INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

SELECTIVE DELIVERY OF THERAPEUTIC agents to targeted hepatobiliary tissues and cell types would make possible safer, more effective treatments by permitting the utilization of optimal therapeutic dosages combined with a reduction in systemic toxicity. Potential strategies for improving the tissue specificity of hepatobiliary treatments include the use of tissue-specific ligands and the focal administration of therapeutic agents. Although a number of ligands tropic for the hepatocyte basolateral membrane have been identified, these agents are not completely tissue specific and can result in widespread systemic distribution following intravenous administration (17). To date there have been no methods reported for selectively targeting the basolateral cell membrane of intra- or extrahepatic cholangiocytes by intravenous administration. Focal delivery of therapeutic agents to the liver has been primarily directed for uptake by the hepatocyte basolateral membrane and has included direct intraparenchymal injection, portal venous infusion, hepatic arterial infusion (28, 32), and surgical systems for temporarily isolating the liver from the vasculature (3, 16, 24). These strategies have shown promise, but problems encountered have included insufficient first-pass uptake, bleeding complications, an inability to maintain the infused drug in contact with the targeted tissue for therapeutically effective periods of time, systemic leakage, or the presence of extrahepatic metastases.

Only minimal attention has been given to using intrabiliary infusion to deliver drugs selectively via the apical surface membrane of cholangiocytes or hepatocytes (31). Recently, retrograde biliary infusion of gene transfer vectors to the liver and bile ducts has been examined using adenoviral vectors in mice (14, 19, 35, 36), rats (37), primates (13, 29), and ex vivo using a cadaveric human liver (33). Retroviral (5) and liposome-mediated gene transfer (12) have also been reported following retrograde biliary infusion in rats. However, these prior studies have primarily focused on the process of gene transfer and have not addressed the physiological, anatomic, or acute histological consequences of vector administration by this route. These issues will need to be better understood to help develop safe and effective methods for focal intrabiliary delivery of drugs and vectors. This will include determining 1) the most appropriate method for intrabiliary infusion (i.e., infusion parameters); 2) the minimal dwell or contact time that cells should be exposed to vectors or drugs; 3) the optimal intraluminal microenvironments for effective binding and internalization of particular agents; 4) the acute histological and pathophysiological effects of intrabiliary delivery; and 5) whether systemic distribution of infusate occurs (and if so, by what mechanism). The existence of gene-specific murine models of human disease makes it particularly attractive to utilize the mouse for the evaluation of the physiological and histological consequences of retrograde biliary infusion.

As an initial step in the creation of a murine system for the comprehensive evaluation of intrabiliary drug and vector delivery, we report the development and application of a novel system for continuous cholangiomanometric monitoring during retrograde biliary infusion in mice. The small size of these animals has previously precluded the development of techniques for cholangiomanometry. With this system, the manometric consequences of retrograde biliary administration were evaluated at different infusion volumes, rates, viscosities, and temperatures. Intrabiliary pressure changed as a function of infusion rate, volume, and viscosity. Higher rates of constant infusion resulted in higher peak intraluminal biliary pressures. The pattern of pressure changes observed was consistent with ductular and/or canalicular filling followed by leakage at a threshold pressure. Following high-pressure retrograde biliary infusion of 5 mM lanthanum chloride, a molecule that cannot transit across intact tight junctions, transmission electron microscopy revealed the presence of electron-dense material in bile canaliculi, interhepatocyte cellular junctions, and the perisinusoidal space of Disse. Retrograde biliary infusion of radiopaque contrast material resulted in the rapid systemic appearance of dye. This leakage from the intrabiliary space was prevented by temporary obstruction of hepatic venous drainage, indicating that systemic redistribution following high-pressure retrograde biliary infusion primarily occurred through sinusoidal and hepatic venous flow, as opposed to lymphatic flow.

Although retrograde biliary infusion resulted in a similar pattern of pressure changes in both normostatic and cholestatic animals, there were significantly smaller initial peak intrabiliary pressures after 4 days of chronic extrahepatic bile duct obstruction, consistent with the appearance of tight junction disruption in cholestasis. [¹⁴C]sucrose was detected in the systemic circulation 5 min after high-pressure retrograde biliary infusion. Since [¹⁴C]sucrose cannot pass across an intact tight junction, its appearance in the systemic circulation following retrograde biliary infusion provides physiological evidence of tight junction disruption and biliary leakage.

These findings indicate that, at elevated intrabiliary volumes/pressures, there is a pressure-dependent physical opening of tight junctions in both normostatic and cholestatic animals, resulting in the leakage of infusate from the intrabiliary space into the subepithelial tissue compartment. This suggests that it may be possible to selectively deliver large macromolecules (>18-20 Å in diameter, the approximate size exclusion for tight junction passage) to specific histological tissue compartments through the control of intraluminal volume and pressure.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animal experiments. All experiments involving animals were approved by the National Heart, Lung, and Blood Institute's Animal Care and Use Committee. CD-1 male mice (20-40 g; Charles River) were used. Anesthesia was induced and maintained with intraperitoneal 2.5% tribromoethanol (Aldrich Chemical, Milwaukee, WI).

Cholangiomanometry. Figure 1 illustrates the experimental procedures utilized for evaluating the effect of retrograde biliary infusion on intrabiliary pressure. Following a midline laparotomy, the gallbladder was manually drained through the cystic duct. A cholecystotomy catheter (Silastic tubing [0.012-in. ID/0.025-in. OD]) was secured within the gallbladder lumen with the catheter tip advanced just proximal to the junction with the cystic duct. Absorbant cellulose (X0-Med, Jacksonville, FL) was used to prevent bile leakage into the peritoneum.

View larger version (54K): [in this window] [in a new window]   Fig. 1.   Schematic of experimental system for determining effect of retrograde biliary infusion on intrabiliary pressure. Infusions were administered through a catheter secured within gallbladder lumen. A second catheter was inserted through lumen of duodenum and advanced retrograde through sphincter of Oddi into common bile duct until tip was rostral to superior pancreatic duct. This catheter was secured in place, thereby creating an obstruction in common bile duct to anterograde movement of bile or infusate. Infused material progressively traveled down cystic duct and then common bile duct until it reached level of pressure catheter. At this point, infused material began to move in a retrograde direction. Since biliary tree is a closed ductular system, pressure recorded from common bile duct pressure transducer accurately reflects intraluminal pressure throughout entire tree. Manometric readings were continuously taken every 0.5-2.0 s through common bile duct catheter using a low-pressure transducer connected to a computer.

Electron microscopy. Animals were infused retrograde with either 5 mM lanthanum chloride (Sigma) or 0.9% NaCl vehicle. Three infusions of 240-µl volume were administered per animal at a rate of 2 or 8 µl/s, with a 10-s pause between infusions. Freshly removed tissue was fixed overnight in 2% glutaraldehyde in 0.2 M cacodylate buffer. Following standard processing and embedding, 0.5-µm-thick sections were stained with uranyl acetate and lead citrate. Sections were then examined using a Philips 201 Electron Microscope.

Experimental system for evaluating the effect of infusion viscosity and temperature on intrabiliary pressure. Solutions of different viscosity were prepared by diluting radiopaque contrast dye with 0.9% NaCl in the following dye-to-saline ratios: undiluted dye, 9:1, 3:1, 1:1, 1:3, and saline without dye. The biliary infusion system was modified to deliver ~37°C infusions. The infusion catheter was preplaced within the lumen of -in. silicone tubing that was continuously perfused with 39°C water. Solutions were preheated to 39°C and drawn up immediately before use. We found that the time required for securing the catheter within the gallbladder lumen resulted in an ~2°C decline in temperature as measured at the catheter tip.

Viscosity measurements. Fluid viscosity was determined using an Ostwald capillary viscometer at 22 and 37°C.

Retrograde biliary infusion of radiopaque dye. A Silastic catheter was placed in the gallbladder as described in Cholangiomanometry. Straight (1 mm × 3 mm) or curved (1 mm × 5 mm) Kleinert-Kutz microvascular clips (Pilling-Weck, Research Triangle, NC) were then placed rostral to the junction of the superior pancreatic duct with the common bile duct. Infusions were administered as noted, and the microvascular clip occlusion caused the infusion to move retrograde. At the end of the administration period (infusion plus dwell time), the clip was removed and a preplaced 6-0 silk tie was used to close the gallbladder opening as the cholecystotomy catheter was withdrawn.

Inferior vena cava occlusion. To evaluate the impact of hepatic venous drainage on the distribution of radiopaque dye following retrograde biliary infusion, the suprahepatic inferior vena cava was temporarily occluded for 5 or 10 min. The liver was gently retracted caudally, and the falciform ligament was identified and divided down to the ventral surface of the vena cava. A curved microvascular clip was used to occlude the vena cava at a level just cephalad to the liver and caudal to the postcaval foramen of the diaphragm.

Digital fluoroscopy. Digital fluoroscopic studies were performed with the radiopaque contrast dye Renograffin-76 (66% diatrizoate meglumine and 10% diatrizoate sodium; Solvay Animal Health, Mendota Heights, MN) and an OEC Series 9400 X-ray imaging system (OEC Diasonics, Salt Lake City, UT).

Mechanical model of biliary infusion system. An "elastic," "leaky" biliary system was simulated using a 6-in. length of -in. diameter silicone tubing that had a 1-in. longitudinal slit cut into it with a scalpel. The proximal end of the silicone tube was joined to a "T" line. One side of the T was connected to a Statham pressure transducer and the other end to a Harvard infusion pump. The distal end of the silicone had a two-way stopcock valve attached, which was used to either seal the end of the tube or to vent it when necessary.

Chronic cholestasis experiments. Following the induction of anesthesia, a midline laparotomy was made and the common bile duct was visualized. A 6-0 silk tie was used to occlude the common bile duct rostral to the junction with the pancreatic ducts. The abdominal incision was closed in two layers with 6-0 silk. Four days later, the animals were reanesthetized and underwent a repeat laparotomy. A microvascular clip was placed above the level of the common bile duct occlusion, and a catheter was secured within the common bile duct. The microvascular clip was then removed, and baseline intrabiliary pressure was determined. A gallbladder catheter was then secured in position, and animals received retrograde biliary infusion as described in Cholangiomanometry.

Measurement of tight junction permeability. Following a midline laparotomy, both ureters were identified and occluded with microvascular clips. A catheter was placed within the gallbladder lumen, and the common bile duct was occluded using a microvascular clip placed above the junction of the common bile duct with the superior pancreatic duct. Retrograde biliary infusions (22°C) of 0.9% NaCl or 2 µCi of [¹⁴C]sucrose (Amersham Pharmacia Biotech, Piscataway, NJ) diluted in 0.9% NaCl were then administered using a range of volumes and rates of infusion. Five minutes after the completion of the infusion, a midsternal incision was rapidly made and blood was obtained by intracardiac puncture using a 27-gauge needle and a syringe. Blood was immediately added to a 1.5-ml microcentrifuge tube containing 10 units of sodium heparin (Elkins-Sinn, Cherry Hill, NJ) and centrifuged at 4,000 rpm for 5 min. Two hundred microliters of plasma were removed and added to a glass vial containing 15 ml of scintillation solution (National Diagnostics, Atlanta, GA). Radioactive counts (cpm) were determined in a scintillation counter (Beckman).

