Core Hippo pathway components act as a brake on Yap and Taz in the development and maintenance of the biliary network.

The development of the biliary system is a complex yet poorly understood process, with relevance to multiple diseases, including biliary atresia, choledochal cysts and gallbladder agenesis. We present here a crucial role for Hippo-Yap/Taz signaling in this context. Analysis of sav1 mutant zebrafish revealed dysplastic morphology and expansion of both intrahepatic and extrahepatic biliary cells, and ultimately larval lethality. Biliary dysgenesis, but not larval lethality, is driven primarily by Yap signaling. Re-expression of Sav1 protein in sav1-/- hepatocytes is able to overcome these initial deficits and allows sav1-/- fish to survive, suggesting cell non-autonomous signaling from hepatocytes. Examination of sav1-/- rescued adults reveals loss of gallbladder and formation of dysplastic cell masses expressing biliary markers, suggesting roles for Hippo signaling in extrahepatic biliary carcinomas. Deletion of stk3 revealed that the phenotypes observed in sav1 mutant fish function primarily through canonical Hippo signaling and supports a role for phosphatase PP2A, but also suggests Sav1 has functions in addition to facilitating Stk3 activity. Overall, this study defines a role for Hippo-Yap signaling in the maintenance of both intra- and extrahepatic biliary ducts.

Postnatal, ontogenic liver growth accomplished by biliary/oval cell proliferation and differentiation.

INTRODUCTION: The liver is well known for its enormous regenerative capacity. If the hepatocytes are compromised the reserve stem cells can regrow the lost tissue by means of oval cells differentiating into hepatocytes. We were curious whether this standby system was able to compensate for ontogenic liver growth arrested by 2-acetylaminofluorene (AAF) treatment or if it can be influenced by cholic acid, known to promote liver growth in several reactions. METHODS: (i) Four weeks-old (60-70g) male F344 rats were kept on standard chow and treated with solvent only, (ii) others were kept on 0,2% cholic acid (CA) enriched diet, (iii) treated with AAF, or (iiii) given a combination of CA diet and AAF treatment (AAF/CA). The proliferative response of epithelial cells was characterized by pulse bromodeoxyuridine labelling. The relative gene expression levels of senescence-related factors and bile acid receptors were determined by quantitative real-time polymerase chain reaction analysis. RESULTS: AAF administration efficiently inhibited the physiological proliferation of hepatocytes in young, male F344 rats after weaning. The activation of stem cells was indicated by the increased proliferation of periportal biliary/oval cells (B/OC). If the rats were fed additionally by cholic acid enriched diet, typical oval cell reaction emerged, subsequently the oval cells differentiated into hepatocytes restituting liver growth. This reaction was mediated by increased production of HGF, IL-6 and SCF by the damaged liver. Moreover, upregulation of FXR expression on B/OC made them competent for bile acids. Our results indicate that endogenous, autocrine mechanisms involved in liver ontogeny are also able to activate the backup regenerative machinery of stem cells.

Directing the growth and alignment of biliary epithelium within extracellular matrix hydrogels.

Three-dimensional (3D) printing of decellularized extracellular matrix (dECM) hydrogels is a promising technique for regenerative engineering. 3D-printing enables the reproducible and precise patterning of multiple cells and biomaterials in 3D, while dECM has high organ-specific bioactivity. However, dECM hydrogels often display poor printability on their own and necessitate additives or support materials to enable true 3D structures. In this study, we used a sacrificial material, 3D-printed Pluronic F-127, to serve as a platform into which dECM hydrogel can be incorporated to create specifically designed structures made entirely up of dECM. The effects of 3D dECM are studied in the context of engineering the intrahepatic biliary tree, an often-understudied topic in liver tissue engineering. Encapsulating biliary epithelial cells (cholangiocytes) within liver dECM has been shown to lead to the formation of complex biliary trees in vitro. By varying several aspects of the dECM structures' geometry, such as width and angle, we show that we can guide the directional formation of biliary trees. This is confirmed by computational 3D image analysis of duct alignment. This system also enables fabrication of a true multi-layer dECM structure and the formation of 3D biliary trees into which other cell types can be seeded. For example, we show that hepatocyte spheroids can be easily incorporated within this system, and that the seeding sequence influences the resulting structures after seven days in culture. STATEMENT OF SIGNIFICANCE: The field of liver tissue engineering has progressed significantly within the past several years, however engineering the intrahepatic biliary tree has remained a significant challenge. In this study, we utilize the inherent bioactivity of decellularized extracellular matrix (dECM) hydrogels and 3D-printing of a sacrificial biomaterial to create spatially defined, 3D biliary trees. The creation of patterned, 3D dECM hydrogels in the past has only been possible with additives to the gel that may stifle its bioactivity, or with rigid and permanent support structures that may present issues upon implantation. Additionally, the biological effect of 3D spatially patterned liver dECM has not been demonstrated independent of the effects of dECM bioactivity alone. This study demonstrates that sacrificial materials can be used to create pure, multi-layer dECM structures, and that strut width and angle can be changed to influence the formation and alignment of biliary trees encapsulated within. Furthermore, this strategy allows co-culture of other cells such as hepatocytes. We demonstrate that not only does this system show promise for tissue engineering the intrahepatic biliary tree, but it also aids in the study of duct formation and cell-cell interactions.

Characterization and spatial relationships of the hepatic vascular-biliary tracts, and their associated pancreocytes and macrophages, in the model fish guppy (Poecilia reticulata): A study of serial sections by light microscopy.

The guppy is a tropical fish that has been used as an experimental model organism in science. It is a species well adapted to the natural environment and that can support adverse environmental conditions, and so, at occasions, its presence can be indicative of environmental disturbances. Moreover, as the liver is very important when studying fish diseases, the knowledge of normal microanatomy is essential to assess histological changes, e.g., related to environmental change or toxic pollutants. The target organ of this histological study is the liver. The main objective is to contribute to the identification of anatomical and structural variations of this organ in different teleost species. We studied the distribution and spatial organization of the different types of blood vessels and biliary ducts and the relationships between them are established. For this, each liver was totally sectioned and the serial sections inspected in detail. The guppy liver presented intra-hepatic pancreatic tissue and so reported its association with the vascular and biliary elements. We observed that the input of afferent vessels (i.e., bringing blood into the liver) occur not only in the hilum but pierce and enter the organ at various points. Within the liver, venous vessels and bile ducts are seen, isolated or associated as venous-arteriolar tracts (VAT), and venous-biliary- arteriolar tracts (VBAT). Sometimes, pancreocytes appear within the liver surrounding isolated veins, forming venous tract with pancreatic acini (VT-P), or dual associations with afferent vessels, forming venous-arteriolar tracts with pancreatic acini (VAT-P). Intrahepatic pancreatic ducts were tiny and rare, putting in question the functional role of the acini. Contrary to other fish species, we did not spot isolated arterioles and associations between these and biliary ducts (BAT).We found aggregates of macrophages, namely associated with afferent and efferent (i.e., draining blood out) venous vessels; the latter fact not commonly reported in other fish species. There was a reduced arterialization of the organ (as arterioles were extremely rare), contrasting with an over predominance of a random distribution of the venous vascularization. The guppy differs to some extent from other previously studied models, highlighting the importance of making this kind of study to offer specific frameworks that can explain specific physiological processes or avoid misinterpretations; for instance about gene expression, as the whole liver specific expression will reflect the presence of hepatocytes and pancreocytes as well.

The importance of membrane microdomains for bile salt-dependent biliary lipid secretion.

Alternative models explaining the biliary lipid secretion at the canalicular membrane of hepatocytes exist: successive lipid extraction by preformed bile salt micelles, or budding of membrane fragments with formation of mixed micelles. To test the feasibility of the latter mechanism, we developed a mathematical model that describes the formation of lipid microdomains in the canalicular membrane. Bile salt monomers intercalate into the external hemileaflet of the canalicular membrane, to form a rim to liquid disordered domain patches that then pinch off to form nanometer-scale mixed micelles. Model simulations perfectly recapitulate the measured dependence of bile salt-dependent biliary lipid extraction rates upon modulation of the membrane cholesterol (lack or overexpression of the cholesterol transporter Abcg5-Abcg8) and phosphatidylcholine (lack of Mdr2, also known as Abcb4) content. The model reveals a strong dependence of the biliary secretion rate on the protein density of the membrane. Taken together, the proposed model is consistent with crucial experimental findings in the field and provides a consistent explanation of the central molecular processes involved in bile formation.

Expression kinetics of hepatic progenitor markers in cellular models of human liver development recapitulating hepatocyte and biliary cell fate commitment.

Due to the limitations of research using human embryos and the lack of a biological model of human liver development, the roles of the various markers associated with liver stem or progenitor cell potential in humans are largely speculative, and based on studies utilizing animal models and certain patient tissues. Human pluripotent stem cell-based in vitro multistage hepatic differentiation systems may serve as good surrogate models for mimicking normal human liver development, pathogenesis and injury/regeneration studies. Here, we describe the implications of various liver stem or progenitor cell markers and their bipotency (i.e. hepatocytic- and biliary-epithelial cell differentiation), based on the pluripotent stem cell-derived model of human liver development. Future studies using the human cellular model(s) of liver and biliary development will provide more human relevant biological and/or pathological roles of distinct markers expressed in heterogeneous liver stem/progenitor cell populations.

Histochemical Analyses of Biliary Development During Metamorphosis of Xenopus laevis Tadpoles.

In mammalian liver development, intrahepatic biliary morphogenesis takes place in periportal, but not in pericentral, regions. Liver progenitor cells transiently form epithelial plate structures and then intrahepatic bile ducts around the portal veins under the influence of the mesenchyme. The present study was undertaken to histochemically examine normal biliary development and its dependence on the action of the thyroid hormone triiodothyronine (T3) in Xenopus laevis tadpoles. In these tadpoles, the development of hepatic ducts and intrahepatic biliary ducts commenced along the portal veins at NF stages 48-50 and stages 50-52, respectively, when the blood concentration of thyroid hormone may be still low. Some periportal hepatocytes expressed carbamoylphosphate synthase I and SOX9, which are hepatocyte and biliary cell markers, respectively, suggesting that periportal hepatocytes give rise to biliary epithelial cells. Periportal biliary cells did not form ductal plates, nor was the periportal mesenchyme well developed as seen in fetal mouse livers. jag1 mRNA was moderately expressed in cells of portal veins and biliary epithelial cells, and notch1 and notch2 mRNAs were weakly detectable in biliary epithelial cells during metamorphosis as seen in developing mammalian livers. These results suggest that Notch signaling plays a decisive role in biliary cell differentiation and morphogenesis of Xenopus tadpoles. Anti-thyroid agent treatment of the tadpoles resulted in delayed biliary morphogenesis, suggesting that biliary development may depend on T3. However, T3 treatment of the tadpoles did not enhance biliary development. Thus, T3 may act positively on biliary development at a very low concentration.

Cholangiocarcinoma: risk factors, environmental influences and oncogenesis.

Cholangiocarcinoma (CCA) is one of the most frequent malignant epithelial liver tumors after hepatocellular carcinoma (HCC). Its incidence seems to be increasing worldwide, although risk factors are heterogeneous and differ globally. Although diagnostic and therapeutic medicine have advanced in several countries, tackling this tumor remains a challenge. The causes of CCA's increasing incidence are likely a differential increment of some factors according to the geographical area, which will be considered in this review. Environment-linked risk factors may play a critical role in the carcinogenesis. Liver flukes may play a major role in East Asia, while exposure to chemical compounds, such as naphthenic acids, has been postulated as a source of the rate increase in Western countries. Carcinogenesis is variable and confounding factors also need to be taken into account. Carcinogenesis depends on a sequential process and most probably involves both cholestasis and chronic inflammation as promoting steps after induction. The release and interaction of interleukin-6 (IL-6), transforming growth factor beta (TGF-beta), tumor necrosis factor alpha (TNF-alpha), and platelet-derived growth factor (PDGF) are at the basis of the proliferation of biliary epithelial cells or cholangiocytes. Additional steps for the final development of CCA may also involve an increase of the mutation rate of tumor suppressor genes, such as TP53, and the evasion of apoptosis.

