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

Allograft Structural Interbody Spacers Compared to PEEK Cages in Cervical Fusion: Benchtop and Clinical Evidence

Cervical degenerative disc disease (CDDD) can lead to radiculopathy and myelopathy, resulting in pain, lack of function, and immobility. Anterior cervical discectomy and fusion (ACDF) is a common surgical treatment modality for advanced CDDD. ACDF involves removal of the affected disc(s) followed by replacement with a bone or synthetic graft. Historically, autograft has been considered the gold standard for interbody fusion. However, it is often associated with limitations, including donor site morbidity and limited quality and supply, prompting surgeons to seek alternatives. Two of the most common alternatives are structural bone allografts and polyetheretherketone (PEEK) synthetic cages. Both, advantageously, have similar mechanical properties to autologous bone, with comparable elastic modulus values. However, a lack of osseointegration of PEEK cages has been reported both pre-clinically and clinically. Reported fusion rates assessed radiographically are higher with the use of structural bone allografts compared to PEEK cages, while having a lower incidence of pseudarthrosis. This book chapter will discuss in detail the pre-clinical and clinical performance of structural allografts in comparison to conventional PEEK cages

Selective Distant Electrostimulation by Synchronized Bipolar Nanosecond Pulses

A unique aspect of electrostimulation (ES) with nanosecond electric pulses (nsEP) is the inhibition of effects when the polarity is reversed. This bipolar cancellation feature makes bipolar nsEP less efficient at biostimulation than unipolar nsEP. We propose to minimize stimulation near pulse-delivering electrodes by applying bipolar nsEP, whereas the superposition of two phase-shifted bipolar nsEP from two independent sources yields a biologically-effective unipolar pulse remotely. This is accomplished by electrical compensation of all nsEP phases except the first one, resulting in the restoration of stimulation efficiency due to cancellation of bipolar cancellation (CANCAN-ES). We experimentally proved the CANCAN-ES paradigm by measuring YO-PRO-1 dye uptake in CHO-K1 cells which were permeabilized by multiphasic nsEP (600 ns per phase) from two generators; these nsEP were synchronized either to overlap into a unipolar pulse remotely from electrodes (CANCAN), or not to overlap (control). Enhancement of YO-PRO-1 entry due to CANCAN was observed in all sets of experiments and reached ~3-fold in the center of the gap between electrodes, exactly where the unipolar pulse was formed, and equaled the degree of bipolar cancellation. CANCAN-ES is promising for non-invasive deep tissue stimulation, either alone or combined with other remote stimulation techniques to improve targeting

The Cytotoxic Synergy of Nanosecond Electric Pulses and Low Temperature Leads to Apoptosis

Electroporation by nanosecond electric pulses (nsEP) is an emerging modality for tumor ablation. Here we show the efficient induction of apoptosis even by a non-toxic nsEP exposure when it is followed by a 30-min chilling on ice. This chilling itself had no impact on the survival of U-937 or HPAF-II cells, but caused more than 75% lethality in nsEP-treated cells (300 ns, 1.8-7 kV/cm, 50-700 pulses). The cell death was largely delayed by 5-23 hr and was accompanied by a 5-fold activation of caspase 3/7 (compared to nsEP without chilling) and more than 60% cleavage of poly-ADP ribose polymerase (compared to less than 5% in controls or after nsEP or chilling applied separately). When nsEP caused a transient permeabilization of 83% of cells to propidium iodide, cells placed at 37 ° C resealed in 10 min, whereas 60% of cells placed on ice remained propidium-permeable even in 30 min. The delayed membrane resealing caused cell swelling, which could be blocked by an isosmotic addition of a pore-impermeable solute (sucrose). However, the block of swelling did not prevent the delayed cell death by apoptosis. The potent enhancement of nsEP cytotoxicity by subsequent non-damaging chilling may find applications in tumor ablation therapies

Electroporation of Mammalian Cells by Nanosecond Electric Field Oscillations and it\u27s Inhibition by the Electric Field Reversal

