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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 PGF2alpha 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.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 1.   Flow diagram of arachidonate metabolism showing the steps where ketorolac and furegrelate inhibit prostaglandin metabolism.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 2.   Timeline for the experimental protocol. See METHODS for description of interventions. PE, pulmonary embolism; BAL, bronchoalveolar lavage.

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), PGF2alpha (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 alpha  = 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 alpha  = 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (18K):
[in this window]
[in a new window]
 
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.

Figure 4, A-C, demonstrates the concentrations of 2,4-dinor TxB2, PGE2, and PGF2alpha 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 PGF2alpha 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 PGF2alpha , as might be predicted based on blockade of thromboxane synthase (site B in Fig. 1).


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 4.   Prostaglandin concentrations in the urine; n = 9 per group. A: 2,3-dinor TxB2, a stable metabolite of TxB2. B: PGE2. C: PGF2alpha . *P < 0.05 vs. control; **P < 0.05 vs. placebo; 1-way ANOVA, Tukey-Kramer post hoc.

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.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   In vivo hemodynamic data and arterial lactate concentrations


                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Respiratory and arterial blood gas measurements

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.


View larger version (17K):
[in this window]
[in a new window]
 
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.

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.


View larger version (20K):
[in this window]
[in a new window]
 
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.

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.


View larger version (53K):
[in this window]
[in a new window]
 
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.


                              
View this table:
[in this window]
[in a new window]
 
Table 3.   Pulmonary angiography data

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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 PGF2alpha , 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 PGF2alpha 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 PGF2alpha 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 PGF2alpha 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Arnold, JH. Measurement of the alveolar deadspace: are we there yet? Crit Care Med 29: 1287-1288, 2001[ISI][Medline].

2.   Bebarta, V, Luyten D, and Heard K. Emergency medicine animal research: does lack of blinding or randomization bias the results (Abstract). Acad Emerg Med 9: 484, 2002[Abstract].

3.   Chida, M, and Voelkel NF. Effects of acute and chronic hypoxia on rat lung cyclooxygenase. Am J Physiol Lung Cell Mol Physiol 270: L872-L878, 1996[Abstract/Free Full Text].

4.   Courtney, DM, and Kline JA. Identification of clinical factors associated with outpatient massive pulmonary embolism. Acad Emerg Med 8: 1136-1142, 2001[Abstract/Free Full Text].

5.   Courtney, DM, Watts JA, and Kline JA. End-tidal CO2 is reduced during hypotension and cardiac arrest in a rat model of massive pulmonary embolism. Resuscitation 53: 83-91, 2002[ISI][Medline].

6.   Daniels, LB, Parker JA, Patel SR, Grodstein F, and Goldhaber SZ. Relation of duration of symptoms with response to thrombolytic therapy in pulmonary embolism. Am J Cardiol 80: 184-188, 1997[ISI][Medline].

7.   Dantzker, DR, Wagner PD, Tornabene VW, Alazraki NP, and West JB. Gas exchange after pulmonary thromboembolization in dogs. Circ Res 42: 92-103, 1978[Abstract].

8.   De Monyé, W, van Strijen MJL, Huisman MV, Kieft GJ, and Pattynama PMT Suspected pulmonary embolism: prevalence and anatomic distribution in 487 consecutive patients. Advances in new technologies evaluating the localisation of pulmonary embolism (ANTELOPE) group. Radiology 215: 184-188, 2000[Abstract/Free Full Text].

9.   Delcroix, M, Melot C, Lejeune P, Leeman M, and Naeije R. Cyclooxygenase inhibition aggravates pulmonary hypertension and deteriorates gas exchange in canine pulmonary embolism. Am Rev Respir Dis 145: 806-810, 1992[ISI][Medline].

10.   Friedrich, T, Lichey J, Nigam S, Maiga M, Schulze G, Wegscheider K, and Priesnitz M. Prostaglandin production in patients with pulmonary embolism. Biomed Biochim Acta 43: 409-412, 1984.

11.   Goldhaber, SZ. Pulmonary embolism thrombolysis improves survival in massive pulmonary embolism. J Thromb Thrombolysis 2: 219-220, 1995[Medline].

