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J Thorac Cardiovasc Surg 2003;126:1867-1874
© 2003 The American Association for Thoracic Surgery
Cardiopulmonary support and physiology |
a Laboratoire d'Etudes des Greffes et Prothèses Cardiaques, Hôpital Broussais, Paris, France
b Hôpital Européen Georges Pompidou, Paris, France
c Hôpital Marie-Lannelongue, Le Plessis-Robinson, France
Received for publication May 10, 2002; revisions received May 15, 2003; accepted for publication June 18, 2003.
* Address for reprints: Dr Rachid Zegdi, Hôpital Européen Georges Pompidou, Service de Chirurgie Cardiovasculaire, 20, rue Leblanc 75908 Paris France
rzegdi{at}hotmail.com
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Abstract |
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METHODS: Wistar rats underwent either a partial femoro-femoral extracorporeal circulation in normothermia for 3 hours (n = 5) or a sham procedure (n = 5). Exhaled carbon monoxide concentration was monitored with an infrared analyzer. After the rats were killed, lungs were harvested for determination of heme oxygenase activity and inducible heme oxygenase expression (by Western blot and immunohistochemistry). Lung injury was also assessed by arterial blood gas analysis and microscopic study.
RESULTS: Extracorporeal circulation was responsible for a lung injury characterized by decreased arterial blood oxygen saturation and typical morphologic findings (marked alveolar neutrophil infiltration; interstitial edema). Exhaled carbon monoxide concentration remained stable throughout the experiment in all sham rats, whereas it increased after extracorporeal circulation (from 0.16 ± 0.05 ppm at baseline to 0.7 ± 0.2 ppm at end of experiment; P = .0001). Pulmonary heme oxygenase activity and inducible heme oxygenase content (assessed by Western blot) were increased within lungs of rats that underwent extracorporeal circulation. Immunohistochemistry revealed that the expression of inducible heme oxygenase was mainly localized to inflammatory cells.
CONCLUSIONS: Post–extracorporeal circulation acute lung injury in rats was associated with an increased expression of inducible heme oxygenase, the functional significance of which remains to be determined.
Carbon monoxide is synthesized in mammalians, mainly from heme degradation.5 Endogenously produced CO does not accumulate within the body and is excreted in exhaled breath.6 Heme oxygenase (HO) is the step-limiting enzyme from heme catabolism. Three isoforms of HO have been described, 1 inducible (HO1) and 2 constitutive (HO2 and HO3). These isoforms differ by their cellular location and the regulation of their expression.7 HO1 isoform has been shown to be primarily induced during oxidative injury.8 Increased expression of HO1 has also been described in a few experimental models of ALI, which was associated with a protective role.9
The primary purpose of this study was to determine the influence of extracorporeal circulation on the expression of HO1 in postperfusion ALI by using a previously described rat model of partial bypass.10 We also evaluated the impact of extracorporeal circulation on the endogenous CO production by measuring blood carboxyhemoglobin concentration and monitoring exhaled CO concentration.
