Selective effect of irreversible electroporation on parenchyma of the pancreas and its vascular structures-an in vivo experiment on a porcine model

Irreversible electroporation is a local, non-thermal ablation method, where short electrical pulses of high voltage lead to changes in cell membrane permeability and cell death. Recent experimental studies have shown that it does not lead to damage of blood vessels, nerves, bile duct or ureters. The aim of our experimental study was to evaluate the negative effect of irreversible electroporation regarding damage to the vascular wall and porcine pancreatic tissue. Irreversible electroporation of the pancreas was performed in 6 pigs after medial laparotomy. Irreversible electroporation was applied to each pig to the splenic lobe of the pancreas in order to assess damage to the pancreatic tissue and to the duodenal lobe of the pancreas to assess damage to the vascular structure of the pancreatic tissue. Higher ablation electric intensity (minimum 500 V/cm – maximum 1,750 V/cm, step 250 V/cm) in 90 μs pulses was utilized on each pig. After 7 days, macroscopic and microscopic evaluations of en bloc resected specimen (pancreas with duodenum) were performed. During 7 post-ablation days, no deaths or clinical worsening occurred in any of the pigs. Necrotic changes in the pancreatic tissue were recorded at an electric intensity of 750 V/cm. Changes in the outer layers of the wall of the arteries and veins occurred at 1,000 V/cm. Transmural vascular wall damage was not recorded in any case. Irreversible electroporation allows for relatively efficient cell death in the target tissues. Our independent experimental work confirms the safety of this method towards vascular structures located in the ablation zone. IRE, NanoKnife, ablative methods, vessel, damage Tumour destructive (ablative) methods are used in cases where the solid tumour cannot be removed with radical resection or by post resection residual tumour tissue treatment. For ablation various physical modalities are used such as the electric current, ultrasound or heat. Irreversible electroporation (IRE) is an ablation method using short electrical pulses (~ microseconds) of high voltage. Their effect leads to changes in cell membrane permeability, to the formation of small (nano) pores, and subsequently, to a disorder of cellular homeostasis and apoptosis of exposed cells (Weaver 1995). Unlike electrothermal ablation methods such as radiofrequency ablation (RFA), microwave ablation and conventional electric heating, during IRE the thermal effect of electricity is applied. The result is also not negatively affected by cooling the blood flow in large vessels situated close to the ablated tissues (heat sink effect). At the same time there should be no damage to the ligament structures of the blood vessels, nerves, bile ducts or ureters. (Rubinsky et al. 2007; Charpentier et al. 2010; Li et al. 2011). ACTA VET. BRNO 2016, 85: 133-137; doi:10.2754/avb201685020133 Address for correspondence: MUDr. Jan Hlavsa, Ph.D. Department of Surgery University Hospital and Masaryk University