Power Doppler Ultrasound Phenotyping of Expanding versus Collapsed Popliteal Lymph Nodes in Murine Inflammatory Arthritis Echoe M. Bouta 1,2. , Yawen Ju 1,3. , Homaira Rahimi 1,4 , Karen L. de Mesy-Bentley 4 , Ronald W. Wood 5,6 , Lianping Xing 1,3 , Edward M. Schwarz 1,2,3,5,6 * 1 Center for Musculoskeletal Research, University of Rochester School of Medicine and Dentistry, Rochester, New York, United States of America, 2 Department of Biomedical Engineering, University of Rochester School of Medicine and Dentistry, Rochester, New York, United States of America, 3 Department of Pathology and Laboratory Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York, United States of America, 4 Department of Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, New York, United States of America, 5 Department of Obstetrics and Gynecology, University of Rochester School of Medicine and Dentistry, Rochester, New York, United States of America, 6 Department of Urology, University of Rochester School of Medicine and Dentistry, Rochester, New York, United States of America Abstract Rheumatoid arthritis is a chronic inflammatory disease manifested by episodic flares in affected joints that are challenging to predict and treat. Longitudinal contrast enhanced-MRI (CE-MRI) of inflammatory arthritis in tumor necrosis factor- transgenic (TNF-Tg) mice has demonstrated that popliteal lymph nodes (PLN) increase in volume and contrast enhancement during the pre-arthritic ‘‘expanding’’ phase of the disease, and then suddenly ‘‘collapse’’ during knee flare. Given the potential of this biomarker of arthritic flare, we aimed to develop a more cost-effective means of phenotyping PLN using ultrasound (US) imaging. Initially we attempted to recapitulate CE-MRI of PLN with subcutaneous footpad injection of US microbubbles (DEFINITYH). While this approach allowed for phenotyping via quantification of lymphatic sinuses in PLN, which showed a dramatic decrease in collapsed PLN versus expanding or wild-type (WT) PLN, electron microscopy demonstrated that DEFINITYH injection also resulted in destruction of the lymphatic vessels afferent to the PLN. In contrast, Power Doppler (PD) US is innocuous to and efficiently quantifies blood flow within PLN of WT and TNF-Tg mice. PD-US demonstrated that expanding PLN have a significantly higher normalized PD volume (NPDV) versus collapsed PLN (0.55360.007 vs. 0.00860.003; p,0.05). Moreover, we define the upper (.0.030) and lower (,0.016) quartile NPDVs in this cohort of mice, which serve as conservative thresholds to phenotype PLN as expanding and collapsed, respectively. Interestingly, of the 12 PLN phenotyped by the two methods, there was disagreement in 4 cases in which they were determined to be expanding by CE-MRI and collapsed by PD-US. Since the adjacent knee had evidence of synovitis in all 4 cases, we concluded that the PD-US phenotyping was correct, and that this approach is currently the safest and most cost- effective in vivo approach to phenotype murine PLN as a biomarker of arthritic flare. Citation: Bouta EM, Ju Y, Rahimi H, de Mesy-Bentley KL, Wood RW, et al. (2013) Power Doppler Ultrasound Phenotyping of Expanding versus Collapsed Popliteal Lymph Nodes in Murine Inflammatory Arthritis. PLoS ONE 8(9): e73766. doi:10.1371/journal.pone.0073766 Editor: David Douglass Brand, Veterans Affairs Medical Center, United States of America Received May 1, 2013; Accepted July 30, 2013; Published September 9, 2013 Copyright: ß 2013 Bouta et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by research grants from the National Institutes of Health PHS awards (T32 AR053459; R01s AR048697, AR053586 and AR056702; P01 AI078907; DP2OD006501; and P30 AR061307). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Edward_Schwarz@URMC.Rochester.edu . These authors contributed equally to this work. Introduction Rheumatoid arthritis (RA) is a debilitating immune-mediated inflammatory disorder characterized by recurrent arthritic flares that lead to joint inflammation and destruction, and cause significant morbidity in RA patients [1]. Murine models of chronic RA, such as the TNF transgenic (TNF-Tg) mouse [2,3], have proven useful in elucidating the pathophysiology of inflammatory-erosive arthritis and evaluating novel interventions. Contrast enhanced (CE)-MRI has emerged as a longitudinal outcome measure to quantify synovial and draining lymph node volume in murine models of inflammatory arthritis [4–11]. These studies have found that TNF-Tg mice with frank ankle arthritis had both larger popliteal lymph node (PLN) volume (PLNvol) and greater LN contrast enhancement (LNCE) when compared to their wild type (WT) littermates. To quantify this as a metric, LN capacity (LNCap = LNCE*PLNvol) was developed as a primary outcome measure to study PLN as a biomarker of inflammatory arthritis in the lower limbs of mice [4]. Subsequent CE-MRI studies demonstrated that arthritic knee flare was associated with the expansion and subsequent collapse of the PLN [8,9,11]. Thus, formal associations between altered PLNvol, LNCE and the onset of arthritic flare have been established. MRI is commonly used to study synovium, tendons, bone and LN in RA pathogenesis [12–15]. By quantifying the amount of synovium and synovial fluid in the joint, CE-MRI can be used to identify patients with early RA [16,17]. PLN have also been used PLOS ONE | www.plosone.org 1 September 2013 | Volume 8 | Issue 9 | e73766