Cholestasis results from impairment in the excretion of bile, which may be due to mechanical obstruction of bile flow or impairment of excretion of bile components into the bile canaliculus. When present, cholestasis warrants prompt diagnosis and treatment. The differential diagnosis of cholestasis beyond the neonatal period is broad and includes congenital and acquired etiologies. It is imperative that the clinician differentiates between intrahepatic and extrahepatic origin of cholestasis. Treatment may be supportive or curative and depends on the etiology. Recent literature shows that optimal nutritional and medical support also plays an integral role in the management of pediatric patients with chronic cholestasis. This review will provide a broad overview of the pathophysiology, diagnostic approach, and management of cholestasis beyond the neonatal and infancy periods.
Alagille Syndrome ; Bile ; Bile Canaliculi ; Cholelithiasis ; Cholestasis* ; Diagnosis ; Diagnosis, Differential ; Hepatitis ; Hepatolenticular Degeneration ; Humans ; Jaundice ; Pruritus

OBJECTIVETo regain bile excretory function of hepatocytes cultured in vitro.

METHODSSandwich configuration was used to culture hepatocytes and the structure of bile canaliculi as well as the function of bile excretion was observed by immunocytochemistry and the test of FDA metabolism with a single collagen configuration as a control.

RESULTSFirst, the formation of bile canaliculi was observed by immunocytochemistry. In sandwiched hepatocytes, the gradual development of bile canaliculi-like structures into an anastomotic network was observed. At 24 h after cell culture, the formation of bile canaliculi was observed. With time progressed, the bile canaliculi became more clear and the network was established at 120 h. In contrast, hepatocytes in single collagen configuration showed almost no network of bile canaliculi. Second, the ability for hepatocytes to internalize, metabolize and excrete compounds into bile was indicated by FDA metabolized in the hepatocytes. In sandwiched hepatocytes, the bile excretory function was shown at 96 h, but in single collagen system, no bile excretion was observed.

CONCLUSIONCultured hepatocytes are able to regain bile excretion in a given certain condition. Sandwich-cultured hepatocytes can reestablish bile canalicular structure and regain bile excretory function.

Animals ; Bile ; secretion ; Bile Canaliculi ; physiology ; Cells, Cultured ; Female ; Hepatocytes ; secretion ; Immunohistochemistry ; Rats ; Rats, Sprague-Dawley

The microfilaments of hepatocyte are distributed throughout the vicinity of cell membranes, especially numerous around the region of bile canaliculus, and provide the maintenance of cell shape, cellular wall tension, canalicular motility, the secretion for bile, etc. To evaluate the relationship between the microfilament and alteration of cell shape, we examined the morphological changes of cultured rat hepatocytes, following treatments with phalloidin or cytochalasin D with fluorescent and electron microscopes. 1. In the fluorescent micrographs, actin microfilament was distributed near the plasma membrane and bile canaliculus. 2. Both drugs, phalloidin or cytochalasin D, produce the cytoplasmic protrusions from the surface. Their shapes were pedunculated with narrow neck or bulged with broad base, respectively. 3. In the phalloidin treated group, cytoplasmic protrusion was seperated from the internal cytoplasm by microfila-ments networks at the narrow base. In contrast, in the cytochalasin D treated group, cytoplasm was bulged with broad base and kept in direct continuity with the canalicular ectoplasm. 4. Pericanalicular ectoplasm of phalloidin treated group was widened and accumulated with microfilaments. But, bile canaliculus of cytochalasin D treated group was markedly dilated and devoid of microvilli, and the ectoplasm was almost disappeared. Considering above results, dysfunction of microfilaments leads to the structural changes and inhibition of bile secretion of hepatocytes.
Actin Cytoskeleton ; Animals ; Bile ; Bile Canaliculi ; Cell Membrane ; Cell Shape ; Cytochalasin D* ; Cytoplasm ; Hepatocytes* ; Microvilli ; Neck ; Phalloidine* ; Rats*

