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Department of Cardiovascular and Thoracic Surgery, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China
Accepted for publication March 26, 2008.
* Address correspondence to Dr Yang, Cardiac Studies, Institute for Biodiagnostics, National Research Council of Canada, 435 Ellice Ave, Winnipeg, Manitoba, R3B1Y6, Canada (Email: victor.yang{at}nrc-cnrc.gc.ca).
Presented at the Forty-fourth Annual Meeting of The Society of Thoracic Surgeons, Fort Lauderdale, FL, Jan 28–30, 2008.
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Abstract |
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 Top Abstract IntroductionMaterial and MethodsResultsCommentDiscussionAcknowledgmentsReferences |
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Methods: Sixteen piglets underwent 90-minute circulatory arrest at 18°C before rewarming. During circulatory arrest, antegrade infusion of Ringer's lactate solution alone (n = 8) or containing L-arginine (1 mg/kg/min, n = 8) was initiated into the pulmonary circulation. Disaturated phosphatidylcholine, total phospholipids, and total proteins from tracheal aspirates were measured serially until the experiment ended (4 hours after rewarming). Various variables of pulmonary function were also monitored.
Results: L-arginine led to less decrement of disaturated phosphatidylcholine/total phospholipids and disaturated phosphatidylcholine/total proteins after DHCA. At 4 hours after rewarming, L-arginine had significantly mitigated the deterioration of pulmonary static compliance (3.6 ± 0.5 vs 3.3 ± 0.3 mL/cm H2O) and partial pressure of arterial oxygen/fraction of inspired oxygen (330 ± 48 vs 296 ± 32 mm Hg). Pulmonary retention of water (6.2 ± 1.0 vs 5.5 ± 1.2) was significantly reduced. The L-arginine-treated group showed an increase in NO metabolites (NO2 –/NO3 –) from the pulmonary circulation, the extent of which is correlated to PS content.
Conclusions: Continuous L-arginine supplementation during DHCA attenuated PS depletion and, therefore, ameliorated postoperative pulmonary dysfunction.
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Introduction |
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TopAbstractIntroductionMaterial and MethodsResultsCommentDiscussionAcknowledgmentsReferences |
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Recent studies indicated that ischemia, followed by reperfusion, played an important role in the development of pulmonary dysfunction due to endothelial injury and consequential abnormal production and secretion of nitric oxide (NO) [3]. It has also been suggested that the intrinsic physiologic level of NO is important in maintaining normal lung surfactant secretion by alveolar type II epithelial cells [4]. As a result, one strategy aimed at maintaining NO production during CPB began to emerge as some other pioneer studies, based on non-CPB induced pulmonary injury, suggested that supplementation of extraneous NO precursor, L-arginine, could attenuate impaired endothelium-dependent pulmonary vasorelaxation [5, 6]. Its efficiency in the field of cardiovascular surgery requiring CPB with or without DHCA is under investigation. For example, it has been reported that initiation of L-arginine after reperfusion could reduce pulmonary vascular resistance (PVR) in animals receiving DHCA [7].
Because lung begins to sustain ischemic injury during DHCA, we asked (1) whether continuous antegrade pulmonary infusion of L-arginine during DHCA could mitigate the impairment of pulmonary function, and (2), if so, whether use of L-arginine during this stage could provide protective effects on the integrity of pulmonary surfactant metabolism. To answer these questions, we used a neonatal piglet DHCA model established in our laboratory [2].
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Material and Methods |
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TopAbstractIntroductionMaterial and MethodsResultsCommentDiscussionAcknowledgmentsReferences |
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Preoperative anesthesia was induced intramuscularly with midazolam (0.3 mg/kg), ketamine (22 mg/kg), and atropine (0.022 mg/kg). After endotracheal intubation, the pig was ventilated mechanically with 60% oxygen and 40% air. The ventilator rate and tidal volume were adjusted to maintain the arterial PaCO 2 between 35 and 45 mm Hg. Anesthesia was maintained with 1.5% to 2.0% isoflurane. A temperature probe was placed in the esophagus to monitor the core temperature. Urine output was collected through a bladder catheter.
