Experimental Bile Duct Ligation Research Articles

Induction of Obstructive Cholestasis in Mice.

Experimental bile duct ligation (BDL) in rodents causes cholestatic liver injury characterized by structural and functional alterations that include periportal biliary fibrosis. These changes are time-dependent and based on excess accumulation of bile acids in the liver. This in turn causes damage of hepatocytes and functional loss, leading to recruitment of inflammatory cells. Liver resident pro-fibrogenic cells facilitate extracellular matrix synthesis and remodeling. The proliferation of bile duct epithelial cells provokes a ductular reaction characterized by bile duct hyperplasia. Experimental BDL surgery is technically simple and quick to perform and reliably generates progressive liver damage with a predictable kinetics. The cellular, structural, and functional alterations induced in this model are similar to that in humans suffering from diverse forms of cholestasis including primary biliary cirrhosis (PBC) or primary sclerosing cholangitis (PSC). Therefore, this extrahepatic biliary obstruction model is used in many laboratories worldwide. Nevertheless, BDL can result in significant variations and high mortality rates when surgery is carried out by untrained or inexperienced personnel. Here we present a detailed protocol to achieve a robust experimental obstructive cholestasis in mice.

Single versus double experimental bile duct ligation model for inducing bacterial translocation

BackgroundDouble common bile duct ligation plus section in rats is used as a model for bacterial translocation, a phenomenon that has been correlated with the degree of liver damage. This study analyzes whether a simpler variant of the technique is also a valid model to study bacterial translocation. MethodsFifty-six male Sprague Dawley rats underwent one of three surgical interventions: a) proximal double ligation and section of the common bile duct; b) proximal simple ligation of the bile duct; and c) sham operation. Bacterial translocation was measured by cultures of mesenteric lymph nodes, blood, spleen and liver. Stool culture and histological analysis of liver damage were also performed. ResultsThe incidence of bacterial translocation in SBL and DBDL groups was 23,5% and 25% respectively. Mortality was similar between ligation groups (11.2% versus 10%). Liver cirrhosis developed in the group of double ligation and section (100% of the animals at 4 weeks), while portal hypertension appeared starting at week 3. None of the animals submitted to simple ligation developed liver cirrhosis. ConclusionsSimple bile duct ligation is associated with a similar incidence of bacterial translocation as double ligation, but without cirrhosis or portal hypertension.

Interleukin-33 drives hepatic fibrosis through activation of hepatic stellate cells.

Liver fibrosis is a consequence of chronic liver disease, causing morbidity and mortality. Interleukin-33 (IL-33) is a critical mediator of inflammation, which may be involved in the development of liver fibrosis. Here, we investigated the role of IL-33 in human patients and experimental bile-duct ligation (BDL)-induced fibrosis in mice. We report increased hepatic IL-33 expression in the murine BDL model of fibrosis and in surgical samples obtained from patients with liver fibrosis. Liver injury, inflammatory cell infiltration and fibrosis were reduced in the absence of the IL-33/ST2 receptor, and the activation of hepatic stellate cells (HSCs) was decreased in ST2-deficient mice. Recombinant IL-33 activated HSCs isolated from C57BL/6 mice, leading to the expression of IL-6, TGF-β, α-SMA and collagen, which was abrogated in the absence of ST2 or by pharmacological inhibition of MAPK signaling. Finally, administration of recombinant IL-33 significantly increased hepatic inflammation in sham-operated BL6 mice but did not enhance BDL-induced hepatic inflammation and fibrosis. In conclusion, BDL-induced liver inflammation and fibrosis are dependent on ST2 signaling in HSCs, and therefore, the IL-33/ST2 pathway may be a potential therapeutic target in human patients with chronic hepatitis and liver fibrosis.

Efficacy of diffusion-weighted magnetic resonance imaging in the evaluation of extrahepatic cholestasis-related hepatic fibrosis.

To investigate the efficacy of diffusion-weighted magnetic resonance imaging (DWI) in the diagnosis and staging of fibrosis induced by experimental bile duct ligation (BDL). Twenty-four rats were divided randomly into four groups: control, BDL--3 days, BDL--2 weeks, and BDL--4 weeks. DWI was performed with b-values of 100 and 500 on the rats from control group at day zero, on the rats from the BDL--3 days group at the end of day 3, on the rats from the BDL--2 weeks group at the end of day 14, and on the rats from the BDL--4 weeks at the end of day 28. When fibrosis scores generated in all groups were evaluated together, a strong negative correlation was detected between fibrosis scores and apparent diffusion coefficient (ADC) values measured using b 100 and b 500. ADC values obtained using b 100 were found to be significantly higher compared to the fibrosis observed in both the BDL--2 weeks and BDL--4 weeks groups (P < 0.003 and P < 0.001, respectively). We think that DWI may be an alternative to liver biopsy for the diagnosis and staging of hepatic fibrosis with underlying extrahepatic cholestasis.

