1 Molecular Hematology Branch and 2 Laboratory of Animal Medicine and Surgery, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda 20892; 3 Laboratory of Cell and Molecular Structure, National Cancer Institute-Frederick Cancer Research Development Center, Frederick 21270; and 4 Molecular Genetics and 5 Pathology Service, Diagnostic and Surgery Section, Veterinary Resources Program, National Center for Research Resources, 6 Bioengineering and Physical Sciences Program, Office of Research Services, Office of the Director, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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The manometric, ultrastructural, radiographic, and physiological consequences of retrograde biliary infusion were determined in normostatic and cholestatic mice. Intraluminal biliary pressure changed as a function of infusion volume, rate, and viscosity. Higher rates of constant infusion resulted in higher peak intraluminal biliary pressures. The pattern of pressure changes observed was consistent with biliary ductular and/or canalicular filling followed by leakage at a threshold pressure. Retrograde infusion with significant elevations in pressure led to paracellular leakage of lanthanum chloride, radiopaque dye, and [14C]sucrose with rapid systemic redistribution via sinusoidal and subsequent hepatic venous drainage. Chronic extrahepatic bile duct obstruction resulted in significantly smaller peak intrabiliary pressures and lower levels of paracellular leakage. These findings indicate that under both normostatic and cholestatic conditions elevated intrabiliary volumes/pressures result in an acute pressure-dependent physical opening of tight junctions, permitting the movement of infusate from the intrabiliary space into the subepithelial tissue compartment. Control of intraluminal pressure may potentially permit the selective delivery of macromolecules >18-20 Å in diameter to specific histological compartments.
drug delivery; polarized epithelia; tight junction; cholestasis
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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.
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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.
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RESULTS |
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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).
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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).
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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 cm
1 · 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).
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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.
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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.
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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].
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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.
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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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We thank Katherine Anders, Gabrielle Kotler, Melody Lowe, Erin Stewart, Karen Lipovsky, Kelly Cole, and Brian Van Den Woude for assistance with viscosity experiments, Howard Bartner for medical illustrations, and Michael Juhn for help with computer graphics.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. M. Wiener, Massachusetts General Hospital, Gastrointestinal Unit-GRJ 722, 55 Fruit St., Boston, MA 02114 (E-mail: SWiener{at}Partners.org).
Received 19 August 1998; accepted in final form 28 December 1999.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Anderson, JM,
Glade JL,
Stevenson BR,
Boyer JL,
and
Mooseker MS.
Hepatic immunohistochemical localization of the tight junction protein ZO-1 in rat models of cholestasis.
Am J Pathol
134:
1055-1062,
1989[Abstract].
2.
Anderson, JM,
and
Van Itallie CM.
Tight junctions and the molecular basis for regulation of paracellular permeability.
Am J Physiol Gastrointest Liver Physiol
269:
G467-G475,
1995
3.
Barker, WC,
Andrich MP,
Alexander HR,
and
Fraker DL.
Continuous intraoperative external monitoring of perfusate leak using iodine-131 human serum albumin during isolated perfusion of the liver and limbs.
Eur J Nucl Med
22:
1242-1248,
1995[ISI][Medline].
4.
Boyer, JL,
LaGarde S,
Ng O-C,
and
Groszman R.
Enhanced biliary regurgitation of 14C-sucrose (14C-S) and Lanthanum (La+++) in ethinyl estradiol (EE) treated rats following retrograde bile duct infusionsa possible mechanism for intrahepatic cholestasis (Abstract).
Hepatology
1:
498,
1981.
5.
Cabrera, JA,
Wilson JM,
and
Raper SE.
Targeted retroviral gene transfer into the rat biliary tract.
Somat Cell Mol Genet
22:
21-29,
1996[ISI][Medline].
6.
Dammann, HG,
and
Essen J.
Influence of retrograde volume and shorttime cholestasis on the biliary reabsorption of sulfobromophthalein sodium (BSP) and phenol 3,6 dibromphthalein disulfonate (DBSP) from the rat biliary tree after retrograde biliary injection (RII).
Res Exp Med (Berl)
168:
187-198,
1976[ISI][Medline].
