Anesth Analg 2001;92:590-595
© 2001 International Anesthesia Research Society
CARDIOVASCULAR ANESTHESIA
The Protective Effect of Acadesine on Lung Ischemia-Reperfusion Injury
Idit Matot, MD*, and
Oded Jurim, MD
*Department of Anesthesiology and CCM and the Laboratory of Experimental Surgery; and the Department of Surgery, Hadassah University Medical Center, The Hebrew University of Jerusalem, Jerusalem, Israel
Address correspondence and reprint requests to Idit Matot, MD, Department of Anesthesiology and Critical Care Medicine, Hadassah Hebrew University Medical Center, PO Box 12000, Jerusalem 91120, Israel. Address e-mail to matoth{at}cc.huji.ac.il
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Abstract
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The purine precursor acadesine is highly effective in preventing ischemia-reperfusion (I-R) injury of the heart and intestine. The aim of this study was to test the effect of acadesine on I-R–induced lung injury. The lobar artery of the left lower lung lobe in intact-chest, spontaneously breathing cats was occluded for 2 h (Group 1, ischemia) and reperfused for 3 h (Group 2, I-R). Animals were subjected to one of the following three protocols: acadesine administered IV 15 min before ischemia (Group 3), 15 min before reperfusion (Group 4), or 30 min after reperfusion (Group 5). Acadesine was administered at an initial dose of 2.5 mg · kg-1 · min-1 for 5 min, followed by 0.5 mg · kg-1 · min-1 until the end of reperfusion. Injury was assessed by histologic examination. The right lower lobe served as control. Compared with the right lower lobe, which showed no abnormal findings in any group (percentage of injured alveoli, 2% ± 1% to 4% ± 2%), the left lower lung lobe in the I-R group revealed a disrupted alveolar structure with 63% ± 9% injured alveoli. Ischemia alone did not produce alterations in alveolar structure. Acadesine significantly reduced the number of injured alveoli when given before ischemia (4% ± 1%) or reperfusion (6% ± 2%) but not when administered after reperfusion (62% ± 8%). In conclusion, acadesine, when administered before ischemia or reperfusion, can blunt I-R–induced lung injury. The mechanism underlying the protection remains to be elucidated.
Implications: Acadesine reduces ischemia-reperfusion–induced lung injury in spontaneously breathing cats when administered before ischemia or reperfusion, but not after reperfusion.
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Introduction
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Exposure of the lung to periods of ischemia and reperfusion causes injury to the organ. The pathogenesis of this injury remains unclear (1). Numerous attempts have been made to preserve lung viability by decreasing the effects of ischemia-reperfusion (I-R) injury occurring after lung transplantation, pulmonary thromboembolectomy, or cardiopulmonary bypass (2–5). Acadesine (5-amino-4-imidazolecarboxamide riboside) is a nucleoside that belongs to a novel class of drugs called adenosine-regulating drugs (6). Acadesine increases the concentration of extracellular tissue adenosine during conditions of net adenosine triphosphate (ATP) breakdown, such as in ischemia (7). The advantage of acadesine is that its effect is localized only to the ischemic tissues and, unlike adenosine, it is safe and well tolerated IV (8). In preclinical studies, acadesine inhibited platelet aggregation and neutrophil adherence to endothelium in ischemic myocardium and decreased free radical- and oxidant-induced cardiac damage (9). In addition to its cardioprotective properties in both experimental (10–14) and human (15–17) studies, acadesine also attenuates mucosal lesions seen during intestinal ischemia and reperfusion in rats and cats (18). Furthermore, it reduces the pulmonary dysfunction associated with postraumatic endotoxemia in pigs (19). The role of acadesine in I-R injury of the lung has not been reported. Therefore, this study was designed to assess the effect of acadesine on I-R lung injury when administered IV before ischemia, before reperfusion, or after reperfusion.
