Assessment of Hyperalgesia

Mice were acclimatized to each test protocol in a quiet room at constant temperature, and tested for mechanical (von Frey) and spontaneous musculoskeletal/deep (grip force) as previously described (7, 23). Behavioral tests were performed consecutively at a 5-min interval between tests, in a double-blind manner.

Mechanical hyperalgesia was measured by applying a 1.0 g (4.08 mN) von Frey (Semmes-Weinstein) monofilament (Stoelting Co, Wood Dale, IL) to the mid-plantar surface of each hindpaw for a total of 10 trials per hindpaw with a 5-s inter-stimulus interval, and paw withdrawal frequency (PWF) was recorded. Deep tissue/musculoskeletal hyperalgesia was assessed by placing mice on a wire-mesh gauge by their forepaws, and the peak grip force exerted in grams was recorded by a computerized grip-force meter (SA Maier Co, Milwaukee, WI).