Anti-inflammatory effect of low intensity ultrasound (LIUS) on complete Freund's adjuvant-induced arthritis synovium J.-I. Chung, S. Barua, B.H. Choi,

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Anti-inflammatory effect of low intensity ultrasound (LIUS) on complete Freund's adjuvant-induced arthritis synovium J.-I. Chung, S. Barua, B.H. Choi, B.-H. Min, H.C. Han, E.J. Baik Osteoarthritis and Cartilage Volume 20, Issue 4, Pages 314-322 (April 2012) DOI: 10.1016/j.joca.2012.01.005 Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 LIUS reduced knee circumference in CFA-induced arthritic rats. CFA (150 μg) was injected into the left knee joint of each rat (CFA group) and after 24 h LIUS was applied everyday for 4 days (post-US group). The sham group was injected with vehicle. Knee circumference was measured each day after treatment. The data represent observations from 9 to 11 animals per group (sham; n = 9, CFA; n = 11, post-US; n = 11) and displayed as means ± 95% confidence intervals. ∗P < 0.05 vs sham and #P < 0.05 vs CFA. Osteoarthritis and Cartilage 2012 20, 314-322DOI: (10.1016/j.joca.2012.01.005) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 CFA induced mRNA expression of inflammatory mediators in synovial membrane. (A) Expression of inflammatory mediators including COX-1, COX-2, TNF-α and IL-1β mRNA was measured in the synovial membrane 3 days after CFA injection. The mRNA levels were normalized to GAPDH (n = 7) and represented relative to control. Except TNF-α, expression of all inflammatory genes was increased in arthritic synovial membranes, which were significantly reduced by LIUS. (B) Correlation between ipsilateral knee circumference and COX-1, COX-2, IL-1β and TNF-α mRNA relative expression in CFA-induced arthritic synovium. Statistical correlation analysis was performed using Spearman correlation analyses. Data are displayed as means ± 95% confidence intervals. ∗P < 0.05 vs sham and #P < 0.05 vs CFA. Osteoarthritis and Cartilage 2012 20, 314-322DOI: (10.1016/j.joca.2012.01.005) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 LIUS attenuated the expression of inflammatory mediators in synovial membrane. Western blot analysis was performed with the tissue collected 3 days (A) and 5 days (B) after CFA injection. Blots are representative immunoblots of sham, CFA and post-US group. The relative integrated density values of COX-1, COX-2, iNOS and CD11b protein levels were normalized to β-actin (Day 3; n = 9, Day 5; n = 12 for each group). Data are displayed as means ± 95% confidence intervals. ∗P < 0.05 vs sham and #P < 0.05 vs CFA. Osteoarthritis and Cartilage 2012 20, 314-322DOI: (10.1016/j.joca.2012.01.005) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 CFA induced CCR5 mRNA expression of inflammatory mediators in synovial membrane. (A) Expression of chemoattractant chemokine receptor CCR5 mRNA was measured in the synovial membrane 3 days after CFA injection. (B) The mRNA level was normalized to GAPDH (n = 7) and reported relative to the control. (C) Correlation between ipsilateral knee circumference and CCR5 mRNA relative expression in CFA-induced arthritic synovium. mRNA levels were normalized to GAPDH (n = 7). Statistical correlation analysis was performed by using Spearman correlation analyses. Data are displayed as means ± 95% confidence intervals. ∗P < 0.05 vs sham and #P < 0.05 vs CFA. Osteoarthritis and Cartilage 2012 20, 314-322DOI: (10.1016/j.joca.2012.01.005) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Immunohistochemical analysis of inflamed synovium. The localization of MPO and Iba1 on days 3 and 5. (A) At days 3 and 5 after CFA injection, sections were labeled with MPO and Iba1 antibodies and visualized with Alexa Fluor 488 Dye conjugated secondary antibodies. Nuclei were stained with Hoechst. Scale bars = 500 μm. (B) Higher magnification of box in (A). Scale bars = 100 μm. The MPO+ neutrophils (green) were infiltrating the inflamed synovium on day 3 and disappeared on day 5. Iba1+ macrophages/monocytes were located around the MPO core on day 3. Almost all cells of the MPO+ core were replaced by highly activated macrophages on day 5. LIUS attenuated neutrophil and macrophage infiltration on both day 3 and 5. Images were acquired with a Zeiss LSM 710 confocal microscope (Carl Zeiss). Osteoarthritis and Cartilage 2012 20, 314-322DOI: (10.1016/j.joca.2012.01.005) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions