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Ann Thorac Surg 2001;72:149-155
© 2001 The Society of Thoracic Surgeons
Accepted for publication March 14, 2001.
Address reprint requests to Dr Sawa, Department of Surgery, Course of Interventional Medicine (E1), Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita City, Osaka 565-0871, Japan
e-mail: sawa{at}surg1.med.osaka-u.ac.jp
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Abstract |
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Methods. Adult Sprague-Dawley rats underwent 60 minutes of CPB (100 mL · kg-1 · min-1, 34°C). Group A (n = 10) received 100 mg/kg of aminoguanidine intraperitoneally 30 minutes before the initiation of CPB, and group B (n = 10) served as controls.
Results. There were significant time-dependent changes in plasma interleukin (IL)-6, IL-8, nitrate + nitrite, the percentage ratio of nitrotyrosine to tyrosine (%NO2-Tyr, an indicator of peroxynitrite formation), and respiratory index (RI). Three hours after CPB termination, IL-6, IL-8, and RI were significantly higher in group A (IL-6, 397.5 ± 80.6 pg/mL; IL-8, 26.99 ± 6.57 ng/mL; RI, 1.87 ± 0.31) than in group B (IL-6, 316.5 ± 73.9 pg/mL, p <0.05; IL-8, 17.21 ± 3.12 ng/mL, p < 0.01; RI, 1.57 ± 0.24, p < 0.05) although nitrate + nitrite (31.8 ± 4.1 µmol/L) and %NO2-Tyr (1.15% ± 0.20%) were significantly lower in group A than in group B (nitrate + nitrite, 50.2 ± 5.0 µmol/L, p < 0.01; %NO2-Tyr, 1.46% ± 0.21%, p < 0.01). Western immunoblot analysis from lung tissue of group A identified marked iNOS inhibition without inhibiting endothelial-constitutive NOS, and neutrophil accumulation in the lung specimens was significantly greater in group A (6.5 ± 0.7/alveoli) than in group B (4.4 ± 0.4/alveoli, p < 0.01).
Conclusions. These results suggest that NO production from iNOS may be an adaptive response for attenuating the CPB-induced inflammatory response.
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Introduction |
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TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
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NO is known to have both cytoprotective and cytotoxic effects on various pathologic conditions including inflammation [5–8]. The major cytoprotective effect is known to have inhibitory action toward neutrophil-endothelial cell adhesion [5]. By blocking neutrophil accumulation in the inflammatory sites, the subsequent vicious cycle by released superoxide or inflammatory cytokines can be attenuated [5, 6, 9]. As for the cytotoxic effect of NO, the inhibition of cellular metabolism by direct reaction of heme and nonheme enzyme has been shown [7]. Nitration of tyrosine to form nitrotyrosine, which is considered an indicator of peroxynitrite formation, is also one of the NO-induced deleterious reactions to tissue [8]. Although an increase in NO production appears to be cytoprotective in the CPB-induced inflammatory response and hemodynamics after CPB was reported to be improved by NO [10–12], a role of excessive NO from iNOS in the CPB-induced inflammatory response remains to be investigated. The determination of its role is of significant importance from the therapeutic aspect. Thus, we performed this study to examine whether the selective inhibition of NO production from iNOS attenuates the CPB-induced inflammatory response or not by use of aminoguanidine, a relatively selective iNOS inhibitor, in a rat-CPB model.
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Material and methods |
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TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
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Experimental protocol
Forty adult male Sprague-Dawley rats weighing 400 to 450 g were used in this study, and were randomly divided into four groups. As the pretreatment for iNOS inhibition, 1 mL of saline containing 100 mg/kg body weight of aminoguanidine hemisulfate (iNOS inhibitor; Sigma Chemical Co., St. Louis, MO) was administered intraperitoneally 30 minutes before the initiation of CPB, and only saline was added in the same way for control. The relative selectivity of 100 mg/kg of aminoguanidine to iNOS was shown in previous studies [13, 14].
Twenty rats underwent CPB for 60 minutes; group A (n = 10) was administered 100 mg/kg of aminoguanidine, and group B (n = 10) was administered only saline. To exclude the influence of operation-induced inflammation without CPB, group C (n = 10) was given 100 mg/kg of aminoguanidine but did not undergo CPB to show that the effects of aminoguanidine really only occur after CPB, and group D (n = 10) was used as sham operated animals.
Rat-CPB was initiated and terminated according to the method we previously described [15]. The rats were anesthetized by intraperitoneal administration of sodium pentobarbital (50 mg/kg) and placed in the supine position. The lungs were ventilated with 100% oxygen through a 18-G tracheotomy tube at a tidal volume of 10 mL/kg and a respiratory rate of 60 breaths per minute. After exposure of the cannulation sites, heparin (300 U/kg) was injected intraperitoneally. A 16-G catheter was placed into the right atrium through the right jugular vein and another 16-G catheter was placed directly into the left femoral vein. Arterial cannulation was performed directly into the left femoral artery using a 20-G catheter. The CPB circuit was composed of a roller pump (Perista Bio-Minipump AC-2120; Atto Co, Ltd, Tokyo, Japan), a membrane oxygenator (Senko Medical Co, Ltd, Osaka, Japan), a venous reservoir, and tubing lines. None of the materials in the CPB circuit were heparin-coated. The bypass circuit was primed with the following solution without blood components: 12 mL plasma expander containing hydroxyethyl-amylum (Hespander; Kyorin Pharmaceutical, Tokyo, Japan), 8 mL lactate Ringer’s solution, 2 mL 7% sodium bicarbonate, 2 mL mannitol, 100 U heparin, and 1.5 mg tobramycin. Perfusion flow rate was maintained at 100 mL · kg-1 · min-1. The perfusate temperature was maintained at 34°C with a heat exchanger. No blood components were transfused throughout the experiment. Routinely, CPB termination was aided only by the continuous administration of dobutamine (3 µg · kg-1 · min-1). The remaining priming solution was infused gradually after the termination of CPB.
Measurements
In groups A and B, 1.5 mL of arterial blood and exhaled air were sampled at the following three times: before the initiation of CPB, at the termination of CPB, and 3 hours after the termination of CPB. In groups C and D, these were sampled at the following three times: at the end of arterial and venous cannulation after systemic heparinization, and 1 hour and 4 hours after arterial and venous cannulation (Fig 1).
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Plasma nitrate and nitrite
Blood samples were centrifuged at 1,200g, and the plasma fraction was diluted 1:1 with nitrite- and nitrate-free distilled water. Subsequently, 400 µL of diluted plasma was ultrafiltered at 2,000g (Ultrafree MC microcentrifuge device, UFC3 LGC; Millipore, Bedford, MA). The filtrates were analyzed by an automated procedure based on the Griess reaction [16].
Peroxynitrite formation
Nitrotyrosine was measured as an index of nitration reaction of NO, which is used as an indicator of peroxynitrite formation, using a high-pressure liquid chromatography (HPLC) method previously described [17]. Briefly, blood samples were centrifuged at 1,200g for 15 minutes, and the filtrates were hydrolyzed for 24 hours. The supernatant fluid was analyzed by HPLC with a C-18 reverse-phase column (Jasco, Tokyo, Japan) and the peak concentrations were measured with an ultraviolet detector set at 274 nm (UV-970; Jasco). The peak was identified on the basis of the retention time of authentic 3-nitro-L-tyrosine or tyrosine. Peroxynitrite formation was expressed as the percentage ratio of nitrotyrosine to tyrosine (%NO2-Tyr) [18].
Exhaled NO concentration
Mandatory ventilation consisting of 5 mL of room air was performed 10 times with a 10 mL syringe. The last 5 mL of exhaled air underwent NO analysis. Three successive 1 mL exhaled air samples were measured with a chemiluminesence analyzer (Sievers model 270B, Boulder, CO), sensitive to NO at concentrations of 5 to 5 x 105 ppb within 30 minutes after the collection of exhaled air. The average value from the three successive samples was used for analysis [19].
Inflammatory cytokines
We measured plasma levels of IL-6 and IL-8 as markers of the development of CPB-induced inflammatory response. Blood samples were centrifuged at 1,200g for 10 minutes. The plasma IL-6 level was measured by enzyme-linked immunosorbent assay (ELISA) using a commercially available kit (Rat ELISA Kits; Biosource International, Camarillo, CA). The IL-8 level was measured by enzyme immunoassay (EIA) using a commercially available kit (Rat IL-8 Kit; Panafarm Laboratory, Tokyo, Japan).
Respiratory index
As RI is an index of oxygenation function of lung and its increase reflects the presence of pulmonary shunting in a variety of conditions including atelectasis, pulmonary contusion, and pulmonary thromboembolism [20], we used RI as a marker of lung damage. RI was calculated by arterial blood gas assay as follows: respiratory index = alveolar - arterial oxygen tension gradient/arterial oxygen tension (AaDO2/PaO2).
