Preliminary report on nitric oxide-mediated oxidative damage in adolescent varicocele.

BACKGROUND: The current study was designed to characterize the process of nitric oxide (NO) and peroxynitrite generation through the determination of nitrotyrosine concentration in the dilated veins of varicoceles in adolescents. METHODS: Ten adolescents with a median age of 13 years (range 12-17) affected by a left idiopathic varicocele (grade II and III) were studied. Whole blood samples were withdrawn from a peripheral vein at time of induction of anaesthesia, and from a dilated spermatic vein before ligation. Peripheral blood samples from five adolescents undergoing minor surgical procedures were used as controls. The nitrotyrosine concentration was evaluated by a sandwich enzyme-linked immunosorbent assay (ELISA), using a monoclonal anti-nitrotyrosine antibody and Western blot analysis. RESULTS: Plasma nitrotyrosine concentrations were significantly greater in the spermatic vein when compared with the peripheral vein (P = 0.031). Nitrotyrosine in plasma of controls did not show any significant difference in comparison with peripheral samples from varicocele patients. Western blot analysis confirmed the above data. CONCLUSIONS: In adolescents with a varicocele, there is an increase in nitrotyrosine concentration within the spermatic vein that can cause protein nitration and cytotoxicity via its reaction with various molecular targets. This could have repercussions on both sperm and testis function. We conclude that an oxidative stress status is present and should be considered as an indication for varicocele treatment in the adolescent.

Apoptosis and necrosis occurring in excitotoxic cell death in isolated chick embryo retina.

Excitotoxic studies using isolated chick embryo retina indicated that such an in vitro model provides a valid tool to characterize the effect of different agonists for subtypes of glutamate ionotropic receptors. In retinas maintained for 24 h in a Krebs medium, after a brief exposure (30 min) to glutamate agonists, we compared the effects produced by NMDA and non-NMDA-agonists, such as kainic acid (KA) or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Delayed retinal damage was assessed by measuring lactate dehydrogenase (LDH) present in the medium after exposure to the previously named agonists. Although at high concentrations, both KA and AMPA produced more relevant release than NMDA, 7-8% of total retinal LDH was released after exposure to a 50 microM concentration of non-NMDA agonists. These values were similar to those obtained after 100 microM NMDA. In this regard, retinal tissue appeared to be less sensitive to excitotoxicity based on the activation of NMDA receptor subtype. All three agents produced histopathological lesions typical for excitotoxic damage. A delayed form of excitotoxicity observed in retina segments was predominated by necrotic features. However, the activation of apoptotic machinery early during the incubation period subsequent to brief exposure to NMDA (100 microM) was also present. The activation of caspase enzymes was studied by a fluorometric protease activity assay as well as by western blot analysis. Caspase-3-like activity reached the highest value within 3 h of incubation after exposure to excitotoxin, then the level of enzyme activity declined to lower values. As confirmed by a time-related appearance of TUNEL-positive nuclei, apoptotic features appeared to be specific for retina response to NMDA. In contrast, the exposure to a 50 microM concentration of KA or AMPA induced necrotic cell damage which was evident through the incubation, leading to a delayed mechanism of excitotoxicity. These observations provide evidence that in the retinal model, with regard to agonist concentrations and subtype of glutamate receptors, the cascade of events leading to excitotoxicity may result in either apoptotic or necrotic neuronal cell damage.