Overall, this project aimed to evaluate and advance the use of two techniques in the study of structural changes brought about by in vitro glycation of mouse tail tendon type I collagen. Firstly, using transmission electron microscopy with positive staining, study of collagen glycation by glucose, galactose, fructose and ribose was conducted. Site specificity of glycation along the fibril was seen and it was demonstrated for the first time using this method that there was variation in the progression of the process between the monosaccharides, suggesting different reaction mechanisms and determinants of site specificity for each sugar. From the early changes after 1 week galactose treatment, early-preferred glycation sites for this sugar were indicated. Using sorbitol, broad non-glycation-related sugar binding along the fibril was identified and this was also seen following galactose, fructose and ribose treatment. Secondly, using X-ray diffraction with isomorphous replacement, a 1D electron density map for collagen fibrils in mouse tail was constructed for the first time as a baseline for this and future intended glycation studies in mice. Use of this technique to study tendon incubated for 2 weeks in ribose and galactose showed electron density changes that appeared consistent with the corresponding electron microscopy glycation data. Using HPLC with fluorescence detection, it was shown that the four sugars produced different “fingerprint” profiles of products of glycation. Galactose produced a similar profile to glucose as hypothesised. However despite its chemical similarity to glucose, galactose also produced glycation products that were absent from the glucose profile. The biochemical information produced was invaluable for interpretation of the data from the structural techniques, inasmuch as providing representations of the relative extents of glycation occurring during each monosaccharide and control treatment. In conclusion, this thesis has demonstrated for the first time that transmission electron microscopy with positive staining, and X-ray diffraction with isomorphous replacement techniques together are useful complementary methods for studying glycation-related changes in type I collagen structure. It is anticipated that these techniques together will be of value in further in vitro and in vivo studies of type I collagen glycation, contributing to a better understanding of the structural bases of the deleterious effects of this process during diabetes and ageing.

The research work described in this thesis embodies a number of studies designed to investigate human vision with emphasis on aspects of the pupil response and chromatic mechanisms in relation to the perceived chromatic afterimages. The aim of the first study was to establish the relationship between the perception of chromatic afterimages and the corresponding involuntary pupil responses. We started by designing and developing a new, computer-based, psychophysics program and employed it to measure the strength and duration of perceived chromatic afterimages in normal trichromats and in colour deficient observers. The dynamic luminance noise technique was used to isolate colour signals and to elicit pupil responses to coloured stimuli of known photoreceptor contrast. A model was developed to explain the afterimage results obtained in the normal trichromats and in colour deficient subjects. The model and the pupil colour responses provided an understanding of luminance and colour processing in dichromats that also helped to explain previously reported pupil colour responses. The model also predicts the colour confusion lines and the characteristics of pupil colour responses in dichromats at any given background chromaticity. In the second study, we investigated and compared pupil responses to visual stimuli that isolate photopic luminance and colour in both the sighted and blind region of the visual fields on subjects with either acquired or congenital homonymous hemianopia. The measured pupil responses in the blind hemifield of patients with acquired cortical damage are either absent or of reduced amplitude when compared to those measured in the corresponding regions of the sighted field, whereas the patients with congenital loss of visual field show similar and even enhanced pupil responses when compared to their sighted hemifield. These results suggest that in the absence of normal functioning of the direct geniculostriate projection, other projections to midbrain nuclei or to extrastriate regions can be enhanced and these include the pupillary pathways. These findings suggest that early damage to the brain might be partly compensated for by reorganising the strength of neural projections to the remaining, non-compromised visual areas. The purpose of the last study was to examine whether melanopsin contributes to the dynamic pupil light reflex responses in humans. A light source containing of four primary components was employed to generate pupillary stimuli that isolate luminance, colour or combined rod and melanopsin. Normal trichormats, rod deficient subjects, one subject with retinitis pigmentosa, one rod monochromat, three subjects with Leber’s Hereditary Optic Neuropathy (LHON) and one subject with Optic Neuritis were investigated using this approach. The results from the LHON subjects suggest not all classes of ganglion cells are affected uniformly in LHON, and that the pupil light reflex responses mediated through rod photoreceptors were affected the least. The characteristics of the pupil responses to the rod/melanopsin stimulus from the rod monochromat and the retinis pigmentosa subjects suggest that melanopsin does not contribute to dynamic pupil light reflex response in humans.

