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

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Vol. 56, No. 12, December 2007, pp 4024–4037
DOI 10.1002/art.23128
© 2007, American College of Rheumatology
Longitudinal Assessment of Synovial, Lymph Node, and
Bone Volumes in Inflammatory Arthritis in Mice by
In Vivo Magnetic Resonance Imaging and
Microfocal Computed Tomography
Steven T. Proulx,1 Edmund Kwok,1 Zhigang You,1 M. Owen Papuga,1 Christopher A. Beck,1
David J. Shealy,2 Christopher T. Ritchlin,1 Hani A. Awad,1 Brendan F. Boyce,1
Lianping Xing,1 and Edward M. Schwarz1
measures were used to assess the natural history of
erosive inflammatory arthritis. We also performed antiTNF versus placebo efficacy studies in TNF-Tg mice in
which treatment was initiated according to either age
(4–5 months) or synovial volume (3 mm3 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 ⴝ 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 (R2 ⴝ 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.
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 (TNFTg) 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
Supported by research grants from Centocor and by grants
from the NIH (USPHS grants AR-43510, AR-46545, AR-48697,
AR-51469, AR-54041, and DE-17096).
Steven T. Proulx, MS, Edmund Kwok, PhD, Zhigang You,
MS, M. Owen Papuga, MS, Christopher A. Beck, PhD, Christopher T.
Ritchlin, MD, Hani A. Awad, PhD, Brendan F. Boyce, MD, Lianping
Xing, PhD, Edward M. Schwarz, PhD: University of Rochester,
Rochester, New York; 2David J. Shealy, PhD: Centocor Research and
Development, Radnor, Pennsylvania.
Dr. Shealy owns stock or stock options in Johnson & Johnson,
of which Centocor is a subsidiary. Dr. Ritchlin has received grants
from Centocor for research related to disease mechanisms of inflammatory arthritis. Dr. Boyce has received consulting fees, speaking fees,
and/or honoraria from Merck and Amgen (less than $10,000 each) and
from Ariad Pharmaceuticals (more than $10,000) and has provided
expert testimony for Ariad Pharmaceuticals. Dr. Schwarz has received
consulting fees, speaking fees, and/or honoraria (less than $10,000
each) from Centocor and Amgen, owns stock or stock options in
Amgen, and has provided expert testimony for Amgen.
Address correspondence and reprint requests to Edward M.
Schwarz, PhD, The Center for Musculoskeletal Research, University
of Rochester Medical Center, 601 Elmwood Avenue, Box 665, Rochester, NY 14642. E-mail:
Submitted for publication April 9, 2007; accepted in revised
form August 31, 2007.
Although murine models of inflammatory arthritis have significantly advanced our understanding of
erosive inflammatory arthritis (1,2), they are limited by a
lack of longitudinal translational outcome measures of
disease progression or interventional therapy. This issue
presents 3 problems for prototypical preclinical investigations of drug effects on inflammation, erosion, and
healing (3,4). First, most cross-sectional studies in mice
with “established arthritis” do not include an objective
assessment of disease severity prior to treatment. Thus,
neither rates of change nor healing responses can be
assessed. Second, the commonly used end points (i.e.,
histology and ex vivo molecular analyses) require the
death of the mice, thus markedly increasing the number
of animals needed to assess efficacy at multiple time
points. Third, although the incidence and severity of
arthritis vary among genetically identical littermates,
there are no established scoring criteria to stratify
different groups of mice based on disease activity in an
intervention study. Therefore, the development of imaging techniques that could assess disease activity and
progression in vivo would greatly enhance the utility of
these preclinical studies.
Magnetic resonance imaging (MRI) has become
the “gold standard” for the assessment of joint inflammation and damage in inflammatory arthritis (5–8).
Several studies have demonstrated the value of MRI in
the detection of synovial inflammation and bone marrow
edema before irreversible joint damage occurs (9–14).
While quantitative measures of these imaging biomarkers have been developed, their validation has been
difficult to perform during clinical studies (15–18). Conversely, while histologic analysis of arthritis in animal
models is readily available to validate imaging biomarkers, initial attempts to use this longitudinal in vivo
imaging modality in mice have fallen short of the desired
3-dimensional (3-D) quantitative outcome measure (19–
The purpose of the current study was to develop
and validate in vivo quantitative 3-D imaging biomarkers
of erosive inflammatory arthritis in mice. Although
many different animal models exist (1,2), we chose the
human tumor necrosis factor–transgenic (TNF-Tg)
mouse largely because it is a model of chronic disease of
known etiology that is completely ameliorated by antiTNF therapy (23). Furthermore, a critical role of TNF in
rheumatoid arthritis (RA) has been firmly established by
the success of treatment with TNF antagonists (24).
Thus, achievement of predicted outcomes in this wellestablished model serves as a validation of the novel 3-D
imaging biomarkers and has important translational
value as well, given the histologic findings that are
similar to those in RA in humans.
Using a custom-designed murine knee coil for
MRI, we have developed volumetric quantifications for
2 outcome measures: synovial inflammation and popliteal lymph node enlargement. We demonstrate the
progression of these biomarkers in the disease, as well as
the reversal of their progression after anti-TNF therapy.
We also validate these measurements with histologic
analyses and demonstrate significant correlations between MRI measurements and novel microfocal computed tomography (micro-CT) measurements of bone
Animals and anti-TNF treatment. The 3647 line of
TNF-Tg mice were originally obtained from Dr. G. Kollias
(Institute of Immunology, Biomedical Sciences Research Center Alexander Fleming, Vari, Greece) and are maintained as
heterozygotes in a CBA ⫻ C57BL/6 background (25). Experiments were performed with sex-matched TNF-Tg and wildtype (WT) littermate controls. The University of Rochester
Committee for Animal Resources approved all animal studies.
An initial natural history study examined TNF-Tg mice
and their WT littermates (n ⫽ 5 per group) from the age of 2
months until the age of 5 months. MRI scans were performed
twice a month at ⬃2-week intervals, except at 4 months of age
because of temporary technical problems with the MR scanner. At 5 months of age, mice were killed, and the knee joints
and popliteal lymph nodes were harvested for histology.
