It is estimated that there are millions of corneal operations performed annually in the United Kingdom and United States following pathology and/or acute injury. Equally, millions elect to have corneal refractive surgery to remedy visual disorders such has myopia, prespreopia, astigamatism and associated keratoconus. Following all of these procedures, there is a risk of complications from scar formation, cell loss, and corneal oedema which may adversely affect the surgical outcome and resulting best vision. Through research into how the cornea heals, it is hoped that these factors could be better understood and ultimately used to increase patient satisfaction. The projects are outlined as followed: 1) Using X-ray scattering techniques to investigate the role of Pax 6 on the collagen ultra- structure of murine corneas 2) Oral mucosal fibroblasts as a novel wound healing treatment of LASIK injured ovine cornea 3) Using X-ray scattering techniques to investigate the ultra-structural changes of a 12 year post-operative penetrating keratoplasty for keratoconous 4) Using small angle X-ray scattering techniques to investigate limbal collagen ultra-structure of keratoconic and normal human tissue In this wound healing study, a range of different pathological and acutely injured mammalian corneas (human, sheep, and mouse) were evaluated using a range of quantitative techniques. These techniques encompass small and wide angle X-ray scattering, mechanical analysis, immunofluorescence, spectrophotometry, and cell/organ culture model. We feel that investigating various species and disorders has provided a more comprehensive and broad overview of the various maladies which can occur during the wound healing process.

Cataracts are the commonest cause of preventable blindness in the world. During surgery the natural lens is replaced with a polymeric intraocular lens (IOL), leaving the capsular bag in situ. The most common postoperative complication is scarring which is known as posterior capsule opacification (PCO). PCO occurs when residual lens epithelial cells (LECs) dedifferentiate and migrate onto the previously cell free posterior capsule. By modifying the IOL surface properties we can manipulate the cellular response. BioInteractions Ltd. is an innovative supplier of biomaterials, which aim to minimise, the host response, and provided the materials for this study. The aim of this study was to evaluate potential IOL coatings to reduce PCO. This can either be achieved by enabling a monolayer of LECs to attach to the posterior surface of the IOL, thus sandwiching the IOL to the capsular bag, or prohibiting cell attachment to the IOL entirely. Materials and Methods: Various coatings were investigated incorporating functional groups of poly ethylene glycol (PEG), sulphates, sulfonates, glycosaminoglycans (Heparin, (HEP) hyaluronic aid, (HA) and chondroitin sulphate (CS)) and zwitterionic monomers (10-30%). Ways to prevent dedifferentiation was also evaluated. LECs were seeded onto all coatings and monitored for a period of 7 – 14 days in cell culture. LECs were examined morphologically, cell nuclei were counted and growth curves were plotted. Water contact angle (CA) measurements were taken to measure the wettability of the coatings. Scanning electron microscope (SEM) analysis was performed to examine the topography of the coating. White light interferometry (WLI) analysis was conducted to analysis the surface roughness. Dedifferentiation of LECs and the use of TGFβ3 to neutralise or prevent dedifferentiation were also investigated. Results and Discussions: Coatings with a greater number of water-based layers were the most hydrophilic, and did not offer the appropriate cell binding sites required to promote cell attachment. In general, little cell attachment was observed on HEP and HA coatings provided by BioInteractions Ltd., cell attachment varied on CS coatings provided by BioInteractions Ltd. When HA and CS were covalently bound onto amine coated coverslips a reduction in cell attachment was observed. The LEC response varied across different ratios of zwitterionic monomer within the coatings. Zwitterionic coatings were not cytotoxic to LECs and surface analysis demonstrated no clear link between wettability and roughness compared to cell attachment. Addition of transforming growth factor beta 2 (TGFβ2) was chosen as a successful dedifferentiation model. Addition of TGFβ3 had little influence at reversing dedifferentiation however it may offer some protection against differentiation. PCR analysis showed a change in regulation of collagens, integrins, matrix metallopeptidase and fibronectin 1 genes, when LECs were incubated with TGFβ2, TGFβ3 or untreated (control LECs). These genes may play important roles in PCO. Conclusions Incorporation of functional groups influenced the cellular response, however the coatings with more water-based layers prohibit cell attachment. The cellular response varied depending on GAG type and the conformation of GAG on the surface coating. HA and CS bound to amine-coated coverslips prohibited cell attachment at higher concentrations, indicating their potential to prohibit LEC attachment. There was no clear link between wettability and cell attachment on the novel zwitterionic coatings. The ratio of zwitterionic-component:arylic-based monomer(s) influenced cell attachment. TGFβ2 successfully dedifferentiated LECs. Further work is required to understand the influence of TGFβ3 on dedifferentiation.

