Analysis of Oxidative Stress in Diabetic Rat Retina and in Cultured Retinal Cells Under Hyperglycemic Conditions

Abstract

Purpose: : Numerous studies show that hyperglycemia leads to oxidative stress in the diabetic retinas, but the mechanisms that generate oxidative stress have not been resolved. Increased electron pressure on the mitochondrial electron transfer chain, increased generation of cytosolic NADH, and decreases in cellular NADPH have all been cited as possible sources of reactive oxygen species (ROS) and nitric oxide. The purpose of this study was to determine the level of oxidative stress in the ex vivo diabetic retina and in retinal endothelial and Muller cells under hyperglycemic conditions.

Methods: : Catalase activity and the level of H₂O₂ were determined in ex vivo control and 2 months streptozotocin-induced diabetic retina using Fluro H₂O₂^TM kit. Cultured bovine retinal endothelial cells and Muller cells (TR-MUL) were incubated either with 5 or 30 mM glucose for 1 to 5 days and generation of ROS measured as oxidized fluorescent product of CM-H₂DCFDA. ROS was also measured in endothelial cells treated with pyruvate (5mM) and glutamine (5mM). TR-MUL cells were also treated with diamide (200 µM), CuSO₄ (20 µM), pyruvate (5 mM) and glutamine (5-10 mM) in presence and absence of high glucose and the level of ROS measured. Diamide and CuSO₄ cause oxidative stress in cells.

Results: : Catalase activities were found to be the similar in both control and diabetic retina. An increased level of H₂O₂ was detected in the diabetic retina compared to non-diabetic when catalase activity was inhibited with CuSO₄. In contradiction to previous studies by others, our study demonstrated that high glucose exposure to both endothelial and Muller cells for 1 to 5 days, the level of ROS significantly reduced (P < 0.05). Treatments with pyruvate could not influence the increase in ROS generation in these cells, however, glutamine treatments caused a significant increase in ROS generation in both endothelial and Muller cells but more dramatically in endothelial cells.

Conclusions: : High levels of glucose may not provide the mitochondria with excess reducing pressure that would generate superoxide anions by reduction of molecular oxygen at intermediate steps in the electron transport chain. However, diabetes influences an increase in oxidative stress in the retina.

Keywords: oxidation/oxidative or free radical damage • diabetic retinopathy • retina