Mutations in OPA1 are the leading cause of autosomal dominant optic atrophy, a disease in which a progressive loss of retinal ganglion cells (RGCs) leads to blindness. In the B6;C3-Opa1Q285STOP (het) mouse, an Opa1 mutation causes a decrease in ATP production and a progressive loss in visual acuity, which coincides with pruning of the predominantly ON-centre RGC dendrites. As 670 nm light can increase ATP production and provide neuroprotective effects, we hypothesise that protection from dendritic pruning will be observed in the ON-centre RGCs of the het mouse with 670 nm light. By Sholl analysis, and other measures of dendritic complexity, we found that ex vivo delivery of 670 nm light to retinal explants provides partial protection against the ex vivo retinal ganglion cell dendropathy, triggered by axotomy, in young wild-type mice. By the same methods of analyses, in vivo delivery of 670 nm light to aged wild-type and het mice partial provided protection against ex vivo RGC dendropathy in RGCs from wild-type mice and partial protection in RGCs from het mice. By immunohistochemistry, the transcription factor NFκB was found to be activated in RGCs from aged het mice treated with in vivo 670 nm light. The oxidative stress sensor, DJ1, was upregulated in RGCs from aged wild-type and het mice, by in vivo 670 nm light. The activation of the serine/threonine protein kinase, AKT, which plays a pivotal role in controlling cell survival and apoptosis, was decreased following in vivo 670 nm light treatment in aged wild-type RGCs.

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Despite being considered the current reference standard for perimetric testing in glaucoma, standard automated perimetry has several cardinal limitations, including an unacceptably high test-retest variability, which increases with increasing depth of defect, and a limited useable dynamic range, with test-retest variability spanning almost the entire instrument range in advanced glaucomatous damage. Prior studies have shown that spatial summation, the mechanism by which the visual system integrates light energy across the area of a stimulus, differs in disease, with an enlarged Ricco’s area (the limit of complete spatial summation) found in individuals with glaucoma. The aim of this work was to investigate whether a perimetric stimulus designed to exploit these changes in spatial summation would enable a greater signal/noise ratio (SNR) than that of the current standard stimulus, by directly measuring the displacement of the spatial summation function in glaucoma. Three stimulus forms were developed; one varying in area alone, one varying in both area and contrast simultaneously, and one varying in contrast alone, all operating within the local Ricco’s area. These novel stimuli were compared with the standard Goldmann III stimulus, in terms of disease signal, noise, and SNR. The experiments presented in this thesis indicate that a stimulus modulating in area alone may offer greater benefits for measuring glaucomatous changes in spatial summation in a clinical setting, in the form of a greater disease signal, more uniform response variability with depth of defect, and greater SNR, when compared with the standard Goldmann III stimulus. Additionally, there is some indication that this stimulus is more robust to the effects of intraocular straylight than the Goldmann III stimulus, although test-retest variability and robustness to optical defocus are largely similar. As this work represents the early investigations of this stimulus, further work is required to examine its translation into a clinical environment.

The research described in this thesis presents the development and evaluation of a prototype multispectral, imaging densitometer. Ultimately, the aim was to develop a method of simultaneously isolating the contributions from the three main photoreceptors, across the retina. Thus, providing the clinician with a viable tool for assessing outer retinal function both efficiently and directly. The construction and use of the prototype multispectral imaging densitometer was described in detail. Healthy participants were recruited and imaged to evaluate the baseline capability of the device. The results were validated through comparison to published values for optical density, regeneration rates and photopigment distribution as obtained through in vivo, in vitro and in situ techniques. A novel ocular reflection model was developed to characterise and mathematically remove the pre-receptoral stray light from the images. Whilst the preliminary investigation returned regeneration rates in line with values from the literature, a ‘red shift’ of approximately 30nm was highlighted in the experimental absorption spectra of the visual pigments, hindering their isolation. Investigation into this phenomenon suggested that the cause was pre-receptoral stray light, with both the crystalline lens and the inner retinal layers contributing to the effect. Attempts to remove the crystalline lens component showed a partial reduction of the ‘red shift’. Greater success was achieved through the modelling of the experimental ocular reflection data to simultaneously characterise the contribution from both components. This research has shown that mapping of visual pigment optical density across the retina is possible with the retinal densitometer described. With further development, the technique is likely to become a useful tool in the diagnosis and monitoring of outer retinal disease.