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Methods
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With the approval of the Institutional Animal Care and Use Committee, adult cats weighing 2.5–4.0 kg were anesthetized with pentobarbital sodium, 20 mg/kg IV, and were strapped in the supine position to a fluoroscopic table. Additional pentobarbital sodium was given hourly (7 mg/kg). The cats spontaneously breathed room air through an endotracheal tube. Polyethylene catheters were inserted into both femoral veins and femoral arteries. With the aid of fluoroscopy and continuous pressure recording, a specially designed 6F triple-lumen catheter was advanced from the left external jugular vein into the lobar artery of the left lower lung lobe (LLL). After heparinization, the LLL was perfused at 35 mL/min with blood withdrawn from the aorta through a catheter in the femoral artery by use of a Harvard peristaltic pump. The LLL was isolated by distending a balloon with contrast dye on the LLL arterial catheter. After a 1-h period of stabilization, ischemia of the LLL was induced by discontinuing the Harvard peristaltic pump for 2 h (ischemia period), and the perfusion circuit was then attached to a femoral vein catheter. After 2 h of ischemia, the perfusion circuit was reattached to the arterial catheter in the LLL, and the LLL was reperfused (reperfusion period) for 3 h at a rate of 35 mL/min by using a Harvard peristaltic pump, as described above, with blood withdrawn from the aorta. Because flow to the lobe was constant, changes in lobar arterial pressure (LAP) reflect changes in vascular resistance. These procedures have been described by Neely and Keith (20). Mean LAP and femoral arterial pressure were measured online. Arterial blood gas tensions and pH (178pH, blood gas analyzer; Ciba-Corning, Medfield, MAAQ) were measured at frequent intervals.
Cats were divided into six experimental groups (n = 5 each): 1) ischemia; 2) ischemia and reperfusion; 3) acadesine administered IV 15 min before ischemia; 4) acadesine administered IV 15 min before reperfusion; 5) acadesine administered IV 30 min after reperfusion; and 6) nonischemic control group with the LLL perfused for 4 h. Drug treatment groups were given an initial dose of acadesine (dissolved in saline; Metabasis Therapeutics, Inc, San Diego, CA), 2.5 mg · kg-1 · min-1 IV for 5 min, followed by IV infusion of 0.5 mg · kg-1 · min-1 until the end of reperfusion. Groups 1, 2, and 6 received saline IV at the same rate. The dosing regimen for acadesine used in these experiments was that used in previous acadesine studies in which cardiac and small intestine protection was reported (6,11,18). At the end of the reperfusion period, the cats were killed, and the lungs were fixed by injection of 10% formalin through the trachea while the lungs were inflated to end-expiratory pressure of 20 cm H2O.
Lung tissue samples were obtained from the LLL and the right lower lobe (RLL), which served as a control. The samples were embedded in paraffin, cut into 4-µm slices, and stained with hematoxylin and eosin. The slides were coded and examined in a blinded manner by a single examiner. Fifty microscopic fields at 40x magnification were examined in each section, and the total number of alveoli in the 50 microscopic fields was scored. Each alveolus was examined for the presence of alveolar exudate (cellular debris and fluid in the intraalveolar space), the number of neutrophils and mononuclear cells, and the number of erythrocytes. An alveolus was defined as injured if it contained exudate, more than two leukocytes (macrophages or neutrophils), or more than two erythrocytes, as previously described (21). The severity of alveolar injury was assessed according to the percentage of injured alveoli (number of injured alveoli divided by the total number of alveoli in the 50 microscopic fields).
At the end of the experiment, LLL and RLL were excised and weighed. Lobes were dried at 80°C until lungs no longer changed their weight on successive weighing.
The results are presented as mean ± SD. Comparisons were made by using Student’s t-test and one-way analysis of variance with Newman-Keuls multiple comparison test as the post hoc test. A P value <0.05 was considered significant.
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Results
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The surgical interventions and perfusion of the LLL with a peristaltic pump had no effect on lung morphology. Neither the macroscopic nor the microscopic findings in the LLL of Group 6 (nonischemic control group) were significantly different from those observed in the RLL, in which no manipulations were performed and which served as the control lobe in all groups. We therefore compared LLL findings in each group with those observed in the corresponding RLL. In addition, the administration of acadesine had no significant effect on lung morphology of the normal, noninjured lung; the percentage of injured alveoli in the RLL of animals that received acadesine (Groups 3–5) was not significantly different from that observed in animals that did not receive acadesine (RLL, Groups 1, 2, and 6) (Table 1).