Brno Jihlavská 20, 625 00 Brno, Czech Republic Tel: +420 532 232 983 Email: jhlavsa@fnbrno.cz http://actavet.vfu.cz/ Thanks to the preservation of vascular stroma, faster tissue regeneration occurs with less scarring, and a positive cellular immune response at the site of ablation was also reported (Li et al. 2012). The aim of our independent in vivo experimental study was to evaluate the effect of IRE regarding damage to artery walls and pancreatic tissue in pigs. Materials and Methods We performed an experimental prospective in vivo study on a group of 6 laboratory pigs weighing 35 ± 2 kg. The experimental project (No. 51-2014) was approved by the expert committee on animal welfare of the University of Veterinary and Pharmaceutical Sciences Brno according to the law on the protection of animals against cruelty, as amended by §17, paragraph 15 g), Act no. 246/1992 Coll. The pigs were housed in stone hutches under controlled environmental conditions (20 to 22 °C room temperature, 50% to 60% relative humidity, 12 h light and 12 h dark cycle). Each animal underwent median laparotomy whereby the duodenal and splenic lobe of pancreas was exposed. To perform IRE we used the NanoKnife® device (Plate I, Fig. 1) and unipolar electrodes with a diameter of 1 mm. Two electrodes were inserted into the pancreas of each pig in the following areas: 1) The duodenal lobe (Plate I, Fig. 2) – the distance between the electrodes was 20 mm, right in the middle between the electrodes were the artery and veins at least 2 mm in diameter 2) The splenic lobe (Plate I, Fig. 3) the distance between the electrodes was 20 mm. We used 90 pulses, each with a duration of 90 μs and each pig was ablated with higher electric intensity in the steps described in Table 1.see Finally, the abdominal wall was closed in three layers. The whole procedure was carried out under general anaesthesia with deep myorelaxation, and the animals were intubated and artificially ventilated. All pigs were sedated by intramuscular administration of a mixture of tiletamine-zolazepam (2 mg/kg, Zoletil 100, Virbac, France), xylazine (0.5 mg/kg, Xylazin 2%, Riemser, Germany) and ketamine (2 mg/kg, Narketan, Vetoquinol, France). All substances were administered together in a single syringe. Anaesthesia was induced by intravenous administration of propofol (1–2 mg/kg, Propofol 1%, Fresenius, Austria) and maintained by continuous intravenous administration of propofol (0.1–0.3 mg/ kg/min). We used atracrium (1.5 mg/kg, Tracurium, GlaxoSmithKline, Italy) for myorelaxation. After surgery, the animals were awakened and pain was efficiently controlled by meloxicam (0,.2 mg/kg, Metacam 5 mg/ ml, Boehringer Ingelheim, Germany). The animals were euthanized 7 days after surgery. In each pig, an en bloc specimen of pancreas with duodenum was excised. All specimens were fixed in formalin and embedded in paraffin. Histological analysis was performed using acid fuchsin orange (AFOG), Sirius red elastic (SRel) and periodic acid-schiff (PAS) staining and evaluated by one experienced pathologist on the macroscopic and microscopic levels.

