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
