Assessment OF aged mdx mice by electrical impedance myography and magnetic resonance imaging

Introduction: Similar to magnetic resonance imaging (MRI), electrical impedance myography (EIM) is dependent on the presence and location of water in muscle to assess neuromuscular diseases. We compared the 2 technologies in mdx mice to better understand their relationship. Methods: EIM and MRI, using T2 relaxation and diffusion‐weighted imaging (DWI), were performed on the gastrocnemius of 10 mdx and 10 wild‐type mice. Muscle function and tissue composition measurements were compared with the EIM and MRI data. Results: EIM reactance and T2 relaxation mapping can discriminate healthy from diseased mice (P < 0.001 for both), but DWI could not. Both T2 relaxation and EIM reactance also correlated closely with muscle function/composition and with each other. Conclusion: Given the low cost of EIM and the simplicity of application, it may be a valuable alternative to muscle MRI in Duchenne muscular dystrophy, where simple cumulative indices of muscle health are being sought. Muscle Nerve 52: 598–604, 2015     

Sonography in carpal tunnel syndrome with normal nerve conduction studies

Introduction: Electrical impedance myography (EIM) is a non‐invasive, painless, objective technique to quantify muscle pathology. Methods: We measured EIM in 8 arm and leg muscles in 61 boys with Duchenne muscular dystrophy (DMD) and 31 healthy boys, ages 3–12 years, at 5 centers. We determined the reliability of EIM and compared results in boys with DMD to controls and to 6‐minute walk distance (6MWD), North Star Ambulatory Assessment (NSAA), timed functional tests (TFTs), and strength (hand‐held dynamometry). Results: EIM was well tolerated and had good inter‐ and intrarater reliability (intraclass correlation coefficient 0.81–0.96). The averaged EIM phase value from all muscles was higher (P < 0.001) in controls (10.45 ± 2.29) than boys with DMD (7.31 ± 2.23), and correlated (P ≤ 0.001) with 6MWD (r = 0.55), NSAA (r = 0.66), TFTs (r = –0.56), and strength (r = 0.44). Conclusion: EIM is a reliable and valid measure of disease severity in DMD. Longitudinal studies comparing EIM with other assessments over time in DMD are warranted. Muscle Nerve 52: 592–597, 2015     

Use of evans blue dye to compare limb muscles in exercised young and old mdx mice

Evans blue dye (EBD) is used to mark damaged and permeable muscle fibers in mouse models of muscular dystrophy and as an endpoint in therapeutic trials. We counted EBD‐positive muscle fibers and extracted EBD from muscles sampled throughout the hindlimbs in young adult and old mdx mice to determine if the natural variability in morphology would allow measurement of a functional improvement in one limb compared to the contralateral limb. Following one bout of rotarod or treadmill exercise that greatly increased serum creatine kinase levels, the number of EBD⁺ muscle fibers in 12–19‐month‐old mdx mice increased 3‐fold, EBD in the muscles increased, and, importantly, contralateral pairs of muscles contained similar amounts of EBD. In contrast, the intra‐ and interlimb amounts of EBD in 2–7‐month‐old mdx mice were much too variable. A therapeutic effect can more readily be measured in old mdx mice. These results will be useful in the design of therapy protocols using the mdx mouse. Muscle Nerve, 2010     

Actin sliding velocity on pure myosin isoforms from dystrophic mouse muscles

Duchenne muscular dystrophy (DMD) is a genetic disease characterized by skeletal muscle wasting and atrophy. Recent evidence suggests that the impaired skeletal muscle performance in DMD is not solely dependent on a loss of contractile muscle mass. In this study the myosin motor function of mdx and control (wildtype, WT) mice was compared using pure myosin isoforms in an “in vitro motility assay” (IVMA). Actin sliding velocity (Vf) on myosin 2B extracted from single muscle fibers of gastrocnemius muscles was significantly lower in mdx mice (3.48 ± 0.13 μm/s, n = 18) than in WT mice (4.02 ± 0.19 μm/s, n = 10). No difference in Vf was found between myosin 1 extracted from soleus muscles of mdx (0.84 ± 0.04 μm/s, n = 13) and of WT (0.89 ± 0.04 μm/s, n = 10). The results suggest that the dystrophic process alters myosin molecular function, and this contributes to the functional impairment in dystrophic muscles. Muscle Nerve 40: 249–256, 2009     

Electrical impedance myography in facioscapulohumeral muscular dystrophy: A 1‐year follow‐up study

