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Ann Thorac Surg 1998;66:1732-1738
© 1998 The Society of Thoracic Surgeons
a Division of Thoracic and Cardiovascular Surgery, Department of Surgery, University of Virginia Health Sciences Center, Charlottesville, Virginia, USA
Address reprint requests to Dr Kron, Division of Thoracic and Cardiovascular Surgery, Department of Surgery, University of Virginia Health Sciences Center, Box 310, Charlottesville, VA 22908
Presented at the Thirty-fourth Annual Meeting of The Society of Thoracic Surgeons, New Orleans, LA, Jan 26–28, 1998.
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Abstract |
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Methods. All lungs from New Zealand White rabbits (weight, 3 to 3.5 kg) were harvested en bloc, flushed with EC solution, and reperfused with whole blood for 30 minutes. Group 1 lungs (immediate control) were immediately reperfused. Group 2 lungs (control) were stored inflated at 4°C for 18 hours before reperfusion. Groups 3 and 4 lungs were flushed with EC solution containing 200 µmol/L 8-bromo-cGMP and stored at 4°C for 18 and 30 hours, respectively. Fresh, nonrecirculated venous blood was used to determine single-pass pulmonary venous–arterial oxygen gradients at 10-minute intervals. Assays for cGMP, cyclic adenosine monophosphate, nitric oxide synthase activity, and myeloperoxidase were performed on all lung tissue samples. Wet to dry weight ratios were determined after 2 weeks of passive desiccation.
Results. Oxygenation (venous-arterial oxygen gradient), pulmonary artery pressure, pulmonary vascular resistance, and edema formation were significantly improved in groups 3 and 4 (addition of 8-bromo-cGMP to EC plus 18 or 30 hours of hypothermic ischemia). Hypothermic storage (groups 2, 3, and 4) decreased both nitric oxide synthase activity and myeloperoxidase levels compared with immediate reperfusion (group 1).
Conclusions. These results suggest that the addition of a membrane-permeable cGMP analogue to an EC pulmonary flush solution improves pulmonary function after prolonged storage compared with EC and prostaglandin (E1) preservation alone. The finding of myeloperoxidase reduced levels after hypothermic storage and subsequent reperfusion may suggest a more important role for pulmonary hemodynamic control in mitigating pulmonary reperfusion injury.
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Introduction |
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The process of pulmonary ischemia-reperfusion injury is multifactorial [4]. Neutrophil- and cytokine-mediated endothelial cell disruption has been shown to contribute substantially to pulmonary dysfunction after ischemia-reperfusion injury [5–7]. However, adequate preservation of the ability of the endothelial cell to produce nitric oxide (NO) is essential for maintaining endothelial cell integrity and is responsible for the regulation of vascular tone during reperfusion [8, 9].
Nitric oxide is produced in the endothelium by a constitutive form of nitric oxide synthase, cNOS, and then rapidly diffuses across the adjacent smooth-muscle lipid bilayer and increases concentrations of cytosolic cyclic guanosine monophosphate (cGMP) by promoting soluble guanylate cyclase activity. Increased cGMP–dependent protein kinase activity ultimately decreases intracellular calcium (Ca) concentrations through the inhibition of both phospholipase C activity and voltage-operated Ca channels while inhibiting inositol phosphate receptor–mediated Ca release from the sarcoplasmic reticulum. Increased cGMP–dependent protein kinase activity also promotes Ca-ATPase and Na/Ca exchanger activity and hyperpolarizes the cell membrane through activation of Ca-activated K channels [10].
We [11] have previously demonstrated that the administration of sodium nitroprusside during pulmonary reperfusion improves pulmonary hemodynamics and oxygenation and reduces edema formation after prolonged hypothermic ischemia. Others [12] have shown that the addition of a cGMP analogue during ischemic storage improves pulmonary function better than inhaled NO. Even though high-potassium solutions are believed to provide better endothelial cell preservation, we [13] have demonstrated the superiority of low-potassium solutions in reducing reperfusion injury after prolonged hypothermic ischemia. We therefore hypothesized that the addition of a cGMP analogue (8-bromo [Br]-cGMP) to a high-potassium preservation solution (Euro-Collins [EC] solution) would improve pulmonary reperfusion injury after prolonged hypothermic ischemia by maintaining endothelial cell integrity while overcoming potassium-induced vasoconstriction more effectively than prostaglandin alone.
