Diffusion tensor MRI to assess damage in healthy and dystrophic skeletal muscle after lengthening contractions

Abstract

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 7T 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.

Figure 1

Mice lacking dystrophin are more susceptible to injury. Producing the injury, the fibular (aka peroneal) nerve was used to stimulate the dorsiflexor muscles supramaximally while moving the plate forced the foot into plantar flexion. Injury was induced by 10 large strain lengthening (“eccentric”) contractions through a 70° arc of motion. Maximal isometric torque was measured before and after injury in wild type (WT) and mice lacking dystrophin (mdx). The dorsiflexors were maximally activated isometrically for 200 ms prior to movement and then forcibly stretched through a 70° arc of plantarflexion at 900°/s. (a) Trace recordings of torque lengthening contractions (superimposed on a maximal lengthening contraction for 200 ms) for repetitions 1, 5, and 10 (black, pink, and red lines, resp.). (b) Maximal isometric torque was recorded at optimal length (L₀) before (black line) and after (red line) injury. Note that mdx muscles generate at least the same absolute force, but they consistently showed a significant drop in torque (yellow arrow) compared to the wild-type muscles, with an average loss of 85% compared to 32% (P < 0.01) in normal mice. Not only do the mdx muscles sustain more force loss, but this usually occurs very early in the protocol (after the first few, resp.). *=P < 0.05.

Figure 2

Histopathology related to injury. (a) cross-section of normal healthy TA muscle from a wild-type mouse. Skeletal muscle fibers are multinucleated and the nuclei stain blue; the sarcoplasm of each cell stains pink. (b) cross-section from a wild-type TA after injury. There was only minimal evidence of perivascular inflammation (arrows) in the wild-type tissue after injury. (c) cross-section from TA muscle of an mdx mouse. Even without injury, there is mild inflammation, slight increase in endomysial connective tissue, heterogeneity in fiber size, and many centrally nucleated fibers (CNFs, open arrows), all indicative of ongoing degeneration/regeneration within the muscle. (d) cross-section from an mdx TA after injury. Even with a protocol that produces mild changes in morphology to healthy muscle, the mdx muscle suffers much more damage, such as myonecrosis, myophagocytosis, and foci of inflammation surrounding individual muscle fibers (closed arrows). Scale bar = 40 μm.

Figure 3

Membrane stability in muscle fibers before and after injury. Evans Blue Dye (EBD) was used to evaluate sarcolemmal integrity within the TA muscles of mice after injury. EBD is detected under fluorescence microscopy (568 nm, insets show high magnification) and the presence of the protein-bound dye inside the muscle fiber indicates damage to the sarcolemma. (a) cryosection of an uninjured wild-type TA muscle from animals injected with EBD. (b) cryosection of an injured wild-type TA; the injury protocol used here was not enough to cause significant membrane damage in wild-type muscle. (c) cryosection of an uninjured mdx TA from animals injected with EBD. (d) cryosection of an injured wild-type TA; intracellular EBD indicates damage to the sarcolemma, which occurred frequently in the mdx injured muscles. (e) Histogram showing quantification of EBD-positive fibers. Without injury, the number of EBD-positive (EBD+) fibers was not significantly different between wild type and mdx. Only the mdx animals showed a significant increase in the number of positively labeled fibers after injury. Scale bar = 200 μm. *: significant difference from noninjured, P < 0.05.

Figure 4

Representative T2-weighted and T2-parametric images. (a) Example of a T2-weighted image (T2) after injury in the wild-type (WT) mouse. The injured TA (dotted red circle) is easily discerned from the TA on the control side (dotted white circle) based on the increased T2 signal, presumably due to edema. (b) Even in noninjured mdx muscle, there are regions of hyperintensity (arrow), a characteristic finding in dystrophic muscle. (c-d) Example of a T2 parametric image (T2-p) after injury in the wild-type and mdx mouse.

Figure 5

Muscle fiber tracking of the tibialis anterior (TA) muscle in vivo. Examples of “fiber tracking” from processed diffusion tensor imaging (DTI). The image shows modeling of fiber tracks based on the DTI data in the TA muscles of a wild-type (wt) mouse and an mdx mouse in the noninjured and injured sides. Even with this mild injury protocol that shows only minimal change in the T2 signal, the injured TA is readily identified by an apparent interruption in the vertical orientation of the tracks (arrows). Tractography is only a visual depiction of the DTI parameters. It is useful in providing accurate representation of the true anatomy of muscles but less so after injury. The colored bands are just where the regions of interest were drawn to outline the TA. Note that the mdx T2 image shows regions of hyperintensity, as seen in Figure 4.

Figure 6

Diffusion tensor imaging (DTI) parameters after injury. Parameters (y-axis, in mm²/s) for the proximal section of TA muscles from wild-type (yellow triangles) and mdx (blue squares) mice are plotted against force loss (x-axis, in % loss compared to pre-injury torque). Parameters include (a) mean diffusivity (MD), (b) axial diffusivity (AD), (c) fractional anisotropy (FA), (d) radial diffusivity (RD). (e) shows T2 (ms) also presented plotted against force loss. Presenting the data in this way, one can see that as there is a loss in force, there is a greater change in the diffusion parameters for the mdx mice than with the wild-type mice.