Quantitation of muscle precursor cell activity in skeletal muscle by Northern analysis of MyoD and myogenin expression: Application to dystrophic (mdx) mouse muscle

The regeneration of skeletal muscle is dependent upon proliferation and fusion of activated mononuclear muscle precursor cells. Early and specific markers of this population of activated cells are the transcription factors MyoD and myogenin. Northern analysis was used to determine levels of MyoD and myogenin mRNA in (i) muscles regenerating after experimental crush injury and (ii) in limb muscles of dystrophic mdx mice at various ages in comparison to controls. In crush-injured muscle, MyoD and myogenin mRNA increased at 24 h, peaked between 2 to 6 days, and returned to uninjured control levels by 15 days after injury. In both mdx and control mice, MyoD and myogenin mRNA levels were high in fetal muscles and decreased rapidly during the 2 weeks after birth. In mdx muscles, the mRNA levels increased significantly from about 21 days, remained high until around 40 days, and then decreased to a relatively constant yet elevated level when compared to control muscles. The elevated levels persisted to 420 days of age. The results show that this technique can be used to provide sensitive quantitative information on the size of the population of activated precursor cells in skeletal muscle. As such, it represents a novel and convenient means of measuring regenerative activity in vivo in whole muscles

Optical coherence tomography can assess skeletal muscle tissue from mouse models of muscular dystrophy by parametric imaging of the attenuation coefficient

We present the assessment of ex vivo mouse muscle tissue by quantitative parametric imaging of the near-infrared attenuation coefficient μt using optical coherence tomography. The resulting values of the local total attenuation coefficient μt (mean ± standard error) from necrotic lesions in the dystrophic skeletal muscle tissue of mdx mice are higher (9.6 ± 0.3 mm-1) than regions from the same tissue containing only necrotic myofibers (7.0 ± 0.6 mm-1), and significantly higher than values from intact myofibers, whether from an adjacent region of the same sample (4.8 ± 0.3 mm-1) or from healthy tissue of the wild-type C57 mouse (3.9 ± 0.2 mm-1) used as a control. Our results suggest that the attenuation coefficient could be used as a quantitative means to identify necrotic lesions and assess skeletal muscle tissue in mouse models of human Duchenne muscular dystrophy

Optical coherence tomography can assess skeletal muscle tissue from mouse models of muscular dystrophy by parametric imaging of the attenuation coefficient

We present the assessment of ex vivo mouse muscle tissue by quantitative parametric imaging of the near-infrared attenuation coefficient μt using optical coherence tomography. The resulting values of the local total attenuation coefficient μt (mean ± standard error) from necrotic lesions in the dystrophic skeletal muscle tissue of mdx mice are higher (9.6 ± 0.3 mm-1) than regions from the same tissue containing only necrotic myofibers (7.0 ± 0.6 mm-1), and significantly higher than values from intact myofibers, whether from an adjacent region of the same sample (4.8 ± 0.3 mm-1) or from healthy tissue of the wild-type C57 mouse (3.9 ± 0.2 mm-1) used as a control. Our results suggest that the attenuation coefficient could be used as a quantitative means to identify necrotic lesions and assess skeletal muscle tissue in mouse models of human Duchenne muscular dystrophy

Identification of muscle necrosis in the mdx mouse model of Duchenne muscular dystrophy using three-dimensional optical coherence tomography

Three-dimensional optical coherence tomography (3D-OCT) was used to image the structure and pathology of skeletal muscle tissue from the treadmill-exercised mdx mouse model of human Duchenne muscular dystrophy. Optical coherence tomography (OCT) images of excised muscle samples were compared with coregistered hematoxylin and eosin-stained and Evans blue dye fluorescence histology. We show, for the first time, structural 3D-OCT images of skeletal muscle dystropathology well correlated with co-located histology. OCT could identify morphological features of interest and necrotic lesions within the muscle tissue samples based on intrinsic optical contrast. These findings demonstrate the utility of 3D-OCT for the evaluation of small-animal skeletal muscle morphology and pathology, particularly for studies of mouse models of muscular dystrophy

Three-dimensional optical coherence tomography of whole-muscle autografts as a precursor to morphological assessment of muscular dystrophy in mice

Three-dimensional optical coherence tomography (3D-OCT) is used to evaluate the structure and pathology of regenerating mouse skeletal muscle autografts for the first time. The death of myofibers with associated inflammation and subsequent new muscle formation in this graft model represents key features of necrosis and inflammation in the human disease Duchenne muscular dystrophy. We perform 3D-OCT imaging of excised autografts and compare OCT images with coregistered histology. The OCT images readily distinguish the necrotic and inflammatory tissue of the graft from the intact healthy muscle fibers in the underlying host tissue. These preliminary findings suggest that, with further development, 3D-OCT could be used as a tool for the evaluation of small-animal muscle morphology and pathology, in particular, for analysis of mouse models of muscular dystrophy

