Asian Cardiovasc Thorac Ann 2007;15:2-8
© 2007 Asia Publishing EXchange Ltd
Antioxidant Attenuates Acute Lung Injury After Cardiopulmonary Bypass in Rats
Li-Zhong Wang, MD,
Shi-Tong Li, MD,
Ying-Tian Wang, PhD,
Zheng-Ping Wang, PhD
Central Laboratory, Shanghai First People’s Hospital, Shanghai, China
For reprint information contact: Li-Zhong Wang, MD Tel: 86 573 220 4805 Fax: 86 573 207 4575 Email: jxlzw{at}56.com, Department of Anesthesiology, Jiaxing Women and Children’s Health Hospital, 42 Jinjian Rd, Jiaxing 314000, Zhejiang Province, China.
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ABSTRACT
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This study tested the effects of the antioxidant pyrrolidine dithiocarbamate on acute lung injury induced by cardiopulmonary bypass in rats. Adult rats were randomly divided into 3 groups of 7 each. The study group was pretreated with pyrrolidine dithiocarbamate before undergoing 60 min of normothermic partial cardiopulmonary bypass, a control group underwent cardiopulmonary bypass only, and a third group underwent a sham operation involving anesthesia and cannulation only. The respiratory index at 60 min after terminating bypass was significantly increased in the study group only. Neutrophil, malondialdehyde, interleukin-8, nuclear factor-
B, and protein levels in bronchoalveolar lavage fluid from the cardiopulmonary bypass group were significantly higher than those in the other two groups, with marked inflammatory changes on lung histopathology. It was concluded that cardiopulmonary bypass can directly induce acute lung injury, and pyrrolidine dithiocarbamate attenuates this injury by inhibiting nuclear factor-B activity.
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INTRODUCTION
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Acute lung injury (ALI) is a common complication and a significant clinical problem in patients undergoing cardiopulmonary bypass (CPB). Among the causative factors, systemic inflammatory response syndrome with neutrophil activation is of major importance. Through the release of reactive oxygen species, proteolytic enzymes, and proinflammatory cytokines, neutrophils can induce lung tissue injury.1,2 Recent studies have revealed that nuclear factor-kappa B (NF-B) is a critical transcription factor in lung injury such as hemorrhage or endotoxemia-induced damage.3 However, there is scant information available about the change in NF-B in the lung after CPB. Release of reactive oxygen species has been proposed as a mechanism in lung injury, and it is a well-established activator of NF-B.4,5 Previous reports have confirmed that the antioxidant pyrrolidine dithiocarbamate (PDTC) improves pulmonary function after ischemia and reperfusion in isolated rabbit lung, and attenuates lipopolysaccharide-induced ALI in the rat.6,7 However, the effects of PDTC on CPB-induced ALI are unknown. In this study, we tested the effects of pretreatment with PDTC on ALI after CPB, using a rat normothermic partial CPB model. We also sought to observe the change in NF-B in the lung after CPB to further explore the possible molecular mechanisms involved.
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MATERIALS AND METHODS
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Adult Sprague-Dawley rats of both sexes weighing 350–450 g were obtained from Shanghai Animal Experimental Center, China. All procedures were conducted in accordance with the National Institutes of Health guidelines for animal use, and approved by the Institutional Animal Care and Use Committee of Fudan University, China. Rats were randomly assigned to one of 3 groups of 7 each. The CPB and PDTC groups underwent 60 min of normothermic partial CPB, and the other group had a sham operation. The extracorporeal circuit consisted of a peristaltic pump (Baoding Longer Precision Pump Co., Ltd., China), a venous reservoir (a 10 mL sterile syringe), a specially designed membrane oxygenator with a heat exchanger and a surface area of 0.1 m2 (Fudan Biological Material Co., Ltd., China) and Longer no. 25 tubing lines. The priming solution included 15 mL of succinylated gelatin (Gelofusine; Braun, China), 5 mL of Ringer’s lactate solution and 1 mL of 5% sodium bicarbonate, of which the oxygenator constituted approximately 15 mL.
