Several studies have investigated the effects of different stimulation methods in modulating human brain plasticity [1–3]. Among these, the paired associative stimulation (PAS) paradigms were shown to modulate the excitability of corticospinal fibers related to the primary motor cortex, as a form of long-term modulation, including long-term potentiation (LTP) or depression (LTD) linked to synaptic plasticity [4, 5]. All these approaches induce electric currents to obtain a depolarization in the stimulated brain regions [6, 7]. However, even non-depolarizing magnetic fields, as in static magnets [8–10] or Low Frequency-Pulsed Electromagnetic Fields (LF-PEMFs) [11, 12], have shown the potential to modulate living structures [8, 13]. In particular, these non-depolarizing approaches seem to influence synaptic activity and ion channels on cellular membranes [8]. Indeed, it has been suggested that LF-PEMF can influence numerous types of changes in cells including migration, cell differentiation, stress response, potentially affecting morphology, migration of embryonic cells, and cell reprogramming [13–17]. Furthermore, it has been reported that LF-PEMF promotes osteogenic and neurogenic differentiation, which has been clinically used to repair bone fractures, promote wound healing [18, 19], and has been shown to have a neuroprotective effect after ischemic stroke in mice during the recovery process [11]. Several trials have also assessed the effects of LF-PEMF on major depressive disorder and unipolar or bipolar depression [20–22]. Moreover, LF-PEMF has been reported to influence brain glucose metabolism, thus affecting local brain activity [23]. Collectively, these studies indicate that LF-PEMF may be involved in neuroprotection.

LF-PEMFs (< 50 Hz) can be considered as a class of non-ionizing radiation with an associate energy < 12 electronvolt (eV), not enough to induce ionization phenomena [24], but with potential effects on biological components [25]. Recently, the CTU Mega 20^® device (see Fig. 1) was presented as a molecular accelerator, using an energy up to 200 J and providing high-power (2 Tesla) pulsating fields with a water-repulsive (diamagnetic) action with a consequent tissue biostimulation [25, 26]. In this research we tested the hypothesis that LF-PEMFs could modulate long-term corticospinal excitability in the healthy brain by applying transcranial pulsed magnetic fields with CTU Mega 20^® (http://www.periso.ch/). Therefore, we employed single-pulse transcranial magnetic stimulation (TMS), which allows an in vivo registration of the amplitude variation of the motor evoked potentials as a tool to explore cortical excitability perturbations of the motor system after CTU Mega 20 application.

Regarding the differences in the subjects’ perception of the stimulation, there was no statistically significant association between the type of stimulation and its perception, as assessed by Fisher’s exact test, p = 1.00, suggesting that real stimulation could not be distinguished from sham stimulation.

Two-way repeated measures ANOVA performed on corticospinal excitability revealed a significant TIME × TREATMENT interaction, F(3, 27) = 0.453, p < 0.001, partial η² = 0.645. A significant main effect of TIME was observed, with significant differences in post hoc tests at + 0, + 15 and + 30 min compared to baseline after real stimulation (all p < 0.05) but not after sham stimulation (all p > 0.05). There was also a significant main effect of TREATMENT, with significant differences at + 0, + 15 and + 30 min versus baseline, for real stimulation but not for sham stimulation (all p < 0.005) (see Table 1, Fig. 2).

There was no significant association between the percentage of increase in average MEP amplitude and baseline rMT in both groups (real stimulation: r_s = 0.10, p = 0.776; sham stimulation: r_s = − 0.46, p = 0.177).

During the treatment phase (application of real or sham CTU Mega 20 protocol), EMG activity at high-gain amplification was monitored to highlight possible depolarizing effects, which were however absent throughout the stimulation.

In this study, we employed a randomized double-blind sham-controlled crossover design (to control for known and unknown factors that could potentially influenced brain activity and TMS registration) to demonstrate that Low Frequency-Pulsed Electromagnetic Fields (LF-PEMFs) induced by CTU Mega 20 were able to modulate cortical excitability in human brains, even after a single-shot application. As described above, these findings were not influenced by subject treatment expectation (real stimulation versus sham), considering its potential effect on brain activity and consequently on TMS parameters [28]. In line with transcranial static magnetic field stimulation [10, 23, 29], cortical excitability enhancement was not directly related to induced electric currents, as is the case for other neuromodulation TMS-based techniques.

In our experiment, by providing 15-min pulsed-magnetic stimulus on the primary motor area, we obtained a persistent increase of more than 60% in corticospinal excitability (as an index of Long-Term Potentiation-Like Cortical Plasticity), recording the MEP from the contralateral first dorsal interosseous muscle. This perturbation lasted for at least for 30 min after the stimulation protocol, potentially maintaining a significant difference (at least 30%), for even a longer time. As reported above, tissue biostimulation provided by CTU Meg 20 was based on non-ionizing LF-PEMFs, which could act primarily at synapse level, altering membrane ion channel function. In particular, it was shown that Ca²⁺ and Na⁺ channel activity can be perturbed by magnetic fields, considering the diamagnetic anisotropic characteristics of membrane phospholipids [8, 30, 31]. Interestingly, CTU Mega 20 is defined as a diamagnetic acceleration system, allowing high-field magnetic impulses potentially capable of inducing magnetic reorientation of membrane phospholipids and consequently inducing a biological effect on nervous system function. From this point of view, there are three points in our proof of concept study that support the idea that non-ionizing LF-PEMFs induced by the CTU Mega 20 could represent a new approach for brain neuromodulation. They are (1) CTU Mega 20 provided a larger stimulation volume (up to 27 cm³ compared to 1–2 cm³ for TMS) allowing the modulation of an extended portion of cortical surface, compared to TMS; (2) as acommercially available device, the CTU Mega 20 is ready to use, with predefined programs to perform stimulation of the nervous system, not requiring specialized staff and can be easily adapted to everyday clinical application; (3) as a completely programmable system, the CTU Mega 20 can be optimized for different types of stimulation and different neurological diseases. In conclusion, the CTU Mega 20 diamagnetic acceleration system may well be of interest in the field of neuromodulation. Further studies to optimize protocol parameters for different neurological and psychiatric conditions are warranted [32, 33].

This proof-of-concept study in healthy subjects supports the idea that non-ionizing LF-PEMFs induced by the CTU Mega 20 diamagnetic acceleration system could represent a new approach for brain neuromodulation. Further studies to optimize protocol parameters for different neurological and psychiatric conditions are warranted.