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Material and methods |
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A median sternotomy and thymectomy were performed. The superior and inferior venae cavae were loosely encircled with ligatures, and the pericardium was opened. Both the pulmonary artery (PA) and the aorta were dissected free and similarly encircled. A pursestring suture was placed in the free wall of the right ventricle, and the rabbit was heparinized (500 U/kg intravenously). After injection of 30 µg of prostaglandin E1 (alprostadil; The Upjohn Company, Kalamazoo, MI) directly into the PA, the venae cavae were ligated, thus initiating pulmonary ischemia.
The PA was then cannulated through a right ventriculotomy in the center of the pursestring suture, and both the right ventricular and PA ligatures were tied around the cannula. After venting of the left ventricle through a left ventriculotomy and ligation of the aorta, 50 mL/kg of cold EC solution with or without the addition of 200 µmol/L 8-Br-cGMP was infused into the PA from a height of 30 cm. Topical cooling was achieved with cold saline slush.
During PA flush, the left atrium was cannulated through the left ventriculotomy, and a second pursestring suture was tied around the cannula. A second catheter was placed in the left atrium to directly transduce left atrial pressures. After the PA flush, the inflow and outflow cannulas were clamped. Care was taken to leave the pleurae intact until the completion of the flush to avoid parenchymal injury. The lungs were stored inflated by clamping the tracheal tube at end-inspiration. The lung-heart block was excised, immersed in cold 0.9% saline solution, and either immediately reperfused or stored at 4°C for up to 30 hours.
All experimental protocols were reviewed and approved by an institutional animal use committee. All animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication 85-23, revised 1985).
Assessment of lung function
After 18 hours of storage at 4°C, the lung-heart block was suspended by a force transducer in a warm, humidified tissue chamber. Ventilation was reestablished with a 95% oxygen and 5% carbon dioxide gas mixture at a tidal volume of 12 mL/kg and a respiratory rate of 20 breaths/min. The lungs were reperfused with homologous fresh whole venous blood from a main reservoir. A second venous blood reservoir was used to determine single-pass oxygenation at 10, 20, and 30 minutes after initiation of reperfusion. Blood was harvested from a single rabbit for each experiment.
The inflow and outflow cannulas were connected to the blood-primed perfusion circuit, care was taken to avoid the introduction of air. The perfusion circuit (Kent Scientific) was designed to recirculate 150 mL of warmed blood through a 270-µm blood filter (model 2C7600; Baxter, Deerfield, IL) using a roller pump (model 7521-40; Cole Palmer Instrument Company, Chicago, IL) at a rate of 60 mL/min. A 270-µm blood filter was chosen so as not to affect leukocyte or platelet counts. Continuous recording of PA pressure, left atrial pressure, lung weight, airway flow, and airway pressure was facilitated by using a dynamic data acquisition program (Workbench PC; Strawberry Tree, Inc, Sunnydale, CA) run on a personal computer (Prolinea 470A, Compaq, Houston, TX). This program automatically calculated and displayed pulmonary vascular resistance (PVR), tidal volume, and dynamic airway compliance. Left atrial pressure was maintained within the physiologic range (4 to 8 mm Hg) by adjusting the height of a small outflow reservoir in the circuit.
Pulmonary venous blood samples were collected for blood gas analysis (Corning 178 pH/blood gas analyzer) at 10, 20, and 30 minutes after the initiation of reperfusion. At each sampling interval, inflow from the main reservoir was interrupted and the circuit was filled with venous blood from the second inflow reservoir. Thirty milliliters of venous blood was passed through the pulmonary vasculature at each interval to ensure accurate measurement of pulmonary venous oxygen content. Oxygen contact with exposed blood surfaces inside the reservoir containers was minimized by continuous infusion of 100% nitrogen. After 30 minutes of reperfusion, specimens of hilar lung tissue were acquired for biochemical assay and weight analysis. Wet to dry ratios were calculated after passive desiccation at room temperature to a stable dry weight.
Experimental protocol
All lungs were flushed with EC solution and reperfused at a physiologic flow rate of 60 mL/min for 30 minutes. Group 1 lungs served as immediate controls (n = 7) and were immediately reperfused after harvest. Group 2 lungs (n = 7) were flushed with EC solution alone and then stored for 18 hours at 4°C before reperfusion. Group 3 (n = 7) and group 4 lungs (n = 7) were flushed with EC solution containing 200 µmol/L 8-Br-cGMP and stored at 4°C for 18 and 30 hours, respectively. Data were recorded every 15 seconds and analyzed at the end of the 30-minute reperfusion period. Oxygenation data were obtained and analyzed at 10-minute intervals.
