A simple in vitro model of ischemia based on hippocampal slice cultures and propidium iodide fluorescence

Research output: Contribution to journalArticle

Abstract

This protocol describes a model of cerebral ischemia based on organotypic hippocampal slice cultures and quantitative assessment of cell death by use of propidium iodide and image analysis. The cultures were made from rat hippocampal slices that were obtained at postnatal day 4-7 and allowed to develop for > 14 days in vitro. For induction of 'in vitro ischemia', the cultures were washed in glucose free buffer and the culture chamber flooded with a nitrogen/carbon dioxide mixture until the oxygen concentration was < 1.0%. The cultures were exposed to this atmosphere for 30-35 min, washed in serum-free medium, and returned to ordinary growth medium. After 24 h, dead cells were quantified by use of propidium iodide. The cell death resulting from the oxygen/glucose deprivation was largely confined to the CA1 region and was blocked by NMDA-receptor antagonists but not by antagonists to AMPA-receptors or metabotropic glutamate receptors. The type of cell death was judged to be necrotic, based on ultrastructural observations. The oxygen/glucose deprived cultures exhibited increased phosphorylation of the MAP kinase cascade. This activation of the MAP kinase cascade was blocked by NMDA-receptor antagonists. The in vitro model described in the present report is simple to use and reproduces many features of in vivo ischemia, including the preferential vulnerability of CA1 cells. The model should be suited to analyses of the mechanisms underlying the regionally selective cell death in the hippocampus and ischemic cell death in general.

Details

Authors
Organisations
External organisations
  • University of Oslo
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Neurosciences

Keywords

  • CA1, CNQX, Electron microscopy, ERK1/2, Fluorescence microscopy, Image analysis, MAP kinase, MEK1/2, MK-801, Necrosis, p44/42 MAP kinase, Propidium iodide
Original languageEnglish
Pages (from-to)173-184
Number of pages12
JournalBrain Research Protocols
Volume4
Issue number2
Publication statusPublished - 1999 Jul 1
Publication categoryResearch
Peer-reviewedYes