Secondary joining of the bile ducts during the hepatogenesis of the mouse embryo

Summary Development of the bile duct system of the mouse embryo was studied histologically and by an immunofluorescent technique. The hepatic primordium consisted of cranial and caudal portions. In the liver of young embryos, the hepatic cords were present in the presumptive cysticduct epithelium, and the histology of the presumptive cystic duct epithelium near the hilus was similar to that of the hilus epithelium. The results suggest that at least a part of the cystic duct epithelium develops from the cranial diverticulum of the hepatic primordium. Lumen structures were precursors of intrahepatic bile ducts and originated from type I (immature) hepatocytes. The lumina of the lumen structures appeared near the hilus area first, but most were discontinuous with those of the hepatic ducts. With the progress of development, the discontinuous lumen structures became distributed around the portal vein branches in the central part of the liver parenchyma, and gradually connected with each other and also with hepatic ducts. the discontinuous laminin immunofluorescence also appeared in the endodermal cells around the portal vein branches at the younger stages. Therefore, it is conceivable that the intrahepatic bile ducts originate from discrete cell populations of type I hepatocytes around the portal vein branches and subsequently become confluent, but not from the cells of hepatic ducts.     

Intrahepatic bile duct development in the rat: a cytokeratin-immunohistochemical study

The development of the intrahepatic bile ducts was studied in rats from day 12 of gestation until 10 days of age using three antibodies directed against cytokeratins in an immunohistochemical procedure on paraffin-embedded liver tissue. In adult rat liver, both hepatocytes and bile ducts were stained by the monoclonal anti-cytokeratin no. 8, whereas two polyclonal antibodies stained bile ducts only. Hepatocytes in developing rat liver were stained by monoclonal anti-cytokeratin no. 8 from day 12 of gestation on. On day 16, cells strongly immunoreactive for cytokeratin no. 8 were observed in a string of pearl-like arrangement around large vascular branches close to the liver hilum. Over the following days, similar structures appeared throughout the liver. Gradually, lumina were formed in these structures, again starting at the liver hilum and resulting in the formation of individual bile ducts. Immunoreactivity with the polyclonal antibodies was first detected in some of the string of pearl-like structures on day 19 and gradually increased until the intensity observed in adult rat liver was reached on day 1 after birth. Even on day 10, portal spaces still revealed more bile duct branches, rings of cells strongly positive for cytokeratin no. 8 and weakly positive with the polyclonal antibodies were present. It is concluded that the intrahepatic bile ducts develop from hepatocytes. The cells closest to large vascular spaces first become strongly positive for cytokeratin no. 8 and only later on acquire additional ("bile duct type") cytokeratins. This process starts at the liver hilum and spreads through the liver. Even at 10 days of age the bile duct system is still immature: around the smaller portal vein branches, rings of cells are still undergoing transformation into bile duct type cells. These data might be useful for reevaluation of pathologic phenomena.     

Morphogenesis of intrahepatic bile ducts of the human fetus

Summary The development of the intrahepatic bile ducts of the human fetus was investigated by light and electron microscopy. Bile canaliculi with microvilli and junctional complexes are already found in the embryo of 7 mm. Some of them are of the intracellular type. At six to seven weeks, large bile canaliculi bounded by four to seven liver cells appear. Subsequently, bile canaliculi are formed predominantly between three to four adjoining liver cells and this arrangement persists throughout later fetal life.The early intrahepatic bile ducts develop around the portal vein as epithelial cell plates derived from the hepatic duct and the branches sprout from the epithelial cell plates in several different places. The epithelial cell plates are separated from each other by primitive connective tissue and they change into a complex network of bile ducts. Formation of the intrahepatic bile ducts is completed by three months.Biliary duct cells at the end of the developing bile ducts are thought to transform into liver cells. Therefore, at the ducts of Hering various transitional cells appear between biliary duct cells and liver cells.The fine structure of the developing liver cells and biliaryduct cells is also described.     

