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Draining lymph node (LN) enlargement has long been recognized as a hallmark of joint inflammation in rheumatoid arthritis (RA) and can be quantified with magnetic resonance imaging (MRI) (1). The importance of this outcome measure as a biomarker of erosive inflammatory arthritis initiation, progression, and response to therapy has recently been demonstrated in murine models (2–5). Despite its potential value, this approach has not gained broad acceptance due to the very high financial, time, and labor costs associated with MRI, and limited access to machines. Thus, investigators have been evaluating ultrasound (US) as a more practical and cost-effective method to study LNs in animal models (6) and RA patients (7). Based on the promising results of such research, we aimed to validate in vivo US measurement of popliteal LN volume in tumor necrosis factor (TNF)–transgenic mice (8) with varying degrees of arthritis, by comparing the results obtained with this imaging modality and those obtained with MRI.

A total of 16 popliteal LNs from 3–8-month-old heterozygous TNF-transgenic mice (3647 line on a C57BL/6 background) were studied. Mice received local anesthesia by intraperitoneal injection of ketamine (60 mg/kg) and xylazine (4 mg/kg), the LNs were scanned by MRI, and their volumes were determined with Amira software (Visage Imaging) as described previously (3).

On the following day, these popliteal LNs were imaged using a high-resolution small-animal US system (VisualSonics 770 with 704 scanhead). For this procedure, mice were anesthetized with ∼2% isoflurane in oxygen, and hair was removed from ankles to hips using a depilatory cream. Each mouse was placed in a supine position on the heated (40°C) imaging platform with paws taped to surface electrodes to monitor heart rate and synchronize respiratory rate (Figure 1A). The popliteal LNs were identified in B-mode by adjusting the scanhead up or down to position the LNs at the plane of focus (Figure 1B), and then scanned in 3-dimension (3-D) mode with a step size of 0.032 mm. The 3-D US image data were used to quantify popliteal LN volume by manual segmentation of the LN and surrounding fat pads. The mean signal intensity of the fat pad was computed using the TissueStatistics module. To eliminate the signal intensity of the fat pad from that of the LN material, selected areas in which the signal intensity was higher than the signal intensity of the fat pad were subtracted, and any resultant empty inclusions (holes) within the LN were filled. However, holes on the surface cannot be filled using this approach. The SurfaceGen module was used to arrange the labeled pixels as a bounded surface for subsequent 3-D visualization and volumetric quantification with SurfaceView (Figure 1C) and TissueStatistics. Linear regression analysis was performed on the volumes generated with both imaging modalities (Figure 1D). We also determined the intra- and interobserver reliability of our US popliteal LN volume measurement, which showed insignificant variability (P = 0.8399 and P = 0.8096, respectively).

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Figure 1. Strong correlation between popliteal lymph node (PLN) volumes determined by magnetic resonance imaging (MRI) versus ultrasound (US). A, An anesthetized mouse was positioned on a heating pad with an electrocardiogram monitor, and a Scanhead 704 was placed above the knee to image the popliteal LN with the US machine. B, The 2-dimensional (2-D) US image of the PLN obtained in B-mode shows a dark popliteal LN (red arrow) surrounded by a white triangular fat pad (green arrow). C, A reconstructed 3-D image of the popliteal LN (green) with surrounding soft tissue was generated with US 3-D mode and Amira analysis software. D, A linear regression analysis was performed by plotting the popliteal LN volume (mm3) measured by MRI (y-axis) versus by US (x-axis). The slope and highly significant R2 value are also presented. E–J, The 3-D images of representative small, medium, and large popliteal LNs generated independently by MRI (E, G, and I) and US (F, H, and J) are presented to illustrate their similarities. The color of each of the popliteal LNs corresponds to the color of the symbols in D. All other PLN measurements are indicated by the blue symbols.

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US proved to be a very facile approach for assessing murine popliteal LNs (Figure 1B), since they were readily identified in B-mode after locating the triangular fat pad. A strong relationship between popliteal LN volumes measured by MRI and by US was found using a linear regression model (R2 = 0.844, P < 0.0001) (Figure 1D). However, vertical placement of popliteal LNs in US images is a potential source of variability in the volumes measured (∼10% difference; see below), and may result from mechanical compression with the scanhead. To reduce this variation, a lower-frequency scanhead could provide greater focal depth and distance from the head. In addition, the scanhead position should be adjusted so that the popliteal LN is centered consistently in the plane of focus (Figure 1B). Of note, smaller popliteal LNs were less susceptible to scanhead compression error, as suggested by the stronger linear relationship between the volume obtained with US and the volume obtained with MRI. To provide a broader illustration of the correlation between MRI and US measurements, 3 popliteal LNs were chosen to represent the small, medium, and large popliteal LNs, and their 3-D rendered images generated by MRI and US are presented in Figures 1E–J.

One surprising result of our study was that the slope in the regression model did not equal 1.0, despite the strong correlation between measurements on the same LNs obtained with the 2 different imaging modalities, in all mice studied (Figure 1D). To test the hypothesis that we might be compressing the LNs during US imaging, we varied the vertical position of the scanhead in an attempt to compress the LN. In a representative test, the popliteal LN showed a volume of 6.86 mm3 at a depth of 7 mm and a volume of 6.39 mm3 at a depth of 5 mm, resulting in a ∼10% difference in LN volume. This is not large enough to account for the differences we observed (slope = 1.45). Thus, other factors must also contribute, and accuracy of the absolute volume measurements attainable with these noninvasive approaches remains a limitation. Another potential source of error with the US measurement is the roughened surface of the LNs (Figures 1F, H, and J), which occurs due to the inability to fill surface holes. This should be addressable in the future with the evolution of superior surface rendering software applications.

