Response to Sodium Current Inhibition by Nanosecond Pulsed Electric Field (nsPEF) - Fact or Artifact? by Verkerk et al

It was nice to learn that our studies of nanosecond pulsed electric field (nsPEF) effects on membrane currents [Nesin et al., 2012; Nesin and Pakhomov, 2012] gained the attention of scientists outside the immediate field of bioelectromagnetics

Inhibition of Voltage-Gated Na+ Current by Nanosecond Pulsed Electric Field (nsPEF) is Not Mediated by NA+ Influx or Ca²+ Signaling

In earlier studies, we found that permeabilization of mammalian cells with nsPEF was accompanied by prolonged inhibition of voltage-gated (VG) currents through the plasma membrane. This study explored if the inhibition of VG Na+ current (INa) resulted from (i) reduction of the transmembrane Na+ gradient due to its influx via nsPEF-opened pores, and/or (ii) downregulation of the VG channels by a Ca2+ -dependent mechanism. We found that a single 300?ns electric pulse at 1.65.3?kV/cm triggered sustained Na+ influx in exposed NG108 cells and in primary chromaffin cells, as detected by increased fluorescence of a Sodium Green Dye. In the whole-cell patch clamp configuration, this influx was efficiently buffered by the pipette solution so that the increase in the intracellular concentration of Na+ ([Na]i) did not exceed 2-3 mM. [Na]i increased uniformly over the cell volume and showed no additional peaks immediately below the plasma membrane. Concurrently, nsPEF reduced VG INa by 30-60% (at 4 and 5.3 kV/cm). In control experiments, even a greater increase of the pipette [Na+] (by 5mM) did not attenuate VG INa, thereby indicating that the nsPEF-induced Na+ influx was not the cause of VG INa inhibition. Similarly, adding 20 mM of a fast Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N\u27,N\u27-tetraacetic acid (BAPTA) into the pipette solution did not prevent or attenuate the inhibition of the VG INa by nsPEF. These findings point to possible Ca2+ -independent downregulation of the VG Na+ channels (e.g., caused by alteration of the lipid bilayer) or the direct effect of nsPEF on the channel

Cellular Regulation of Extension and Retraction of Pseudopod-Like Blebs Produced by Nanosecond Pulsed Electric Field

Recently we described a new phenomenon of anodotropic pseudopod-like blebbing in U937 cells exposed to nanosecond pulsed electric field (nsPEF). In Ca2+ -free buffer such exposure initiates formation of pseudopod-like blebs (PLBs), protrusive cylindrical cell extensions that are distinct from apoptotic and necrotic blebs. PLBs nucleate predominantly on anode-facing cell pole and extend toward anode during nsPEF exposure. Bleb extension depends on actin polymerization and availability of actin monomers. Inhibition of intracellular Ca2+ , cell contractility, and RhoA produced no effect on PLB initiation. Meanwhile, inhibition of WASP by wiskostatin causes dose-dependent suppression of PLB growth. Soon after the end of nsPEF exposure PLBs lose directionality of growth and then retract. Microtubule toxins nocodazole and paclitaxel did not show immediate effect on PLBs; however, nocodazole increased mobility of intracellular components during PLB extension and retraction. Retraction of PLBs is produced by myosin activation and the corresponding increase in PLB cortex contractility. Inhibition of myosin by blebbistatin reduces retraction while inhibition of RhoA-ROCK pathway by Y-27632 completely prevents retraction. Contraction of PLBs can produce cell translocation resembling active cell movement. Overall, the formation, properties, and life cycle of PLBs share common features with protrusions associated with ameboid cell migration. PLB life cycle may be controlled through activation of WASP by its upstream effectors such as Cdc42 and PIP2, and main ROCK activator-RhoA. Parallels between pseudopod-like blebbing and motility blebbing may provide new insights into their underlying mechanisms

Gadolinium Modifies the Cell Membrane to Inhibit Permeabilization by Nanosecond Electric Pulses

