Introduction: Multiple sclerosis is a disabling disease with impact on the social, financial and occupational life of an individual. Diagnosis of Multiple sclerosis even with the McDonald criteria, sometimes can take many years, by which time the patient has already accumulated significant disability. Hence the need for early diagnosis and prompt treatment for a better prognosis highlighted in different MS studies. Visual dysfunction in patients with Multiple sclerosis is a common finding. Asymptomatic visual loss has been highlighted in various studies. In the United Kingdom, studies have focused on genetics, environmental factors, assessment of neurological state (EDSS, MSFC) along with MRI of the brain and spinal cord. There is no single study in the UK that has looked at the visual pathway in Multiple sclerosis and correlated with the neurological outcomes. Aim: Correlation of structure and function of visual pathway in Multiple Sclerosis Methodology: We recruited 55 (110 eyes) patients with relapsing remitting MS and 25 controls (50 eyes) into this project with specific inclusion and exclusion criteria. Power calculations were done based on the parameters used for the study. Coefficient of reliability and variability was tested prior to the study in a subset of control patients. Logistic regression model with odds ratio was used to assess the ability of each test to differentiate disease from control. Subgroup analysis was done for the optic neuritis group and the group receiving disease-modifying therapy. Function of the visual pathway was assessed using Sloan contrast charts along with HRR colour chart and HVF indices. Structure of the visual pathway was mainly assessed using different protocols from Optical Coherence Tomography. Ethical approval was obtained through the North Midlands IRAS and Keele University. Results: The structure of the visual pathway correlates significantly with function of vision. This in turn correlates with neurological function. Visual tests are more sensitive than neurological testing by EDSS and hence can be used in diagnosis and to determine prognosis in RRMS. GCIP index, EDI OCT and posterior pole protocols on OCT have revealed useful and valuable information that can be translated into clinical use to manage patients with MS. Conclusion: Sloan contrast charts and HRR colour testing are sensitive measures of disease progression. Visual field test indices on the Humphrey’s field test closely relate to RNFL thickness on posterior pole OCT. GCIP and EDI-OCT show different clinico-pathological staging in MS. Visual parameters have synergistic power in evaluating the disease process in MS. Visual testing for structure and function is a valuable addition to the routine assessment of MS patients. Visual assessment helps in detection of the spectrum of disease in MS with specifics to the range of manifestations and severities of illness. Multiple sclerosis like many other diseases exhibit the iceberg phenomenon. The iceberg phenomenon describes a situation in which a large percentage of a problem is subclinical or otherwise hidden from view. Thus, only the "tip of the iceberg" is apparent to the clinician. Visual assessments help in uncovering disease below the sea level by early detection and subsequent better disease control. Visual studies in MS using the parameters in this study must be considered for routine use in our day to day practice in diagnosis and managing patients more effectively.

As we navigate the environment, images flow over our retinae and produce complex moving patterns including contraction and expansion. These in turn serve to inform us about our movement in the world, as well as about the positions and relative motions of the objects around us. The processes underlying the perception of radial motion were explored using psychophysics, eye tracking and functional magnetic resonance imaging (fMRI). The initial experiments studied how adaptation to radial motion affects discrimination using partially-coherent dot stimuli. Adaptation increased absolute detection thresholds in the adapted direction, but had no effect on the discrimination of higher pedestals. The change in sensitivity is consistent with a divisive inhibition mechanism, as well as with one based on proportion estimates. An orientation discrimination experiment adds support for the latter. Directed attention to one component of a motion-balanced transparently-moving stimulus impairs sensitivity to a level comparable to that following adaptation, although there were large inter-subject differences. A novel approach using relative velocity adjustments of transparently-moving dot fields was used to investigate the reference frame of motion adaptation, which is shown to be mainly retinotopic and diminished by gaze modulations. Finally, fMRI was used to probe the neural substrates of radial motion perception, including retinotopic mapping, localisation of MT/MST, and selectivity for contraction and expansion within the MT+ complex.

