INTERNATIONAL JOURNAL OF HYPERTHERMIA 2019, VOL. 36, NO. 1, 676–685 https://doi.org/10.1080/02656736.2019.1627585 Development of a catheter-based technique for endoluminal radiofrequency sealing of pancreatic duct Elz_ bieta Ewertowskaa, Anna Andaluzb, Xavier Mollb, Adria Aguilarb, Felix Garciab, Dolors Fondevilab , Rita Quesadac , Macarena Trujillod , Fernando Burdıoe and Enrique Berjanoa a BioMIT, Department of Electronic Engineering, Universitat Politecnica de Valencia, Valencia, Spain; bDepartament de Medicina i Cirurgia Animals, Facultat de Veterinaria, Universitat Autonoma de Barcelona, Barcelona, Spain; cDepartment of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain; dBioMIT, Department of Applied Mathematics, Universitat Politecnica de Valencia, Valencia, Spain; eDepartment of Surgery, Hospital del Mar, Barcelona, Spain ABSTRACT ARTICLE HISTORY Introduction: Endoluminal sealing of the pancreatic duct by glue or sutures facilitates the manage- Received 17 November 2018 ment of the pancreatic stump. Our objective was to develop a catheter-based alternative for endolu- Revised 20 May 2019 minal radiofrequency (RF) sealing of the pancreatic duct. Accepted 31 May 2019 Materials and methods: We devised a novel RF ablation technique based on impedance-guided cath- KEYWORDS eter pullback. First, bench tests were performed on ex vivo models to tune up the technique before Ablation; endoluminal; the in vivo study, after which endoluminal RF sealing of a 10 cm non-transected pancreatic duct was pancreatic duct; conducted on porcine models using a 3 Fr catheter. After 30 days, sealing effectiveness was assessed radiofrequency; sealing by a permeability test and a histological analysis. Results: The RF technique was feasible in all cases and delivered 5 W of power on an initial imped- ance of 308 ± 60 X. Electrical impedance evolution was similar in all cases and provided guidance for modulating the pullback speed to avoid tissue sticking and achieve a continuous lesion. During the follow-up the animals rate of weight gain was significantly reduced (p < 0.05). Apart from signs of exo- crine atrophy, no other postoperative complications were found. At necropsy, the permeability test failed and the catheter could not be reintroduced endoluminally, confirming that sealing had been successful. The histological analysis revealed a homogeneous exocrine atrophy along the ablated seg- ment in all the animals. Conclusions: Catheter-based RF ablation could be used effectively and safely for endoluminal sealing of the pancreatic duct. The findings suggest that a fully continuous lesion may not be required to obtain complete exocrine atrophy. 1. Introduction Radiofrequency ablation (RFA) is one of the recently explored sealing techniques. In this case the action mechan- Sealing of the pancreatic duct is used to manage two main ism does not rely on introducing a sealing agent, but on pancreatic malignancies: pancreatitis and pancreatic ductal inducing epithelial necrosis to trigger fibrosis (cicatrization) adenocarcinoma [1]. Sealing is usually achieved either by and collagen shrinkage and cause ductal atrophy. Our ligation of the affected pancreatic duct to eliminate pancre- research group had already examined the effect of pancre- atic secretions or tumor resection followed by anastomosis atic stump closure by RF-assisted transection in terms of fis- of the remaining pancreatic stump [2,3]. However, the com- tula formation on rat and pig models [5–7]. The results mon problem of leakage of the pancreatic contents into the showed that RFA not only induced exocrine atrophy for duc- abdomen, also known as pancreatic fistula, can affect patient tal occlusion but also reduced the occurrence of fistula. morbidity and mortality during the postoperative period. These findings encouraged us to go beyond the transection Many studies have explored feasible techniques for sealing plane and consider extending RFA endoluminally along a the pancreatic stump in order to replace anastomosis and segment of the pancreatic duct itself. minimize the risk of leakage [4]. Sutures, staplers, clips or The concept of endoluminal thermal ablation is not really endoluminal glue injections are some of the most frequently new since it has already been used in clinical applications for used tools currently used in clinical practice. However, none vascular occlusion such as e.g. hepatic tumor arteries, vari- of these techniques has been proven to effectively reduce cose veins [8,9] and biliary tumors [10]. However, to our the high risk of failure in these cases. knowledge, there is no evidence that this technique has CONTACT Enrique Berjano eberjano@eln.upv.es Department of Electronic Engineering, Universitat Politecnica de Valencia, (Building 7F), Camino de Vera, 46022 Valencia, Spain ß 2019 The Author(s). Published with license by Taylor & Francis Group, LLC This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. INTERNATIONAL JOURNAL OF HYPERTHERMIA 677 been previously employed for pancreatic remnant sealing, as 2.2. Considerations on the catheter design proposed here. As in the case of glue, the main purpose of In general, successful endoluminal ablation should ensure: thermal coagulation of the duct walls is to prevent leakage (1) catheter flexibility to operate inside the duct, (2) shallow by sealing the secretory ducts along its walls and inducing thermal lesions circumscribed exclusively to the tissue ductal atrophy and also ensure the loss of ductal permeabil- around the pancreatic duct (sufficient