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Intraductal thermal injury using a heat probe and radiofrequency ablation electrode in a swine model of biliary stenosis
Clinics and Research in Hepatology and Gastroenterology (2013) 37, 159—165
Available online at
www.sciencedirect.com
ORIGINAL ARTICLE
Intraductal thermal injury using a heat probe and radiofrequency ablation electrode in a swine model of biliary stenosis Jae Uk Shin a, Kwang Hyuck Lee a,∗, Su-A Kim a, Jong Hak Choi a, Kwang Min Kim a, Jong Kyun Lee a, Kyu Taek Lee a, Yoon-La Choi b a
Division of Gastroenterology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50, Irwon-dong, Gangnam-gu, Seoul 135-710, South Korea b Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea Available online 15 June 2012
Summary Background and objective: An animal model for bile duct stenosis using intraductal thermal injury has not yet been established. The aims of the current study were to develop biliary stenosis in a swine model by inducing intraductal thermal injury using a heat probe or radiofrequency ablation electrode and to investigate an effective and safe energy dose. Methods: Intraluminal thermal injury was applied to the common bile duct with a heat probe in three swines and a radiofrequency ablation electrode in the other three swines by either endoscopic retrograde cholangiography or open laparotomy. Cholangiography and histologic evaluation of common bile duct were taken 2 weeks after thermal injury. Results: Thermal injury with a heat probe at 25 J for 40 seconds produced a stricture in all three animals. Application of a radiofrequency ablation electrode produced a stricture in two of three animals. An energy dose of 40 W at 80 ◦ C for 30 seconds produced biliary stenosis without any complications initially and 2 weeks after thermal injury. Conclusions: The application of a heat probe and a radiofrequency ablation electrode for intraductal thermal injury resulted in a reproducible animal model of biliary stenosis. © 2012 Elsevier Masson SAS. All rights reserved.
Introduction Inoperable pancreatobiliary malignancies or benign biliary strictures often require endoscopic management. Currently,
∗ Corresponding author. Tel.: +82 2 3410 3409; fax: +82 2 3410 6983. E-mail address: [email protected] (K. Hyuck Lee).
many of the procedures for adequate biliary drainage over the long term are challenging. Multiple plastic stents and covered self-expandable metal stents have shown promising results for the management of benign biliary strictures. However, the data to support using of multiple plastic stents or self-expandable metal stents are still not enough [1—3]. If investigators could easily develop an animal model of artificial stenosis, it would facilitate the development of equipment or techniques for biliary dilatation. Vorwerk
2210-7401/$ – see front matter © 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.clinre.2012.04.013
160 et al. [4] used a surgically created percutaneous access and a Nd:YAG laser contact probe, and several studies tried bile duct obstruction via ligation [5] or compression by various prostheses [6]. Rumella et al. was the first to use an endoscopic biliary approach with a heat probe and multipolar probe to produce thermal injury to the common bile duct in animals [7]. However, there was no further investigation of biliary stenosis by intraductal thermal injury thereafter. With a radiofrequency ablation (RFA) device, it is easier to control exposure time, energy dose, and heat temperature than with a heat probe. Therefore, an RFA device may more easily control the degree of bile duct injury and could be used in producing artificial bile duct stenosis. RFA has been used for the treatment of various malignancies. Intraductal thermal injury with RFA device might be applied for the treatment of malignant biliary obstruction. However, there were only a few investigations for intra-bile duct application of RFA, and the effective and safe energy dose for intraductal thermal injury with RFA is not established [8,9]. The aims of the current study were to develop a reliable, reproducible animal model of benign biliary stenosis through intraductal thermal injury with a heat probe or RFA electrode and to investigate an effective and safe energy dose for application in producing biliary stenosis.
Material and methods A total of six healthy pigs weighing 40 to 50 kg were divided into two groups according to the device used to induce thermal injury (group A used a heat probe and group B used a RFA needle). This study was approved by the Institutional Animal Care and Use Committee of our institution. All the pigs were fasted for 24 hours before the procedure. On the day of the procedure, animals were sedated by intramuscular injection of tiletamine/zolazepam (Zoletil® ; 6 mg/kg) and xylazine (Rompun® ; 2 mg/kg). After tracheal intubation, anesthesia was maintained by inhalation with 2% isoflurane. Electrocardiogram, heart rate, blood pressure, oxygen saturation, and end-tidal CO2 were monitored by veterinary technicians throughout the procedure. Enrofloxacin (2.5 mg/kg) was administered intramuscularly before and for 2 days after the procedure as a prophylaxis agent against cholangitis. Ketoprofen (2 mg/kg) was administered intramuscularly on the day of procedure for treatment of pain. Endoscopic retrograde cholangiopancreatography (ERCP) with a therapeutic side viewing endoscope (TJF130, Olympus America, Inc., Melville, NY) was performed on the pigs in group A using standard biliary catheters and guidewires under the guidance of fluoroscopy. After obtaining baseline cholangiograms and sphincterotomy using a papillotome, the opening of major papilla was dilated with a Hurricane balloon catheter (Boston Scientific Corp., 10 mm in diameter) to insert a heat probe catheter. Then, a 7 Fr heat probe (Olympus America, Inc) was inserted and positioned in the common bile duct (CBD) under the guidance of fluoroscopy. ERCP was performed by one expert who had previously performed ERCP more than 500 times. If the expert failed to successfully perform ERCP, then open laparotomy was performed for access to the bile duct. Open laparotomy was performed on the pigs in group B by one veterinarian. A median incision was made and