12.   Goldhaber, SZ, Haire WD, Feldstein ML, and Miller M. Alteplase versus heparin in acute pulmonary embolism: randomised trial assessing right-ventricular function and pulmonary perfusion. Lancet 341: 507-511, 1993[ISI][Medline].

13.   Gurewich, V, Cohen ML, and Thomas DP. Humoral factors in massive pulmonary embolism: an experimental study. Am Heart J 76: 784-794, 1968[ISI][Medline].

14.   Hardman, JG, and Aitkenhead AR. Estimation of alveolar deadspace fraction using arterial and end-tidal CO2: A factor analysis using a physiological simulation. Anaesth Intensive Care 27: 452-458, 1999[ISI][Medline].

15.   Kasper, W, Konstantinides S, Geibel A, Olschewski M, Heinrich F, Grosser KD, Rauber K, Iversen S, Redecker M, and Kienast J. Management strategies and determinants of outcome in acute major pulmonary embolism: results of a multicenter registry. J Am Coll Cardiol 30: 1165-1171, 1997[ISI][Medline].

16.   Kleny, R, Charpentler A, and Kleny M. What is the place of pulmonary embolectomy today? J Cardiovasc Surg (Torino) 32: 549-554, 1991[ISI][Medline].

17.   Kline, JA, Kubin AK, Patel MM, Easton EJ, and Seupal RA. Alveolar deadspace as a predictor of severity of pulmonary embolism. Acad Emerg Med 7: 611-617, 1999[ISI].

18.   Levine, M, Hirsh J, Weitz J, Cruickshank M, Neemah J, Turple A, and Gent M. A randomized trial of a single bolus dosage regimen of recombinant tissue plasminogen activator in patients with acute pulmonary embolism. Chest 98: 1473-1479, 1990[Abstract].

19.   McIntyre, KM, and Sasahara AA. The hemodynamic response to pulmonary embolism in patients without prior cardiopulmonary disease. Am J Cardiol 28: 288-295, 1971[ISI].

20.   Meyer, G, Sors H, Charbonnier B, Kasper W, Bassand JP, Kerr IH, Lesaffre E, Vanhove P, and Verstraete M. Effects of intravenous urokinase versus alteplase on total pulmonary resistance in acute massive pulmonary embolism: a European multicenter double-blind trial. J Am Coll Cardiol 19: 239-245, 1992[ISI][Medline].

21.   Murciano, JC, Harshaw D, Neschis DG, Koniaris L, Bdeir K, Medinilla S, Fisher AB, Golden MA, Cines DB, Nakada MT, and Muzykantov VR. Platelets inhibit the lysis of pulmonary microemboli. Am J Physiol Lung Cell Mol Physiol 282: L529-L539, 2002[Abstract/Free Full Text].

22.   Nikodymova, L, Daum S, Stiksa J, and Widimsky J. Respiratory changes in thromboembolic disease. Respiration 25: 51-66, 1968[ISI][Medline].

23.   O'Brien, J, Duncan H, Kirsh G, Allen V, King P, Hargraves R, Mendes L, Perera T, Catto P, Schofield S, Ploschke H, Hefner T, Churchland M, Woolnough S, Wuttke R, Manning M, Jeffries T, Hensley L, Bath P, Bainbridge D, Guinane F, McMahon L, Zavattaro D, and Wilson D. Prevention of pulmonary embolism and deep vein thrombosis with low dose aspirin: pulmonary embolism prevention (PEP) trial. Lancet 355: 1295-1302, 2000[ISI][Medline].

24.   Paajanen, H. The effect of ionic and nonionic contrast media on the metabolism of prostaglandin E2 in rat lungs. Invest Radiol 19: 216-220, 1984[ISI][Medline].

25.   Paoletti, P, Fornai E, Giannella NA, Prediletto R, Ruschi S, Pisani P, and Giuntini C. The assessment of gas exchange by automated analysis of O2 and CO2 alveolar to arterial differences. Int J Clin Monit Comput 3: 89-97, 1986[Medline].

