the muscular dystrophies ( mds ) are a heterogeneous group of inherited disorders characterized by progressive weakness and degeneration of skeletal muscles . duchenne muscular dystrophy ( dmd ) , the most common form of md , is an x - linked disorder that was first described over a century ago . dmd is caused by the absence of dystrophin , a 427  kda protein found on the cytoplasmic surface of the plasma membrane of muscle fibers ( the sarcolemma ) in skeletal and cardiac muscle . it is well established that the absence of dystrophin leads to the impaired linkage between the cytoskeleton and the extracellular matrix protein laminin , which is thought to result in fragility of the sarcolemma and a disruption in the proper transmission of force during contraction . muscle strains are one of the most common complaints treated by physicians .   when an activated muscle lengthens because the external load exceeds the tension generated by the muscle contraction , this is termed a lengthening (  eccentric  ) contraction . the force generated during a maximal lengthening contraction is at least twofold the force developed during a maximal isometric contraction ; therefore lengthening contractions are more likely to produce damage than either isometric or concentric contractions . plain films , or x - rays , are not very useful for imaging muscle pathology , unless heterotopic bone formation has occurred within the muscle . diagnosis of acute muscle strains is still typically made based on physical examination and patient history , but muscle injuries can be detected with mr imaging methods .   as noninvasive technology continues to improve and imaging , such as magnetic resonance imaging ( mri ) , becomes more commonplace , these tools will play a greater role in diagnosis , prognosis , and in rehabilitation planning . unlike x - rays , mri offers superb tissue contrast and has high sensitivity to the hemorrhage and edema that follow muscle injuries . this , together with the capability to evaluate multiple arbitrary anatomic planes , make it the ideal technique to evaluate muscle injures . because of the similarities between muscle injury and muscle disease , mri may yield useful information for both of these conditions . conventionally , muscle strains are revealed best by t2-weighted mri images , which optimize contrast between injured muscles with edema ( increased signal intensity ) and normal uninjured muscles . more recently , diffusion tensor imaging ( dti ) has been explored as a more accurate marker for muscle damage compared to t2-weighted mri . the variables obtained with dti , at least in other tissues such as the brain , show a strong and rapid response to damage , whereas the t2 signal can take a prolonged period to change . self - diffusion of water in tissue is restricted by membranes , resulting in an apparent diffusion coefficient ( adc ) that is lower than the free diffusion coefficient and is orientation dependent for elongated structures .   in short , dti can be used to determine the three - dimensional architecture of skeletal muscle [ 10 , 11 ] as well as provide useful information regarding muscle damage . the purpose of this study was to determine if measurements obtained from dti would serve as a reliable marker of damage after a muscle strain injury in dystrophic ( mdx ) and control mice . we hypothesize that dti biomarkers would provide a more informative assessment of muscle injury than t2 and that these measures will further elucidate the increased susceptibility to injury of the mdx model . all protocols were approved by the university of maryland institutional animal care & use committee ( iacuc ) . the injury model results in a significant and reproducible injury and has been described in detail previously [ 1316 ] . briefly , 5 adult healthy ( c57bl/10scsn ) and 5 dystrophic ( c57blscsn - dmd ) male mice were purchased ( the jackson laboratory , bar harbor , me ) and were approximately 8 weeks old at the start of experiments .   before each injury protocol , mice were anesthetized ( 4 - 5% isoflurane in an induction chamber , then 2% isoflurane via a nosecone for maintenance ) using a precision vaporizer ( cat no . 91103 , vet equip , inc , pleasanton , calif ) .   sterile ophthalmic cream ( paralube vet ointment , pharmaderm , floham park , nj ) was applied to each eye to protect the corneas from drying .   during the procedure , the animal was kept warm by use of a heat lamp .    