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Skeletal Muscle Degeneration and Regeneration after Femoral Artery Ligation in Mice: Monitoring with Diffusion MR Imaging¹

¹ From the Biomedical NMR Group, Department of Biomedical Engineering, Eindhoven University of Technology, Den Dolech 2, 5612 AZ, Eindhoven, the Netherlands (A.M.H., G.J.S., G.S.v.B., K.N.); and the Nutrition and Toxicology Research Institute (NUTRIM), Department of Movement Sciences, Maastricht University, Maastricht, the Netherlands (M.R.D.). Received March 17, 2006; revision requested May 18; revision received July 13; final version accepted September 8. Address correspondence to K.N. (e-mail: k.nicolay{at}tue.nl).

	ABSTRACT

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Purpose: To prospectively evaluate quantitative diffusion magnetic resonance (MR) imaging for monitoring skeletal muscle injury and repair after femoral artery ligation in mice.

Materials and Methods: All experimental procedures were approved by the local institutional animal care and use committee. Muscle degeneration and regeneration were induced in 16 mice by using unilateral ligation of the femoral artery. Diffusion-tensor and T2-weighted MR imaging examinations were performed before, immediately after, and 3, 10, and 21 days after ligation. Histologic analysis was also performed at these time points. The dynamic changes in T2 and in five diffusion-tensor imaging indexes were studied by using histogram analysis. Differences between the ligated and nonligated limbs were assessed with paired t tests, and analysis of variance was used to determine temporal evolutions. Parametric maps were clustered to depict regional differences in the responses of the different MR imaging indexes.

Results: MR indexes in the ligated limb changed over time (P < .007), and temporal evolutions in the ligated and nonligated limbs differed significantly (P < .001). When ischemia was induced, diffusivity and T2 increased, with a maximum change at 3 days, when most muscle damage was observed at histologic analysis. At 10 days, diffusion values were reduced overall, whereas T2 was still increased. At 21 days, parameter values had largely returned to normal. Changes on the diffusion-tensor and T2 maps had spatial differences, which corresponded to the different phases of tissue regeneration observed at histologic analysis. An additional finding was the transient change in direction of the principal eigenvector during the period of maximal muscle damage.

Conclusion: After femoral artery ligation, the diffusion-tensor indexes changed dynamically in association with the severity and location of muscle damage.

© RSNA, 2007

	INTRODUCTION

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Skeletal muscle ischemia-reperfusion injury is frequently observed—for example, in peripheral vascular diseases and after surgery. The processes that underlie this type of muscle injury are commonly investigated by using animal models. These include models of prolonged ischemia (1–3), in which the femoral artery typically is ligated, the muscle tissue degenerates and regenerates over time, and muscle function is temporarily lost. After injury is induced, skeletal muscle has the ability to undergo rapid repair, limiting the loss of muscle mass. The initial phase of muscle repair is characterized by necrosis of the damaged tissue and initiation of an inflammatory response. This phase is followed by activation of myogenic cells, which leads to the formation of new myofibers and the reconstruction of the contractile apparatus (1).

Magnetic resonance (MR) imaging is a noninvasive imaging tool that enables excellent soft-tissue differentiation. Skeletal muscle MR imaging—commonly based on T2 and T1 contrast—can be used to diagnose diseases, evaluate lesions, help guide biopsy, and monitor therapeutic interventions (2). Generally, with muscle injury, an increase in T2 with a homogeneous or heterogeneous regional appearance is reported (3–6). Overall, T2 is sensitive to the presence of edema, necrosis, and inflammation (7). However, these MR findings are usually not specific for a given muscle disorder, and additional clinical or histologic features should be analyzed to establish a specific diagnosis (8).

Diffusion-weighted MR imaging is used to measure the self diffusion of water in tissue, which is affected by the presence and orientation of physical barriers. Cellular damage alters this structural organization and consequently results in changed diffusion pathways. The orientation dependency of the diffusion barriers can be characterized by measuring the diffusion tensor (DT). From this tensor, the mean apparent diffusion coefficient (ADC), eigenvalues, eigenvectors, and indexes of diffusion anisotropy can be deduced. All of these parameters provide information about the local microstructure and geometry of the tissue. Although diffusion-weighted MR imaging and DT imaging have been used to study the dynamics of water content (9–11) and the fiber architecture (12,13) in healthy muscles, diffusion MR imaging has been applied to also assess myopathies relatively recently (14,15).

