Section of Pharmacology, Department of Pharmacy-Drug Sciences, University of Bari "Aldo Moro", Bari, Italy.
Axxam, S.p.A, Bresso, Milan, Italy.
PLoS One. 2021 Jan 12;16(1):e0245397. doi: 10.1371/journal.pone.0245397. eCollection 2021.
The mdx mouse model of Duchenne muscular dystrophy is characterized by functional and structural alterations of the diaphragm since early stages of pathology, closely resembling patients' condition. In recent years, ultrasonography has been proposed as a useful longitudinal non-invasive technique to assess mdx diaphragm dysfunction and evaluate drug efficacy over time. To date, only a few preclinical studies have been conducted. Therefore, an independent validation of this method by different laboratories is needed to increase results reliability and reduce biases. Here, we performed diaphragm ultrasonography in 3- and 6-month-old mdx mice, the preferred age-window for pharmacology studies. The alteration of diaphragm function over time was measured as diaphragm ultrasound movement amplitude. At the same time points, a first-time assessment of diaphragm echodensity was performed, as an experimental index of progressive loss of contractile tissue. A parallel evaluation of other in vivo and ex vivo dystrophy-relevant readouts was carried out. Both 3- and 6-month-old mdx mice showed a significant decrease in diaphragm amplitude compared to wild type (wt) mice. This index was well-correlated either with in vivo running performance or ex vivo isometric tetanic force of isolated diaphragm. In addition, diaphragms from 6-month-old dystrophic mice were also highly susceptible to eccentric contraction ex vivo. Importantly, we disclosed an age-dependent increase in echodensity in mdx mice not observed in wt animals, which was independent from abdominal wall thickness. This was accompanied by a notable increase of pro-fibrotic TGF-β1 levels in the mdx diaphragm and of non-muscle tissue amount in diaphragm sections stained by hematoxylin & eosin. Our findings corroborate the usefulness of diaphragm ultrasonography in preclinical drug studies as a powerful tool to monitor mdx pathology progression since early stages.
mdx 肌营养不良症小鼠模型的膈肌在疾病早期就表现出功能和结构的改变，与患者的情况非常相似。近年来，超声检查已被提议作为一种有用的纵向非侵入性技术，用于评估 mdx 膈肌功能障碍，并随时间评估药物疗效。迄今为止，只有少数临床前研究进行了。因此，需要不同实验室对这种方法进行独立验证，以提高结果的可靠性并减少偏差。在这里，我们对 3 月龄和 6 月龄 mdx 小鼠进行了膈肌超声检查，这是药理学研究的首选年龄窗口。随着时间的推移，膈肌功能的变化通过膈肌超声运动幅度来测量。在相同的时间点，还首次评估了膈肌回声密度，作为进行性收缩组织丧失的实验指标。同时还进行了其他体内和体外与肌肉营养不良相关的读数的平行评估。与野生型（wt）小鼠相比，3 月龄和 6 月龄 mdx 小鼠的膈肌幅度均显著降低。该指数与体内跑步表现或离体等长强直力高度相关。此外，来自 6 月龄的营养不良小鼠的膈肌也极易发生离体偏心收缩。重要的是，我们发现 mdx 小鼠的回声密度随年龄的增加而增加，而 wt 动物则没有观察到这种情况，这与腹壁厚度无关。这伴随着 mdx 膈肌中促纤维化 TGF-β1 水平的显著增加，以及苏木精和伊红染色的膈肌切片中非肌肉组织量的显著增加。我们的发现证实了膈肌超声检查在临床前药物研究中的有用性，因为它是一种从早期监测 mdx 病理进展的强大工具。
