A Method for Collection of Bile in the Conscious Sheep

A method to collect bile directly from the hepatic duct is described for use in the sheep. The technique is a combination of the intestinal re-entrant cannulae and a catheter from the duodenal lumen to the hepatic duct. The cystic duct is ligated near its junction with the common bile duct. The catheter is fixed in the proximal visible end of the hepatic duct. One plastic cannula is fixed to the duodenum opposite to the opening of the common bile duct and the other is fixed in the same way about 15 cm posterior to the first one. The two plastic cannulae fixed together with a plastic tube serve as an extra-abdominal anastomosis. During the collection periods the bile duct catheter is passed through an opening in the wall of the connection tube into a collection bag that is fixed to the plastic cannulae. Between the collection periods the catheter ends in the lumen of the anastomosis.     

The extracorporeal bile duct: a new model for determination of bile flow and bile composition in the intact rat

A new model is described which allows measurement of bile flow and sampling of bile in the intact rat with a physiologically functioning sphincter of Oddi. A number of metabolic parameters have been followed to show that animals with such an "extracorporeal bile duct" (EBD) behave as intact controls. Especially, there was no difference in the increase in body weight or hepatic fatty acid and cholesterol synthesis between EBD animals and intact controls. The amount of bile salts circulating through the biliary tract amounted to 30.5+/-1.5mumol . 100 g body wt-1 . hr-1, when diurnal variations were averaged. Animals adapted to a restricted feeding regimen showed a significant increase of bile flow and of biliary bile salt and cholesterol excretion during feeding (10AM-3 PM); these parameters reached their maximum 3 hours after onset of food intake.     

A self-powered constant infusion device for use in unrestrained rats

We developed a device that delivers fluid through a catheter at a constant rate and can be used in conscious animals to solve a variety of problems. For example, this device can be used for delivering drugs and maintaining intravascular catheter patency. The device provides infusions at low flows (1.0-1.5 ml/day), so that experimental agents may be administered with minimal volume loading of the rat. Arterial and venous catheter patency is maintained by infusion of heparinized saline through indwelling catheters attached to the device. The catheters exit from the rat in the intrascapular area and are routed through a protective spring to the device, which is suspended above the cage. The catheters may be attached to pressure transducers, blood may be sampled, and injections or infusions may be made without disturbing the rat. Because the device is self-contained, it can be suspended by a fluid-free swivel that rotates through 360 degrees, providing minimal restraint. The device has been used successfully to measure arterial and central venous blood pressures in two studies using rats.     

A case of tool use in captive olive baboons (Papio anubis)

Some captive olive baboons can use their tails for picking up objects from the floor. When provided with a short handle and crosspiece, these animals learn to manipulate this tool with their hands, and one of them became proficient in inserting the crosspiece into a small wire loop, attached to a piece of bread and in lifting it up to his cage. Another baboon in the same colony who never used his tail to retrieve objects, did not attempt to use this tool. The psychological aspects of tail-use and tool-use are discussed.     

Pathways of absorption of retinal and retinoic acid in the rat

The chemical and anatomical pathways of absorption of dietary retinal, retinoic acid, and retinol were examined in rats containing lymph, bile, and duodenal cannulae. The experiments were designed to maintain physiological conditions to the greatest possible extent. In each rat an uninterrupted flow of bile into the duodenum was maintained by connecting the duodenal cannula to the bile duct of a second rat. Labeled vitamin A compounds were introduced into the duodenum in very small amounts (7-14 micrograms) in the form of a bile-lipid mixture resembling normal intestinal contents. Under these conditions, most (70-80%) of the radioactivity recovered after the feeding of labeled retinol or retinal was found in the lymph, predominantly in saturated retinyl esters. In contrast, 92-95% of the radioactivity recovered after the feeding of labeled retinoic acid was found in the bile, and was contained in a mixture of polar metabolites, most of them more polar than free retinoic acid. Two-thirds of the small amount of radioactivity found in lymph after retinoic acid-(14)C feeding was in the form of free retinoic acid. The results indicate that under normal conditions the major pathway of retinal absorption involves its reduction to retinol, which is then esterified and transported via the lymphatics in a manner similar to that of dietary retinol. A small proportion of retinal is apparently normally oxidized, and is then transported via the portal vein and excreted in the bile in a manner similar to that of dietary retinoic acid. The relative importance, in quantitative terms, of these two pathways of retinal metabolism can vary, depending on the status of the animal.     