We expected DT MR imaging to be sensitive to not only acute damage development but also prolonged muscle injury and repair processes. Therefore, the goal of our study was to prospectively evaluate quantitative diffusion MR imaging for monitoring skeletal muscle injury and repair in mice after femoral artery ligation.

	MATERIALS AND METHODS

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Animal Model
All experimental procedures were approved by the local institutional animal care and use committee. Experiments were performed on 16 C57BL/6 mice (Charles River, Maastricht, the Netherlands). The mice were anesthetized with 1.0%–1.5% isoflurane (Schering-Plough Animal Health, Maarssen, the Netherlands) (in air), which was administered at a flow rate of 0.4 L/min and controlled by using an SF3 flowmeter system (UNO, Zevenaar, the Netherlands) and an isoflurane vaporizer (Penlon, Abingdon, United Kingdom). The anesthetic was delivered through a face mask. A temperature of 38°C was maintained, and the animal's respiration was monitored by using an electrocardiographic respiratory trigger unit (ECG Trigger Unit HR; Rapid Biomedical, Würzburg, Germany).

Muscle ischemia was induced (research assistant and A.M.H.) in the mouse's right hind limb by blocking blood flow in the right femoral artery, above the origin of the natural collateral vessels (16). An incision was made at the ventral side of the right limb. The femoral artery was exposed and ligated with suture knots. Care was taken to leave the femoral vein and nerve untouched. A similar dermal incision was made in the contralateral (left) limb and served as the sham procedure.

Fifteen mice were examined (A.M.H., G.S.v.B., G.J.S.) immediately, 12 were examined 3 days, seven were examined 10 days, and four were examined 21 days after ligation. The first five mice were also examined before ligation. However, it turned out that examining the animals before ligation, performing the ligation, and then examining them again immediately after the procedure were too stressful for the mice and resulted in unstable anesthesia. Therefore, the remaining mice were not examined before ligation. On each examination day, a number of mice were randomly sacrificed after the MR experiments for histologic analysis (A.M.H., G.S.v.B., M.R.D.): One mouse was sacrificed before ligation; three were sacrificed immediately (approximately 2.5 hours) after ligation; four, 3 days after ligation; three, 10 days after ligation; and four, 21 days after ligation. Two mice died: one during ligation and one after 8 days. We assessed limb functionality (A.M.H., G.S.v.B.) daily by looking at behavioral changes and at the shape and color of the limbs.

MR Imaging
MR measurements were performed by using a horizontal 9.5-cm bore, 270-MHz unit with a Varian imaging console (Varian, Palo Alto, Calif) equipped with 380 mT/m shielded gradients with a 150-µsec rise time to maximum and a 3.2-cm-diameter birdcage coil. A diffusion-weighted spin-echo MR sequence with fat suppression was used. The diffusion gradients were applied in six noncolinear directions (17), and one reference image was obtained without diffusion weighting. Imaging parameters were as follows: 1500/30 (repetition time msec/echo time msec), 20 x 20-mm² field of view, 128 x 64 matrix, 1.5-mm section thickness, 0.5-mm intersection gap, four sections, two acquired signals, diffusion gradient separation time of 13 msec, diffusion gradient duration of 8 msec, and b value of 0 sec/mm² or 572 sec/mm². Fat suppression was achieved by means of frequency-selective excitation followed by gradient spoiling. To calculate T2 maps, a multiecho spin-echo MR sequence with fat suppression was used with the same spatial resolution as the DT imaging measurements, 4000/13.3–79.8, an echo train length of six, and two acquired signals. To determine changes in limb volume, a high-spatial-resolution spin-echo MR sequence was performed by using 2000/21, a 20-mm² field of view, a 256 x 128 matrix, a 1.5-mm section thickness, an intersection gap of 0.5 mm, 11 sections, and four acquired signals.

