Recent studies have indicated that avian magnetoreception is based on vision, being both wavelength and luminance sensitive. Using this novel behavioural assay, the ability to respond to changes in magnetic field conditions was tested in the dark and the light. Darkness was found to have an adverse impact on birds' abilities to perceive magnetic field changes. Magnetoreception as measured by reactivity was virtually eliminated in the dark as compared with the same birds' behaviour in normal light conditions.

The current understanding of anterior eye shape in humans is limited due to available technology and its accessibility. Accurate curvature metrics of specific areas of the peripheral cornea, corneo-limbal junction and anterior sclera have remained obscured by the limits of the palpebral aperture, since the upper and lower eyelids cover most of the vertical aspect. This thesis starts by comparing the ‘gold standard’ keratometry measurements to commonly used topographic systems. Keratometric analogues were found to be significantly different and in addition provided spurious vertical anterior ocular surface (AOS) profiles. These findings revealed a need to establish an accurate model. Magnetic resonance imaging (MRI) potentially offers the best opportunity to image the entire AOS structure. However, preliminary studies in this thesis demonstrated that the use of a 3-Tesla MRI scanner was unable to obtain sufficiently resolute data to meet requirements. As an alternative, ocular impression taking techniques were adopted during the remainder of this work to acquire the AOS data. Eye casts from impression moulds were scanned using active laser triangulation and virtual 3-dimensional surfaces rendered. Further investigations defined the most suitable material for impression taking and the amount of deformation of the AOS caused by the procedure. The ocular impression casting and scanning process was examined for accuracy and reliability. This protocol was used to sample a population of normal white European eyes in order to establish a database and define wide-field AOS variability. Volumetric and 2-dimensional topographic profiles were extracted from the digital 3-dimensional representation obtained, allowing for the analysis of point-to-point curvature differences. For the first time, the entire AOS shape has been defined with known accuracy. In addition, effects of myopic refractive error and gender are presented. This data is of potential importance to ophthalmic surgeons, ocularists, contact lens practitioners, vision scientists and researchers, in the form of a digital archive of normal white European wide-field AOS topography as a reference source.

Age-related macular degeneration (AMD) is the principle cause of visual loss and blindness in the developed world. As new treatments and therapies are developed the need to better diagnose and then monitor outcomes of treatment has become more important. This thesis evaluates both structural and functional changes that occur in the early stage of AMD, known as age-related maculopathy (ARM), with the aim of determining their diagnostic potential. This thesis also explores the relationship between structural and functional parameters. Twenty four participants with ARM and 26 control participants were recruited. Retinal function was probed using four focal electroretinography (ERG) techniques: the focal cone ERG, focal flicker ERG, ERG photostress test and focal rod ERG. Long wavelength optical coherence tomography (OCT) was used to assess retinal structure, specifically retinal, choroidal and four intra-retinal layer thicknesses at 21 macular locations. These techniques were initially developed and optimised for the detection of AMD related changes. The ability of each parameter to diagnose ARM was assessed. Correlation and linear regression analyses were carried out to identify any relationships between retinal structure and function in healthy controls. Retinal thickness was reduced in participants with ARM at parafoveal locations (~2° eccentricity), but choroid thickness was unaffected. Diagnostically, focal ERG parameters provided better sensitivity and specificity to ARM than OCT measures, with the ERG photostress test providing the best diagnostic potential. No strong relationships were shown between any ERG parameter and any retinal or choroidal layer volume in control participants. Three ERG parameters were shown to be related to specific retinal features of ARM, but the strongest associations were between ERG photostress test recovery and focal cone ERG b-wave implicit time and a diagnosis of wet AMD in the contralateral eye. In conclusion the structural and functional parameters assessed appeared to provide independent information regarding disease status and severity. ERG parameters showed better diagnostic potential than OCT measures. The single most diagnostic parameter was the recovery time constant of the ERG photostress test.

