ANNALS OF SURGERY
Vol. 232, No. 2, 202–207
© 2000 Lippincott Williams & Wilkins, Inc.
Effect of Endotoxin on Opossum Gallbladder
Motility: A Model of Acalculous Cholecystitis
Joseph J. Cullen, MD, Earl B. Maes, BS, Surrinder Aggrawal, Jeffrey L. Conklin, MD, Kimberly S. Ephgrave, MD, and
Frank A. Mitros, MD
From the Departments of Surgery and Internal Medicine, University of Iowa College of Medicine and Veterans Affairs Medical
Center, Iowa City, Iowa
Objective
To determine whether endotoxin causes histologic changes in
the gallbladder consistent with acalculous cholecystitis, and
to determine the effects of endotoxin on gallbladder motility.
Summary Background Data
Acute acalculous cholecystitis is frequently seen in critically ill,
septic patients, after prolonged fasting and gallbladder stasis.
The pathogenesis of acalculous cholecystitis is unknown;
however, previous studies have suggested that ischemia may
play a role.
Methods
Adult opossums received Escherichia coli lipopolysaccharide.
The gallbladder was removed for histologic examination or for
physiologic studies 4 hours to 2 weeks later. For histologic
examination, gallbladder strips underwent standard hematox-
ylin-and-eosin processing. For physiologic studies, they were
mounted in a tissue bath to determine responses to cholecys-
tokinin octapeptide or electrical field stimulation.
Acute acalculous cholecystitis is a common accompani-
ment to multiple system organ failure and may be an im-
portant source of sepsis that is reversible.1 Lipopolysaccha-
ride impairs bile acid and organic anion hepatic transport,2
but alterations in gallbladder function that may contribute to
Supported by Merit Review awards (to JJC, JLC) from the Department of
Veterans Affairs.
Presented at the Annual Meeting of the American Gastroenterological
Association, May 16 –19, 1999, Orlando, Florida, and published in
abstract form in Gastroenterology 1999; 116:A981.
Correspondence: Joseph J. Cullen, MD, 4622 JCP, University of Iowa
Hospitals and Clinics, Iowa City, IA 52242.
Accepted for publication December 9, 1999.
202
Results
Intravenous endotoxin at a dose of 0.005 mg/kg resulted in
disrupted mucosal surfaces and areas of hemorrhage; higher
doses of endotoxin resulted in coagulation necrosis, hemor-
rhage, areas of fibrin deposition, and extensive mucosal loss,
consistent with an acute ischemic insult. Endotoxin abolished
the contractile response to cholecystokinin octapeptide in
gallbladder strips 4 hours after endotoxin administration. The
0.005-mg/kg dose of endotoxin decreased the contractile
response to cholecystokinin octapeptide for up to 96 hours
after endotoxin administration and decreased the contractile
response to electrical field stimulation for 48 hours after ad-
ministration. Inhibition of nitric oxide synthase reversed the
decreased contractile response to cholecystokinin octapep-
tide.
Conclusions
Endotoxin causes an ischemic insult to the gallbladder similar
to that seen in acalculous cholecystitis. Also, endotoxin may
lead to gallbladder stasis by decreasing gallbladder contractile
responses to hormonal and neural stimuli.
the jaundice and the cholestasis of sepsis are unknown.
Although the upper gastrointestinal tract is relatively bac-
teria-free, bacterial overgrowth could potentially occur in
the setting of impaired gallbladder motility. Changes in the
biliary tract flora, coupled with impairment of the normal
barrier function, allow the gallbladder to serve as a reservoir
for pathogens that can enter the portal and systemic circu-
lations. Enteral nutrition decreases the rates of nosocomial
infections3,4; however, the gastrointestinal function of crit-
ically ill patients is frequently not adequate to permit enteral
nutrition because of gastric and small intestinal ileus result-
ing from sepsis. Total parenteral nutrition, which has been
found to be a contributing factor for acalculous cholecysti-
tis,5,6 is then needed to support the patient.
Vol. 232 ● No. 2
Acalculous cholecystitis is being diagnosed with increas-
ing frequency in critically ill, septic patients and typically
occurs in conditions associated with biliary stasis, including
major surgery, severe trauma and sepsis, long-term total
parenteral nutrition, and prolonged fasting.7 The pathogen-
esis of acute acalculous cholecystitis is unknown; however,
some authors have suggested that ischemia or reperfusion
injury may play a role.8 Impaired gallbladder emptying has
been thought to be a key factor leading to inspissation of
bile and sludge formation.9,10 The marked gallbladder hy-
pomotility in these conditions may make diagnosis of the
acalculous cholecystitis difficult. Serial ultrasonography in
septic patients has demonstrated a characteristic ultra-
sonographic appearance of gallbladder enlargement, boggy
thickening of the wall, and biliary sludge.1
The aims of our study were to determine whether endo-
toxin causes histologic changes in the gallbladder similar to
the ischemic insult that occurs during acalculous cholecys-
titis and to determine the effects of endotoxin on gallbladder
motility. We hypothesized that endotoxemia causes histo-
logic changes in the gallbladder consistent with an ischemic
injury, which may lead to changes in motor function. We
also hypothesized that endotoxemia results in impaired neu-
romuscular changes in vitro that may lead to diminished
gallbladder motility and impaired emptying. Finally, we
hypothesized that increased nitric oxide production in re-
sponse to the endotoxemia may be a mechanism that pro-
duces gallbladder smooth muscle relaxation.
METHODS
Preparation of Animals
Adult opossums (Didelphis virginia) of either sex weigh-
ing 3 to 6 kg were fasted overnight and then anesthetized
with ketamine (30 mg/kg)/acepromazine (0.3 mg/kg) given
intramuscularly and sodium pentobarbital (50 mg/kg) given
intraperitoneally. Intravenous access was obtained by
means of the right internal jugular vein, and the animals
received either 0.9% NaCl (1 mL given intravenously) or
E. coli lipopolysaccharide (serotype 055:B5, Sigma Chem-
ical, St. Louis, MO) in a dose of 0.005, 0.05, or 0.50 mg/kg
given intravenously. The animals were then allowed to
recover and were given free access to food and water.
Animals receiving 0.005 mg/kg were anesthetized at 4 hours
or 1, 2, 3, 4, or 14 days later with ketamine (30 mg/kg)/
acepromazine (0.3 mg/kg) given intramuscularly and so-
dium pentobarbital (50 mg/kg) given intraperitoneally. The
gallbladder was removed for physiologic studies and the
animal was killed. In additional studies, opossums receiving
saline or any of the three doses of lipopolysaccharide were
anesthetized 4 hours later and the gallbladder was removed
for histologic examination. All procedures were carried out
according to the protocol approved by the Department of
Veterans Affairs and the University of Iowa Animal Care and
Use Committees. Surgical procedures and experiments were
Acute Acalculous Cholecystitis 203
performed in accordance with the Guide for the Care and Use
of Laboratory Animals of the Public Health Service.