Anesthesia was induced with intraperitoneal administration of midazolam 4 mg·kg–1 (Roche, Switzerland) and fentanyl 150 µg·kg–1 (Yichang Renfu, China), and maintained with additional intravenous doses of 
of the induction dose every 30 min until the end of the study. Rats in the PDTC group were injected intraperitoneally with PDTC 100 mg·kg–1 (Sigma) dissolved in 1 mL saline 30 min before the initiation of CPB. After insertion of a 16-gauge cannula into the trachea, mechanical ventilation was undertaken at 60 breaths·min–1 and a fraction of inspired oxygen (FiO2) of 100%. The tidal volume was adjusted to maintain arterial carbon dioxide tension (PaCO2) at 35–45 mm Hg. Both femoral arteries were exposed and cannulated with 22-gauge intravenous catheters (BD Medical, Sandy, UT, USA). The left femoral artery was used for monitoring mean arterial pressure (MAP) and the right served as the inflow for the CPB circuit. Anticoagulation consisted of 300 IU·kg–1 heparin, which was administered through the arterial cannula.
Via a small neck incision, the right internal jugular vein was exposed and a 14-gauge cannula with several side holes for venous drainage was inserted and gently advanced 3 cm towards the heart. Preliminary experiments had shown that at this distance the tips of the cannulas were placed mostly in the right atrium. The position of the venous cannula was further adjusted and confirmed by sufficient central venous drainage during perfusion. Cardiopulmonary bypass was established between the right atrium and the right femoral artery with a perfusion flow rate of 100 mL·kg–1·min–1. During perfusion, gas flow (100% O2) through the oxygenator was maintainedat 0.05–0.1 L·min–1 and the rat heart continued to beat. Rectal temperature was monitored continuously and maintained at 37°C by pumping water at 38°C through the heat exchanger of the oxygenator. After 60 min of CPB, the rat was weaned off CPB without the need for vasopressors and protamine. However, if the MAP was less than 60 mm Hg, the remaining priming solution was infused.
After terminating CPB, the venous cannula was withdrawn to the superior vena cava and clamped. The rats remained on mechanical ventilation and were observed for another 60 min. Rats in the sham group underwent the same surgical procedures and observation time as the CPB group. In addition, 5-mL blood samples were withdrawn and the equivalent volume of Gelofusine was infused through the venous cannula to maintain a similar hematocrit to that in the other two groups. No blood component was transfused throughout the experimental period in any group. At the end of the study, the rats were sacrificed and the lungs were immediately excised via a median thoracotomy following evaluation of the central venous pressure through the jugular venous cannula. After washing with ice-cold saline to remove excess blood, the right upper lung lobe was used for histological examination and the rest of the lung tissue was snap-frozen in liquid nitrogen and subsequently kept at –80°C. All surgical procedures were performed in a sterile fashion.
Samples of arterial blood (200 µL) were taken before the initiation of CPB (T1), at the end of CPB (T2), and 60 min after CPB (T3) in the CPB and PDTC groups, and at the corresponding time points in the sham group, for analyses of blood gases and hemoglobin using an AVL 995 blood gas analyzer (AVL, Schaffhausen, Switzerland). The respiratory index (RI) at T1 and T3 was calculated from the formula:
where AaDO2 is the difference between the arterial and alveolar oxygen tensions.
