*3.2. Confirmation of Periportalization in Further Mouse Models of Liver Fibrosis*

Bile duct ligation (BDL). This mouse model was investigated because it represents a periportal fibrosis model, in contrast to the CCl4 model described above, where pericentral fibrosis is induced. Ligation of the common bile duct leads to the formation of bile infarcts due to the rupture of the apical hepatocyte membrane in the acute phase up to day three [22]. In the chronic phase after approximately seven days, the liver adapts to the obstruction of the bile duct, bile infarcts do no longer occur and the infarct regions regenerate. However, a slowly progressing periportal fibrosis occurs in the chronic phase. In the present study, mice at day 21 after BDL were compared to sham-operated controls (Figure 6A). Macroscopically, BDL mice showed a strongly distended gallbladder with transparent, so-called 'white bile' (Figure 6B). Histologically, a strong ductular response was observed accompanied by periportal fibrosis (Figure 6B).

**Figure 5.** *Cont*.

**Figure 5.** Spatio-temporal analysis of periportalization of selected pericentral and periportal enzymes. (**A**) Immunostaining of the pericentral proteins cytochrome P450 3A, 1A, 2C, 2E and glutamine synthetase (GS) as well as the periportal proteins arginase 1 and carbamoyl phosphate synthetase 1 (CPS1). The left margin indicates the time of treatment with CCl4. Scale bars: 200 μm. (**B**) 3D-Reconstructions of CYP1A immunostained liver tissue showing normal pericentral zonation in control (left), and central-to-central bridging at month six of CCl4 intoxication. (**C**) Whole slide scans of CYP1A-immunostained liver lobules at 2, 6 and 12 months after CCl4 treatment with segmentation (green) and quantification of the fraction of the CYP1A positive area. The data are means ± standard errors of 3 mice per time point. \* *p* < 0.05; \*\*\* *p* < 0.001 compared to the untreated controls (0). (**D**) Whole slide scans of CYP1A and arginase1 positive liver tissue.

**Figure 6.** Periportal fibrosis after bile duct ligation (BDL). (**A**) Experimental schedule. (**B**) Macroscopic appearance and visualization of fibrosis by Sirius red staining. Scale bars: 100 μm.

Immunostaining for CYP2E1 and GS showed a massive decrease 21 days after BDL (Figure 7A). While the CYP2E1 positive area amounted to approximately 50% of the total tissue area in controls, this value fell to only approximately 14% after BDL (Figure 7C,D). Corresponding analysis of the periportal enzymes arginase 1 and CPS1 showed that the negative pericentral regions in controls become positive ('periportalized') after BDL (Figure 7B). Whole slide scans immunostained for CYP2E1 and CPS1 confirmed the results shown in Figure 7 (Figure S1). Therefore, BDL associated fibrosis was accompanied by similar changes in zonation as fibrosis induced by chronic administration of CCl4. Mdr2−/<sup>−</sup> mice represent a further model of periportal fibrosis. Similar to the CCl4 model and BDL, also eight- and 64-week-old knockout mice showed reduced expression of CYP2E1 (Figure S2).

**Figure 7.** Periportalization of lobular zonation after BDL. (**A**) Immunostaining of the pericentral proteins CYP2E1 and GS. Scale bars: 100 μm. (**B**) Immunostaining of the periportal proteins arginase1 and CPS1. Scale bars: 200 μm. (**C**) CYP2E1-immunostained whole slide scans of liver lobules of BDL mice, and segmentation of the positive area (green). (**D**) Quantification of the fraction of CYP2E1 positive tissue 21 days after BDL and in controls. The data are means ± standard errors of 3 mice per group. \*\*\* *p* < 0.001 compared to the sham controls (0).
