Ischemia-reperfusion (I/R) pulmonary edema probably reflects a leukocyte-dependent, oxidant-mediated mechanism. Nitric oxide (NO) attenuates leukocyte-endothelial cell interactions and I/R-induced microvascular leak. Cyclic adenosine monophosphate (cAMP) agonists reverse and prevent I/R-induced microvascular leak, but reversal by inhaled NO (INO) has not been tested. In addition, the role of soluble guanylyl cyclase (sGC) activation in the NO protection effect is unknown.
Rat lungs perfused with salt solution were grouped as either I/R, I/R with INO (10 or 50 ppm) on reperfusion, or time control. Capillary filtration coefficients (Kfc) were estimated 25 min before ischemia (baseline) and after 30 and 75 min of reperfusion. Perfusate cell counts and lung homogenate myeloperoxidase activity were determined in selected groups. Additional groups were treated with either INO (50 ppm) or isoproterenol (ISO-10 microM) after 30 min of reperfusion. Guanylyl cyclase was inhibited with 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ-15 microM), and Kfc was estimated at baseline and after 30 min of reperfusion.
(1) Inhaled NO attenuated I/R-induced increases in Kfc. (2) Cell counts were similar at baseline. After 75 min of reperfusion, lung neutrophil retention (myeloperoxidase activity) and decreased perfusate neutrophil counts were similar in all groups. (3) In contrast to ISO, INO did not reverse microvascular leak. (4) 8-bromoguanosine 3',5'-cyclic monophosphate (8-br-cGMP) prevented I/R-induced microvascular leak in ODQ-treated lungs, but INO was no longer effective.
Inhaled NO attenuates I/R-induced pulmonary microvascular leak, which requires sGC activation and may involve a mechanism independent of inhibition of leukocyte-endothelial cell interactions. In addition, INO is ineffective in reversing I/R-induced microvascular leak.
Lung transplantation is the only therapeutic option for many patients with end-stage lung disease. In patients undergoing lung transplantation, radiographic evidence of reperfusion pulmonary edema is nearly universal, although the magnitude of injury varies.  A recent report of 40 patients who underwent single-lung transplantation indicated that 40% developed severe hypoxemia and had clinical, radiographic, and hemodynamic abnormalities that were identical to adult respiratory distress syndrome.  The most severe cases of edema after single-lung transplantation may require independent lung ventilation and extracorporeal lung oxygenation. 
Pulmonary edema after lung transplantation may largely reflect increased microvascular permeability secondary to ischemia-reperfusion (I/R) and is generally believed to be due to a leukocyte-dependent, oxidant-mediated mechanism. [4–6] There is increasing evidence that nitric oxide (NO), via direct and indirect effects, may modulate microvascular barrier function. [7–12] Decreased NO levels have been found after I/R,  and NO donors attenuate I/R-induced microvascular dysfunction.  There is conflicting evidence as to whether inhaled NO (INO) confers protection in IR-induced microvascular dysfunction. [13,14] We tested the effects of INO in the salt solution-perfused rat lung because leukocyte-endothelial cell interactions have been identified as a significant mechanism for I/R-induced microvascular leak in this preparation. [4,15]
Materials and Methods
Isolated Perfused Lung Preparation
The protocol was approved by the University of Colorado Health Sciences Center Animal Care and Use Committee. Adult male Sprague-Dawley rats (weighing 234–360 g; purchased from SASCO, Indianapolis, IN) were anesthetized with pentobarbital (60 mg/kg given intraperitoneally), and a tracheotomy catheter was inserted. The lungs were ventilated at inspiratory and expiratory pressures of 5.5 and 2.0 cm H2O, respectively, with room air until the heart was cannulated, after which they were ventilated with 21% oxygen, 5% carbon dioxide, and 74% nitrogen. After the rats had a median sternotomy, heparin (200 units) was administered via the right ventricle and allowed to circulate for 3 min. Pulmonary artery and double-lumen left ventricular catheters were inserted and secured with sutures. The lungs were perfused (Gilson Minipuls 3; Gilson, Middleton, WI) at a constant flow of 0.04 ml/g body wt/min with a bicarbonate-buffered physiologic salt solution (Krebs-Henseleit, K-3753; Sigma Chemical Co., St Louis, MO) that contained 11.1 mM D-glucose, 1.2 mM MgSO4, 1.2 mM KH2PO4, 4.7 mM KCL, 118.1 mM NaCl, and 1.5 mM CaCl2; and was osmotically stabilized with 4% bovine serum albumin (A-7906, Sigma Chemical Co.). The lungs and heart were removed en bloc, suspended from a force transducer (FT03; Grass Instruments, Quincy, MA) and placed in a chamber with 100% humidity at 37 degrees Celsius. The first 20 ml of perfusate was used to flush the lungs; an additional 60 ml was used for recirculation. Pulmonary artery and pulmonary venous pressures were continuously monitored (TSD 104; Biopac, Goelta, CA), captured, and stored on a Macintosh computer (Apple Computers, Cupertino, CA) using an analog to digital converter (MP100A, Biopac). Zone 3 conditions were maintained in all experiments [arterial > venous > alveolar pressures]. At the end of the experiment, the right lung was stored at -70 degrees Celsius for later determination of myeloperoxidase. The left lung was dried in a desiccator for 48 h.
