Non-Invasive MRI and Spectroscopy of <i>mdx</i> Mice Reveal Temporal Changes in Dystrophic Muscle Imaging and in Energy Deficits

<div><p>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 <i>mdx</i> 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 <i>mdx</i> 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 <i>mdx</i> mice. Using these techniques, we studied dystrophic <i>mdx</i> muscle in mice from 6 to 12 weeks of age, examining both the peak disease phase and natural recovery phase of the <i>mdx</i> disease course. T2 and fat-suppressed imaging revealed significant levels of tissue with elevated signal intensity in <i>mdx</i> hindlimb muscles at all ages; spectroscopy revealed a significant deficiency of energy metabolites in 6-week-old <i>mdx</i> mice. As the <i>mdx</i> 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 <i>mdx</i> 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.</p></div

Changes in T2 imaging and cross-sectional area of dystrophic <i>mdx</i> thighs over time.

<p>A) Representative T2-weighted images of thigh muscle from the right hindlimb of one <i>mdx</i> 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 <i>mdx</i> and wild-type mice at all time points. D) The absolute volume of tissue with an elevated signal within the thigh of <i>mdx</i> and wild-type mice. E) CSA_max shows growth of the muscle size over time, and an increase in the cross-sectional area of the thigh muscle in <i>mdx</i> mice as compared to wild-type mice from 8 weeks onward (n = 5 wild-type and 6 <i>mdx</i> mice; data are means ±SEM; *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001).</p

Statistical sample size calculations to detect intervention effects in <i>mdx</i> mice.

<p><i>Abbreviations:</i> NMR Spec, Nuclear Magnetic Resonance spectroscopy; PCr, phosphocreatine; tATP, total adenosine triphosphate; Vol., Volume; WT, wild-type.</p><p>Statistical sample size calculations to detect intervention effects in <i>mdx</i> mice.</p

T2 imaging and histology of the <i>mdx</i> leg.

<p>Additional mice were assayed by T2 imaging at 6 months of age, followed by immediate collection of the whole leg for histology. A) Representative T2 images are provided of <i>mdx</i> (top two rows) and wild-type (bottom row) mice. The region of interest outlined in white is shown enlarged in (B). C) H&E stained cross section images corresponding to MRI slices in panels A and B. A montage image of the full leg is provided, with the inset area displayed in (D) at higher magnification (Rectangles in B and C represent the approximate areas presented in higher magnification images in D; Scale bars  = 2 mm in C and 0.5 mm in D).</p

Longitudinal fat-suppressed MRI of dystrophic <i>mdx</i> thigh muscles.

<p>A) Representative fat-suppressed images of thigh muscle from the right hindlimb of one <i>mdx</i> 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 <i>mdx</i> and wild-type mice at all time points (n = 5 wild-type and 6 <i>mdx</i> mice; data are means ±SEM; ***<i>p</i><0.001).</p

T2 of <i>mdx</i> leg shows changes in dystrophic muscle and cross-sectional area over time.

<p>A) Representative T2-weighted images from one <i>mdx</i> 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 <i>mdx</i> 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 <i>mdx</i> disease. D) The absolute volume of tissue with elevated signal intensity detected within the leg of mice. E) The CSA_max values over time show the growth of muscle, and an increase for the <i>mdx</i> mice as compared to wild-type mice (n = 5 wild-type and 6 <i>mdx</i> mice; data are means ±SEM; *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001).</p

Longitudinal fat-suppressed imaging of dystrophic <i>mdx</i> leg muscles.

<p>A) Representative fat-suppressed images of leg muscle from the left hindlimb of one <i>mdx</i> (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 <i>mdx</i> and wild-type mice at all time points and illustrates a change between the peak disease (6 week) and recovery phases of <i>mdx</i> disease (n = 5 wild-type and 6 <i>mdx</i> mice; data are means ±SEM; *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001).</p

Magnetic Nanobeads as Potential Contrast Agents for Magnetic Resonance Imaging

Metal-oxo clusters have been used as building blocks to form hybrid nanomaterials and evaluated as potential MRI contrast agents. We have synthesized a biocompatible copolymer based on a water stable, nontoxic, mixed-metal-oxo cluster, Mn₈Fe₄O₁₂(L)₁₆(H₂O)₄, where L is acetate or vinyl benzoic acid, and styrene. The cluster alone was screened by NMR for relaxivity and was found to be a promising <i>T</i>₂ contrast agent, with <i>r</i>₁ = 2.3 mM^–1 s^–1 and <i>r</i>₂ = 29.5 mM^–1 s^–1. Initial cell studies on two human prostate cancer cell lines, DU-145 and LNCap, reveal that the cluster has low cytotoxicity and may be potentially used <i>in vivo</i>. The metal-oxo cluster Mn₈Fe₄(VBA)₁₆ (VBA = vinyl benzoic acid) can be copolymerized with styrene under miniemulsion conditions. Miniemulsion allows for the formation of nanometer-sized paramagnetic beads (∼80 nm diameter), which were also evaluated as a contrast agent for MRI. These highly monodispersed, hybrid nanoparticles have enhanced properties, with the option for surface functionalization, making them a promising tool for biomedicine. Interestingly, both relaxivity measurements and MRI studies show that embedding the Mn₈Fe₄ core within a polymer matrix decreases <i>r</i>₂ effects with little effect on <i>r</i>₁, resulting in a positive <i>T</i>₁ contrast enhancement