All values were expressed as the mean ± the standard error of the mean. The experimental groups were compared using analysis of variance. Differences were considered significant if the p value was less than 0.05.
NOS enzyme assay
Lung tissue taken from the pulmonary hilum (0.2 g) was homogenized in 1 mL of buffer containing 0.05 mmol/L Tris-HCl pH 7.4, 0.1 mmol/L EDTA (ethylenediaminetetraacetic acid), 1 mol/L dithiothreitol, and 10 mg/mL of protease inhibitor cocktail (Sigma P8340). Samples were centrifuged at 4°C for 15 minutes at 10,000 rpm, and the supernatant was assayed for total NOS enzyme activity as determined by the conversion of 14C-L-arginine to 14C-L-citrulline as previously described [14]. Reactions contained 50 mmol/L HEPES pH 7.5, 200 mmol/L NADPH (nicotinamide adenine dinucleotide phosphate [reduced form]), 1 mmol/L dithiothreitol, 10 mmol/L FAD (flavin adenine dinucleotide), 100 mmol/L tetrahydrobiopterin, 10 mmol/L L-arginine, 3 mmol/L Ca2+, and 2.9 mmol/L 14C-L-arginine (New England Nuclear NEC-267E) in a total volume of 50 mL. Reactions were started by adding 100 mg of protein homogenate and incubated at 37°C for 60 minutes. Reactions were then applied to columns containing 0.5 mL of AG 50W-X8 cation-exchange resin (Na+ form; Bio-Rad, Hercoles, CA). The column was eluted with 1.5 mL of water, and 12 mL of scintillation fluid was added to the effluent from the column before loading into the liquid scintillation counter. Units of NOS activity were expressed as picomoles of citrulline per minute per milligram of protein.
cGMP and cyclic adenosine monophosphate assays
To evaluate tissue levels of cGMP and cyclic adenosine monophosphate (cAMP), 0.5 g of hilar lung tissue was homogenized at 4°C in 5 mL of 6% trichloroacetic acid, followed by centrifugation for 15 minutes at 4°C. The supernatant was then removed, washed four times with water-saturated ethyl ether, and vacuum-dried at 60°C for 3 to 4 hours. The resulting pellet was resuspended in 1 mL 0.1N HCl, and the solution was subjected to radioimmunoassay as previously described [15]. The results were reported as picomoles of cGMP per gram of tissue (wet weight).
Myeloperoxidase assay
To elucidate the role of neutrophils in ischemia-reperfusion injury, myeloperoxidase was studied as a marker of neutrophil accumulation. Frozen hilar lung samples (0.5 g) were allowed to thaw and then were homogenized (Polytron model PCV 11; Kinnematica AG, Littau, Switzerland) in 2.5 mL of 0.05 mol/L NaPO-4 at 4°C for 30 seconds. The samples were centrifuged (Sorvall RC-5B refrigerated superspeed centrifuge; DuPont Instruments, Newtown, CT) at 4°C, the supernatant was discarded, and the pellet was resuspended in 2.5 mL of 0.5% HTAB (hexadecyltrimethylammonium bromide). This solution was homogenized and sonicated (Bransonic Ultrasonic Cleaner 3210; Branson Ultrasonics Co, Danbury, CT) for 2 minutes at room temperature. The samples were then centrifuged at 4°C for 10 minutes, and the supernatant was removed. Of the supernatant, 0.1 mL was added to a solution of 0.7% H2O2 and 0.1 mg/mL of ODD (o-dianisidine dihydrochloride) (Sigma Chemical Co, St. Louis, MO) and placed immediately into the spectrophotometer (LKB Biochrom model 4050; Cambridge, England); the change in absorbance at 460 nm over 2 minutes at room temperature was recorded. Myeloperoxidase activity was reported as change in absorbance per gram of tissue (wet weight).
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Results |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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0.000004). Similarly, PVR was significantly reduced in group 3 (40,269 ± 6,464 dynes · s · cm-5) and group 4 (30,329 ± 853 dyne · s · cm-5) compared with group 2 (53,429 ± 8,779 dyne · s · cm-5 and group 1 (53,722 ± 5,877 dyne · s · cm-5) (p 0.000004). Lungs stored for 30 hours with the addition of 8-Br-cGMP (group 4) demonstrated improved hemodynamics compared with lungs stored for 18 hours (group 3); however, only differences in PVR were significant (p 0.000004).
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0.011). Group 3 lungs (1.81 ± 0.14 mL/mm Hg) were not significantly less compliant than those undergoing immediate reperfusion (group 1).