Western blot analysis and histologic analysis
All rats were killed 3 hours after the termination of CPB. Lung specimens were immediately frozen in liquid nitrogen and stored at -80°C.
Western immunoblot analysis was performed to confirm ecNOS and iNOS induction 3 hours after CPB termination. Protein extracts (100 µg), prepared according to the method of Cucchiaro and associates [21], were loaded onto a 10% SDS-PAGE system. The blots were transferred onto a PVDF membrane, and then incubated in Tris-buffered saline/Tween-20 (20.0 mmol/L Tris-HCL, pH 7.5, 150.0 mmol/L NaCl, 0.1% Tween-20) containing 3% bovine serum albumin to block the nonspecific absorption. The membrane was immunoblotted with the monoclonal mouse antiendothelial NOS antibody (Transduction Laboratories, N30020) at 1:2500 (vol/vol) dilution or the monoclonal antiinducible NOS antibody (Transduction Laboratories, N39120) at 1:500 (vol/vol) dilution for detection of ecNOS and iNOS. Protein bands were visualized by use of the enhanced chemiluminescence substrate system (Amersham).
Naphthol AS-D chloroacetate esterase was used to evaluate neutrophil accumulation to lung endothelial cells. Accumulated neutrophils and alveoli were counted under microscopy using magnification x100 in 10 fields in each 10 serial sections. The results were corrected by number of alveoli [15].
Statistical analysis
All data are expressed as mean ± standard deviation (SD). Comparisons among the groups were analyzed by two-way repeated-measures analysis of variance and the unpaired Student’s t test. Coefficient correlations between plasma nitrate + nitrite, %NO2-Tyr and RI were analyzed by multiple regression analysis. All analysis was performed using the Statview v5.0 statistical package (Abacus Concepts Inc, Berkeley, CA). A p value of less than 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
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Plasma nitrate and nitrite
Plasma level of nitrate + nitrite before the initiation of CPB did not differ significantly among the four groups. There were significant time-dependent changes in plasma nitrate + nitrite in group A and B (p < 0.0001, analysis of variance, treatment effect), although there were no significant time-dependent changes either in group C or D. At the termination of CPB, plasma nitrate + nitrite was significantly higher than before CPB (p < 0.01) without any significant difference between the groups. In group A, plasma nitrate + nitrite 3 hours after CPB termination was significantly lower than that at CPB termination (p < 0.05). In group B, however, it was significantly higher (p < 0.01), and there was a significant difference in plasma nitrate + nitrite 3 hours after CPB termination between group A and B (Table 1).
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Exhaled NO concentration
All four groups showed similar exhaled NO concentrations before CPB. There were significant time-dependent changes in exhaled NO concentrations in group A and B (p < 0.0001, ANOVA, treatment effect), though there were no significant time-dependent changes either in group C or D. In both group A and B, exhaled NO was significantly lower at the termination of CPB than before CPB (p < 0.01), without any significant difference between the two groups. Three hours after the termination of CPB, group B showed significantly higher exhaled NO concentration than before CPB (p < 0.01). In group A, however, exhaled NO concentration 3 hours after CPB termination was not significantly different from that at CPB termination, and was significantly lower than in group B (Table 1).
Inflammatory cytokines
Before the initiation of CPB, plasma level of IL-6 was below the minimum detectable level in all four groups. Only in group A and B, plasma IL-6 was detected after the termination of CPB, and there were significant time-dependent changes in plasma IL-6 in group A and B (p < 0.0001, ANOVA, treatment effect). Plasma IL-6 was significantly higher 3 hours after the termination of CPB than at the termination of CPB (p < 0,01). Although there was no significant difference between the groups at the termination of CPB, plasma IL-6 in group A was significantly higher than that in group B 3 hours after the termination of CPB (Table 2).
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Respiratory index
Before CPB, RI did not differ significantly among the 4 groups. There were significant time-dependent changes in RI value in group A and B (p < 0.0001, ANOVA, treatment effect). Group A and B showed significantly higher RI value at the termination of CPB than that before CPB (p < 0.01), and RI 3 hours after the termination of CPB was significantly higher than that at the termination of CPB (p < 0.01). RI value 3 hours after CPB termination was significantly higher in group A than in group B. Similar to the pattern of other variables, there were no time-dependent changes either in group C or D.
Multiple regression analysis demonstrated a significant correlation between RI, plasma nitrate + nitrite level and %NO2-Tyr in each group (Table 3). Low nitrate + nitrite and high %NO2-Tyr increase RI value.