Data Analysis
The MR images were analyzed (A.M.H., G.S.v.B., G.J.S.) by using Mathematica (Wolfram Research, Champaign, Ill) software. The pixel intensities of the DT imaging data set were fitted by using the b matrix to obtain the six elements of the DT (D). All imaging gradients were taken into account in the calculation of the b matrix (18). The following parameters were calculated: three eigenvalues (₁, ₂, and ₃), eigenvectors, mean ADC, and fractional anisotropy (FA), where ADC = trace(D)/3 and

T2 maps were derived by means of monoexponential fitting of the six echoes.

For each animal and time point, a frequency distribution of the parametric images for all pixels in the three sections through the muscles was created. The fourth image section was not used because it contained mainly bone. The histogram had 60 frequency bins ranging from 0–60 msec, (0–3) x 10^–3 mm²/sec, and 0–1 for T2, diffusivity, and FA, respectively. The modus of these frequency distributions—that is, the position of the bin with the highest counts—was analyzed over time.

Histologic Analysis
After the final MR examinations were performed, the mice were perfusion fixed with 3.5% formaldehyde. Both hind limbs were excised, preserved in formaldehyde (for approximately 1 month), and then embedded in plastic (Technovit 7100; Heraeus Kulzer, Duiven, the Netherlands). The limbs were cut into 3-µm cross sections and stained with Gomori trichrome. The histologic slices were compared (A.M.H., G.S.v.B., M.R.D., G.J.S.) as closely as possible with the same area on the MR images, as determined by means of visual inspection of the limb contour and the location of the bones. Damaged areas with swollen cells, inflammatory cells, or large amounts of collagen that were visible on the histologic slices were compared with the MR indexes.

Statistical Analyses
SPSS, version 12.0.1 (SPSS, Chicago, Ill), was used to perform the statistical analyses (A.M.H., G.S.v.B., M.R.D.). Differences in MR index values between the ligated and nonligated limbs were assessed by using paired t tests at each time point. The temporal evolution of the MR indexes was tested by using repeated-measures one-way analyses of variance (with time as the factor); the ligated and nonligated limbs were tested at separate analyses of variance. To address the variation in the number of animals that were measured as a function of time, four analyses of variance were performed for each MR index and condition—that is, before and immediately after ligation for three mice; immediately and 3 days after ligation for 12 mice; immediately and 3 and 10 days after ligation for seven mice; and immediately and 3, 10, and 21 days after ligation for seven mice. Repeated-measures two-way analyses of variance (with time and condition [ie, ligated vs nonligated] as factors) were also performed. Both the main effect condition and the product of interaction time multiplied by condition indicated the physiologically relevant differences; the effect of time was tested at one-way analyses of variance. In all cases, P < .05 indicated significance.

The DT and T2 data were clustered to capture regional differences in the MR indexes on one image. k-Means clustering was performed on the basis of ₃, FA, and T2 values for the image section obtained through approximately the middle of the belly through the tibialis anterior. ₃ Was used because it showed the highest correlation with tissue damage, while FA was chosen since it reports on tissue anisotropy (15). Clustering was applied to the combined parametric images obtained in all of the mice independently on days 3 and 10 after ligation. Mean index values for each data cluster were determined. The final number of clusters used was based on multiple runs. We increased the number of clusters until we observed no significant differences in mean T₂, ₃, or FA between the clusters.

	RESULTS

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Limb Function and Histologic Changes
Immediately after ligation, the right limb turned purple and became stiff, while the left limb, which was exposed to the sham intervention, remained normal. In the following days, the mice with a ligated limb limped severely or dragged the affected limb. The mice started to use the injured limb again within 4–7 days. Ten days after ligation, the effects of the procedure were still noticeable: The animals moved with a slight limp. In the following week, limb functionality recovered such that at day 21, no difficulties in movement were noted. The volume of the ligated limb, as deduced from the MR image findings, had increased by 35% (P < .001) by day 3, compared with the volume of the nonligated limb, and was normal by day 10.

