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Glaucoma is a leading cause of visual disability across the world and when diagnosed the glaucoma patient will spend the rest of their life receiving treatment in managed clinical care. In the glaucoma clinic, retinal and optic nerve head (ONH) imaging can be used to help the clinician to manage patient treatment appropriately. By providing high resolution images of the optic nerve head structures and identifying changes therein related to disease onset and progression, an objective measure can be obtained as to how well or badly treatment is preventing further disease damage. This thesis contributes to the field of glaucoma progression detection by the analysis of clinical imaging data using confocal scanning laser tomography (CSLT). Primarily it is an investigation of how best to appraise and optimise current algorithms which aim to detect these glaucomatous structural changes in the optic nerve head. This is done by addressing how the performance of these methods can be best assessed in the absence of a gold standard for glaucomatous structural progression. Glaucoma expert assessment of photographs of the optic disc is the current clinical standard of assessing glaucomatous damage evident in the ONH. This is used in this thesis to act as a reference standard by which these algorithms can be compared. In addition, the statistical principles underpinning trend detection techniques are also investigated along with the performance of these techniques to detect trends in CSLT data in the presence of different types of measurement noise and image quality. A new computer model is developed and validated to simulate stable series of CSLT images, with realistic variability, which can be used to benchmark the false-positive rates of current and future progression algorithms. In conclusion, the main results reported in this thesis show that uncertainties involved in expert assessment of change in ONH photographs limits this as a reference standard for structural change in glaucoma. In addition, since stability in clinical datasets is uncertain, simulation using modelled series is shown to provide a new benchmark for comparing methods of progression detection.

The application of multifocal techniques to the visual evoked cortical potential permits objective electrophysiological mapping of the visual field. The multifocal visual evoked cortical potential (mfVECP) presents several technical challenges. Signals are small, are influenced by a number of sources of noise and waveforms vary both across the visual field and between subjects due to the complex geometry of the visual cortex. Together these factors hamper the ability to distinguish between a mfVECP response from the healthy visual pathway, and a response that is reduced or absent and is therefore representative of pathology. This thesis presents a series of methodological investigations with the aim of maximising the information available in the recorded electrophysiological response, thereby improving the performance of the mfVECP. A novel method of calculating the signal to noise ratio (SNR) of mfVECP waveform responses is introduced. A noise estimate unrelated to the response of the visual cortex to the visual stimulus is created. This is achieved by cross-correlating m-sequences which are created when the orthogonal set of m-sequences are created but are not used to control a stimulus region, with the physiological record. This metric is compared to the approach of defining noise within a delayed time window and shows good correlation. ROC analysis indicates a small improvement in the ability to distinguish between physiological waveform responses and noise. Defining the signal window as 45-250ms is recommended. Signal quality is improved by post-acquisition bandwidth filtering. A wide range of bandwidths are compared and the greatest gains are seen with a bandpass of 3 to 20Hz applied after cross-correlation. Responses evoked when stimulation is delivered using a cathode ray tube (CRT) and a liquid crystal display (LCD) projector system are compared. The mode of stimulus delivery affects the waveshape of responses. A significantly higher SNR is seen in waveforms is shown in waveforms evoked by an m=16 bit m-sequence delivered by a CRT monitor. Differences for shorter m-sequences were not statistically significant. The area of the visual field which can usefully be tested is investigated by increasing the field of view of stimulation from 20° to 40° of radius in 10° increments. A field of view of 30° of radius is shown to provide stimulation of as much of the visual field as possible without losing signal quality. Stimulation rates of 12.5 to 75Hz are compared. Slowing the stimulation rate produced increases waveform amplitudes, latencies and SNR values. The best performance was achieved with 25Hz stimulation. It is shown that a six-minute recording stimulated at 25Hz is superior to an eight-minute, 75Hz acquisition. An electrophysiology system capable of providing multifocal stimulation, synchronising with the acquisition of data from a large number of electrodes and performing cross-correlation has been created. This is a powerful system which permits the interrogation of the dipoles evoked within the complex geometry of the visual cortex from a very large number of orientations, which will improve detection ability. The system has been used to compare the performance of 16 monopolar recording channels in detecting responses to stimulation throughout the visual field. A selection of four electrodes which maximise the available information throughout the visual field has been made. It is shown that a several combinations of four electrodes provide good responses throughout the visual field, but that it is important to have them distributed on either hemisphere and above and below Oz. A series of investigations have indicated methods of maximising the available information in mfVECP recordings and progress the technique towards becoming a robust clinical tool. A powerful multichannel multifocal electrophysiology system has been created, with the ability to simultaneously acquire data from a very large number of bipolar recording channels and thereby detect many small dipole responses to stimulation of many small areas of the visual field. This will be an invaluable tool in future investigations. Performance has been shown to improve when the presence or absence of a waveform is determined by a novel SNR metric, when data is filtered post-acquisition through a 3-20Hz bandpass after cross-correlation and when a CRT is used to deliver the stimulus. The field of view of stimulation can usefully be extended to a radius of 30° when a 60-region dartboard pattern is employed. Performance can be enhanced at the same time as acquisition time is reduced by 25%, by the use of a 25Hz rate of stimulation instead of the frequently employed rate of 75Hz.

