Neuromuscular Ultrasound as a Biomarker in the SOD1 Mouse Model of Amyotrophic Lateral Sclerosis

Abstract

A progression marker that indicates early disease-related changes and treatment responses in the to date incurable neurodegenerative disease amyotrophic lateral sclerosis (ALS) is highly desirable. Translation of therapeutics that have been successful in in vivo models into trials in human patients has proven difficult in recent decades. This failure can be attributed, at least in part, to the lack of specific biomarkers for ALS diagnosis and progression in human ALS patients as well as in in vivo models. Neuromuscular ultrasound is an easily accessible, non-invasive tool to support diagnosis of ALS in humans.

Introduction

Amyotrophic lateral sclerosis (ALS) is the most common, adult-onset motor neuron disorder. Clinical manifestations include muscle weakness and atrophy, dysarthria, dysphagia, weight loss and respiratory insufficiency, leading to death within 3–5 years after onset.

To date, disease-modifying treatment options are limited: Riluzole, a glutamate antagonist, has shown modest efficacy, while the benefits of edaravone, a free radical scavenger approved in a few countries, have not been confirmed in a phase 3 study. Recently, promising treatment strategies have emerged for familial ALS with mutations in the SOD1 (superoxide dismutase 1), C9orf72 and FUS (fused in sarcoma) genes (NCT03626012, NCT04768972), though they are suitable for up to 10% of ALS patients only.

Materials and methods

Animals

Transgenic SOD1G93A mice (B6.Cg-Tg(SOD1*G93A)1Gur/J; RRID:IMSR_JAX:004435) overexpressing the human SOD1 mutation were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). By mating transgenic males with wild-type (C57BL/6J; RRID:IMSR_JAX:000664) females, transgenic hemizygous mice were bred and genotyped by polymerase chain reaction (PCR).

Ultrasound techniques

We used the Vevo® 2100 Imaging system (Visual Sonics Inc., Toronto, Canada) with a 40 MHz (22–55 MHz) real time linear array transducer to visualize the sciatic nerve and hind limb muscles. It offers spacial resolution down to 30 µm. Imaging was performed with the transducer fixed with the equipment provided by Visual Sonics. System-settings such as frequency (40 MHz), gain (15 dB), depth (8 mm for longitudinal plane, 5 mm for axial plane), time gain compensation and focus were kept unchanged.

Histomorphology

Following electroneurography, animals were euthanized and samples for histological evaluation were collected. In detail, the right sciatic nerve, right gastrocnemius muscle and lumbar part of the spinal cord were removed.

Motor neuron survival, astrocytosis and microgliosis in the spinal cord
As previously described [49], lumbar spinal cord tissue was fixed overnight in 4% paraformaldehyde (PFA) and then cryopreserved at least overnight in 30% sucrose. After embedding in cryoprotection compound (Sakura Finetek Germany GmbH, Staufen, Germany), the tissue was stored at −80°C until the sections were prepared using a cryotome (10x 12 µm serial sections per slide).

Neuromuscular junction (NMJ) immunostaining

For endplate (NMJ) analysis, gastrocnemius muscle tissue was harvested and fixed overnight in 4% PFA and then cryopreserved at least overnight in 30% sucrose. After embedding in cryoprotection compound, the tissue was stored at −80°C until further processing. A series of 20 µm longitudinal sections of the muscle were prepared using a cryotome (3 slides with 6 sections each).

Results

Changes in the sciatic nerve and muscles of the hind limbs in mutant SOD1G93A mice can be visualized successfully using Neuromuscular ultrasound Neuromuscular ultrasound was well tolerated by all animals; no side effects other than the effects associated with anesthesia or depilation of the hind leg occurred. After awakening all animals recovered and showed no new motor impairments especially of the examined hind limb. 

Discussion

SOD1G93A mice develop a rapidly progressive motor neuron disease. Current outcome measures (weight loss, severity of paralysis, motor testing and nerve conduction studies) are only moderately satisfactory markers to assess disease progression and effects of experimental drugs. In this study, we were able to show 1. that neuromuscular ultrasound is feasible in the SOD1G93A mouse model, 2. that changes in nerve and muscle morphometry are similar to the changes described in human ALS, and 3.

Citation: Wohnrade C, Thau-Habermann N, Gschwendtberger T, Rückoldt J, Huang Z, Schreiber S, et al. (2026) Neuromuscular ultrasound as a biomarker in the SOD1 mouse model of amyotrophic lateral sclerosis. PLoS One 21(7): e0353397. https://doi.org/10.1371/journal.pone.0353397

Editor: Atsushi Asakura, University of Minnesota Medical School, UNITED STATES OF AMERICA

Received: September 12, 2025; Accepted: June 23, 2026; Published: July 14, 2026

Copyright: © 2026 Wohnrade et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and the supporting information.

Funding: Deutsche Gesellschaft für Muskelkranke.

Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Camilla Wohnrade has received travel reimbursement from ITF Pharma GmbH, outside of the submitted work. Susanne Petri has received grants from the German Neuromuscular Society, the Federal Ministry of Education and Research, the German Israeli Foundation for Scientific Research and Development, and the EU Joint Programme for Neurodegenerative Disease Research, and received consultant/speaker fees from Biogen, Roche, Amylyx, Zambon, Cytokinetics, Desitin Pharma, and Italfarmaco, outside of the submitted work. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. This does not alter our adherence to PLOS ONE policies on sharing data and materials.