The studies comprising this thesis were conducted to further understand how structural changes to the corneal extracellular matrix can affect the cornea’s unique properties, with the ultimate goal of improving novel treatments and their outcomes. Following refractive surgery, changes in matrix structure can cause loss of structural integrity and transparency, which may adversely affect the surgical outcome. The first objective was to define what governs corneal shape and ultrastructural organisation by analysing the abnormal post-hatch corneal collagen arrangement in an avian model (beg). Structural information was also obtained post-in vivo microwave keratoplasty to assess the treatment as a suitable alternative to more invasive correction procedures. In addition, modifying the resident cell type as a means of improving post-treatment wound healing following LASIK was also investigated using corneal stromal stem cells. Finally, the efficiency of transepithelial riboflavin/UVA collagen cross-linking in terms of riboflavin uptake and post-treatment corneal stiffness, was evaluated. X-ray scattering studies revealed that corneal flattening in beg chickens is related to biomechanical changes brought about by an alteration in collagen arrangement at the corneal periphery. This highlights the importance of the limbal fibril annulus in corneal shape preservation. X-ray studies also revealed that microwave keratoplasty may impact on peripheral vision by introducing spatial disruption of stromal collagen, resulting in localised corneal opacity in the treatment area. Loss of fibrillar structure and order could also have further implications for corneal biomechanics and shape. The application of human corneal stromal stem cells under LASIK-like flaps was revealed to be a promising approach for increasing flap adherence strength whilst maintaining corneal clarity. Introducing these cells in the early stages of flap-healing appears to improve the repair process, conceivably through an embryonic-like mechanism. Lastly, stress-strain and riboflavin uptake results for transepithelial riboflavin delivery during corneal cross-linking were encouraging, however refinements to the testing procedure are required to fully assess the treatment efficacy. Collectively these findings accentuate the importance of the precise stromal collagen fibril arrangement and composition for maintaining corneal transparency, shape and general functionality. All these factors must be taken into consideration when implementing novel correction procedures or modifying existing treatments for corneal defects.

The purpose of the studies described herein was to better understand the impact of involuntary eye movements on oculomotor control and perception in infantile nystagmus. Therapeutic interventions that result in slowed nystagmus oscillations often fail to elicit significant quantifiable improvements in visual function, despite patients reporting subjective benefits. It is difficult to justify surgical or pharmacological intervention when the only outcome measures are subjective. Objective quantification of nystagmus eye movements per se usually involves time-consuming manual marking of recordings to both calibrate and analyse data. As a result, analyses are rarely (if ever) performed in the clinical setting. Software was therefore developed to automate calibration and assessment. Psychophysical experiments were undertaken to quantify the spatiotemporal constraints of vision in infantile nystagmus. Visual acuity was measured in the absence of retinal image motion to reveal the maximum improvement to spatial vision that might be expected if nystagmus were halted altogether. The results indicate that poor spatial vision underlies infantile nystagmus, even in cases without comorbid pathology. Gaze acquisition time was compared to stimulus recognition time. The results indicate that infantile nystagmus does not increase visual processing time; rather, redeploying gaze takes longer. An incidental finding revealed a temporal relationship between voluntary saccades and involuntary nystagmus quick phases. Both typically occur together, presumably to maximise efficiency and minimise saccadic suppression. Clinical tests of gaze acquisition time must now be developed, to be used in conjunction with the software developed here, as objective outcome measures of therapeutic interventions.

Purpose: This thesis aimed to characterise and analyse the 3D micro- and nanoarchitecture of connective tissue within the lamina cribrosa (LC). Methods: The microarchitecture of load-bearing connective tissue components, elastin and fibrillar collagen, of the young, elderly and glaucomatous optic nerve head (ONH) was analysed following two-photon excited fluorescence, second harmonic generation and small angle light scattering. Microfocus and conventional small angle X-ray scattering were used to analyse ONH nanoarchitecture and the potential of X-ray microtomography (XMT) as a 3D imaging technique was evaluated. Results: Fibrillar collagen and elastic fibres stretched radially across the optic nerve (ON) canal in the LC, encircled the central retinal vessels and were absent in the prelamina. In the postlaminar ON septae, collagen was perpendicular to that in the LC. Differences in young and elderly ONH tissue included; wavy collagen bundles exclusively within the young ONH and distinct elastic fibres found in the elderly ONH. Analysis of ONH reconstructions of 3D SHG datasets revealed that elderly LCs contained higher fibrillar collagen content when compared to the young LCs. Interestingly, the connective tissue beams of the inferior-temporal LC quadrant were significantly more aligned in glaucoma when compared to age-matched controls. Distinct X-ray reflections, potentially elastin in the peripapillary sclera and representative of CNS myelin in the postlaminar ON were identified. XMT enabled quantification of the regional variation in LC thickness, connective tissue content, pore area and pore count, showing potential for 3D quantification without the need for tissue sectioning. Conclusion: Differences in the young and elderly ONH microarchitecture and nanoarchitecture include fibrillar collagen content, alignment and packing and the presence of elastic fibres. These data will be important for the development of finite element models that can predict ONHs at risk of developing glaucomatous optic neuropathy.

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.

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.