The RLLs in all groups, as well as the LLLs in the control group (Group 6), did not show any abnormal findings. The LLLs in the ischemic group were pale. In contrast, the LLLs of the I-R group showed hemorrhagic lesions extending throughout the entire lobe. No abnormal macroscopic findings were observed in the lungs subjected to I-R with IV administration of acadesine before ischemia or before reperfusion. In contrast, lungs treated with acadesine after reperfusion exhibited gross hemorrhagic foci.
The total number of alveoli examined in the LLLs was 252 ± 12, 239 ± 10, 247 ± 13, 245 ± 12, 251 ± 14, and 243 ± 16 in Groups 1–6, respectively. The corresponding number of alveoli in the control RLLs was 239 ± 13, 256 ± 15, 244 ± 14, 253 ± 18, 241 ± 13, and 250 ± 15, respectively. The dry weights of the LLLs were not significantly different among the groups: 6.1 ± 0.5, 5.8 ± 0.6, 6.5 ± 0.4, 6.4 ± 0.3, 5.9 ± 0.5, and 6.4 ± 0.4 g in Groups 1–6, respectively. Compared with Group 6 (no ischemia), the alveoli in the control lobes (RLLs) in all groups ( Fig. 1a) and in the LLLs in Group 1 (ischemia only) did not contain a significantly larger amount of injured alveoli (Table 1). In the I-R group (Fig. 1b, Table 1), the percentage of injured alveoli and alveoli with exudate and the number of leukocytes and erythrocytes in a single alveolus were significantly more compared with those in the corresponding RLL (control lobe) and those in Group 1 (ischemia only) and Group 6 (no ischemia). I-R caused marked lung edema as assessed by wet weight/dry weight ratios of the lobes (Table 1).
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Figure 1. Representative light micrographs showing structural alteration of alveolar parenchyma. a, Right lower lobe (control). b, Left lower lobe with ischemia-reperfusion injury (Group 2). c, Left lower lobe subjected to ischemia and reperfusion with acadesine administered before ischemia. Magnification 40x. Infiltration with leukocytes and red blood cells with thickening of alveolar septa is observed in tissue samples from lungs subjected to ischemia and reperfusion.
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The effects of acadesine treatment on indicators of lung injury are summarized in Table 1. IV administration of acadesine before ischemia (Group 3) prevented the changes observed during I-R–induced lung injury (Fig. 1c); variables of lung injury were similar to those observed in the uninjured RLLs and in biopsy tissue obtained from LLL Groups 1 and 6. IV administration of acadesine before reperfusion (Group 6) significantly attenuated variables of lung injury. The morphological appearance and wet/dry ratio of tissue obtained from animals given acadesine after reperfusion (Group 5) was similar to that of biopsy tissue from animals undergoing I-R without acadesine (Group 2).
There were no significant differences within or between groups in mean systemic arterial pressure at baseline, after 2 h of ischemia, or after 1 h and 3 h of reperfusion ( Table 2).
LAP remained stable over the 60 min of LLL perfusion. After reperfusion, the LAP increased significantly in all groups compared with the pressures present in the same lung before ischemia or with the LAP measured in the time-matched Group 6 lung (nonischemic), with the maximum LAP reached within 5 min of reperfusion (Table 2). This increase in LAP was followed by a gradual decrease toward the baseline value with time. At the end of reperfusion, LAP was still significantly increased (compared with baseline LAP) in Groups 2 and 5. The mean LAP, however, was not significantly different among the groups at the end of reperfusion.
In all groups, no acidosis, no significant increase in partial pressure of carbon dioxide, and no decrease in partial pressure of oxygen were observed during ischemia or reperfusion ( Table 3).
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Discussion
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The present study demonstrates that 1) the administration of acadesine before ischemia can protect the lung from reperfusion-induced injury; 2) acadesine, given before reperfusion, markedly attenuates I-R–induced lung injury, although the protection was not complete; 3) acadesine administered after reperfusion fails to attenuate lung injury caused by reperfusion; and 4) there was no evidence of drug-induced changes in vital signs. A number of aspects of our results warrant further discussion.
