Alpha-1-antitrypsin ameliorates features of oxidative stress and structural retinal damage in diabetic mice

POTILINSKI, MC; SALICA, JP; ORTIZ, G; GALLO, JE

Baltimore

Congreso; 2017 ARVO Annual Meeting; 2017

ASSOCIATION FOR RESEARCH IN VISION AND OPHTHALMOLOGY

Purpose : Oxidative stress has been implicated in the etiology of several diseases and in aging, including the pathogenesis of diabetic retinopathy (DR). Indeed nitric oxide (NO) was found higher in patients with proliferative DR. It is known that Muller cells (MC) are responsible for the secretion of several molecules that are involved in the regulation of retinal homeostasis and play key role in the interaction between endothelium and neural retina. The aim of our work was to evaluate the levels of 2,7-dichlorofluorescein (DCFC) on supernatant of MC and human microvascular endothelial cells (Hmec-1) exposed to hyperglycemia. Besides, we evaluated the retinal thickness and the loss of ganglion cells (GC) in c57BL6J mice with 8 weeks of diabetes treated with hAAT.Methods : MC were obtained from retinas of c57BL6J mice. Isolated cells were used in immunofluorescence for CRALBP, GFAP and Vimentin markers. Cells were growth in standard conditions and were exposed to different concentration of hAAT for 16hs. Intracellular oxidants were detected by flow cytometry after incubating with 5 μm 2,7-dichlorodihydrofluorescein (DCFH) 45 min.Mice c57BL6J of 8 weeks old were injected with 2 doses of 100mg/kg of streptozotocin spaced 48 hours. Blood glucose test was performed and levels of 200 mg/dl or higher were considered diabetic (DBT). Animals were injected weekly, with a single dose of 60mg/kg of hAAT. Cells of the ganglion cell layer (GCL) were quantified by counting cells in the middle part of the retina and thickness measurements of the entire retina were taken.Results : The oxidation of DCFH originates DCFC, a fluorescent compound in presence of H2O2 and other ROS, such as HO. and ROO. DCFH fluorescence contributed by ROS generation in MC exposed to high glucose is notably decreased by hAAT. GC count was significantly lower in untreated DBT animals while DBT animals treated with hAAT had results close to control values. In turn the retinal thickness was also decreased in DBT animals treated but without treatment. Furthermore, a decrease in mRNA levels of iNOS was found in DBT mice treated with hAAT as well as an increased in mRNA levels of Arg1.Conclusions : A better understanding on how the oxidative stress induced by chronic hyperglycemia occurs in DR is necessary. The use of hAAT as an anti-oxidative stress molecule in early DR could be a promising approach for this potentially blinding disease.