Chest ACCP Member Benefits
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Guest Access | Sign In via User Name/Password
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Article Archive
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via ISI Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by El Kebir, D.
Right arrow Articles by Blaise, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by El Kebir, D.
Right arrow Articles by Blaise, G.
(Chest. 2005;128:2910-2917.)
© 2005 American College of Chest Physicians

Effects of Inhaled Nitric Oxide on Inflammation and Apoptosis After Cardiopulmonary Bypass*

Driss El Kebir, PhD; Bernard Hubert, MD; Rame Taha, MD; Eric Troncy, PhD; Tianlong Wang, MD; Dominique Gauvin, BS; Marius Gangal, MD, PhD and Gilbert Blaise, MD

* From the Laboratory of Anesthesia (Drs. El Kebir, Taha, Troncy, Wang, Gangal, and Blaise, and Ms. Gauvin), Department of Anesthesia and Research Center, Center Hospitalier de Ùniversitie de Montreal, Hopital Notre-Dame, Montreal, QC, Canada; and Department of Anesthesia and Intensive Care Medicine (Dr. Hubert), CHU Liege, Belgium.

Correspondence to: Gilbert Blaise, MD, Center Hospitalier de Ùniversitie de Montreal–Hopital Notre-Dame Hospital, Laboratory of Anesthesia, Deschamps Pavilion, Room FS-1136, 1560 Sherbrooke St East, Montreal, QC, Canada, H2L 4M1; e-mail: blaisegil{at}sympatico.ca


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Cardiopulmonary bypass (CPB), a procedure often used during cardiac surgery, is associated with an inflammatory process that leads to lung injury. We hypothesized that inhaled nitric oxide (INO), which has anti-inflammatory properties, possesses the ability to modulate lung cell apoptosis and prevent CPB-induced inflammation.

Methods: Twenty male pigs were randomly classified into four groups: sham, sham plus INO, CPB, and CPB plus INO. INO (20 ppm) was administered for 24 h after anesthesia. CPB was performed 90 min into INO treatment. BAL fluid and blood were collected at time 0 (before CPB), at 4 h after beginning CPB, and 24 h after beginning CPB (T24).

Results: At T24, BAL interleukin (IL)-8 levels and neutrophil percentages were elevated significantly in the CPB group. At T24, INO reduced IL-8 concentrations and attenuated the increase of neutrophil percentage in the CPB-plus-INO group. Nitrite-plus-nitrate (NOx) concentrations were decreased significantly in groups without INO. Moreover, animals treated with INO showed higher rates of pulmonary apoptosis compared to their respective control groups except for the sham-plus-INO group, in which they were diminished.

Conclusion: These results demonstrate that NOx production is reduced after CPB, and that INO acts as an anti-inflammatory agent by decreasing neutrophil numbers and their major chemoattractant, IL-8. INO also increases cell apoptosis in the lungs during inflammatory conditions, which may explain, in part, how it resolves pulmonary inflammation.

Key Words: cardiopulmonary bypass • endothelial nitric oxide synthase • inhaled nitric oxide


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cardiopulmonary bypass (CPB) induces a systemic inflammatory response via many factors, including the exposure of blood to nonphysiologic surfaces and conditions, surgical trauma, ischemia-reperfusion of the organs, changes in body temperature, and release of endotoxin.

The effects of CPB on proinflammatory tumor necrosis factor (TNF)-{alpha}, interleukin (IL)-6, IL-8, and anti-inflammatory IL-10 cytokines have been measured, with many studies12 showing an increase in plasma and BAL cytokines after CPB. IL-8, a potent chemoattractant for neutrophils, is involved in neutrophil influx and in the release of their cytotoxic products, which include free radical species, proteolytic enzymes, and eicosanoids that subsequently cause acute lung injury (ALI). Neutrophils normally have a short half-life (< 24 h), with aging cells undergoing apoptosis (programmed cell death). This mechanism is the most efficient way of removing these cells without release of their cytotoxic products, limiting damage to surrounding tissues. Conversely, inhibition of apoptosis of these cells increases their life span, leading to amplification of the inflammatory process.

