High-frequency irreversible electroporation (H-FIRE) is a tissue ablation modality employing bursts of electrical pulses in a positive phase-interphase delay (d ₁ )-negative phase-interpulse delay (d ₂ ) pattern. Despite accumulating evidence suggesting the significance of these delays, their effects on therapeutic outcomes from clinically-relevant H-FIRE waveforms have not been studied extensively. Objective: We sought to determine whether modifications to the delays within H-FIRE bursts could yield a more desirable clinical outcome in terms of ablation volume versus extent of tissue excitation. Methods: We used a modified spatially extended nonlinear node (SENN) nerve fiber model to evaluate excitation thresholds for H-FIRE bursts with varying delays. We then calculated non-thermal tissue ablation, thermal damage, and excitation in a clinically relevant numerical model. Results: Excitation thresholds were maximized by shortening d ₁ , and extension of d ₂ up to 1,000 μs increased excitation thresholds by at least 60% versus symmetric bursts. In the ablation model, long interpulse delays lowered the effective frequency of burst waveforms, modulating field redistribution and reducing heat production. Finally, we demonstrate mathematically that variable delays allow for increased voltages and larger ablations with similar extents of excitation as symmetric waveforms. Conclusion: Interphase and interpulse delays play a significant role in outcomes resulting from H-FIRE treatment. Significance: Waveforms with short interphase delays (d ₁ ) and extended interpulse delays (d ₂ ) may improve therapeutic efficacy of H-FIRE as it emerges as a clinical tissue ablation modality.

Treatment of intracranial disorders suffers from the inability to accumulate therapeutic drug concentrations due to protection from the blood–brain barrier (BBB). Electroporation-based therapies have demonstrated the capability of permeating the BBB, but knowledge of the longevity of BBB disruption (BBBD) is limited. In this study, we quantify the temporal, high-frequency electroporation (HFE)-mediated BBBD in an in vivo healthy rat brain model. 40 male Fisher rats underwent HFE treatment; two blunt tipped monopolar electrodes were advanced into the brain and 200 bursts of HFE were delivered at a voltage-to-distance ratio of 600 V/cm. BBBD was verified with contrast enhanced T1W MRI (gadopentetate dimeglumine) and pathologically (Evans blue dye) at time points of 1, 24, 48, 72, and 96 h after HFE. Contrast enhanced T1W scans demonstrated BBBD for 1 to 72 h after HFE but intact BBB at 96 h. Histologically, tissue damage was restricted to electrode insertion tracks. BBBD was induced with minimal muscle contractions and minimal cell death attributed to HFE. Numerical modeling indicated that brief BBBD was induced with low magnitude electric fields, and BBBD duration increased with field strength. These data suggest the spatiotemporal characteristics of HFE-mediated BBBD may be modulated with the locally applied electric field.

Objective: Tissue electroporation is achieved by applying a series of electric pulses to destabilize cell membranes within the target tissue. The treatment volume is dictated by the electric field distribution, which depends on the pulse parameters and tissue type and can be readily predicted using numerical methods. These models require the relevant tissue properties to be known beforehand. This study aims to quantify electrical and thermal properties for three different tissue types relevant to current clinical electroporation. Methods: Pancreatic, brain, and liver tissue were harvested from pigs, then treated with IRE pulses in a parallel-plate configuration. Resulting current and temperature readings were used to calculate the conductivity and its temperature dependence for each tissue type. Finally, a computational model was constructed to examine the impact of differences between tissue types. Results: Baseline conductivity values (mean 0.11, 0.14, and 0.12 S/m) and temperature coefficients of conductivity (mean 2.0, 2.3, and 1.2 % per degree Celsius) were calculated for pancreas, brain, and liver, respectively. The accompanying computational models suggest field distribution and thermal damage volumes are dependent on tissue type. Conclusion: The three tissue types show similar electrical and thermal responses to IRE, though brain tissue exhibits the greatest differences. The results also show that tissue type plays a role in the expected ablation and thermal damage volumes. Significance: The conductivity and its changes due to heating are expected to have a marked impact on the ablation volume. Incorporating these tissue properties aids in the prediction and optimization of electroporation-based therapies.

Objective: Electroporation-based therapies (EBTs) employ high voltage pulsed electric fields (PEFs) to permeabilize tumor tissue; this results in changes in electrical properties detectable using electrical impedance spectroscopy (EIS). Currently, commercial potentiostats for EIS are limited by impedance spectrum acquisition time ( ~ 10 s); this timeframe is much larger than pulse periods used with EBTs ( ~ 1 s). In this study, we utilize rapid EIS techniques to develop a methodology for characterizing electroporation (EP) and thermal effects associated with high-frequency irreversible EP (H-FIRE) in real-time by monitoring inter-burst impedance changes. Methods: A charge-balanced, bipolar rectangular chirp signal is proposed for rapid EIS. Validation of rapid EIS measurements against a commercial potentiostat was conducted in potato tissue using flat-plate electrodes and thereafter for the measurement of impedance changes throughout IRE treatment. Flat-plate electrodes were then utilized to uniformly heat potato tissue; throughout high-voltage H-FIRE treatment, low-voltage inter-burst impedance measurements were used to continually monitor impedance change and to identify a frequency at which thermal effects are delineated from EP effects. Results: Inter-burst impedance measurements (1.8 kHz - 4.93 MHz) were accomplished at 216 discrete frequencies. Impedance measurements at frequencies above ~ 1 MHz served to delineate thermal and EP effects in measured impedance. Conclusion: We demonstrate rapid-capture (<; <; 1 s) EIS which enables monitoring of inter-burst impedance in real-time. For the first time, we show impedance analysis at high frequencies can delineate thermal effects from EP effects in measured impedance. Significance: The proposed waveform demonstrates the potential to perform inter-burst EIS using PEFs compatible with existing pulse generator topologies.

Objective: The use of high voltage, high frequency bipolar pulses (HFBPs) is an emerging electroporation-based therapy for the treatment of solid tumors. In this study, we quantify the extent of nonlinearity and dispersion during HFBP treatment. Methods: We utilize flat-plate electrodes to capture the impedance of porcine liver tissue during the delivery of a burst of HFBPs of widths 1μs and 2μs at different pulse amplitudes. Next, we fit the impedance data to a frequency dependent parallel RC network to determine the conductivity and permittivity of the tissue as a function of frequency, for different applied electric fields. Finally, we present a simple model to approximate the field distribution in tissue using the conductivity function at a frequency that could minimize the errors due to approximation with a nondispersive model. Results: The conductivity/permittivity of the tissue was plotted as a function of frequency for different electric fields. It was found that the extent of dispersion reduces with higher applied electric field magnitudes. Conclusion: This is the first study to quantify dispersion and nonlinearity in tissue during HFBP treatment. The data has been used to predict the field distribution in a numerical model of liver tissue utilizing two needle electrodes. Significance: The data and technique developed in this study to monitor the electrical properties of tissue during treatment can be used to generate treatment-planning models for future high frequency electroporation therapies as well as provide insights regarding treatment effect.

Background: Irreversible electroporation (IRE) has been previously investigated in preclinical trials as a treatment for intracranial malignancies. Here, we investigate next generation high-frequency irreversible electroporation (H-FIRE), as both a monotherapy and a combinatorial therapy, for the treatment of malignant gliomas.Methods: Hydrogel tissue scaffolds and numerical modeling were used to inform in-vivo H-FIRE pulsing parameters for our orthotopic tumor-bearing glioma model. Fischer rats were separated into five treatment cohorts including high-dose H-FIRE (1750V/cm), low-dose H-FIRE (600V/cm), combinatorial high-dose H-FIRE + liposomal doxorubicin, low-dose H-FIRE + liposomal doxorubicin, and standalone liposomal doxorubicin groups. Cohorts were compared against a standalone tumor-bearing sham group which received no therapeutic intervention. To further enhance the translational value of our work, we characterize the local and systemic immune responses to intracranial H-FIRE at the study timepoint.Results: The median survival for each cohort are as follows: 31 days (high-dose H-FIRE), 38 days (low-dose H-FIRE), 37.5 days (high-dose H-FIRE + liposomal doxorubicin), 27 days (low-dose H-FIRE + liposomal doxorubicin), 20 days (liposomal doxorubicin), and 26 days (sham). A statistically greater overall survival fraction was noted in the high-dose H-FIRE + liposomal doxorubicin (50%, p = 0.044), high-dose H-FIRE (28.6%, p = 0.034), and the low-dose H-FIRE (20%, p = 0.0214) compared to the sham control (0%). Compared to sham controls, brain sections of rats treated with H-FIRE demonstrated significant increases in IHC scores for CD3+ T-cells (p = 0.0014), CD79a+ B-cells (p = 0.01), IBA-1+ dendritic cells/microglia (p = 0.04), CD8+ cytotoxic T-cells (p = 0.0004), and CD86+ M1 macrophages (p = 0.01).Conclusions: H-FIRE may be used as both a monotherapy and a combinatorial therapy to improve survival in the treatment of malignant gliomas while also promoting the presence of infiltrative immune cells.