Clinics in Surgery Research Article Published: 15 Feb, 2019 Hypersaline Infusion Protocol through the Portal Vein may Focus Electroporation on Tumor Tissue, but is it really Safe? Preliminary Results Pañella C1*, Moll X2, Quesada R1, Villanueva A3, Iglesias M4, Andaluz A2, Lucia O5, Sánchez- Velázquez P1, Grande L1 and Burdío F1 1 Department of General Surgery, Hospital Del Mar Medical Research Institute (IMIM), Spain 2 Department of Animal Medical and Surgery, Autonomous University of Barcelona, Spain 3 Chemoresistance and Predictive Factors Group, Program against Cancer Therapeutic Resistance (ProCURE), Catalan Institute of Oncology (ICO), Bellvitge Biomedical Research Institute (IDIBELL), Spain 4 Department of Pathological Anatomy, Pompeu Fabra University, Spain 5 Department of Electronic Engineering and Communications, Universidad de Zaragoza, Spain Abstract Introduction: Irreversible Electroporation (IRE) is highly dependent on the electrical conductivity of the tissue and the high conductivity of tumor tissue, which leads to a lower field than in the surrounding healthy tissue. Hypersaline Infusion (HI) through the portal vein focuses IRE on scattered liver tumors, by creating a differential conductivity between the different types of tissue. The aim of this study is to determine the effects of the HI protocol on the hepatic and histological biochemical results. OPEN ACCESS Methods: Ten male Sprague Dawley rats were used for HI protocol. Blood samples were collected at *Correspondence: pre-, immediately post-, 24-hrs, 72-hrs, 1-week and 3-weeks post-HI. All the animals were sacrificed Clara Pañella, Department of General after one-month follow-up in order to collect histological samples. Surgery, Unit of Hepato-biliary Results: The mortality rate in this procedure reached 30% (3/10). Only the pH and transaminases and Pancreatic Surgery, Service at 24-hrs were significantly and directly linked to mortality (p=0.036 and p=0.004, respectively). of General and Digestive Surgery, The three non-surviving animals had a four-time higher AST level at 24-hrs. Natremia normalized Hospital Del Mar Medical Research at 24-hrs post-HI. Statistically significant differences were found in hepatic necrosis between the Institute, Passeig Marítim 25-29,08003 non-surviving (n=3) and surviving rats (n=7) (30.67 ± 10.97 vs. 2.86 ± 7.56% respectively, p=0.01). Barcelona, Spain, Tel: + 34 93 2483207/3490; Fax: + 34 93 2483433; Discussion: HI through the portal system involves a significant risk of possibly lethal cytolysis E-mail: cpvilamu@gmail.com and acidosis. Therefore, compensatory measures and a reduced saline overload are warranted to improve the survival rates. Received Date: 14 Jan 2019 Accepted Date: 11 Feb 2019 Introduction Published Date: 15 Feb 2019 Citation: Electroporation (EP) has become a popular non-thermal ablation technique because of its many applications, ranging from the cancer treatment field (irreversible electroporation-IRE) [1-4] to in Pañella C, Moll X, Quesada R, vivo and in vitro gene transfection or electrochemotherapy (reversible electroporation-RE) [5,6]. EP Villanueva A, Iglesias M, Andaluz A, applies a high-pulsed electric field to the targeted biological cells, which increases cell membrane et al. Hypersaline Infusion Protocol permeability and leads to either cell apoptosis or necrosis [4,7]. IRE has been reported to have two through the Portal Vein may Focus main advantages over other thermal ablation techniques such as radiofrequency or microwave: 1) Electrooration on Tumor Tissue, but is the results are not influenced by the so-called heat sink effect in the vicinity of large vessels, which it really Safe? Preliminary Results. Clin may help to avoid tumour recurrences, and 2) the extracellular matrix of the supportive connective Surg. 2019; 4: 2335. tissue and adjacent vital structures are spared during ablation [8]. However, IRE is also highly Copyright © 2019 Pañella C. This dependent on tissue electrical conductivity and as tumor tissue is normally highly conductive, this is an open access article distributed leads to a lower electrical field than in the surrounding healthy tissue [9]. under the Creative Commons We recently published a preliminary study in which the conductivity of the healthy tissue was Attribution License, which permits selectively increased by means of hypersaline infusion (HI) through the portal vein. In this study we unrestricted use, distribution, and found that healthy tissue conductivity could be up to 1.4 times higher (1.04 - 1.76 range) than tumor reproduction in any medium, provided tissue [9]. These different conductivities could be used to focus IRE application on scattered liver the original work is properly cited. tumors. In this regard, Qasrawi et al. [10] highlighted the impact of conductivity heterogeneities Remedy Publications LLC., | http://clinicsinsurgery.com/ 1 2019 | Volume 4 | Article 2335 Pañella C, et al., Clinics in Surgery - Gastroenterological Surgery The authors consider that HI could be a valuable therapeutic tool when used to increase the conductivity of healthy hepatic tissue in order to protect it against tumor tissue (without portal irrigation) due to its high conductivity and the fact that it is easily eliminated by the kidney. In a pioneering study [16] we assumed that the contribution of sodium (Na+) during HI is exclusively to the extracellular liquid and can determine electrolyte imbalance, such as hyponatraemia [17,18], but we did not assess HI side effects. More importantly, we did not assess the post-HI histological changes over time or the ionic disturbances it could cause. In this context, the purpose of the present study was to determine the effects of the HI protocol on the hepatic biochemical results, as well as its histological effects on the liver and other organs. Materials and Methods Figure 1: Relationship between AST at 24-hrs with the dead or alive animals, in which AST was four-times higher in dead rats. The Government of Catalonia’s Ethics Committee on Animal Research approved this study on a small animal model to analyze the electrolytic changes after HI (FBP-13-1474P2 procedure, DAAM: 7016). This animal research protocol was conducted following Directives 2010/63/EU of the European Parliament and Council of 22 September 2010, for the protection of animals for experimental and scientific purposes. Animal model Ten six-week-old male Sprague Dawley rats (Charles River Laboratories, Kingston, NY, USA) were used to carry out blood tests during and after the HI protocol. All the animals were maintained under standard conditions with the appropriate diet and water ad libitum. All the animals were supervised following Morton and Griffiths’ guidelines on the recognition of pain, distress and discomfort [19] and sacrificed one month after the HI protocol. Hypersaline infusion protocol The same HI protocol was used as previously described in [9]. Intravenous furosemide (loop diuretic, 2 mL/Kg, Seguril Sanofi®, Spain) was administered pre and post-HI protocol to compensate the Figure 2: Linear regression underlining the correlation between acidosis fluid overload. The procedure entails the administration of DiH2O at 24-hrs with cytolisis (AST at 24-hrs), hepatic necrosis and survival. A) Represents negative linear relationship between pH at 24-hrs and cytolisis (Agua para preparaciones inyectables, Grifols®, Spain) and NaCl 20% (AST 24-hrs). B) Also shows an inverse relationship between acidosis (pH (Cloruro sódico, 20% Grifols®, Spain). The analgesic regimen and at 24-hrs) and percentage of necrosis at autopsy. C) Shows the impact of antibiotic were administered according to the protocol previously acidosis on the number of days of survival (4 ± 2.65 for dead rats (n=3)). D) described. Reflects the relationship between the percentage of hepatic necrosis of dead rats and days of survival. Blood tests Each animal was given six blood tests during the protocol. The and the electric field distribution on treatment outcomes. In fact, samples pre- and immediately post-HI were obtained through the other authors have also underlined the importance of the differential jugular catheter during HI, and then at 24-hrs, 72-hrs, 1-week and electrical conductivity of the liver parenchyma and blood vessels, 3-weeks post-HI, blood samples were also collected from the lateral since vessels have higher conductivity that could have an impact on vein tail. The electrolytes (Na+ and K+), Hemoglobin (Hb), haematocrit the distributed electrical field [8,10,11]. It therefore seems possible and blood gases (HCO3−, BE, pH, pCO2, pO2 and SatO2) from all the to modify the baseline electric field around large vessels, especially animals were analyzed immediately with an i-STAT® device (Abbott when the aim is a systemic treatment over an entire area with Point of Care Inc, Princeton, NJ, USA) at pre, post-HI and at 24-hrs multiple metastatic lesions. Many studies have been based their post-HI (0.1mL). Five sixths of the samples (0.5 mL) were kept to therapies on homogenizing the local electrical field; for example, analyze liver biochemistry (aspartate aminotransferase-AST, alanine better electroporation outcomes have been obtained in cutaneous aminotransferase-ALT) and kidney function (creatinine). The 24-hrs tumors using plate electrodes between the treated tissue to increase to 3-weeks post-HI samples were kept in tubes with an anticoagulant the surface contact [12] and conductive gels have been used to (0.5 mL). The centrifuged serum was carefully removed using a homogenize the electric field for superficial cutaneous tumors [13]. pipette and kept at -4ºC for biochemical analysis after the post- However, in deeper tumors such as those found in metastatic livers surgical follow-up period. the heterogeneity of the local field has to be taken into account, and not only the shape and position of the electrodes and voltages used Histopathological samples [14,15], but also the conductivity of the tissue to be treated. Autopsies were performed on the sacrificed or dead animals. Liver, Remedy Publications LLC., | http://clinicsinsurgery.com/ 2 2019 | Volume 4 | Article 2335 Pañella C, et al., Clinics in Surgery - Gastroenterological Surgery Figure 3: Box plots present ALT and AST pre- and post-HI, 24 and 72-hours Figure 4: A) and B) represent a normal liver after HI protocol with minimum post-HI and 1 and 3-weeks post-HI. Severe hepatocytolisis was observed: vascular congestion at 4xs and 10x of surviving rats sacrificed at 30 days. ALT at 24-hrs was 2286.53 ± 2971.77 vs. 50.71 ± 54.32 UI/L at 3-weeks post- Areas of sinusoidal vascular congestion are indicated by ( ← ). C) and D) HI, p=0.018. AST at 24-hrs was 7795.33 ± 8074.62 vs. 159.29 ± 100.64 UI/L represent hepatic parenchyma with hepatocytes zonal necrosis at 4x and at 3-weeks post-HI, p=0.028. (#) indicates the most important parameters 10x of dead rat at 48-hrs post-HI (area of hepatic necrosis indicated by dotted that reached statistical significance. line). spleen, kidneys, lungs and heart were collected, 4% formalin-fixed and and p=0.004, respectively). In fact, the rats with poor evolution had paraffin-embedded. Sections of 3 μm were cut for haematoxylin-eosin four-time higher AST at 24-hrs (Figure 1), often associated with staining. The histological analyses were performed by pathologists lower pH at 24-hrs. Furthermore, a negative linear relationship was with no prior knowledge of the animals’ history. found between pH at 24-hrs and cytolysis (R2=0.61; p=0.023) (Figure 2A and Figure 3) and also in the rate of necrosis at autopsy (R2=0.5; The histological assessment procedures were the following: p=0.034) (Figure 2B). Not surprisingly, the three dead rats had preservation of parenchyma from different organs (qualitative extended hepatic necrosis and again a negative linear relationship was analysis), percentage of liver vessel congestion (semi-quantitative); observed with survival (R2=0.7; p=0.003) (Figure 2C). As expected, quantity of tissue necrosis in liver and spleen (mean percentage of 10 pH at 24-hrs was also related to survival (R2=0.79; p=0.001) (Figure fields chosen at random at 200 xs); sinusoidal obstruction syndrome 2D). or sinusoidal dilatation in liver (assessed semi-quantitatively according to the Rubbia-Brandt classification [20]. Review of other biochemical parameters Statistical analyses Despite the compensation with hyposaline infusion, the mean post-HI natremia (155.4 ± 5.02 mmol/L) was higher than preoperative All the statistics were processed by the SPSS statistical software values (p=0.005) but normalized at 24-hrs post-HI (124.63 ± 18.22 package (SPSS, Version 21, IBM, and Armonk, NY, USA) and mmol/L) and mortality was not related to natremia. On the contrary, expressed as mean ± standard deviation. post-HI kalemia did not remain stable and no relation to either The Mann-Whitney U test was used for non-parametric data to acidosis or mortality, nor there were significant signs of renal failure compare blood test results and death/sacrifice events. The Friedman (Table1). test was used for non-parametric data