Cellular and stromal elements organization in the liver of grass carp, Ctenopharyngodon idella (Cypriniformes: Cyprinidae).

Fish liver is considered as a key organ that controls various life functions. The cellular and stromal elements of liver of eighteen specimens of adult grass carp were investigated by light- and electron- microscopy and enzyme histochemistry. The liver was formed of two lobes with a long process extended from the right lobe. Serial paraffin section of the liver identified different kinds of vascular- biliary structures as follows: 1) pancreatic-venous-biliary-arteriolar tracts (P-VBAT); 2) venous-biliary-arteriolar tracts, (VBAT); 3) pancreatic-venous-biliary tracts (P-VBT); 4) venous-biliary tracts (VBT); 5) venous-arteriolar tracts (VAT); 6) isolated veins named as venous tracts (VT); 7) isolated bile ducts, named as biliary tracts (BT); 8) biliary-arteriolar tracts (BAT); 9) pancreatic-biliary tracts (P-BT); 10) pancreatic- venous tracts (P-VT). Macrophages aggregates were associated with VBT and P-BT. The hepatic parenchyma was consisted of many populations of cells. Histochemically, the hepatocytes were strongly reacted with PAS, and Best's carmine. Moreover, strong staining patterns for acid phosphatase, ATPase, and alkaline phosphatase were demonstrated in hepatocytes. The hepatic satellate (Ito) cells were observed in the space of Disse and between hepatocytes. Rodlet cells and eosinophilic granular/ mast cells were encountered in the liver of grass carp. The sinusoids were lined by fenestrated endothelial cells and Kupffer cells. Moreover, dendritic-like cells were demonstrated in the sinusoids and perisinusoidal connective tissue. The biliary duct system was constituted of bile canaliculi, ductules, and bile ducts. Telocytes with their characteristics telopodes were located around bile ducts. The current findings are offering fundamental data on histology of grass carp liver.

Pathological changes in primary cilia: A novel mechanism of graft cholangiopathy caused by prolonged cold preservation in a rat model of orthotopic liver transplantation.

To study the impairment of cholangiocyte primary cilia caused by prolonged cold preservation and its correlation with graft cholangiopathy after orthotopic liver transplantation (OLT). Subjects were 60 male Wistar rats that were divided into 2 groups: a control group (n = 30) receiving a donor liver preserved for 1 h and a study group (n = 30) receiving a donor liver preserved for 12 h. A two-cuff method was used to establish the OLT model, and the hepatic artery and bile ducts were reconstructed using stents. Samples were collected 2, 8, and 16 weeks after surgery, and 5 samples were collected from each group at each time point. Serum biochemical indicators were measured, morphological changes in intrahepatic bile ducts and cholangiocyte primary cilia were observed using an optical microscope and scanning electronic microscope, respectively, and the ciliary marker (α-tubulin) and membrane proteins (PC-1, TRPV4, and P2Y12) were detected using immunofluorescence analysis and Western blotting. In the study group, phlogocytes infiltrated around bile ducts and bile ducts proliferated markedly at 8 weeks. At 16 weeks, the biliary structures were indistinct and some bile ducts disappeared, a large amount of collagen was deposited, numerous phlogocytes infiltrated around ducts, some biliary epithelial cells (BECs) were deformed or dead, and primary cilia disappeared. In the control group, the intrahepatic bile ducts and BECs were nearly intact and the primary cilia were present. In the study group, the expression of α-tubulin, polycystin-1 (PC-1), TRPV4, and P2Y12 in bile ducts disappeared completely after 8 weeks. In the control group, expression of the marker and proteins decreased at 2 weeks and increased slightly after 8 weeks. These results suggest that the study group had dysfunctional primary cilia at the start of OLT and that this dysfunction was irreversible. In the control group, the primary cilia defects and subsequent biliary injury were temporary. Thus, prolonged cold preservation of a donor liver may cause graft cholangiopathy by altering the integrity and functions of cholangiocyte primary cilia.