In the drug-intervention studies, mice received either
murine monoclonal anti-human TNF IgG1 antibody or an
irrelevant murine IgG1 placebo control (Centocor R&D,
Radnor, PA) at a dosage of 10 mg/kg/week by intraperitoneal
injection, as previously described (3). Two studies of anti-TNF
versus placebo were performed. Study 1 controlled for the age
of the mice, and study 2 controlled for MRI-based evidence of
The age-controlled study consisted of 4 groups of
5–6-month-old mice (n ⫽ 4 per group) at baseline. Groups 1
and 2 were WT littermates that received placebo or anti-TNF,
respectively. Groups 3 and 4 were TNF-Tg mice that received
placebo or anti-TNF, respectively. Treatments were administered for 16 weeks. MRI scans were performed at baseline and
every 4 weeks thereafter in the TNF-Tg groups, and at baseline
and 16 weeks in the WT groups. At 16 weeks, mice were killed,
and the knee joints and popliteal lymph nodes were harvested
for histologic assessment.
The synovitis-controlled anti-TNF study consisted of 2
groups, each containing 4 female TNF-Tg mice. These mice
were scanned every 2 weeks starting at 3 months of age and
were entered into the study when it was determined that they
had synovial volumes ⬎3 mm3 (see below). The mean ⫾ SD
age at initiation of treatment was 3.88 ⫾ 0.79 months. One
group received weekly anti-TNF injections, while the second
group was given placebo, as described above. MRI scans were
performed at baseline and every 2 weeks for 8 weeks. In vivo
micro-CT scans of the knees and ankles were performed at
baseline and at 8 weeks. At 8 weeks, mice were killed, and the
knee joints, ankle joints, and popliteal lymph nodes were
harvested for histologic assessment.
Contrast-enhanced MRI (CE-MRI). MR scans were
performed with a 3T Siemens Trio MRI (Siemens Medical
Solutions, Erlangen, Germany). Mice were anesthetized by
intraperitoneal injection of a ketamine/xylazine mixture at a
dosage of 110 ␮g/ml. Mice were then positioned on an imaging
platform with the right leg inserted through the custom
Figure 1. Contrast-enhanced magnetic resonance imaging (CE-MRI) of the mouse knee. A,
Anesthetized mouse positioned in the knee surface coil prior to MRI scanning. B and C,
Sagittal MR images of a 5-month-old tumor necrosis factor–transgenic mouse obtained
precontrast (B) and postcontrast (C). Note the high-resolution of the tibia (t), femur (f),
synovium (s), popliteal lymph node (ln), and gastrocnemius muscle (m). D and E, Threedimensional (3-D) imaging using Amira 3.1 software. For 3-D imaging and volumetric
quantitation, the precontrast image is first registered to and subtracted from the postcontrast
image (D). Then a limit line surrounding the synovium is manually drawn around the region
of interest (ROI) on the postcontrast image, and copied to the subtracted image (yellow line).
The lymph node is manually segmented as it is clearly visualized on postcontrast 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. F and G, Determination
of the threshold value used to quantify synovial volume in the ROI based on the delivered
dose of gadodiamide (Gd-DTPA-BMA). A dosage study was performed to establish a direct
linear relationship between the Gd-DTPA-BMA dose and contrast enhancement of the
gastrocnemius muscle (F). At constant synovial volume, the threshold used to segment the
synovial volume was curve-fitted to the contrast enhancement of the muscle (G). This curve
is used to threshold and segment the enhanced synovial area in the drawn ROI from the
surrounding tissues, using muscle as a normalization tissue to determine the delivered dose
of contrast agent.
designed knee coil (Figure 1A). The coil is composed of a
1.5-cm–diameter circular loop consisting of 2 parallel 14-gauge
copper wires. This design was found to give optimal signal-tonoise ratio (SNR) while providing sufficient volume coverage
of the joint. An imaging template assisted in reproducible
positioning between scans and between animals.
After a series of three 10-second localization scans, a
fat-suppressed T1-weighted high-resolution scan was performed (sagittal T1-weighted fast low-angle shot (FLASH)
sequence, consisting of a recovery time of 45 msec, an echo
time of 9.03 msec, a 192 ⫻ 192–pixel matrix, with a 20 ⫻
20–mm field of view, 32 slices of 0.16-mm slice thickness, a flip
angle of 25°, 1 signal average, and a scan time of 8 minutes 28
seconds). Gadodiamide (Gd-DTPA-BMA) contrast agent
(Omniscan; Amersham Health, Oslo, Norway) at a dose 0.500
ml/kg diluted in sterile saline was injected via the retroorbital
venous plexus. After 3 minutes to allow for Gd-DTPA-BMA to
equilibrate with the joint fluid, a second high-resolution scan
was performed to image the knee with contrast enhancement.
Imaging sessions took ⬃30 minutes per mouse.
MRI analysis. Amira 3.1 software (TGS/Mercury
Computer Systems, San Diego, CA) was used for image
segmentation and volume computations of synovium and
popliteal lymph nodes. The 3-D stacks of images for the
precontrast (Figure 1B) and postcontrast (Figure 1C) scans
were loaded into the software. An automatic registration
module (Registration 3 LineSearch 3 Correlation) was used
to align the images in 3-D. The precontrast scan was then
subtracted from the postcontrast scan using the arithmetic
module, resulting in a 3-D stack of images of contrast enhancement (Figure 1D).
A segmentation and threshold procedure using the
Amira Segmentation Editor was used to determine synovial
and lymph node volumes. The editor allows the user to create
3-D “labels” of each tissue of interest. First, limit lines
corresponding to the regions of interest (ROIs) enclosing the
synovium, but excluding enhancing tissues such as the subcutaneous layer and the popliteal vessels, were drawn on each
slice. For visualization purposes, these lines were drawn on the
postcontrast image stack, and the labels were copied onto the
subtracted image. Next, a section (⬎15 mm3) of muscle tissue
was labeled on the subtracted image stack. The TissueStatistics
module was used to determine the contrast enhancement of
the muscle as an estimate of the delivered dosage of GdDTPA-BMA.
