title: Graded sensitiveness of the various retinal neuron populations on the glyoxal-mediated formation of advanced glycation end products and ways of protection.
abstract: 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.
authors.affiliation: Institute of Anatomy, Medical Faculty Carl Gustav Carus, Dresden University of Technology, Fetscherstrasse 74, FRG.
authors.forename: Friedemann
authors.initials: F
authors.lastname: Reber
authors.forename: Romy
authors.initials: R
authors.lastname: Geffarth
authors.forename: Michael
authors.initials: M
authors.lastname: Kasper
authors.forename: Andreas
authors.initials: A
authors.lastname: Reichenbach
authors.forename: Erwin D
authors.initials: ED
authors.lastname: Schleicher
authors.forename: Axel
authors.initials: A
authors.lastname: Siegner
authors.forename: Richard H W
authors.initials: RH
authors.lastname: Funk
mesh_terms: 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
chemical_list: 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