Measurement of the Capillary Filtration Coefficient
The capillary filtration coefficient is a measure of hydraulic conductivity (permeability) and was estimated using a modified gravimetric method.  In an isolated perfused lung, perfusion and colloid oncotic pressure are constant. When lung weight is stable (weight equilibrium), hydrostatic forces are equally opposed by oncotic forces. In our experiments, the weight equilibrium was disturbed by increasing venous pressure from 3.5 to 9.0 mmHg for 15 min. Edema formation (net filtration) was estimated by lung weight gain, and pulmonary capillary pressure (Pc) was estimated by double occlusion.  To determine the change in Pc(Delta Pc), Pcwas estimated before and after pulmonary venous pressure was increased. A rapid increase in weight (vascular distension) was followed by gradual weight gain (net filtration). It was assumed that changes in interstitial pressure and oncotic forces were minimal during the period of measurement. Because the rate of gradual weight gain between 5 and 15 min of pressure challenge increases in linearly, it was analyzed by linear regression. The initial rate of weight change at time = 0 (Delta W/Delta T0) is estimated by the slope of the linear regression. The Kfc was estimated by determining the ratio of Delta W/Delta T0to Delta Pc. Capillary filtration coefficient measurements were normalized to left lung dry weight and expressed as ml [center dot] min sup -1 [center dot] mmHg sup -1*[center dot] 10 g lung tissue.
Absolute and Differential Leukocyte Cell Counts
Perfusate samples were obtained 5 min before ischemia and after the Kfc determinations at 30 and 75 min of reperfusion. Absolute counts were determined from an undiluted 10-micro liter aliquot using a Neubauer hemocytometer (Buffalo, NY). Counts are expressed as cells per milliliter of perfusate. Differential counts were obtained by centrifuging undiluted 0.5-ml samples of perfusate at 600 rpm for 8 min onto microscope slides (n = 14; Shandon Cytospin, Pittsburgh, PA). After drying, slides were stained with Wright's stain and differential counts were performed. Leukocytes were grouped as either neutrophil or mononuclear (lymphocytes, monocytes, basophils, or eosinophils). Peripheral blood samples (n = 6) were analyzed with an automated blood count analyzer (Coulter STKS; Coulter Corp., Miami, FL). Differential counts were verified manually.
Measurement of Myeloperoxidase Activity
Myeloperoxidase activity of the right lung homogenate was measured using a modified quantitative assay.  Briefly, the lung was homogenized in 4 ml of 20 mM potassium phosphate buffer (pH 7.4) for 30 s and centrifuged at 40,000g for 30 min. The supernatant was removed and the pellet was resuspended in 4 ml of 50 mM potassium phosphate buffer (pH 6) with 0.5% hexadecyltrimethyl ammonium bromide buffer and sonicated on ice for 90 s. After incubation at 60 degrees Celsius for 2 h, the sample was vortexed and centrifuged for 5 min. One hundred microliters of sample supernatant was added to 2.9 ml O-diansidine buffer (0.0168 g O-diansidine, 100 ml of 50 mM potassium phosphate buffer [pH 6], and 0.5 ml of 30 mM H2O2). The change in absorbance was measured at 460 nM with a spectrophotometer (DU7; Beckman Instruments, Fullerton, CA). One unit of myeloperoxidase activity was defined as Delta absorbance [center dot] min sup -1 [center dot] g right lung dry weight sup -1 (calculated dry weight). All reagents were obtained from Sigma Chemical Corporation.
Inhaled Nitric Oxide Administration
Nitric oxide at 800 ppm (Scott Medical Products, Plumsteadville, PA) was blended to concentrations of either 10 or 50 ppm with 21% oxygen, 5% carbon dioxide, and 74% nitrogen with a low flow air/oxygen blender (Bird, Palm Springs, CA) and placed in a reservoir bag. Nitric oxide concentrations were verified using a hand-held NO monitor (sealed electrochemical sensor, model EC 90; Bedfont Scientific, Upchurch, Kent, UK).
