Selective Biliary Obstruction Research Articles

Unique reciprocal changes of hepatocellular membrane transporter expression and fluidity in rats with selective biliary obstruction

Background and aims: The localized occlusion of bile ducts by intrahepaticlithiasis and intrahepatic neoplasms dose not cause severe jaundice, although the reason is unclear. The aim of this study was to clarify the molecular change of the liver in partial cholestasis, especially with respect to transporter expression (Mrp2, Mrp3, Bsep, P-gps) and membrane fluidity. Methods and results: After 3 days of selective bile duct ligation in Sprague–Dawley rats, the obstructed lobe (OL) and non-OL were analyzed. Canalicular and sinusoidal membrane fluidity were decreased in the OL compared with the non-OL. Mrp2 protein expression was significantly lower in the OL (by 62.9±6.9%) when compared with the non-OL. Mdr1b and Mdr2 mRNA were up-regulated in the OL when compared with the non-OL. Interestingly, Mrp3 protein and mRNA were induced in both the non-OL and OL of rats without hyperbilirubinemia. Conclusions: It seems that the reduction of membrane fluidity in the OL is a local response to localized cholestasis, while the induction of Mrp3 observed in both the OL and non-OL may be a response of the whole liver to localized impairment of bile secretion, although the regulatory mechanism is yet to be established.

Potential role of bile duct collaterals in the recovery of the biliary obstruction: Experimental study in rats using microcholangiography, histology, serology and magnetic resonance imaging

Obstructive cholestasis induced in animals at the level of the lobar and common bile ducts is known to be reversible with time. This study was conducted not only to test the hypothesis that formation of bile duct collaterals is responsible for the recovery of biliary obstruction but also to assess the potential of hepatobiliary agent-enhanced magnetic resonance imaging for visualizing cholestasis. A total of 52 rats were divided into three groups with selective biliary obstruction, total biliary obstruction and sham surgery. We studied the evolution of cholestasis by correlating microcholangiographic, histological findings with the results of liver tests and hepatobiliary agent-enhanced magnetic resonance imaging. Lobar cholestasis undetected by liver tests but seen on magnetic resonance imaging as a difference between ligated and unligated lobes, occurred in 15 out of 20 rats subjected to selective biliary obstruction within 48 hr after ligation, and recovered later on as a result of the development of bile duct collaterals. Five rats failed to show local cholestasis as a result of the existence of interlobar accessory bile channels. All 18 total biliary obstruction-treated rats were cholestatic soon after ligation, as confirmed by high serum bilirubin and alkaline phosphatase levels and as documented by poor liver enhancement on magnetic resonance imaging. Cholestasis recovered within 4 wk with normalization of liver enhancement on magnetic resonance imaging as a result of the formation of bile duct collaterals (as demonstrated by microcholangiographic and histological study). Bile duct collateral formation is responsible for the recovery from obstructive cholestasis in rats. A similar mechanism might be present in conditions of bile duct obstruction without cholestasis. Hepatobiliary agent-enhanced magnetic resonance imaging is more sensitive than blood tests in detecting local cholestasis and can be used to monitor noninvasively the evolution of biliary obstruction.

Mechanisms of liver adaptation to prolonged selective biliary obstruction (SBO) in the rat

To determine changes which occur in selective biliary obstruction (SBO), we studied male adult Fischer rats after one month of either SBO or selective biliary cannulation (SBC) of the median lobe duct (MLD). In rats with SBC the ML was obstructed with a sealed PE-10 catheter placed in the MLD. One month later at laparotomy the ML was either drained or not drained for 30 min before the injection of 200 microCi [99Tc]DIDA (2,6-diethylacetoanilido-imino-diacetic acid). Bile was collected and biopsies of the obstructed ML and non-obstructed right lobe (RL) were taken at 1, 3, 10 and 30 min. Serum bile acid concentrations were higher in SBC not drained rats than in control as were hepatic bile acid concentrations. The latter, however, did not achieve statistical significance. In SBC-drained rats biliary bile acid secretion from the obstructed lobe was lower than that from the non-obstructed lobe for 30 min after the release of obstruction but was thereafter the same. Hepatic DIDA levels in both the obstructed and non-obstructed portions of liver from SBO animals were higher than in liver from controls, despite normal DIDA biliary excretion. This is in part explained by increased cytosolic binding of DIDA. In rats with SBO the MLD was simply ligated and transected. After one month uptake kinetics of [14C]taurocholate in freshly isolated hepatocytes from obstructed and non-obstructed lobes were similar suggesting that no major impairment of BA uptake occurs. We conclude that cholestasis is still present after 30 days of SBO in spite of the presence of interlobular biliary connections. The observed increased hepatic storage capacity for DIDA is probably an adaptive mechanism in mild chronic cholestasis.

Failure to induce selective cholestasis in the rat after long-term extrahepatic selective biliary obstruction.

