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M. S. Ola, K. F. LaNoue; Analysis of Oxidative Stress Under in vivo Conditions in Diabetic Rat Retina. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5926. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
An increase in the generation of reactive oxygen species (ROS) has been detected in tissues of experimental animals with diabetes, in cultured retinal cells and vascular endothelial cells exposed to high glucose concentration. Excess ROS may induce oxidative damage to retinal cells, causing vascular leakage, neuronal cell death and other symptoms of diabetic retinopathy. ROS has been detected by others using dichlorofluorescein (DCF) derivatives applied to cultured cells or to frozen tissues but measurement of ROS production in vivo has not been reported. The purpose of this study was to establish a method to measure in vivo ROS production and then investigate the effect of various potentially therapeutic drugs such as the anti-inflammatory agent, minocycline and memantine on ROS generation in diabetic retinas.
The measurement of ROS is based on fluorogenic marker CM-H2DCFDA, a chloromethyl derivative of H2DCFDA (Molecular Probe). A fresh stock solution (2.16 mM) of CM-H2DCFDA was made and 3 µl from the stock was injected intravitreally into the eye cavity of anesthetized rats. After 6-8 hours, the retina was dissected, rinsed in cold saline and then immersed in 300 µl of 50 mM Hepes buffer, pH 7.4 containing 0.1% SDS. Retinas were sonicated, centrifuged and 100 µl supernatant assayed fluorometrically at excitation and emission wavelengths of 485 and 538 nm respectively. ROS values in retinas of control, diabetic and diabetic rats treated with minocycline (40mg/kg/day) or memantine (30mg/kg/day) are reported.
Intravitreal injection of lipopolysacharide (LPS), an endotoxin and diamide, an oxidizing agent of glutathione increased ROS levels several fold in control rat retina. ROS levels in diabetic rat retinas were 2 fold higher than control retinas 8 hours following H2DCFDA injection. After 10 days treatment of 3 month diabetic rats with minocycline (40 mg/kg/day), the ROS levels in the diabetic retina were reduced to control levels. Memantine treatment (30 mg/kg/day) for 10 days also significantly lowered ROS levels in 3 months diabetic rat retinas compared to untreated diabetic retina.
Measurements of in vivo ROS generation in rat retinas are established. As expected LPS and diamide increases the generation of ROS in control retina. ROS levels increase in diabetic retina and minocycline, a known anti-inflammatory agent and memantine (an NMDA receptor blocker) lower ROS levels in diabetic rat retina. The data demonstrate the presence of oxidative stress in the diabetic retina which can be reduced therapeutically.
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