Pocket and portable electronic low vision aids (PELVAs) are relatively new devices designed to enhance vision of people with visual impairment. Therefore, the aim of this thesis was to evaluate their use and prescribing patterns for PELVAs for patients with visual impairment and to inform clinicians of the functions or attributes that are most important when considering their prescription. Firstly, the parameters (including magnification screen size, luminance contrast, and resolution) of PELVAs were assessed. Magnification and screen size were also compared with the manufacturers’ data. Secondly, data (age, gender, ocular condition, visual acuity, living situation, registration of visual impairment, type of low vision aid prescribed) from 6,668 patients who attended the Low Vision Service for Wales were analyzed to assess the clinicians’ prescribing patterns for PELVAs. Thirdly, reading frequency and duration, and self-reported effectiveness of PELVAs and optical low vision aids for patients with visual impairment were evaluated by using a telephone interview based upon the Manchester Low Vision Questionnaire. Finally, reading speed was measured for normally sighted subjects with simulated visual impairment who used a PELVA and an optical low vision aid. The factors that could predict reading speed were investigated. PELVAs enhanced the luminance contrast of high and low contrast letters which may be beneficial for patients with contrast reduction, for example due to cataracts. Variations were found between reported and measured magnification of the PELVA. Only 10% of adult patients were prescribed a PELVA, and younger males were more likely to use them. A larger proportion of children (36.5%) were prescribed a PELVA. Patients who used PELVAs rated them highly for near vision tasks and were more likely to use PELVAs for reading once a day but for a long duration, whereas optical low vision aids were used more frequently and for a shorter duration. It was found that the reading speed was significantly improved using PELVA and optical low vision for subjects with simulated visual impairment; Bailey-Lovie near visual acuity, high and low contrast Colenbarnder near visual acuity, and number of visible characters were significant predictors of reading speed.

