Poster AA3 - Electrostimulation with nanosecond-duration electric pulses: a novel rehabilitation tool to enhance skeletal muscle force

Introduction: Electrical stimulation is commonly used in rehabilitation clinics and research labs to reduce pain, stimulate tissue healing, and strengthen muscles. One such form of electrostimulation is neuromuscular electrical stimulation (NMES) that involves sending electrical impulses through the skin to nerves and/or muscles causing the muscles to contract. It is often used to “re-train” or “re-educate” a muscle to function and to build strength after a surgery or period of disuse. A major challenge in the use of NMES is the rapid fatigue (i.e., reduction in force generation capability of muscle over time) caused by the stimulation itself, which limits the duration that NMES can be applied. Pulsed power technologies have produced a new type of electrical stimulus, nanosecond-duration electric pulses (NEP), which are capable of evoking peripheral nerve stimulation without damage [Casciola et al., Sci Rep. 2017, 7(1):10453]. Because of their very short duration, these pulses have significantly less power compared to the conventional (millisecond) duration pulses used in NMES. This begs the question: do NEP present any advantages compared to the conventional-duration electric pulses (CEP) currently used? To answer this question, we used a well-established murine ex vivo neuromuscular preparation, phrenic nerve and attached diaphragm muscle, and compared the effects of high frequency nerve stimulation with NEP vs. CEP on muscle fiber shortening.

Materials and

Methods: Animal studies were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and the University of Nevada IACUC. Briefly, 4- to 8-week-old male and female wild-type mice were anesthetized by isoflurane inhalation and sacrificed by cervical decapitation. An abdominal incision was made to provide access to the phrenic nerve and the diaphragm, which were then removed and perfused with oxygenated Krebs-Ringer’s solution. The whole diaphragm was pinned onto Sylgard-coated dishes and the phrenic nerve was drawn into a suction, cuff-like electrode made of borosilicate glass and platinum wires. After 30 min perfusion, the left phrenic nerve was stimulated with a square pulse from an S88 stimulator once to ensure the muscle contracted before treatment with high-frequency trains of CEP or NEP. Five trains of CEP (0.3 ms) or NEP (30 ns) were applied 15 min apart at 40 Hz for 30 s. Brightfield recordings were performed on a Nikon Eclipse FN1 upright microscope using a 4x lens, an Andor Neo sCMOS camera and Nikon NIS Elements 4.1. Image sequences were recorded at 25 frames per second and analyzed by custom software Volumetry G8d, GWH. To measure contractile force by fiber shortening, two contrasted regions were identified in the stimulated hemi-diaphragm. Spatiotemporal maps were constructed with the axis of the regions selected for imaging spatially perpendicular to the long axis of the muscle fibers. The movement of the two regions was tracked in the spatiotemporal maps using edge detection and the distance between them calculated.

Results, Conclusions, and Discussions: In initial series of experiments, we identified the suprathreshold CEP and NEP voltage values that stimulate the phrenic nerve to a similar extent, i.e., that generate a similar peak contractile force in diaphragm muscle cells. For this determination, we increased the voltage of the pulses (CEP and NEP) incrementally and measured peak contractile force optically by fiber shortening at every voltage. We found that for a pulse duration of 0.3 ms, the suprathreshold electric pulse amplitude required to evoke muscle contraction was 6 V, whereas for a 30 ns pulse, the suprathreshold electric pulse amplitude required to evoke muscle contractions of similar magnitude to those evoked by CEP was 1.1 kV. Next, we compared the effects of high frequency trains of nerve stimulation with NEP with those of CEP. In preliminary findings, a significant reduction in peak shortening was observed in response to the fifth train of CEP but not NEP stimulation, indicating that CEP but not NEP caused muscle fatigue. Specifically, peak shortening induced by the fifth train of CEP displayed a significant reduction from that induced by the first train (average decrease of 30%), whereas peak shortening induced by the fifth train of NEP failed to change at all from that induced by the first train. Another striking difference between CEP and NEP applied at 40 Hz is that trains of CEP but not NEP induced fatigue within the train as observed by a failure to maintain peak shortening within an individual train. Fig. 1 shows a 28% vs. 3% reduction of initial peak shortening vs. end peak shortening for CEP vs. NEP, respectively. Together, these results indicate that, although trains of NEP and CEP delivered to the phrenic nerve generate equal contractile force of diaphragm muscle fibers, NEP trains produce significantly less fatigue at the 40-Hz frequency that is known to induce fatigue when conventional-duration pulses are used, suggesting that NEP may represent a more effective form of electrostimulation than CEP. Our future studies are investigating Ca2+ responses in diaphragm muscle cells in response to phrenic nerve stimulation with NEP and CEP.

Acknowledgements (Optional): This work was supported by the Air Force Office of Scientific Research (AFOSR Grant FA9550-23-1-0724) and the National Institutes of Health (NIH Grant P20GM103650).