Nitric oxide (NO) has numerous physiologic and pathophysiologic functions. It can be inhaled as a therapeutic agent. Inhaled NO (INO) is capable of decreasing high pulmonary artery pressure, improving hypoxemia by reducing intrapulmonary shunt, and optimizing ventilation-perfusion matching. INO can also inhibit the inflammatory process by lowering cytokine synthesis and inactivating nuclear factor (NF)-{kappa}B,3 as several cytokines contain a binding site for NF-{kappa}B in their promoter regions. NO can also decrease the expression of adhesion molecules, preventing neutrophil adhesion and migration. INO can exert its effect on the lungs, on leukocytes trapped in the pulmonary area, but as it is transported by RBC to the general circulation, INO could have extrapulmonary outcomes.

Studies456 have demonstrated that NO can inhibit apoptosis induced in several cell types by proinflammatory agents. NO also exhibits a proapoptotic role, which involves inducible NO synthase (iNOS) activation. The source and dose of NO, cell type, and local environment appear to be important determinants of cellular fate.7 All this knowledge of NO has stimulated much interest in INO as a therapeutic agent against inflammation and to improve pulmonary function. In our study, we assessed whether 20 ppm of INO administered preventively can modulate both inflammation and apoptosis in the lungs of pigs submitted to CPB for 90 min.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
The following experimental protocol was performed with the approval of the institutional animal care committee in compliance with the guidelines of the Canadian Council on Animal Care. The anesthetic method and CPB technique have been described extensively elsewhere.8 Briefly, healthy male pigs (mean weight, 37.15 ± 2.48 kg [± SD]) were premedicated IM with atropine (0.04 mg/kg), azaperone (4 mg/kg), and ketamine (25 mg/kg), and then anesthetized with 5 µg/kg of fentanyl and 5 mg/kg of thiopental. After intubation with an 8-mm endotracheal tube (Mallinckrodt Company; Mexico City, Mexico), the pigs were placed in the supine position. Anesthesia was maintained by continuous infusion of 5 mg/kg/h of thiopental and 20 µg/kg/h of fentanyl. Muscle relaxation was induced with 0.2 mg/kg of pancuronium followed by intermittent reinjections of 0.1 mg/kg to achieve optimal surgical and ventilatory conditions.

After induction of anesthesia and tracheal intubation, 20 ppm of NO gas was injected cyclically into the inspiratory line during the inspiratory phase by a NO injector for 24 h, and 1,000 ppm NO (Balanced N2 Cylinder; VitalAire Santé Ltée; Montreal, QC, Canada) NO and NO2 concentrations delivered to the animals were monitored with an electrochemical device (Polytron NO/NO2; Drager A.G.; Lubeck, Germany). During CPB, NO was also added directly to the gas mixture delivered to the oxygenator.

Two groups of pigs underwent 90 min of CPB with cardioplegic cardiac arrest for 75 min. After 90 min, the animals were weaned from CPB (Table 1 ). Homeostasis was performed after removal of the CPB cannula, and the chest was closed.


View this table:
[in this window]
[in a new window]

  		Table 1.. CPB Time Course

 
The CPB circuit consisted of a membrane oxygenator with a cardiotomy reservoir (Affinity NT541; Medtronic; Minneapolis, MN), filter (Affinity 351; Medtronic), and tubing. A Sarns roller pump (Sarns; Ann Arbor, MI) was used. The circuit was primed with 1,500 mL of lactated Ringer solution (Baxter Corporation; Toronto, ON, Canada), 500 mL of colloidal fluid (Pentaspan; DuPont Pharma; Mississauga, ON, Canada), 1 mEq/kg of natrium bicarbonate, 5,000 IU of heparin, and 200 mL of mannitol.

The animals were ventilated and monitored in an intensive care set-up for 24 h after the beginning of CPB, and then killed. Lung samples were obtained as described below. BAL fluid was collected at time before CPB (T0), 4 h after the beginning of CBD (T4), and 24 h after the beginning of CPB (T24). Animals not submitted to CPB underwent sternotomy alone for 90 min and chest closure without bypass (sham operation).

Protocol
The animals were randomized into four groups: (1) the sham group (n = 5) was subjected to sternotomy for 90 min, followed by chest closure without CPB, and monitored until T24; (2) the sham-plus-INO group (n = 5) was submitted to INO administration (20 ppm), 90-min sternotomy, chest closure without CPB, and monitored until T24; (3) the CPB group (n = 5) was subjected to 90 min of CPB and monitored until T24; and (4) the CPB-plus-INO group (n = 5) underwent 90 min of CPB with INO therapy (20 ppm) throughout the experiment and was monitored until T24.

