4.3. Formalin-Induced Pain Test

The formalin test was carried out in an observation chamber made of transparent Plexiglas material. The mice received the drug treatment (time—0 min) 30 min prior to the formaldehyde injection. In response to the noxious stimuli of formaldehyde injection, the mice demonstrated a nocifensive behavior of licking/biting of the injected paw. The formaldehyde-induced paw-licking behavior occurs in two phases [43]. The early-phase behavioral response to pain is mainly due to the activation of C-fibers by formaldehyde injection acting as a stimulus [44]. The late-phase behavioral response to pain is due to the inflammation induced by formaldehyde and to changes in the dorsal horn of the spinal cord [44]. The mice received the formaldehyde injection 30 min after drug treatment (time—30 min). Then, 1 min later, the mice were introduced in the observation chamber to quantify the early-phase response (time—31 to 36 min). Both early-phase response and late-phase response were measured for a period of 5 min. At the end of the early phase observation, the mice were returned to their home cages. The measurement of the late-phase response (time—56 to 61 min) began 25 min after the start of the early observation, with the introduction of the mice in the observation chamber for a second time. During each observation phase, the number of times a mouse licked or bit the injected paw was counted as a measure of pain.