Cholangiocyte primary cilia are chemosensory organelles that detect biliary nucleotides via P2Y12 purinergic receptors.

Cholangiocytes, the epithelial cells lining intrahepatic bile ducts, contain primary cilia, which are mechano- and osmosensory organelles detecting changes in bile flow and osmolality and transducing them into intracellular signals. Here, we asked whether cholangiocyte cilia are chemosensory organelles by testing the expression of P2Y purinergic receptors and components of the cAMP signaling cascade in cilia and their involvement in nucleotide-induced cAMP signaling in the cells. We found that P2Y(12) purinergic receptor, adenylyl cyclases (i.e., AC4, AC6, and AC8), and protein kinase A (i.e., PKA RI-beta and PKA RII-alpha regulatory subunits), exchange protein directly activated by cAMP (EPAC) isoform 2, and A-kinase anchoring proteins (i.e., AKAP150) are expressed in cholangiocyte cilia. ADP, an endogenous agonist of P2Y(12) receptors, perfused through the lumen of isolated rat intrahepatic bile ducts or applied to the ciliated apical surface of normal rat cholangiocytes (NRCs) in culture induced a 1.9- and 1.5-fold decrease of forskolin-induced cAMP levels, respectively. In NRCs, the forskolin-induced cAMP increase was also lowered by 1.3-fold in response to ATP-gammaS, a nonhydrolyzed analog of ATP but was not affected by UTP. The ADP-induced changes in cAMP levels in cholangiocytes were abolished by chloral hydrate (a reagent that removes cilia) and by P2Y(12) siRNAs, suggesting that cilia and ciliary P2Y(12) are involved in nucleotide-induced cAMP signaling. In conclusion, cholangiocyte cilia are chemosensory organelles that detect biliary nucleotides through ciliary P2Y(12) receptors and transduce corresponding signals into a cAMP response.

Telomere shortening in the damaged small bile ducts in primary biliary cirrhosis reflects ongoing cellular senescence.

UNLABELLED: Telomere shortening is a trigger of cellular senescence. Biliary epithelial cells in damaged small bile ducts in primary biliary cirrhosis (PBC) show senescent features such as the expression of senescence-associated beta-galactosidase and the increased expression of p16(INK4a) and p21(WAF1/Cip1). We investigated whether the telomere shortening is involved in the pathogenesis of biliary cellular senescence in PBC. We analyzed the telomere length of biliary epithelial cells using quantitative fluorescence in situ hybridization in livers taken from the patients with PBC (n = 13) and control livers (n = 13). We also assessed immunohistochemically the prevalence of DNA damage and the expression of p16(INK4a) and p21(WAF1/Cip1). The study showed a significant decrease in telomere length in biliary epithelial cells in the damaged small bile ducts and bile ductules in PBC compared with normal-looking bile ducts and bile ductules in PBC, chronic viral hepatitis, and normal livers (P < 0.01). gammaH2AX-DNA-damage-foci were detected in biliary epithelial cells in damaged small bile ducts and bile ductules in PBC but were absent in biliary epithelial cells in chronic viral hepatitis and normal livers. The expression of p16(INK4a) and p21(WAF1/Cip1) was increased corresponding to telomere shortening and gammaH2AX-DNA-damage-foci in the damaged small bile ducts in PBC. CONCLUSION: Telomere shortening and an accumulation of DNA damage coincide with increased expression of p16(INK4a) and p21(WAF1/Cip1) in the damaged bile ducts, characterize biliary cellular senescence, and may play a role in the following progressive bile duct loss in PBC.

A light and transmission electron microscope study of hepatic portal tracts in the rhesus monkey (Macacus rhesus).

