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