Magnetic stimulation supports muscle and nerve regeneration after trauma in mice

Abstract

Introduction: Magnetic stimulation (MS) has the ability to induce muscle twitch and has long been proposed as a therapeutic modality for skeletal muscle diseases. However, the molecular mechanisms underlying its means of action have not been defined.

Methods: Muscle regeneration after trauma was studied in a standard muscle injury mouse model. The influence of MS on the formation of motor units, posttrauma muscle/nerve regeneration, and vascularization was investigated.

Results: We found that MS does not cause systemic or muscle damage but improves muscle regeneration by significantly minimizing the presence of inflammatory infiltrate and formation of scars after trauma. It avoids posttrauma muscle atrophy, induces muscle hypertrophy, and increases the metabolism and turnover of muscle. It triples the expression of muscle markers and significantly improves muscle functional recovery after trauma.

Conclusions: Our results indicate that MS supports muscle and nerve regeneration by activating muscle-nerve cross-talk and inducing the maturation of neuromuscular junctions.

Keywords: magnetic stimulation therapy; muscle contraction; nerve regeneration; neuromuscular junction; rehabilitation outcome.

Conflict of interest statement

The authors declare no conflicts of interest with respect to the authorship and/or publication of this article.

© 2015 Wiley Periodicals, Inc.

Figures

Figure 1. MS causes no systemic damage, accelerates the regeneration process, and reduces scar width and inflammatory infiltrates.

(A, B) The degree of injury to the quadriceps was reduced after MS treatment. (C) The levels of systemic myoglobin after MS stimulation were comparable with normal parameters. (D) Conversely, the systemic values of creatine kinase increased up to 5-fold. Dashed lines indicate normal values. (E, F, G) Hematoxylin and eosin-stained sections demonstrated that the crush injury scar was significantly reduced after 5 days of MS treatment. (H) Simultaneously, the extension of the inflammatory infiltrate, as assessed by the presence of lymphocytes and macrophages on the interface of injury, was reduced to half. (I, J) Staining with smoothelin/Cy3 (red), von Willebrand factor/FITC (green) and DAPI (blue) demonstrated a lower density of new vessels at day 5 in the MS-treated samples. (K, L) While the control samples still displayed at least a 150% higher number of micro vessels when compared to intact quadriceps , MS-treated samples demonstrated values similar to native tissue. +MS indicates magnetic stimulation treatment, whereas -MS represents the non-stimulated controls (*P<0.05, **P<0.001).

Figure 2. MS reduces post-trauma muscle atrophy, boosts muscle turn-over, and induces hypertrophy of the injury interface.

Muscle fiber cross-section measurements were performed in study animals after muscle crush with or without MS within the central zone of the injury and at the border. The values were then normalized with muscle fiber cross-section of the control unscathed muscle and expressed as percentage of it. The isolated effect of MS on the injury center (A-D) and on the interface (E-H) was analyzed. (A, B, C) In the center of the injury site, MS induced hypertrophy and doubled the cross-sectional diameter of the fibers. (D) This effect was associated with a remarkable increase in the regeneration ratio, as represented by muscle fibers with central nuclei. (E, F) Similarly, in the injury interface, the inflammatory infiltrate was reduced, and (G) the fiber cross-sections were again hypertrophic, with fiber cross-sections that were approximately 40% larger than in control and MS-untrained quadriceps. (H) The regeneration ratio, as assessed by the percentage of myofibers with central nuclei, was almost 3 times higher in stimulated samples. +MS indicates the presence of magnetic stimulation treatment, whereas -MS represents the unstimulated controls (**P<0.001)

Figure 3. MS induces a muscle type switch to slow-twitch fibers

After crush injury and MS treatment, the quadriceps was retrieved and analyzed by western blot or histology. We found that the expression of muscle proteins was increased after MS treatment. (A) Desmin nearly doubled its expression levels, and (B) a specific increase in MyH type 1 could be detected. (C) No significant difference in MyH type 2 was found by WB. (D, E, F) Staining was performed with anti-myosin-heavy-chain-slowtwitch/FITC-anti-mouse-IgM (green), anti-myosin-heavy-chain-fast-twitch/Cy3-antimouse-IgG (red), and DAPI (blue). When compared with intact control quadriceps, no fiber type change was found in injured muscle without MS stimulation. (E) On the other hand, a shift to type 1 fibers was verified in MS-treated samples. (F) MyH type 1 expression was up to 3-fold higher in MS-treated samples than in native muscle. (G) As expected, a relative decrease in MyH type 2 was detected. +MS indicates magnetic stimulation treatment, whereas -MS represents the unstimulated controls (*P<0.05, **P<0.001)

Figure 4. MS improves muscle contractile strength of native control muscle tissue and aids in the recovery of muscle contractile strength after trauma

After 5 days of magnetic stimulation, the muscles were retrieved and assessed. Organ bath of tibialis muscle strips was performed using 40-80V and 40-80Hz. (A) In native control muscle tissue, MS increased the contractile strength to 150% of non-stimulated samples. (B) Representative organ bath contractions at 40 V/40 Hz. The induced muscle crush injury was sufficient to reduce muscle contractile strength to approximately one-half (dashed line) of that of control muscles (continuous line). (C) MS treatment after injury significantly promoted the recovery of lost muscle strength after trauma. +MS indicates magnetic stimulation treatment, whereas -MS represents the non-stimulated controls (*P<0.05, **P<0.001).

Figure 5. MS intensifies muscle-nerve cross-talk, increases nerve ingrowth, and promotes AChR clustering.

Analyses of the nerve component within the injured muscle were performed by WB and immunostaining. (A) Agrin expression was increased, (B, C) along with specific neurofilament 68 (NF68) and the PGP9.5-neuronal marker. (D, E) Immunostaining confirmed the WB results, demonstrating an increased number of nerves in the tissues after MS. Staining was performed with neurofilament 68/488-anti-mouse-IgG (green), α-BTX (red), and DAPI (blue). (F) Surprisingly, the amount of nerves detected after MS was approximately 50% higher compared to normal tissue. (G, H, I) NMJ staining of acetylcholine receptors (AChR) with α-bungarotoxin (red) demonstrated that they were more clustered and had a significantly higher density after MS. (J, K) Analysis of single fibers isolated from the entire injury area after collagenase digestion demonstrated the total ratio of AChR cluster per muscle fiber. (L) The total number of clusters per fiber was 4-fold higher in MS-treated samples, demonstrating the positive effect of MS in reorganizing the neuromuscular junction after trauma. +MS indicates magnetic stimulation treatment, whereas -MS represents non-stimulated controls (*P<0.05, **P<0.001)