Statistical treatment of results. Data are presented as means ± SE. Actual volumes administered are shown. Mean pressure curves were generated by taking the mean pressure at a specific infusion time point or volume for those animals administered an infusion at that respective rate and volume. Pressure changes were determined relative to the preinfusion pressure for each animal. One-way ANOVA was utilized for comparisons between groups. Repeated-measures ANOVA was performed when a group of animals received multiple infusions at the same rate and volume. Post hoc group and treatment comparisons were performed using a Bonferroni t-test or a Student-Newman-Keuls test. Linear regression was carried out using the method of least squares. The relationship between viscosity and pressure was evaluated by determining the Pearson product-moment correlation coefficient. Comparison between regression lines was performed using an overall test for coincidence. P values <0.05 were considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Retrograde biliary infusion leads to intraluminal pressure changes that are dependent on infusion rate and volume. Baseline measurements of intrabiliary pressure were continuously recorded for 25 min during common bile duct occlusion with no retrograde biliary infusion. Since bile was still being formed, the intrabiliary pressure gradually rose from a baseline of 0.8 ± 0.2 mmHg (n = 5), reaching a mean pressure of 10.0 ±1.4 mmHg by 10 min (Fig. 2). This pressure remained fairly constant for at least 25 min, when recording was discontinued. The small-amplitude episodic fluctuations in intrabiliary pressure may be indicative of intrabiliary contraction. In several animals, baseline pressures were recorded for an additional 35-50 min. These pressures did not markedly differ from those recorded at 25 min (data not shown).

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Effect of common bile duct occlusion on intrabiliary pressure. With use of system shown in Fig. 1, baseline intrabiliary pressures were recorded during common duct occlusion. Intrabiliary pressures were monitored for up to 75 min after occlusion in some animals and were not different from pressures recorded at 25 min. A: typical manometric tracing from a single animal. Inset is an enlarged tracing of an ~9-min period of continuous intrabiliary recording and reveals the presence of pulsatile waves of contraction. These pressure waves are consistent with contractile elements participating in movement of bile in an anterograde direction toward sphincter of Oddi. B: mean control tracing from a group of 5 mice during common bile duct occlusion and no retrograde biliary infusion.

View larger version (34K): [in this window] [in a new window]   Fig. 3.   Temporal pattern of pressure changes during retrograde biliary infusion. Following occlusion of common bile duct, intrabiliary pressures were recorded while infusing 0.9% NaCl through gallbladder catheter. This resulted in retrograde biliary delivery of infusate along with a distinct pattern of changes in intrabiliary pressure. Pressures were recorded every 0.5-2.0 s using method shown in Fig. 1. A: typical pressure tracing from a single mouse (240 µl administered at 2 µl/s). Five phases are evident as a result of retrograde biliary infusion: phase 1 (segment PQ), progressive rise in intraluminal pressure; phase 2 (Q), peak pressure; phase 3 (QR), decline in pressure; phase 4 (RS), sustained, plateau pressure; and, immediately after the infusion is discontinued, phase 5 (ST), rapid decline in pressure. B: changes in mean intrabiliary pressure as a function of time at different infusion rates and volumes. Mean pressure curves for each infusion volume and rate were generated by taking mean pressure at each time point. Intrabiliary pressure changes during retrograde biliary infusion were dependent on both volume and rate of infusion used. C: changes in intrabiliary pressure as a function of infusion volume. Data shown in B are presented as mean change in biliary pressure (mmHg) from preinfusion pressure as a function of volume infused (µl).

View larger version (40K): [in this window] [in a new window]   Fig. 4.   Effect of infusion rate and volume on peak intrabiliary pressure and recovery pressure. A: histogram showing effect of infusion rate and volume on both maximal (peak) intrabiliary pressure during retrograde biliary infusion and pressure when infusion was completed (end pressure). All animals underwent common bile duct occlusion. Control animals underwent common bile duct occlusion without a retrograde biliary infusion. * P < 0.05 vs. control. NS, nonsignificant difference. B: effect of infusion rate and volume on recovery pressure (pressure in period immediately following completion of a retrograde biliary infusion). Following completion of an infusion, intrabiliary pressure rapidly declined toward preinfusion level. Larger-volume, more rapid rate infusions tended to lead to lower recovery pressures.

Repeat infusions result in lower peak intrabiliary pressures. In some studies, a single animal underwent a sequence of up to four repeat infusions at the same infusion volume and rate. Pressure was continuously monitored and each infusion was separated by ~3 min from the next infusion. Figure 5A shows a typical time course of three infusions. Figure 5B shows that repeat infusions resulted in significantly smaller rises in pressure than were produced by the initial infusion for a particular infusion rate and volume. This finding was statistically significant except at the largest volume and fastest rate evaluated (240 µl infused at 16 µl/s). At any given infusion volume and rate, the pressure changes produced by the second, third, and fourth infusions were not significantly different from each other, even if the second infusion had resulted in a pressure change significantly smaller than that achieved by the first infusion. At faster infusion rates, repeat infusions tended to lead to postinfusion pressures below the initial preinfusion pressure (not shown). Figure 5C shows that the difference between the first and subsequent infusions was apparent early in the repeat infusion. An initial infusion of 80 µl of 0.9% NaCl at an infusion rate of 0.66 µl/s (n = 4) produced a rise in pressure of 16.6 ± 1.3 mmHg after only 16 µl had been infused. In contrast, infusion of 16 µl during a second infusion at the identical infusion rate resulted in a significantly smaller pressure rise (9.6 ± 2.1 mmHg; P < 0.05).