Introduction: Electrical impedance myography (EIM) is a noninvasive technique for measuring muscle composition and a potential physiological biomarker for facioscapulohumeral muscular dystrophy (FSHD). Methods: Thirty‐two participants with genetically confirmed and clinically affected FSHD underwent EIM in 7 muscles bilaterally. Correlations between EIM and baseline clinical measures were used to select EIM variables of interest in FSHD, and EIM and clinical measures were followed for 1 year. Results: There were no significant changes in the EIM variables. Although 50‐kHZ reactance correlated the strongest with clinical measures at baseline, the 50–211‐kHZ phase ratio demonstrated lower within‐subject 12‐month variability, potentially offering sample size savings for FSHD clinical trial planning. Discussion: EIM did not identify significant disease progression over 12 months. It is currently unclear whether this is because of limitations of the technology or the slow rate of disease progression in this cohort of FSHD patients over this period of time. Muscle Nerve 58 : 213–218, 2018     

Electrical impedance myography in the diagnosis of radiculopathy

Introduction: We sought to determine whether electrical impedance myography (EIM) could serve as a diagnostic procedure for evaluation of radiculopathy. Methods: Twenty‐seven patients with clinically and radiologically diagnosed cervical or lumbosacral radiculopathy who met a “gold standard” definition underwent EIM and standard needle electromyography (EMG) of multiple upper or lower extremity muscles. Results: EIM reactance values revealed consistent reductions in the radiculopathy‐affected myotomal muscles as compared with those on the unaffected side; the degree of asymmetry was associated strongly with the degree of EMG abnormality (P < 0.001). EIM had a sensitivity of 64.5% and a specificity of 77.0%; in comparison, EMG had a sensitivity of 79.7% but a specificity of 69.7%. Conclusions: These findings support the potential for EIM to serve as a new non‐invasive tool to assist in diagnosis of radiculopathy; however, further refinement of the technique is needed for this specific application. Muscle Nerve 48:800–805, 2013     

Electrophysiological subtypes and prognosis of Guillain–Barré syndrome in Northeastern China

Introduction: The dystrophic features in hindlimb skeletal muscles of female mdx mice are unclear. Methods: We analyzed force‐generating capacity and force decline after lengthening contraction‐induced damage (fragility). Results: Young (6‐month‐old) female mdx mice displayed reduced force‐generating capacity (?18%) and higher fragility (23% force decline) compared with female age‐matched wild‐type mice. These 2 dystrophic features were less accentuated in young female than in young male mdx mice (?32% and 42% force drop). With advancing age, force‐generating capacity decreased and fragility increased in old (20 month) female mdx mice (?21% and 57% force decline), but they were unchanged in old male mdx mice. Moreover, estradiol treatment had no effect in old female mdx mice. Conclusions: Female gender–related factors mitigate dystrophic features in young but not old mdx mice. Further studies are warranted to identify the beneficial gender‐related factor in dystrophic muscle. Muscle Nerve, 2013     

Clinical trial readiness in non‐ambulatory boys and men with duchenne muscular dystrophy: MDA–DMD network follow‐up

Introduction: Outcomes sensitive to change over time in non‐ambulatory boys/men with Duchenne muscular dystrophy (DMD) are not well‐established. Methods: Subjects (n = 91; 16.8 ± 4.5 years old) were assessed at baseline and 6‐month intervals for 2 years. We analyzed all subjects using an intent‐to‐treat model and a subset of stronger subjects with Brooke Scale score ≤4, using repeated measures. Results: Eight patients (12–33 years old) died during the study. Sixty‐six completed 12‐month follow‐up, and 51 completed 24‐month follow‐up. Those taking corticosteroids performed better at baseline, but rates of decline were similar. Forced vital capacity percent predicted (FVC% predicted) declined significantly only after 2 years. However, Brooke and Egen Klassifikation (EK) Scale scores, elbow flexion, and grip strength declined significantly over both 1 and 2 years. Conclusion: Brooke and EK Scale scores, elbow flexion, and grip strength were outcomes most responsive to change. FVC% predicted was responsive to change over 2 years. Corticosteroids benefited non‐ambulatory DMD subjects but did not affect decline rates of measures tested here. Muscle Nerve 54 : 681–689, 2016     

Inflammation in muscular dystrophy and the beneficial effects of non‐steroidal anti‐inflammatory drugs

