Inhibition of prostaglandin synthesis during polystyrene
microsphere-induced pulmonary embolism in the rat
Alan E.
Jones1,
John A.
Watts1,
Jacob P.
Debelak1,
Lisa R.
Thornton1,
John G.
Younger2, and
Jeffrey A.
Kline1
1 Department of Emergency Medicine, Carolinas
Medical Center, Charlotte, North Carolina 28203; and
2 Department of Emergency Medicine, University of
Michigan, Ann Arbor, Michigan 48109
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ABSTRACT |
Our objective
was to test the effect of inhibition of thromboxane synthase versus
inhibition of cyclooxygenase (COX)-1/2 on pulmonary gas exchange and
heart function during simulated pulmonary embolism (PE) in the rat. PE
was induced in rats via intrajugular injection of polystyrene
microspheres (25 µm). Rats were randomized to one of three
posttreatments: 1) placebo (saline), 2)
thromboxane synthase inhibition (furegrelate sodium), or 3)
COX-1/2 inhibition (ketorolac tromethamine). Control rats received no
PE. Compared with controls, placebo rats had increased thromboxane
B2 (TxB2) in bronchoalveolar lavage fluid and
increased urinary dinor TxB2. Furegrelate and ketorolac
treatments reduced TxB2 and dinor TxB2 to
control levels or lower. Both treatments significantly decreased the
alveolar dead space fraction, but neither treatment altered arterial
oxygenation compared with placebo. Ketorolac increased in vivo mean
arterial pressure and ex vivo left ventricular pressure (LVP) and right
ventricular pressure (RVP). Furegrelate improved RVP but not LVP.
Experimental PE increased lung and systemic production of
TxB2. Inhibition at the COX-1/2 enzyme was equally as
effective as inhibition of thromboxane synthase at reducing alveolar
dead space and improving heart function after PE.
thromboembolism/treatment; cyclooxygenase; thromboxane; leukotriene; ketorolac; heart failure; Langendorff; animal
model
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INTRODUCTION |
PULMONARY EMBOLISM
(PE) continues to be a major cause of morbidity and mortality in the
United States. In one large autopsy-based study, massive PE was the
second leading cause of sudden death in adults aged <65 yr
(4). The primary treatment strategy for massive PE is the
recanalization of occluded pulmonary vasculature by fibrinolytic agents
(11), catheter fragmentation (32), or
surgical removal of clot (16). However, up to one-half of patients with massive PE have contraindications to fibrinolysis (15), and few hospitals have facilities for invasive
treatment of PE. Even under optimal conditions (e.g., immediate bolus
infusion of a fibrinolytic agent), these interventions require >2 h to effect a significant reduction in pulmonary vascular resistance (20). PE may also cause pulmonary vasoconstriction through
the liberation of vasoconstrictive agents, including
PGF2
and thromboxane (Tx) A2 and
B2 (10). PE can cause hypoxemia and increased
pulmonary arterial pressure in previously healthy patients (19). Both hypoxemia and increased shear forces in the
pulmonary vascular bed have been found to increase expression of the
cyclooxygenase (COX)-2 gene (3). In humans with PE, blood
concentrations of thromboxane have been found to be elevated for up to
7 days after onset of symptoms (10). Both the mechanical
vascular occlusion and release of vasoconstrictive agents from massive
PE appear to produce a synergistic effect that causes acute pulmonary
hypertension, worsened gas exchange, impaired right ventricular (RV)
function that can culminate in acute cor pulmonale, circulatory shock, and even death (26, 30, 39).
In the present work, we inhibited two enzymes in the prostaglandin
pathway shown in Fig. 1. Site
A, the COX-1/2 enzyme, was inhibited with ketorolac, and
site B, the thromboxane synthase enzyme, was inhibited with
furegrelate. Figure 1 shows that one could rationalize that proximal
inhibition at the COX-1/2 enzyme might result in decreased substrate
that is available to produce vasodilatory prostaglandins
(PGI2 and PGE2), resulting in worsened pulmonary hypertension (34) and ventilation-perfusion
relationships (7, 33). Also, the location of COX-1/2
suggests that COX-1/2 inhibition might cause arachidonate to be shunted
toward the lipoxygenase pathway, resulting in increased synthesis of
vasoconstrictive and bronchoconstrictive leukotrienes (28,
38). We sought to test the hypothesis that thromboxane synthase
inhibition would provide greater improvement than COX-1/2 inhibition in
alveolar dead space (VD/VTalv) and RV function
after pulmonary vascular occlusion.

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Fig. 1.
Flow diagram of arachidonate metabolism showing the steps
where ketorolac and furegrelate inhibit prostaglandin metabolism.
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METHODS |
Experiments were performed in Sprague-Dawley rats weighing
between 415 and 543 g. The study had three phases. 1)
Assessment of treatment efficacy: four groups of animals were used to
compare the efficacy of the drugs of interest. These groups include
animals treated with placebo, furegrelate sodium, or ketorolac
tromethamine and a control group. A sufficient number of animals were
studied to allow n = 9 in each four groups.
