Preclinical Ultrasonography in Rodent Models of Neuromuscular Disorders: The State of the Art for Diagnostic and Therapeutic Applications

Abstract

Ultrasonography is a safe, non-invasive imaging technique used in several fields of medicine, offering the possibility to longitudinally monitor disease progression and treatment efficacy over time. This is particularly useful when a close follow-up is required, or in patients with pacemakers (not suitable for magnetic resonance imaging). By virtue of these advantages, ultrasonography is commonly used to detect multiple skeletal muscle structural and functional parameters in sports medicine, as well as in neuromuscular disorders, e.g., myotonic dystrophy and Duchenne muscular dystrophy (DMD). The recent development of high-resolution ultrasound devices allowed the use of this technique in preclinical settings, particularly for echocardiographic assessments that make use of specific guidelines, currently lacking for skeletal muscle measurements. In this review, we describe the state of the art for ultrasound skeletal muscle applications in preclinical studies conducted in small rodents, aiming to provide the scientific community with necessary information to support an independent validation of these procedures for the achievement of standard protocols and reference values useful in translational research on neuromuscular disorders.

Keywords: neuromuscular disorders; skeletal muscle; translational research; ultrasonography.

Figure 1

Sample image of soleus skeletal muscle longitudinal acquisition by ultrasonography. Image acquisition was performed in B-mode operating with a linear probe working at a frequency of 40 MHz. Lateral and axial resolutions were 80 and 40 µm, respectively. Soleus muscle is well distinguishable by the hyperechoic white structures of epimysium (blue arrow). The direction of muscle bundles is also clearly visible (yellow arrow).

Figure 2

Main ultrasound views for skeletal muscle acquisition and evaluation of structural and functional parameters. (a) Rat placed in prone position (up) for GAS and SOL muscle acquisitions (down) used for volume PA, FL, and MT measurements. (b) Rat placed in supine position (up) for FDL muscle acquisitions (down) used for volume measurements. For both (a,b), image acquisition was performed in B-mode operating with a linear probe working at a frequency of 40 MHz. Lateral and axial resolutions were 80 and 40 µm, respectively. (c) Mouse placed in prone position (up) for 3-dimensional acquisitions of hind limb muscles in power Doppler mode (down) used for volume and percentage of vascularization measurements. Multiple 2-dimensional (2D) images were acquired at regular intervals in power Doppler mode by using an MS250 linear probe working at a frequency of 21 MHz, characterized by lateral and axial resolutions of 165 and 75 mm, respectively. At the end of the procedure, 3D images were reconstructed by Vevo2100 software 1.7.1. (d) Mouse placed in supine position (up) for DIA acquisitions (down) used for the evaluation of DIA movement amplitude and DIA echodensity. B-mode image acquisition was performed by using an MS250 linear probe working at a frequency of 21 MHz, characterized by lateral and axial resolutions of 165 and 75 mm, respectively.

Figure 3

Representative ultrasonographic images of gastrocnemius, soleus, and diaphragm muscles and related measurements. (a) Proximal (left) and distal (right) acquisitions showing in cyan the measurements used to calculate the GAS volume by using the truncated cone method. (b) Proximal and distal acquisitions showing in cyan the measurements used to calculate the GAS volume by using the innovative sinusoidal method. (c) B-mode (left) and M-mode diaphragm acquisition. Diaphragm amplitude was measured in M-mode as the distance between the baseline and the peak of contraction. The B-mode images were used for echodensity evaluation. For methodological details, see Figure 2.