High-frequency irreversible electroporation (H-FIRE) for non-thermal ablation without muscle contraction

Abstract

Background: Therapeutic irreversible electroporation (IRE) is an emerging technology for the non-thermal ablation of tumors. The technique involves delivering a series of unipolar electric pulses to permanently destabilize the plasma membrane of cancer cells through an increase in transmembrane potential, which leads to the development of a tissue lesion. Clinically, IRE requires the administration of paralytic agents to prevent muscle contractions during treatment that are associated with the delivery of electric pulses. This study shows that by applying high-frequency, bipolar bursts, muscle contractions can be eliminated during IRE without compromising the non-thermal mechanism of cell death.

Methods: A combination of analytical, numerical, and experimental techniques were performed to investigate high-frequency irreversible electroporation (H-FIRE). A theoretical model for determining transmembrane potential in response to arbitrary electric fields was used to identify optimal burst frequencies and amplitudes for in vivo treatments. A finite element model for predicting thermal damage based on the electric field distribution was used to design non-thermal protocols for in vivo experiments. H-FIRE was applied to the brain of rats, and muscle contractions were quantified via accelerometers placed at the cervicothoracic junction. MRI and histological evaluation was performed post-operatively to assess ablation.

Results: No visual or tactile evidence of muscle contraction was seen during H-FIRE at 250 kHz or 500 kHz, while all IRE protocols resulted in detectable muscle contractions at the cervicothoracic junction. H-FIRE produced ablative lesions in brain tissue that were characteristic in cellular morphology of non-thermal IRE treatments. Specifically, there was complete uniformity of tissue death within targeted areas, and a sharp transition zone was present between lesioned and normal brain.

Conclusions: H-FIRE is a feasible technique for non-thermal tissue ablation that eliminates muscle contractions seen in IRE treatments performed with unipolar electric pulses. Therefore, it has the potential to be performed clinically without the administration of paralytic agents.

Figure 1

Characteristic waveforms of IRE and H-FIRE with the corresponding TMP development across the plasma membrane (Φ_pm). The 1500 V/cm unipolar pulse (A) causes the TMP to rise above the critical threshold for IRE (1 V, dashed line). The 1500 V/cm bipolar burst without a delay (B) and with a delay (C) causes the TMP to oscillate around the same critical threshold.

Figure 2

Schematic diagram of the pulse generation system. The example output is a portion of the bipolar burst delivered during in vivo H-FIRE of the brain.

Figure 3

Comparison of time above the critical threshold (Φ_cr) for IRE at various center frequencies. Bipolar bursts were simulated with an electric field of 1000 V/cm and 1500 V/cm and an on-time of 20 μs. As the frequency of the applied field is increased, the time above the critical threshold diminishes. This characteristic dispersion is shifted towards higher frequencies for bursts that have a delay between the positive and negative polarity pulses. A conventional IRE pulse is depicted by the 0 kHz data point.

Figure 4

Schematic diagram of the FEM alongside the predicted electric field (E) and temperature (T) distributions in brain tissue. The 3D mesh (A) consisted of 23989 elements. An energized electrode (red) and ground electrode (black) with a 0.45 mm diameter were spaced 1 mm apart (edge-to-edge) and had an exposure length of 1 mm (not including the blunt tip). (B) The resulting electric field distribution in the x-z plane for an applied voltage of 400 V along the energized electrode. (C) The resulting temperature distribution in the x-z plane following the simulation of 180, 200 μs pulses. Upper and lower triangles in the legends depict maximums and minimums within the entire subdomain, respectively.

Figure 5

Snapshot of acceleration (a) versus time during IRE and H-FIRE treatments. Acceleration at the cervicothoracic junction was detected by the accelerometer based recording system during all IRE protocols. None of the H-FIRE protocols resulted in detectable acceleration of the cervicothoracic junction (e.g. shown, 400 V/250 kHz).

Figure 6

Peak acceleration (a) during IRE protocols averaged over the first 90 pulses. Mean peak acceleration during IRE treatments at the cervicothoracic junction for each applied voltage was significantly different from each other. H-FIRE resulted in no detectable acceleration of the cervicothoracic junction.

Figure 7

MRI appearance of H-FIRE and IRE lesions in rat brain. In all panels, IRE and H-FIRE induced lesions appear as focal hyper-intense regions (white) compared to adjacent untreated cerebrocortical tissue (gray). Top Panels (A-C) obtained from Rat #3, in which both the left and right cerebral hemispheres were treated with H-FIRE at 300 V/250 kHz and 400 V/250 kHz, respectively. Bottom Panels (D-F), Rat #4, which underwent H-FIRE in the right cerebrum at 400 V/500 kHz, and IRE at 50 V in the left cerebrum. Panels A and D, post-gadolinium T1-weighted MRI sequences in the axial plane. Panel B, post-gadolinium T1-weighted MRI sequences in the right parasagittal plane. Panels C and F, post-gadolinium T1-weighted MRI sequences in the dorsal plane. Panel D, T2-weighted MRI sequence in the transverse plane. In all panels, the right side of the brain is on the left side of the panel.

Figure 8

Histopathology of rat brain tissue. Untreated (A and B) and H-FIRE treated at 200 V/250 kHz (C and D, Rat #2, right hemisphere). Hematoxylin and eosin stain. The delineation between treated and untreated tissue is shown in Panel C (black, dotted line)

Figure 9

MRI and corresponding neuropathology of rat brain tissue lesioned with H-FIRE and IRE. Top Panels (A-C) obtained from Rat #3, in which both the right (Panel B) and left (Panel C) cerebral hemispheres were treated with H-FIRE at 400 V/250 kHz and 300 V/250 kHz, respectively. Bottom Panels (D-F), Rat #4, which underwent H-FIRE in the right cerebrum at 400 V/500 kHz (Panel E), and IRE in the left cerebrum at 50 V (Panel F). Panels A and D are the same as those presented in Figure 7A and D, with an outline of the lesions that are further represented in Panels B, C, E, and F (Hematoxylin and eosin stain, bar = 1 mm). In Panels B, C, E, and F, the delineation between treated and untreated tissue is shown (black, dotted line). In Panels A and D, the right side of the brain is on the left side of the panel.