2) Pulmonary angiography: four groups of rats (placebo,
furegrelate, ketorolac, and control, n = 3 per group)
were studied by pulmonary angiography. 3) Measurement of
pleural effusion, lung water, and histology after PE: two groups (control and placebo, n = 6 per group) were designed to
allow measurements of lung wet-to-dry weight and to measure pleural effusion volumes. Studies were conducted according to the National Institutes of Health guidelines on the use of experimental animals. The
Institutional Animal Care and Use Committee (IACUC) of Carolinas Medical Center approved all methods. Before experimentation, rats had
ad libitum access to standard Teklad rat diet (Harlan Teklad, Madison, WI).
Pulmonary embolization protocol.
Experimental animals were anesthetized with an intramuscular injection
of 100 mg/kg of ketamine and 3 mg/kg of xylazine. The neck was shaved
and prepared with aseptic technique. The left jugular vein was
dissected and cannulated with PE-90 tubing. Undiluted polystyrene
microsphere beads (mean diameter 24 ± 1 µm, catalog no. 7525A;
Duke Scientific, Palo Alto, CA) totaling 0.15 ml/100 g body wt with 0.1 ml/100 g body wt of saline flush were given at a rate of 0.1 ml/min to
induce fixed pulmonary obstruction. We previously demonstrated in an
acute model that this dose of microspheres produced approximately a
peak reduction in mean arterial blood pressure (MAP) of 25% from basal
measurements followed by partial recovery of arterial blood pressure to
~10% below basal level (5). This dose also causes the
in vivo RV systolic blood pressure to increase from 30 ± 1 at
baseline to 55 ± 1.8 mmHg measured 30 min after embolization,
suggesting ~75% pulmonary vascular occlusion (19). In
the present experiment, we extend the duration of the exposure of the
PE to 16 h. After PE induction, the jugular vein was ligated with
2-0 silk ligature, and the incision was closed with 2-0 silk suture.
Rats were placed back in cages to recover with ad libitum access to
food and water.
Measurement of treatment efficacy.
The treatments were placebo (1 ml saline), furegrelate sodium (15 mg/kg
in 1 ml saline; Cayman Chemical, Ann Arbor, MI), and ketorolac
tromethamine (10 mg/kg in 1 ml saline; Abbott Laboratories, Chicago,
IL). A technician prepared fresh treatment solutions each day according
to a computer-generated (Excel Visual Basic Macro; Microsoft, Seattle,
WA) random schedule of treatments. This technician did not otherwise
participate in the in vivo portion of the experiments but was aware of
the outcome of each rat and had instructions to stop using a treatment
when nine rats survived to completion of data collection. Two identical
treatment injections were given intraperitoneally to awake rats, the
first at 5 h and the second at 14 h after induction of PE.
The first treatment was timed to correspond with the clinical
observation that massive PE is usually diagnosed at least 5 h
after symptom onset (4, 37). The investigators who
performed the injections and physiological and biochemical measurements
were blinded to the treatment group. Animals were studied at 16 h,
because in pilot studies, we recognized this as the time when rats
usually began to develop visible evidence of respiratory distress. A
timeline of the experimental protocol is shown in Fig.
2. Controls rats were not randomized and
were studied after the treatment efficacy experiment was completed. Control rats were not subjected to any stress other than anesthesia for
instrumentation.

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Fig. 2.
Timeline for the experimental protocol. See
METHODS for description of interventions. PE, pulmonary
embolism; BAL, bronchoalveolar lavage.
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At 16 h after PE induction, surviving rats were again anesthetized
by blinded technicians in the above-described fashion. Rats were placed
on a warming pad filled with recirculating water warmed to 105°F
(Gaymar solid-state T-pump; Orchard Park, NY). The rat's neck was
shaved, and a tracheostomy was performed by cannulating the trachea
with PE-240 tubing. Both the right carotid artery and right internal
jugular vein were dissected and cannulated with Millar Mikro-Tip
micromanometer catheter transducers (Millar Instruments, Houston, TX).
A 2-French Millar catheter (SPR-249-A) monitored arterial blood
pressure in the carotid artery. A 2-French bent Millar catheter
(SPR-513) was advanced through the internal jugular vein to monitor
right atrial pressure. The right femoral artery was dissected and
cannulated with PE-50 tubing filled with saline for arterial blood sampling.