The primary outcome measure using popliteal LNs as a biomarker is prediction of RA progression by popliteal LN volume enlargement, and we conclude that popliteal LNs found to be larger on MRI will also be shown to be larger on US imaging. The 3 largest and the 3 smallest LNs (Figure 1D) clearly differed from one another, and the rank order of size derived with each modality was the same for these 6 observations. The LNs in the middle of the range were close in size; therefore, the rank order of size may not correlate as consistently between MRI and US.

Although palpable draining LNs have long been recognized as a sign of RA, their value as a quantitative biomarker of disease initiation, arthritis flare, and response to therapy has only recently been recognized (7). However, if this biomarker is to be broadly utilized, a practical means for its assessment is needed, such as US, which can be readily performed during an office visit. For this reason, US imaging has recently been evaluated as an alternative to MRI to assess various musculoskeletal conditions. In some cases, such as detection of psoriatic arthritis of fingers and toes in patients with psoriasis (9) and detection of bone erosions in patients with gouty arthritis (10), US has been shown to be just as effective as MRI. However, in other cases, such as predicting the development of RA from undifferentiated peripheral inflammatory arthritis, MRI assessment of bone edema, synovitis, and erosion pattern has proved to be more useful (11).

In summary, US is comparable to MRI for determining relative popliteal LN volume in mice with inflammatory arthritis. Since it can be performed at less than 10% of the financial cost of MRI, we conclude that US is a quick, inexpensive, and reliable method to examine this biomarker of RA pathogenesis and response to therapy.

Acknowledgements

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  2. Acknowledgements

Supported by research grants from the NIH Public Health Service (grant AR-48697 to Dr. Xing and grants AI-78907, AR-54041, and AR-61307 to Dr. Schwarz). Dr. Schwarz has received consulting fees, speaking fees, and/or honoraria from Regeneron, MedImmune, Abbott, and Pfizer (less than $10,000 each) and DePuy and Smith & Nephew (more than $10,000 each), owns stock or stock options in Laget, and has served as an expert witness on behalf of Smith & Nephew.

  • 1
    Huh YM, Kim S, Suh JS, Song H, Song K, Shin KH. The role of popliteal lymph nodes in differentiating rheumatoid arthritis from osteoarthritis by using CE 3D FSPGR MR imaging: relationship of the inflamed synovial volume. Korean J Radiol 2005; 6: 11724.
  • 2
    Proulx ST, Kwok E, You Z, Beck CA, Shealy DJ, Ritchlin CT, et al. MRI and quantification of draining lymph node function in inflammatory arthritis. Ann N Y Acad Sci 2007; 1117: 10623.
  • 3
    Proulx ST, Kwok E, You Z, Papuga MO, Beck CA, Shealy DJ, et al. Longitudinal assessment of synovial, lymph node, and bone volumes in inflammatory arthritis in mice by in vivo magnetic resonance imaging and microfocal computed tomography. Arthritis Rheum 2007; 56: 402437.
  • 4
    Li J, Kuzin I, Moshkani S, Proulx ST, Xing L, Skrombolas D, et al. Expanded CD23(+)/CD21(hi) B cells in inflamed lymph nodes are associated with the onset of inflammatory-erosive arthritis in TNF-transgenic mice and are targets of anti-CD20 therapy. J Immunol 2010; 184: 614250.
  • 5
    Li J, Zhou Q, Wood R, Kuzin I, Bottaro A, Ritchlin C, et al. CD23+/CD21hi B cell translocation and ipsilateral lymph node collapse is associated with asymmetric arthritic flare in TNF-Tg mice. Arthritis Res Ther 2011; 13: R138.
  • 6
    Clavel G, Marchiol-Fournigault C, Renault G, Boissier MC, Fradelizi D, Bessis N. Ultrasound and Doppler micro-imaging in a model of rheumatoid arthritis in mice. Ann Rheum Dis 2008; 67: 176572.
  • 7
    Manzo A, Caporali R, Vitolo B, Alessi S, Benaglio F, Todoerti M, et al. Subclinical remodelling of draining lymph node structure in early and established rheumatoid arthritis assessed by power Doppler ultrasonography. Rheumatology (Oxford) 2011; 50: 1395400.
  • 8
    Keffer J, Probert L, Cazlaris H, Georgopoulos S, Kaslaris E, Kioussis D, et al. Transgenic mice expressing human tumour necrosis factor: a predictive genetic model of arthritis. EMBO J 1991; 10: 402531.
  • 9
    De Simone C, Caldarola G, D'Agostino M, Carbone A, Guerriero C, Bonomo L, et al. Usefulness of ultrasound imaging in detecting psoriatic arthritis of fingers and toes in patients with psoriasis. Clin Dev Immunol 2011; 2011: 390726.
  • 10
    Schlesinger N, Thiele RG. The pathogenesis of bone erosions in gouty arthritis. Ann Rheum Dis 2010; 69: 190712.
  • 11
    Machado PM, Koevoets R, Bombardier C, van der Heijde DM. The value of magnetic resonance imaging and ultrasound in undifferentiated arthritis: a systematic review. J Rheumatol Suppl 2011; 87: 317.