Characteristics and time course of acute and chronic myocardial lesion formation after electroporation ablation in the porcine model

Electroporation ablation creates deep and wide myocardial lesions. No data are available on time course and characteristics of acute lesion formation.


| INTRODUCTION
Electroporation ablation is a highly promising ablation modality for catheter ablation in patients with cardiac arrhythmias. In our previous animal studies, large myocardial lesions could be created safely and effectively. [1][2][3][4] However, the time course and characteristics of lesion formation, highly relevant for the electrophysiological endpoint and waiting period after catheter ablation, has not been studied yet. The purpose of the present study is to investigate the development of myocardial lesions during the first 60 min after epicardial electroporation ablation, after 3 weeks and 3 months follow-up.

| METHODS
All pigs in the present study were also used in other (main) studies, performed after prior approval from the Animal Experimentation Committee of Utrecht University, and in compliance with the Guide for Care and Use of Laboratory Animals. Ablation lesions that were analyzed for the present study were either created directly before scheduled killing (acute phase) or collected as part of the treatment protocol (chronic phase).
Anesthesia was induced with 4 mg/kg thiopental sodium. During the rest of the procedure, .5 mg/kg/h midazolam, 2.5 mg/kg/h sufentanil, and .1 mg/kg/h pancuronium bromide were administered.

| Creation and collection of lesion samples
Lesions obtained ≤60 min after ablation were created especially for the present study at 0, 10, 20, 30, 40, 50, and 60 min before killing.
After medial sternotomy, epicardial ablation was performed with a custom-made linear suction device, which has been used in previous studies. 1,2,5 In short, the custom linear suction device comprised a 35 mm-long and 6 mm-wide linear noninsulated electrode inside a 42 mm-long and 7 mm-wide insulated plastic suction cup. The suction device was sucked with a constant vacuum of 50-60 cm H 2 O on the epicardium. The constant underpressure ensured a good and stable electrode-to-myocardium contact. After opening the chest, the suction device was positioned on the epicardial side of the right ventricle (RV), perpendicular to the left anterior descending artery. Coronary arteries were not targeted.
A single cathodal 200 J application was delivered at four different nonoverlapping sites between the RV base and RV apex. In each of the seven pigs, energy was delivered at four different time points in accordance with a predefined tight time schedule comprising fixed time intervals in the 60 min before killing. This schedule resulted in a set of four epicardial lesions for every predefined 10 min time interval (Table 1).
For investigation of myocardial lesion development in the chronic phase, lesion samples were selected from our collection of samples taken 3 weeks and 3 months after electroporation ablation. [1][2][3][4] These lesions had been created on the epicardial side of the left ventricle, either with a circular multi-electrode catheter under at least 1 cm of blood, inside the pericardial space using a 12 mm diameter circular catheter, or using a linear suction device.

| Energy delivery
The ablation energy was generated using a monophasic external defibrillator (Lifepak 9, Physio-Control, Inc.). A large skin patch (7506, Valleylab, Inc.) on the lower back of the animal served as an indifferent electrode. A single, 6 ms cathodal application was delivered.
A cathodal shock polarity was chosen, because that has the highest threshold for arcing in a blood environment. All lesions <60 min had been created using a single 200 J application. The other lesions had been created with single applications ranging from 30 to 200 J.
Voltage and current waveforms of all applications were recorded as previously described. 4

| Histological evaluation
All animals were killed by exsanguination. The heart was removed en bloc, the area with ablation lesions was excised integrally, and lesions were separated and fixed in formalin. After fixation, 3-4 mm-thick segments were taken. All paraffin-embedded segments were sectioned and stained using hematoxylin-eosin and/or Elastic-van Gieson (EvG).

