On-line version ISSN 1678-2674
Acta Cir. Bras. vol.16 no.2 São Paulo Apr./May/June 2001
3 ORIGINAL ARTICLE
A NEW METHOD FOR THE EXPERIMENTAL INDUCTION OF SECUNDARY BILIARY CIRRHOSIS IN WISTAR RATS1
Jorge GL, Leonardi LS, Boin IFSF, Silva Jr OC, Escanhoela CAF. A new method for the experimental induction of secundary biliary cirrhosis in Wistar rats. Acta Cir Bras [serial online] 2001 Apr-Jun;16(2). Available from URL: http://www.scielo.br/acb.
ABSTRACT: The aim of this study was to describe a method for the induction of experimental secondary biliary fibrosis (SBF). Forty-seven Wistar rats were submitted to hepatic duct obstruction (OB group) for thirty days without ligature, section or cannulization causing interruption of biliary flow. This technique was carried out by simple traction of the bile duct passing it through the xiphoid appendix. Nine rats were submitted to a sham operation for bile duct stricture and seven rats comprised the control group. Blood samples were collected for the measurement of total bilirubin (TB), alkaline phosphatase (AP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Liver fragments were removed for morphological study. Thirty days after surgery TB, AP, ALT and AST levels were significantly increased in the hepatic duct ligation group compared to the sham operated group and the presence of SBF in the OB group was confirmed by morphological study of the liver. There was technical failure in 31.92% cases. The survival was 100% at fifteen days and 82.97% at the end of the experiment. We concluded that this simple surgical technique may be used to study the consequence of bile duct obstruction which could be a reversible process depending on the obstruction time. This technique can be carried out from cholestasis to fibrosis.
KEY WORDS: 1. Secondary biliary cirrhosis. 2. Bile duct obstruction. 3. Cholestasis.
Obstructive jaundice has been induced in rats, but the method and place of hepatic duct ligation greatly affects the intensity and duration of jaundice and also histological changes in the liver (1) . Some authors have demonstrated secondary biliary cirrhosis(2-4) and others secondary biliary fibrosis (3, 5). Recannulization of the hepatic duct after simple and single ligation and ligation and transection of the duct is a frequent cause of unsuccessful experimental secondary biliary cirrhosis (2, 3, 5-8) . The recannulization can occur either by invagination of the hepatic duct, located just below the ligature, or above the ligature (8) entering the distended sac, or by proliferation of clusters of collateral ductules from the hepatic duct below the ligature to reach the distended portion of the duct (9). The aim of this article was to describe a simple and efficient method for the induction of experimental secondary biliary cirrhosis, avoiding cannulization or section and mantaining the extrahepatic biliary tract easily accessible . The dilated bile duct can be submitted to other procedures whenever necessary.
Sixty-three male Wistar rats weighing from 256g to 340g (average: 300g) were distributed in three groups. The animals were from the Vivarium Center (CEMIB) and the experiment was carried out in the Experimental Liver Transplantation Laboratory at the Medicine and Experimental Surgery Nucleus at the State University of Campinas Medical Science Faculty (NMCE/UNICAMP-SP/BRAZIL). Forty-seven rats underwent biliary obstruction (OB group), nine underwent a sham operation (OS group) and seven were kept as a control group (OC group). After eight hours fasting with water "ad libitum", the animals were anesthetized with diethyl-ether by continuous inhalation. The abdominal wall was opened through a 1.5 cm in length median incision from the xiphoid appendix. The bile duct was isolated at the upper margin of the pancreas and one cm of free duct was repaired with 4-0 polypropylene thread. After, the xiphoid appendix was exposed and one hole was made in it with an "rat ear machine". The polypropylene thread (2.0 cm in length) was pulled through this hole with a hemostatic clamp and brought with it a bile duct loop over the xiphoid appendix. A nº 08 silicone catheter was passed through the loop having been sectioned and exposed between muscle and skin (figure 01). The abdominal wall was closed with an ininterrupted suture using a 4-0 polypropylene stitch.