Based on the level of muscle enhancement, a threshold
value corresponding to synovial enhancement was found using
the equation determined by a dosage study (see next section).
All voxels above the threshold value within the limit lines were
labeled as synovium. Enhanced voxels above this threshold
that were within the bone marrow were then subtracted from
the label.
The lymph node, which is much easier to visualize than
synovium, was segmented by manually drawing ROIs on
postcontrast images and were thresholded based on signal
intensity ⱖ1,500 arbitrary units (AU) to define the boundary
between the lymph node and the fat pad surrounding the node.
These labels were also copied onto the subtracted image.
Rendering as a 3-D image (Figure 1E) was performed by
applying the SurfaceGen module to reconstruct the tissue
labels into volumes, and the SurfaceView module was used for
visualization. The TissueStatistics module was used to quantify
the volumes of the tissues. Total time for image analysis by an
experienced operator was ⬃20 minutes per mouse.
To determine the threshold parameters for longitudinal analyses of synovial volume, we performed a dose-response
study in which 4 TNF-Tg mice (3–5 months old) were scanned
on 3 consecutive days using incremental dosages of Gd-DTPABMA (dose 1 ⫽ 0.167 ml/kg, dose 2 ⫽ 0.333 ml/kg, and dose
3 ⫽ 0.500 ml/kg). The concentrations of contrast agent in
saline were adjusted, such that the bolus injected into each
animal was at a constant volume between doses. After 24
hours, no trace of contrast agent was detected on the precon-
trast scans obtained on day 2 or day 3 in the animals. A strong
linear relationship between muscle enhancement and dosage
was found using a linear mixed-effects regression model (R2 ⫽
0.87) (Figure 1F). Data from dose 2 were analyzed with the
above-described method; however, an initial synovial threshold
equal to muscle enhancement plus 1,000 AU was used to
generate a synovial volume. The data from day 1 and day 3
were then analyzed, and the threshold value needed to generate the same synovial volume was recorded. The threshold
values versus muscle enhancement values for all 4 mice at all 3
doses fit the increasing form of a 2-phase exponential-decay
curve (Figure 1G), with R2 ⫽ 0.98. The values from this curve
were used to determine synovial threshold values that were
adjusted for Gd-DTPA-BMA dosage as measured by contrast
enhancement of muscle.
Reproducibility for synovial and lymph node volume
measurements was assessed for intrareader, interreader, and
inter-MRI variations. For determining intrareader variation,
10 CE-MRI scans (5 TNF-Tg and 5 WT mice) were analyzed
by the same operator (STP) on 2 different days in random
order. The same 10 scans were analyzed by a second operator
(MOP) after a training period of 2 hours to determine
interreader variation. For inter-MRI variation, 5 TNF-Tg mice
with disease of varying severity were scanned twice within 48
hours, and the same operator analyzed the scans in random
Volumetric assessment of bone erosion via micro-CT.
High-resolution in vivo micro-CT was used to scan the mouse
knee and ankle joints (VivaCT 40; Scanco, Southeastern, PA).
Animals were anesthetized with isoflurane. Each joint was
scanned at an isotropic resolution of 17.5 ␮m in a custom
sample holder at 55 keV, with cone beam mode. The data were
reconstructed via Scanco software into Dicom files for analysis.
Amira 3.1 software was used to segment and visualize the
bones of the knee and ankle on the micro-CT scans. For the
knee, the Segmentation Editor was used to label the femur,
tibia, patella, and menisci (Figure 2A). A density threshold
⬎11,000 AU was set as representing “bone,” and the labels
were reconstructed using the SurfaceGen module to visualize
the joint (Figure 2B). The threshold was kept constant
throughout the study. Since the entire patella is scanned, the
volume of this bone, as determined from the TissueStatistics
module, was used as a quantitative measure of bone erosion
for the knee (Figure 2C). For the ankle, the talus was
specifically labeled, and the volume of this bone was used as
the measure of erosion in this joint (Figure 2D). Analysis
typically takes an experienced operator ⬃15 minutes per joint.
Histologic assessment. The right knee joint was removed and fixed in 10% neutral buffered formalin. The joints
were then decalcified in 14% EDTA at room temperature (pH
adjusted to 7.2) for 21 days. The joint was then carefully
embedded in paraffin for sectioning into 3-␮m slices. Sections
were then stained with orange G–Alcian blue for histologic
examination. The popliteal lymph node was also harvested and
prepared in a similar manner, omitting the decalcification step.
The lymph node area was quantified using ImageJ software
(NIH Image, National Institutes of Health, Bethesda, MD;
online at: at the slice found to have
maximal cross-sectional area.
Statistical analysis. Linear mixed-effects regression
models, with mouse as a random effect and time (treated as a
analysis of variance models. All underlying assumptions of the
parametric methods were checked, and no serious violations
were detected. P values less than 0.05 were considered significant, and P values less than 0.01 were considered highly
Figure 2. Three-dimensional reconstruction and quantification of
bone volume from microfocal computed tomography (micro-CT)
imaging. A, A representative sagittal slice from a micro-CT scan of the
knee of a wild-type mouse, demonstrating the density-based segmentation that is performed on the bone to generate labels for the patella
(yellow), femur (light blue), tibia (dark blue), and menisci (red), as
described in Materials and Methods. B, Reconstruction of the bones in
3 dimensions. The labels generated from the micro-CT sagittal slice
are then used to reconstruct the bones in 3 dimensions. C, Threedimensional image of the patella. Due to its ease of reconstruction and
proximity to inflamed synovium, the volume of the patella is used as a
quantitative measure of bone volume at the knee joint. D, Threedimensional image of the ankle joint. The ankle joint is reconstructed
in a manner similar to that of the knee. The volume of the talus
(yellow) is used as a quantitative measure of bone volume in the ankle
joint. Inset, Three-dimensional view of the talus, rotated and enlarged.
continuous covariate) as a fixed effect, were used to assess
changes over time based on longitudinal data. Similar models
used age or dose instead of time to assess changes over age or
linear dose-response relationships based on repeated measurements. Analyses based on cross-sectional data used standard
linear regression models. A nonlinear mixed-effects model,
with a random effect for mouse to account for the repeated
measures design, was used to fit the 2-phase exponential-decay
curve. Two-sided t-tests assuming unequal variances were used
to make comparisons with the micro-CT data or with the
CE-MRI data between groups at the same time point or age.