to completely seal the ity. In order to efficiently perform endoluminal ablation of secondary ducts draining into the main duct), and (3) the the pancreas, a thermal coagulation zone should meet the ability to estimate a suitable level of applied voltage from following characteristics: (1) it should be long enough to suit the initial impedance measurement. Potential catheter the remnant pancreas after a resection; (2) be as contiguous designs for endoluminal ablation should also take the follow- as possible, i.e. with the minimum number and length of ing issues into account: number of electrodes (single or mul- gaps; (3) there should be sufficient margin across the duct tiple), function mode (mono-, bi-, multi-polar), electrode type wall, i.e. ablate the entire epithelium and ensure loss of per- (dry or wet), and application procedure (point-to-point or meability, which requires a depth of 1–2 mm; and (4) it must continuous pullback). be safe, i.e. no steam pops or tissue disruption, which means The first tentative test was on a single long RF electrode keeping tissue temperature below 80–90  C and avoiding tis- based on a 3 Fr metallic guide whose length was tailored to sue sticking to the electrode. the lesion length in order to create one long lesion by Additional technical requirements for a device aimed at ‘segmental’ ablation. This first idea was discarded since the endoluminal surgery of the pancreatic duct should also edge effect prevented the formation of a long uniform ther- include: (1) a suitable diameter to fit the duct along the mal lesion around one RF electrode. Regardless of the abla- entire route; (2) flexible enough to pass easily through the tion time (10–20 s), the lesion surrounding the electrode was duct, which is not usually straight; and (3) be long enough nonhomogeneous and much deeper at the edges, which to cover the entire target segment. The electrodes used to was associated with overheating, audible steam pops (regis- create the thermal lesion should be on the catheter itself. In tered at 2–4 s) and tissue sticking. Also, the long metal wire this context, our objective was thus to develop a new cath- was not sufficiently flexible to operate inside soft delicate tis- eter-based technique for endoluminal RF sealing of the pan- sue. Another consideration was that a single long RF elec- creatic duct and to assess its safety and performance on an trode must be operated in monopolar mode, making it in vivo porcine model. difficult to predict the thermal lesion geometry, since the exact power delivered to the target site depends on the unpredictable electrical characteristics of all the tissues 2. Materials and methods involved in the electrical circuit. The bipolar mode employs at least two closely spaced The first phase, based on an ex vivo model, was used to tune electrodes to create shallow thermal lesions confined to the up the experimental setup, which consisted of an RF catheter inter-electrode space. The applied power depends almost combined with impedance-guided pullback. The aim was to exclusively on the characteristics of the tissue between the determine the suitability of the catheter in terms of diam- electrodes. Less power is required in bipolar mode to ablate eter, size, flexibility, and ablation protocol. The second phase the same lesion volume, which is appropriate in terms of was based on an in vivo porcine model and assessed the safety, especially when working on small or fragile organs, safety and feasibility of the novel technique with a 30-day such as the pancreas, which is surrounded by other deli- follow-up. In both phases the electrodes were powered by a cate organs. Radionics Cosman Coagulator CC-1 (Radionics, Burlington, A final issue to be considered is the possible internal cool- MA, USA), a versatile generator previously used with endo- ing of the electrodes. Although cooled electrodes have an vascular RF catheters for arterial occlusion in liver cancer [8]. advantage over dry electrodes and avoid tissue sticking (e.g. Impedance evolution was recorded during the ablations by a CelonProSurge Applicator, Olympus, Germany), they are also USB data acquisition module connected to the RF generator. known to create deeper thermal lesions than the dry type. As the goal in endoluminal ablation is to create relatively shallow thermal lesions around the pancreatic duct (1–2 mm 2.1. Tissue samples for ex vivo and in vivo models depth) and avoid damage to the pancreatic parenchyma or adjacent organs, dry electrodes seem to be the bet- The in vivo study was conducted on 8 Landrace-Large White ter choice. pigs (27.3 kg mean weight), according to the protocol For all the procedures we employed commercially avail- approved by Ethical Commission of the Universitat able flexible catheters, which include a pair of metal electro- noma de Barcelona (Authorization Number CEEAH 3487 Auto des separated by plastic insulation. The catheter would be and DMAH 9583) and the Government of Catalonia’s Animal introduced into the pancreatic duct through the papilla and Care Committee. The ex vivo study was conducted on two would create thermal lesions around the electrodes, as illus- types of sample: bovine liver acquired from a local slaughter- trated in Figure 1. The lesions could be created either point- house, and porcine pancreas obtained from 3 pigs, which to-point (i.e. positioning the catheter at one point and then underwent necropsy under the same authorized protocol as repositioning it to a contiguous point after having created a that used in the in vivo study. lesion) or continuously by the pullback technique. 