After instrumentation, animals were ventilated with a small-animal,
pressure-regulated, mechanical ventilator (model 2094; Kent Scientific,
Litchfield, CT). Ventilator settings were as follows: respiratory rate
30-35, peak inspiratory pressure 10-14 cmH20,
flow 1.0 l per min, and partial pressure of oxygen was at room
air. To measure ventilation parameters, we attached a gas flow
transducer (model TSD 137C; Biopac Systems, Santa Barbara, CA) to the
inspiratory limb of the ventilator circuit. End-tidal CO2
was measured by a side stream quantitative CO2 capnometer (model CO2100A; Biopac Systems) attached to the expiratory
limb. End-tidal O2 was measured by a side stream
paramagnetic oxygen sensor attached to the expiratory limb (model
O2100A; Biopac Systems). All pressure transducers as well as the flow
transducer, oxygen sensor, and CO2 capnometer supplied data
that were visualized in real time and recorded via a commercially
available data acquisition software program (AcqKnowledge, version
3.5.6; Biopac Systems). After instrumentation, animals in all groups
were given succinylcholine (1 mg/kg iv) to relax breathing efforts,
allowing end-tidal CO2 measurements to be obtained from
flat-topped, steady-state expirograms, during controlled, constant
mechanical ventilation and without interference from interposed
spontaneous breathing efforts. Injection of succinylcholine marked the
beginning of data collection (time 0). Parameters measured
included: end-tidal expired carbon dioxide (etCO2),
end-tidal expired oxygen (etO2), minute ventilation (MV), MAP, right atrial pressure (RAP), pH, partial pressure of carbon dioxide in arterial blood (PaCO2), partial pressure of
oxygen in arterial blood, and lactate. Arterial blood gas and
lactate results were obtained using a Med/Ultra II (Staf
Profile Ultra; Nova Biomedical, Waltham, MA).
Pulmonary gas exchange was estimated primarily by the
VD/VTalv, calculated using etCO2 in
the Severinghaus equation (1, 14, 25). Use of the
etCO2 to estimate the alveolar dead space fraction as
opposed to the mixed-expired CO2 was chosen because the
end-tidal equation provides a more specific estimate of the alveolar
dead space fraction compared with the physiological dead space
fraction, which measures both airway and alveolar dead space (25). VD/VTalv correlates with
pulmonary vascular resistance (22), and perfusion
defects in humans with PE (17) decreases in parallel with
reduction in perfusion defects during treatment (25) and
correlates with the plasma concentration of TxB2 in dogs
with PE (39).
Measurements of MAP, RAP, MV, etCO2, etO2, and
arterial blood gases were performed at 15 and 45 min after injection of
succinylcholine. Total body CO2 production
(VCO2) was calculated at body temperature, ambient pressure, and saturated with water vapor. In pilot work, we
found that the side stream pumps that aspirate a portion of the expired
breath volume for the oximeter and capnometer affected the MV, MAP, and
arterial blood gas measurements. Therefore, the pumps were turned on
only long enough to obtain steady-state measurements of
etCO2 and etO2. After the 45-min gas exchange
measurements were completed, bronchoalveolar lavage (BAL) was performed
with 8 ml normal saline (23°C) using four washes via the tracheostomy (average return volume 5.5 ml). The lavage fluid was stored at
70°C. The abdomen was then opened, and urine was aspirated from the
bladder and stored at
70°C. Hearts were then removed via midline
thoracotomy for ex vivo perfusion.
To measure intrinsic RV and left ventricular (LV) heart function, we
isolated and perfused hearts in Langendorff mode as previously described (36). Hearts were rapidly excised and
immediately placed in ice-cold, modified Krebs-Henseleit-bicarbonate
buffer made with distilled, deionized water and containing (in mM) 118 NaCl, 4.7 KCl, 21 NaHCO3, 1.25 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, and 11 D-glucose. Total Na+ concentration was 140 mM,
and total K+ concentration was 5.6 mM. Buffer was filtered
through Millipore (Millipore, Bedford, MA) paper before use. Buffer was
saturated by bubbling with 95% O2 and 5% CO2,
which produced PO2 = 600-650 Torr and
PCO2 = 35-40 Torr. Within 30 s
of removal, hearts were perfused with Krebs-Henseleit-bicarbonate
buffer (37°C) using the Langendorff technique and 60-mmHg retrograde
aortic perfusion pressure. Immediately after perfusion was initiated,
the pulmonary artery was incised to allow free ejection from the RV,
and a stab incision was made in the LV apex to allow the LV thebesian
venous drainage. Initial coronary flow was determined immediately after the incisions. This measurement was performed 1 min after perfusion began and before placement of balloons in the ventricles. Hearts were
instrumented to measure LV and RV pressures. Briefly, latex balloons,
attached to PE-60 tubing, were then placed via the mitral valve and
pulmonary valve into the LV and RV, respectively. Both balloons were
simultaneously filled with water until end-diastolic pressure equaled
zero in both ventricles. Each balloon was pretested to determine its
threshold distension volume (i.e., volume that would raise the static
pressure of a balloon >0 mmHg). A proper-sized balloon was used to
ensure that the balloon was not filled over its distension volume.
Balloon pressures were measured with a Gould P28 pressure transducer
(Gould Electronics, Millersville, MD). Approximately 15 min after
unpaced measurements, a platinum needle was inserted into the LV apex,
and hearts were electrically paced (300 beats/min, using 5-ms duration,
and voltage set at two times the pacing capture threshold) using a
Grass SD9 simulator (Astro-Med, West Warwick, RI).
