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

Laake, Jon Henrik; Haug, Finn Mogens; Wieloch, Tadeusz LU and Ottersen, Ole Petter (1999) In Brain Research Protocols 4(2). p.173-184
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... (More)

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.

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author
organization
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Contribution to journal
publication status
published
subject
keywords
CA1, CNQX, Electron microscopy, ERK1/2, Fluorescence microscopy, Image analysis, MAP kinase, MEK1/2, MK-801, Necrosis, p44/42 MAP kinase, Propidium iodide
in
Brain Research Protocols
volume
4
issue
2
pages
12 pages
publisher
Elsevier
external identifiers
  • scopus:0032861177
ISSN
1385-299X
DOI
10.1016/S1385-299X(99)00021-5
language
English
LU publication?
yes
id
8101893b-a302-42d8-9f42-367f784e5ec6
date added to LUP
2019-06-13 16:56:58
date last changed
2019-09-26 04:37:43
@article{8101893b-a302-42d8-9f42-367f784e5ec6,
  abstract     = {<p>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 &gt; 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 &lt; 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.</p>},
  author       = {Laake, Jon Henrik and Haug, Finn Mogens and Wieloch, Tadeusz and Ottersen, Ole Petter},
  issn         = {1385-299X},
  keyword      = {CA1,CNQX,Electron microscopy,ERK1/2,Fluorescence microscopy,Image analysis,MAP kinase,MEK1/2,MK-801,Necrosis,p44/42 MAP kinase,Propidium iodide},
  language     = {eng},
  month        = {07},
  number       = {2},
  pages        = {173--184},
  publisher    = {Elsevier},
  series       = {Brain Research Protocols},
  title        = {A simple in vitro model of ischemia based on hippocampal slice cultures and propidium iodide fluorescence},
  url          = {http://dx.doi.org/10.1016/S1385-299X(99)00021-5},
  volume       = {4},
  year         = {1999},
}