CME JOURNAL OF MAGNETIC RESONANCE IMAGING 00:00–00 (2014)
Original Research
Diffusion Tensor Imaging Assesses Triceps Surae Dysfunction After Achilles Tenotomy in Rats Yusuke Hara, MD,1 Kazuya Ikoma, MD, PhD,1* Masamitsu Kido, MD, PhD,1 Tsuyoshi Sukenari, MD,1 Yuji Arai, MD, PhD,1 Hiroyoshi Fujiwara, MD, PhD,1 Mitsuhiro Kawata, MD, PhD,2 and Toshikazu Kubo, MD, PhD1 tissues, which have high water molecule anisotropy, anisotropic disturbances that accompany pathological changes can be imaged noninvasively (2); accordingly, this method has been commonly used in the central nervous system area (2). Recently, DTI has been used outside the central nervous system and has been reported to be a highly sensitive method for imaging skeletal muscles (3–5). As muscle fibers and other components bestow muscle tissue with high anisotropy, DTI has been used to evaluate internal microstructure and pathology in skeletal muscle (6) and cardiac muscle (7) fibers. Moreover, calculating various DTI parameters allows qualitative evaluation of these tissues. DTI parameters have been reported to be related to internal structural changes due to active motion in healthy muscle and internal structural changes in atrophied muscle after denervation (8,9). Rupture of the Achilles tendon is often encountered in routine medical care. Problems that can arise after Achilles tendon rupture include reduced triceps surae function and poor performance in the heel-raise test (10). In the process of treating the Achilles tendon, evaluating triceps surae function is important for the recovery of motor function. Methods of evaluating skeletal muscle function involve measuring muscle strength by using a manual muscle test or an apparatus; however, these can be problematic as evaluations cannot be performed until the tendon is restored, without an apparatus, or if other technical problems are encountered (10). There is a need for a clinical imaging method that can quantitatively evaluate skeletal muscle morphology and function in detail during the acute postinjury stage. After Achilles tendon rupture, we hypothesized that the internal structure of the triceps surae and the anisotropy of water molecules in the muscle tissue change. By analyzing the relationship of changes in DTI parameters in posttenotomy muscle tissue with changes in triceps surae microstructure and motor function, we show that DTI may be used in the evaluation of muscle function. Changes occur in the internal structure of the triceps surae, and the anisotropy of water molecules in the tissue decrease. We hypothesized that DTI values might change in response to this decrease in anisotropy. As muscle strength is highly dependent on internal structure, we
Purpose: To elucidate the relationship of diffusion tensor imaging (DTI) with structural changes and muscle function in skeletal muscle. Materials and Methods: The subjects were 21 12-weekold male rats. Achilles tenotomy was performed on the right legs. Proton density-weighted images and DTI (7.04T) of the triceps surae at 2 and 4 weeks posttenotomy were obtained. Eigenvalues (l) and fractional anisotropy (FA) were calculated from the muscle images. After imaging, histological specimens of the triceps surae were prepared. The long axis and cross-sectional area of the triceps surae were measured 2 and 4 weeks postoperatively. The strength of ankle plantar flexion was measured. Correlations of DTI parameters with morphological values and muscle strength were analyzed. Results: l1 declined after tenotomy (P < 0.01), and l2 and l3 declined at 4 weeks posttenotomy (P < 0.01). FA increased at 4 weeks posttenotomy (P < 0.01). Atrophy of the triceps surae was observed in the tissues images of the treatment group. The muscle belly significantly shortened postoperatively, and a decline in plantar flexion force was observed after tenotomy. Positive correlations were observed between l1 and muscle strength (r ¼ 0.89) and between l1 and the length of the long axis of the muscle belly (r ¼ 0.81). Conclusion: DTI may serve as a marker of muscle function. Key Words: Achilles tendon; DTI; MRI; muscle function J. Magn. Reson. Imaging 2014;00:000–000. C 2014 Wiley Periodicals, Inc. V
DIFFUSION TENSOR IMAGING (DTI) uses magnetic resonance imaging (MRI) to visualize the diffusion anisotropy of water molecules within tissues (1). In fibrous 1 Department of Orthopaedics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan. 2 Department of Anatomy and Neurobiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan. *Address reprint requests to: K.I., Department of Orthopaedics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto, Japan. E-mail: [email protected] Received May 7, 2014; Accepted June 30, 2014. DOI 10.1002/jmri.24707 View this article online at wileyonlinelibrary.com. C 2014 Wiley Periodicals, Inc. V
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Table 1 Scheme of the Study Protocol Study In vivo imaging and histology of the muscle Motor function test and morphological measurements Correlation analysis
Numbers 6 6 9
The number of rats used in each study.
also hypothesized that changes in DTI may correlate with changes in muscle strength. The purpose of the present study was to investigate changes in rat triceps surae after Achilles tenotomy based on the relationships between DTI parameters and functions and to examine the usefulness of DTI in evaluating muscle function. MATERIALS AND METHODS Rat Model The subjects included a total of 21 12-week-old male Sprague–Dawley rats. This study used rats that differed in terms of in vivo imaging and histological findings, motor function test and morphological measurements, and correlation analysis (Table 1). The rats were reared in individual cages. All treatments, surgeries, and MRI were performed under isoflurane inhalation anesthesia. Anesthesia was induced using 4% isoflurane in an anestheticinduction chamber and then maintained with continuous inhalation of 1.5% isoflurane using a facemask; body temperature and respiratory rates were monitored. All experimental procedures in this study were approved by the Animal Care and Use Committee of our university. The approval number of this study is M25–204.