There is considerable dissatisfaction with the reliability and sensitivity of the methods used to assess the glaucomatous visual field. Two types of visual field test, which have been proposed as having potential in diagnosing glaucomatous visual field defects, have been modified and tested on a group of patients from a glaucoma clinic, a group of age-matched control subjects and a younger control group. 1. A grating pattern was generated using a laser interferometer which projected a large diameter image onto the retina independent of the subject's refractive error. The experimental set up which produced the most reliable and consistently low contrast threshold values in normal subjects was sought. The display characteristics which were examined included different orientations for the field quadrants as projected to the subject; stationary and flickering patterns using a variety of flicker generation methods; red and green light sources; and concentric or vertical sinusoidal grating patterns. Ultimately the optimal display was found to be a stationary image consisting of a green, vertical sinusoidal grating pattern. Arcuate regions of the visual field (at 10 to 20° from fixation) were stimulated in 4 distinct, obliquely oriented quadrants and a low spatial frequency (one cycle per degree) was chosen. 2. Normal limits were obtained from age-matched control subjects for comparison with the results for the patients from the glaucoma clinic. In the patient group, of the 13 who completed the test, 9 individuals were identified as abnormal with one or more of their contrast threshold scores exceeding that limit. The patients' Friedmann visual field plots were analysed and the amount of loss in each quadrant was quantified. There was a positive correlation between the quantified visual field loss and contrast threshold scores in 6 patients, a statistically borderline correlation in 2 patients and the absence of a correlation was found in 5 patient's results. The results for a subgroup of 6 visually abnormal eyes (not affected by glaucoma) excluded from the age-matched control group are also described. Their visual defects included mild cataract, amblyopia and retinal detachment. There were no clear abnormal results in 5 of the eyes in this group; however, in one subject with retinal scarring due to an infection, there was a distinct elevation of contrast threshold in the affected eye. Humphrey visual field plots were obtained for all but one of the age-matched control subjects. 3. Simultaneous brightness ratios (SBR) have previously been shown to provide an indication of'glaucomatous damage. The same subject groups as described above were tested. SBRs were obtained for central vision in both eyes of subjects (inter-ocular ratio). This technique was now extended for the first time to paired regions within each eye (intra-ocular ratios) producing 'nasal I temporal' and 'upper I lower' ratios. In each test the subject controlled the brightness ratio which was changed in a smoothly graduated and continuou8 way. The most effective procedure for recording repeatable SBRs was first explored, and it was determined that these could be best obtained by alternating the start point of the graduated filter position. For each subject, 5 ratios were obtained: inter-ocular SBR; upper I lower intra-ocular SBR for right eye and left eye; and nasal I temporal intra-ocular SBR for right eye and left eye. 4. Normal limits were obtained from age-matched control subjects for comparison with the results for the patients from the glaucoma clinic. In each of the 5 SBR tests carried out, these limits were wide, reflecting considerable variation in the normal results. Of the 14 patients who completed the tests, 5 were identified as abnormal by one or more of their SBRs being outside normal limits. Three of these were identified as abnormal by their inter-ocular SBRs alone, one was abnormal according to his upper I lower intra-ocular SBR alone and one patient had an abnormal inter-ocular SBR and an abnormal intra-ocular SBR. The corresponding regions of the patients' Friedmann visual fields were quantified, and these values were used to calculate visual field loss ratios. There was a positive correlation between the visual field loss ratios and SBRs in 3 patients, but no correlation in 11 patients. In the sub-group of 6 visually abnormal eyes without glaucoma, mild cataract appeared not to adversely affect SBR. Mean SBRs were normal in the subject with retinal detachment but there was evidence of an enhanced amount of variation in the readings. Two subjects with a damaged retina and one with an amblyopic eye did produce abnormal inter-ocular SBRs, with the normal eye being significantly more sensitive in both cases.

