1. Volume 42, Issue 2, Pages 257-265 (February 2005)
Adaptative bile duct proliferative response in experimental bile duct ischemia 
Marc Beaussier, Dominique Wendum, Laura Fouassier, Colette Rey, Véronique Barbu, Elisabeth Lasnier, André Lienhart, Jean-Yves Scoazec, Olivier Rosmorduc, Chantal Housset 
Journal of Hepatology 
Volume 42, Issue 2, Pages (February 2005)
DOI: /j.jhep
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

2. Fig. 1
Model of hepatic arterial deprivation. All the rats underwent the same initial surgical procedure, in which the liver was isolated from peripheral vascular connections (dotted line). In Sham (A), no additional vascular intervention was performed. In the HA group (B), the animals underwent division of the main hepatic artery. In the EP group (C), the animals underwent double ligation of the extrahepatic peribiliary vascular plexus under cover of common bile duct canulation. In the HA/EP group (D), the animals underwent both division of the main hepatic artery and double ligation of the extrahepatic peribiliary vascular plexus.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

3. Fig. 2
Body weight and biochemical liver tests in experimental groups. Body weight (A) and serum concentrations of total bilirubin (B) and of liver enzymes (C–F) were assessed immediately before (time 0) and weekly after surgery, in rats submitted to Sham operation (O); HA, i.e. section of the main hepatic artery (▵); EP, i.e. ligation of the extrahepatic peribiliary vascular plexus (□); HA/EP, i.e. combined HA and EP procedures (♦), as described in methods. Serum concentrations of bile acids (G) are shown in HA/EP (♦) as compared with Sham (O). Results of at least 4 animals are shown as means ±SEM. Animals in the HA/EP group are significantly different from other groups for bilirubin and for liver enzymes, and from Sham for bile acids (P<0.05 ANOVA with repeated measures). Differences between groups at each time point were assessed with Mann Whitney U test (*P<0.05).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

4. Fig. 3
Induction of VEGF expression by ischemia/hypoxia in bile ducts. In vivo (A, B), VEGF expression was assessed by immunohistochemistry in the liver of Sham and HA/EP-operated rats. Portal tract-centered photomicrographs of liver tissue sections from rats on post-operative day 2 are shown. In Sham (A), no VEGF immunolabeling is detectable; in HA/EP (B), VEGF immunoreactivity is visible in the epithelium lining an interlobular bile duct (open arrow), and with less intensity in the hepatic arteriole vascular wall (closed arrow). Original magnification ×200. In vitro (C–E), VEGF expression was assessed by immunofluorescence (C, D) and RT-PCR (E) in bile duct preparations maintained under normoxia or exposed to 4h-hypoxia. Results of RT-PCR are expressed as a number of copies of all VEGF transcripts per microgram 18S RNA. They represent means ±SEM of 8 bile duct preparations. *P<0.01 vs normoxia.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

5. Fig. 4
Bile duct damage induced by ischemia. Representative photomicrograph of bile duct damage in a HA/EP-operated rat, 4h after the induction of ischemia. Bile duct epithelial cells show nuclear clarification and cytoplasmic vacuolization (open arrowheads), cytoplasmic protrusions in the bile duct lumen (closed arrowheads) and apoptotic figures (arrow). A cell plug illustrating epithelial cell desquamation, is visible in the bile duct lumen. Semi-thin liver tissue section stained with toluidine blue. Original magnification ×520.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

6. Fig. 5
Bile duct proliferation induced by ischemia. Portal tract-centered photomicrographs of liver tissue sections at different post-operative time points are shown (A–F). On day 2 in HA/EP (A), mitotic figures are visible in bile duct epithelial cells (arrow). On week 1 in HA/EP (B), bile ducts are surrounded and occasionally infiltrated by inflammatory cells (the arrow points to a polymorphonuclear leukocyte infiltrating the bile duct epithelium). As compared with normal portal tract in a Sham-operated rat (C), in a HA/EP-operated rat on week 1 (D), a ductular reaction made of well-formed bile duct structures confined to enlarged portal tract has formed. At later time points, in HA/EP rats, on week 2 (E) and week 6 (F), the ductular reaction shows ongoing progression, while lobular extension remains limited. Original magnifications: A, B ×400; C–F ×200. (G) shows the progression of ductular reaction, as assessed by the count of bile duct sections. Results are expressed as a number of bile duct sections per field. They represent means ±SEM of 20 fields in tissue sections from 2 animals in each condition. (*) P<0.05 vs all other conditions.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

7. Fig. 6
Time course of cholangiocyte proliferation following bile duct ischemia. Cell proliferation was assessed by Ki67 immunolabeling in the liver of Sham- and HA/EP-operated animals. (A,B) Portal tract-centered photomicrographs of liver tissue sections from HA/EP-operated rats show, on post-operative week 1 (A), a large number of Ki67-labeled cholangiocytes; on post-operative week 6 (B), the vast majority of cholangiocytes are Ki67-negative, one labeled hepatocyte is shown (arrow) as an internal positive control. Original magnification: A, B×100. Cholangiocyte proliferation was measured as the proportion of Ki67-positive cholangiocytes: (C) in bile ducts, irrespective of their anatomical location, on liver tissue sections from Sham (open bars) and HA/EP (solid bars) rats on post-operative day (D) 2, weeks (W) 1, 2 and 6; (D) in periportal ductules and in interlobular bile ducts, separately, on liver tissue sections from Sham (open bars) and HA/EP (solid bars) rats on post-operative day 2. Ki67-labeled cholangiocytes in a small periportal ductule (open arrow) and in an interlobular duct (closed arrow) at day 2, are shown in inset. In (C,D), proliferation was assessed by the count of Ki67-positive and -negative cholangiocytes in 20 random portal tract-centered fields from two animals in each condition. Results represent means ±SEM. (*P<0.05 vs Sham).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

8. Fig. 7
Progression of fibrosis associated with bile duct proliferative response to ischemia. Fibrosis was assessed by Sirius red staining. Liver tissue sections are shown from a Sham-operated rat (A) and from HA/EP-operated rats on post-operative day D2 (B); weeks W1 (C), W2 (D), and W6 (E). In (A, B) Sirius red staining is detected mostly around central veins, vascular and ductal structures in portal tracts, as in normal rats. In (C–E), thin fibrotic deposits are detected around proliferative bile ducts of the ductular reaction. Original magnification ×50. In (F), morphometric analysis of Sirius red staining in HA/EP-operated rats on post-operative day D2, weeks W1, W2 and W6, is shown, in comparison with normal rats. Results represent the means ±SEM of Sirius red-stained areas from 10 consecutive fields in tissue sections from 2 animals in each condition. (*P<0.05 vs normal).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

9. Fig. 8
Changes in bile secretion associated with bile duct ischemia and proliferation. (A) Bile flow and (B) bicarbonate output were measured in Sham-operated rats (n=3), and in HA/EP rats within 24h (D1; n=3) or 3 weeks (W3; n=5) after the induction of ischemia. All animals underwent an infusion of secretin (total amount of 0.2nmol) through the jugular vein during 50min, as indicated. Results represent means ±SEM. The group of rats within 24h of ischemia, is significantly different from other groups for bile flow and bicarbonate output. The group of rats at 3 weeks after the induction of ischemia was significantly different from Sham, only for bicarbonate output (P<0.05 ANOVA with repeated measures). Differences between groups at each time point were assessed with Mann Whitney U test (*P<0.05 vs Sham and Ischemia W3; **P<0.05 vs Sham).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions