Irreversible electroporation (IRE) is one of the newer minimally-invasive tissue ablation modalities that holds considerable promise for focal treatment of solid organ malignancies.^1–5 In fact, great enthusiasm has been generated by IRE because due to its non-thermal nature. It overcomes many of the limitations associated with more conventional thermal minimally-invasive tumor ablation technologies such as radiofrequency ablation, cryoablation, and microwave ablation.⁶ Besides benefits such as short procedure times, ablation without being affected by the heat-sink phenomenon, and being amenable to real-time monitoring with ultrasound; the most notable advantage of IRE lies in its capability to effectively destroy tumor cells while sparing the extracellular matrix and vital structures (e.g., blood vessel and bile ducts) which allows for rapid regeneration of normal tissue with minimal to no scarring.^1–13

However, because IRE relies on creation of highly intense electrical fields across the cell membrane for destruction of tumor cells, high voltage pulses need to be generated and applied to the target tissue by means of inserting conducive electrodes.¹⁴ Originally, IRE was performed using 2,000- to 3,000-volt pulses at a high rate of 240 consecutive pulses per minute (PPM). This protocol elicited some safety concerns in regard to application of IRE for treatment of tumors located near the heart (e.g., perihilar masses, lung mass or hepatic tumors at the dome of the liver) because such strong electrical pulses could potentially cause arrhythmias such as ventricular flutter and fibrillation; especially when the pulses were delivered during the “vulnerable period” of the heart cycle or concurrent with underlying arrhythmias.^15–18

To address this safety issue and reduce the risk of arrhythmia in IRE ablation near the heart or in patients with structural or functional heart problems, recent IRE ablation has been performed during the synchronization with electrocardiogram (ECG) tracings and delivered only during the “invulnerable phase” of the cardiac cycle.^15–18 To achieve this, Mali et al¹⁵ developed an algorithm based on early detection of the QRS complex and delivery of IRE pulses in the interval between the QR junction and R wave peak; which is prior to the vulnerable period and the safest time for pulse delivery. This method was also shown to be an effective and reliable means for preventing pulse application in case of abnormalities in the heart rate.^15,16 Subsequently, several clinical investigations on human liver, lung, prostate and kidney showed that this strategy significantly reduces the risk of IRE-induced arrhythmia.^18–25

However, it is still without clear evidence whether the synchronization compromises the efficacy of IRE in cell death or is capable of inducing complete cell death in the ablation zone. In this study, we evaluated the ablation efficiency of ECG-synchronized IRE (esIRE) and compared its efficacy to non-synchronized IRE (nsIRE) ablation in normal swine liver.

Animal care

All animals were studied under the supervision of the Division of Laboratory Animal Medicine at our institution. All animals received appropriate humane care in compliance with guidelines set by National Institutes of Health. All experiments were performed after the study protocol was approved by our institution’s Ethical and Animal Research Committees (ARC 2006-054-41A).

Irreversible electroporation of liver

A total of five Yorkshire swine underwent IRE ablation of liver using an IRE pulse generator (NanoKnife®; Angiodynamics), single bipolar or two monopolar IRE electrodes, and an ECG trigger monitor. Six ablations (3 in right lobe using monopolar probes and 3 in left lobe using bipolar probes) were performed in each animal under ultrasound guidance (LOGIQ P6; GE Company). The probe was purposely placed closer to the heart in the central upper part of the each lobe. During ablation, continuous ECG monitoring was performed in all animals. Ablations were performed according to 1 of the following 3 protocols. In the nsIRE group, IRE pulses were administered at a conventional, non-gated rate of 240 PPM in 9 sets of 10 pulses each, with a 3-second capacitor recovery time in between each set, for a total ablation time of 47 seconds. There were 2 esIRE groups, a medium rate group in which 90 pulses were administered at 50 PPM for a total ablation time of 108 seconds, and a low rate group in which 90 pulses were administered at 20 PPM for a total ablation time of 273 seconds. In each group, either 2,250 or 3,000 V was applied per pulse, for a total of 90 pulses per ablation procedure. Table 1 summaries the specific parameters for each ablation method.

Table 1 . nsIRE vs. esIRE Ablation Characteristics.

	nsIRE		esIRE
	240 PPM		50 PPM	20 PPM
Number of ablation	12		12	6
Locations of ablation
Right lobe	6		6	3
Left lobe	6		6	3
Voltage (V)
2,250	6		6
3,000	6		6	6
Probe spacing (cm)
1.5	6		6
2.0	6		6	6
Number of pulses	90		90	90
Average ablation time (sec)	47		108	273
Cardiac dysrhythmias	Yes (5/12)		None (0/12)	None (0/6)
Post-ablation complications	None		None	None

Tissue collection and immunohistochemistry

The animals were euthanized 24 hours after the procedure with an overdose of pentobarbital sodium. The livers were harvested and sectioned transverse to the probe insertion track at 5-mm intervals (Fig. 1). Gross ablation zones were measured and photographed for comparison with ultrasound measurements obtained prior to euthanasia. The sections were fixed in 10% formalin and preserved at 4°C until further processed. Each section was then stained with Hematoxylin and Eosin for histopathological evaluation.

Figure 1. Schematic presentation of irreversible electroporation ablation zone in pathological analysis (A) and ultrasound analysis (B).

Data collection/analysis and statistical analysis

IBM SPSS v. 22 (IBM Corp.) were used for statistical analysis. Measurements of the IRE-ablated zones from histopathological specimens and US images were obtained from and compared between nsIRE and esIRE groups. Specifically, one way ANOVA and Dunnett’s t-test were used for comparison of gross measurements and US measurements of IRE ablation zones between groups. P < 0.05 was considered statistically significant.

Five animals underwent a total of 30 ablation procedures using nsIRE (n = 12), esIRE medium rate (n = 12), or esIRE low rate (n = 6) protocols.

Of note, no esIRE group animals (0%) had any significant cardiac dysrhythmia. However, five sessions (41.7%) in the nsIRE had an ECG evidence of cardiac dysrhythmia (P = 0.003, confidence interval = 13.4872 to 68.3219) which was converted to a normal sinus rhythm at the end of IRE ablation.

No significant statistical difference was observed in comparison of the mean ablation dimensions measured by ultrasound between nsIRE, esIRE medium rate, and esIRE low rate groups (P = 0.93, 240 PPM vs. 50 PPM and P = 0.89, 240 PPM vs. 20 PPM). Table 2 shows the P-values of mean dimensions of height, width, and depth in the nsIRE group compared against the esIRE medium rate and esIRE low rate groups. Again, no significant differences were observed between groups in any of these size dimensions (P > 0.05) (Fig. 2).

Table 2 . Radiological and Histological Ablation Size Correlation between esIRE and nsIRE in Three Dimensions (Unit: cm).

Parameters	Radiological correlation (ultrasound)				Pathological correlation (gross histology)
	Height	Width	Depth		Height	Width	Depth
L240	2.53	3.15	2.31		2.75	2.99	1.83
L50	2.36	3.03	2.12		3.18	3.60	2.15
L20	2.58	3.33	2.49		2.53	3.50	2.25
L240 vs. L20	0.87	0.94	0.83		0.75	0.53	0.52
L240 vs. L50	0.92	0.96	0.87		0.66	0.83	0.61
L50 vs. L20	0.55	0.55	0.96		0.72	0.62	0.70

Figure 2. Ultrasound images of nsIRE (A) and esIRE (B, C) ablation with different frequencies demonstrate identical hypoechogenic changes of immediately after IRE ablation. IRE, irreversible electroporation; PPM, pulses per minute; nsIRE, non-synchronized irreversible electroporation; esIRE, electrocardiogram-synchronized irreversible electroporation.

The comparison of the mean ablation dimensions measured in gross specimens demonstrated no significant statistical difference between nsIRE and medium and low rate esIRE groups (P = 0.55, 240 PPM vs. 50 PPM and P = 0.56, 240 PPM vs. 20 PPM). Table 2 shows P-values for the comparison of ablation zone measurements on gross pathology on the specific dimensions of height, width, and depth. No significant differences were observed between nsIRE and medium and low rate esIRE (all P > 0.05) (Fig. 3).

Figure 3. Gross specimen images of nsIRE (A) and esIRE (B, C) ablation with different frequencies demonstrate comparable hyperemic changes with preservation of vital structures such as portal vein, hepatic vein and bile ducts. IRE, irreversible electroporation; PPM, pulses per minute; nsIRE, non-synchronized irreversible electroporation; esIRE, electrocardiogram-synchronized irreversible electroporation.

The results of gross histological examination are depicted in Fig. 3. Macroscopic examination of the gross specimens of IRE-ablated tissue demonstrated well-demarcated margin of ablation. Uniform hyperemic changes were noted within the ablation zone without evidence of thermal injury. The entire hepatic morphology was grossly intact with preservation of vascular and biliary scaffoldings in all three groups. Microscopic evaluation demonstrated evidence of vascular congestion and markedly increased influx of immune cells (Fig. 4). Early evidence of cell death including karyorrhexis, karyopyknosis, and cell shrinkage are noted in all three groups. Microscopic examination confirmed well-demarcated margins of ablation, complete cell death within the ablation zone and preservation of scaffoldings of large blood vessels and bile ducts in all samples.