This study reports on morphological features of hepatic portal tracts in the liver of a rhesus monkey. The light microscope shows that the number of each type of principal component comprising a portal tract varies but that there are usually one to five lymphatics, one bile ductule, one bile duct, one arteriolar and one arterial branch of the hepatic artery, and one hepatic portal vein. Bile ductules, in cross section, have 6-10 cells (mostly low pyramidal, but with a few cuboidal) bordering the lumen, an outside diameter of from about 20 to 25 microm, and a luminal diameter of from 2 to 10 microm. Bile ducts, in cross section, have more than 10 cells (about equal numbers of low pyramidal and cuboidal) bordering the lumen, an outside diameter greater than 25 microm and a luminal diameter of greater than 10 microm. The term "pyramidal" has not previously been applied to the cells of the ductules and ducts. The monkey tracts show several cytological features previously undescribed, viz., abortive cilia and basal bodies in the duct cells, abortive cilia in the ductule cells, and an occasional aggregation of ribosomes in arterial endothelial cells. They also show a major histological feature previously mentioned but not illustrated, viz., bundles of nerve processes which exhibit a preferential location, i.e., proximity to the arterioles and arteries.

Clinicopathological study on cholangiolocellular carcinoma suggesting hepatic progenitor cell origin.

UNLABELLED: Cholangiolocellular carcinoma (CLC), a subtype of cholangiocellular carcinoma (CC), is thought to originate from the ductules/canals of Hering, where hepatic progenitor cells (HPCs) are located. We investigated the clinicopathological features of 30 CLCs and their relationship to HPCs. We evaluated the expression of hepatocytic markers (hepatocyte paraffin-1, canalicular polyclonal carcinoembryonic antigen, and CD10), biliary/HPC markers (keratin [K]7, K19, and neural cell adhesion molecule), the adenosine triphosphate binding cassette transporters: multidrug resistance protein 1, multidrug resistance-associated protein (MRP)1, MRP3, and breast cancer resistance protein, using immunohistochemistry and electron microscopy. In addition, gene expression profiling of CLC was performed and compared with the profile of hepatocellular carcinoma (HCC) with or without HPC features (K19 expression). In surrounding nontumoral tissue, K7-positive and K19-positive HPCs/ductular reaction were observed. More than 90% of the tumor was composed of CLC areas that showed small monotonous and/or anastomosing glands, strongly positive for K7 and K19. Especially at the tumor boundary, all cases showed a HCC-like trabecular area characterized by canalicular CD10/polyclonal carcinoembryonic antigen expression, and submembranous K7 expression, similar to intermediate hepatocytes. K7-positive/K19-positive HPCs were also seen. Out of 30 cases, 19 showed papillary and/or clear glandular formation with mucin production, representing CC areas. These three different areas showed transitional zones with each other. We observed an increased expression of MRP1, MRP3, and breast cancer resistance protein in the tumor. Electron microscopy findings in HCC-like trabecular areas confirmed the presence of HPCs and intermediate hepatocytes. HPC markers, K7, K19, prominin-1, receptor for stem cell factor c-kit, octamer-4 transcription factor, and leukemia inhibitory factor were upregulated (P < 0.05), while albumin was downregulated in CLC (P = 0.007) toward K19-negative HCCs. Comparison of CLC with K19-positive HCCs indicated a high homology. CONCLUSION: All these findings highly suggest a progenitor cell origin of CLC.

Non invasive high resolution in vivo imaging of alpha-naphthylisothiocyanate (ANIT) induced hepatobiliary toxicity in STII medaka.