Tumour destructive (ablative) methods are used in cases where the solid tumour cannot be removed with radical resection or by post resection residual tumour tissue treatment.For ablation various physical modalities are used such as the electric current, ultrasound or heat.Irreversible electroporation (IRE) is an ablation method using short electrical pulses (~ microseconds) of high voltage.Their effect leads to changes in cell membrane permeability, to the formation of small (nano) pores, and subsequently, to a disorder of cellular homeostasis and apoptosis of exposed cells (Weaver 1995).
Unlike electrothermal ablation methods such as radiofrequency ablation (RFA), microwave ablation and conventional electric heating, during IRE the thermal effect of electricity is applied.The result is also not negatively affected by cooling the blood flow in large vessels situated close to the ablated tissues (heat sink effect).At the same time there should be no damage to the ligament structures of the blood vessels, nerves, bile ducts or ureters.(Rubinsky et al. 2007;Charpentier et al. 2010;L i et al. 2011).
Thanks to the preservation of vascular stroma, faster tissue regeneration occurs with less scarring, and a positive cellular immune response at the site of ablation was also reported (L i et al. 2012).
The aim of our independent in vivo experimental study was to evaluate the effect of IRE regarding damage to artery walls and pancreatic tissue in pigs.

Materials and Methods
We performed an experimental prospective in vivo study on a group of 6 laboratory pigs weighing 35 ± 2 kg.The experimental project (No. 51-2014) was approved by the expert committee on animal welfare of the University of Veterinary and Pharmaceutical Sciences Brno according to the law on the protection of animals against cruelty, as amended by §17, paragraph 15 g), Act no.246/1992 Coll.The pigs were housed in stone hutches under controlled environmental conditions (20 to 22 °C room temperature, 50% to 60% relative humidity, 12 h light and 12 h dark cycle).Each animal underwent median laparotomy whereby the duodenal and splenic lobe of pancreas was exposed.To perform IRE we used the NanoKnife ® device (Plate I, Fig. 1) and unipolar electrodes with a diameter of 1 mm.
Two electrodes were inserted into the pancreas of each pig in the following areas: 1) The duodenal lobe (Plate I, Fig. 2) -the distance between the electrodes was 20 mm, right in the middle between the electrodes were the artery and veins at least 2 mm in diameter 2) The splenic lobe (Plate I, Fig. 3) -the distance between the electrodes was 20 mm.
We used 90 pulses, each with a duration of 90 μs and each pig was ablated with higher electric intensity in the steps described in Table 1.seeFinally, the abdominal wall was closed in three layers.The whole procedure was carried out under general anaesthesia with deep myorelaxation, and the animals were intubated and artificially ventilated.All pigs were sedated by intramuscular administration of a mixture of tiletamine-zolazepam (2 mg/kg, Zoletil 100, Virbac, France), xylazine (0.5 mg/kg, Xylazin 2%, Riemser, Germany) and ketamine (2 mg/kg, Narketan, Vetoquinol, France).All substances were administered together in a single syringe.Anaesthesia was induced by intravenous administration of propofol (1-2 mg/kg, Propofol 1%, Fresenius, Austria) and maintained by continuous intravenous administration of propofol (0.1-0.3 mg/ kg/min).We used atracrium (1.5 mg/kg, Tracurium, GlaxoSmithKline, Italy) for myorelaxation.After surgery, the animals were awakened and pain was efficiently controlled by meloxicam (0,.2 mg/kg, Metacam 5 mg/ ml, Boehringer Ingelheim, Germany).The animals were euthanized 7 days after surgery.In each pig, an en bloc specimen of pancreas with duodenum was excised.All specimens were fixed in formalin and embedded in paraffin.Histological analysis was performed using acid fuchsin orange (AFOG), Sirius red elastic (SRel) and periodic acid-schiff (PAS) staining and evaluated by one experienced pathologist on the macroscopic and microscopic levels.

Results
During the seven days after the application of IRE on the porcine pancreas, no death or clinical worsening occurred in any of the six pigs.Depending on the electric field intensity applied, macroscopic and microscopic changes in the blood vessels and pancreatic tissue were observed which are presented in Table 2 and Figs 4a-15

Discussion
IRE is a new local electro-ablative, non-thermal method.Short electrical pulses of high voltage affect transmembrane potential, induce instability polarized phospholipid double layer, resulting in the formation of small (nano) pores in the cell membranes.Pore formation is reversible or irreversible (Freeman et al. 1994).
Table 1.Distribution of electric intensity.

