Author Affiliations: Department of Hepatobiliary Surgery (Shrestha A, Zhou Y, Li FY, Ma WJ, Cheng NS, Xu RH, Zhang YQ, Jiang T, Feng H, Li W and Han Q), and Department of Medicine (Mao H), West China Hospital of Sichuan University, Chengdu 610041, China

 

Corresponding Author: Hui Mao, MD, Department of Medicine, West China Hospital of Sichuan University, Chengdu 610041, China (Tel: 86-28- 85422465; Fax: 86-28-85422468; Email: lfy_74@vip.163.com)

 

© 2014, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(14)60007-2

 

 

Contributors: MH and LFY designed the research. SA and ZY established the animal model and performed histological observation. MWJ, CNS, XRH, ZYQ and JT performed the biochemistry. FH, LW and HQ performed Western blotting. SA and ZY contributed equally to this paper. LFY is the guarantor.

Funding: This work was supported by grants from the National Nature Science Foundation of China (30801111 and 30972923); Science & Technology Support Project of Sichuan Province (10SZ0166, 14ZC1337 and 14ZC1335).

Ethical approval: Not needed.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

 

 

BACKGROUND: The high recurrence rate of hepatolithiasis and the high operative risk of right posterior, caudate or multiple lobe hepatectomy are the unsettled problems in hepatobiliary surgery. The present study was to investigate the efficacy of chemical hepatectomy performed via applying sequential embolization of the branches of the bile duct and portal vein to the targeted hepatic lobe.

 

METHODS: The bile duct and portal vein branches of the median hepatic lobe of rats were treated with: 1) bile duct embolization followed by portal vein ligation (BDE+PVL) and 2) portal vein ligation followed by bile duct embolization (PVL+BDE). The efficacy of chemical hepatectomy in BDE+PVL and PVL+BDE groups was compared with that of sole BDE by histology and Western blotting analysis of collagen I expression.

 

RESULTS: After six weeks of the chemical hepatectomy, rats in the BDE group showed hepatocyte damages, fibrosis and "self-cut" only in the periphery of the embolized lobe. In contrast, rats in the PVL+BDE and BDE+PVL groups exhibited complete necrosis of hepatocytes and replacement with proliferative ductules and collagen fibers, leading to complete fibrosis and "self-cut" phenomenon in the whole targeted lobe. Collagen I expression in the PVL+BDE group was slightly higher than that in the BDE+PVL group; however, no statistically significant difference was noted.

 

CONCLUSION: The sequential embolization of the bile duct and portal vein branches to the targeted hepatic lobe may be a feasible and effective approach to acheive the ideal effect of chemical hepatectomy in a short period of time.

 

(Hepatobiliary Pancreat Dis Int 2014;13:55-59)

 

KEY WORDS: hepatolithiasis; bile duct; portal vein; sequential embolization; chemical hepatectomy

The high recurrence rate of hepatolithiasis together with the high operative risk of hepatectomy is one of the most intractable and unresolved problems in hepatobiliary surgery. In recent years, application of choledochoscopy has partly improved hepatolithiasis treatment. However, 33.9% of the calculi could be complicated with stenosis of secondary or even smaller subsidiary biliary ducts. This kind of calculi are not easy to remove completely, and have a recurrence rate of 4.2%-40.0% in 4-10 years after operation, thus triggering the reoperation rate as high as 37.1%-74.4%.^{[1, 2]} There is no effective method for the prevention of intrahepatic bile duct stenosis and stone recurrence available. Application of conventional hepatectomy for the treatment of stones and biliary duct stenosis localized in one segment or in one lobe can achieve good result in 91.2% of the patients. However, hepatectomy alone cannot eradicate hepatolithiasis, as about 16.0% of the patients undergoing hepatectomy may have recurrence of stones at other site in the liver. Moreover, conventional hepatectomy, especially of the right posterior lobe and caudate lobe carries a significant operative risk. Thus, new therapeutic approach for the treatment and prevention of hepatolithiasis and its recurrence is urgently required.^{[3, 4]}