Measurements
BAL: The BAL method consisted of instilling three separate aliquots (25 mL, 25 mL, and 25 mL) of isotonic sterile saline solution (0.9% NaCl) into a segment of the cranial lobe of either lung, via a flexible fiberoptic bronchoscope introduced through the endotracheal tube and wedged in an airway. During BAL sampling, fraction of inspired oxygen was increased to 100%.

BAL Analysis: BAL cells were harvested by centrifugation at 800 revolutions per minute for 8 min at 4°C. The supernatant was aliquoted and preserved at – 80°C for further analysis of IL-8 and TNF-{alpha}. The pellet was resuspended in 10 mL of RPMI-1640 plus 10% fetal calf serum, and viability was determined by trypan blue exclusion. Cell counts were then adjusted to 1 x 106/mL. Total cell count was achieved by the hemocytometer method; differential cytospin slides were produced and stained with Wright-Giemsa for cell differentiation. Cells were counted under a microscope at original x 100 magnification.

Cytokine Measurements: BAL IL-8 and TNF-{alpha} concentrations were measured by enzyme-linked immunoassay (Biosource; Camarillo, CA; Pierce Endogen, Woburn, MA), according to the manufacturer protocols.

Plasma and Lung NO Metabolite Measurements: Heparinized blood samples were centrifuged at 1,500 revolutions per minute for 10 min at 4°C, and the plasma was stored at – 80°C. At T24, the pigs were killed, and their lungs were harvested and stored at – 80°C. Plasma and lung tissue nitrite-plus-nitrate (NOx) levels were quantified by chemiluminescence NO analyzer (270B; Sievers Instruments; Boulder, CO) and a data acquisition system (Dataq; Dataq Instruments; Akron, OH) as described previously.9

Apoptosis in Lung Tissue: Apoptosis in lung tissue samples was measured by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) technique with fluorescein in situ apoptosis detection kits (S7110 ApopTag; Intergen Company; Purchase, NY), as described in the manufacturer protocol. Briefly, lung sections were mounted on immunologic slides, deparaffinized, and pretreated with proteinase K. Free termini of the 3'-OH end of lung DNA were labeled with digoxigenin-deoxynucleoside triphosphate. A peroxidase-binding antibody conjugate (anti-digoxigenin) was added, followed by its staining with fluorescein (substrate). The slides were mounted under glass coverslips and read with a fluorescence microscope. A positive control was pretreated with deoxyribonuclease, and a negative control was incubated without terminal transferase. At least 10 fields per tissue section were analyzed per pig. Apoptosis was calculated as the percentage of fluorescence of each field divided by total tissue surface fluorescence. TUNEL labeling was defined as positive when tissue fluorescence intensity was as high as or higher than that of the positive control. All slides were analyzed with Metamorph 4.6 software (Roper Scientific; Tucson, AZ).

Western Blot Analysis: Pig lung tissues were snap frozen in liquid nitrogen and stored at – 80°C. Protein extracts were prepared by Dounce homogenization of lung tissue in 50 mmol/L Tris (pH 7.6)-HCl lysis buffer containing protease inhibitors (5 mmol/L ethylenediamine tetra-acetic acid; 1 mmol/L phenylmethylsulfonyl fluoride; 5 µg/mL pepstatin; 5 µg/mL aprotinin, 5 µg/mL leupeptin). Extracts were clarified through centrifugation (14,000g for 10 min at 4°C). The supernatants were quantified for protein concentration by protein assay (Bio-Rad Laboratories; Hercules, CA). Equal amounts (50 µg) of protein were subjected to (10 to 15%) sodium dodecylesulfate-polyacrylamide gel electrophoresis. The proteins were transferred to nitrocellulose membranes (Amersham Biosciences; Buckinghamshire, UK) that were blocked with 5% non-fat dry milk in Tris buffered saline solution containing 0.1% Tween 20, and then washed with Tris buffered saline solution-Tween. The membranes were incubated overnight at 4°C with gentle shaking with primary polyclonal rabbit anti-endothelial NOS (eNOS) antibody, or polyclonal rabbit anti-iNOS antibody (1:1,000 and 1:3,000) dilutions, respectively; Transduction Laboratories) or polyclonal rabbit anti-caspase-3 antibody (5 µg/mL; NeoMarker, supplied by Medicorp; Montreal, QC, Canada), which recognize the native form (32 kd) and processed (20 kd and 18 kd) forms of caspase-3.