This study shows that occlusion of the pulmonary artery alone without interfering with ventilation in an intact-chest spontaneously breathing cat, a condition which simulates pulmonary embolism, does not produce necrosis. Lung damage, however, occurs with restoration of flow after removal of pulmonary arterial obstruction. In addition, the significant protection afforded by acadesine administered after the onset of ischemia confirms that those changes are generated by injury during reperfusion. Our results are consistent with those of previous studies indicating the ventilated I-R injury (1,21–23). Murata et al. (21) demonstrated that ischemia followed by reperfusion but with continuous oxygenation resulted in the destruction of the alveolar structure, with edema and leukocyte accumulation. Moreover, Khimenko et al. (22) found, in a model of isolated rat lung subjected to 45 minutes of nonventilated ischemia or to air-ventilated ischemia followed by 90 minutes of reperfusion and ventilation, that ventilation during ischemia exacerbated the injury in the ventilated lung. The increase in vascular permeability and tumor necrosis factor-
concentration were significantly more in the ventilated I-R group than in the nonventilated I-R group.
In this study, exposure of the lung to ischemia followed by reperfusion caused a prompt increase in LAP followed by a gradual decrease, which remained increased during the remainder of lung perfusion, compared with the baseline value. These data are consistent with previous studies reporting a rapid increase in lobar (20) and pulmonary (2,4,5,24,25) artery pressure when restoration of blood flow was introduced. Zamora et al. (25) found an increase in the thromboxane A2 levels of lung effluent after I-R lung injury in isolated perfused rabbit lungs. Moreover, the thromboxane receptor antagonist attenuated this pressor response, suggesting an etiologic role for thromboxane in the generation of pulmonary hypertension. Similarly, Jiang et al. (2) reported that pretreatment with the stable prostaglandin I2 analog inhibited the production of thromboxane and the increase in pulmonary artery pressure in blood-perfused rabbit lungs subjected to ischemia and reperfusion. Adkins and Taylor (26) demonstrated, in a model of isolated perfused rabbit lungs, that the increase in pulmonary artery pressure during reperfusion was not prevented by the addition of catalase, inhibition of xanthine oxidase, or the prevention of neutrophil adherence to endothelium, interventions that were found to protect the lung from reperfusion injury. However, they showed that compounds that increase cyclic adenosine monophosphate level (4) could prevent the transient increase in pulmonary artery pressure. In our experiments, acadesine did not prevent the increase in LAP with reperfusion. LAPs, however, returned to baseline only when treatment with acadesine was started before ischemia or before reperfusion, suggesting that the changes generated by injury during reperfusion could have contributed to the sustained increase in pressure.
The specific mechanism by which acadesine protects against lung reperfusion injury in our model is not clear. Acadesine is a nucleoside analog that enters the cell and interacts with enzymes involved in the formation and use of adenosine (6), thereby increasing local adenosine levels during conditions of net ATP breakdown, such as during ischemia (6,7). In isolated perfused rat lung subjected to ventilated ischemia and reperfusion, Fisher et al. (1) confirmed that the injury was not initiated by decreased levels of ATP. They showed that the lung tissue ATP content was essentially the same as that of controls. Therefore, it seems unlikely that the beneficial effects of acadesine observed in this study depended on the repletion of tissue ATP. Similarly, the cardioprotective properties of acadesine are not mediated through enhancement of ATP repletion in the ischemic myocardium (7,27). Acadesine may, however, protect from reperfusion lung injury by modifying the complicated cascade leading to injury (28). Because reperfusion injury is mediated, in part, by cell-adhesion molecules and by the release of oxidants, acadesine’s beneficial effect can be attributed to its ability to inhibit upregulation of adhesion receptors (9), its ability to cause a reduction in CD18 expression on lipopolysaccharide-stimulatedAQ neutrophil (19), and its ability to protect against oxidant injury (7). In the cat intestine, acadesine treatment significantly attenuated the mucosal damage seen after intestinal ischemia and reperfusion (18). The improvement was caused by the inhibition of neutrophil accumulation and activation during the reperfusion phase. In addition, others reported that acadesine is effective in reducing myocardial injury induced by ischemia and reperfusion in a variety of settings in different species (10–14) and in patients undergoing coronary artery bypass graft operations (15–17). Gruver et al. (13) reported that in the heart, acadesine inhibited platelet aggregation and neutrophil adherence to endothelium and decreased neutrophil accumulation in ischemic myocardium. Further studies are necessary to evaluate the apparent decrease in tissue injury during reperfusion of the in vivo isolated cat lung.
