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Comparison of Systemic Responses of Radiofrequency Ablation, Cryotherapy, and Surgical Resection in a Porcine Liver Model

From the Departments of Surgery (KKN, CML, RTP, JYT, YHW, DWH, STF) and Pathology (TWS) and the Centre for the Study of Liver Disease, The University of Hong Kong, Pokfulam, Hong Kong, China.

Correspondence: Address correspondence and reprint requests to: Kelvin K. Ng, MBBS (HK), FRCSEd (Gen), Department of Surgery, University of Hong Kong Medical Centre, Queen Mary Hospital, 102 Pokfulam Road, Hong Kong, China; Fax: 852-28-17-5475; E-mail: kcng66{at}yahoo com.

	ABSTRACT

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Background: The degree of systemic response after hepatic radiofrequency ablation (RFA) has not been well investigated.

Methods: An in vivo study was conducted on 23 domestic swine. Different hepatic procedures (RFA, cryotherapy, hepatic pedicle ligation, and hepatectomy) were performed on the medial lobe of the liver (30% of the liver volume). Systemic responses in terms of systemic inflammatory marker changes and end-organ functions were determined.

Results: During the early postoperative period, the systemic inflammatory marker concentrations (tumor necrosis factor- and interleukin-1ß) in the RFA group were significantly lower than in the cryotherapy group but significantly higher than in the control group. The corresponding concentrations in the hepatectomy group remained similar to those in the control group. The pattern of changes of serum inflammatory marker concentrations in the pedicle ligation group followed the pattern in the RFA group. The serum intracellular content concentrations (lactate dehydrogenase and urate) of the cryotherapy group peaked at 6 hours after operation, which was significantly later than in the other groups. Liver function, renal function, and coagulation profiles remained normal in the RFA group. However, the renal function deteriorated in the cryotherapy group on day 1. Both platelet count and activated clotting time showed significant derangement in the cryotherapy group compared with the control group. There was more severe interstitial pneumonitic change of the porcine lung after cryotherapy than after RFA.

Conclusions: The systemic responses of RFA were significantly less severe than those of cryotherapy in this porcine model. However, the increase in serum inflammatory markers and pneumonitis after RFA was substantial when compared with hepatectomy.

Key Words: Systemic • Responses • Radiofrequency • Ablation • Cryotherapy

	INTRODUCTION

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With recent advances in localized thermal ablative therapies for malignant liver tumors, much interest has been developed regarding the safety and efficacy of these treatment modalities for unresectable malignant liver tumors.^1–4 Hepatic cryotherapy, a local ablative therapy that uses subfreezing temperatures, carries a potentially lethal complication known as cryoshock phenomenon. This is a syndrome of multiorgan failure and severe coagulopathy without evidence of sepsis. It was postulated that the cryoshock phenomenon was related to the release of toxic substances from the cryolesion, which caused a systemic inflammatory reaction after the thawing process of the cryoablation.⁵ Clinical studies have shown that cryoablation of 30% to 35% of the liver volume is associated with this cryoshock phenomenon, which carries a high mortality rate (18%).^6,7 Radiofrequency ablation (RFA) uses thermal energy to induce cellular destruction of the liver tumor in situ. Although the complication rate after RFA is lower than that after cryotherapy in clinical series,^8,9 there are scarce data in the literature on the systemic effects of hepatic RFA. It has been shown in a rat model that hepatic cryotherapy, but not RFA, is associated with lung inflammation.¹⁰ Nevertheless, other possible components of the systemic inflammatory reaction, such as coagulopathy and inflammatory changes in the liver remnant and the kidney, were not studied in detail.

It is believed that the systemic inflammatory responses of thermal ablative therapies could be related to the presence of necrotic tissue left in the liver remnant. However, there is insufficient scientific evidence to support this hypothesis. Because RFA is being increasingly used in patients with unresectable liver tumors, its potential systemic inflammatory effects need to be evaluated before we can vigilantly adapt this therapy. This study investigated the systemic responses of hepatic RFA in comparison to cryotherapy and surgical resection of the same liver volume by using a porcine liver model.