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Materials and methods |
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Extracorporeal circulation
Circuit preparation
The ECC circuit consisted of a nonpulsatile roller pump (Jouvelet, Simal, Paris, France) and sterile Tygon tubing with an inner diameter of 2.4 mm for the venous line and 0.8 mm for the arterial line (Masterflex R3603, Bioblock Scientific, Illkirch, France). ECC was established at a flow rate of 100 mL/kg/min. Venous return was drained by gravity into a 20-mL sterile reservoir. The membrane oxygenator obtained from Capiox 308 (Terumo, Osaka, Japan) is commonly used in pediatric cardiac surgery. To reduce the prime volume of the oxygenator (110 mL), we removed the heat exchanger and placed at each extremity of the membrane 2 silicone "cones" specifically designed for the experiment. These cones excluded approximately 75% of the total membrane surface area from circulation. The total prime volume of the circuit was 35 mL of which the oxygenator constituted 15 mL. The priming was done with 20 mL of fresh blood obtained from 2 homologous donor rats and with 15 mL of synthetic colloid (Plasmion, Frésénius, France). Gas flow (100% O2) was maintained between 50 and 75 mL/min. Preliminary experiments have shown that this gas flow was sufficient to achieve adequate oxygenation.10
Surgical procedure
Male Wistar rats (475-550 g) were used for all experiments. They were anesthetized with an intramuscular administration of ketamine hydrochloride (80 mg/kg) and chlorpromazine hydrochloride (2 mg/kg). Anesthesia was maintained throughout the experiment with additional doses of intravenous ketamine. Sustained muscle relaxation was achieved with an intravenous injection of 1 mg/kg of pancuronium bromide. Rats were continuously ventilated through a tracheostomy with air at 80 breaths/min and 8 mL/kg tidal volume under 4 cm H2O positive end-expiratory pressure (Harvard Rodent Respirator, model 680, SARL Ealing, Ulis, France). Central temperature was monitored with an esophageal probe and maintained between 36°C and 38°C by a heating lamp placed above the animal and the oxygenator. The tail artery was cannulated (25-gauge Teflon catheter; Ethicon, Issy les Moulineaux, France) to monitor systemic arterial pressure (Hewlett Packard, model 78342A; Hewlett Packard Company, Palo Alto, Calif). The right femoral vein and artery were exposed and cannulated with 16- and 25-gauge Teflon heparinized catheters, respectively. Anticoagulation consisted of heparin (500 IU/kg), which was administered in the following manner: half the dosage was given to the blood donor rats and a quarter of the dosage was added to the priming volume and to the study rat.
Protocol
Rats were assigned to either an ECC group or a sham group. Both the ECC and sham groups consisted of 5 rats. Anesthesia, ventilation, cannulation, and heparinization were identical in both groups throughout the experiment. Connection to the extracorporeal circuit was only performed in the ECC group. Ventilation was not discontinued during the ECC period. ECC lasted for 3 hours and rats were further monitored for another 3 hours before they were killed. After cessation of the ECC, rats were infused with the remaining priming medium when diastolic pressure was less than 50 mm Hg. Arterial blood gas analysis was performed before the start of ECC (baseline) and at the end of the experiment.
Rats were killed by exsanguination. They were then perfused with 500 mL of cold saline (4°C). Lungs were harvested without the trachea and the proximal bronchi. The left lung was immersed in 4% neutral buffered formalin for 48 hours. The right lung was homogenized in a buffer containing 50 mmol/L Tris-HCl (pH 7.4), 250 mmol/L sucrose, and 1 tablet (per 50 mL) of an antiprotease mixture (Complete; Boehringer Mannheim, Meylan, France). The homogenate was centrifuged at 10,000g for 20 minutes and the supernatant was collected and stored at -80°C. Protein concentration was determined by the modified Peterson method.
Measurement of exhaled carbon monoxide concentration
Exhaled CO was measured using an infrared CO analyzer (CO 2000; Sérès, La Duranne, France). The CO analyzer was calibrated prior to each experiment with a CO calibration gas at a concentration of 10.3 ppm (Air Liquide, Bonneuil, France). Its sensitivity was 0.1 ppm and its sampling rate was set at 1L/min.
All rats were ventilated with CO-free rendered air (CO concentration 
0.1 ppm) by its passage through a heated hopcalite filter (Sérès). Expired breath was collected into a 6-L polyethylene bag that did not react with CO. The bag was placed at the expiratory outlet of the ventilator and was rapidly connected to the CO analyzer when filled. Each gas collection lasted approximately 20 minutes. Therefore, measurements indicated at each time point represent the mean exhaled CO concentration of gas collected over the preceding 20 minutes. Exhaled CO concentration was determined before (baseline), during (30, 90, and 180 minutes), and after cessation (1, 2, and 3 hours) of ECC.
