AJR 2005; 184:1215-1219
© American Roentgen Ray Society
Micro-CT Arthrography: A Pilot Study for the Ex Vivo Visualization of the Rat Knee Joint
Frank W. Roemer1,2,
Andreas Mohr1,3,
John A. Lynch1,
Margarita D. Meta1,
Ali Guermazi1 and
Harry K. Genant1
1 Osteoporosis and Arthritis Research Group, Department of Radiology, University
of California at San Francisco, San Francisco, CA 94117.
Received April 15, 2004;
accepted after revision August 19, 2004.
2 Present address: Department of Radiology, Klinikum Augsburg,
Stenglinstrasse 2, Augsburg 86156, Germany. Address correspondence to F. W.
Roemer
(f.w.roemer{at}gmx.de).
3 Present address: Department of Radiology, University of Schleswig-Holstein,
Campus Kiel, Kiel 24105, Germany.
Abstract
OBJECTIVE. In our study, we evaluated the potential of micro-CT for
the assessment of the rat knee joint using ex vivo micro-CT arthrography. The
aims of the study were to introduce the technique of micro-CT arthrography and
to visualize the normal anatomy of the rat knee. The secondary aims were the
quantification of retropatellar cartilage thickness and the analysis of
microstructural cancellous bone parameters within the tibial epiphysis.
CONCLUSION. Micro-CT arthrography is a novel technique for the
indirect visualization of the distinct features and structural analysis of the
rat knee joint. This technique represents an additional imaging and analysis
tool in small-animal research.
Introduction
Aprerequisite for drug development in rheumatoid arthritis (RA) and
osteoarthritis (OA) research is the establishment and analysis of animal
models that mimic the pathologic process in humans.
The major applications of micro-CT to date have been the analysis of bone
and vascular microstructure and the characterization of the phenotype of
transgenic and knockout animal models during preclinical investigations. This
method allows the use of extremely high-resolution nondestructive imaging and
quantitative analysis because of a high matrix (up to 2,048 voxels) and small,
isotropic voxel size (as small as 5 mm). The morphology of osseous changes of
arthritic joints in small-animal models has been well visualized on micro-CT
[1]. CT arthrography of the
human knee has been shown to be able to indirectly image the articular surface
and other joint structures including soft-tissues in an excellent manner
[2]. Concerning structural bone
analysis, micro-CT has advantages over the destructive and 2D approach of
histomorphometry [3]. Analysis
of the periarticular cancellous bone microstructure using micro-CT showed
alterations in experimental OA and RA animal models
[4,
5].
The goals of this preliminary ex vivo study in four healthy rats were to
introduce the technique of micro-CT arthrography and to visualize the normal
anatomy of the rat knee. Secondary aims were the measurement of retropatellar
cartilage thickness at defined locations and the structural bone analysis of
the proximal cancellous tibial epiphysis. These analyses may serve as a
reference for possible follow-up studies in arthritic knees, where alterations
of cartilage and of periarticular bone are to be expected.
Materials and Methods
This pilot study included the right hind limbs of four healthy rats (female
mature Sprague-Dawley rats; age, 3 months; weight, 320-340 g). After
euthanizing the animals, we excised the knee joints by separating the mid
tibia and mid femur. The institutional committee on animal research approved
the experimental protocol.
A suspension of oil (Supreme 5E-30, Chevron Texaco) and barium sulfate (96%
weight/volume; E-Z-PAQUE, E-Z-EM) was used as contrast material. We injected
0.1 mL of contrast material. Flexion and extension of the knee joint were
performed to provide a homogeneous distribution of the contrast agent.
The micro-CT system used in this study was a cone-beam tomograph (µCT
40, Scanco Medical). The specimens were scanned in the transverse plane
mounted in a cylindric sample holder at an isotropic resolution of 30 µm
(144 µA; 55 kV; image matrix, 1,024 x 1,024 pixels; field of view,
30.7 mm; slice thickness, 30 µm). Scanning times varied between 2.8 and 3.2
hr depending on the degree of flexion of the specimen, which resulted in
slight differences in the number of slices needed for complete coverage of the
anatomy. Image reconstruction times were 5.6-6.4 hr.
