Antioxidants are required during the early critical period, but not later, for neuronal survival.
Methods for growing primary neuronal cultures rely on the inclusion of antioxidants in the culture medium, but no studies have determined precisely if or when antioxidants are required for neuronal survival, despite the significance this information would have for understanding neurodevelopment and studying oxidative stress. We show that cortical neurons grown in Neurobasal media with B27 supplement required antioxidants for only the first 24 hr post-explantation, after which the antioxidants could be removed permanently without noticeable loss of neuronal survival over the normal lifespan. Cortical cultures never exposed to antioxidants did not survive. These findings represent a novel method for substantially antioxidant-free neuronal culture, whereby antioxidants can be removed permanently from the cultures after only 1 day. This method may prove critical for studies of oxidative stress, because B27 antioxidants significantly diminished pro-oxidative effects of the excitatory neurotransmitter glutamate and hydrogen peroxide on cortical cultures, even if antioxidants were removed before the oxidizing treatment. Together, these findings suggest a brief window of high vulnerability to reactive oxygen species, and have important implications for studies of oxidative stress and developmental neuroscience.
Perry
Seth W
SW
Center for Aging and Developmental Biology, University of Rochester Medical Center, Rochester, New York 14642, USA. Seth_Perry@urmc.rochester.edu
Norman
John P
JP
Litzburg
Angela
A
Gelbard
Harris A
HA
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D002452:Cell Count / Q000379:methods; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology*; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004622:Embryo, Mammalian; D005455:Fluorescent Antibody Technique / Q000379:methods; D018698:Glutamic Acid / Q000633:toxicity; D006861:Hydrogen Peroxide / Q000494:pharmacology; D009474:Neurons / Q000166:cytology / Q000187:drug effects*; D018384:Oxidative Stress / Q000187:drug effects; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D013778:Tetrazolium Salts; D013844:Thiazoles; D013997:Time Factors; D014404:Tubulin / Q000378:metabolism
D000975:Antioxidants; D016895:Culture Media, Serum-Free; D017382:Reactive Oxygen Species; D013778:Tetrazolium Salts; D013844:Thiazoles; D014404:Tubulin; D018698:Glutamic Acid; D006861:Hydrogen Peroxide; C022616:thiazolyl blue