Figure 5. CCl₄ increases hydropic degeneration and DNA damage in NS-depleted hepatocytes and enhances A6⁺ cell expansion and biliary hyperplasia in albNS^cko livers.

(A) Mice are injected with CCl₄ at 2 weeks of age and analyzed on the first, second, and fourth day post injection. H&E staining shows that CCl₄ causes acute pericentral necrosis and leukocyte infiltration (indicated by white arrows) in both NS^flx/flx (A1) and albNS^cko livers (A3) during the first 2 days of the injection. CCl₄ triggers hydropic degeneration (indicated by black arrows) of hepatocytes specifically in the peri-nodular regions of albNS^cko livers. Bars, 100um. (B) In response to CCl₄ treatment, albNS^cko livers also display a notable increase of CK-19⁺ ducts (B1, arrows) and scattered progenitor-like cells (B2), both of which are not seen in CCl₄-treated NS^flx/flx livers (B3). (C) Immunostaining on serial sections of 4um thickness shows that A6 and CK-19 double-positive progenitor cells are increased significantly in CCl₄-treated albNS^cko livers (C1) compared to oil-treated albNS^cko livers (C2). (D) qRT-PCR assays showed that CCl₄-induced damage upregulates the transcript levels of both EpCAM and AFP in albNS^cko livers but not in NS^flx/flx livers. Asterisks represent p values (described in Fig. 1). (E) In oil-treated albNS^cko livers (grey bars), most Ki67⁺ cells are found in the regenerative nodules and the bile duct epithelium, and only a small percentage of the non-regenerative hepatocytes in peri-nodular regions are Ki-67⁺. Following CCl₄ treatment, mitotic cells are increased most significantly on the second day post injection in the peri-nodular areas, regenerative nodules, and bile duct epithelium. (F) Most γ-H2AX⁺ cells (indicated by white arrows) in oil-treated albNS^cko livers are found in the peri-nodular areas. On the second day post injection, CCl₄ increases γ-H2AX⁺ cells only in the peri-nodular regions of albNS^cko livers where NS is deleted but not in the regenerative nodules or the bile duct epithelium (indicated by yellow arrows). The CCl₄ treatment itself does not cause any DNA damage in NS^flx/flx livers. Scale bars, 50um in (A, B, and C) and 20um in (F).