Glaucoma is a progressive optic neuropathy that causes irreversible vision loss and is the second leading cause of blindness worldwide. Glaucoma is characterised by loss of retinal ganglion cells (RGC) and the proposed site of primary damage is the lamina cribrosa (LC), where RGC axonal transport is disrupted causing subsequent RGC damage and eventual cell death. Current detection for primary open angle glaucoma (POAG) is based upon clinical measures such as intraocular pressure (IOP), visual field loss and changes to the optic nerve head (ONH). However, for there to be an indication that there is a problem using these measures, often RGC damage has already occurred. Therefore it is crucial to determine ocular parameters that alter in the earliest stage of disease, prior to vision loss occurring. In this thesis optical coherence tomography (OCT) was used to assess the optic nerve heads and maculae of control eyes and eyes with preperimetric, early and advanced glaucoma in order to characterise changes that could potentially be used as biomarkers for the earliest stages of the disease. A custom built 1050 nm research OCT was used to acquire datasets from the macula and optic nerve heads of eyes glaucomatous and control eyes in vivo. Analysis of the inner retinal layers at the macula was performed to indirectly assess RGC integrity. At the ONH the prelamina and LC volume and regional depth and thicknesses were investigated. Additionally, nerve fibre layer and Bruch’s membrane parameters were assessed. Finally, LC beam coherence and orientation were probed in order to determine whether regional or glaucomatous changes ould be detected at the LC connective tissue microstructure. Prelamina depth and thickness was shown to be an indicator of early and preperimetric glaucoma (p < 0.01), although the volume of the prelamina did not change with increasing stage of glaucoma (p > 0.01). Border nerve fibre layer revealed significant thinning in early glaucoma compared to control, and the superior peripapillary nerve fibre layer was thinner in preperimetric glaucoma than control. The ratio of inner plexiform layer (IPL) : ganglion cell layer (GCL) showed significant differences between control eyes and preperimetric glaucoma, and as such has potential to be a useful biomarker for indicating the earliest stages of disease. Both the GCL and IPL were thinner in early glaucoma than control (p < 0.01), a hypothesis that cell body shrinkage and death occurs in preperimetric glaucoma and dendritic loss occurs in early glaucoma, when vision loss is first apparent, is suggested. Additionally, LC beams showed greater coherence in the superior and inferior poles than the temporal region, indicating that the shows regional variation but that further research is required to characterise changes. In conclusion, 1050 nm OCT was used to probe microstructural parameters of the optic nerve head in vivo to characterise changes that could be used as a potential biomarker for early glaucoma. ONH and retinal parameters have been identified that, with further research, may be used to differentiate between control eyes and those with preperimetric and early glaucoma. These have the potential to help identify those ONHs at risk of glaucoma damage.

Keratan sulfate (KS) is an elongated glycosaminoglycan (GAG) chain found throughout the cornea, the clear tissue at the front of the eye. It is thought that KS plays a specialized role in maintaining the ordered spatial arrangement of collagen fibrils that comprise the thickest layer of the cornea, the stroma. Repeating N-­‐acetylglucosamine (GlcNAc) and galactose monosaccharides make up the fine structure of KS, and sulfate groups frequently modify their C-­‐6 positions. Recent studies have linked improper sulfation of GlcNAc residues to macular corneal dystrophy, a vision condition characterized by a progressive loss of corneal transparency. Since then, in vitro experiments have implicated four enzymes in highly sulfated KS biosynthesis. It is currently believed that β-­‐1,3-­‐N-­‐ acetylglucosaminyltransferase 7 (β3GnT7), corneal GlcNAc 6-­‐O sulfotransferase (CGn6ST), and β-­‐1,4-­‐ lactosyltransferase 4 (β4GalT4) sequentially catalyze the addition of GlcNAc, transfer sulfate to its C-­‐6 position, and link galactose to the growing KS backbone, respectively. The fourth enzyme, KS galactose 6-­‐O sulfotransferase (KSGal6ST) is thought to act last, sulfating galactose residues. Mutant mice deficient in CGn6ST, KSGal6ST, or β3GnT7 were recently developed to investigate the consequences of dysfunctional sulfo-­‐ or glycosyltransferases on corneal morphology. Electron microscopy and immunohistochemistry data in this thesis showed that the systemic absence of CGn6ST or β3GnT7 resulted in a corneal stroma devoid of KS, but mutation of only KSGal6ST led to a KS phenotype similar to that of wild type controls. Western blot analysis conducted on β3GnT7-­‐null corneas indicated that KS assumed an unusually truncated form, as compared to wild type controls. A secondary result evident in electron micrographs was that in cases where KS levels dropped below the detectable threshold (i.e. in CGn6ST and β3GnT7 mutant corneas), a concurrent appearance of atypically elongated GAGs was visible, suggesting a compensatory mechanism to preserve corneal organization. Since the unusual GAGs were susceptible to chondroitinase ABC digestion, it is thought they are comprised of chondroitin/dermatan sulfate (CS/DS). Studies using high performance liquid chromatography were also undertaken to establish protocols in which future work could quantify the change in CS/DS expression observed in CGn6ST and β3GnT7 mutant corneas.

