Short-term serum supplementation improves glucose-oxygen deprivation in primary cortical cultures grown under serum-free conditions.
Brain ischemia can be studied in vitro by depriving primary neurons of oxygen and glucose by replacing oxygen with argon and glucose with its antimetabolite 2-deoxy-D-glucose. In this contribution, we explain how to construct a reliably functioning ischemia chamber and use it to study neuronal cell death in neuron-enriched fetal primary cortical cultures grown under serum-free conditions. We observed that these cultures exhibited a significant cell death even during exposure to oxygenated balanced salt solution used as control for oxygen-glucose deprivation. We show that addition of only 2% fetal calf serum 24 h prior, during, and after treatment almost abolished this undesirable cell loss and proportionally increased cell death induced by oxygen-glucose deprivation. Western blots and immunocytochemistry showed that these effects were mainly due to an increase in neuronal viability under control conditions accompanied by a limited glial proliferation independent of the treatment condition. Under these modified conditions, the cultures could also still be effectively preconditioned by a short-term oxygen-glucose deprivation. In summary, this modified protocol combines the advantages of serum-free neuronal culture, where potentially toxic antimitotic substances can be omitted, with a serum-mediated protection of neurons against unspecific factors and concomitant sensitization for oxygen-glucose deprivation.
Lewerenz
Jan
J
Research Group Protective Signaling, Zentrum für Molekulare Neurobiologie and Department of Neurology, University Hospital Hamburg-Eppendorf, Hamburg, Germany.
Thomsen
Susanne
S
Steinbeck
Julius A
JA
Methner
Axel
A
D000818:Animals; D001128:Argon / Q000378:metabolism; D002545:Brain Ischemia / Q000378:metabolism / Q000473:pathology; D016923:Cell Death / Q000502:physiology*; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free / Q000737:chemistry; D003847:Deoxyglucose / Q000378:metabolism; D005260:Female; D005947:Glucose / Q000378:metabolism*; D009457:Neuroglia / Q000166:cytology* / Q000378:metabolism; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D011247:Pregnancy; D051381:Rats; D017208:Rats, Wistar
D016895:Culture Media, Serum-Free; D001128:Argon; D003847:Deoxyglucose; D005947:Glucose; D010100:Oxygen