Bronchoalveolar lavage fluid (BALF) was collected at the end of the study for neutrophil count, IL-8 (rat cytokine-induced neutrophil chemoattractant-1), and protein concentrations; 20 mL of ice-cold phosphate-buffered saline at pH 7.4 was instilled via the tracheal cannula in 10 mL aliquots, and gently withdrawn. Each withdrawn volume was approximately 8 mL, which indicated a similar dilution among the animals. The BALF was centrifuged at 1500 g for 10 min at 4°C, and the cell-free supernatant of the first aliquot was assayed for IL-8 and protein concentrations. The protein concentration was determined by a modification of the Bradford method.8 The IL-8 level was measured by an enzyme-linked immunosorbent assay (R&D Systems, USA) with a minimum detection level of 0.7–1.3 pg·mL–1. The pellets from two aliquots were combined and neutrophils were counted with an automated counter (Coulter Electronics, UK). For evaluation of lipid peroxidation, approximately 1 g of right lower lung tissue was homogenized in 9 volumes of ice-cold saline using a glass homogenizer to produce a 10% homogenate. Lipid peroxidation was determined with a malondialdehyde (MDA) test kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions. The absorbance was measured at 525 nm and standardized for protein content as determined by the Bradford method.8
The right upper lung lobe was fixed in 10% formaldehyde solution and 4-µm sections were cut with a Leica 2135 microtome (Germany). The sections were further embedded in paraffin and stained with hematoxylin and eosin. Histologic examination was performed with an Olympus BX51 optical microscope (Japan). An electrophoretic mobility shift assay (EMSA) of the NF-B in lung was carried out as described previously.9 Nuclear protein of left upper lung tissue was extracted with a nuclear extraction reagent (NE-PER, Pierce, USA) and quantified by the Bradford method.8 Biotin end-labeled and non-labeled double-stranded oligonucleotide probes containing a putative binding site for NF-B, of sequence 5'-AGTTGAGGGGACTTTCCCAGGC-3', were synthesized and labeled by Shanghai Sangon Biological Engineering Technology and Service Co., Ltd. (China). The assay is based on the fact that DNA-protein complexes migrate more slowly than unbound oligonucleotides in a native polyacrylamide gel, resulting in a shift in migration of the labeled DNA band. The band was detected using the LightShift chemiluminescent EMSA kit (Pierce, USA). The relative intensity of the NF-B band was quantified using ImageQuant version 5.2 software (GE Healthcare).
All statistical analyses were carried out using SPSS 10.0 for Windows (SPSS, Inc., Chicago, IL, USA). Data are expressed as mean ± standard deviation. Statistical analyses were performed using analysis of variance followed by the Bonferroni post-hoc test. Comparisons of RI at T1 and T3 within each group were performed by the paired-samples t test. A value of p < 0.05 was considered significant.
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RESULTS
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There was no technical failure or operative death in this study. Rectal temperature remained stable throughout the study. Central venous pressure at the end of the study was similar among the 3 groups (data not shown). However, CPB induced a significant reduction of MAP; MAP at T2 and T3 was significantly decreased compared to T1 in all groups (Table 1
). The blood gases were unchanged in the sham group. In the other two groups, PaO2 was significantly increased at T2 and decreased at T3, although the differences between T3 and T1 were not significant. However, RI at T3 (2.8 ± 0.6) was significantly increased compared with T1 (1.8 ± 0.5; p < 0.01) in the CPB group. No significant difference was found in the other groups (PDTC group: T1 = 1.3 ± 0.6, T3 =1.6 ± 0.7; sham group: T1 = 1.5 ± 0.5, T3 = 1.7 ± 0.5). The hemoglobin at both T2 and T3 was lower than T1 within each group ( p < 0.05), but not significantly different among groups (Table 1).
The neutrophil counts and concentrations of protein and IL-8 in BALF obtained from the CPB group were higher than those from the other groups ( p < 0.05). There was no significant difference between the PDTC and sham groups (Table 2). Lung lipid peroxidation MDA in each group was: 1.94 ± 0.62 (CPB group), 1.05 ± 0.20 (PDTC group), and 0.92 ± 0.14 nmoL·mg–1 protein (sham group). Lipid peroxidation MDA in the CPB group was significantly increased compared to the other groups ( p < 0.01), but the difference between the PDTC and sham groups was not significant.
Light microscopic findings included more neutrophil infiltration and accumulation in the interstitium and intra-alveolar space, with evident exudation in the alveoli in the CPB group. In contrast, neutrophils mainly presented within the capillary lumen, and almost normal lung architecture was seen in the sham group. The changes in the PDTC group were intermediate between those in the CPB and sham groups; although neutrophils and exudation presented in the interstitium and alveoli, the degree was much less than that in the CPB group (Figure 1).
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Figure 1. Histologic examination of lung from (A) CPB group, (B) PDTC group, and (C) sham group by light microscopy (hematoxylin and eosin stain, original magnification x 400).
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NF-B analyses showed NF-B DNA binding activity significantly increased in lung tissue from the CPB group compared to the other two groups ( p < 0.05). PDTC pretreatment significantly suppressed the activation of NF-B (Figure 2).