 

Diseased bile duct is the location where lithogenesis is inevitable and blockage of this bile duct leads to atrophy, fibrosis and "self-cut" of that liver segment. Li et al^[5] explored the feasibility and efficacy of biliary embolization agent in the lumen of diseased bile duct to prevent stone recurrence and found that application of biliary embolization agent into the lumen of diseased bile duct not only eradicates the pathological cause of stone recurrence, i.e. chronic proliferative cholangitis, but also results in atrophy, fibrosis and "self-cut" of diseased segment of the liver, achieving the targeted "chemical hepatectomy" which avoids the risk and trauma of traditional liver resection.^{[2, 5]} The embolized bile duct obviously distends and thereby directly compresses the adjacent blood vessels, causing decreased blood flow to the targeted lobe. However, incomplete compression may slow the process of atrophy, fibrosis and "self-cut". In view of this, sequential embolization of the branches of the bile duct and portal vein to the target lobe might accelerate the process of chemical hepatectomy.^[6-9] In this study, we intended to explore the efficacy and feasibility of: (1) bile duct embolization followed by portal vein ligation (BDE+PVL); and (2) portal vein ligation followed by bile duct embolization (PVL+BDE) compared with simple BDE on the desired effect of chemical hepatectomy for the targeted hepatic lobe in a short period of time.

 

 

Fifty-one Sprague-Dawley rats, weighing 220-250 g provided by the Experimental Animal Center of Sichuan University (Chengdu, China), were randomly divided into the following four groups:

 

(i) Bile duct embolization followed by portal vein ligation (BDE+PVL, n=15): The biliary duct and portal vein branches of the median hepatic lobe were identified. Stay suture was applied to the portal vein and finally fixed to the anterior abdominal wall. The median biliary duct was doubly ligated. Subsequently, the median biliary duct was injected with 0.15 mL of N-butyl α-cyanoacrylate (Tissue adhesive glue, Jingya Co., Beijing, China) rapidly under pressure via a T26G PTFE catheter (Albboth Co., Chicago, IL, USA). One week after the procedure, the abdomen was re-opened, and the portal vein was ligated.

 

(ii) Portal vein ligation followed by bile duct embolization (PVL+BDE, n=15): The first portal branch of the median hepatic lobe was ligated, the abdomen was re-opened after one week, and then median biliary duct was ligated and injected with 0.15 mL of N-butyl α-cyanoacrylate.

 

(iii) Biliary duct embolization (BDE, n=15): The median biliary duct was doubly ligated and injected with 0.15 mL of N-butyl α-cyanoacrylate.

 

(iv) Sham-operated (SO, n=6): the median biliary duct was dissected.

 

Blood samples were collected from the tail veins of rats weekly for five weeks. Alanine transaminase (ALT) and total bilirubin (TB) were measured using commercially available kit (Nanjing Bioengineering Co., Nanjing, China).

 

On the 6th week, all rats were sacrificed. The liver samples of the rats were taken and fixed in liquid nitrogen and 10% formaldehyde. Five micrometer thick formalin-fixed liver tissue slides were prepared for HE staining.

 

One hundred mg of extracted protein was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and then electrophoretically transferred to a polyvinylidine difluoride (PVDF) membrane and hybridized. The membrane was blocked with 5% non-fat dry milk for 1 hour then incubated with collagen I primary antibody (Zymed Co., South San Francisco, CA, USA) overnight at 4 ��. After rinsing with Tris-buffered saline and Tween 20 (TBST), the membrane was incubated for 2 hours with horseradish peroxidase (HRP)-conjugated secondary antibody. Immunoreactive bands were visualized with enhanced chemiluminescence and captured on X-ray film. The result was expressed as the ratio of the expression of target gene to that of β-actin.