26.   Perlman, M, Johnson A, and Malik A. Ibuprofen prevents thrombi-induced lung vascular injury: mechanism of effect. Am J Physiol Heart Circ Physiol 252: H605-H614, 1987[Abstract/Free Full Text].

27.   PIOPED Investigators. Tissue plasminogen activator for the treatment of acute pulmonary embolism. Chest 97: 528-533, 1990[Abstract].

28.   Piper, PJ. Formation and actions of leukotrienes. Physiol Rev 64: 744-761, 1985[ISI].

29.   Poullis, M. Aspirin for the treatment of pulmonary embolism: vasoconstriction versus physical obstruction. Am Heart J 140: E22, 2000[Medline].

30.   Reeves, WC, Demers LM, Wood MA, Skarlatos S, Copenhaver G, Whitesell L, and Luderer JR. The release of thromboxane A2 and prostacyclin following experimental acute pulmonary embolism. Prostaglandins Leukot Med 11: 1-10, 1983[ISI][Medline].

31.   Sasahara, AA, Hyers TM, and Cole CM. The urokinase pulmonary embolism trial: a national cooperative study. Circulation 47, Suppl 2: II66-II89, 1973.

32.   Schmitz-Rode, T, Janssen U, Duda S, Erley C, and Gunther RW. Massive pulmonary embolism: percutaneous emergency treatment by pigtail rotation catheter. J Am Coll Cardiol 36: 375-380, 2000[ISI][Medline].

33.   Schulman, LL, Lennon PF, Ratner SJ, and Enson Y. Meclofenamate enhances blood oxygenation in acute oleic acid lung injury. J Appl Physiol 64: 711-718, 1988.

34.   Schuster, DP, Stephenson AH, Holmberg S, and Sandiford P. Effect of eicosanoid inhibition on the development of pulmonary edema after acute lung injury. J Appl Physiol 80: 915-923, 1996[Abstract/Free Full Text].

35.   Smulders, Y. Pathophysiology and treatment of haemodynamic instability in acute pulmonary embolism: the pivotal role of pulmonary vasoconstriction. Cardiovasc Res 48: 23-33, 2000[ISI][Medline].

36.   Sullivan, DM, Watts JA, and Kline JA. Biventricular cardiac dysfunction after massive pulmonary embolism in the rat. J Appl Physiol 90: 1648-1656, 2001[Abstract/Free Full Text].

37.   Susec, O, Boudrow D, and Kline J. The clinical features of acute pulmonary embolism in ambulatory patients. Acad Emerg Med 4: 891-897, 1997[Abstract].

38.   Tegeder, I, Neupert W, Guhring H, and Geisslinger G. Effects of selective and unselective cyclooxygenase inhibitors on prostanoid release from various rat organs. J Pharmacol Exp Ther 292: 1161-1168, 2000[Abstract/Free Full Text].

39.   Utsonomiya, T, Krausz MM, Levine L, Shepro D, and Hechtman HB. Thromboxane mediation of cardiopulmonary effects of embolism. J Clin Invest 70: 361-368, 1982[ISI][Medline].

40.   Wagenvoort, CA. Pathology of pulmonary thromboembolism. Chest 107: 10S-17S, 1995[Abstract/Free Full Text].

41.   Wegenius, G, Wegener T, Ruhn G, Saldeen T, and Erikson U. Videodensitometry in rats with pulmonary damage due to microembolism. Acta Radiol Diagn (Stockh) 26: 785-788, 1985[Medline].

42.   Westcott, JY, Chang S, Stenc D, Pradelles P, Maclouf J, Voelkel NF, and Murphy RC. Analysis of 6-keto PG1a, 5-HETE, and LTC4 in rat lung: comparison of GC/MS, RIA, and EIA. Prostaglandins 32: 857-873, 1986[Medline].


Am J Physiol Lung Cell Mol Physiol 284(6):L1072-L1081
1040-0605/03 $5.00 Copyright © 2003 the American Physiological Society




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
284/6/L1072    most recent
00283.2002v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Jones, A. E.
Articles by Kline, J. A.
Articles citing this Article
PubMed
PubMed Citation
Articles by Jones, A. E.
Articles by Kline, J. A.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2003 by the American Physiological Society.