Biliary Duct Obstruction Efficacy
The biliary duct obstruction efficacy or technical failure was observed after 30 days. The animals were anesthetized as before and the wall abdomen was opened according to the same, careful technique already described previously. Catheter position; biliary duct dilation diameter in centimeters; presence or absence of biliary tract ruptures and abdominal free collections; liver coloration and macroscopic aspects were systematically observed. Blood samples were withdrawn by aortic punction for plasma enzyme determination and the liver was immediately taken out. The median left lobe was sectioned and fragmented for formalin fixation and posterior microscopic evaluation.
Plasma Enzyme Determination
Thirty days after surgery, the surviving animals were sacrificed and blood samples were withdrawn from the inferior vena cava of each animal for the measurement of total bilirubin (BT) by Jendrassik/Grof reaction (10), alkaline phosphatase (AP) by kinetic colorimetric test (11), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) by kinetic UV test (12) of Cobas Mira Instruments .
Liver fragments were removed and fixed by immersion in 10% formalin solution for 24 hours and conserved in 70% alcohol for later paraffin embedding. Sections, 5 µm thick, were stained with H&E and Masson's trichrome and examined under a light microscope. The parameters evaluated were: fibrous septa, portal fibrosis, ductal proliferation, cholangitis and presence of regenerative liver cell nodules. The grade of intensity of liver parenchyma lesions were classified as normal, mild, moderate and severe. The OC group rats were used as histological normal control and were compared with the sham (OS) and biliary obstructive groups (OB).
Results are reported as means (± SD); the data were analyzed statistically by the Mann-Whitney and Wilcoxon tests . A p < 0,05 value was accepted as significant.
Initially, 47 rats were submitted to bile duct obstruction. In the OB group 15 rats were lost; eight died before the 30th day after surgery and in seven (31.92%) rats technical failure was observed. This was due to errors in catheter traction in four animals and biliary tract rupture in another three. For this reason only 32 rats were analyzed in this group. All OS group rats showed good results and 100% survival rate. The operating time was 12-15 minutes.
After the laparotomy, the position of the catheter above the xiphoid appendix was easily verified. The median dilation diameter observed in the bile duct of the OB group was 4.5 cm with a range of 2 to 8.5cm increasing abdominal circumference due to a great tract biliar dilation regarding the choledochous cyst (figure 02).
Fluid aspect was liquid, serous or hemorrhagic, greenish or yellow-whitish with grumous. Bilious ascitis, due to bile duct rupture, was observed in those three animals which died.
The liver in the OB group was pale, with micronodulation distributed evenly with some free parenchyma. Histological analysis in the OB group showed mild/moderate portal fibrosis in 96.9% them; with moderate to severe fibrous septa spreading out to link portal tracts in 87.4%; extensive ductal proliferation in 81.3% and moderate in 18.7% (figure 03). Thick fibrous septa separating well-demarcated nodules and ductal proliferation could be seen in 68.7% of them.
Biochemical analysis of the OB group showed high levels of TB, AP, ALT and an AST when compared to the sham-operated group ( Table 1).
Experimental biliary cirrhosis developed in animals has been influenced by several factors including the kind of animals. The animals that had already been used for these experiments were dogs and rats (12-14) . In this study we used rats due to easy manipulation and more easily reproduced results.
The experimental biliary cirrhosis pattern to obtain biliary obstruction in the interruption of the bile flow into the duodenum due to bile duct ligature with non-absorbable stitches in rats has already been cited. Single or double ligatures were usually used followed by bile duct cannulization, double ligature and bile duct section or using a suture around the bile duct with or without a silicone tube (14-16) .
Of these models the extrahepatic bile obstruction induced with a suture wound around the hepatic duct was more effective in reducing the chance of recannulization of the hepatic duct and induced secondary biliary cirrhosis after 30 days of observation in approximately 80% of the rats operated on (17) . Similarly, the use of the silicone tube (ST) impaired or prevented BD recannulization from the dilated portion above the ST to the duodenum below the ST (8) or prevented the proliferation of collateral ductule from the hepatic duct immediately below the ST from reaching the distended portion of the duct (9) . The goal of this surgical procedure was achieved without the need for more aggressive operations such as transection with or without resection of a bile duct segment ( 1) .
