Impact of Nanosecond Pulsed Electric Fields on

Impact of Nanosecond Pulsed Electric Fields on Primary Hippocampal Neurons Caleb C. Roth1, Jason A. Payne2, Marjorie A. Kuipers2, Gary L. Thompson3, Gerald J. Wilmink2, Bennett L. Ibey2 1

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General Dynamics Information Technology, Fort Sam Houston, TX, USA Radio Frequency Bioeffects Branch, Human Effectiveness Directorate, Air Force Research Laboratory, Fort Sam Houston TX, USA 3 National Research Council, Fort Sam Houston, TX USA ABSTRACT

Cellular exposure to nanosecond pulsed electric fields (nsPEF) are believed to cause immediate creation of nanopores in the plasma membrane. These nanopores enable passage of small ions, but remain impermeable to larger molecules like propidium iodide. Previous work has shown that nanopores are stable for minutes after exposure, suggesting that formation of nanopores in excitable cells could lead to prolonged action potential inhibition. Previously, we measured the formation of nanopores in neuroblastoma cells by measuring the influx of extracellular calcium by preloading cells with Calcium Green-AM. In this work, we explored the impact of changing the width of a single nsPEF, at constant amplitude, on uptake of extracellular calcium ions by primary hippocampal neurons (PHN). Calcium Green was again used to measure the influx of extracellular calcium and FM1-43 was used to monitor changes in membrane conformation. The observed thresholds for nanopore formation in PHN by nsPEF were comparable to those measured in neuroblastoma. This work is the first study of nsPEF effects on PHN and strongly suggests that neurological inhibition by nanosecond electrical pulses is highly likely at doses well below irreversible damage. Keywords: Nanopores, nanosecond electrical pulses, membrane damage, primary hippocampal neurons, calcium green, FM1-43

1. INTRODUCTION Nanosecond pulsed electric fields (nsPEF) are high voltage square wave pulses with duration (τ) under 1 µs. Originally engineered for plasma formation, various biological applications of nsPEF are being pursued including electromuscular incapacitation, cancer therapy, gene transfection, and pain suppression.1-7 When applied directly to mammalian cells, multifarious effects have been observed including nuclear granulation, cellular swelling, bleb formation, and apoptosis.1,2 Recent studies have shown that when nsPEF are applied directly to cells small pores, termed nanopores, are preferentially formed in the plasma membrane.8 In contrast to classical electroporation, these nanopores are believed to have a diameter of only a few nanometers and therefore do not readily allow large molecules such as propidium iodide to enter the cell.8,9 Therefore, unlike longer duration pulses, nsPEF enable the manipulation of cellular function without a high degree of mortality. Further distinguishing nsPEF inducednanopores, many unique properties have been identified using a variety of techniques including patch clamp, fluorescent microscopy, direct ion measurement in bulk solution, and flow cytometry.10,11,12 Specifically, the direction of ion flow appears predominantly inward, they open and close regularly at a slower rate than protein ion channels, and they have a lifetime at room temperature over many minutes.8 Interestingly, nsPEFs may provide a unique technique for controlling the activity of neurons within deep tissue by causing either stimulation (at low dose) or inhibition (at high dose). Previous work by Rogers has shown that low voltage nsPEFs can trigger action potentials (AP) leading to contractions in isolated frog muscle. 13 It was believed that nsPEF can cause AP generation by activating the release of acetylcholine into the synaptic cleft.6 Jiang and Cooper demonstrated that a single, 12 ns pulse at 403 V/cm was capable of activating skin nociceptors. They demonstrated this same effect at 100 pulses delivered at 4000 Hz with very low voltages (16.7 V/cm) without any Photonic Therapeutics and Diagnostics VIII, edited by Nikiforos Kollias, et al., Proc. of SPIE Vol. 8207, 820763 © 2012 SPIE · CCC code: 1605-7422/12/$18 · doi: 10.1117/12.911802