Propitious role of bone marrow-derived mononuclear cells in an experimental bile duct ligation model: potential clinical implications in obstructive cholangiopathy

To evaluate the role of bone marrow-derived mononuclear cells (BMC) in rat bile duct ligation (BDL) model. Wistar rats were categorized into four Groups A-D. Normal liver biopsy was taken from Group A. BDL model was created in Groups B and C (15 each). Normal saline and BMC were injected through portal vein (PV) in Groups B and C, respectively. In Group D (healthy rat), only BMC were infused through PV. Groups B and C were compared for body weight, liver functions, survival, and histopathological changes. Serum bilirubin was lower in Group C at day 6 (p=0.0010). Median survival time was 5 (4, 6) and 13 (9, 17) days in Groups B and C (p=0.0147), respectively. Portal edema (p=0.013) and portal inflammation (p=0.025) were less in Group C vs Group B. On post hoc subgroup analysis of rats surviving 8-26days, portal inflammation (p=0.004), bile duct proliferation (p=0.016) and portal fibrosis (p=0.038) were less in Group C vs Group B. Hepatocyte regeneration was found in four rats in Group C. CD34-positive cells were prominent in sinusoids and portal tracts in the BDL rat model. BMC have shown to delay fibrosis, facilitate hepatocyte regeneration and improve survival in an experimental BDL model, with potential clinical implication in obstructive cholangiopathy.

Factor VII activating protease (FSAP) exerts anti-inflammatory and anti-fibrotic effects in liver fibrosis in mice and men

Factor VII activating protease (FSAP) is a circulating serine protease produced in the liver. A single nucleotide polymorphism (G534E, Marburg I, MI-SNP) in the gene encoding FSAP (HABP2) leads to lower enzymatic activity and is associated with enhanced liver fibrosis in humans. FSAP is activated by damaged cells and its substrates include growth factors and hemostasis proteins. We have investigated the progression of liver fibrosis in FSAP deficient mice and FSAP expression in human liver fibrosis. Serum FSAP concentrations declined in patients with end-stage liver disease, and hepatic FSAP expression was decreased in patients with advanced liver fibrosis and liver inflammation. Moreover, there was an inverse correlation between hepatic FSAP expression and inflammatory chemokines, chemokine receptors as well as pro-fibrotic mediators. Upon experimental bile duct ligation, FSAP(-/-) mice showed enhanced liver fibrosis in comparison to wild type mice, alongside increased expression of α-smooth muscle actin, collagen type I and fibronectin that are markers of stellate cell activation. Microarray analyses indicated that FSAP modulates inflammatory pathways. Lower FSAP expression is associated with enhanced liver fibrosis and inflammation in patients with chronic hepatic disorders and murine experimental liver injury. This strengthens the concept that FSAP is a "protective factor" in liver fibrosis and explains why carriers of the Marburg I SNP have more pronounced liver fibrosis.

Adenoviral CCN3/NOV gene transfer fails to mitigate liver fibrosis in an experimental bile duct ligation model because of hepatocyte apoptosis

CCN3/NOV, a matricellular protein of the CYR61-CTGF-NOV (CCN) family, comprises six secreted proteins that associate specifically with the extracellular matrix. CCN proteins lack specific high-affinity receptors; instead, they regulate crucial biological processes, such as fibrosis, by signalling via integrins and proteoglycans. Recent studies have linked overexpression of CCN3/NOV to mitigate kidney fibrosis. This study aims to investigate CCN3/NOV overexpression in liver fibrogenesis in vivo. The biological efficacy of adenoviral expressed CCN3/NOV directed under transcriptional control of the constitutively active Cytomegalovirus promoter (Ad-NOV) was analysed in a bile duct ligation model and in cultured primary hepatocytes. Even though Ad-NOV gene transfer in a 3-week bile duct ligation mouse model showed the expected high levels of CCN3/NOV in both mRNA and protein, it failed to reduce liver fibrogenesis, but instead enhanced hepatocyte apoptosis. Furthermore, overexpressed CCN3/NOV in cultured primary hepatocytes resulted in decreased levels of CCN2/CTGF, the profibrotic marker protein in liver fibrosis. Both Ad-NOV and Ad-CTGF induced reactive oxygen species production, enhanced p38 and JNK activation. Therefore, we conclude that CCN3/NOV overexpression in vivo is insufficient to mitigate liver fibrogenesis because of the induction of hepatocyte injury and apoptosis.