Electric intensity
The effect of the reversible formation of pores (reversible electroporation) is used to facilitate the transport of substances (cytostatic, enzymes, antibodies, DNA, fluorescent or radioactive agents) into the cell (Yao et al. 2013;Teissie 2014).
During irreversible electroporation, permanent defects in cell membranes are generated, which causes the disruption of cellular homeostasis and cell death.Given that, during IRE in contrast with radiofrequency ablation (RFA), microwave ablation or conventional electric heating, the thermal effect of electric current is not applied, and the ablation result is not negatively affected by a possible cooling of the blood flowing in the vessels running near the electrodes (the heat sink effect).At the same time, there should be no damage to the ligament structures of blood vessels, nerves, ducts, bronchi or ureters (Maor et al. 2007;Philips et al. 2013;Martin 2015), (see Table 3).This ablation technique is used in human medicine for the treatment of primary and secondary tumours of the liver, kidney, lung, prostate, pancreas, eyes, and brain.(Phillips et al. 2013).For example, after IRE of local advanced pancreatic adenocarcinoma, an increase was recorded in local progression -free survival (14 vs. 6 months, P = 0.01), distant progression -free survival (15 vs. 9 months, P = 0.02), and over all survival (20 vs. 13 months, P = 0.03) compared with standard chemoradiotherapy (Martin et al. 2013).
Procedures should be performed under general anaesthesia with deep neuromuscular blockade.During the ablation itself, a different number of uni-or bi-polar electrodes placed into the target tissue is used.Electrodes can be inserted percutaneously with ultrasound or CT navigation, as well as laparoscopically or by open approach, including a combination with resection surgery (Lee et al. 2007;Martin et al. 2015).The range of ablation is dependent on the length of the electrode's active parts (0.5-4 cm), their mutual distance (0.5-4 cm), electrical intensity (1,500-3,000 V/cm), the number (70 to 90) and the duration of individual electric pulses (70-100 μs) (Silk et al. 2014).In our experiment, we used two unipolar electrodes with a diameter of 1 mm and they were spaced 20 mm.The electric intensity of ablation was a gradual increase within the range of 500-1,750 V/cm in steps of 250 V/cm.Electrodes insertion into ablated tissue may be associated with bleeding, biliary or pancreatic fistula.In the case of the ablation of pancreatic tissue pancreatitis and portal vein thrombosis have been described.The electrical pulses cause muscle contraction; deep myorelaxation is therefore necessary.There is also the risk of cardiac arrhythmias, and the frequency of the electrical pulses must be synchronized with the heart rhythm (Mali et al. 2008).In our in vivo experiment no intraoperative complications, death or clinical deterioration of used animals occurred.It should be noted that only clinical conditions (body temperature, blood pressure, heart rate, saturation, mind, abdominal and wound state) were evaluated.Because it was not the aim of the experiment, paraclinical examinations were not performed.
The IRE ablation is contraindicated in intrathoracic lesions with the presence of a pacemaker or defibrillator, in lesions near the implanted electrical devices or objects with metallic parts, in eye with ocular lens, in patients with recent myocardial infarction, cardiac arrhythmia or epilepsy (Månsson et al. 2014).
Focusing on IRE of the pancreas, the experiments with porcine models demonstrate the efficacy and safety of this method.Particularly interesting is the ability of this method to preserve intact vessels of ligamentous structures (Charpentier et al. 2010;Bower et al. 2011).In agreement with previous experiments, our independent study also did not record transmural vascular disorders.Nevertheless, changes in the outer layers of the wall artery and vein occurred at an electric intensity of 1,000 V/cm.At the same electric intensity, we also observed intraluminal venous thrombosis.Arterial thrombosis was not observed in our experiment.Necrosis of the pancreatic tissue was recorded from the electric field intensity of 750 V/cm; i.e. at a lower intensity than for which we recorded the least minimal changes of the outer layers vascular wall.
The question is, whether the tumour cells respond to IRE as well as healthy tissues.Presuming the known resistance of tumour cells to apoptotic phenomena, we expect the need to use higher energies or extension of the number and duration of pulses.This is also not an absolute declaration of the absence of thermal damage with IRE.Based on physical laws, it is known that the passage of electric current through a conductor (electrode) releases at least minimal joule heat.It has been experimentally proven that in cases of high currents, and in combination with a high number of pulses, thermal damage also occurred (> 270 pulses and voltage settings > 2,900 V) (Faroja et al. 2013;Silk et al. 2014).The behaviour of only partially ablated tumour tissue was also interesting.In an experimental model recurrent tumours had a steeper growth curve (slope = 0.73) compared to primary tumors (0.60, P = 0.02) and had a significantly higher percentage of EpCAM expression that could be more resistant to the standard care of chemotherapy (Philips et al. 2015).
In conclusion, the IRE is a new non-thermal, electro-ablative method which allows relatively efficient cell death in the target tissue.As documented in our independent experiments, energies fully sufficient to induce necrosis of the pancreatic tissue do not cause transmural damage of the vascular wall.

Table 2 .
The effect of IRE on the artery, vein, and pancreatic tissue for each electric intensity.

Table 3 .
Thermal ablative methods vs. IRE according to Martin 2015.