A total of 28 Male Sprague-Dawley rats (250–300 g were used) were housed (2/cage) under standard conditions (12:12-h light:dark cycle with ad libitum access to food and water). The study was conducted in compliance with the Animal Welfare Act, the implementing Animal Welfare Regulations and the principles of the Guide for the Care and Use of Laboratory Animals.

On the day before administering ʎ-carrageenan (Sigma-Aldrich, St. Louis, MO, USA), it was dissolved in saline to create a 3.5% solution and stored at 4°C. AG490 (Sigma-Aldrich) was dissolved in 3.5% dimethylsulfoxide (DMSO) prior to the start of an experiment on each study. Naloxone methiodide (Sigma-Aldrich) was dissolved in water, and DMSO was diluted with water.

Unilateral hind paw inflammation in the rat was induced by intraplantar (i.pl.) injection of 100 µl 3.5% ʎ-carrageenan in the left hind paws (9). Inflammation was evident at 48 h post-injection, as indicated by redness and swelling of the affected paw.

The experiments were performed in rats 48 h after ʎ-carrageenan injection. A total of 4 groups (n=6) of rats were randomly included in the dose-response study. Group 1 was the vehicle control, which received 100 µl i.pl. injection of 3.5% DMSO in saline. Groups 2–4 were injected with 3 different doses of AG490 (1, 5 or 10 µg). To study the effects of naloxone on AG490-induced antinociception, an additional group of rats (group 5; n=4) was observed. Group 5 was co-administered with AG490 (10 µg) and naloxone (10 µg). The drugs were administered i.pl. in a volume of 100 µl. As reported earlier, the in vivo pharmacological effects of AG490 were observed 4 h after treatment (5,6). Thus, the behavioral tests were performed before (baseline assessment) and 4 h after treatment. First, the rats were subjected to the thermal hyperalgesia test; 10 min later, the paw pressure test was performed on the same set of rats. All the experiments were performed between 8:00 a.m. and 2:00 p.m. to reduce the confounding influence of diurnal variations, and all the procedures were performed in a blinded fashion.

The thermal hyperalgesia test was performed using a plantar analgesia instrument (IITC Life Science, Woodland Hills, CA, USA), as described previously (6). Briefly, rats were acclimated to the Plexiglas chambers for 30 min prior to testing. A radiant heat source was focused at the mid-plantar surface of the hind paw, and the paw withdrawal latency (PWL) was recorded. Both paws were tested alternatively at 2–3 min intervals for a total of 3 trials. A mean count and latency were used for analysis. The percentage of maximal possible effect (MPE) (%) was calculated according to the following formula: % MPE = (post-drug latency - pre-drug latency)/[cut-off (20 sec) - pre-drug latency] × 100.

The nociceptive thresholds were assessed using a modified digital Randall-Selitto device (IITC Life Science). Briefly, each rat was gently restrained and an increasing mechanical pressure was exerted on the mid-plantar surface of the hind paw until a paw withdrawal response was observed. A total of 3 trials were performed on the inflamed and contralateral paws, and the average was taken for analysis. The paw withdrawal threshold (PWT) was expressed in grams, and a cut-off value of 300 g was used to prevent tissue injury (14). PWT was calculated for each rat using ΔPWT = tested PWT - baseline PWT (5).

The data are expressed as mean ± standard error of the mean. The difference in PWL and PWT between ipsilateral (inflamed) and contralateral (non-inflamed) paws were compared using a paired t-test. The results of dose-response studies and naloxone effects were evaluated by analysis of variance followed by Bonferroni post hoc test (P<0.05 was considered to indicate a statistically significant difference).

As reported previously (9), ʎ-carrageenan treatment to the left hind paw produced localized inflammation, as evidenced by redness and swelling. In comparison to the contralateral paws, the inflamed paws showed a significant decrease in PWL and PWT to the application of thermal and mechanical stimuli (P<0.05; data not shown).

An i.pl. injection of AG490 (1, 5 and 10 µg) to the inflamed paw produced a dose-dependent, statistically significant anti-hyperalgesic effect in the thermal test (Fig. 1A). Bonferroni post-hoc analysis showed a significant difference between control and 5 µg (P<0.05), and control and 10 µg of the AG490-treated groups (P<0.01). Although the 1 µg AG490-treated group showed a trend of increased PWL compared to the control group, it did not reach significance (P>0.05). The most potent effect was observed for 10 µg of AG490 in comparison to the control group. AG490 treatment did not alter the PWL of the contralateral paws (P>0.05, data not shown). These results clearly indicate that blocking JAK2 signaling attenuates thermal hypersensitivity in an inflamed condition. In the mechanical hyperalgesia test, AG490 treatment resulted in a trend towards increased PWT when compared to the control group (Fig. 1B), but it was not of statistical significance (P=0.0504). No difference in PWT was observed on contralateral paws (P>0.05, data not shown).

Co-administration of naloxone and AG490 to the inflamed paw increased the PWL in comparison to AG490 and control groups (P<0.01) (Fig. 2A). This suggests that naloxone had no effect on the antinociceptive activity of AG490. Notably, the animals that received AG490 and naloxone exhibited greater nociceptive effects compared to the animals treated with AG490 alone; however, the difference between the groups was not significant (P>0.05). A similar trend was observed in the mechanical hyperalgesia test (Fig. 2B) without a significant difference between groups (P>0.05). From these data, the opioid signaling system appears to not be involved in AG490-mediated antinociception.