	MATERIALS AND METHODS

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An in vivo study was conducted on 23 domestic swine (weight, 35–40 kg). Approval of the study protocol was obtained from the Ethics Committee on the Use of Live Animals for Teaching and Research of the University of Hong Kong. The animals were divided into five groups that received different treatments. The swine were kept fasting 8 hours before the experiment. During the experiment, intramuscular injections of 50 mg/kg of ketamine and .12 mg of atropine sulfate were given to each swine for anesthetic induction. The swine were anesthetized by intravenous injection of .5 mg/kg of midazolam and 4 mg of pancuronium. General anesthesia was maintained by endotracheal intubation with inhalation of 1% isoflurane with oxygen and nitric oxide at a ratio of 1:3. Cardiac and respiratory parameters were monitored throughout the experiment. Insertion of a hemodialysis catheter by a right external jugular vein cut-down technique was performed in all swine for subsequent blood sample collection. The hemodynamic status, oxygen saturation, and body temperature were monitored closely.

Dynamic Liver Function Test
Before laparotomy, the indocyanine green (ICG) clearance test (expressed as the percentage of retention at 15 minutes after injection) was performed for each swine as follows. ICG is a dye specifically excreted by the liver, and the ICG clearance test is commonly used in assessing liver function before hepatic surgery.¹¹ The freeze-dried ICG powder (25 g) was mixed with 5 mL of sterile water. A serum sample (5 mL) was taken for baseline measurement. Then .1 mL/kg of ICG solution was injected into the swine through the jugular vein catheter. Serial serum samples (5 mL) were taken at various time points (5, 10, 15, and 20 minutes) after the ICG solution was given. All of the collected serum samples were centrifuged at 3000 rpm for 15 minutes. The ICG concentration of the supernatant was determined by using a spectrophotometer at a wavelength of 805 nm. The ICG concentration for serum samples at 5, 10, 15, and 20 minutes was divided by the denominator (baseline ICG concentration). The percentage of ICG retention at 15 minutes was used as the reference value to evaluate the dynamic liver function of all swine before and after different hepatic procedures.

Operative Procedures
Laparotomy was performed via a midline incision. The nonvascular attachment to the porcine liver was divided. The left medial lobe of the porcine liver (~30% of liver volume by weight; mean volume, 66.4 ± 2.5 mL) was isolated,¹² and the adjacent organs were packed away from the operative field (Fig. 1A). RFA with a clustered cooled-tip radiofrequency (RF) electrode (Cool-tip RF System; Radionics, Burlington, MA) was performed in group 1 (n = 5). The Cool-tip RFA system consists of an RF generator to produce an RF current of approximately 500 kHz at a maximal power of 200 W, an RF electrode of either single or clustered probe design, a water-pumping machine, and return ground pads. The RF electrode is 17 gauge, with internal dual channels for chilled water to be pumped through by the water pumping machine. The resulting cooling effect around the tip of the electrode (maximal exposed length of 3 cm) can reduce charring of the surrounding tissue, which would decrease tissue conductivity and block the RF current. The clustered electrode comprises three RF electrodes placed in close proximity to each other (.5 cm apart). The synergistic effect among the triple electrodes can effectively increase the final ablative volume. Repeated cycles of RFA procedures (12 minutes in each cycle) were performed in the selected liver lobe of each swine until the entire liver lobe, in which there was overlapping of ablative zones, was completely ablated (Fig. 1B).

View larger version (91K): [in this window] [in a new window]   FIG. 1. (A) The left medial lobe of porcine liver was selected for hepatic procedures (left, head end; right, foot end). Different hepatic procedures were performed with (B) radiofrequency ablation (Cool-tip radiofrequency system; A, ablated liver; E, radiofrequency electrode), (C) cryotherapy (Cryotech LC System 2000; A, ablated liver; C, cryoprobe), and (D) hepatic pedicle ligation (L, ischemic liver segment).

Outcome Measures
All swine were allowed to recover from anesthesia and were observed for 7 days. A normal diet was resumed on postoperative day 1, and all swine were examined once daily for general health status. Serum samples were taken for systemic inflammatory marker measurements (serum tumor necrosis factor [TNF]- and serum interleukin [IL]-1ß), intracellular content measurements (lactate dehydrogenase [LDH] and urate concentration), and assessment of organ function (liver biochemistry, creatinine concentration, platelet count, and activated coagulation time) before the operation and at various time points after the operation (6 hours, 1 day, 3 days, and 7 days). Serum TNF- and IL-1ß concentrations were measured with the swine TNF- colorimetric enzyme-linked immunosorbent assay kit (Pierce Endogen, Rockford, IL) and the swine IL-1ß enzyme-linked immunosorbent assay kit (BioSource International, Camarillo, CA), respectively. On postoperative day 7, all swine were anesthetized as described previously. The ICG clearance test was repeated before laparotomy. Representative tissue samples were collected from the liver remnant, kidney, and lung for paraffin sectioning and histological study with hematoxylin and eosin staining. All swine were killed by intravenous injection of 100 mg/kg of pentobarbitone.

