Prolonged nociceptive behaviors in formalin mice model. (A) Baseline thermal withdrawal latency (TWL) was determined for each mouse prior to treatment. This value was used to establish 100% baseline for that subject. Control groups (saline and naïve) mice showed habituation when retested at 3 and 6 hours. Thermal hypersensitivity (lower TWL relative to baseline as 100%) is observed in the formalin treated mice (n = 15) compared to the control groups (saline-treated n = 11; naïve n = 10) at 1 and 3 but not 6 hours post injection. The formalin treated group TWL was significantly different at 1 hr compared to naïve (p < 0.001) and (p < 0.005) saline groups; at 3 hr compared to saline (p < 0.02) and naïve (p < 0.05) groups. TWL returns to baseline values at 24 hours in the treated groups. #p ≤ 0.05 across group comparison for TWL values by two way RM-ANOVA followed by pairwise Tukey t-test post hoc analysis. (B) Mechanical allodynia, measures as paw withdrawal in response to force of probe in grams, was assessed in the injected hindpaws (ipsilateral = injected paw, IP) and opposite (contralateral, CL) hindpaws. There was a significant difference between the ipsilateral and contralateral footpad (p < 0.02) in the formalin mice. Ipsilateral hindpaw allodynia was significant at 1, 6 and 24 hours post-inflammation relative to the baseline values (n = 15). At 24 hours post-inflammation, the contralateral threshold was significantly (p < 0.05) lower compared to the baseline values in the contralateral footpad. ZJ43 (150 mg/kg; IP, n = 10) administered 30 prior to and 8 hours post formalin injection significantly reduced allodynia in the ipsilateral footpad (∞p ≤ 0.05, ZJ43 (formalin)-IP vs. Formalin-IP mechanical withdrawal threshold values at 24 hr post inflammation). The difference between ZJ43 (formalin)-CL and Formalin-CL did not reach statistical significance (p = 0.13). Two way RM-ANOVA was used followed by pairwise Tukey t-test post hoc analysis. Baseline (B) = probe force that induced paw withdrawal in mice prior to injection of formalin or saline. Data presented as mean ± SEM, *p ≤ 0.05 vs. baseline values; was used followed by pairwise Tukey t-test post hoc analysis.