Introduction: Glucocorticoids are the only drugs available for the treatment of Duchenne muscular dystrophy (DMD), but it is unclear whether their efficacy is dependent on their anti‐inflammatory activity. Methods: To address this issue, mdx mice were treated daily with methylprednisolone and non‐steroidal anti‐inflammatory drugs (NSAIDs: aspirin, ibuprofen, parecoxib). Results: NSAID treatment was effective in ameliorating muscle morphology and reducing macrophage infiltration and necrosis. The percentage of regenerating myofibers was not modified by the treatments. The drugs were effective in reducing COX‐2 expression and inflammatory cytokines, but they did not affect utrophin levels. The effects of the treatments on contractile performance were analyzed. Isometric tension did not differ in treated and untreated muscle, but the resistance to fatigue was decreased by treatment with methylprednisolone and aspirin. Conclusions: NSAIDs have a beneficial effect on mdx muscle morphology, pointing to a crucial role of inflammation in the progression of DMD. Muscle Nerve, 2012     

Expression of intercellular adhesion molecule‐1 by myofibers in mdx mice

Introduction: We investigated the extent to which intercellular adhesion molecule‐1 (ICAM‐1), a critical protein of the inflammatory response, is expressed in skeletal muscles of mdx mice (a murine model of Duchenne muscular dystrophy). Methods: Muscles were collected from control and mdx mice at 2‐24 weeks of age and analyzed for ICAM‐1 expression by means of Western blot and immunofluorescence. Results: Western blot revealed higher expression of ICAM‐1 in mdx compared with control muscles through 24 weeks of age. In contrast to control muscles, ICAM‐1 was expressed on the membrane of damaged, regenerating, and normal myofibers of mdx mice. CD11b+ myeloid cells also expressed ICAM‐1 in mdx muscles, and CD11b+ cells were closely associated with the membrane of myofibers expressing ICAM‐1. Conclusions: These findings support a paradigm in which ICAM‐1 and its localization to myofibers in muscles of mdx mice contributes to the dystrophic pathology. Muscle Nerve 52: 795–802, 2015     

Gender differences in contractile and passive properties of mdx extensor digitorum longus muscle

Introduction: Duchenne muscular dystrophy (DMD) is a severe, muscle‐wasting disease caused by mutations in the dystrophin gene. The mdx mouse is the first and perhaps the most commonly used animal model for study of DMD. Both male and female mdx mice are used. However, it is not completely clear whether gender influences contraction and the passive mechanical properties of mdx skeletal muscle. Methods: We compared isometric tetanic forces and passive forces of the extensor digitorum longus muscle between male and female mdx mice. Results: At age 6 months, female mdx mice showed better‐preserved specific tetanic force. Interestingly, at 20 months of age, female mdx muscle appeared stiffer. Conclusions: Our results suggest that gender may profoundly influence physiological measurement outcomes in mdx mice. Gender should be considered when using the mdx model. Muscle Nerve, 2012     

A novel and simple method for genotyping the mdx mouse using high‐resolution melt polymerase chain reaction

The mdx mouse mutation arises from a C‐to‐T point mutation, which terminates the translation of dystrophin and results in the loss of a functional dystrophin protein. mdx mice are used widely in studies of the role of dystrophin and of potential treatments for Duchenne muscular dystrophy, thus accurate genotyping is essential. Current methods require labor‐intensive efforts and can often lead to misconstrued results. This study describes a simple and highly reliable, sensitive, and user‐friendly, high‐resolution melt (HRM) assay that is able to utilize DNA obtained from a variety of sources in order to genotype the known sequence variant of the mdx mouse. Muscle Nerve 39: 603–608, 2009     

An octaguanidine-morpholino oligo conjugate improves muscle function of mdx mice

Introduction: Skeletal muscles of mdx mice lack functional levels of dystrophin due to a mutation in Dmd exon 23. Morpholino antisense oligomers can induce expression of a truncated dystrophin by redirecting splicing to skip processing of exon 23. Methods: We tested whether systemic administration of Vivo‐Morpholino, an octaguanidine delivery moiety–Morpholino conjugate that targets exon 23 (VMO23), restored function to muscles of mdx mice. Results: Extensor digitorum longus (EDL) muscles of mdx mice were weaker, less powerful, and showed greater functional deficits after eccentric contractions than normal. VMO23 treatment normalized EDL force and power of mdx mice and eliminated their exaggerated sensitivity to eccentric contractions. Diaphragm muscle strips from mdx mice also produced lower‐than‐normal force and power, and these variables were restored to normal, or near‐normal, levels by VMO23 treatment. Conclusion: These results provide a functional basis for continuing development of VMO23 as a treatment for Duchenne muscular dystrophy. Muscle Nerve, 2011     

Suramin affects metalloproteinase‐9 activity and increases beta‐dystroglycan levels in the diaphragm of the dystrophin‐deficient mdx MOUSE