678 E. EWERTOWSKA ET AL. Figure 1. (A) Overview of the catheter-based technique for endoluminal radiofrequency sealing of pancreatic duct. The catheter is introduced into the pancreatic duct through the papilla. (B) Detail of catheter tip in which RF power is applied between two electrodes. The thermal lesion causes the duct to seal (the illustration is that of a human pancreas). (C) 5 Fr bipolar RF catheter used for ex vivo ablations on bovine liver (the most proximal electrodes were used for ablations). (D) 3 Fr bipolar RF catheter used for ex vivo and in vivo ablations on porcine pancreas. 2.3. Protocol for ex vivo experiments who moved the catheter according to impedance progress as reflected in an audible signal provided by the RF gener- Three sets of ex vivo ablations were conducted, two on ator. In this second set of ex vivo ablations the applied volt- bovine liver and one on pancreatic tissue. Bovine liver was age was slightly lower (43 V instead of 50 V), since selected for its accessibility, homogeneity, and the ease of preliminary trials had shown that better pullback control identifying the shape of the thermal lesions. could be obtained at this value. The liver ablations were con- The first set of ex vivo ablations on liver was performed ducted with a 5 Fr catheter as used in cardiac electrophysi- without pullback in order to tentatively establishing the rela- ology (see Figure 1(C)). Their durations varied with the tionship between the applied power, duration and created length of the liver fragment. thermal lesion. This also allowed us to determine the Once the impedance-guided procedure was seen to be approximate potential starting point for pullback, i.e., when feasible, a third set of ablations was conducted on porcine the catheter pullback should start to avoid overheating and pancreas. Sample size in this set was small (n ¼ 3) due to a roll-off (abrupt rise in impedance). The applied voltage was limited supply of ex vivo pancreatic specimens. Since it was 50 V and the ablation durations varied according to the roll- off time. not possible to introduce a 5 Fr catheter into the porcine The second set of liver ablations was conducted with duct, this set was finally conducted by a 3 Fr catheter catheter pullback and provided information on the relation (Minitrode, Bioampere Research, Conselve, Italy) with two 3 between pullback speed and lesion continuity. Both the first and 4 mm long 10 mm interspaced electrodes (Figure 1(D)). and second sets were conducted with the catheter between In this case the applied voltage was also 43 V, which implied two fragments of liver (sandwich structure). The pullback a power of 5 W. sequence was guided by the impedance progress as follows: catheter pullback only started when impedance reached a 2.4. Protocol for in vivo experiments plateau (ensuring sufficient heating of the tissue in contact with the electrodes) and with early signs of an imminent cut- The ablation protocol established in the ex vivo trials was out, i.e. when the impedance attempted to rise (implying then tested on 8 Landrace-Large White pigs. This sample size overheating). In other words, the goal was to keep the was used in a previous study with a similar objective [7]. impedance as steady as possible, since it is reasonable to Prior to surgery, all the animals underwent a 12 h-fast. All assume that this implies that the same amount of power is were premedicated with azaperone (4 mg/kg intramuscularly (IM)) (StresnilV, Ecuphar veterinaria, Spain), ketamine (10 mg/ R being applied along the entire duct, hence ensuring the con- kg IM) (KetamidorV, Richter Pharma, Austria) and morphine R tinuity of the thermal lesion without the need for overlap- (0.2 mg/kg IM) (Morfina B.BraunV, B.Braun Medical, Spain). R ping. Pullback was carried out manually by the operator, INTERNATIONAL JOURNAL OF HYPERTHERMIA 679 Induction was performed with propofol 2–4 mg kg1 intra- initial impedance drop toward a steady minimum value, 2) venously (IV) (Propofol lipuroV, B.Braun Vetcare, Spain). After R plateau phase with a fairly steady minimum value, and 3) induction, all the animals were intubated with an orotracheal abrupt impedance rise. The beginning of the plateau phase tube, and anesthesia was continued during the surgical pro- occurred at 12 ± 3 s and coincided with the start of visible cedure with isoflurane 2% (IsovetV, Piramal Critical Care R and audible signs of tissue overheating, such as steam pops, Limited, UK) in 100% oxygen through a rebreathing circuit. and also with a visible thermal lesion (tissue whitening). Lactated Ringer’s solution was infused at a rate of 10 ml/kg/h Impedance during the plateau was 384 ± 20 X. The abrupt during the peri-operative period. Antibiotic therapy was impedance rise occurred at 21 ± 6 s and was preceded by administered with cephazoline 20 mg/kg IV (cefazolina audible pops 5 s before ablation ended. normonV, Laboratorios Normon, Spain) via the cephalic vein. R The second set of ex vivo ablations employed the pull- Heart rate, respiratory rate, pulse oximetry, electrocardiog- back technique and an attempt to keep a constant imped- raphy, noninvasive blood pressure and capnography were ance value as long as possible, which meant keeping the monitored during anesthesia using a multi-parameter moni- plateau for as long as possible. The applied voltage was tor (VetCareV, B Braun Vetcare, Spain). R reduced to 43 V, since 50 V led to rapid tissue overheating, Thermal