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observable loss of neuron function.14 Vernier et al. have shown that neuro-secretory chromaffin cells, when exposed to a 4 ns 4 MV/m pulse, uptake calcium and release catecholamines.15 This data suggests that directed nsPEF stimulation of adrenal glands could cause controlled catecholamine release. It has been hypothesized that higher voltage nsPEF exposure resulting in the formation of stable nanopores will inhibit AP by depolarizing the plasma membrane. Previous work in non-excitable cell lines using patch clamp has shown prolonged depolarization of the plasma membrane at high doses.16 Modeling work suggests that at higher voltages (100 kV/cm at 10 ns or 2 kV/cm at 600 ns) nsPEFs can cause the inhibition of AP by forming a conductance block.6 If proven effective, nsPEF delivery may provide researchers with a noncontact, nonchemical, nondestructive, and scalable technique to cause both stimulation and inhibition of AP in nerves.9 This paper describes experiments performed on isolated PHN to determine the thresholds for nanopore formation following nsPEF exposure. We monitored both the change in membrane symmetry and the movement of ions across the plasma membrane. Changes in membrane asymmetry were monitored with FM1-43 dye. FM1-43 is a fluorescent molecule that integrates into phospholipid membrane and increases its quantum efficiency causing an observable increase in fluorescent emission. It is commonly used to detect/track vesicle migration in neurons by identifying the arrival of a new membrane at the surface of the plasma membrane.17 Previous work has shown that the emission of FM1-43 greatly increases in the plasma membrane following exposure to nsPEF.15,18 Although the reason for this change remains unknown, various explanations have been proposed including induced exocytosis of vesicles, later diffusion of phospholipids through nanopores, or induced changes in membrane arrangement.19 In addition to detecting changes in membrane symmetry, the tracking of ion movements into a cell have been used in detecting nanopore formation. Pakhomov et al. showed that cells exposed to nsPEF will immediately uptake thallium ions (similar size as calcium) in the presence of a channel blocking cocktail.8 Previous work by our laboratory and others has shown that when nsPEFs are applied to a cell a dramatic increase in intercellular calcium occurs immediately after exposure. These results suggest that the burst of intracellular calcium is likely caused by passive ion diffusion through nanopores created during stimulation.8,20,21 Other studies have shown positive activation of L-type calcium channels and disruption of intracellular membranes, which may also cause the observed increase in intracellular calcium. In this paper, we use calcium uptake and changes in plasma membrane symmetry as an experimental endpoint to mark the formation of nanopores. This endpoint was chosen because of the physiological relevance of calcium uptake and its critical role in AP propagation. We believe that determination of thresholds for calcium uptake and changes in membrane asymmetry will correlate strongly with thresholds for AP inhibition in PHN.

2. MATERIALS AND METHODS 2.1 Primary Hippocampal Neuron Culture To promote growth and adhesion, glass bottom 35 mm culture dishes (Cat# P35G-1.5-20C, MatTek Corp., Ashland, MA) were coated with 50 µg/mL concentration of poly-D-lysine (Sigma, St. Louis, MO) solution for approximately 24 hr at 0.15 mL/cm2. Prior to use, the dishes were rinsed with sterile 18 MΩ deionized water and dried. PHN prepared from embryonic day 18 (E18) rat hippocampi tissues were acquired from a commercial vendor (Brainbits LLC, Springfield IL). To culture PHN, the cells were dissociated from the tissue by repeated triturating with 1 mL pipette tip. Following a resting period, the supernatant, containing dispersed cells, was transferred to a 15 mL tube, spun at 200 G for 1 min, and removed. The remaining cell pellet was resuspended with 1 mL of NbActiv1™ (Brainbits LLC, IL) with 25 µM glutamate (Sigma, MO). Cells were plated at approximately 16 x 10 3 cells/cm2, and incubated in 37°C, 5% CO2. After 4 days, one half of the medium was exchanged with fresh, warm NbActiv4® (Brainbits LLC, IL) medium with 25 µM glutamate. This process was repeated every 3 to 5 days throughout the culture lifetime. 2.2 Fluorescent Staining Plated neurons were washed twice with calcium and magnesium free PBS. Outside buffer comprised of MgCl2 at 2 mM, KCl at 5 mM, HEPES at 10 mM, Glucose at 10 mM CaCl2 at 2 mM, and NaCl at 135 mM was prepared and 2mL was added to culture dish containing the PHN. The buffer was adjusted to an osmolarity of 290-310 mOsm and a pH of 7.4. To stain the cells for calcium measurements, 2 μL of 3 mM Calcium Green 1-AM (CaGr) ester was