Heme oxygenase activity
The method was derived from Tenhunen and colleagues.12 Briefly, as a source of biliverdin reductase, livers from fasted rats were harvested and immediately placed in cold 0.9% NaCL. The livers were then weighed and homogenized in 4 volumes of 2 mmol/L MgCl2–100 mmol/L phosphate buffer (pH 7.4). The homogenate was centrifuged at 105,000g for 1 hour at 4°C and the supernatant (liver cytosol) was used as a source of biliverdin reductase.
The reaction mixture consisted of 200 µL of lung supernatant, 50 µL of liver cytosol, 20 µL of 1 mmol/L heme b solution, 200 µL of 2.75 mmol/L ß-nicotinamide adenine dinucleotide phosphate (NADPH) solution, and 530 µL of 2 mmol/L MgCl2–100 mmol/L phosphate buffer (pH 7.4). The samples were incubated in a 37°C water bath in the dark for 1 hour. The reaction was stopped by placement on ice. An NADPH-free reaction mixture provided a baseline against which the measured concentrations were compared. The absorbance of the samples was measured by spectrophotometry (model DU-70; Beckman, Villepinte, France) at 464 and 530 nm. The amount of bilirubin formed was calculated from the difference in optical density at 464 and 530 nm, assuming an extinction coefficient of 40 mmol/L-1 · cm-1. The values are expressed as picomoles of bilirubin formed per milligram of protein per hour.
HO1 detection by Western blot
An aliquot of homogenate was suspended in sodium dodecylsulphate (SDS) sample buffer (125 mmol/L Tris-HCl [pH = 7.4], 10% glycerol, 6% SDS, 0.01% ß-mercaptoethanol, and 0.1% bromophenol blue). Equal amounts of protein per lane (150 µg) were subjected to electrophoresis in a 12% SDS-polyacrylamide gel. Gels were transferred onto nitrocellulose membranes by electroblotting. The membranes were blocked by incubation in phosphate-buffered saline solution (PBS) 1X, 5% nonfat dry milk, and 0.2% Tween 20 for 1 hour at room temperature. Blots were then incubated overnight at 4°C in the same buffer containing a rabbit polyclonal antibody against rat HO1 (1:1,000; Tebu, France). Blots were washed 4 times for 15 minutes in PBS 1X and 0.2% Tween 20 and thereafter incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) secondary antibody (1:15,000; Amersham, Orsay, France). Blots were then washed and immunoreactivity was assessed using an enhanced Western blot detection system according to the manufacturer's protocol (Amersham). Spleen homogenates were used as positive controls for HO1.
Histology
After fixation, the left lung was embedded in paraffin. Two sections of 3-µm thickness were obtained from each block and stained with hematoxylin-eosin.
Immunohistochemistry
Tissue sections were deparaffinized in xylene and then rehydrated in graded alcohol solutions. The sections were blocked in a solution of 5% nonfat milk, 1% bovine serum albumin (BSA) before incubation overnight at 4°C with a rabbit polyclonal antibody to HO1 (Tebu, Le Perray, France) in 1% milk and 1% BSA in PBS 1X (1:1,000 dilution). The sections were washed 3 times with PBS for 5 minutes each and incubated for 1 hour with secondary antibody (biotinylated goat anti-mouse IgG; Jackson Laboratories, L’arbresle, France) at a dilution of 1:500 in 1% milk, PBS 1X at room temperature. The signal was detected with peroxidase-conjugated avidin and diaminobenzidine. The slides were counterstained with 1% hematoxylin. For negative controls, sections were processed as above except that the primary incubation was performed with nonimmune rabbit serum (Jackson Laboratories) instead of primary antibody.
Blood gas analysis
Arterial blood gas analysis, pH, hematocrit, and carboxyhemoglobin were determined with a calibrated blood gas analyzer (ABL 505; Radiometer, Copenhagen, Denmark) and a CO oximeter (OSM 3, Radiometer).
