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Slice cultures as a model for cerebral ischemia

Jon Laake jon.laake at basalmed.uio.no
Sun Nov 13 10:58:27 EST 1994


Using the slice culture technique described by Gähwiler (e.g. Trends 
Neurosci. 11, 484-489, 1988) I have done some in vitro "ischemia" 
experiments using oxygen and glucose deprivation. The problem with 
this approach is that it is difficult to obtain exactly the same 
conditions from one experiment to another. Here is what I do: The 
medium of the cultures is changed to a Ringer solution without glucose
and with or without magnesium and/or calcium. This Ringer solution has 
been prebubbled with 95% N2/5% CO2 and kept at 37 degrees. Because the 
change of medium takes place in a normal atmosphere the Ringer will 
probably contain quite a lot of oxygen and have lost a lot of CO2 during 
this procedure. After the change of medium I put the tissue culture tubes
in the incubator and switch to 95% N2/5% CO2. According to the oxygen 
analyzer O2 is reduced to less than 1% in 5 min. Temperature is somewhat 
lowered due to the rapid infusion of compressed gas and typically falls to 
35.5 degrees. 30 min. of this treatment is enough to kill all neurons in a 
hippocampal culture. 20 min. gives highly variable results, from total 
damage via "selective" CA1 damage to nothing at all. Damage is assessed 
using propidium iodide and thionin staining. 

If anybody has experience with this technique or ideas about how to 
standardize this approach, please contact me or post response to the 
newsgroup.

Jon Henrik Laake, MD
Institute of Basic Medical Sciences,
University of Oslo
Norway



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