Alkal2

Thermal hyperalgesia was determined by measuring the latency to withdrawal of the right hind paws in response to a focused beam of radiant heat (IR = 30) of a Plantar Test apparatus (UgoBasile). Animals were placed individually in a small, enclosed testing arena (20 cm × 18.5 cm × 13 cm, length × width × height) on top of a wire mesh floor. Mice were allowed to acclimate for a period of at least 90 minutes. The device was positioned beneath the animal, so that the radiant heat was directly under the plantar surface of the ipsilateral hind paw. Three trials for each mouse were performed. The apparatus was set at a cut-off time of 30 seconds to avoid tissue damage. Thermal hyperalgesia was evaluated immediately prior to the treatments (time 0) and 15, 45, 90, and 180 minutes after i.t. treatment with crizotinib (Sigma-Aldrich) or after 30, 60, 120, and 180 minutes of lorlatinib (Sigma-Aldrich) administration. For ALKAL-2–induced hyperalgesia, mice were treated with ALKAL2 (MyBioSource LLC, MBS1425538) (0.01; 0.1; 1 μM, i.t.) or vehicle after lorlatinib administration (Sigma-Aldrich) (1 mg/kg, i.g., 30 minutes prior to ALKAL2 administration), and thermal hyperalgesia was evaluated 1, 3, 6, 24, 48, and 72 hours after ALKAL2 treatment.