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Ann Thorac Surg 1996;62:1632-1638
© 1996 The Society of Thoracic Surgeons
Laboratoire de Chirurgie Expérimentale and Department of Thoracic and Vascular Surgery and Heart-Lung Transplantation, Centre Chirurgical Marie-Lannelongue, Paris-Sud University, Le Plessis Robinson, France
Accepted for publication July 9, 1996.
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Abstract |
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Methods. A blood-perfused, isolated rat lung model was used. Lungs were flushed and harvested from non–heart-beating donors after 30 minutes of in situ warm ischemia. The lung was then stored for 2 hours at 4°C. Inhaled NO at 30 ppm was given either during the period of warm ischemia, during reperfusion, or during both periods. Lung ischemia-reperfusion injury was assessed after 1 hour of reperfusion by measuring pulmonary vascular resistance, coefficient of filtration, wet-to-dry lung weight ratio, and myeloperoxidase activity.
Results. A severe IR injury occurred in lungs undergoing ischemia and reperfusion without NO as evidenced by high values of pulmonary vascular resistance (6.83 ± 0.36 mm Hg•mL-1•min-1), coefficient of filtration (3.02 ± 0.35 mL•min-1•cm H2O-1•100 g-1), and wet-to-dry lung weight ratio (8.07 ± 0.45). Lower values (respectively, 3.31 ± 0.44 mm Hg•mL-1•min-1, 1.49 ± 0.34 mL•min-1•cm H2O-1•100 g-1, and 7.44 ± 0.43) were observed when lungs were ventilated with NO during ischemia. Lung function was further improved when NO was given during reperfusion only. All measured variables, including myeloperoxidase activity were significantly improved when NO was given during both ischemia and reperfusion. Myeloperoxidase activity was significantly correlated with coefficient of filtration (r = 0.465; p < 0.05).
Conclusions. These data suggest that inhaled NO significantly reduces ischemia-reperfusion injury in lungs harvested from non–heart-beating donors. This effect might be mediated by inhibition of neutrophil sequestration in the reperfused lung.
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Introduction |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Experimental NHBD lung transplantation has been pioneered by Egan and co-workers [2–4]. In a canine lung transplantation model, lungs harvested 1 hour after cessation of circulation showed an adequate gas exchange function. However, lungs harvested after more than 2 hours of warm in situ ischemia demonstrated a significantly poorer pulmonary function [2]. Evidence is accumulating that ischemia is not a period where cell functions are uniformly inhibited [5]. A study performed by our group showed that warm ischemia in the rabbit lung is associated with severe endothelial dysfunction [6]. In this model, as in other studies [7], cold ischemia was less harmful than warm ischemia.
Some progress has been made in reducing the cellular damage that occurs during warm ischemia. The tolerable period of ischemia has been increased by different methods, such as maintaining the lungs ventilated during in situ ischemia [4] or the use of oxygen free radical scavengers in the flush solution [3]. However, the obligatory period of warm ischemia provides the setting for a severe reperfusion injury and seems to be the major limiting factor in NHBD lung transplantation.
Recent studies have identified nitric oxide (NO) as an important cytoprotective modulator [8]. In our previous studies, we showed that inhaled NO at reperfusion protected against the development of increased capillary permeability and pulmonary resistance after ischemia-reperfusion (IR) lung injury in neonatal piglets [9]. L-Arginine was able to significantly reverse the endothelial dysfunction occurring after warm ischemia [6]. The protective effects of NO are consistently related to prevention of the pulmonary sequestration of polymorphonuclear neutrophils (PMNs) [8]. Indeed, PMNs play a pivotal role in IR injury by becoming activated, adhering to the endothelium, and releasing reactive oxygen species into the surrounding tissues [9, 10]. Thus, an increasing amount of experimental and clinical data support a protective role for NO in IR injury [8]. Furthermore, it is advantageous that NO can be directly supplied to the pulmonary cell by ventilating the lung even after cessation of circulation.