After washing, the membranes were incubated with gentle shaking for 2 h at room temperature with goat anti-rabbit IgG-horseradish peroxidase antibody (1:5,000 dilution; Bio-Rad). The membranes were visualized with enhanced chemiluminescence kit (Amersham Biosciences) and detected by photographic film. Bands were quantified with using software (ImageQuant; Molecular Dynamics; Sunnyvale, CA).

Statistical Analysis
All results are expressed as means ± SEM. Comparisons between experimental groups were performed with one-way analysis of variance (ANOVA), followed by the post hoc Tukey test, and comparisons over time within each group were made by ANOVA for repeated measures, followed by the Bonferroni post hoc test. Nonparametric Kruskal-Wallis ANOVA was followed by Dunn post hoc test, where appropriate. Correlations between variables were analyzed by Pearson correlation test. p < 0.05 was considered statistically significant.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of INO on BAL Cell Count, Neutrophil Percentage, and IL-8 Levels
BAL cell count and neutrophil percentage showed a trend to increase from T0 to T24 in all groups. This increment was significant in the CPB group (p < 0.05). However, its magnitude was attenuated in groups receiving INO (Table 2 ). Neutrophil count was correlated with IL-8 concentration in the CPB group at T24 (r = 0.66, p < 0.05).


View this table:
[in this window]
[in a new window]

  		Table 2.. Change in WBC Count and Neutrophil Percentage in BAL*

 
At T24, IL-8 levels were significantly elevated in the CPB group and reduced in the CPB-plus-INO group (Fig 1 ). No significant variation in the IL-8 between T0 and T4 was noted in all groups. BAL TNF-{alpha} concentrations were not detected.



View larger version (10K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1.. Effect of INO on BAL IL-8 concentrations. IL-8 was increased in the CPB group and reduced significantly in the CPB-plus-INO group. *p < 0.05 compared to T0. {dagger}p < 0.05 vs the sham-plus-INO group at T24.

 
NOx Concentrations
In plasma, NOx levels decreased significantly in the sham group at T24, and at T4 in the CPB group (Table 3 ); however, they were significantly higher at T24 in INO groups (171.77 ± 40.77 µmol/L) than T0 (131.9 ± 12.02 µmol/L). In lung tissue, NOx concentration was similar in groups without INO. It was increased in the CPB-plus-INO group compared to the control animals (p < 0.05) [Fig 2 ].


View this table:
[in this window]
[in a new window]

  		Table 3.. Plasma NOx Levels*

 


View larger version (21K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2.. Lung NOx changes measured per milligram of lung tissue. *p < 0.05 vs the corresponding control group.

 
Effect of INO on Lung Tissue Apoptosis
The percentage of apoptosis was significantly decreased in the sham-plus-INO group vs the sham group (p < 0.05). In the CPB group, the percentage of apoptosis was lower but not statistically significant in comparison to the sham group. Conversely, the percentage of apoptotic cells increased significantly in the CPB-plus-INO group (p < 0.05) compared to the CPB group (Fig 3 , 4 ).



View larger version (30K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3.. In situ TUNEL assay of fragmented DNA in lung sections. Top left, A: Negative control; top right, B: positive control; center left, C: sham; center right, D: sham plus INO; bottom left, E: CPB; and bottom right, F: CPB plus INO. This result is presented as a histogram in Figure 4.

 


View larger version (17K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4.. Effect of INO on lung apoptosis. INO significantly reduced apoptosis in the sham-plus-INO group; conversely, apoptosis was significantly increased in the CPB-plus-INO group. *p < 0.05 vs the corresponding control groups.

 
Western blot analysis with an anti-caspase-3 antibody, which recognizes procaspase-3 and the active form, showed the expression of the processed form of caspase-3 in all groups. The expression of activated caspase-3 revealed that the cells were in apoptosis (Fig 5 ), confirming the results obtained with the TUNEL technique.



View larger version (23K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5.. Immunoblot of caspase-3 protein using an antibody that recognizes a native form (32 kd) and an active form (18 to 20 kd). Protein extracts were prepared from lung tissues of each group (n ≥ 3). The active form is present in all groups. Apoptotic Jurkat cells served as positive control.

 
eNOS
Western blot analysis was performed to identify the mechanism of decreased NOx production. eNOS protein demonstrated similar levels in all groups (Fig 6 ). No iNOS protein was detected in the lungs of all pigs.



