Anatomy and Neuroscience - Theses
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ItemThe role of microglia in regulation of vasculature and blood flow in the healthy and diabetic retina( 2022)Diabetic retinopathy is a common vascular complication of diabetes and a leading cause of blindness in those of working age. Prior to overt vascular pathology, the retina displays subtle changes to neurons, glia, and blood vessels that are likely important for disease progression. However, current treatments for diabetic retinopathy are only effective at targeting late-stage pathology. Treatments that target the early cellular changes in the diabetic retina have the potential to halt disease progression before vision is threatened. One of the earliest changes observed in the diabetic retina is a reduction in blood flow. This early vascular dysfunction has been observed in the absence of any other signs of retinopathy, suggesting it may be a key early driver of disease and a promising target for intervention. It has been suggested that the underlying cause of reduced blood flow is dysfunction of the mechanisms that regulate blood flow in the retina. However, our current understanding of these mechanisms is largely incomplete. The central aim of this thesis was, therefore, to explore how blood flow is regulated in the normal retina, and to determine how this function is altered in the diabetic retina. Recent work from our group and others have identified that microglia, the resident immune cells of the central nervous system, may play a role in regulation of blood flow. Based on this emerging evidence, our hypothesis was that microglia regulate vascular function in the retina, and that hyperglycaemia leads to changes in microglia that impair this function and result in reduced blood flow. To explore this hypothesis, we first performed RNA sequencing of retinal microglia isolated from mice lacking Cx3cr1, a chemokine receptor specific to microglia and an important regulator of many microglial functions. This revealed a role for Cx3cr1 in several possible functions related to vasculature, including vascular development, microglial-vascular adhesion, and vascular tone, which were further assessed with in vitro and in vivo imaging techniques. Imaging data revealed the Cx3cr1null retina showed increased vascular density, reduced microglial-vascular contact, and most interestingly, dilation of capillaries. This loss of vascular tone may have been due to reduced expression of angiotensin converting enzyme, a component of the renin angiotensin system (RAS), which promotes vasoconstriction. The ability of microglia to dynamically alter blood vessel diameter and hence control blood flow was then assessed by live cell imaging of the ex vivo retina. We observed frequent spontaneous calcium transients in microglia which appeared to induce vasoconstriction, which may have been mediated by purinergic signalling. Microglia also evoked vasoconstriction via a calcium-independent mechanisms, which was promoted by addition of fractalkine, the ligand for Cx3cr1. Transcriptomic data suggested FKN-Cx3cr1 signalling may promote vasoconstriction via modulation of the RAS. This was confirmed by inhibition of the RAS in the ex vivo retina, which abolished FKN-evoked vasoconstriction. As earlier work from our group has shown FKN-Cx3cr1 signalling and the microglial RAS are upregulated in the diabetic retina concurrent with reduced blood flow, we postulated that this vascular dysfunction may be caused by aberrant microglia-mediated vasoregulation. To test this, we trialled pharmacological blockade of the RAS in an animal model of type 1 diabetes. Without treatment, diabetic animals exhibited constriction of retinal capillaries, reduced blood flow, and dysfunction of inner retinal neurons. Microglia did not display classical signs of activation but did show increased accumulation on capillaries. RAS blockade successfully restored capillary diameter in the diabetic retina, but surprisingly failed to improve blood flow or neuronal function. Finally, while RAS blockade did not affect the number of microglia accumulating on capillaries, it did increase the extent to which individual microglia contacted vasculature, further alluding to the importance of the microglial RAS in regulation of retinal vascular function. In summary, our findings indicate that microglia and Cx3cr1 are important for vascular function in the retina, in particular for vascular development and maintenance of capillary tone. We also established that microglia can dynamically alter blood vessel diameter in multiple ways, suggesting these cells may be important for regulating retinal blood flow. Finally, restoration of capillary diameter by RAS blockade in the diabetic retina supports the theory that aberrant microglia-mediated vasoregulation contributes to early vascular dysfunction in diabetic retinopathy. These findings may form the basis for new treatments that can prevent vascular dysfunction in diabetic retinopathy and other CNS diseases.
