitin ligase Parkin are associated with autosomal recessive juvenile Parkinsonism. Furthermore, Parkin is recruited to Lewy bodies in brains of patients with sporadic Parkinson’s disease (PD), suggesting a role for Parkin in the pathogenesis of PD. This study investigated the effect of the DnaJ/Hsp40-like protein HSJ1 (DnaJB2) on Parkin biology. HSJ1 proteins are neuronal cochaperones that regulate Hsp70 activity through their J domain and exploit ubiquitin interaction motifs (UIM) to promote sorting of misfolded ubiquitylated proteins to the proteasome for degradation. Disease-related mutant forms of Parkin formed inclusions when overexpressed in neuronal cells. Co-expression of mutant Parkin with HSJ1a significantly reduced inclusion formation and altered inclusion characteristics. The effect of HSJ1a on Parkin inclusions required a functional J domain. Mutant Parkin could be co-immunoprecipitated with HSJ1a. Wild-type Parkin was also co-immunoprecipitated with HSJ1a, suggesting a potential interaction with normal Parkin. Importantly, HSJ1a enhanced the E3 ubiquitin ligase activity of wild-type Parkin, dependent on the HSJ1 UIM domain. HSJ1 and Parkin promoted the degradation of the Parkin substrate Synphilin-1. Finally, it was confirmed that Parkin relocates to mitochondria upon mitochondrial membrane depolarization. Moreover, Parkin induced the selective elimination of impaired mitochondria by mitophagy. Interestingly, HSJ1 rescued the relocation of Parkin mutants to damaged mitochondria and partially restored mutant Parkin’s ability to induce mitophagy. These data show that HSJ1 proteins may affect Parkin aggregation and function, which may have a broader relevance to PD.

Ocular inflammatory diseases, which include uveitis, are a leading cause of visual impairment. A number of systemic immunosuppressive agents (ISAs) are available, as well as biological therapies. However, these therapies may not always be effective and can be limited by toxicity. This thesis aimed to evaluate established and emerging therapies in ocular inflammatory disease. The reasons for changing ISAs and the success of subsequent ISA regimes have not previously been investigated for ocular inflammatory diseases. A review of a case series of 64 uveitis patients on ISAs revealed that the commonest reason for a treatment failing was lack of efficacy. A comparison between the most commonly used strategies after treatment failure, such as switching strategies (eg switch-to mycophenolate vs switch-to methotrexate) and add-on strategies (eg add-on azathioprine vs mycophenolate) revealed no significant difference in the time-to-success and retention times. The long-term efficacy and effects of repeat therapy with rituximab (anti-B cell therapy) in ocular Wegener’s granulomatosis (WG) was evaluated in the largest case series reported to date. Rituximab was effective in inducing remission, but relapse occurred in 33% of patients at about 12 months, and could be predicted by rising anti-PR3 titres. Retreatment with rituximab was effective. The work presented in the final part of this thesis utilised murine models of experimental autoimmune uveitis (EAU) to evaluate novel tumour necrosis factor (TNF) inhibitory therapies. EAU was initially characterised in two strains of mice, C57B/6 and B10.RIII, to identify key therapeutic windows and key target inflammatory cytokines. When testing the TNF inhibitor therapies, the EAU model failed due to possible genetic contamination of the B10.RIII strain from the supplier, this is still an on-going issue. However, some of the TNF inhibitory therapies administered by intravitreal injection increased the degree of inflammation compared to those injected with a placebo control.

The adult human retina has a population of Müller glia that exhibit stem cell characteristics. The study investigated the feasibility of obtaining Müller stem cells from small samples of human, rat, rabbit and nonhuman primate retinae in order to provide proof of concept for the development of patient specific therapies. It also explored the role of human Müller stem cells in providing a source of rod photoreceptor precursors that may be applied to experimental photoreceptor replacement. Müller glia with stem cell characteristics were readily obtained from small samples of fresh cadaveric human retina and retinae from rats and non-human primates. However derivation of cells from human retinectomy specimens was not possible, with the marked inflammatory environment likely to be inhibiting proliferation of these cells in vitro. As determined by quantitative PCR and immunocytochemistry, in vitro culture of Müller stem cells in the presence of FGF2, taurine, retinoic acid and IGF-1 led to increased expression of the photoreceptor markers CRX, NR2E3 and rhodopsin, associated with increased gene expression of components of the phototransduction cascade. The expression of NRL was not modified, suggesting that the differentiation of adult human Müller stem cells towards photoreceptors may occur through a pathway independent of this transcription factor. In vitro functional analysis suggested an increased responsiveness of differentiated cells to cGMP analogues and light stimulation as judged by calcium imaging and patch clamp techniques. Following subretinal transplantation into rodent models of photoreceptor degeneration, Müller stem cell derived photoreceptor precursors migrated and integrated into the outer nuclear of degenerate rodent retina, demonstrating expression of photoreceptor markers and local synapse formation. Grafting of photoreceptor differentiated but not undifferentiated cells led to a significant increase in rod photoreceptor function as shown by the scotopic electroretinogram (ERG). The present observations show that human Müller stem cells have the capacity to differentiate into photoreceptor precursors and that these have the potential to be used in the development of therapies for human photoreceptor disease.

