A novel murine model of reversible bile duct obstruction demonstrates rapid improvement of cholestatic liver injury

Abstract

There are limited murine models of cholestatic liver diseases characterized by chronic biliary obstruction and resumption of bile flow. While murine bile duct ligation (BDL) is a well-established model of obstructive cholestasis, current models of BDL reversal (BDLR) alter biliary anatomy. We aimed to develop a more physiologic model of BDLR to evaluate the time course and mechanism for resolution of hepatic injury after biliary obstruction. In the present study, we restored bile flow into the duodenum without disruption of the gall bladder after murine BDL using biocompatible PE-50 tubing. After establishing the technique, overall survival for BDLR at 7 or 14 days after BDL was 88%. Sham laparotomy was performed in control mice. Laboratory data, liver histology, and hepatic gene expression were compared among BDL, BDLR, and controls. Laboratory evidence of cholestatic liver injury was observed at day 7 after BDL and rapid improvement occurred within 48 hr of BDLR. After BDLR there was also enhanced gene expression for the bile acid transporter Abcb11, however, bile duct proliferation persisted. Assessment of the immune response showed increased gene and protein expression for the general immune cell marker Cd45 in BDLR versus BDL mice suggesting a reparative immune response after BDLR. In summary, we have established a novel murine model of BDLR that allows for the investigation into bile acid and immune pathways responsible for hepatic repair following obstructive cholestasis. Future studies with our model may identify targets for new therapies to improve outcome in pediatric and adult cholestatic liver disease.

Keywords: biliary obstruction; cholestasis; liver injury; liver repair; macrophages.

FIGURE 1

Experimental murine model of reversible bile duct ligation (BDL). (a) BDL surgery was performed by placing two knots on the common bile duct. Reversal was achieved by using a PE‐50 biocompatible tubing to connect the bile duct to the small intestine. (b) Experimental groups included BDL and BDLR mice with sham surgeries performed in parallel with each surgery in the experimental groups (B—bile duct ligation surgery, R—reperfusion surgery, X—sham laparotomy). (c) After establishing the technique of BDLR, overall survival after 7‐day and 14‐day for BDLR surgery reached 88%

FIGURE 2

Serum liver chemistries after BDL and BDLR. (a and b) Serum alanine aminotransferase (ALT) and alkaline phosphatase (ALP) values rapidly improved following BDLR in mice. (c and d) Total serum bilirubin and bile acid levels also quickly normalized. Serum bilirubin levels increased between day 2 (D2) and day 7 (D7) with p < .001. All serum liver chemistries were significantly improved at reversal day 2 (R‐D2) and 7 (R‐D7) compared to D7. Mean laboratory values after sham surgery were normal for all values except serum bile acids. Upper limit of normal is depicted by a dotted line for each biochemical test (ALT = 77 IU/L, ALP = 96 IU/L, total bilirubin = 0.9 mg/dl, and bile acid < 1 μmol/L). ⁺Indicates comparison to D2; *indicates comparison to D7. **p < .01, ***p < .001, ****p < .0001; ⁺⁺⁺ p < .001

FIGURE 3

Improvement in acute histologic changes after BDLR. (a) Total Ishak score for H&E slides across all groups demonstrated significantly worse inflammatory liver injury in BDL mice as compared to both their sham controls and BDLR mice. (b) Individual scores for portal inflammation, focal lytic necrosis, confluent necrosis, and periportal inflammation were all significantly worse in BDL mice compared to BDLR. **p < .01, ***p < .001

FIGURE 4

Histologic changes after BDL and BDLR. (a) Hematoxylin & Eosin (H&E) and immunohistochemistry of livers in BDL mice demonstrated hepatocyte injury with a portal infiltrate and increased numbers of CK19‐positive bile ducts. (b) In contrast, BDLR mice demonstrated minimal hepatocyte injury, portal infiltrate, and bile duct proliferation. Overall, Ki67 staining for cell proliferation was sparse across all groups and Sirius red staining showed minimal fibrosis in all mice

FIGURE 5

Characterization of immune cell infiltrate by immunohistochemistry. (a) Prominent staining for CD45‐positive immune cells and macrophages (α‐CD68 and α‐F4/80) was observed in the portal tracts in BDL mice rather than the parenchyma as seen in sham controls. (b) The portal infiltration of immune cells appeared reduced in BDLR mice. Ly6g‐positive neutrophils were sparse across all samples, although increased in BDLR mice compared to sham (a and b)

FIGURE 6

Increased numbers of CD45‐positive cells in BDLR mice. (a) Quantification of the percent area of CK19‐positive stain was significantly increased in BDL mice versus sham controls. There was no difference in percent of positive Sirius Red stain between groups. (b) Cell quantification demonstrated increased numbers of CD45‐positive cells in BDLR mice compared to BDL. Ly6g‐positive cells were increased only in BDLR mice versus their sham controls. *p < .05

FIGURE 7

Changes in inflammatory gene expression. (a) CD45 and Ly6g gene expression were significantly increased in BDLR mice compared to BDL and sham controls. However, Cd68 was increased in both BDL and BDLR mice compared to their controls, respectively, whereas Emr1 gene expression was not different between groups. (b) The proinflammatory genes Ccl2, Tnf‐α, and Il33 were significantly increased in BDL mice, whereas Ifn‐γ was increased in BDLR mice. *p < .05, **p < .01, ***p < .001, ****p < .0001

FIGURE 8

Gene expression of bile acid metabolism genes and mediators of fibrosis. (a) BDL mice had significantly decreased gene expression for Cyp7a1 and Abcc2 compared to their sham controls. In contrast, Abcb11 was significantly increased after BDLR versus BDL. (b) Increased gene expression for Collagen 1 (Col1a1) was present in BDL mice compared to both their sham controls and BDLR mice. Increased expression of Acta2 was observed only in BDL mice. *p < .05, **p < .01, ***p < .001, ****p < .0001