Effects of local cryotherapy on systemic endothelial activation, dysfunction, and vascular inflammation in adjuvant-induced arthritis (AIA) rats

Abstract

Aim: This study explored the systemic vascular effects of local cryotherapy with a focus on endothelial changes and arterial inflammation in the model of rat adjuvant-induced arthritis (AIA).

Methods: Cryotherapy was applied twice a day on hind paws of AIA rats from the onset of arthritis to the acute inflammatory phase. Endothelial activation was studied in the aorta by measuring the mRNA levels of chemokines (CXCL-1, MCP-1 (CCL-2), MIP-1α (CCL-3)) and adhesion molecules (ICAM-1, VCAM-1) by qRT-PCR. Endothelial dysfunction was measured in isolated aortic and mesenteric rings. Aortic inflammation was evaluated via the mRNA expression of pro-inflammatory cytokines (TNF-α, IL-6) by qRT-PCR and leucocyte infiltration analysis (flow cytometry). Plasma levels of TNF-α, IL-6, IL-1β, IL-17A, and osteoprotegerin (OPG) were measured using Multiplex/ELISA.

Results: AIA was associated with an increased aortic expression of CXCL-1 and ICAM-1 as well as an infiltration of leucocytes and increased mRNA expression of IL-6, IL-1β, and TNF-α. Local cryotherapy, which decreased arthritis score and structural damages, reduced aortic mRNA expression of CXCL-1, IL-6, IL-1β, and TNF-α, as well as aortic infiltration of leucocytes (T lymphocytes, monocytes/macrophages, neutrophils) and improved acetylcholine-induced vasorelaxation in the aorta and mesenteric arteries. Plasma levels of IL-17A and OPG were significantly reduced by cryotherapy, while the number of circulating leucocytes was not. IL-17A levels positively correlated with endothelial activation and dysfunction.

Conclusion: In the AIA model, local cryotherapy reduced systemic endothelial activation, immune cell infiltration, and endothelial dysfunction. Mechanistically, the reduction of circulating levels of IL-17A appears as the possible link between joint cooling and the remote vascular effects.

Keywords: Arthritis; Endothelial activation; Endothelial dysfunction; Local cryotherapy; Vascular inflammation.

Fig. 1

Effect of local cryotherapy on arthritis score and joint damage in AIA rats. Experiments were conducted in AIA rats from the “endothelial activation” series treated or not with local cryotherapy. A Time-course of arthritis score. B Radiographic score and C representative X-ray radiography of hind paws at the end of experiment. D–I Effect of cryotherapy on the different items of the radiographic score: swelling, cartilage destruction, osteoporosis, bone erosion and new bone formation. Results are expressed as means ± SEM (n=number of rats/group). *(p<0.05), **(p<0.01)

Fig. 2

Expression of endothelial activation markers in AIA rats and effect of cryotherapy. A–G Effect of AIA on aortic mRNA expression of markers of endothelial activation as compared to control rats on day 24 post-immunization. H–N Effect of a daily treatment with local cryotherapy in AIA rats. Endothelial activation was assessed in thoracic aorta by measuring by qRT-PCR the mRNA expression of IL-6 (A, H), TNF-α (B, I), CXCL-1 (C, J), ICAM-1 (D, K), VCAM-1 (E, L), MCP-1 (CCL-2) (F, M) and MIP-1α (CCL-3) (G, N). Results are expressed as means ± SEM (n=number of rats/group). *(p<0.05), **(p<0.01). NS, non-significant

Fig. 3

Aortic expression of endothelial activation markers at the preclinical stage and at onset of arthritis in AIA rats. mRNA expression of ICAM-1 (A, F), VCAM-1 (B, G), CXCL-1 (C, H), MCP-1 (CCL-2) (D, I), and MIP-1α (CCL-3) (E, J) was studied in thoracic aorta of control and AIA rats at the preclinical stage (day 4 post-immunization, A–E) and at the onset of arthritis (day 11 post-immunization, F–J) by qRT-PCR. Results are expressed as means ± SEM (n=number of rats/group). *(p<0.05), ***(p<0.001). NS, non-significant

Fig. 4

Leucocyte infiltration in aorta from AIA rats and effect of cryotherapy. A–H Flow cytometry analyses of CD45⁺ populations on rat thoracic aorta from control and AIA rats at the acute inflammatory phase (day 24 post-immunization). I–P Effect of local cryotherapy in AIA rats treated from day 11 to day 24 post-immunization. Different leucocyte subpopulations were studied: monocytes/macrophages CD11b/c⁺ RP-1⁻ (B, J), neutrophils CD11b/c⁺ RP-1⁺ (C, K), T lymphocytes CD3⁺ (D, L), CD4⁺ (E, M), CD8⁺ (G, O), CD4⁺ IL-17A⁺ (F, N) and CD8⁺ IL-17A⁺ (H, P) T cells. Gating strategies are shown in Supplementary Fig. 1A–J. Data are presented as the number of stained cells per mg of the aorta. Results are expressed as means ± SEM (n=10 rats/group). *(p<0.05), **(p<0.01), ***(p<0.001). NS, non-significant

Fig. 5

Effect of local cryotherapy on endothelial dysfunction in AIA rats. Experiments were conducted in AIA rats treated or not with local cryotherapy. Expression of endothelial pathways involved in AIA-associated endothelial dysfunction including COX-2 (A), Arg-2 (B), p22phox (C), and p47phox (D) was studied in aorta by qRT-PCR. Results are expressed as means ± SEM (n=number of rats/group). E, F Endothelial function was studied in rat on aortic rings and on mesenteric arteries by the measurement of acetylcholine (Ach)-induced vasorelaxation in endothelium-intact artery segments preconstricted with phenylephrine 10⁻⁶ moles/L. Results are expressed as means ± SEM (n=number of arterial rings, 6–7 rats/group). *(p<0.05), **(p<0.01), ***(p<0.001)

Fig. 6

Circulating leucocytes in AIA rats and effect of cryotherapy. A–G Effect of AIA at the acute inflammatory phase (day 24 post-immunization) on leucocytes populations in the blood as compared to controls. H–N Effect of local cryotherapy on blood leucocytes and leucocyte subpopulations in AIA rats. Flow cytometry analysis assessed the absolute number of blood leucocytes (CD45⁺ cells, A, H) and of the different leucocyte subpopulations: CD3⁺ T lymphocytes (B, I), CD11b/c⁺ monocytes/macrophages (C, J), CD4⁺ (D, K), CD8⁺ (F, M) T cells, and intercellular IL-17A⁺ CD4⁺ (E, L) or CD8⁺ (G, N) T cells. Gating strategies are shown in Supplementary Fig. 1K-R. Data are expressed as number of stained cells per μl of blood. Results are expressed as means ± SEM (n=10 rats/group). *(p<0.05), **(p<0.01), ***(p<0.001). NS, non-significant

Fig. 7

Correlations between clinical scores, plasma, and aortic cytokines expression, aortic adhesion molecule expression, aortic and blood count of leucocytes, and endothelial function in AIA rats. A–X Correlations were determined using Spearman correlation test in AIA rats treated or not with local cryotherapy. Endothelial function was expressed as the Emax of acetylcholine (Ach). Data in bold are significant (p<0.05)