Acadesine in the dose and schedule tested had lung protective properties in the feline lung when administered before ischemia or reperfusion. Moreover, in this model, with the exception of bronchial blood flow, there was no blood flow to the LLL during ischemia. Because of similarities in the percentage of cardiac output carried by the feline and human bronchial circulation (4%–7%), acadesine could also be useful in preventing I-R injury of the lung when administered IV to patients before reperfusion. Nevertheless, it should be emphasized that variables such as the choice of species, the choice of dose, and the duration of ischemia, as well as the timing and the frequency of administration, may affect the results.
Our data point to several clinical implications. Reperfusion might play a role in acute pulmonary embolism and after thrombolytic therapy (29). Also, reperfusion injury might increase inflammation in adult respiratory distress syndrome, in which perfusion of the pulmonary vasculature is occluded by vasoconstriction, microemboli, or in situ thromboses (30). Although many questions remain unresolved, acadesine treatment may prove to be a promising new concept for treatment of lung I-R disorders.
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Acknowledgments
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This work was supported by a grant from the Joint Research Fund of the Hebrew University and Hadassah and by the Chief Scientist, Israel Ministry of Health, Grant 4300.
We are grateful to Simion Breitman, MD, PhD, Department of Experimental Medicine, for his evaluation of the pulmonary histological findings; to Nachum Navot for his outstanding technical assistance; and to Metabasis Therapeutics, Inc, San Diego, CA, for providing the acadesine.
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References
|
|---|
-
Fisher AB, Dodia C, Ayene I, Al-Mehdi A. Ischemia-reperfusion injury to the lung. Ann N Y Acad Sci 1994; 723: 197–207.[ISI][Medline]
-
Jiang XW, Kambara K, Gotoh N, et al. Effect of low-dose beraprost sodium, a stable prostaglandin I2 analogue, on reperfusion injury to rabbit lungs. Am J Respir Crit Care Med 1998; 158: 1669–75.[Abstract/Free Full Text]
-
Wickersham NE, Johnson JJ, Meyrick BO, et al. Lung ischemia-reperfusion injury in awake sheep: protection with verapamil. J Appl Physiol 1991; 71: 1554–62.[Abstract/Free Full Text]
-
Adkins WK, Barnard JW, May S, et al. Compounds that increase cAMP prevent ischemia-reperfusion pulmonary capillary injury. J Appl Physiol 1992; 72: 492–7.[Abstract/Free Full Text]
-
Ljungman AG, Grum CM, Deeb GM, et al. Inhibition of cyclooxygenase metabolite production attenuates ischemia-reperfusion lung injury. Am Rev Respir Dis 1991; 143: 610–7.[ISI][Medline]
-
Mullane K. Acadesine: the prototype adenosine regulating agent for reducing myocardial ischaemic injury. Cardiovasc Res 1993; 27: 43–7.[Free Full Text]
-
Gruber HE, Hoffer ME, McAllister DR, et al. Increased adenosine concentration in blood from ischemic myocardium by AICA riboside: effects on flow, granulocytes and injury. Circulation 1989; 80: 1400–11.[Abstract/Free Full Text]
-
Dixon R, Gourzis J, McDermott D, et al. AICA-riboside: safety, tolerance, and pharmacokinetics of a novel adenosine-regulating agent. J Clin Pharmacol 1991; 31: 342–7.[Abstract]
-
Mathew JP, Rinder CS, Tracey JB, et al. Acadesine inhibits neutrophil CD11B up-regulation in vitro and during in vivo cardiopulmonary bypass. J Thorac Cardiovasc Surg 1995; 109: 448–56.[Abstract/Free Full Text]
-
Zhao ZQ, Williams MW, Sato H, et al. Acadesine reduces myocardial infarct size by an adenosine mediated mechanism. Cardiovasc Res 1995; 29: 495–505.[ISI][Medline]
-
Kingma JG, Simard D, Rouleau JR. Timely administration of AICA riboside reduces reperfusion injury in rabbits. Cardiovasc Res 1994; 28: 1003–7.[Abstract/Free Full Text]
-
Galinanes M, Bullough D, Mullane KM, Hearse DJ. Sustained protection by acadesine against ischemia- and reperfusion-induced injury: studies in the transplanted rat heart. Circulation 1992; 86: 589–97.[Abstract/Free Full Text]
-
Gruver EJ, Toupin D, Smith TW, Marsh JD. Acadesine improves tolerance to ischemic injury in rat cardiac myocytes. J Mol Cell Cardiol 1994; 26: 1187–95.[ISI][Medline]
-
Tsuchida A, Yang XM, Burckhartt B, et al. Acadesine extends the window of protection afforded by ischaemic preconditioning. Cardiovasc Res 1994; 28: 379–83.[Abstract/Free Full Text]
-
Menasche P, Jamieson E, Flameng W, Davies MK. Acadesine: a new drug that may improve myocardial protection in coronary artery bypass grafting. J Thorac Cardiovasc Surg 1995; 110: 1096–106.[Abstract/Free Full Text]
-
Leung JL, Stanley T, Mathew J, for the McSPI Research Group. Initial multicenter randomized controlled trial on the safety and efficacy of acadesine in patients undergoing CABG surgery. Anesth Analg 1994; 78: 420–34.[Abstract/Free Full Text]
-
Mangano DT, for the Multicenter Study of Perioperative ischemia (McSPI) Research Group. Effect of acadesine on myocardial infarction, stroke, and death following surgery: a meta-analysis of 5 international randomized trials. JAMA 1996; 277: 325–32.