Glaucoma describes a group of optic neuropathies characterised by progressive irreversible loss of visual function. Within this thesis, a health economic model was constructed to map service provision from diagnosis considering two competing strategies: the current practice of annual visual field (VF) monitoring against the proposed guidelines of performing 6 VFs in the first two years. The constructed model found the proposed practice to be cost effective at a willingness to pay ceiling ratio of £30,000 per quality adjusted life year (QALY), identifying an incremental cost effectiveness ratio (ICER) of £21,679. The findings of the model however were potentially sensitive to the modelled infrastructure improvement costs required to undertake the proposed guidelines and a costing study to more accurately ascertain these costs was recommended. Following this study, statistical analysis of 473,252 VFs was undertaken to investigate trends in initial identification and progression rates whilst also narrowing their parameters within the health economic model. Consequently, the average level of glaucomatous vision loss at diagnosis was found to be improving by 0.11 dB per year on average whilst proportions of patients with ‘advanced’ loss at diagnosis fell significantly from 30% to 21%. Average progression rates were found to have fallen from -0.11 dB per year to -0.06 dB per year whilst average rates of loss in older eyes ( > 70 years) were found to progress faster than in younger eyes ( < 60 years). Furthermore, testing frequency was found not to vary by visual impairment risk factors. The constructed health economic model was subsequently updated to incorporate the more narrowly defined parameter distributions whilst also being re-specified to incorporate societal costs of visual impairment to count the true costs of the disease. This resulted in an improved ICER of £11,382. In conclusion, it is likely that implementing the proposed guidelines of 6 VFs in the first two years is more cost-effective than annual monitoring. This argument is further reinforced once societal costs are accounted for however a scoping study to examine the required costs of improving the glaucoma monitoring infrastructure is required.

This study involved the determination of the effects of binocular viewing on contrast sensitivities in 11 normal subjects and in different categories of amblyopes. These were simple anisometropic amblyopes (n=9), micro-esotropic amblyopes with anomalous BSV (n=6), esotropic amblyopes with anomalous BSV (n=3) esotropic without BSV 9n=5), exotropic amblyopes without BSV (n=2) and a group of non-amblyopic strabismics (non-amblyopic esotropes without BSV (n=4); non-amblyopic exotropes without BSV (n=2).An ophthalmic examination was carried out on all individuals. The examination procedures undertaken comprised determination of the visual acuity, subjective refraction, the results of which were confirmed by retinoscopy, and assessment of uniocular fixation patterns. The state of BSV, the direction and magnitude of the angle of deviation, the amplitude of accommodation and pupillary diameter were also determined. The subjects were accordingly placed into the appropriate groups on the basis of the basis of the results of the ophthalmic examination. Measurement of uniocular and binocular contrast sensitivities in response to stationary vertical sinusoidal grating patterns were undertaken. The stimulus display consisted of a Tektronix 5103 cathode ray tube (CRT) with a screen subtense of 2 degrees. Mean contrast threshold values were measured for monocular and binocular viewing over the range of spatial frequencies studied which varied between 8c/deg to 40c/deg depending on the group being examined. The conclusions reached were, first, in individuals with BSV (normal or anomalous), binocular enhancement of contrast sensitivities occurred. However, strabismic amblyopes without BSV and non-amblyopic strabismics without BSV did not exhibit enhanced binocular contrast sensitivities; on the contrary, binocular contrast sensitivities were reduced compared to those obtained through the better eye. Furthermore, when bifoveal stimulation was effected, a further reduction in binocular contrast sensitivity occurred. This study has thus shown that binocular contrast sensitivities are augmented compared with monocular contrast sensitivities when BSV is present, but are decreased when BSV is absent. Furthermore, correction of the angle of squint in strabismics, whether BSV is present or not, further reduces the binocular contrast sensitivities.