Statistical Analysis
All continuous variables are expressed as mean ± SD. The SPSS statistical package version 10 (SPSS Inc., Chicago, IL) was used to analyze the results. The unpaired t-test and paired t-test were used to compare continuous variables between and within groups, respectively. A P value of <.05 was considered statistically significant.

	RESULTS

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All swine survived after the operation, and there was no major intraoperative or postoperative complication. There was evidence of tissue necrosis involving the entire selected liver lobe in groups 1, 2, and 3 on histological examination.

Systemic Inflammatory Responses
Changes in the mean serum TNF- concentration after different hepatic procedures (groups 1 to 5) are described in Fig. 2A. During the early postoperative period (6 hours after the operation), the mean serum TNF- concentration of group 1 (96.4 ± 12.3 pg/mL) was significantly lower than that of group 2 (220.7 ± 16.7 pg/mL; P = .021), but it was significantly higher than that of the control group (40.2 ± 8.8 pg/mL; P = .013). At this time point, the mean serum TNF- concentration of group 1 was similar to that of group 3 (P = .52). There was no significant change in the mean serum TNF- concentration of group 4 (55.8 ± 10 pg/mL) when compared with that of the control group (P = .65). Only group 2 had a significantly increased serum TNF- concentration on day 1 (130 ± 19.5 pg/mL) compared with the control group (35 ± 9.9 pg/mL; P < .001) and on day 3 (90.6 ± 17.8 pg/mL) compared with the control group (15.8 ± 5.4 pg/mL; P < .001), whereas all swine in the other groups (groups 1, 3, and 4) had serum TNF- concentrations similar to those of the control group. The pattern of changes in the mean serum IL-1ß concentration after different hepatic procedures was similar to that of changes in the mean serum TNF- concentration (Fig. 2B). At 6 hours after operation, the mean serum IL-1ß concentration of group 1 (88.3 ± 13.3 pg/mL) was significantly lower than that of group 2 (180.4 ± 18.7 pg/mL; P = .015), but it was significantly higher than that of the control group (37.8 ± 14 pg/mL; P = .021). The pattern of changes in the mean concentration of serum IL-1ß in group 3 followed that of the changes in group 1. There was no significant difference in the mean serum IL-1ß concentration between group 4 (45.6 ± 9.8 pg/mL) and the control group (P = .58).

View larger version (25K): [in this window] [in a new window]   FIG. 2. Changes in (A) serum tumor necrosis factor (TNF)- concentration and (B) serum interleukin (IL)-1ß concentration after different hepatic procedures. RFA, radiofrequency ablation; Gp, group; Preop, before surgery; conc, concentration.

View larger version (25K): [in this window] [in a new window]   FIG. 3. Changes in (A) serum lactate dehydrogenase (LDH) concentration and (B) serum urate concentration after different hepatic procedures. RFA, radiofrequency ablation; Gp, group; Preop, before surgery; conc, concentration.

View larger version (24K): [in this window] [in a new window]   FIG. 4. Changes in (A) serum bilirubin concentration, (B) serum creatinine concentration, (C) platelet count, and (D) activated clotting time after different hepatic procedures. RFA, radiofrequency ablation; Gp, group; Preop, before surgery; conc, concentration.

View larger version (95K): [in this window] [in a new window]   FIG. 5. Microscopic examination of porcine lung parenchyma showing (A) moderate pneumonitis with thickened alveolar septum (S) and preservation of alveolar space after hepatic radiofrequency ablation, (B) severe interstitial pneumonitis and airspace edema (E) after hepatic cryotherapy, and (C) normal histological appearance after hepatic resection.