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
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Although the induction of iNOS has been shown to contribute to the development of myocardial damage through its negative inotropic effect or induction of apoptosis in reperfusion injury [24, 25], it cannot be simply extended to CPB-induced inflammatory response. The pathophysiology of CPB-induced inflammatory response differs from that of myocardial ischemia-reperfusion injury in many aspects. Firstly, organs are not subjected to ischemic insults during CPB. Secondly, whole blood is forced to have contact with artificial surfaces of the bypass circuit. Thirdly, the vascular wall is exposed to a nonphysiologic perfusion pattern. Therefore, the major injurious events of CPB reside in activated circulating blood components such as platelets and leukocytes as well as in mechanical damage of endothelial cell surface.
CPB-induced chemotactic mediators, such as complement activation, free radical generation, protease release, and cytokine release, activate ecNOS soon after CPB initiation as well as induce endothelial damage. The ecNOS activation enhances endogenous NO production and contributes to attenuate neutrophil accumulation to endothelial cells. However, these inflammatory responses continue to progress and enhance endothelial damage several hours after CPB terminated, which results in reducing NO production from ecNOS. Namely, even though NO production from ecNOS increased through mechanical stimulation of CPB to endothelial cells in the early stage of CPB, its increase cannot cover progressive endothelial cell damage after CPB termination. The reduction in NO production loses its inhibitory effect on P-selectin upregulation, which is thought to be a key process in neutrophil-endothelial cell adhesion, and accelerates endothelial damage [26, 27]. On the other hand, iNOS expression is induced several hours after the initiation of inflammatory response [2–4]. This subsequent iNOS expression induces excessive NO production after the termination of CPB, which can compensate the reduction of NO from ecNOS resulting from the prolonged endothelial damage. Therefore, iNOS expression may be an adaptation to late stage of CPB-induced inflammatory response, and our results can support this hypothesis. This concept is in agreement with a recent study performed in iNOS-deficient mice in which transplant arteriosclerosis was exacerbated [28].
Regarding exhaled NO concentration, its reduction at the end of CPB, observed in both group A and B, is considered to result from pulmonary endothelial damages during CPB because exhaled NO production in the preinflammatory state is generally attributed to ecNOS of pulmonary endothelial cells as well as neural NOS of lung epithelial cells [29, 30]. The marked increase at 3 hours after CPB termination in the control group and almost complete block of the increase by iNOS inhibition suggest that the main sources of exhaled NO in the late stages are iNOS of macrophages, lung epithelial cells, and pulmonary endothelial cells, previously described [29, 30]. Although the role of exhaled NO in CPB-induced inflammatory response remains unclear, augmented exhaled NO production appears not to be a simple marker of lung inflammation and the increase in exhaled NO can also be considered an adaptive response to inflammation, as well as that in plasma NO. This study may account for the current clinical observation that inhaled NO attenuates lung damage by reducing pulmonary vasoconstriction and right ventricular dysfunction [31].
Concerning the results of peroxynitrite formation, CPB-induced inflammation appeared not to be attenuated but to develop, although iNOS inhibition attenuated the increase in %NO2-Tyr 3 hours after CPB termination. These results may suggest that peroxynitrite is not a causative factor in the development of CPB-induced inflammatory response. Generally, peroxynitrite is thought to be a potent oxidant playing a cytotoxic role in the development of inflammation. As for the reason iNOS inhibition aggravated CPB-induced inflammation, several metabolic and functional pathways through NO and peroxynitrite can be speculated. Rubbo and associates demonstrated a scavenging effect of high NO on nitration reaction [32]. On the other hand, current experimental studies have demonstrated that peroxynitrite is bioconverted to nitrosoglutathione by interactions with glutathione in a blood environment and plays a cytoprotective role in the development of myocardial ischemia-reperfusion injury [33–35]. This study suggests that iNOS expression may have a cytoprotective effect on CPB-induced inflammatory response. With respect to clinical implications, however, further investigations are needed to clarify the detail mechanism of the development of CPB-induced inflammatory response in relation to peroxynitrite formation.
In conclusion, the decrease in iNOS-derived NO production by the pretreatment with aminoguanidine aggravated CPB-induced inflammatory response manifested by increased lung dysfunction, cytokine release, and neutrophil accumulation. These data demonstrate that selective iNOS inhibition does not attenuate CPB-induced inflammatory response, and excessive NO from iNOS may have a possible cytoprotective effect on CPB-induced inflammatory response. Thus, iNOS expression may be an adaptive response in view of attenuating CPB-induced inflammatory response in the late stage.
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TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
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