Specific cloning of DNA fragments unique to the dog Y chromosome

A novel technique that enabled the specific cloning of a DNA fragment unique to the dog Y chromosome is described. The method involves competitive hybridization of DNA prepared from male dog lymphocytes with biotin-labeled DNA prepared from female dog lymphocytes. The biotinylated female-female and male-female hybrid DNA fragments were removed by capture with streptavidin-coated paramagnetic particles. Full-length double-stranded DNA was generated from the remaining fragments by using the Klenow fragment of DNA polymerase I, followed by direct cloning using a low-background ligation technique. Analysis of putative recombinant clones derived by this method has led to the identification of a fragment that hybridizes specifically to male dog DNA. The clones were selected initially on the basis of a differential signal obtained when hybridized to dilutions of male and female dog DNA immobilized on neutral nylon membrane. To evaluate its suitability as a probe for trans-sexually grafted cells in transplantation studies, the fragment was labeled with digoxigenin and hybridized in situ to male and female dog tissue sections. The clone designated number 6.2 hybridized strongly to male dog nuclei. The cloning strategy employed could be extended to other studies in which competitive reassociation can be used to identify unique DNA sequences

Three-dimensional optical coherence tomography of whole-muscle autografts as a precursor to morphological assessment of muscular dystrophy in mice

Three-dimensional optical coherence tomography (3D-OCT) is used to evaluate the structure and pathology of regenerating mouse skeletal muscle autografts for the first time. The death of myofibers with associated inflammation and subsequent new muscle formation in this graft model represents key features of necrosis and inflammation in the human disease Duchenne muscular dystrophy. We perform 3D-OCT imaging of excised autografts and compare OCT images with coregistered histology. The OCT images readily distinguish the necrotic and inflammatory tissue of the graft from the intact healthy muscle fibers in the underlying host tissue. These preliminary findings suggest that, with further development, 3D-OCT could be used as a tool for the evaluation of small-animal muscle morphology and pathology, in particular, for analysis of mouse models of muscular dystrophy

New horizons in the pathogenesis, diagnosis and management of sarcopenia

Sarcopenia is the age-related loss of skeletal muscle mass and function. It is now recognised as a major clinical problem for older people and research in the area is expanding exponentially. One of the most important recent developments has been convergence in the operational definition of sarcopenia combining measures of muscle mass and strength or physical performance. This has been accompanied by considerable progress in understanding of pathogenesis from animal models of sarcopenia. Well-described risk factors include age, gender and levels of physical activity and this knowledge is now being translated into effective management strategies including resistance exercise with recent interest in the additional role of nutritional intervention. Sarcopenia is currently a major focus for drug discovery and development although there remains debate about the best primary outcome measure for trials, and various promising avenues to date have proved unsatisfactory. The concept of ‘new tricks for old drugs’ is, however, promising, for example, there is some evidence that the angiotensin-converting enzyme inhibitors may improve physical performance. Future directions will include a deeper understanding of the molecular and cellular mechanisms of sarcopenia and the application of a lifecourse approach to understanding aetiology as well as to informing the optimal timing of interventions. <br/

Quantitative assessment of muscle damage in the mdx mouse model of Duchenne muscular dystrophy using polarization-sensitive optical coherence tomography

Minimally invasive, highresolution imaging of muscle necrosis has the potential to aid in the assessment of diseases such as Duchenne muscular dystrophy. Undamaged muscle tissue possesses high levels of optical birefringence due to its anisotropic ultrastructure, and this birefringence decreases when the tissue undergoes necrosis. In this study, we present a novel technique to image muscle necrosis using polarization-sensitive optical coherence tomography (PS-OCT). From PS-OCT scans, our technique is able to quantify the birefringence in muscle tissue, generating an image indicative of the tissue ultrastructure, with areas of abnormally low birefringence indicating necrosis. The technique is demonstrated on excised skeletal muscles from exercised dystrophic mdx mice and control C57BL/10ScSn mice with the resulting images validated against colocated histological sections. The technique additionally gives a measure of the proportion (volume fraction) of necrotic tissue within the three-dimensional imaging field of view. The percentage necrosis assessed by this technique is compared against the percentage necrosis obtained from manual assessment of histological sections, and the difference between the two methods is found to be comparable to the interobserver variability of the histological assessment. This is the first published demonstration of PSOCT to provide automated assessment of muscle necrosis