This thesis considers the question: What roles do the tear film and corneal nerves play in the normal blink mechanism. The hypothesis proposed is that tear film thinning, which occurs prior to full break-up, allows increased evaporation of the tear film. The evaporation produces a localised reduction in the tear film temperature, which is then detected by the temperature sensitive nerves in the corneal epithelium. To test this hypothesis, a series of experiments was completed. The first study, which investigated the pattern of diurnal change in corneal sensitivity, revealed that corneal sensitivity increases during the day following post-sleep eyelid opening to reach a plateau approximately five hours after eye opening (Kruskall-Wallis, p<0.05). The second study assessed corneal nerve function under anaesthesia produced by 0.5% Proxymetacaine Hydrochloride. Onset of anaesthesia was observed within 2 minutes (Wilcoxon matched pairs test, p<0.05), with a maximum of anaesthesia reached at 15 minutes post-instillation (Wilcoxon matched pairs test, p<0.05). Corneal sensitivity did not return to pre-instillation levels at 60 minutes post-instillation (Wilcoxon matched pairs test, p<0.05). The third study assessed the effect of iris colour and ethnic origin on corneal sensitivity, skin sensitivity, tear film stability and blink rate. The study showed that corneal sensitivity for a cooling stimulus was affected by iris colour and ethnic origin (Kruskall-Wallis, p<0.05): as iris pigmentation increases, corneal sensitivity decreases. Although statistically tear film stability was found to be influenced by iris colour, no clear pattern of change was associated with iris colour (Kruskall-Wallis, p>0.05). Tear film stability was not affected by ethnic origin (Kruskall-Wallis, p>0.05). Blink rate was significantly correlated to tear film stability (Spearman, r = -0.536, p<0.05). The fourth study considered the relationship between blink rate, corneal sensitivity, tear film stability, anaesthesia, ocular surface temperature and ocular surface evaporation. Tear film stability was strongly correlated to blink rate, with the blink rate increasing as tear film stability decreases (Spearman, r = 0.926, p<0.05). Corneal sensitivity was significantly correlated to corneal sensitivity, but only at low corneal sensitivity levels. Blocking corneal sensitivity by anaesthesia, the blink rate was significantly reduced, suggesting that corneal sensitivity is involved in the mechanism controlling normal involuntary blinking (Wilcoxon matched pairs test, p<0.05). Tear film evaporation from the ocular surface was not correlated to the blink frequency (Spearman, r = -0.381, p>0.05). The amount of temperature cooling at the inter-blink interval was not correlated to blink rate (Spearman, r = 0.241, p>0.05). The final experimental study examined involuntary blinking with contact lens wear discomfort. There was a significant increase in the blink rate with increasing discomfort (Kruskall-Wallis, p<0.05). Subjects experiencing discomfort had a less stable tear-film, both with (Mann Whitney test, p<0.05) and without (Mann Whitney test, p<0.05) contact lens wear, and had an elevated blink-rate compared to subjects experiencing comfort (Mann Whitney test, p<0.05). Ocular surface discomfort was not related to an elevated corneal sensitivity (Mann-Whitney test, p>0.05). These series of studies showed that tear film stability and corneal sensitivity are involved in the blink mechanism, providing strong evidence that normal involuntary blinking is affected by sensory stimuli arising from the exposed ocular surface.