Interferon-gamma directly mediates developmental biliary defects.

Biliary atresia (BA) is the most common identifiable hepatobiliary disease affecting infants, in which there are defects in intra- and extrahepatic bile ducts and progressive fibrosis. Activation of interferon-gamma (IFNγ) appears to be critical in both patients with BA and in rodent models of BA. We have recently reported a zebrafish model of biliary disease that shares features with BA, in which inhibition of DNA methylation leads to intrahepatic biliary defects and activation of IFNγ target genes. Here we report that ifng genes are hypomethylated and upregulated in zebrafish larvae treated with azacytidine (azaC), an inhibitor of DNA methylation. Injection of IFNγ protein into developing zebrafish larvae leads to biliary defects, suggesting that activation of the IFNγ pathway is sufficient to cause developmental biliary defects. These defects are associated with decreased cholangiocyte proliferation and with a decrease in the expression of vhnf1 (hnf1b, tcf2), which encodes a homeodomain protein with previously reported roles in biliary development in multiple models. These results support an importance of IFNγ in mediating biliary defects, and also demonstrate the feasibility of direct injection of intact protein into developing zebrafish larvae.

Ursodeoxycholic acid stimulates the formation of the bile canalicular network.

Ursodeoxycholic acid (UDCA) is a hepatoprotective bile acid used in the treatment of chronic liver diseases. Although several pharmacological effects, including choleresis and inhibition of apoptosis, have been proposed, the impact of UDCA on hepatic structure is not well understood. Here, the influence of UDCA on bile canalicular (BC) morphology was evaluated in vitro in immortalized rat hepatocytes (McA-RH 7777 cells) and primary rat hepatocytes. Cells cultured for 3 days in the presence of UDCA, the BC lumen was enlarged and the bile canaliculi were surrounded by multiple cells (≥5) with a continuous canal-like structure, reminiscent of the in vivo BC network. The effects were dependent on p38MAPK and conventional PKC in McA-RH cells, and partially dependent on p38MAPK, MAPK/ERK kinase, and conventional PKC in primary rat hepatocytes. These findings were then studied in vivo in a rat model of dimethylnitrosamine-induced hepatic injury, in which the BC network is significantly disrupted. In accordance with the in vitro observations, administration of UDCA (40 mg/kg/day) to the injured rats for 18 days improved the BC network compared with the vehicle control. Serum hepatic markers were not altered by UDCA treatment, suggesting that the morphological effects were due to the direct actions of UDCA on network formation. Our data provide new evidence of the pharmacological potential of UDCA in accelerating or regenerating BC network formation in vitro, in hepatic cell culture models, and in vivo in a rat model of hepatic injury, and provide a basis for understanding its hepatoprotective effects.

Intrahepatic biliary anomalies in a patient with Mowat-Wilson syndrome uncover a role for the zinc finger homeobox gene zfhx1b in vertebrate biliary development.

BACKGROUND: zfhz1b is the causative gene for Mowat-Wilson syndrome, in which patients demonstrate developmental delay and Hirschsprung disease, as well as other anomalies. MATERIALS AND METHODS: We identified a patient with Mowat-Wilson syndrome who also developed cholestasis and histopathologic features consistent with biliary atresia, suggesting that mutations involving zfhz1b may lead to biliary developmental anomalies or injury to the biliary tract. We used the zebrafish model system to determine whether zfhx1b has a role in vertebrate biliary development. RESULTS: Using zebrafish we determined that zfhx1b was expressed in the developing liver during biliary growth and remodeling, and that morpholino antisense oligonucleotide-mediated knockdown of zfhx1b led to defects in biliary development. These findings were associated with decreased expression of vhnf1, a transcription factor known to be important in biliary development in zebrafish and in mammals. CONCLUSIONS: Our studies underscore the importance of genetic contributions in the etiology of infantile hepatobiliary disorders, including biliary atresia.

