Model 390

To evaluate nociception, thermal withdrawal latencies were assayed as previously described.³⁶ The Hargreaves test was performed to evaluate heat sensitivity thresholds, measuring latency of withdrawal to a radiant heat source (IITC Life Science, Model 390). We applied the radiant heat source to both the hind paws sequentially and measured the latency to evoke a withdrawal. Two to five replicates were acquired per hind paw per mouse, and values for both paws were averaged.

Thermal PWL to radiant heat stimuli was measured using a plantar stimulator analgesia meter (IITC model 390). Animals were placed under separate plastic boxes on a heated glass floor. Radiant heat was applied from below to the plantar surface of the ipsilateral hind paw, and an electronic timer measured the time until the animal removed its paw from the thermal stimulus. PWL was defined as the average latency in three sets of a single stimulation. We waited at least 10 minutes between each set to prevent sensitization. We used a maximum exposure of 20 seconds to prevent any potential tissue damage. %PWL was calculated by dividing the measured PWL by the pre-surgery basal PWL. Hyperalgesia was characterized as an enhanced withdrawal of the paw (i.e., shorter PWL) to this the radiant heat.

Mechanical allodynia was evaluated using an electronic von Frey anesthesiometer (IITC Life Science, model Alemo 2390–5, Woodland Hills, CA) as described previously [22 (link),36 (link),86 ,87 ]. In brief, animals were habituated to 10 cm x 10 cm acrylic holding containers placed on an elevated mesh platform 1 h prior to testing. Black dividers prevented mice from seeing other mice being tested simultaneously. Pressure was applied to the plantar surface of the hindpaw using the anesthesiometer, and the maximum force applied before the animal withdrew its paw was recorded. Each paw was stimulated twice with at least 7 min between stimulations. The two withdrawal thresholds were averaged for each data point.
Responsivity to heat stimulation was measured using a thermal plantar test apparatus (IITC Life Science, model 390) as described previously [6 ]. In brief, animals were habituated on a glass surface heated to 30°C in the same acrylic containers with black dividers as used for mechanical allodynia. A focused beam of light was applied to the hindpaw, and the latency for the animal to withdraw its paw was recorded. Each hindpaw of the animal was tested 2–3 times with at least 5 min between each measurement. A cutoff latency of 20 s per stimulation was applied to avoid tissue damage. The withdrawal latencies for all stimulations on a single paw were averaged for each data point.