J.U. Shin et al. the duodenum was incised longitudinally. After inspection of the papilla, a biliary catheter and guidewire were inserted through the papilla under the guidance of fluoroscopy. Balloon dilatation with a Hurricane balloon catheter (Boston Scientific Corp., 10 mm in diameter) was performed after obtaining a baseline cholangiogram. A RFA electrode (CoATherm CTS electrode S1709, 17-gauge, 1 cm tip exposure, 90 mm total length; APRO KOREA Inc., Korea) was then inserted and positioned in the common bile duct under the guidance of fluoroscopy. Energy was delivered by an RFA generator (CoATherm RF-G200 generator; APRO KOREA Inc., Korea) operating at 480 kHz and a maximum power output of 200 W. After thermal injury to the common bile duct, the duodenum and abdominal wall were closed with a suture (Fig. 1). The energy doses used to induce thermal injury were based on a study which showed that the application of a heat probe at 10 or 15 J (1 J = 1 W per second) produced a stricture in all pigs, whereas the application of a multipolar probe at 12, 16, 20, or 24 J produced a stricture in only one of six pigs [7]. Therefore, we decided to use an energy dose of 25 J for 40 seconds with a heat probe and energy doses of 20 W at 60 ◦ C for 30 seconds, 40 W at 80 ◦ C for 30 seconds, or 40 W at 100 ◦ C for 30 seconds with a radiofrequency catheter. During the 2 weeks (4 weeks for two of the pigs) following the procedure, animals were maintained on their usual diet. Clinical signs including weight loss, daily food intake, jaundice, and fever were monitored daily by a veterinary technician. Biochemical tests of liver function were assessed at baseline and 2 weeks after thermal injury. Follow-up ERCP with a heat probe was performed 2 weeks after thermal injury on the animals in group A. An uncovered metal stent was inserted in cases of bile duct stenosis, and the animal was sacrificed by potassium chloride overdose 4 weeks after thermal injury. For the animals in group B (thermal injury by open surgical RFA) and those animals in group A which had open bile duct surgery, euthanization by potassium chloride overdose was carried out 2 weeks after thermal injury. Before sacrifice, a veterinarian performed general anesthesia and opened the abdomen and duodenal wall. A final cholangiogram was obtained under fluoroscopy in all animals to identify bile duct strictures, then the liver and common bile duct were excised from each animal for gross examination. The common bile duct was opened longitudinally and examined for damage and tissue response. The duct was preserved in formalin and stained with hematoxylin-eosin (H&E) and Masson’s trichrome. An experienced gastrointestinal pathologist evaluated the extent of inflammatory cell infiltration, fibrous changes, and epithelial injury.
Results Induction of thermal injury and clinical follow-up Thermal injuries with a heat probe were administered to the three pigs of group A by ERCP (two pigs) or by open surgery (one pig) (Table 1). For the first pig, thermal injury by ERCP with a heat probe was successful. However, 2 weeks later, the pig showed poor oral intake and decreased general activity. Dilatation of the common bile duct was confirmed on the cholangiogram obtained by ERCP (Fig. 2A).
Intraductal thermal injury using heat probe and radiofrequency electrode
161
Figure 1 A. A heat probe was inserted into the common bile duct under fluoroscopy. B. A picture of a heat probe showing the plastic catheter with an electrode at the tip. C. Balloon dilatation of papilla was performed by open laparotomy. D. Bile duct cannulation was performed by endoscopy. E. A picture of a radiofrequency ablation electrode showing the 90 mm needle and 1 cm electrode at the tip. F. A radiofrequency ablation electrode was inserted into the common bile duct under fluoroscopy after open laparotomy.
A covered metal stent (80 mm in length, 10 mm in diameter; Hanarostent® , M.I. Tech, Korea) was inserted into the CBD and intramuscular antibiotics were administered. Laboratory data showed elevated total bilirubin (2.8 mg/dL) (Table 2). The day before the 4-week time point after thermal injury, the pig died. An autopsy was performed on the pig for gross and microscopic examination of the common bile duct. Thermal injury by ERCP failed in the second pig, therefore, open surgery with a heat probe was performed. Two weeks later, the pig was euthanized despite no abnormal
symptoms. The cholangiogram taken just prior to euthanization confirmed CBD dilatation (Fig. 2B). However, laboratory tests showed only a mild elevation of alkaline phosphatase (ALP) (Table 2). For the third pig, thermal injury by ERCP with a heat probe was successful. Two weeks after thermal injury, the pig did not show any abnormal symptoms. ERCP was performed to obtain a cholangiogram but failed. There were no abnormal findings in the laboratory tests except for mild elevation of ALP. Four weeks after thermal injury, the pig developed symptoms of poor oral intake and
Table 1 Summary of thermal injury method and the result. All of three cases with heat probe resulted in bile duct stenosis. Radiofrequency ablation (RFA) with low energy dose (20 W 60 ◦ C 30 sec) failed to make bile duct stenosis, and RFA with high energy and high temperature (40 W 100 ◦ C 30 sec) developed bile duct stenosis with complication of bile duct perforation and abscess formation.
Pig Pig Pig Pig Pig Pig
1 2 3 4 5 6
Biliary approach
Thermal injury
Stenosis
Cholangitis
Progress
ERCP Surgery ERCP Surgery Surgery Surgery
Heat probe (25 J 40 sec) Heat probe (25 J 40 sec) Heat probe (25 J 40 sec) RFA (20 W 60 ◦ C 30 secs) RFA (40 W 80 ◦ C 30 sec) RFA (40 W 100 ◦ C 30 sec)
Success Success Success Failure Success Success
Develop Develop Develop None Develop Abscess
Death at 4 wks Autopsy at 2 wks Autopsy at 4 wks Autopsy at 2 wks Autopsy at 2 wks Autopsy at 2 wks
ERCP: endoscopic retrograde cholangiopancreatography.
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J.U. Shin et al.