-
Schoenberg MH, Poch B, Moch D, et al. Effect of acadesine treatment on postischemic damage to small intestine. Am J Physiol 1995; 269: H1752–H1759.[Abstract/Free Full Text]
-
Fabian TC, Fabian MJ, Yockey JM, Proctor KG. Acadesine and lipopolysaccharide-evoked pulmonary dysfunction after resuscitation from traumatic shock. Surgery 1996; 119: 302–15.[ISI][Medline]
-
Neely CF, Keith IM. A1 adenosine receptor antagonists block ischemia-reperfusion injury of the lung. Am J Physiol 1995; 268: L1036–L1046.[Abstract/Free Full Text]
-
Murata T, Nakazawa H, Mori I, et al. Reperfusion after two-hour period of pulmonary artery occlusion causes pulmonary necrosis. Am Rev Respir Dis 1992; 146: 1048–53.[ISI][Medline]
-
Khimenko PL, Bagby GJ, Fuseler J, Taylor AE. Tumor necrosis factor- in ischemia and reperfusion injury in rat lungs. J Appl Physiol 1998; 85: 2005–11.[Abstract/Free Full Text]
-
Zhao G, Al-Mehdi AB, Fisher AB. Anoxia-reoxygenation versus ischemia in isolated rat lungs. Am J Physiol 1997; 273: L1112–L1117.[Abstract/Free Full Text]
-
Kennedy TP, Rao NV, Hopkins C, et al. Role of reactive oxygen species in reperfusion injury of the rabbit lung. J Clin Invest 1989; 83: 1326–35.
-
Zamora CA, Baron DA, Heffner JE. Thromboxane contributes to pulmonary hypertension in ischemia-reperfusion lung injury. J Appl Physiol 1993; 74: 224–9.[Abstract/Free Full Text]
-
Adkins WK, Taylor AE. Role of xanthine oxidase and neutrophils in ischemia-reperfusion injury in rabbit lung. J Appl Physiol 1990; 69: 2012–8.[Abstract/Free Full Text]
-
Smits GJ, McVey M, Cox BF, et al. Cardioprotective effects of the novel adenosine A1/A2 receptor agonist AMP 579 in a porcine model of myocardial infarction. J Pharmacol Exp Ther 1998; 286: 611–8.[Abstract/Free Full Text]
-
Al-Mehdi AB, Fisher AB. Invited editorial on "Tumor necrosis factor- in ischemia and reperfusion injury in rat lungs". J Appl Physiol 1998; 85: 2003–4.[Free Full Text]
-
Ward BJ, Pearse DB. Reperfusion pulmonary edema after thrombolytic therapy of massive pulmonary emboli. Am Rev Respir Dis 1998; 138: 1308–11.
-
Greene R, Zapol WM, Snider MT, et al. Early bedside detection of pulmonary vascular occlusion during acute respiratory failure. Am Rev Respir Dis 1981; 124: 593–60.[ISI][Medline]
Accepted for publication November 1, 2000.
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Abstract
Introduction