Correlations between measures were estimated using Pearson’s correlation coefficient and were tested for significance
using a 2-sided t-test. Interreader, intrareader, and inter-MRI
reliability was estimated using coefficients of variation and
intraclass correlation coefficients based on random-effects
Longitudinal CE-MRI biomarkers of TNFinduced arthritis in mice. High-resolution MR images
of mouse knees clearly defined contrast-enhanced inflamed synovium and enlarged lymph node in TNF-Tg
mice (Figure 3A) and permitted volumetric quantification (Figure 3B). These findings were absent in WT
littermates (Figures 3E and 3F). The remarkable differences between the joints of the TNF-Tg and WT mice
were confirmed histologically (Figures 3C and D and
Figures 3G and H, respectively).
Before we used these novel imaging outcome
measures in prospective studies, we assessed the validity
of our lymph node volume calculations by demonstrating
correlations with the maximum lymph node area as
determined by MRI and histology. (Figures illustrating
the validation of the CE-MRI lymph node volume
measurements by correlation with the 2-D MRI and
histology are available upon request from the corresponding author). Linear regression analyses demonstrated significant relationships (P ⬍ 0.0001) between
the 2-D and 3-D measurements and substantiated the
validity of our volumetric CE-MRI measurements for
use in outcomes studies.
Intrareader, interreader, and inter-MRI reliability was assessed with intraclass correlation coefficient
(ICC) analysis and found to be excellent for all variables.
Synovial volume measurements were found to have ICC
values of 0.996 for intrareader, 0.984 for interreader,
and 0.968 for inter-MRI reliability. Lymph node volume
quantifications had ICC values of 0.999, 0.997, and
0.994, respectively. Additionally, the inter-MRI reliability was assessed with a coefficient of variation and found
to be 4.53% for synovial volume and 3.95% for lymph
node for n ⫽ 5 TNF-Tg mice.
In our first longitudinal study of 3-D CE-MRI
biomarkers of inflammatory arthritis in mice, we assessed the progression of disease in 2 month-old
TNF-Tg versus WT littermates over a 3-month period.
Figure 3I shows that TNF-induced knee synovitis began
at ⬃3 months of age and steadily increased thereafter
(slope ⫽ 0.60 mm3/month; P ⬍ 0.0001). In contrast, the
synovial volume in the WT mice significantly decreased
over time (slope ⫽ –0.26 mm3/month; P ⫽ 0.01), likely
Figure 3. Identification, quantification, and validation of synovial and lymph node volume
as longitudinal outcome measures of inflammatory knee arthritis in mice. A–D, A
representative 5-month-old transforming growth factor–transgenic (TNF-Tg) mouse. E–H,
A 5-month-old wild-type (WT) littermate. Postcontrast magnetic resonance images show
enhancing synovium (arrows). Bone marrow edema is present in the TNF-Tg mouse, as
indicated by the high signal intensity in the bone marrow space (A), but is absent in the WT
mouse (E). Corresponding 3-dimensional reconstructions and calculated synovial (yellow)
and popliteal lymph node (red) volumes (B and F) are also shown. The dramatic differences
in these quantitative imaging biomarkers are validated in the corresponding orange
G–Alcian blue–stained histology sections shown at 40⫻ magnification (C and G); boxed
areas of C and G are shown at 200⫻ magnification (D and H, respectively). The TNF-Tg
mouse displays thickened synovial lining (#) and infiltration into subchondral bone
(arrowhead). I and J, Disease progression in TNF-Tg and WT mice as a function of synovial
volume (I) and lymph node volume (J). Mice were scanned bimonthly from the age of 2
months until the age of 5 months (see Materials and Methods), and the synovial and lymph
node volumes for each scan were calculated. Values are the mean ⫾ SD of 5 mice per group.
ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01, versus WT mice of the same age, by 2-sided t-test. Linear
mixed-effects regression analysis revealed a highly significant difference in the slopes for
both the synovial and lymph node volumes in TNF-Tg mice versus WT mice (P ⬍ 0.0001 for
both comparisons).
due to growth effects. This resulted in a highly significant difference in slope for the TNF-Tg mice versus the
WT mice (P ⬍ 0.0001). While a highly significant
difference in synovial volume between TNF-Tg and WT
mice was observed at 4.5 months (mean ⫾ SD 3.32 ⫾
0.93 mm3 versus 1.44 ⫾ 0.42 mm3; P ⬍ 0.01), the
significance of this difference decreased at 5 months due
to the increased variability in the TNF-Tg group (4.26 ⫾
1.57 mm3 versus 1.89 ⫾ 0.41 mm3; P ⬍ 0.05).
Interestingly, TNF-induced changes in popliteal
lymph node volumes preceded knee synovitis (Figure
3J), since the steadily significant increase (slope ⫽ 2.95
mm3/month; P ⬍ 0.0001) in TNF-Tg animals began at
the age of 2 months (2.43 ⫾ 0.76 mm3) and plateaued at
the age of 4.5 months (10.17 ⫾ 5.69 mm3). Despite the
large variability at the end of the study, we still observed
a highly significant 10-fold difference in lymph node
volume between the TNF-Tg mice and the WT mice at
5 months (10.41 ⫾ 5.57 mm3 versus 1.20 ⫾ 0.34 mm3;
P ⬍ 0.01). As expected, the popliteal lymph node
volume in the WT mice did not change throughout the
study (slope ⫽ –0.10 mm3/month; P ⫽ 0.71). However,
we detected a highly significant difference in slope for
the lymph node volume in TNF-Tg mice versus WT mice
(P ⬍ 0.0001).