ablations were performed with the same 3 Fr electrode sticking and a non-uniform lesion. As in the first catheter as used on the ex vivo pancreas introduced into the set, the initial impedance and plateau impedance were duct through the pancreatic papilla by means of laparotomy 568 ± 48 X and 334 ± 37 X, respectively. In this case, due to and enterotomy. Once the catheter was placed in the end the voltage reduction, applied power was 5 W. In this duct (10 cm insertion depth), RF power (37 V) was applied. second set we identified two main factors associated with As in the ex vivo experiments, the catheter was pulled back pullback that affected lesion geometry and size: speed along the entire duct until one of the electrodes was no lon- (slow vs. fast) and continuity (intermittent vs. uninterrupted ger inside it (RF generator automatically shut down). To con- –continuous–). A fast and continuous pullback caused an ini- trol the pullback speed, the operator continuously monitored tial minor impedance drop and led to greater number of impedance visually on a laptop and by following the audible abrupt impedance rises, although without visible lesions. signal emitted by the RF generator. When pullback was slow, two different types of behavior After this process, enterotomy and laparotomy were were seen that depended on pullback continuity: closed in the conventional manner. The animals were kept alive for a 30-day follow-up, after which all were again anes- 1. When pullback was slow and intermittent, impedance thetized, intubated and ventilated for laparotomy and dissec- followed a sawtooth waveform with small abrupt rises tion of the entire pancreas, which was then immediately (see black arrow in Figure 2(A)) each time the catheter placed in 10% buffered neutral formalin. The animals were was moved, so that the evolution matched well with the then euthanized using a commercial euthanasia solution. The lesion pattern. The intermittency involved tissue charring principal duct of the dissected pancreas was identified and and sticking just before the end of pullback, or at cannulated and the following tests were conducted: a feasi- extremely low speed, while gaps appeared (absence of bility test, consisting of reintroducing the catheter into the thermal lesion) with a fast pullback. pancreatic duct through the duodenal papilla, and a perme- 2. When pullback was slow and continuous, the impedance ability loss test consisting of injecting saline into the ablated waveform smoothed out (see Figure 2(B)) and avoided duct. The pancreas was divided into 3 mm–thick sections for tissue sticking. The lesions thus produced were also uni- further histological analysis and 2 or 3 5 mm samples from form in depth, with a whitish zone extending continu- each section were stained with hematoxylin and eosin and ously 1 mm around the electrode along the entire then evaluated by light microscopy. Pathologists unaware of ablation line (see Figure 2(D)). In these cases the plateau the experimental design assigned the degree of exocrine was reached in 23 s, after which pullback was applied pancreas atrophy from 0 to 5. at a mean speed of 0.07 cm/s. When the speed All the surgical procedures were performed by the same became too slow, impedance gradually rose (black arrow surgical team (AA and XM). To analyze sealing effectiveness, in Figure 2(C)), which was interpreted as an early sign of alterations of the animals’ weight increments at 30 days after an imminent impedance jump (implying overheating) surgery, feasibility of reintroducing the catheter into the pan- and therefore pullback was speeded up. creatic duct, and duct permeability were considered. The degree of atrophy was determined by histological analysis. The third set of ex vivo ablations was conducted on pan- creatic tissue with a 3 Fr instead of a 5 Fr catheter, since the 3. Results pancreatic lumen was narrower than expected. The initial impedance was 523 ± 29 X, a value slightly lower than that 3.1. Ex vivo experiments obtained by a 5 Fr catheter on bovine liver. The applied volt- In the first set of ex vivo ablations on bovine liver (without age was kept at the same level as in the previous set pullback) initial impedance was 606 ± 14 X, and the mean (43 V). Impedance followed the three-phase pattern applied power was 5 W (50 V applied voltage). In all the observed in the first two sets of ex vivo ablations. The time ablations we observed a similar impedance pattern, which to reach plateau was more than 100 s, which was three times went through the following consecutive phases: 1) gradual longer than in the two previous sets. The impedance value 680 E. EWERTOWSKA ET AL. Figure 2. Results of the second set of ex vivo experiments. (A–C) Examples of impedance evolution (A). Small-amplitude sawtooth associated with slow and inter- mittent catheter pullback; (B and C). Smooth patterns associated with slow and continuous catheter pullback. (D) Thermal lesion created on bovine liver by slow and continuous pullback (impedance shown in B). Figure 3. Results of the third set of ex vivo experiments. (A) Typical impedance during ablation. (B) Lesion created along the pancreatic duct in a pancreas treated with 43 V and pullback as continuous as possible. Gray bands show power cut off instants after abrupt impedance increases associated with tissue sticking and car- bonization (black arrow in B). After this event pullback was always speeded up in order to relocate the catheter, which in turn led to some visible gaps (white arrow in B). at the plateau was 302 ± 40 X (see Figure 3(A)). Abrupt contributed to a more intermittent pullback and to irregular impedance increases were associated with tissue sticking and lesions in terms of depth and continuity (i.e. with gaps). carbonization points (see black arrow in Figure 3(B)), just after which the catheter was relocated. Unfortunately, this 3.2. In vivo experiments movement caused visible gaps (see white arrow in Figure 3(B)), since they were associated exactly with the zones Since the tissue lesions of the in vivo group could not be where the electrodes did not apply power as the catheter seen until 30 days after the procedure, the evolution of the was moved. As in the previous sets, we also observed that electrical variables was the only indicator for creating uni- pulling back too fast impeded the creation of visible thermal form and continuous thermal lesions. The mean initial lesions. Overall, the lesions created in this third set showed impedance was 308 ± 60 X, considerably lower than in the ex larger margins (2–3 mm) than the two previous sets on liver vivo specimens. The applied voltage was reduced to 37 V, with a 5 Fr catheter. During the ablations, more abrupt which implied a power of 5 W (comparable to the value impedance changes and more roll-offs occurred, which measured in ex vivo ablations). A time of 30 s was required INTERNATIONAL JOURNAL OF HYPERTHERMIA 681 to reach the impedance plateau (220 ± 35 X), at which time the treated samples revealed complete acinar atrophy and pullback started. Impedance followed the same three-phase that only the intralobular ducts remained after ductal abla- pattern as in the ex vivo ablations. However, the in vivo abla- tion (Figure 5(D,E)). The epithelium of the interlobular and tions showed a higher incidence of roll-offs, estimated as some of the intralobular ducts showed a metaplasia from one every 40 s of ablation, and the impedance rises usually columnar or cuboidal to squamous and was lost in other involved tissue sticking, which forced us to relocate the cath- areas (Figure 5(F)). All the samples showed marked periductal eter rapidly in order to complete the ablation, which often and interlobular fibrosis (Figure 5(D–F)) and scattered inflam- required vigorous action. This meant the impedance evolu- matory infiltrate (Figure 5(F)). tion was more irregular than in the ex vivo experiments. Total ablation duration varied slightly in the trials in relation 4. Discussion to specific duct length and roll-off occurrence. An average of 11 cm of pancreatic duct was ablated at a mean pullback Sealing the pancreatic duct has always been challenging due speed of 0.1 cm/s. to the risk of pancreatic juice leaking into the peritoneal cav- During the 30-day follow-up the animals experienced ity, which continues to be the main complication contribu- alterations of stool consistency. After this period, their mean ting to the high mortality rate [7]. The endoluminal RFA with weight was 31 kg, which meant a slower than normal rate of pullback proposed here could potentially reduce this risk. weight gain (4 kg per 30 days). At necropsy, the feasibility Moreover, being a minimally-invasive technique, it offers the test to verify the presence of ductal occlusion failed, since it advantages of a likely faster recovery and fewer side effects was impossible to re-insert more than a few centimeters of than injecting a sealing agent or tissue suturing. To our the catheter into the duct, due to the lumen being too nar- knowledge, this is the first study to examine the feasibility of row. This also meant we could not perform the permeability the catheter-based RF sealing technique for the pancreatic test, as the liquid could not be injected as planned through duct. As this was a development study, it had several phases the distal end of the duct. All this suggested that sealing to progressively fine-tune the proposed technique (catheter was completely effective after 30 days at the level of the and ablation protocol) and finally obtain relevant information duodenal papilla. Beyond the papilla, a macroscopic view of on its safety and performance on an in vivo porcine model. cross-sections of pancreatic duct revealed different degrees of ductal dilation (diameter ranging widely from <1 mm to 4.1. Initial steps fine-tuning technique >10 mm), although at the level of the head the lumen seemed completely sealed in all cases (Section A in Figure 4). The first phases conducted on liver ex vivo fragments Figure 5 shows histological images of normal pancreatic showed how the different variables involved interact with tissue and pancreas after treatment. The histological study of each other during ablation with pullback. What we learned Figure 4. Results of the in vivo experiments. Macroscopic images of one of the treated pancreas and cross sections at the level of the head (A) and body (B). Red circles indicate the position of the main duct. Lumen was not completely closed in all cases and ductal dilation was observed, particularly in the body (units in cm). Gray dashed line indicates the main duct position in pancreas. 