Non-Invasive MRI and Spectroscopy of mdx Mice Reveal Temporal Changes in Dystrophic Muscle Imaging and in Energy Deficits
Magnetic resonance imaging Legs NMR spectroscopy Muscle tissue Muscle analysis Inflammation Cardiac muscles Mouse models
Alexandru Korotcov Stephen Lin Heather Gordish-Dressman Stanley Fricke Raymond W. Sze Eric P. Hoffman Paul Wang Kanneboyina Nagaraju Christopher R. Heier Alfredo D. Guerron
In Duchenne muscular dystrophy (DMD), a genetic disruption of dystrophin protein expression results in repeated muscle injury and chronic inflammation. Magnetic resonance imaging shows promise as a surrogate outcome measure in both DMD and rehabilitation medicine that is capable of predicting clinical benefit years in advance of functional outcome measures. The mdx mouse reproduces the dystrophin deficiency that causes DMD and is routinely used for preclinical drug testing. There is a need to develop sensitive, non-invasive outcome measures in the mdx model that can be readily translatable to human clinical trials. Here we report the use of magnetic resonance imaging and spectroscopy techniques for the non-invasive monitoring of muscle damage in mdx mice. Using these techniques, we studied dystrophic mdx muscle in mice from 6 to 12 weeks of age, examining both the peak disease phase and natural recovery phase of the mdx disease course. T2 and fat-suppressed imaging revealed significant levels of tissue with elevated signal intensity in mdx hindlimb muscles at all ages; spectroscopy revealed a significant deficiency of energy metabolites in 6-week-old mdx mice. As the mdx mice progressed from the peak disease stage to the recovery stage of disease, each of these phenotypes was either eliminated or reduced, and the cross-sectional area of the mdx muscle was significantly increased when compared to that of wild-type mice. Histology indicates that hyper-intense MRI foci correspond to areas of dystrophic lesions containing inflammation as well as regenerating, degenerating and hypertrophied myofibers. Statistical sample size calculations provide several robust measures with the ability to detect intervention effects using small numbers of animals. These data establish a framework for further imaging or preclinical studies, and they support the development of MRI as a sensitive, non-invasive outcome measure for muscular dystrophy.
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MRI and NMR spectroscopy

In vivo monitoring of mouse hindlimbs and muscle damage was performed using a 9.4 T, 89-mm vertical bore NMR spectrometer (Bruker Biospin MRI, Billerica, MA) with a 25-mm inner diameter dual nucleus (31P/1H) birdcage coil. For anatomical positioning, a pilot image set of three orthogonal imaging planes were used. MRI pulse sequences used for data acquisition used T2-weighted imaging (T2) and fat-suppressed T2-weighted imaging (FS) sequences optimized for imaging of muscle inflammation. The imaging sequence used was a rapid acquisition with relaxation enhancement (RARE) sequence: echo time (TE) = 7.4 ms; repetition time (TR) = 3,000 ms; RARE factor 16; flip angle α = 90°; field of view = 2.56 cm×2.56 cm; slice thickness = 1 mm with no gap between slices; matrix size = 256×256; number of averages = 12.

Spectra were processed using TopSpin v1.5 software (Bruker Biospin MRI, Billerica, MA). For 31P spectroscopy studies, un-localized single-pulse spectroscopy was performed with 4k transients and a band width of 50 ppm. Integral areas of spectral peaks corresponding to inorganic phosphorous (Pi), phosphocreatine (PCr), and the three phosphate groups of adenosine triphosphate (α-ATP, β-ATP, and γ-ATP) were measured. Pi peaks were not detectable in several of the mice assayed (3 mdx and 5 wild-type); for these mice Pi values were uniformly omitted from the analyses. The presence of phosphomonoester (PME) or phosphodiester (PDE) peaks was also recorded; however, the signal-to-noise ratio of these peaks was not always adequate for accurate quantification. Levels of PCr and Pi were normalized by dividing by either the total ATP present in that spectrum or by the amount of β-ATP present in that spectrum. The results were consistent between both of these normalization methods; data are presented as the ratio of each parameter to total ATP.