Our biliary fibrosis pattern showed the possibility of obtaining from the cholestasis to fibrosis depending on the time used for obstruction (2-5 weeks). Neither stitches nor bile duct cannulations or transections were performed. The gold standard was the bile duct traction through and over the xiphoid appendix. This obstructive jaundice technique can allow new surgical models such as choledochojejunoanastomosis after choledochous dilation as long as this has been maintained intact whereas biliary tract dilation occurs if the obstruction time was short. Another advantage is that the method is easily reproducible. The technical failure was around 30% due to collection secondary biliary duct rupture leading to animal death or due to failure in the traction. The great dilation has been the reason for bile duct rupture and less time should be used avoiding biliary peritonitis. The correct traction can improve the results and decrease the technical failure rate.
Generally the animals after biliary obstruction developed secondary biliary fibrosis or cirrhosis. This should be diagnosed when there was disorganized hepatic architecture with well-demarcated alterations such as parenchyma degeneration, fibrous septa and regenerating liver cell nodules (7, 18) .This was reported in rats subjected to bile duct ligation for 3-5 weeks (3) , whereas other authors were unable to find evidence of SBF after 14 days in bile duct ligated rats (7, 9, 19) . Furthermore, the animals' age is important because old rats showed less cirrhosis rate than young rats (20) . We used 12- week-old rats (adults) and 96.9% portal fibrosis and regenerative liver cell nodules in 68.7% animals was obtained. Similar rates were obtained in literature (16, 21-23) .
Two factors are important in the experimental biliary cirrhosis induction: death rate in the first two weeks and the bile duct recannulization (5, 21) . Our model showed 100% survival rate in the first two weeks. That survival rate was due to the non-opening of the bile duct, absence of extrahepatic lesions and bleeding, short operating time (maximum of 15 minutes) and a small (1.5cm) laparotomy minimizing wound bleeding and secondary infection and facilitating wound cicatrization. The recannulization can usually take place after hepatic duct invagination with the occurrence of biliary duct epithelialization from the distal duct to the dilation duct (24, 25) . This could have happened due to two mechanisms already cited above or other unknown factors (14) . These authors related the use of a suture around the bile duct with or without a silicone prosthetic tube avoiding recannulization but the mortality rate was still important and they have obtained some success (50-80% survival rate after 2 weeks) following the use of subcutaneous administration of vitamin K . The polyethilene catheter introduction in the bile duct has shown good results but it is necessary to open the bile duct and this raises the mortality rate after two weeks (16, 26) .
From a biochemical point of view , the serum values of total bilirubin, alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase, 30 days after the surgical procedure, were higher than those reported by other authors (6, 27-30) .
Unbelievably, the bile tract dilation showed a great diameter (2.0-8.5) cm. The aspect of biliary fluid was liquid, serous or hemorrhagic, greenish or yellow-whitish with grumous. Histological alterations suggestive of cholangitis were not found in our study and similar results were obtained by other authors (23, 31) .
The liver in the obstructive group (OB) was pale, with micronodulation distributed evenly with some free parenchyma. Histological analysis in the OB group has shown portal fibrosis mild/moderate in 96.8% them; with moderate to severe fibrous septa spreading out to link portal tracts in 87.4%; extensive and ductal proliferation, portal fibrosis and fibrous septa in 18.7% (figure 03). Thick fibrous septa separating well-demarcated nodules and ductal proliferation can be seen in 68.7% of them. Fibrous formation secondary biliary obstruction appeared after 3 weeks and this could be a critical biliary obstruction stage because the liver lesions render in irreversible structural disorganization (32) . This question is important because a high survival rate to observe these results is necessary. Biliary fibrosis was defined by Weinbren et al. (1985) but with irreversible behavior according to Koyama et al. (1975). In this experimental biliary cirrhosis pattern, fibrous septa, intense and severe ductal proliferation and generative liver cell nodules were observed.
The results indicate that the present method will effectively reduce the chance of bile duct recannulization, thus facilitating long-term from extrahepatic cholestasis to biliary fibrosis or secondary biliary cirrhosis.
This alternative and experimental biliary obstruction pattern led to bilious-duodenal flow interruption without ligature, cannulization or biliary tract section. Biochemical analysis has shown high levels of aminotransferases (ALT and AST), alkaline phosphatase and total bilirubin after 30th day of obstruction when compared to the sham group. There was technical failure in 31.92% cases. The survival was 100% at fifteen days and 82.97% at the end of the experiment. We concluded that this simple surgical technique may be used to study the consequence of bile duct obstruction which could be a reversible process depending on the obstruction time. This technique can lead from cholestasis with portal fibrosis of biliary pattern with well-demarcated nodules.