Chronic bile duct cannulation in laboratory rats.

To our knowledge this is the first report of rat bile duct cannulations in which the distal cannula is hemisected but extends to the sphincter of Oddi. It is minimally invasive and requires only about 45 minutes preparation time. In contrast to studies described in the literature, enterohepatic recirculation remains intact but bile can always be separated from pancreatic secretions at investigator discretion in the model. In addition, biliary flow and pressure can be measured without compromise. Acute biliary secretory pressure, under anesthesia, was 17 cm water. Bile flow, averaging 9.6 microliters/min/100 g was measured in unanesthetized rats surviving for 2 weeks (60% of animals monitored). Gross necropsy findings indicated that animals dying in less than 7 days usually suffered bile peritonitis subsequent to catheter rupture of the bile duct or loss from the ligature restraint. Deaths after 2 weeks were usually related to cholestasis due to blockage of the catheter with mineral debris and/or duct tissue. A detailed literature review of bile duct cannulation in rats has been made.     

Technique for long-term infusion into the inferior mesenteric artery of unrestrained rats

A technique for long-term infusion into the inferior mesenteric artery was developed which allows simple and reliable regional infusion into the colorectal segment of unrestrained rats. The cannulation system consists of an injection port 'In Stoppers' as a flow swivel, connected to an injection needle, which is inserted into a polyethylene tube protected by a steel spiral. During infusion the animals are free to move in the cage with access to food and water ad libitum. The method is suitable for regional chemotherapy as well as for studies of colorectal tumours in rats. In this study 73% of the cannulae remained functional for continuous infusion over a 15 day period.     

Physical provocation potentiates aggression in male rats receiving anabolic androgenic steroids

Anabolic androgenic steroids (AAS) have been linked to indiscriminant and unprovoked aggression and violence. We employed a brief tail pinch to examine the effects of different AAS on intermale aggression in gonadally intact male rats in response to a mild physical provocation. Animals received 5 mg/kg testosterone propionate (TP), nandrolone (ND), or stanozolol (ST) 5 days/week. Controls received vehicle injections. After 12 weeks, rats were tested for aggression while treatments continued. Animals were paired with either gonadally intact or castrated opponents and were tested in the subject rat's home cage, the opponents's home cage, and a neutral cage. Aggression was tested during tail pinch of the subject rat and during tail pinch of the opponent rat. In TP-treated males, tail pinch significantly enhanced aggression in all social and environmental conditions compared to intact controls. TP treatment also significantly enhanced aggression when the opponents were tail pinched. Tail pinch did not increase aggression in ND-treated males, and aggression was significantly lower than controls in ST-treated males. As expected, cell nuclear androgen receptor binding was significantly elevated by the high dose of TP. Our results show that while AAS alone does not induce the indiscriminate and unprovoked aggression characteristic of 'roid rage, TP heightens the animals sensitivity to     

A novel method for establishment and characterization of extrahepatic bile duct epithelial cells from mice

Culture of extrahepatic bile duct epithelial cells is a useful model to investigate physiology of extrahepatic bile duct epithelia and hepatobiliary disease mechanisms. The aim of this work was to establish and characterize a primary murine extrahepatic bile duct epithelial cell culture. Epithelial cells were isolated from extrahepatic bile ducts of BALB/c mice that were intraperitoneally injected with newborn bovine serum to induce the proliferation of extrahepatic bile ducts’ epithelial cells and cultured on rat tail type I collagen-coated plastic culture flask containing DMEM/HamF12 with 10% FBS and 10 ng/ml epidermal growth factor at 37°C in an incubator with 5% humidified CO₂. The cells showed typical morphologic characteristics of epithelial phenotypes with cobblestone appearance in monolayer within 5–6 d after culture; they were positive against anticytokeratin-19 immunostaining. Transmission electron microscopy showed typical bile duct epithelia with microvilli on the cytomembrance, Golgi complex, massive mitochondria, and rough endoplasmic reticulum in the cytoplasmic. The growth curve of the epithelial cells was determined by a MTT assay which showed a normal sigmoidal growth curve. This culture technique might be a reliable method for isolation, purification, and primary culture of extrahepatic bile duct epithelial cells that can serve as a model for in vitro studies on the pathophysiology of hepatobiliary diseases as well as pharmacological and toxicological targets relevant to hepatobiliary diseases.     