Urine and BAL from all animals were collected and stored at
70°C
before use. Samples were used after one thaw. BAL samples were
centrifuged at 10,000 rpm for 10 min after thawing, and supernatants were used for enzyme immunoassay testing. Prostaglandins and
leukotrienes were measured by commercially available enzyme-linked
immunoassays (Cayman Chemical), which have shown excellent
correlation with gas chromatography/mass spectrometry
measurements in Sep-Pak-purified rat lung homogenates
(42). Samples were acidified to a pH of 4.0 with 1.0 M
acetate buffer. We activated a SEP C18 (Peninsula Laboratories,
Belmont, CA) column by rinsing with 5 ml of methanol and then with 5 ml
of ultrapure water. We first purified the samples to remove cross
contaminants through a SEP C18 by passing them through the column then
rinsing the column with 5 ml of ultrapure water and 5 ml of hexane
(Peninsula Laboratories), using materials and procedures described in
detail in the pamphlet provided with each kit by Cayman Chemical.
Elution was performed with vacuum assistance if necessary. Samples were
dried under nitrogen stream. Dried samples were stored in covered tubes
for a maximum of 24 h before final analysis. Urine was
tested for the concentration of bicyclo-PGE2 (catalog no.
514531), PGF2
(catalog no. 516011), and 2,3-dinor
TxB2 (catalog no. 519051). 2,3-Dinor TxB2 was
measured in the urine because it is a stable metabolite that gives a
better indication of systemic thromboxane production than measurement of TxA2 or -B2. Purified BAL samples were
tested for concentrations of TxB2 (catalog no. 519031) and
cysteinyl-leukotriene C4, D4, and
E4 concentrations (catalog no. 520501). The leukotriene
assay was selected for its specificity for leukotrienes C4,
D4, and E4, because these compounds are more
potent constrictors of vascular smooth muscle than is leukotriene
B4 (28).
Pulmonary angiography.
Pulmonary vascular occlusion and right heart function was assessed by
real-time pulmonary angiography on 12 rats separate from the 36 rats
that underwent physiological and biochemical analyses. This was done
because pulmonary angiography required a large volume of intrapulmonary
contrast, which could affect physiological measurements and prostanoid
production in the rat lung (24). Contrast-enhanced
pulmonary arteriography was performed with real-time C-arm fluoroscopy
with cineangiography (Optimus M-200; Phillips Medical Systems, Shelton,
CT) and incorporating a modification of the technique described by
Wegenius et al. (41). PE was induced, and treatments were
given with the same protocol described above. Sixteen hours after
induction of PE, rats were anesthetized, and the right jugular vein was
dissected and cannulated with PE-60 tubing. Spontaneously breathing,
anesthetized rats were taken to the fluoroscopy suite. The tip of the
PE-60 tubing was advanced under fluoroscopic guidance to touch but not
traverse the tricuspid valve. One milliliter of iopamidol contrast
agent (Isovue 250; Bracco Diagnostics, Princeton, NJ) was injected over 5 s while high resolution cineangiographic images were obtained in
the anteroposterior views. The animal was repositioned, and repeat
injections were performed to obtain lateral and then posterior-anterior views. Developed films were viewed on a cineangiographic projector (Vanguard XR-350; Vanguard Instrument, New York, NY) by a
blinded observer to determine the single frame at which maximal
opacification was observed and to obtain images for the manuscript and
to grade the impairments in right heart function and lung perfusion. On the basis of pilot work, the observer graded the severity on a scale of
1-3 (1 = unchanged, 2 = moderately impaired, and 3 = severely impaired compared with images from healthy rats) of the following parameters: 1) RV impairment (dilation, reduced
filling, and hypokinesis), 2) degree of reversed flow
(reflux) of contrast across the tricuspid valve into the inferior vena
cava, 3) relative reduction in maximal lung opacification,
and 4) delay in return of contrast to the LV after injection.
Measurement of pleural effusion, lung tissue water content, and
pulmonary histology.
Six rats were subjected to the PE procedure as described above, but no
treatment was given. Six healthy rats were used for comparison. Animals
were anesthetized, and a celiotomy was performed. We used a
battery-operated electrocautery device to cauterize a 3-mm hole in the
midline of the ventral diaphragm and took care not to touch the lung. A
flexible polyvinyl catheter attached to a suction bulb was passed
through the hole to aspirate fluid found in the thorax. The volume of
the fluid was then measured. The chest was then opened via midline
thoracotomy, and the lungs were freeze-clamped in situ with aluminum
tongs cooled in liquid nitrogen. Powdered frozen wet lung tissue was
weighed and dried overnight at 90°C and reweighed to calculate the
wet-to-dry ratio from which the total lung water content was measured.
For histology, six additional rats underwent the PE protocol, and after
anesthesia, both lungs and the heart were removed and placed in 10%
buffered formalin solution. The organs were sent to a commercial
histology laboratory (Histoserve, Vienna, VA) for fixation and slide
preparation with hematoxylin and eosin staining. Hearts were sectioned
in two planes in the long axis to show a four-chamber view, and lungs were sectioned in two planes in the long axis.