For analysis of MRI images, two-dimensional sequential (2dseq) files were converted to digital imaging and communications in medicine (DICOM) files and analyzed using ImageJ v1.48 (NIH) software. To obtain volumetric data and account for possible variability between individual MRI slices, multiple consecutive MRI slices of each leg and thigh were assembled into image stacks encompassing 5- or 3-mm sections of the mouse hindlimb. Each stack was analyzed individually, and values for the two legs or thighs of each mouse were then averaged to obtain a single value for that mouse. For the leg, five consecutive slices along the long axis of the tibia were assayed, beginning 2 mm distal from the tibial plateau as a reference point to ensure mice were assayed at the same anatomical location. For the thigh, three consecutive slices along the long axis of the femur were assayed, beginning 6 mm proximal from the femoral condyles. Within each slice, regions of interest were digitally traced in ImageJ for each leg such that they were defined as the total area internal to the leg or thigh. Within regions of interest in each individual MRI image, we measured the total cross-sectional area, as well as the volumetric area (in voxels, or volume pixels) of bone, of muscle (with bone subtracted), and of tissue exceeding threshold signal intensity. For cross-sectional area, the highest value for each leg or thigh was recorded as the maximal cross-sectional area (CSAmax). Bone was measured by digitally tracing the dark outline shape of the tibia or femur in MRI images, and measuring the area outlined. Muscle area was measured by subtracting bone from the combined muscle and bone area making up the full region of interest. Elevated signal intensity was measured using ImageJ software in a semi-automated manner by measuring the volumetric area in voxels that exceeded background threshold within the regions of interest. Percent of tissue with elevated signal intensity was calculated by dividing this measurement by the area in voxels measured for muscle.

Longitudinal MRI of mdx muscle detects effects of mdx genotype and age

To image dystrophic muscle in live mice during the peak necrosis and recovery phases of the mdx condition, we performed T2-weighted imaging and fat-suppressed imaging of leg and thigh muscles every 2 weeks, from 6 to 12 weeks of age. Heterogeneous areas of elevated intensity were visible in all mdx mice and at all time points, in contrast to the more uniform and dark signal for healthy control muscle tissue (Figure 2A). Orientation and relevant anatomy are provided (Figure 2B). By aligning anatomically matched MRI slices using the tibial plateau as a reference point across successive time points, we observed qualitative changes in the sites and patterns of hyperintense foci within mdx leg muscles between two-week intervals.

A) Representative T2-weighted images from one mdx mouse (left) and one wild-type mouse (right) over time, each imaged from 6 to 12 weeks of age. The full MRI image of each mouse is provided on the outside column, with the leg of the left hindlimb for each mouse outlined in white and a magnified version of the leg muscles provided in the center columns. The black arrows mark a region of mdx muscle that showed a reduction in intensity between time points, while the gray arrows mark a region that showed an elevation of intensity between time points. The tibia, visible as a triangular structure in the upper right corner of each leg section, was used to orient the muscle slices. B) Orientation and anatomy of the leg cross sections. Anterior muscle groups (A, yellow) include tibialis anterior and extensor digitorum longus. Medial muscle groups (M, orange) include flexor hallucis and flexor digitorum. Posterior muscle groups (P, red) include gastrocnemius, soleus, and plantaris. The tibia bone is also marked (T). C) The percentage of tissue within the leg muscle that showed signal intensity elevated over the threshold that separates healthy muscle from affected tissue illustrates a change between the necrotic (6 week) and recovery phases of mdx disease. D) The absolute volume of tissue with elevated signal intensity detected within the leg of mice. E) The CSAmax values over time show the growth of muscle, and an increase for the mdx mice as compared to wild-type mice (n = 5 wild-type and 6 mdx mice; data are means ±SEM; *p<0.05, **p<0.01, ***p<0.001).