The functional assessment of patients with primary open angle glaucoma (POAG) is important both for quantifying glaucomatous damage and for providing the clinician with an indication of the level of visual impairment from glaucoma. In its commonest form, perimetry is carried out using automated computerised techniques to bracket visual sensitivity (differential light sensitivity, DLS) at multiple test locations. Test parameters for contemporary standard automated perimetry (SAP) have largely been translated from the Goldmann kinetic parameter with minimal modification, with the use of an achromatic circular stimulus of a fixed size and perimetric strategies which utilise logarithmic (decibel, dB) increments of sensitivity. Perimetry is not without its problems: tests can often be unreliable and learning and fatigue effects contribute to test variability. Electrodiagnostic tests offer an alternative and objective means of functional assessment. In this regard, the photopic negative response (PhNR) is a technically easy test to perform and is not dependent on precise fixation stability or refractive correction. Following the introductory chapter (Chapter 1), the thesis consists of two broad areas of study: psychophysical (visual field) changes in POAG (Chapters 2 and 3) and electrophysiological changes in POAG (Chapter 4). Chapter 2 explores the use of linear units for the measurement of DLS, with the development of new algorithms which yielded lower perimetric bias and variability compared to conventional dBincrement algorithms. Two new stimuli for perimetry were subsequently developed with the aim of improving correlation and agreement of DLS with structural parameters in POAG (Chapter 3). The electrophysiological investigations demonstrated the PhNR to be as sensitive as global visual field measures and existing electrophysiological tests for the identification of functional loss associated with glaucomatous optic neuropathy (Chapter 4) and further refinement of this response for the diagnosis of glaucoma has been recommended (Chapter 5).

Diabetes affects around 2.3 million people in the UK and can lead to both acute and chronic complications. One of the chronic effects of diabetes is the damage that occurs to the vascular endothelium, affecting multiple organ systems, collectively termed diabetic vascular disease. Many proteins have been identified as being altered in diabetes, one of which is annexin 2, a calcium dependent phospholipid binding protein involved in many cellular processes. In response to diabetes annexin 2 is enriched on the surface of the endothelium where it functions as a co-receptor for tissue plasminogen activator and plasminogen to form plasmin, creating a pro-fibrinolytic environment. This thesis aimed to further examine the role of annexin 2 in diabetes, utilising the annexin 2 knockout mouse in both in vitro and in vivo experiments. As part of our in vitro experiments, we identified a hyperglycaemic translocation of annexin 2 to alternative cellular compartments, using sucrose density centrifugation, and an increased susceptibility of VE-cadherin to destabilisation upon the action of vascular endothelial growth factor in annexin 2 knockout endothelial cells. In the in vivo experiments we utilised streptozotocin to induce diabetes in the annexin 2 knockout mouse, and examined the progression of both diabetic retinopathy and diabetic nephropathy. Annexin 2 knockout mice developed more severe symptoms of diabetic nephropathy with increased microalbuminuria, mesenchymal matrix expansion and histological changes indicative of renal disease. In contrast to this, symptoms pertaining to diabetic retinopathy were mild in all mice. Non-diabetic annexin 2 knockout mice also exhibited mild hypoglycaemia, potentially implicating defects in the insulin signalling pathway, and suggesting a novel role for annexin 2 in glucose homeostasis.