Statistical analyses and sample size calculation.
Data are presented as means with standard error. Data were compared for
significance between the three groups using a one-way ANOVA with
Tukey-Kramer post hoc test with
= 0.05. All statistical tests
were performed with StatsDirect software, version 1.2.2. In accordance
with IACUC requirements, we calculated the minimum sample size required
to test the hypothesis that pharmacological reduction in production of
vasoconstrictor prostaglandins would improve
VD/VTalv and RV function following PE. In a
pilot study, we found that rats subjected to PE (0.15 ml/100 g of 10%
polystyrene microspheres of mean diameter 24 µm) demonstrated a mean
increase in VD/VTalv to 0.30 ± 0.15 (SD)
compared with healthy rats 0.08 ± 0.06 (SD). Assuming that a 50%
improvement in VD/VTalv represented an
important change, at an 80% power to detect this difference with
= 0.05, we estimated the sample size at n = 9. Rats were studied according to a preset randomization schedule. Rats
were assigned to treatments based on a computer-generated random
sequence. An independent monitor determined when nine surviving rats
from each treatment group were studied to completion of data collection.
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RESULTS |
The survival rate 16 h after induction of PE in rats treated
with saline was 9/15 (60%, 95% CI: 32-84%), and the survival rate after furegrelate and ketorolac treatment was identical, at 9/11
(82%, 95% CI: 48-98%).
Effect of PE and treatment on prostanoid production.
BAL samples from surviving rats subjected to PE 16 h before
demonstrated a significant increase in TxB2 and leukotriene
C4, D4, and E4 concentrations
compared with lavage samples from healthy controls (Fig. 3,
A and B). Treatment
with both furegrelate and ketorolac significantly reduced the
TxB2 concentrations compared with placebo. However, neither
treatment was associated with a significant change in
cysteinyl-leukotriene C4, D4, and
E4 concentrations, indicating that blockade of
prostaglandin synthesis at either the COX-1/2 or the thromboxane
synthase enzyme did not lead to a significant shunt in arachidonate
toward the synthesis of leukotriene C4, D4, and
E4.

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Fig. 3.
A: BAL concentration of thromboxane (Tx)
B2. B: BAL concentrations of
cysteinyl-leukotrienes (C4, D4, and
E4). Measurements were obtained in 6 healthy rats (Control)
and rats subjected to PE 16 h previously and treated with 2 injections of saline (Placebo), furegrelate sodium (Furegrelate), or
ketorolac tromethamine (Ketorolac). All rats were instrumented with
catheters for hemodynamic monitoring and were mechanically ventilated
via tracheostomy at the time of measurements (n = 9 per
group). *P < 0.05 vs. control; **P < 0.05 vs. placebo; 1-way ANOVA, Tukey-Kramer post hoc.
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Figure 4, A-C,
demonstrates the concentrations of 2,4-dinor TxB2,
PGE2, and PGF2
found in urine aspirated from
the bladder 16 h after induction of PE. Rats subjected to PE
demonstrated approximately a threefold increase in urinary 2,4-dinor
TxB2 concentration but no increase in PGE2.
With both furegrelate and ketorolac treatments, the urinary 2,4-dinor
TxB2 concentrations were decreased. Ketorolac, but not
furegrelate, caused a decrease in urinary PGE2 and
PGF2
concentration. These data reflect the fact that
ketorolac causes proximal inhibition of prostaglandin synthesis.
Furegrelate treatment also caused a decrease in 2,4-dinor
TxB2 concentration but did not lead to an increase in
either PGE2 or PGF2
, as might be predicted
based on blockade of thromboxane synthase (site B in Fig.
1).

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Fig. 4.
Prostaglandin concentrations in the urine;
n = 9 per group. A: 2,3-dinor
TxB2, a stable metabolite of TxB2.
B: PGE2. C: PGF2 .
*P < 0.05 vs. control; **P < 0.05 vs.
placebo; 1-way ANOVA, Tukey-Kramer post hoc.
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Table 1 demonstrates in vivo hemodynamic
data measured in surviving rats 16 h after induction of PE
(n = 9 per group). Means from two 1-min sample periods
(the first obtained at 15 min and the second at 45 min after injection
of succinylcholine) were averaged to give one number for each parameter
in Table 2. Compared with healthy
controls, rats subjected to PE and treated with saline demonstrated
significant arterial hypotension (P = 0.007), increased arterial lactate concentration, but no significant change in RAP. Treatment with both furegrelate and ketorolac increased the MAP to a
level that was not different from control, but only rats treated with
ketorolac demonstrated a significant improvement in MAP compared with
placebo. Treatments did not significantly alter arterial lactate
concentrations. These data suggest that neither treatment completely
ameliorated the total-body perfusion defect from the PE insult, but
ketorolac treatment was associated with a slightly higher peripheral
vascular resistance, leading to the higher arterial pressures.
