Alemo 2390 5

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.

Paw withdrawal thresholds in response to mechanical stimulation were measured in grams (g) using an electronic von Frey anesthesiometer (IITC model Alemo 2390–5, Woodland Hills, CA) as described in our previous work (Li et al., 2019 (link); Slivicki et al., 2020 ; Slivicki et al., 2018 (link)). Mice were allowed to habituate under individual, inverted plastic cages on a mesh table for at least 30 min prior to testing. Following the cessation of exploratory behaviors, a force was applied to the midplantar region of the hind paw with a semiflexible tip connected to the anesthesiometer. Mechanical stimulation was removed when the mouse withdrew its paw from contact with the mechanical stimulator, and the value of the applied force was recorded in grams. Mechanical paw withdrawal thresholds were obtained in duplicate for each paw and are reported as the mean of duplicate determinations from each animal, averaged across animals, for each group.