ARA290 in a Rat Model of In fl ammatory Pain - Tissue-Protective Cytokines: Methods and Protocols - page 213

30. Allow animal to recover either on a heated blanket or in a warm
air recovery chamber.
31. Instruments should be sterilized (using ethanol and the hot
bead sterilizer) between animals.

3.3 Assessment
of Pain: Heat
Hyperalgesia

1. Habituate rats for 1 h in their home cage in the testing room
on day one, followed by 1 h in the individual Perspex animal
enclosures of the Hargreaves rig for at least three consecutive
days (see Notes 9 and 10).
2. Calibrate the intensity of the radiant heat source according to
manufacturer's instructions using the Radiometer.
3. Set the radiant heat source intensity such that the mean with-
drawal latency is 10-15 s for the size and strain of rats being
tested.
4. Testing to establish baseline withdrawal latencies should be
performed on three separate, preferably consecutive, days prior
to surgery (see Note 11).
5. On each test day, allow rats to acclimate in rig for 15 min prior
to testing.
6. If rat urinates, remove animal and dry glass floor as well as the
paws of the rat. Do not test for at least 2 min after drying (see
Note 12).
7. Aim the moveable radiant heat source at the glabrous skin on
the mid-plantar surface of the hind limb (see Note 13). Do
not start the test when the rat is grooming or sleeping
(see Note 14).
8. When the radiant heat source is switched on, it will activate a
timer that will automatically stop when the animal withdraws
their foot. It is important to set the cutoff of the radiant light
source to 20 s to avoid damage to skin.
9. Record the withdrawal latency or 20 s if the cut off for the
radiant light source is reached.
10. If the rat moves away from heat source rather than rapidly
withdrawing the paw, wait for 10 min and then retest.
11. Repeat the test on the first hind paw of each rat before testing
the other paw (or allow at least 10 min between sides).

3.4 Assessment
of Pain: Mechanical
Allodynia

1. Habituate rats for 1 h in their cage in the testing room on day
one, followed by 1 h in the individual Perspex animal enclo-
sures for at least three consecutive days (see Notes 9 and 10).
2. Baseline testing should then be performed on three separate,
preferably consecutive, days prior to surgery (see Note 11).
3. On each test day, allow rats to acclimate in the rig for 15 min
prior to testing.