Bacterial keratitis and bacterial endophthalmitis are two of the most devastating sight threatening eye infections. It is currently unclear whether the organisms isolated from these infections represent specialised members of these species or whether all strains are equally likely to cause infection. One method of differentiating strains genotypically is by a typing technique known as multilocus sequence typing (MLST). Using this technique, a better understanding of the molecular epidemiology of eye infections can be achieved. Propionibacterium acnes (P. acnes) is an important anaerobic organism causing several types of eye infections. Although it is now beginning to be recognised as a serious opportunistic pathogen, few studies have been done to investigate the population biology of P. acnes at the molecular level. Our continuing inability to distinguish between strains of P. acnes means that we still do not fully understand how antibiotic-resistant strains spread, nor whether certain strains, or clonal complexes, of P. acnes are associated with certain infections. These are key issues that can now be understood with our development of an MLST system for P. acnes. A diverse culture collection of 125 P. acnes isolates have been analysed using the MLST scheme developed. Sequence analysis shows that there are phylogenetically distinct groups within P. acnes and identified a novel cluster not previously described. Analysis of recombination using several methods suggests that frequent recombination occurs within these subgroups. There appears to be no association between these subgroups and clinical manifestation of P. acnes infection or geographical location. The P. acnes MLST scheme was validated against 16S rRNA gene and complete recA gene typing as well as immunofluorescence microscopy (IFM) and random amplification of polymorphic DNA (RAPD) analysis. P. acnes is a slow growing organism and is difficult to culture from ocular samples. A real-time PCR assay was developed in order to overcome the low culture positive rate and delay associated with conventional culture methods. Primers targeting one of the seven housekeeping genes used in the MLST scheme (gmk), specific for P. acnes were selected. The real-time PCR assay was both specific to P. acnes and highly sensitive. Pseudomonas aeruginosa (P. aeruginosa) is another important organism in the development of serious eye infections, and is the commonest cause of contact lens related microbial keratitis. Infections with this organism can lead to rapid deterioration of vision and possible blindness. A previously developed MLST scheme has been applied to 117 eye isolates from around the UK and China. This typing data was compared to 166 P. aeruginosa isolates from other clinical and environmental sources. Overall, MLST data supports previous findings that P. aeruginosa has a nonclonal population with epidemic clones. Sequence analysis showed that eye isolates do not cluster away from isolates from other clinical infection sites.

