Retinitis pigmentosa (RP) is a group of hereditary eye diseases which involve degeneration to the retina of the eye. GARP2 (glutamic acid-rich protein) is one of the retinal proteins expressed solely in the rod photoreceptor. This thesis focuses on both the genetic and proteomic profile of GARP2 and its possible role in the aetiology of RP.

Neovascularisation occurs in response to tissue ischemia in conditions including ischaemic retinopathy, with new vessels often infiltrating the transparent vitreous by a poorly understood mechanism. Nitric oxide produced by eNOS is a downstream mediator of VEGF function and plays a crucial role in vascular formation and maturation; therefore, we investigated the role of NOS using transgenic models in the murine model of oxygen induced retinopathy (aiR). Using eNOS over-expressing eNOS-GFP mice, we investigated vascular growth during retinal development, during aiR and also using an in-vitro model and found that eNOS over-expression increased angiogenesis. Over-expression of eNOS in the eNOS-GFP mice resulted in an increase in vaso-obliteration following hyperoxia exposure, which was followed by an increased neovascular response and improved revascularisation of the ischaemic retina. There was evidence of dysfunction of NOS in the eNOS-GFP animals, with an increase in oxidative stress which may contribute to the neovascular formation. Using the BH4 deficient hph-l mouse model we investigated the role of this NOS co-factor in the development of the retinal vasculature and the pathology of ischaemic retinopathy. We found that there was a reduced severity of both the vaso-obliterative . and neovascular phases of ischaemic retinopathy. A reduction in NOS activity due to the BH4 deficiency was demonstrated in-vitro in microglial cells from the hph-l animals and could explain the reduced angiogenesis seen in the hph-l animals. Our results demonstrate that the pathology of ischaemic retinopathy is altered by changes in NOS expression and function with eNOS over-expression resulting in more severe vaso-obliteration, followed by improved retinal revascularisation, while a deficiency in BH4 results in reduced severity of both the vaso-obliterative and neovascular phases of ischaemic retinopathy, but with a delayed revascularisation. These results indicate that selective alteration of NOS expression at different stages of ischaemic retinopathy may reduce the severity of the vaso-obliterative stage and help promote recovery of the ischaemic retina.

BEST1, the gene encoding bestrophin-1, was first identified associated with Best disease, an early onset autosomal dominant macular dystrophy characterised by an abnormal electrooculargram (EOG) and vitelliform lesions in the macula (Petrukin etal. 1998 and Marquardt et al. 1998). Since its discovery over 100, mostly missense, mutations in BEST1 have been associated with Best disease. A second dominant retinal dystrophy is caused by mutations in BEST1 (Yardley et al 2004). Autosomal dominant vitreoretinochoroidopahty (ADVIRC) is characterised by a hyperpigmented band around the periphery of the retina and punctate retinal and vitreous opacities.

The aim was to investigate the effect of streptozotocin-induced diabetes and rosuvastatin on retinal leukocyte-endothelial cell interactions in rodents. Leukocytes, isolated from the spleens of donor animals and fluorescently-labelled with calcein-AM, were transferred between control, diabetic, rosuvastatin treated control and diabetic, and co-treated rosuvastatin and mevalonate diabetic, mice. Fluorescent leukocytes were imaged in the retinas of syngeneic recipients using a scanning laser ophthalmoscope (SLO) and recorded. After imaging animals were infused with 2% Evans Blue, killed and eyes removed and fixed in 2% paraformaldehyde. Retinas were flat mounted and examined for fluorescent cells under a confocal microscope. 2 weeks of STZ-diabetes caused an increase in retinal leukocyte adhesion <i>in vivo</i> and in <i>ex vivo</i> whole mounts, due to alterations to the leukocytes and to the endothelium. Treatment of donor and recipient diabetic animals with rosuvastatin prevented and reversed this increase, as did prevention treatment of recipient diabetic animals only. However, treatment of donor diabetic animals only had no effect. Co-treatment with mevalonate abrogated the effect of rosuvastatin. Rosuvastatin treatment had no effect on plasma cholesterol or triglyceride levels. These results confirm previous reports indicating that leukocyte adhesion is, in part at least, responsible for the capillary occlusions that occur in DR. Rosuvastatin may be effective in the treatment of DR by preventing leukocyte adhesion to the vascular endothelium in retinal vessels. Rosuvastatin appears to exert its effects on the endothelium and its effects are independent of lipid-lowering but dependent upon cholesterol biosynthesis inhibition.