To assess the extent of microfilaments in cholestatic liver diseases we examined the cytoplasmic microfilaments in intrahepatic and extrahepatic cholestasis in man by electron microscopy. Study subjects were two patients with drug-induced intrahepatic cholestasis, three patients with intrahepatic cholestasis due to viral hepatitis, four patients with extrahepatic cholestasis due to stones of the common bile duct and two patients with primary biliary cirrhosis. Two biopsied specimens from patients without clinical or histological evidence of liver disease served as noncholestatic controls. The microfilaments in hepatocytes and biliary ductular cells were significantly increased in cholestasis compared with those in non-cholestatic controls. Well developed bundles of microfilaments were noted around the pericanalicular ectoplasm and seemed to be parallel to plasma membrane of the hepatocytes in cholestasis. In cholestasis, there were increased bundles of microfilaments around the periluminal region, lateral cell wall, and nucleus of biliary ductular cells. Two patterns of microfilaments bundles (fine microfilamentous network and spindle-shaped dense or clusters of microfilaments) were associated with cholestasis. The clustered form of microfilaments also seemed to be clearly associated with intracytoplasmic vacuoles containing bile salts. In conclusion, the increase of microfilaments in hepatocytes and biliary ductular cells may be the consequence of various forms of cholestasis. Further studies are needed to clarify the functional significance of increased microfilaments in cholestasis.
Bile Canaliculi/*pathology/ultrastructure ; Biopsy ; Cholestasis, Intrahepatic/*pathology ; Hepatocytes/*pathology/ultrastructure ; Humans ; Microfilaments/*pathology/ultrastructure ; Microscopy, Electron

BACKGROUND: Intrahepatic cholestasis can occur early after living donor liver transplantation (LDLT). We investigated the changes in the expressions of the bile acid transporters and the liver histology in the patients who suffered with early cholestasis (EC) following LDLT. METHODS: The histological differences between 15 graft livers with EC after LDLT and 5 graft livers with biliary stricture following LDLT were evaluated. The hepatic mRNA levels of the bile canaliculi transporters (BSEP, MRP2, MRP3, MDR1, MDR3, NTCP) in 40 (20 graft livers, 20 matched donor livers) liver biopsy tissues were analyzed by performing real-time reverse-transcription polymerase chain reaction (RT-PCR). RESULTS: Microscopic examination revealed hepatocellular and/or bile canalicular cholestasis around acinar zone 3 in the livers of the patients with EC. In the livers with biliary stricture, the cholestasis was dominantly observed in the hepatocytic cytoplasm and in the bile ductules around the portal area rather than around acinar zone 3. The BSEP and MRP2 mRNA levels in the EC livers were significantly reduced by 44% and 23%, respectively (p=0.000), compared to the matched donor livers. The levels of MDR3 and NTCP mRNA in the EC livers increased by 738% (p=0.000) and 281% (p<0.01), respectively. The change of the expressions of the bile acid transporters in the patients with biliary stricture was less significant than that in the EC group. CONCLUSIONS: These results suggest that the altered expressions of the bile acid transporters may play a role in the pathogenesis of EC following LDLT.
Bile ; Bile Canaliculi ; Biopsy ; Carrier Proteins ; Cholestasis ; Cholestasis, Intrahepatic ; Constriction, Pathologic ; Cytoplasm ; Humans ; Liver ; Liver Transplantation ; Living Donors ; Membrane Glycoproteins ; Polymerase Chain Reaction ; RNA, Messenger ; Tissue Donors ; Transplants

PURPOSE: The authors evaluated the morphological and proliferative properties of embryonic stem (ES) cells following intrasplenic transplantation. The results were compared with those obtained following intrasplenic transplantation of cultured hepatocytes. METHODS: ES cells of blastocysts were collected from superovulated Sprague Dawley rats. Hepatocytes were collected from the liver of 7-week old rats by perfusion of collagenase. The ES cells and hepatocytes were cultured for 6 days and transplanted into the rat spleen. The properties of the ES cells and cultured hepatocytes following transplantation were investigated by morphological methods. RESULTS: ES cells in the culture proliferated faster than hepatocytes, and differentiated to various shaped cells. Following transplantation, ES cells were distributed near the periarterial lymphatic sheath. Cultured hepatocytes gathered chiefly around the trabeculae. On PCNA stain of transplanted ES cells, positive cells appeared on day 7 and became distinct on days 10 and 14. Transplanted hepatocytes showed no PCNA positive cells on day 14. On electron microscopic examination, ES cells differentiated to hepatocyte-like structures on day 10, and became functioning hepatocytes on day 14. Transplanted hepatocytes formed bile canaliculi on day 10, although development of organelles was insufficient on day 14. CONCLUSION: ES cells proliferated faster than cultured hepatocytes. Intrasplenic ES cells proliferated at the germinal center and hepatocytes around the trabecula. ES cells differentiated to cells that had the function of hepatocytes.
Animals ; Bile Canaliculi ; Blastocyst ; Collagenases ; Embryonic Stem Cells* ; Germinal Center ; Hepatocytes ; Liver ; Organelles ; Perfusion ; Proliferating Cell Nuclear Antigen ; Rats ; Rats, Sprague-Dawley ; Spleen