Conduct of Tests
For physiologic studies, the gallbladder was opened as a
flat sheet and full-thickness strips approximately 2 ⫻ 7 mm
were cut. Care was taken to cut strips of the same length and
width. The strips were placed in 5-mL organ baths and
mounted to record smooth muscle contractions. Gallbladder
strips were stimulated by either electrical field stimulation
(EFS), to determine their response to intrinsic nerve stimu-
lation, or the addition of cholecystokinin-8. The organ baths
contained Krebs solution (consisting of [in mmol/L] 138.5
sodium, 4.6 potassium, 2.5 calcium, 1.2 magnesium, 125
chloride, 21.9 bicarbonate, 1.2 phosphate, 1.2 sulfate, and
11.5 glucose) and were maintained at 37°C and gassed with
95% O2/5% CO2. Specimens were stretched to an initial
tension of 0.4 mg, and the strips were allowed to equilibrate
for 60 minutes. EFS was delivered by using 8-Hz stimula-
tion with 0.1-msec pulses for 5 seconds delivered at
1-minute intervals using 70 volts from a Grass S48 stimu-
lator (Grass Instruments, Quincy, MA). The response to
EFS was quantified by measuring the amplitude of the off
response. This electrical stimulus is known to produce near-
maximal nerve-mediated response.11 It releases nitric oxide
from intrinsic gut motor nerves and produces a nerve-
mediated contraction, the off response, after the end of each
train.12,13
In other smooth muscle strips from the gallbladder, a
dose-response curve was generated by adding cholecysto-
kinin-8 (Sigma Chemical) stepwise to the organ bath to
obtain final concentrations of 1.5 ⫻ 10⫺11 to 3 ⫻ 10⫺5
mol/L. In additional experiments, the contribution of nitric
oxide to decreased responsiveness of cholecystokinin-8 was
determined. Dose-responses to cholecystokinin-8 were de-
termined; if there was no response or a blunted contractile
response, even at the highest doses, NG-nitro-L-arginine
(L-NNA), 0.1 to 1.0 mol/L, was added to the tissue bath for
5 minutes, and dose-responses to cholecystokinin-8 were
repeated. After determining the dose-response relationship,
each strip was measured, blotted lightly, and weighed. The
cross-sectional area of each strip was calculated from length
and weight data by assuming that the density of smooth
muscle was 1.05 g/cm3. All active contractile force was
normalized to the cross-sectional area and was expressed as
force (g/cm3).14 Such normalization can correct for any
differences in tension development that are due simply to
differences in the size of the strips, even though in the
gallbladder, smooth muscle bundles intermingle and are not
oriented in any preferential direction.15
For both the EFS experiments and the dose-response to
cholecystokinin-8 experiments, the output of the force dis-
placement transducers was processed through a MacLab 8
analog-digital converter (ADInstruments, Inc., Milford,
MA), recorded on a Macintosh IIcx computer (Apple Com-
204 Cullen and Others Ann. Surg. ● August 2000

Figure 1. Transverse sections of opossum gallbladder before and after administration of Escherichia coli
lipopolysaccharide (⫻50). (S, serosa; SM, smooth muscle; M, mucosa.) (A) Control. (B) Four hours after
administration of 0.005 mg/kg. Areas of hemorrhage are seen in the smooth muscle layer, but the mucosal
layer is relatively intact. (C) Four hours after administration of 0.05 mg/kg. There are increased areas of
coagulation necrosis and hemorrhage in the serosal, muscle, and mucosal layers, as well as areas of fibrin
deposition and increased mucosal loss. (D) Four hours after administration of 0.5 mg/kg. Abundant areas
of hemorrhage and coagulation necrosis are seen. There is extensive mucosal loss and edema in all layers
of the gallbladder wall.

puter, Inc., Cupertino, CA), and analyzed with MacLab
software.
Histologic Examination
Gallbladders from each group of animals (n ⫽ 4 per
group: control, lipopolysaccharide 0.005, 0.05, and 0.5 mg/
kg) were removed at 4 hours for histologic examination.
Briefly, the gallbladders were sectioned into longitudinal
strips and placed in formaldehyde. The strips were embed-
ded in paraffin, sectioned, and stained with hematoxylin and
eosin for viewing under light microscopy by an experienced
pathologist (F.A.M.).
Statistical Analysis
Analysis of variance with the Tukey test and the Wil-
coxon rank-sum test for nonparametric data were used to
determine differences in the treatment groups. Statistical
significance of differences between groups was tested using
the computer program SYSTAT (Evanston, IL). All data are
expressed as means ⫾ standard error of the mean.
RESULTS
Histologic Examination
Endotoxin produced histologic changes consistent with
an ischemic insult, similar to what is seen with acalculous
cholecystitis. Four hours after administration of the larger
doses of endotoxin (0.50 and 0.05 mg/kg), coagulation
necrosis and hemorrhage, consistent with acute ischemic
necrosis, were seen in histologic sections of the gallbladder
(Fig. 1). Also, there were areas of fibrin deposition and
mucosal loss, also consistent with an ischemic insult. Four
hours after administration of the lower dose of endotoxin
Vol. 232 ● No. 2 Acute Acalculous Cholecystitis 205

Figure 2. Effect of cholecystokinin-8 (1.5 ⫻ 10⫺8

mol/L) on gallbladder isometric tension before and
after administration of 0.005 mg/kg endotoxin). Con-
trols, n ⫽ 16; 4 to 96 hours, n ⫽ 8; 2 weeks, n ⫽ 4.