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Hepatic expression profile of forkhead transcription factor genes in normal Balb/c mice and their dynamic changes after bile duct ligation

Dysregulation of Forkhead box (Fox) transcription factor family genes was previously shown to lead to congenital disorders, diabetes mellitus, and carcinogenesis, and recent reports suggested that several Fox genes play important roles in the pathogenesis of liver fibrosis. The present study was initiated to determine the expression profiles of the Fox genes in normal Balb/c mouse liver and their dynamic expression changes during fibrogenesis induced by experimental bile duct ligation (BDL). RT-PCR was employed to detect 18 FOX family members including FOXO1, FOXO3, FOXM1, and FOXL1 in normal mouse liver. FQ-PCR was performed to analyze the dynamic mRNA expression changes of nine inflammation- or proliferation-related FOX family genes in BDL mice. Results showed that all the 18 Fox genes were expressed in the normal mouse liver, among which the expression of FOXO1 and FOXO3 were found to be the highest. The inflammation and proliferation-related FOX family genes were found to be dynamically changed during BDL-induced liver injury with reduced FOXO1 and enhanced FOXOL1 and FOXM1, indicating their potential involvement in the pathogenesis of liver fibrosis. This is the first systematic evaluation of hepatic expression of FOX genes in both normal and BDL mice.

Oxidized Low-Density-Lipoprotein Accumulation is Associated with Liver Fibrosis in Experimental Cholestasis

OBJECTIVEThe aim of the present study was to examine the probable relationship between the accumulation of oxLDL and hepatic fibrogenesis in cholestatic rats.INTRODUCTIONThere is growing evidence to support the current theories on how oxidative stress that results in lipid peroxidation is involved in the pathogenesis of cholestatic liver injury and fibrogenesis. One of the major and early lipid peroxidation products, OxLDL, is thought to play complex roles in various immuno-inflammatory mechanisms.METHODSA prolonged (21-day) experimental bile duct ligation was performed on Wistar-albino rats. Biochemical analysis of blood, histopathologic evaluation of liver, measurement of the concentration of malondialdehyde (MDA) and superoxide-dismutase (SOD) in liver tissue homogenates, and immunofluorescent staining for oxLDL in liver tissue was conducted in bile-duct ligated (n = 8) and sham-operated rats (n = 8).RESULTSSignificantly higher levels of MDA and lower concentrations of SOD were detected in jaundiced rats than in the sham-operated rats. Positive oxLDL staining was also observed in liver tissue sections of jaundiced rats. Histopathological examination demonstrated that neither fibrosis nor other indications of hepatocellular injury were found in the sham-operated group, while features of severe hepatocellular injury, particularly fibrosis, were found in jaundiced rats.CONCLUSIONOur results support the finding that either oxLDLs are produced as an intermediate agent during exacerbated oxidative stress or they otherwise contribute to the various pathomechanisms underlying the process of liver fibrosis. Whatever the mechanism, it is clear that an association exists between elevated oxLDL levels and hepatocellular injury, particularly with fibrosis. Further studies are needed to evaluate the potential effects of oxLDLs on the progression of secondary biliary cirrhosis.

Characterization of time-related changes after experimental bile duct ligation

Although bile duct ligation (BDL) in mice is used to study cholestasis, a detailed description of this animal model is lacking. The aim of this study was to define specific phases of acute and chronic injury and repair in the different cellular compartments of the liver. C57BL/6 mice underwent BDL or sham laparotomy, and serum and liver tissue were analysed between 8 h and 6 weeks later. Biliary infarcts and alanine aminotransferase levels revealed acute hepatocellular injury peaking at days 2-3, paralleled by enhanced transcription of pro-proliferative mediators and followed by a distinct peak of hepatocellular proliferation at day 5. Cholangiocellular proliferation occurred in large bile ducts on days 2-3 and in small bile ducts on day 5. Neutrophil infiltration occurred within 8 h, with neutrophils remaining the predominant immune cell type until day 3. Acute injury was followed by continuous tissue repair, lymphocyte and Kupffer cell infiltration, and accumulation of collagen during the second week. Thereafter, the number of alpha-smooth muscle actin-positive cells and the expression of transforming growth factor beta1, tissue inhibitor of metalloproteinases 1 and procollagen (I) decreased, and liver fibrosis stabilized. BDL elicits dynamic changes in mouse liver. The chronological dissection and quantification of these events identified specific phases of acute and chronic cholestatic liver injury.