Changes in longitudinal 3-D biomarkers of erosive inflammatory arthritis following effective anti-TNF
therapy. In order to validate the CE-MRI and micro-CT
3-D outcome measures as in vivo biomarkers of erosive
inflammatory arthritis in an intervention study, we used
a model of known etiology (TNF-Tg) (23,25), and a
proven treatment (anti-TNF) whose efficacy in this
model has been demonstrated by several groups of
investigators (3,23,26). Since we observed that the onset
of knee synovitis in TNF-Tg mice varies from 3 months
to 5 months of age (Figure 3I), we aimed to determine
whether entering the mice into the study based on
synovial volume rather than age would improve the
statistical outcome of a small number of animals.
Initiation of therapy based on age. In a traditional
intervention study based on age, we randomized 5–6month-old TNF-Tg and WT mice to receive either
anti-TNF or placebo treatment (Figure 4). The baseline
CE-MRI data for the TNF-Tg mice demonstrated remarkable variability in synovial volume (5.66 ⫾ 2.46
mm3) and lymph node volume (9.14 ⫾ 3.12 mm3). Due
to this variability, we did not observe a significant
decrease in synovial volume with anti-TNF treatment
(slope ⫽ –0.17 mm3/week; P ⫽ 0.06), despite a 44%
reduction from baseline values at 16 weeks (Figure 4A).
However, in comparison with placebo treatment
(slope ⫽ 0.20 mm3/week; P ⫽ 0.02), the difference in
slope was highly significant (P ⬍ 0.01).
In contrast, we observed a significant difference
in lymph node volume at the first time point following
treatment (3.71 ⫾ 2.11 mm3 in the anti-TNF group
versus 8.91 ⫾ 1.50 mm3 in the placebo group; P ⬍ 0.01).
Overall, anti-TNF therapy demonstrated a significant
67% reduction from baseline (slope ⫽ –0.33 mm3/week;
P ⬍ 0.0001) by the end of the study (Figure 4B).
Consistent with the lymph node volume plateau observed in the natural history study after 4.5 months
(Figure 3H), no changes were observed in placebotreated TNF-Tg mice over the course of this study
(slope ⫽ 0.08 mm3/week; P ⫽ 0.30), but the slopes for
placebo versus anti-TNF treatment were highly significantly different (P ⬍ 0.001). No changes or drug effects
were observed in the WT groups.
One surprising outcome of this study was the
synovial volume peak at 12 weeks (122% increase from
baseline), which decreased to a 52% change from baseline at 16 weeks (Figure 4A). To better understand this,
we investigated the areas of decreased Gd-DTPA-BMA
Figure 4. Effects of anti–tumor necrosis factor (anti-TNF) therapy in
5–6-month-old TNF-transgenic (TNF-Tg) and wild-type (WT) mice.
Mice underwent contrast-enhanced magnetic resonance imaging (CEMRI) at baseline and were then randomized to receive anti-TNF or
placebo treatment. TNF-Tg mice underwent CE-MRI every 4 weeks
thereafter; WT mice underwent CE-MRI at 16 weeks after treatment.
A and B, Synovial and lymph node volumes, respectively. Data from
each scan of the TNF-Tg placebo-treated (pink line) versus anti-TNF–
treated (blue line) groups and of the WT placebo-treated (dark green
line) versus anti-TNF–treated (red line) groups were calculated and
plotted. Note the decrease in synovial volume between 12 and 16
weeks in the placebo-treated TNF-Tg group. Anti-TNF treatment had
no effects in WT mice, and no changes in synovial or lymph node
volumes were detected in these animals after 16 weeks. Values are the
mean ⫾ SD of 4 mice per group. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01, versus
placebo-treated TNF-Tg mice at the same time point, by 2-sided t-test.
Linear mixed-effects regression analysis revealed a highly significant
difference in the slopes for both the synovial (P ⬍ 0.01) and lymph
node (P ⬍ 0.001) volumes in anti-TNF–treated versus placebo-treated
TNF-Tg mice. C–F, CE-MR image (C) and tissue sections (D–F) from
the knee of a representative placebo-treated TNF-Tg mouse. The
linear progression of inflammatory arthritis was limited by tissue
fibrosis, as shown by nonenhancing synovial regions on CE-MRI
(arrows) and on the corresponding histology section (#) shown at 40⫻
magnification (D); boxed areas at the bottom and top of C are shown
at 200⫻ magnification (E and F, respectively), illustrating pannus
fibrosis during the end-stage of arthritis in this animal.
enhancement (Figure 4C), with corresponding histologic
assessment of the knees from these mice, and we found
large regions of pannus fibrosis (Figures 4D–F). This
Figure 5. Effects of anti–tumor necrosis factor (anti-TNF) therapy in TNF-transgenic
(TNF-Tg) mice with established synovitis. Mice underwent contrast-enhanced magnetic
resonance imaging (CE-MRI) bimonthly from 3 months of age until a synovial volume ⬎3
mm3 was achieved. They then underwent in vivo microfocal computed tomography
(micro-CT) scanning and were randomized to receive anti-TNF or placebo treatment. Mice
underwent CE-MRI every 2 weeks for 8 weeks, when a followup micro-CT scan was
performed. A and B, Synovial and lymph node volumes, respectively. Data from each scan
were calculated and plotted. Values are the mean ⫾ SD of 4 mice per group. ⴱ ⫽ P ⬍ 0.05;
ⴱⴱ ⫽ P ⬍ 0.01, versus placebo-treated mice at the same time point, by 2-sided t-test. Linear
mixed-effects regression analysis revealed a highly significant difference in the slopes for
both the synovial (P ⬍ 0.001) and lymph node (P ⬍ 0.0001) volumes in anti-TNF–treated
versus placebo-treated mice. C and D, Three-dimensional (3-D) reconstructions and
calculated synovial (yellow) and popliteal lymph node (red) volumes at baseline (C) and
after 8 weeks of anti-TNF therapy (D). The protective effects of anti-TNF therapy are also
apparent from these 3-D reconstructions of images from a representative mouse. E and F,
Treatment effect evaluated by a measure of the difference in the slopes between treatment
groups. When treatment was initiated based on synovial volume rather than age as the entry
criterion, there was a significantly larger treatment effect on the synovial volume (E) (0.92
mm3/week versus 0.37 mm3/week; P ⫽ 0.04) and the lymph node (LN) volume (F) (1.26
mm3/week versus 0.41 mm3/week; P ⫽ 0.04). Values are the mean ⫾ SD of 4 mice per group.