The chest was opened with a median sternotomy. The ascending aorta was cannulated with an 8F aortic cannula and the right atrium with a 14F vena cava cannula for CPB in all the animals. The main pulmonary artery (PA) was cannulated with a modified 6F retrograde cardioplegic catheter used for continuous antegrade pulmonary infusion (large lumen) and mean PA pressure (mPAP) monitoring (small lumen) during DHCA. A 12F vena cava cannula and a 5F polyurethane sheath were placed into the left atrium to drain the infused solution during DHCA and to monitor the left atrial pressure (LAP), respectively. An electromagnetic flow probe (FR-100T, Nihon Kohden, Tokyo, Japan) was placed on the main PA for the measurement of cardiac output (CO). The right internal mammary artery was cannulated to monitor aortic blood pressure.
The CPB circuit consisted of a Sarns 8000 nonpulsatile roller pump (Sarns, Terumo CVS, Ann Arbor, MI) and a Medtronic Minimax Plus infant membrane oxygenator (Medtronic, Minneapolis, MN) with venous reservoir forming the CPB circuit. Circuit blood gases were monitored using a CDI-300 (CDI; 3M Healthcare, Irvine, CA) continuous in-line blood gas analyzer. A pediatric arterial filter (20 µm, Dideco, Mirandola, Italy) was incorporated in the bypass circuit. The circuit was primed with fresh donor pig whole blood, Ringer's lactate solution, Pentaspan (B. Braun Medical, Irvine, CA), sodium bicarbonate, and heparin to maintain a hematocrit of about 25%. Arterial blood gas was maintained within normal ranges. Sodium bicarbonate was added to adjust the pH to 7.40, when necessary. Alpha-stat blood gas management was used throughout the experiment.
Groups and Protocol
Sixteen piglets were randomly assigned to two groups. During 90 minutes of DHCA, the 8 piglets in group RL, serving as control, received a continuous pulmonary infusion of Ringer's lactate solution, and the 8 piglets in group Arg received continuous pulmonary infusion of Ringer's lactate solution containing L-arginine. The protocol (Table 1) for both experimental groups consisted of 10 minutes of normothermic CPB (baseline at 37°C), about 35 minutes of cooling, 10 minutes of hypothermic CPB at 18°C, 90 minutes of DHCA with continuous pulmonary infusion of Ringer's lactate solution without (group RL) or with L-arginine (group Arg), 10 minutes of hypothermic reperfusion CPB at 18°C, about 40 minutes of rewarming, and 4 hours of subsequent observation after successful weaning from bypass. The body temperature was maintained about 37.5°C.
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Measurements
Chemical analysis of pulmonary surfactant
Measurement of pulmonary surfactant has also been detailed in our previous study [2]. Briefly, tracheal aspirates containing pulmonary surfactant were suctioned using 10 mL of normal saline at room temperature. The time point set for aspiration was before CPB (baseline), at the end of hypothermic CPB, 90-minute DHCA and rewarming, as well as at 2 and 4 hours after weaning from bypass. Aliquots of aspirates were extracted with threefold volumes of chloroform-methanol (2:1, vol/vol) to isolate the phospholipids in the chloroform phase. Disaturated phosphatidylcholine (DSPC), the major surface-tension lowering component of lung surfactant, was separated from other phospholipids as described by Mason and colleagues [8]. Briefly, samples from the chloroform phase were dried under nitrogen gas, oxidized with a small volume of osmium tetroxide in carbon tetrachloride for 15 minutes, and dried again under nitrogen, dissolved in chloroform-methanol (20:1, vol/vol), and passed through a neutral aluminum column. The DSPC fraction was collected by adding a mobile phase of chloroform-methanol-7M ammonium hydroxide (70:30:2, vol/vol/vol) to the column.
Amounts of DSPC and total phospholipids (TPL) were determined according to the methods described by Bartelett [9] and corrected by the total volume of the aspirates. The ratio of DSPC and TPL (DSPC/TPL, %), an indicator reflecting the extent of surfactant change due to potentially injurious factors, was calculated. Total proteins (TP) in the aspirates were measured according to the method of Lowry and associates [10] with bovine serum albumin as the standard and total aspirated fluid as the normalization volume. Also calculated was the ratio of DSPC and TP (DSPC/TP, µg/mg), a variable reflecting the balance between alveolar surfactant and plasma proteins leaking into the airspaces due to impaired vascular integrity.