added to the solution and incubated at room temperature for 30 minutes to allow for cellular uptake. To stain for membrane conformation changes, 12.2 μL FM1-43 (T-3163, Molecular Probes, Invitrogen, Eugene, OR) was added to the solution and gently mixed for 5 minutes. Propidium iodide (4 mM) was added to the buffer solution prior to imaging to mark dead or injured cells, which were avoided. Additionally, this dye was monitored before and after nsPEF exposure. Following the loading of the fluorescent dye, the dish was placed onto an inverted Zeiss 710 LSM Confocal (Carl Zeiss MicroImaging GmbH, Germany) microscope. All experiments outlined in this paper were performed at room temperature, roughly 24-26ºC. 2.3 Exposure and Imaging

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High voltage pulses with discrete pulse widths of 600, 400, 200, 60, 30, and 10 ns were delivered to the cells via a custom pulser built by Old Dominion University. The exposure configuration has been described in depth in previous publications.22 In short, we utilized a Stanford DG535 (Stanford Research Systems, Sunnyvale, CA) digital delay generator to trigger the microscope to begin image acquisition. After a preset delay, a signal is sent to the HP 8112A pulse generator which then triggers the nanosecond electrical pulses (Figure 1). The microscope is programmed to acquire 1, 40x image per second (512x512) for 30 seconds (90 total images 30 T-PMT, 30 FL1, and 30 FL2). A delay of 5 seconds is set before a single nsPEF is triggered giving a baseline level of fluorescence prior to exposure. Characteristics of the nanosecond pulse (pulse width and amplitude) are monitored on a Tektronix TDS3052 500-MHz oscilloscope (Tektronix Inc, Beaverton, OR) for each exposure to ensure accurate pulse delivery. Pulses were delivered to the cells using a custom micro-electric probe comprised of two 50 µm diameter tungsten electrodes positioned in parallel with a gap spacing of approximately 100 µm.

Figure 1. Custom nsPEF exposure system setup for microscopy.

2.4 Modeling An accurate calculation of electric field values was crucial for analyzing the dose-response relationship of the cells to high peak power pulses. Finite Difference Time Domain (FDTD) was used to calculate the electric field amplitude delivered to the cells. The model was constructed by assuming a homogeneous 0.9% saline solution (permittivity 75.3, conductivity 1.55 S/m), and a pair of electrodes modeled as perfect conductors (50 µm diameter, 120 µm separation) placed 50 µm above the 180 µm thick glass coverslip (permittivity 3.8, conductivity 0 S/m) at a 42o angle.23,24 A voxel size of 2 x 2 x 2 µm was chosen with a time-step of 3.84 x 10-15 s as required by courant stability criteria. A long trapezoidal waveform consisted of a 0.5 ns duration linear ramp followed by a DC component for the remainder of the simulation and was truncated once all field values reached a steady-state. The resulting field values should therefore be regarded as quasi-static and represent the fields incident upon the cells during the DC portion of the nanosecond pulse. 2.5 Data Analysis A numerical value for each fluorescent channel was extrapolated from the virtual stack of images (cells body) for each frame using Image J software.25 Fluorescent intensity change for the CaGr response was calculated as a percent change from the average of the 3 frames prior to exposure to the average of 3 frames taken 2 seconds after exposure (peak response) to avoid the deleterious effect of photobleaching. For FM1-43, due to the more transient response of the dye, three baseline images were averaged, and the final 3 images (30 seconds post) were averaged.


