The focus of this thesis is on how light is scattered on its passage through the optics of the human eye, and the consequences for visual performance under different lighting conditions. A number of visual psychophysical measurement techniques were employed to investigate the impact of light scatter on various aspects of visual performance. The preliminary experiments carried out were designed to explore the physical properties of scattered light in the eye. Scattered light varies in both amount and angular dependence, outcomes that relate directly to the number and size of particles involved. In this respect, scattered light is estimated using two methods: one that measures both the amount and the angular dependence of scattered light and the other that assumes constant angular dependence in all observers. The findings show that there are significant differences in angular dependence between observers and that the size of the differences correlates with errors in the estimation of the overall amount of scatter. Experiments were then carried out to investigate the effects of increased scattered light on visual performance and whether these can explain any aspects of age-related visual degradation. To disentangle increased scattered light from the innumerable other changes that occur with ageing, the amount of scattered light in young, healthy eyes was increased using fogging filters. Increased scatter is shown to have only a small effect on chromatic sensitivity and the ability to recognise letters or other high contrast optotypes that are commonly used to assess visual acuity. Contrast sensitivity, on the other hand, can be much reduced in the presence of increased light scatter.

The research contained in this thesis describes three studies designed to investigate the ability of the observer to detect stimuli defined by changes in luminance in space and/or time in mesopic conditions, including contrast sensitivity, temporal flicker sensitivity and visual acuity. The first two studies determined the effect of the aging of the retina on spatial and temporal contrast sensitivity at photopic and mesopic light levels. The literature states that older people experience losses of retinal neurons including rods, cones and ganglion cells. Furthermore, older people tend to have particular difficulties with vision at low light levels which can be attributed to greater loss of rods than cones, particularly at parafoveal eccentricities. Spatial and temporal contrast sensitivity was measured separately in two groups of participants, aged 20-73 (n=74) and 20-74 (n=80) years of age, respectively. Measures were taken to ensure that thresholds largely reflected age-related changes to the retina rather than the optics of the eye. Spectral content of the stimuli was restricted to the middle and long wavelength regions of the visual spectrum and the pupil was measured continuously so as to obtain participant-specific retinal illuminances for each condition. The HRindex was derived and calculated for each participant as a single number which summarized performance from photopic to mesopic light levels. As age increased both spatial and temporal contrast vision worsened and older participants showed particularly elevated thresholds at lower light levels when compared to younger participants. Spatial contrast thresholds show a steady linear decline with age, whereas temporal modulation thresholds were relatively stable up to 50 years of age and then demonstrated a rapid decline. These different trends of changes in performance with increasing age suggests that contrast and temporal HRindex may be measuring the aging of different retinal mechanisms. The normal limits of HRindex values were calculated which could be used in the future to detect abnormal performance. A secondary aim of the first two studies was to determine if binocular summation of spatial and temporal contrast thresholds declined with age, while accounting for differences in retinal illuminance between monocular and binocular conditions. For spatial contrast vision, binocular summation declined significantly with age and 18% showed binocular inhibition. However, the binocular summation of flicker signals did not change significantly with age and only 1% of participants showed binocular inhibition. Interocular differences cannot explain our results. The third study determined whether altering the scotopic/photopic luminous efficiency ratio could improve spatial acuity at mesopic light levels. This was achieved by altering the spectral power distribution of illuminating lights to increase the contribution of rods to vision at constant levels of photopic illumination. It was found that visual acuity at the fovea was improved by low levels of increased scotopic luminance, but peripheral acuity was improved by larger increases of scotopic luminance. The three studies demonstrate that the detection of luminance defined stimuli can be compromised in a number of external conditions such as low light levels, as well as due to internal changes caused by aging to the optics of the eye, retina and/or the central visual system.