In all experiments, lungs were isolated and allowed to equilibrate and attain isogravimetric conditions for 20 min. In selected experiments, 15 micro Meter 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (495320 ODQ; Calbiochem, La Jolla, CA), a potent and specific inhibitor of soluble guanylyl cyclase, was added to the perfusate after the baseline Kfc value was determined. Lungs were separated into four groups:
Time Control +/- 15 micro Meter ODQ (n = 17).
After the 20-min equilibration period, lungs were ventilated and perfused for 120 to 180 min. Two to three Kfc determinations were made. The first immediately after equilibration (baseline) and the next two at 30 and 75 min of reperfusion.
Ischemia Reperfusion +/- 15 micro Meter ODQ (n = 24).
Ten minutes after measuring baseline Kfc, ventilation and perfusion were interrupted for 45 min (warm ischemia). The inflow and outflow cannulae were clamped and the lungs were allowed to become atelectatic. Ventilation and flow were reinstituted to the original settings. After full flow was established, Kfc was determined at 30 and 75 min of reperfusion.
Ischemia Reperfusion, Treatment +/- 15 micro Meter ODQ (n = 24).
Lungs were subjected to I/R, and Kfc determinations were performed as previously described. At the time of reperfusion, lungs were ventilated with either 10 or 50 ppm INO for the rest of the experiment. Three lungs, pretreated with ODQ, were treated with 250 micro Meter 8-bromoguanosine 3'5'-cyclic monophosphate (8-br-cGMP) on reperfusion.
Ischemia Reperfusion, Delayed Treatment (n = 8).
This protocol followed that described for I/R treatment except treatment consisted of either 50 ppm INO or 10 micro Meter isoproterenol, which was initiated after the Kfc determination at 30 min of reperfusion.
All results are expressed as means +/- SEM. Comparisons were made with Student's t tests, paired and unpaired, and one-way analysis of variance with repeated measures as appropriate. Student-Newman-Keuls post hoc procedures were applied as appropriate. Significance was considered at P less or equal to 0.05.
Ischemia Reperfusion Induces Pulmonary Microvascular Leak
Ischemia-reperfusion has been reported to induce an increase in pulmonary microvascular permeability, and we verified this observation in our preparation. In time-control experiments, repeated Kfc determinations were unchanged in 180 min (Figure 1). The I/R group was subjected to 45 min of ischemia and 90 min of reperfusion, and Kfc was increased 90%+/- 21% and 119%+/- 23% at 30 and 75 min of reperfusion, respectively (P <0.05). Increased Kfc after I/R was independent of changes in pulmonary artery, venous, or capillary pressures (Table 1).
Inhaled Nitric Oxide Prevents Ischemia-Reperfusion-induced Microvascular Dysfunction
We tested the hypothesis that INO would reduce I/R-induced microvascular permeability. Lungs were subjected to 45 min of ischemia and treated with either 10 or 50 ppm INO at the time of reperfusion. Inhaled NO attenuated the I/R-induced increase in Kfc at both 30 and 75 min of reperfusion (Figure 1). Inhaled NO did not influence pulmonary artery, venous, or capillary pressures (Table 1). These data show that INO attenuates the development of I/R-induced permeability when administered at the time of reperfusion.
Inhaled Nitric Oxide Does Not Prevent Leukocyte Retention
Nitric oxide reduces leukocyte-endothelial interactions.  Therefore we wanted to determine if INO reduced lung leukocyte retention in association with the attenuation of microvascular leak. Absolute leukocyte counts, peripheral and baseline perfusate, were 5.8 +/- 1 x 106and 5.7 +/- 0.5 +/- 103cells/ml, respectively. Differential leukocyte counts in peripheral blood (poly 19%+/- 3%; mono 81%+/- 3%) and in baseline perfusate (poly 25%+/- 3%; mono 71%+/- 4%) were not different. During perfusion, a time-dependent decline in counts was due largely to a decrease in neutrophils. Neutrophil counts at 75 min of reperfusion were decreased by 77%+/- 1%, 71%+/- 6%, and 55%+/- 9% in the control, I/R, and INO (50 ppm) groups, respectively (P < 0.05;Figure 2(A)). To determine if the decrease in circulating neutrophils was due to lung retention, lung tissue myeloperoxidase activity was measured and found not to be different in either the I/R or NO (50 ppm)-treated group compared with control (Figure 2(B)), suggesting that lung retention of neutrophils was independent of treatments.