A novel transparent stock of medaka (Oryzias latipes; STII), homozygous recessive for all four pigments (iridophores, xanthophores, leucophores, melanophores), permits transcutaneous, high resolution (<1 microm) imaging of internal organs and tissues in living individuals. We applied this model to in vivo investigation of alpha -naphthylisothiocyanate (ANIT) induced hepatobiliary toxicity. Distinct phenotypic responses to ANIT involving all aspects of intrahepatic biliary passageways (IHBPs), particularly bile preductular epithelial cells (BPDECs), associated with transitional passageways between canaliculi and bile ductules, were observed. Alterations included: attenuation/dilation of bile canaliculi, bile preductular lesions, hydropic vacuolation of hepatocytes and BPDECs, mild BPDEC hypertrophy, and biliary epithelial cell (BEC) hyperplasia. Ex vivo histological, immunohistochemical, and ultrastructural studies were employed to aid in interpretation of, and verify, in vivo findings. 3D reconstructions from in vivo investigations provided quantitative morphometric and volumetric evaluation of ANIT exposed and untreated livers. The findings presented show for the first time in vivo evaluation of toxicity in the STII medaka hepatobiliary system, and, in conjunction with prior in vivo work characterizing normalcy, advance our comparative understanding of this lower vertebrate hepatobiliary system and its response to toxic insult.

Cholangiocyte cilia detect changes in luminal fluid flow and transmit them into intracellular Ca2+ and cAMP signaling.

BACKGROUND & AIMS: Cholangiocytes have primary cilia extending from the apical plasma membrane into the ductal lumen. While the physiologic significance of cholangiocyte cilia is unknown, studies in renal epithelia suggest that primary cilia possess sensory functions. Here, we tested the hypothesis that cholangiocyte cilia are sensory organelles that detect and transmit luminal bile flow stimuli into intracellular Ca2+ ([Ca2+]i) and adenosine 3',5'-cyclic monophosphate (cAMP) signaling. METHODS: Scanning electron microscopy, transmission electron microscopy, and immunofluorescent confocal microscopy of rat isolated intrahepatic bile duct units (IBDUs) were used to detect and characterize cholangiocyte cilia. The fluid flow-induced changes in Ca2+ and cAMP levels in cholangiocytes of microperfused IBDUs were detected by epifluorescence microscopy and a fluorescence assay, respectively. RESULTS: In microperfused IBDUs, luminal fluid flow induced an increase in [Ca2+]i and caused suppression of the forskolin-stimulated cAMP increase. The fluid flow-induced changes in [Ca2+]i and cAMP levels were significantly reduced or abolished when cilia were removed by chloral hydrate or when ciliary-associated proteins polycystin-1 (a mechanoreceptor), polycystin-2 (a Ca2+ channel), and the Ca2+-inhibitable adenylyl cyclase isoform 6 were individually down-regulated by small interfering RNAs. CONCLUSIONS: Cholangiocyte cilia are sensory organelles containing polycystin-1, polycystin-2, and adenylyl cyclase isoform 6 through which luminal fluid flow affects both [Ca2+]i and cAMP signaling in the cell. The data suggest a new model for regulation of ductal bile secretion involving cholangiocyte cilia.

An immunohistochemical study of human fetal liver in the Meckel-Gruber syndrome.

AIMS: The ductal plate abnormality of the liver in fetuses with the Meckel-Gruber syndrome has been well characterised, but its aetiology remains unknown. We have analysed liver structure in six fetuses with this syndrome, using routine histology, immunocytochemistry, and electron microscopy. METHODS: Liver tissue from six fetuses of 11-27 weeks gestational age was examined by immunoperoxidase staining with antigens to cyokeratin (AE1/3) and polyclonal CEA. We also examined the ultrastructure of the syndromic fetal liver. The findings were compared with livers of control fetuses obtained from miscarriages, of similar size and gestational age but without dysmorphic features or developmental anomalies. RESULTS: The ductal plate abnormality was present in all the fetuses with the Meckel-Gruber syndrome. There were abnormalities of biliary excretion in all syndromic fetuses. Ultrastructural studies of the portal tract revealed abnormal collagen bundles in the Meckel-Gruber syndrome. CONCLUSIONS: Our findings, in conjunction with other reports in the literature, suggest that the ductal plate abnormality may be caused by failure of anastomosis of the intra- and extrahepatic biliary systems, perhaps in association with abnormalities of the portal tract stroma and biliary excretion.

Morphological characterization of ductular reactions in canine liver disease.