with the animal supine , the hindlimb was stabilized , and the foot was secured onto a plate , the axis of which was attached to a stepper motor ( model t8904 , nmb technologies , chatsworth , calif ) and a torque sensor ( qwfk-8 m , sensotec ) to measure torque . the fibular nerve was stimulated via subcutaneous needle electrodes ( j05 needle electrode needles , 36btp , jari electrode supply , gilroy , calif ) , and proper electrode position was determined by a series of isometric twitches . impulses generated by an s48 square pulse stimulator ( grass instruments , west warwick , ri ) were 1  ms in duration and passed through a psiu6 stimulator isolation unit ( grass instruments , west warwick , ri ) . in addition to visual confirmation of isolated dorsiflexion , an increase in twitch torque in response to increasing voltage indicated that opposing plantarflexor muscles   were not being simultaneously stimulated . the majority of torque produced by the dorsiflexors is from the tibialis anterior muscle ( ta ) , and we have shown previously that this model results in injury to this muscle [ 15 , 16 , 19 , 20 ] .   a custom program based on commercial software ( labview version 8.5 , national instruments , austin , tex ) was used to synchronize contractile activation and the onset of ankle rotation . injury resulted from 10 forced lengthening ( plantarflexion ) contractions through a 070 arc of motion ( with the foot orthogonal to the tibia , considered 0 ) . a sham procedure was performed whereby the identical protocol was performed ( including stimulation ) , but without lengthening . a maximal isometric contraction ( 200  ms duration ) of the dorsiflexors was used to measure maximal torque before injury .   for each animal , maximal isometric torque was also measured 5 minutes after injury ( to measure force lost due to injury ) . all isometric contractions were performed with the ankle at 20 into plantarflexion , a position that results in optimal force production . high resolution mri ( magnetic resonance imaging ) was performed on a bruker biospin ( billerica , mass ) 7.0 tesla mr system equipped with a 12  cm gradient insert ( 660  mt / m maximum gradient , 4570  t / m / s maximum slew rate ) running paravision 5.0 software to assess muscle damage on the day of injury . an mr - compatible small - animal monitoring and gating system ( sa instruments , inc . ) was used to monitor respiration rate and body temperature . a custom - made holder was used to position the mouse in the supine position with both legs parallel to the bore of the magnet from knee to foot . two hours after injury , animals were placed inside the scanner , with a four - channel receive - only surface coil placed anterior to the ta . anesthesia was maintained using 2% isofluorane and was adjusted to maintain a safe respiration rate . after imaging localizers , the following mr scans were performed : t1-weighted rapid acquisition with relaxation enhancement ( rare ) with the following parameters : te = 9.52  ms , tr = 1800 , echo train length = 4 , na = 8 , in - plane resolution 100  100  m , and slice thickness = 750  m ; dual echo pd / t2 rare : te = 19.0/57.1  ms , tr = 5000  ms , echo train length = 4 , na = 1 , in - plane resolution 100  100  m , and slice thickness = 750  m . spin echo ( se ) diffusion tensor image data was acquired using 12 noncolinear directions : b - value = 350  s / mm , te = 26  ms , tr = 4500  ms , na = 1 , in - plane resolution 150  150  m , and slice thickness = 750  m . multislice multiecho ( msme ) t2 mapping image data was acquired in the same slice positions as the dti data using 16 tes = 11.4  ms to 182.5  ms with te = 11.4  ms , tr = 10000  ms , na = 1 , in - plane resolution 150  150  m , and slice thickness = 750  m . t2 mapping was performed using custom software written in matlab ( the mathworks ; natick , mass ) using nonlinear least squares to fit the measured data at each pixel to the canonical t2 signal equation . diffusion tensor reconstruction and tractography was performed using trackvis ( martinos center for biomedical imaging ; massachusetts general hospital ; boston , mass ) and matlab to calculate mean diffusivity ( md ) , fractional anisotropy ( fa ) , radial diffusivity ( rd ) , and axial diffusivity ( ad ) images . a region of interest ( roi ) in the ta and tractography was used to guide bilateral segmentation of the ta . diffusion tensor tractography was calculated using the fiber assignment by continuous tracking ( fact ) method   with a termination criteria of an angle greater than 35 , followed by subsequent spline filtering . tracts were restricted to those traveling through several transverse slices of manually traced regions of interest within each of the left and right tas . this was used to create an image mask for each muscle , which was then divided into proximal , middle , and distal sections of approximately equal length . finally , the masks were used to calculate average measurements of md , fa , rd , ad , and t2 within each section of the injured and uninjured muscle . these measurements were compared between normal and dystrophic mice for both the uninjured and the injured side using a wilcoxon rank sum test . after mr and functional data were collected , anesthetized animals were fixed with 4% paraformaldehyde via perfusion through the left ventricle to preserve tissue morphology . tas were harvested , weighed , snap frozen in liquid nitrogen , and then stored at 80c . animals not perfused fixed were euthanized by carbon dioxide inhalation followed by decapitation .   