Lanthanide ions are the only known blockers of permeabilization by electric pulses of nanosecond duration (nsEP), but the underlying mechanisms are unknown. We employed timed applications of Gd3+ before or after nsEP (600-ns, 20 kV/cm) to investigate the mechanism of inhibition, and measured the uptake of the membrane-impermeable YO-PRO-1 (YP) and propidium (Pr) dyes. Gd3+ inhibited dye uptake in a concentration-dependent manner. The inhibition of Pr uptake was always about 2-fold stronger. Gd3+ was effective when added after nsEP, as well as when it was present during nsEP exposure and removed afterward. Pores formed by nsEP in the presence of Gd3+ remained quiescent unless Gd3+ was promptly washed away. Such pores resealed (or shrunk) shortly after the wash despite the absence of Gd3+. Finally, a brief (3 s) Gd3+ perfusion was equally potent at inhibiting dye uptake when performed either immediately before or after nsEP, or early before nsEP. The persistent protective effect of Gd3+ even in its absence proves that inhibition by Gd3+ does not result from simple pore obstruction. Instead, Gd3+ causes lasting modification of the membrane, occurring promptly and irrespective of pore presence; it makes the membrane less prone to permeabilization and/or reduces the stability of electropores

Primary Pathways of Intracellular Ca2+ Mobilization by Nanosecond Pulsed Electric Field

Permeabilization of cell membranous structures by nanosecond pulsed electric field (nsPEF) triggers transient rise of cytosolic Ca2+ concentration ([Ca2+]i), which determines multifarious downstream effects. By using fast ratiometric Ca2+ imaging with Fura-2, we quantified the external Ca2+ uptake, compared it with Ca2+ release from the endoplasmic reticulum (ER), and analyzed the interplay of these processes. We utilized CHO cells which lack voltage-gated Ca2+ channels, so that the nsPEF-induced [Ca2+]i changes could be attributed primarily to electroporation. We found that a single 60-ns pulse caused fast [Ca2+]i increase by Ca2+ influx from the outside and Ca2+ efflux from the ER, with the E-field thresholds of about 9 and 19 kV/cm, respectively. Above these thresholds, the amplitude of [Ca2+]i response increased linearly by 8-10 nM per 1 kV/cm until a critical level between 200 and 300 nM of [Ca2+]i was reached. If the critical level was reached, the nsPEF-induced Ca2+ signal was amplified up to 3000 nM by engaging the physiological mechanism of Ca2+-induced Ca2+-release (CICR). The amplification was prevented by depleting Ca2+ from the ER store with 100 nM thapsigargin, as well as by blocking the ER inositol-1,4,5-trisphosphate receptors (IP3R) with 50 μM of 2-aminoethoxydiphenyl borate (2-APB). Mobilization of [Ca2+]i; by nsPEF mimicked native Ca2+ signaling, but without preceding activation of plasma membrane receptors or channels. NsPEF stimulation may serve as a unique method to mobilize [Ca2+]i and activate downstream cascades while bypassing the plasma membrane receptors. (C) 2012 Elsevier B.V. All rights reserved.

Electroporation by Subnanosecond Pulses

Electropermeabilization of cell membranes by micro- and nanosecond-duration stimuli has been studied extensively, whereas effects of picosecond electric pulses (psEP) remain essentially unexplored. We utilized whole-cell patch clamp and Di-8-ANEPPS voltage-sensitive dye measurements to characterize plasma membrane effects of 500 ps stimuli in rat hippocampal neurons (RHN), NG108, and CHO cells. Even a single 500-ps pulse at 190kV/cm increased membrane conductance and depolarized cells. These effects were augmented by applying brief psEP bursts (5–125 pulses), whereas the rate of pulse delivery (8Hz–1kHz) played little role. psEP-treated cells displayed large inward current at negative membrane potentials but modest or no conductance changes at positive potentials. A 1-kHz burst of 25 pulses increased the whole-cell conductance in the range (−100)–(−60) mV to 22–26nS in RHN and NG108 cells (from 3 and 0.7 nS, respectively), but only to 5 nS in CHO (from 0.3nS). The conductance increase was reversible within about 2min. Such pattern of cell permeabilization, with characteristic inward rectification and slow recovery, was similar to earlier reported effects of 60- and 600-ns pulses, pointing to the similarity of structural membrane rearrangements in spite of a different membrane charging mechanism