Representative calcium images and a temporal response of both FM1-43 and CaGr following nsEP exposure is depicted in Figure 2. To quantify the electric field amplitude for each cell, a transparent 2-D image of the FDTD model described above was overlaid on each confocal image, and an approximate E-field was assigned to each cell based upon its position between the electrodes.

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Time (seconds) Figure 2. Images represent the pre (top) and post (bottom) nsPEF exposure in CaGr labeled PHN. The graph displays a representative temporal response of FM1-43 and CaGr in PHN following a 600 ns nsPEF at 16.2 kV/cm. Error bars represent the s.e. of the mean fluorescence change for 8-10 cells.

The raw fluorescent data was processed into a binary dataset by setting a threshold at double the highest shamexposed change observed (5%). Probit analysis was chosen due to the inherent variability in fluorescent response of cells at or near the threshold exposure point where both positive and no responses can be seen. In direct analysis, this type of variability can lead to large population variation and inaccurate estimation of exposure thresholds. The binary data was fed into a Probit model and an estimated ED50 (point at which 50% of cells would be expected to show a 5% increase in fluorescence) was calculated.

3. RESULTS All available discrete pulse widths (600, 400, 200, 60, 30, and 10 ns) were tested and compared to sham exposures at the maximum charging voltage of the power supply, 999 V (16.2 kV/cm electric field at the cell). Probit analysis of the raw data for CaGr predicted a pulse width ED50 of 64 ns (Figure 3). This data compares very well with neuroblastoma (NG-108) data collected that showed a predicted pulse width ED50 for CaGr of 44 ns.22 At the longer pulse widths (600 and 400), 100% of cells responded. At 10 ns, we had 100% of cells not responding. At 60 ns pulse width, roughly 40% of cells responded positively. The upper fiducial limits (UFL) and lower fiducial limits (LFL) of the ED50 prediction were 20 ns. To determine if nsPEF caused any measureable intracellular calcium release, calcium was omitted from the recipe and any residual calcium was chelated by the addition of 2 mM KEGTA. The grey circles in Figure 3 show the results of nsPEF in the absence of extracellular calcium. This lack of a response does not rule out the possible release of intracellular calcium from mitochondria or endoplasmic reticulum, but shows that such a release was not observed using our system. Therefore, we conclude that the calcium rises observed within the cells following nsPEF are indeed the influx of calcium from the extracellular solution. Although this supports the hypothesis that nanopores were formed in the plasma membrane, we cannot conclude that ion channels are not involved from this data set.


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Pulse Width (nsec) 1 Pulse, 16.2 kV/cm Figure 3. Relative percent increase in the fluorescent levels of CaGr dye 5 seconds after a single pulse exposure at 16.2 kV/cm at varying pulse widths from 600-10 ns (left). Resultant prediction curve from Probit analysis and the corresponding FL predictions (right). Error bars represent the s.e. of the mean fluorescent change of 10-20 cells. Pulse Width (nsec) 1 Pulse, Amplitude 999V

FM1-43 was used to show a change in membrane confirmation following nsPEF as had been previously described.18 Figure 4 shows the fluorescent change observed for 10-20 cells exposed to nsPEF at each pulse width. This data shows a rather small response at 30 and 60 ns, followed by an amplified response at longer pulse widths. This suggests that longer pulse widths are indeed increasing the changes monitored in the membrane by this dye. Although less obvious, this appears to mirror that seen in the CaGr raw data. Probit analysis of the raw data for FM1-43 predicted a pulse width ED50 of 65 ns. This data matches well with the CaGr data generated within this same cell type (pulse width ED50 of 64 ns). For this dataset, the LFL was 50.9 ns and the UFL was 89.6 ns, overlapping that seen with CaGr. 60

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