Identification of lipoprotein X-like particles in rat plasma following Intralipid infusion.

Fasting rats were infused with 10% Intralipid for 24 h (0.33 mL/h per 100 g body weight) and the plasma lipoproteins isolated and compared with those of fed animals and animals with bile duct ligatures as controls. There was a 6- to 10-fold increase in the free cholesterol and phospholipid content of total plasma in animals infused with Intralipid or with ligated bile ducts. The changes were largely restricted to the low density lipoproteins (d=1.019--1.063 g/mL) where free cholesterol and phospholipid increased 30- to 60-fold compared with fed control animals. Hydroxylapatite chromatography of the low density lipoprotein fractions of both Intralipid-infused and bile duct ligated animals yielded a subfraction which was rich in free cholesterol (27%), phosphatidylcholine (66%), and protein (6%); the latter was composed primarily of albumin and apo C proteins. The electrophoretic mobility and polyanionic precipitation properties of the abnormal lipoprotein were indistinguishable from those of lipoprotein X isolated from the animals with bile duct ligatures. The albumin in the abnormal lipoprotein from both groups of experimental animals was detected immunochemically only after delipidation of the lipoprotein. Twice as much of the lipoprotein X accumulated in Intralipid-infused than in the bile duct ligated animals. On rechromatography of the residual low density lipoprotein other subfractions could be isolated which possessed lipid and protein proportions intermediate between those of the lipoprotein X and of normal rat plasma low density lipoprotein. The activity of lecithin cholesterol acyl transferase was increased twofold in the Intralipid-infused animals when compared with control animals, but it decreased by 50% in the animals with bile duct ligatures. It is concluded that the unusual lipoprotein X accumulates in the plasma of Intralipid-infused animals owing to incomplete clearance of the exogenous phospholipid, which mobilized tissue cholesterol and in the form of vesicular particles serves as a lipid phase for apo C proteins. A comparable mechanism is suggested for the formation of lipoprotein X in the animals with bile duct ligature.     

Cholecystokinin and gastrointestinal cancer

The gut hormone cholecystokinin exerts various actions on the gastrointestinal tract, including the regulation of growth. The hormone has been reported to induce hypertrophy and hyperplasia of the pancreas and to enhance chemically-induced pancreatic carcinogenesis in animals. Stimulation of endogenous cholecystokinin secretion through the induction of deficiency of intraintestinal proteases and bile salts by trypsin-inhibiting nutrients, bile salt-binding drugs or surgical intervention is also capable of stimulating growth and tumour development in the rat. In man, factors suggested to increase the risk of pancreatic cancer, such as a high-fat and high-protein diet or gastrectomy, are known to stimulate plasma cholecystokinin secretion. Receptors for cholecystokinin have been demonstrated on human pancreatic adenocarcinomas, and cholecystokinin has been demonstrated to enhance the growth of xenografted pancreatic cancer and to inhibit growth of gastric and bile duct cancer. The recently developed cholecystokinin-receptor antagonists inhibit not only pancreatic growth but also pancreatic carcinogenesis in animals. These new drugs may be valuable new tools for inhibiting pancreatic cancer growth in humans.     

胆汁酸代谢与肠道微生物

肠道微生物与胆汁酸代谢密切相关,肠道微生物参与了胆汁酸在肠道中的修饰过程;肠道微生物通过法尼醇受体影响胆汁酸的合成;肠道微生物通过调节胆汁酸的代谢影响机体健康,反过来胆汁酸也可以通过调节肠道微生物菌群的组成影响机体健康.肠道微生物与胆汁酸代谢间的稳态影响着机体健康,现对肠道微生物与胆汁酸代谢及其相互影响做一综述.     