Infectious keratitis is a sight threatening disease which can cause permanent visual loss if not diagnosed and treated at an early stage. It can be caused by different types of microbes which are either commensals or transferred from the environment. To fight against these threats, the ocular surface (OS) has developed innate and adaptive immune mechanisms. Antimicrobial peptides (AMPs) are natural effectors on the OS with actions that range from microbicidal effects to cell signalling. Human beta defensin (hBD) 1-3 and 9, Liver expressed antimicrobial peptide (LEAP) 1 and 2, human cathelicidin (LL37), ribonuclease7 (RNase7) are the main AMPs on the OS. In this work, the pattern of ocular AMPs gene expression in human OS cells treated with Acanthamoeba castellanii, Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) was studied and established. This was examined by quantitative real-time PCR (RT-PCR) using the Taqman assay. Among the studied AMPs, hBD3 gene showed the most significant increase in human OS cells infected with Acanthamoeba. LL37 demonstrated the highest level of gene expression in the samples infected with bacteria. In a different study, the gene expression of two AMPs (hBD3 and 9) was studied in OS samples taken from patients with different types of infectious keratitis both during and after the infection. This was compared with the expression in healthy subjects. Impression cytology (IC) was used to obtain samples of OS epithelium from recruited subjects. An optimized method for RNA extraction of IC samples was developed. Corresponding to the results of the in vitro study, hBD3 showed an overall up-regulation in all categories whereas hBD9 was down-regulated. These changes were most significant in patients with acute Acanthamoeba keratitis. The gene expression of both hBD3 and 9 showed a tendency towards returning to the levels found in healthy subjects when healing of the corneal infection was complete. In another study carried out to examine the antimicrobial activity of hBD3 we were surprised to find that we could not replicate this. We were unable to reproduce the previously reported antimicrobial activity of hBD3 but were able to demonstrate that the antimicrobial effect could be attributed to the acidic solvent used in preparing the hBD3 protein. The clinical significance of application of corneal densitometry as measured by the Pentacam system was assessed for the first time in patients with infectious keratitis. We demonstrated that corneal densitometry varied with levels of inflammation and was not confined to the site of infection only. It affected the whole cornea and reverted towards normal values as the inflammation settled when the infection was brought under control. We were able to demonstrate that densitometry can be used as a measure of the corneal response to infection and inflammation and could be used to monitor response to therapy. Finally, separate comprehensive prospective and retrospective studies of the clinical profile of severe infectious keratitis in Nottinghamshire were conducted. These two studies covered a total period of 7 years. The results of both studies were similar. Indeed, OS disease, CL wear and previous ocular surgery were found to be the most common risk factors. Positive results of corneal scraping were obtained only in about 40% of cases. Acanthamoeba, S. aureus, and P. aeruginosa were the most frequent causative organisms. Fortified topical antibiotics were effective in treating most cases. Therapeutic corneal grafting was found to be an effective and safe procedure in refractive infectious keratitis.

Primary open angle glaucoma is mostly prevalent in patients over 60 years. However, studies examining the role of laser scanning tomography in detecting glaucoma hitherto have employed non-population based data drawn from younger subjects. This study employed an elderly, Caucasian, population-based cohort (minimum age 65 years, n=721). All subjects underwent ophthalmological examination including Goldmann applanation tonometry, suprathreshold automated visual field test and Heidelberg retina tomography. Normality was defined as normal visual acuity, visual field and intraocular pressure. Perimetrically normal males were found to have significantly larger optic cups than females. Normative data were used to construct three diagnostic tests: Linear regression of rim area, RADAAR (rim area/disc area asymmetry ratio) and the optic disc hemifield test (comparison of the superior and inferior rim/disc area ratios in the temporal and nasal sectors). Specificities at the 99th limit of normality were 91.4%, 95.1% and 98.3% respectively. Sensitivity was assessed using a cohort of patients (n=58) with a new diagnosis of open angle glaucoma at the Queen's Medical Centre, Nottingham within the last two years. Sensitivities at the 99th limit of normality were 72.4%, 55.6% and 27.6% respectively. Applying the regression analysis bilaterally and RADAAR simultaneously generated a specificity of 83.0% and sensitivity of 88.9%. Inclusion of the optic disc hemifield test did not further increase sensitivity. Linear regression of rim area/disc area was found to be susceptible to non-linearity and heteroscedasticity, causing reduced specificity in bigger optic discs. Modeling the relationship separately for each disc area quartile overcame this limitation to produce a diagnostic test with constant accuracy. Whilst laser scanning ophthalmoscopy can discriminate glaucoma with reasonable accuracy, it is limited by disagreement in contour placement and poor image quality in the elderly. The combination of multiple statistical tests is not ideal - application of statistical shape analysis techniques may better employ the normative data.

Intraocular angiogenesis is associated with a number of common, blinding conditions including wet age-related macular degeneration, proliferative diabetic retinopathy, retinopathy of prematurity and rubeotic glaucoma. The pathogenesis of these disorders is centered on choroidal, retinal and iris microvascular endothelial cells (ECs) respectively. When studying these conditions, workers have applied conclusions from in vitro studies of human umbilical vein EC (HUVEC) and microvascular endothelial cells, derived from different species or organs. However it is now widely accepted that endothelial cells are very heterogeneous and .extrapolation of results from these cells may not provide reliable data applicable to human eye disease. I have successfully isolated and cultured matched human retinal, choroidal and iris endothelial cells (and sourced HUVECs) and have examined their gene expression profiles. I found wide differences in gene expression between HUVEC and ocular ECs and in addition, between matched human retinal and choroidal endothelial cells. Taken together, these results suggest that HUVECs are not suitable surrogates for studying human ocular disease and secondly. It has implications for our understanding and treatment of retinal and choroidal vascular diseases. To further define the heterogeneity of endothelial cells within a vascular bed, I developed a technique for the isolation of human inner choroidal ECs. After the cells were characterized by surface marker expression, the response of these unique cells to various growth factors was defined and gene expression microarray analysis was performed and compared with matching outer choroidal ECs. This demonstrated differences in genes involved in fenestration formation and growth factor expression. These findings may have implications for our understanding of macular diseases. Overall, this body of work provides an insight into endothelial cell heterogeneity within the eye and may provide clues as to the reasons for ocular vascular bed disease susceptibility and helps identify potential future selective anti-angiogenic treatments.