(*P ⬍ .05 vs. control, Wilcoxon rank-sum test.)

(0.005 mg/kg), smaller areas of hemorrhage were seen and
the mucosal surface was lost.
Physiologic Studies
Endotoxemia impaired the contractile response to chole-
cystokinin-8 (Fig. 2). Compared with controls, animals re-
ceiving endotoxin had no response to cholecystokinin-8
until the highest doses of cholecystokinin-8 (1.5 ⫻ 10⫺5
mol/L) were used. For example, cholecystokinin-8 (1.5 ⫻
10⫺7 mol/L) increased isometric tension in control gallblad-
der strips to 29.9 ⫾ 5.5 g/cm3 but had little effect on muscle
strips from endotoxemic animals, producing an increase in
tension of less than 1.0 g/cm3 (P ⬍ .01 vs. control). The
lowest dose of endotoxin (0.005 mg/kg) disrupted the gall-
bladder contractile response to cholecystokinin-8 for 96
hours. Cholecystokinin-8 (1.5 ⫻ 10⫺8 mol/L) increased
isometric tension in control gallbladder strips to 14.2 ⫾ 2.9
g/cm3. For 96 hours, the same dose of cholecystokinin 8 had
little effect on isometric tension, increasing it to only 2.1 ⫾
1.1 g/cm3. At 14 days after receiving endotoxin (0.005
mg/kg), the gallbladder muscle strips’ response to cholecys-
tokinin-8 increased to 10.4 ⫾ 6.2 g/cm3, which was similar
to that found in the controls.
L-NNA (1 mol/L) reversed the decreased contractile re-
sponse to cholecystokinin-8 48 hours after the endotoxin
injection (0.005 mg/kg). In controls, cholecystokinin-8
(1.5 ⫻ 10⫺6M) resulted in a large contraction of the gall-
bladder strips (Fig. 3). As mentioned above, endotoxin
diminished this response for up to 4 days. However, in the
presence of L-NNA, a competitive inhibitor of nitric oxide
synthase, the contractile response to cholecystokinin-8 re-
turned to control values. L-NNA had no effect on the
gallbladder contractile response to cholecystokinin-8 during
the rest of the endotoxemic study days and had no effect on
cholecystokinin-8 contractile response in animals given the
higher doses of endotoxin (0.5 or 0.05 mg/kg). Thus, in-
creased nitric oxide production may play a role in the
diminished gallbladder motility seen during endotoxemia.
Also, EFS-induced gallbladder contractions were de-
creased for 4 hours and 24 hours after endotoxin adminis-
tration (0.005 mg/kg) (Fig. 4). EFS-induced changes in
isometric tension were 22.5 g/cm3 ⫾ 2.1 in controls but
decreased to 5.5 g/cm3 ⫾ 0.8 at 4 hours and 12.0 g/cm3 ⫾
2.1 at 24 hours (P ⬍ .05 vs. control). Nerve-mediated
changes in isometric tension in endotoxemic animals at 48
hours were similar to controls.
DISCUSSION
Our study results demonstrate that endotoxin disrupts
mucosal surfaces and causes areas of hemorrhage through-
out the gallbladder. With increased doses of endotoxin,
coagulation necrosis, hemorrhage, areas of fibrin deposi-
tion, and extensive mucosal loss are seen, consistent with an
acute ischemic insult. Endotoxin abolishes the contractile
response to cholecystokinin-8, whereas a low dose of en-
dotoxin decreases the contractile response to cholecystoki-
nin-8 for up to 96 hours and decreases the contractile
response to EFS for 48 hours. Inhibition of nitric oxide

Figure 3. Effect of cholecystokinin-8 (CCK-8) on

gallbladder isometric tension before and after endo-
toxin administration. First tracing: gallbladder
smooth muscle response to CCK-8 (1.5 ⫻ 10⫺6
mol/L). Second tracing: blunted gallbladder con-
tractile response to CCK-8 48 hours after endo-
toxin administration (0.005 mg/kg). Third tracing:
effect of NG-nitro-L-arginine (L-NNA) (1 mol/L) on
the baseline tone of the gallbladder strip during endo-
toxemia. Fourth tracing: reversal of the blunted con-
tractile response to CCK-8 during endotoxemia by
pretreating the strips with L-NNA.