Inhaled Nitric Oxide Does Not Reverse Established I/R-induced Microvascular Dysfunction
Agonists that elevate endothelial cell intracellular cyclic adenosine monophosphate (cAMP) reverse established increases in microvascular permeability. [20–22] Because cyclic guanosine monophosphate (cGMP) has been shown to decrease permeability in pulmonary artery endothelial cells,  we tested the effect of INO on established pulmonary microvascular leak. Isoproterenol, a potent cyclic adenosine monophosphate agonist, was administered in an additional treatment group as a positive control. Lungs were subjected to 45 min of ischemia followed by reperfusion. Microvascular leak was established and Kfc was found to be increased at 30 min of reperfusion (90%+/- 33% and 94%+/- 31%; NO delayed treatment vs. isoproterenol delayed treatment, P < 0.05;Figure 3). Nitric oxide did not reduce Kfc at 75 min of reperfusion, whereas isoproterenol reduced Kfc to baseline values (89%+/- 20% vs. -19%+/- 21%; NO delayed treatment vs. isoproterenol delayed treatment;Figure 3).
Activation of Soluble Guanylyl Cyclase by Inhaled Nitric Oxide Attenuates Ischemia-Reperfusion-induced Microvascular Leak
We tested the effect of INO in lungs treated with ODQ after the baseline Kfc was determined (Figure 4). Over time, ODQ had no effect on Kfc. Inhaled NO was no longer effective at attenuating I/R-induced increases in Kfc at 30 min of reperfusion. However, treatment with 250 micro Meter 8-br-cGMP at reperfusion effectively prevented an I/R-induced increase in Kfc (data not shown).
Reperfusion pulmonary edema is a common complication of lung transplantation and may result in life-threatening hypoxemia. [1,2] In neutrophil-dependent forms of oxidant-mediated microvascular leak, there is evidence that NO may have cytoprotective effects by various mechanisms, including prevention of leukocyte adhesion, [19,24] inhibition of leukocyte activation,  and augmentation of cGMP.  The protective effect of NO has been observed in isolated lung preparations subjected to various chemical forms of oxidant-mediated injury. [11,26,27] Recently Barbotin-Larrieu et al.  reported a beneficial effect of INO in the setting of I/R using neonatal pigs. In addition, clinical anecdotes suggest that INO may be beneficial in the setting of I/R. [28,29] Large decreases in endogenous NO levels have been found on reperfusion in an orthotopic lung transplant preparation.  The decrease in NO was associated with the generation of O2sup -, decreased tissue cGMP, graft function, and recipient survival. Interestingly, NO synthase activity was preserved, suggesting that increased NO degradation was responsible for the decline in NO levels. Supplementation with 8-br-cGMP had beneficial effects on graft function, leukocyte retention, and recipient survival. Given the findings of Pinsky et al.  and others, [8,30] it appears that supplementation of the NO/ cGMP pathway enhances graft function independent of vasodilation. Therefore we wanted to determine if INO administered at the time of reperfusion would attenuate I/R-induced increases in microvascular permeability and whether increases in microvascular permeability could be reversed by INO. Because NO may have effects on leukocyte adhesion, we also determined if lung leukocyte retention was decreased by INO. Because the effectiveness of INO in the presence of soluble guanylyl cyclase (sGC) inhibitors has not been tested, studies were conducted using the sGC inhibitor ODQ.
We observed a protective effect of INO in the setting of I/R in a salt solution-perfused isolated lung. Inhaled NO was effective if administered at the time of reperfusion but was ineffective in reversing established microvascular leak. Barbotin-Larrieu et al.  suggested that this protective effect could be explained by inhibition of lung leukocyte retention as assessed by determination of perfusate neutrophil counts. They observed a correlation between a decrease in circulating neutrophils and an increase in Kfc. Myeloperoxidase tissue activity was not determined by this group. This association is only suggestive of a mechanism for the effects of INO and is not conclusive. In our preparation, in contrast to that of Barbotin-Larrieu et al.,  the protective effect of INO could not be explained by inhibition of lung leukocyte retention as assessed by determination of perfusate counts and lung tissue myeloperoxidase activity. Our data suggest that INO may have protective effects independent of inhibition of leukocyte retention. The studies differ in that we used a salt solution-perfused preparation in contrast to a blood-perfused preparation. Other investigators have shown that endogenously retained neutrophils play an important role in I/R-induced increases in microvascular permeability in the salt solution-perfused rat lung. [4,15] Endogenously retained leukocytes probably represent a dilution because whole-blood and perfusate samples only differ by their absolute counts (5.8 +/- 1 x 106cells/ml vs. 5.7 +/- 0.5 x 104cells/ml) but have similar differential counts. It is noteworthy that despite a dramatic reduction of circulating neutrophils, I/R-induced increases in microvascular leak are still readily observed. We speculate that INO may have effects on neutrophil activation that are independent of adhesion, but limitations of our preparation prevent us from determining this mechanism.