Portal tract fibrogenesis in the liver

The portal area is the 'main entrance' and one of the two main exits of the liver lobule. Through the main entrance portal and arterial blood reach the liver sinusoids. Through the exit the bile flows towards the duodenum. The three main structures, portal vein and artery with their own wall (and vascular smooth muscle cells) and bile duct with its basal membrane, are surrounded by loose myofibroblasts and by the first layer of hepatocytes and non-parenchymal cells. Chronic diseases of the liver can lead to development of liver cirrhosis, characterized by formation of fibrotic septa which can be portal-portal in the case of the chronic biliary damage or portal-central in the case of the chronic viral hepatitis. Central-central septa can also be observed under other pathological conditions. When damaging noxae are introduced to the liver, inflammatory cells are first recruited to the portal field, the first layer of hepatocytes may be destroyed (enlargement of the portal field) and portal (myo)fibroblasts become activated. A similar reaction may take place when the target of inflammation is the bile duct with consecutive reduction of the bile flow, activation of the portal (myo)fibroblasts, proliferation of bile ducts and destruction of the hepatocytes around the portal field. Increased matrix deposition may be the consequence. During the past years several publications dealt with the pathomechanisms of portal fibrogenesis as well as with its resolution. One of the most intriguing observations was that it is not hepatic stellate cells of the hepatic sinusoid, but portal (myo)fibroblasts which rapidly acquire the phenotype of 'activated' (myo)fibroblasts in the early stages of cholestatic fibrosis. These may also become the main mesenchymal cells of the porto-portal or porto-central fibrotic septa. This article reviews the similarities as well as differences between the mesenchymal cells of the portal tract and of the fibrotic septa vs 'activated' stellate cells of the hepatic sinusoids, and discusses the debate over their relative contributions to liver fibrogenesis.     

Phylogenetic analyses of the hepatic architecture in vertebrates

The mammalian liver has a structural and functional unit called the liver lobule, in the periphery of which the portal triad consisting of the portal vein, bile duct and hepatic artery is developed. This type of hepatic architecture is detectable in many other vertebrates, including amphibians and birds, whereas intrahepatic bile ducts run independently of portal vein distribution in actinopterygians such as the salmon and tilapia. It remains to be clarified how the hepatic architectures are phylogenetically developed among vertebrates. The present study morphologically and immunohistochemically analyzed the hepatic structures of various vertebrates, including as many classes and subclasses as possible, with reference to intrahepatic bile duct distribution. The livers of vertebrates belonging to the Agnatha, Chondrichthyes, Amphibia, Aves, Mammalia, and Actinopterygii before Elopomorpha, had the portal triad‐type architecture. The Anguilliformes livers developed both periportal bile ducts and non‐periportal bile ducts. The Otocephala and Euteleostei livers had independent configuration of bile ducts and portal veins. Pancreatic tissues penetrated the liver parenchyma along portal veins in the Euteleostei. The liver of the lungfish, which shares the same origin with amphibians, did not have the portal triad‐type architecture. Teleostei and lungfish livers had ductular development in the liver parenchyma similar to oval cell proliferation in injured mammalian livers. Euteleostei livers had penetration of significant numbers of independent portal veins from their intestines, suggesting that each liver lobe might receive a different blood supply. The hepatic architectures of the portal triad‐type changed to non‐portal triad‐type architecture along the evolution of the Actinopterygii. The hepatic architecture of the lungfish resembles that of the Actinopterygii after Elopomorpha in intrahepatic biliary configuration, which may be an example of convergent evolution.     

FGF signaling segregates biliary cell-lineage from chick hepatoblasts cooperatively with BMP4 and ECM components in vitro.

Intrahepatic bile ducts (IHBDs) are indispensable for transporting bile secreted from hepatocytes to the hepatic duct. The biliary epithelial cells (BECs) of the IHBD arise from bipotent hepatoblasts around the portal vein, suggesting the portal mesenchyme is essential for their development. However, except for Notch or Activin/TGF-beta signaling molecules, it is not known which molecules regulate IHBD development. Here, we found that FGF receptors and BMP4 are specifically expressed in the developing IHBD and the hepatic mesenchyme, respectively. Using a mesenchyme-free culture of liver bud, we showed that bFGF and FGF7 induce the hepatoblasts to differentiate into BECs, and that BMP4 enhances bFGF-induced BEC differentiation. The extracellular matrix (ECM) components in the hepatic mesenchyme induced BEC differentiation. Forced expression of a constitutively active form of the FGF receptor partially induced BEC differentiation markers in vivo. These data strongly suggest that bFGF and FGF7 promote BEC differentiation cooperatively with BMP4 and ECMs in vivo.     