to analyze differences between Histopathological evaluation the blood test results over time. The Wilcoxon test for pairwise The hepatic parenchyma exhibited a normal architecture in seven comparisons was carried out on all the analytical parameters. Linear of the ten rats after the HI protocol, with polygonal hepatocytes and regression was used to correlate acidosis and hypertransaminasemia preserved portal triads. Some dispersed congestion was observed with hepatic necrosis and survival. A p-value of <0.05 was considered in vessels throughout healthy hepatic tissue, mostly in two-thirds statistically significant. of the parenchyma (Grade 2) [20]. However, the sacrificed or dead Results animals had wide areas of variously distributed hepatic coagulating necrosis and a sparsely-preserved liver architecture. The differences Cytolisis and acidosis: key elements in the postoperative in hepatic necrosis between the non-surviving (n=3) and surviving period rats (n=7) were statistically significant (30.67 ± 10.97 vs. 2.86 ± 7.56% The mortality rate in this procedure reached 30% (3/10). Two of respectively), p=0.01 (Figure 4). the ten animals were sacrificed at 72-hrs and 1-week post-HI, both due to respiratory difficulties and lethargic behavior. One animal that There was no significant damage of the kidney and lung had shown similar behavior was found dead at 48-hrs post-HI. parenchyma or myocardial tissue. From all the analytical results, only pH and transaminases at the Discussion 24-hrs were significantly and directly linked to mortality (p=0.036 IRE is a promising and relatively new technique that is considered Remedy Publications LLC., | http://clinicsinsurgery.com/ 3 2019 | Volume 4 | Article 2335 Pañella C, et al., Clinics in Surgery - Gastroenterological Surgery Table 1: Mean ± standard deviation of blood test results from 10 Sprague Dawley rats. Blood samples extracted from jugular vein for the pre and post-HI protocol and from the lateral tail vein for the 24-hr and 72-hr post-HI, 1-week and 3-weeks after surgery. Normal values for blood test and venous blood gases [32,33]. P H P C02 P O2 BE HCO3 TCO2 Sat O2 Na+ K+ Hct Hb ALT AST Creat Normal values 7.24 70.8 70.11 3.8 30.99 33.1 88.78 137 4.27 41.3 14.04 187.35 330.3 0.38 PH 7.38 ± 0.12 Pre ± 0.05 ± 11.82 ± 14.33 ± 2.14 ± 2.41 ± 2.66 ± 5.16 ± 2.19 ± 0.41 ± 2.41 ± 0.81 ± 473.57 ± 805.1 ± 0.29 PCO2 37.17 ± 13.93mm 7.18 62.36 84.1 5.4 23.05 25.1 90.5 155.4 4.35 27.5 9.36 233.84 480.7 0.31 PO2 43.42 ± 15.94mm Post ± 0.09 ± 14.19 ± 31.31 ± 2.42 ± 2.15 ±2.59 ± 5.14 ± 4.76 ± 0.65 ± 2.8 ± 0.94 ± 202.83 ± 303.67 ± 0.05 BE -1.29 ± 2.87 m Eq/L 7.42 35.03 94.78 -1 23.37 24.56 85 124.75 7.4 29 9.84 2286.53 7795.33 0.53 HCO3 24.53 ± 3.33 m Eq/L 24-hours ± 0.12 ± 11.78 ± 67.39 ± 8.51 ± 7.21 ± 7.6 ± 16.1 ± 16.86 ± 1.92 ± 5.1 ± 1.73 ± 2971.77 ± 8074.62 ± 0.25 Sat O2 70.6 ± 17.33% 6.84 256.38 634.57 0.49 72-hours Na+ 150 ± 3.4 mEq/L ± 1.2 ± 417.27 ± 875.49 ± 0.27 K+ 7 ± 0.65 mEq/L 36.89 156.38 0.87 1-Week Hct 41.6 ± 5% ± 35.79 ± 86.97 ± 1.27 50.71 159.29 0.44 Hb 14.2 ± 0.5 g/dl ± 54.32 ± 100.64 ± 0.19 ALT 49 ± 24.1 UI/L 3-weeks AST 95 ± 31.7 UI/L Creat 2.5 ± 1.25 mg/dL to preserve the tissue scaffold, which would be advantageous when intrahepatic HI compensation by infusing deionised serum into the treating tumors next to vessels or biliary ducts [21,22], although it hepatic artery. This new approach should improve the differential does not preferentially ablate tumor over healthy tissue. In fact, due conductivity between healthy and tumor tissue, given the preferential to the higher conductivity of tumor tissue IRE may ablate healthier arterial perfusion of liver tumors [31]. than tumor tissue [8,23-25]. This is especially true when a sandwich In conclusion, as HI in the portal system leads to a significant risk electrode position is applied to the scattered tumor tissue deep inside of possibly lethal cytolysis and acidosis, compensatory measures and healthy liver tissue [13,26,27]. A possible approach to overcome this a reduced saline overload are warranted to improve the survival rates. shortcoming would be to increase the conductivity of the healthy tissue by means of hyperconductive solutions such as hypersaline References serum, as has already been demonstrated [9]. 1. Martin RC 2nd, McFarland K, Ellis S, Velanovich V. 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