View larger version (37K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 6.. Top: Western blot analysis of eNOS protein in porcine lung tissue from each group (n ≥ 3). Pig endothelial cell protein (20 µg) was used as a positive control. The same membranes were probed with an antibody against actin (Calbiochem) to assure equal loading of the gel. Bottom: Densitometric values of relative eNOS protein normalized to sham (taken as 100%). Values are expressed as mean ± SEM from at least three blots. *p < 0.05

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study showed a CPB-induced decrease of endogenous NO production, and the data indicate that INO delivered early and at a low dose (20 ppm) had a beneficial effect on inflammation. This occurs by reducing cytokine synthesis and by attenuating neutrophil activation and migration into the alveolar spaces. However, INO promotes apoptosis in inflammatory conditions. Increasing apoptosis could be one of the mechanisms by which INO leads to the resolution of lung inflammation.

The positive aspect of NO is that plays an important role in the physiologic modulation of coronary artery tone and myocardial function. However, increased formation of NO within the myocardium can also have detrimental and negative effects, contributing to the pathophysiology of myocardial dysfunction in ischemic heart diseases. If the increased production of NO is well in balance with a moderate increase in oxygen radicals, then NO will exert beneficial effects. However, if the oxygen radicals are produced in excess of NO as in prolonged ischemic injury, then deleterious effects will be induced.

Inhaled NO is the first vasodilator to produce truly selective, potent pulmonary vasodilation that is independent of endothelial cell function. Therefore, it has been employed in the preoperative, perioperative, and postoperative assessment of pulmonary hypertension. Although many studies67 demonstrate a clear benefit in patient outcome with INO use, several safety concerns remain, including unpredictable and nonsustained responses to INO and a clinically significant rapid increase in pulmonary vascular resistance on its acute withdrawal.

ALI after CPB has been attributed to the CPB-associated pulmonary and systemic inflammatory response syndrome.1011 Several studies1213 have already demonstrated that CPB can induce ALI and may also cause ARDS. These effects are neutrophil related. A significant relationship between neutrophil influx and IL-8, a potent chemoattractant mainly released by neutrophils, has been noted in many inflammatory conditions.13

In our study, CPB caused neutrophil activation and infiltration, reflected by an increase in BAL IL-8 levels and neutrophil percentage in comparison to the control group. Our results are consistent with previous data1415 that showed heightened in TNF-{alpha}, IL-6, and IL-8 production after CPB. The effects of INO in ALI and ARDS have been indicated in many studies,1617 which suggest that INO reduces pulmonary hypertension and improves oxygenation. INO can also inhibit transendothelial migration of activated neutrophils, and preserve alveolar-capillary integrity after acute insults.18 The inflammatory process associated with CPB has been observed in conjunction with a decrease of arterial oxygen. A study by Kotani and associates19 has demonstrated a significant correlation between an increase in the number of neutrophils, IL-8, and elastase concentrations in BAL fluid, with changes in PaO2/fraction of inspired oxygen and intrapulmonary shunt. This may explain at least in part how INO improves oxygenation after CPB.

In INO groups, BAL analysis revealed a decrease of IL-8 concentration and a trend toward reduction of neutrophil number. This effect could occur by inhibiting NF-{kappa}B, a multiprotein complex that regulates a variety of genes, including those of IL-8, TNF-{alpha}, and several adhesion molecules. Accordingly, INO may prevent the production of proinflammatory cytokines because the genes of these molecules contain, in their promoter regions, the binding sites for NF-{kappa}B.20

In our study, we have also observed a decrease of plasma NOx concentrations in the sham and CPB groups. Our findings are similar to those of other studies2122 that have demonstrated a reduction of endogenous NO production after CPB or in swine models of endotoxemia. This alteration in NO production may reflect endothelial dysfunction, even if we did not find any significant difference in eNOS protein expression between the sham and CPB groups. The dysfunction probably affects eNOS activity but not gene expression.23 Furthermore, during CPB, substrate or cofactor availability may play a role in lowering NO production.24 This result implies that INO may replace the loss of endogenous NO.

