j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 7 ( 2 0 1 4 ) 6 9 0 e6 9 8
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
A novel model for orthotopic liver transplantation in rats using hepatic rearterialization and biliary extradrainage system Jilin Lu, PhD,a,1 Shuyun Wang, PhD,a,1 Peihao Wen, PhD,a Shuang Liu, MD,a Junwei Fan, PhD,a Chongzhi Zhou, PhD,a Xing Sun, PhD,a Huamei Tang, MD,b and Zhihai Peng, PhDa,* a
Department of General Surgery, Shanghai First People’s Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China b Department of Pathology, Shanghai First People’s Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China
article info
abstract
Article history:
Background: Although the rat orthotopic liver transplantation (OLT) model has existed for
Received 1 September 2013
many years, only a few models can be applied for dynamic bile collection. The aim of this
Received in revised form
study was to introduce a dependent rat OLT model with hepatic rearterialization and an
5 November 2013
expediently dynamic bile collection system.
Accepted 11 November 2013
Methods: Forty-five male SpragueeDawley rats were divided into the following three groups
Available online 18 November 2013
(n ¼ 15 each): group A, OLT without hepatic rearterialization; group B, OLT with hepatic rearterialization; group C, OLT with hepatic rearterialization and a biliary extradrainage
Keywords:
system. In groups B and C, a modified sleeve anastomosis between the donor common
Hepatic rearterialization
hepatic artery and the recipient proper hepatic artery was performed to restore the hepatic
Biliary extradrainage
artery blood flow. In group C, after hepatic rearterialization, biliary extradrainage and
Liver transplantation
jejunum stoma were performed to reestablish the bile flow, and a waistcoat-like external
Rat
fixator was introduced to protect this system. Results: The surgical success rates in groups A, B, and C were 100% (15/15), 93% (14/15), and 93% (14/15), respectively. In groups B and C, the hepatic artery patency rates were 93% and 86% on postoperative day 3 and postoperative day 21, respectively. Also, the liver function and bile duct integrity were preserved better than that in group A. In group C, the biliary extradrainage system was well preserved and bile collection was easily performed. Conclusions: The rat OLT model with hepatic rearterialization and a convenient biliary extradrainage system was satisfactory in maintaining the survival rate, hepatic artery patency rate, and recovery of graft function, so it can be applied in various studies after transplantation. ª 2014 Elsevier Inc. All rights reserved.
* Corresponding author. Department of General Surgery, Shanghai Jiao Tong University Affiliated First People’s Hospital, 100 Haining Road, Shanghai 200080, China. Tel.: þ86 21 63240090x3136; fax: þ86 21 65262903. E-mail address: [email protected] (Z. Peng). 1 The authors contributed equally to this work. 0022-4804/$ e see front matter ª 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2013.11.1080
j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 7 ( 2 0 1 4 ) 6 9 0 e6 9 8
1.
Introduction
Bile analysis plays an important role not only in evaluating the function, immune status, and bile duct injury of the graft [1e3], but also in the study of toxicology and pharmacology of the hepatocyte after orthotopic liver transplantation (OLT) in rat. Regardless of the fact that the rat OLT model has existed for many years, only a few models can be applied for dynamic bile collection. The model for dynamic bile collection after OLT must satisfy the following two conditions: hepatic artery reconstruction and a convenient biliary extradrainage system. Hepatic arterialization, which is considered to be necessary to maintain the integrity of the bile duct system [4], has limited applications in the traditional rat OLT model, because of the stringent requirement of microsurgical skill. In addition, continuous bile drainage, bile collection, and how to avoid disturbing the fluid and electrolyte balance, and digestive function are also very difficult to solve. The purpose of this study was to present a stable and dependent rat OLT model with hepatic artery reconstruction and a facilitated bile collection system. The OLT model was based on the classic “two-cuff” technique with some modifications, and introduction of our invention of a waistcoat-like external fixator for the rat (CN Patent No. 2007201748710) [5], which was designed to protect the biliary extradrainage tube and bile collection apparatus from damage by the rat. This novel OLT model with hepatic rearterialization and the biliary extradrainage system could provide technical support for the dynamic bile collection, and can be applied in various studies after OLT in rat.
2.
Materials and methods
2.1.