Background: People with macular disease often face difficulties using their preferred retinal locus (PRL) during visual tasks. These difficulties are due to impaired oculomotor control, amongst other causes. The aim of this work was to investigate whether stabilizing the visual target at the PRL is beneficial for visual acuity and reading. Methods: Control of retinal image instability at the PRL was achieved using an eyetracker that moved the target according to the eye movements. Crowded and uncrowded visual acuity was measured at the PRL in people with macular disease and in healthy peripheral retina of control subjects. RSVP reading speed was also measured using the same method of stabilization at the PRL and healthy peripheral retina. Results: Results of a series of experiments showed that stabilizing the visual target can improve visual performance in most cases. In healthy peripheral retina crowded visual acuity improved when the image was stabilized and reduced when fixation instability was over-compensated. At the PRL, in patients, no improvement in visual acuity was obtained under stabilized conditions and again visual acuity reduced for over-compensated fixation instability. However, reading speed improved under stabilized conditions, by 20% in healthy peripheral retina of control subjects, and by up to 40% at the PRL of people with macular disease. Discussion: Good oculomotor control is critical for complex crowded tasks like reading. The improvement in reading speed found whilst compensating for oculomotor instability at the PRL is encouraging. These results indicate that training programs which aim to improve fixation control are likely to bring benefits for visual tasks. The observed increase in reading speed might be clinically relevant but the technique used to control instability needs simplification to be implemented outside the laboratory.

Many patients with Behcet’s disease (BD) have disease that is severe and do not respond to the conventional treatment of systemic corticosteroids and immunosuppressive agents. Recently, IFN-α therapy has shown promise as an effective treatment that may also improve long-term outcome. This study aims to provide an insight into the mechanisms of action and disease-modifying ability of IFN-α by focussing on the impact on T cell subsets. In this study, I describe my investigation into the effects of an additional 6-month course of IFN-α therapy in modulating T cell subsets and their cytokine expression in BD patients ex vivo over the first 12 months. Investigation of T cell responses confirms previous findings on Th1 and Th2 cells and their associated cytokines following IFN-α treatment. Analysis of Th17 cells shows these cells are downregulated by IFN-α therapy in peripheral blood. On the other hand, Foxp3+ T cells are upregulated by IFN-α therapy which is a novel mechanism that may contribute substantially towards the disease-modifying ability of IFN-α treatment. Notably, most effects of IFN-α on T cells, including Th17 and Treg cells, persist even after cessation of treatment. Also, I report in details the responses of CD4+, CD8+ and γδ T cell subsets in vitro following treatment of healthy donor PBMCs with IFN-α. The findings are largely in agreement with the ex vivo study. Fopx3-expressing and IFN-γ-, IL-10-, and TGF-β- producing CD4+ and CD8+ T cells are all increased by IFN-α treatment. Whereas, IL-17-producing CD4+, CD8+ and γδ T cell are decreased following treatment with IFN-α. Our data may provide new inroads into elucidating the immunomodulatory mechanisms involved in the disease-modifying ability of IFN-α therapy. Which of the above mechanisms plays the most important role in the observed beneficial effects of IFN-α in the treatment of BD remains to be elucidated.

The ocular surface is continually exposed to the outside environment and is a common site of inflammation. Conjunctival epithelial cells are thought to play a role in innate responses at the ocular surface. The hypothesis of my study is that conjunctival epithelial cells also contribute to T cell and mast cell effector mechanisms in chronic allergic eye disease via secretion of cytokines. In this study we initially demonstrate that the conjunctiva expresses TLRs, and that the TLR3 ligand (poly I:C) activates conjunctival epithelial cells in vitro to secrete inflammatory mediators as part of the innate immune response. Conjunctival tissues were also shown to express the Th2 associated cytokine, IL-13 as well as TSLP – a cytokine thought to be involved in Th2 differentiation. Conjunctival tissues from chronic allergic eye disease subjects were found to have increased IL-13 and TSLP expression compared to normal controls. Using a human conjunctival epithelial cell line, cells could be induced to express increased levels of TSLP following exposure to poly I:C or pro-inflammatory cytokines. Th17 cells, identified by coexpression of CD4 and IL-17, were also detected in CAED tissues and a high level of expression of IL-17A was localised to the epithelium. However, although capable of secreting IL- 25, IL-17A was not secreted by conjunctival epithelial cells, indicating that the IL-17 observed histologically may have been IL-17 binding to the surface of the epithelium. IL-17 receptor C (IL-17RC) expression was found to be increased in CAED tissues whilst IL-17RA was upregulated when conjunctival epithelial cells were stimulated with pro-inflammatory cytokines together with poly I:C. Blockade of IL-17RA and subsequent stimulation with IL-17 led to increased IL-8 and decreased TGF-β secretion. Although being implicated in the immunopathogenesis of certain diseases, IL-17 and its other family members may potentially serve to play an immunoregulatory role in immunity at the ocular surface.

No description available.