Acanthamoeba may cause a painful, potentially blinding, opportunistic infection of the cornea. The incidence of Acanthamoeba keratitis has increased in line with contact lens usage. Only in the last five years, however, has it been recognised that amoebae other than Acanthamoeba may also be associated with keratitis. Several reports implicating Vahlkampfia and Hartmannella now exist. These relatively unknown genera of free-living amoebae had not previously been recognised as causal agents of disease. This thesis investigated the pathogenicity of Acanthamoeba for corneal cells and tissue in vitro, compared, both quantitatively and qualitatively, to that of Vahlkampfla and Hartmannella isolates from a case of amoebal keratitis, termed V-EYE and H-EYE (Aitken et al, 1995). Problems pertinent to the diagnosis of Acanthamoeba, Vahlkampfta and Hartmannella species (sp) in human corneal tissue were also addressed.

This thesis describes the developm ent o f Dynamic Noise Perimetry (DNP), a novel method based on the equivalent noise input technique. The method, specifically targeted to an early stage o f OAG, used a 0.5 cycle per degree sine-wave grating presented at 8Hz in conjunction with an external noise m ask that was optimised for the stimulus. Equivalent noise and sampling efficiency w ere determ ined at various locations within the visual field to identify a stage o f the disease that was analogous to ganglion cell shrinkage, a stage which is believed to precede conventional methods o f detection. A pilot study initially determ ined w hether the spatial parameters o f the mask, in terms o f noise check size, were dependent on the spatial and temporal param eters o f the grating stimulus. The results show ed that the m axim al dimensions o f each check, i.e. the critical check size, were correlated w ith the drift frequency o f the stimulus. In a second and preliminary study, the variation in the critical check size with grating spatial and temporal frequency was investigated as a function o f eccentricity. Critical noise check size, in terms o f noise checks per cycle, decreased w ith increasing spatial frequency and drift frequency o f the stimulus, and with eccentric viewing. These results were used to optim ise the critical parameters for the noise mask. Temporal contrast sensitivity, equivalent noise and sampling efficiency were determined at various locations in the visual field, in 20 normal individuals and in 10 individuals with OAG. Temporal contrast sensitivity was reduced, and equivalent noise levels were elevated in early OAG, w hen com pared with normal individuals. Derivative measures o f sampling efficiency and equivalent noise declined with glaucomatous field loss. DNP was able to identify individuals w ith OAG, at locations which exhibited abnormal Pattern Deviation values and/ or abnorm al retinal nerve fibre layer thickness. DNP clearly warrants further development

The optical and biomechanical properties of the cornea are largely governed by the collagen-rich stroma, a layer that represents approximately 90% of the total thickness. It has been postulated that a novel corneal layer exists in the posterior stroma, immediately above Descemet’s membrane, termed ‘pre-Descemet’s layer’. The main aim of this thesis was to determine if this region has different structural properties to the overlying stroma. A second aim was to examine the elastic fibre distribution throughout the depth of the stroma in healthy and diseased corneas, with focus on pre-Descemet’s layer. Techniques used include serial block face scanning electron microscopy, transmission electron microscopy, and X-ray diffraction, amongst various other imaging techniques. Depth analysis revealed that centre-to-centre interfibrillar spacing was significantly lower in the first ~10µm of stroma distal to Descemet’s membrane compared to overlying regions in central cornea. Three-dimensional analysis revealed the presence of long elastic fibres running throughout the stroma, parallel to the surface of the cornea, which were concentrated in pre-Descemet’s layer. This elastic material seemed to originate from the limbus as fenestrated sheets before travelling radially into the cornea as small fibres. This data provides evidence for pre-Descemet’s layer containing altered biomechanical properties that may contribute to the formation of a variable cleavage plane observed during pneumodissection. Additionally, the elastic fibre network is likely to play an important role in the deformation and recovery of the cornea. Furthermore, the presence of elastic fibres in foetal cornea suggests a potential role in development. The distribution of elastic fibres was very different in keratoconic buttons. No fibres were located above Descemet’s membrane, whereas the elastic fibres appeared concentrated below the epithelium in thinned coned regions, potentially as a biomechanical response to prevent rupture. Attempts were made to elucidate a functional role for elastic fibres by studying the corneas from a mouse model for Marfan syndrome, where there was a ~50% reduction in elastic fibre quantity. These corneas were significantly thinner and flatter than wild types suggesting that elastic fibres play a role in maintaining the shape of the cornea. Overall, this thesis has characterised pre-Descemet’s layer, demonstrating that structural differences are present. It is likely that the network of elastic fibres described in this thesis play a multi-functional role in the cornea.