7.
De Vos, R,
and
Desmet VJ.
Morphological changes of the junctional complex of the hepatocytes in rat liver after bile duct ligation.
Br J Exp Pathol
59:
220-227,
1978[ISI][Medline].
8.
Fallon, MB,
Brecher AR,
Balda MS,
Matter K,
and
Anderson JM.
Altered hepatic localization and expression of occludin after common bile duct ligation.
Am J Physiol Cell Physiol
269:
C1057-C1062,
1995
9.
Fallon, MB,
Mennone A,
and
Anderson JM.
Altered expression and localization of the tight junction protein ZO-1 after common bile duct ligation.
Am J Physiol Cell Physiol
262:
C1439-C1447,
1992.
10.
Gaudi, E,
Onori P,
Pannarale L,
and
Alvaro D.
Hepatic microcirculation and peribiliary plexus in experimental biliary cirrhosis: a morphological study.
Gastroenterology
11:
1118-1124,
1996.
11.
Hampton, JC.
Electron microscopic study of extrahepatic biliary obstruction in the mouse.
Lab Invest
10:
502-513,
1961[ISI].
12.
Hayashi, N,
Takehara T,
and
Kamada T.
In vivo transfection of rat liver with hepatitis C virus cDNA using cationic liposome-mediated gene delivery.
Princess Takamatsu Symp
25:
143-149,
1995[Medline].
13.
Hofland, HE,
Nagy D,
Liu JJ,
Spratt K,
Lee YL,
Danos O,
and
Sullivan SM.
In vivo gene transfer by intravenous administration of stable cationic lipid/DNA complex.
Pharm Res
14:
742-749,
1997[ISI][Medline].
14.
Hoyt, RF, Jr,
DeLeonardis J,
Clevenger R,
and
Wiener SM.
Gallbladder catheterization: a common portal for selectively delivering therapeutic agents to murine hepatobiliary and pancreatic tissues (Abstract).
Contemp Topics Lab Animal Sci
35:
59,
1996.
15.
Koga, A,
and
Todo S.
Morphological and functional changes in the tight junctions of the bile canaliculi induced by bile duct ligation.
Cell Tissue Res
195:
267-276,
1978[ISI][Medline].
16.
Ku, Y,
Iwasaki T,
Fukumoto T,
Tominaga M,
Muramatsu S,
Kusunoki N,
Sugimoto T,
Suzuki Y,
Kuroda Y,
Saitoh Y,
Sako M,
Matsumoto S,
Hirota S,
and
Obara H.
Induction of long-term remission in advanced hepatocellular carcinoma with percutaneous isolated liver chemoperfusion.
Ann Surg
227:
519-526,
1998[ISI][Medline].
17.
Meijer, DK,
and
Molema G.
Targeting of drugs to the liver.
Semin Liver Dis
15:
202-256,
1995[ISI][Medline].
18.
Metz, J,
Aoki A,
Merlo M,
and
Forssmann WG.
Morphological alterations and functional changes of interhepatocellular junctions induced by bile ligation.
Cell Tissue Res
82:
299-310,
1997.
19.
Peeters, MJ,
Patijn GA,
Lieber A,
Meuse L,
and
Kay MA.
Adenovirus-mediated hepatic gene transfer in mice: comparison of intravascular and biliary administration.
Hum Gene Ther
7:
1693-1699,
1996[ISI][Medline].
20.
Peterson, RE,
and
Fujimoto JM.
Retrograde intrabiliary injection: absorption of water and other compounds from the rat biliary tree.
J Pharmacol Exp Ther
185:
150-162,
1973[ISI][Medline].
21.
Phillips, MJ,
Oshio C,
Miyairi M,
and
Smith CR.
Intrahepatic cholestasis as a canalicular motility disorder. Evidence using cytochalasin.
Lab Invest
48:
205-211,
1983[ISI][Medline].
22.
Powell, DW.
Barrier function of epithelia.
Am J Physiol Gastrointest Liver Physiol
241:
G275-G288,
1981
23.
Rahner, C,
Stieger B,
and
Landmann L.
