The autonomic innervation of the liver and gallbladder of Rana ridibunda

1--The innervation of the liver and gallbladder of Rana ridibunda has been studied by the following methods: (a) demonstration of cholinesterase activity; (b) FIF method for catecholamines; (c) immunohistochemistry for VIP and (d) electron microscopy. 2--The hepatocytes are arranged in regular rows of hepatic cords, very little connective tissue is distributed in the parenchyma, the innervation being restricted to the big branches of blood vessels. 3--Well defined cholinergic and adrenergic plexuses surround the hepatic arteries, portal veins and biliary ducts. The VIPergic innervation is scarce in the liver but a richly branched plexus spreads in the wall of the gallbladder. 4--Cholinesterase-positive cells are widely distributed accompanying the nerve trunks of the gallbladder. The innervation distribution is prominent in the portion of the gallbladder next to the hepatic hilus. 5--A population of melanin-storing cells besides free melanin granules are present in the liver parenchyma and are prominent in the gallbladder where the melanocytes are disposed in close contact with blood vessels and nerve structures. We have observed that the number of these visceral melanocytes considerably increases in winter, particularly in the liver.     

Changes in the acinar distribution of some enzymes involved in carbohydrate metabolism in rat liver parenchyma after experimentally induced cholestasis

Extrahepatic cholestasis induced by ligation and transsection of the common bile duct caused a change in the parenchyma/stroma relationship in rat liver. Two weeks after ligation, the periportal zones of the parenchyma were progressively invaded by expanding bile ductules with surrounding connective tissue diverging from the portal areas. Parenchymal disarray developed and small clumps of hepatocytes or isolated hepatocytes were scattered within the expanded portal areas. These cells showed normal activity of lactate, succinate and glutamate dehydrogenase and may, therefore, be considered to be functionally active. After cholestasis the remainder of the liver parenchyma showed adaptational changes with respect to glucose homeostasis, as demonstrated by histochemical means. Glycogen stores disappeared completely whereas glycogen phosphorylase activity increased about ten fold. The increased glycogen phosphorylase activity and glycogen depletion indicate a greater glycogenolytic capacity in liver parenchyma after bile duct ligation to maintain as far as possible a normal plasma glucose concentration. The parenchymal distribution pattern of glucose-6-phosphatase activity did not change significantly after bile duct ligation. The isolated hepatocytes within the expanded portal tracts showed a high activity of this enzyme whereas the pericentral parenchyma was only moderately active. The distribution patterns of glucose-6-phosphate dehydrogenase and lactate dehydrogenase activity in the liver parenchyma were also largely unchanged after bile duct ligation, but the histochemical reaction for glucose-6-phosphate dehydrogenase activity demonstrated infiltration of the remainder of the parenchyma by non-parenchymal cells, possibly Küpffer cells and leucocytes as part of an inflammatory reaction. Under normal conditions the mitochondrial enzymes succinate and glutamate dehydrogenase show an opposite heterogenous distribution pattern in liver parenchyma. Following cholestasis both enzymes became uniformly distributed. The underlying regulatory mechanism for these different changes in distribution patterns of enzyme activities is not yet understood.     

The autonomic innervation of the liver and gallbladder of Podarcis hispanica

The innervation of the liver and gallbladder of the lizard Podarcis hispanica has been studied by the following methods: a) demonstration of cholinesterase activity; b) FIF method for catecholamines; and c) immunohistochemistry for VIP. The hepatic parenchyma of the reptile's liver show hepatocytes arranged in regular rows of hepatic cords, the portal triad being typical of higher vertebrates (birds and mammals). Nerve fibers are found in the scarce connective tissue distributed among the hepatocytes. The innervation is restricted to the big branches of blood vessels and biliary ducts. It is represented by cholinergic, noradrenergic and VIPergic fibers. The gallbladder shows a well developed cholinergic plexus with pyramidal cells in the interconnection points of the fiber network. The noradrenergic and VIPergic plexuses are also more widely distributed in the gallbladder than in the liver.     