Before ligation (Fig 1a), the myocytes had their well-known typical angular shape and peripheral nuclei. Immediately after ligation (Fig 1b), the cells appeared to be slightly rounder and the volume of the extracellular compartment was somewhat increased. At day 3, most of the myocytes were round and swollen (Fig 1c), almost no nuclei were visible, and signs of inflammation were present at the periphery of the muscles (Fig 1d). The recovery progressed inward (ie, from muscle periphery toward inner region); at 10 days (Fig 1e), three zones were visible: a zone with round and swollen myocytes in the center of the muscles (similar to findings in Fig 1c), a zone with inflammation (similar to findings in Fig 1d), and a zone of both small myocytes with a central nucleus and white spots (presumably fat infiltrations). At day 21, almost complete tissue recovery was seen (Fig 1f), with the exception that the myocytes were slightly smaller than normal.

View larger version (155K): [in this window] [in a new window] [Download PPT slide]   Figure 1a: Histologic specimens obtained from different regions of the tibialis anterior at different time points in the ligation model illustrate changes in tissue over time: (a) central region before ligation; (b) central region immediately after ligation; (c) central region 3 days after ligation; (d) peripheral region 3 days after ligation; (e) central region 10 days after ligation, with three zones—degenerating zone (near tendon sheet [*]), inflammatory zone, and regenerating zone (small dotted lines); and (f) central region 21 days after ligation. Muscle recovery generally progressed from the outer regions to the central regions. Given examples (from different mice) are representative of all mice and all muscles, although the time course of recovery can vary between mice and between muscles. Muscles with a large cross-sectional area needed more time to recover than did smaller muscles. Arrows in a point to peripheral nuclei. In b–e, long black arrows point to rounder and/or swollen myocytes. In d and e, short black arrows point to inflammatory cells. In e and f, white arrows point to central nuclei. All slices were stained simultaneously with Gomori trichrome; therefore, differences in color did not originate from staining artifacts. Green color indicates collagen. (Original magnification, x10.)

View larger version (155K): [in this window] [in a new window] [Download PPT slide]   Figure 1b: Histologic specimens obtained from different regions of the tibialis anterior at different time points in the ligation model illustrate changes in tissue over time: (a) central region before ligation; (b) central region immediately after ligation; (c) central region 3 days after ligation; (d) peripheral region 3 days after ligation; (e) central region 10 days after ligation, with three zones—degenerating zone (near tendon sheet [*]), inflammatory zone, and regenerating zone (small dotted lines); and (f) central region 21 days after ligation. Muscle recovery generally progressed from the outer regions to the central regions. Given examples (from different mice) are representative of all mice and all muscles, although the time course of recovery can vary between mice and between muscles. Muscles with a large cross-sectional area needed more time to recover than did smaller muscles. Arrows in a point to peripheral nuclei. In b–e, long black arrows point to rounder and/or swollen myocytes. In d and e, short black arrows point to inflammatory cells. In e and f, white arrows point to central nuclei. All slices were stained simultaneously with Gomori trichrome; therefore, differences in color did not originate from staining artifacts. Green color indicates collagen. (Original magnification, x10.)

View larger version (156K): [in this window] [in a new window] [Download PPT slide]   Figure 1c: Histologic specimens obtained from different regions of the tibialis anterior at different time points in the ligation model illustrate changes in tissue over time: (a) central region before ligation; (b) central region immediately after ligation; (c) central region 3 days after ligation; (d) peripheral region 3 days after ligation; (e) central region 10 days after ligation, with three zones—degenerating zone (near tendon sheet [*]), inflammatory zone, and regenerating zone (small dotted lines); and (f) central region 21 days after ligation. Muscle recovery generally progressed from the outer regions to the central regions. Given examples (from different mice) are representative of all mice and all muscles, although the time course of recovery can vary between mice and between muscles. Muscles with a large cross-sectional area needed more time to recover than did smaller muscles. Arrows in a point to peripheral nuclei. In b–e, long black arrows point to rounder and/or swollen myocytes. In d and e, short black arrows point to inflammatory cells. In e and f, white arrows point to central nuclei. All slices were stained simultaneously with Gomori trichrome; therefore, differences in color did not originate from staining artifacts. Green color indicates collagen. (Original magnification, x10.)

