Assessment of posterior eye cup respiration in mouse models of oxidative stress, age-related macular degeneration and diabetic retinopathy

Abstract

Age-related macular degeneration (AMD) is a complex multifactorial disease that primarily affects the central retina. It is characterized by the development of drusen and is a leading cause of blindness. Mitochondrial abnormalities are reported in patients with AMD and diabetic retinopathy. These abnormalities include macular mitochondrial DNA mutations, respiratory chain protein dysfunction and gross structural changes in mitochondria size and cristae. The goal of this thesis is to develop an ex vivo assay to assess mitochondrial metabolism function by analyzing mitochondrial respiratory parameters of the mouse posterior eye cup (PEC) tissue, using a Seahorse real-time metabolic analyzer. The optimal tissue size and assay conditions were identified, and a novel method of analyzing mitochondrial respiratory parameters of mouse posterior eye tissues was developed. Furthermore, we have validated these methods to assess PEC tissue respiration in several mouse models of ocular disease: in PolG mice that age prematurely due to excessive mitochondrial DNA damage, in a RPE oxidative stress model induced by intravascular injection of sodium iodate, and a streptozotocin (STZ)-induced hyperglycemic mouse model. Respiratory parameters of PEC from PolG mice are decreased compared to wild type mice. Systemic delivery of the RPE toxin sodium iodate decreases oxygen consumption rates (OCR) and increases the extracellular acidification rate (ECAR) suggesting a switch from oxidative to glycolytic metabolism. STZ induced hyperglycemia increases basal respiration and decreases glycolysis compared to normoglycemic mice, suggesting a shift from glycolysis to oxidative phosphorylation. The newly developed explant Seahorse XF model can be used to assess metabolic dysregulation of PEC tissues in mouse models of ocular disease.