Dynamics of the development of oval cell population induced in the liver by dipin and partial hepatectomy

It has been shown that dipin in combination with partial hepatectomy (model of dipin hepato-carcinogenesis) induces massive oval cell proliferation. The main stages of oval cells development examined by means of light microscopy of half-thin slices are the following: 1) appearance around portal tracts--1-3 weeks; 2) migration into parenchyma along terminal branches of portal vessels--3-8 weeks; 3) maximum development--8-10 weeks; 4) induction focuses of growth of new formed hepatocytes around portal tracts--8-10 weeks; 5) forcing of oval cells towards the centre of liver lobules and their elimination. There was close correlation between oval cells development, fibrosis and inflammation. Oval cells remained around portal tracts until the development of numerous hepatomas in 10 months after treatment.     

A 3-dimensional presentation of the functional liver unit]

The reconstruction of the liver parenchyma of a golden hamster after poisoning with allyl formate is described. Allyl formate primarily destroys the periportal areas and leads, following the microvascularisation of the liver parenchyma, to a necrosis of the hepatocytes progressing towards the terminal hepatic venule. The still intact parenchymal zones can be characterized by the positive PAS reaction. In this study the preterminal and the terminal portal branches as well as zone 3, situated in the vasculatory periphery, were reconstructed. By this method, a three-dimensional presentation of the acinar functional zones was possible for the first time.     

The differentiation of oval cells into hepatocytes during induced hepatic carcinogenesis in mice. An electron microscopic study

An electron microscopic study of murine oval cells, induced by a single injection of genotoxic agent dipin and by a partial hepatectomy, has shown that their ultrastructure and direction of differentiation depend on localization in the liver lobule. Oval cells around portal tracts go through three stages of development: low differentiated cells 4.40 +/- 0.51 mu in diameter with ovoid nuclei 3.43 +/- 0.44 mu, intermediate cells, and young hepatocytes. They form common ducts surrounded by a basal lamina, and produce bile canaliculi-like structures and intermediate junctions between them. Another part of the oval cell population is organized similar to the bile duct epithelium. It consists of cells 9.37 +/- 1.1 mu in diameter with nuclei 7.28 +/- 1.16 mu in diameter and form a system of branching and anastomosing ducts widespread along the parenchyma from the portal to the central veins. Our data indicate that the oval cells can differentiate into hepatocytes, and support a hypothesis according to which the cells of terminal bile ductules are liver epithelial stem cells which can differentiate into a hepatocyte or a bile duct cell lineage in periportal microenvironment.     

Common antigens of oval cells and cholangiocytes in the mouse. Their detection by using monoclonal antibodies

Monoclonal antibodies (MAb) were produced against antigens (Ag) of oval cells isolated from the preneoplastic murine liver. To suppress the immune response to major antigens common with hepatocytes, the principle of anti-idiotype immunization was employed. Characteristics of three MAb reacting selectively with the foci of oval cell proliferation are described. MAb A6 and G7 detected two different antigens (Ag A6 and Ag G7, respectively) common for oval cells and cholangiocytes. Ag A6 was also found in normal parenchyma (in membranes of single hepatocytes adjacent to portal veins), in the preneoplastic liver (in hepatocytes formed de novo) and in some hepatoma cells. Ag G7 was not detected in hepatocytes. MAb E5 stained the matrix in the areas adjacent to oval cells and large bile ducts. All the three Ag were widely distributed in normal tissues of mice. The significance of the detected Ag as markers of murine liver epithelial cell lines and stages of their differentiation is discussed as well as the possible relationship between Ag A6 and Ag of human blood groups.     

Liver stem/progenitor cells: their characteristics and regulatory mechanisms

Liver stem cells give rise to both hepatocytes and bile duct epithelial cells also known as cholangiocytes. During liver development hepatoblasts emerge from the foregut endoderm and give rise to both cell types. Colony-forming cells are present in the liver primordium and clonally expanded cells differentiate into either hepatocytes or cholangiocytes depending on culture conditions, showing stem cell characteristics. The growth and differentiation of hepatoblasts are regulated by various extrinsic signals. For example, periportal mesenchymal cells provide a cue for bipotential hepatoblasts to become cholangiocytes, and mesothelial cells covering the parenchyma support the expansion of foetal hepatocytes by producing growth factors. The adult liver has an extraordinary capacity to regenerate, and after 70% hepatectomy the liver recovers its original mass by replication of the remaining hepatocytes without the activation of liver stem cells. However, in certain types of liver injury models, liver stem/progenitor-like cells, known as oval cells in rodents, proliferate around the portal vein, while the roles of such cells in liver regeneration remain a matter of debate. Clonogenic and bipotential cells are also present in the normal adult liver. In this minireview we describe recent studies on liver stem/progenitor cells by focusing on extracellular signals.     