Age-related macular degeneration (AMD) is predicted to affect 196 million people by 2020 (Wong et al. 2014). To date there is no clear pathogenesis for the condition however, hypoxia has been implicated (Stefánsson et al. 2011). Currently, treatment is only available for neovascular AMD. To develop treatments targeted for early AMD a better understanding of the pathogenesis is required. There is also a need for sensitive functional biomarkers to improve diagnosis and monitoring and to expedite the evaluation of therauptics in clinical trials. The aim of this research was to investigate the hypothesis that hypoxia is involved in the pathogenesis of early AMD. Studies were carried out exploring the effect of transient systemic hypoxia (14% oxygen) and hyperoxia (60% oxygen) on scotopic thresholds and electroretinograms (ERGs) of participants with early AMD. It was hypothesised that the visual function of participants with AMD, but not age-matched controls, would improve during the hyperoxic episode and that hypoxia would have a greater detrimental effect on visual function in people with early AMD. There were no significant differences in scotopic thresholds within each group when breathing 60% or 14% oxygen compared to medical air (21% oxygen). There were also no significant differences in full-field ERG parameters between gas conditions or groups, apart from the amplitude of the b-wave which was significantly reduced under hypoxia in the control group. The amplitude of the focal flicker ERG was significantly higher in the control group than the AMD group when breathing both 14% and 21% oxygen. However, there were no significant differences in the parameters of the focal ERG within each group. These findings suggest that hypoxia is not responsible for the elevation of scotopic thresholds reported in AMD. There is also no evidence that ERG changes are attributable to hypoxia. This thesis provides no evidence to support the role of hypoxia in the pathogenesis of early AMD.

Glaucoma is a leading cause of blindness. As a progressive condition, it is important to monitor how the visual field (VF) changes over time with perimetry in preventing vision from deteriorating to a stage where quality of life is affected. However, there is little evidence of how clinical measurements correlate with meaningful quality of life landmarks for the patient or, by extension, the proportion of patients in danger of progressing to these landmarks. Further, measurement variability associated with visual fields make it difficult to monitor true change over time. The purpose of this thesis was to use large-scale clinical data (almost 500,000 VFs) to address some of these issues. The first study attempted to relate clinical measurements of glaucoma severity to UK legal fitness to drive status. Legal fitness to drive (LFTD) was estimated using the integrated visual field as a surrogate of the Esterman test, which is the approved method by the UK DVLA of defining LFTD, while the mean deviation (MD) was used to represent defect severity. An MD of -14dB or worse in the better eye was found to be associated with a 92% (95% Confidence Interval [CI]: 87-95%) probability of being legally unfit to drive. The second study used a statistical model to estimate the number of patients progressing at rates that could lead to this landmark of significant visual impairment or blindness in their predicted remaining lifetime. A significant minority of patients were progressing at rates that could lead to statutory blindness, as defined by the US Social Security Administration, in their predicted remaining lifetime (5.2% [CI: 4.5-6.0%]) with a further 10% in danger of becoming legally unfit to drive (10.4% [CI: 9.4-11.4%]). More than 90% (CI: 85.7-94.3%) of patients predicted to progress to statutory blindness had an MD worse than -6dB in at least one eye at presentation, suggesting an association between baseline VF damage and risk of future impairment. The next section investigated whether choice of testing algorithm, SITA Standard or SITA Fast, affected the time taken to detect progression in VF follow-up. The precision of the tests was measured using linear modelling techniques and the impact of these differences was analysed using simulations. Though SITA Fast was found to be slightly less precise, no evidence was found to suggest that this resulted in progression being detected later. The final study evaluated a validated and published risk calculator, which utilised baseline risk factors to profile risk of fast progression. A simpler model using baseline VF data was developed to have similar statistical properties for comparison(including equivalent R2 statistics). The results suggested that risk calculators with low R2 statistics had little utility for predicting future progression rate in clinical practice. Together these results contribute a variety of novel findings and demonstrate the benefit of using large quantities of data collected from the everyday clinical milieu to extend clinical knowledge.

