Nutritional antioxidants as antidegenerative agents

In this study, primary cultures of cerebellar granule neurons were prepared from eight-day-old Wistar rats, and maintained in an appropriate medium co ntaining a high (25 mM) concentration of KCl. All experiments were performe d with fully differentiated neurons (eight days). To induce apoptosis, cult ure medium was replaced with a serum-free medium (containing 5 mM KCl) eigh t days after plating. In another series of experiments, apoptosis was induc ed by application of glutamate (50 mu M) to the cell cultures. Apoptosis wa s measured by flow cytometry, the TUNEL (terminal deoxynucleotidyl transfer ase-mediated dUTP-fluorescein nick end-labeling) method, and by the classic al method of DNA fragmentation. Since there is evidence that an increased f ormation of reactive oxygen species (ROS) is involved in the apoptosis indu ced by both low K+ concentrations and glutamate, a series of natural antiox idants and a red wine lyophilized extract (which is rich in antioxidant com pounds) were tested in our experimental model. It was found that ascorbic a cid (30 mu M) and a red wine lyophilized extract (5 mu g/ml) were capable o f blocking the apoptotic process. Addition of the following natural antioxi dants did not have any protective effect on apoptosis induced by low K+ con centrations: trans- and cis-resveratrol (5-200 mu M), alpha-tocopherol (100 -200 mu M), reduced glutathione (100-400 mu M), 3-hydroxytirosol (25-100 mu M), epicatechin (25-100 mu M), or quercetin (25-50 mu M). It is concluded that only a limited number of natural antioxidants are provided with antiap optotic activity in cultured cerebellar granule neurons. This effect is pro bably exerted by reducing ROS formation, and by blocking caspase-3 activity . (C) 2000 ISDN. Published by Elsevier Science Ltd. All rights reserved.