In vivo confocal microscopy (IVCM) offers a unique real time non-invasive imaging method to explore live tissues at cellular and subcellular levels of histological detail with magnifications very much comparable to conventional ‘ex vivo’ light microscopy. Therefore it has been widely used over the past two decades to investigate the ocular surface and cornea in health, disease and following surgical procedures. One of the main challenges in understanding IVCM is to get a proper interpretation of the images that present various figures and patterns of tissue structures all in black and white with variable degree of reflectivity, being whiter (or brighter) when they are more (or hyper) reflective. The lack of good correlation between IVCM and corresponding light microscopy for the same tissue samples has lead to speculative interpretations of IVCM images in the literature. In this work we tried to fill that gap through performing IVCM to patients with various corneal and ocular surface disorders just few days prior to obtaining the tissue samples (corneal graft, excisional biopsy or alcohol delamination) for histopathological examination to make sure that IVCM images were truly representative to the tissue details that might change if more time was left to elapse between IVCM and tissue sampling. In ocular surface disease group (chapter three) we contrasted IVCM criteria in conjunctival epithelial overgrowth onto the cornea in limbal stem cell deficiency and Pterygium-like disorders with those seen in Corneal/Conjunctival Intraepithelial neoplasia (CIN) to confirm reliable diagnostic criteria of CIN with IVCM. In corneal oedema (chapter four) IVCM viewing of (histologically confirmed) subepithelial fibroblasts without clinically visible corneal scarring has been reported for the first time in IVCM literature particularly in Fuchs endothelial dystrophy. Sub-basal corneal nerve reduction as well as stromal keratocytes and endothelial changes were clearly illustrated with IVCM. In keratoconus cases (chapter five) morphological epithelial changes related to regenerative atypia have been studied for the first time and compared to those seen in CIN through light microscopy and IVCM in addition to other epithelial changes associated with keratoconus. Novel IVCM criteria for Bowman’s zone breaks have been described and compared carefully with histological sections. In corneal dystrophies (chapter six) IVCM criteria in Thiel-Behnke’s corneal dystrophy (CDBII) as well as in macular, granular and lattice dystrophy correlated well with light microscopic findings with a novel IVCM pattern in one Macular dystrophy case. In Acanthamoeba keratitis (chapter seven) a new form of Acanthamoeba cysts has been described for the first time through IVCM. Moreover this study presented for the first time IVCM diagnostic criteria in corneal intraepithelial neoplasia, keratoconus, Acanthamoeba keratitis and some corneal dystrophies. A comprehensive IVCM illustration of various types and stages of corneal fibrosis has been achieved as well.

Infantile Nystagmus Syndrome (INS) is defined as a constant involuntary movement of the eyes, affecting 0.12% of the population. Previous research shows that eye movements in patients with nystagmus increase in stressful conditions, and they report that their vision gets worse. However, there have been no definitive conclusions as to the effect of stress on visual acuity (VA). The aim of the studies described here was to assess visual function, including VA, during periods of stress. The results showed that there was no difference in VA measured with horizontally and vertically oriented Landolt C’s, but, in agreement with published research, VA was found to be poorer when using vertically oriented gratings as compared to horizontal gratings. Using a Trans-Cutaneous Electrical Nerve Stimulation (TENS) machine, an effective clinical stressor, we confirmed that the intensity of nystagmus increased when under stress; however VA, as measured using Landolt Cs, was not affected. Patient response time also increased during stressful periods and was significantly longer in INS than in control subjects. Using a questionnaire, we identified the most stressful situations for patients with nystagmus as being: “finding a person in a crowd” and “crossing a road in heavy traffic”. Under stress, rather than vision becoming blurred, patients with nystagmus reported that they “take longer to see things” and “have difficulty with seeing facial features and small detail”. The results reported here have important implications for patients with nystagmus in the real world, strongly suggesting that, although maximum acuity is unaffected by stress, more time should be allowed for tasks at both work and school. Further research is required to fully understand the changes in other aspects of visual function with stress.