Differential immunohistochemical localization of cytokeratins and collagen types I and III in experimentally-induced cirrhosis

Liver cirrhosis was induced in male Wistar rats by subcutaneous injection (1 ml of 30 g/l) of an aqueous solution of thioacetamide. Using the indirect immunoperoxidase technique, high molecular weight keratins were localized in bile ducts and ductules. Low molecular weight cytokeratins were present in regenerating hepatocytes in active cirrhosis; bile ducts were unstained. These results suggest that cytokeratin staining may be useful in distinguishing bile duct epithelium and hepatocytes in hepatobiliary diseases. Anticollagen type III antibody stained hepatocytes and thin connective tissue fibres, while anticollagen type I antibody stained thicker fibres and some sinusoidal cells but not hepatocytes. Collagens were usually undetectable in normal liver cells. It is suggested, therefore, that hepatocytes may play a major role in collagen type III production which precedes the deposition of collagen type I. By contrast, collagen type I may be produced by fibroblasts and some cells along sinusoids (e.g. perisinusoidal fat-storing cells) after liver injury.     

Bile duct adenomas in heterozygous (MZ) deficiency of alpha 1-protease inhibitor

A case of bile duct adenomas in association with heterozygous (MZ) deficiency of alpha 1-proteinase inhibitor (API) is presented. The salient features were the presence of large API-containing globules in the adenomatous tissue and only minimally, in granular form, in hepatocytes. alpha 1-Proteinase inhibitor was not demonstrated in portal bile ducts entrapped in the adenomatous tissue or in bile ducts present in the liver parenchyma. Bile duct markers such as cytokeratin and carcinoembryonic antigens were present in the adenomatous tissue and also in the normal bile ducts, but not in the hepatocytes, suggesting that the adenomatous structures are ductular in origin, and probably not from ductular metaplasia of liver cells. Accumulation of API is postulated to be a triggering factor in neoplastic transformation.     

Embryology of Extra‐ and Intrahepatic Bile Ducts,the Ductal plate

In the human embryo, the first anlage of the bile ducts and the liver is the hepatic diverticulum or liver bud. For up to 8 weeks of gestation, the extrahepatic biliary tree develops through lengthening of the caudal part of the hepatic diverticulum. This structure is patent from the beginning and remains patent and in continuity with the developing liver at all stages. The hepatic duct (ductus hepaticus) develops from the cranial part (pars hepatica) of the hepatic diverticulum. The distal portions of the right and left hepatic ducts develop from the extrahepatic ducts and are clearly defined tubular structures by 12 weeks of gestation. The proximal portions of the main hilar ducts derive from the first intrahepatic ductal plates. The extrahepatic bile ducts and the developing intrahepatic biliary tree maintain luminal continuity from the very start of organogenesis throughout further development, contradicting a previous study in the mouse suggesting that the extrahepatic bile duct system develops independently from the intrahepatic biliary tree and that the systems are initially discontinuous but join up later. The normal development of intrahepatic bile ducts requires finely timed and precisely tuned epithelial–mesenchymal interactions, which proceed from the hilum of the liver toward its periphery along the branches of the developing portal vein. Lack of remodeling of the ductal plate results in the persistence of an excess of embryonic bile duct structures remaining in their primitive ductal plate configuration. This abnormality has been termed the ductal plate malformation. Anat Rec, 291:628–635, 2008. © 2008 Wiley‐Liss, Inc.     

