C3H/He Mice as an Incompatible Cholangiocarcinoma Model by Clonorchis sinensis, Dicyclanil and N-Nitrosodimethylamine

Abstract

Clonorchis sinensis is a Group-I bio-carcinogen, associated with cholangiocarcinoma (CCA). The hamster is the only experimental model of C. sinensis-mediated CCA, but we oblige another animal model. The present study intended to develop a C. sinensis (Cs) mediated CCA model using C3H/He mice, co-stimulated with N-nitrosodimethyl-amine (NDMA) and dicyclanil (DC). The mice were divided into 8 groups with different combinations of Cs, NDMA, and DC. Six months later the mice were sacrificed and subjected to gross and histopathological examination. The body weights were significantly reduced among the groups treated with 2 or more agents (eg. Cs+NDMA, Cs+DC, NDMA+DC, and Cs+NDMA+DC). In contrast, liver weight percentages to body weight were increased in above groups by 4.1% to 4.7%. A Change of the spleen weight was observed only in Cs+NDMA group. Though C. sinensis infection is evident from hyperplastic changes, only 1 worm was recovered. T wo mice, 1 from Cs and the other from Cs+DC group, showed mass forming lesions; 1 (281.2 mm(3)) from the Cs group was a hepatocellular adenoma and the other (280.6 mm(3)) from the Cs+DC group was a cystic mass (peliosis). Higher prevalence of gray-white nodules was observed in Cs group (42.9%) followed by Cs+NDMA+DC group (21.4%). The mice of the Cs+NDMA+DC group showed hyper-proliferation of the bile duct with fibrotic changes. No characteristic change for CCA was recognized in any of the groups. In conclusion, C3H/He mice produce no CCA but extensive fibrosis when they are challenged by Cs, NDMA, and DC together.

Keywords: C3H/He mice; Clonorchis sinensis; NDMA; cholangiocarcinoma; dicyclanil; fibrosis; hepatocellular adenoma; peliosis.

Fig. 1.

Experimental scheme for the development cholangiocarcinoma (CCA) in C3H/He mice.

Fig. 2.

Gross findings in different groups of mice at necropsy. (A) Body weight. (B) Liver weight. (C) Liver weight percentage to body weight. (D) Spleen weight. (E) Spleen weight percentage to body weight. (F) Length of spleen. Bar diagrams are mean±SEM. ^*P<0.05 and ^†P<0.01.

Fig. 3.

Gross photos and histopathology of mice liver from different groups. (A) Gross appearance of liver showing normal appearance among most of the groups (scale bar=5 mm). (B) Representative H-E staining of liver section showing typical biliary triad in most of the mice groups (scale bar=200 μm). a, control; b, Cs; c, NDMA; d, DC; e, Cs+NDMA; f, Cs+DC; g, NDMA+DC; h, Cs+NDMA+DC.

Fig. 4.

Gross and histopathological section of abscess from Cs group liver. (A) Gross enlarged view of abscess (scale bar=5 mm). (B-D) Representative H-E staining of abscess (B-C: scale bar=100 μm; D: scale bar=50 μm).

Fig. 5.

Hyperplastic and fibrotic changes in Cs+NDMA+DC group mice. (A) H-E staining of liver section showing hyper-proliferative bile ducts surrounded by a fibrotic zone. (B) A portion of extensive fibrosis (scale bar=50 μm).

Fig. 6.

Glycogen rich clear cells in hepatocellular adenoma of a mouse in Cs group and peliosis (cystic lesion) in a Cs+DC group mouse. (A) H-E staining of adenoma mass (scale bar=400 μm). (B) Periodic acid Schiff staining (scale bar=100 μm). (C) Diastase periodic acid Schiff staining (scale bar=50 μm). (D) H-E staining of cystic lesions (scale bar=1 mm). (E) Pooled erythrocytes lined by hepatocytes (scale bar=100 μm). (F) Enlarged view showed no accumulation of inflammatory cells (scale bar=100 μm).