206 Cullen and Others
synthase reverses the decreased contractile response to cho-
lecystokinin-8. Endotoxin causes an ischemic insult to the
gallbladder similar to that seen in acalculous cholecystitis.
Also, the motility changes produced by endotoxemia may lead
to gallbladder stasis by temporarily decreasing gallbladder
contractile responses to hormonal and neural stimuli.
These data correspond with the results of previous studies
from our laboratory16,17 and others18,19 regarding the effects
of sepsis and endotoxemia on gastrointestinal motility. Es-
kandari et al18,19 demonstrated that endotoxin caused a 62%
reduction in jejunal spontaneous circular muscle activity
and a 91% suppression of bethanechol-stimulated contrac-
tions in the jejunum. Thus, endotoxin administration seems
to result in smooth muscle impairment, even though the
effects of endotoxin on gastrointestinal motility may not be
due to decreased splanchnic perfusion.16 The smallest dose
of endotoxin (0.005 mg/kg) used in the current study is
2.5% the dose we have given to dogs16 and 0.0005% the
dose we have given to rats.17 Our present study suggests
that the gallbladder may be more sensitive to the effects of
endotoxin than other areas of the gut, because a low dose of
endotoxin produces profound changes in biliary tract mo-
tility for an extended period. In general, the opossums
tolerated the lowest dose of endotoxin (0.005 mg/kg) even
for up to 2 weeks. At the higher doses of endotoxin (0.05 to
0.5 mg/kg), the animals appeared more listless and had
more areas of gross hemorrhage in the gallbladder lumen.
Calabuig et al20 demonstrated a decrease in gallbladder
emptying and a prolongation of the migrating myoelectric
complex after hemorrhagic shock. Also, impairment of gall-
bladder smooth muscle contractility after hemorrhagic
shock has been demonstrated in vitro.21 In their study,
decreased responses to cholecystokinin octapeptide and cho-
linergic agonists occurred in gallbladder smooth muscle strips
after hemorrhagic shock. Thus, shock may impair gallblad-
der motility, leading to stasis and decreased emptying.
We recently demonstrated decreased bile acid secretion
in dogs after an endotoxin bolus.22 Bile secretion decreased
for 1 day after the endotoxin bolus, although there were no
changes in plasma cholecystokinin levels. These studies
suggest that during endotoxemia, plasma levels of chole-
cystokinin change little; however, the gallbladder may be
unresponsive to the effects of the gut hormone. Previous
Ann. Surg. ● August 2000
Figure 4. Effect of electrical field stimulation-induced
gallbladder contractions after endotoxin administra-
tion (0.005 mg/kg). Electrical field stimulation param-
eters: 8 Hz, 0.1-msec pulses, 5-second duration,
1-minute intervals, 70 V. Controls, n ⫽ 16; 4 to 96
hours, n ⫽ 8; 2 weeks, n ⫽ 4. (*P ⬍ .05 vs. control,
Wilcoxon rank-sum test.)
studies have demonstrated decreased bile acid synthesis
during endotoxemia as a result of decreased transport of bile
acids in hepatic cells.23 Bolder et al2 demonstrated de-
creased bile flow during endotoxemia, which was attributed
to decreased secretion of bile acids. However, impaired
canalicular bile formation, sphincter of Oddi dysfunction, or
gallbladder hypomotility may also delay emptying of bile
into the duodenum, which could potentially predispose to
bacterial proliferation. Decreased bile flow and the subse-
quent decreased bile acid concentration in the small intes-
tine may also occur, which may lead to translocation24 and
endotoxin absorption.25 Thus, a vicious cycle may result,
with increased endotoxin absorption caused by impaired
biliary secretion and stasis in the gallbladder, which could in
turn cause more endotoxin absorption.