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DISCUSSION
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The main advantage of the rat CPB model is reduced costs of animals and equipment and the convenience of a study model that does not require a full-scale operating environment.10 To minimize effects not related to CPB, such as ischemia/reperfusion and hypothermia, and to optimize the investigation of systemic inflammatory responses, we used a normothermic partial CPB model and did not apply donor blood as the priming solution. We found that this CPB model was easily established. The main difficulties experienced were with cannulation of the internal jugular vein and hemodynamic instability that occurred after weaning from CPB. In preliminary experiments, some rats died due to perforation of the jugular vein or right atrium and hypotension after CPB. We prevented these problems by careful handling and by minimizing the circuit length to reduce the priming volume that led to excessive hemodilution. In addition, we administered the remaining priming solution after CPB, as required. With these measures, all the rats in this study survived the experimental procedure.
Pulmonary dysfunction is a common complication after heart surgery and may be the result of multiple insults from the combined effects of anesthesia, CPB, and surgical trauma.1 In a prospective clinical study, Cox and colleagues11 demonstrated in patients undergoing coronary revascularization with or without CPB that alveolar-arterial oxygen gradients increased progressively but with no significant differences at any time between the two groups of patients. Therefore, it is questionable whether CPB itself is directly responsible for the lung injury. We found significant increases in RI and in neutrophils, protein, and IL-8 in BALF after CPB. Respiratory index is an index of the oxygenation function of the lung and its increase reflects pulmonary shunting in a variety of conditions. The protein content of BALF is a marker of the permeability of the bronchoalveolar-capillary barrier.12 IL-8 is a potent chemoattractant that induces neutrophil chemotaxis and accumulation in inflamed airways, and the IL-8 level in BALF, but not in plasma, correlates significantly with the changes in arterial oxygenation (PaO2/FiO2) and an intrapulmonary shunt (Qs/Qt) after CPB.13,14 The inflammatory changes seen by light microscopy and the significant increase of MDA in the lung indicate oxidative stress. Taken together, these results imply CPB itself can directly result in some degree of lung injury, at least partly caused by neutrophil infiltration and oxidative stress in response to CPB.
The dosage of PDTC was based upon a previous study.15 As expected, pretreatment with PDTC attenuated all changes in the lung after CPB. This agrees with other studies showing PDTC mitigated lung injury induced by ischemia/reperfusion or lipopolysaccharide.6,7 The protective effect of PDTC appears to be due to its antioxidant property. As a direct oxygen scavenger, PDTC can limit the availability of superoxide and hydrogen peroxide. PDTC is also a chelator of heavy metals, and probably prevents the formation of hydroxyl radicals produced through the Haber-Weiss reaction.7 The in vivo antioxidant property of PDTC was confirmed by its attenuation of the increase of MDA in this study. To further understand the molecular mechanisms underlying the pathogenesis of CPB-related ALI and the protective effect of PDTC, we evaluated the activity of NF-B and found a significant increase of NF-B in parallel with the severity of lung injury. NF-B is known to regulate the expression of a wide range of genes whose products are critical in the initiation and progression of ALI, including IL-8 and intercellular adhesion molecule-1.3 Therefore, NF-B activation may play an important role in the pathogenesis of CPB-related ALI. Moreover, as in other cases, we found PDTC pretreatment attenuated this NF-B activation.15,16 This suggests that inhibition of NF-B activation may be an important molecular mechanism by which PDTC exerts protective effects against CPB-related ALI. NF-B is normally sequestered in the cytoplasm through association with an inhibitor protein, IB. On exposure of the cell to activation signals, such as reactive oxygen species, the IB protein is degraded. On being freed from association with IB, the NF-B complex moves to the nucleus where it binds to specific sequences in the promoter/enhancer regions of related genes.3,5 As a result of the role of reactive oxygen species in NF-B activation, the inhibitory effect of PDTC on NF-B activation is usually thought to be due to its antioxidant properties. However, Hayakawa and colleagues17 reported that NF-B inhibition by PDTC was through inhibition of IB ubiquitin ligase activity and independent of its antioxidative function. The exact mechanism by which PDTC inhibits NF-B activation in CPB warrants further investigation.
It is noteworthy that the hypotension and hemodilution that occurred in this study might have affected lung function. However, both phenomena are inherent to CPB and no significant difference existed between the CPB and PDTC groups. We do not think these factors affected the overall findings of this study. It was concluded that CPB itself can induce lung injury, and pretreatment with the antioxidant PDTC attenuates this injury in rats. The protective effect of PDTC is mediated through its antioxidant property. Furthermore, suppression of NF-B activation by PDTC may be an important underlying molecular mechanism.