Methods: Fresh blood samples were collected from 69 age-matched subjects consisting of 45 patients with diabetes (with or without retinopathy) and 24 controls.  <i>In vitro</i> hyperglycaemic incubations were performed to detect any changes in surface expression of markers of activation (CD69 and CD25 / IL-2 receptor).  Patient and control samples underwent flow cytometry for the same markers of activation and also 4 adhesion molecules: LFA1, VLA4, ICAM-1and L-selectin. Results: <i>In vitro</i> hyperglycaemic incubations did not induce a significant change in CD69 or CD25 levels.  Significantly reduced L-selectin expression was found on lymphocytes from patients with diabetes compared to controls (p=0.004).  The lowest levels of expression were found in those with proliferative diabetic retinopathy (p=0.001).  There were no significant differences in surface expression of the markers of activation and the other adhesion molecules studied.  Increased nuclear production of L-selectin was indicated by the finding of significantly higher mRNA levels (p=0.007) in patients with DR than in those with no retinopathy.  Similarly, serum L-selectin levels were significantly higher (p=0.04) in those with DR compared to controls.  Lymphocyte adhesion relative to control was essentially unchanged for diabetic patients with no retinopathy but was markedly increased for those with retinopathy (p=0.0001). Conclusion: Lymphocyte activation, reduced surface LO-selectin, increased circulating and nuclear L-selectin levels, and a corresponding increase in adhesion to endothelial cells is evident in people with diabetic retinopathy.  This suggests a role for lymphocyte activation in the pathogenesis of diabetic retinopathy.

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Visual perception is dependent on both light transmission through the eye and neuronal conduction through the visual pathway. Advances in clinical diagnostics and treatment modalities over recent years have increased the opportunities to improve the optical path and retinal image quality. Higher order aberrations and retinal straylight are two major factors that influence light transmission through the eye and ultimately, visual outcome. Recent technological advancements have brought these important factors into the clinical domain, however the potential applications of these tools and considerations regarding interpretation of data are much underestimated. The purpose of this thesis was to validate and optimise wavefront analysers and a new clinical tool for the objective evaluation of intraocular scatter. The application of these methods in a clinical setting involving a range of conditions was also explored. The work was divided into two principal sections: 1. Wavefront Aberrometry: optimisation, validation and clinical application The main findings of this work were: • Observer manipulation of the aberrometer increases variability by a factor of 3. • Ocular misalignment can profoundly affect reliability, notably for off-axis aberrations. • Aberrations measured with wavefront analysers using different principles are not interchangeable, with poor relationships and significant differences between values. • Instrument myopia of around 0.30D is induced when performing wavefront analysis in non-cyclopleged eyes; values can be as high as 3D, being higher as the baseline level of myopia decreases. Associated accommodation changes may result in relevant changes to the aberration profile, particularly with respect to spherical aberration. • Young adult healthy Caucasian eyes have significantly more spherical aberration than Asian eyes when matched for age, gender, axial length and refractive error. Axial length is significantly correlated with most components of the aberration profile. 2. Intraocular light scatter: Evaluation of subjective measures and validation and application of a new objective method utilising clinically derived wavefront patterns. The main findings of this work were: • Subjective measures of clinical straylight are highly repeatable. Three measurements are suggested as the optimum number for increased reliability. • Significant differences in straylight values were found for contact lenses designed for contrast enhancement compared to clear lenses of the same design and material specifications. Specifically, grey/green tints induced significantly higher values of retinal straylight. • Wavefront patterns from a commercial Hartmann-Shack device can be used to obtain objective measures of scatter and are well correlated with subjective straylight values. • Perceived retinal stray light was similar in groups of patients implanted with monofocal and multi focal intraocular lenses. Correlation between objective and subjective measurements of scatter is poor, possibly due to different illumination conditions between the testing procedures, or a neural component which may alter with age. Careful acquisition results in highly reproducible in vivo measures of higher order aberrations; however, data from different devices are not interchangeable which brings the accuracy of measurement into question. Objective measures of intraocular straylight can be derived from clinical aberrometry and may be of great diagnostic and management importance in the future.