View larger version (38K): [in this window] [in a new window]   Fig. 5.   Effect of repeat infusion on intrabiliary pressure. Animals received up to 4 sequential retrograde biliary infusions. Each infusion was separated by ~3 min from next infusion. * P < 0.05 vs. the initial infusion. ** P < 0.05 for comparison among 2nd, 3rd, and 4th infusions. A: typical experimental tracing from an animal that was administered 3 sequential infusions of 80 µl over 2 min (0.66 µl/s). Arrows denote onset of each infusion. B: effect of repeat infusion on maximal pressure change for different volumes and rates of infusion. C: effect of repeat infusion on change in intrabiliary pressure as a function of volume infused. Data are shown for first and second infusions in animals that were administered 80 µl at 0.66 µl/s; 3rd infusion curve is not presented because it was virtually identical to 2nd infusion curve.

Intraluminal pressure changes are determined by infusion viscosity. To evaluate the impact of infusate viscosity on intraluminal pressure, animals underwent retrograde biliary administration using a range of fluid viscosities at different rates and volumes of infusion. As infusion viscosity was increased, intrabiliary pressure was similarly elevated. The graph shown in Fig. 6A presents the temporal pattern of intrabiliary pressure changes at different infusion viscosities. The effect of viscosity on intrabiliary pressure became more apparent at later stages of the infusion. Following 11.5 s of infusion (240 µl; 16 µl/s), intrabiliary pressure was significantly greater with the higher-viscosity infusion (27.1 ± 2.7 mmHg at infusion viscosity of 0.0070 cm¹ · g · s vs. 56.8 mmHg ± 8.8 mmHg at infusion viscosity 0.0642 cm¹ · g · s; P < 0.05). The relationship between fluid viscosity and intraluminal biliary pressure held over a range of infusion rates and volumes but was increasingly evident at larger volumes and faster rates of infusion (Fig. 6B). Figure 6C is a linear regression analysis of peak intrabiliary pressure as a function of infusion viscosity at two different infusion temperatures. Intrabiliary pressure was dependent on infusion viscosity at both 22 and 37°C (correlation coefficients: 22°C, r = 0.82; 37°C, r = 0.74). Although higher-viscosity infusions tended to result in greater increases in pressure at 37°C than at 22°C, the linear regression lines shown in Fig. 6C were not significantly different (P > 0.05). Repeat infusions with solutions of different viscosities followed the same pattern as was seen in Fig. 5B with saline infusion, i.e., repeat infusions resulted in significantly lower peak pressure changes than were produced by the initial infusion (data not shown).

View larger version (36K): [in this window] [in a new window]   Fig. 6.   Effect of infusion viscosity on intrabiliary pressure. Animals were administered retrograde biliary infusions using a range of fluid viscosities. Fluid was administered at both room temperature (22°C) and 37°C. A: temporal pattern of pressure changes as a function of infusion viscosity. Mean pressure tracings are shown for animals that received infusions of 37°C fluid at 240 µl over 15 s. Higher-viscosity infusions led to increasingly elevated intrabiliary pressures compared with lower-viscosity infusions. Similar patterns were observed with other rates, volumes, and temperatures. B: Bar graph showing means ± SE of peak intrabiliary pressures observed at 37°C as a function of infusion rate and viscosity. Numbers on bars refer to number of experiments per condition. Peak pressure was elevated in a viscosity-dependent fashion, with this effect increasingly evident as infusion rate and volume were increased. * P < 0.05 compared with 0.0070 cm1 · g · s viscosity infusion for that volume and rate of infusion. ** P < 0.05 compared with 0.0366 cm1 · g · s viscosity infusion for that volume and rate of infusion. C: Linear regression analysis of relationship between viscosity and peak intrabiliary pressure at 2 infusion temperatures (22 and 37°C).

High-pressure retrograde biliary infusion results in the acute disruption of interhepatocyte tight junctions. The pattern of pressure changes observed following retrograde biliary infusion, in conjunction with the finding that repetitive infusions lead to significantly lower peak intrabiliary pressures, suggest that retrograde biliary infusion leads to biliary ductular and/or canalicular filling followed by leakage at a threshold pressure. Movement of fluid from the intraluminal space may conceivably occur either directly across physically opened tight junctions or indirectly by altered rates of transepithelial transcytosis. However, the rapidity of the pressure changes observed above suggest that tight junction disruption is a more likely mechanism. We evaluated the ability of retrograde biliary infusion to disrupt tight junctions by qualitative ultrastructural examination of the intrahepatic distribution of lanthanum chloride, a heavy metal normally impermeant to structurally intact tight junctions (Figs. 7, 8, and 9). Electron-dense deposits consistent with the presence of lanthanum chloride were found within bile ducts but not in their adjacent subepithelial tissue compartments following retrograde biliary infusion of 720 µl of 5 mM lanthanum chloride administered at a rate of 2 µl/s or 8 µl/s (Fig. 7). In contrast, electron-dense deposits were found within biliary canaliculi, interhepatocyte cell spaces, and the perisinusoidal space of Disse (Fig. 8). Figure 9 consists of electron micrographs that reveal the probable overall pathway taken by high-pressure retrograde biliary infusate: from biliary canaliculi to their adjacent subepithelial compartments, i.e., through cell junctions, then coursing in the lateral intercellular spaces before ultimately reaching the perisinusoidal space of Disse. The presence of lanthanum chloride within interhepatocyte cell spaces and the perisinusoidal space of Disse immediately following high-pressure retrograde biliary infusion are consistent with an acute alteration of tight junction permeability. It is theoretically possible (but extremely unlikely within the time frame of these experiments) that active transport (including transcytosis) resulted in the movement of lanthanum chloride from the intraluminal space to the lateral intercellular space and the space of Disse. An alteration in tight junction permeability rather than transcytosis is also supported by the failure to find ultrastructural evidence of lanthanum chloride particles intracellularly within either hepatocytes or cholangiocytes.

View larger version (195K): [in this window] [in a new window]   Fig. 7.   Lanthanum chloride remains confined to lumen of intrahepatic bile ducts following high-pressure retrograde biliary infusion. Transmission electron microscopy (TEM) was used to look for evidence of tight junction disruption in intrahepatic bile ducts following high-pressure retrograde biliary infusion. Animals were administered 5 mM lanthanum chloride or vehicle by high-pressure retrograde biliary infusion (3 infusions of 240 µl/animal, administered at a rate of 2 or 8 µl/s, with a 10-s pause between infusions). Immediately following completion of final infusion, liver was removed and prepared for TEM. Areas with typical electron-dense deposits consistent with presence of lanthanum chloride are circled. Lanthanum chloride deposits were detected within lumen of bile ducts but were not found in immediately adjacent subepithelial tissue compartments (lamina propria or peribiliary capillary plexus). Panels A-D are representative sequentially higher magnifications of a typical intrahepatic bile duct from experimental animals.

View larger version (138K): [in this window] [in a new window]   Fig. 8.   Lanthanum chloride is not confined to lumen of biliary canaliculi following high-pressure retrograde biliary infusion. Tissue samples from animals described in Fig. 7 were evaluated for disruption of canalicular tight junctions. Electron-dense deposits, consistent with presence of lanthanum chloride, were detected within bile ducts, biliary canaliculi, interhepatocyte cellular junctions, and perisinusoidal space of Disse. Since tight junctions are normally impermeable to lanthanum chloride, presence of electron-dense deposits within cellular junctions and perisinusoidal space of Disse most likely occurred by tight junction disruption. Representative images are presented. A: negative (vehicle) control. B-E: experimental tissues. Arrows denote interpreted pathway of paracellular leakage.

View larger version (145K): [in this window] [in a new window]   Fig. 9.   Overall pathway of paracellular leakage of lanthanum chloride following high-pressure retrograde biliary infusion. Electron micrographs are presented illustrating overall pattern of lanthanum chloride deposition following high-pressure retrograde biliary administration. Electron-dense deposits, consistent with lanthanum chloride, can be seen throughout these areas on micrographs shown. A-C are experimental tissues. Arrows denote interpreted pathway of paracellular leakage, i.e., movement from canalicular lumen across disrupted canalicular tight junctions into lateral intercellular space and subsequently into perisinusoidal space of Disse.

High-pressure retrograde biliary infusion results in the systemic distribution of radiopaque contrast dye through hepatic venous drainage. Since high-pressure retrograde biliary infusion appeared to result in the transit of retrograde biliary infusate from canaliculi through the lateral intercellular space to the space of Disse, we sought to determine the ultimate redistribution pathway taken following retrograde biliary infusion. In some animals radiopaque contrast dye (infusion temperature 22°C, fluid viscosity 0.0285 or 0.1216 cm¹ · g · s) was rapidly infused retrograde (80 µl or 240 µl, 2-16 µl/s), and simultaneous digital fluoroscopy was utilized to determine if, and when, dye entered the systemic circulation. Digital images captured at 30 frames/s revealed the rapid appearance of dye in the systemic circulation. Figure 9 shows a typical example of these studies. Dye appeared to travel up the suprahepatic inferior vena cava (Fig. 10A) before being seen in the heart (Fig. 10B). The identification of this structure as the inferior vena cava was confirmed by temporarily preventing hepatic venous return during an identical experiment. Temporary obstruction of the suprahepatic inferior vena cava prevented systemic distribution during and after high-pressure retrograde biliary infusion (Fig. 10, C and D). Accordingly, although some degree of lymphatic drainage may possibly have occurred, high-pressure retrograde biliary infusion of radiopaque dye appears to have primarily resulted in systemic delivery of infusate via hepatic venous drainage.

View larger version (52K): [in this window] [in a new window]   Fig. 10.   Effect of suprahepatic inferior vena cava occlusion on systemic distribution of radiopaque dye delivered by high-pressure retrograde biliary infusion. Digital fluoroscopy was used to determine whether high-pressure retrograde biliary infusion would lead to systemic leakage and, if so, by what route. A and B: radiopaque contrast material was administered (240 µl, 8 µl/s), and images were recorded 30 times/s. A: fluoroscopic image showing dye having filled liver and subsequently entering systemic circulation. IVC, suprahepatic inferior vena cava. B: masked image showing dye in liver and heart. C and D: effect of occlusion of suprahepatic inferior vena cava on systemic distribution of dye. This animal was administered dye under same high-pressure conditions except that suprahepatic inferior vena cava was occluded using a microvascular clip before infusion and for 5 min afterwards. Dye was not seen in systemic circulation until IVC clip was removed. C: preinfusion masked image. CBD, common bile duct. D: postinfusion masked image taken 2 min after completion of infusion. Dye remains confined to liver.

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View larger version (104K): [in this window] [in a new window]   Fig. 11.   Relationship between intrabiliary pressure and onset of systemic leakage of radiopaque dye. To determine when systemic leakage occurred, animals underwent high-pressure retrograde biliary infusion of radiopaque contrast dye while simultaneously having intrabiliary pressure measured, and digital fluoroscopic images were recorded. Radiopaque dye initially appeared in systemic circulation as intrabiliary pressure was rapidly rising and was greatest once peak pressure was reached. Shown are results from infusion of dye at 8 µl/s. Systemic leakage was initially detected after only 24 µl had been infused (3 s).

View larger version (12K): [in this window] [in a new window]   Fig. 12.   Mechanical model of retrograde biliary infusion. Intraluminal pressure was monitored during constant-rate infusion into a closed silicone tube with a closed, longitudinal slit. This provided a mechanical analog of retrograde biliary infusion with features of both elasticity and leakiness. Shown are pressure recordings from a typical experiment. Manometric recording during constant infusion rates resulted in patterns of pressure changes very similar to those obtained with retrograde biliary infusion in vivo (compare initial infusion with Fig. 3A and repeat infusion with Fig. 5). Peak intraluminal pressure and leakage of infusate were dependent on both infusion volume and rate (data not shown).

Chronic cholestasis reduces the peak intrabiliary pressure response to retrograde biliary infusion. To determine the effect of chronic cholestasis on the dynamic response of the biliary system to retrograde biliary infusion, a group of animals underwent 4 days of chronic extrahepatic bile duct obstruction. Biliary manometry was then performed using a range of retrograde biliary volumes and rates of infusion (Fig. 13). Intrabiliary pressure after 4 days of cholestasis was compared with the values previously shown in Fig. 2 (t = 0 min for unobstructed and t = 10 min for 10 min of common bile duct obstruction). After 4 days of chronic extrahepatic bile duct obstruction, baseline (preinfusion) intrabiliary pressure remained significantly elevated [normostasis baseline = 0.8 ± 0.2 mmHg (n = 5); 4 days of cholestasis, 8.2 ± 1.0 mmHg (n = 10); P < 0.05]. The preinfusion intrabiliary pressure after 4 days of cholestasis was not significantly different from the pressure level after only 10 min of biliary tree obstruction [10 min of cholestasis = 10.0 ± 1.4 mmHg (n = 5); 48 h of cholestasis = 8.2 ± 1.0 mmHg (n = 10); P > 0.05].

View larger version (36K): [in this window] [in a new window]   Fig. 13.   Manometric changes during retrograde biliary infusion in chronically cholestatic mice. A: pattern of pressure changes observed in chronically cholestatic animals following retrograde biliary infusion. Data are presented as change in pressure (mmHg) from preinfusion value as a function of infusion time(s). Cholestatic animals had patterns of pressure changes similar to those seen in normostatic animals following retrograde biliary infusion (for comparison with normostatic animals, see Fig. 3), i.e., 5 phases consisting of 1) a progressive rise in intraluminal pressure; 2) a peak pressure; 3) a decline in pressure; 4) a sustained, plateau pressure; and, immediately after infusion is discontinued, 5) a rapid decline in pressure. B: effect of chronic cholestasis on maximal change in pressure during retrograde biliary infusion. Both normostatic (N) and chronically cholestatic (C) animals (4 days of chronic extrahepatic bile duct obstruction) were evaluated for effect of repeat retrograde biliary infusion on intraluminal pressure. Numbers on bars represent number of experiments per condition. Retrograde biliary infusion resulted in significantly smaller rises in intrabiliary pressure in cholestatic animals than in normostatic animals. In both normostatic and cholestatic animals, repeat infusions tended to result in smaller changes in pressure than achieved with an initial infusion. However, this effect became significant in cholestatic animals only at higher rates and volumes of infusion. * P < 0.05 for comparison between first and subsequent infusions at a particular infusion rate. ** P < 0.05 for comparison between normostatic and cholestatic animals for an identical infusion condition. NS, nonsignificant difference.

Retrograde biliary infusion results in tight junction disruption in both normostatic and cholestatic animals. To determine the effect of retrograde biliary infusion on tight junction permeability under both normostatic and cholestatic conditions, [¹⁴C]sucrose was infused retrograde using a range of infusion rates and volumes in naive mice and after 4 days of chronic extrahepatic bile duct obstruction. Plasma samples were obtained 5 min later by intracardiac puncture. Since physically intact tight junctions are impermeant to sucrose, the appearance of [¹⁴C]sucrose in the systemic circulation under these experimental conditions would signify that tight junction disruption had occurred. Figure 14 shows the results from this experiment. In normostatic animals, systemic leakage of [¹⁴C]sucrose at 5 min after infusion was detected at approximately equivalent levels across a range of infusion rates and volumes. In cholestatic animals, the amount of leakage tended to be lower than for normostatic animals at a given infusion volume and rate. In cholestatic animals, increasing infusion volume or infusion duration tended to lead to greater amounts of paracellular leakage.