Injury to optic nerve (ON) axons plays a major role in glaucoma progression. ON crush is an established model of axonal injury which results in retrograde degeneration and death of retinal ganglion cells (RGCs). However, it is unknown how signal transmission to higher visual structures such as primary visual cortex (V1) is affected after ON crush. In human glaucoma, visual function is assessed using visual field (VF) tests, but it is also not clear how the test results relate to the disease progression in the retina. Unilateral ON crush was performed on the left eyes of adult C57BL/6 mice. V1 function of the right hemisphere was assessed longitudinally by optical imaging (OI) and in vivo calcium two-photon imaging under anaesthesia before and at 7 days, 14 days and 30 days after ON crush. Human retinas from glaucoma patients were investigated for changes in RGC density and compared to the score from the VF data obtained prior to the patients’ death. ISI and 2P experiments demonstrate a significant shift in OD towards the ipsilateral eye and significant reduction of signal magnitude in V1 in response to contralateral eye stimulation in all ON crush animals. Additionally, response magnitude to ipsilateral eye stimulation was significantly increased after ON crush. While there was significant RGC loss in human glaucoma compared to age matched controls that was correlated to mean VF loss, the scores from the individual VF test points were uncorrelated to RGC density in anatomically equivalent areas. This work demonstrates that unilateral ON crush results in immediate loss of signal transmission from the retina to V1 via a crushed ON. A significant increase of responsiveness in V1 to non-crushed eye stimulation was observed, which indicates that injury of the ON in adulthood may evoke compensatory plasticity in V1.

Autosomal dominant optic atrophy (ADOA) is an inherited optic neuropathy due to mutation in the OPA1 gene. Patients present with bilateral optic nerve head pallor and loss of visual function. Patients also show a reduction in the P50:N95 ratio of the pattern electroretinogram (PERG) and thinning of the retinal nerve fibre layer (RNFL) and macula. In a mouse model of ADOA, previously generated in this laboratory, the defect first manifested as a dendritic pruning of RGCs, which appeared to be ON-centre specific. Electrophysiological evidence in both humans and the mutant mice showed a reduction in the photopic negative component (PhNR) of the brief flash electroretinogram (ERG). The separation of the photopic ERG into ON- and OFF-pathway components using long-duration monochromatic (red) flash on a rod suppressing blue background, provided an opportunity to assess ON- and OFF-RGC function in ADOA, which had not been previously investigated in humans. This study therefore aimed to assess the effect of ADOA on the red-on blue PhNR-ON and PhNR-OFF components to determine whether the PhNR-ON was a selective marker for ADOA. In this thesis, a protocol was developed for recording long duration red-on-blue PhNRs from the macula (focal) and entire retina (full-field). A comparison of the retinotopic characteristics of the PhNRs (brief and long-duration) with the N95 of the PERG and the distribution of RGCs in the retina showed that the PhNR was capable of assessing RGC function. Retinal function was then probed in twelve participants with ADOA and sixteen controls using focal and full-field long duration ERG, full-field brief flash ERG and PERG. Retinal structure was also assessed using optical coherence tomography. In conclusion, this thesis found that the PhNR-ON and PhNR-OFF amplitudes were equally affected with no evidence of a preferential ON-pathway loss.