	DISCUSSION

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Serum IL-1, IL-6, and TNF- were shown to be the possible cytokine mediators of the cryoshock syndrome after cryotherapy.¹⁵ In addition, the activation of transcription factor complex nuclear factor-B, a transcription factor for TNF-, IL-1, IL-2, IL-6, and IL-8, was found to be responsible for the acute lung injury after cryoablation.^5,10 In our experiment, hepatic cryotherapy was associated with a significantly higher level of serum IL-1ß and TNF- than hepatic RFA or resection during the early postoperative period (from 6 hours to 3 days after surgery). Moreover, the increase in serum intracellular contents (LDH and urate) after cryotherapy reached the peak at 6 hours after the cryoablative process, and this suggests a release of cellular products at this early postoperative period. Our results showed that the resulting systemic inflammatory responses mostly affected the porcine lung and coagulation cascade, as evidenced by the acute pneumonitic change and deranged coagulation profile (thrombocytopenia and prolonged activated clotting time). The lung parenchyma, which is rich in tissue macrophages, is the major organ involved in this inflammatory response, and this is compatible with the clinical observation of acute respiratory distress syndrome. Likewise, the coagulation derangement may be the result of disseminated intravascular coagulation triggered by the inflammatory cascade. After hepatic cryotherapy, there was no significant deterioration of liver function in terms of liver biochemistry or the ICG clearance test. Nevertheless, the renal function worsened on postoperative day 1. The exact mechanism of this renal impairment is unknown. It might be related to the accumulation of cellular products in the renal tubules causing acute tubular necrosis, although there was no significant histological kidney change in this experiment. In fact, myoglobinemia and the resulting myoglobinuria were suggested to be the possible causes of transient renal failure after cryotherapy.¹⁶

In contrast, hepatic RFA of 30% to 35% of the porcine liver volume did not induce severe organ injuries in the form of lung inflammation or coagulation disorders. Although there was no significant change in organ function after hepatic RFA, a significant increase in systemic inflammatory markers (serum IL-1ß and TNF-) was detected at 6 hours after the procedure. Chapman et al.¹⁰ demonstrated that there was no evidence of acute lung inflammation in rats that underwent 35% liver ablation with RFA, as compared with severe acute lung injury after hepatic cryotherapy of the same liver volume. However, our experiment showed that the pneumonitic change of the porcine lung after hepatic RFA was substantial when compared with the liver resection and control groups, although it was less severe than that after cryotherapy. To our knowledge, this is the first report to demonstrate systemic inflammatory responses after hepatic RFA procedures of 30% to 35% of the liver volume. The reasons for the different inflammatory responses after RFA and cryotherapy could be related to the underlying ablative mechanisms. There was much similarity between the hepatic RFA group and the hepatic pedicle ligation group in terms of changes in the serum IL-1ß and TNF- concentrations. Unlike with cryotherapy, the thermal injury by RFA results in coagulation, cellular protein denaturation, and subsequent apoptosis. There was destruction of intracytoplasmic organelles with relatively intact cytoplasmic membranes after RFA, compared with the intact cytoplasmic organelles with disruption of the plasma membrane after cryoablation under electron microscopy.¹⁰ Hence, it would be expected that cryotherapy could result in a release of intracellular products through the leaking plasma membrane into the systemic circulation, and this may not be the case in RFA. Moreover, Kupffer cells within the ablated area, which was involved in the release of systemic cytokines, would be subjected to thermal injury by RFA and rendered inactive. In addition, immediate thrombosis of small and medium-sized blood vessels induced by RFA might prevent the further release of breakdown cellular products into the systemic circulation.¹⁷ With these similar mechanisms, hepatic pedicle ligation might result in an infarcted liver segment, which could mimic the RF-ablated area in this experiment.

Hepatic resection of 30% to 35% of the porcine liver volume seemed safe in our study as far as the systemic inflammatory responses were concerned. Nevertheless, one should be cautious when interpreting this observation, because hepatectomy in clinical practice involves more complicated procedures than those in our experiment, such as hilar dissection and mobilization of liver lobes. Hepatic resection and the resulting surgical trauma may induce more severe systemic inflammatory reactions than those estimated in this experiment.¹⁸

The main limitation of this study was the use of healthy liver tissue for different hepatic procedures. Cirrhotic liver is commonly encountered in patients with unresectable liver tumors who are undergoing local ablative therapy. Hence, experiments that use similar approaches in cirrhotic liver and liver tumor models will be the next step to further evaluate systemic inflammatory reactions after different ablative therapies. Furthermore, the dose-response change of systemic inflammation after hepatic RFA has not been studied in this experiment, and the maximal host tolerance to large-volume RFA remains to be investigated.

In conclusion, this in vivo experiment has demonstrated that the systemic responses of hepatic RFA were significantly less severe than those of cryotherapy, which caused severe interstitial pneumonitis and coagulation derangement. However, the increase in serum inflammatory markers and, hence, pneumonitic changes after hepatic RFA was substantial when compared with that after hepatic resection and sham operation.

	ACKNOWLEDGMENTS

 
The acknowledgments are available online in the full-text version at www.annalssurgicaloncology.org. They are not available in the PDF version.