Biliary differentiation and bile duct morphogenesis in development and disease.

The biliary tract consists of a network of intrahepatic and extrahepatic ducts that collect and drain the bile produced by hepatocytes to the gut. The bile ducts are lined by cholangiocytes, a specialized epithelial cell type that has a dual origin. Intrahepatic cholangiocytes derive from the liver precursor cells, whereas extrahepatic cholangiocytes are generated directly from the endoderm. In this review we discuss the mechanisms of cholangiocyte differentiation and bile duct morphogenesis, and describe how developing ducts interact with the hepatic artery. We also present an overview of the mechanisms of biliary dysgenesis in humans.

Wound healing in the biliary tree of liver allografts.

An increasing need for liver transplantation requires evaluation and triage of organs harvested from "extended criteria" donors. Although there is currently no widely accepted definition, most would agree that "extended criteria" includes organs donated by individuals that are old (>65 years), obese, infected with HBV or HCV, non-heart beating (NHBD), or had an unstable blood pressure before harvesting or the organ experienced a long cold ischemic time. These organs carry a statistical risk of dysfunction early after transplantation, but in the majority of recipients, hepatic parenchymal function recovers. Later, however, a small but significant percentage of extended criteria donors develop biliary strictures within several months after transplantation. The strictures occur primarily because of preservation injury that leads to "ischemic cholangitis" or deep wounding of the bile duct wall. Subsequent partial wound healing and wound contraction, but failed restitution of the biliary epithelial cell (BEC) lining, result in biliary tract strictures that cause progressive biliary fibrosis, increased morbidity, and decreased organ half-life. Better understanding of the pathophysiologic mechanisms that lead to biliary strictures in extended criteria donors provides an ideal proving ground for regenerative medicine; it also can provide insights into other diseases, such as extrahepatic biliary atresia and primary sclerosing cholangitis, that likely share certain pathogenic mechanisms. Possible points of therapeutic intervention include limiting cold and warm ischemic times, donor and/or donor organ treatment, ex vivo, to minimize the ischemic/preservation injury, maximize blood flow after transplantation, promote BEC wound healing, and limit myofibroblasts activation and proliferation in the bile duct wall. The pathobiology of biliary wound healing and therapeutic potential of interleukin-6 (IL-6) are highlighted.

Endogenous opioids modulate the growth of the biliary tree in the course of cholestasis.

BACKGROUND & AIMS: There is poor knowledge on the factors that modulate the growth of cholangiocytes, the epithelial cell target of cholangiopathies, which are diseases leading to progressive loss of bile ducts and liver failure. Endogenous opioids are known to modulate cell growth. In the course of cholestasis, the opioidergic system is hyperactive, and in cholangiocytes a higher expression of opioid peptide messenger RNA has been described. This study aimed to verify if such events affect the cholangiocyte proliferative response to cholestasis. METHODS: The presence of the delta opioid receptor (OR), muOR, and kappaOR was evaluated. The effects on cholangiocyte proliferation of the in vitro and in vivo exposure to their selective agonists, together with the intracellular signals, were then studied. The effects of the OR antagonist naloxone on cell growth were also tested both in vivo and in vitro. RESULTS: Cholangiocytes express all 3 receptors studied. deltaOR activation strongly diminished the proliferative and functional response of cholangiocytes to cholestasis, whereas muOR resulted in a slight increase in cell growth. The deltaOR signal is mediated by the IP3/CamKIIalpha/PKCalpha pathway, which inhibits the cAMP/PKA/ERK1/2/AKT cascade. In contrast, muOR activation stimulates the cAMP/PKA/ERK1/2/AKT cascade but does not affect the IP3/CamKIIalpha/PKCalpha pathway. The blockage of endogenous opioid peptides by naloxone further enhanced cholangiocyte growth both in vivo and in vitro. CONCLUSIONS: The increase in opioid peptide synthesis in the course of cholestasis aims to limit the excessive growth of the biliary tree in the course of cholestasis by the interaction with the deltaOR expressed by cholangiocytes.