Animal
Inbred male SpragueeDawley rats, weighing 220e260 g, were purchased from Shanghai SLAC Laboratory Animal Co, Ltd (number of certificate of conformity: SCXK [Shanghai] 20070005; Shanghai, China). The body weight of donors was lower than that of corresponding recipients. The rats were housed in light and temperature-controlled individually ventilated cages. All experimental procedures involving animals were performed in compliance with the Animal Care and Use Committee of Shanghai Jiao Tong University and conformed to the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health (NIH Publication 86/23, 1985).
separated and divided to free the suprahepatic vena cava and infrahepatic vena cava (IHVC). The bile duct was inserted with a 22G stent at a distance of 1 cm away from the hepatic hilum. The portal vein (PV) was freed and divided from the pyloric vein. Then the liver was flushed with 20 mL of cold Ringer lactate through the PV. The donor graft was bathed in 0 C flushing solution for cuff preparation and storage before implantation. After the removal of the original liver, the suprahepatic vena cava anastomosis was performed with a running 7-0 suture and the two-cuff anastomosis technology was used to reestablish the blood flow of the PV and the IHVC during the recipient operation. Bile duct was connected with the splint technique. Group B (n ¼ 15)dthe OLT with hepatic artery reconstruction: during the donor operation, the preparation of the IHVC, bile duct, and PV was similar to that in group A. However, the whole lengths of the common hepatic artery (CHA) and the proper hepatic artery (PHA) were dissected and carefully preserved after all the branches were ligated and divided. After 200 IU of heparin was injected through the dorsal vein and the liver was flushed with 20 mL of cold Ringer lactate through the PV, the CHA was divided at the bifurcation from the celiac artery. The recipient operation was similar to that in group A, but in this group, the PHA of the recipient was ligated and divided at the bifurcation of the left hepatic artery. The procedure of liver implantation was similar to that in group A. However, after reconstruction of the inferior vena cava and the PV perfusion and rejoining of the bile duct, the hepatic artery perfusion was reconstructed by the sleeve anastomosis of the donor CHA and the recipient PHA (Fig. 1). Patency of the hepatic artery was confirmed twice (immediately after rearterialization and before the abdomen is closed) according to the arterial pulsation, engorgement, and positive filling test (Fig. 2). Group C (n ¼ 15)dthe OLT with hepatic artery reconstruction and biliary extradrainage: the procedures of donor and recipient operation were similar to that in group B. However, after venous and arterial perfusion was restored, one epidural catheter was inserted into the donor bile duct and another catheter was implanted into the high jejunum. These two catheters were inserted into the subcutaneous layer of the right lateral abdomen and connected with a connecting tube, which was placed outside the abdominal wall (Fig. 3A). The waistcoat-like external fixator was used for preventing the biliary extradrainage tube from damage by the rat (Fig. 3B).
2.3. 2.2.
691
Postoperative treatment
Operational procedures
All rats in this study were subjected to ether anesthesia and divided into the following three groups: Group A (n ¼ 15)dthe OLT without hepatic artery reconstruction: the procedures of the donor operation were almost similar to that described by Kamada et al. [6] except for some modifications. After a midline abdominal incision was made, all ligaments of the liver were dissected and divided. The left diaphragmatic vein, right renal vein, and suprarenal vein were
The animal that received liver transplantation was caged individually for 24 h. Both normal saline and food were allowed freely from the time of awakening. Piperacillin sodium, at a daily dose of 200 mg/kg, was injected intramuscularly for 3 d. In the recipient animal of group C, the catheter that was implanted into the high jejunum was washed every day with 1 mL of normal saline. The clinical condition of the recipient was observed daily and assessed according to the physiological condition, activity and signs of jaundice, or
692
j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 7 ( 2 0 1 4 ) 6 9 0 e6 9 8
Fig. 1 e Modified sleeve anastomosis between the donor CHA and the recipient PHA. (A) The donor CHA was penetrated from outside to inside 2 mm above the stump. After piercing the adventitia at the edge of the recipient PHA in the corresponding position, the same suture penetrated the donor CHA inside to outside close to the first suture point. (B) Pulled the thread gently to make the whole recipient PHA slipped into the donor CHA and the suture tied to create the gluing loop. (C) Four sutures passed through all layers of the donor CHA, but only pierced the adventitia of the recipient vessel at the location of the bifurcation of CHA and GDA. (D) The sutures were tied to create the fixing loop.
bleeding. On the basis of these conditions, the rats were regarded as healthy, weak, or severely ill.
2.4.
