The multifocal electroretinogram (mfERG) provides spatial and temporal information on the retina’s function in an objective manner, making it a valuable tool for monitoring a wide range of retinal abnormalities. Analysis of this clinical test can however be both difficult and subjective, particularly if recordings are contaminated with noise, for example muscle movement or blinking. This can sometimes result in inconsistencies in the interpretation process. An automated and objective method for analysing the mfERG would be beneficial, for example in multi-centre clinical trials when large volumes of data require quick and consistent interpretation. The aim of this thesis was therefore to develop a system capable of standardising mfERG analysis. A series of methods aimed at achieving this are presented. These include a technique for grading the quality of a recording, both during and after a test, and several approaches for stating if a waveform contains a physiological response or no significant retinal function. Different techniques are also utilised to report if a response is within normal latency and amplitude values. The integrity of a recording was assessed by viewing the raw, uncorrelated data in the frequency domain; clear differences between acceptable and unacceptable recordings were revealed. A scale ranging from excellent to unreportable was defined for the recording quality, first in terms of noise resulting from blinking and loss of fixation, and secondly, for muscle noise. 50 mfERG tests of varying recording quality were graded using this method with particular emphasis on the distinction between a test which should or should not be reported. Three experts also assessed the mfERG recordings independently; the grading provided by the experts was compared with that of the system. Three approaches were investigated to classify a mfERG waveform as ‘response’ or ‘no response’ (i.e. whether or not it contained a physiological response): artificial neural networks (ANN); analysis of the frequency domain profile; and the signal to noise ratio. These techniques were then combined using an ANN to provide a final classification for ‘response’ or ‘no response’. Two methods were studied to differentiate responses which were delayed from those within normal timing limits: ANN; and spline fitting. Again the output of each was combined to provide a latency classification for the mfERG waveform. Finally spline fitting was utilised to classify responses as ‘decreased in amplitude’ or ‘not decreased’. 1000 mfERG waveforms were subsequently analysed by an expert; these represented a wide variety of retinal function and quality. Classifications stated by the system were compared with those of the expert to assess its performance. An agreement of 94% was achieved between the experts and the system when making the distinction between tests which should or should not be reported. The final system classified 95% of the 1000 mfERG waveforms correctly as ‘response’ or ‘no response’. Of those said to represent an area of functioning retina it concurred with the expert for 93% of the responses when categorising them as normal or abnormal in terms of their P1 amplitude and latency. The majority of misclassifications were made when analysing waveforms with a P1 amplitude or latency close to the boundary between normal and abnormal. It was evident that the multilayered system has the potential to provide an objective and automated assessment of the mfERG test; this would not replace the expert but can provide an initial analysis for the expert to review.

The use of amniotic membrane (AM) as a dressing for ocular surface injuries has attracted the interest of the military ophthalmological community. First applied in the 1930s, the tissue is widely used today, although clinical indications for treatment are incompletely defined. While AM is most commonly stored frozen and thawed before use, dried AM is preferred for logistical reasons. Optimal preservation of the tissue is necessary to preserve its quality. The effect of drying on the physical and biological properties of the tissue are unknown. A systematic review of the evidence of AM treatment of acute chemical injuries was conducted. A framework was proposed for optimising the dried tissue through thermal, moisture sorption and surface analytical techniques. The physical properties of AM preparations were compared by mechanical testing and mathematical modelling, and an attempt was made to cross-link the AM collagen. Inflammatory aspects of the tissue were assessed by immunological techniques, zymography and macrophage assays. There is a lack of high quality evidence to support the clinical application of AM for acute burns. Complex interactions were demonstrated between the dried tissue, its excipients and moisture, suggesting novel ways of optimising the product. The mechanical properties of the dried membrane indicated that the process adversely affected the tissue, and artificial cross-linking could not be achieved. While the presence of antimicrobial peptides was not clearly established, the elution of collagenolytic enzymes was shown in therapeutic preparations of AM. The production of tumour necrosis factor by macrophages, which adhere to the spongy layer of AM, was suppressed. This project makes original contributions relevant to the use of dried AM as a biomaterial in ophthalmic surgery. Further refinements of this work, animal model experimentation and clinical trials may support its future acceptance as a clinical application.

