Laboratory of Biochemistry of Lipids, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093, Warsaw, Poland.
Laboratory of Molecular Basis of Behavior, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093, Warsaw, Poland.
Skelet Muscle. 2018 Jul 19;8(1):21. doi: 10.1186/s13395-018-0168-8.
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.
杜氏肌营养不良症（DMD）是一种致命的 X 连锁遗传疾病。尽管 DMD 是最常见的肌肉营养不良形式，但仅开发了两种 FDA 批准的药物来延缓其进展。为了评估治疗 DMD 的疗法，最近引入了几种鼠模型。由于多种鼠模型揭示了 DMD 病理学的机制，因此如何在整个肌肉骨骼系统中监测疾病的进展仍然是一个悬而未决的问题。监测这种进展的一种重要方法是对肌肉活检中的钙沉积进行组织学评估。尽管组织学是准确的，但它仅限于小组织区域，不能用于评估 DMD 的系统性进展。因此，我们旨在开发一种适用于快速和高分辨率筛选整个鼠体内钙沉积的方法。
在成年雄性 C57BL/10-mdx 和成年雄性 C57BL 小鼠上进行了程序。动物被处死、灌注、多聚甲醛固定，并使用优化的基于灌注的 CUBIC 方案进行全身清除。接下来，用茜素红 S 对清除的生物体进行染色以可视化钙沉积并进行成像。
研究表明，C57BL/10-mdx 小鼠整个机体的退化肌肉中存在钙沉积。在前肢、膈肌、腰部、骨盆区和后肢的骨骼肌中观察到钙化沉积物。对股四头肌、肱三头肌和脊柱 pars lumborum 中的钙化沉积物进行了特征描述。累积频率分布分析表明，股四头肌中钙化沉积物的分布特征与肱三头肌和脊柱 pars lumborum 肌肉不同（p<0.001），股四头肌与脊柱 pars lumborum 也不同（p<0.001）。所选肌肉中钙化沉积物的数量、体积和平均体积之间存在统计学差异。
总之，我们提出了一种新的方法来监测 Duchenne 肌肉营养不良症鼠模型中的原位钙沉积。使用提出的设置进行样本成像是可行的，适用于整个器官/身体成像。结合定制的 LSFM 设备的开发，它允许对清除肌肉中的钙沉积进行靶向和精确表征。因此，所提出的方法可广泛用于监测整个机体肌肉受坏死影响的程度以及动物的治疗或运动如何改变这种影响。我们相信，这将是研究广泛的肌肉营养不良和骨骼相关疾病动物模型中器官变化的有价值的工具。