Therefore, we hypothesized that inhaled NO might be beneficial in reducing the IR injury that occurs in lungs harvested from NHBDs. An isolated rat lung model was used, in which lung function was assessed by measuring pulmonary vascular resistance (PVR), coefficient of filtration (Kfc), wet-to-dry lung weight ratio (W/D), and myeloperoxidase activity (MPO). Nitric oxide was given either during ischemia, during reperfusion, or during both.
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Material and Methods |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Non–Heart-Beating Cadaver Preparation
The rats were anesthetized with sodium thiopental (50 mg/kg intraperitoneally). After median laparotomy and heparinization (200 IU intravenously), the animals were exsanguinated to death through the abdominal aorta. Immediately after sacrifice, the animals were tracheostomized and ventilated with a Harvard rodent ventilator (model 680; Harvard Apparatus, South Natick, MA) at 60 breaths/min, a tidal volume of 3 mL, and a positive end-expiratory pressure of 1 cm H2O. Non–heart-beating cadaver animals were maintained in the supine position and left at room temperature for 30 or 60 minutes while cadaver ventilation was performed with humidified room air, supplemented or not with NO at 30 ppm.
Lung Harvest
After sternotomy, a polyethylene cannula was inserted into the pulmonary artery through the right ventricle, and the catheter was secured using a tie placed around the pulmonary artery. A second cannula was placed in the left atrium through a left ventricular incision. Both lungs were flushed with 20 mL of an extracellular type of preservation solution (Celsior; Pasteur-Mérieux, Lyon, France; cooled to 4°C) through the pulmonary arterial cannula with a pressure of 20 cm H2O. Reduced glutathione was stocked in a separate air-proof syringe and added to the preservation solution immediately before use. The trachea was clamped to keep the lungs partially inflated, and the heart and lungs were removed en bloc. The heart-lung block was then stored at 4°C for 2 hours in a container filled with saline solution. The average time of the harvest was 12 ± 2 minutes.
Isolated Perfused Lung
The technique described in a previous report [10, 11] was modified so that the heart-lung block could be suspended from a Statham force-displacement transducer to monitor weight changes of the lung. Lungs were reventilated with a gas mixture (20% O2, 5% CO2, 75% N2), supplemented or not with NO at 30 ppm. The lungs were perfused through the pulmonary artery cannula with 20 mL of heparinized blood obtained from two other donor rats. Pulmonary effluent blood was collected into a plastic reservoir through the cannula placed in the left atrium, and blood was recirculated using a peristaltic pump (Ismatec; Bioblock, Paris, France). The flow rate was maintained at 0.03 mL/g body weight/min. Zone 3 conditions (arterial > venous > alveolar pressures) were maintained throughout all experiments.
Hemodynamic Measurements
Pulmonary arterial pressure and pulmonary venous pressure were continuously monitored with a P23 ID transducer (Statham, Oxnard, CA). Pulmonary vascular resistance was calculated as pulmonary arterial pressure - pulmonary venous pressure/flow rate.
Coefficient of Filtration
Coefficient of filtration was used as the index of endothelial permeability to fluid with the method described by Drake and associates [12]. In brief, after an isogravimetric period of 45 minutes, pulmonary venous pressure was rapidly increased by 8 cm H2O by raising the outflow end of the left atrial cannula and maintained at that level for 15 minutes. The rapid increase in lung weight gain caused by vascular recruitment was followed by a phase of slow weight gain reflecting filtration of fluid into the pulmonary interstitium. Hydrostatic capillary pressure was measured by using the double vascular occlusion method, performed before and after the increase in venous outflow pressure [13]. Coefficient of filtration was calculated by dividing the weight increase per minute by the change in hydrostatic capillary pressure that occurred after the increase in venous outflow pressure [13]. Coefficient of filtration was normalized for the lung weight and expressed in milliliters per minute per centimeter of water per 100 g of lung tissue. The lung weight was estimated by measuring the weight of the heart, mediastinal tissue, and lungs at the beginning of the experiment and substracting from this value the weight of the extrapulmonary tissue at the end of the experiment.