Figure 2 A. Cholangiogram of pig 1 shows bile duct dilatation and a metal stent (between black arrows). B. Cholangiogram of pig 2 shows a stricture of the common bile duct (dark arrow) and bile duct dilatation. C. Cholangiogram of pig 3 shows abrupt stenosis of the common bile duct (dark arrow) and severe bile duct dilatation. D. Gross morphology of the common bile duct in pig 1 shows a dark pigmented fibrosis band (white arrow) with an uncovered metal stent. E. A picture of the common bile duct of pig 2 showing a whitish fibrotic area (white arrow) and hyperemic mucosal inflammation above the level of stenosis. F. A picture of the common bile duct of pig 3 showing a whitish fibrotic area (white arrow) and severe bile duct dilatation with mucosal inflammation.
vomiting. The cholangiogram obtained under open laparotomy with general anesthesia confirmed CBD dilatation (Fig. 2B), and laboratory tests showed marked abnormalities (Table 2). The pig was then euthanized. Thermal injuries with a radiofrequency electrode were administered to the three pigs of group B (Table 1). The electrode was prepared for RFA of hepatocellular carcinoma and could not pass through the working channel of an endoscope. Therefore, all procedures were performed by
Table 2
The results of laboratory test after intraductal thermal injury. T-bilirubin (mg/dL)
Pig Pig Pig Pig Pig Pig
1 2 3 4 5 6
open laparotomy under general anesthesia. Three different energy doses were tested for the induction of thermal injury. Thermal injury was administered to the fourth pig with an energy dose of 20 W at 60 ◦ C for 30 seconds. Two weeks later, a cholangiogram was taken and the pig was euthanized. The cholangiogram showed no definite signs of stenosis or dilatation (Fig. 3B). For the other two pigs in group B (fifth and sixth pigs), energy doses of 40 W at 80 ◦ C for 30 seconds and 40 W at 100 ◦ C for 30 seconds were applied, respectively. Two
AST (IU/L)
ALT (IU/L)
ALP (IU/L)
0 wk
2 wks
4 wks
0 wk
2 wks
4 wks
0 wk
2 wks
4 wks
0 wk
2 wks
4 wks
NA NA NA 0.2 0.3 0.2
2.8 0.5 0.3 0.5 0.5 0.2
NA NA 2.7 NA NA NA
NA NA NA 22 24 20
52 22 30 25 36 25
NA NA 46 NA NA NA
NA NA NA 51 29 29
14 21 21 36 29 26
NA NA 20 NA NA NA
NA NA NA 243 303 216
137 446 408 350 314 258
NA NA 392 NA NA NA
T-bilirubin: total bilirubin (normal range: 0.1—0.5 mg/dL); AST: aspartate transaminase (normal range, 17—44 IU/L); ALT: alanine transaminase (normal range, 17—78 IU/L); ALP: alkaline phosphatase (normal range, 69—333 IU/L); NA: not applicable; wk: week.
Intraductal thermal injury using heat probe and radiofrequency electrode
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Figure 3 A. Baseline cholangiogram of pig 4 shows a normal bile duct. B. Cholangiogram of pig 4, 2 weeks after thermal injury, shows no bile duct dilatation. C. Gross morphology of pig 4 shows linear hyperemic mucosal inflammation without fibrosis or stenosis. D. Microscopic findings of pig 4 show mildly increased fibrosis with regenerating mucosa and inflammation in the mucosal layer. E. Baseline cholangiogram of pig 5 shows a normal bile duct. F. A linear stenosis line (white arrow) with mild common bile duct dilatation is seen on the cholangiogram of pig 5, 2 weeks after thermal injury. G. Gross morphology of pig 5 shows a fibrotic lesion (white arrow) and diffuses mucosal hyperemic inflammation. H. Microscopic findings of pig 5 show an increase in diffuse fibrosis through the entire wall and denudated mucosa with inflammation. I. Baseline cholangiogram of pig 6 shows a normal bile duct. J. Cholangiogram of pig 6, 2 weeks after thermal injury, shows bile duct stenosis (white arrow) with severe dilatation. K. Gross findings of pig 6 show a distal CBD stricture with complications of bile duct perforation and periductal abscess formation (white arrow). L. Microscopic findings of pig 6 show extensive necrosis (white arrow) driven by perforation of the bile duct.
weeks after thermal injury, CBD stenosis with dilatation was confirmed in each of the pigs (Figs. 3F and J). Both pigs were euthanized after cholangiography and showed no abnormal symptoms or laboratory test results (Table 2).
Gross and microscopic findings During the gross examination of extrahepatic bile ducts, the CBD of the first pig was filled with a pus-like yellowish sludge, and there was a dark stricture-like band on the mid-CBD (Fig. 2D). The CBD of the second pig showed a whitish fibrous band with less severe diffuse mucosal hyperemia above the level of stenosis (Fig. 2E). The third pig showed extensive yellowish sludge inside of the CBD and a whitish fibrous band and diffuse reddish mucosal inflammation inside of the extrahepatic bile duct (Fig. 2F). The CBD of the fourth pig showed a hyperemic band, resembling mucosal inflammation, without stenosis or scar tissue. (Fig. 3C). Fibrosis at the mid-level of the CBD and diffuse hyperemic mucosal inflammation were seen inside of the CBD in the fifth pig (Fig. 3G). There was a stricture with
perforation and abscess formation on the lower level of the CBD in the sixth pig (Fig. 3K). Microscopic findings of thermal injury using RFA were compared in the three pigs in group B. Microscopic findings in the fourth pig, which received the lowest energy dose, showed mildly increased fibrosis with regenerating mucosa and inflammation in the mucosal layer (Fig. 3D). The fifth pig showed an increase of diffuse fibrosis through the entire wall and denudated mucosa with inflammation (Fig. 3H). The microscopic findings of the sixth pig, which received the highest energy dose and temperature, showed extensive necrosis driven by perforation of the bile duct (Fig. 3L).