| Inspection of all lesions
Visual inspection of the ablation area and the electrodes directly after the energy application never revealed any perforation, blood clots, or charring. There were no differences between the ablation devices.
The suction device did cause some local epicardial hematoma due to bursting of superficial tiny epicardial blood vessels ( Figure 1).
After every energy application, a light purplish colorization around the bruised area was visible. In addition, in contrast to the adjacent unaffected myocardium, the ablated area did not contract during ventricular systole. A sharp zone between lesion and undamaged myocardium could be observed, as a cross-section of the lesion was obtained ( Figure 2).

| Acute phase lesion histology (<60 min)
Lesions were readily identified from normal myocardium. In comparison with control myocardium, all sections with ablated myocardium showed interstitial edema, recognizable as empty space between cardiomyocytes and also contraction band necrosis ( Figure 3A-D). All lesions were transmural. Other well-known characteristics of myocardial necrosis, such as nuclear condensation, cardiomyocyte swelling, inflammatory cells, and hemorrhage were not present. The histological features were present from the very beginning (t = 0 min) with no changes in the next hour. Adverse mechanical complications, such as myocardial rupture, were not noticed.
The ablated myocardium was well demarcated from the unaffected myocardium. However, extensions containing contraction bands (eosinophilic staining cross-bands reflecting cardiomyocyte hypercontraction) created an irregular border zone between lesion and unaffected myocardium ( Figure 4).
F I G U R E 1 Electroporation ablation of the right ventricular free wall: using a custom-made suction device, a single cathodal 200 J application was delivered at four sites of the right ventricle between the base and apex, perpendicularly and just aside from the left anterior descending coronary artery (green arrow). (A) The suction device brought into position for the second energy application; the first energy application was delivered 10 min earlier, more apically. (B) The situation preceding the fourth energy application from a slightly different angle, three energy applications were delivered 50, 40, and 20 min earlier, respectively. The ablated myocardium is greyish colored. A linear hematoma at previous ablation sites, due to the negative pressure of the suction device, can be identified. LV, left ventricle; RV, right ventricle. Green arrow, left anterior descending artery; white arrows, edges of the pericardium; 1, 2, 3, 4 (in blue), ablation application sites F I G U R E 2 Cross-section of the lesion in the right ventricular free wall, created with a single 200 J application, using a custom-made suction device. A sharp border zone between lesion and normal myocardium can be observed. Blue arrows, lesion at the pericardial sites; dashed green lines, demarcation of the lesion

| Chronic phase lesion histology
Lesions were clearly distinguishable from normal myocardium.

| Electrical pulses and lesion formation
Electroporation ablation provides tissue ablation without thermal damage and has been widely used for the treatment of malignant tumors. 8 Cell death is established by injury of the cell membrane due found after a few weeks, pure fibrosis with nearly inflammatory cell infiltration was found after a few months. 14 Only two older studies report on lesion formation due to lowenergy (nonarcing) DC ablation, however, not immediately after energy delivery but after 2-7 days. 15,16 These authors found that low-energy DC and high-energy DC created equal amounts of necrosis and similar lesion characteristics, supporting the hypothesis that electroporation is the main lesion mechanism rather than thermal effects and barotrauma.
Lavee et al. 17 reported the first study in which contemporary irreversible electroporation (multiple electrical pulses per energy application) was used for cardiac ablation. After 24 h, a clear demarcation between the epicardially ablated area and unaffected atrial tissue was observed. All lesions were transmural, measuring a mean depth of 0.9 cm. 17 In another study, Hong et al. 18 reported on histological features of myocardial lesions 1 h after epicardial electroporation ablation: again, demarcated lesions were observed, displaying contraction band formation and cardiomyocyte swelling surrounded by interstitial edema and hemorrhage.
In our study, myocardial lesions were visible immediately. However, histologically a sharp border between ablated and unaffected myocardium was not always found, neither in acute nor chronic lesions. Irregular penetrating extensions disturbing the border zone may represent preexisting strands of connective tissue, as they can also be detected in nonablated myocardium (Figures 5 and 6). On the other hand, concentration of these strands is clearly higher at the border zone and may also be caused by electroporation ablation.