Adenovirus-mediated human pancreatic lipase gene transfer to rat bile: gene therapy of fat malabsorption

This study explored the potential of using the gene therapy approach, based on adenovirus-mediated expression of pancreatic lipase in the hepatobiliary tract, to increase lipid digestion in the intestinal lumen and promote lipid absorption through the gastrointestinal tract. Recombinant adenovirus containing the human pancreatic lipase cDNA (AdPL) was shown to transduce and mediate pancreatic lipase biosynthesis in rat IEC-6 epithelial cells in vitro. Retrograde infusion of recombinant adenovirus (3 x 10(8) plaque-forming units) containing the bacterial LacZ gene (AdLacZ) into the bile duct of rats resulted in positive X-gal reaction products in the periportal liver cells 7 days after AdLacZ infusion. A high level of human pancreatic lipase was detected in bile after retrograde bile duct infusion of rats with AdPL but not in the bile of animals infused with AdLacZ. Triglyceride hydrolytic activity in the bile of AdPL-infused rats was equivalent to that present in pancreatic juice. In contrast, serum obtained from these animals did not contain any detectable pancreatic lipase activity. These results suggest that ectopic expression of pancreatic enzymes in the hepatobiliary tract may be an alternative therapeutic strategy for treating fat malabsorption due to pancreatic insufficiency.     

A surgical model for studying biliary bile acid and cholesterol metabolism in swine.

Techniques were developed in young growing pigs to simultaneously collect and reinfuse bile. Silastic cannulae were designed and surgically implanted in the common bile duct and the duodenum. Direct sampling of the hepatic bile was achieved by bypassing the gallbladder. The techniques allowed for steady-state studies of hepatic function to be conducted in conscious swine in two different studies. Pigs, thus surgically modified, can serve as an appropriate model for physiologic, pharmacologic, and nutritional research that involves bile sampling.     

Betaine homocysteine methyltransferase: gene cloning and expression analysis in rat liver cirrhosis

It has been known for over half a century that homocysteine levels are elevated in liver cirrhosis, but the basis for it is not fully understood. Using differential display, we identified betaine homocysteine methyltransferase (BHMT) as a gene down-regulated in rat liver cirrhosis and most likely involved in this dysregulation. A partial BHMT clone was isolated by screening of a cDNA library with the differential display fragment. The full-length gene was generated by primer extension of cDNA. Expression levels of BHMT in cirrhotic livers of bile duct ligated rats were compared to controls by Northern and Western blotting as well as by enzyme activity measurements. BHMT mRNA levels were reduced to 29+/-23% in established liver cirrhosis induced by bile duct ligation (BDL) as compared to controls. Enzyme assays in crude liver homogenates showed a similar reduction in BHMT activity in bile duct ligated rat livers. By Western blotting, BHMT could be detected in crude liver homogenates of control animals, but was reduced to below the limit of detection in cirrhotic livers. In conclusion, these findings establish a reduced BHMT enzyme activity in cirrhotic rat livers, which may explain the elevated plasma homocysteine levels in cirrhosis.     

Fasciola hepatica: Azetidine inhibition of bile duct hyperplasia in the infected rat

In controlled experiments the luminal bile duct perimeters were compared between rats infused with l-azetidine-2-carboxylic acid (AZC) (120 μmole/rat/day) and rats infused with saline after both groups had been implanted with adult Fasciola. The average bile duct lumen in the group receiving AZC did not enlarge relative to controls, and was about one-half the average perimeter of ducts from rats infused with saline that had worm implants. These results indicate that azetidine can inhibit bile duct hyperplasia induced by Fasciola which inhabit the duct. The results also support the hypothesis that the hyperplasia of fascioliasis is mediated through the release of free proline from the worms where it is present in high concentrations.     