https://doi.org/10.1371/journal.pone.0112477.g002

Quantitatively, we detected significant effects of the mdx genotype on measures of muscle damage and size by both T2-weighted and fat-suppressed imaging, in both the leg and thigh muscles. No significant differences were observed in volumetric bone area between mdx and wild-type, for either the tibia or femur, for any of the imaging methods (Figure S1). In T2 imaging of mdx leg muscles, we found a significant increase in the percentage of tissue with elevated intensity (p<0.001) that changed over time (p<0.01), without a significant interaction effect being present. In T2 images of leg muscle, 6-week-old mdx mice had 21±3% tissue with elevated signal, as compared to 4±2% for wild-type mice (p<0.001; Figure 2C). As the mdx mice entered the recovery phase of disease, they showed a 38% reduction in the levels of affected tissue, to a mean of 13±2% of volume pixels (voxels) with elevated intensity at 8 weeks of age (p<0.001). These values then remained fairly steady, with no significant decrease from 8 to 12 weeks of age. These data illustrate the ability of MRI to detect significant levels of affected tissue in dystrophic legs in 6- to 12-week old mdx mice.

We also wanted to gain insight into whether affected muscle in mdx is either being eliminated or being “diluted” as the muscle grows larger and enters the recovery phase. To do this, we assayed the absolute volume of tissue with elevated intensity in the legs as well as the cross-sectional area of the legs. We detected a significant effect of the mdx genotype (p<0.001), with increased volume of elevated signal over wild-type at all ages (Figure 2D). We detected no significant effects of time on the volume of elevated signal in mdx mice and no significant interaction effect. Examining the sizes of muscles throughout the 6- to 12-week period assayed, we detected significant effects of the mdx genotype (p<0.001) and of time (p<0.001) on CSAmax, without a significant interaction effect. Initially, the mdx mice showed a smaller but significant increase over wild-type in CSAmax (p<0.05), with values of 27.7±1.3 mm2 versus 24.7±2.6 mm2, respectively (Figure 2E). In contrast to the results for elevated signal, as the mdx mice progressed into the recovery phase, differences in CSAmax between the mdx and wild-type mice became larger, with the difference increasing by 113% from week 6 to week 8. At 8 weeks, mdx calves had CSAmax values of 34.7±2.2 mm2, versus 28.4±2.5 mm2 for wild-type (p<0.01), and this difference was maintained through 12 weeks of age, at which point the mdx mice had CSAmax values of 40.0±3.9 mm2, versus 33.5±1.3 mm2 for wild-type (p<0.01). Together, these MRI data show that as mdx mice recover from the necrotic phase and assume a milder phenotype [20], [22], [23], they show a decrease in the percentage of affected tissue driven by an increase in muscle size, without a complete resolution of phenotype.

To enhance the visualization of signal from possible edema and inflammation, given the reduced signal likely from possible fatty infiltration of the muscle, we performed fat-suppressed imaging immediately following the standard T2-weighted imaging (Figure 3). Here again, we saw significant phenotypes in 6-week old mdx mice, with an average of 9±3% tissue with elevated intensity present in mdx mice, versus 1±1% for wild-type mice (p<0.001). Following week 6, mdx mice again showed a 40% reduction in affected leg tissue as they entered the recovery phase of disease, to 5±4% versus 0.6±0.3% for wild-type (p<0.05) at 8 weeks. Values for both genotypes were then maintained at similar levels through 12 weeks of age. These data are consistent with the T2 imaging results.