PURPOSE: Primary mammalian hepatocytes largely retain their liver-specific functions when they are freshly derived from donors. However, long-term cultures of functional hepatocytes are difficult to establish. To increase the longevity and maintain the differentiated functions of hepatocytes in primary culture, cells can be cultured in a sandwich configuration of collagen. In such a configuration, hepatocytes can be cultured for longer periods compared with cultures on single layers of collagen. However, research regarding mouse hepatocytes in sandwich culture is lacking. METHODS: Primary mouse hepatocytes were sandwiched between two layers of collagen to maintain the stability of their liver-specific functions. After gelation, 2 mL of hepatocyte culture medium was applied. RESULTS: After 24 hours, 5, 10 days of culture, the collagen gel sandwich maintained the cellular border and numbers of bile canaliculi more efficiently than a single collagen coating in both high and low density culture dishes. Reverse transcription-polymerase chain reaction analysis of alpha-1-antitrypsin (AAT), hepatocyte nuclear factor 4 alpha (HNF4A), alphafetoprotein, albumin, tryptophan oxygenase (TO), the tyrosine aminotransferase gene, glucose-6-phosphatase, glyceraldehyde-3-phosphate dehydrogenase for mouse primary hepatocytes cultured on collagen coated dishes and collagen gels showed superior hepatocyte-related gene expression in cells grown using the collagen gel sandwich culture system. AAT, HNF4A, albumin, TO were found to be expressed in mouse hepatocytes cultured on collagen gels for 5 and 10 days. In contrast, mouse hepatocytes grown on collagen-coated dishes did not express these genes after 5 and 10 days of culture. CONCLUSION: The collagen gel sandwich method is suitable for primary culture system of adult mouse hepatocytes.
Adult ; Animals ; Bile Canaliculi ; Collagen ; Gels ; Gene Expression ; Glucose-6-Phosphatase ; Hepatocyte Nuclear Factor 4 ; Hepatocytes ; Humans ; Longevity ; Mice ; Oxidoreductases ; Tissue Donors ; Tryptophan Oxygenase ; Tyrosine Transaminase

To examine the role of actin microfilaments which are located at beneath the plasma membrane, we observed the ultrastructural changes of rat hepatocyte induced by alteration of the microfilamentous integrity. The isolated hepatocytes from Sprague-Dawley were cultured in the L-15 medium containing phalloidin (agent that cause polymerization of actin) or cytochalasin D (agent that cause depolymerization of actin) for 30 min, 1 hour, 2 hours, 4 hours, 10 hours and 20 hours, respectively. The results observed with scanning and transmission electron microscope were as follows. 1. Following the alteration of actin microfilaments, bile canaliculi were dilated and devoid of microvilli. In phalloidin treated group, the thickening of microfilamentous ectoplasm was more marked than that of cytochalasin D treated group. Whereas, the dilation of bile canaliculi was more marked in cytochalasin D group. 2. Both drugs, phalloidin or cytochalasin D, produced the alteration of cell shape to form cytoplasmic protrusions at the cell surface. In the phalloidin treated group, protrusions were pedunculated, and the microfilament networks were accumulated at the narrow neck region. 3. In cytochalasin D treated group, no microfilament barrier was seen at the broad base of protrusion which exhibit direct continuity with the internal cytoplasm. 4. Single hepatocyte tend to recover their structural integrity as those in vivo. The new bile canaliculus was sealed off at the intercellular space by tight junctions, and intercellular contacts were established by the junctional complexes. The results demonstrated that excessive accumulation or depletion of microfilaments induced by phalloidin or cytochalasin D altered the cell shape different, respectively. The microfilaments of ectoplasm play an important role in the maintenance of the structural integrity of cultured hepatocytes.
Actin Cytoskeleton* ; Animals ; Bile Canaliculi ; Cell Membrane ; Cell Shape ; Cytochalasin D ; Cytoplasm ; Extracellular Space ; Hepatocytes* ; Microvilli ; Neck ; Phalloidine ; Polymerization ; Polymers ; Rats ; Rats, Sprague-Dawley ; Tight Junctions