Human liver model systems in a dish

The human adult liver has a multi‐cellular structure consisting of large lobes subdivided into lobules containing portal triads and hepatic cords lined by specialized blood vessels. Vital hepatic functions include filtering blood, metabolizing drugs, and production of bile and blood plasma proteins like albumin, among many other functions, which are generally dependent on the location or zone in which the hepatocyte resides in the liver. Due to the liver's intricate structure, there are many challenges to design differentiation protocols to generate more mature functional hepatocytes from human stem cells and maintain the long‐term viability and functionality of primary hepatocytes. To this end, recent advancements in three‐dimensional (3D) stem cell culture have accelerated the generation of a human miniature liver system, also known as liver organoids, with polarized epithelial cells, supportive cell types and extra‐cellular matrix deposition by translating knowledge gained in studies of animal organogenesis and regeneration. To facilitate the efforts to study human development and disease using in vitro hepatic models, a thorough understanding of state‐of‐art protocols and underlying rationales is essential. Here, we review rapidly evolving 3D liver models, mainly focusing on organoid models differentiated from human cells.     

Microbiochemical approach to liver cell heterogeneity around terminal hepatic venules

Using lyophilized cryostat sections of liver the activities of alanine aminotransferase, lactate dehydrogenase, and pyruvate kinase were measured using a Lowry technique in the first layer of hepatocytes adjacent to terminal hepatic venules and in the residual parenchymal of the perivenous zone of the acinus in normally fed adult male Wistar rats. Alanine aminotransferase was homogeneously distributed in the two areas measured (ratio hepatocytes adjacent to terminal hepatic venules/residual parenchyma of the perivenous zone: 1.05). Enzyme activities of the lactate dehydrogenase were significantly lower in the hepatocytes adjacent to terminal hepatic venules (ratio: 0.65) and those of the pyruvate kinase significantly higher (ratio: 1.12) than in the residual parenchyma of the perivenous zone indicating liver cell heterogeneity in this zone of the liver acinus.     

High zone-selectivity of cell permeabilization following digitonin-pulse perfusion of rat liver

Summary The plasma membrane permeabilization obtained by exposure of hepatocytes to digitonin is utilized in the so-called digitonin-pulse perfusion of rat liver (Quistorff and Grunnet 1987). Brief pulses of digitonin applied with antegrade and retrograde perfusion of the liver caused selective elution of cytosolic enzymes and metabolites from the periportal and the perivenous zone of the same liver. In the present study a light microscopical examination of the liver fixed immediately after the digitonin pulse confirmed the very high zonal selectivity of the method inferred from the marker enzyme pattern of the eluates: Only cells around the port of entry of digitonin were affected and the borderline between affected and non-affected cells was always sharp. The typical periportal lesion was triangular in shape, enclosing the portal space, while the perivenous lesion was roughly circular, concentric with the hepatic vein. Assuming that the digitonin lesion reflects the microcirculatory flow pattern these findings seem to be at variance with the acinar model of Rappaport (Rappaport et al. 1954). The lesion in the lobuli near the surface of the liver as reflected by the discoloration pattern observed on the surface was the same as the lesion of deeper lobuli. The conducting vessels of the liver were only insignificantly affected by digitonin. At the cellular level only the sinusoidal luminal surface of the hepatocytes was affected. The cytoplasmic matrix of the cells including glycogen appeared thinned. All cell types of the liver parenchyma seemed to be equally affected by the digitonin treatment.     

