Hargreaves apparatus

To assess nociceptive heat sensitivity, we used a conventional hot-plate test or a radiant heat paw withdrawal test (Hargreaves method). The temperature of the hot plate was adjusted to 42 or 52 ± 0.1°C. The latency to the first hind paw licking or withdrawal was taken as an index of nociceptive threshold. The cutoff time was set at 120 seconds at 42°C and 20 seconds at 52°C, to avoid damage to the paw. For the Hargreaves method, the mice were placed in clear Plexiglas boxes (22 × 19 × 14 cm) on a glass surface and allowed to habituate for 15 minutes before testing. A thermal stimulus was pointed at the plantar surface of each hind paw through the glass using a Hargreaves apparatus (Stoelting, Wood Dale, IL) until the animal lifted its paw away from the heat source. The latency to their paw withdrawal was automatically recorded with the apparatus to the nearest 0.1 seconds, and a cutoff latency of 20 seconds was used to avoid tissue damage. Three to 5 trials were conducted with at least 5 minutes between each trial. Mean values per paw were calculated and used for statistical analysis.

The surgery was done as described previously (Dimitrov et al., 2014 (link)). Briefly, the mice were anesthetized and the shaved skin of the upper left thigh was treated with antiseptic. A pair of small forceps was used to expose the main branch of the sciatic nerve and a 4 mm long piece of sterile polyethylene tubing or “cuff” (PE 90, i.d. 86 mm and o.d. 1.27 mm, Becton Dickinson Intramedic, Franklin Lakes, NJ), split lengthwise was placed onto the nerve. The surgical incision was closed with wound clips and analgesia with NSAID was given for the following three days. The sham mice underwent the same general anesthesia as the experimental mice. The upper thigh was shaved and a skin incision was made, but the sciatic nerve was only exposed and not exteriorized. The sham mice received the same pain treatment for three days after the surgery. The development of neuropathic pain was assessed by testing the mice for thermal hypersensitivity with the Hargreaves apparatus (Stoelting, Wood Dale, Il) and mechanical allodynia with von Frey filaments (North Coast Medical, Morgan Hill, CA) every tenth day from the fifth day to the end of the experiments, as described previously (Dimitrov et al., 2014 (link)).