View larger version (174K): [in this window] [in a new window] [Download PPT slide]   Figure 1d: Histologic specimens obtained from different regions of the tibialis anterior at different time points in the ligation model illustrate changes in tissue over time: (a) central region before ligation; (b) central region immediately after ligation; (c) central region 3 days after ligation; (d) peripheral region 3 days after ligation; (e) central region 10 days after ligation, with three zones—degenerating zone (near tendon sheet [*]), inflammatory zone, and regenerating zone (small dotted lines); and (f) central region 21 days after ligation. Muscle recovery generally progressed from the outer regions to the central regions. Given examples (from different mice) are representative of all mice and all muscles, although the time course of recovery can vary between mice and between muscles. Muscles with a large cross-sectional area needed more time to recover than did smaller muscles. Arrows in a point to peripheral nuclei. In b–e, long black arrows point to rounder and/or swollen myocytes. In d and e, short black arrows point to inflammatory cells. In e and f, white arrows point to central nuclei. All slices were stained simultaneously with Gomori trichrome; therefore, differences in color did not originate from staining artifacts. Green color indicates collagen. (Original magnification, x10.)

View larger version (168K): [in this window] [in a new window] [Download PPT slide]   Figure 1e: Histologic specimens obtained from different regions of the tibialis anterior at different time points in the ligation model illustrate changes in tissue over time: (a) central region before ligation; (b) central region immediately after ligation; (c) central region 3 days after ligation; (d) peripheral region 3 days after ligation; (e) central region 10 days after ligation, with three zones—degenerating zone (near tendon sheet [*]), inflammatory zone, and regenerating zone (small dotted lines); and (f) central region 21 days after ligation. Muscle recovery generally progressed from the outer regions to the central regions. Given examples (from different mice) are representative of all mice and all muscles, although the time course of recovery can vary between mice and between muscles. Muscles with a large cross-sectional area needed more time to recover than did smaller muscles. Arrows in a point to peripheral nuclei. In b–e, long black arrows point to rounder and/or swollen myocytes. In d and e, short black arrows point to inflammatory cells. In e and f, white arrows point to central nuclei. All slices were stained simultaneously with Gomori trichrome; therefore, differences in color did not originate from staining artifacts. Green color indicates collagen. (Original magnification, x10.)

View larger version (143K): [in this window] [in a new window] [Download PPT slide]   Figure 1f: Histologic specimens obtained from different regions of the tibialis anterior at different time points in the ligation model illustrate changes in tissue over time: (a) central region before ligation; (b) central region immediately after ligation; (c) central region 3 days after ligation; (d) peripheral region 3 days after ligation; (e) central region 10 days after ligation, with three zones—degenerating zone (near tendon sheet [*]), inflammatory zone, and regenerating zone (small dotted lines); and (f) central region 21 days after ligation. Muscle recovery generally progressed from the outer regions to the central regions. Given examples (from different mice) are representative of all mice and all muscles, although the time course of recovery can vary between mice and between muscles. Muscles with a large cross-sectional area needed more time to recover than did smaller muscles. Arrows in a point to peripheral nuclei. In b–e, long black arrows point to rounder and/or swollen myocytes. In d and e, short black arrows point to inflammatory cells. In e and f, white arrows point to central nuclei. All slices were stained simultaneously with Gomori trichrome; therefore, differences in color did not originate from staining artifacts. Green color indicates collagen. (Original magnification, x10.)

View larger version (60K): [in this window] [in a new window] [Download PPT slide]   Figure 2: Representative parametric images obtained in one mouse at different times (immediately [A] and days 3 [B], 10 [C], and 21 [D]) after right femoral artery ligation. On all images, the ligated limb is on the left. Image sections were obtained approximately centrally through the limb. ADC (values in x10–3 mm2/sec) and FA maps were derived from diffusion-weighted spin-echo MR (1500/30, b = 0 sec/mm2 or 572 sec/mm2) measurements. T2 maps (values in milliseconds) were derived from multiecho spin-echo MR (4000/13.3–79.8) measurements. The limbs appear flattened as a consequence of positioning, and the cross-sectional position of both limbs varied.