The aim of this PhD thesis was to develop segmentation algorithm adapted and optimized to retinal OCT data that will provide objective 3D layer thickness which might be used to improve diagnosis and monitoring of retinal pathologies. Additionally, a 3D stack registration method was produced by modifying an existing algorithm. A related project was to develop a pre-apoptotic retinal monitoring based on the changes in texture parameters of the OCT scans in order to enable treatment before the changes become irreversible; apoptosis refers to the programmed cell death that can occur in retinal tissue and lead to blindness. These issues can be critical for the examination of tissues within the central nervous system. A novel statistical model for segmentation has been created and successfully applied to a large data set. A broad range of future research possibilities into advanced pathologies has been created by the results obtained. A separate model has been created for choroid segmentation located deep in retina, as the appearance of choroid is very different from the top retinal layers. Choroid thickness and structure is an important index of various pathologies (diabetes etc.). As part of the pre-apoptotic monitoring project it was shown that an increase in proportion of apoptotic cells in vitro can be accurately quantified. Moreover, the data obtained indicates a similar increase in neuronal scatter in retinal explants following axotomy (removal of retinas from the eye), suggesting that UHR-OCT can be a novel non-invasive technique for the in vivo assessment of neuronal health. Additionally, an independent project within the computer science department in collaboration with the school of psychology has been successfully carried out, improving analysis of facial dynamics and behaviour transfer between individuals. Also, important improvements to a general signal processing algorithm, dynamic time warping (DTW), have been made, allowing potential application in a broad signal processing field.

The heterozygous mutation, B6;C3-Opa1Q285STOP, leads to a 50% reduction in Opa1 transcript and protein in the mouse retina and neural tissues and models autosomal dominant optic atrophy and presents with visual dysfunction and structural changes in the retina and optic nerve. This thesis explores the intimate relationship between retinal ganglion cell dendritic architecture, health and synaptic connectivity as influenced by the mitochondrial fusion protein Opa1. Using a range experimental paradigms it is reported here retinal ganglion cell dendritic atrophy which is exacerbated with age and localised exclusively to sublamina b of the inner plexiform layer. There is a marked reduction in the number of glutamatergic synaptic sites and PSD95 levels on ON-centre retinal ganglion cells. In addition, there is a significant increase in synaptic vesicle number and density in both ON and OFF bipolar cells. These processes cast light on the intimate relationship between normal mitochondrial fusion and fission balances, as influenced by the OPA1 protein, in neural cell connectivity in the mammalian retina and the changes shown here serve as an exciting biomarker for disease and rescue and recovery therapeutics.

The overall objective this work is to contribute to the understanding of how the precise structure of the corneal stroma is achieved during development, and to apply this knowledge to the latest attempts at engineering effective stromal constructs for use in transplantation. The cornea is the major refractive element of the human eye, accounting for two-thirds of total focusing power. Representing around 85% of corneal thickness, the stroma possesses the mechanical strength needed to protect intraocular tissues, whilst still achieving the high level of transparency necessary for light transmission. This is chiefly due to the small, uniform diameter collagen fibrils arranged into a precisely ordered series of orthogonal lamellae. Proteoglycans in the stroma are thought to regulate the arrangement and diameter of the collagen fibrils, although the mechanism by which this occurs is not fully understood. The deceptively complex organisation of the stroma may be responsible for the relatively little progress that has been made in engineering constructs that can reproduce the structural and functional characteristics of the cornea. Further study into the embryonic development of the cornea may aid attempts to recapitulate in vivo mechanisms for corneal construction. Of particular relevance would be the method of collagen organisation and deposition in the developing avian corneal stroma and the interactions that occur within the collagen fibril bundles as development progresses. Initially, en face sections were used to study the organisation and arrangement of collagen fibrils in the developing stroma. It is hypothesized that in tendon, the formation of parallel arrays of collagen fibrils occurs via fibroblast surface recesses and invaginations. It was evident through transmission electron microscopy that this process also occurs in the developing corneal stroma via surface recesses on stromal keratocytes. Analysis of the interactions between the collagen and proteoglycans within fibril bundles demonstrated that the developing cornea is less well structured than often considered and is possibly a much more fluid and dynamic system than originally thought. Proteoglycan size and orientation show a degree of variety and disorder and appear to follow no set organisation or positioning. The data suggests that proteoglycans were seen forming aggregates that were capable of bridging the gap between more distant neighbouring fibrils. Following the study of the developing corneal stroma, collagen gel based constructs were engineered and their structural and functional characteristics were analysed to assess their potential as stromal equivalents for use in tissue engineering. Manipulating the assembly of collagen fibrils by varying the pH and cross-linker concentration had a dramatic effect on the structure and functionality of the final gel construct. A range of collagen gels were then implanted into intra-stromal pockets to determine their biocompatibility and in vivo properties.

