Whole-body clearing, staining and screening of calcium deposits in the mdx mouse model of Duchenne muscular dystrophy

Lukasz Bozycki, Kacper Łukasiewicz, Paweł Matryba, Slawomir Pikula

Duchenne muscular dystrophy (DMD) is a fatal, X-linked genetic disorder. Although DMD is the most common form of muscular dystrophy, only two FDA-approved drugs were developed to delay its progression. In order to assess therapies for treating DMD, several murine models have recently been introduced. As the wide variety of murine models enlighten mechanisms underlying DMD pathology, the question on how to monitor the progression of the disease within the entire musculoskeletal system still remains to be answered. One considerable approach to monitor such progression is histological evaluation of calcium deposits within muscle biopsies. Although accurate, histology is limited to small tissue area and cannot be utilized to evaluate systemic progression of DMD. Therefore, we aimed to develop a methodology suitable for rapid and high-resolution screening of calcium deposits within the entire murine organism. Procedures were performed on adult male C57BL/10-mdx and adult male C57BL mice. Animals were sacrificed, perfused, paraformaldehyde-fixed, and subjected to whole-body clearing using optimized perfusion-based CUBIC protocol. Next, cleared organisms were stained with alizarin red S to visualize calcium deposits and subjected to imaging. Study revealed presence of calcium deposits within degenerated muscles of the entire C57BL/10-mdx mouse organism. Calcified deposits were observed within skeletal muscles of the forelimb, diaphragm, lumbar region, pelvic region, and hindlimb. Calcified deposits found in quadriceps femoris, triceps brachii, and spinalis pars lumborum were characterized. Analysis of cumulative frequency distribution showed different distribution characteristics of calcified deposits in quadriceps femoris muscle in comparison to triceps brachii and spinalis pars lumborum muscles (p < 0.001) and quadriceps femoris vs spinalis pars lumborum (p < 0.001). Differences between the number of calcified deposits in selected muscles, their volume, and average volume were statistically significant. In aggregate, we present new methodology to monitor calcium deposits in situ in the mouse model of Duchenne muscular dystrophy. Sample imaging with the presented setup is feasible and applicable for whole-organ/body imaging. Accompanied by the development of custom-made LSFM apparatus, it allows targeted and precise characterization of calcium deposits in cleared muscles. Hence, presented approach might be broadly utilized to monitor degree to which muscles of the entire organism are affected by the necrosis and how is it altered by the treatment or physical activity of the animal. We believe that this would be a valuable tool for studying organs alternations in a wide group of animal models of muscle dystrophy and bone-oriented diseases.