Diabetic retinopathy: AGE–induced neuronal degeneration in retinal explants.

Abstract

Abstract: : Purpose:Neuronal degeneration was reported to occur in diabetic retinopathy before the onset of detectable microvascular abnormalities. To investigate whether advanced glycation end products (AGE) generated during the disease are directly responsible for retinal degeneration, AGE were applied on retinal explants. Methods: Retinal explants were obtained from non–diabetic adult rats by maintaining the retina attached to the retinal pigment epithelium. Retinal explants were incubated 4 days in the presence or absence of 200 µg/ml of AGE resulting from the glycation of bovine serum albumin with fructosamine (AGE–BSA). Neural apoptosis was quantified using in situ DNA terminal dUTP nick end fluorescein labeling (TUNEL–FITC) and immunostaining with antibodies to anti–cleaved caspase–3. Expressions of the glial fibrillary acidic protein (GFAP), the glutamate transporter (GLAST) and the glutamine synthetase were localized by immunofluorescence. Results:TUNEL–positive cells and cleaved caspase–3–positive cells were increased by 2.2 and 2.5 in AGE–incubated retinal explants with respect to controls (p<0.05). The ganglion cell layer was the most sensitive retinal layer to AGE–induced apoptosis, with a 4–fold increase of TUNEL–positive cells in AGE–incubated retinal explants with respect to controls (p<0.05). Neuronal degeneration was confirmed by the increased and extensive GFAP labeling in Müller glial cells from AGE–treated retinal explants, by contrast to the Müller cell end feet labeling in control explants. No differences were observed for GLAST and glutamine synthetase between controls and AGE–treated retinal explants. Conclusions: These observations indicated that AGE can induce retinal neurodegeneration in the absence of blood perfusion. Neuronal degeneration was observed in the ganglion cell layer as previously reported in diabetic retinopathy and its animal models. These results validated retinal explants as a model for the study of diabetic retinopathy pathogenesis. They showed further that AGE production could be an early pathological mechanism in the induction of diabetic retinopathy.

Keywords: neuroprotection • diabetic retinopathy • retinal culture