Determination of bilirubin and hemoglobin plasmatic concentration
Measurement of total bilirubin plasmatic concentration was performed by using an automated colorimetric method (Synchron CX7; Beckman Coulter, Villepinte, France). Hemoglobin concentration was assessed spectrophotometrically by measuring the absorbance of the samples at 561 and 576 nm (DU70, Beckman Coulter).
Statistics
Results are expressed as mean ± standard deviation. Comparisons of continuous data between sham and ECC groups were performed with the Mann-Whitney test.
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There was no difference in baseline arterial blood gas values between ECC and sham groups (Table 1). ECC was associated with a significant decrease in arterial oxygenation (SaO2 = 80.8% ± 11.9% in the ECC group vs SaO2 = 95.5% ± 3.7% in the sham group at the end of the experiment; P = .009).
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HO activity was significantly higher in the rat lungs from the ECC group (485 ± 64.3 pmol/mg protein/h in the ECC group vs 178.8 ± 29.1 pmol/mg protein/h in the sham group; P = .009). Likewise, there was a statistically significant difference between the 2 groups regarding the spleen HO activity (Figure 3).
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Discussion |
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Based on the histological study (Figure 1), ECC in rats was clearly responsible for a diffuse inflammatory disorder of the lungs, which resembles the lung injury of other models of ALI. Moreover, the morphological findings in this rat model of post-ECC ALI were close to those previously described in larger animals (pig, dog, sheep) and in humans.3,4 In a previous study, we also observed an increase in lung water content (reflecting pulmonary edema) and in Evans blue dye content (an index of microvascular permeability) in lungs of rats that underwent a 90-minute ECC.13
We chose to cannulate the femoral vessels to prevent the lung injury that might have occurred with a thoracic approach. Our rat model of ECC was a partial bypass, with a pump flow approximately equivalent to half of the theoretical rat cardiac output. Ventilation, therefore, could not be discontinued during ECC. Rats that underwent ECC survived without any evident sequela.10 We believe that our rat model of partial bypass is appropriate to investigate the pathophysiology and the natural course of postperfusion ALI.
Carbon monoxide in the body originates from an endogenous source and an exogenous source. Ambient CO represents the exogenous source. Mammalian cells synthesize CO (mainly from heme catabolism), which represents the endogenous source.6 Endogenously produced CO does not accumulate within the body. A small amount of CO is oxidatively transformed into carbon dioxide at a rate of 0.1% to 0.2% of the CO body content per hour. The bulk of synthesized CO is transported by blood (as carboxyhemoglobin) to the lungs where it is excreted in exhaled breath. Lung excretion is considered as the quasi-exclusive way of CO elimination from the body. This pulmonary excretion depends on many variables such as alveolar ventilation, CO lung diffusing capacity, lung capillary oxygen partial pressure, carboxyhemoglobin concentration, and endogenous CO production.14
In the present study, we demonstrated for the first time an increase in exhaled CO concentration after ECC. The concomitant increase in carboxyhemoglobin concentration (the largest CO store within the body) suggests that the increased exhaled CO reflects an increased endogenous CO production. Systemic hemolysis is known to increase the endogenous CO production and the exhaled CO concentration.15,16 Hemolysis occurred in our rat model of bypass (Table 1). In addition to hemolysis, other processes may explain the increased endogenous CO production during ECC, such as an exaggerated turnover of hemoproteins other than hemoglobin (such as cytochromes, peroxidases) or the production of CO secondary to lipid peroxidation.5
Elevated CO concentrations in exhaled breath have recently been reported in various bronchopulmonary inflammatory disorders such as asthma,17 bronchiectasis,18 or cystic fibrosis.19 This increased exhaled CO has been demonstrated to reflect the severity of the inflammatory process in some instances.17 We are not aware of any study dealing with exhaled CO measurement in experimental or clinical ALI. Based on our study, it seems unlikely that monitoring exhaled CO will help noninvasive assessment of the severity of the inflammatory process in postperfusion ALI, as long as hemolysis persists.