Intrahepatic bile duct proliferation (ductular reaction) was examined histologically, immunohistochemically and ultrastructurally in four cases of canine liver disease, diagnosed as chronic hepatitis, liver fibrosis, cirrhosis and cholangiocellular carcinoma. Ductular reaction was a common finding in all cases. Most of the proliferated bile ducts were similar to normal bile ducts. In addition, duct-like structures occurred, consisting of hepatocytes and of intermediate cells that had phenotypic characteristics of both cholangiocytes and hepatocytes. The proliferated bile ducts were immunohistochemically negative for proliferating cell nuclear antigen (PCNA) and stem cell factor (SCF). The proliferated bile ducts in these four cases of canine liver disease thus showed both typical ductular reactions, such as elongation and tortuosity of the existing bile ducts, and atypical ductular reactions resulting from metaplasia of hepatocytes.

Does a betaretrovirus infection trigger primary biliary cirrhosis?

Patients with primary biliary cirrhosis develop progressive ductopenia associated with the production of antimitochondrial antibodies that react with a protein aberrantly expressed on biliary epithelial cells and peri-hepatic lymph nodes. Although no specific microbe has been identified, it is thought that an infectious agent triggers this autoimmune liver disease in genetically predisposed individuals. Previous serologic studies have provided evidence to suggest a viral association with primary biliary cirrhosis. Here we describe the identification of viral particles in biliary epithelium by electron microscopy and the cloning of exogenous retroviral nucleotide sequences from patients with primary biliary cirrhosis. The putative agent is referred to as the human betaretrovirus because it shares close homology with the murine mammary tumor virus and a human retrovirus cloned from breast cancer tissue. In vivo, we have found that the majority of patients with primary biliary cirrhosis have both RT-PCR and immunohistochemistry evidence of human betaretrovirus infection in lymph nodes. Moreover, the viral proteins colocalize to cells demonstrating aberrant autoantigen expression. In vitro, we have found that lymph node homogenates from patients with primary biliary cirrhosis can induce autoantigen expression in normal biliary epithelial cells in coculture. Normal biliary epithelial cells also develop the phenotypic manifestation of primary biliary cirrhosis when cocultivated in serial passage with supernatants containing the human betaretrovirus or the murine mammary tumor virus, providing a model to test Koch's postulates in vitro.

Agonist-induced coordinated trafficking of functionally related transport proteins for water and ions in cholangiocytes.

We previously proposed that ductal bile formation is regulated by secretin-responsive relocation of aquaporin 1 (AQP1), a water-selective channel protein, from an intracellular vesicular compartment to the apical membrane of cholangiocytes. In this study, we immunoisolated AQP1-containing vesicles from cholangiocytes prepared from rat liver; quantitative immunoblotting revealed enrichment in these vesicles of not only AQP1 but also cystic fibrosis transmembrane regulator (CFTR) and AE2, a Cl- channel and a Cl-/HCO3- exchanger, respectively. Dual labeled immunogold electron microscopy of cultured polarized mouse cholangiocytes showed significant colocalization of AQP1, CFTR, and AE2 in an intracellular vesicular compartment; exposure of cholangiocytes to dibutyryl-cAMP (100 microm) resulted in co-redistribution of all three proteins to the apical cholangiocyte plasma membrane. After administration of secretin to rats in vivo, bile flow increased, and AQP1, CFTR, and AE2 co-redistributed to the apical cholangiocyte membrane; both events were blocked by pharmacologic disassembly of microtubules. Based on these in vitro and in vivo observations utilizing independent and complementary approaches, we propose that cholangiocytes contain an organelle that sequesters functionally related proteins that can account for ion-driven water transport, that this organelle moves to the apical cholangiocyte membrane in response to secretory agonists, and that these events account for ductal bile secretion at a molecular level.

Evaluation of differential gene expression by microarray analysis in small and large cholangiocytes isolated from normal mice.