The Draize eye irritation test has been widely adopted as the "gold standard" to evaluate the potential eye irritation of a wide range of chemicals and formulations, including; pharmaceuticals, cosmetics, and their raw ingredients. The rationale for pursuing the development of human ocular based in vitro alternatives is to provide greater confidence in the prediction of human reactions to mild and moderate chemicals. This is particularly important with the implementation of the EC White Paper, "Strategy for a future chemicals policy" (2001) that is estimated to require the testing of approximately 30,000 'existing' chemicals by 2012. The development of in vitro alternatives for toxicity testing has mainly focused upon tests for quantitative measurement of acute toxicity following a single high-dose exposure. However, the degree of toxicity of any exposure is a function of; the dose which target cells receive, the duration of the exposure and the ability of the exposed cells to recover from the exposure. However, little account is taken of the potential role of long-term effects in modulating the toxic response. This study aims to generate an in vitro model utilizing a human corneal cell line monolayer to investigate the effects chronic exposure to exogenous chemicals has upon toxicity of a subsequent acute challenge. Surfactants are ubiquitous within our daily environment, being significant active components in both household and personal care products, cosmetics and pharmaceuticals. Initially the effects of four representative surfactants (sodium dodecyl sulphate; anionic, tween 20; non-ionic, cocamidopropylbetaine; amphoteric and benzalkonium chloride; cationic) were examined following chronic exposure. Although the measured endpoints (neutral red uptake, resazurin reduction, fluorescein leakage and total protein content) revealed no alterations in J-HCET morphology, barrier function or biochemistry as a consequence of chronic exposure, it was determined that pre-exposure modulated the toxicity of subsequent acute exposures. The observed modulation in toxicity could have significant health implications for personal care products, cosmetics and pharmaceuticals intended for use near or within the eye. However, the mechanism(s) by which the toxicity of subsequent surfactant exposures was modulated remains to be elucidated. Whilst standard surfactants are good indicators of the effects following chronic exposure, there are pharmaceuticals designed for repeat use in the eye that have been associated with long term ocular irritancy, and a discontinuation of use i.e. timolol maleate. J-HCET cultures exposed to BSO in vitro confirm that the toxicity of timolol to the human corneal cells line was enhanced by suppressing the activity of γ-glutamylcysteine synthetase through irreversible inhibition, resulting in a decrease in intracellular glutathione (GSH) levels. In addition, chronic exposures to timolol maleate were also associated with an increase in toxicity of subsequent acute challenges. The long term use of Timolol maleate in the treatment of glaucoma in vivo may result in similar alterations in the intracellular GSH concentrations, resulting in discontinuation of treatment as consequence of ocular irritation through the generation of reactive oxidation species beyond the threshold that depleted intracellular GSH can respond. Since in vitro methods have been, and are being developed as alternatives to animal experiments, the use of bovine serum as a source of growth factors can seem to be contradictory to the purposes of the Three R's concept of Russell and Burch (1959). This study was conducted using culture media that contain animal derived growth supplements and a media where these had been substituted for plant derived materials. Comparisons were made between the effects these two supplements had upon a number of biological factors including morphology, biochemistry, barrier function and the response to exogenous chemicals. No alterations were observed in these parameters as a consequence of using culture medium containing plant derived materials compared to those containing animal derived growth supplements. This study has demonstrated that the development of a reliable and reproducible in vitro assay in keeping with the principles of the Three R's for modeling chronic – repeat ocular irritation is possible. However, the mechanistic relevance of the endpoints chosen and cell layer ultrastructure is considered to be an essential component. The further development of cell based in vitro systems to predict human responses to chronic/repeat ocular irritation is required.