左外叶活体肝移植的解剖学研究

目的：模拟左外叶活体肝移植门静脉、肝动脉和胆管的切取方法。方法：解剖正常人肝脏标本30具，观察肝脏铸型标本30具，测量门静脉、肝动脉及胆管长度、管径及属支或分支分布情况。结果：左外叶门静脉的血供来自门静脉左支，主要为左外叶上段门静脉支、左外叶下段门静脉支；动脉主要来源于肝固有动脉、肝左动脉、肝中动脉，偶有迷走动脉支；胆道引流属支有左外叶上段胆管支、左外叶下段胆管支。结论：左外叶解剖变异较多，活体取肝前应仔细研究其结构特点，设计合理的切取模式；对门静脉、肝动脉和胆管支需行必要的整形，以便与受体相应的管道进行吻合。     

Quantification of the intrahepatic biliary tree during human fetal development

Development and differentiation of bile ducts have been studied for the understanding of pathogenesis of biliary atresia and other diseases of the intrahepatic biliary tree. The aim of this study is to correlate the type of biliary structure with the size of the portal tract and the gestational age. Twenty-four human livers were studied. Fetuses were assigned to four gestational age groups: group I, up to 20 postfecundation weeks (PFW); group II, from 21–26 PFW; group III, from 27–32 PFW; and group IV, from 33–38 PFW. In each specimen, 30 portal tracts were classified as small, medium, or large according to the diameter of the portal vein. In order to identify the bile duct cells, the sections were immunolabeled with anti-cytokeratin antibody, and the biliary structure was classified as absent (bile ducts (BD) = 0), presence of bile duct cells without lumen (BD = 1), or presence of bile duct with lumen (BD = 2). In the small portal tracts, either there were no biliary structures or just a few. There was a substantial increase in the number of medium portal tracts that included a bile duct as a function of gestational age. The majority of large portal tracts exhibited a bile duct. In human fetus up to 20 PFW, it is possible to find 70% of portal tracts without bile ducts, and at 38 PFW it is expected that more than 50% of the portal tract has a BD > 0. We suggest the use of the diameter of the portal vein and the gestational age for the quantification of biliary structures and the evaluation of maturity of intrahepatic biliary tree. Anat. Rec. 251:297–302, 1998. © 1998 Wiley-Liss, Inc.     

Differentiation of functional hepatocytes and biliary epithelial cells from immature hepatocytes of the fetal mouse in vitro

Differentiation of functional hepatocytes and biliary epithelial cells from immature hepatocytes was analysed in vitro. When fetal mouse liver fragments containing immature hepatocytes but no bile ducts were cultured organotypically, the immature hepatocytes differentiated into large hepatocytes. Some of these expressed bile duct markers such as cytokeratin and Dolichos biflorus agglutinin-binding sites, though only to a small extent, and typical intrahepatic bile duct cells failed to differentiate. Dexamethasone stimulated immature hepatocytes to differentiate into both mature hepatocyte and biliary epithelial cell lineages. Especially in the liver fragments cultured on Matrigel, dexamethasone stimulated the expression of bile duct markers (such as cytokeratin and binding sites for two types of lectin) in the immature hepatocytes. These results support the idea that immature hepatocytes can differentiate into both mature hepatocytes and biliary epithelial cells during normal development of the mouse liver, and suggest that glucocorticoids stimulate both these differentiation pathways. It also seems that basal laminar components may play a role in bile duct differentiation.     

Three-dimensional computer-assisted reconstruction of ductal plate in the rat embryo (Carnegie stages 19–23)