to study the integrity of the muscle fiber membrane , additional mice ( n = 3 each group ) received an intraperitoneal injection of 1% evans blue dye ( ebd ; sigma , st . louis , mo ) ( wt / vol ) in phosphate - buffered saline ( pbs , ph 7.4 ) at a volume of 1% body mass ( bm ) ( 1  mg ebd/0.1  ml pbs/10  g  bm ) . this solution was sterilized by passage through a millex - gp 0.22  m filter ( millipore , bedford , mass ) and administered 24  h before death of the animal to assure a good signal . ebd binds to albumin and is detected by fluorescence microscopy ( at 568  nm ) in the extracellular space . presence of the protein - bound dye inside the muscle fiber indicates damage to the sarcolemma .   here , the sections were assessed blindly , and the myofibers were judged in a binary fashion , as positive or negative for intracellular ebd . transverse sections were cut on a cryostat ( 10  m thickness ) and collected onto glass slides ( superfrost plus ; vwr , west chester , pa ) .   for ebd - injected tissue , myofibers with dye - labeled sarcoplasm were quantified in cross - sections under fluorescence optics [ 16 , 24 ] . at least 400 fibers in 10 optical fields were assessed in 3 mice ; results are expressed as percent labeled fibers .   in sections from muscles without ebd , sections were stained with hematoxylin and eosin ( h&e ) for tissue evaluation . sections were randomized and viewed at 100x magnification in a zeiss axioskop light microscope , and pictures were taken with a digital camera ( axiocam hr using axiovision 3.0 ) . each optical field contained an average of 38  7 fibers , and more than 45 fields were counted per muscle . to induce injury in the tibialis anterior muscle ( ta ) , we superimposed a lengthening contraction onto a maximal isometric contraction ( figure 1(a ) ) .   to assess the amount of injury , we measured maximal torque before and after injury ( figure 1(b ) ) .   while the mechanism of injury was identical between animals , the injury was much more severe in dystrophic mice , with an average force loss of 85% , compared to 42% ( p < 0.05 ) in normal mice . for h&e staining , there was only minimal evidence of perivascular inflammation in the wild - type tissue after injury ( figure 2(b ) ) compared to undisturbed healthy muscle ( figure 2(a ) ) .   even without injury , centrally nucleated fibers , fiber size variation , mild inflammation , and slight increases in endomysial connective tissue in the uninjured mdx mice are all consistent with ongoing muscle damage and regeneration ( figure 2(c ) ) . in contrast , the myonecrosis , myophagocytosis , and foci of inflammation surrounding individual muscle fibers seen in the injured mdx muscle ( figure 2(d ) ) are all consistent with acute muscle injury . the amount of membrane damage , as evidenced by the number of fibers with intracellular ebd , also reflected the magnitude of functional injury ( figures 3(a)3(e ) ) .   in wild - type tissue sections ( figure 3(a ) ) , there were few fibers that could be found to have intracellular ebd ( 1% ) ; however after the relatively mild injury protocol ( figure 3(b ) ) , the number of fibers with membrane damage did not increase significantly ( 13  2% , p < 0.06 ) . the uninjured mdx tissue ( figure 3(c ) ) already had a small amount of ebd - positive fibers ( 11  4% , p < 0.05 ) , but there was almost a 5-fold increase in the number of fibers with membrane damage after injury ( 49  8% , p < 0.05 , figure 3(d ) ) . mri revealed a clear increase on the t2 signal after injury ( figures 4(a ) and 4(b ) ) . quantitative changes in t2 are apparent in figures 4(c ) and 4(d ) , where the expected t2 signal was fit across multiple echo times in t2-weighted images . there was no significant difference in parameters between the injured and uninjured leg in the normal mice . further , there were no significant differences in any of the measured parameters for the ta between normal and dystrophic mice in the uninjured side . however , there were marked differences in parameters in the injured side between normal and dystrophic mice and between the injured and uninjured leg in the dystrophic mice ( table 1 ) . as in prior studies , the sham animals did not have an increase in t2 signal by the time imaging was performed . when comparing parameter of diffusion on the injured side , dystrophic mice showed significantly increased md and ad and decreased fa ( p < 0.05 ) in the proximal and middle ta compared to wild - type uninjured mice . these differences were much more marked than the more limited differences we measured with t2 .   