Fig. 3

4R reduces inflammation-induced paw edema in male but not female mice (a) Experimental timeline for paw thickness measurements. In vivo thickness measurements of CFA-injected and non-injected hind paws before and 1–8 days after 4R (1, 6, and 15 mg/kg) or vehicle systemic administration of (b) male and (c) female mice. All values are expressed as mean ± SEM. n = 6–16 animals per treatment and sex. Two-way ANOVA (b) or Two-way RM ANOVA (c) followed by posthoc Tukey’s multiple comparisons test: CFA-injected paw vs non-injected paw in pre-drug vehicle (veh) conditions, p < 0.0001 (####); 4R CFA-injected paw vs non-injected vehicle conditions day 7, p < 0.0001 (§§§§), Two-way ANOVA followed by posthoc Dunnett’s multiple comparisons test: vehicle vs 4R systemic treatment in CFA-injected paws of the same time-point, p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***). Results of multiple comparisons between vehicle (veh) and 4R 1 mg/kg, 6 mg/kg or 15 mg/kg are shown in purple, teal and orange asterisks, respectively. (d) Experimental timeline for histology samples. e Representative H&E images of dermal (D) and subdermal (S) skin, open arrows point to polymorphonuclear immune cell infiltration. Scale Bar: 200 µm. Histological analysis of hind paw skin 8 days post local CFA and 7 days post 4R (f) glabrous skin thickness of the hind paws and (g) subdermal immune cell count. All values are expressed as mean ± SEM. n = 5–6 mice per treatment and sex. Two-way ANOVA followed by Šidák’s multiple comparisons test: Vehicle vs 4R, p < 0.05 (*), p < 0.01 (**)