Longitudinal assessment of synovial, lymph node, and bone volumes in inflammatory arthritis in mice by in vivo magnetic resonance imaging and microfocal computed tomography

Abstract

Objective: To develop longitudinal 3-dimensional (3-D) measures of outcomes of inflammation and bone erosion in murine arthritis using contrast-enhanced magnetic resonance imaging (CE-MRI) and in vivo microfocal computed tomography (micro-CT) and, in a pilot study, to determine the value of entry criteria based on age versus synovial volume in therapeutic intervention studies.

Methods: CE-MRI and in vivo micro-CT were performed on tumor necrosis factor-transgenic (TNF-Tg) mice and their wild-type littermates to quantify the synovial and popliteal lymph node volumes and the patella and talus bone volumes, respectively, which were validated histologically. These longitudinal outcome measures were used to assess the natural history of erosive inflammatory arthritis. We also performed anti-TNF versus placebo efficacy studies in TNF-Tg mice in which treatment was initiated according to either age (4-5 months) or synovial volume (3 mm(3) as detected by CE-MRI). Linear regression was performed to analyze the correlation between synovitis and focal erosion.

Results: CE-MRI demonstrated the highly variable nature of TNF-induced joint inflammation. Initiation of treatment by synovial volume produced significantly larger treatment effects on the synovial volume (P = 0.04) and the lymph node volume (P < 0.01) than did initiation by age. By correlating the MRI and micro-CT data, we were able to demonstrate a significant relationship between changes in synovial and patellar volumes (R(2) = 0.75, P < 0.01).

Conclusion: In vivo CE-MRI and micro-CT 3-D outcome measures are powerful tools that accurately demonstrate the progression of erosive inflammatory arthritis in mice. These methods can be used to identify mice with arthritis of similar severity before intervention studies are initiated, thus minimizing heterogeneity in outcome studies of chronic arthritis seen between genetically identical littermates.

Figures

Figure 1. CE-MRI of the mouse knee

An anesthetized mouse positioned in the knee surface coil is shown prior to MRI scan (A). A pre-contrast (B), and post-contrast (C), image from a 5-month-old TNF-Tg mouse is shown demonstrating the high-resolution of the tibia (t), femur (f), synovium (s), popliteal lymph node (ln), and gastrocnemius muscle (m) from a sagital MR view. For 3D imaging and volumetric quantitation with Amira 3.1, the pre-contrast image is first registered to and subtracted from the post-contrast image (D). Then a limit line surrounding the synovium is manually drawn around the region of interest (ROI) on the post-contrast image, and copied to the subtracted image (D, yellow). The lymph node is manually segmented as it is clearly visualized on post-contrast images. This is performed on all slices encompassing the knee and lymph node and the segmented labels are reconstructed as volumes (E) for visualization and quantification. The threshold value to quantify synovial volume in the ROI is determined from the delivered dosage of Gd-DTPA. A dosage study was performed to establish a direct linear relationship between dosage and gastrocnemius muscle contrast enhancement (F). At constant synovial volume, the threshold used to segment synovial volume was curve fit to muscle contrast enhancement (G). This curve is used to threshold and segment enhanced synovial area in the drawn ROI from the surrounding tissues using muscle as a normalization tissue to determine delivered dosage of contrast agent.

Figure 2. 3D reconstruction and quantification of bone volume from micro-CT

A representative sagittal slice from a micro-CT scan of a wild type knee is shown to demonstrate the density-based segmentation that is performed on the bone to generate labels for patella (yellow), femur (light blue), tibia (dark blue) and menisci (red), as described in Materials and Methods (A). These labels are then used to reconstruct the bones in 3D (B). The patella (C), due to its ease of reconstruction and proximity to synovitis, is used as a quantification of bone volume at the knee joint. The ankle joint (D) is reconstructed in a similar manner as the knee with the talus (yellow and inset) as measure of bone volume in this joint.