while not significant , rd trended toward increased values . dti tractography ( see figure 5 , e.g. ) was used to visualize the tas . since x - rays reveal little regarding skeletal muscle anatomy or function , muscle injury is still typically diagnosed by taking a history and performing a physical examination . however , modalities such as mri are becoming more affordable and more commonplace , and there is a surge of interest in using them to learn more about both muscle injury and muscle disease . acute muscle strain injury is a stochastic event , and that would not be ethical or desirable to induce in human subjects ; therefore most human studies involve imaging after  eccentric exercise  or  exertional muscle injury  ( i.e. , overuse type injuries ) .   by using an established animal model to induce an acute contraction - induced injury , we were able to compare changes in muscle function , in vivo imaging , and histology shortly after injury . our injury method clearly resulted in muscle damage , as indicated by the measurable loss of force , changes in h&e staining , and an uptake of ebd by injured muscle fibers , as well as marked changes in mri parameters , especially dti . there is a growing number of animal studies that use mri to assess muscle injury ; however , these include such methods as overuse ( running downhill ) , myotoxins , denervation , hindlimb suspension reloading , and ischemia - reperfusion [ 8 , 12 , 2631 ] , rather than by acute muscle strain injury . our in vivo animal model of injury provides several advantages , such as the ability to study exclusively one type of contraction ( eccentric ) , to measure force directly from an individual muscle , and to characterize the biochemical and morphological state of the muscle at defined times following injury and during recovery .   because the animal is anesthetized , we can reliably induce damage to a single muscle group with a known velocity , arc of motion , and contractile level . effort , such as in a maximal voluntary contraction ( mvc ) , and pain are not confounding factors when assessing changes in function . downhill treadmill running is an exercise that is sometimes used as an injury model , but there are problems with compliance . the in vivo injury model provides a known dose of injury to a specific muscle group in all animals . regardless of the method used to induce damage , the t2 signal in mri is typically increased significantly after muscle injury , but after some protocols there is a long - term persistence of this signal [ 33 , 34 ] , with a time scale that is not in line with the temporal changes of inflammation and edema . although some studies have shown that t2 values gradually peak hours or days after injury [ 26 , 30 ] , these employed a myotoxin to injure the muscle . myotoxins provide a model to study necrosis , inflammation , and massive degeneration and regeneration but do not provide a physiological model of injury .   in a recent paper , mathur et al .   used downhill running to study the changes in t2 after eccentric exercise of mdx mice . they used older animals ( 515 months ) but still found that mdx mice were more susceptible to damage , with t2 returning to preexercise levels 10 days after exertion . interestingly , they noted a strong relationship between the percent of ebd - positive area and the percentage of pixels with elevated t2 ( r = 0.79 ) . ebd binds to albumin , a blood plasma protein , and is detected by fluorescence microscopy in the extracellular space . presence of the protein - bound dye inside a muscle fiber indicates damage to the sarcolemma . since albumin is a relatively large protein ( 67  kda ) , their findings suggest that downhill running results in relatively large sarcolemmal tears of damaged myofibers . mcintosh et al .   provided one of the first studies to use mri to assess skeletal muscle in mdx mice . these foci of high intensity were subsequently confirmed by walter et al . , who used more advanced techniques to obtain higher resolution images as well to quantify the changes in the t2 signal that occurred in the mdx ( and sg/ ) mice compared to controls . walter et al . also showed that delivery of sg by aav ( adenoassociated virus ) into hindlimb muscles was not only able to rescue to phenotype ( i.e. , return of normal histology ) but also restored the t2 properties of skeletal muscle . dti parameters have been compared to longitudinal sections of the rat ta , the results of which show that dti directions actually represent the local orientation of fibers in the rat ta muscle . have used dti fiber tracking successfully to measure pennation angles