at an angle of 20 flexion, and the ankle was placed in the neutral position. The scanning sequence parameters for PDWI were as follows: relaxation time (TR), 2000 msec; echo time (TE), 45 msec; echo train length, 4; field of view (FOV), 60 60 mm; matrix; 512 512; and slice thickness, 2 mm. The scanning parameters for DTI were as follows: TR, 3000 msec; TE, 14 msec; echo train length, 4; FOV, 60 mm 60 mm; pixel matrix, 128 128; slice thickness, 2 mm; and b-values, 0 s/mm2 and 350 s/ mm2. Motion probing gradient orientations were applied along six directions: [Gx,Gy,Gz] ¼ [1,1,0], [1,0,1], [0,1,1], [-1,1,0], [0,-1,1], and [1,0,-1]. The entire scanning time for each subject was 120 minutes. Image Analysis The anatomical position of the triceps surae was confirmed in the sagittal and axial PDWI images, and the axial slice in which the muscle belly area was largest was determined. The regions of interest for the soleus, medial, and lateral heads of the gastrocnemius were established in the axial images (12). Eigenvalues (l1, l2, and l3) and fractional anisotropy (FA), which is an index of anisotropic strength, were calculated in these areas of interest. These were measured using ImageJ software (13). FA values were calculated using eigenvalues (l1, l2, and l3) in the following formulas: qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 2 2 pffiffiffiffiffiffiffiffiffi ðl1 -DÞ þ ðl2 -DÞ þ ðl3 -DÞ qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi FA¼ 2=3 ðl21 þl22 þ l23 Þ D¼ðl1 þl2 þl3 Þ=3
In Vivo Imaging and Histology of the Muscle In Vivo Imaging
Achilles Tenotomy Operation To model triceps surae dysfunction, Achilles tenotomy was performed on the right hindlegs (11). Using aseptic techniques, a longitudinal skin incision was created directly above the Achilles tendon to visually confirm the Achilles and plantaris tendons. A scalpel was used to cut only the Achilles tendon about 5 mm proximal to its attachment to the calcaneal bone; after that, discontinuity of the Achilles tendon was confirmed. The Achilles tendon was left unsutured, while the plantaris tendon was left intact as an internal splint. The wound was washed with physiological saline solution and closed using 4-0 monofilament nylon. MRI Measurement MR images were obtained using a high magnetic field MRI unit designed for animal experiments (Varian MRI system 7.04T, horizontal orientation; Agilent Technology, Palo Alto, CA) with a 210 mm gradient coil (Agilent Technology); vnmrJ MRI software was used (Agilent Technology). The MRI protocol included fast spin-echo proton density-weighted imaging (PDWI) and DTI. Before running the DTI sequence, PDWI was obtained to confirm the muscle belly of triceps surae. The knee joint was fixed
Changes in postoperative DTI were evaluated in six rats that underwent Achilles tenotomy. We performed imaging of the hindlegs on both sides before tenotomy and 2 and 4 weeks posttenotomy; we then compared the unaffected and affected sides at each point. Histology of the Muscle After undergoing MRI, six rats were sacrificed by intraperitoneal injection of a pentobarbital overdose at 4 weeks postoperatively. The triceps surae bellies from the proximal attachment to the muscle-tendon junction were removed with a scalpel. The 20-mm-thick fresh-frozen slices of muscle bellies were made using liquid nitrogen and nhexane. Hematoxylin and eosin (H&E) staining and Azan staining were performed. Motor Function Test and Morphological Measurements Motor Function Test For evaluation of motor function, three rats were used at 2 weeks posttenotomy and three rats at 4 weeks posttenotomy. Under inhalation anesthesia, the rats were placed in a lateral recumbent position and the
DTI for Assessing Muscle Function
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Table 2 Results of DTI Parameters
l1 l2 l3 FA
Unaffected side Affected side Unaffected side Affected side Unaffected side Affected side Unaffected side Affected side
Pre
2 Weeks
4 Weeks
1.814 6 0.070 1.820 6 0.076 1.333 6 0.008 1.389 6 0.025 1.092 6 0.029 1.089 6 0.075 0.238 6 0.042 0.238 6 0.037
1.823 6 0.088 1.737 6 0.120 1.316 6 0.077 1.337 6 0.079 1.065 6 0.029 1.050 6 0.029 0.271 6 0.022 0.265 6 0.040
1.939 6 0.050 1.793 6 0.051 1.473 6 0.169 1.303 6 0.112 1.214 6 0.049 1.019 6 0.083 0.237 6 0.022 0.282 6 0.043
Mean values 6 standard deviation of the eigenvalues and the fractional anisotropy (l1, l2, l3)[103 mm2/s].
knee joint and trunk were fixed. The knee and ankle joint were fixed at the same leg position as that for MRI. Hair on the hindlegs was removed. Surface stimulation electrodes (NM-410S; Nihon Kohden, Tokyo, Japan) were placed on the posterior surface of the lower legs and electrical stimulation was applied at 20 mA, 1 Hz. A digital force gauge (FGP-20; NidecShimpo, Kyoto, Japan) was attached to the sole to measure plantar flexion force (14).
Motor Function Tests
Morphological Measurements
The measured values 2 weeks and 4 weeks posttenotomy on the left and right sides were compared using a paired t-test. Mean values were calculated for the ratio of the long axis length of the muscle belly to the full long axis length (muscle belly length / full length) of the triceps surae at each timepoint. The anteroposterior and horizontal diameters were used to approximate the cross-sectional area, and mean values for each timepoint were calculated.
The rats that underwent motor function tests (three rats at 2 weeks posttenotomy and three rats at 4 weeks posttenotomy) were used to evaluate morphological changes after tenotomy. After euthanasia, we harvested the triceps surae, still attached to the bone. The length of the triceps surae belly, its anteroposterior diameter, and horizontal diameter were measured using digital calipers. The length of the muscle belly was measured from the distal muscle-tendon junction to the proximal attachment of the triceps surae to the femur. The maximum anteroposterior and horizontal diameters were measured. The knee and ankle joint were fixed at the same leg position as that for MRI. Correlation Analysis Finally, we investigated the correlations between posttenotomy DTI parameters and the results of motor function tests and morphological measurements. Three rats that did not undergo tenotomy, three rats at 2 weeks posttenotomy, and three rats at 4 weeks posttenotomy were used. MRI was performed with the methods previously described, and motor function tests were conducted afterward. The full length of the long axis of the triceps surae and the length of the long axis of the muscle belly of the triceps surae were measured in the sagittal PDWI images. The long axis of the muscle belly was measured from the distal muscle-tendon joint to the proximal attachment of the triceps surae to the femur.
The values measured on the affected side were divided by those on the unaffected side at 2 weeks and 4 weeks posttenotomy. With the control value as 1, an unpaired t-test was used to compare the control with the affected/unaffected (A/U) ratio at each timepoint. Morphological Measurements
Correlation Analysis We evaluated correlations between changes in long axis length and l1, and between changes in muscle strength and DTI parameters. A/U ratios were calculated for muscle belly length / full length, muscle strength, FA, l1, l2, and l3 as the degree of change; then correlation coefficients were computed. JMP10 (SAS Institute, Cary, NC) was used for statistical analysis. Differences were considered significant at P < 0.05. RESULTS In Vivo Imaging The values of l1 declined significantly on the affected side at 2 and 4 weeks posttenotomy (P < 0.01). The values of l2 and l3 declined significantly on the affected side at 4 weeks posttenotomy (P < 0.01). The FA values increased significantly on the affected side at 4 weeks posttenotomy (P < 0.01) (Table 2, Fig. 1a–d).
Statistical Analysis In Vivo Imaging
Histology of the Muscle
We compared the eigenvalues and FA values at each imaging timepoint, and compared the unaffected and affected sides using paired-t-test.
Tissue and cross-sectional images are shown in Fig. 2. H&E staining revealed indistinct muscle bundle boundaries, and dilations between muscle bundles
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Figure 1. Changes in sides for l1 (a), l2 (b), l3 (c), and FA (d) for each imaging timepoint. a: l1 significantly declined at 2 weeks and 4 weeks posttenotomy (*P < 0.01) (white box, unaffected side; lined box, affected side). b: l2 significantly declined at 4 weeks posttenotomy (*P < 0.01). c: l3 significantly declined at 4 weeks posttenotomy (*P < 0.01). d: FA significantly increased at 4 weeks posttenotomy (*P < 0.01).
(Fig. 2a,b), which suggest muscle atrophy. Furthermore, Azan staining showed slight proliferation of connective tissue outside the perimysium (Fig. 2c,d).
