4.4. Capsaicin-Induced Spontaneous Pain Test and Drug Injection

To investigate the effect of the TRPV1 antagonist capsazepine on pain induced by TRPV1 channel activation, several doses of capsazepine were administered in a capsaicin-induced spontaneous pain model. The capsaicin (Sigma-Aldrich, St. Louis, MO, USA) test was performed as described previously [42]. Briefly, mice received intraplantar injections of 20 μL of capsaicin solution (1.6 μg/paw) into the right hind paw. Immediately after capsaicin injection, animals were placed into clear observation chambers (15 × 15 × 15 cm), and nociceptive responses were evaluated as the time spent licking the injected paw for 15 min. The vehicle group consisted of animals injected with intraplantar physiological saline solution.

Capsazepine (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in physiological saline solution and intrathecally administered at three doses (0.1, 1, and 10 μg per mouse) 5 min prior to capsaicin injection. The control group consisted of animals that received intrathecal physiological saline solution. The experimenter was blinded to treatment conditions.

Intrathecal (i.t.) injections of capsazepine were performed as reported previously [16], using a 10 μL Hamilton syringe with a 30-gauge needle. Briefly, the mouse was held tightly between the thumb and middle finger at the level of both iliac crests, and the fifth lumbar spinous process was palpated with the index finger. The needle was inserted through the vertebral column into the lumbar 5 to lumbar 6 intervertebral space, and a tail flick response was considered indicative of a successful i.t. injection. Capsazepine was slowly injected over a 10 s period. The needle was carefully removed from the spinal cord. Following injection, the animals were immediately returned to the observation chamber.