Our study demonstrates that increased nitric oxide pro-
duction may play a role in the decreased response of gall-
bladder smooth muscle to cholecystokinin-8 during endo-
toxemia. Application of a nonspecific nitric oxide synthase
inhibitor restored the contraction response to cholecystoki-
nin-8. The role of nitric oxide in mediating the smooth
muscle dysfunction during endotoxemia has been demon-
strated in several studies. Eskandari et al26 demonstrated a
reduction in circular smooth muscle activity after an endo-
toxin bolus that was reversed with in vitro application of a
specific inducible nitric oxide synthase inhibitor. Weisbrodt
et al27 also reported that competitive inhibition of nitric
oxide synthase reversed the lipopolysaccharide-induced
suppression of rat longitudinal muscle activity. In gallblad-
der tissues not treated with endotoxin, Mourelle et al28
demonstrated that competitive inhibition of nitric oxide
synthase increased the gallbladder strips’ contractile re-
sponse to cholecystokinin-8.
Our study suggests that endotoxin causes changes in
gallbladder motility and histology similar to what is seen
during acute acalculous cholecystitis. Other experimental
preparations have been proposed to mimic the clinical sce-
nario of acalculous cholecystitis. Parkman et al29,30 have
demonstrated gallbladder smooth muscle dysfunction after
common bile duct ligation. This experimental preparation
has been proposed to demonstrate the effects of acute acal-
culous cholecystitis because it reliably produces acute in-
flammation of the gallbladder. Also, the response of gall-
Vol. 232 ● No. 2
bladder strips to potassium and bethanechol is decreased.
Our experimental preparation of acalculous cholecystitis
differs because bile flow is not impeded, but inflammation
and motility changes occur. Clinical studies of acalculous
cholecystitis have suggested the absence of obstruction of
either the cystic duct or the common bile duct.7,9
The results of our histologic studies suggest that endo-
toxin causes an acute ischemic insult in the gallbladder,
leading to coagulation necrosis, hemorrhage, areas of fibrin
deposition, and extensive mucosal loss. At increased doses
of endotoxin, coagulation necrosis, hemorrhage, areas of
fibrin deposition, and extensive mucosal loss are seen, sug-
gesting a dose-response effect of endotoxin on the gallblad-
der. Although we studied the effects of the larger doses of
endotoxin (0.05– 0.5 mg/kg) on the gallbladder contractile
response to cholecystokinin-8 and EFS at only 4 hours after
endotoxin administration, our results suggest that these
larger doses of endotoxin may lead to an even greater
physiologic impairment of cholecystokinin-8 or EFS than
what was seen at the lower dose (0.005 mg/kg). The results
of previous histologic studies in acalculous cholecystitis
correlate with our findings. In a study comparing histologic
findings with evidence of small vessel occlusion in gallblad-
ders removed for acute acalculous cholecystitis, Warren9
suggested that decreased splanchnic flow led to intravascu-
lar coagulation and necrosis in acalculous cholecystitis.
In conclusion, a single dose of endotoxin causes coagu-
lation necrosis, hemorrhage, fibrin deposition, and extensive
mucosal loss in the gallbladdder consistent with an acute
ischemic insult similar to that seen in acalculous cholecys-
titis. Endotoxin abolishes the contractile response to chole-
cystokinin-8 for up to 96 hours and decreases nerve-medi-
ated responses for 48 hours, which may lead to gallbladder
stasis by temporarily decreasing the gallbladder contractile
responses to hormonal and neural stimuli. Finally, nitric
oxide may play a role in the motility changes in the gall-
bladder during endotoxemia, because inhibition of nitric
oxide synthase reverses the decreased contractile response
to cholecystokinin-8.
Acknowledgments
The authors thank Marilyn Hinkhouse for technical assistance and Sally
Blackmon for manuscript preparation.
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