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   Figure 3: Changes in MR indexes before and as a result of femoral artery ligation. Data are mean values (for all mice) of the modus of the frequency distributions for three sections through the limb. ADC, 1, 2, and 3 (in x10–3 mm2/sec); T2 (in milliseconds); and FA values are shown. = ligated limb, = nonligated limb. Error bars represent standard deviations. * = P < .05, ** = P < .01, and *** = P < .001 for comparison of ligated versus nonligated limbs. The temporal evolution of the MR indexes in each limb indicated significant effects in the ligated limb (P < .001 for T2, 1, 2, and 3; P = .007 for ADC). In the nonligated limb, the temporal evolution was significant for 1 (P < .001), 2 (P < .001), 3 (P = .047), and ADC (P < .001), but not for T2 (P = .815) or FA (P = .284). The temporal evolutions of all indexes were different between the ligated and nonligated limbs (P < .001).

View larger version (19K): [in this window] [in a new window] [Download PPT slide]   Figure 4a: Color plot of the orientation of the eigenvectors, for the mouse and image section shown in Figure 2, (a) immediately after femoral artery ligation; (b) 3 days after ligation, when all eigenvalues were increased; (c) 10 days after ligation; and (d) 21 days after ligation. A change in eigenvector orientation after 3 days was observed in half the mice. The effects were more pronounced for some animals, which had more pixels with changed orientation. At day 10, a region of changed orientation was still present in the gastrocnemius. This region corresponded to the location of increased ADC (Fig 2). The eigenvector orientations were color coded as follows: Blue indicated out of plane; green, top to bottom; and red, left to right.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Figure 4b: Color plot of the orientation of the eigenvectors, for the mouse and image section shown in Figure 2, (a) immediately after femoral artery ligation; (b) 3 days after ligation, when all eigenvalues were increased; (c) 10 days after ligation; and (d) 21 days after ligation. A change in eigenvector orientation after 3 days was observed in half the mice. The effects were more pronounced for some animals, which had more pixels with changed orientation. At day 10, a region of changed orientation was still present in the gastrocnemius. This region corresponded to the location of increased ADC (Fig 2). The eigenvector orientations were color coded as follows: Blue indicated out of plane; green, top to bottom; and red, left to right.

View larger version (20K): [in this window] [in a new window] [Download PPT slide]   Figure 4c: Color plot of the orientation of the eigenvectors, for the mouse and image section shown in Figure 2, (a) immediately after femoral artery ligation; (b) 3 days after ligation, when all eigenvalues were increased; (c) 10 days after ligation; and (d) 21 days after ligation. A change in eigenvector orientation after 3 days was observed in half the mice. The effects were more pronounced for some animals, which had more pixels with changed orientation. At day 10, a region of changed orientation was still present in the gastrocnemius. This region corresponded to the location of increased ADC (Fig 2). The eigenvector orientations were color coded as follows: Blue indicated out of plane; green, top to bottom; and red, left to right.

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   Figure 4d: Color plot of the orientation of the eigenvectors, for the mouse and image section shown in Figure 2, (a) immediately after femoral artery ligation; (b) 3 days after ligation, when all eigenvalues were increased; (c) 10 days after ligation; and (d) 21 days after ligation. A change in eigenvector orientation after 3 days was observed in half the mice. The effects were more pronounced for some animals, which had more pixels with changed orientation. At day 10, a region of changed orientation was still present in the gastrocnemius. This region corresponded to the location of increased ADC (Fig 2). The eigenvector orientations were color coded as follows: Blue indicated out of plane; green, top to bottom; and red, left to right.