 

All the analyses were made using SPSS 10.0 software (SPSS Inc., Chicago, IL, USA). The quantitative data were expressed as mean±SD. One-way analysis of variance (ANOVA) was used for comparisons among means, and Fisher's least significant difference (LSD) analysis was used for within group comparisons. P<0.05 was considered statistically significant.

 

 

During the first 4 weeks, ALT and TB levels in the BDE+PVL and PVL+BDE groups were increased when compared with those in the SO group. However, by weeks 4-5, ALT and TB levels in the BDE+PVL and PVL+BDE groups returned to normal or were slightly higher than normal values, with no significant difference from those in the SO group (P>0.05, Fig. 1).

 

In the PVL+BDE and BDE+PVL groups, hepatocytes disappeared completely and were replaced by proliferative ductules and collagen fibers, leading to complete fibrosis and "self-cut" in almost the whole embolized lobe (Fig. 2A, B). In the BDE group, hepatocytes vanished almost completely and bile duct hyperplasia with fibrosis was seen only in the peripheral region giving effect of "self-cut" in the peripheral region of the target lobe. Large islands of residual liver cells were still visible in the target lobe except in the peripheral area (Fig. 2C). In the PVL+BDE and BDE+PVL groups, the degree of fibrosis of embolized target liver lobe was similar but there was slight difference in histological appearance. In the BDE+PVL group, hyperplasia of bile ducts was obvious and its degree of proliferation was greater than that of collagen fiber proliferation along with the presence of cholestasis in the targeted liver segment. In the PVL+BDE group the degree of collagen fiber proliferation was more obvious than that of bile duct hyperplasia, and no obvious cholestasis was seen. No liver abscess was seen during the entire experiment.

 

Collagen I protein expression in the BDE+PVL and PVL+BDE groups was found to be 5-6 times higher than that in the BDE group, and about 20-25 times higher than that in the SO group (P<0.05). Collagen I expression in the PVL+BDE group was slightly higher than that in the BDE+PVL group, but no statistically significant difference was noted (P>0.05, Fig. 3).

 

 

Hepatolithiasis is a commonly encountered condition in the Asia-Pacific region, but it is still regarded intractable as the long-term effects of current available therapeutic approaches are far from satisfactory.^{[1, 10]} Calculi in secondary or tertiary subsidiary branches of the bile duct are usually complicated with biliary duct stenosis. Therefore, even after successful stone clearance with choledochoscopy, stone recurrence is inevitable in these areas. Presently, traditional hepatectomy seems to be the treatment of choice for such cases. But it is limited to localize hepatolithiasis and biliary strictures to one segment or lobe. Unfortunately, 40.0% of hepatolithiasis patients have calculi distributed throughout the liver and most patients cannot tolerate resection of multiple lobes/segments. Due to these reasons, application of hepatectomy to treat hepatolithiasis is greatly restricted. Li et al^[5] explored the feasibility and efficacy of filling the diseased bile duct with chemical embolization agent to prevent stone recurrence and perform chemical resection of the diseased bile duct and hepatic segment. Thus, chemical embolization of the bile duct is likely an innovative and less invasive approach in treating hepatolithiasis, preventing stone recurrence and conducting chemical hepatectomy.^{[4-7, 11]}

 

