Fig. 5

MCC950 dose dependently mitigates nociceptive sensitization and decreases activated (p20 fragment) caspase-1 protein at 24 h post-injury paradigm Hypersensitive hind paw withdrawal to tactile stimulus, ipsilateral to injured hemisphere (A, left hindpaw) and contralateral to injured hemisphere (B, right hindpaw), in injured animals at baseline, 24 h after three injuries, and 24 h after each injection dosage of MCC950. Statistically significant decrease in response thresholds (i.e., more pain) were observed at 24 h after three injuries when compared with baseline. There was no significant change in response threshold with 0.1 and 1 mg/kg treatment of MCC950 compared to before treatment. Conversely, significant increase was observed in response threshold (i.e., less pain) with 10 and 20 mg/kg MCC950 treatments (n = 10/group; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, repeated-measures ANOVA, Tukey’s HSD) with no significant improvement at 20 mg/kg compared to 10 mg/kg. Mouse grimace scoring (MGS) was performed in animals at baseline, 24 h after last injury, and 24 h after each MCC950 injection dose. C Statistically significant increase in MGS (i.e., more pain) at 24 h after three injuries when compared with baseline in animals (n = 10/group; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, repeated-measures ANOVA, Tukey’s HSD). No significant change in MGS scores with 0.1 and 1 mg/kg treatment of MCC950 compared to before treatment. Significant decrease in MGS scores (i.e., less pain) with 10 and 20 mg/kg MCC950 treatments (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, repeated-measures ANOVA, Tukey’s HSD). No significant improvement at 20 mg/kg compared to 10 mg/kg. Western blot analyses of cortical caspase-1 levels revealed increased relative p20 caspase-1 expression after mTBI, which was decreased in the MCC950 treatment group (D and E). Mice were injected with MCC950 intraperitoneally at 10 mg/kg (5 mg/mL) at 1 h and 24 h after each injury. Cortical brain tissues were collected 24 h after the third injury. D Shown is a sample image of p20 caspase-1 activated protein levels in the sham, 3 × mTBI group, and 3 × mTBI group treated with MCC950. E Significant increases were detected in cortical activated p20 caspase-1 levels at 24 h after third mTBI in the injury group compared to sham. Injury animals treated with MCC950 showed significant attenuation of caspase-1 level (n = 4–5 mice per group; *p < 0.05, ***p < 0.001, two-way ANOVA, Tukey’s HSD)