High zone-selectivity of cell permeabilization following digitonin-pulse perfusion of rat liver. A re-interpretation of the microcirculatory zones

The plasma membrane permeabilization obtained by exposure of hepatocytes to digitonin is utilized in the so-called digitonin-pulse perfusion of rat liver (Quistorff and Grunnet 1987). Brief pulses of digitonin applied with antegrade and retrograde perfusion of the liver caused selective elution of cytosolic enzymes and metabolites from the periportal and the perivenous zone of the same liver. In the present study a light microscopical examination of the liver fixed immediately after the digitonin pulse confirmed the very high zonal selectivity of the method inferred from the marker enzyme pattern of the eluates: Only cells around the port of entry of digitonin were affected and the borderline between affected and non-affected cells was always sharp. The typical periportal lesion was triangular in shape, enclosing the portal space, while the perivenous lesion was roughly circular, concentric with the hepatic vein. Assuming that the digitonin lesion reflects the microcirculatory flow pattern these findings seem to be at variance with the acinar model of Rappaport (Rappaport et al. 1954). The lesion in the lobuli near the surface of the liver as reflected by the discoloration pattern observed on the surface was the same as the lesion of deeper lobuli. The conducting vessels of the liver were only insignificantly affected by digitonin. At the cellular level only the sinusoidal luminal surface of the hepatocytes was affected. The cytoplasmic matrix of the cells including glycogen appeared thinned. All cell types of the liver parenchyma seemed to be equally affected by the digitonin treatment.     

Zonal expression of hepatocytic marker enzymes during liver repopulation

Hepatocytes are metabolically specialised cells displaying distinctive gene expression patterns within the liver lobule. Here, we investigate whether pre-cultured adult rat hepatocytes adopt periportal and pericentral enzyme expression following their transplantation into the regenerating rat liver. Isolated primary rat hepatocytes, representing a mixture of both periportal and pericentral origin, lost expression of carbamoyl phosphate synthetase I (CPS I) and cytochrome P450 subtype 2B1 (CYP2B1) in culture as shown by immunofluorescence and Western blot analysis. Accordingly, urea synthesis and CYP2B1 enzyme activity decreased. Hepatocytes from DPPIV (CD26) wild type rats were cultured for 4 and 7 days, and then transplanted into the livers of CD26 deficient rats following prior treatment with retrorsine and partial hepatectomy to drive selective donor cell proliferation. CD26 positive donor cells engrafted in the periportal regions and grew in clusters expanding into the parenchyma as time proceeded. Ten weeks after transplantation, cells derived from donors surrounding the portal veins expressed CPS I, but not CYP2B1. The reverse was true for CD26 positive cells in close proximity to the central veins displaying immunoreactivity to CYP2B1, but no longer to CPS I. Hepatocytes lose their specific marker enzyme expression in culture. After transplantation, donor hepatocytes proliferate in the host parenchyma whilst acquiring the position-specific enzyme expression of the surrounding periportal and pericentral host hepatocytes. These results indicate the high degree of plasticity of gene expression in hepatocytes subjected to a change in microenvironment.     

Histology and ultrastructure of the hepatopancreas of the tigerfish,Hydrocynus forskahlii

Study of the histology and ultrastructure of the hepatopancreas of the tigerfish, Hydrocynus forskahlii shows that the liver parenchyma is divided into irregularly shaped lobules, separated by the exocrine pancreas and associated connective tissue. The hepatocytes are arranged in interconnecting cords or laminae, two to three cell layers in thickness. Sinusoids separate the laminae. The spherical to oval-shaped hepatocytes contain large, round, centrally situated nuclei with prominent nucleoli. The cytoplasm of the hepatocytes contains abundant rough and smooth endoplasmic reticulae, free polysomes, and mitochondria. Exocrine pancreatic tissue is scattered throughout the liver. This tissue is encapsulated by an endothelium resting on a thin layer of connective tissue and is separated from the liver parenchyma by a sinusoid. The nuclei of the exocrine pancreas cells are spherical, basally situated within the cells, and contain dark nucleoli. Vesicular rough endoplasmic reticulae and secretory granules lie in the apical regions of the exocrine pancreas cells. © 1996 Wiley-Liss, Inc.     

Sinusoidal profiles of lactate dehydrogenase activity in rat liver

Lactate dehydrogenase activities were measured along two sinusoidal paths (1) between small portal tracts and central veins and (2) between regions of adjoining septal branches and central veins in the livers of male Wistar rats, using a Lowry technique. The established profiles of enzyme activity provide further support of functional heterogeneity of liver sinusoids and their abutting hepatocytes related to morphological differences of the sinusoidal bed. Within the hepatocytes a pronounced heterogeneity in enzyme activity was recorded surrounding small portal tracts and central veins. The lowest values of activity were determined in those cells located in close proximity to the vessels, which emphasizes their exceptional morphological and functional position.     