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REFERENCES
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- Asimakopoulos G, Smith PL, Ratnatunga CP, Taylor KM. Lung injury and acute respiratory distress syndrome after cardiopulmonary bypass. Ann Thorac Surg 1999;68:1107–15.[Abstract/Free Full Text]
- Dreyer WJ, Michael LH, Millman EE, Berens KL, Geske RS. Neutrophil sequestration and pulmonary dysfunction in a canine model of open heart surgery with cardiopulmonary bypass. Evidence for a CD18-dependent mechanism. Circulation 1995;92:2276–83.[Abstract/Free Full Text]
- Fan J, Ye RD, Malik AB. Transcriptional mechanisms of acute lung injury. Am J Physiol Lung Cell Mol Physiol 2001;281:L1037–50.[Abstract/Free Full Text]
- Shenkar R, Schwartz MD, Terada LS, Repine JE, McCord J, Abraham E. Hemorrhage activates NF-kappa B in murine lung mononuclear cells in vivo. Am J Physiol 1996;270(5 Pt 1):L729–35.[Medline]
- Schmidt KN, Amstad P, Cerutti P, Baeuerle PA. The roles of hydrogen peroxide and superoxide as messengers in the activation of transcription factor NF-kappa B. Chem Biol 1995;2:13–22.[Medline]
- Long SM, Laubach VE, Tribble CG, Kaza AK, Fiser SM, Cassada DC, et al. Pyrrolidine dithiocarbamate reduces lung reperfusion injury. J Surg Res 2003;112:12–8.[Medline]
- Nathens AB, Bitar R, Davreux C, Bujard M, Marshall JC, Dackiw AP, et al. Pyrrolidine dithiocarbamate attenuates endotoxin-induced acute lung injury. Am J Respir Cell Mol Biol 1997;17:608–16.[Abstract/Free Full Text]
- Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–54.[Medline]
- Matata BM, Galinanes M. Peroxynitrite is an essential component of cytokines production mechanism in human monocytes through modulation of nuclear factor-kappaB DNA binding activity. J Biol Chem 2002;277:2330–5.[Abstract/Free Full Text]
- Ballaux PK, Gourlay T, Ratnatunga CP, Taylor KM. A literature review of cardiopulmonary bypass models for rats. Perfusion 1999;14:411–7.[Abstract/Free Full Text]
- Cox CM, Ascione R, Cohen AM, Davies IM, Ryder IG, Angelini GD. Effect of cardiopulmonary bypass on pulmonary gas exchange: a prospective randomized study. Ann Thorac Surg 2000;69:140–5.[Abstract/Free Full Text]
- Kang JL, Lee HW, Lee HS, Pack IS, Castranova V, Koh Y. Time course for inhibition of lipopolysaccharide-induced lung injury by genistein: relationship to alteration in nuclear factor-B activity and inflammatory agents. Crit Care Med 2003;31:517–24.[Medline]
- Richman-Eisenstat JB, Jorens PG, Hebert CA, Ueki I, Nadel JA. Interleukin-8: an important chemoattractant in sputum of patients with chronic inflammatory airway diseases. Am J Physiol 1993;264(4 Pt 1):L413–8.[Medline]
- Kotani N, Hashimoto H, Sessler DI, Muraoka M, Wang JS, O’Connor MF, 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;90:1046–51.[Abstract/Free Full Text]
- Liu SF, Ye X, Malik AB. Inhibition of NF-kappaB activation by pyrrolidine dithiocarbamate prevents in vivo expression of proinflammatory genes. Circulation 1999;100:1330–7.[Abstract/Free Full Text]
- Chandrasekar B, Streitman JE, Colston JT, Freeman GL. Inhibition of nuclear factor kappa B attenuates proinflammatory cytokine and inducible nitric-oxide synthase expression in postischemic myocardium. Biochim Biophys Acta 1998;1406:91–106.[Medline]
- Hayakawa M, Miyashita H, Sakamoto I, Kitagawa M, Tanaka H, Yasuda H, et al. Evidence that reactive oxygen species do not mediate NF-kappaB activation. EMBO J 2003;22:3356–66.[Medline]
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ABSTRACT
INTRODUCTION