NO was found to be involved in regulation of the apoptotic process; in fact, NO can promote apoptosis25 as well as inhibit it.456 We observed, in this study, both antiapoptotic and proapoptotic roles of INO. These opposing effects seem to depend on the local environment in which NO acts. Hyperoxia and mechanical ventilation have the potential to cause cellular damage,2627 and investigations2829 have shown that hyperoxia can also induce apoptosis. When INO is administered at low concentrations (10 to 20 ppm), it has a protective effect against pulmonary injury.30 As such, NO can protect against apoptosis induced by proinflammatory agents in cultured endothelial cells.31 It may, therefore, be able to protect the lungs in vivo against the apoptotic process. Hyperoxia has already been shown to be associated with increased NF-{kappa}B activity in vivo.32 As NO inhibits NF-{kappa}B transcription, it can consequently suppress apoptosis. In addition, INO can also subdue apoptosis through inactivation of caspase-3 by S-nitrosylation.33

Many studies have disclosed that during CPB, apoptosis of neutrophils is delayed by proinflammatory cytokines3435 or by modified C-reactive protein.36 In our experiments, the increase of proinflammatory mediators released during CPB probably inhibits apoptosis in these cells.

In the CPB-plus-INO group, INO, with its anti-inflammatory properties, decreased IL-8 concentrations, and this reduction suppressed the potential to inhibit the apoptotic process. Our findings are consistent with a previous study by Chello and associates,35 who demonstrated that the decline of neutrophil apoptosis is mainly a consequence of increased plasma IL-8 concentrations.

NO possesses the ability to act directly (leading to DNA damage) or indirectly (through reactive nitrogen species). Indeed, NO can interact with superoxide anion produced by inflammatory cells, leading to peroxynitrite formation, a potent oxidant that can induce apoptosis.37

The apoptosis process could participate in the removal of inflammatory cells and may be one of the mechanisms limiting tissue injury. Even if our study did not allow us to identify which kind of cells undergo apoptosis, neutrophils are a major source of IL-8 and the principal cause of inflammation. Inducing apoptosis of neutrophils would reduce the inflammation, prevent tissue lesions, and facilitate recovery. Our limitation of this study is that we did not establish in which cells the apoptotic process occurs, but the outcome of pigs receiving inhaled NO was better than those without NO (reflected by hemodynamics parameters data not shown). And this argues on apoptosis ongoing on inflammatory cells rather than on endothelial cells or pneumocytes.

The TUNEL technique could be associated to false-positive findings. Nevertheless the TUNEL technique is still reliable and the most common technique widely used in tissue apoptosis detection. Prochazkova et al38 demonstrated a positive correlation between the TUNEL technique and others techniques.

INO has been shown to evoke selective pulmonary vasodilation and contrasts markedly with IV-administered vasodilators that provoke dilation also in nonventilated areas of the lung and systemic circulation. NO diffusing into the bloodstream can rapidly react with oxyhemoglobin to form methemoglobin and nitrate. The presence of molecules that could conserve and stabilize NO bioactivity, which might regulate a regional blood flow and oxygen delivery, has been reported.39

It is clear that the mechanism by which INO leads to inflammation resolution remains to be clarified, and additional investigations are needed to discover if weaning from INO will cause rebound inflammation. Answering these questions is a crucial step before proceeding to clinical trials.


   Acknowledgements
 
The authors thank Ovid Da Silva (Editor, Research Support Office, Research Center, Center Hospitalier de Ùniversitie de Montreal) for editorial assistance, and Abdelhamid Liacini, Wissal El Assaad, and Serge Séguin for technical work.


   Footnotes
 
Abbreviations: ALI = acute lung injury; ANOVA = analysis of variance; CPB = cardiopulmonary bypass; eNOS = endothelial nitric oxide synthase; IL = interleukin; INO = inhaled nitric oxide; iNOS = inducible nitric oxide synthase; NF = nuclear factor; NO = nitric oxide; NOx = nitrate plus nitrite; T0 = time before cardiopulmonary bypass; T4 = 4 h after beginning cardiopulmonary bypass; T24 = 24 h after beginning cardiopulmonary bypass; TNF = tumor necrosis factor; TUNEL = terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling

This work was supported by Canadian Institutes of Health Research grant 6537.

