Ultrasonography validation for early alteration of diaphragm echodensity and function in the mdx mouse model of Duchenne muscular dystrophy.

Stephen E Alway,Antonietta Mele,Jean-Francois Rolland,Paola Mantuano,Adriano Fonzino,Annamaria De Luca,Ornella Cappellari,Francesco Rana,Roberta Francesca Capogrosso,Francesca Sanarica

doi:10.1371/journal.pone.0245397

Stephen E Alway, Antonietta Mele + Show 8 more

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https://doi.org/10.1371/journal.pone.0245397

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Abstract

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.

Highlights

The dystrophic mdx mouse is a widely used animal model for Duchenne muscular dystrophy (DMD) in preclinical studies, which shares the same biochemical defect of DMD patients for an X-linked stop codon mutation in the DMD gene encoding for dystrophin protein [1,2,3]
In 2016, Whitehead and colleagues described the possibility to monitor diaphragm function in vivo in mdx mice by means of ultrasonography, a non-invasive technique that had been mainly used to detect late changes in cardiac function in this murine model [5]. Such an ultrasonographic alteration of diaphragm function was well-correlated with the impairment of diaphragm force measured ex vivo, supporting the value of this approach for translational studies [5]. It is well-known that mdx diaphragm shows a progression of dystrophic pathology which closely resembles the one observed in humans, and its functional and structural alteration contributes to respiratory impairment in dystrophic subjects [1, 6]
We found a good correlation between diaphragm amplitude and e-test values obtained in vivo at M3 and M6, indicating that the impaired running performance may be, at least in part, attributable to the progressively impaired respiratory function in addition to the main involvement of an increased fatigability of hind limb muscles

Summary

Introduction

The dystrophic mdx mouse is a widely used animal model for Duchenne muscular dystrophy (DMD) in preclinical studies, which shares the same biochemical defect of DMD patients for an X-linked stop codon mutation in the DMD gene encoding for dystrophin protein [1,2,3]. In 2016, Whitehead and colleagues described the possibility to monitor diaphragm function in vivo in mdx mice by means of ultrasonography, a non-invasive technique that had been mainly used to detect late changes in cardiac function in this murine model [5]. Such an ultrasonographic alteration of diaphragm function was well-correlated with the impairment of diaphragm force measured ex vivo, supporting the value of this approach for translational studies [5]. A morphological and functional ultrasound evaluation of skeletal muscles, including diaphragm, has been promisingly conducted in several clinical studies on DMD patients [7,8,9,10]

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