It has been demonstrated repeatedly, over a period of many years that some animals can perceive magnetic field parameters and use this sensory information to their advantage. The sensory mechanisms involved are, however, still poorly understood. Due to a general lack of understanding of magnetoreception and its properties, experiments to date have been somewhat disjointed with many, often very different, investigative approaches being employed. As a result, direct corroboration of results is uncommon and progress is often slow. Advancement of this field is important for numerous reasons, such as improving knowledge of factors affecting animal migration in a time of concern over climate change and the possible imminent change in earth’s magnetic field parameters. Perhaps most significantly, such studies might provide a potential keystone in the study of the function of quantum mechanical processes in biological systems. The current study utilises a novel approach, designed to ensure improved reproducibility. Reported here for the first time, prepulse inhibition of a startle response is used to investigate magnetoreception in the homing pigeon (Columba livia). This powerful, well-characterised paradigm relies on reflexive behaviours and therefore ensures an objective method for demonstrating the perception of a stimulus. Visual psychophysics and polarised light vision are also explored. Proof of concept is provided for the use of prepulse inhibition to investigate magnetoreception. The value of using liquid crystal displays in an investigation of pigeon head movements, in magnetoreception and more generally, is demonstrated. A new, low invasiveness technique for tracking head movements is described. The need to eliminate anthropogenic electromagnetic signals in order to carry out reliable magnetoreception experiments is highlighted. Suggestions are also made regarding possible future directions of research in this area.

Glaucoma is an optic neuropathy characterised by the loss of retinal ganglion cells (RGC). Dendritic atrophy occurs early in the disease, prior to soma and axonal degeneration. RGCs exhibit reduced branching density and dendritic field size. This thesis seeks to further characterise dendritic atrophy in glaucoma in the context of two external factors that may contribute to the disease pathology – immune system effects mediated via complement and the influence of the perineuronal net (PNN), a specialised extracellular matrix that surrounds RGCs. RGC morphology was investigated in a rat bead model of experimental glaucoma using ballistic labelling techniques; morphological changes were related to synaptic loss and PNN composition using immunohistochemistry. A model was derived for the classification of diseased RGCs in order to prevent labelling bias in subsequent investigations. The immune system was modulated using a complement inhibitor (using a transgenic mouse and pharmacological agent in rats) and PNNs disrupted using the bacterial enzyme Chondroitinase ABC. Experimental glaucoma caused significant dendritic loss, with partial protection conferred by both complement inhibition and PNN digestion. Analysis of retinal sections also revealed partial protection of synapses. PNNs did not show any changes in their composition in the rat in experimental glaucoma but human glaucoma eyes showed increased glycosaminoglycan sulphation in the RGC layer which was correlated with visual deficit. Manipulation of the RGC external environment therefore proved successful in protecting from dendritic atrophy.

The purpose of this thesis was to assess long-term outcomes of the visual dysfunction arising from the ocular toxicity associated with the anti-epileptic drug vigabatrin (VGB). The risk of vigabatrin-associated visual field loss (VAVFL) with increasing exposure to VGB was modelled from retrospectively collected data from a cohort of 147 individuals (median exposure 7.9 years; IQR 3.6, 11.0). The modelled frequency of VAVFL increased with increasing exposure and plateaued at 75-80% after approximately 6 years duration and 5kg cumulative dose. The relationship between the numbers of retinal ganglion cell soma and axons, derived by standard automated perimetry and time-domain optical coherence tomography (TDOCT), respectively, was evaluated in 24 individuals with VAVFL and in 16 exposed to vigabatrin but with normal fields (VGBN). A strong linear association was present between the two outcomes, which was suggestive of an optic neuropathy, and was similar to the association for a control group of 18 individuals with open angle glaucoma. A follow-up visual field, after a median interval of 7.0 years (IQR 6.5, 7.6) was determined in 19 individuals with VAVFL and in 8 with VGBN, after a median withdrawal from VGB of 7.1 years (IQR 5.4, 8.4). No consistent trend was noted for either a deterioration or improvement in the field. A follow-up scan of the peripapillary retinal nerve fibre layer (RNFL) thickness, by TDOCT, after a median interval of 6.5 years (IQR 5.8, 6.9) was obtained in 13 individuals with VAVFL and in 4 with VGBN, after a median withdrawal from VGB of 8.0 years (IQR 5.3, 10.2). No consistent trend was noted for either a deterioration or improvement of the RNFL thickness. The macular thickness was evaluated by TDOCT in 32 individuals with VAVFL and in 14 with VGBN. No difference in thickness was noted between the two groups.