Kupffer cell blockade improves liver microcirculation in endotoxemia following experimental bile duct ligation

Kupffer cell blockade improves liver microcirculation in endotoxemia following experimental bile duct ligation

Bile acids: from genomics to disease and therapyEdited by G. Paumgartner, D. Keppler, U. Leuschner, and A. Stiehl. 320 pp. $181.00. Kluwer Academic Publishers: the Netherlands, 2003. ISBN 0-79238-781-3. Web address for ordering: www.wkap.nl

Bile acids: from genomics to disease and therapyEdited by G. Paumgartner, D. Keppler, U. Leuschner, and A. Stiehl. 320 pp. $181.00. Kluwer Academic Publishers: the Netherlands, 2003. ISBN 0-79238-781-3. Web address for ordering: www.wkap.nl

Impaired renal function in obstructive jaundice: roles of the thromboxane and endothelin systems.

Patients with intra- or extrahepatic bile-duct obstruction are susceptible to acute renal failure (ARF) especially when undergoing major surgery. We observed in jaundiced rats 4 days after bile-duct ligation (BDL) a decrease in GFR accompanied by polyuria which is associated with increased urinary thromboxane (TX) excretion and glomerular TXB2 synthesis. The TXA2/ PGH2 receptor antagonist daltroban normalized GFR but not urine concentration. There was also a rise in plasma and urinary endothelin (ET-1) with increased papillary ET synthesis. The ETA/ETB receptor blocker bosentan restored GFR and the renal concentrating ability. Since we showed previously that ET-induced decreases in renal perfusion and ultrafiltration coefficient Kf are mediated by other autacoids such as TX, this may explain why both bosentan and daltroban normalized GFR. These results suggest that increased renal glomerular TX and vascular and inner medullary collecting duct ET synthesis contribute to defective GFR and distal tubular function in experimental BDL. Similar alterations may also predispose the human kidney to ARF in patients with obstructive jaundice.

Coexpression of periportal and perivenous enzymes in rat hepatocytes after experimental bile duct ligation: comparison with intrasplenically transplanted hepatocytes.

The coexpression of normally periportal and perivenous markers has been described in heterotopically transplanted hepatocytes. To determine whether such a coexpression might also occur in hepatocytes retaining their original intrahepatic location, we compared in bile-duct-ligated livers and intrasplenically transplanted hepatocytes, the expression and distribution of the predominantly periportal glucose-phosphatase, succinate dehydrogenase, and lactate dehydrogenase, the predominantly perivenous glutamate dehydrogenase, NADPH-dehydrogenase, and beta-hydroxybutyrate dehydrogenase, and the strictly perivenous glutamine synthetase. The coexpression of high levels of the two periportal markers glucose-6-phosphatase and lactate dehydrogenase and of the perivenous marker NADPH dehydrogenase was observed in two situations: in clusters of hepatocytes isolated within the ductular proliferation in bile-duct-ligated livers and the majority of intrasplenically transplanted hepatocytes. The expression of glutamine synthetase was different according to the site. The protein was observed in certain intrasplenically transplanted hepatocytes bordering the splenic vessels but was never detected in hepatocyte clusters found in bile-duct-ligated livers. Our study therefore suggests that the coexpression of periportal and perivenous markers in the same hepatocytes is likely to be a non-specific consequence of the loss of the normal connections of hepatocytes with the normal liver microcirculation.

Experimental biliary fibrosis correlates with increased numbers of fat-storing and Kupffer cells, and portal endotoxemia