Motor Function Test Plantar flexion force on the affected side decreased significantly to 82.1 6 0.04% on the unaffected side at 2 weeks posttenotomy (P < 0.05; Fig. 3) and to
Figure 2. Histological changes in the triceps surae belly. An H&E-stained 20-mm slice after tenotomy (a) and control (b). Dilation is observed between muscle bundles at 4 weeks posttenotomy. An Azanstained 20-mm slice after tenotomy (a) and control (b). No particular growth of connective tissue is evident in the areas of dilation between muscle bundles.
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Figure 3. Plot of affected/unaffected (A/U) ratios of posttenotomy ankle plantar flexion force. Plantar flexion force decreased significantly at 2 weeks and 4 weeks posttenotomy (*P < 0.05, **P < 0.01).
76.3 6 0.12% on the unaffected side at 4 weeks posttenotomy (P < 0.01; Fig. 3). Morphological Measurements The triceps surae muscle belly length / full length was 70.1 6 0.02% on the affected side and 79.2 6 0.04% on the unaffected side at 2 weeks posttenotomy, and 70.9 6 0.02% on the affected side and 76.9 6 0.03% on the unaffected side at 4 weeks posttenotomy. Muscle belly length on the affected side significantly shortened at each timepoint (P < 0.01; Fig. 4a). The crosssectional area of the triceps surae belly at 2 weeks posttenotomy was 92.3 6 6.6 mm2 on the affected side and 101.9 6 4.7 mm2 on the unaffected side, and at 4 weeks posttenotomy was 97.7 6 4.0 mm2 on the affected side and 121.4 6 6.0 mm2 on the unaffected side. Cross-sectional areas on the affected side significantly decreased at each timepoint (P < 0.01; Fig. 4b). Correlation Coefficients A positive correlation was observed between the A/U ratios for muscle strength and l1, with a correlation coefficient r ¼ 0.89 (P < 0.01; Fig. 5a). Correlations were not observed between the A/U ratio for muscle strength and the A/U ratios for l2, l3, and FA, with correlation coefficients r ¼ 0.44, 0.64, and 0.27, respectively (Fig. 5b–d). A positive correlation was observed between the A/U ratios for muscle belly length / full length and l1, with correlation coefficient r ¼ 0.81 (P < 0.01; Fig. 6).
DISCUSSION Skeletal muscle has a fibrous structure and is regarded as possessing anisotropy due to its structural characteristics. In research on muscle injury, DTI has been shown to be useful in diagnosis, as changes in anisotropy occur at the site of injury (15). Sinha et al (16) used tractography to visualize the path of healthy muscle fibers. Galban et al (12) reported sex-based differences in the anisotropic diffusion of water molecules in the triceps surae. Zarais-
Figure 4. Triceps surae on the affected and unaffected sides were removed 2 weeks and 4 weeks posttenotomy. These graphs show the length of the long axis of the triceps surae belly divided by the length of the whole muscle and the cross-sectional area. a: The long axis of the triceps surae belly significantly shortened 2 weeks and 4 weeks posttenotomy (*P < 0.01). b: The cross-sectional area of the triceps surae significantly decreased 2 weeks and 4 weeks posttenotomy (*P < 0.01) (white box, unaffected side; lined box, affected side).
kaya et al (17) used DTI to investigate triceps surae injury and intramuscular hematoma, finding lower FA values than in healthy muscle. Therefore, DTI is useful and is being used to depict structural changes in skeletal muscle. However, the interrupted continuity of muscle fibers as seen in muscle injury is not present in posttendon rupture muscle tissue. DTI has also been used to examine muscle atrophy after denervation. Saotome et al (9) employed DTI to evaluate denervated skeletal muscle in rats. FA was significantly higher in atrophied muscle after denervation compared to healthy muscle. Regarding eigenvalues, while they reported no major change in l1, there were significant decreases in l2 and l3. They concluded that reductions in l2 and l3 are related to the density of muscle fibers in the axial plane, but the relationship between l1 and muscle fiber diameter was weak. Zhang et al (18) created a denervation muscle atrophy model by performing unilateral sciatic nerve ligation on the lower leg and evaluated DTI parameters chronologically. The muscle atrophy results of the denervation model showed internal structural changes in cross-sections, significant reductions in l2 and l3, and an increase in FA,
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Figure 5. Correlations between the affected/unaffected (A/U) ratio of muscle strength and the A/U ratios of l1 (a), l2 (b), l3 (c), and FA (d). a: A positive correlation between muscle strength A/U ratio and l1 A/U ratio was observed, with the correlation coefficient r ¼ 0.89 (P < 0.01). b: A correlation was not observed between muscle strength A/U ratio and l2 A/U ratio, with the correlation coefficient r ¼ 0.44 (P ¼ 0.23). c: A correlation was not observed between muscle strength A/U ratio and l3 A/U ratio, with the correlation coefficient r ¼ 0.64 (P ¼ 0.06). d: A correlation was not observed between muscle strength A/ U ratio and FA A/U ratio, with the correlation coefficient r ¼ –0.27 (P ¼ 0.48).
indicating that DTI is useful in detecting skeletal muscle atrophy. In functional evaluations of skeletal muscle, DTI has been used to examine models during passive relaxation and contraction of skeletal muscle. Schwenzer et al (8) evaluated DTI of the triceps surae in healthy humans while passively moving the ankle from 10 dorsiflexion to 40 plantar flexion using a device they developed. An increase in mean diffusivity was observed when the muscle was contracted, which was consistent with a physiological increase in muscle fibers on the axial plane. Moreover, l1 did not significantly vary when shortened or elongated, but l2 and l3 significantly decreased when relaxed. Hatakenaka et al (19,20) reported that l1 decreased while l2 and l3 increased during skeletal muscle changes in length and during passive changes to the articular range of motion. Furthermore, Okamoto et al (21) reported increases in FA, l1, and l2 due to contractions of skeletal muscle during active articular motion. Maganaris et al (22) investigated the relationships between the length of triceps surae fibers and the angle of the muscle’s pinnate structure with angles of ankle plantar/dorsiflexion. From their results, Schwenzer et al (8) also concluded that muscle fiber length greatly affects proton diffusivity.
Past studies have shown that DTI parameters are strongly related to both the causes of pathology and the resultant changes in internal tissue structure. From Hatakenaka et al and Schwenzer et al’s results, we surmised that in skeletal muscle changes in
Figure 6. Correlation between the affected/unaffected (A/U) ratio of muscle-belly length/full length and the A/U ratio of l1. A positive correlation was observed between muscle belly length / full length A/U ratio and l1 A/U ratio, with the correlation coefficient r ¼ 0.81 (P < 0.01).