Zebrafish vps33b, an ortholog of the gene responsible for human arthrogryposis-renal dysfunction-cholestasis syndrome, regulates biliary development downstream of the onecut transcription factor hnf6.

Arthrogryposis-renal dysfunction-cholestasis syndrome (ARC) is a rare cause of cholestasis in infants. Causative mutations in VPS33B, a gene that encodes a Class C vacuolar sorting protein, have recently been reported in individuals with ARC. We have identified a zebrafish vps33b-ortholog that is expressed in developing liver and intestine. Knockdown of vps33b causes bile duct paucity and impairs intestinal lipid absorption, thus phenocopying digestive defects characteristic of ARC. By contrast, neither motor axon nor kidney epithelial defects typically seen in ARC could be identified in vps33b-deficient larvae. Biliary defects in vps33b-deficient zebrafish larvae closely resemble the bile duct paucity associated with knockdown of the onecut transcription factor hnf6. Consistent with this, reduced vps33b expression was evident in hnf6-deficient larvae and in larvae with mutation of vhnf1, a downstream target of hnf6. Zebrafish vhnf1, but not hnf6, increases vps33b expression in zebrafish embryos and in mammalian liver cells. Electrophoretic mobility shift assays suggest that this regulation occurs through direct binding of vHnf1 to the vps33b promoter. These findings identify vps33b as a novel downstream target gene of the hnf6/vhnf1 pathway that regulates bile duct development in zebrafish. Furthermore, they show that tissue-specific roles for genes that regulate trafficking of intracellular proteins have been modified during vertebrate evolution.

Autocrine/paracrine regulation of the growth of the biliary tree by the neuroendocrine hormone serotonin.

BACKGROUND & AIMS: The biliary tree is the target of cholangiopathies that are chronic cholestatic liver diseases characterized by loss of proliferative response and enhanced apoptosis of cholangiocytes, the epithelial cells lining the biliary tree. The endogenous factors that regulate cholangiocyte proliferation are poorly understood. Therefore, we studied the role of the neuroendocrine hormone serotonin as a modulator of cholangiocyte proliferation. METHODS: The presence of the serotonin 1A and 1B receptors on cholangiocytes was evaluated. We then tested whether the activation of such receptors by the administration of the selective agonists modifies cholangiocyte proliferation and functional activity both in vivo and in vitro. In addition, the intracellular signal mediating the serotonin receptor action in cholangiocytes was characterized. We studied the expression and secretion of serotonin by cholangiocytes and the effects of the neutralization of the secreted hormone on the growth of the biliary tree. RESULTS: Cholangiocytes express the serotonin 1A and 1B receptors. Their activation markedly inhibits the growth and choleretic activity of the biliary tree in the bile duct-ligated rat, a model of chronic cholestasis. Such changes are mediated by enhanced d -myo-inositol 1,4,5-triphosphate/Ca 2+ /protein kinase C signaling and the consequent inhibition of the adenosine 3',5'-cyclic monophosphate/protein kinase A/Src/extracellular signal-regulated kinase 1/2 cascade. Cholangiocytes secrete serotonin, the blockage of which enhances cholangiocyte proliferation in the course of cholestasis. CONCLUSIONS: We observed the existence of an autocrine loop based on serotonin that limits the growth of the biliary tree in the course of chronic cholestasis. Our novel findings might open new approaches for the management of cholangiopathies.

Biliary motility: postnatal changes in guinea pigs.