Discussion This study showed that heat probe and RFA were both effective at producing bile duct strictures, and we found effective and safe energy dose of heat probe and RFA to produce benign biliary stenosis. Rumella et al. reported that application of a heat probe at 15 J could produce bile duct stenosis in all animals, but
164 application of a multipolar probe at 12 W for 2 seconds failed to produce stenosis in most animals [7]. In the current study, we investigated whether development of an animal model for bile duct stenosis by thermal injury with a heat probe was reproducible and if higher energy doses would be safe. Higher energy doses with a heat probe (25 J for 40 seconds) produced bile duct stenosis and cholangitis in all three pigs of group A, confirmed 2 and 4 weeks after thermal injury. However, the first pig in group A died 4 weeks after thermal injury, despite stent insertion. The lack of proper antibiotics and intravenous hydration might have been related to the death. Therefore, no further biliary stenting was attempted. RFA is generally used for the treatment of hepatocellular carcinoma (HCC). Energy doses for the treatment of HCC range from 60 to 120 W for 6 to 12 minutes [10,11]. Endoscopic RFA has been applied for the treatment of Barrett’s esophagus with dysplasia [12—14]. Energy delivery typically lasts less than 1.5 seconds, and an energy dose of 2 × 15 J/cm2 is the most effective regimen for circumferential, full-thickness ablation of the epithelium without injuring the submucosa [15,16]. Recently, an endobiliary RFA device (HabibTM EndoHPB, EMcision Ltd, UK, London) that is compatible with standard side viewing endoscopes and passes over 0.035-inch guidewires was developed and investigated in an animal model [8]. This study used energy doses of 5−10 W for a maximum of 2 minutes. At higher power settings and longer ablation times, difficulties were encountered reintroducing endoHPB into the biliary tree. Also, a pilot study that used this device in patients with malignant biliary obstruction reported a 90-day biliary patency. The energy dose used in this study was set at 400 kHz and ranged from 5−10 W for 2 minutes [9]. The energy doses and ablation times delivered by the RFA device in our study were different from ones used in the above two studies [8,9]. Current study used a monopolar RFA electrode, which is commonly used in the treatment of HCC. The following energy doses were used in our study: 20 W at 60 ◦ C for 30 seconds, 40 W at 80 ◦ C for 30 seconds, and 40 W at 100 ◦ C for 30 seconds. These energy doses were based on those reported for the treatment of primary and secondary hepatic tumors. Results from our study showed that an energy dose of 20 W at 60 ◦ C for 30 seconds produced mucosal injury without bile duct stenosis, whereas a dose of 40 W at 100 ◦ C for 30 seconds caused complications including perforation of the common bile duct and periductal abscess formation. Therefore, the optimal energy dose for bile duct stenosis using RFA was 40 W at 80 ◦ C for 30 seconds (total energy: 120 J = 40 W/sec × 30 seconds). The other two studies (mentioned above) applied a lower dose of energy (5−10 W) for a longer time (2 minutes) (total energy: 120 J = 10 W/sec × 120 seconds) [8,9]. The mode of energy delivery was different between our study and the other studies, but the total cumulative energy was similar. Macatula et al. reported that RF ablation with low power (less than 120 W for 12 minutes) for the treatment of HCC could achieve comparable clinical outcomes with fewer adverse effects compared with maximal power (more than 120 W) [17]. Also, Komorizino et al. demonstrated successful treatment of HCC after a single session of RF ablation using a maximum power of 90 W [18]. Therefore, for the treatment of intra-bile duct malignancies, energy doses lower than the doses required for bile duct stenosis may be sufficient.
J.U. Shin et al. However, further research on the energy dose required for treatment of bile duct malignancies by RF ablation is still needed. Short observation time and small number of pigs are limitations of this study. The animals were observed only for 2 weeks. That was because in cases of surgical stricture and cholangitis, it was almost impossible to keep them alive for more than 2 weeks by making them have food or maintain IV fluid. Nowadays endobiliary RFA devices were developed but it was available in few centers only for research purpose. We couldn’t help but perform CBD coagulation through surgical approach with conventional RFA devices, which cannot be inserted into endoscopic working channel. The swines had to be sacrificed 2 weeks after formation of biliary strictures. This study was planned as a pilot study; therefore, we performed experiments with a minimum number of animals. This study showed reproducible animal model for benign biliary stenosis and proper energy dose. Reliable methods for artificial biliary stenosis could help the development of new treatments for benign biliary strictures. Also, application of RFA within the bile duct might be promising for the treatment of malignant biliary obstruction, however more investigations are needed.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Funding: This study had financial supports by M.I. Tech, Korea.
References [1] Poley JW, van Tilburg AJ, Kuipers EJ, Bruno MJ. Breaking the barrier: using extractable fully covered metal stents to treat benign biliary hilar strictures. Gastrointest Endosc 2011;74:916—20. [2] Perri V, Familiari P, Tringali A, Boskoski I, Costamagna G. Plastic biliary stents for benign biliary diseases. Gastrointest Endosc Clin N Am 2011;21:405—33 [viii]. [3] Gupta R, Reddy DN. Stent selection for both biliary and pancreatic strictures caused by chronic pancreatitis: multiple plastic stents or metallic stents? J Hepatobiliary Pancreat Sci 2011;18:636—9. [4] Vorwerk D, Guenther RW, Kuepper W, Kissinger G. Thermal laser-induced stenosis of the common bile duct. An alternative model for experimental research. Invest Radiol 1989;24:758—61. [5] Hirazawa K, Oka M, Ogura Y, Miyahara M, Hazama S, Suzuki T. New technique for inducing reversible obstructive jaundice in the rat. Eur Surg Res 1997;29:195—201. [6] Posner MC, Burt ME, Stone MD, Han BL, Warren RS, Vydelingum NA, et al. A model of reversible obstructive jaundice in the rat. J Surg Res 1990;48:204—10. [7] Rumalla A, Petersen BT, Baron TH, Burgart LJ, Herman LJ, Wiersema MJ, et al. Development of a swine model for benign stenosis of the bile duct by endoscopic application of intraluminal thermal injury. Gastrointest Endosc 2003;57:73—7. [8] Khorsandi SE, Vavra P, Navarra G, Kysela P, Habib N. The modern use of radiofrequency energy in surgery, endoscopy and interventional radiology. Eur Surg 2008;40:204—10.