682 E. EWERTOWSKA ET AL. Figure 5. Results of in vivo experiment. Histological images of normal pancreas (A, B, C) and samples with sealing-induced pancreatic atrophy (D, E, F). Compared to normal pancreatic tissue, the treated samples show evident atrophy of the acinar component (purple dye) with only intralobular ducts remaining (D, E); the epi- thelium of the interlobular ducts is flattened or has been lost (F, black arrow); marked interlobular fibrosis (D, E; F); scattered inflammatory infiltrate (E, F). H&E  5 (A, D) and 20 (B, C, E, F). was that: (1) a few watts (5 W) are enough to create shrinkage of the collagen fibers around the main duct and 1–2 mm deep lesions; (2) pullback must begin when the subsequent reduction of its lumen, making it less permeable. impedance plateau is reached, which may coincide with aud- Initially we thought that in order for these phenomena to ible pops; (3) it is possible to modulate the rate of pullback occur efficiently, a continuous thermal lesion had to be cre- to keep the impedance as steady as possible, i.e. to try to ated along a segment of the main duct. This was probably maintain the plateau phase during the entire procedure; (4) over-optimistic, since although thermal ablation produces this can be done by monitoring impedance and/or by an immediate tissue shrinkage (due to protein denaturation and audible signal associated with impedance progress; and (5) if collagen contraction) this could only create a partial occlu- all these conditions are observed, the thermal lesion can be sion, as opposed to other mechanical methods (such as continuous (without gaps) along the entire path. suture or stapler). When we tried to transfer these conditions to the case of What the in vivo study revealed is that it is very difficult the pancreatic duct (pancreas ex vivo ablations), we learned to create continuous lesions along the pancreatic duct, pos- that: (1) a narrower catheter diameter is required (3 Fr sibly due to the irregular anatomy of the main duct, but also instead of 5 Fr); (2) catheter pullback through the pancreatic due to the technical limitations of the impedance-guided protocol. In fact, gaps were observed in the ex vivo pancreas duct is less straightforward than the simple movement just after ablation. In the same way, the sudden impedance between two slices of liver tissue; (3) this means that the rises during in vivo ablations suggest the possible presence impedance-guided lesion is not so effective in the pancreas, of gaps in the lesion and the possibility of the diameter not with sticking, a need for overlap, and possible gaps; (4) sud- being reduced along the whole ablation length. This was den impedance rises are possibly related to tissue sticking later confirmed in the macroscopic analysis, where the main and require moving the catheter, with the possibility of gaps; duct was not completely occluded in all zones. Complete (5) gaps can be also produced by excessive pullback speed; duct occlusion at the pancreas head was also evident from and (6) when present, thermal lesions are 2–3 mm deep. This the impossibility of reinserting the catheter through the duo- information was used to carry out a 30-day survival in denal papilla. We still do not have a convincing explanation vivo study. of why the proximal duct (head zone) was more occluded than the distal duct. The images in Figure 4 also show that 4.2. Clinical assessment of the technique the ablated duct was usually circular in shape, regardless of the dilation diameter or location, which suggests that the On planning the in vivo study we expected thermal ablation energy deposition occurred symmetrically around the elec- inside the pancreatic main duct to produce the following trodes and was limited in depth. phenomena: (1) occlusion of the small exocrine ducts Interestingly, despite the existence of lesion gaps and branching from the main duct and subsequent acinar atro- presence of non-occluded segments, clinical observations phy, causing the suppression of exocrine secretions; and (2) during follow-up and histological findings were compatible INTERNATIONAL JOURNAL OF HYPERTHERMIA 683 with complete exocrine atrophy. The atrophic pancreas was allows working in a specific impedance range that cuts out deprived of acini to secrete digestive enzymes, which caused when exceeded. We had to adapt this technique to the pre- the failure of the digestive functions. All the animals experi- sent study, since endoluminal RFA had never been used enced digestive alterations and a notably reduced rate of before to seal the pancreatic duct. The delicate pancreatic weight gain after the 30-day follow-up. tissue structure required a flexible applicator and the right In view of all the results, the complete occlusion of the catheter to fit a 1 mm duct diameter, which ruled out the entire main duct may not be required to generate complete use of the commercial product of 5 Fr. exocrine atrophy. In fact, it is possible that the partially In addition, impedance monitoring has already been pro- occluded duct segments also contributed to the exocrine posed to estimate the temperature and lesion size during RF atrophy. Since RF ablation also severs duct walls and produ- ablation due to the inverse relationship of both parameters ces thermal necrosis and apoptosis of the ablated zone, it is [14]. Tissue heating enhances ion mobility and current flow likely that atrophy and acinar cell loss are also a result of and thus produces a characteristic impedance drop in the thermally-induced apoptosis. It is thus reasonable to assume early stages of ablation. Although thermal lesions in high- that a fully continuous lesion along the main duct might not temperature ablative techniques require temperatures in the be necessary to achieve exocrine atrophy, which would be range 50–100  C, it is also known that lesion evolution is triggered not only by an immediate RF-induced effect, but closely connected to the temperature-time