Intravital microscopy, a new in vivo technique, documented age-dependent changes in choledochoduodenal junction motility in male guinea pigs. In the guinea pig, the choledochoduodenal junction served as a pump that actively emptied its luminal contents into the duodenum. In the neonates (less than or equal to 1 wk old), this choledochoduodenal junction pump was not fully developed. Unlike the older guinea pigs, some neonates had an incompetent sphincter ductus choledochi (SDC) allowing retrograde flow of bile during ampullary contractions. While fasting, neonates had decreased frequency of SDC (1.2 +/- 0.4 contractions/min) and ampullary (0.1 +/- 0.1 contractions/min) contractions as compared to juveniles (4-6 wk old) (SDC = 6.4 +/- 1.0; ampulla = 1.2 +/- 0.2 contractions/min) and adults (greater than 1 yr old) (SDC = 6.7 +/- 1.6; 0.8 +/- 0.2 contractions/min). Following a meal (Ensure), unlike older guinea pigs, the neonate did not have a significant increased duration and decreased frequency of SDC contractions. Altered neonatal SDC motility correlated with an incompletely developed SDC including decreased muscle mass and mucosal thickness. By 4 wk of age, choledochoduodenal junction motility was similar to that of the adult. These developmental alterations in junctional motility and structure may affect the flow of bile into the duodenum contributing to physiologic cholestasis and decreased intraduodenal bile acids seen in neonates.

Effects of growth and maturation on biliary structures of guinea pigs.

The biliary system and major duodenal papilla of male guinea pigs were examined in vivo using intravital microscopy. Study of the photomicrographs and videotape recordings indicated that biliary organs changed with age and growth of the guinea pigs. Physiological data including the frequency of sphincteric contractions were recorded and efforts were made to correlate these data with changes in structure thought to be due to maturation. The volume of smooth muscle in the bile duct sphincter (measured using histological sections) increased with age as did surface area of the mucosa. The bile duct was separate from the pancreatic duct in each of 28 guinea pigs. Study of the guinea pig extrahepatic biliary system (EBS) allowed the isolated examination of effects of maturation on bile duct capacity. The anatomical reason for this is that the ampulla of the bile duct emptied into the duodenum (independently of the pancreatic duct) at the major duodenal papilla. The duct of the pancreas entered the duodenum distally (caudally) to the major duodenal papilla on the minor duodenal papilla. The separation of biliary and pancreatic ducts simplified the making of casts of the EBS. It is important to consider this feature in selection of a model for studies of bile duct function in living animals. Three anatomical techniques were selected to focus from differing perspectives on growth-related changes that occurred among the EBS of neonatal, juvenile and adult animals. These techniques were: The capacity (volume) of bile contained within the EBS was determined using plastic casts of the system.(ABSTRACT TRUNCATED AT 250 WORDS)

Postnatal development of bile secretory physiology in the dog.

To determine whether bile formation in the dog is an immature process at birth, several determinants of bile secretion were studied in anesthetized, bile duct-cannulated puppies of 0-42 days of age and adult dogs. Basal canalicular bile flow rate, estimated by 14C-erythritol biliary clearance, averaged 0.182 microliter/min/g liver in 0-3 day-old puppies and increased to 0.324 and 0.461 microliter/min/g in puppies 7-21 and 28-42 days of age, respectively. The latter two values were not significantly different from that observed in adult dogs (0.348 microliter/min/g). This age-related increase in canalicular bile flow could be accounted for primarily by an increase in bile acid independent bile flow (0.103, 0.277, 0.375, and 0.252 microliter/min/g in 0-3, 7-21, and 28-42 day-old puppies and adult dogs, respectively). Calculated ductular bile water reabsorption (14C-erythritol biliary clearance-bile flow) was virtually absent in 0-3 day-old puppies (0.005 microliter/min/g), and averaged 0.017 and 0.092 microliter/min/g in puppies of 7-21 and 28-42 days of age, respectively. In adult dogs, ductular bile water reabsorption was 0.132 microliter/min/g. These functional deficiencies of the newborn dog were associated with an increased biliary permeability to 3H-inulin which could not be accounted for solely by an increased solute diffusion due to the lower rate of canalicular bile flow. Administration of taurocholate up to 2000 nmol/min/kg produced in all animals a similar increase in canalicular bile flow and bile acid excretion, and was not associated with changes in ductular bile water reabsorption rate. These findings are interpreted to indicate that, in the dog, bile secretory function is immature at birth and develops during postnatal life.