Eye cosmetics usage in the UK is commonplace. Despite its popularity, there is a lack of published literature exploring the ocular effects of eye cosmetic usage. The influence of eye cosmetics upon symptoms of dry eye and contact lens discomfort has been suggested but these links have not been established. Consequently, this thesis aims to establish any link between ocular comfort with eye cosmetic usage. This was achieved by conducting a survey which showed the use of eye cosmetics, particularly eyeliner, significantly reduced ocular comfort. Amongst cosmetic users, contact lens wearers did not experience significantly reduced ocular comfort compared to non-contact lens wearers. Eye care practitioners often report observations of eye cosmetics contaminating the tear film, even when the products are applied to peri-ocular skin, although these reports are anecdotal. This thesis demonstrates that cosmetic pencil eyeliner migrates most readily and maximally contaminates the tear film when applied along the inner lid margin. After two hours post-application, contamination of the tear film from pencil eyeliner was negligible. This finding led to a subsequent study which examined the clinical and immunological responses of the ocular surface following migration of cosmetic pencil eyeliner. Clinically, eyeliner pencil did not appear to induce signs of ocular surface inflammation. However seven consecutive days of eyeliner application along the inner lid margin increased lipid layer thickness and dry eye symptoms compared to when the eyeliner was applied to peri-ocular eyelid skin. Subclinical signs of ocular surface inflammation were investigated by examining the concentration of inflammatory cytokines, IL-6 and IL-8, in tear fluid. A small reduction of cytokine concentration was exhibited after one day of eyeliner use although concentrations returned to near baseline levels after seven days of use. In conclusion, pencil eyeliner is safe to use and does not appear to induce clinical or subclinical signs of inflammation when used for up to seven days consecutively. The causes of increased dry eye symptoms are undetermined and the longer term effects of eyeliner application remain unknown.

Glaucoma is an umbrella term for a number of related optic neuropathies which have the common pathology of a progressive, irreversible vision loss associated with atrophy of retinal ganglion cells. Together, the various forms of glaucoma constitute the second leading cause of vision loss in the developed world. Current therapies for the treatment of glaucoma focus on alleviating the primary risk factor, an elevation in intraocular pressure. These treatments are effective at mitigating the progression of vision loss however they cannot recover vision and do not completely halt vision loss, limiting their use as treatments. To better understand the biology underlying the loss of retinal ganglion cells in glaucoma, I have examined the role of complement in retinal ganglion cell loss. Complement is a network of cross-reacting serine proteases which form part of the humoral immune system and are primarily responsible for clearance of apoptotic cells and defence against pathogens. To understand the role played by complement in glaucoma I used an inducible model of glaucoma to establish the complement activation occurs in the glaucomatous retina. I then used the inhibitor of the classical complement cascade, C1 inhibitor to protect the dendrites and cell bodies of retinal ganglion cells and found that this protection did not prevent axonal degeneration. Using in vitro and in vivo animal models of complement deficiency I established that deficiency in complement components C3 and C6 which are further down the cascade, exacerbates damage suffered in hypertensive glaucoma. This study adds to the existing evidence that the role of complement in central nervous system degeneration is a complex, multifactorial process, with elements of the complement system being variously protective and damaging. It does, however, add hope to the prospect of developing a treatment for glaucomatous optic neuropathy based on manipulation of the complement system.

Cystic fibrosis (CF) is caused by defective function of CF Transmembrane Conductance Regulator (CFTR), an epithelial ion channel that facilitates chloride secretion. Previous research has identified impaired dark adaptation (DA) in CF, which has been attributed to concomitant vitamin A deficiency or CF-related diabetes (CFRD). However, CFTR has been localised to the retinal pigment epithelium (RPE) and it is proposed that abnormal DA could be a primary manifestation of CF. DA is similarly impaired in individuals with type 1 and 2 diabetes and is thought to be caused by retinal hypoxia as oxygen inhalation ameliorates abnormal thresholds. The aim of this thesis was to investigate DA during oxygen inhalation in CF subjects with and without CFRD to gain further insight about the aetiology of this abnormal DA. The work also aimed to examine retinal structure using optical coherence tomography (OCT) to determine the consequences of CFTR dysfunction at the RPE. Final DA thresholds were not impaired in CF subjects as a whole during the inhalation of air. However, when grouped according to diabetic status, CFRD subjects showed a significantly elevated final rod threshold which was ameliorated following oxygen inhalation. This suggests that the retina is hypoxic in CFRD subjects and that impaired DA in CF is secondary to CFRD rather than a primary manifestation of CFTR malfunction at the RPE. Contrary to the proposed hypothesis, retinal and RPE/photoreceptor layer thickness was significantly thinner in CF subjects. These results suggest that impaired CFTR function at the RPE does not directly affect retinal structure. · In conclusion, this is the first study to determine that retinal structural and functional abnormalities are not caused directly by CFTR dysfunction but are a secondary manifestation of the disease. Further research is necessary to understand the impact of these findings.

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