Venous-arterial oxygen gradient
A 30-mL venous blood challenge was administered every 10 minutes during reperfusion from a separate, noncirculating venous blood reservoir. Single-pass pulmonary venous blood gas analysis was carried out on aspirated samples immediately after reperfusion of the lungs. The oxygenation gradient was determined by subtracting the oxygen tension of the venous reservoir blood from the oxygen tension of the blood returning from the pulmonary venous catheter. Significant improvement in oxygenation occurred for all groups compared with 18-hour control lungs (group 2) after 30 minutes of reperfusion: 60.40 ± 25.4 mm Hg in group 2 versus 387.8 ± 119.1 mm Hg in group 1, 520.7 ± 63.2 mm Hg, in group 3, and 461.9 ± 41.9 mm Hg in group 4 (p 0.000001). Similar significant improvements in the venous-arterial oxygen gradient were observed at 10 and 20 minutes of reperfusion (Fig 2). Both groups receiving 8-Br-cGMP followed by a period of hypothermic ischemia (groups 3 and 4) oxygenated as well as lungs undergoing immediate reperfusion.
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0.0003). The addition of 8-Br-cGMP during 18 hours of hypothermic ischemia significantly reduced edema formation: 6.55 ± 0.16 in group 3 versus 8.54 ± 0.74 in group 2 (p 0.0003). Increasing the interval of hypothermic ischemia from 18 to 30 hours significantly increased edema formation compared with that in lungs undergoing immediate reperfusion: 7.67 ± 0.44 in group 4 versus 5.41 ± 0.12 in group 1 (p 0.0003) (see Table 1).
NOS enzyme assay
Lungs that were reperfused immediately and did not undergo hypothermic ischemia (group 1) maintained the highest NOS activity (Table 2). There were no significant differences in NOS enzyme activity with the addition of 8-Br-cGMP during hypothermic ischemia. However, there was a trend toward declining NOS activity associated with 8-Br-cGMP administration, especially after 30 hours of hypothermic ischemia: 302.6 ± 40.1 pmol · min-1 · mg-1 in group 4 versus 492.0 ± 27.5 pmol · min-1 · mg-1 in group 1 (p 0.052).
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0.022) (see Table 2).
Myeloperoxidase assay
Lungs undergoing immediate reperfusion had significantly higher myeloperoxidase levels than lungs undergoing periods of hypothermic ischemia: 12.7 ± 2.6 pmol/0.5 g in group 1 versus 2.69 ± 1.04 pmol/0.5 g in group 2, 2.78 ± 0.60 pmol/0.5 g in group 3, and 3.55 ± 1.36 pmol/0.5 g in group 4 (p 0.0004)]. There were no significant differences in myeloperoxidase levels between groups receiving 8-Br-cGMP and those not receiving 8-Br-cGMP during periods of prolonged hypothermic ischemia (see Table 2).
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Nitric oxide synthase activity is not decreased during reperfusion after brief periods of ischemia. However, the bioavailability of NO on reperfusion is markedly decreased as a result of the formation of reactive metabolites, as NO rapidly combines with the increased oxygen intermediates produced during the ischemic insult [12]. We have also demonstrated that periods of prolonged ischemia may contribute to decreased NO availability by an absolute reduction in NOS activity. This latter activity declined with increasing intervals of hypothermic ischemia irrespective of cGMP administration. This phenomenon could be attributed to an increase in endothelial cell dropout, metabolic dysfunction resulting from ischemic injury alone, or both. The administration of cGMP appeared to bypass this shortage of NO to provide adequate vascular relaxation throughout pulmonary reperfusion. However, cGMP did not increase NOS activity through a positive feedback mechanism as demonstrated in previous experiments performed in endothelial cell cultures [18]. Again, this could possibly be explained by increased endothelial cell dropout or metabolic dysfunction, not necessarily a negative feedback mechanism.
Activation of guanylate cyclase has been shown to decrease intracellular Ca concentrations resulting in decreased macromolecule permeability of the vascular endothelium [19, 20]. Cell cultures of endothelial monolayers have confirmed this effect of cGMP by demonstrating decreased albumin flux with exposure to agents that increase the activity of either particulate or soluble guanylate cyclase [21]. Wet to dry weight ratios in this experiment demonstrated a significant decrease in edema formation in lungs undergoing hypothermic ischemia for 18 hours with added 8-Br-cGMP. There was no significant difference in wet to dry weight ratio between immediately reperfused lungs (group 1) and lungs treated with 8-Br-cGMP during 18 hours of hypothermic storage (group 3).