The ability to examine the appearance of the retina is of paramount importance for the diagnosis and monitoring of ophthalmic disease and for the evaluation of treatment outcomes. Direct cross-sectional imaging of retinal structure could be useful for early diagnosis and more sensitive monitoring of a variety of retinal conditions such as macular oedema and glaucoma. The view of the fundus given by ophthalmoscopy provides very limited depth information and clinicians will often have to resort to additional techniques such as flourescein angiography or visual field testing for information on structural abnormalities within the retina. Other currently available imaging techniques do not provide sufficient depth resolution to produce useful cross-sectional images of retinal structure. Optical coherence tomography (OCT) is a new imaging technique which is capable of producing cross-sectional images of the retina with a resolution that surpasses that of conventional imaging techniques. This new technique has axial resolution of around 1 O.tm and can resolve individual retinal layers, thus providing information on retinal structure. In principle, OCT is very similar to ultrasound however it makes use of a light source rather than an acoustic one. The technique is non-contact and non-invasive and is generally well tolerated by patients. This thesis describes the evaluation of this new imaging technique with regards to its potential within routine clinical practice. A number of investigations were performed to fuffil this evaluation. Tests were carried out to experimentally measure the system's resolution and the accuracy and precision of measurements made from the OCT scans. A number of factors that could affect the quality of the scans were identified and their effects were minimised wherever possible. The software provided with the system was rigorously tested and potential sources of error were identified. Various studies were undertaken to quantify the repeatability and reproducibility of measurements made from scans and normative values were established. These results were used to assess the ability of the technique to detect and quantify several retinal disorders. The potential of the technique for corneal imaging was investigated - a scanning protocol was established and customised software for processing cornea! scans was developed. The relationship between OCT bands and retinal morphology was investigated by correlating scans from canine retina with corresponding light microscopy images and by observing the position of retinal abnormalities on scans from patients with a variety of conditions that affected different parts of the retina. Finally the clinical potential of OCT was investigated by carrying out various studies on a number of retinal conditions. Further clinical studies which combine anatomical information from OCT with functional information from electrophysiology are currently underway. Current developments are aimed at improving the imaging processing features and user interface so as to provide a more robust, user-friendly system for routine clinical use.

Proliferative vitreoretinopathy, a potentially blinding condition, involves excessive proliferation of retinal pigment epithelium (RPE) cells and is the main complication following retinal detachment (RD). Complicated cases of RD are treated with silicone oil (SiO) tamponades which can potentially be used as drug reservoirs. The aim of this study was to investigate different methodologies to develop a sustained and controlled drug release of anti-proliferative and anti inflammatory drugs from SiO tamponades using all-trans retinoic acid (atRA) and ibuprofen (Ibu). In detailed studies of atRA and Ibu, including atRA degradation behaviour, the drugs were found to be non-toxic to an adult RPE cell line (ARPE 19) below 10-5 M. The solubility of both drugs in SiO was assessed using radioisotope techniques. Prodrugs of atRA and Ibu were synthesised via conjugation to polyethylene oxide (PEO), and cleavage of the resulting ester bond, toxicity towards RPE cells, solubility in SiO and release into media were assessed. Prodrug cleavage was successful in vitro for Ibu but not achieved in the case of atRA due to its highly conjugated nature. Cytotoxicity assays showed PEO attachment had no effect on cytotoxicity and PEO prodrug solubility in SiO followed the expected trend of decreasing solubility with increased PEO chain length. Overall the saturation concentration of drug in SiO achieved through the use of PEO-prodrugs was too low for an effective therapy. Lipophilic prodrugs with a poly(dimethylsiloxane) (PDMS) pro-moiety were synthesised and investigated. Their cleavage was problematic due to PDMS being highly hydrophobic and cleavage could only be achieved in vitro when a small hydrophilic spacer was added between PDMS and the drug. The effects of PDMS prodrugs as additives in SiO were investigated and the presence of PDMS-atRA in SiO was shown to have a positive effect on both atRA solubility and longevity of release. The clinically-relevant release period (6-8 weeks) was independent of atRA starting concentration but dependant on the PDMS-atRA concentration within the blend. This has potential for further development into tamponade drug reservoirs for future patient benefits. A series of linear and branched amphiphilic copolymer architectures were also evaluated as additives for SiO. Monomer selection included oligoethylene oxide methacrylate (OEGMA), 2-hydroxyethyl methacrylate, PDMS-methacrylate (PDMSMA) and the brancher PDMS-dimethacrylate (PDMSDMA). SiO solubility of p(OEGMA-co-PDMSDMA) was investigated and copolymers which contained the smallest hydrophilic and largest lipophilic components only achieved small solubility (0.1 % v/v). To overcome these solubility issues, hydrophobic PDMSMA monomer was utilised. Both linear p(PDMSMA-co-OEGMA) and branched p(PDMSMA-co-OEGMA-co-PDMSDMA) were successfully synthesised and displayed high solubility within SiO, up to 40-50 % v/v. The potential for SiO tamponades as long-acting drug reservoirs has been demonstrated after inclusion of a novel end-modified PDMS additive leading to long term release of atRA. The formation of novel polymer architectures that show considerable miscibility with SiO also shows the scope of the opportunity for further additive development to tailor release profiles.