Determination of Myeloperoxidase Activity
The method described by Mullane and associates [14] was used to measure MPO activity in the lungs. After 60 minutes of reperfusion, the left lung was frozen in liquid nitrogen and stored at -80°C. It was then pulverized and homogenized in 10% wt/vol of hexadecyltrimethyl ammonium bromide buffer (0.5% hexadecyltrimethyl ammonium bromide in 50 mmol/L phosphate buffer at pH 6.0) with a Polytron homogenizer (Kinematica, Luzern, Switzerland). The homogenate was sonicated on ice for 15 seconds, frozen at -70°C, and thawed three times, then centrifuged at 40,000 g for 15 minutes. Supernatant was assayed for MPO activity spectrophotometrically. Twenty microliters of supernatant was combined with 12 µL of 25 mmol/L H2O2, 30 µL of 40 mmol/L O-dianisidine hydrochloride, and 1.938 mL of 50 mmol/L phosphate buffer (pH 6.0). The change in absorbance was measured at 460 nm on a Beckman spectrometer (model 25 spectrometer; Beckman, France). One unit of MPO activity was defined as the activity degrading 1 µmol of peroxide per minute at 25°C.
Measurements of Wet and Dry Lung Weights
The right lung was excised at the end of the experiment and weighed for determination of the wet lung weight. The lungs were then dried in an oven at 60°C for 30 days and weighed again to allow determination of the W/D ratio.
Specific Protocols
The rats were divided into six groups as follows (n = 6 in each group). Cold ischemia was always 2 hours, and reperfusion was always 60 minutes (Fig 1
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GROUP 2 (NON–HEART-BEATING DONORS/CONTROL).
Non–heart-beating rats were ventilated without NO for 30 minutes (warm ischemia). After 2 hours of cold ischemia, the lungs were mounted in the perfusion chamber and ventilated without NO.
GROUP 3 (NITRIC OXIDE/ISCHEMIA).
Non–heart-beating rats were ventilated with addition of inhaled NO for 30 minutes. After 2 hours of cold ischemia, lungs were mounted in the perfusion chamber and ventilated without NO.
GROUP 4 (NITRIC OXIDE/REPERFUSION).
Non–heart-beating rats were ventilated without NO for 30 minutes. After 2 hours of cold ischemia, lungs were mounted in the perfusion chamber and ventilated with additional inhaled NO.
GROUP 5 (NITRIC OXIDE/ISCHEMIA-REPERFUSION).
Non–heart-beating rats were ventilated with addition of inhaled NO for 30 minutes. After 2 hours of cold ischemia, lungs were mounted in the perfusion chamber and ventilated with additional inhaled NO.
GROUP 6 (NITRIC OXIDE FOR 60 MINUTES/ISCHEMIA-REPERFUSION).
Non–heart-beating rats were ventilated with addition of inhaled NO for 60 minutes. After 2 hours of cold ischemia, the lungs were mounted in the perfusion chamber and ventilated with additional inhaled NO.
Statistical Analysis
All results are expressed as mean ± standard error of the mean. Comparisons were performed using analysis of variance; the Newman-Keuls test was used as a post hoc test. A p value less than 0.05 was considered significant.
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Results |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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). Thirty minutes of warm ischemia in the NHBD/control group resulted in a significant increase in PVR (6.83 ± 0.36 mm Hg•mL-1•min-1; p < 0.05), Kfc (3.02 ± 0.35 mL•min-1•cm H2O-1•100 g-1; p < 0.05), and W/D ratio (8.07 ± 0.45; p < 0.05) as compared with the HBD/control group. Polymorphonuclear neutrophil sequestration at the end of the 1-hour reperfusion period was not significantly different (MPO activity, 0.25 ± 0.03 U/100 mg lung) from that of the NHBD/control group.