Table 2 presents arterial blood gas and end-tidal partial pressure data
for the four groups. Rats subjected to PE demonstrated a significant
decrease in peak partial pressure of carbon dioxide measured at
end-tidal respiration (PetCO2) and an increase in the nadir
end-tidal partial pressure of oxygen measurement (PetO2), coincident with increased VD/VTalv (Fig.
5A) and significantly increased the difference between the PetO2 and the arterial
PO2 (Fig. 5B). Rats treated with
ketorolac and furegrelate after PE did not show significant change in
these parameters when compared with control rats. All rats with PE
tended to have a lower VCO2, and this effect
reached statistical significance in the placebo and furegrelate groups.
This reduction in VCO2 may have offset any
increase in VD/VTalv, preventing a significant
increase in PaCO2. Treatment with both furegrelate and
ketorolac decreased the VD/VTalv significantly
compared with placebo. Both treatments tended to improve the
PetCO2, PetO2, and the
PetO2-to-arterial PO2 gradient,
although none of the changes were statistically significant when
compared with placebo rats. Neither furegrelate nor ketorolac treatment
significantly altered arterial blood PO2 or
SaO2% compared with placebo.

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Fig. 5.
Indexes of gas exchange; n = 9 per group.
A: the alveolar dead space fraction
(VD/VTalv). Both furegrelate and ketorolac
significantly decreased this measurement. B: the
PetO2-arterial PO2 gradient.
*P < 0.05 vs. control; **P < 0.05 vs.
placebo; 1-way ANOVA, Tukey-Kramer post hoc.
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Figure 6 shows the RV and LV systolic
pressure data from ex vivo beating hearts. Hearts from rats treated
with placebo demonstrated a significant reduction in both RV and LV
systolic function. Both furegrelate and ketorolac treatments were
associated with significantly improved RV systolic function. Ketorolac,
but not furegrelate treatment, was associated with improved LV systolic
function.

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Fig. 6.
Measurements of intrinsic cardiac function in hearts that
were removed from rats and perfused ex vivo; n = 9 per
group. A: right ventricular systolic pressure (RVSP).
B: left ventricular systolic pressure (LVSP).
*P < 0.05 vs. control; **P < 0.05 vs.
placebo; 1-way ANOVA, Tukey-Kramer post hoc.
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Pulmonary angiographic data.
Photographs in Fig. 7
show typical results of pulmonary angiography
performed on a healthy control rat (Fig. 7A) and three treatment rats, each studied 16 h after induction of PE. The
photographs shown are from the frame that demonstrated maximal lung
opacification in the anteroposterior view. Table
3 summarizes the results of the pulmonary
angiography. Rats treated with placebo showed significant impairment in
all parameters graded in reference to controls. The images from placebo
rats demonstrated severe RV hypokinesis with severe regurgitation of
contrast into the vena cavae and slowed forward flow of contrast into
the lungs. As a result of the dilution of the contrast from the
tricuspid regurgitation as well as the slow forward flow, the maximal
opacification of the lungs in placebo rats was reduced when compared
with controls. Rats treated with furegrelate demonstrated a tendency to
have greater lung opacification, mostly observed in the "return" or capillary phase during the period when contrast was returning to the
LV. However, furegrelate-treated rats demonstrated moderate-to-severe reflux of contrast into the inferior vena cava and delayed LV opacification. Rats treated with ketorolac demonstrated mild RV dilation, slightly reduced peripheral lung opacification, no visible reflux of contrast into the inferior vena cava, and normal return of
contrast to the LV.

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Fig. 7.
Images are digitized single frames of anteroposterior
views photographed from real-time cineangiofluoroscopy of rats that
received 1.0 ml of iodinated contrast injected into the right atrium.
Each image corresponds to the frame of maximal lung opacification
within 60 s after contrast injection. A: from a
healthy, anesthetized rat. The right ventricle is fully opacified and
appears crescent-shaped. B: from a placebo rat that received
pulmonary vascular occlusion 16 h previously with polystyrene
microspheres as described in METHODS. Photograph
B demonstrates 3 of 4 key abnormalities induced by untreated PE,
including regurgitation of contrast into the inferior vena cava
(arrow), impaired right ventricular filling, with a globular appearance
to the right ventricle (arrowheads), and slightly decreased peripheral
lung opacification (bracket). The photograph does not adequately
demonstrate the abnormality of delayed opacification of the left
ventricle. C: effect of furegrelate treatment, which
produced bright opacification that was best observed during the return
of contrast to the left ventricle, during the so-called venous return
phase. D: effect of ketorolac treatment, including a
relatively normal right ventricle and the absence of tricuspid
regurgitation but with slightly diminished peripheral lung
opacification.
|
|
Assessment of pleural effusion, lung water content, and histology.
In six control rats, no measurable pleural effusion volume was
recovered. In six rats subjected to the PE protocol and no treatment,
the volume of pleural effusion after 16-h duration was 8.5 ± 0.9 ml. Lung water content was not different in control rats (left lungs
79.4 ± 0.34%, right lungs 77.9 ± 0.70%) vs. rats subjected to PE (left lungs 79.1 ± 0.2%, right lungs 79.2 ± 0.32%). Lung sections demonstrated homogenous distribution of the
polystyrene microspheres in the prealveolar arterioles in both lungs.