Sample collection procedures
Bile was collected on postoperative day (POD) 3 (seven recipients in each group) and on POD 21 (eight recipients in each group). In groups A and B, after anesthetizing the OLT rats, celiotomy was performed. Bile was collected through a catheter implanted into the common bile duct. In group C rats with
extradrainage, because of the existence of a waistcoat-like external fixator, which can protect the biliary extradrainage tube and bile collection apparatus, the bile could be collected easily without limiting the daily activity of rats. After bile collection, patency of the hepatic artery was checked again, a blood sample was collected through the inferior vena cava, and the grafted liver was dissected quickly for further analysis. The grafted liver tissue was fixed using 4.5% buffered formalin. Paraffin embedding, sectioning, and Masson trichrome staining were performed according to the standard histologic technique.
2.5.
Evaluation of bile duct injury
The bile duct injury was evaluated semiquantitatively by using the bile duct injury severity score (BDISS) method [3]. The BDISS of liver was based on the following three parameters: bile duct damage, ductular proliferation, and cholestasis. All the three parameters were graded as absent, mild, moderate, and severe with scores of 0, 1, 2, and 3, respectively. The bile duct injury was regarded as either mild (BDISS < 5) or severe (BDISS 5).
2.6.
Fig. 2 e Illustration of hepatic rearterialization with modified sleeve anastomosis technique between the donor CHA and the recipient PHA. (Color version of figure is available online.)
Liver function and bile biochemical analysis
The function of the graft after OLT was evaluated using the parameters of aspartate transaminase (AST) and alanine aminotransferase (ALT), which were measured through an automated chemical analyzer (Advia 1650; Siemens-Bayer, Basingstoke, UK). Total bile acid concentration, biliary
693
j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 7 ( 2 0 1 4 ) 6 9 0 e6 9 8
Fig. 3 e The extrahepatic bile duct that connected the stoma in high jejunum with two catheters placed outside the abdominal wall (A). The waistcoat-like external fixator was used for preventing the biliary extradrainage tube from damage by the rat (B). (Color version of figure is available online.)
alkaline phosphatase (ALP), and biliary gamma-glutamyl transpeptidase (g-GT) activity were measured by an automated chemical analyzer(Advia 1650; Siemens-Bayer, Basingstoke, UK).
2.7.
Statistical analysis
All data are expressed as the mean standard deviation. All statistical analyses were performed using SPSS 10.0 for Windows (SPSS Inc, Chicago, IL). Unpaired t-test or c2-test was used to analyze statistically significant difference. A P value of
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
A novel model for orthotopic liver transplantation in rats using hepatic rearterialization and biliary extradrainage system Jilin Lu, PhD,a,1 Shuyun Wang, PhD,a,1 Peihao Wen, PhD,a Shuang Liu, MD,a Junwei Fan, PhD,a Chongzhi Zhou, PhD,a Xing Sun, PhD,a Huamei Tang, MD,b and Zhihai Peng, PhDa,* a
Department of General Surgery, Shanghai First People’s Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China b Department of Pathology, Shanghai First People’s Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China
article info
abstract
Article history:
Background: Although the rat orthotopic liver transplantation (OLT) model has existed for
Received 1 September 2013
many years, only a few models can be applied for dynamic bile collection. The aim of this
Received in revised form
study was to introduce a dependent rat OLT model with hepatic rearterialization and an
5 November 2013
expediently dynamic bile collection system.
Accepted 11 November 2013
Methods: Forty-five male SpragueeDawley rats were divided into the following three groups
Available online 18 November 2013
(n ¼ 15 each): group A, OLT without hepatic rearterialization; group B, OLT with hepatic rearterialization; group C, OLT with hepatic rearterialization and a biliary extradrainage
Keywords:
system. In groups B and C, a modified sleeve anastomosis between the donor common
Hepatic rearterialization
hepatic artery and the recipient proper hepatic artery was performed to restore the hepatic
Biliary extradrainage
artery blood flow. In group C, after hepatic rearterialization, biliary extradrainage and
Liver transplantation
jejunum stoma were performed to reestablish the bile flow, and a waistcoat-like external
Rat
fixator was introduced to protect this system. Results: The surgical success rates in groups A, B, and C were 100% (15/15), 93% (14/15), and 93% (14/15), respectively. In groups B and C, the hepatic artery patency rates were 93% and 86% on postoperative day 3 and postoperative day 21, respectively. Also, the liver function and bile duct integrity were preserved better than that in group A. In group C, the biliary extradrainage system was well preserved and bile collection was easily performed. Conclusions: The rat OLT model with hepatic rearterialization and a convenient biliary extradrainage system was satisfactory in maintaining the survival rate, hepatic artery patency rate, and recovery of graft function, so it can be applied in various studies after transplantation. ª 2014 Elsevier Inc. All rights reserved.