Pulmonary function and pulmonary vascular resistance
Measurement of pulmonary static compliance (Cstat), oxygenation index measured by partial pressure of arterial oxygen/fraction of inspired oxygen (PaO
2/FIO
2), and PVR had been detailed in our previous study [2]. Briefly, a Servo 900C ventilator (Siemens-Elema AB, Solna, Sweden) was used to assess Cstat. Its measurement was done before CPB, at the end of hypothermic CPB, every 30 minutes during 90-minute DHCA, at the end of rewarming, and at 2 and 4 hours after CPB. The peak inspiratory pressure and positive end-expiratory pressure were kept constant at 25 cm H2O and 2 cm H2O, respectively, while Cstat was assessed. Static compliance was measured as a tidal volume divided by the applied pressure (end-inspiratory plateau pressure minus end-expiratory pressure) and expressed in mL/cm H2O. A mean value of Cstat was calculated for a minimum of six breaths at each measurement. Measurements of both oxygenation index and PVR were scheduled before, and at 1, 2, and 4 hours after CPB. Blood was sampled from the arterial catheter to determine the level of PaO
2. PaO
2/FIO
2 was used to reflect pulmonary oxygenation change before and after CPB involving DHCA. In addition, PVR was calculated using the formula: PVR (dyn · s · cm–5) = 80 x [mPAP (mm Hg) – LAP (mm Hg)]/CO (L/min).
Myeloperoxidase activity
At the end of the experiment, lung tissue was preserved for measurement of myeloperoxidase, which indicates the extent of neutrophil infiltration [2]. Briefly, after homogenization of the frozen lung tissue samples (about 50 mg) in 0.5% hexadecyltrimethylammonium bromide dissolved in 10 mmol/L 3-[N-morpholino] propane sulfonic acid, centrifugation was set at 21,000g for 20 minutes at 4°C. The supernatant was mixed with sodium phosphate (80 mmol/L, pH 5.5) and tetramethyl benimide (16 mmol/L) and incubated at 25°C for 5 minutes. After hydrogen peroxide (1 mmol/L) was added, the samples were incubated exactly 3 minutes at 25°C. A blank without hydrogen peroxide was also analyzed for each tissue. The reaction was stopped by adding aliquots of cold acetic acid (2M). The optical density was measured at 650 nm on a spectrophotometer. Myeloperoxidase activity was the quantity of enzyme degrading 1 µmol of hydrogen peroxide per minute at 37°C.
Plasma nitrite/nitrate levels
Nitrite/nitrate (NO2
–/NO3
–) is the stable end products of NO in the plasma. Blood was serially sampled before and at 1, 2, and 4 hours after bypass from the sheath placed in the left atrium. They were measured using a commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Briefly, nitrate was converted to nitrite with Aspergillus nitrite reductase, and the total nitrite was measured with the Griess reagent. The absorbance was determined at 550 nm with a spectrophotometer.
Wet-to-dry lung weight ratio
A piece of lung tissue (about 1 g) from the posterior part of the left lower lobe was cut, and its wet weight was determined in an automatic electric balance (AP250D, Ohaus, Florham, NJ). The piece of lung tissue was then dried in an oven at 80°C for 48 hours and weighed again to obtain its dry weight for calculation of the wet-to-dry weight ratio.