Forty-eight hours after extra-hepatic selective biliary obstruction (SBO), there is evidence of cholestasis in the obstructed lobes (OL). However, some major ultrastructural features of cholestasis are missing. The aim of this work was to investigate the long-term effect of SBO. One month after surgery, and in comparison with sham-operated rats, bile flow, liver weight, and liver weight ratio of obstructed/nonobstructed lobes were normal. Furthermore, there was no evidence of cholestasis in OL by light and electron microscopy. Bile duct communications between obstructed and non-obstructed lobes were evidenced by Indian ink injection. In sham-operated rats, bile duct communications between ducts of the different lobes were involved in bile drainage. It appears, therefore, that the main reason for the lack of cholestasis 1 month after SBO is the drainage of bile from OL through accessory bile ducts.

Study of the long-term effects of selective biliary obstruction (SBO).

Selective biliary obstruction (SBO) is a model of partial cholestasis in the rat in which the bile duct draining the median lobe is ligated and transected; the remaining biliary tree remains intact. Other authors introduced this experimental model and studied morphological and biochemical modifications in the liver after 2 days from surgery. They suggest that an adaptation may occur. Choosing some markers of cholestasis and some other markers of various cytoplasmic organelles, we studied the long-term effects that occur in serum and in total liver homogenate of selectively obstructed rats as compared to controls. Alkaline phosphatase activity and bile acids content, which were significantly higher than controls in serum and in total liver homogenate of the median lobe after 2 and 8 days from SBO, returned to normal range values after 30 days. Cytochrome-oxidase and glucose-6-phosphatase activity in total homogenate of the SBO median lobe remains perfectly similar to the control values in time. Results, together with morphological observations, suggest that cholestasis is present immediately after operation, then decreases gradually and disappears finally. The obstructed median lobe seems to cope with cholestasis.

Effect of cycloheximide on bile flow adaptive response in selective biliary obstruction.

The adaptive increase in bile flow was studied up to 24 h after selective biliary obstruction (SBO) in the rat. Between 6 and 12 h after SBO bile flow increased significantly, such that 12 and 24 h after SBO bile flow did not differ significantly from control. The prior administration of cycloheximide (300 micrograms/100 g) resulted in a significant impairment of the increase in bile flow normally occurring between 6 and 12 h after SBO. These data indicate that the adaptive response in bile flow is dependent on de novo protein synthesis and suggest that the protein composition of the plasma membrane may determine the adaptive increase in bile flow that occurs after SBO in the rat.

A quantitative analysis of hepatic ultrastructure in rats during enhanced bile secretion.

The ultrastructural changes in hepatocytes of rats subjected to selective biliary obstruction (SBO), wherein the biliary system draining approximately two-thirds of the liver is obstructed, were evaluated by quantitative electron microscopy or stereology. The remaining unobstructed portion of the organ compensates for this loss of bile secretion by functioning in a hypersecretory mode. This animal model permits the comparison of hepatocellular fine structure associated with the conditions of nonsecretion and hypersecretion of bile with that found in normal secreting sham-operated rats. Since recent evidence suggests the presence of lobular gradients in hepatic structure and function, both centrolobular and periportal hepatocytes were examined. The low incidence of Golgi membrane profiles in high magnification electron micrographs results in a low confidence level of sampling and, thus, necessitates the application of a novel parameter for estimating the amount of Golgi complex, i.e., the Golgi-rich area. For the most part, the lobular variation in hepatic fine structure in the sham-operated animals was similar to that described by Loud ('68). However, the periportal parenchyma contained approximately twice the volume of Golgi-rich area as the centrolobular tissue. The amount of cytoplasmic lipid increased significantly in the SBO unobstructed lobes, although there were few or no changes in the other intracellular organelles or inclusions except those related to the Golgi complex. The volume of Golgi-rich area increased significantly in the centrolobular tissue of the SBO unobstructed (hypersecretory) lobes to the extent that both intralobular zones contained similar amounts of this component. These data suggest that the Golgi complex is a dynamic unit which responds to changes in hepatocellular activity and may be involved in bile secretion.

Bile Secretion in Selective Biliary Obstruction: Adaptation of taurocholate transport maximum to increased secretory load in the rat