Multiplanar reconstructions were performed on a desktop computer using
customized software for image analysis. The images were evaluated for the
appearance and detectability of bone, cartilage, menisci, and soft-tissue
structures such as ligaments, plicae, and tendons. After exact reconstruction
of the patella within the axial and sagittal planes, we measured the
retropatellar cartilage thickness three times at three different sites
(labeled UP, MID, and LOW). The mean value and SD of these three measurements
were calculated (Figs. 1A and
1B).
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Fig. 1A. —Measurement of retropatellar cartilage thickness and
visualization of intrinsic joint structures in rat using micro-CT
arthrography. Micro-CT arthrographic image shows three locations for
measurement of retropatellar cartilage thickness in axial plane.
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Fig. 1B. —Measurement of retropatellar cartilage thickness and
visualization of intrinsic joint structures in rat using micro-CT
arthrography. Micro-CT arthrographic image shows three locations for
measurement of retropatellar cartilage thickness in axial plane that are
labeled UP, MID, and LOW.
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Structural analysis of the periarticular cancellous bone of the proximal
tibial epiphysis was performed using the micro-CT software. The volume of
interest was semiautomatically drawn in the original axial images adapted to
the anatomy of the epiphysis. A fixed threshold was applied to extract the
mineralized bone phase. The morphometric indexes of bone volume density (bone
volume/total volume [BV/TV]), trabecular number, trabecular thickness,
trabecular separation, bone surface density, structure model index, and degree
of anisotropy were calculated. The structure model index provides an
estimation of the plate or rodlike nature of trabecular bone
[6]. The mean intercept length
method was applied to quantify the degree of anisotropy. High values for the
degree of anisotropy indicated that the orientation of the trabecular
structure had become more anisotropic
[7]. A plate model of the
cancellous bone was assumed in the derivations of the equations used to
calculate the quantities
[3].
Results
Cortical and trabecular bone is depicted with intermediate attenuation
compared with intraarticular contrast material and soft-tissue or fat. Despite
the tibial, femoral, and fibular osseous structures, the pyramid-shaped
meniscal ossicles and the fabellae at the heads of the gastrocnemius muscles
were regularly visualized.
Ligaments are visualized with soft-tissue attenuation on micro-CT images.
The arrangement of the anterior and posterior cruciate ligaments was
comparable to those of humans (Fig.
1C). The meniscotibial ligaments that attach the anterior horns of
both menisci to the contralateral tibial intercondylar area were regularly
visualized and were best analyzed in the axial plane
(Fig. 1D).
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Fig. 1C. —Measurement of retropatellar cartilage thickness and
visualization of intrinsic joint structures in rat using micro-CT
arthrography. Sagittal reconstruction of micro-CT arthrographic images shows
anterior cruciate ligament (black arrow), infrapatellar plica
(arrowhead), and infrapatellar fat pad (white arrow).
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Fig. 1D. —Measurement of retropatellar cartilage thickness and
visualization of intrinsic joint structures in rat using micro-CT
arthrography. Axial reconstruction of micro-CT arthrographic images shows
distinct meniscofemoral ligaments (white arrows). Popliteal tendon
(black arrow) is visualized laterally
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Cartilage was indirectly visualized as a band of low attenuation between
the subchondral bone and the intraarticular contrast material. Because of the
anatomy of the rat's knee, the chondral surface of the central parts of the
femoral condyles and tibial plateaus could not be well differentiated; the
contrast material coating was usually insufficient or the cartilage of the
meniscal ossicles could not be differentiated from femoral or tibial
cartilage. However, retropatellar cartilage of the osseous patella could be
well seen in the sagittal and axial reconstructions.
