Nitrosative Stress in the Rat Retina at the Onset of Streptozotocin-Induced Diabetes

Cell Physiol Biochem. 2017;42(6):2353-2363. doi: 10.1159/000480007. Epub 2017 Aug 18.


Background/aims: Nitric oxide is a multifunctional molecule that can modify proteins via nitrosylation; it can also initiate signaling cascades through the activation of soluble guanylate cyclase. Diabetic retinopathy is the leading cause of blindness, but its pathogenesis is unknown. Multiple mechanisms including oxidative-nitrosative stress have been implicated. Our main goal was to find significant changes in nitric oxide (NO) levels and determine their association with nitrosative stress in the rat retina at the onset of diabetes.

Methods: Diabetes was induced by a single intraperitoneal administration of streptozotocin. The possible nitric oxide effects on the rat retina were evaluated by the presence of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), a specific marker for NO-producing neurons, detected by histochemistry performed on whole retinas and retina sections. Immunohistochemistry was also performed on retina sections for iNOS, 3-nitrotyrosine (3-NT) and glial fibrillary acidic protein (GFAP). Retinal nitric oxide levels were assessed by measuring total nitrate/nitrite concentrations. Retinal cGMP levels were determined by radioimmunoassay. Western blots for nitrotyrosine (3-NT) and oxidized proteins were performed.

Results: In the present study, we found increased activity of NADPH-diaphorase and iNOS immunoreactivity in the rat retina at the onset of diabetes; this increase correlated with a remarkable increase in NO levels as early as 7 days after the onset of diabetes. However, cGMP levels were not modified by diabetes, suggesting that NO did not activate its signaling cascade. Even so, Western blots revealed a progressive increase in nitrated proteins at 7 days after diabetes induction. Likewise, positive nitrotyrosine immunolabeling was observed in the photoreceptor layer, ganglion cell layer, inner nuclear layer and some Müller cell processes in the retinas of diabetic rats. In addition, levels of oxidized proteins were increased in the retina early after diabetes induction; these levels were reduced by the administration of L-NAME. In addition, stress in Müller cells was determined by immunoreactivity to the glial fibrillary acidic protein.

Conclusions: Our findings indicated the occurrence of nitrosative stress at the onset of diabetes in the rat retina and emphasized the role of NO in retinal function and the pathogenesis of retinopathy.

Keywords: CGMP; Diabetic retina; GFAP; INOS; Müller cells; NADPH-d; Nitric oxide; Nitrotyrosine.

MeSH terms

  • Animals
  • Cyclic GMP / metabolism
  • Diabetes Mellitus, Experimental / chemically induced
  • Diabetes Mellitus, Experimental / metabolism
  • Diabetes Mellitus, Experimental / pathology*
  • Glial Fibrillary Acidic Protein / metabolism
  • Immunohistochemistry
  • Microscopy, Fluorescence
  • NADPH Dehydrogenase / metabolism
  • NG-Nitroarginine Methyl Ester / pharmacology
  • Nitric Oxide / metabolism*
  • Nitric Oxide Synthase Type II / metabolism
  • Oxidative Stress* / drug effects
  • Protein Carbonylation / drug effects
  • Rats
  • Rats, Long-Evans
  • Retina / drug effects
  • Retina / metabolism*
  • Retina / pathology
  • Streptozocin / toxicity
  • Tyrosine / analogs & derivatives
  • Tyrosine / metabolism


  • Glial Fibrillary Acidic Protein
  • Nitric Oxide
  • 3-nitrotyrosine
  • Tyrosine
  • Streptozocin
  • Nitric Oxide Synthase Type II
  • NADPH Dehydrogenase
  • Cyclic GMP
  • NG-Nitroarginine Methyl Ester