A literature review revealed a lack of systematic research investigating standards of clinical practice within optometry. Three main approaches have been used to evaluate the content of clinical consultations: abstraction of clinical records, clinical vignettes and unannounced standardised patients. The aim of this thesis was to obtain an objective insight into the content of optometric eye examinations using these three approaches. In the first scenario, the SP presented for a private eye examination as a 20 year-old myope, complaining of recent onset headaches. The presence of headache was detected in 98% of cases. 22% asked at least four of the eight questions appropriate for primary care headache investigation and 69% of practitioners asked the patient to seek a medical opinion regarding the headaches. The second SP presented as a 44 year-old patient of African racial origin for a private eye examination having experienced recent difficulty with her near vision. 95% of optometrists visited carried out optic disc assessment and tonometry and 35% of optometrists carried out all of these tests. 6% advised the SP of the increased POAG risk in those of African racial descent. The third SP presented for a private eye examination as a 59 year-old patient, with recent onset flashing lights in one eye in the dark. The presence of photopsia was proactively detected in 87% of cases. 35% asked four of the seven questions appropriate for identifying the nature of the flashing lights. 66% recommended dilated fundoscopy to be carried out by either themselves or by another eyecare practitioner. 29% of optometrists asked the patient to seek a second opinion regarding the photopsia. SP encounters are an effective way of measuring clinical care within optometry. Substantial differences exist between different practitioners in the duration and depth of their clinical investigations. This is not surprising, since practitioners are individuals with different levels of experience and therefore variations in approach are inevitable. This highlights the fact that not all eye examinations are the same. The findings of optometric consultations for record abstraction mirror the findings in other healthcare disciplines: clinical records are an imperfect representation of the content of a clinical consultation. Clinical records are subject to a recording bias leading to both under- and over-estimation of the care provided due to the presence of false negatives and false positives. It was proven that clinical vignettes can be easily administered and are a cost-effective way of assessing levels of clinical care and can therefore be used in a great variety of settings. Different methods of measuring clinical care capture different elements of clinical practice and are prone to different biases. A three-way comparison of standardised patient, clinical record cards and computerised vignettes showed that clinical records are an imperfect representation of the content of an optometric clinical consultation as they tend to under-estimate actual care provided, while vignette scores tend to over-estimate clinical performance.

In the UK, 20-50% of older people have undetected reduced vision and in most of these cases the poor vision can be readily corrected by new spectacles and/or cataract surgery. It is often assumed that older people with vision loss will have regular eye examinations so that these problems can be detected, but for many older people this assumption is wrong. One approach to improving the take-up of eyecare services is to carry out vision screening of older people in the community to raise awareness of the need for professional eyecare. The present study aimed to investigate which tests would be most appropriate to screen for correctable visual loss in the older population and to incorporate these tests in a screening tool that would be effective yet simple to administer. The present research sought to investigate whether computerised techniques would be an effective method to screen vision in older people. In Study 1, a computerised vision screener was used to test 180 older people in South London. All participants also received a full, ‘gold standard’, eye examination. Significant cataract was present in 32%, correctable refractive error in 39%, and overall 58% had at least one of these forms of correctable visual problems. The computerised vision screener was able to detect these conditions in about 80% of cases. In Study 2, 200 participants were screened using a revised version of the computerised vision screener. A new flipchart vision screener including the main tests from the computer vision screener was also investigated. 31% of participants in Study 2 had significant cataracts, 30% had correctable refractive errors, and 51% had at least one of these conditions. The computer screener and flipchart tool were both good at detecting significant cataract and refractive errors. About 80% of cases of visual loss due to these problems or due to AMD could be detected with either of the screening tools. Using a pragmatic operational criterion, the screening tools detect about 94% of cases who might be considered by an optometrist to be in need of an eye examination (either overdue or reduced visual acuity). Glaucoma is a difficult disease to diagnose and it was found, as expected, that neither screening instrument was very good at detecting glaucoma. The results showed that the best single test to use for screening of visual loss is HCVA which provides both a high sensitivity (77%) and specificity (73%). Greater sensitivity (80%) is achieved when high contract acuity, low contrast acuity and near acuity are used in combination. Greater specificity (77%) can be achieved by using low contrast acuity alone. It is concluded that vision screening does not replace the need for professional eyecare, but acts as a tool to better inform the public of the need for regular eyecare.