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ItemChanges in Retinal Ganglion Cells during Disease and Aging( 2020)Healthy retinal ganglion cell (RGC) function is vital for vision, and diseases that cause RGC degeneration can be debilitating. RGC degeneration features prominently during age-related diseases such as glaucoma and diabetic retinopathy. This thesis investigates how RGCs change in models of these diseases and if similar changes can be observed in normal aging. Exploring and comparing functional, structural, and molecular changes that develop in rodent RGCs during pathological states and normal aging can expand our understanding of how they degenerate during disease. Glaucoma is characterised by gradual RGC degeneration and is usually accompanied by increased intraocular pressure (IOP). We first examined the effect of an acute, non-ischemic IOP insult on RGC activity in wildtype mice. In wildtype mice, OFF-RGCs alone showed reduced spontaneous and light-elicited activity after IOP elevation. Next, we explored the role of the P2X7-receptor (P2X7-R) following IOP elevation, as it has been suggested to contribute to RGC death in glaucoma. After IOP elevation in P2X7-R knockout mice, both ON- and OFF-RGCs exhibited reduced light-elicited activity. Staining for P2X7-R in Thy1-YFP-H mice showed greater expression on ON-RGC dendrites than in other RGC cell subtypes. This study demonstrated the dysfunction of OFF-RGCs after acute, non-ischemic IOP elevation was not prevented by P2X7-R ablation. P2X7-R knockout seemed to worsen the effects of IOP elevation as it also caused ON-RGC dysfunction. In early stages of diabetic retinopathy, there is increasing evidence for RGC degeneration, prior to perturbation of other retinal neurons. Examining individual RGC function, we found that after 4 weeks of STZ-induced diabetes OFF-RGCs showed an increase in spiking activity at a single light intensity (220 photoisomerisations/sec/rod) compared to control. No changes in RGC density, synaptic protein puncta counts or Muller glia gliosis were identified. Microglia, however, showed a reduction in volume. These changes early in diabetes, though subtle, suggest dysfunction of the retinal circuitry alongside the development of inflammation. Aging can exacerbate RGC degeneration and contribute to the development of diseases such as glaucoma and diabetes. To probe the effects of age on RGCs in greater detail we used a combination of functional, structural, and molecular techniques. By examining the transcriptomes of isolated RGCs from young and aged mice using RNA-sequencing, we found upregulated genes in the pathways for oxidative stress, protein degradation and synaptic function. The upregulation of these genes may be a defensive strategy against age-related stresses during normal, healthy aging. This appears to be supported by our finding that RGCs were not as susceptible to structural or functional loss with age in comparison to photoreceptors and other cell classes. Overall, we found RGCs were dysfunctional prior to death by using early-stage disease models. The results of this thesis provide evidence that age- and disease-related stressors may invoke divergent responses in RGCs, despite aging being a risk factor for retinopathies. Stressors like increased IOP and hyperglycaemia worsen RGC function in a subtle manner; yet aging itself does not seem to pose a threat to RGC survival or function as RGCs seem more robust when compared to other retinal neurons. Future explorations could consider whether an additive effect of aging and disease may cause RGC defence mechanisms to become compromised
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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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ItemThe role of the purinergic system in the normal and diseased retina( 2015)Extracellular adenosine 5’-triphosphate (ATP), its breakdown products and other related nucleotides are chemical transmitters mediating purinergic signaling within the central and peripheral nervous systems. In the retina, ATP has been shown to act as a neuro- and glio-transmitter that is important for visual processing and maintaining tissue homeostasis. However, the cellular expression and potential role of different components in the purinergic signaling pathway remains to be characterized in the retina. The mechanism by which purinergic signaling mediates the pathogenesis of retinal degeneration also remains ill-defined. The fundamental aim of this study was to investigate the role of purinergic signaling in the normal mammalian retina and to determine how this signaling pathway is involved in the mechanisms of retinal degeneration. The cellular localization of the vesicular nucleotide transporter (VNUT), the synaptic vesicles in which ATP is stored, was explored in the mammalian retina. With the use of a novel polyclonal antibody directed against VNUT, specific expression in dopaminergic interplexiform cells (IPCs) in the retina was demonstrated by fluorescence immunohistochemistry and confocal microscopy. Further investigations by three-dimensional reconstructions revealed that VNUT-expressing IPC distal varicosities were in close contact with