: Infantile nystagmus (IN) is a pathological condition of continuous, horizontal eye oscillations. Despite ongoing eye movements, those with IN do not experience oscillopsia, i.e. the illusory perception that the environment is moving to-and fro. The correct use of neural compensation for their eye movements, just as in typical individuals, is hypothesised to account for this lack of oscillopsia. This neural mechanism requires that an estimate of eye velocity (‘extra-retinal signal’) be compared to an estimate of the motion of the retinal image (‘retinal signal’), and any difference due to object motion. Despite this hypothesis, there have been no previous investigations on how accurately those with IN can estimate object motion. Even in typical adults, eye movement compensation is not perfect, which underlies a number of pursuit-based illusions. One such illusion, the Filehne illusion can be used to investigate the relative mismatch between the extra-retinal and retinal signals as individuals judge their perception of stationarity while attempting to follow a moving target. This illusion was used to investigate, through comparison with typical individuals, whether those with IN correctly recover object motion. Experiments were conducted to characterise the ability of those with IN to fixate as well as follow targets, using a novel two-dimensional eye movement measure. Under repeated testing, the fixation gaze angle at which the IN oscillation is minimum (a null zone) was not consistent. Moreover, those with IN were unable to accurately or precisely follow targets using their slow phase. Results from a comparison of fixation and pursuit performance in those with IN suggested no difference in either the accuracy or precision. Importantly, the psychophysical judgements of those with IN pursuit eye movements are prone to similar compensation errors as typical controls. However, the mechanisms by which this is achieved will require further exploration.

Volitional target-selecting eye movements, such as saccades or smooth pursuit, are frequently considered distinct and separate from automatic gaze-stabilising eye movements like optokinetic nystagmus or the vestibulo-ocular reflex. This difference is regularly mapped onto brain anatomy, with distinctions made between subcortical, automatic processes; and cortical, volitional ones. However gaze-stabilising and target-selecting eye movements must work together when a moving observer views natural scenes. Yet such co-ordination would not be possible if automatic and volitional actions are sharply divided. This thesis focuses upon interactions between gaze-stabilising and target-selecting eye movements, and how these interactions can aid our understanding of the relationship between automatic and volitional processes. For a saccade executed during optokinetic nystagmus to accurately land on target, it must compensate for the ongoing optokinetic movement. It was found that targeting saccades can partially compensate for concomitant optokinetic nystagmus. The degree of compensation during optokinetic nystagmus was indistinguishable from compensation due to voluntary smooth pursuit displacements. A subsequent experiment found that locations are similarly misperceived during optokinetic nystagmus and smooth pursuit. Furthermore, saccade end-points are subject to the same perceptual mislocalisations. The next experiment established that fast-phases of optokinetic nystagmus can act like competitive saccades and cause curvature in targeting saccades. Moreover, optokinetic nystagmus fast-phases are delayed by irrelevant visual distractors in the same way as saccades (the saccadic inhibition effect). Lastly, it was established that the fast-phases of Infantile Nystagmus Syndrome also show the saccadic inhibition effect. In conclusion, target-selecting and gaze-stabilising eye movements show substantial co-ordination. Furthermore these results demonstrate considerable commonalties between ‘automatic’ and ‘volitional’ eye movements. Such commonalities provide further evidence there is no sharp distinction between automatic and volitional processes. Instead it is likely there are substantial interconnections between automatic and volitional mechanisms, and volition has a graded influence upon behaviour.