Age-related cataract is the commonest cause of treatable blindness in the world today. It is an ever-increasing problem with prolonged life-expectancy and a burgeoning elderly population. Although successfully treated by modem, sophisticated cataract surgery, the resources needed to provide a surgical set-up and the prolonged training time required to produce a competent surgeon can challenge the available finances of developed economies and result in unacceptable waiting times while they may simply be unaffordable in developing economies and result in high prevalence of blindness. Surgical treatment, although effective in dramatically reversing blindness, is associated with a small risk of sight-threatening complications. Against this background, it is worth considering an alternative, simpler, medical means of treating cataracts that is easily administered and equally effective. Calpains are Ca2+dependent intracellular proteases and several of these enzymes are believed to participate in cataractogenesis. Calpain 2 is the major isoform of calpain involved in human cataractogenesis and its activation appears to be investigate the mechanism of lipid activation of calpains as an essential link towards understanding the molecular and cellular dynamics of cataractogenesis in vivo. A clear picture of the enzymatic events in cataractogenesis will form the basis of drug development to cause selective enzyme inhibition. In chapter 2, atomic absorption spectroscopy shows that the progressive uptake of extra-lenticular Ca2+ by porcine lenses correlates with increases in levels of lens opacification with 8.0 umoles Ca2+ (gm wet lens weight)-1 corresponding to cataract occupying approximately 70% of the lens cell volume. This degree of cataract was reduced by approximately 40%, when a calpain inhibitor, SJA6017 (final concentration 0.8 uM), was included in the extra-lenticular medium, therefore suggesting that the observed porcine opacifications involve the Ca2+ mediated activation of calpains and that cataract could be retarded by the topical administration of calpain inhibitors. In chapter 3, DWIH (Depth weighted insertion hydrophobicity) analysis shows the small subunit of several calpain 2 isozymes to each posess a segment with the potential to form a lipid / membrane interactive a-helix (DWIH values circa 7.0). Extended hydrophobic moment analysis shows these segments to be potential oblique orientated a-helix formers (< uH > circa 0.5). This potential is confirmed by hydropathy plot and graphical analyses, which show each a-helix to possess a significant N -> C hydrophobicity gradient. It is suggested that the lipid / membrane activation of calpain 2 may involve oblique angled membrane penetration by an ahelical segment in domain V of the enzyme. In chapter 4, VP1, a peptide homologue of this domain V segment, is shown to be strongly haemolytic (half-maximal lyric dose = 1.45 mM). FTIR conformational analysis shows VP1 to adopt a-helical structure (20% - 65% of primary structure) in the presence of lipids. These levels are maximal in the presence of anionic lipid (65% of primary structure) and monolayer studies show the peptide to exhibit high levels of anionic lipid monolayer penetration with surface pressure changes (A SP) of 5-6 mN m-1 at 30 mN m-1, which are reduced by approximately 40% ± 15% in the presence of 100 mM NaCl. It is suggested that membrane penetration by the domain V a-helix of calpain 2 may proceed via electrostatic interactions and snorkelling, involving associations between an arginine residue located in the polar face of this a-helix and anionic membrane lipid. It has been suggested that lipid / membranes may modulate calpain 2 activity by lowering the enzyme's in vivo Ca2 requirements. In chapter 5, colorimetric assay of calpain 2 shows that the enzyme requires 4 mM Ca2+ for 100% proteolytic activity, as defined by this assay. In the presence of 1 mM Ca2+, negligible calpain 2 proteolysis is detected but at this level of Ca2+, in the presence of either Dimyristoyl phosphatidylinositol(DMPI), Dimyristoyl phosphatidylserine(DMPS), Dimyristoyl phosphatidylcholine(DMlPC) or Dimyristoyl phosphätidylethanolamine(DMPE), calpain 2 shows proteolytic activity which ranges between 37% and 77% of the protein's full enzymatic activity. The large subunit of calpain 2 (LS-calpain 2) is proteolytical]y active in the absence of the calpain 2 small subunit and it has been suggested that this latter subunit is dispensable for the lipid activation of calpain 2. LS-calpain 2 is assayed under conditions corresponding to those used here for calpain 2 assay and is shown to require the presence of 6 mM Ca 2 for 100% enzymatic