In the present study, we have investigated the correlation between hepatic fibrosis in rats subjected to bile duct ligation, the numbers of Kupffer and fat-storing cells, and the level of endotoxin in both the portal and systemic circulation. The extent of hepatic fibrosis was measured by morphometry. Kupffer cells were identified by indirect immunoperoxidase staining using ED-2 anti-macrophage antibody. Fat-storing cells were stained with DE-B-5 anti-desmin antibody. Endotoxin levels were determined by the Limulus Lysate test. Following bile duct ligation, connective tissue septa rapidly developed in periportal areas. After 1 week, the volume density of connective tissue had increased from 0.6±0.1% in control animals to 3.8±1.1%. After 2 weeks, this volume increased to 19.9±1.3%, and after 3 weeks to 34.3±2.7%. The number of periportal fat-storing cells increased 2.8-fold during the first 2 weeks, whereas pericentral fat-storing cells increased only 1.7-fold. After 2 weeks, no further increase was observed. During the first week of bile duct ligation, the number of Kupffer cells increased nearly two-fold. Thereafter, no further increase was detected. In control rats, only two of ten rats showed low amounts of endotoxin in the portal blood. Portal endotoxemia increased with time after bile duct ligation. After 3 weeks, all rats were positive. The measured endotoxin levels were approximately 7 times higher than in control rats. We conclude that the development of fibrosis secondary to experimental bile duct ligation is accompanied by portal endotoxemia, and increases in the numbers of Kupffer and periportal fat-storing cells. We found a significant correlation between portal endotoxemia, the number of Kupffer and fat-storing cells, and the extent of fibrous septa, supporting the view that high endotoxemia levels coincide with Kupffer cell activation and fibrogenesis.

Cytokines and progression of liver damage in experimental bile duct ligation

Cytokines and progression of liver damage in experimental bile duct ligation

Morphology of the liver of the brook lamprey, Lampetra lamottenii before and during infection with the nematode, Truttaedacnitis stelmioides, hepatocytes, sinusoids, and perisinusoidal cells

Routine light microscopy and transmission and scanning electron microscopy were used to describe and compare the livers of larval lampreys, Lampetra lamottenii before and during infection of the bile ducts by the nematode, Truttaedacnitis stelmioides. The hepatocytes of uninfected animals differ from other lamprey species in that they contain abundant glycogen, smooth endoplasmic reticulum (SER), and lipoprotein indicating that the liver may be involved in glucose metabolism. Infestation of the biliary tree by T. stelmioides coincides with alterations to the hepatocytes. These changes include dilation of the bile canaliculi, smooth endoplasmic reticulum (SER), rough endoplasmic reticulum (RER), and Golgi apparatus, swollen mitochondria in cells showing a high degree of hypertrophy, and an abundance of dense bodies. Following infection, the sinusoidal lumina became dilated and contain a moderate electron-dense precipitate, an abundance of melanomacrophages, lipocytes, and mononuclear cells. There is also a widening of the fenestrae of the sinusoidal endothelium following infection. Many of the changes in hepatocytes and sinusoids following parasite infections closely resemble those observed in hepatocytes in various pathologies and following experimental bile duct ligation and, therefore, are likely a consequence of increased biliary pressure due to bile duct obstruction.

Cryonecrosis of the liver after experimental bile duct ligation

Cryonecrosis of the liver after experimental bile duct ligation

Significance of liver canalicular changes after experimental bile duct ligation

An ultrastructural morphologic, histochemical, and morphometric study was made on the canalicular structures in rat liver after experimental bile duct ligation (BDL). Four different canalicular structures are distinguished, which are proved by morphometry to correspond to the different types observed during perinatal maturation. Canaliculus type 4, corresponding to the excretory pole of a normal adult hepatocyte, is characterized by a lumen filled with regular, slender microvilli. Canaliculus type 3, corresponding to the canalicular structure described in cholestasis, has a wide lumen devoid of microvilli and is found 2–3 days after BDL. A transitional canalicular structure between type 3 and 4, partly provided with microvilli, is found from the first day after BDL. After 3 days of BDL an increasing number of other canalicular types is observed. Canaliculi type 2, showing an irregular lumen with irregular microvillus-like projections, and canaliculi type 1, representing intracytoplasmic invaginations of two adjacent cell membranes, are found together with canaliculi type 3 and type 3–4. After incubation for alkaline phosphatase, the enzyme activity is detected in canaliculi type 1, 3–4, and type 4. The striking analogy of the sequential changes in canalicular structures after BDL and during perinatal maturation leads to the following interpretation: (1) Canaliculi type 1 and 2, more frequently observed in longstanding cholestasis correspond to newly formed excretory poles of the hepatocytes. (2) Canaliculus type 3, or the so-called cholestatic canalicular type, reflects an inactive secretory function. Morphometrically this canalicular type shows a very low S/V relation which may be partly responsible for this secretory inactivity. (3) Canaliculus type 3–4 reflects a disturbed secretory function after BDL and a beginning secretory activity during perinatal maturation.