	FOOTNOTES

 
An in vivo experiment showed severe systemic inflammatory responses after hepatic cryotherapy when compared with radiofrequency ablation (RFA). However, the increase in serum systemic inflammatory marker concentrations and pneumonitis after RFA was substantial compared with that after hepatic resection.

Received for publication October 29, 2003. Accepted for publication March 29, 2004.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Shiina S, Teratani T, Obi S, Hamamura K, Koike Y, Omata M. Nonsurgical treatment of hepatocellular carcinoma: from percutaneous ethanol injection therapy and percutaneous microwave coagulation therapy to radiofrequency ablation. Oncology 2002; 62 (Suppl 1): 64–8.
2. Primrose JN. Treatment of colorectal metastases: surgery, cryotherapy, or radiofrequency ablation. Gut 2002; 50: 1–5.[Free Full Text]
3. Curley SA. Radiofrequency ablation of malignant liver tumors. Ann Surg Oncol 2003; 10: 338–47.[Abstract/Free Full Text]
4. Bleicher RJ, Allegra DP, Nora DT, Wood TF, Foshag LJ, Bilchik AJ. Radiofrequency ablation in 447 complex unresectable liver tumors: lessons learned. Ann Surg Oncol 2003; 10: 52–8.[Abstract/Free Full Text]
5. Blackwell TS, Debelak JP, Venkatakrishnan A, et al. Acute lung injury after hepatic cryoablation: correlation with NF-kappa B activation and cytokine production. Surgery 1999; 126: 518–26.[Medline]
6. Sarantou T, Bilchik A, Ramming KP. Complications of hepatic cryosurgery. Semin Surg Oncol 1998; 14: 156–62.[CrossRef][Medline]
7. Seifert JK, Morris DL. World survey on the complications of hepatic and prostate cryotherapy. World J Surg 1999; 23: 109–13.[CrossRef][Medline]
8. Pearson AS, Izzo F, Fleming RY, et al. Intraoperative radiofrequency ablation or cryoablation for hepatic malignancies. Am J Surg 1999; 178: 592–9.[CrossRef][Medline]
9. Adam R, Hagopian EJ, Linhares M, et al. A comparison of percutaneous cryosurgery and percutaneous radiofrequency for unresectable hepatic malignancies. Arch Surg 2002; 137: 1332–9.[Abstract/Free Full Text]
10. Chapman WC, Debelak JP, Wright PC, et al. Hepatic cryoablation, but not radiofrequency ablation, results in lung inflammation. Ann Surg 2000; 231: 752–61.[CrossRef][Medline]
11. Lau H, Man K, Fan ST, Yu WC, Lo CM, Wong J. Evaluation of preoperative hepatic function in patients with hepatocellular carcinoma undergoing hepatectomy. Br J Surg 1997; 84: 1255–9.[CrossRef][Medline]
12. Court FG, Wemyss-Holden SA, Morrison CP, et al. Segmental nature of the porcine liver and its potential as a model for experimental partial hepatectomy. Br J Surg 2003; 90: 440–4.[CrossRef][Medline]
13. Bischof JC, Rubinsky B. Large ice crystals in the nucleus of rapidly frozen liver cells. Cryobiology 1993; 30: 597–603.[Medline]
14. Brown NJ, Bayjoo P, Reed MW. Effect of cryosurgery on liver blood flow. Br J Cancer 1993; 68: 10–2.[Medline]
15. Seifert JK, Stewart GJ, Hewitt PM, Bolton EJ, Junginger T, Morris DL. Interleukin-6 and tumor necrosis factor-alpha levels following hepatic cryotherapy: association with volume and duration of freezing. World J Surg 1999; 23: 1019–26.[CrossRef][Medline]
16. Weaver ML, Atkinson D, Zemel R. Hepatic cryosurgery in treating colorectal metastases. Cancer 1995; 76: 210–4.[CrossRef][Medline]
17. Schell SR, Wessels FJ, Abouhamze A, Moldawer LL, Copeland EM III. Pro- and antiinflammatory cytokine production after radiofrequency ablation of unresectable hepatic tumors. J Am Coll Surg 2002; 195: 774–81.[Medline]
18. Namekata K, Takamori S, Kojima K, Beppu T, Futagawa S. Significant changes in the serum levels of IL-6, h-HGF, and type IV collagen 7S during the perioperative period of a hepatectomy: relevance to SIRS. Surg Today 2000; 30: 403–9.[Medline]

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