Determining Bile Duct Density in the Mouse Liver

Analysis of BD development and repair in mice is an important tool in studying the pathogenesis and mechanism of cholestatic disorders. In addition, development of new therapies is in part dependent upon establishing a reproducible and preferably quantifiable phenotype. Current phenotyping in mouse models usually involves serum chemistry, liver histology and immunostaining for cell-type specific markers. Although these techniques generate valuable information about the structure and function of the biliary system, they do not provide a direct measure of the effects of a given genetic manipulation on the number of BDs. In anatomic pathology, BD paucity in human patients is determined through the analysis of BD to PV ratio in a biopsy section⁹. While clinicians employ serum chemistry analysis to determine the severity of cholestasis and liver disease in ALGS and other cholestatic diseases^18,19, histological assessment in mouse models is critical to both understanding the effects of disease modifiers on BD development and the effectiveness of therapies on restoring normal duct development. This is in part because mice can have a severe decrease in the number of patent BDs but still show only a modest increase in the serum bilirubin level⁸, likely due to the highly efficient bile drainage in the mouse liver. Our previous work has shown that Jag1 heterozygosity results in impaired BD maturation but not the absence of cholangcioytes⁸. Thus, to analyze BD development and assess disease severity in mouse models, it is not sufficient to merely examine the absence or presence of cholangiocytes. To address this issue, here we have presented a simple method for objective measurement of patent BD numbers in the mouse liver.

Our analysis is dependent on proper fixation and embedding of the mouse liver. Livers are embedded ventral side up to ensure similar sectioning from one sample to another. This is the most stable position. The sections used for staining must be deep enough in the liver lobes, as there are differences in the number of BDs and the size of PVs in the periphery versus the hilum of the liver. We occasionally see PVs that are cut longitudinally. In those cases, there is usually a neighboring BD that is also cut longitudinally and appears like a long open tube. To ensure reproducibility, we count these long tubes as a single BD. Identification of patent BDs is unambiguous for the most part. However, some biliary structures appear lumenized but do not show the round to ellipsoid morphology typically seen in normal BDs¹⁴. In our hands, this can sometimes result in one structure being called a BD by an investigator but not by another colleague. Therefore, to ensure consistency and reproducibility in data analysis and presentation, we recommend that all samples related to a specific project be analyzed by two investigators independently.

Anti-CK8 has been shown to mark immature and mature biliary cells, while anti-CK19 only marks mature biliary cells^12,20. Therefore, even if a PV is not associated with a mature BD, it can be readily differentiated from central veins because of the presence of CK8+ cells. Using these two antibodies in combination with anti-αSMA ensures complete coverage of portal tracts in our quantification. Moreover, in our hands, the individual CK19 or CK8 staining of the biliary cells generates a relatively weak signal and is associated with some background staining. Mixing the two CK antibodies results in a consistently strong signal in biliary cells and therefore facilitates the quantification.

Maturation of the intrahepatic biliary tree occurs in a hilar-to-peripheral direction and continues postnatally²¹. Indeed, there are many more immature cholangiocytes in early postnatal livers, especially in peripheral areas, which are at the leading front of postnatal liver expansion. Moreover, we observed a change in BD numbers as the animals age⁸, with more ducts in older animals. In addition, some portal structures contain multiple BDs while others have one or no ducts, particularly along the liver periphery. We use a microscopy slide covering all liver lobes for each animal and systematically quantify the BD to PV ratio across the whole slide. While this is not essential, it ensures that ample portal tracts are analyzed for each animal regardless of age and liver size (60-90 PVs per liver depending on the genotype and age). Moreover, by analyzing all PVs on the slide, we ensure that both more mature hilar and less mature peripheral areas are counted at all stages of liver development. Covering all liver lobes for each animal can also decrease variability in measurements if a given mutation does not affect BD development uniformly across the liver.

The method is limited in distinguishing smaller bile conduits from groups of unincorporated biliary cells. The bile ductules⁴ are usually too small to be recognized as a lumenized structure in the magnification that we use to analyze these stainings. Therefore, they are likely to be excluded from our quantifications, and thus the method is skewed towards identifying medium to large ducts. Despite this limitation, the method described here readily distinguishes between the Jag1+/– and Jag1+/+ livers. Moreover, it is sensitive enough to detect the partial rescue of the Jag1+/– BD paucity upon simultaneous loss of one copy of Polgut1+/– and the modest increase in BD density in Jag1+/+ animals with conditional loss of Poglut1 in vascular smooth muscle cells⁸. These observations indicate this method’s usefulness in determining alterations in BD density in various genetic backgrounds, even when the changes in BD number are modest.

In recent years, visualization of the 3D structure of the biliary tree has been used by several groups to analyze BD development^22,23,24,25. These elegant methods rely on filling the biliary tree from the common BD by ink or resin, and therefore examine the patency of the biliary system. Moreover, they provide information about the 3D structure of the biliary tree and its formation in the liver periphery as the liver grows, which cannot be assessed by 2D assessment used in protocols like the one presented here. However, successful performance of these 3D visualization techniques requires considerable expertise²⁶. In contrast, the technique presented here is rather straightforward and can be executed by any group with access to routine equipment for histological and imaging analysis. Moreover, analysis of the sections double-stained for wsCK and αSMA will also show whether ductular reactions or vascular smooth muscle cell abnormalities exist in the liver^8,27,28. We suggest that quantifying the BD to PV ratio in the whole liver sections can provide a sensitive and reproducible measure of biliary development in mice and can serve as a relatively easy technique to help the investigators decide whether they should consider more sophisticated techniques like 3D visualization of the biliary tree.