4.7. Complete Freund’s Adjuvant (CFA)-Induced Chronic Inflammatory Pain

Unilateral CFA-induced inflammation of the hind limb with development of hyperalgesia was used as a model of chronic inflammatory pain in mice [31]. Mice were injected with 20 µL emulsified CFA (1 mg/mL) into the plantar surface of the right hindpaw under brief isoflurane anesthesia. Nociceptive testing was performed 72 h after inoculation with CFA, where mice were accustomed to each testing condition for 2–3 days prior to the inoculation with CFA, using the Hargreaves and von Frey tests. Hindpaws withdrawal latencies to thermal and mechanical stimulation were measured before inoculation with CFA into the mouse right hindpaw (basal latencies, BL), 72 h post-inoculation (pre-treatment values, defined as 0 h), and at 30 min and every hour up to 8 h and 24 h after s.c. administration of saline (control), KGNOP1 (0.49 and 1.22 µmol/kg), or morphine (15.5 µmol/kg).

Thermal sensitivity was assessed using the Hargreaves test [67] with an analgesiometer (Ugo Basile S.R.L., Varese, Italy). Mice were placed in individual Plexiglas boxes positioned on a glass surface (Ugo Basile S.R.L., Varese, Italy). Measurements of paw withdrawal latency to heat stimuli started after a period of habituation of 30 min. A movable infrared generator (30% intensity) located under the glass floor was focused onto the plantar surface of the hindpaw and switched on to heat. Onset of the radiant stimulus triggered a timer which was stopped by subsequent paw movement. Three measurements were carried out and the average value was calculated. To prevent tissue damage, a cut-off time of 20 s was imposed. The latency to withdraw the hindpaw from the stimulus was expressed as paw withdrawal latencies in seconds.

Mechanical sensitivity was assessed by measuring the paw withdrawal threshold in response to probing of the plantar surface of the hindpaw with von Frey filaments on the basis of the up-down method [68]. A series of calibrated von Frey monofilaments with bending forces between 0.04 and 4 g (Ugo Basile S.R.L., Varese, Italy) were used. Mice were placed in individual Plexiglas boxes placed on an elevated platform with a mesh floor. The von Frey filament was inserted perpendicularly from below the platform, up through the wire mesh, and applied to the plantar surface of the hindpaw for 3 s until a behavioral reaction was observed. The behavioral assessment was initiated with a force of 0.6 g and the response as paw withdrawal, shaking, lifting, and licking was recorded. Data were calculated using the Up-Down Reader Software and plotted as the 50% threshold (g) [69].

Antinociceptive effects were determined as percentages (% reversal of thermal/mechanical sensitivity) according to the following formula: 100 × [(T₁ − T₀)/(T_BL − T₀)], where T₀ is the nociceptive value at 72 h post-inoculation with CFA (defined as 0 h), T₁ is the value obtained following drug administration, and T_BL is the basal value before CFA inoculation.