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of O
2
in the range of the experimental design (e.g., 21% O
2
, 5%
CO
2
, balance N
2
) to calibrate the span.
5. Calibrate the ventilation volume by injection of 1 ml of air in
about 2-3 s into the animal chamber.
3.2 Effect of Epo on
Basal Ventilatory State
1. Weight the mouse (see Note 20).
2. Put the mouse into the plethysmography chamber, close the
chamber, and let the animal acclimatized until it reach a calm and
quiet state reflected by a regular respiratory signal (typically
between 30 and 60 min).
3. Open the plethysmography chamber and gently manipulate
the animal to take body temperature with rectal probe (this is
the initial body temperature) (see Note 16).
4. Put the mouse back to the plethysmography chamber and let it
acclimatized again.
5. Once the mouse is calm, start to register the acquired data
during 10 min with the animal exposed to room air (or to a
cylinder containing 21% O
2
, 5% CO
2
, balance N
2
for more
accuracy). These data provide the baseline state to which the
experimental state (severe hypoxia in this example) will be
compared with.
1. In continuity with the basal state registering, turn open the gas
cylinder containing 15% O
2
, 5% CO
2
, and balance N
2
.
2. At this moment, a hand-made tag could be added on the
recording stream to help to localize the O
2
regimen modi fication
at the time of analysis.
3. Thereafter, a gradual decrease in O
2
exposure is generated by
sequentially changing at each 5 min for a cylinder containing
10 and 6% O
2
, respectively (with 5% CO
2
and balance N
2
in
each case) (see Note 22). Tag each modification of O
2
regimen
on the recording stream.
4. Continue to register the acquired data during 10 min with the
animal exposed to hypoxia at 6% O
2
. When compared with
basal state, hypoxia should increase both frequency and ampli-
tude of respiratory signal.
5. When hypoxia exposure is ended, turn off the gas cylinder
generating the hypoxic air. This moment could be tagged on
the stream as the end of hypoxia.
6. Continue to register the signal during 10 min with the animal
exposed to room air (or to a cylinder containing 21% O
2
, 5%
CO
2
, and balance N
2
). These data characterized which is called
the recovery period.
3.3 Effect of Epo on
Ventilatory Response
to Severe Hypoxia
( See Note 21 )
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