Figure 3. Identification, quantification, and validation of synovial and lymph node volume as longitudinal outcome measures of inflammatory knee arthritis in mice

Data are presented from a representative 5-month-old TNF-Tg (A–D) and a WT (E–H) mouse. The post-contrast MRI are shown with enhancing synovium denoted by yellow arrows (A and E). Also of note in these MRI scans is the bone marrow edema in the TNF-Tg mouse, as seen by the high signal intensity in the bone marrow space (A), which is absent in the WT (E). Corresponding reconstructions and calculated synovial (yellow) and popliteal lymph node (red) volumes (B and F) are presented. The dramatic differences in these quantitative imaging biomarkers are validated in corresponding orange G/alcian blue stained histology sections at 40X (C and G) and 200X (D and H). The TNF-Tg mouse displays thickened synovial lining (#) and infiltration into subchondral bone (arrow head). TNF-Tg and WT mice (n=5) were scanned bimonthly from 2 months until 5 months of age, as described in Materials and Methods. The synovial (I) and LN (J) volumes for each scan were calculated, and the data from the TNF-Tg (dashed line) and WT (solid line) are plotted as the mean +/− standard deviation. Two-sided t-tests revealed significant (*, p

Figure 4. Effects of anti-TNF therapy in 5–6 month-old WT and TNF-Tg mice

WT and TNF-Tg mice were MR imaged at baseline and were randomized into anti-TNF and placebo treatment groups (n=4 per group) at 5–6 months of age. TNF-Tg mice were scanned every 4 weeks thereafter. WT mice received a further follow-up MRI 16-weeks after treatment. The synovial (A) and LN (B) volumes for each scan were calculated, and the data from the TNF-Tg placebo (pink long-dashed) vs. anti-TNF (blue solid) groups, and WT placebo (green intermittent-dashed) vs. anti-TNF (red short-dashed) groups, are plotted as the mean +/− standard deviation. Anti-TNF treatment had no effects in WT mice, and no changes in synovial or LN volume were detected in these animals after 16 weeks. In contrast, two-sided t-tests revealed significant (*, p<0.05) and highly significant (**, p<0.01) differences between anti-TNF vs. placebo TNF-Tg groups of the same time point after therapy. Linear mixed-effects regression analysis revealed a highly significant difference in slopes for anti-TNF versus placebo in both synovial volume (p<0.01) and LN volume (p<0.001). The linear progression of inflammatory arthritis (note decrease in synovial volume from 12 to 16 weeks in placebo group) was limited by tissue fibrosis, as shown by non-enhancing synovial regions on MRI (C, yellow arrows) and corresponding 40X histology (D, indicated by #) of a representative placebo TNF-Tg mouse. The degeneration of the pannus in the end-stage of arthritis in this animal is further illustrated by the decreased cellularity of the synovium (boxes in D) shown at 200X (E and F).

Figure 5. Effects of anti-TNF therapy in TNF-Tg mice with established synovitis

TNF-Tg mice (n=4) received bimonthly CE-MRI from 3-months of age until they reached a synovial volume above 3mm3. At this time they received an in vivo micro-CT scan and were randomized into anti-TNF and placebo treatment groups (n=4 per group). The mice were then scanned every 2 weeks until sacrifice at 8 weeks, when they received a follow-up micro-CT scan. The synovial (A) and LN (B) volumes for each scan were calculated, and the data from the placebo (dashed line) vs. anti-TNF (solid line) groups, are plotted as the mean +/− standard deviation. Two-sided t-tests revealed significant (*, p<0.05) and highly significant (**, p<0.01) differences between anti-TNF vs. placebo groups at the same time point after therapy. Linear mixed-effects regression analysis revealed a highly significant difference in slopes for anti-TNF versus placebo in both synovial (p<0.001) and LN (p<0.0001) volumes. The protective effects of anti-TNF therapy were also apparent from the Amira 3D reconstructions of pannus and LN, as shown in baseline (C) and 4 weeks (D) images from a representative mouse. Treatment effect was evaluated by a measure of the difference in slopes between anti-TNF and placebo groups. When treatment was initiated using synovial volume rather than age as the entrance criterion, there was a significantly larger treatment effect in synovial volume (E, 0.92 versus 0.37 mm3/week, p=0.04) and in lymph node volume (F, 1.26 versus 0.41 mm3/week, p=0.04).

Figure 6. Bone erosion is arrested with anti-TNF therapy and correlates with sustained synovial inflammation during TNF-induced arthritis

The change in cortical bone volume determined from the baseline and 8 week micro-CT scans for the anti-TNF (gray bars) and placebo (black bars) treated mice is presented as the mean +/−SD for the patella and talus (A). While there was no significant difference in delta patellar bone volume between groups (p=0.14), anti-TNF treatment had a significant effect on talus bone loss vs. placebo (*p