Hypothesis: The hypothesis of this research is that changes in the sulphation patterns of the glycosaminoglycans are directly related to changes in ultrastructure, and hence transparency of the cornea. Aims: The aims of this research were to investigate glycosaminoglycan sulphation patterns and collagen fibril ultrastructure from central to peripheral regions of the cornea, and to gain a greater understanding in the effects of keratan sulphate and its reliance on oxygen supply. Methods: The bovine corneal extracellular matrix composition and collagen fibril parameters (fibril diameter and interfibrillar spacing) were biochemically and biophysically evaluated. This involved taking measurements of corneal thickness and hydration, as well as the amount of hydroxyproline and sulphated glycosaminoglycan. Immunolocalization of proteoglycan protein cores (lumican and keratocan) and specific glycosaminoglycans, particularly their sulphation distribution were studied using specific antibodies. Sulphation patterns of keratan sulphate were also quantified using specific antibodies. Transmission electron microscopy coupled with synchrotron small angle x-ray fibre diffraction was also employed to gain a greater understanding of the corneas' collagen fibril architecture and its interaction with glycosaminoglycans across the depths of the cornea. Results: The bovine cornea is thicker in the outer peripheral regions of the cornea and accordingly an increased amount of hydroxyproline is found at this region of the tissue. Keratan sulphate is predominantly found in the bovine cornea and is particularly heavily sulphated across the cornea. The degree of sulphation of keratan sulphate decreases at the outer peripheral regions of the cornea, which, interestingly, is where a transition of collagen fibrils occurs in that fibrils become less uniformly arranged, changes in fibril diameter are seen, and interfibrillar spacing values alter. Depth- profiled synchrotron microbeam analyses show that at different radial positions, from the corneal centre outwards, fibril diameter is greater superficially than in deep stromal regions. This does not include Bowman's layer which is below the spatial resolution of the analysis. The mid-depth stroma has higher interfibrillar spacing than is seen in posterior regions of the stroma, where fibril spacing appeared more compact. Previous work has pointed to a link between glycosaminoglycan content and oxygen availability. Work presented here indicates that in rabbit corneas, after 24 hr in 2% atmospheric O2, the glycosaminoglycan sulphation pattern changes significantly, with a significant increase of the high sulphated epitope of keratan sulphate. Conclusion: My data reveal that collagen fibrils in the central regions of the cornea are more closely packed and uniform in diameter than those in the outer peripheral cornea, and this may have potential implications for the transparency of the tissue. Stromal architecture is likely governed by sulphated proteoglycans, and changes in the types and sulphation patterns of corneal glycosaminoglycans from the corneal centre to periphery might be linked to differences in collagen ultrastructure. Moreover, the findings of differences in collagen fibril ultrastructure with depth through the cornea are possibly linked to biochemical alterations in proteoglycans. Finally, it is hypothesised that detrimental conditions, such as hypoxia after contact lens wear, might have an effect on the type and sulphation status of glycosaminoglycan synthesized and in vitro evidence for this is presented and discussed. (Abstract shortened by UMI.).

After birth the brain adapts to characteristics in the environment in order to optimise its resources with respect to the individual's circumstances. For instance, early monocular deprivation results in reduced cortical representation and visual acuity of the deprived eye. However, such a loss of visual function in one eye after only transient periods of compromised vision through injury or infection would seem to be maladaptive. I examined here whether cortical deprivation effects can be counteracted by daily periods of normal experience. Cats received variable daily regimens of monocular deprivation (by wearing a mask) and binocular exposure. Visual cortex function was subsequently assessed with optical imaging of intrinsic signals, visually evoked potentials, and extracellular electrophysiological recordings. Regardless of the overall length of visual experience, daily binocular vision for as little as 30 minutes, but no less, allowed normal ocular dominance and visual responses to be maintained despite several times longer periods of deprivation. Thus, the absolute amount of daily binocular vision rather than its relative share of the total daily exposure determined the outcome. When 30 minutes binocular exposure were broken up into two 15-minute blocks flanking the deprivation period, ocular dominance resembled that of animals with only 15 minutes binocular vision, suggesting that binocular experience must be continuous to be most effective. My results demonstrate that normal experience is clearly more efficacious in maintaining a binocular visual cortex than abnormal experience is in shifting the ocular dominance distribution. These findings con tribute to the larger debate about how much nature and nurture, respectively, contribute to the development of the brain they suggest that while experience plays a significant role, for some functions there may be an intrinsic bias towards a state that is optimally adapted to the most probable environment.

Diabetic retinopathy (DR) is the major cause of registerable blindness in the working population in Western countries. It has been proposed that the retina is subject to sub-clinical levels of tissue hypoxia prior to the development of DR, and that a rod-driven hypoxia during darkness may be a significant causal factor in its development. The aim of this study was firstly to use the scotopic electroretinogram (ERG) in order to gain an objective measure of retinal function in subjects with diabetes mellitus (DM). Should there be a level of inner retinal hypoxia present this may be indirectly demonstrated by reduced oscillatory potential (OP) amplitudes, thought to arise predominantly from amacrine cells and known to be sensitive to vascular changes within the retina, and reduced b- wave amplitudes thought to arise predominantly from the bipolar cells of the inner retina. If hypoxia were present and reversable in the short term an increase in amplitude would be expected with oxygen (O2) inhalation. ERGs were recorded from subjects with Type 2 DM both with and without retinopathy before, during and following O2 inhalation and compared to age- matched control subjects. No significant difference in amplitude was observed between subjects with DM and control subjects before O2 inhalation, however both b-wave and summed OP amplitudes were significantly increased following O2 inhalation in diabetic subjects with retinopathy, and OP3 significantly increased in subjects without retinopathy, yet remained unchanged in the control group. The retinal O2 demand has been reported to halve in light conditions compared to darkness and it has therefore been proposed that patients with DM may benefit from sleeping with night-time illumination in order to reduce the level of rod activity, and thus metabolic demand on the retinal tissue, to reduce inner retinal hypoxia. The amount of light required to significantly reduce rod activity was investigated by means of a simultaneous cone-rod ERG in DM subjects with no retinopathy and control subjects. It was found that a background illumination level as little as 3.4lux was sufficient to significantly reduce rod activity in all subjects.