* Corresponding author. Department of General Surgery, Shanghai Jiao Tong University Affiliated First People’s Hospital, 100 Haining Road, Shanghai 200080, China. Tel.: þ86 21 63240090x3136; fax: þ86 21 65262903. E-mail address: [email protected] (Z. Peng). 1 The authors contributed equally to this work. 0022-4804/$ e see front matter ª 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2013.11.1080
j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 7 ( 2 0 1 4 ) 6 9 0 e6 9 8
1.
Introduction
Bile analysis plays an important role not only in evaluating the function, immune status, and bile duct injury of the graft [1e3], but also in the study of toxicology and pharmacology of the hepatocyte after orthotopic liver transplantation (OLT) in rat. Regardless of the fact that the rat OLT model has existed for many years, only a few models can be applied for dynamic bile collection. The model for dynamic bile collection after OLT must satisfy the following two conditions: hepatic artery reconstruction and a convenient biliary extradrainage system. Hepatic arterialization, which is considered to be necessary to maintain the integrity of the bile duct system [4], has limited applications in the traditional rat OLT model, because of the stringent requirement of microsurgical skill. In addition, continuous bile drainage, bile collection, and how to avoid disturbing the fluid and electrolyte balance, and digestive function are also very difficult to solve. The purpose of this study was to present a stable and dependent rat OLT model with hepatic artery reconstruction and a facilitated bile collection system. The OLT model was based on the classic “two-cuff” technique with some modifications, and introduction of our invention of a waistcoat-like external fixator for the rat (CN Patent No. 2007201748710) [5], which was designed to protect the biliary extradrainage tube and bile collection apparatus from damage by the rat. This novel OLT model with hepatic rearterialization and the biliary extradrainage system could provide technical support for the dynamic bile collection, and can be applied in various studies after OLT in rat.
2.
Materials and methods
2.1.
Animal
Inbred male SpragueeDawley rats, weighing 220e260 g, were purchased from Shanghai SLAC Laboratory Animal Co, Ltd (number of certificate of conformity: SCXK [Shanghai] 20070005; Shanghai, China). The body weight of donors was lower than that of corresponding recipients. The rats were housed in light and temperature-controlled individually ventilated cages. All experimental procedures involving animals were performed in compliance with the Animal Care and Use Committee of Shanghai Jiao Tong University and conformed to the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health (NIH Publication 86/23, 1985).
separated and divided to free the suprahepatic vena cava and infrahepatic vena cava (IHVC). The bile duct was inserted with a 22G stent at a distance of 1 cm away from the hepatic hilum. The portal vein (PV) was freed and divided from the pyloric vein. Then the liver was flushed with 20 mL of cold Ringer lactate through the PV. The donor graft was bathed in 0 C flushing solution for cuff preparation and storage before implantation. After the removal of the original liver, the suprahepatic vena cava anastomosis was performed with a running 7-0 suture and the two-cuff anastomosis technology was used to reestablish the blood flow of the PV and the IHVC during the recipient operation. Bile duct was connected with the splint technique. Group B (n ¼ 15)dthe OLT with hepatic artery reconstruction: during the donor operation, the preparation of the IHVC, bile duct, and PV was similar to that in group A. However, the whole lengths of the common hepatic artery (CHA) and the proper hepatic artery (PHA) were dissected and carefully preserved after all the branches were ligated and divided. After 200 IU of heparin was injected through the dorsal vein and the liver was flushed with 20 mL of cold Ringer lactate through the PV, the CHA was divided at the bifurcation from the celiac artery. The recipient operation was similar to that in group A, but in this group, the PHA of the recipient was ligated and divided at the bifurcation of the left hepatic artery. The procedure of liver implantation was similar to that in group A. However, after reconstruction of the inferior vena cava and the PV perfusion and rejoining of the bile duct, the hepatic artery perfusion was reconstructed by the sleeve anastomosis of the donor CHA and the recipient PHA (Fig. 1). Patency of the hepatic artery was confirmed twice (immediately after rearterialization and before the abdomen is closed) according to the arterial pulsation, engorgement, and positive filling test (Fig. 2). Group C (n ¼ 15)dthe OLT with hepatic artery reconstruction and biliary extradrainage: the procedures of donor and recipient operation were similar to that in group B. However, after venous and arterial perfusion was restored, one epidural catheter was inserted into the donor bile duct and another catheter was implanted into the high jejunum. These two catheters were inserted into the subcutaneous layer of the right lateral abdomen and connected with a connecting tube, which was placed outside the abdominal wall (Fig. 3A). The waistcoat-like external fixator was used for preventing the biliary extradrainage tube from damage by the rat (Fig. 3B).
