Thermal nociceptive test. Hypersensitivity to thermal nociceptive stimuli was assessed using the Hargreaves test (23). Rats were placed individually in an open Plexiglas cylindrical chamber (20 cm in diameter, 35 cm high) on a 3-mm-thick transparent glass floor and allowed to habituate for at least 20 min before testing. A movable radiant heat source (Model 7370; Ugo Basile Plantar Test, Italy) was positioned under the glass floor directly beneath the plantar surface of the right hind paw, and the time (in seconds) that elapsed from switching on the radiant heat until paw withdrawal was measured automatically. A cutoff time of 20 s was established to prevent tissue damage. Each trial was repeated three times with 5-min intervals for basal threshold and two times spaced (2-min intervals) after NP treatments 3 hours after λ-carrageenan injection. The average of PWLs was calculated and expressed as means ± SEM.

Experimental design for algesimetry test. Basal responses to thermal stimuli were obtained on the day before the λ-carrageenan injection. On the basis of previous studies, acute pharmacological treatments were performed 3 hours after carrageenan injection, which corresponded to the peak inflammatory response. The efficacy of these treatments on thermal hyperalgesia was evaluated by measurement of PWLs using the Hargreaves test at regular time intervals after drug or vehicle administration, first at 10 min and then each at 30 min for a period of 4 hours (Fig. 4).

Acute pharmacological treatments. Morphine and LENK were dissolved in 5% dextrose solution, whereas Nal and Nal-M were dissolved in physiological saline (NaCl, 0.9%). All these drugs and NP suspensions were prepared just before administration. All acute treatments were performed 3 hours after λ-carrageenan intraplantar injection according to Fig. 4. Nal and Nal-M were injected subcutaneously, whereas the intravenous route in the tail vein was used for LENK-SQ NPs, LENK, and their controls. The antagonist (Nal or Nal-M) was administered 15 min before the agonist (morphine or tested NPs). A single dose of morphine (1 mg/kg), Nal (0.5 mg/kg), and Nal-M (0.5 mg/kg) was administered on the basis of literature data (40). A single intravenous dose of LENK-SQ NPs (20 mg/kg, equivalent to 11.48 mg/kg of LENK) or control unconjugated SQ NPs (8.28 mg/kg) was used based on the maximal volume of LENK-SQ NPs that could be injected.

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