of myofibers in human skeletal muscle [ 10 , 11 , 38 , 39 ] , and subsequent studies have also used dti in mice , both for three - dimensional reconstruction of fiber tracks   and as a measure of damage induced by ischemia reperfusion . provide an excellent description of measured changes in dti parameters after ischemia - reperfusion type of injury in mouse skeletal muscle . they found that dti indices changed in response to ischemia - reperfusion and that the indices correlated with histopathology , although they caution against overinterpretation since their study was not designed to achieve exact correlation . such correlation requires optimal mri protocols , an exact spatial correspondence between histology and mri slices , and specific immunohistochemical labeling to identify specific cell types . one can use dti to compare muscle architecture of dystrophic and healthy hindlimb muscles of mice in vivo . this could be extremely valuable to muscle physiologists who need to obtain the cross - sectional area ( csa ) of a muscle to calculate the  specific force  ( force normalized to csa ) without removing the muscles . in addition to the computer - generated models of fiber tracks , we were able to collect quantifiable data . using our injury model , the diffusion parameters ( decrease in fa and increases in md and ad ) and t2 findings are consistent with increased edema . however , the lack of significant changes in rd may suggest increased diffusion along the sarcolemma as a result of cell swelling . interestingly , significant changes in dti parameters were evident in the middle and proximal sections of the ta , and significant t2 changes were evident in the proximal ta . such findings support earlier work suggesting that damage to the myofibers is not limited to the muscle - tendon junction , where soreness often occurs . in addition to technical issues involved with imaging , position of the limb , and the lack of any single biological marker to explain the loss of force after injury [ 43 , 44 ] , one must still use caution when interpreting t2 intensity or dti findings . the exact meaning of t2 changes are still to be elucidated [ 28 , 32 , 45 ] . while tractography corresponds to normal anatomy , there is a tendency after injury for the more superficial fascicles to appear abnormal ( figure 5 , arrows ) . this three - dimensional reconstruction can appear to have fibers that  punch through  the strong connective tissue ( epimysium ) that surrounds that muscle and enter into the space occupied by another muscle , when indeed this is not the case . this is likely to be even more of a problem when using dti on patients with dmd , as they have significant progressive damage over time and a gradual replacement of muscle tissue by fat , something that does not occur in the mdx mouse model of dmd . however , this change in normal direction of myofibers is clearly indicative of damage , and tractography in healthy muscle is still a useful tool to obtain volume in order to calculate  specific force ( force per unit cross - sectional area ) .   in summary , we have used mri to study injured muscle by observing edema and muscle fiber diffusion changes with t2 and dti sequences , respectively . dti exhibited significant changes in both the middle and proximal sections of the ta , whereas t2 changes were only significant in the proximal section . this is , to the best of our knowledge , the first animal study to use dti in a reliable and valid animal model of eccentric injury . the results suggest that dti is a reliable marker in the assessment of acute muscle injury , even at early time points where the mr signal changes are dominated by local edema .
the purpose of this study was to determine if variables calculated from diffusion tensor imaging ( dti ) would serve as a reliable marker of damage after a muscle strain injury in dystrophic ( mdx ) and wild type ( wt ) mice . 
 unilateral injury to the tibialis anterior muscle ( ta ) was induced in vivo by 10 maximal lengthening contractions . 
 high resolution t1- and t2-weighted structural mri , including t2 mapping and spin echo dti was acquired on a 7 t small animal mri system . 
 injury was confirmed by a significant loss of isometric torque ( 85% in mdx versus 42% in wt ) . 
 greater increases in apparent diffusion coefficient ( adc ) , axial , and radial diffusivity ( ad and rd ) of the injured muscle were present in the mdx mice versus controls . 
 these changes were paralleled by decreases in fractional anisotropy ( fa ) . 
 additionally , t2 was increased in the mdx mice , but the spatial extent of the changes was less than those in the dti parameters . 
 the data suggest that dti is an accurate indicator of muscle injury , even at early time points where the mr signal changes are dominated by local edema .