activity. These Ca2+ levels are unaffected by the presence of either: DMPI, DMPS, DMPC or DMPE, and based on these combined results, it is suggested that the lipid activation of calpain 2 requires the presence of the small subunit. In addition, it is shown that when compared to zwitterionic lipid under corresponding conditions anionic lipid induces an approximate twofold enhancement of calpain 2 proteolytic activity (70% - 77% as compared to 37% - 49%) and a similar enhancement in its average rates of lipid monolayer interaction (ASP = 1.5 x 10 mN M-1 sec-1 at 10 mN M-1 as compared to ASP = 5.0 x 10-4 mN M-1 sec-1 at 10 mN M-1). It is suggested that calpain 2 may posess an electrostatically driven preference for anionic lipid, which contributes to lowering the enzyme's Ca2+ requirement for activation. In chapter 6, these combined data are discussed in relation to a model for the lipid activation of calpain 2 and proposals for future work are presented.

Aims: The cornea is a tough, transparent tissue providing the primary refractive element of the eye. The stroma consists of specially arranged collagen required for corneal transparency. Correct stromal hydration is important in the maintenance of transparency, a feature controlled by the endothelial cells on the posterior surface of the cornea. The aims of this research were firstly to investigate the morphology of corneal endothelial cells and their expression of the sodium bicarbonate cotransporter during avian embryonic development and secondly, to clarify the effect of disease, surgery and drugs on the posterior cornea including in particular the corneal endothelium. Methods: The corneal endothelial cell morphology and posterior stroma were examined using transmission electron microscopy to determine the ultrastructure of the cells and collagen fibril arrangement in the stroma in all results chapters. Immunohistochemistry and A-scan ultrasonography were employed to identify the presence of the Na+HCO3- cotransporter and to determine the thickness changes in embryonic chick cornea, respectively. Electron tomography was also used to determine the collagen arrangement in Descemet’s membrane. Results: The expression of the Na+HCO3- cotransporter was identified in the endothelial layer of the embryonic chicks at all stages imaged. Central corneal thickness increased in the initial stages of development before a plateau between the E12-E15 developmental period followed by a steady thickness decrease. The ultrastructure of Descemet’s membrane was determined using electron tomography of transverse and en face resin embedded sections from which a model was produced. Polygonal and elongated structures were observed with proteoglycans present at the intermodal regions of the collagenous structures. The polygonal lattice visualised in en face sections appeared to be composed of stacked globular domains which were integrated into the collagen type VIII model. Predominant changes in the Col8a2 knock-in mouse models were observed in the posterior cornea. Differences included increased proteoglycans at the Descemet’s endothelial interface, dilated rough endoplasmic reticulum and focal posterior oedema. This animal model exhibits features similar to those seen in the human form of early-onset Fuchs’ endothelial corneal dystrophy, unlike previous models reported. The final chapter is concerned with regeneration of the corneal endothelial cells. Tissue from posterior corneal surgery examined using electron microscopy revealed the presence of the host endothelial cells and fibrous tissue at the interface in non-Descemet’s membrane stripping automated endothelial keratoplasty and interface haze in Descemet’s membrane stripping automated endothelial keratoplasty. However, these features did not appear to interfere with the adhesion of the graft nor the clarity. Finally, ultrastructural analysis of Rho-kinase inhibited cells showed cells with typical morphology when compared with the untreated group Conclusions: 1) The Na+HCO3- cotransporter is present in the embryonic cornea. It is possible that the cotransporter is involved in the developmental stages and probably the thickness changes we observe during this period. 2) The ultrastructure of Descemet’s membrane appears to be composed of stacked globular domains arranged in a polygonal lattice alongside more elongated structures interspersed with proteoglycans within the internodal regions. 3) Our studies have helped validate Col4a2 mice as a promising Fuchs’ endothelial corneal dystrophy model. 4) Our investigation into posterior corneal surgery revealed ultrastructural changes that occur post-surgery at the graft interface.