The eye is a glucocorticoid target tissue which orchestrates expression of target genes through the glucocorticoid receptor (GR). The classical function of GR involves its interaction with glucocorticoid to influence transcription of genes involved in numerous physiological processes which include inflammation. The first line of defence in the ocular tissues includes the mucosal barrier and expression of receptors that recognise pathogen. These mechanisms activate the innate immune response during inflammation, however, in the ‘normal’ eye, immunomodulatory components exist to promote immune privilege. 11beta hydroxysteroid dehydrogenase 1 (11β-HSD1) regulates cortisol locally in tissues and has already been localised to to some ocular surface and intraocular tissues. The aim of this thesis is to evaluate the functional role of 11β-HSD1 in the eye and the orbit and whether the 11β-HSD1 can be targeted to modify various disease processes in the eye. An animal model was used to characterise the pre-receptor regulation of glucocorticoids and this was further characterised in human ocular and orbital tissues and cells. The results showed that 11β-HSD1 is functional in certain ocular (corneal epithelial, fibroblast and conjunctival fibroblast) and orbital (orbital preadipocyte) cells. The data therefore emphasises the putative role of 11β-HSDs in the ocular and orbital microenvronment.

In the antisaccade task participants are required to saccade to the mirror image location of a sudden onset target. As such, the task provides a powerful tool with which to investigate the cognitive processes underlying goal-directed behaviour. In healthy participants antisaccade errors (prosaccades directed towards the target) occur on approximately 20% of trials, and increased antisaccade error rate is widely used as a measure of “cognitive disinhibition” in clinical settings. One aspect of antisaccade performance that has received relatively little attention is the large variability in error rate typically observed within healthy participants. Whilst there are many studies describing increased antisaccade error rates in patient populations, there has been comparatively little research into what individual differences might underlie the dramatic variations that are observed within healthy participants. This thesis presents five papers, each of which explores potential sources of variability in antisaccade performance in healthy participants. The first paper used a cueing manipulation to explore the extent to which individual differences in the ability to maintain the task goal in mind will influence antisaccade error rate. The second paper addressed the potential role of differences in motivation, by determining the extent to which antisaccade performance is moderated by a range of incentives. In paper 3, the role of strategic influences was investigated by altering the task instructions that participants were given. In paper 4, task instructions and working memory load were manipulated in order to determine their effect on antisaccade error awareness. The final paper, based on data gathered across the preceding experiments, explored the extent to which individual differences in factors such as working memory capacity, processing speed, and personality measures (schizotypy and impulsivity) correlated with antisaccade performance. Across all studies, the data is used to test predictions made by current parallel programming models of antisaccade performance. The data suggests that a range of “top-down” factors can influence antisaccade performance, but that the most important individual difference in explaining antisaccade error rate in healthy participants is prosaccade latency.

Aims The aim of this PhD research project was to investigate the application of dental pulp stem cells (DPSC) as a treatment for traumatic and degenerative eye diseases. The accuracy and reliability of counting retinal ganglion cells (RGC) in radial retinal section was also assessed. Methods Numbers of RGC in radial retinal sections were compared to numbers in retinal wholemounts. DPSC were cultured with RGC and survival and neuritogenesis were quantified. DPSC were also transplanted intravitreally into rat models of optic neuropathy (optic nerve crush) and glaucoma and surviving RGC and regenerated axons were quantified in radial retinal sections. Results Quantifying RGC in radial retinal sections was as reliable and accurate as the current gold standard Thus, retinal wholemounts with Brn3a proved to be the most reliable marker for RGC. DPSC protected RGC from optic nerve crush-/glaucoma-induced death, promoting significant regeneration of RGC axons in the former and preserving visual function (as measured by electroretinography) in the latter. The mechanism of action, as determined in vitro, appeared to be through the secretion of multiple neurotrophic factors (NTF). Conclusions In conclusion, DPSC is a potent cell therapy in the treatment of traumatic and degenerative eye disease.