Introduction: In Duchenne muscular dystrophy and in the mdx mouse, muscle fiber degeneration and subsequent fibrosis lead to cardiorespiratory failure. Previously, we demonstrated that the anti‐fibrotic agent suramin was effective in decreasing fibrosis in mdx muscles. In this study, we were interested to see whether suramin could affect metalloproteinases (MMP) and improve the functional activity of the mdx diaphragm muscle. Methods: Zymography was performed to evaluate MMP‐2 and MMP‐9 activity. Western blotting was used to analyze the levels of beta‐dystroglycan. Muscle function was assessed in hemidiaphragm in vitro preparations. Results: We found that suramin affects metalloproteinase‐9 activity and increases beta‐dystroglycan. Furthermore, suramin also protects against diaphragm muscle fatigue over time. Conclusions: These results show the potential benefits of suramin in maintaining the structure of the dystrophin‐glycoprotein complex. Muscle Nerve, 46:810–813, 2012     

Functional in situ assessment of muscle contraction in wild‐type and mdx mice

Introduction: Reports of muscle testing are frequently limited to maximal force alone. The experiments reported here show that force generation and relaxation rates can be obtained from the same experiments and provide a more complete functional characterization. Methods: Partial in situ testing was performed on the tibialis anterior of young wild‐type (WT) mice, young mdx mice, and old mdx mice. Force, force generation rate, and relaxation rates were measured during a fatigue test, 2 frequency–force tests, and a passive tension test. Results: We measured increased force but decreased force generation rate in WT compared with mdx muscles, and increased force but decreased relaxation rate of old compared with young mdx muscles. Young mdx muscles were the most sensitive to increases in passive tension. Conclusions: These measurements offer an improved understanding of muscle capability and are readily acquired by further analysis of the same tests used to obtain force measurements. Muscle Nerve 53: 260–268, 2016     

A new method of genotyping MDX4CV mice by PCR‐RFLP analysis

Introduction: The mdx^4cv mouse is a common model to study Duchenne muscular dystrophy. The most used methodology to identify the genotype of these mice is Sanger DNA sequencing. Methods: Here, we provide a simple, cost‐effective alternative approach to identify the wild‐type, heterozygous, or homozygous/hemizygous genotypes of these mice, using commonly available laboratory equipment and reagents. Results: Our technique exploits a restriction fragment length polymorphism that is generated by the point mutation found in exon 53 of mdx^4cv mice. Conclusions: This technique can benefit laboratories that require complex breeding strategies involving mdx^4cv mice. Muscle Nerve 56 : 522–524, 2017     

Evidence of mdx mouse skeletal muscle fragility in vivo by eccentric running exercise

Duchenne muscular dystrophy is an X-linked devastating disease due to the lack of expression of a functional dystrophin. Unfortunately, the dystrophin-deficient mdx mouse model does not present clinical signs of dystrophy before the age of 18 months, and the role of dystrophin in fiber integrity is not fully understood. The fragility of the skeletal muscle fibers was investigated in transgenic mice expressing β-galactosidase under the control of a muscle specific promoter. Adult mdx/β-galactosidase (dystrophin-negative) and normal/β-galactosidase (dystrophin-positive) mice were submitted to one short session of eccentric, downhill running exercise. The leakage of muscle enzymes creatine kinase and β-galactosidase was investigated before, 1 h after, and 3 days after the running session. A significant and transient rise in the level of these enzymes was noted in the serum of mdx mice following the exercise session. Thus, the lack of dystrophin in the mdx model led to local microdamages to the exercised muscle allowing leakage of proteins from the fibers. The peak leakage was transient, suggesting that muscle fiber lesions were rapidly repaired following this short, noninvasive eccentric running session. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:567–576, 1998.     

Understanding the beneficial effects of doxycycline on the dystrophic phenotype of the mdx mouse

Introduction: The purpose of this study was to better understand the beneficial effects of doxycycline on the dystrophic muscles of the mdx mouse. Methods: Doxycycline (DOX) was administered for 36 days, starting on postnatal day 0, via drinking water. Untreated mdx mice received plain water for the same period and served as a control group. Results: DOX decreased the levels of metalloproteinase‐9 and tumor necrosis factor‐alpha in the biceps brachii and diaphragm of the mdx mice. It also reduced the total amount of calcium in the muscles studied, concomitant with an increase in the levels of calsequestrin 1. Conclusions: The results show that DOX can affect factors that are important in dystrophic pathogenesis and highlight its potential as a readily accessible therapy in clinical trials for treatment of Duchenne muscular dystrophy. Muscle Nerve 50:283–286, 2014