Intraductal thermal injury using heat probe and radiofrequency electrode [9] Steel AW, Postgate AJ, Khorsandi S, Nicholls J, Jiao L, Vlavianos P, et al. Endoscopically applied radiofrequency ablation appears to be safe in the treatment of malignant biliary obstruction. Gastrointest Endosc 2011;73:149—53. [10] Nishikawa H, Inuzuka T, Takeda H, Nakajima J, Sakamoto A, Henmi S, et al. Percutaneous radiofrequency ablation therapy for hepatocellular carcinoma: a proposed new grading system for the ablative margin and prediction of local tumor progression and its validation. J Gastroenterol 2011;46: 1418—26. [11] Morimoto M, Numata K, Nozawa A, Kondo M, Nozaki A, Nakano M, et al. Radiofrequency ablation of the liver: extended effect of transcatheter arterial embolization with iodized oil and gelatin sponge on histopathologic changes during follow-up in a pig model. J Vasc Interv Radiol 2010;21:1716—24. [12] Shaheen NJ, Overholt BF, Sampliner RE, Wolfsen HC, Wang KK, Fleischer DE, et al. Durability of radiofrequency ablation in Barrett’s esophagus with dysplasia. Gastroenterology 2011;141:460—8. [13] Shaheen NJ, Sharma P, Overholt BF, Wolfsen HC, Sampliner RE, Wang KK, et al. Radiofrequency ablation in Barrett’s esophagus with dysplasia. N Engl J Med 2009;360:2277—88.
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[14] van Vilsteren FG, Alvarez Herrero L, Pouw RE, ten Kate FJ, Visser M, Seldenrijk CA, et al. Radiofrequency ablation for the endoscopic eradication of esophageal squamous high grade intraepithelial neoplasia and mucosal squamous cell carcinoma. Endoscopy 2011;43:282—90. [15] Dunkin BJ, Martinez J, Bejarano PA, Smith CD, Chang K, Livingstone AS, et al. Thin-layer ablation of human esophageal epithelium using a bipolar radiofrequency balloon device. Surg Endosc 2006;20:125—30. [16] Ganz RA, Utley DS, Stern RA, Jackson J, Batts KP, Termin P. Complete ablation of esophageal epithelium with a balloon-based bipolar electrode: a phased evaluation in the porcine and in the human esophagus. Gastrointest Endosc 2004;60:1002—10. [17] Macatula TC, Lin CC, Lin CJ, Chen WT, Lin SM. Radiofrequency ablation for hepatocellular carcinoma: use of low versus maximal radiofrequency power. Br J Radiol 2011 [Epub ahead of print]. [18] Komorizono Y, Oketani M, Sako K, Yamasaki N, Shibatou T, Maeda M, et al. Risk factors for local recurrence of small hepatocellular carcinoma tumors after a single session, single application of percutaneous radiofrequency ablation. Cancer 2003;97:1253—62.
Available online at
www.sciencedirect.com
ORIGINAL ARTICLE
Intraductal thermal injury using a heat probe and radiofrequency ablation electrode in a swine model of biliary stenosis Jae Uk Shin a, Kwang Hyuck Lee a,∗, Su-A Kim a, Jong Hak Choi a, Kwang Min Kim a, Jong Kyun Lee a, Kyu Taek Lee a, Yoon-La Choi b a
Division of Gastroenterology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50, Irwon-dong, Gangnam-gu, Seoul 135-710, South Korea b Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea Available online 15 June 2012
Summary Background and objective: An animal model for bile duct stenosis using intraductal thermal injury has not yet been established. The aims of the current study were to develop biliary stenosis in a swine model by inducing intraductal thermal injury using a heat probe or radiofrequency ablation electrode and to investigate an effective and safe energy dose. Methods: Intraluminal thermal injury was applied to the common bile duct with a heat probe in three swines and a radiofrequency ablation electrode in the other three swines by either endoscopic retrograde cholangiography or open laparotomy. Cholangiography and histologic evaluation of common bile duct were taken 2 weeks after thermal injury. Results: Thermal injury with a heat probe at 25 J for 40 seconds produced a stricture in all three animals. Application of a radiofrequency ablation electrode produced a stricture in two of three animals. An energy dose of 40 W at 80 ◦ C for 30 seconds produced biliary stenosis without any complications initially and 2 weeks after thermal injury. Conclusions: The application of a heat probe and a radiofrequency ablation electrode for intraductal thermal injury resulted in a reproducible animal model of biliary stenosis. © 2012 Elsevier Masson SAS. All rights reserved.
Introduction Inoperable pancreatobiliary malignancies or benign biliary strictures often require endoscopic management. Currently,
∗ Corresponding author. Tel.: +82 2 3410 3409; fax: +82 2 3410 6983. E-mail address: [email protected] (K. Hyuck Lee).
many of the procedures for adequate biliary drainage over the long term are challenging. Multiple plastic stents and covered self-expandable metal stents have shown promising results for the management of benign biliary strictures. However, the data to support using of multiple plastic stents or self-expandable metal stents are still not enough [1—3]. If investigators could easily develop an animal model of artificial stenosis, it would facilitate the development of equipment or techniques for biliary dilatation. Vorwerk
2210-7401/$ – see front matter © 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.clinre.2012.04.013
160 et al. [4] used a surgically created percutaneous access and a Nd:YAG laser contact probe, and several studies tried bile duct obstruction via ligation [5] or compression by various prostheses [6]. Rumella et al. was the first to use an endoscopic biliary approach with a heat probe and multipolar probe to produce thermal injury to the common bile duct in animals [7]. However, there was no further investigation of biliary stenosis by intraductal thermal injury thereafter. With a radiofrequency ablation (RFA) device, it is easier to control exposure time, energy dose, and heat temperature than with a heat probe. Therefore, an RFA device may more easily control the degree of bile duct injury and could be used in producing artificial bile duct stenosis. RFA has been used for the treatment of various malignancies. Intraductal thermal injury with RFA device might be applied for the treatment of malignant biliary obstruction. However, there were only a few investigations for intra-bile duct application of RFA, and the effective and safe energy dose for intraductal thermal injury with RFA is not established [8,9]. The aims of the current study were to develop a reliable, reproducible animal model of benign biliary stenosis through intraductal thermal injury with a heat probe or RFA electrode and to investigate an effective and safe energy dose for application in producing biliary stenosis.
