TEMPORAL‐SPATIAL REGULATION OF APOPTOSIS IN EXPERIMENTAL BILIARY ATRESIA

Abstract

Background: Biliary atresia results from a fibrosing cholangitis that leads to bile duct obstruction. Recent studies using a mouse model of rhesus rotavirus (RRV)-induced biliary atresia demonstrate increased TNF-alpha levels in liver. Based on the established role of TNF-alpha in apoptosis, we hypothesized that RRV triggers bile duct apoptosis in experimental biliary atresia. Methods: Balb/c mice were inoculated with RRV or saline on day 1 of life, and the liver and EHBD were studied at days 2-18. Hepatic mononuclear cells were isolated for gene expression analysis by real-time PCR. TUNEL and active caspase-3 assays were performed on paraffin-embedded liver and EHBD sections, while mRNA expression of caspases was assessed using snap-frozen samples. Results: RRV infection resulted in obstruction of the EHBD after day 6. At this time, there was a marked increase in mRNA expression of FasL, perforin, and granzyme B in hepatic mononuclear cells compared to controls (p < 0.02). From days 6-18, intrahepatic portal tracts consistently showed higher numbers of TUNEL-positive cells than controls (p < 0.02). To investigate temporal dynamics of apoptosis in the EHBD, we counted TUNEL-positive cells at days 2-14. TUNEL labeling was localized to duct epithelial cells at day 4 and peaked in cells occluding the duct lumen at day 5 (mean ± SD: 20.9 ± 4.3 vs. 0.53 ± 0.29; p < 0.01). The active caspase-3 assay confirmed these results, showing maximal cell staining at day 5. mRNA expression of caspases-1 and -4 was higher in both liver and EHBD, with fold changes ranging from 2.0-7.4. Conclusions: RRV challenge induces: 1) the expression of pro-apoptotic molecules in hepatic mononuclear cells, 2) infiltration of portal tracts with TUNEL-labeled cells, and 3) a peak in TUNEL and active caspase-3 labeling in the EHBD at the time of obstruction. These results support a pathogenic role for apoptosis in experimental biliary atresia.