AIMS: We have shown that large and small cholangiocytes, which reside primarily in large and small intrahepatic bile ducts, respectively, have different functions and responses to injuries. However, there are no systematic studies of the molecular differences between small and large cholangiocytes, which would explain cholangiocyte heterogeneity. To evaluate the differential gene expression between small and large cholangiocytes, microarray analysis was performed. METHODS: Primary cultures of small and large cholangiocytes were isolated from normal mice (BALB/c), and immortalized by the introduction of the SV40 large T antigen gene. After cloning, small and large cholangiocyte cell lines were established. Their characteristic features were confirmed by electron microscopy (EM) and measurement of transepithelial electrical resistance (TER), and secretin-stimulated cAMP levels. Isolated total RNAs were hybridized with microarrays (Atlas Glass Array Mouse 1.0 and 3.8), which detects 4850 cDNA expressions. After hybridization, the fluorescent signals were scanned by a GenePix fluorescent scanner and analyzed using ArrayGauge software. RESULTS: EM, TER and secretin-stimulated cAMP synthesis are consistent with the concept that small and large immortalized cholangiocytes originate from small and large ducts, respectively. When a cut-off value at the expression signal difference of 3.0 times was employed, 230 cDNAs among 4850 cDNAs (4.74%) were differentially expressed between small and large cholangiocytes. Of these 230 cDNAs, aquaporin 8, IL-2 receptor beta chain and caspase 9 were more strongly expressed by large cholangiocytes. CONCLUSIONS: Microarray successfully displayed characteristic differential cDNA expression between small and large cholangiocytes. This technique provides molecular information, which further supports our hypothesis that small and large bile ducts have different functions.

The PICM-19 cell line as an in vitro model of liver bile ductules: effects of cAMP inducers, biopeptides and pH.

The PICM-19 fetal liver cell line was isolated from the primary culture and spontaneous differentiation of pig epiblast cells, i.e. embryonic stem cells. PICM-19 cells were induced to differentiate into mostly ductular formations by culturing at pH 7.6-7.8. The ductules were functionally assayed by treatment with cAMP inducing agents and bioactive peptides reported to influence the secretory activity of liver bile ductules. The secretory response of the cells was assessed by qualitative or quantitative measurement of the cross-sectional area of the ductal lumens and the appearance of biliary canaliculi in between PICM-19 cells that had formed monolayers instead of ducts. Forskolin (10 microM) and 8-bromoadenosine 3':5'-cyclic monophosphate (bcAMP; 2 mM) stimulated fluid transport and expansion of ductal structures in 15-20 min and stimulated the appearance and expansion of biliary canaliculi in 30-60 min. Cholera toxin (50 ng/ml) stimulates fluid transport in both ductules and canaliculi in 1-2 h, while 8-bromoguanosine 3':5'-cyclic monophosphate (bcGMP; 2 mM) stimulated only biliary canaliculi in 2 h. Glucagon (1.4 nM) produced a similar response in 5-10 min in ductal structures only, but the response was transitory and was almost completely reversed within 30 min. Secretin (100 pM) and vasoactive intestinal peptide (75 pM) produced a sustained response with maximal ductal lumen expansion occurring in 5-10 min and neither had an immediate effect on canaliculi. Somatostatin (0.5 microM) and gastrin (1 microM) caused marked reduction or disappearance of ductal lumens in 30-60 min, but was ineffective in reversing secretin (100 nM)-induced duct distension. Application of the adrenergic agonists, epinephrine, isoproterenol, and phenylephrine (100 microM), resulted in the complete shrinkage of ductal lumens in 20-30 min. A shift to pH 7.0-7.2 resulted in almost complete reduction of ductal lumens, while a shift to pH 7.8-8.0 resulted in expansion, although not full expansion, of the ductal lumens. PICM-19 bile duct cultures were positive for cytokeratin-7, aquaporin-1 and aquaporin-9 by Western blot analysis. The amounts of these proteins increased in the cultures as differentiation proceeded over time. Transmission electron microscopy revealed that the ductal structures were usually sandwiched between SIM mouse, thioguanine- and ouabain-resistant (STO) feeder cells that had produced a collagen matrix. Also, the ductular PICM-19 cells possessed cilia, probably occurring as a single cilium in each cell, that projected into the lumens of the ducts. The results indicated that the in vitro-produced ductal structures of the PICM-19 cell line are a functional model for biliary epithelium.