Material and methods A total of six healthy pigs weighing 40 to 50 kg were divided into two groups according to the device used to induce thermal injury (group A used a heat probe and group B used a RFA needle). This study was approved by the Institutional Animal Care and Use Committee of our institution. All the pigs were fasted for 24 hours before the procedure. On the day of the procedure, animals were sedated by intramuscular injection of tiletamine/zolazepam (Zoletil® ; 6 mg/kg) and xylazine (Rompun® ; 2 mg/kg). After tracheal intubation, anesthesia was maintained by inhalation with 2% isoflurane. Electrocardiogram, heart rate, blood pressure, oxygen saturation, and end-tidal CO2 were monitored by veterinary technicians throughout the procedure. Enrofloxacin (2.5 mg/kg) was administered intramuscularly before and for 2 days after the procedure as a prophylaxis agent against cholangitis. Ketoprofen (2 mg/kg) was administered intramuscularly on the day of procedure for treatment of pain. Endoscopic retrograde cholangiopancreatography (ERCP) with a therapeutic side viewing endoscope (TJF130, Olympus America, Inc., Melville, NY) was performed on the pigs in group A using standard biliary catheters and guidewires under the guidance of fluoroscopy. After obtaining baseline cholangiograms and sphincterotomy using a papillotome, the opening of major papilla was dilated with a Hurricane balloon catheter (Boston Scientific Corp., 10 mm in diameter) to insert a heat probe catheter. Then, a 7 Fr heat probe (Olympus America, Inc) was inserted and positioned in the common bile duct (CBD) under the guidance of fluoroscopy. ERCP was performed by one expert who had previously performed ERCP more than 500 times. If the expert failed to successfully perform ERCP, then open laparotomy was performed for access to the bile duct. Open laparotomy was performed on the pigs in group B by one veterinarian. A median incision was made and
J.U. Shin et al. the duodenum was incised longitudinally. After inspection of the papilla, a biliary catheter and guidewire were inserted through the papilla under the guidance of fluoroscopy. Balloon dilatation with a Hurricane balloon catheter (Boston Scientific Corp., 10 mm in diameter) was performed after obtaining a baseline cholangiogram. A RFA electrode (CoATherm CTS electrode S1709, 17-gauge, 1 cm tip exposure, 90 mm total length; APRO KOREA Inc., Korea) was then inserted and positioned in the common bile duct under the guidance of fluoroscopy. Energy was delivered by an RFA generator (CoATherm RF-G200 generator; APRO KOREA Inc., Korea) operating at 480 kHz and a maximum power output of 200 W. After thermal injury to the common bile duct, the duodenum and abdominal wall were closed with a suture (Fig. 1). The energy doses used to induce thermal injury were based on a study which showed that the application of a heat probe at 10 or 15 J (1 J = 1 W per second) produced a stricture in all pigs, whereas the application of a multipolar probe at 12, 16, 20, or 24 J produced a stricture in only one of six pigs [7]. Therefore, we decided to use an energy dose of 25 J for 40 seconds with a heat probe and energy doses of 20 W at 60 ◦ C for 30 seconds, 40 W at 80 ◦ C for 30 seconds, or 40 W at 100 ◦ C for 30 seconds with a radiofrequency catheter. During the 2 weeks (4 weeks for two of the pigs) following the procedure, animals were maintained on their usual diet. Clinical signs including weight loss, daily food intake, jaundice, and fever were monitored daily by a veterinary technician. Biochemical tests of liver function were assessed at baseline and 2 weeks after thermal injury. Follow-up ERCP with a heat probe was performed 2 weeks after thermal injury on the animals in group A. An uncovered metal stent was inserted in cases of bile duct stenosis, and the animal was sacrificed by potassium chloride overdose 4 weeks after thermal injury. For the animals in group B (thermal injury by open surgical RFA) and those animals in group A which had open bile duct surgery, euthanization by potassium chloride overdose was carried out 2 weeks after thermal injury. Before sacrifice, a veterinarian performed general anesthesia and opened the abdomen and duodenal wall. A final cholangiogram was obtained under fluoroscopy in all animals to identify bile duct strictures, then the liver and common bile duct were excised from each animal for gross examination. The common bile duct was opened longitudinally and examined for damage and tissue response. The duct was preserved in formalin and stained with hematoxylin-eosin (H&E) and Masson’s trichrome. An experienced gastrointestinal pathologist evaluated the extent of inflammatory cell infiltration, fibrous changes, and epithelial injury.
Results Induction of thermal injury and clinical follow-up Thermal injuries with a heat probe were administered to the three pigs of group A by ERCP (two pigs) or by open surgery (one pig) (Table 1). For the first pig, thermal injury by ERCP with a heat probe was successful. However, 2 weeks later, the pig showed poor oral intake and decreased general activity. Dilatation of the common bile duct was confirmed on the cholangiogram obtained by ERCP (Fig. 2A).
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Figure 1 A. A heat probe was inserted into the common bile duct under fluoroscopy. B. A picture of a heat probe showing the plastic catheter with an electrode at the tip. C. Balloon dilatation of papilla was performed by open laparotomy. D. Bile duct cannulation was performed by endoscopy. E. A picture of a radiofrequency ablation electrode showing the 90 mm needle and 1 cm electrode at the tip. F. A radiofrequency ablation electrode was inserted into the common bile duct under fluoroscopy after open laparotomy.