Figure 4. Histological images of nsIRE (A) and esIRE (B, C) ablation with different frequencies demonstrate comparable (1) post-ablation changes of vascular congestion and markedly increased influx of immune cells, early evidence of cell death including karyorrhexis, karyopyknosis, and cell shrinkage, (2) well-demarcated margins of ablation with complete cell death within the ablation zone and (3) preservation of scaffoldings of large blood vessels and bile ducts (H&E stain, ×5). IRE, irreversible electroporation; PPM, pulses per minute; nsIRE, non-synchronized irreversible electroporation; esIRE, electrocardiogram-synchronized irreversible electroporation.

IRE is one of the newer minimally-invasive ablation technologies that has major advantages over conventional heat-based ablation techniques. Development of non-arrhythmogenic IRE protocols that conserve these favorable properties has been crucial for widespread utilization of IRE in tumor ablation. However, non-arrhythmogenic, synchronized IRE’s tumoricidal or cytocidal effects has not been completely validated yet.

In this study, we used radiological and histological analyses to show that no histopathological or radiological differences are observed between esIRE and nsIRE ablated areas. Furthermore, we demonstrated that esIRE ablation maintains its ability to create complete, focal cell death in the ablation zone while preserving tissue scaffolding and adjacent critical structures such as large blood vessels and bile ducts. The improved safety profile of esIRE in conjunction with its apparent bioequivalence in ablation efficacy demonstrated in the current study may make esIRE the technique of choice for ablation of lesions in the vicinity of the heart, or in patients with underlying conduction disease. As suggested by Mali et al,¹⁷ the esIRE group had no ECG evidence of cardiac dysrhythmias in all 18 ablations.

Our finding of equivalent ablation areas in the nsIRE and esIRE groups implies that within the 20 to 240 PPM range, the frequency of IRE pulsation may be relatively unimportant as a determinant of ablation zone extent. Furthermore, histological findings confirmed that total cellular demise within the ablation zone is preserved over these frequency ranges. In contrast, number and voltage of the applied pulses may be more significant factors in determining the magnitude of ablation. In fact, and in consistence with our results, it has been demonstrated that equivalent electroporation efficacy can be achieved by inversely varying the number and amplitude of the pulses;²⁶ indicating that these variables have a complementary effect on the target tissue. More recently, high-frequency IRE has been studied and demonstrated similar ablation efficacy, producing similar or larger ablation zone without requiring paralytic or cardiac synchronization.^27,28 These findings with our results suggest that cardiac safety of IRE can be obtained using both very slower frequency or very high frequency without compromising the ablation outcome.

It is also important to note several limitations of our study. First, given our relatively small sample size of 30 ablation procedures, it is possible that our study was statistically underpowered to detect meaningful differences in ablation zone volumes. Also, having a different number of ablation between 20 PPM esIRE vs. 50 PPM esIRE or nsIRE could introduce statistical bias. The lack of prior studies directly comparing the expanse of nsIRE- vs. esIRE-induced ablation zones made a prior estimation of sample size difficult. However, negligible absolute differences observed in ablation zones’ measurements between nsIRE and esIRE groups suggest that a statistically significant difference would be very unlikely to emerge if sample sizes are increased. Second, although histologic confirmation of complete cell death was obtained in normal tissue here, it is possible that nsIRE and esIRE protocols show different ablation efficacy when applied to a malignant lesion. Therefore, further studies using animal tumor models are necessary to confirm equivalent efficacy of nsIRE and esIRE in eradication of tumor cells. To address this concern, experiments are under way to validate our findings in a liver tumor model and test the hypothesis that esIRE is capable of complete tumor cell death as an up-and-coming tool for tumor ablation by interventional radiologists.

In this study, we used radiological and histological analyses to evaluate and compare the tumoricidal or cytocidal efficacy of tissue ablation in ECG-synchronized and non-synchronized IRE ablation using swine liver. In conclusion, (1) no histopathological or radiological differences were observed between esIRE and nsIRE ablated areas, (2) esIRE ablation maintains its ability to create complete, focal cell death without damaging extracellular matrix or adjacent critical structures (e.g., vessels and bile ducts) and (3) in comparison to pulse frequency, the number and voltage of the applied pulses play a more significant role in determining the extent and magnitude of cell death.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

None.

No potential conflict of interest relevant to this article was reported.