Statistical Analysis
Statistical analysis was performed using SAS software (SAS Institute, Cary, NC). All data are presented as mean ± standard deviation (SD). Pulmonary surfactant (DSPC/TPL and DSPC/TP), PVR, and NO2
–/NO3
– were normalized by comparison with baseline values and expressed as a percentage of the baseline values. A repeated-measures analysis of variance and the Duncan multiple-range test were used for comparison between different time points within a group, and the Student t test was used for comparison between the two groups. The Pearson test was used for correlation study between changes of pulmonary surfactant and plasma NO2
–/NO3
– levels. A value of p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterial and MethodsResultsCommentDiscussionAcknowledgmentsReferences |
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Chemical Analysis of Pulmonary Surfactant
Disaturated phosphatidylcholine was expressed as the ratio of disaturated phosphatidylcholine to total phospholipids (DSPC/TPL) and disaturated phosphatidylcholine to total proteins (DSPC/TP) from the tracheal aspiration. The level obtained before CPB was used as the baseline level (100%). A gradual decrease in both DSPC/TPL and DSPC/TP were observed during and after CPB (including DHCA; Fig 1). Decrement of both variables was more significant in group RL than in group Arg, with statistical difference beginning to appear at 2 hours after weaning from bypass.
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Plasma Nitrite/Nitrate Levels
The percentage change of NO2
–/NO3
–, sampled from the left atrium, increased significantly within 2 hours after weaning from CPB in group Arg, but no significant change was observed in group RL (Fig 3). Comparison between groups at respective time points revealed that a significant difference appeared at 1 hour after weaning from CPB between the two groups.
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Relationship of Pulmonary Surfactant to Plasma Nitrite/Nitrate Levels
The correlation coefficients between pulmonary surfactant in the tracheal aspirates (2 hours after rewarming) and plasma NO2
–/NO3
– (1 hour after rewarming) were determined separately for the two groups (Fig 4). In group Arg, significant correlation was noted between DSPC/TPL and NO2
–/NO3
–. However, no significant correlation was observed in group RL.
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Comment |
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TopAbstractIntroductionMaterial and MethodsResultsCommentDiscussionAcknowledgmentsReferences |
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The initial evidence indicating an interaction of pulmonary surfactant and L-arginine comes from research focusing on understanding of the relationship between NO and surfactant metabolism. Sun and colleagues [4] examined the effects of NO on lung surfactant secretion using an isolated perfused rat lung model and primary culture of alveolar type II cells, which synthesize and secrete pulmonary surfactant. They found that administration of L-NG-nitroarginine methyl ester (L-NAME), an inhibitor of both constitutive and inducible NO synthases, through the pulmonary circulation led to significantly decreased surfactant secretion. Further tests revealed that such inhibition mainly involved specific blockage of constitutive NO synthase. Their subsequent investigation focusing on NO signaling pathway revealed that NO-mediated surfactant secretion was related to cyclic guanosine monophosphate and protein kinase G in alveolar type II cells [4]. As a result, lack of sufficient NO production, which could be induced by pulmonary ischemia and reperfusion injury during DHCA, will inevitably play some role in inducing disturbed metabolism of pulmonary surfactant and pulmonary function.
Inversely, lack of surfactant production in alveolar type II cells will further aggravate its abnormality because surfactant is also an important signal for stimulation of constitutive NO synthase within the cell's own cytoplasm. Pulmonary vascular endothelium is a major source of NO production and its impairment would inevitably impair surfactant production and secretion. Of course, leaking of serum proteins into the alveolar caused by perfusion-induced lung injury could further aggravate surfactant dysfunction due to direct inhibition [11]. As a result, DSPC/TP is also a valuable indicator in assessing the extent of surfactant integrity.
Of course, the beneficial effect with the use of L-arginine is not only centered around the provision of NO inside the lung but is also possibly related to its inhibiting effects on neutrophils infiltrating into the lung tissue during CPB. It has been proposed that pulmonary dysfunction related to the use of CPB only is mainly due to systemic inflammatory response syndrome, the results of which include massive infiltration of neutrophils cells into the lung tissue. Ultimately, the lung would be injured due to secretion of enzymes, such as various proteinases and oxygen free radicals. Several studies had indicated that L-arginine could attenuate pulmonary infiltration of neutrophils. For example, Sheridan and colleagues [12] found that L-arginine could reduce endotoxin-induced neutrophils accumulation in the lung by more than 50%. Our results also supported this notion that L-arginine could help reduce the extent of neutrophil infiltration in the lung tissue. This inhibitory affects of L-arginine are related to attenuation of lung chemokine production, which is mediated by nuclear factor-
B (NF-B), an inducible transcription factor under the control of inhibitory factor B-a (IB-a).