Received for publication December 17, 2004. Accepted for publication May 10, 2005.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Jansen, NJ, van Oeveren, W, Gu, YJ, et al (1992) Endotoxin release and tumor necrosis factor formation during cardiopulmonary bypass. Ann Thorac Surg 54,744-747[Abstract]
  2. Wan, S, LeClerc, JL, Vincent, JL Cytokine responses to cardiopulmonary bypass: lessons learned from cardiac transplantation. Ann Thorac Surg 1997;63,269-276[Abstract/Free Full Text]
  3. Raychaudhuri, B, Dweik, R, Connors, MJ, et al Nitric oxide blocks nuclear factor-{kappa}B activation in alveolar macrophages. Am J Respir Cell Mol Biol 1999;21,311-316[Abstract/Free Full Text]
  4. Dimmeler, S, Haendeler, J, Nehls, M, et al Suppression of apoptosis by nitric oxide via inhibition of interleukin-1beta-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like proteases. J Exp Med 1997;185,601-607[Abstract/Free Full Text]
  5. Mannick, JB, Asano, K, Izumi, K, et al Nitric oxide produced by human B lymphocytes inhibits apoptosis and Epstein-Barr virus reactivation. Cell 1994;79,1137-1146[CrossRef][ISI][Medline]
  6. Beauvais, F, Michel, L, Dubertret, L The nitric oxide donors, azide and hydroxylamine, inhibit the programmed cell death of cytokine-deprived human eosinophils. FEBS Lett 1995;361,229-232[CrossRef][ISI][Medline]
  7. Chung, HT, Pae, HO, Choi, BM, et al Nitric oxide as a bioregulator of apoptosis. Biochem Biophys Res Commun 2001;282,1075-1079[CrossRef][ISI][Medline]
  8. Hubert, B, Salazkin, I, Desjardins, J, et al Cardiopulmonary bypass surgery in swine: a research model. J Exp Anim Sci 2003;43,135-149
  9. Yang, F, Troncy, E, Francoeur, M, et al Effects of reducing reagents and temperature on conversion of nitrite and nitrate to nitric oxide and detection of NO by chemiluminescence. Clin Chem 1997;43,657-662[Abstract/Free Full Text]
  10. Peek, GJ, Firmin, RK The inflammatory and coagulative response to prolonged extracorporeal membrane oxygenation. ASAIO J 1999;45,250-263[ISI][Medline]
  11. Wan, S, LeClerc, JL, Vincent, JL Inflammatory response to cardiopulmonary bypass: mechanisms involved and possible therapeutic strategies. Chest 1997;112,676-692[Abstract/Free Full Text]
  12. Milot, J, Perron, J, Lacasse, Y, et al Incidence and predictors of ARDS after cardiac surgery. Chest 2001;119,884-888[Abstract/Free Full Text]
  13. Boutten, A, Dehoux, MS, Seta, N, et al Compartmentalized IL-8 and elastase release within the human lung in unilateral pneumonia. Am J Respir Crit Care Med 1996;153,336-342[Abstract]
  14. Cremer, J, Martin, M, Redl, H, et al Systemic inflammatory response syndrome after cardiac operations. Ann Thorac Surg 1996;61,1714-1720[Abstract/Free Full Text]
  15. Casey, LC Role of cytokines in the pathogenesis of cardiopulmonary-induced multisystem organ failure. Ann Thorac Surg 1993;56,S92-S96[ISI][Medline]
  16. Troncy, E, Collet, JP, Shapiro, S, et al Inhaled nitric oxide in acute respiratory distress syndrome: a pilot randomized controlled study. Am J Respir Crit Care Med 1998;157,1483-1488
  17. Dellinger, RP, Zimmerman, JL, Taylor, RW, et al Effects of inhaled nitric oxide in patients with acute respiratory distress syndrome: results of a randomized phase II trial: Inhaled Nitric Oxide in ARDS Study Group. Crit Care Med 1998;26,15-23[CrossRef][ISI][Medline]
  18. Bloomfield, GL, Holloway, S, Ridings, PC, et al Pretreatment with inhaled nitric oxide inhibits neutrophil migration and oxidative activity resulting in attenuated sepsis-induced acute lung injury. Crit Care Med 1997;25,584-593[CrossRef][ISI][Medline]
  19. Kotani, N, Hashimoto, H, Sessler, DI, et al Neutrophil number and interleukin-8 and elastase concentrations in bronchoalveolar lavage fluid correlate with decreased arterial oxygenation after cardiopulmonary bypass. Anesth Analg 2000;90,1046-1051[Abstract/Free Full Text]
  20. Manning, AM, Bell, FP, Rosenbloom, CL, et al NF-{kappa}B is activated during acute inflammation in vivo in association with elevated endothelial cell adhesion molecule gene expression and leukocyte recruitment. J Inflamm 1995;45,283-296[ISI][Medline]