In comparison with the intra-hepatic vascular system, the distribution of the intra-hepatic biliary system is clearer. Because of the presence of the communicating branches of the portal vein-hepatic artery, embolization of the hepatic artery or portal vein is difficult to achieve complete fibrosis, "self-cut" and chemical hepatectomy of the targeted hepatic lobe. Therefore, chemical embolization of the bile duct to a specific hepatic lobe seems to be safer and more accurate and has better clinical results. The bile duct, portal vein and hepatic artery are enclosed in a dense and fibrous Glisson sheath. After embolization of the bile duct, it obviously distends and directly compresses both the portal vein and hepatic artery which reduce blood supply to the target lobe.^{[8, 12-14]} This sort of blockage which is usually incomplete could slow the process of liver atrophy, fibrosis and "self-cut". Therefore, considering sequential embolization of the branches of the bile duct and portal vein to the targeted segment of the liver might be able to increase the efficacy and accelerate the process of chemical hepatectomy. Few studies^{[5, 6, 9]} also suggest that sequential embolization of the branches of the bile duct and portal vein of the targeted hepatic lobe not only leads to complete necrosis of hepatocytes, but also prompts liver atrophy and improves fibrosis. Sequential obstruction of the branches of the bile duct and portal vein, reservation of the hepatic artery, and the secondary establishment of collateral circulation all help to prevent the occurrence of liver abscess.^{[9, 15]}

 

Based on these hypotheses, the preliminary findings of our study also confirmed that sequential embolization of the branches of the bile duct and portal vein to the targeted hepatic lobe (BDE+PVL or PVL+BDE groups) can accelerate the process of chemical hepatectomy. Simple BDE on the 6th week showed disappearance of hepatocytes, fibrosis and effect of "self-cut" in the peripheral area, thus conducting chemical hepatectomy only in the periphery of the embolized lobe. Large islands of residual liver cells were still visible in the targeted hepatic lobe except in the peripheral area. In contrast, sequential embolization of the branches of the bile duct and portal vein (PVL+BDE and BDE+PVL groups) resulted in disappearance of hepatocytes in 6 weeks and the liver tissue was completely replaced by proliferative ductules and collagen fibers and thus, leading to fibrosis and "self-cut" in the whole embolized lobe. In comparison between different groups, collagen I expression in the BDE+PVL and PVL+BDE groups were found to be 5-6 times higher than that in the BDE group, and about 20-25 times higher than that in the SO groups. Collagen I expression in the PVL+BDE group was slightly higher than that in the BDE+PVL group, but the difference was not statistically significant. Histological findings suggested that in the BDE+PVL group, liver cells disappeared and were mainly replaced by proliferative bile ductules, whereas in the PVL+BDE group liver cells were mainly replaced by collagen fibers. The reason behind this mechanism may be due to the embolization of the bile duct, triggering the proliferation of small bile ductules. When the portal vein was ligated at first, it mainly triggered an increase in collagen fibers. No liver abscess or altered liver functions were observed during the whole experimental process. Therefore, this suggests that sequential embolization of the branches of the bile duct and portal vein is a safer, faster and more reliable way to perform an ideal chemical hepatectomy.

 

Successful implication of chemical cholecystectomy opens the gateway for chemical hepatectomy which could be the new minimal invasive treatment for hepatolithiasis. Chemical biliary embolization may be a breakthrough for the intractable problem of hepatolithiasis and its recurrences by reducing the re-operative rate, risk and trauma of traditional hepatectomy, also expanding the scope of treatment (such as patients who are under high risk for surgical treatment). Furthermore, chemical hepatectomy of target liver segment is more selective than the traditional hepatectomy and therefore, chemical hepatectomy could be used accurately for sub-segmental hepectomy and preserve significant functional hepatocytes. In comparison with simple BDE, sequential embolization of the branches of the bile duct and portal vein of the targeted hepatic lobe (either BDE+PVL or PVL+BDE group) can accelerate the apoptosis of target liver cells and liver atrophy and improve the degree of fibrosis, thus resulting in the ideal effect of chemical hepatectomy of targeted hepatic lobe in a short period of time. For special parts such as the caudate and right posterior lobes, which are difficult to remove surgically, chemical hepatectomy is expected to replace traditional surgery to some extent. Studies^{[9, 11, 15-19]} have shown that sequential embolization of the branches of the bile duct and portal vein is still in its preliminary stage. It requires further in-depth discussion and validation about its clinical significance, indications, contraindications and complications before its clinical application.

 

 

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