A) Representative fat-suppressed images of leg muscle from the left hindlimb of one mdx (top) and one wild-type (bottom) mouse over time, each imaged from 6 to 12 weeks of age. Black arrows mark a region of muscle that showed a reduction in intensity between time points, while gray arrows mark a region that showed an increased intensity between time points. The tibia is present as a triangular structure in the upper right corner of the leg sections. B) The percentage of tissue within the leg that has an elevated signal intensity shows a difference between mdx and wild-type mice at all time points and illustrates a change between the peak disease (6 week) and recovery phases of mdx disease (n = 5 wild-type and 6 mdx mice; data are means ±SEM; *p<0.05, **p<0.01, ***p<0.001).

https://doi.org/10.1371/journal.pone.0112477.g003

For the thigh muscles, results were qualitatively consistent with those found for the leg muscles; we again observed changes in the patterns of affected tissue over time within the same mouse in anatomically aligned MRI slices (Figure 4A). Orientation and relevant anatomy are provided (Figure 4B). In standard T2 images, 6-week old mdx mice showed a significant increase in the percentage of tissue with elevated signal, with values of 22±5% versus 7±1% for wild-type mice (p<0.001; Figure 4C). This parameter decreased steadily over time for the mdx thigh, to 14±5% for mdx and 5±1% for wild-type mice at 12 weeks (p<0.01). Examining the absolute volume of tissue with increased signal independent of muscle size, we found a significant effect of the mdx genotype at all time points (p<0.01), but no significant effect of time over the ages assayed (Figure 4D). The size of the thigh muscle, as measured by CSAmax, was not significantly different between genotypes at 6 weeks of age (Figure 4E). However, as with the leg, CSAmax increased for the mdx thighs when compared to wild-type, beginning at 8 weeks of age with values of 57.9±2.4 mm2 for mdx versus 49.3±6.4 mm2 for wild-type (p<0.05). This size differential continued to increase through 12 weeks, to 73.0±7.5 mm2 for mdx versus 57.0±3.2 mm2 for wild-type mice (p<0.01).

A) Representative T2-weighted images of thigh muscle from the right hindlimb of one mdx and one wild-type mouse over the study period. The black arrows mark a region of muscle that showed a reduction in intensity over time, while the gray arrows mark a region that showed an increased intensity over time. The femur is visible as an elliptical structure towards the center of the thigh. B) Orientation and anatomy of thigh cross sections. Anterior muscle groups (A, yellow) include vastus intermedius, vastus lateralis, and rectus femoris. Lateral muscle groups (L, orange) include biceps, semitendinosus and semimembranosus muscles. Medial muscle groups (M, red) include gracilis and adductor muscles. The femur bone (F) is also marked. C) The percentage of tissue within the thigh muscle that showed a signal intensity elevated over the threshold that separates healthy muscle from affected tissue shows a difference between mdx and wild-type mice at all time points. D) The absolute volume of tissue with an elevated signal within the thigh of mdx and wild-type mice. E) CSAmax shows growth of the muscle size over time, and an increase in the cross-sectional area of the thigh muscle in mdx mice as compared to wild-type mice from 8 weeks onward (n = 5 wild-type and 6 mdx mice; data are means ±SEM; *p<0.05, **p<0.01, ***p<0.001).

https://doi.org/10.1371/journal.pone.0112477.g004

In fat-suppressed imaging of the thigh, mdx mice again showed a significant increase in the percentage of tissue with elevated signal at 6 weeks, with 17±6% as compared to 2±1% for wild-type (p<0.001; Figure 5). By 10 weeks of age, this decreased to 9±1% for mdx and 2±1% for wild-type (p<0.001) as the mice progressed to the recovery stage of disease. Together, thigh data are in agreement with the leg. These data illustrate that mdx mice show a substantial decrease in the percentage of affected tissue and an increase in muscle area as they progress from the peak disease phases [20], [22], [23] to the recovery phase of the disease.

A) Representative fat-suppressed images of thigh muscle from the right hindlimb of one mdx and one wild-type mouse over the course of the study. Black arrows mark a region of muscle that showed a reduction in intensity over time, while gray arrows mark a region that showed an increase in intensity over time. The femur is visible as an elliptical structure in the central area of the thigh. B) The percentage of tissue with an elevated signal intensity within the thigh shows a difference between mdx and wild-type mice at all time points (n = 5 wild-type and 6 mdx mice; data are means ±SEM; ***p<0.001).

https://doi.org/10.1371/journal.pone.0112477.g005