  21. Strand, OA, Leone, AM, Giercksky, KE, et al N(G)-monomethyl-L-arginine improves survival in a pig model of abdominal sepsis. Crit Care Med 1998;26,1490-1499[CrossRef][ISI][Medline]
  22. Morita, K, Ihnken, K, Buckberg, GD, et al Pulmonary vasoconstriction due to impaired nitric oxide production after cardiopulmonary bypass. Ann Thorac Surg 1996;61,1775-1780[Abstract/Free Full Text]
  23. Sheehy, AM, Burson, MA, Black, SM Nitric oxide exposure inhibits endothelial NOS activity but not gene expression: a role for superoxide. Am J Physiol 1998;274,L833-L841
  24. McMullan, DM, Bekker, JM, Parry, AJ, et al Alterations in endogenous nitric oxide production after cardiopulmonary bypass in lambs with normal and increased pulmonary blood flow. Circulation 2000;102,III172-III178
  25. Albina, JE, Cui, S, Mateo, RB, et al Nitric oxide-mediated apoptosis in murine peritoneal macrophages. J Immunol 1993;150,5080-5085[Abstract]
  26. Davis, WB, Rennard, SI, Bitterman, PB, et al Pulmonary oxygen toxicity: early reversible changes in human alveolar structures induced by hyperoxia. N Engl J Med 1983;309,878-883[Abstract]
  27. Sanders, SP, Zweier, JL, Kuppusamy, P, et al Hyperoxic sheep pulmonary microvascular endothelial cells generate free radicals via mitochondrial electron transport. J Clin Invest 1993;91,46-52[ISI][Medline]
  28. Otterbein, LE, Chin, BY, Mantell, LL, et al Pulmonary apoptosis in aged and oxygen-tolerant rats exposed to hyperoxia. Am J Physiol 1998;275,L14-L20
  29. Waxman, AB, Einarsson, O, Seres, T, et al Targeted lung expression of interleukin-11 enhances murine tolerance of 100% oxygen and diminishes hyperoxia-induced DNA fragmentation. J Clin Invest 1998;101,1970-1982[ISI][Medline]
  30. McElroy, MC, Wiener-Kronish, JP, Miyazaki, H, et al Nitric oxide attenuates lung endothelial injury caused by sublethal hyperoxia in rats. Am J Physiol 1997;272,L631-L638
  31. Shen, YH, Wang, XL, Wilcken, DE Nitric oxide induces and inhibits apoptosis through different pathways. FEBS Lett 1998;433,125-131[CrossRef][ISI][Medline]
  32. Pardo, A, Barrios, R, Maldonado, V, et al Gelatinases A and B are up-regulated in rat lungs by subacute hyperoxia: pathogenetic implications. Am J Pathol 1998;153,833-844[Abstract/Free Full Text]
  33. Li, J, Billiar, TR, Talanian, RV, et al Nitric oxide reversibly inhibits seven members of the caspase family via S-nitrosylation. Biochem Biophys Res Commun 1997;240,419-424[CrossRef][ISI][Medline]
  34. Biffl, WL, Moore, EE, Moore, FA, et al Interleukin-6 delays neutrophil apoptosis via a mechanism involving platelet-activating factor. J Trauma 1996;40,575-578[ISI][Medline]
  35. Chello, M, Mastroroberto, P, Quirino, A, et al Inhibition of neutrophil apoptosis after coronary bypass operation with cardiopulmonary bypass. Ann Thorac Surg 2002;73,123-129[Abstract/Free Full Text]
  36. Khreiss, T, Jozsef, L, Hossain, S, et al Loss of pentameric symmetry of C-reactive protein is associated with delayed apoptosis of human neutrophils. J Biol Chem 2002;277,40775-40781[Abstract/Free Full Text]
  37. Ghafourifar, P, Schenk, U, Klein, SD, et al Mitochondrial nitric-oxide synthase stimulation causes cytochrome c release from isolated mitochondria: evidence for intramitochondrial peroxynitrite formation. J Biol Chem 1999;274,31185-1188[Abstract/Free Full Text]
  38. Prochazkova, J, Kylarova, D, Vranka, P, et al Comparative study of apoptosis-detecting techniques: TUNEL, apostain, and lamin B. Biotechniques 2003;35,528-534[Medline]
  39. McMahon, TJ, Moon, RE, Luschinger, BP, et al Nitric oxide in the human respiratory cycle. Nat Med 2002;8,711-717[ISI][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Article Archive
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via ISI Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by El Kebir, D.
Right arrow Articles by Blaise, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by El Kebir, D.
Right arrow Articles by Blaise, G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS