SUPPLEMENTARY MATERIALS AND METHODS
Sensory Nerve Functional assays. von Frey analyses were performed using an
electronic von Frey apparatus (Model 2390; IITC Inc., Woodland Hills, CA) and standard
procedures. Animals were placed in elevated, clear-plastic, wire mesh-bottomed cages
and their paws accessed from the underside of the mesh. All animals were tested after a
20 minute acclimation period. The von Frey filament attached to a sensor was applied
perpendicularly to the plantar surface of the hind paw with increasing force for up to 6
seconds. The force provoking an abrupt paw withdrawal was recorded. Each animal
was tested three times with at least a 10 min interval between tests. Unlike STZ rats, it
was very difficult to detect hypoalgesia in mice; the von Frey test was therefore only
used to detect hyperalgesia.
A cold water test was based on a previous report48 with modification. The system
contained two water baths, thermocirculator (Model GD120; Harvard Apparatus,
Holliston, MA), and a cage with wire bottom that allowed the animal’s feet to touch the
water when the cage was placed in the water bath. First, a single animal was placed in
the cage for 15 min to allow it to acclimate; the cage was then transferred to a water
bath containing water at room temperature and the animal acclimated for 5 to 15 min.
Finally, the cage was moved to another water bath containing cold water (10oC).
Nociceptive responses (maintaining a rearing position for > 10 s) were observed and
their latency recorded. The cutoff time (60 s) was recorded if the animal did not respond
prior to the cutoff time. Six weeks after STZ treatment, the treated animals began to
show rearing activity even in room temperature water. Therefore this test was used only
within 6 weeks post STZ to detect hyperalgesia and initial hypoalgesia.