Bile canaliculi is closely related to the cytoskeleton; actin filament web, microtubules and cytokeratin intermediate filaments. To understand how cytoskeletal alteration affects bile canalicular structure, the investigators injected cytochalasin B and colchicine into mice intraperitoneally to inhibit the polymerization of actin filaments and microtubules respectively, and observed the structural changes of bile canaliculi and hepatocytes with transmission and scanning electron microscopes. Bile canaliculi were dilatated and microvilli were decreased in number and length after injection of cytochalasin B and colchicine. Some bile canaliculi branched irregularly after colchicine treatment. Actin filament web in the canalicular ectoplasm was disrupted leaving granular zone after cytochalasin B treatment, but was intact after colchicine treatment. Intermediate filament bundles located at angles to the canalicular membrane appeared after colchicine treatment. Intercellular junctions delimiting bile canaliculi were intact after colchicine treatment, however were disrupted after cytochalsin B treatment. Focal junctions resembling desmosome were formed between microvilli after colchicine treatment. In both cytochalasin B and colchicine treated groups, lumen of rough endoplasmic reticulum were dilated, Golgi apparatus became prominent, and lipid droplets were appeared in the cytoplasm. These results suggest that both intact actin filaments and microtubules are necessary to keep the structural integrity of bile canaliculi.
Actin Cytoskeleton ; Animals ; Bile Canaliculi* ; Bile* ; Colchicine* ; Cytochalasin B* ; Cytoplasm ; Cytoskeleton ; Desmosomes ; Endoplasmic Reticulum, Rough ; Golgi Apparatus ; Hepatocytes* ; Humans ; Intercellular Junctions ; Intermediate Filaments ; Keratins ; Liver* ; Membranes ; Mice* ; Microtubules ; Microvilli ; Polymerization ; Polymers ; Research Personnel

Opisthorchis viverrini infection causes inflammation and liver injury leading to periductal fibrosis. Little is known about the pathological alterations in bile canaliculi in opisthorchiasis. This study aimed to investigate bile canalicular alterations in O. viverrini-infected hamsters and to examine the chemopreventive effects of curcumin on such changes. Hamsters were infected with O. viverrini and one group of animals was fed with 1% dietary curcumin supplement. Animals were examined during the acute infection phase, days 21 and 30 post-infection (PI) and chronic infection phase (day 90 PI). Scanning electron microscopy revealed that in the infected group fed with a normal diet, bile canaliculi became slightly tortuous by 30 day PI and more tortuous at day 90 PI. Transmission electron microscopy showed a reduction in microvilli density of canaliculi starting at day 30 PI, with a marked loss of microvilli at day 90 PI. These ultrastructral changes were slightly seen at day 21 PI, which was similar to that found in infected animals fed with 1% curcumin-supplemented diet. Notably, curcumin treatment prevented the reduction of microvilli density, reduced the dilation of bile canaliculi, and decreased the tortuosity of the bile canaliculi relative to non-infected animals on a normal diet at days 30 and 90 PI. These results suggest that curcumin reduces alteration of bile canaliculi and may be a promising agent to prevent the onset of bile duct abnormalities induced by O. viverrini infection.
Animals ; Anthelmintics/*administration & dosage ; Bile Canaliculi/*pathology/ultrastructure ; Chemoprevention/methods ; Cricetinae ; Curcumin/*administration & dosage ; Disease Models, Animal ; Electrons ; Liver/pathology/ultrastructure ; Male ; Mesocricetus ; Microscopy, Electron, Scanning ; Microscopy, Electron, Transmission ; Opisthorchiasis/parasitology/*pathology/*prevention & control ; Opisthorchis/*growth & development