Calkins and colleagues [13] examined the effect of L-arginine on chemokine production, IB-a degradation, and NF-B DNA binding in the lung after systemic lipopolysaccharide-induced inflammation. To block NO production, an NO synthase inhibitor was given before L-arginine was applied. Lipopolysaccharide induced production of chemokines as reflected by increased expression of messenger RNA (mRNA) and protein. L-arginine attenuated such increased expression, prevented reduction of intracellular IB-a levels, and inhibited NF-B DNA binding. NO synthase inhibition abolished the effects of L-arginine on all measured variables. As such, they concluded that L-arginine could abrogate chemokine protein and mRNA production during systemic inflammation, which is dependent on NO and is mediated by stabilization of IB-a levels and inhibition of NF-B DNA binding [13].
This study has some intrinsic limitations. First, as a result of anatomic differences between humans and animals, and the use of an acute swine model, our data may not be completely translated into clinical situations. However, animal models provide controlled experimental conditions and allow measurements that often are not feasible in humans. Second, our study did not test the use of NO synthases specific inhibitors such as L-NAME to confirm that increased production of NO is a major mechanism leading to improved pulmonary surfactant integrity, pulmonary function, and PVR after CPB involving the use of DHCA. However, our results clearly suggested that better preservation of pulmonary surfactant was related to increased plasma NO2 –/NO3 – level, the result of which is also in agreement with other similar studies [7, 14]. The present study provides a detailed report on acute change of important pulmonary parameters after intrapulmonary continuous infusion of L-arginine during DHCA. A chronic animal model would be ideal to evaluate delayed pulmonary changes that cannot be measured in an acute study.
In conclusion, in our pig model, intrapulmonary supplementation of L-arginine during DHCA significantly reduced the impairment of pulmonary surfactant integrity, pulmonary function, and vascular resistance, leading to less pulmonary tissue injury. Further studies are necessary to determine the optimal conditions such as dosage and timing for its administration.
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Discussion |
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DR YANG: Our group previously published a paper comparing DHCA with deep hypothermic low-flow perfusion without use of any extra medications [2]. We found that DHCA alone could help, to some extent, retain the integrity of the pulmonary surfactant. When it was compared with the new strategy using L-arginine during DHCA, we found that its continuous pulmonary infusion with Ringer's lactated solution as solvent could provide additional protective effects in terms of surfactant integrity and pulmonary function.
DR CARL L. BACKER (Chicago, IL): Can I ask, what is the relationship or mechanism of action comparing nitric oxide and surfactant? I think the Vanderbilt group has published some data using L-arginine showing that it increases nitric oxide and decreases pulmonary vascular resistance. What is the relationship between surfactant and nitric oxide?
DR YANG: Several research groups assessed the function of L-arginine in the alveolar type II epithelium and pulmonary vascular endothelium, respectively. They found that L-arginine is not only necessary for the endothelium to keep the vascular smooth muscle cell relaxing, it also plays a very important role in the maintenance of normal function by epithelial type II cells as these cells also contains nitric oxide synthase (constitutive form) [4]. The content of nitric oxide is a very important signaling factor for the control of surfactant synthesis and final excretion into the alveolar cavity. As a result, we believed that the mechanisms of the protection provided by L-arginine included its effects on both endothelium and epithelium (ie, maintaining surfactant synthesis and secretion during and after surgery). As such, our next stage of research will focus on in vitro study to answer this question.
DR BACKER: How far do you think we are from trying to use this clinically for this purpose? Do you think this is going to be used clinically within the next 5 or 10 years?
DR YANG: Actually, a group from another hospital (ie, Fu Wai hospital) in China had reported intraoperative pulmonary use of solution containing L-arginine for patients with tetralogy of Fallot; however, the solution contained several other drugs [15]. As a result, we don't know which component in their solution plays a key role in maintaining normal pulmonary function after surgery. In this study, we decided to observe the effects of L-arginine alone instead of multiple components altogether. A bench-to-bedside study will be carried out in the future.
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Acknowledgments |
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TopAbstractIntroductionMaterial and MethodsResultsCommentDiscussionAcknowledgmentsReferences |
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