The present study compared electroporation efficiency of bipolar and unipolar nanosecond electric field oscillations (NEFO). Bipolar NEFO was a damped sine wave with 140 ns first phase duration at 50% height; the peak amplitude of phases 2-4 decreased to 35%, 12%, and 7% of the first phase. This waveform was rectified to produce unipolar NEFO by cutting off phases 2 and 4. Membrane permeabilization was quantified in CHO and GH3 cells by uptake of a membrane integrity marker dye YO-PRO-1 (YP) and by the membrane conductance increase measured by patch clamp. For treatments with 1-20 unipolar NEFO, at 9.6-24 kV/cm, 10 Hz, the rate and amount of YP uptake were consistently 2-3-fold higher than after bipolar NEFO treatments, despite delivering less energy. However, the threshold amplitude was about 7 kV/cm for both NEFO waveforms. A single 14.4 kV/cm unipolar NEFO caused a 1.5-2 times greater increase in membrane conductance (p \u3c 0.05) than bipolar NEFO, along with a longer and less frequent recovery. The lower efficiency of bipolar NEFO was preserved in Ca2+ free conditions and thus cannot be explained by the reversal of electrophoretic flows of Ca2+. Instead, the data indicate that the electric field polarity reversals reduced the pore yield

Activation of the Phospholipid Scramblase TMEM16F by Nanosecond Pulsed Electric Field (nsPEF) Facilitates Its Diverse Cytophysiological Effects

Nanosecond pulsed electric fields (nsPEF) are emerging as a novel modality for cell stimulation and tissue ablation. However, the downstream protein effectors responsible for nsPEF bioeffects remain to be established. Here we demonstrate that nsPEF activate TMEM16F (or Anoctamin 6), a protein functioning as a Ca2+-dependent phospholipid scramblase and Ca2+-activated chloride channel. Using confocal microscopy and patch clamp recordings, we investigated the relevance of TMEM16F activation for several bioeffects triggered by nsPEF, including phosphatidylserine (PS) externalization, nanopore-conducted currents, membrane blebbing, and cell death. In HEK 293 cells treated with a single 300-ns pulse of 25.5 kV/cm, Tmem16f expression knockdown and TMEM16F-specific inhibition decreased nsPEF-induced PS exposure by 49 and 42%, respectively. Moreover, the Tmem16f silencing significantly decreased Ca2+-dependent chloride channel currents activated in response to the nanoporation. Tmem16f expression also affected nsPEF-induced cell blebbing, with only 20% of the silenced cells developing blebs compared with 53% of the control cells. This inhibition of cellular blebbing correlated with a 25% decrease in cytosolic free Ca2+ transient at 30 s after nanoporation. Finally, in TMEM16F-overexpressing cells, a train of 120 pulses (300 ns, 20 Hz, 6 kV/cm) decreased cell survival to 34% compared with 51% in control cells (*, p \u3c 0.01). Taken together, these results indicate that TMEM16F activation by nanoporation mediates and enhances the diverse cellular effects of nsPEF

Electrosensitization Increases Antitumor Effectiveness of Nanosecond Pulsed Electric Fields In Vivo

Nanosecond pulsed electric fields are emerging as a new modality for tissue and tumor ablation. We previously reported that cells exposed to pulsed electric fields develop hypersensitivity to subsequent pulsed electric field applications. This phenomenon, named electrosensitization, is evoked by splitting the pulsed electric field treatment in fractions (split-dose treatments) and causes in vitro a 2- to 3-fold increase in cytotoxicity. The aim of this study was to show the benefit of split-dose treatments for in vivo tumor ablation by nanosecond pulsed electric field. KLN 205 squamous carcinoma cells were embedded in an agarose gel or grown subcutaneously as tumors in mice. Nanosecond pulsed electric field ablations were produced using a 2-needle probe with a 6.5-mm interelectrode distance. In agarose gel, splitting a pulsed electric field dose of 300, 300-ns pulses (20 Hz, 4.4-6.4 kV) in 2 equal fractions increased cell death up to 3-fold compared to single-train treatments. We then compared the antitumor effectiveness of these treatments in vivo. At 24 hours after treatment, sensitizing tumors by a split-dose pulsed electric field exposure (150 + 150, 300-ns pulses, 20 Hz, 6.4 kV) caused a 4- and 2-fold tumor volume reduction as compared to sham and single-train treatments, respectively. Tumor volume reduction that exceeds 75% was 43% for split-dose-treated animals compared to only 12% for single-dose treatments. The difference between the 2 experimental groups remained statistically significant for at least 1 week after the treatment. The results show that electrosensitization occurs in vivo and can be exploited to assist in vivo cancer ablation