Chemotaxis and cell migration are important processes for life, involved in organism development and homeostasis and implicated in a number of disease states. Dictyostelium discoideum, an amoeba, is a useful model for investigation of chemotaxis and development, due to its ability to undergo chemotactic aggregation and development upon starvation. Although cell migration has been well described on planar transparent surfaces, it is uncertain how well these conditions replicate the natural environment of a cell. However, attempts to better replicate these environments generally make use of opaque substrates and 3D matrices, in which it is more challenging to image cell migration. Protocols were developed to enable optical coherence tomography, a 3D structural imaging technique which requires no sample processing or staining, to be successfully employed in imaging Dictyostelium cell migration in time-lapse on non-transparent substrata and within an agarose gel. I compared the effects of two substrates, a nitrocellulose filter and a polystyrene Petri dish on aggregating cells and found differences in speed but not persistence. Extension of this to include cells within agarose revealed that these cells exhibited less directed migration, but their velocity was unaffected. I showed that cells lacking myosin II failed to complete development within an agarose gel and had significantly reduced velocity and directional migration when compared to their parent strain. Furthermore, the velocities of cells migrating within agarose gel were bimodally distributed, potentially indicating two distinct cell populations, fast and slow, and fast movement was shown to be largely myosin II dependent. Great potential therefore exists for cell-substrate and cell-matrix interactions to affect the migration character of cells, even those, such as Dictyostelium, which do not form strong focal adhesions. Moreover a properly ordered cytoskeleton is implicated in enabling cells to effectively utilise different modes of cell motility.

Age-related macular degeneration (AMD) is the largest cause of blindness for those over 65 in the developed world. There is currently no treatment for the retinal cellular loss associated with the disease. One potential therapy is to implant retinal stem cells into the eye using a biodegradable polymer scaffold. Blends of the biodegradable polymers, poly(L-lactic acid) (PLLA) and poly(D,L-lactic-co-glycolic acid) (PLGA) have been formulated into microspheres. The influence of changing processing parameters on the size and morphology of the microspheres has been studied. A human retinal pigment epithelial (APRE-19) cell line was shown to adhere, survive and proliferate on the surface of the microspheres in vitro. Assays have demonstrated that the nature of the blend influenced cell behaviour. Transplantation of retinal pigment epithelial (RPE) cells on a supportive matrix has also been investigated as a therapy for AMD. In view of AMD related pathology of the native RPE support, Bruch’s membrane (BM), transplanted RPE cells require a scaffold to reside on. Copolymers based on methyl methacrylate (MMA) and poly(ethylene glycol) methacrylate (PEGM) have been synthesised and chemically modified at the PEG terminus. These polymers were subsequently manufactured into a fibrous scaffold using an electrospinning technique and investigated as an artificial BM. RPE cells were shown to attach and proliferate successfully on the surface of the fibrous scaffold in vitro. Cell adhesion was significantly enhanced on scaffolds with the PEG chain terminus modification. Significantly less apoptotic cell death was also observed on these surfaces. The diffusion properties of these artificial membranes have also been investigated. In addition, the novel gelation of the produced copolymers under certain conditions has been studied

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the developed world, but the mechanisms leading to AMD are poorly understood. Circulating retinal autoantibodies and antibody deposits in the retina are associated with AMD but despite this relationship, immune complex (IC)-mediated responses and underlying mechanisms of inflammation in the retina have not been characterised. IgG antibodies can activate immune effector function through formation of IC and their interaction with Fcγ receptors (FcγR) expressed by immune cells. This study aims to test the hypothesis that IC formed in the retina induce an inflammatory response following interaction with activating FcγRs expressed on microglia and/or macrophages, which may contribute to the pathogenesis of AMD. To study the biological effect of IC formation in the retina a model of IC injury was developed and fully characterised. The involvement of mouse FcγRs (mFcγRs) was first studied using Fc gamma chain deficient (γ-/-) mice, lacking cellular expression of activating mFcγRs, and further characterised using FcγRI-/-, FcγRIII-/- and FcγRIV-/- mice. The presence of IC and human FcγR (hFcγR) expression was investigated in human donor eyes from early and wet AMD patients and healthy controls. Finally the effect of inflammatory mediators on human retinal pigmented epithelium (RPE) function was investigated by direct stimulation with cytokines or indirect stimulation using conditioned medium of polarised human macrophages. IC deposition in the mouse retina led to an inflammatory response that depended on the presence of activating mFcγRs, particularly mFcγRI and mFcγRIII, but not on mFcγRIV. Immune complex deposition and increased numbers of immune cells expressing hFcγRIIa and hFcγRIIb were found in the choroid of early AMD donors and microglia in the retina of wet AMD donor eyes. Finally, macrophage activation differentially impacted on RPE cell function, with regards to barrier function and VEGF secretion. The results in this thesis support the hypothesis that immune complex-mediated inflammation could play a role in the pathogenesis of AMD.