Local ice cryotherapy decreases synovial interleukin 6, interleukin 1β, vascular endothelial growth factor, prostaglandin-E2, and nuclear factor kappa B p65 in human knee arthritis: a controlled study

Abstract

Background: The aim of this study was to assess the anti-inflammatory effects of local cryotherapy in human non-septic knee arthritis.

Methods: In the phase I of the study, patients were randomized to receive either ice (30 min; N = 16) or cold CO₂ (2 min; N = 16) applied twice during 1 day at an 8-h interval on the arthritic knee. In phase II, 16 other ice-treated arthritic knees according to the same protocol were compared to the contralateral non-treated arthritic knees (N = 16). The synovial fluid was analyzed just before the first cold application, then 24 h later. IL-6, IL-1β, TNF-α, IL-17A, VEGF, NF-kB-p65 protein, and PG-E2 levels were measured in the synovial fluid and compared before/after the two cold applications.

Results: Forty-seven patients were included (17 gouts, 11 calcium pyrophosphate deposition diseases, 13 rheumatoid arthritides, 6 spondyloarthritides). Local ice cryotherapy significantly reduced the IL-6, IL-1β, VEGF, NF-kB-p65, and PG-E2 synovial levels, especially in the microcrystal-induced arthritis subgroup, while only phosphorylated NF-kB-p65 significantly decreased in rheumatoid arthritis and spondyloarthritis patients. Cold CO₂ only reduced the synovial VEGF levels. In the phase II of the study, the synovial PG-E2 was significantly reduced in ice-treated knees, while it significantly increased in the corresponding contralateral non-treated arthritic knees, with a significant inter-class effect size (mean difference - 1329 [- 2232; - 426] pg/mL; N = 12).

Conclusions: These results suggest that local ice cryotherapy reduces IL-6, IL-1β, and VEGF synovial protein levels, mainly in microcrystal-induced arthritis, and potentially through NF-kB and PG-E2-dependent mechanisms.

Trial registration: Clinicaltrials.gov, NCT03850392-registered February 20, 2019-retrospectively registered.

Keywords: Cytokines; Knee arthritis; Local cryotherapy; NF-kB; PG-E2.

Fig. 1

Study design. In the first phase of the study, patients were randomized to receive either ice (30 min; N = 16) or cold CO₂ (2 min; N = 16) twice at an 8-h interval. In the second phase, 16 patients with arthritides of both knees were included. One of the knees was treated with local ice (N = 16) while the contralateral arthritic knees were used as paired controls (N = 16) for the synovial fluid analyses. One of these 16 patients had been previously included in the first phase of the study (ice group). Therefore, a total of 47 patients were included. “⍉” indicates no cryotherapy treatment

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)

Fig. 3

a–h Pro-inflammatory mediator evolution after two cold applications in the synovial fluid of ice-treated knees (N = 16) compared to contralateral non-treated arthritic knees (N = 16) in knee bi-arthritic patients (phase II). Sixteen patients with knee bi-arthritis were then included (phase II). One knee was treated with ice according to the study protocol (N = 16). The contralateral knees were used as paired controls (N = 16—on the right part of the graphs). The synovial fluid was analyzed before and after treatment as previously described, on both knees for each patient. The results of synovial fluid analyses of all the ice-treated patients (phase I + II; N = 31) are also shown on the left part of the graphs, for comparison. Data are presented as means ± SEM. 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); controls: contralateral non-treated arthritic knees. 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 applications (IL-6: N = 3 in ice-treated phase I + phase II patients (1 at H0, 2 at both H0 and H24), N = 0 in ice-treated phase II and contralateral non-treated knees; IL-1β: N = 2 in ice-treated phase I + phase II patients (1 at H0, 1 at H24), N = 0 in ice-treated phase II and contralateral non-treated knees; TNF-α: N = 0; IL-17A: N = 0; VEGF: N = 0; NFkB-p65: N = 5 in ice-treated phase I + phase II patients (2 at H0, 3 at H24), N = 0 in ice-treated phase II, N = 4 in contralateral non-treated knees (2 at H0, 1 at H24, 1 at both H0 and H24); NFkB-p65P: N = 5 in ice-treated phase I + phase II patients (2 at H0, 3 at H24), N = 0 in ice-treated phase II, N = 4 in contralateral non-treated knees (2 at H0, 1 at H24, 1 at both H0 and H24); PG-E2: N = 5 in ice-treated phase I + phase II patients (2 at H0, 3 at H24), N = 0 in ice-treated phase II, N = 4 in contralateral non-treated knees (2 at H0, 1 at H24, 1 at both H0 and H24))

Fig. 4

a–h Pro-inflammatory mediator evolution after two cold applications in the synovial fluid in the microcrystal-induced arthritis subgroup (N = 28; ice: N = 19). The synovial fluid of all cryotherapy-treated patients suffering from microcrystal-induced arthritis (N = 27 including 18 ice-treated patients) was analyzed just before the first cold application, then 24 h later (after two cold application). 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); microcrystals: patients suffering from microcrystal-induced knee arthritis. 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 applications (IL-6: N = 0; IL-1β: N = 2 in ice-treated patients (1 at H0, 1 at H24); TNF-α: N = 1 (1 CO₂-treated patient both at H0 and H24); IL-17A: N = 0; VEGF: N = 0; NFkB-p65: N = 6 in ice + CO₂-treated patients (3 at H0, 2 at H24, 1 CO₂-treated patient at both H0 and H24), N = 3 in ice-treated patient (2 at H0, 1 at H24); NFkB-p65P: N = 6 in ice + CO₂-treated patients (3 at H0, 2 at H24, 1 CO₂-treated patient at both H0 and H24), N = 3 in ice-treated patient (2 at H0, 1 at H24); PG-E2: N = 6 in ice + CO₂-treated patients (3 at H0, 2 at H24, 1 CO₂-treated patient at both H0 and H24), N = 3 in ice-treated patient (2 at H0, 1 at H24))