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Jorge GL, Leonardi LS, Boin IFSF, Castro e Silva Jr O, Escanhoela CAF. Novo modelo experimental de obstrução biliar em ratos Wistar. Acta Cir Bras [serial online] 2001 Abr-Jun;16(2). Disponível em URL: http://www.scielo.br/acb.
RESUMO: O objetivo desse experimento foi o desenvolvimento de um modelo de obstrução do ducto biliar, através de procedimento cirúrgico sem ligadura ou secção do mesmo, que permitisse a evolução para cirrose e mantivesse a via biliar extra-hepática facilmente acessível a eventuais manuseios. Foram utilizados 48 ratos Wistar, distribuídos em três grupos: 32 animais foram submetidos à obstrução do ducto hepático comum (grupo OB), 9 foram submetidos à operação simulada (grupo OS) e 7 foram adotados como controle para análise histológica (grupo OC). No desenvolvimento da técnica, o ducto hepático foi isolado logo após sua emergência justa-pancreática, deixando-se 1 cm de ducto livre, reparado com fio de polipropileno. Em seguida procedeu-se à exposição do apêndice xifóide e, através de marcador de orelha de rato, foi feito um pequeno orifício circular. Passou-se, por este, o fio de polipropileno e tracionou-se até o ducto biliar formar uma alça acima do orificio do apendice xifóide. Deste modo um cateter de silicone foi colocado sob esta alça. O ducto biliar permaneceu, portanto, tracionado e exposto entre a musculatura e a pele. O fechamento da parede muscular e da pele foi feito com sutura contínua. A avaliação histopatológica do fígado destes animais e os efeitos bioquímicos séricos foram realizados após 30 dias da obstrução. Nos ratos do grupo OB ocorreu aumento significativo nas dosagens séricas da bilirrubina, das aminotransferases (ALT e AST), da fosfatase alcalina, da gamaglutamiltransferase e redução significativa da albumina sérica quando comparados aos valores das dosagens dos ratos do grupo OS. A análise histológica demonstrou a formação de nódulos regenerativos em 68,7% dos casos do grupo OB, com presença de fibrose portal (de grau leve e moderado) em 96,8% dos animais, acompanhada pela formação de septos fibrosos (de grau moderado e intenso) em 87,4 %. Houve ainda intensa proliferação de ductos biliares em 81,2%, e moderada em 18,7% dos casos. Conseguiu-se desta maneira, o desenvolvimento de um modelo experimental alternativo que provoca a interrupção do fluxo bíleo-duodenal, semelhante à oclusão das vias biliares, sem necessidade de ligadura, canulação ou secção do ducto biliar. A obstrução biliar provocada pela tração do ducto biliar oferece excelentes condições de pesquisa desde o momento da interrupção do fluxo biliar até o desenvolvimento de colestase, acompanhada pela distorção da arquitetura hepática, caracterizada por fibrose portal com formação de septos e transformação nodular multi-focal.
DESCRITORES: 1. Obstrução biliar experimental. 2. Cirrose biliar secundária. 3. Colestase.
Conflito de interesses: nenhum
Fontes de financiamento: nenhuma
Address for correspondence:
Dra. Ilka de Fátima Santana Ferreira Boin
Rua Aldo de Oliveira Barbosa, 184
Data do recebimento: 17/01/2001
Data da revisão: 20/02/2001
Data da aprovação: 11/03/2001
1. From Campinas State University Faculty of Medical Sciences - São Paulo/Brazil.
2. Biologist at Campinas State University Faculty of Medical Sciences Liver Transplantation Laboratory - São Paulo/Brazil.
3. Chairman at Campinas State University Faculty of Medical Sciences Liver Transplantation Laboratory - São Paulo/Brazil.
4. Coordinator at Campinas State University Faculty of Medical Sciences Liver Transplantation Laboratory - São Paulo/Brazil.
5. Coordinator of Hepatology Laboratory Faculty of Medicine, Ribeirão Preto, University of São Paulo/Brazil.
6. Coordinator at at Campinas State University Faculty of Medical Sciences Liver Transplantation Pathology - São Paulo/Brazil.