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). The W/D ratio (7.44 ± 0.43) and MPO activity (0.22 ± 0.05 U/100 mg lung) were not significantly affected.
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The best results were obtained in the group ventilated with NO during both ischemia and reperfusion (PVR, Kfc, W/D, and MPO activity were 1.04 ± 0.10 mm Hg • mL-1•min-1, 0.598 ± 0.128 mL•min-1•cm H2O-1•100 g-1, 6.10 ± 0.36, and 0.14 ± 0.02 U/100 mg lung, respectively). In this group, even MPO activity was significantly decreased as compared with the NHBD/control group (p < 0.05). Furthermore, values obtained for all variables in the NO/IR group were very close to those obtained in the HBD/control group.
Within those four groups, PVR was significantly correlated with Kfc (r = 0.881; p < 0.001), and MPO activity was significantly correlated with Kfc (r = 0.465; p < 0.05) (Fig 4).
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). Polymorphonuclear neutrophil sequestration was not significantly different (MPO activity: 0.14 ± 0.02 versus 0.23 ± 0.02 U/100 mg lung). Both groups were ventilated with inhaled NO during ischemia and during reperfusion.
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Comment |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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The model used in the present study has been routinely used in the assessment of lung preservation and IR injury [11, 12]. It is simple, and minimizes interferences with surgical technique, rejection, and immunosuppression that can obscure results in in vivo models. It provides more accurate estimates of the variables relevant to measurement of hydraulic conductivity (Kfc) as an index of pulmonary microvascular permeability. Coefficient of filtration control values presented for the HBD group are greater than those we observed in a previous work [11] with freshly prepared lungs because they were obtained in lungs after 2 hours of cold preservation followed by a 60-minute reperfusion period.
Limitations of this model are the short period of reperfusion after ischemia and the absence of gas exchange studies that could be done only if the lung was fed with desaturated blood. Preliminary experiments with longer reperfusion periods had demonstrated overwhelming lung injury. Therefore, the reperfusion period was reduced to 1 hour. The experimental schedule was set in a attempt to reproduce the clinical conditions to be encountered in lung transplantation using NHBD. These conditions include a warm ischemic period followed by cold ischemia and warm reperfusion with blood. To further approximate clinical conditions, we decided to keep the lungs ventilated during the period of in situ ischemia [4].
It is a simple method to provide oxygen to pulmonary cells, thus improving cell milieu homeostasis [15] and improving distribution of the preservation solution when lungs are harvested [4]. It is becoming evident that ischemia and reperfusion each produce different patterns of injury [16]. Warm ischemia-induced endothelial cell dysfunction in the pulmonary artery is well documented [6, 17] and seems to occur earlier than the alterations of pulmonary cell metabolism [18]. Alessandrini and associates [19] documented that nearly all lung cells were still viable 4 hours after death under oxygen ventilation, and D'Armini and colleagues [15] reported that O2 ventilation of the nonperfused lung preserves adenosine nucleotides up to 4 hours after death. We were unable to match these long ischemic times in our study, because 60 minutes of postmortem in situ ischemia, even under NO ventilation, resulted in severe reperfusion injury.