The mean cartilage thickness at the patella midline was 250 mm (range,
220-270 mm; SD, ± 24.49 mm) for the MID location. At the UP location,
the midline thickness averaged 242.5 mm (range, 230-250 mm; SD, ± 9.57
mm), and for the LOW location at midline, the mean thickness averaged 205 mm
(range, 180-240 mm; SD, ± 35 mm) (Figs.
1A and
1B).
A representative 3D micro-CT image of the tibial cancellous epiphysis is
shown in Figure 2. The mean
total volume of the cancellous tibial epiphysis was 15.5 mm3, and
the mean BV/TV was 0.24. The average bone surface density was 21.1/mm; mean
trabecular thickness, 96 µm; mean trabecular number, 2.56/mm; and mean
trabecular separation, 0.30 mm. The structure model index averaged 1.91,
indicating a more rodlike structure for the normal tibial epiphysis of the rat
[6]. The degree of anisotropy
averaged 1.50 [7].
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Fig. 2. —Micro-CT arthrography was used to produce 3D surface
reconstruction of structural analysis of cancellous bone within tibial
epiphysis of rat viewed from above. Virtual light source is located at right
upper corner of image.
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Discussion
In this pilot study, previously described anatomic features were indirectly
visualized in accordance with the literature
[8]. Cartilage was depicted as
a low-attenuation band between the high attenuation of intraarticular contrast
medium and the intermediate attenuation of bone, which correlates with the
results of CT arthrography in the human knee
[2]. Quantification of
cartilage thickness was limited to the retropatellar cartilage. To our
knowledge, no literature is available dealing with retropatellar cartilage
thickness in small-animal models. Loeuille et al.
[9] reported cartilage
thickness at the femoral condyles in rats of approximately 250 µm using
micro-MRI. However, these micro-MRI studies used nonisotropic voxels and had a
minimal spatial resolution of 60 µm
[9,
10]. In our study, the
resolution of 30-µm isotropic voxel size proved to be a satisfactory
compromise among resolution, scanning time, and contrast homogeneity.
Additional advantages of micro-CT compared with micro-MRI are its
widespread accessibility in research institutions, its superior
cost-efficiency, its well-known capacities for analysis and visualization of
bone structure, and the possibility of multiplanar reconstructions with the
same resolution as the original axial data set. Limitations of the technique
include inferior soft-tissue contrast and relatively long scanning
periods.
We used only commonly available and inexpensive clinical material such as
barium sulfate or standard injection needles. The procedure itself can be
performed in approximately 10 min including placement of the specimen in the
sample holder. Nonionic iodinated contrast material could not be used in this
study because diffusion of the contrast medium from the joint cavity appeared
to be relevant. This had been shown in a preliminary test of different
contrast agents including iodinated contrast material and gadolinium
solutions.
The use of just four specimens allows us to make only a preliminary
evaluation of the method, and only healthy knees of control animals were
evaluated. Experience with newly developed in vivo micro-CT systems will show
whether the technique may be applicable in longitudinal evaluations of the
same animal over time. Such studies would necessitate a modification of
contrast material because the oil-barium suspension used for this study is not
suitable for in vivo research. Intrinsic early cartilaginous changes such as
chondral swelling and edema cannot be visualized on micro-CT arthrography
because the indirect imaging technique depicts only surface lesions
corresponding to lesions that are recognizable from experiences gathered with
CT arthrography of the human knee
[2]. Diffusion of contrast
material into the cartilage was not observed.
Another drawback of our study is the lack of correlation of our morphologic
data with histology or other cross-sectional imaging methods. However, reports
on the anatomy of the rat knee are available and our results correlate well
with these descriptions [8].
Structural analysis of cancellous bone using micro-CT correlates well with the
destructive approach of histomorphometry
[3].
In conclusion, micro-CT arthrography represents a novel nondestructive,
multiplanar, and easily applicable additional imaging and analysis tool in
small-animal research.
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Abstract
Introduction