Cholestatic effect of large bilirubin loads and cholestasis protection conferred by cholic acid co-infusion: a molecular and ultrastructural study.

BACKGROUND: Large intravenous bilirubin loads block biliary phospholipid secretion, produce canalicular membrane lesions and cause canalicular cholestasis. Cholic acid co-infusion forestalls these untoward effects. The aim of this study was first to determine whether bilirubin overload causes cholestasis through reducing the activity or the hepatic expression of the bile salt export pump (bsep) or Na-taurocholate co-transporting polypeptide (ntcp) and, secondly, whether cholic acid co-infusion forestalls cholestasis by upregulating bsep, ntcp or phosphoglycoprotein 3 (pgp3) expressions or activities. A further aim was to determine whether large bilirubin infusions also produce ultrastructural changes inside hepatocytes. METHODS: The effects of intravenous infusion of 2 g bilirubin over 150 min on hepatic expression of bsep, ntcp and pgp3 were studied in bile acid-depleted and cholic acid co-infused pigs, and related to canalicular bile acid transport and bile secretion. Effects on hepatocyte ultrastructural morphology were analysed by electron microscopy. RESULTS: Bilirubin-induced cholestasis reflected marked diminution of bsep and pgp3 transport activities and not reduced hepatic expression of these transporters. Hepatocyte ultrastructural abnormalities were predominantly confined to the hepatocyte canalicular membrane in cholestatic livers. Cholic acid co-infusion with bilirubin conferred complete cholestasis protection through enhancing pgp3 and bsep transporter activities and not through upregulating their expression. Bilirubin infusion did not change ntcp expression. CONCLUSION: Bilirubin-induced cholestasis is due to markedly impaired activity of the membrane-embedded bsep transporter consequent upon ultrastructural injury to the canalicular membrane. Cholic acid co-infusion with bilirubin enhances bsep and pgp3 activities and confers protection against canalicular membrane injury and bilirubin-induced cholestasis.

Study of experimentally induced lesions in sheep by grazing Brachiaria decumbens.

A histologic and ultrastructural study of the alterations found in the lymph nodes and livers of nine sheep with experimental cholangiohepatopathy by grazing on Brachiaria decumbens has been performed. Sheep were euthanized in three groups, on the 77th, 89th, and 150th days of the experimental feeding. The main gross lesions were whitish spots of multifocal distribution scattered throughout the hepatic parenchyma from all B. decumbens-grazed animals and whitish foci surrounded by reddened halos in the mesenteric and hepatic lymph nodes of sheep necropsied on the 150th. The principal histologic findings included hepatocellular cloudy swelling, marked multifocal cholangitis in the portal triads with bile duct proliferation and infiltration of macrophages and lymphocytes. Crystals were observed within bile ducts and surrounded by macrophages. Ultrastructurally, there were criytaloid structures within the macrophages and hepatocytes, which also presented hyperplasia of smooth endoplasmic reticulum. These findings suggest that hepatocytes were the initial target of the toxic effects, which depending on the degree of severity developed would cause both, subsequent cholangiopathy or occasional photosensitization. Additionally, the developmental stages of the hepatic lesions observed in this study have been presented.

Bile canalicular barrier function and expression of tight-junctional molecules in rat hepatocytes during common bile duct ligation.