View larger version (21K): [in this window] [in a new window]   Fig. 14.   Effect of retrograde biliary infusion on tight junction permeability in normostatic and chronically cholestatic animals. Tight junction permeability was assessed by retrograde biliary infusion of [14C]sucrose at a range of volumes and rates of infusion in both normostatic and chronically cholestatic animals. Heparinized blood samples were obtained by cardiac puncture 5 min later, and 200-µl aliquots of plasma were assayed in a scintillation counter for 14C content. Since tight junctions are normally impermeable to sucrose, presence of [14C]sucrose in systemic circulation following retrograde biliary infusion suggests that tight junction disruption occurred in both normostatic and cholestatic animals. However, in cholestatic animals retrograde biliary infusion tended to result in lower levels of paracellular leakage of [14C]sucrose than in normostatic animals under identical infusion conditions.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

These experiments indicate that retrograde biliary infusion beyond a critical filling volume/pressure results in the acute opening of tight junctions in both normostatic and chronically cholestatic mice, leading to the progressive movement of infusate from the canalicular lumen to the lateral intercellular space and then into the perisinusoidal space of Disse. Redistribution of infusate from the space of Disse appears to occur via hepatic sinusoidal and subsequent hepatic venous drainage rather than lymphatic drainage. Intrabiliary pressure changes were dependent on the infusion volume, rate, and viscosity. Digital fluoroscopic evaluation (Fig. 11) revealed that the murine biliary tree was filled to the point of systemic leakage after ~24-48 µl of infusate had been administered at 8 µl/s. Delivery at other rates can result in filling at different volumes (see Fig. 3).

During constant-rate retrograde biliary infusion in mice, a consistent pattern of pressure changes occurs as shown in Fig. 3. This consists of a gradual pressure rise, a threshold peak pressure, and then a variable decline to a steady-state level. Once the infusion is discontinued, intrabiliary pressure rapidly declines toward the preinfusion pressure. The amplitude and chronology of this pattern may be altered by varying the infusion rate, volume, or viscosity. Repeat infusions result in peak intraluminal pressure changes significantly lower than are produced by an initial infusion using the same volume and rate of infusion.

Retrograde biliary infusion increases the total fluid volume within a confined luminal space (the biliary tree) and will, therefore, raise intrabiliary pressure. The pattern and degree of pressure changes will be determined by whether biliary ductular structures can adapt to increased wall tension and/or have leakage sites through which fluid may potentially exit at elevated intraluminal pressures. Contractile elements are found around bile ducts and canaliculi, and their rhythmic activity may play an important role in normal bile flow (21, 34). Total canalicular and ductular diameter can increase in response to chronic extrahepatic bile duct obstruction (11, 27). It is, therefore, possible that these elements may function in some ways analogous to the role of smooth muscle cells in vascular capacitance vessels, i.e., capable of slow adaptation to increased intraluminal volume. However, whether the caliber (i.e., diameter and tone) of biliary structures can acutely change in response to intraluminal stimuli is less clear. Loss of fluid from the intraluminal space in response to elevated pressure could conceivably occur either directly by causing paracellular movement across disrupted tight junctions or indirectly by enhancing rates of fluid internalization and movement via apical membrane endocytosis, macrocytosis, or epithelial transcytosis.

Figure 3 indicates that biliary structures do have some component of acute capacitance because incremental increases in the volume of infusate initially resulted in gradual changes in intrabiliary pressure. A rigid or saturated ductular system would have demonstrated a much larger pressure change in response to the addition of only small increments of fluid volume. It is unclear whether this component of capacitance represents the filling of void space in flaccid tubular structures or the distension (or relaxation) of viscoelastic elements in response to the increase in intrabiliary volume. That the initial slopes of the curves shown in Fig. 3C vary with infusion rate suggests that biliary ductular structures may also have some component of acute distensibility that is in the nature of a viscoelastic tube. Viscoelasticity of bile ducts and canaliculi is histologically plausible since an extracellular matrix is present in the space of Disse, deep to the basement membrane of biliary epithelial cells, as well as throughout the hepatic parenchyma (26). However, a more appropriate future analysis of distensibility could be obtained with steady-state (i.e., constant-pressure) conditions. This would also make it possible to more fully evaluate whether biliary capacitance can acutely change in response to intrabiliary infusion (by, for example, relaxation of contractile elements and/or changes in biliary ductular diameter).

The mechanical model shown in Fig. 12, although only a simple representation of a complex anatomic system, does provide a didactic tool to investigate potential explanations for the pressure curves in Figs. 3, 4, and 5. There are notable similarities between the mechanical model and the actual biliary pressure curves obtained from in vivo experiments. The elasticity of the silicone tube may mimic the distensibility of the bile ducts, ductules, and canaliculi. The "leaky slit" may be analogous to disruption of tight junctions within the hepatobiliary tree. Once disrupted, the leakage of infusate can occur into the space outside the tube (space of Disse and/or subepithelial space), allowing the pressure to fall to a lower steady-state value necessary to maintain that rate of leakage. With subsequent infusions, the threshold pressure for leakage is lowered since the slit (tight junction) has been weakened by previous disruption. Very large-volume, rapid-rate repeat infusions can mask this defect, resulting in peak intraluminal pressures similar to those seen with the initial infusion.

Tight junctions in the hepatobiliary tree maintain cellular polarity by keeping the luminal space physically (and, therefore, functionally) separated from the subepithelial tissue compartment (2, 22). Biliary canalicular (2) and probably ductular (30) tight junctions play a critical role in maintaining selective biliary tree permeability to different solutes. In rats, retrograde biliary administration has been reported as leading to increased absorption of infusate as a function of infusate volume (6, 20). This suggests that an acute increase in biliary permeability occurred, consistent with the hypothesis that increasing intrabiliary pressure may result in a structural change in canalicular and/or ductular tight junctions.

