Neuromuscular magnetic stimulation counteracts muscle decline in ALS patients: results of a randomized, double-blind, controlled study

Abstract

The aim of the study was to verify whether neuromuscular magnetic stimulation (NMMS) improves muscle function in spinal-onset amyotrophic lateral sclerosis (ALS) patients. Twenty-two ALS patients were randomized in two groups to receive, daily for two weeks, NMMS in right or left arm (referred to as real-NMMS, rNMMS), and sham NMMS (sNMMS) in the opposite arm. All the patients underwent a median nerve conduction (compound muscle action potential, CMAP) study and a clinical examination that included a handgrip strength test and an evaluation of upper limb muscle strength by means of the Medical Research Council Muscle Scale (MRC). Muscle biopsy was then performed bilaterally on the flexor carpi radialis muscle to monitor morpho-functional parameters and molecular changes. Patients and physicians who performed examinations were blinded to the side of real intervention. The primary outcome was the change in the muscle strength in upper arms. The secondary outcomes were the change from baseline in the CMAP amplitudes, in the nicotinic ACh currents, in the expression levels of a selected panel of genes involved in muscle growth and atrophy, and in histomorphometric parameters of ALS muscle fibers. The Repeated Measures (RM) ANOVA with a Greenhouse-Geisser correction (sphericity not assumed) showed a significant effect [F(3, 63) = 5.907, p < 0.01] of rNMMS on MRC scale at the flexor carpi radialis muscle, thus demonstrating that the rNMMS significantly improves muscle strength in flexor muscles in the forearm. Secondary outcomes showed that the improvement observed in rNMMS-treated muscles was associated to counteracting muscle atrophy, down-modulating the proteolysis, and increasing the efficacy of nicotinic ACh receptors (AChRs). We did not observe any significant difference in pre- and post-stimulation CMAP amplitudes, evoked by median nerve stimulation. This suggests that the improvement in muscle strength observed in the stimulated arm is unlikely related to reinnervation. The real and sham treatments were well tolerated without evident side effects. Although promising, this is a proof of concept study, without an immediate clinical translation, that requires further clinical validation.

Figure 1

Study design. BV (baseline visit) represents the screening of ALS patients; T0 is the first recording of clinical strength and neurophysiological parameters before stimulation, T1 after one week, and T2 after two weeks of stimulation; T3 is thirty days after the end of stimulation.

Figure 2

Flowchart diagram of the study.

Figure 3

Significant improvement in muscle strength of the flexor carpi radialis muscle. (a) Muscle strength was tested by means of the MRC Muscle Scale and (b) handgrip dynamometer after NMMS. No significant improvement in muscle strength was observed in the control group. (c) No significant difference in cMAP amplitudes was observed before and after NMMS in the APB and (d) flexor carpi radialis muscles after median motor nerve stimulation at the elbow (Data represent mean ± SEM).

Figure 4

Morphologic and morphometric analysis of untreated and NMMS treated muscles. (a) Hematoxylin and eosin staining of muscle biopsies in sNMMS- and (b) rNMMS-treated muscles display grouped atrophy and scattered hypertrophic fibers (scale bar 50 µm). (c) Immunohistochemical staining for slow and fast myosin show that the majority of the fast fibers are atrophic in the representative sNMMS muscle (upper panel). The contralateral rNMMS-treated muscle (lower panel) is characterized by a prevalence of fast fibers. (d) Density estimation of the diameter for fast and slow twitch fibers across 4 patients, showing a significant shift in the peak fast fiber diameter towards higher values in rNMMS (red line) if compared with sNMMS (black line) (p < 0.001).

Figure 5

Electrophysiological analysis of ACh responses in sNMMS- and rNMMS. (a) The bar graphs show the mean ACh current amplitude (mean ± SEM) in sNMMS vs rNMMS injected oocytes. (Inset) Representative current traces from sNMMS (left) and rNMMS (right) injected oocytes. [ACh] = 500 μM; 4 s applications. *p < 0.05. Note the increase in current amplitude after rNMMS. (b) The bar graphs show the mean ACh current decay time (mean ± SEM) in sNMMS vs rNMMS injected oocytes. (Inset) Representative current traces from sNMMS (upper) and rNMMS (lower) injected oocytes. [ACh] = 500 μM; 30 s applications. *p < 0.05. Note the faster current decay time induced by rNMMS. (c) Averaged EC₅₀ values in oocytes injected with membranes from sNMMS and rNMMS patients, showing an increase in ACh affinity after rNMMS treatment.

Figure 6

NMMS treatment counteracts muscle atrophy and enhances robustness and resistance to fibrosis in ALS muscles. (A) Real-time PCR analysis of IGF-1EA, (B) myostatin, (C) MuRF-1, (D) atrogin-1, (E) SREBP-1, (F) MiR-24, (G) MiR-1 transcript in rNMMS- and sNMMS-treated samples (**p < 0.01, *p < 0.05. The number above the graph indicates the percentage decrease in transcript expression in treated vs control samples. Data are represented as mean ± SEM).

Figure 7

NMMS treatment stabilizes the neuromuscular end plate and preserves muscle fiber composition during disease progression. (A) Real-time PCR analysis of MiR-206, (B) HDAC4, (C) Myogenin, (D) AChRγ, (E) AChRα, (F) Mef2c transcripts in rNMMS- and sNMMS-treated samples (*p < 0.05 **p < 0.01. Data represent mean ± SEM).

Figure 8

Schematic representation of NMMS effect on ALS muscle decline.