Next generation CANCAN focusing for remote stimulation by nanosecond electric pulses.

Focusing electric pulse effects away from electrodes is a challenge because the electric field weakens with distance. Previously we introduced a remote focusing method based on bipolar cancellation, a phenomenon of low efficiency of bipolar nanosecond electric pulses (nsEP). Superpositioning two bipolar nsEP into a unipolar pulse canceled bipolar cancellation ("CANCAN" effect), enhancing bioeffects at a distance despite the electric field weakening. Here, we introduce the next generation (NG) CANCAN focusing with unipolar nsEP packets designed to produce bipolar waveforms near electrodes (suppressing electroporation) but not at the remote target. NG-CANCAN was tested in CHO cell monolayers using a quadrupole electrode array and labeling electroporated cells with YO-PRO-1 dye. We routinely achieved 1.5-2 times stronger electroporation in the center of the quadrupole than near electrodes, despite a 3-4-fold field attenuation. With the array lifted 1-2 mm above the monolayer (imitating a 3D treatment), the remote effect was enhanced up to 6-fold. We analyzed the role of nsEP number, amplitude, rotation, and inter-pulse delay, and showed how remote focusing is enhanced when re-created bipolar waveforms exhibit stronger cancellation. Advantages of NG-CANCAN include the exceptional versatility of designing pulse packets and easy remote focusing using an off-the-shelf 4-channel nsEP generator.

Sub-MHz bursts of nanosecond pulses excite neurons at paradoxically low electric field thresholds without membrane damage.

Neuromodulation applications of nanosecond electric pulses (nsEP) are hindered by their low potency to elicit action potentials in neurons. Excitation by a single nsEP requires a strong electric field which injures neurons by electroporation. We bypassed the high electric field requirement by replacing single nsEP stimuli with high-frequency brief nsEP bursts. In hippocampal neurons, excitation thresholds progressively decreased at nsEP frequencies above 20-200 kHz, with up to 20-30-fold reduction at sub-MHz and MHz rates. For a fixed burst duration, thresholds were determined by the duty cycle, irrespective of the specific nsEP duration, rate, or number of pulses per burst. For 100-µs bursts of 100-, 400-, or 800-ns pulses, the threshold decreased as a power function when the duty cycle exceeded 3-5 %. nsEP bursts were compared with single "long" pulses whose duration and amplitude matched the duration and the time-average amplitude of the burst. Such pulses deliver the same electric charge as bursts, within the same time interval. High-frequency nsEP bursts excited neurons at the time-average electric field 2-3 times below the threshold for a single long pulse. For example, the excitation threshold of 139 ± 14 V/cm for a single 100-µs pulse decreased to 57 ± 8 V/cm for a 100-µs burst of 100-ns, 0.25-MHz pulses (p < 0.001). Applying nsEP in bursts reduced or prevented the loss of excitability in multiple stimulation attempts. Stimulation by high-frequency nsEP bursts is a powerful novel approach to excite neurons at paradoxically low electric charge while also avoiding the electroporative membrane damage.

Peculiarities of Neurostimulation by Intense Nanosecond Pulsed Electric Fields: How to Avoid Firing in Peripheral Nerve Fibers.

Intense pulsed electric fields (PEF) are a novel modality for the efficient and targeted ablation of tumors by electroporation. The major adverse side effects of PEF therapies are strong involuntary muscle contractions and pain. Nanosecond-range PEF (nsPEF) are less efficient at neurostimulation and can be employed to minimize such side effects. We quantified the impact of the electrode configuration, PEF strength (up to 20 kV/cm), repetition rate (up to 3 MHz), bi- and triphasic pulse shapes, and pulse duration (down to 10 ns) on eliciting compound action potentials (CAPs) in nerve fibers. The excitation thresholds for single unipolar but not bipolar stimuli followed the classic strength-duration dependence. The addition of the opposite polarity phase for nsPEF increased the excitation threshold, with symmetrical bipolar nsPEF being the least efficient. Stimulation by nsPEF bursts decreased the excitation threshold as a power function above a critical duty cycle of 0.1%. The threshold reduction was much weaker for symmetrical bipolar nsPEF. Supramaximal stimulation by high-rate nsPEF bursts elicited only a single CAP as long as the burst duration did not exceed the nerve refractory period. Such brief bursts of bipolar nsPEF could be the best choice to minimize neuromuscular stimulation in ablation therapies.

Interference targeting of bipolar nanosecond electric pulses for spatially focused electroporation, electrostimulation, and tissue ablation.

Stimulation and electroporation by nanosecond electric pulses (nsEP) are distinguished by a phenomenon of bipolar cancellation, which stands for a reduced efficiency of bipolar pulses compared to unipolar ones. When two pairs of stimulating electrodes are arrayed in a quadrupole, bipolar cancellation inhibits nsEP effects near the electrodes, where the electric field is the strongest. Two properly shaped and synchronized bipolar nsEP overlay into a unipolar pulse towards the center of the electrode array, thus canceling the bipolar cancellation (a "CANCAN effect"). High efficiency of the re-created unipolar nsEP outweighs the weakening of the electric field with distance and focuses nsEP effects to the center. In monolayers of CHO, BPAE, and HEK cells, CANCAN effect achieved by the interference of two bipolar nsEP enhanced electroporation up to tenfold, with a peak at the quadrupole center. Introducing a time interval between bipolar nsEP prevented the formation of a unipolar pulse and eliminated the CANCAN effect. Strong electroporation by CANCAN stimuli killed cells over the entire area encompassed by the electrodes, whereas the time-separated pulses caused ablation only in the strongest electric field near the electrodes. The CANCAN approach is promising for uniform tumor ablation and stimulation targeting away from electrodes.