The aim of this study was to obtain a better understanding in the healing of LASIK-like flaps using an in vitro organ culture method in bovine corneas. At early stages of the PhD, during protocol optimisation, a 5mm trephine was used to injure bovine corneas. At later stages a custom-made eye holder was used to induce LASIK-like incisions in corneas. Immunohistochemistry for a-smooth muscle actin (aSMA) and cytokeratin was used to monitor myofibroblast and epithelial cell expression, respectively, during the wound healing process. Additionally, the effect of certain cytokines (i.e. TNFa, Fas ligand, TGF-Pi and IL-la) was tested in terms of corneal transparency, myofibroblast expression and tissue mechanical strength during the healing process. The later series of experiments was an attempt to manipulate and improve wound healing after LASIK. Healing in this in vitro system closely followed the effects that are already known from the literature. In addition, preliminary evidence on the cytokine corneas proved that there is a correlation between cytokine type and concentration with the effect in tissue transparency, extend of wound healing response and tissue mechanical strength. X-ray diffraction also provided important information about collagen ultrastructural changes in the corneas during the healing process. Parameters such as fibrillar diameter, spacing, distribution and orientation were studied. Collagen fibrillar diameter and spacing remained constant for control corneas during the organ culture time-span, indicating that this in vitro system does not induce any swelling effects on the tissue. However, injured corneas became significantly swollen (p<0.05) during culture. Swelling effects were more severe in trephined corneas than in LASIK-like injured ones. However, collagen fibrillar diameter remained normal in the periphery of injured corneas, but it increased significantly in areas within and around the wound in trephined samples and in the flap incision site for LASIK-like ones. In both types of wounding, collagen orientation changes were observed and were associated with the process of creating the injury. However, in the case of trephine wounded corneas, tissue swelling and changes in collagen orientation reflected the processes of tissue repair. These differences will determine corneal stability and strength follow trauma and, possibly, refractive surgery. The transparency of the cornea depends on both the collagen and the interstitial proteoglycans. In order to obtain a better insight in ultrastructural changes during the wound healing process molecular modelling techniques were used in order to construct a theoretical model for the core protein of biglycan. This molecule is a dermatan sulphate proteoglycan and its numbers increase up to seven times during wound healing. It is considerably larger than the rest of proteoglycans and molecular modelling also revealed numerous potential collagen interaction sites.

Prostaglandin derivatives form the most widely used medicinal treatments given to glaucoma patients to lower intraocular pressure. Prostaglandins are believed to increase matrix metalloproteinase (MMP) and tissue inhibitor of matrix metalloproteinase (TIMP) activity, leading to increased in aqueous outflow, via uveoscleral outflow pathway. However, the direct impact of MMPs on the tissues within uveoscleral pathway has not been determined. The aim of this project was to compare the direct effect of prostaglandins and MMPs on the tissues within the uveoscleral outflow pathway. To determine the effect of known inducers of MMP activity, scleral fibroblasts and ciliary muscle cells were cultured in the presence of interleukin-1a, tumour necrosis factor, transforming growth factor p and prostaglandin F2a (PGF2a). The effect of prostaglandin F2a and MMPs on the uveoscleral pathway tissue i.e. sclera, was assessed as a measure of permeability, molecular and supramolecular scleral collagen integrity and proteoglycan composition. A significant induction of MMP 1, 2, 3 and 9 secretion and activity with cytokines and PGF2a, within human scleral fibroblast and ciliary muscle cell cultures (p<0.05). A 3-fold increase in scleral permeability was observed within 24 hour of incubation in PGF2a, whereas upto 10-fold increase was observed in MMP treated. The helical rise per residue (at 1.5nm), lateral packing (at 0.29nm) and D-spacing (at 66nm) of scleral collagen was unaffected by MMP and PGF2a incubation. Significant change in aggrecan degradation was observed within scleral tissue incubated in MMP and PGF2a (p<0.05). However, no significant change in small leucine rich proteoglycans i.e. biglycan, decorin and lumican, within sclera occurred within sclera incubated in MMP or PGF2a.