In bile duct morphogenesis it has been established that the extrahepatic bile ducts in human originate from hepatic diverticulum while intrahepatic bile ducts arise from the ductal plate (DP), a network of primitive biliary epithelium that develops in the periportal connective tissue. The aim of this work was to reconstruct in rat embryos, stages 19–23, the three-dimensional (3D) distribution of the DP by means of a computer-assisted method. Six specimens, stages 19–23, fixed, dehydrated and paraffin-embedded, were submitted to serial histological sections and stained by hematoxylin-eosin and Heidenhain techniques. The images were directly digitalized with a CCD camera. The serial views were aligned anatomically by software and the data were analyzed following segmentation and thresholding. At stage 19, the DP was not yet organized. The periportal mesoderm (M) was gaining ground with some cords of cubic cells evoking primitive ductal cells. At stage 20, a row of ductal cubic cells went around the transverse portal sinus at the junction between M and liver cells. At stage 21, the DP developed at the periphery of periportal connective tissue and appeared in direct continuity with the hepatic duct (HDu). Four evaginations emerged from the DP and were growing up in the hepatic parenchyma. At stage 23, the DP appeared as a large network in continuity with the HDu located at the periphery of periportal M and presenting several evaginations radiating in the liver parenchyma. This work in the rat embryo permits the clear visualization of the development of the junctional zone in the hepatic hilum. Three phenomena are observed: (1) proximal left and right hepatic ducts and their segmental branches are derived from DP and not from the HDu; (2) the extrahepatic biliary system is in contact with the developing hilar ducts; (3) ductal maturation begins at the hilum and proceeds centrifugally. These observations are of great relevance in explaining pathological changes appearing at the hepatic hilum of neonates: hepatic polycystic disease, intrahepatic bile duct agenesis or atresia, and cyst of the extrahepatic bile duct.     

Morphometric and immunohistochemical characterization of human liver regeneration.

Regeneration in human liver is characterized in part by the formation of ductular structures, so-called ductular hepatocytes in massive hepatic necrosis and bile ductules in mechanical biliary obstruction. In an attempt to characterize the liver regenerative process, we performed image analysis and immunohistochemical staining of the ductular structures in these well defined human liver disorders, 13 cases of massive hepatic necrosis and 9 cases of mechanical biliary obstruction. The proliferation index was determined and the expression of several antigens was localized by immunohistochemical staining using antibodies to alpha-fetoprotein, alpha-1-antitrypsin, albumin, and cytokeratin 19. The ductular structures in adult human liver were compared with the developing ductal plates in 11 fetal livers, ranging in age from 9 to 36 weeks of gestation. Image analysis demonstrated that the mean total area, mean nuclear area, and mean cell size of ductular hepatocytes were significantly larger than those of bile ductules (p < 0.05). The proliferation index of ductular hepatocytes and bile ductules was significantly higher than that of hepatocytes of normal livers (p < 0.02). Bile ducts, bile ductules in mechanical biliary obstruction, ductular hepatocytes in massive hepatic necrosis, and the ductal plate cells in fetal liver showed strong staining for cytokeratin 19, which characterizes intermediate filaments associated with bile duct epithelial cells. Albumin, a liver-specific protein, and alpha-1-antitrypsin, a protease inhibitor, were strongly expressed in ductal plate cells of fetal liver, hepatocytes, and ductular hepatocytes, whereas bile duct cells and bile ductules were negative for albumin. In summary, ductular hepatocytes demonstrate morphometric and immunophenotypic features of both hepatocytes and biliary epithelial cells, whereas bile ductules share characteristics primarily with fetal ductal plates and mature bile ducts. These findings suggest that ductular hepatocytes in massive hepatic necrosis may serve as bipotential progenitor cells, and bile ductules in mechanical biliary obstruction are related to ductal plates of fetal liver.     

Development of intrahepatic bile ducts in humans. Possible role of laminin

Laminin, a major extracellular matrix-attachment glycoprotein, may play an important role in the differentiation and migration of epithelial cells during normal development. Therefore, the morphogenesis of bile ducts in human liver of fetuses at sequential gestational ages, neonates, children, and adults was examined by single and double immunohistochemical staining for laminin and for cytokeratins. The latter served as a marker for developing and mature bile duct epithelial cells. A close association was observed between laminin deposition and the differentiating ductal plate cells at the epithelial-mesenchymal interface of portal tracts and during the subsequent migration of ductular structures into the center of portal tracts. Simultaneously, laminin disappeared from the margins of portal tracts, but scattered ductal plate-like structures with laminin remained demonstrable in neonates, children, and even adults. These observations were substantiated by semiquantitative evaluation of laminin at the periphery of portal tracts. Thus, clear evidence is provided that laminin accompanies bile duct epithelial cells during all successive stages of differentiation and migration during the development of the human hepatobiliary system. The persisting ductal plate cells may represent a common stem cell for proliferation of bile ductules and hepatocytes.     