fibrosis is consistent with a loss of CE-MRI signal
enhancement as a result of the loss of vascularity.
Initiation of therapy based on synovial volume. To
determine if a more significant therapeutic effect of
anti-TNF therapy on synovial and lymph node volumes
could be observed in TNF-Tg mice with established
arthritis, we repeated the placebo-controlled study by
randomizing the animals to the treatments at the point
Figure 6. Arrested bone erosion after anti–tumor necrosis factor (anti-TNF) therapy and
correlation with sustained synovial inflammation in TNF-transgenic (TNF-Tg) mice. A,
Change in cortical bone volume of the patella and talus, as determined from the baseline
and 8-week microfocal computed tomography (micro-CT) scans in the anti-TNF and
placebo treatment groups. While there was no significant difference in the change in patellar
bone volume between groups (P ⫽ 0.14), anti-TNF treatment had a significant effect on
bone loss in the talus (ⴱ ⫽ P ⬍ 0.02). Values are the mean ⫾ SD of 4 mice per group. B and
C, Area under the curve (AUC) measurement as a function of joint inflammation and time
(B) and regression analysis of change in patellar volume versus synovial AUC (C). The
contrast-enhanced magnetic resonance imaging data for the synovial volume in a representative placebo-treated TNF-Tg mouse (Figure 5) were plotted to show the AUC measurement as a function of joint inflammation and time. This measurement was then used to
perform regression analyses of the change in patellar volume versus the synovial volume
AUC in all 8 mice in the study described in Figure 5. D–I, Micro-CT reconstructed images
of the patella at baseline (D) and at 8 weeks (E), with accompanying histology section (F)
(at 10⫻ magnification) and of the talus at baseline (G) and at 8 weeks (H), with
accompanying histology section (I) (at 4⫻ magnification). Boxed area in I shows the area of
severe synovitis in the talus, which had reduced the volume of bone to 20% of its original
size. Erosion data from the representative placebo-treated TNF-Tg mouse shown in B were
used to reconstruct the images shown in D, E, G, and H.
when CE-MRI first showed a knee synovium volume of
⬎3 mm3. This value represents a 50% increase over that
in WT mice at 3–5 months of age, confirming the
presence of synovitis (Figures 3I and 4A). In this study,
anti-TNF therapy had dramatic effects on synovial volume (Figure 5A) and lymph node volume (Figure 5B)
over time as compared with placebo.
Synovial volumes did not demonstrate a significant decrease during the course of the 8-week study with
anti-TNF treatment (slope ⫽ –0.20 mm3/week; P ⫽
0.21). However, this was due to the rapid response in
these animals to anti-TNF therapy, reaching levels in the
WT mice within 4 weeks, as visualized in volumetric
reconstructions of the baseline (Figure 5C) and 4-week
(Figure 5D) data from a representative animal. A significant decrease (–49%) from baseline to 4 weeks
(slope ⫽ –0.50 mm3/week; P ⫽ 0.01) was found, and
there was no further change from 4 weeks to 8 weeks
(3%) (slope ⫽ 0.03; P ⫽ 0.91). In contrast, synovial
volumes in the placebo-treated mice showed a highly
significant increase (169%) throughout the 8 weeks of
study (slope ⫽ 0.72 mm3/week; P ⬍ 0.0001). This
resulted in highly significantly different slopes for placebo versus anti-TNF treatment (P ⬍ 0.001). Another
advantage of this study design is that no pannus fibrosis
effects were seen in any of the mice.
Lymph node volume significantly decreased
(–73%) with anti-TNF therapy (slope ⫽ –0.84 mm3/
week; P ⬍ 0.0001) (Figure 5B). Lymph node volumes
also showed a highly significant increase (60%) with
placebo therapy (slope ⫽ 0.43 mm3/week; P ⬍ 0.01).
This resulted in highly significantly different slopes for
placebo versus anti-TNF treatment (P ⬍ 0.0001). Interestingly, the lymph node volume had wide variability in
placebo-treated mice throughout the study (Figure 5B),
suggesting that synovial inflammation and lymph node
volume are not directly linked.
More significant treatment effect with therapy initiated based on constant synovial volume rather than with
therapy initiated based on age. The difference in slopes of
volume versus time between anti-TNF and placebo
treatment groups is a measure of the treatment effect of
anti-TNF therapy. Initiation of treatment based on
synovial volume resulted in a significantly larger treatment effect on synovial volumes than initiation based on
age alone (difference in slopes of 0.92 mm3/week versus
0.37 mm3/week; P ⫽ 0.04) (Figure 5E). The treatment
effect on lymph node volume was also significantly
larger when the entry criterion was based on synovial
volume as opposed to age (difference in slopes of 1.26
mm3/week versus 0.41 mm3/week; P ⬍ 0.01) (Figure 5F).