Reperfusion syndrome of the lung is characterized by increased pulmonary capillary permeability, pulmonary edema, and an acute increase in pulmonary vascular resistance. Several experimental and clinical reports support a beneficial role for NO in this setting [8, 9, 20, 21]. Nitric oxide is an important endogenous biological mediator of vascular tone, platelet function, neurotransmission, inflammation, and immune responses [8]. It not only is a potent vasodilator, but also inhibits interaction of the vessel wall with circulating PMNs [8]. Indeed, activated PMNs play a essential role in reperfusion injury. A previous study from this group [9] showed that inhaled NO, applied during reperfusion, prevented endothelial dysfunction and PMN accumulation in a neonatal piglet model of IR injury. In the present model, excellent lung function indexes were obtained when NO was applied during both ischemia and reperfusion. Neutrophil sequestration was also significantly decreased in this group. After pulmonary reperfusion, warm ischemia-induced endothelial dysfunction is exacerbated, endogenous NO levels plummet, and PMNs are activated and adhere to the endothelial surface [8, 22]. Supplying the lung with exogenous NO was a logical step in an attempt to maintain endothelial homeostasis during ischemia and reduce PMN adherence during reperfusion. Indeed, a significant correlation between MPO activity and Kfc was evident in our study. Moreover, it has been recently shown that in isolated rat lung NO does not directly influence fluid loss associated with endothelial lung injury [23]. Therefore, prevention of PMN sequestration is likely to be the key mechanism by which NO exerts its beneficial effect in reperfusion injury. Production of non–PMN-derived oxygen radicals is considered the initial proinflammatory event that leads to adhesion of PMNs to the endothelium and to PMN activation. Inhaled NO at 24 ppm was recently shown to prevent the increase in pulmonary vascular permeability caused by hydrogen peroxide in an isolated, buffer-perfused lung preparation [24]. This observation suggests that besides the inhibition of PMN lung sequestration, alternative pathways exist by which NO may decrease lung reperfusion injury.
Nitric oxide administration has also been reported to have adverse effects, depending on the timing of administration, the dose, or the model [25, 26]. During reperfusion, a massive burst of oxygen free radicals occurs. Nitric oxide promptly reacts with the superoxide anion to form the potent oxidant peroxynitrite, a strong oxidant, which would be expected to worsen IR injury [25, 26]. In a recent study, Naka and associates [27] reported that inhaled NO, given at reperfusion, did not have beneficial effects on the immediate survival of rat lung transplant recipients. In contrast, stimulation of the distal NO pathway with cyclic guanosine monophosphate during the preservation period was associated with better results [27]. Although no adverse effects from the administration of NO during reperfusion were noted, Naka and associates concluded that buttressing the NO pathway during ischemia is a better strategy to reduce IR injury. Eppinger and colleagues [28] reported that inhaled NO, started at reperfusion, was toxic after 30 minutes, but protective at 4 hours. In their study, the initial alteration could be prevented by delaying NO inhalation until after the beginning of reperfusion, or by pretreating the animals with superoxide dismutase, which suggests that the interaction of NO with superoxide was responsible for the toxicity. Although we did not measure lung function 30 minutes after reperfusion, we did not notice adverse effects at 60 minutes in the group treated with NO during reperfusion only. This group still fared better than the group subjected to NO during ischemia only, although not significantly. Furthermore, we used much lower concentrations of NO (30 ppm) compared with Eppinger and colleagues' study (80 ppm). In addition, the preservation solution we used (Celsior) contains reduced glutathione, a powerful antioxidant. Indeed, we demonstrated in a previous study [29] that the use of Celsior significantly prevented the IR-induced increase in pulmonary microvascular permeability. If NO administration during initial reperfusion resulted in a significant increase in peroxynitrite, the reduced glutathione contained in Celsior might have contributed to neutralizing it.
In conclusion, in lungs harvested from NHBD, IR injury was significantly reduced by administration of inhaled NO during ischemia and reperfusion. Beneficial effects were also observed when NO was applied only during reperfusion. This effect might be related to a homeostatic effect in the pulmonary vascular bed during ischemia, and to prevention of PMN sequestration during reperfusion. Nitric oxide ventilation might be a useful tool in the management of patients with reperfusion injury after NHBD lung transplantation.
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Acknowledgments |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Footnotes |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Address reprint requests to Dr Mazmanian, Centre Chirurgical Marie-Lannelongue, 133 Ave de la Résistance, 92350 Le Plessis Robinson, France (e-mail: gmmazman{at}pratique.fr).
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References |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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