We [13] have previously shown that the use of low-potassium–dextran solution during prolonged ischemia preserves pulmonary hemodynamics and oxygenation better than EC solution. Increased intracellular potassium has been shown to serve as a direct antagonist to the hyperpolarizing effects of Ca [22]. Hyperpolarization of the endothelial cell is believed to play an important role in NO generation and subsequent smooth muscle cell relaxation. In fact, increased hyperpolarization caused by increased cytosolic Ca levels and calmodulin activity are thought to be an integral process for increasing the activity of ecNOS. This is especially true with increasing shear stresses experienced in the immediate reperfusion interval [23, 24]. This would implicate the use of high-potassium solutions such as EC solution as inhibitory to the process of hemodynamic accommodation during reperfusion. Euro-Collins solution may contribute to the overall endothelial cell integrity as demonstrated in solid-organ preservation by decreasing endothelial cell macromolecule permeability and metabolic activity. However, this endothelial cell quiescence comes at the cost of decreased NOS activity and adequate hemodynamic regulation after prolonged periods of hypothermic ischemia.
Increased activity of guanylate cyclase has been shown to reduce cAMP by activating cGMP–stimulated phosphodiesterase [10]. However, some [25] think that 8-Br-cGMP may not be as effective as native cGMP in activating phosphodiesterase. Our data suggest that 8-Br-cGMP may have some effect on regulating cAMP levels and subsequent endothelial cell permeability. As levels of cGMP increased in the pulmonary vasculature, corresponding levels of cAMP were reduced. However, this conclusion may be confounded by a time-dependent phenomenon. Our data also demonstrate that cAMP levels decreased with increasing intervals of pulmonary ischemia.
Prostaglandins are thought to exert their vasodilatory effects through stimulation of particulate cGMP activity. Both prostaglandin E2 and prostacyclin have been shown to inhibit the secretion of endothelin-1 and facilitate vascular relaxation through particulate guanylate cyclase activation [26]. Traditionally, a continuous infusion of prostaglandin has been used to dilate the pulmonary vascular tree during perfusion with EC solution. Supplementation of a membrane-permeable analogue of cGMP during hypothermic ischemia appears to produce a more profound and lasting effect on preserving pulmonary hemodynamic accommodation than prostaglandin E1 alone. Clinically, the addition of cGMP to pulmonary perfusates could possibly prevent the need of systemic exposure to intravascular vasodilators during pulmonary reperfusion after recipient implantation. The uptake of cGMP by the pulmonary vasculature during storage with 200 µmol/L cGMP and EC solution appears to continue throughout 30 hours of hypothermic ischemia. On reperfusion, the intracellular cGMP should not enter the circulation and produce untoward systemic vascular effects.
Myeloperoxidase levels were significantly higher in lungs undergoing immediate reperfusion (group 1). This finding points to an increased sequestration of functioning neutrophils in the pulmonary parenchyma with immediate reperfusion. However, there is little corresponding physiologic dysfunction demonstrated with this phenomenon. In fact, lungs stored for 18 hours without cGMP had the lowest level of myeloperoxidase activity (group 2), yet functioned significantly worse than those stored for 18 or 30 hours with the addition of 200 µmol/L 8-Br-cGMP (groups 3 and 4). Perhaps a longer reperfusion period would allow increased leukosequestration in this model. These findings demonstrate the multifactorial nature of ischemia-reperfusion injury and highlight the importance of adequate hemodynamic control during the initial reperfusion interval after prolonged ischemia.
The bioavailability of NO after pulmonary ischemia is reduced in the face of preserved NOS activity. The reaction of reactive metabolites with NO is rapid and may account for the decrease in available NO seen with reperfusion. For this reason, NO supplementation during reperfusion may be less effective than cGMP augmentation in reducing PVR during reperfusion. The presence of a high-potassium preservation solution such as EC solution may decrease the macromolecule permeability of the endothelial cell layer and result in decreased edema formation. The administration of 8-Br-cGMP during ischemic storage provides distal stimulation of the NO/cGMP pathway, and this leads to decreased PVR and improved pulmonary function. Supplementation with cGMP directly affects smooth muscle relaxation and bypasses the antagonistic effects of potassium on endothelial Ca levels and NOS activity.
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Acknowledgments |
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The technical advice and support of Anthony J. Herring is acknowledged.
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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); group 2 = 18-hour control (
); group 3 = cyclic guanosine monophosphate analogue (cGMP) treatment and 18-hour storage (
); and group 4, cGMP treatment and 30-hour storage (
).
); group 2 = 18 hour control (