Effect of anti-TNF therapy on bone erosion. By
incorporating longitudinal micro-CT analysis into this
study, in which focal erosions were assessed by subtracting the baseline bone volumes from the 8-week bone
volumes, we were able to evaluate changes in bone
volume of the patella and talus in TNF-Tg mice treated
with anti-TNF or placebo (Figure 6A). While half of the
placebo-treated mice had a decreased patella volume
due to erosions and all of the anti-TNF–treated mice
showed an increase or no change in patellar volume,
these changes were small and failed to demonstrate a
significant drug effect (P ⫽ 0.14). However, all of the
placebo-treated mice had a markedly decreased talus
volume, and 75% of the anti-TNF–treated mice showed
an increase in the size of this bone, which resulted in a
significant difference between these groups (P ⬍ 0.02).
Correlation of longitudinal 3-D biomarkers of
inflammation and bone erosion. As a final validation of
our 3-D biomarkers, we assessed the relationship between synovial inflammation and bone erosion using the
data from the study of the initiation of treatment based
on synovial volume (n ⫽ 8 mice). Since the erosion data
are a function of the change in bone volume over time,
we first had to convert the synovial volume data to
reflect the severity of synovitis over the study period.
This was done by computing the area under the curve
from the plot of the synovial volume versus time for each
animal (Figure 6B). Using this approach, we found a
highly significant correlation between our measures of
synovial volume and patellar erosion (R2 ⫽ 0.7469, P ⬍
0.01) (Figure 6C). The impact of unchecked synovitis on
bone was demonstrated by a longitudinal analysis of the
patella (Figures 6D and E). The dramatic loss of bone as
quantified by micro-CT was associated with widespread
synovial infiltration into the bone as visualized in the
corresponding histology section (Figure 6F). An even
more dramatic loss of bone was present at the talus
(Figures 6G and H), in which severe synovitis, as visualized histologically (Figure 6I), had reduced the volume
of bone to 20% of its original size.
In vivo imaging measurements have emerged as
the outcome of choice for translational research in
preclinical studies, based on their potential to objectively
quantify change and their compatibility with modalities
used in clinical trials (27). Since MRI and micro-CT are
widely accepted as the “gold standards” for the assessment of soft tissue and bone volumes, respectively, we
aimed to adapt these methods as longitudinal outcome
measures of erosive inflammatory arthritis in mice. In
the case of CE-MRI for the mouse knee, several innovations were required (Figure 1). These included first,
the generation of a mouse knee–specific coil that can
interface with a clinical 3T MRI; second, the establishment of pulse sequences that produce high-resolution
images (105 ␮m) with minimal slice thickness (160 ␮m)
to reduce partial volume effects; third, a method by
which to normalize for the variability of Gd-DTPABMA administration using muscle contrast enhancement; and fourth, standardization for thresholding and
segmentation of biomarkers for longitudinal quantitative 3-D analyses. Although commercial small-animal
MRI instruments may become more popular in the
future, we chose to use a clinical MRI to ensure that all
of the biomarkers we identified could be used to study
arthritis in humans with readily available pulse sequences. In addition, the quantification methods developed could easily be adapted to data collected with
small-animal scanners.
Several quantification methods have been developed to assess synovial volumes in humans by CE-MRI
(28,29). However, no quantification methods have been
established for clinical trials, for which there is a great
demand. The currently accepted evaluation determined
by the Outcome Measures in Rheumatology Clinical
Trials Group is the RA MRI Scoring system RAMRIS,
which consists of a semiquantitative global scoring system (scores of 0–3) based on synovial tissue thickness
and Gd-DTPA enhancement (30). The current gold
standard for quantification of synovitis is a manual
segmentation technique (14,31). Although this technique has been validated in intervention studies and has
demonstrated correlation with an aspirated volume of
synovial fluid, the time required for analysis (0.75–2
hours) and the technical expertise necessary have limited its use in clinical trails. To address these limitations,
automated segmentation methods have been evaluated
(32,33). This 2-step segmentation process consists of a
limited manual segmentation to remove enhancing vessels and skin (similar to the limit lines used in the current
study) and application of a threshold on subtracted
Methods by which to threshold enhancing synovial tissue remain a subject of controversy. Østergaard et
al (32) evaluated several different thresholds based on
the percentage of enhancement of synovium compared
with the manual segmentation technique and determined that a 45% enhancement threshold was optimal.
Although this reduced the time required for analysis to
20 minutes, there was increased inter-MRI variation as
compared with manual segmentation techniques, particularly when misalignment occurred between precontrast
and postcontrast scans. Palmer et al (33) used a threshold value based on the signal intensity differences between several regions of nonenhancing tissue (suppressed fat) and enhancing pannus. This method has an
advantage in that the threshold is determined after each
individual scan; however, the time required for analysis
is ⬃45 minutes. The threshold is also sensitive to misregistration artifacts and the consistency of fat suppression.
To our knowledge, the current study is the first to
use adjacent muscle as a normalization tissue with which
to determine the synovial threshold. This approach is
attractive because muscle is an enhancing tissue that is
not implicated in the pathologic changes of inflammatory arthritis and because its tissue properties do not
change significantly between scans; variations in values
in muscle will reflect the signal variations that are
present between scanning sessions. In small-animal studies, this approach is especially warranted due to the
inconsistencies in dosage delivery that are inherent
during administration via intravenous injection. During
our dosage study, we found that muscle was linearly
enhanced with increasing dosages of Gd-DTPA-BMA.
Even more importantly, there was a direct relationship
between muscle contrast enhancement and the synovial
threshold that was used to maintain a constant synovial
volume between doses. The use of this optimized threshold, combined with the ability to minimize motion
artifacts with anesthesia and standardized positioning,
has allowed reproducible measurements of synovial volume in mice (4.5% coefficient of variation) that far
exceed those reported in humans (22% median relative
variation as measured by the manual segmentation
method [34]). Whether a similar threshold approach
based on muscle enhancement could be adapted to
clinical studies warrants further investigation.
Considering that micro-CT has been used to
examine focal erosions in murine arthritis models for
several years (35), it is somewhat surprising that this
approach has yet to evolve into a longitudinal quantitative outcome measure. Based on our experience, we
found that this is likely due to the difficulty in registering
the baseline and outcome 3-D micro-CT images so that
the erosion as a negative change in volume could be
accurately assessed. It is also clear from our work
(Figure 2), that segmentation can only be readily performed on small bones that are clearly defined by
soft-tissue boundaries (e.g., the patella and talus). This is
because minor imperfections in the registration of large
bones and subjective segmentation of bone parts (e.g.,
the distal femur and proximal tibia) can result in significant measurement errors.
