Fig. 2

Pro-inflammatory mediator evolution after two cold applications in the synovial fluid of treated knees: randomized comparison of ice- (N = 16) versus cold CO₂- (N = 16) treated patients (phase I). Thirty-two patients were first randomized to receive either local ice (30 min twice; N = 16) or cold CO₂ (2 min twice; N = 16) on an arthritic knee. The synovial fluid was analyzed before and after treatment as previously described. Synovial IL-6 (a), IL-1β (b), TNF-α (c), IL-17A (d), and VEGF (e) assessed by multiplex flow cytometry (CBA® BD Bioscience, Franklin Lakes, NJ, USA); NF-KB-p65 (f)/NF-kB-p65-P (g) assessed by ELISA (85-86083-11®, Thermofisher, Waltham, MA, USA); and PG-E2 (h) assessed by ELISA (KGE004B®, Bio-Techne, Minneapolis, MN, USA) levels were all measured at 9 a.m. (just before the first cold application, then 24 h later). Data are presented as means ± SEM. Paired Wilcoxon-Mann-Whitney tests were performed. *p < 0.05, **p < 0.01, ***p < 0.001. H0: first evaluation (9 a.m., before the first cold application); H24: second evaluation (9 a.m., 24 h later, after the two cold applications). Missing values were due to the fact that some cytokines and mediators could not be detected—or with out-of-range values—in some synovial fluid samples before and/or after cold application (IL-6: N = 1 in the CO₂ group at H24; IL-1β: N = 1 at H24 in the ice group and N = 1 both at H0 and H24 in the CO₂ group; TNF-α: N = 1 both at H0 and H24 in the CO₂ group; IL-17A: N = 0; VEGF: N = 0; NFkB-p65: N = 4 in the CO₂ (1 at H0, 1 at H24, 1 at both H0 and H24) and ice (2 at H0, 2 at H24) groups; NFkB-p65P: N = 4 in the CO₂ (1 at H0, 1 at H24, 1 at both H0 and H24) and ice (2 at H0, 2 at H24) groups; PG-E2: N = 4 in the CO₂ (1 at H0, 1 at H24, 1 at both H0 and H24) and ice (2 at H0, 2 at H24) groups)