Tight junctions of hepatocytes form the intercellular barrier between the blood circulation and bile flow. We focused on early stages of common bile duct ligation to observe changes in tight junctions without the irreversible changes seen after lengthy ligation. Common bile ducts of 12-week-old male rats were ligated for 6 h because, at this time point, no histological changes were observed. Serum bilirubin and bile acid levels began to increase 3 h after ligation and were restored to the control level immediately after surgical removal of the ligation. To examine the barrier of hapatocytes, horseradish peroxidase was injected via the femoral vein, and bile was collected for the first 10 min. A four-fold elevation of the secretion and concentration was observed in the bile of ligated rats compared with that of control animals. We next examined lanthanum permeability by perfusion fixation of the liver. At 6 h after ligation, both dilation of the bile canaliculi and partial loss of microvilli were commonly observed. There were dense deposits of lanthanum in almost all bile canaliculi of ligated rats. In control animals, neither dilation of the bile canaliculi nor loss of microvilli was detected, and only 44% of bile canaliculi exhibited deposits. An apparent increase of occludin mRNA expression was detected in livers after 6 h ligation, whereas the expression of claudin-1, -2, and -3 was not influenced by ligation. These results indicate that regulation of occludin gene expression is different from that of claudin-1, -2, and -3. The early phase of bile stasis employed in this study is thought to be an indispensable approach for understanding the precise regulation of tight junctions.

Combined hepatocellular and cholangiocellular carcinoma in a dog.

A transitional type of combined hepatocellular and cholangiocellular carcinoma developed in a 12-year-old male Yorkshire terrier dog. The tumor was histologically composed of both hepatocellular carcinoma and cholangiocellular carcinoma components, and both elements were closely intermingled. Intraluminal mucin accumulation in cytokeratin-positive tubular/glandular structures was observed within the cholangiocellular carcinoma components and this feature was useful histological marker for a differential diagnosis between combined hepatocellular and cholangiocellular carcinoma and a pseudoglandular type of hepatocellular carcinoma. This primary hepatic tumor is extremely rare in dogs.

Isolation of functional polarized bile duct units from mouse liver.

The development of genetically altered murine animals has generated a need for in vitro systems in the mouse. We have now characterized a novel isolated bile duct unit (IBDU) preparation from the mouse to facilitate such studies. The mouse IBDU is isolated by portal perfusion of collagenase, blunt dissection, further enzymatic digestions, filtering through sized mesh, and culturing on Matrigel for 16-72 h. This mouse IBDU forms a central, enclosed lumen lined by polarized cytokeratin-19-positive cholangiocytes with numerous microvilli on the apical membrane. The IBDU responds to secretory stimuli, including secretin, vasoactive intestinal peptide, IBMX, and forskolin, resulting in expansion of the central lumen from secretion as quantified by videomicroscopy. The secretory response to secretin is dependent on Cl- and HCO3-in the perfusate. These findings indicate that mouse IBDUs are intact, polarized, functional bile duct secretory units that permit quantitative measurements of fluid secretion from mouse bile duct epithelium for the first time. This method should facilitate studies of cholangiocyte secretion in genetically altered murine animal models.

Biliary tract of the rat as observed by scanning electron microscopy of cast samples.

The three-dimensional distribution of the biliary tract in the rat was studied by scanning electron microscopy of biliary casts. The casts were prepared by a retrograde infusion of a low viscosity or monomeric methacrylate resin mixture into the common bile duct. No resin flow from the bile canaliculi to sinusoidal capillaries was ever noted. Bile canaliculi formed intricate meshworks and drained via the Hering's canals into the bile ductules. The bile canalicular meshworks of adjacent lobules intercommunicated with each other. The bile ductules formed a marked periportal plexus around the portal vein branch, and drained into the intrahepatic bile duct running along the portal vein branch. The junctional zone of the Hering's canal and bile ductule usually showed an ampullary dilation. When the Hering's canal directly drained into a thick bile ductule or into a periportal plexus of bile ductules, such an ampullary dilation at the origin of the bile ductule was never replicated. The extrahepatic bile duct protruded many crypt-like projections which presumably corresponded to parietal glands. It is suggested that the periportal plexus of bile ductules may store the bile as a substitute for the gallbladder.