Anatomy and Neuroscience - Theses
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ItemThe neurovascular unit in early diabetic retinopathy: cellular changes and an emerging role for microglia( 2018)In the developed world the leading cause of blindness in working age adults is diabetic retinopathy (DR). The risk of vision loss due to diabetes increases with disease duration, and most frequently affects those who have lived with diabetes for one to two decades. For individuals with diabetes, there is a need to develop therapeutic interventions that are capable of halting or delaying degeneration of the retina as early as possible to preserve vision throughout the life of the patient. DR ultimately leads to vision loss via the death of retinal neurons, which occurs after severe dysregulation of the retinal vasculature. Retinal oedema and/or the proliferation of blood vessels both signal the advancement to a high-risk stage of vision-threatening DR, and our understanding and management of these late stages is comprehensive. However, the initial cellular changes that occur soon after the onset of diabetes and contribute to this end-stage vascular pathology are not completely understood. These include changes to the neurovascular unit: retinal blood flow, neuronal function, and glial / microglial cell reactivity. It is these early signs that are investigated in this thesis, for it is these changes at this early timepoint that hold the most promise for intervention to prevent onset of clinical vasculopathy. Using an animal model of diabetic retinopathy, the relative contribution of neurons, blood vessels, and glia/microglia to early DR pathology was assessed at two early time-points after diabetes onset. In vivo measurement of retinal blood flow, blood vessel structure, and neuronal function was undertaken; in conjunction with histological analysis of pericytes, Müller glia, astrocytes, and microglia, as well as the interactions of these cells with the vasculature. Additionally, microglia were investigated for their potential to assist in vaso-regulation using ex vivo live cell imaging and single cell population RNA-Seq. Functional measurements revealed that retinal blood flow and retinal ganglion cell function were both reduced after 4 weeks of hyperglycaemia. In depth analysis of retinal vessel structure showed capillaries to be constricted at this time-point and were also unresponsive to hyperoxic challenge. Neuronal function was further degraded by 12 weeks of diabetes, although there were no signs of cell death. Glial cells showed no difference in structure, or signs of gliosis after 4 weeks of diabetes, and did not alter contact with the vasculature. Microglia were found to be in a resting phenotype and increased their contact with retinal capillaries after 4 weeks of diabetes. The microglia specific fractalkine signalling axis was shown to initiate vasoconstriction on retinal blood vessels, a response which was absent in the diabetic retina. Vaso-active genes were identified in microglial populations, several of which were upregulated in the diabetic retinae. In summary there are two main findings to report. Firstly, this project expands our understanding of the chronopathology of cell-specific dysfunction in the early stages of DR, and indicates that capillary constriction may be a new biomarker for early stage disease. Secondly, the discovery that microglia contribute to blood flow maintenance opens new avenues for research into vascular diseases, including DR, which may unlock novel therapeutic targets.
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ItemRetinal dysfunction in an animal model of retinopathy of prematurity( 2012)Retinopathy of prematurity (ROP) is characterised by retinal neovascularisation and visual impairment following high oxygen treatment of premature infants. While attempts to develop new therapies to reduce the severity of ROP are underway, the main method of treatment involves laser photocoagulation of the peripheral retina. This treatment is invasive and can lead to further visual impediments, highlighting the necessity of developing new, less invasive therapeutic treatments to effectively manage ROP. One potential treatment of emerging interest involves blockade of the renin-angiotensin system (RAS). Blockade of the RAS in animal models of ROP have been shown to reduce retinal neovascularisation, inflammatory responses, and, to a degree, neuronal degeneration. However the effects of RAS blockade on neuronal function in animal models of ROP remain unknown. Therefore, the aim of this study was to investigate the functional and structural changes in an animal model of ROP and to determine whether RAS inhibition prevented vascular anomalies and neuronal dysfunction. The rat model of ROP, known as oxygen-induced retinopathy (OIR), is a widely used animal model that closely mirrors the characteristic vascular, glial and neuronal abnormalities of human ROP. This study examined whether the putative prorenin receptor antagonist, handle region peptide (HRP), and the angiotensin II type-1 receptor (AT1R) inhibitor, valsartan, prevents neural, glial and vascular anomalies in the OIR rat retina. Immunohistochemical techniques were used with established cell markers to assess whether HRP and valsartan reduced the development of pathological neovascularisation and altered the microglial response in OIR. The functional deficits in OIR rats with and without treatment with RAS inhibitors were assessed using the electroretinogram. Retinal morphology following treatment was evaluated histologically, including retinal layer thicknesses and synapse morphology. To evaluate why RAS inhibition did not improve the neuronal deficits, the effects of high oxygen rearing on retinal development was assessed. Histological techniques were used to examine the morphological development of retinal vasculature, glia and neurons during the high oxygen phase in the OIR model. The results presented highlighted the anti-angiogenic nature of RAS inhibitors in the developing retina and provided further insight into the role of retinal microglia in OIR. This study indicated that OIR rats have deficits in both rod and cone pathway function and that treatment with RAS inhibitors does not improve this functional loss, despite the advantageous effects of RAS blockade upon reducing glio-vascular pathology. In addition, the results from this study showed that the course of retinal development was altered during high oxygen rearing, with changes in vessels, microglia and some neurons observed. In summary, the findings shown in this thesis demonstrate abnormal vascular, glial and neuronal changes during and on removal from the high oxygen phase in the OIR rat model. Despite the beneficial effects RAS inhibitors had on reducing the vascular pathology, visual function in these rats was not improved following treatment. The retinal changes that occur during the high oxygen phase, prior to pathological neovascularisation and RAS inhibition, may contribute to these deficits. These findings have implications for the treatment of ROP. Notably, future therapies need to target pathological angiogenesis, whilst promoting physiological growth of vessels into the retina. Moreover, preventing the neuronal changes that occur during high oxygen exposure may need to be considered in order to maximise visual outcomes for infants with ROP.