In this study, we focused our attention on the
synovial and lymph node volume as the primary outcome measures of inflammation, based on their facile
segmentation and quantification from CE-MR images.
Interestingly, our findings indicated that these tissues
behave differently during the onset of inflammatory
arthritis and its amelioration following effective therapy.
We found that the popliteal lymph node volume was the
most sensitive biomarker of lower limb arthritis. This
conclusion comes from the observation that lymph node
volumes significantly increased when TNF-Tg mice were
2.5 months-old (Figure 3H), which correlated with the
point at which increased TNF serum levels and changes
in peripheral blood mononuclear cell populations are
first detected in this model (36).
Our finding that increases in lymph node volume
precede the occurrence of knee synovitis is consistent
with the function of popliteal lymph nodes in draining
both the knee and ankle joint tissues and the fact that
arthritis first occurs in the ankle joint in this model (23).
This conclusion is corroborated by similar results in the
K/BxN serum-transfer model of arthritis (37), in which
the popliteal lymph node volume increased concurrently
with ankle inflammation in the absence of knee synovitis. (Figures illustrating the assessment of K/BxN seruminduced synovitis and lymph node inflammation by
longitudinal CE-MRI are available upon request from
the corresponding author.) Thus, the introduction of
proinflammatory mediators into the joint alone is not
sufficient for the initiation of pannus formation.
Our future efforts will focus on understanding
the mechanism that initiates this destructive process.
This issue is paramount, since synovitis correlates with
joint destruction in inflammatory arthritis (Figure 6C).
In addition, many group of investigators have generated
transgenic animals that can be used to dissect these
pathologies (35,38–41), and these transgenic animals
can now be assessed with our novel longitudinal outcome measures.
By using a validated drug therapy in our model,
we were able to address the major concern with all
preclinical intervention studies of arthritis that are performed with a single cross-sectional end point, namely,
whether or not the differences observed are due to drug
effects or to variability between the initiation and/or
progression of disease in the individual mice. Using our
previous study of anti-TNF treatment of TNF-Tg mice
as an example (3), we were unable to draw definitive
conclusions about the healing effects on bone and
cartilage lesions since there were no baseline assessments. When we repeated this experiment using age as
the enrollment criterion (Figure 4), these concerns
regarding interanimal variability were realized based on
the broad range of baseline synovitis levels (3.00–9.85
mm3). Furthermore, during this study, we saw a dramatic decrease in synovial volume in the placebo group
from age 8 months to age 9 months that was due to
pannus tissue fibrosis.
Thus, by modifying the study design to include an
entry criterion based on synovial volume (Figure 5), we
were able to make several remarkable observations.
First, we found that the treatment effect of anti-TNF
therapy on synovial volume and lymph node volume is
significantly greater than when the entry criterion is
based on age. Second, we noted that synovial volumes
were significantly reduced to levels in the WT mice after
only 4 weeks of anti-TNF therapy. Third, we found that
initiation of the study earlier in the disease process
combined with an 8-week time course avoided the
pannus tissue fibrosis effects previously observed.
Fourth, a healing response was noted in which the mean
lymph node volumes in the TNF-Tg mice treated with
anti-TNF therapy were reduced but were still 2.5 times
higher than those in the WT mice at the end of the study.
Fifth, we found that bone erosion was arrested, as
demonstrated by a significant difference in the change in
talus volume between anti-TNF–treated and placebotreated animals.
In summary, we found that CE-MRI and
micro-CT are very useful longitudinal outcome measures of the severity of inflammatory arthritis since they
demonstrated significant results with as few as 4 mice
per group. We are also pursuing additional outcomes
with the CE-MRI, including quantifications of bone
marrow edema, which appears as a bright signal with this
MR pulse sequence in the bones of arthritic animals as
compared with a dark signal in the bones of WT
controls. (Figures illustrating bone marrow edema as
determined by CE-MRI and its correspondence to osteitis as determined histologically are available upon request from the corresponding author.) This biomarker
has previously been demonstrated to be a faithful predictor of focal erosions in RA (15), and its nature has
recently been demonstrated to be osteitis (42). Moreover, bone marrow edema measured by CE-MRI has
been effectively used to assess disease severity and
response to therapy in patients with ankylosing spondylitis (43,44). Thus, the hope is that an edema outcome
measure could be developed as the long sought-after
surrogate for arthritic pain in animal models and/or a
predictor of joint destruction in humans.
The authors would like to thank Patricia Weber for
technical assistance with the MRI, Laura Yanoso for technical
assistance with the micro-CT, Colleen Hock for assistance with
animal breeding and genotyping, and Krista Scorsone for
technical assistance with the histology.
Mr. Proulx and Dr. Schwarz had full access to all of the data
in the study and take responsibility for the integrity of the data and the
accuracy of the data analysis.
Study design. Proulx, Kwok, You, Shealy, Ritchlin, Awad, Boyce,
Xing, Schwarz.
Acquisition of data. Proulx, Kwok, You, Awad, Boyce.
Analysis and interpretation of data. Proulx, Kwok, Papuga, Beck,
Shealy, Ritchlin, Awad, Boyce, Xing, Schwarz.
Manuscript preparation. Proulx, Kwok, You, Papuga, Beck, Shealy,
Ritchlin, Awad, Boyce, Xing, Schwarz.
Statistical analysis. Proulx, Beck, Schwarz.
Centocor facilitated the study design, writing of the manuscript, and the decision to submit the manuscript for publication.
Centocor had no role in the data collection or interpretation of the
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node, tomography, inflammatory, longitudinal, lymph, bones, volume, mice, magnetic, imagine, arthritis, vivo, synovial, assessment, resonance, computer, microfocus
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