View larger version (96K): [in this window] [in a new window] [Download PPT slide]   Figure 5: Comparison of MR and histologic findings in a mouse that still had considerable femoral artery damage after 10 days. A, T2 (0–60 msec), B, 3 ([0–2.1] x 10–3 mm2/sec), and, C, FA (0.1–0.7) maps, and, D, clustered MR image based on 3, FA, and T2 values are shown. Red represents high diffusivity; blue, high T2; and light blue, the highest T2. E, Histologic specimen from the same limb at an approximately corresponding cross section. For display purposes, the red and blue channels of the red/green/blue image (E) were enhanced (with image-processing tools) relative to the green channel. F, Histologic specimen from central region of tibialis anterior (area outlined by dotted-line rectangle in E) has high ADC and swollen cells. The locations of the blue regions in D are similar to the locations of the blue areas in E. G, Histologic specimen from the gastrocnemius (area outlined by solid-line rectangle in E) has high T2 and multiple inflammatory cells and collagen. Other mice had similar corresponding clustered image and histologic findings. (Gomori trichrome stain; original magnification, x40.)

View larger version (45K): [in this window] [in a new window] [Download PPT slide]   Figure 6a: Clusters of MR index data obtained (a) immediately, (b) 3 days, (c) 10 days, and (d) 21 days after ligation. The clusters are based on 3, FA, and T2 values and were obtained from the data presented in Figure 2. The clusters are representative of the data sets for the other mice. Mean values for all mice are given in the Table. In general, green, pink, and yellow represent normal values; red represents high diffusivity; blue represents high T2; light blue represents the highest T2; and orange represents reduced diffusivity.

View larger version (46K): [in this window] [in a new window] [Download PPT slide]   Figure 6b: Clusters of MR index data obtained (a) immediately, (b) 3 days, (c) 10 days, and (d) 21 days after ligation. The clusters are based on 3, FA, and T2 values and were obtained from the data presented in Figure 2. The clusters are representative of the data sets for the other mice. Mean values for all mice are given in the Table. In general, green, pink, and yellow represent normal values; red represents high diffusivity; blue represents high T2; light blue represents the highest T2; and orange represents reduced diffusivity.

View larger version (47K): [in this window] [in a new window] [Download PPT slide]   Figure 6c: Clusters of MR index data obtained (a) immediately, (b) 3 days, (c) 10 days, and (d) 21 days after ligation. The clusters are based on 3, FA, and T2 values and were obtained from the data presented in Figure 2. The clusters are representative of the data sets for the other mice. Mean values for all mice are given in the Table. In general, green, pink, and yellow represent normal values; red represents high diffusivity; blue represents high T2; light blue represents the highest T2; and orange represents reduced diffusivity.

View larger version (48K): [in this window] [in a new window] [Download PPT slide]   Figure 6d: Clusters of MR index data obtained (a) immediately, (b) 3 days, (c) 10 days, and (d) 21 days after ligation. The clusters are based on 3, FA, and T2 values and were obtained from the data presented in Figure 2. The clusters are representative of the data sets for the other mice. Mean values for all mice are given in the Table. In general, green, pink, and yellow represent normal values; red represents high diffusivity; blue represents high T2; light blue represents the highest T2; and orange represents reduced diffusivity.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ADVANCES IN KNOWLEDGE References

The above-mentioned processes were observed in our study. Functional recovery of the ligated limb, as was the time course of tissue damage and repair determined at histologic analysis, was comparable to findings in the literature (16,19). The histologic findings were similar to the results obtained by Carlson and Gutmann (20), who studied the morphology of regeneration in the free grafts of lower limb rat muscles. They found that the muscle regeneration process followed a centripetal gradient—that is, it progressed from the outer regions to the inner regions—which resulted in the formation of successive zones of degeneration and regeneration in regenerating muscles. This centripetal gradient was also observed in the ligation experiments performed in our study. With time, the regions with swollen cells became smaller and were located centrally in the muscles, and the area with inflammatory cells moved from the periphery inward.

We observed this same centripetal gradient on the DT and T2 maps, with an increase in T2 gradually moving toward the inner regions. The regional differences between DT and T2 responses showed that these indexes are measures of different processes—for example, areas with swollen cells were characterized by high ADC, while high T2 corresponded to inflamed areas. Our study results indicate that regional differences between quantitative DT MR findings and T2-weighted MR findings might enable the discrimination between degenerative and regenerative areas of the response of skeletal muscle to arterial occlusion.