The metabolism of sodium cortisone 27-[35S]-sulphate in the rat

1. The metabolism of sodium cortisone 21-[(35)S]sulphate was investigated in rats. 2. Quantitative and qualitative experiments showed that substantial amounts of (35)SO(4) (2-) appeared in the urine of free-ranging rats receiving the ester. 3. Whole-body radioautograms indicated considerable biliary elimination of (35)S and also pointed to the liver as the site of metabolism. 4. When female rats with bile-duct cannulae received sodium cortisone 21-[(35)S]sulphate approx. 70% of the dose appeared in the bile as a doubly conjugated steroid (metabolite I). This metabolite was identified as 3alpha-(beta-d-glucopyranuronosido)- 17alpha-hydroxy-21-[(35)S]sulpho-oxy-5alpha-pregnane-11,20-dione. 5. When metabolite I was administered to a rat with a bile-duct cannula 90% of the dose appeared in the bile unchanged. After the administration (intraperitoneally or orally) of metabolite I to free-ranging rats considerable amounts of (35)SO(4) (2-) appeared in the urine. 6. The route by which (35)SO(4) (2-) might be produced from cortisone [(35)S]sulphate in free-ranging animals is discussed.     

Migration of gall stones.

The factors influencing the migration of gall stones are ill understood. Altogether 331 patients undergoing cholecystectomy were studied prospectively. The diameters of the cystic and common bile ducts and of stones in the gall bladder and bile ducts were measured. Increasing pressure was applied to the freshly excised gall bladder in an attempt to evacuate stones through the cystic duct. Stones passed in 33 (60.0%) of patients with choledocholithiasis, 45 (67.2%) of patients with pancreatitis, and 7 (3.2%) of patients without either pancreatitis or choledocholithiasis. Stones migrated in 6 (3.0%) who had a normal cystic duct diameter (less than or equal to 4 mm) and in 46 (32.5%) with a duct over 4 mm diameter. Common bile duct stones were often larger than the diameter of the cystic duct and when reintroduced into the gall bladder would not migrate. The passage of debris (less than or equal to 1 mm) through the cystic duct bore no relation to the presence or absence of choledocholithiasis or a dilated cystic duct. Small stones (1-4 mm diameter) must migrate to initiate and facilitate further migration; some must increase in size in the common bile duct. Increased biliary pressure consequently dilates the duct system retrogradely, allowing larger stones to follow. Patients at risk of stone migration and thereby pancreatitis and jaundice have large ducts that can be detected by ultrasound assessment.     

The brush cells of the common bile duct of the rat

Summary Two different fixative procedures (immersion and perfusion) and four different fixative solutions were used in order to obtain the best preservation of the brush cells of the common bile duct of the rat. The results indicate that only perfusion fixation through the common bile duct is suitable, independent of the fixative solutions and their osmolarity.Numerous brush cells were seen in the proximal and distal regions of the common bile duct. In these locations, they could be implicated in a registration and/or regulation of intraluminal pressure variations.     

Evidence for fluid-phase pinocytosis of extrahepatic bile duct cells isolated from normal rats in culture.

In order to elucidate the physiological function of extrahepatic bile duct cells, we isolated epithelial cells from the rat extrahepatic bile duct by digesting resected segments of the extrahepatic bile duct with 0.15% trypsin in ice-cold Ca(2+)-free Hanks' balanced salt solution supplemented with 0.25 mM EDTA overnight. As a result, the epithelial cells were collected as aggregates and attached to culture dishes coated with type I collagen. Approximately 95% of the cells cultured for 24 hrs were found to be positive for gamma-glutamyl transpeptidase and cytokeratin-19, but negative for vimentin. These characteristics were identical to the features of rat extrahepatic biliary epithelial cells in situ. Ultrastructurally, the cells were long and columnar in configuration on the 2nd day in culture, and possessed numerous microvilli at the apical surface and well-developed junctional complexes at the lateral surface. These findings also indicate that the cells maintain an epithelial nature and are morphologically polarized. When the cells were exposed to a low dose of horseradish peroxidase (HRP) on the 2nd day in culture, which was followed by fixation and treatment with 3-3'-diaminobenzidine, HRP was found preferentially in the cytoplasmic vesicles near the apical surface. HRP was then observed in the intercellular spaces; however, the electron-dense tracer, ruthenium red, did not permeate into the intercellular spaces, and HRP was found in neither cytoplasms nor intercellular spaces when the cells were incubated in HRP-containing medium at 4 degrees C for 30 min. These results suggest that the extrahepatic bile duct epithelial cells are involved in the reabsorption of bile constituents.