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.