Short-term serum supplementation improves glucose-oxygen deprivation in primary cortical cultures grown under serum-free conditions

Methods Cell Sci. 2003;25(3-4):227-36. doi: 10.1007/s11022-004-9121-9.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Argon / metabolism
  • Brain Ischemia / metabolism
  • Brain Ischemia / pathology
  • Cell Death / physiology*
  • Cells, Cultured
  • Culture Media, Serum-Free / chemistry
  • Deoxyglucose / metabolism
  • Female
  • Glucose / metabolism*
  • Neuroglia / cytology*
  • Neuroglia / metabolism
  • Neurons / cytology*
  • Neurons / metabolism
  • Oxygen / metabolism*
  • Pregnancy
  • Rats
  • Rats, Wistar

Substances

  • Culture Media, Serum-Free
  • Argon
  • Deoxyglucose
  • Glucose
  • Oxygen