DTI for Assessing Muscle Function
muscle belly length along the long axis due to changes in articular range of motion would change anisotropy. We believe the length of the long axis of the muscle belly is an important part of perpendicular anisotropy. Achilles tenotomy has been used to model reduced muscle activity (23). After tenotomy, atrophic changes such as reduced muscle mass and growth of connective tissue between muscle bundles are observed (24). Furthermore, there is a decrease in tensile force after tenotomy (23). In this study, we confirmed elongation of the tendon and shortening of the long axis of the muscle belly from the second week posttenotomy. The muscle cross-sectional area also decreased from the second week. Plantar flexion force decreased from the second week, exhibiting a difference between the affected and unaffected sides. With regard to DTI parameters, l1 and l3 decreased from 2 weeks posttenotomy, whereas l2 decreased and FA increased from 4 weeks posttenotomy. The l1 A/U ratio strongly correlated with the A/U ratios of muscle strength and length of the long axis of the muscle belly. We believe that l1 may useful to evaluate posttenotomy muscle strength. l1 is thought to show anisotropy horizontal to the muscle fibers, whereas l2 and l3 show crosssectional anisotropy. Reduced l1 reflects the shortening of the triceps surae, which is considered an important factor in decreased motor function after Achilles tenotomy. A decrease in the cross-sectional area was observed in triceps surae cross-sections. Qualitative histological evaluations may be necessary to consider changes in l2 and l3. The proliferation of connective tissue was mild, which might be related to a variety of factors besides muscle tissue density, such as connective tissue density. One limitation of this study was the short observation period. The Achilles tendon recovers relatively quickly after rupture and muscle atrophy has been reported to progress over time (25). By observing this model over a longer period, we would expect to see an even stronger correlation between DTI and muscle function. Another limitation involves the b-value. The b-value used here was 350 s/mm2, which has been used frequently in research studies on skeletal muscle. Appropriate b-values for skeletal muscle have been reported to be 300–900 s/mm2 (26). Setting a high b-value allows for more detailed evaluations of intracellular anisotropy, but this can decrease the S/ N ratio and harm the reliability of the test. Moreover, since we desired to evaluate not only muscle cell anisotropy but also connective tissue inside the perimysium, we used a relatively low b-value. In the future, it will be necessary to vary the b-value to determine the most appropriate value for muscle dysfunction. We found that during the initial stage after Achilles tenotomy, muscle function and the degree of tendon elongation were correlated with l1. Muscle dysfunction due to tendon elongation is one complication of Achilles tendon rupture. In the early stage before the tendon recovers, there is a possibility of rerupture, and the evaluation of muscle dysfunction is problematic. The use of our method allows for estimation of muscle function and assessment of tendon elongation
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during the initial phase of treatment for ruptured tendon, potentially preventing muscle dysfunction. In conclusion, tendon elongation and shortening of the long axis of the muscle belly occur after Achilles tenotomy. In DTI, decreased l1, l2, and l3 of the triceps surae and increased FA were observed. l1 exhibited strong positive correlations with muscle strength and length of the long axis of the muscle belly. DTI could be potentially used in the evaluation of muscle function. REFERENCES 1. Basser PJ, Mattiello J, LeBihan D. MR diffusion tensor spectroscopy and imaging. Biophys J 1994;66:259–267. 2. Mamata H, Jolesz FA, Maier SE. Characterization of central nervous system structures by magnetic resonance diffusion anisotropy. Neurochem Int 2004;45:553–560. 3. Heemskerk AM, Sinha TK, Wilson KJ, Ding Z, Damon BM. Quantitative assessment of DTI-based muscle fiber tracking and optimal tracking parameters. Magn Reson Med 2009;61:467–472. 4. Shorten PR, Sneyd J. A mathematical analysis of obstructed diffusion within skeletal muscle. Biophys J 2009;96:4764–4778. 5. Aliev MK, Tikhonov AN. Random walk analysis of restricted metabolite diffusion in skeletal myofibril systems. Mol Cell Biochem 2004;256:257–266. 6. Okamoto Y, Okamoto T, Yuka K, Hirano Y, Isobe T, Minami M. Correlation between pennation angle and image quality of skeletal muscle fibre tractography using deterministic diffusion tensor imaging. J Med Imaging Radiat Oncol 2012;56:622–627. 7. Sosnovik DE, Wang R, Dai G, Reese TG, Wedeen VJ. Diffusion MR tractography of the heart. J Cardiovasc Magn Reson 2009;11: 47. 8. Schwenzer NF, Steidle G, Martirosian P, et al. Diffusion tensor imaging of the human calf muscle: distinct changes in fractional anisotropy and mean diffusion due to passive muscle shortening and stretching. NMR Biomed 2009;22:1047–1053. 9. Saotome T, Sekino M, Eto F, Ueno S. Evaluation of diffusional anisotropy and microscopic structure in skeletal muscles using magnetic resonance. Magn Reson Imaging 2006;24:19–25. 10. Kearney RS, McGuinness KR, Achten J, Costa ML. A systematic review of early rehabilitation methods following a rupture of the Achilles tendon. Physiotherapy 2012;98:24–32. 11. Bring D, Reno C, Renstrom P, Salo P, Hart D, Ackermann P. Prolonged immobilization compromises up-regulation of repair genes after tendon rupture in a rat model. Scand J Sci Sports 2010;20: 411–417. 12. Galb an CJ, Maderwald S, Uffmann K, de Greiff A, Ladd ME. Diffusive sensitivity to muscle architecture: a magnetic resonance diffusion tensor imaging study of the human calf. Eur J Appl Physiol 2004;93:253–262. 13. Rasband WS. ImageJ, U.S. National Institutes of Health, Bethesda, MD. http://imagej.nih.gov/ij/, 1997–2012. 14. Takagi T, Nakamura M, Yamada M, et al. Visualization of peripheral nerve degeneration and regeneration: monitoring with diffusion tensor tractography. Neuroimage 2009;44:884–892. 15. McMillan AB, Shi D, Pratt SJ, Lovering RM. Diffusion tensor MRI to assess damage in healthy and dystrophic skeletal muscle after lengthening contractions. J Biomed Biotechnol 2011;2011: 760726. 16. Sinha U, Yao L. In vivo diffusion tensor imaging of human calf muscle. J Magn Reson Imaging 2002;15:87–95. 17. Zaraiskaya T, Kumbhare D, Noseworthy MD. Diffusion tensor imaging in evaluation of human skeletal muscle injury. J Magn Reson Imaging 2006;24:402–408. 18. Zhang J, Zhang G, Morrison B, Mori S, Sheikh KA. Magnetic resonance imaging of mouse skeletal muscle to measure denervation atrophy. Exp Neurol 2008;212:448–457. 19. Hatakenaka M, Yabuuchi H, Matsuo Y, et al. Effect of passive muscle length change on apparent diffusion coefficient: detection with clinical MR imaging. Magn Reson Med Sci 2008;7:59–63. 20. Hatakenaka M, Matsuo Y, Setoguchi T, et al. Alteration of proton diffusivity associated with passive extension and contraction. J Magn Reson Imaging 2008;27:932–937.