2.3. 2.2.
691
Postoperative treatment
Operational procedures
All rats in this study were subjected to ether anesthesia and divided into the following three groups: Group A (n ¼ 15)dthe OLT without hepatic artery reconstruction: the procedures of the donor operation were almost similar to that described by Kamada et al. [6] except for some modifications. After a midline abdominal incision was made, all ligaments of the liver were dissected and divided. The left diaphragmatic vein, right renal vein, and suprarenal vein were
The animal that received liver transplantation was caged individually for 24 h. Both normal saline and food were allowed freely from the time of awakening. Piperacillin sodium, at a daily dose of 200 mg/kg, was injected intramuscularly for 3 d. In the recipient animal of group C, the catheter that was implanted into the high jejunum was washed every day with 1 mL of normal saline. The clinical condition of the recipient was observed daily and assessed according to the physiological condition, activity and signs of jaundice, or
692
j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 7 ( 2 0 1 4 ) 6 9 0 e6 9 8
Fig. 1 e Modified sleeve anastomosis between the donor CHA and the recipient PHA. (A) The donor CHA was penetrated from outside to inside 2 mm above the stump. After piercing the adventitia at the edge of the recipient PHA in the corresponding position, the same suture penetrated the donor CHA inside to outside close to the first suture point. (B) Pulled the thread gently to make the whole recipient PHA slipped into the donor CHA and the suture tied to create the gluing loop. (C) Four sutures passed through all layers of the donor CHA, but only pierced the adventitia of the recipient vessel at the location of the bifurcation of CHA and GDA. (D) The sutures were tied to create the fixing loop.
bleeding. On the basis of these conditions, the rats were regarded as healthy, weak, or severely ill.
2.4.
Sample collection procedures
Bile was collected on postoperative day (POD) 3 (seven recipients in each group) and on POD 21 (eight recipients in each group). In groups A and B, after anesthetizing the OLT rats, celiotomy was performed. Bile was collected through a catheter implanted into the common bile duct. In group C rats with
extradrainage, because of the existence of a waistcoat-like external fixator, which can protect the biliary extradrainage tube and bile collection apparatus, the bile could be collected easily without limiting the daily activity of rats. After bile collection, patency of the hepatic artery was checked again, a blood sample was collected through the inferior vena cava, and the grafted liver was dissected quickly for further analysis. The grafted liver tissue was fixed using 4.5% buffered formalin. Paraffin embedding, sectioning, and Masson trichrome staining were performed according to the standard histologic technique.
2.5.
Evaluation of bile duct injury
The bile duct injury was evaluated semiquantitatively by using the bile duct injury severity score (BDISS) method [3]. The BDISS of liver was based on the following three parameters: bile duct damage, ductular proliferation, and cholestasis. All the three parameters were graded as absent, mild, moderate, and severe with scores of 0, 1, 2, and 3, respectively. The bile duct injury was regarded as either mild (BDISS < 5) or severe (BDISS 5).
2.6.
Fig. 2 e Illustration of hepatic rearterialization with modified sleeve anastomosis technique between the donor CHA and the recipient PHA. (Color version of figure is available online.)
Liver function and bile biochemical analysis
The function of the graft after OLT was evaluated using the parameters of aspartate transaminase (AST) and alanine aminotransferase (ALT), which were measured through an automated chemical analyzer (Advia 1650; Siemens-Bayer, Basingstoke, UK). Total bile acid concentration, biliary
693
j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 7 ( 2 0 1 4 ) 6 9 0 e6 9 8
Fig. 3 e The extrahepatic bile duct that connected the stoma in high jejunum with two catheters placed outside the abdominal wall (A). The waistcoat-like external fixator was used for preventing the biliary extradrainage tube from damage by the rat (B). (Color version of figure is available online.)
alkaline phosphatase (ALP), and biliary gamma-glutamyl transpeptidase (g-GT) activity were measured by an automated chemical analyzer(Advia 1650; Siemens-Bayer, Basingstoke, UK).
2.7.
Statistical analysis
All data are expressed as the mean standard deviation. All statistical analyses were performed using SPSS 10.0 for Windows (SPSS Inc, Chicago, IL). Unpaired t-test or c2-test was used to analyze statistically significant difference. A P value of
