First evidence of long-term effects of transcranial pulse stimulation (TPS) on the human brain

Background With the high spatial resolution and the potential to reach deep brain structures, ultrasound-based brain stimulation techniques offer new opportunities to non-invasively treat neurological and psychiatric disorders. However, little is known about long-term effects of ultrasound-based brain stimulation. Applying a longitudinal design, we comprehensively investigated neuromodulation induced by ultrasound brain stimulation to provide first sham-controlled evidence of long-term effects on the human brain and behavior. Methods Twelve healthy participants received three sham and three verum sessions with transcranial pulse stimulation (TPS) focused on the cortical somatosensory representation of the right hand. One week before and after the sham and verum TPS applications, comprehensive structural and functional resting state MRI investigations and behavioral tests targeting tactile spatial discrimination and sensorimotor dexterity were performed. Results Compared to sham, global efficiency significantly increased within the cortical sensorimotor network after verum TPS, indicating an upregulation of the stimulated functional brain network. Axial diffusivity in left sensorimotor areas decreased after verum TPS, demonstrating an improved axonal status in the stimulated area. Conclusions TPS increased the functional and structural coupling within the stimulated left primary somatosensory cortex and adjacent sensorimotor areas up to one week after the last stimulation. These findings suggest that TPS induces neuroplastic changes that go beyond the spatial and temporal stimulation settings encouraging further clinical applications.

The role of reactive oxygen species in the immunity induced by nano-pulse stimulation

Reactive oxygen species (ROS) are byproducts of tumor cells treated with Nano-Pulse Stimulation (NPS). Recently, ROS have been suggested as a contributing factor in immunogenic cell death and T cell-mediated immunity. This research further investigated the role of NPS induced ROS in antitumor immunity. ROS production in 4T1-luc breast cancer cells was characterized using three detection reagents, namely, Amplex Red, MitoSox Red, and Dihydroethidium. The efficiency of ROS quenching was evaluated in the presence or absence of ROS scavengers and/or antioxidants. The immunogenicity of NPS treated tumor cells was assessed by ex vivo dendritic cell activation, in vivo vaccination assay and in situ vaccination with NPS tumor ablation. We found that NPS treatment enhanced the immunogenicity of 4T1-luc mouse mammary tumor, resulted in a potent in situ vaccination protection and induced long-term T cell immunity. ROS production derived from NPS treated breast cancer cells was an electric pulse dose-dependent phenomenon. Noticeably, the dynamic pattern of hydrogen peroxide production was different from that of superoxide production. Interestingly, regardless of NPS treatment, different ROS scavengers could either block or promote ROS production and stimulate or inhibit tumor cell growth. The activation of dendritic cells was not influenced by blocking ROS generation. The results from in vivo vaccination with NPS treated cancer cells suggests that ROS generation was not a prerequisite for immune protection.

Phrenic afferent activation modulates cardiorespiratory output in the adult rat

Phrenic afferents project to brainstem areas responsible for cardiorespiratory control and the mid-cervical spinal cord containing the phrenic motor nucleus. Our purpose was to quantify the impact of small and large diameter phrenic afferent activation on phrenic motor output. Anesthetized and ventilated rats received unilateral phrenic nerve stimulation while contralateral phrenic motor output and blood pressure were recorded. Twelve currents of 40Hz inspiratory-triggered stimulation were delivered (20 seconds on, 5 minutes off) to establish current response curves. Stimulation pulse width was varied to preferentially activate large diameter phrenic afferents (narrow pulse width) and recruit small diameter fibers (wide pulse width). Contralateral phrenic amplitude was elevated immediately post-stimulation at currents above 35 µA for wide, and 70 µA for narrow pulse stimulation when compared to animals not receiving stimulation (time controls). Wide pulse width stimulation also increased phrenic burst frequency at currents ≥35 µA, caused a transient decrease in mean arterial blood pressure at currents ≥50 µA, and resulted in a small change in heart rate at 300 µA. Unilateral dorsal rhizotomy attenuated stimulation-induced cardiorespiratory responses, indicating phrenic afferent activation is required. Additional analyses compared phrenic motor amplitude to output before stimulation and showed that episodic activation of phrenic afferents with narrow pulse stimulation can induce short-term plasticity. We conclude that activation of phrenic afferents: 1) enhances contralateral phrenic motor amplitude when large diameter afferents are activated, and 2) when small diameter fibers are recruited the amplitude response is associated with changes in burst frequency and cardiovascular parameters.

