Metabolic and functional approaches to investigate the effects of cytokines on the physiology of in-vitro and in-vivo models of diabetic retinopathy
Diabetic retinopathy (DR) is the leading cause of vision impairment in working age adults worldwide. It is clinically described as hyperglycemia-induced retinal blood vessel damage and is categorized into early non-proliferative and late proliferative stages. Although DR becomes clinically evident only when vascular damage has occurred to the retina, the onset of pathology occurs much earlier than the clinical manifestation. Several studies have reported that metabolic and functional dysregulation in the retina precedes the development of vascular damage in DR. However, current treatment strategies are aimed at treating the late stage sight-threatening vascular problems and do not prevent DR progression. A better understanding of the underlying molecular mechanisms will enable the development of newer therapeutic interventions to modulate these pathways early on and delay the onset of vascular problems. The latest evidence from our lab suggests that in addition to hyperglycemia, retinal inflammation plays an important role in DR development. However, it is still unclear how co-occurrence of hyperglycemia and inflammation alters retinal homeostasis leading to vascular damage. Therefore, the aim of the thesis was to investigate the metabolic and functional effects of pro-inflammatory cytokines on the hyperglycemic retina in an in-vitro and in-vivo DR model. The findings from the in-vitro DR model confirmed that the combined exposure of the retina to high glucose and pro-inflammatory cytokines affected key metabolites including glucose, lactate, ATP and glutamate, and elicited re-distribution of glutamate and glutamine in the retina. These changes suggested that glucose and glutamate metabolic pathways are affected early on in DR. To determine the contribution of glycolysis and glutamate recycling pathways to the metabolic imbalance in DR, the key regulatory enzymes, glyceraldehyde 3-phosphate dehydrogenase and glutamine synthetase were pharmaceutically inhibited, and metabolite levels were evaluated. Inhibition of glycolysis was found to play a major role in the metabolic imbalance observed in DR, while inhibition of glutamate recycling to glutamine had a smaller contribution. The activity of glutamate ionotropic receptors was further probed to determine whether pro-inflammatory cytokines regulate cation channel permeability in DR. Activation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor but not the other ionotropic receptors, resulted in increased cation permeation into amacrine and ganglion cells in DR conditions, suggesting that these cells are specifically involved in the progress of DR. Finally, to confirm the findings from the in-vitro model and assess DR pathogenesis, an in-vivo mouse DR model incorporating hyperglycemia and intraocular inflammation was employed. These mice showed signs of vascular damage, reduced retinal functional and metabolic imbalance within two days of administering pro-inflammatory cytokines to the eye. Taken together, this thesis provides a thorough understanding of the early retinal changes occurring in DR when hyperglycemia and inflammation are present together. The results contribute to the pool of knowledge that suggest an early intervention to prevent the onset of inflammation in the eye can prevent sight-threatening stages of the DR condition.
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