No polystyrene microspheres were observed in the coronary vasculature.
 |
DISCUSSION |
This study was conducted to determine whether inhibition of
prostaglandin synthesis at either site A or B in
Fig. 1 would have a beneficial effect on pulmonary gas exchange and
cardiac function during fixed, prolonged pulmonary vascular occlusion in the absence of infused autologous clot.
We tested the hypothesis that inhibition of the thromboxane synthase
enzyme would be more beneficial than inhibition of the COX-1/2 enzyme
during pulmonary vascular occlusion.
The urine and BAL prostaglandin concentrations indicate that both
treatments successfully inhibited their pharmacological targets.
Ketorolac treatment produced significant reduction in PGF2
, PGE2, and TxB2 in the
urine but did not cause leukotriene C4, D4, and
E4 concentrations to increase significantly in BAL samples.
Furegrelate also decreased TxB2 concentrations in BAL but
did not increase PGF2
concentrations in urine. Our
hypothesis was that thromboxane synthase inhibition (see Fig. 1) might
produce a beneficial pattern of decreased thromboxane concentrations
and increased concentrations of vasodilatory prostaglandins
PGE2 and PGI, whereas ketorolac might uniformly decrease
all prostaglandin concentrations and, therefore, be less efficacious.
This hypothesis was formed primarily on theoretical grounds, because
prior experimental evidence in a prolonged model of pulmonary vascular
occlusion was lacking (9, 26, 39). On the basis of
analysis of the physiological and angiographic data in this study, it
appears that COX-1/2 inhibition demonstrated at least equal efficacy to thromboxane synthase inhibition comparing the end points of pulmonary gas exchange and RV function and possibly better efficacy on LV function and MAP.
The second question was whether inhibition of prostaglandin synthesis
at either site A or B (Fig. 1) could exert a
beneficial effect in the setting of nonthrombotic, fixed pulmonary
vascular occlusion. Prior experimental models of PE that have either
measured or pharmacologically altered prostaglandin concentrations
included autologous clot infusion (9, 26, 30, 39). These
animal models have found short-lived increase in plasma thromboxane
concentrations (30, 39) and conflicting results as to
whether pretreatment with COX-1/2 inhibition is beneficial (26,
30) or detrimental (9) to gas exchange and
pulmonary vascular resistance (9). The use of autologous
clot introduces two variables that are difficult to control. First,
fresh clots, per se, produce vasoactive metabolites (13).
Thus with fresh clots, it is difficult to determine if pharmacological
inhibition of prostaglandin synthesis is affecting production of
prostaglandins from the clot or production from the lung tissue.
Second, in the rat, fresh clots are dissolved within a few hours by
endogenous fibrinolysis (21). This effect could account
for the observation of an ephemeral increase in plasma thromboxane
concentrations with autologous clot models. In humans, thromboembolism
to the lung almost always occurs from mature, organized thromboses that
produce more persistent pulmonary vascular occlusion (40).
In humans with major pulmonary thromboembolism (mean baseline perfusion
defect
40%), anticoagulation with heparin relieves only 0-5%
of the perfusion defect after 24 h, whereas fibrinolytic treatment
relieves only 5-15% of the perfusion defect after 24 h
(6, 12, 18, 27, 31). Moreover, the diagnosis of PE is
often not discovered until 24 h after symptom onset
(37). These data may help explain why humans diagnosed
with PE maintain a significant increase in plasma TxB2 and
PGF2
concentrations for up to 7 days after symptom onset
(10).
For these reasons, we used polystyrene microspheres rather than
infusing autologous clots. The concentration and size of the polystyrene microspheres were constant, whereas in prior work, we found
that the concentration and size of blood-derived clots were difficult
to standardize (36). Microscopic examination revealed that
the microspheres were uniformly distributed in precapillary lung
arterioles 16 h after dosing and that no microspheres were seen in
the heart vasculature, suggesting the absence of embolization to the
coronary arteries (as could be speculated if a patent foramen ovale
were present). Microscopic examination of lungs containing the
microspheres demonstrated that the pulmonary arteries were free of
thrombotic material. The present model allows us to conclude that
inhibition of COX-1/2 and thromboxane synthase after the induction of
PE improved VD/VTalv and RV function by action
on nonembolized vasculature, as opposed to recanalization of embolized vasculature.