A covered metal stent (80 mm in length, 10 mm in diameter; Hanarostent® , M.I. Tech, Korea) was inserted into the CBD and intramuscular antibiotics were administered. Laboratory data showed elevated total bilirubin (2.8 mg/dL) (Table 2). The day before the 4-week time point after thermal injury, the pig died. An autopsy was performed on the pig for gross and microscopic examination of the common bile duct. Thermal injury by ERCP failed in the second pig, therefore, open surgery with a heat probe was performed. Two weeks later, the pig was euthanized despite no abnormal
symptoms. The cholangiogram taken just prior to euthanization confirmed CBD dilatation (Fig. 2B). However, laboratory tests showed only a mild elevation of alkaline phosphatase (ALP) (Table 2). For the third pig, thermal injury by ERCP with a heat probe was successful. Two weeks after thermal injury, the pig did not show any abnormal symptoms. ERCP was performed to obtain a cholangiogram but failed. There were no abnormal findings in the laboratory tests except for mild elevation of ALP. Four weeks after thermal injury, the pig developed symptoms of poor oral intake and
Table 1 Summary of thermal injury method and the result. All of three cases with heat probe resulted in bile duct stenosis. Radiofrequency ablation (RFA) with low energy dose (20 W 60 ◦ C 30 sec) failed to make bile duct stenosis, and RFA with high energy and high temperature (40 W 100 ◦ C 30 sec) developed bile duct stenosis with complication of bile duct perforation and abscess formation.
Pig Pig Pig Pig Pig Pig
1 2 3 4 5 6
Biliary approach
Thermal injury
Stenosis
Cholangitis
Progress
ERCP Surgery ERCP Surgery Surgery Surgery
Heat probe (25 J 40 sec) Heat probe (25 J 40 sec) Heat probe (25 J 40 sec) RFA (20 W 60 ◦ C 30 secs) RFA (40 W 80 ◦ C 30 sec) RFA (40 W 100 ◦ C 30 sec)
Success Success Success Failure Success Success
Develop Develop Develop None Develop Abscess
Death at 4 wks Autopsy at 2 wks Autopsy at 4 wks Autopsy at 2 wks Autopsy at 2 wks Autopsy at 2 wks
ERCP: endoscopic retrograde cholangiopancreatography.
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J.U. Shin et al.
Figure 2 A. Cholangiogram of pig 1 shows bile duct dilatation and a metal stent (between black arrows). B. Cholangiogram of pig 2 shows a stricture of the common bile duct (dark arrow) and bile duct dilatation. C. Cholangiogram of pig 3 shows abrupt stenosis of the common bile duct (dark arrow) and severe bile duct dilatation. D. Gross morphology of the common bile duct in pig 1 shows a dark pigmented fibrosis band (white arrow) with an uncovered metal stent. E. A picture of the common bile duct of pig 2 showing a whitish fibrotic area (white arrow) and hyperemic mucosal inflammation above the level of stenosis. F. A picture of the common bile duct of pig 3 showing a whitish fibrotic area (white arrow) and severe bile duct dilatation with mucosal inflammation.
vomiting. The cholangiogram obtained under open laparotomy with general anesthesia confirmed CBD dilatation (Fig. 2B), and laboratory tests showed marked abnormalities (Table 2). The pig was then euthanized. Thermal injuries with a radiofrequency electrode were administered to the three pigs of group B (Table 1). The electrode was prepared for RFA of hepatocellular carcinoma and could not pass through the working channel of an endoscope. Therefore, all procedures were performed by
Table 2
The results of laboratory test after intraductal thermal injury. T-bilirubin (mg/dL)
Pig Pig Pig Pig Pig Pig
1 2 3 4 5 6
open laparotomy under general anesthesia. Three different energy doses were tested for the induction of thermal injury. Thermal injury was administered to the fourth pig with an energy dose of 20 W at 60 ◦ C for 30 seconds. Two weeks later, a cholangiogram was taken and the pig was euthanized. The cholangiogram showed no definite signs of stenosis or dilatation (Fig. 3B). For the other two pigs in group B (fifth and sixth pigs), energy doses of 40 W at 80 ◦ C for 30 seconds and 40 W at 100 ◦ C for 30 seconds were applied, respectively. Two
AST (IU/L)
ALT (IU/L)
ALP (IU/L)
0 wk
2 wks
4 wks
0 wk
2 wks
4 wks
0 wk
2 wks
4 wks
0 wk
2 wks
4 wks
NA NA NA 0.2 0.3 0.2
2.8 0.5 0.3 0.5 0.5 0.2
NA NA 2.7 NA NA NA
NA NA NA 22 24 20
52 22 30 25 36 25
NA NA 46 NA NA NA
NA NA NA 51 29 29
14 21 21 36 29 26
NA NA 20 NA NA NA
NA NA NA 243 303 216
137 446 408 350 314 258
NA NA 392 NA NA NA
T-bilirubin: total bilirubin (normal range: 0.1—0.5 mg/dL); AST: aspartate transaminase (normal range, 17—44 IU/L); ALT: alanine transaminase (normal range, 17—78 IU/L); ALP: alkaline phosphatase (normal range, 69—333 IU/L); NA: not applicable; wk: week.
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163
Figure 3 A. Baseline cholangiogram of pig 4 shows a normal bile duct. B. Cholangiogram of pig 4, 2 weeks after thermal injury, shows no bile duct dilatation. C. Gross morphology of pig 4 shows linear hyperemic mucosal inflammation without fibrosis or stenosis. D. Microscopic findings of pig 4 show mildly increased fibrosis with regenerating mucosa and inflammation in the mucosal layer. E. Baseline cholangiogram of pig 5 shows a normal bile duct. F. A linear stenosis line (white arrow) with mild common bile duct dilatation is seen on the cholangiogram of pig 5, 2 weeks after thermal injury. G. Gross morphology of pig 5 shows a fibrotic lesion (white arrow) and diffuses mucosal hyperemic inflammation. H. Microscopic findings of pig 5 show an increase in diffuse fibrosis through the entire wall and denudated mucosa with inflammation. I. Baseline cholangiogram of pig 6 shows a normal bile duct. J. Cholangiogram of pig 6, 2 weeks after thermal injury, shows bile duct stenosis (white arrow) with severe dilatation. K. Gross findings of pig 6 show a distal CBD stricture with complications of bile duct perforation and periductal abscess formation (white arrow). L. Microscopic findings of pig 6 show extensive necrosis (white arrow) driven by perforation of the bile duct.
weeks after thermal injury, CBD stenosis with dilatation was confirmed in each of the pigs (Figs. 3F and J). Both pigs were euthanized after cholangiography and showed no abnormal symptoms or laboratory test results (Table 2).