Effects of In Vitro Muscle Contraction on Thermogenic Protein Levels in Co-Cultured Adipocytes

The crosstalk between the exercising muscle and the adipose tissue, mediated by myokines and metabolites, derived from both tissues during exercise has created a controversy between animal and human studies with respect to the impact of exercise on the browning process. The aim of this study was to investigate whether co-culturing of C2C12 myotubes and 3T3-L1 adipocytes under the stimuli of electrical pulse stimulation (EPS) mimicking muscle contraction can impact the expression of UCP1, PGC-1a, and IL-6 in adipocytes, therefore providing evidence on the direct crosstalk between adipocytes and stimulated muscle cells. In the co-cultured C2C12 cells, EPS increased the expression of PGC-1a (p = 0.129; d = 0.73) and IL-6 (p = 0.09; d = 1.13) protein levels. When EPS was applied, we found that co-culturing led to increases in UCP1 (p = 0.044; d = 1.29) and IL-6 (p = 0.097; d = 1.13) protein expression in the 3T3-L1 adipocytes. The expression of PGC-1a increased by EPS but was not significantly elevated after co-culturing (p = 0.448; d = 0.08). In vitro co-culturing of C2C12 myotubes and 3T3-L1 adipocytes under the stimuli of EPS leads to increased expression of thermogenic proteins. These findings indicate changes in the expression pattern of proteins related to browning of adipose tissue, supporting the use of this in vitro model to study the crosstalk between adipocytes and contracting muscle.

Chronic Pain Alters Somatosensory Evoked Potentials and Paired-pulse Inhibition in Athlete

Injuries are inevitable for athletes, and when injuries end up causing chronic pain, they usually force athletes to withdraw from training. Chronic pain is known to be caused by plastic changes in the brain; thus, the purpose of this study was to assess the somatosensory evoked potential (SEP) and the paired-pulse inhibition (PPI) in athletes suffering from chronic pain as compared to pain-free athletes. Twenty track and field (T&F) athletes, that were also undergraduate students, were recruited for this study. These athletes (12 men; 8 women) were divided into two groups of 10 based on their self-reporting of actively experiencing chronic pain (defined as pain that persisted for more than 3 months) or not. Both SEP and PPI in the primary somatosensory cortex (SI) were elicited by constant current square-wave pulses (of 0.2 ms duration) that were delivered to the right median nerve by an electrical stimulator through a surface bar electrode with a cathode proximal. Paired-pulse stimulation was set at interstimulus intervals of 30 and 100 ms. Subjects were randomly presented with 1,500 single- and paired-pulse stimuli at 2 Hz. Our measurements demonstrated a trend toward a lower N20 and P25 amplitude as well as a disinhibition of the PPI_30 ms in the athletes suffering from chronic pain. These findings suggest that chronic pain may modulate excitatory and inhibitory function of the SI in athletes as well as in patients suffering from complex regional pain syndrome or fibromyalgia.

Transcriptome Analyses of In Vitro Exercise Models by Clenbuterol Supplementation or Electrical Pulse Stimulation

Exercise has beneficial effects on human health and is affected by two different pathways; motoneuron and endocrine. For the advancement of exercise research, in vitro exercise models are essential. We established two in vitro exercise models using C2C12 myotubes; EPS (electrical pulse stimulation) for a motoneuron model and clenbuterol, a specific β2 adrenergic receptor agonist, treatment for an endocrine model. For clenbuterol treatment, we found that Ppargc1a was induced only in low glucose media (1 mg/mL) using a 1-h treatment of 30 ng/mL clenbuterol. Global transcriptional changes of clenbuterol treatment were analyzed by RNA-seq and gene ontology analyses and indicated that mitogenesis and the PI3K-Akt pathway were enhanced, which is consistent with the effects of exercise. Cxcl1 and Cxcl5 were identified as candidate myokines induced by adrenaline. As for the EPS model, we compared 1 Hz of 1-pulse EPS and 1 Hz of 10-pulse EPS for 24 h and determined Myh gene expressions. Ten-pulse EPS induced higher Myh2 and Myh7 expression. Global transcriptional changes of 10-pulse EPS were also analyzed using RNA-seq, and gene ontology analyses indicated that CaMK signaling and hypertrophy pathways were enhanced, which is also consistent with the effects of exercise. In this paper, we provided two transcriptome results of in vitro exercise models and these databases will contribute to advances in exercise research.