There is evidence that NO may play a role in the homeostatic regulation of leukocyte adhesion in the microcirculation.  Leukocyte adhesion is primarily regulated by the CD11/CD18 [19,31] glycoprotein complex found on the surface of leukocytes, and increased adhesion through a mechanism that involves this complex has been observed after inhibition of NO. Furthermore, cGMP may be important for the endothelial surface expression of P-selectin, a glycoprotein important for leukocyte rolling,  and pretreatment with anti-p-selectin antibodies attenuates I/R-induced microvascular leak. [33,34] Interestingly, P-selectins can “tether” neutrophils to endothelial cells without neutrophil activation.  Therefore it is possible to have leukocyte adhesion without the formation of cell-damaging oxygen-derived radicals. We found leukocyte retention in all groups despite observing clear protection by the administration of INO at the time of reperfusion. Although prevention of leukocyte adhesion may be an important step in the development of neutrophil-mediated microvascular leak, our results suggest it may not be the only mechanism by which NO protects against I/R-induced microvascular leak.
There are several ways through which NO may impart cytoprotection without influencing leukocyte tethering to endothelium. Nitric oxide has a high affinity for O2sup - and is an efficient scavenger of O2sup -.  But quenching of O2sup - by NO forms OONO sup -, which may cause cell injury. Two alternative mechanisms may explain our observations:(1) direct inhibition of neutrophil reduced nicotinamide adenine dinucleotide phosphate oxidase by NO, or (2) augmentation of cGMP by NO. Nitric oxide has been shown to directly inactivate neutrophil reduced nicotinamide adenine dinucleotide phosphate oxidase,  an enzyme important for the respiratory burst and the generation of O2sup -. Therefore it is plausible that NO may protect by preventing the generation of oxygen-derived radicals. This paradigm would result in minimal OONO sup - formation because NO and O2sup - interactions would be reduced. However, if this were a significant mechanism, we would expect to see a protective effect from INO independent of soluble guanylyl cyclase activity. We have clearly established that activation of soluble guanylyl cyclase is required for the protective effect of INO (Figure 4) This mechanism was not explored by Barbotin-Larrieu et al.  Pinsky et al.  convincingly showed that augmentation of cGMP attenuates I/R-induced microvascular dysfunction, and this is supported by our observations.
A practical approach in the clinical setting would be to supplement the NO-cGMP pathway by administering INO. Nitric oxide reacts with superoxide anion (O2sup -) to form peroxynitrite (OONO sup -), which ultimately forms [center dot] OH sup - and nitrogen dioxide ([center dot] NO sub 2), metabolites that may cause DNA strand breaks and lipid peroxidation.  Thus there has been considerable debate as to whether INO can lead to cell injury due to the rapid formation of OONO sup -. [35,36] Hemoglobin in whole blood rapidly clears NO,  but hemoglobin was largely absent from our preparation and therefore INO may be expected to form OONO sup - at even higher rates in our system. However, given our observation that INO protected against the development of microvascular leak, it seems unlikely that formation of peroxynitrite from INO is physiologically significant during short periods of administration.
In addition to cAMP, endothelial cell cGMP has been implicated as a fine regulator of microvascular permeability ; therefore we examined the efficacy of INO for reversing established microvascular leak. In contrast to isoproterenol, which increases cAMP, INO was ineffective in reversing established microvascular leak. Clinically, INO therapy may be effective only before the development of I/R-induced microvascular leak; it may be less effective in the setting of preexisting microvascular dysfunction.
We have clearly established that INO initiated at the time of reperfusion attenuates microvascular permeability changes after I/R. We have explored the role of NO in leukocyte retention and found that it is not a significant mechanism of cytoprotection in our preparation. In the salt solution-perfused rat lung, INO attenuates I/R-induced pulmonary microvascular leak by a mechanism that requires soluble guanylyl cyclase activation and may involve a mechanism independent of inhibition of leukocyte-endothelial cell interactions. In addition, INO is ineffective in reversing established I/R-induced microvascular leak.