Structure-function correlation of tight junctional impairment following intrahepatic and extrahepatic cholestasis in rat liver.
Gastroenterology
110:
1564-1578,
1996[ISI][Medline].
24.
Ravikumar, TS,
Pizzorno G,
Bodden W,
Marsh J,
Strair R,
Pollack J,
Hendler R,
Hanna J,
and
D'Andrea E.
Percutaneous hepatic vein isolation and high dose hepatic arterial infusion chemotherapy for unresetable liver tumors.
Clin Oncol (R Coll Radiol)
12:
2723-2736,
1994.
25.
Robenek, HI,
Herwig J,
and
Thermann H.
The morphologic characterizaton of intercellular junctions between normal human liver cells and cells from patients with extrahepatic cholestasis.
Am J Pathol
100:
93-103,
1980[Abstract].
26.
Rojkind, M,
and
Greenwel P.
The extracellular matrix in the liver.
In: The Liver, Biology and Pathobiology (3rd ed.), edited by Arias IM,
Boyer JL,
Fausto N,
Jakoby WB,
Schachter DA,
and Shafritz DA.. New York: Raven, 1994, p. 843-868.
27.
Schweizer, W,
Duda P,
Tanner S,
Balsiger D,
Hoflin F,
Blumgart LH,
and
Zimmerman A.
Experimental atrophy/hypertrophy complex (AHC) of the liver: portal vein, but not bile duct obstruction, is the main driving force for the development of AHC in the rat.
J Hepatol
23:
71-78,
1995[ISI][Medline].
28.
Simonetti, RG,
Liverati A,
Angiolini C,
and
Pagliaro L.
Treatment of hepatocellular carcinoma: a systematic review of randomized controlled trials.
Ann Oncol
8:
117-136,
1997[Abstract].
29.
Sullivan, DE,
Dash S,
Du H,
Hiramatsu N,
Aydin F,
Kolls J,
Blanchard J,
Baskin G,
and
Gerber MA.
Liver-directed gene transfer in non-human primates.
Hum Gene Ther
1:
1195-1206,
1997.
30.
Tavolini, N.
The intrahepatic biliary epithelium: an area of growing interest in hepatology.
Semin Liver Dis
7:
280-292,
1987[ISI][Medline].
31.
Van den Bogaerde, JB,
and
Jordaan M.
Intraductal administration of albendazole for biliary ascariasis.
Am J Gastroenterol
92:
1531-1533,
1997[ISI][Medline].
32.
Venook, AP,
and
Warren RS.
Regional chemotherapy approaches for primary and metastatic liver tumours.
Surg Oncol Clin N Am
5:
411-427,
1996[Medline].
33.
Vickers, SM,
Phillips JO,
Kerby JD,
Bynon JS, Jr,
Thompson JA,
and
Curiel DT.
In vivo gene transfer to the human biliary tract.
Gene Ther
3:
824-828,
1996.
34.
Watanabe, N,
Tsukada N,
Smith CR,
and
Phillips MJ.
Motility of bile canaliculi in the living animal: implications for bile flow.
J Cell Biol
113:
1069-1080,
1991[Abstract].
35.
Wiener SM, Hoyt RF Jr, DeLeonardis J, Clevenger R, Eckhaus M,
Owens J, Jambou R, Trapnell B, Weaver L, Mech C, Bartner H, Kotin RM,
and Safer B. Organ-selective in vivo gene transfer in mice
(Abstract). Sixth International Parvovirus Workshop,
Montpellier, France, 1995, p. 32.
36.
Wiener, SM,
Hoyt RF, Jr,
DeLeonardis JR,
Clevenger RR,
Eckhaus M,
Owens J,
Kotin RM,
and
Safer B.
Organ-specific vector delivery and gene transfer in vivo (Abstract).
Gastroenterology
110:
A1360,
1996[ISI].
37.
Yang, Y,
Raper SE,
Cohn JA,
Engelhardt JF,
and
Wilson JM.
An approach for treating the hepatobiliary disease of cystic fibrosis by somatic gene transfer.
Proc Natl Acad Sci USA
90:
4601-4605,
1993[Abstract].
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