Behavior and Origin of DT Imaging Changes
It has been established that ADC changes can be due partly to changes in cell size (21–26). In brain tissue, the ADC reflects mainly extracellular diffusion and decreases with cell swelling (21). Furthermore, intracellular diffusion increases, as observed by Trouard et al (26) after ischemia-induced cell swelling in rat glioma cell culture. Damon et al (12) argued that for skeletal muscle, depending on the measurement setting, ₁, ₂, and ₃ values largely reflect the diffusion of water in the intracellular space. Therefore, in contrast to brain cell swelling, myocyte swelling is expected to cause an increase in ADC. This increase in diffusivity was observed in a previous study of acute ischemia in skeletal muscle (27).

A relationship between ADC and cell size was also indicated by our study findings. Three days after ligation, myocytes were swollen (16,19), and this swelling coincided with increases in ₂ and ₃. At day 10, areas with smaller cells and slightly reduced eigenvalues were present. We observed that the orientation of the principal eigenvector changed during the course of the damage. In healthy tissue, the primary eigenvector is known to be along the fiber direction (28,29). In our study, however, at day 3, the orientation of the principal eigenvector was perpendicular to the fiber direction in half of the ligated limbs. This means that the diffusion in the direction perpendicular to the fibers was higher than the diffusion parallel to the fibers. By definition, without the orientation of the eigenvectors taken into account, ₁ is greater than or equal to ₂, which is greater than or equal to ₃ in the calculation of eigenvalues. Therefore, it is possible that the original ₂ becomes larger than the original ₁. This effect is not caused by systemic errors in the estimation of the eigenvectors: We observed no preferential direction of the eigenvectors when we analyzed DT imaging measurements in water phantoms (unpublished results). In the calculation of ADCs, the direction of the eigenvectors is irrelevant. However, in studies in which the eigenvalues are of interest, it is important to verify the direction of the eigenvectors.

Potential shortcomings of this study include the use of parametric t tests with a low number (four to 12) of observations and the imperfect spatial alignment between the MR images and the histologic slices. In future studies, the MR index maps should be carefully coregistered with the histologic slices. This will facilitate quantitative analysis of the correlation between the MR indexes and both the morphologic (eg, cell size and shape) and the histochemical (eg, fiber type dependence and inflammatory activity) features of ischemia-induced muscle injury.

Practical application: Diffusion MR imaging can be used as an in vivo marker and as a diagnostic tool for assessment of ischemia-induced muscle damage. Combined use of DT and T2-weighted MR imaging might enable discrimination between muscle disorders, which up to now have been indistinguishable with MR imaging, and thereby facilitate improved monitoring of the healing of muscle over time. Our study results also indicate that DT imaging and T2-weighted imaging are useful for longitudinally studying the effects of therapeutic interventions aimed at limiting muscle degeneration and/or promoting tissue repair. To our knowledge, no other techniques are available for noninvasive discrimination of the effects of tissue damage and repair.

	ADVANCES IN KNOWLEDGE

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ADVANCES IN KNOWLEDGE References

 

• Parametric values derived by using diffusion-tensor imaging dynamically increase and decrease in response to ischemia-induced skeletal muscle injury.
  
• In healthy muscle tissue, the principal diffusion eigenvector is known to be along the fiber direction; however, in damaged tissue, the orientation of this eigenvector may be perpendicular to the long axis of the fibers.
  

	FOOTNOTES

 

Abbreviations: ADC = apparent diffusion coefficient • DT = diffusion tensor • FA = fractional anisotropy

Authors stated no financial relationship to disclose.

Author contributions: Guarantors of integrity of entire study, A.M.H., K.N.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; manuscript final version approval, all authors; literature research, A.M.H., G.S.v.B.; experimental studies, all authors; statistical analysis, A.M.H., M.R.D.; and manuscript editing, all authors

	References

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ADVANCES IN KNOWLEDGE References

 

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