8 21. Okamoto Y, Kunimatsu A, Kono T, Nasu K, Sonobe J, Minami M. Changes in MR diffusion properties during active muscle contraction in the calf. Magn Reson Med Sci 2010;9: 1–8. 22. Maganaris CN, Baltzopoulos V, Sargeant AJ. In vivo measurements of the triceps surae complex architecture in man: implications for muscle function. J Physiol 1998;512:603– 614. 23. Buller AJ, Lewis DM. Some observations on the effects of tenotomy in the rabbit. J Physiol 1965;178:326–342.
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Original Research
Diffusion Tensor Imaging Assesses Triceps Surae Dysfunction After Achilles Tenotomy in Rats Yusuke Hara, MD,1 Kazuya Ikoma, MD, PhD,1* Masamitsu Kido, MD, PhD,1 Tsuyoshi Sukenari, MD,1 Yuji Arai, MD, PhD,1 Hiroyoshi Fujiwara, MD, PhD,1 Mitsuhiro Kawata, MD, PhD,2 and Toshikazu Kubo, MD, PhD1 tissues, which have high water molecule anisotropy, anisotropic disturbances that accompany pathological changes can be imaged noninvasively (2); accordingly, this method has been commonly used in the central nervous system area (2). Recently, DTI has been used outside the central nervous system and has been reported to be a highly sensitive method for imaging skeletal muscles (3–5). As muscle fibers and other components bestow muscle tissue with high anisotropy, DTI has been used to evaluate internal microstructure and pathology in skeletal muscle (6) and cardiac muscle (7) fibers. Moreover, calculating various DTI parameters allows qualitative evaluation of these tissues. DTI parameters have been reported to be related to internal structural changes due to active motion in healthy muscle and internal structural changes in atrophied muscle after denervation (8,9). Rupture of the Achilles tendon is often encountered in routine medical care. Problems that can arise after Achilles tendon rupture include reduced triceps surae function and poor performance in the heel-raise test (10). In the process of treating the Achilles tendon, evaluating triceps surae function is important for the recovery of motor function. Methods of evaluating skeletal muscle function involve measuring muscle strength by using a manual muscle test or an apparatus; however, these can be problematic as evaluations cannot be performed until the tendon is restored, without an apparatus, or if other technical problems are encountered (10). There is a need for a clinical imaging method that can quantitatively evaluate skeletal muscle morphology and function in detail during the acute postinjury stage. After Achilles tendon rupture, we hypothesized that the internal structure of the triceps surae and the anisotropy of water molecules in the muscle tissue change. By analyzing the relationship of changes in DTI parameters in posttenotomy muscle tissue with changes in triceps surae microstructure and motor function, we show that DTI may be used in the evaluation of muscle function. Changes occur in the internal structure of the triceps surae, and the anisotropy of water molecules in the tissue decrease. We hypothesized that DTI values might change in response to this decrease in anisotropy. As muscle strength is highly dependent on internal structure, we
Purpose: To elucidate the relationship of diffusion tensor imaging (DTI) with structural changes and muscle function in skeletal muscle. Materials and Methods: The subjects were 21 12-weekold male rats. Achilles tenotomy was performed on the right legs. Proton density-weighted images and DTI (7.04T) of the triceps surae at 2 and 4 weeks posttenotomy were obtained. Eigenvalues (l) and fractional anisotropy (FA) were calculated from the muscle images. After imaging, histological specimens of the triceps surae were prepared. The long axis and cross-sectional area of the triceps surae were measured 2 and 4 weeks postoperatively. The strength of ankle plantar flexion was measured. Correlations of DTI parameters with morphological values and muscle strength were analyzed. Results: l1 declined after tenotomy (P < 0.01), and l2 and l3 declined at 4 weeks posttenotomy (P < 0.01). FA increased at 4 weeks posttenotomy (P < 0.01). Atrophy of the triceps surae was observed in the tissues images of the treatment group. The muscle belly significantly shortened postoperatively, and a decline in plantar flexion force was observed after tenotomy. Positive correlations were observed between l1 and muscle strength (r ¼ 0.89) and between l1 and the length of the long axis of the muscle belly (r ¼ 0.81). Conclusion: DTI may serve as a marker of muscle function. Key Words: Achilles tendon; DTI; MRI; muscle function J. Magn. Reson. Imaging 2014;00:000–000. C 2014 Wiley Periodicals, Inc. V
DIFFUSION TENSOR IMAGING (DTI) uses magnetic resonance imaging (MRI) to visualize the diffusion anisotropy of water molecules within tissues (1). In fibrous 1 Department of Orthopaedics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan. 2 Department of Anatomy and Neurobiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan. *Address reprint requests to: K.I., Department of Orthopaedics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto, Japan. E-mail: [email protected] Received May 7, 2014; Accepted June 30, 2014. DOI 10.1002/jmri.24707 View this article online at wileyonlinelibrary.com. C 2014 Wiley Periodicals, Inc. V
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Table 1 Scheme of the Study Protocol Study In vivo imaging and histology of the muscle Motor function test and morphological measurements Correlation analysis
Numbers 6 6 9
The number of rats used in each study.
also hypothesized that changes in DTI may correlate with changes in muscle strength. The purpose of the present study was to investigate changes in rat triceps surae after Achilles tenotomy based on the relationships between DTI parameters and functions and to examine the usefulness of DTI in evaluating muscle function. MATERIALS AND METHODS Rat Model The subjects included a total of 21 12-week-old male Sprague–Dawley rats. This study used rats that differed in terms of in vivo imaging and histological findings, motor function test and morphological measurements, and correlation analysis (Table 1). The rats were reared in individual cages. All treatments, surgeries, and MRI were performed under isoflurane inhalation anesthesia. Anesthesia was induced using 4% isoflurane in an anestheticinduction chamber and then maintained with continuous inhalation of 1.5% isoflurane using a facemask; body temperature and respiratory rates were monitored. All experimental procedures in this study were approved by the Animal Care and Use Committee of our university. The approval number of this study is M25–204.