Although patient anxiety is known to be a problematic feature within many areas of healthcare, the subject has been largely neglected within optometry. Therefore, this thesis addresses the issue of patient anxiety within optometric practice. The initial study reported in Chapter 3 is a comparative study of patient anxiety. Results show that there is a distribution of anxiety levels within optometric practice which overlap with dental and general medical practice. A significant association between patient anxiety with patient satisfaction (rs=-0.19 p<0.001) and compliance (rs=-0.19 p<0.001) is reported. Furthermore, an ordinal regression analysis identifies the predictors of patient anxiety as trait anxiety, expectancy of bad news and non-spectacle wear. The development of a new 10-item questionnaire to identify anxious patients in optometric practice is reported in Chapter 4 (the OPAS). A 4- item contact lens anxiety subscale is also described. The most anxiety-provoking parts of a contact lens fitting examination are reported in Chapter 5, in a study which measured patient anxiety with both questionnaires and physiological methods (skin conductance and pulse rate). Heightened anxiety was identified during periods of communicative interaction and contact lens insertion and removal. The ability of interventions to reduce anxiety is the focus of the final study reported in Chapter 6. There was no significant reduction in OPAS scores between patients who received an information leaflet, listened to music or received neither intervention prior to their eye examination. However, there was a significant association between OPAS scores and post-examination patient satisfaction (rs = -0.32 p<0.001) and compliance (rs = -0.47 p<0.001). The results from these studies increase our understanding about the presence, causes and effects of patient anxiety within optometric practice. This thesis shows, for the first time, that heightened patient anxiety is associated with decreased satisfaction, compliance and is likely to be a contributing factor in determining healthcare outcomes.

Introduction: The lens is an unusual and transparent tissue. This transparency of the lens depends on a precise and fine regulation of cell proliferation and differentiation when this control is disrupted cataract arises. Previous studies have identified regulators of the cell cycle in lens epithelial and fibre cells or in the identification of the main components of fibre cell differentiation. However, few studies have been carried out in order to understand how proliferation and differentiation are regulated and coordinated in the vertebrate lens. Survivin, also called Birc5, is the smallest member of the inhibitor of apoptosis protein (IAP) family. Survivin functions at a pivotal junction of the cell cycle/apoptosis balance and is vital in maintaining normal tissue homeostasis. Purpose: The purpose of the present study was the analysis of the expression of Survivin in the lens during development in order to provide evidence that Survivin may have an important role as a regulator of cell proliferation and differentiation during lens development. Methods: In order to clarify the role of Survivin in vertebrate lens development three models were used: embryonic chick lens, chick epithelial dissociated primary cell culture and postnatal mouse lens. A thorough spatio-temporal analysis of Survivin expression in the developing lens was carried out. RT-PCR, RT-QPCR, Western Blot and immunocytochemistry were used to study the expression of Survivin during lens development. Proliferation (PCNA) and denucleation pattern (TUNEL) were correlated with dynamic changes in the pattern of expression of Survivin during lens development. Results: Survivin expression was detected in the three models studied. In embryonic chick lens model, Survivin expression was developmentally regulated with high peak at early embryonic lens stages of development (ED6). Finally, Survivin protein expression was absent of the lens before the denucleation in the fibre cells were observed at ED 16. In the chick lens epithelial dissociated cell culture, Survivin expression was associated with the increase of proliferation in cell culture. Survivin was detected in presence of denucleation and down regulation of Survivin was in coincidence of increase of denucleation at day 8 of cell culture. In postnatal mouse lens development, gene expression analysis confirmed the results observed in the other two models. The main difference was observed at protein level. The WB showed an additional band to Survivin wild type at NB and Pn7. Also, an increase of Survivin wild type was observed at Pn7, in coincidence with decrease in proliferation. Conclusions: Overall, the results presented suggest that Survivin is an essential survival factor in vivo and in vitro during the development of the vertebrate lens and suggest that Survivin expression is critical to maintaining cell survival in cells that are continuously proliferating and in those younger lens fibre cells that withdraw from the cell cycle, but not in cells in quiescence i.e. the central lens epithelial cells, after ED 12 in chicken and at Pnl4 in mouse lenses.

An aim of this thesis is to map a genetic factor that is related to myopia progression. A genome-wide association study of myopia, refractive error and two ocular determinants of refractive error, axial length and corneal curvature was undertaken. A number of genetic locations were identified and extra genotyping and replication in an independent cohort is underway. A further aim of this thesis is investigation of an environmental risk factor of myopia. Epidemiological analyses of two myopia risk factors were undertaken; one in the ALSPAC cohort, another in a cohort from Northern England. In both cases, a relationship with myopia was identified and a plausible mechanism for the relationship is discussed.

Transmission electron microscopy of a human cornea with excess chondroitin sulphate/dermatan sulphate glycosaminoglycan showed that changes in proteoglycan structure, content and sulphation lead to the formation of abnormally large collagen fibrils. The lack of sulphation of keratin sulphate in MCD also led to abnormally large fibrils, which are present in the deep stromal layers. These findings suggest overlapping roles of the two proteoglycan populations in the corneal stroma with possible feedback mechanisms, too. Taken together, the findings of this thesis indicate the central role played by proteoglycan-collagen interactions in the development and maintenance of properly formed corneal stroma.