Damage-free peripheral nerve stimulation by 12-ns pulsed electric field

Abstract

Modern technologies enable deep tissue focusing of nanosecond pulsed electric field (nsPEF) for non-invasive nerve and muscle stimulation. However, it is not known if PEF orders of magnitude shorter than the activation time of voltage-gated sodium channels (VGSC) would evoke action potentials (APs). One plausible scenario requires the loss of membrane integrity (electroporation) and resulting depolarization as an intermediate step. We report, for the first time, that the excitation of a peripheral nerve can be accomplished by 12-ns PEF without electroporation. 12-ns stimuli at 4.1–11 kV (3.3–8.8 kV/cm) evoked APs similarly to conventional stimuli (100–250 μs, 1–5 V, 103–515 V/m), except for having higher selectivity for the faster nerve fibers. Nerves sustained repeated tetanic stimulations (50 Hz or 100 Hz for 1 min) alternately by 12-ns PEF and by conventional pulses. Such tetani caused a modest AP decrease, to a similar extent for both types of stimuli. Nerve refractory properties were not affected. The lack of cumulative damages even from tens of thousands of 12-ns stimuli and the similarities with the conventional stimulation prove VGSC activation by nsPEF without nerve membrane damage.

Excitation of murine cardiac myocytes by nanosecond pulsed electric field

“…Optical recordings of the membrane potential were accomplished with FluoVolt dye (Thermo Fisher Scientific, Waltham, MA), which was previously validated for the recording of neuronal APs in our group 25 and for cardiac APs by others. 36 The procedures for field stimulation of individually selected cells on a microscope stage were similar to those described previously.…”

“…Such stimuli may be too brief to elicit APs directly by membrane depolarization, since the process of opening of voltage-gated sodium channels, as evidenced by the time course of gating currents, takes tens to hundreds of microseconds. Experimental findings suggest that some electroporative damage can already take place at the nsPEF threshold for cell excitation 25,31,32 (with the exception of peripheral nerve stimulation), 33,34 although the causal connection has not been proven and remains questionable. It suggests that there might be a coupling mechanism to link nsPEF and AP generation.…”

“…27,28 Although gating at the single molecule level might be a "jump process" between discrete states, 28,29 with probability increased by depolarization, nsPEF-induced depolarization may be too brief to open the critical number of channels to trigger an AP. 25 The objective of the present study was to evaluate the mechanism of AP induction by nsPEF in murine cardiac myocytes, and specifically to assess the role of nanoelectroporation in myocyte excitation. For conventional (long) stimuli, it was demonstrated that electroporation caused by a strong shock leads to a prolonged depolarization, which can result in excitation of cardiac muscle.…”

“…The underlying mechanisms have not been explored yet and may involve permeabilization of the sarcoplasmic reticulum by nsPEF, [18][19][20] damage to voltage-gated Ca 2+ channels, 15 alteration of phosphoinositide signaling, [21][22][23] and nanoelectroporation of the cell membrane (which can be detected by the loss of membrane potential and transport of water and small ions, but not by propidium uptake). 11,12,19,[24][25][26] The present study was aimed at testing the latter mechanism by exploring if there are any abnormal responses upstream from Ca 2+ handling, namely at the level of the action potential (AP) induction by nsPEF.…”

“…30 In a similar manner, electroporation may be involved in nsPEF-induced AP generation. Experimental findings suggest that some electroporative damage can already take place at the nsPEF threshold for cell excitation 25,31,32 (with the exception of peripheral nerve stimulation), 33,34 although the causal connection has not been proven and remains questionable. 25 The objective of the present study was to evaluate the mechanism of AP induction by nsPEF in murine cardiac myocytes, and specifically to assess the role of nanoelectroporation in myocyte excitation.…”