Graded sensitiveness of the various retinal neuron populations on the glyoxal-mediated formation of advanced glycation end products and ways of protection

Background: The accumulation of advanced glycation end products (AGEs) in retinal cells is known to be associated with the risk of diabetic retinopathy. To develop a model of AGE-related metabolic stress in retinal organ cultures, we investigated the accumulation of a typical glycoxidation product (Nepsilon-[carboxymethyl] lysine [CML]) and its possible pro-apoptotic effects on different retinal cell populations. Methods: Retinal organ cultures (rat) were kept for 9 h in the Ames medium containing 0 (control), 5, 25, 50, 150, 300 and 800 muM glyoxal. The expression of bax, active caspase-3, and the accumulation of CML were studied by using immunohistochemistry after the paraffin embedding of retinal explants. Apoptosis was studied using the terminal deoxynucleotidyl transferase-mediated dUTP digoxigenin nick end labeling (TUNEL) test and electron microscopy. Alpha lipoic acid (alpha-LA), sodium metavanadate (NaVO3), N-acetylcysteine (NAC), aminoguanidine (AG), and nicotinamide (NA) were used to influence glyoxal effects in organ cultures. Results: In cultured normal non-diabetic retinae, small amounts of CML and the apoptosis-promoting factors bax and active caspase-3 were present. CML, bax and active caspase-3 increased after incubation with glyoxal. Incubation with glyoxal (<300 mu M, 9 h) increased apoptotic events in all layers. At low glyoxal concentrations, we found a graded sensitiveness of the different layers: at 25 mu M 39.4% in GCL, 28.2% in INL, 11.9% in ONL. After 800 mu M glyoxal, approximately 50% of the cells in all layers of the retina were apoptotic. In the ONL, this ratio was reduced by NaVO3 (17%), by AG (27%), by NA (24.8%), by NAC (25.2%), and by alpha-LA (33.5%). In the INL, AG (25.9%) produced the best result. In the GCL, NAC, NaVO3 and AG reduced apoptosis. A-LA had no significant protective effect. Conclusion: The glyoxal-induced rapid formation of CML shows the ability of our retina model to simulate AGE-related effects in vitro. The dose-dependent expression of apoptosis-promotor molecules indicates that the apoptosis-inducing machinery starts in most retinal cells within 9 h. The neurotoxicity of glyoxal-induced AGE formation was shown by the significantly increased rate of cell death in the retina. The significant decrease of apoptotic events (P<0.01) indicates that antioxidants and AGE formation blocker can exert a differentiated cytoprotection for each of the retinal cell layers.