Hepatic fibrosis and cirrhosis: the (myo)fibroblastic cell subpopulations involved

Fibrosis, defined as the excessive deposition of extracellular matrix in an organ, is the main complication of chronic liver damage. Its endpoint is cirrhosis, which is responsible for significant morbidity and mortality. The accumulation of extracellular matrix observed in fibrosis and cirrhosis is due to the activation of fibroblasts, which acquire a myofibroblastic phenotype. Myofibroblasts are absent from normal liver. They are produced by the activation of precursor cells, such as hepatic stellate cells and portal fibroblasts. These fibrogenic cells are distributed differently in the hepatic lobule: the hepatic stellate cells resemble pericytes and are located along the sinusoids, in the Disse space between the endothelium and the hepatocytes, whereas the portal fibroblasts are embedded in the portal tract connective tissue around portal structures (vessels and biliary structures). Differences have been reported between these two fibrogenic cell populations, in the mechanisms leading to myofibroblastic differentiation, activation and "deactivation", but confirmation is required. Second-layer cells surrounding centrolobular veins, fibroblasts present in the Glisson capsule surrounding the liver, and vascular smooth muscle cells may also express a myofibroblastic phenotype and may be involved in fibrogenesis. It is now widely accepted that the various types of lesion (e.g., lesions caused by alcohol abuse and viral hepatitis) leading to liver fibrosis involve specific fibrogenic cell subpopulations. The biological and biochemical characterisation of these cells is thus essential if we are to understand the mechanisms underlying the progressive development of excessive scarring in the liver. These cells also differ in proliferative and apoptotic capacity, at least in vitro. All this information is required for the development of treatments specifically and efficiently targeting the cells responsible for the development of fibrosis/cirrhosis.     

Unusual structure of the biliary system in the liver of the grass carp and the silver carp

It is known that the bile canaliculus in the liver of almost all vertebrates is made up of membranes of two or more adjacent liver cells. Studying the liver cell ultrastructure of lasting and fed grass carp and silver carp, it was demonstrated that a bile canaliculus is formed by deep invagination of a cell membrane of one hepatocyte. The membrane forms microvilli along the bile canaliculus. The bile canaliculus is seen in the centre of liver cell cytoplasm on the cross section and stretches from the centre of the liver cell cytoplasm to the cell membrane on the longitudinal section. The bile canaliculus is connected with a small duct cell, which is distinct from a liver cell in its small size, little amount of cell organelles and the presence of cytoplasmic filaments. The terminal part of the biliary tract consists of one liver cell and one bile duct cell. The part of the tract adjacent to the terminal one is composed of two or three small bile duct cells devoid of basal membrane. Thus, the liver parenchyma is constituted of a net of numerous bile ducts. In the portal tract, there is a large bile duct, consisting of 12-13 bile duct cells, surrounded by basal membrane and connective tissue cells.     

Liver cell polyploidization: a pivotal role for binuclear hepatocytes

Polyploidy is a general physiological process indicative of terminal differentiation. During liver growth, this process generates the appearance of tetraploid (4n) and octoploid (8n) hepatocytes with one or two nuclei. The onset of polyploidy in the liver has been recognized for quite some time; however, the cellular mechanisms that govern it remain unknown. In this report, we observed the sequential appearance during liver growth of binuclear diploid (2 x 2n) and mononuclear 4n hepatocytes from a diploid hepatocyte population. To identify the cell cycle modifications involved in hepatocyte polyploidization, mitosis was then monitored in primary cultures of rat hepatocytes. Twenty percent of mononuclear 2n hepatocytes failed to undergo cytokinesis with no observable contractile movement of the ring. This process led to the formation of binuclear 2 x 2n hepatocytes. This tetraploid condition following cleavage failure did not activate the p53-dependent checkpoint in G1. In fact, binuclear hepatocytes were able to proceed through S phase, and the formation of a bipolar spindle during mitosis constituted the key step leading to the genesis of two mononuclear 4n hepatocytes. Finally, we studied the duplication and clustering of centrosomes in the binuclear hepatocyte. These cells exhibited two centrosomes in G1 that were duplicated during S phase and then clustered by pairs at opposite poles of the cell during metaphase. This event led only to mononuclear 4n progeny and maintained the tetraploidy status of hepatocytes.