Experimental studies of chronic extrahepatic cholestasis also support a connection between elevated intrabiliary pressure and biliary tree leakiness. A preliminary study in ethinyl estradiol-induced cholestatic rats found that retrograde biliary infusion resulted in evidence of paracellular leakage (4). Chronic extrahepatic biliary obstruction results in structural alterations in canalicular tight junctions (1, 7-10, 15, 18, 23, 25) and accumulation in the space of Disse of ZO-1 and occludin proteins, structural components of these junctions (1, 8, 9). These changes appear to correlate with increased functional leakiness across the canalicular tight junction (7, 8, 9, 18). Canalicular changes are detectable within 48 h of the onset of obstruction (23). However, it is unknown whether chronic extrahepatic obstruction directly effects canalicular tight junctions through elevations in intrabiliary pressure or through some other mechanism. Our data support the hypothesis that at sufficiently high intraluminal pressures, tight junctions may undergo acute, mechanical alteration, acting as a pressure release valve that removes excess intrabiliary volume from the luminal compartment. Tight junctions vary in terms of their structure and functional leakiness (22), and we speculate that they may also have different pressure thresholds for physical opening.

In cholestatic animals, retrograde biliary infusion resulted in significantly lower peak intrabiliary pressures during a first infusion than were observed under first infusion conditions in normostatic animals. This suggests that chronic extrahepatic bile duct obstruction resulted in some degree of tight junction disruption independent of any induced by retrograde biliary infusion. However, cholestatic animals also tended to have smaller amounts of [¹⁴C]sucrose in the bloodstream 5 min after retrograde biliary infusion than did normostatic animals. One possible explanation for this apparently contradictory finding is that peak intrabiliary pressure may directly affect the diameter to which tight junctions are opened and/or the driving force for paracellular movement and thereby determine the amount of paracellular leakage of molecules of a particular diameter. If this possibility is correct, then in the present experiments the tight junction disruption known to be caused by cholestasis was sufficient for there to be some amount of leakage of molecules smaller than sucrose (e.g., water) during a retrograde biliary infusion. This fluid leakage would have thereby minimized the extent of the peak intrabiliary pressure rise achieved during a retrograde biliary infusion. This smaller peak intrabiliary pressure in turn could have diminished the number of tight junctions that were acutely widened to the degree that molecules of the diameter of sucrose would acutely pass through or alternatively may have reduced the driving force (pressure gradient) driving the paracellular movement of sucrose across disrupted tight junctions. Future experiments using a range of different diameter molecules and infusion pressures will be necessary to more precisely determine the correlation between the absolute level of intrabiliary pressure, the degree of tight junction disruption, and the amount of paracellular leakage.

Obstruction of hepatic venous return increases hepatic sinusoidal hydrostatic pressure, thereby increasing the rate and volume of lymphatic drainage. Accordingly, if retrograde biliary infusate enters the sinusoidal space, it would be predicted that obstruction of hepatic venous return would lead to increased lymphatic drainage of any leaked material and, therefore, continued systemic redistribution. However, Fig. 10 indicates that hepatic venous obstruction prevented the appearance in the systemic circulation of radiopaque contrast material administered by high-pressure retrograde biliary infusion. This does not rule out some component of lymphatic drainage but does imply that under these particular experimental conditions retrograde biliary infusion leads to systemic distribution of leaked infusate primarily by venous rather than lymphatic drainage. These findings also suggest that hepatic venous obstruction may have prevented or reduced movement of infusate across disrupted tight junctions. Increased sinusoidal pressure may have been sufficient to oppose and overcome the pressure force driving the movement of infusate through this pathway.

In conjunction, the lanthanum chloride and [¹⁴C]sucrose studies indicate that high-pressure retrograde biliary infusion is associated with an acute disruption of interhepatocyte canalicular tight junctions and the subsequent movement of molecules in the size range of 7,000-11,000 Da into the lateral intercellular space and then into the perisinusoidal space of Disse. Since the transmission electron micrograph studies were done on a qualitative rather than a quantitative basis, it is also conceivable that at sufficiently high intraluminal pressures, tight junctions between adjacent epithelial cells in larger intra- or extrahepatic bile ducts may be similarly disrupted. This could result in the entry of infusate into venous and/or lymphatic capillaries located within the lamina propria, the subepithelial tissue compartment of these structures. Such venous drainage would ultimately be distributed in a vascular pattern (i.e., sinusoidal distribution) within the liver.

In summary, we have developed a novel system for cholangiomanometry in mice and have used this to evaluate the acute physiological consequences of retrograde biliary infusion at different infusion rates, volumes, viscosities, and temperatures. Our data suggest that under both normostatic and cholestatic conditions elevated intrabiliary volumes/pressures result in a physical opening of tight junctions, leading to the leakage of infusate from the intrabiliary space into the subepithelial tissue compartment. Control of intrabiliary infusion pressure may, therefore, potentially provide a novel method for the selective delivery of therapeutic agents to either the intrabiliary or subepithelial tissue compartment(s). These results may also be applicable to other luminal structures lined by polarized epithelial cells. Further experimentation will be required (using macromolecules having a range of sizes and charges) to more precisely determine the relationship between different levels of intraluminal pressure and the acute histological distribution of macromolecules normally excluded by an intact paracellular barrier.