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 (N(epsilon)-[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 micro M 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 (NaVO(3)), 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 micro M, 9 h) increased apoptotic events in all layers. At low glyoxal concentrations, we found a graded sensitiveness of the different layers: at 25 micro M 39.4% in GCL, 28.2% in INL, 11.9% in ONL. After 800 micro M glyoxal, approximately 50% of the cells in all layers of the retina were apoptotic. In the ONL, this ratio was reduced by NaVO(3) (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, NaVO(3) and AG reduced apoptosis. A-LA had no significant protective effect.


CONCLUSIONS
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.
Reber
Friedemann
F
Institute of Anatomy, Medical Faculty Carl Gustav Carus, Dresden University of Technology, Fetscherstrasse 74, FRG.
Geffarth
Romy
R
Kasper
Michael
M
Reichenbach
Andreas
A
Schleicher
Erwin D
ED
Siegner
Axel
A
Funk
Richard H W
RH
D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005260:Female; D017127:Glycation End Products, Advanced / Q000378:metabolism*; D006037:Glyoxal / Q000633:toxicity*; D006146:Guanidines / Q000494:pharmacology; D007124:Immunoenzyme Techniques; D020287:In Situ Nick-End Labeling; D008239:Lysine / Q000031:analogs & derivatives* / Q000378:metabolism; D008297:Male; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D009536:Niacinamide / Q000494:pharmacology; D009924:Organ Culture Techniques; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051381:Rats; D017207:Rats, Sprague-Dawley; D012160:Retina / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008063:Thioctic Acid / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology; D051028:bcl-2-Associated X Protein
C494828:Bax protein, rat; D017127:Glycation End Products, Advanced; D006146:Guanidines; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D009536:Niacinamide; D014638:Vanadates; D006037:Glyoxal; C048496:N(6)-carboxymethyllysine; D008063:Thioctic Acid; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D008239:Lysine; C004479:pimagedine; D000111:Acetylcysteine