P.321Diaphragm echodensity and function in mdx mice by non-invasive ultrasonography: validation and correlation with in vivo and ex vivo functional readouts

Abstract

In the mdx mouse model of Duchenne muscular dystrophy (DMD), diaphragm (DIA) alterations occur at early age, with a progression which closely resembles patients' condition. High frequency ultrasound has been recently proposed as a longitudinal, non-invasive technique to monitor the progression of mdx DIA dysfunction. At present, data available are still limited, particularly in mdx mice at early disease stages. Moreover, no evidence has ever been provided about the usefulness of ultrasonography to measure echodensity, as an index of contractile tissue loss in mdx DIA. In light of this, and considering the importance assumed by an independent validation of new experimental procedures in preclinical studies on DMD, our study was aimed to perform an ultrasonography evaluation of DIA amplitude and echodensity, in 3- and 6-month-old mdx mice (the age window typically used for pharmacological studies), compared to wild type (wt). In parallel, in vivo (forelimb force, fatigability) and ex vivo (DIA contractile recordings, TGF-β1 levels) dystrophy-relevant outcomes were assessed. Ultrasonography showed a significant decrease of DIA amplitude in 3- and 6-month-old mdx mice compared to wt, which correlated with the isometric force reduction observed ex vivo. An age-dependent increase of mdx DIA echodensity was disclosed; this paralleled the high levels of muscular pro-fibrotic TGF-β1. DIA from 6-month-old mdx mice also showed a consistent force drop after eccentric contraction and a change in stiffness compared to wt, likely in relation to an increased fibrosis. Notably, no alteration of left ventricular function was found in mdx hearts, supporting the view that respiratory complications occur independently from cardiac ones. Our findings corroborate the usefulness of DIA ultrasonography as a tool to evaluate mdx mouse pathology progression since early stages, and this possibility opens important new perspectives to improve translational research on DMD (Supported by PRIN-MIUR n. 20108YB5W3_004).