The data show that both COX-1/2 inhibition and thromboxane synthase
inhibition reduced VD/VTalv without a
significant change in arterial blood oxygenation compared with
placebo-treated rats. This finding has mechanistic and therapeutic
implications specific to PE. The present model produces primarily
pulmonary vascular occlusion without an increase in lung water content,
suggesting the absence of pulmonary edema. In contrast, most
inflammatory models of acute lung injury are designed to produce
increased transcapillary leak, resulting in alveolar edema, and
increased shunt fraction. In an edematous model of lung injury produced by infusion of oleic acid, Schulman et al. (33) showed
that meclofenamate improved oxygenation by promoting redistribution of
lung perfusion away from injured lung toward normal lung. By enhancing
perfusion redistribution, meclofenamate improved hypoxemia by
decreasing the fraction of the cardiac output that perfused the poorly
ventilated, edematous lung (i.e., decreased the shunt fraction). In
contrast, Dantzker et al. (7) demonstrated that induction
of PE in dogs causes arterial hypoxemia by redistribution of pulmonary
blood flow away from the occluded vasculature, producing regions of
nonoccluded lung with low ventilation-perfusion relationships, leading
to increased venous admixture and hypoxemia. With this in mind, we were
concerned that either treatment might reduce the effect of
TxB2 in nonoccluded lung regions, thus producing a
trade-off of increased flow through nonoccluded vasculature, but at the
expense of increased venous admixture and worsened hypoxemia. In fact,
neither treatment worsened blood oxygenation. Both treatments
reduced VD/VTalv and the degree of RV dilation (and tricuspid regurgitation was observed on pulmonary angiography) and
improved RV function during ex vivo perfusion. Both treatments ameliorated the increase in TxB2 concentration found in the
BAL. Together, these findings suggest that, after pulmonary vascular occlusion in our model, TxB2 produced mostly a detrimental
effect on the balance of pulmonary vasoconstriction and dilation and that inhibition of its synthesis did not cause worsened hypoxemia.
Several authors have suggested that a controlled trial of a COX
inhibitor to treat humans with PE can be justified on the basis of
previously published literature (23, 29, 35). We believed
that a randomized, blinded treatment study of treatment efficacy in
animals was needed before further clinical testing. Because bias can
occur in drug testing even in animal models (2), we used a
study protocol that employed blinding and randomization to reduce this
potential bias. We interpret the present findings as firm
evidence that neither ketorolac nor furegrelate treatment was harmful
to rats subjected to fixed, pulmonary vascular occlusion.
Several aspects of this model warrant critical consideration. The size
of the microspheres was relatively small, causing occlusion of
precapillary arterioles, rather than segmental or lobar vascular occlusion, which is the usual circumstance in humans (8).
We were also unable to provide any direct evidence of pulmonary
vasospasm in nonoccluded vasculature. In pilot work, we attempted to
measure pulmonary arterial pressure and pulmonary vascular resistance but found that these measurements were extremely labile, and the rats
with PE developed arterial hypotension with introduction of a
micromanometer into the RV. For this reason, we used pulmonary angiography to demonstrate visual evidence of increased pulmonary vascular resistance. The finding that RAP 16 h after induction of
PE did not increase in vivo in placebo rats might appear somewhat contrary, given that severe tricuspid regurgitation was observed on
pulmonary angiography. When measured immediately after the infusion of
polystyrene beads, the RAP increased by ~50% (5). In
view of the severe depression in ex vivo function in placebo rats
studied 16 h after PE induction, together with the observation of
RV hypokinesis during angiography, we speculate that the RAP data in
the placebo rats represent pseudonormalization secondary to the
inability of the damaged myocardium to generate enough systolic
contraction to compress the right atrium in dehydrated animals. Animals
in all treatment groups were also pretreated with succinylcholine to
facilitate accurate collection of physiological parameters. The effect
of succinylcholine on prostaglandin metabolism in this model remains
uncertain, but we can find no evidence to suggest that succinylcholine
affects the production of eicosanoids. Finally, this model produced
large pleural effusions that could have compromised ventilation. The
etiology of the pleural effusions and the role of treatment in reducing
pleural effusion as a possible mechanism of efficacy in this model are
currently under study.
In summary, this experiment presents data from a randomized, blinded
animal study that demonstrates the presence of increased concentrations
of TxB2 in the BAL and 2,3-dinor TxB2 in the
urine 16 h after induction of pulmonary vascular occlusion with
polystyrene microspheres. Posttreatment with a nonselective COX-1/2
inhibitor, ketorolac, decreased the concentrations of TxB2
in BAL and PGF2
and PGE2 in urine; it was
associated with a significant decrease in measured
VD/VTalv and improved in vivo MAP and ex vivo
RV function. Selective inhibition of thromboxane synthase with
furegrelate decreased BAL TxB2 concentrations and
significantly decreased the VD/VTalv and
improved ex vivo RV function. These data show that neither treatment
was detrimental to the treatment of PE and that COX-1/2 inhibition was
at least as effective as thromboxane synthase inhibition in a rat model
of fixed pulmonary vascular occlusion.
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
J. A. Kline, Dept. of Emergency Medicine, Carolinas Medical
Center, 1000 Blythe Blvd., Charlotte, NC 28203 (E-mail:
jkline{at}carolinas.org).
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. Section 1734 solely to indicate this fact.
First published March 14, 2003;10.1152/ajplung.00283.2002
Received 15 August 2002; accepted in final form 29 January 2003.
 |
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