The work presented in this thesis is a collection of papers from research spanning over 25 years. The research commenced whilst the candidate was employed in the Tennent Institute of Ophthalmology and later the Department of Anatomy at the University of Glasgow and continued in the School of Anatomy & Human Biology, The University of Western Australia. The research focuses on the biology of immune cells, primarily dendritic cells (DC), macrophages and mast cells, in the context of various components of the eye, including the aqueous outflow pathways, iris, cornea, ciliary body, choroid and retina, and the supporting tissues (lids and conjunctiva). The studies are broad in the sense that they deal with the role of these cells in development (such as removing the vascular networks around the developing lens), their normal homeostasis and function (distribution, phenotype, turnover and functional activity) and their role in models of a number of eye diseases. The findings are important in understanding the pathogenesis of diseases including bacterial keratitis, anterior uveitis, autoimmune uveoretinitis (endogenous posterior uveitis), toxoplasmic retinochoroiditis, age-related macular degeneration and ocular allergic responses, namely any ocular disease with an immune-mediated pathology. Many of the findings in the enclosed papers were firsts in the field and have shaped our understanding of ocular inflammatory disease. In part the success of some these studies was due to the novel method of performing immunostaining on tissue wholemounts dissected from small rodent eyes. These preparations provided unique ‘plan’ views of the complex networks of DCs and macrophages in the iris and choroid which previously had not been appreciated. In addition, the wholemount approach used in the characterisation and study of immune cells in delicate eye tissues, were applied to related studies of the meninges and choroid plexus of the brain. These studies were amongst the first to fully characterise distinct DC and macrophage networks in the pia, arachnoid, dura and choroid plexus. The research presented in the more recent publications have utilised a range of transgenic, knock-out, congenic and chimeric mouse models to elucidate the function of immune cells in the eye.

Glaucoma is a progressive disease of the optic nerve that can result in irreversible loss of visual function and impairment in everyday visual tasks. The experimental studies described in this thesis primarily aim to investigate the performance of people with glaucoma on search and other visual tasks whilst simultaneously monitoring eye movements, making comparison with age-related visually healthy people. In an experiment focussing on visual search, a patient group (n=30) took significantly longer on average to find a target in images of everyday scenes than controls (n=30). Furthermore, comparison of eye movements made by the participants during this task revealed there was a statistically significant reduction (6%) in saccade rate in the patients compared to the controls, and that saccade rate correlated with performance. Similar differences in eye movements were observed when the same groups passively viewed a selection of images in a slideshow. A bivariate contour ellipse (BCE) analysis revealed that, on average, patients viewed smaller regions of the images compared to the controls. Eye movement differences between patients and controls were also examined in a different cohort of people with glaucoma (n=14) and visually healthy controls (n=22) whilst they watched a selection of Hazard Perception Test driving films. Saccade rate of the patients was found to increase by 9%, though results from the BCE analysis suggested the average size of viewing area was similar in both groups. Finally, a novel interview-based study of 50 people with glaucoma provides evidence that patients do not perceive their visual field defect as a black ‘tunnel’ effect, or as ‘black patches’, but more like blurred regions: this finding may, for example, impact on how glaucomatous visual field loss is depicted in patient information about the condition. In conclusion, the results from this thesis show how visual loss from glaucoma influences how patients perceive and react to their visual environment. The principal findings from the studies described in this thesis also show, for the first time, that eye movement analysis could provide a window into the functional deficits associated with glaucoma.