Gross and microscopic findings During the gross examination of extrahepatic bile ducts, the CBD of the first pig was filled with a pus-like yellowish sludge, and there was a dark stricture-like band on the mid-CBD (Fig. 2D). The CBD of the second pig showed a whitish fibrous band with less severe diffuse mucosal hyperemia above the level of stenosis (Fig. 2E). The third pig showed extensive yellowish sludge inside of the CBD and a whitish fibrous band and diffuse reddish mucosal inflammation inside of the extrahepatic bile duct (Fig. 2F). The CBD of the fourth pig showed a hyperemic band, resembling mucosal inflammation, without stenosis or scar tissue. (Fig. 3C). Fibrosis at the mid-level of the CBD and diffuse hyperemic mucosal inflammation were seen inside of the CBD in the fifth pig (Fig. 3G). There was a stricture with
perforation and abscess formation on the lower level of the CBD in the sixth pig (Fig. 3K). Microscopic findings of thermal injury using RFA were compared in the three pigs in group B. Microscopic findings in the fourth pig, which received the lowest energy dose, showed mildly increased fibrosis with regenerating mucosa and inflammation in the mucosal layer (Fig. 3D). The fifth pig showed an increase of diffuse fibrosis through the entire wall and denudated mucosa with inflammation (Fig. 3H). The microscopic findings of the sixth pig, which received the highest energy dose and temperature, showed extensive necrosis driven by perforation of the bile duct (Fig. 3L).
Discussion This study showed that heat probe and RFA were both effective at producing bile duct strictures, and we found effective and safe energy dose of heat probe and RFA to produce benign biliary stenosis. Rumella et al. reported that application of a heat probe at 15 J could produce bile duct stenosis in all animals, but
164 application of a multipolar probe at 12 W for 2 seconds failed to produce stenosis in most animals [7]. In the current study, we investigated whether development of an animal model for bile duct stenosis by thermal injury with a heat probe was reproducible and if higher energy doses would be safe. Higher energy doses with a heat probe (25 J for 40 seconds) produced bile duct stenosis and cholangitis in all three pigs of group A, confirmed 2 and 4 weeks after thermal injury. However, the first pig in group A died 4 weeks after thermal injury, despite stent insertion. The lack of proper antibiotics and intravenous hydration might have been related to the death. Therefore, no further biliary stenting was attempted. RFA is generally used for the treatment of hepatocellular carcinoma (HCC). Energy doses for the treatment of HCC range from 60 to 120 W for 6 to 12 minutes [10,11]. Endoscopic RFA has been applied for the treatment of Barrett’s esophagus with dysplasia [12—14]. Energy delivery typically lasts less than 1.5 seconds, and an energy dose of 2 × 15 J/cm2 is the most effective regimen for circumferential, full-thickness ablation of the epithelium without injuring the submucosa [15,16]. Recently, an endobiliary RFA device (HabibTM EndoHPB, EMcision Ltd, UK, London) that is compatible with standard side viewing endoscopes and passes over 0.035-inch guidewires was developed and investigated in an animal model [8]. This study used energy doses of 5−10 W for a maximum of 2 minutes. At higher power settings and longer ablation times, difficulties were encountered reintroducing endoHPB into the biliary tree. Also, a pilot study that used this device in patients with malignant biliary obstruction reported a 90-day biliary patency. The energy dose used in this study was set at 400 kHz and ranged from 5−10 W for 2 minutes [9]. The energy doses and ablation times delivered by the RFA device in our study were different from ones used in the above two studies [8,9]. Current study used a monopolar RFA electrode, which is commonly used in the treatment of HCC. The following energy doses were used in our study: 20 W at 60 ◦ C for 30 seconds, 40 W at 80 ◦ C for 30 seconds, and 40 W at 100 ◦ C for 30 seconds. These energy doses were based on those reported for the treatment of primary and secondary hepatic tumors. Results from our study showed that an energy dose of 20 W at 60 ◦ C for 30 seconds produced mucosal injury without bile duct stenosis, whereas a dose of 40 W at 100 ◦ C for 30 seconds caused complications including perforation of the common bile duct and periductal abscess formation. Therefore, the optimal energy dose for bile duct stenosis using RFA was 40 W at 80 ◦ C for 30 seconds (total energy: 120 J = 40 W/sec × 30 seconds). The other two studies (mentioned above) applied a lower dose of energy (5−10 W) for a longer time (2 minutes) (total energy: 120 J = 10 W/sec × 120 seconds) [8,9]. The mode of energy delivery was different between our study and the other studies, but the total cumulative energy was similar. Macatula et al. reported that RF ablation with low power (less than 120 W for 12 minutes) for the treatment of HCC could achieve comparable clinical outcomes with fewer adverse effects compared with maximal power (more than 120 W) [17]. Also, Komorizino et al. demonstrated successful treatment of HCC after a single session of RF ablation using a maximum power of 90 W [18]. Therefore, for the treatment of intra-bile duct malignancies, energy doses lower than the doses required for bile duct stenosis may be sufficient.
J.U. Shin et al. However, further research on the energy dose required for treatment of bile duct malignancies by RF ablation is still needed. Short observation time and small number of pigs are limitations of this study. The animals were observed only for 2 weeks. That was because in cases of surgical stricture and cholangitis, it was almost impossible to keep them alive for more than 2 weeks by making them have food or maintain IV fluid. Nowadays endobiliary RFA devices were developed but it was available in few centers only for research purpose. We couldn’t help but perform CBD coagulation through surgical approach with conventional RFA devices, which cannot be inserted into endoscopic working channel. The swines had to be sacrificed 2 weeks after formation of biliary strictures. This study was planned as a pilot study; therefore, we performed experiments with a minimum number of animals. This study showed reproducible animal model for benign biliary stenosis and proper energy dose. Reliable methods for artificial biliary stenosis could help the development of new treatments for benign biliary strictures. Also, application of RFA within the bile duct might be promising for the treatment of malignant biliary obstruction, however more investigations are needed.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Funding: This study had financial supports by M.I. Tech, Korea.
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