relationship, also by biological processes within 30 days after ablation. which depends on the amount of power applied. In particu- lar, it is known that a few minutes of exposure at 50  C trig- gers progressive tissue shrinkage and irreversible tissue 4.3. Safety issues damage [15], while only a few seconds at 60  C produce tis- Regarding the safety issues involved in the ablative tech- sue coagulative necrosis seen as a whitish area in the abla- nique, we must keep in mind that bipolar RFA has already tion zone [16,17]. Once thermal equilibrium has been been clinically used for endoluminal ablation of vessels and reached, the impedance value stabilizes at the minimum and biliary duct, so the concept of endoluminal ablation itself is lesion size remains static. Further ablation leads to tissue well-known and assumed to be safe. The risks of using RFA overheating and vaporization at temperatures between 100 instead of another mechanical method, such as staplers, and 105  C and is characterized by audible steam pops, an sutures or clips, could include overheating of the target tis- abrupt impedance rise, tissue carbonization and sticking [17]. sue and unintentional ablation of the neighboring tissue Once focused on impedance monitoring, our findings identi- (e.g. intestine). In our study these risks were minimized by fied three different phases in its progress during ablation: 1) establishing a ‘safe ablation environment’ by trying to keep a drop associated with initial heating, 2) a plateau phase, the pancreas as far as possible from other viscera during and 3) occasional abrupt increases associated with overheat- ablation by simple mechanical deviation. In addition, all the ing. All the phases could easily be distinguished in our ex procedures were accomplished by personnel experienced in vivo findings, in which whitish and carbonization zones using RF-based devices. Finally, the absence of postoperative appeared, suggesting that the tissue was in fact exposed to complications associated with RF power suggests that the the 60–100  C range. Overheating incidents also occurred in proposed ablation method keeps risks at a reasonably the in vivo study and suggest that the tissue was exposed to low level. temperatures of up to 100  C. The longer the plateau phase, the longer the ablation with sufficient power delivery and without carbonization. It has also been suggested that keep- 4.4. Technical feasibility ing the plateau impedance at a target value for a specific The ablation control proposed in this study is entirely based time should lead to the desired lesion depth [9]. The value on impedance progress. We discarded temperature monitor- of the plateau phase depends on tissue type and other con- ing due to it being technically complex to include tempera- ditions that affect its electrical properties, such as hydration ture sensors in such a small catheter. Moreover, while a or blood irrigation [18,19]. For instance, in our study the dif- temperature sensor would only provide information on the ferences between the ex vivo and in vivo models were pos- temperature in the tissue in contact with it (not necessarily sibly related to additional heat loss by convective blood the maximum tissue temperature), impedance gives a more perfusion cooling (also known as heat sink effect), which is complete picture of the characteristics of tissue affected by only present under in vivo conditions. Other factors could ablation through the direct relationship between electrical also have affected these differences, such as a smaller conductivity and temperature. In fact, this technique has impedance drop and a higher threshold temperature for been used previously in the management of biliary tumors coagulative necrosis, finally producing smaller lesion depths and venous obstruction or other venous insufficiencies in the in vivo case. Accordingly, we observed that in vivo [10–13]. While we designed our own endoluminal RFA proto- samples had nearly half as low initial impedance values col, other studies used commercial devices designed for (308 ± 60 X vs 523 ± 29 X) and a smaller impedance drop treating varicose veins (e.g. Celon ProCurve, Olympus, (220 ± 35 X vs 302 ± 40 X at plateau) than ex vivo specimens. Germany). In both cases the method is based on impedance We were not able to assess the differences in lesion depth feedback for catheter guidance by visualizing impedance between ex vivo and in vivo cases since in the latter case the and power evolution and attending to audible signals. It also lesion dimensions were assessed after a 30-day follow-up 684 E. EWERTOWSKA ET AL. period. However, our findings can be discussed in the light the appropriate powers and speeds to obtain continuous of previous liver studies (with a similar pancreas blood perfu- lesions and avoid overheating, the inherent tissue heterogen- sion rate, 860 ± 170 ml/min/kg vs. 767 ± 357 ml/min/kg [20]). eity impedes creating a uniform lesion along the entire path. These studies showed that in vivo lesions have a 36–44% The operator’s skill thus plays a vital role in varying pullback smaller diameter than ex vivo lesions [21–23], which sug- speed according to the impedance variations encountered. gests that the in vivo pancreatic lesions created in our study The amount of delivered power and pullback speed will also could be around 1 mm deep (since lesion depth was 2–3 mm vary according to the desired outcome and may differ from in the ex vivo case and similar power levels were used in the manufacturer’s recommendations [27,28]. For all these both cases). reasons it is important to first estimate the relationship Additionally, different studies have reported that tissue between impedance, delivered power and the required ther- shrinkage (which starts at 50  C) produces up to 20–30% of mal lesion before applying endoluminal RFA with pullback. tissue constriction of the initial margin and therefore the real ablation zone is actually greater than that measured [15,24,25], while a limited degree of tissue shrinkage could 4.5. Limitations of the study also reduce the chances of complete mechanical occlusion of Although our final clinical objective is the management of the duct, although, as seen in our study, it was sufficient to the pancreatic stump, the in vivo study was conducted on a reduce the duct diameter to less than the catheter diameter non-transected pancreatic duct. A transected pancreas model and ruled out catheter reintroduction. would be more complex and would entail a higher risk of Due to variations in the electrical properties of ex and in pancreatic leakage. Further research on this matter should vivo tissue, a lower voltage value was used in the in vivo therefore be conducted involving endoluminal RF sealing of study (43 V vs. 38 V). This change also affected the pullback the transected pancreatic duct in order to confirm the feasi- speed (0.03 cm/s vs. 0.1 cm/s), but in all cases a power of bility of the technique on a more realistic model. 5 W could be delivered, suggesting that the delivered As regards the clinical implications, it is important to note power, as determined by applied voltage and pullback that the porcine model used in this study is anatomically dif- speed, is the vital parameter in achieving successful RFA. The ferent to the human pancreas in certain respects, as it has a power and pullback speed values used in our study were secondary duct branching out from the main pancreatic duct noticeably lower than those usually reported for venous (see Figure 1 from [7]). In our study this duct was not ablation (18–25 W and 0.5–1 cm/s, respectively) ablated and therefore maintained its enzymatic secretory [9,12,13,26,27]. Numerous studies have reported that the rec- function, which could have reduced the effectiveness of the ommended clinical power and pullback speed values can proposed technique. For this very reason, the technique increase the incidence of tissue carbonization and sticking could be even more effective in the human pancreas. [12]. An additional issue in obtaining permanent sealing is the need to reapply the treatment (overlapping) to provide sufficient energy for effective sealing [28]. Interestingly, 5. Conclusions Badham et al. [12] suggested that values of 6 W and 0.08 cm/ The impedance-guided endoluminal RFA technique designed s are the most optimal to obtain a successful vein lesion by to seal the pancreatic duct seems to be a promising, feasible single RF ablation and avoid tissue overheating and sticking. and safe alternative for management of the pancreatic stump These values are very similar to these employed by us (5 W, and reducing the risk of leakage. Although power and pull- 0.03–0.1 cm/s). It is important to note that while we aimed back speed still need to be optimized, the findings obtained for a slow continuous pullback, Badham et al. used a discon- suggest that a fully continuous RFA lesion along the entire tinuous pullback, so that 0.08 cm/s was really an approximate duct may not in fact be required to achieve pancre- value. This similarity could be owing to the electrical proper- atic atrophy. ties of both tissues. In the in vivo study we found that a large number of abrupt impedance increases and tissue sticking events were inevitable. This problem could pose a Disclosure statement serious risk of soft tissue rupture during the treatment. The All authors have no conflict of interest or financial ties to disclose. relationship between the applied power and pullback speed determines the required energy level and the rate at which it is delivered to the tissue, and thus the final thermal dose Funding to cause tissue damage. The optimization of these parame- This work was supported by the Spanish Ministerio de Economıa, ters is still pending. n, Desarrollo Industria y Competitividad under “Plan Estatal de Investigacio The outcomes obtained from this technique are undoubt- e Innovacio n Orientada a los Retos de la Sociedad” (Grant edly affected by the additional factor of the operator’s skill TEC2014–52383–C3–R) and by the Spanish Ministerio de Ciencia, in controlling catheter pullback [26]. As already mentioned, Innovacion y Universidades under “Programa Estatal de I þ D þ i Orientada a los Retos de la Sociedad" (Grant: RTI2018-094357-B-C21 and an excessively slow pullback implies greater power deposited –C22). El_zbieta Ewertowska has a Predoctoral Grant (BES-2015–073285) in the target area together with carbonization and sticking, from the Ministry of Economy, Industry and Competitiveness while an exceedingly fast pullback produces gaps or even no (Government of Spain). Also funding was received from Secretarıa de lesion at all. Although the manufacturers usually recommend n, Desarrollo e Innovacio Estado de Investigacio n. INTERNATIONAL JOURNAL OF HYPERTHERMIA 685 ORCID concentrating on occlusion rates, side-effects and clinical outcome. Eur J Dermatol. 2011;21:945–951. Dolors Fondevila http://orcid.org/0000-0001-6651-3173 [14] Hartung WM, Burton ME, Deam AG, et al. 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