Surgical anatomy of the vasculobiliary apparatus at the hepatic hilum as applied to liver transplantations and major liver resections

Introduction

To evaluate the hepatic arterial, bile duct and portal venous anatomy as applicable to major liver resections.

Alcoholic liver disease. Parenchyma to stroma relationship in fibrosis and cirrhosis as revealed by three-dimensional reconstruction and immunohistochemistry.

Severe ethanol-induced liver damage is characterized by fibrous dissociation of liver cell plates leading to many apparently isolated hepatocytes. Three-dimensional reconstruction, however, revealed hepatocytes that were surrounded by connective tissue as endpoints of "parenchymal pillars" or in association with liver cell plates and bile ductules. Double immunofluorescence studies displayed the expression of cytokeratin (CK) 7 in bile ducts, including bile ductules, but also in some hepatocytes still organized in liver cell plates. The other bile duct, typical CK, namely CK 19, was only detectable in few hepatocytes. However, the expression of CK 7 and/or CK 19 was less frequent in hepatocytes that were closely associated with bile ductules. CK 7 and CK 19 were also found in some, but not all, Mallory bodies. These observations indicate that the expression of these two CKs is neither related to a transformation of hepatocytes to bile duct-like structures ("ductal metaplasia") nor to the formation of Mallory bodies. Furthermore, double immunofluorescence studies revealed small groups of hepatocytes and bile ductules that were encircled by basement membrane material, thus suggesting the formation of "secretory units."     

腹腔镜下肝门血流阻断肝切除术的解剖学基础

目的　探讨腹腔镜下肝门血流阻断在肝切除术（LH）的解剖基础及手术路径。 方法　解剖尸体肝脏,分离血流阻断所涉及各肝门结构,观察在二维平面中毗邻,测量在肝外长度及夹角;观察LH视频中肝门结构,总结镜下的位置及特征。 结果　肝动脉平面低于肝管（90%）,肝门静脉分叉位置固定于后方;肝左和肝中静脉在肝外大多共干（90%）,肝右静脉与共干间存在间隙,与肝后下腔静脉(IVC)前方相通;肝短静脉位于IVC两侧,有（7±3）支;IVC韧带在尸体中易忽略,活体中较明显,为包绕IVC的膜性结构,厚度个体差异大;各结构在肝外长度及夹角为肝门血流阻断提供足够空间;镜下各结构位置及特征与实体比较有特殊性。 结论　LH中应用肝门血流阻断有解剖依据及路径遵循。     

Arterial supply and biliary drainage of the dorsal liver: A dissection study using controlled specimens

Liver surgeons favor using the entity called the 'dorsal liver' (i.e. the caudate lobe and other paracavally located liver parenchyme of segments 7 and 8). According to minute dissection of 48 livers, we describe the territories of the left/right portal veins, hepatic ducts and hepatic arteries in the dorsal liver. In the caudate lobe, the right hepatic artery, rather than the left hepatic artery (23/48 vs 19/48 for right vs left, respectively), tended to supply the 'left' portal vein territory. Similarly, paradoxical drainage patterns, such as the right hepatic duct draining the left portal vein territory, were found in seven of 48 livers. In the territory of the hilar bifurcation, right hepatic artery dominance was also evident and various bile drainage patterns were found. These included double drainage by the bilateral hepatic ducts (3/48) and drainage into the confluence of bilateral ducts (6/48). In contrast, the arterial supply and biliary drainage of the paracavally located parenchyme of segments 7 and 8 usually depended on the proper segmental arteries and ducts and their variations were within the range of those found in other parts of the right lobe. Therefore, the dorsal liver concept may not be anatomical but, rather, simply aimed at usefulness in surgery. Nevertheless, clear subdivision of the caudate lobe according to biliary drainage and/or arterial supply seemed difficult because of the paradoxical relatioships among the portal vein, hepatic artery and bile duct. Consequently, the present results support extended surgery based on the dorsal liver concept for carcinomas involving the caudate lobe.