horizontal cell processes and cone photoreceptor terminals in the outer retina, suggesting a role of vesicular ATP release in modulating outer retinal processing. The expression profile of the P2X4 receptor (P2X4-R) was examined in detail with reference to the specific neuronal and glial cell classes in the retina. Fluorescence immunohistochemistry and pre-embedding immuno-electron microscropy together demonstrated post-synaptic expression of the P2X4-R in both plexiform layers of the retina. In the outer retina, P2X4-R expression was identified on horizontal cell somata and processes. In the inner retina, P2X4-R immunoreactivity on amacrine and ganglion cells was observed. Furthermore, P2X4-R expression was detected on all glial cell types in the retina. These data indicate that P2X4-Rs are likely to play a role in the lateral inhibitory pathways, as well as a role in macro- and micro-glial signaling in the retina. The role of purinergic signaling in the cellular and vascular responses of the retino-choroidal complex to laser-induced injury was investigated. Application of the nanosecond laser at suprathreshold energy setting in P2X7-R knockout (P2X7-KO) mice resulted in an attenuated inflammatory response at the RPE/choroidal layer, indicating a role for purinergic signaling in mediating inflammation in response to retinal injury. The safety and efficacy benefits of the nanosecond laser system over standard photocoagulation lasers were also demonstrated by the lack of post-treatment neovascularization. Taken together, these findings indicate a critical involvement of the purinergic system in modulating diverse signal transmissions and maintaining tissue integrity in the normal mammalian retina. This study also provides evidence that the purinergic system has an important role in mediating the inflammatory response to retinal pathology.
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ItemThe role of chemokines in the retina( 2013)This thesis investigates mice with genetic knock-outs in certain chemokines or their receptors which have shown signs of Age Related Macula Degeneration (AMD) by causing dysfunction to the immune response in the retina. We explored two mechanisms of retinal damage in mice lacking the chemokine, CCL2 or the chemokine receptor, CX3CR1. We tested the underlying retinal function and cellular and structural responses during aging and after light-induced oxidative damage to examine the role of these signaling pathways in the retina.
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ItemThe condition of the retina in retinitis pigmentosa during late stages of degeneration( 2012)Retinitis pigmentosa (RP) is a family of inherited retinal degenerations that are characterised by photoreceptor death resulting in blindness. It was once thought that following photoreceptor death, the inner retina remained unchanged. However, studies have begun to show that there are extensive changes at late stages in the disease process. This thesis further extends these studies by examining the inner retina at various time points after photoreceptor death so that the cellular changes that arise throughout the degenerative process can be characterised. A novel transgenic mouse model, the rd1-FTL, was used to document the condition of the retina. This animal model expresses a mutation which results in retinal degeneration as well as containing an axonal targeted transgenic marker gene that labels all neurons that have the c-fos gene activated. A combination of enzyme histochemistry and fluorescence immunohistochemistry was used to reveal an up-regulation of c-fos in the central retina following complete photoreceptor loss that remained throughout the later stages of degeneration. This was also coupled with a major glial dysfunction in this area. Neurochemical analysis prior to and during c-fos upregulation revealed significant changes in the inner retina. Amino acid immunocytochemistry was used to show that many amino acids in the degenerated retina were altered in patches across the retina. The most significant changes were documented in Müller cells and bipolar cells. Additionally, gross remodelling of neural and glial cells occurred in the retina which worsened as animals aged. A comprehensive morphological analysis of retinal ganglion cells in the degenerated retina showed that the cell types which covered the largest area in the retina were altered whilst the smaller cells types remained unchanged. However, these smaller cells did show altered morphology in regions that also expressed c-fos in late stages of degeneration. Furthermore, there were significant changes in the stratification of many ganglion cells in the degenerated retina that included the loss of certain cell types as well as different branching patterns of dendrites. Overall, this thesis shows that there are extensive changes in the inner retina following photoreceptor loss. It provides a thorough examination at different stages in the degenerative process to highlight the varying conditions that can occur in this disease. Approaches aimed at vision restoration rely on a strong understanding of this underlying cellular anatomy and this work will assist with the design of these approaches.