Understanding the biomechanical properties of the cornea is important in order to develop and improve new reliable standard procedures which can be used effectively to assess corneal behaviour in any disease condition, or before/after any ocular surgery. We believe that the Ocular Response Analyzer (ORA) is the only device that can measures the biomechanical properties of the cornea in vivo. However, it has been used for the first time both in vivo and in vitro. This thesis presents a clinical and laboratory investigation of the biomechanical properties of the cornea before/after LASIK and corneal cross-linking to improve our understanding of the knowledge required in both the laboratory and the clinic. Different machines were used in this project, including an ORA, an Oculus Pentacam, a spectrophotometer and a UV-X Illumination system. Laser in situ keratomileusis (LASIK) is, at present, one of the most well-known operations used to correct refractive errors; however, ocular problems arising from corneal thinning have been reported in some previous studies. Therefore, I looked at the effects of surgery on the central/peripheral thickness and the anterior/posterior curvature, and determined to what extent they affect the biomechanical properties of the cornea. During the past decade, much research has focused on improving and developing a new operation called corneal collagen cross-linking with riboflavin and UVA, which is used to stop the progression of keratectasia in the cornea (which occurs in keratoconus and sometimes follows refractive surgery). In the next phase, a range of experiments were conducted on cross-linking to determine to what extent this operation affects the molecular structure and biomechanical properties of the cornea. This thesis has shown for the first time that it is possible to obtain ORA signals in vitro and this opened up the possibility of examining whole eyes as well as excised corneas. It is also confirmed that the values of CH do not represent only a corneal biomechanical property, but rather depend on the presence of the rest of the eye. These in vitro studies have opened up a number of possibilities the future corneal biomechanical studies.

CD4+ T cells can be subdivided on the basis of their lymphokine repertoire produced on activation resulting in TH1 like and TH2 like populations. The purpose of this study was to measure the intraocular expression of cytokines as a means of defining the CD4+ lymphocyte subsets present during the development of uveoretinitis. Lewis rats were immunised with retinal antigen and pertussis toxin resulting in early signs of disease activity evident by day 9 with increasing severity evident by day 12. Extensive clinical and histological damage was observed by day 14 with a reduction in severity through to end stage disease at day 24. In this study, the failure to establish pure populations of retinal antigen specific T lymphocyte cell lines and the observation of the lack of sensitivity of Northern hybridization to signals expressed at low levels resulted in the more sensitive technique of RT-PCR being utilized. Both IL2 and IFN mRNA expression was detected at all stages of disease with highest levels being present early in uveitis. In contrast IL4 mRNA levels increased with disease progression. This study suggests a pathogenic role for TH1 like cells and a protective role for TH2 like cells in this autoimmune disease. In order to provide an insight into alternative treatment strategies of the disease, immunomodulation of EAU was carried out using the immunosuppressive drugs CsA, FK506 and rapamycin and the resultant cytokine mRNA profiles examined. The results indicated that CsA and FK506 downregulated the TH1 response having suppressive effects on the levels of IL2 and IFN mRNA respectively. In contrast rapamycin was found to modulate the TH2 response enhancing IL4 levels. From this data, a drug based strategy employing rapamycin appears to be the most favourable approach.