In a recent study, we observed an increased heme oxygenase activity in lungs from rats that underwent a 90-minute ECC.13 In the present study, we demonstrated an elevated HO1 isoform expression in postperfusion lung injury. Up-regulation of HO1 within lungs has been previously reported in other models of ALI such as those following endotoxemia or prolonged hyperoxia.9,20 Increases in HO1 mRNA and protein content have been detected within lungs 2 to 4 hours and 4 to 8 hours after systemic administration of endotoxin (a condition mimicking sepsis), respectively. Immunohistochemical studies have shown that up-regulation of HO1 occurred in various cells including airway epithelial cells or inflammatory cells. Similar findings have been previously reported in hyperoxic lung injury20 and were also observed in our rat model of post-ECC ALI.
In our rat model of bypass, the pulmonary up-regulation of HO1 in post-ECC ALI was likely the consequence of multiple factors. In vitro and in vivo studies have demonstrated that cellular expression of HO1 can be dramatically induced by hemoglobin.21 Up-regulation of HO1 could reflect, at least in part, the exposure of pulmonary cells to free hemoglobin. Free hemoglobin might have originated from peripheral hemolysis, a well-known complication of prolonged bypass with roller pump as in our rat model, and from intrapulmonary hemorrhage, a common microscopic feature of post-ECC ALI.3,4 The blood concentrations of various cytokines have also been shown to increase during and after ECC.22 Recently, Kotani and colleagues23 reported elevated concentrations of tumor necrosis factor, interleukin 6, and interleukin 8 in patients' bronchoalveolar lavage fluid obtained after ECC. Thus, the systemic and pulmonary production of proinflammatory cytokines might also be responsible for the up-regulation of HO1 in post-ECC ALI.7
There is a large growing body of evidence suggesting that induction of HO1 expression may confer protection to cells and tissues against oxidative injury. Recently, Yamada and colleagues24 observed that up-regulation of HO1 (by hemin or interleukin 1-ß) prevented the hydrogen peroxide–induced increase in permeability of human tracheal epithelium in culture. This protective effect was abolished by competitive inhibitors of HO. Otterbein and associates25 also reported that overexpression of HO1 achieved by gene transfer in rat lungs improved survival and attenuated the lung injury secondary to prolonged hyperoxia. The functional significance of HO1 up-regulation in post-ECC acute lung injury is the subject of an ongoing experimental study in our laboratory.
It has been speculated that the protection conferred by HO1 up-regulation could be related to the byproducts of heme degradation. Bilirubin has potent antioxidant properties in vitro.26 Hayashi and colleagues27 described a bilirubin-induced inhibition of neutrophil adhesion to the endothelium during an oxidative vascular injury. Ferritin, the synthesis of which can be induced by an HO-dependent iron release,28 has also been demonstrated to be a potent antioxidant.29 Finally, CO (usually considered as a potentially toxic waste product of metabolism) has been shown to have a major anti-inflammatory action. Otterbein and coworkers30 recently demonstrated that exogenous CO at low concentration (50-500 ppm) attenuated posthyperoxia ALI by stimulation of the p38-mitogen–activated protein kinase pathway. Fujita and colleagues31 also reported a protective effect of CO in ischemia-reperfusion lung injury through derepression of fibrinolysis. The role of the increased endogenous CO production that we observed in our rat model of bypass should be investigated.
In summary, we demonstrated that a 3-hour ECC in rats was responsible for an ALI, which was associated with an increased expression of HO1. We also observed an increase in exhaled CO concentration, reflecting an elevated endogenous CO production, possibly secondary to an exaggerated hemolysis. The determination of the physiological significance of the increased HO1 expression in lungs following ECC will require further studies.
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Acknowledgments |
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This article has been cited by other articles:
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  Y. Jin and A. M. K. Choi Cytoprotection of Heme Oxygenase-1/Carbon Monoxide in Lung Injury Proceedings of the ATS, October 1, 2005; 2(3): 232 - 235. [Abstract] [Full Text] [PDF]
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