at an angle of 20 flexion, and the ankle was placed in the neutral position. The scanning sequence parameters for PDWI were as follows: relaxation time (TR), 2000 msec; echo time (TE), 45 msec; echo train length, 4; field of view (FOV), 60 60 mm; matrix; 512 512; and slice thickness, 2 mm. The scanning parameters for DTI were as follows: TR, 3000 msec; TE, 14 msec; echo train length, 4; FOV, 60 mm 60 mm; pixel matrix, 128 128; slice thickness, 2 mm; and b-values, 0 s/mm2 and 350 s/ mm2. Motion probing gradient orientations were applied along six directions: [Gx,Gy,Gz] ¼ [1,1,0], [1,0,1], [0,1,1], [-1,1,0], [0,-1,1], and [1,0,-1]. The entire scanning time for each subject was 120 minutes. Image Analysis The anatomical position of the triceps surae was confirmed in the sagittal and axial PDWI images, and the axial slice in which the muscle belly area was largest was determined. The regions of interest for the soleus, medial, and lateral heads of the gastrocnemius were established in the axial images (12). Eigenvalues (l1, l2, and l3) and fractional anisotropy (FA), which is an index of anisotropic strength, were calculated in these areas of interest. These were measured using ImageJ software (13). FA values were calculated using eigenvalues (l1, l2, and l3) in the following formulas: qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 2 2 pffiffiffiffiffiffiffiffiffi ðl1 -DÞ þ ðl2 -DÞ þ ðl3 -DÞ qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi FA¼ 2=3 ðl21 þl22 þ l23 Þ D¼ðl1 þl2 þl3 Þ=3
In Vivo Imaging and Histology of the Muscle In Vivo Imaging
Achilles Tenotomy Operation To model triceps surae dysfunction, Achilles tenotomy was performed on the right hindlegs (11). Using aseptic techniques, a longitudinal skin incision was created directly above the Achilles tendon to visually confirm the Achilles and plantaris tendons. A scalpel was used to cut only the Achilles tendon about 5 mm proximal to its attachment to the calcaneal bone; after that, discontinuity of the Achilles tendon was confirmed. The Achilles tendon was left unsutured, while the plantaris tendon was left intact as an internal splint. The wound was washed with physiological saline solution and closed using 4-0 monofilament nylon. MRI Measurement MR images were obtained using a high magnetic field MRI unit designed for animal experiments (Varian MRI system 7.04T, horizontal orientation; Agilent Technology, Palo Alto, CA) with a 210 mm gradient coil (Agilent Technology); vnmrJ MRI software was used (Agilent Technology). The MRI protocol included fast spin-echo proton density-weighted imaging (PDWI) and DTI. Before running the DTI sequence, PDWI was obtained to confirm the muscle belly of triceps surae. The knee joint was fixed
Changes in postoperative DTI were evaluated in six rats that underwent Achilles tenotomy. We performed imaging of the hindlegs on both sides before tenotomy and 2 and 4 weeks posttenotomy; we then compared the unaffected and affected sides at each point. Histology of the Muscle After undergoing MRI, six rats were sacrificed by intraperitoneal injection of a pentobarbital overdose at 4 weeks postoperatively. The triceps surae bellies from the proximal attachment to the muscle-tendon junction were removed with a scalpel. The 20-mm-thick fresh-frozen slices of muscle bellies were made using liquid nitrogen and nhexane. Hematoxylin and eosin (H&E) staining and Azan staining were performed. Motor Function Test and Morphological Measurements Motor Function Test For evaluation of motor function, three rats were used at 2 weeks posttenotomy and three rats at 4 weeks posttenotomy. Under inhalation anesthesia, the rats were placed in a lateral recumbent position and the
DTI for Assessing Muscle Function
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Table 2 Results of DTI Parameters
l1 l2 l3 FA
Unaffected side Affected side Unaffected side Affected side Unaffected side Affected side Unaffected side Affected side
Pre
2 Weeks
4 Weeks
1.814 6 0.070 1.820 6 0.076 1.333 6 0.008 1.389 6 0.025 1.092 6 0.029 1.089 6 0.075 0.238 6 0.042 0.238 6 0.037
1.823 6 0.088 1.737 6 0.120 1.316 6 0.077 1.337 6 0.079 1.065 6 0.029 1.050 6 0.029 0.271 6 0.022 0.265 6 0.040
1.939 6 0.050 1.793 6 0.051 1.473 6 0.169 1.303 6 0.112 1.214 6 0.049 1.019 6 0.083 0.237 6 0.022 0.282 6 0.043
Mean values 6 standard deviation of the eigenvalues and the fractional anisotropy (l1, l2, l3)[103 mm2/s].
knee joint and trunk were fixed. The knee and ankle joint were fixed at the same leg position as that for MRI. Hair on the hindlegs was removed. Surface stimulation electrodes (NM-410S; Nihon Kohden, Tokyo, Japan) were placed on the posterior surface of the lower legs and electrical stimulation was applied at 20 mA, 1 Hz. A digital force gauge (FGP-20; NidecShimpo, Kyoto, Japan) was attached to the sole to measure plantar flexion force (14).
Motor Function Tests
Morphological Measurements
The measured values 2 weeks and 4 weeks posttenotomy on the left and right sides were compared using a paired t-test. Mean values were calculated for the ratio of the long axis length of the muscle belly to the full long axis length (muscle belly length / full length) of the triceps surae at each timepoint. The anteroposterior and horizontal diameters were used to approximate the cross-sectional area, and mean values for each timepoint were calculated.
The rats that underwent motor function tests (three rats at 2 weeks posttenotomy and three rats at 4 weeks posttenotomy) were used to evaluate morphological changes after tenotomy. After euthanasia, we harvested the triceps surae, still attached to the bone. The length of the triceps surae belly, its anteroposterior diameter, and horizontal diameter were measured using digital calipers. The length of the muscle belly was measured from the distal muscle-tendon junction to the proximal attachment of the triceps surae to the femur. The maximum anteroposterior and horizontal diameters were measured. The knee and ankle joint were fixed at the same leg position as that for MRI. Correlation Analysis Finally, we investigated the correlations between posttenotomy DTI parameters and the results of motor function tests and morphological measurements. Three rats that did not undergo tenotomy, three rats at 2 weeks posttenotomy, and three rats at 4 weeks posttenotomy were used. MRI was performed with the methods previously described, and motor function tests were conducted afterward. The full length of the long axis of the triceps surae and the length of the long axis of the muscle belly of the triceps surae were measured in the sagittal PDWI images. The long axis of the muscle belly was measured from the distal muscle-tendon joint to the proximal attachment of the triceps surae to the femur.
The values measured on the affected side were divided by those on the unaffected side at 2 weeks and 4 weeks posttenotomy. With the control value as 1, an unpaired t-test was used to compare the control with the affected/unaffected (A/U) ratio at each timepoint. Morphological Measurements
Correlation Analysis We evaluated correlations between changes in long axis length and l1, and between changes in muscle strength and DTI parameters. A/U ratios were calculated for muscle belly length / full length, muscle strength, FA, l1, l2, and l3 as the degree of change; then correlation coefficients were computed. JMP10 (SAS Institute, Cary, NC) was used for statistical analysis. Differences were considered significant at P < 0.05. RESULTS In Vivo Imaging The values of l1 declined significantly on the affected side at 2 and 4 weeks posttenotomy (P < 0.01). The values of l2 and l3 declined significantly on the affected side at 4 weeks posttenotomy (P < 0.01). The FA values increased significantly on the affected side at 4 weeks posttenotomy (P < 0.01) (Table 2, Fig. 1a–d).
Statistical Analysis In Vivo Imaging
Histology of the Muscle
We compared the eigenvalues and FA values at each imaging timepoint, and compared the unaffected and affected sides using paired-t-test.
Tissue and cross-sectional images are shown in Fig. 2. H&E staining revealed indistinct muscle bundle boundaries, and dilations between muscle bundles
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Figure 1. Changes in sides for l1 (a), l2 (b), l3 (c), and FA (d) for each imaging timepoint. a: l1 significantly declined at 2 weeks and 4 weeks posttenotomy (*P < 0.01) (white box, unaffected side; lined box, affected side). b: l2 significantly declined at 4 weeks posttenotomy (*P < 0.01). c: l3 significantly declined at 4 weeks posttenotomy (*P < 0.01). d: FA significantly increased at 4 weeks posttenotomy (*P < 0.01).
(Fig. 2a,b), which suggest muscle atrophy. Furthermore, Azan staining showed slight proliferation of connective tissue outside the perimysium (Fig. 2c,d).
Motor Function Test Plantar flexion force on the affected side decreased significantly to 82.1 6 0.04% on the unaffected side at 2 weeks posttenotomy (P < 0.05; Fig. 3) and to
Figure 2. Histological changes in the triceps surae belly. An H&E-stained 20-mm slice after tenotomy (a) and control (b). Dilation is observed between muscle bundles at 4 weeks posttenotomy. An Azanstained 20-mm slice after tenotomy (a) and control (b). No particular growth of connective tissue is evident in the areas of dilation between muscle bundles.
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Figure 3. Plot of affected/unaffected (A/U) ratios of posttenotomy ankle plantar flexion force. Plantar flexion force decreased significantly at 2 weeks and 4 weeks posttenotomy (*P < 0.05, **P < 0.01).
76.3 6 0.12% on the unaffected side at 4 weeks posttenotomy (P < 0.01; Fig. 3). Morphological Measurements The triceps surae muscle belly length / full length was 70.1 6 0.02% on the affected side and 79.2 6 0.04% on the unaffected side at 2 weeks posttenotomy, and 70.9 6 0.02% on the affected side and 76.9 6 0.03% on the unaffected side at 4 weeks posttenotomy. Muscle belly length on the affected side significantly shortened at each timepoint (P < 0.01; Fig. 4a). The crosssectional area of the triceps surae belly at 2 weeks posttenotomy was 92.3 6 6.6 mm2 on the affected side and 101.9 6 4.7 mm2 on the unaffected side, and at 4 weeks posttenotomy was 97.7 6 4.0 mm2 on the affected side and 121.4 6 6.0 mm2 on the unaffected side. Cross-sectional areas on the affected side significantly decreased at each timepoint (P < 0.01; Fig. 4b). Correlation Coefficients A positive correlation was observed between the A/U ratios for muscle strength and l1, with a correlation coefficient r ¼ 0.89 (P < 0.01; Fig. 5a). Correlations were not observed between the A/U ratio for muscle strength and the A/U ratios for l2, l3, and FA, with correlation coefficients r ¼ 0.44, 0.64, and 0.27, respectively (Fig. 5b–d). A positive correlation was observed between the A/U ratios for muscle belly length / full length and l1, with correlation coefficient r ¼ 0.81 (P < 0.01; Fig. 6).
DISCUSSION Skeletal muscle has a fibrous structure and is regarded as possessing anisotropy due to its structural characteristics. In research on muscle injury, DTI has been shown to be useful in diagnosis, as changes in anisotropy occur at the site of injury (15). Sinha et al (16) used tractography to visualize the path of healthy muscle fibers. Galban et al (12) reported sex-based differences in the anisotropic diffusion of water molecules in the triceps surae. Zarais-
Figure 4. Triceps surae on the affected and unaffected sides were removed 2 weeks and 4 weeks posttenotomy. These graphs show the length of the long axis of the triceps surae belly divided by the length of the whole muscle and the cross-sectional area. a: The long axis of the triceps surae belly significantly shortened 2 weeks and 4 weeks posttenotomy (*P < 0.01). b: The cross-sectional area of the triceps surae significantly decreased 2 weeks and 4 weeks posttenotomy (*P < 0.01) (white box, unaffected side; lined box, affected side).
kaya et al (17) used DTI to investigate triceps surae injury and intramuscular hematoma, finding lower FA values than in healthy muscle. Therefore, DTI is useful and is being used to depict structural changes in skeletal muscle. However, the interrupted continuity of muscle fibers as seen in muscle injury is not present in posttendon rupture muscle tissue. DTI has also been used to examine muscle atrophy after denervation. Saotome et al (9) employed DTI to evaluate denervated skeletal muscle in rats. FA was significantly higher in atrophied muscle after denervation compared to healthy muscle. Regarding eigenvalues, while they reported no major change in l1, there were significant decreases in l2 and l3. They concluded that reductions in l2 and l3 are related to the density of muscle fibers in the axial plane, but the relationship between l1 and muscle fiber diameter was weak. Zhang et al (18) created a denervation muscle atrophy model by performing unilateral sciatic nerve ligation on the lower leg and evaluated DTI parameters chronologically. The muscle atrophy results of the denervation model showed internal structural changes in cross-sections, significant reductions in l2 and l3, and an increase in FA,
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Figure 5. Correlations between the affected/unaffected (A/U) ratio of muscle strength and the A/U ratios of l1 (a), l2 (b), l3 (c), and FA (d). a: A positive correlation between muscle strength A/U ratio and l1 A/U ratio was observed, with the correlation coefficient r ¼ 0.89 (P < 0.01). b: A correlation was not observed between muscle strength A/U ratio and l2 A/U ratio, with the correlation coefficient r ¼ 0.44 (P ¼ 0.23). c: A correlation was not observed between muscle strength A/U ratio and l3 A/U ratio, with the correlation coefficient r ¼ 0.64 (P ¼ 0.06). d: A correlation was not observed between muscle strength A/ U ratio and FA A/U ratio, with the correlation coefficient r ¼ –0.27 (P ¼ 0.48).
indicating that DTI is useful in detecting skeletal muscle atrophy. In functional evaluations of skeletal muscle, DTI has been used to examine models during passive relaxation and contraction of skeletal muscle. Schwenzer et al (8) evaluated DTI of the triceps surae in healthy humans while passively moving the ankle from 10 dorsiflexion to 40 plantar flexion using a device they developed. An increase in mean diffusivity was observed when the muscle was contracted, which was consistent with a physiological increase in muscle fibers on the axial plane. Moreover, l1 did not significantly vary when shortened or elongated, but l2 and l3 significantly decreased when relaxed. Hatakenaka et al (19,20) reported that l1 decreased while l2 and l3 increased during skeletal muscle changes in length and during passive changes to the articular range of motion. Furthermore, Okamoto et al (21) reported increases in FA, l1, and l2 due to contractions of skeletal muscle during active articular motion. Maganaris et al (22) investigated the relationships between the length of triceps surae fibers and the angle of the muscle’s pinnate structure with angles of ankle plantar/dorsiflexion. From their results, Schwenzer et al (8) also concluded that muscle fiber length greatly affects proton diffusivity.
Past studies have shown that DTI parameters are strongly related to both the causes of pathology and the resultant changes in internal tissue structure. From Hatakenaka et al and Schwenzer et al’s results, we surmised that in skeletal muscle changes in
Figure 6. Correlation between the affected/unaffected (A/U) ratio of muscle-belly length/full length and the A/U ratio of l1. A positive correlation was observed between muscle belly length / full length A/U ratio and l1 A/U ratio, with the correlation coefficient r ¼ 0.81 (P < 0.01).
DTI for Assessing Muscle Function
muscle belly length along the long axis due to changes in articular range of motion would change anisotropy. We believe the length of the long axis of the muscle belly is an important part of perpendicular anisotropy. Achilles tenotomy has been used to model reduced muscle activity (23). After tenotomy, atrophic changes such as reduced muscle mass and growth of connective tissue between muscle bundles are observed (24). Furthermore, there is a decrease in tensile force after tenotomy (23). In this study, we confirmed elongation of the tendon and shortening of the long axis of the muscle belly from the second week posttenotomy. The muscle cross-sectional area also decreased from the second week. Plantar flexion force decreased from the second week, exhibiting a difference between the affected and unaffected sides. With regard to DTI parameters, l1 and l3 decreased from 2 weeks posttenotomy, whereas l2 decreased and FA increased from 4 weeks posttenotomy. The l1 A/U ratio strongly correlated with the A/U ratios of muscle strength and length of the long axis of the muscle belly. We believe that l1 may useful to evaluate posttenotomy muscle strength. l1 is thought to show anisotropy horizontal to the muscle fibers, whereas l2 and l3 show crosssectional anisotropy. Reduced l1 reflects the shortening of the triceps surae, which is considered an important factor in decreased motor function after Achilles tenotomy. A decrease in the cross-sectional area was observed in triceps surae cross-sections. Qualitative histological evaluations may be necessary to consider changes in l2 and l3. The proliferation of connective tissue was mild, which might be related to a variety of factors besides muscle tissue density, such as connective tissue density. One limitation of this study was the short observation period. The Achilles tendon recovers relatively quickly after rupture and muscle atrophy has been reported to progress over time (25). By observing this model over a longer period, we would expect to see an even stronger correlation between DTI and muscle function. Another limitation involves the b-value. The b-value used here was 350 s/mm2, which has been used frequently in research studies on skeletal muscle. Appropriate b-values for skeletal muscle have been reported to be 300–900 s/mm2 (26). Setting a high b-value allows for more detailed evaluations of intracellular anisotropy, but this can decrease the S/ N ratio and harm the reliability of the test. Moreover, since we desired to evaluate not only muscle cell anisotropy but also connective tissue inside the perimysium, we used a relatively low b-value. In the future, it will be necessary to vary the b-value to determine the most appropriate value for muscle dysfunction. We found that during the initial stage after Achilles tenotomy, muscle function and the degree of tendon elongation were correlated with l1. Muscle dysfunction due to tendon elongation is one complication of Achilles tendon rupture. In the early stage before the tendon recovers, there is a possibility of rerupture, and the evaluation of muscle dysfunction is problematic. The use of our method allows for estimation of muscle function and assessment of tendon elongation
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during the initial phase of treatment for ruptured tendon, potentially preventing muscle dysfunction. In conclusion, tendon elongation and shortening of the long axis of the muscle belly occur after Achilles tenotomy. In DTI, decreased l1, l2, and l3 of the triceps surae and increased FA were observed. l1 exhibited strong positive correlations with muscle strength and length of the long axis of the muscle belly. DTI could be potentially used in the evaluation of muscle function. REFERENCES 1. Basser PJ, Mattiello J, LeBihan D. MR diffusion tensor spectroscopy and imaging. Biophys J 1994;66:259–267. 2. Mamata H, Jolesz FA, Maier SE. Characterization of central nervous system structures by magnetic resonance diffusion anisotropy. Neurochem Int 2004;45:553–560. 3. Heemskerk AM, Sinha TK, Wilson KJ, Ding Z, Damon BM. Quantitative assessment of DTI-based muscle fiber tracking and optimal tracking parameters. Magn Reson Med 2009;61:467–472. 4. Shorten PR, Sneyd J. A mathematical analysis of obstructed diffusion within skeletal muscle. Biophys J 2009;96:4764–4778. 5. Aliev MK, Tikhonov AN. Random walk analysis of restricted metabolite diffusion in skeletal myofibril systems. Mol Cell Biochem 2004;256:257–266. 6. Okamoto Y, Okamoto T, Yuka K, Hirano Y, Isobe T, Minami M. Correlation between pennation angle and image quality of skeletal muscle fibre tractography using deterministic diffusion tensor imaging. J Med Imaging Radiat Oncol 2012;56:622–627. 7. Sosnovik DE, Wang R, Dai G, Reese TG, Wedeen VJ. Diffusion MR tractography of the heart. J Cardiovasc Magn Reson 2009;11: 47. 8. Schwenzer NF, Steidle G, Martirosian P, et al. Diffusion tensor imaging of the human calf muscle: distinct changes in fractional anisotropy and mean diffusion due to passive muscle shortening and stretching. NMR Biomed 2009;22:1047–1053. 9. Saotome T, Sekino M, Eto F, Ueno S. Evaluation of diffusional anisotropy and microscopic structure in skeletal muscles using magnetic resonance. Magn Reson Imaging 2006;24:19–25. 10. Kearney RS, McGuinness KR, Achten J, Costa ML. A systematic review of early rehabilitation methods following a rupture of the Achilles tendon. Physiotherapy 2012;98:24–32. 11. Bring D, Reno C, Renstrom P, Salo P, Hart D, Ackermann P. Prolonged immobilization compromises up-regulation of repair genes after tendon rupture in a rat model. Scand J Sci Sports 2010;20: 411–417. 12. Galb an CJ, Maderwald S, Uffmann K, de Greiff A, Ladd ME. Diffusive sensitivity to muscle architecture: a magnetic resonance diffusion tensor imaging study of the human calf. Eur J Appl Physiol 2004;93:253–262. 13. Rasband WS. ImageJ, U.S. National Institutes of Health, Bethesda, MD. http://imagej.nih.gov/ij/, 1997–2012. 14. Takagi T, Nakamura M, Yamada M, et al. Visualization of peripheral nerve degeneration and regeneration: monitoring with diffusion tensor tractography. Neuroimage 2009;44:884–892. 15. McMillan AB, Shi D, Pratt SJ, Lovering RM. Diffusion tensor MRI to assess damage in healthy and dystrophic skeletal muscle after lengthening contractions. J Biomed Biotechnol 2011;2011: 760726. 16. Sinha U, Yao L. In vivo diffusion tensor imaging of human calf muscle. J Magn Reson Imaging 2002;15:87–95. 17. Zaraiskaya T, Kumbhare D, Noseworthy MD. Diffusion tensor imaging in evaluation of human skeletal muscle injury. J Magn Reson Imaging 2006;24:402–408. 18. Zhang J, Zhang G, Morrison B, Mori S, Sheikh KA. Magnetic resonance imaging of mouse skeletal muscle to measure denervation atrophy. Exp Neurol 2008;212:448–457. 19. Hatakenaka M, Yabuuchi H, Matsuo Y, et al. Effect of passive muscle length change on apparent diffusion coefficient: detection with clinical MR imaging. Magn Reson Med Sci 2008;7:59–63. 20. Hatakenaka M, Matsuo Y, Setoguchi T, et al. Alteration of proton diffusivity associated with passive extension and contraction. J Magn Reson Imaging 2008;27:932–937.
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