<p> Growth is one of the fundamental characteristics of biological systems. The study of eye growth regulation presents an interesting window that allows for the investigation of the role of the visual environment on internal processes. We now know that there is an intricate circuitry within the eye, independent of higher brain processes, that controls the growth of the eye but more needs to be elucidated about these local regulatory circuits. An improved understanding of this circuitry is critical to developing new therapies for abnormalities in eye growth regulation such as myopia, which is impacting more and more individuals around the world each day and in its more severe from, is linked to potentially blinding ocular complications. </p><p> The role of retinal glucagon, a neuropeptide, in the regulation of eye growth and refractive error has attracted the interest of researchers over the past 15 years yet there remain many unresolved questions. The research described in this dissertation aimed to elucidate the respective roles in eye growth regulation of specific subpopulations of retinal glucagonergic amacrine cells, which have been the subject of much speculation as the source of inhibitory growth signals, i.e. stop signals, yet not thoroughly investigated. </p><p> The approach taken to investigate this problem is to ablate glucagonergic amacrine cells in vivo using different neurotoxins, and to examine how this affects the sign-dependent circuitry of eye growth regulation. In addition, with the advent of advancements in high resolution imaging and electrophysiology, we were able to characterize the effects of these neurotoxins on the region-specific and time-sensitive changes in the structure and function of the living retina. </p><p> That the inhibitory response induced by imposed myopic defocus remains intact, in spite of total ablation of glucagon cells (Chapter 5) or elimination of the peripheral glucagon cells (Chapter 3) and other unintended adverse retina effects, compared to findings from previous studies involving QUIS (Chapter 2) of this thesis, is a novel finding. These results point to the same conclusion that glucagon cells themselves are not responsible for the decoding of the sign of optical defocus, but appear to have a role in fine-tuning of compensatory growth responses. The results of our experiments also suggest that the choroid may serve as an intermediate relay, and the altered anterior chamber development raise the further possibility that retina-derived growth modulatory factors also regulate the anterior segment, perhaps reaching this more remote site by diffusion forward through the vitreous chamber or via the uvea.</p>

Autosomal dominant optic atrophy (ADOA) is a mitochondrial disorder caused by a nuclear DNA mutation. The mutation is within the OPA1 gene, which encodes a 120kDa protein product. The protein, OPA1, is a dynamin like GTPase, which is responsible for fusion of mitochondria. It is located in the inner mitochondrial membrane and functions in conjunction with outer membrane fission proteins to maintain mitochondrial integrity. The OPA1 protein is expressed ubiquitously and heterozygous mutations cause atrophy of the optic nerve. The variability in human ADOA phenotype may suggest some level of susceptibility to vision loss whereas other individuals appear symptom free. The Opa1 Q285STOP mouse model of ADOA provides a unique opportunity to examine the disease pathology with respect to mitochondrial haploinsufficiency. The tissue specificity of the disease suggests local control over the OXPHOS environment. The bioenergetic activity of Opa1Q285STOP mouse is currently unestablished. The status of antioxidant activity supporting bioenergetic function may be vital in maintaining stability within fusion deficient mitochondria. Mitochondrial stability could influence the extent of reactive species, and ultimately, ATP production. This thesis combined the study of mitochondrial haploinsufficiency with analysis of OXPHOS and antioxidant levels in Opa1Q285STOP mouse where a bioenergetic deficit was identified in all mitochondria tested. The addition of a plant derived antioxidant and its potential effects within the retinal environment and surrounding central nervous system of Opa1 Q285STOP mouse did not appear to influence the extended phenotypic profile observed in this mouse model. The administration of resveratrol to the wild type population conferred disadvantages both In Vivo and In Vitro.