Damage-Free Peripheral Nerve Stimulation by 12-ns Pulsed Electric Field

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 mus, 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

Facilitation of Electroporative Drug Uptake and Cell Killing by Electrosensitization

Cell permeabilization by electric pulses (EP), or electroporation, is widely used for intracellular delivery of drugs and plasmids, as well as for tumour and tissue ablation. We found that cells pre-treated with 100-mus EP develop delayed hypersensitivity to subsequent EP applications. Sensitizing B16 and CHO cells by splitting a single train of eight 100-mus EP into two trains of four EP each (with 5-min. interval) decreased the LD(50) 1.5-2 times. Sensitization profoundly enhanced the electroporation-assisted uptake of bleomycin, a cell-impermeable cytotoxic agent accepted for killing tumours by electrochemotherapy. EP exposures that were not lethal per se caused cell death in the presence of bleomycin and proportionally to its concentration. Sensitizing cells by a split-dose EP exposure increased bleomycin-mediated lethality to the same extent as a 10-fold increase in bleomycin concentration when using a single EP dose. Likewise, sensitization by a split-dose EP exposure (without changing the overall dose, pulse number, or amplitude) enhanced the electroporative uptake of propidium up to fivefold. Enhancement of the electroporative uptake appears a key mechanism of electrosensitization and may benefit electrochemotherapy and numerous applications that employ EP for cell permeabilization

Four Channel 6.5 kV, 65 A, 100 ns-100 μs Generator with Advanced Control of Pulse and Burst Protocols for Biomedical and Biotechnological Applications

Pulsed electric fields in the sub-microsecond range are being increasingly used in biomedical and biotechnology applications, where the demand for high-voltage and high-frequency pulse generators with enhanced performance and pulse flexibility is pushing the limits of pulse power solid state technology. In the scope of this article, a new pulsed generator, which includes four independent MOSFET based Marx modulators, operating individually or combined, controlled from a computer user interface, is described. The generator is capable of applying different pulse shapes, from unipolar to bipolar pulses into biological loads, in symmetric and asymmetric modes, with voltages up to 6.5 kV and currents up to 65 A, in pulse widths from 100 ns to 100 µs, including short-circuit protection, current and voltage monitoring. This new scientific tool can open new research possibility due to the flexibility it provides in pulse generation, particularly in adjusting pulse width, polarity, and amplitude from pulse-to-pulse. It also permits operating in burst mode up to 5 MHz in four independent channels, for example in the application of synchronized asymmetric bipolar pulses, which is shown together with other characteristics of the generator

Electroporation safety factor of 300 nanosecond and 10 millisecond defibrillation in Langendorff-perfused rabbit hearts

AIMS: Recently, a new defibrillation modality using nanosecond pulses was shown to be effective at much lower energies than conventional 10 millisecond monophasic shocks in ex vivo experiments. Here we compare the safety factors of 300 nanosecond and 10 millisecond shocks to assess the safety of nanosecond defibrillation. METHODS AND RESULTS: The safety factor, i.e. the ratio of median effective doses (ED50) for electroporative damage and defibrillation, was assessed for nanosecond and conventional (millisecond) defibrillation shocks in Langendorff-perfused New Zealand white rabbit hearts. In order to allow for multiple shock applications in a single heart, a pair of needle electrodes was used to apply shocks of varying voltage. Propidium iodide (PI) staining at the surface of the heart showed that nanosecond shocks had a slightly lower safety factor (6.50) than millisecond shocks (8.69), p = 0.02; while PI staining cross-sections in the electrode plane showed no significant difference (5.38 for 300 ns shocks and 6.29 for 10 ms shocks, p = 0.22). CONCLUSIONS: In Langendorff-perfused rabbit hearts, nanosecond defibrillation has a similar safety factor as millisecond defibrillation, between 5 and 9, suggesting that nanosecond defibrillation can be performed safely