Bile secretory function was studied in rats with selective biliary obstruction (SBO), in which ligation of the bile ducts draining the median and left hepatic lobes obstructs about two-thirds of liver mass. Forty-eight hours after SBO serum total bile acid was increased 13-fold, and total bile acid concentration in the obstructed lobes of the liver was 3 times higher than controls, but biliary secretion of water, bile acids, cholesterol, and phospholipids by the nonobstructed lobes was similar to controls when expressed per 100-g rat. To investigate the possibility that this high rate of bile acid secretion by the remaining nonobstructed liver reflected enhanced bile secretory capacity, taurocholate transport maximum (Tm) was measured in selectively obstructed and control rats at 4 and 48 hr after surgery. In the selectively obstructed rats, but not in controls, Tm increased 50% between 4 and 48 hr and, when expressed per gram of nonobstructed liver, was 81% higher than controls at 48 hr (0.69 ± 0.06 versus 0.38 ± 0.02 pmole per min per g of nonobstructed liver). The rise in Tm between 4 and 48 hr suggested that the augmentation of Tm in SBO rats at 48 hr represented an adaptive response rather than recruitment of immediately available secretory reserve. The mechanism for these alterations in Tm was investigated in rats with intact bile ducts subjected to intraduodenal taurocholate infusion for 48 hr. In these rats doubling of bile acid pool size and secretion rate was associated with a 50% increase in taurocholate Tm. These findings suggest that the maximum secretory capacity for bile acids is not fixed, but rather can adapt to a sustained increase in secretory load. This observation may provide the basis for further functional and structural clarification of the process of bile secretion.

Hepatic microsomal mixed function oxidase system response to selective biliary obstruction

Three days after selective biliary obstruction in rats, bile output from the single unobstructed lobe was equivalent to the usual output from three hepatic lobes. There was a significant decrease in the level of the hepatic microsomal mixed function oxidase system components, cytochrome P-450 and NADPH-cytochrome c reductase. The levels observed after three days were reduced to an equivalent extent in both selectively obstructed and unobstructed lobes, and corresponded to the level seen after total biliary obstruction. Hepatic bromosulphothlein T m was significantly reduced three days after selective obstruction, but did not differ from normal after six days. These data indicate that a factor generated in response to biliary obstruction acts to lower hepatic mixed function oxidase system components and increase bile flow in functioning hepatic tissue.

Selective Biliary Obstruction: A Model for the Study of Lipid Metabolism in Cholestasis

The metabolic changes associated with cholestasis are diverse and may be related either directly or indirectly to failure of bile secretion. Thus, abnormalities in lipid and lipoprotein metabolism could result from the effects of: (1) failure of lipids to be secreted into bile; (2) qualitative or quantitative changes in the lipoproteins of plasma or intestinal lymph; or (3) altered liver cell function not directly attributable to the first two factors. In this paper, a method is presented which allows these factors to be analyzed separately by permitting obstructed and unobstructed liver tissue from the same animal to be compared in regard to structure and function. In this model, selective biliary obstruction, light microscopic changes consistent with cholestasis are found in the obstructed median lobe but not in the unobstructed right lobe. By electron microscopy, osmiophilic material is present in the canaliculi on the obstructed side, while on the unobstructed side the prominent Golgi apparatus, moderately dilated canaliculi, and intact microvilli suggest increased bile secretory activity. Tissue concentrations of total and free cholesterol and of phospholipids are similar in unobstructed and obstructed hepatic lobes. In serum, levels of cholesterol, phospholipid, and alkaline phosphatase are significantly elevated; an abnormal lipoprotein, characteristic of biliary obstruction, also appears. It is anticipated that selective biliary obstruction will be useful in the experimental approach to further elucidate the abnormalities in lipid and lipoprotein metabolism in cholestasis, and the process of bile secretion in general.

Studies of Hepatic Cholesterol Synthesis in Experimental Acute Biliary Obstruction

In vitro studies suggest that hepatic cholesterol synthesis is increased in acute biliary obstruction, but the mechanism for this increase, and its relevance to increased plasma cholesterol concentration have not been resolved. We studied hepatic cholesterol synthesis and response to feedback inhibition in rats 48 hr after selective or complete biliary obstruction. After selective obstruction of the median hepatic lobe, cholesterol synthesis in slices from the obstructed lobe was similar to that after complete biliary obstruction, and was 34% greater than in unobstructed right lobe. With homogenates, using both 14C-acetate and 3H2O as precursors, obstructed lobe synthesis was 90% greater than the unobstructed. There were no significant differences in incorporation of 14C-acetate into 14C02, in tissue cholesterol concentrations, or in uptake of lipoprotein 14C-cholesterol by the two lobes. In animals with complete biliary obstruction, a 48-hr intravenous infusion of normal quantities of lymph lipoprotein cholesterol did not prevent the increase in hepatic cholesterol synthesis, whereas identical infusions significantly blunted the increase in bile fistula rats. Furthermore, although a 48-hr intravenous infusion of twice normal amounts of lipoprotein cholesterol suppressed synthesis in both lobes of selectively obstructed rats, it failed to abolish the relative difference between them. In triparanol-treated rats, complete biliary obstruction caused a greater increase in blood desmosterol than in control rats, both absolutely and relative to blood cholesterol. These studies provide evidence that in acute biliary obstruction, increased precursor incorporation in vitro is associated with accelerated net synthesis of cholesterol in vivo, and reflects an abnormality in the control of hepatic cholesterol synthesis.