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J Thorac Cardiovasc Surg 2004;128:109-116
© 2004 The American Association for Thoracic Surgery
Cardiopulmonary support and physiology |
a Research Center and Department of Surgery, Montreal Heart Institute, Montreal, Quebec, Canada
b Department of Anesthesiology, Montreal Heart Institute, Montreal, Quebec, Canada
c Cardiovascular Surgery Unit, Arnaud de Villeneuve Teaching Hospital, Montpellier, France
Received for publication April 1, 2003; revisions received September 25, 2003; accepted for publication September 29, 2003.
* Address for reprints: Louis P. Perrault, MD, PhD, Research Center, Montreal Heart Institute, 5000 Belanger Street East, Montreal, Quebec H1T 1C8, Canada
lpperrau{at}icm.umontreal.ca
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Abstract |
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METHODS: Three groups of Landrace swine were compared: control without cardiopulmonary bypass (control group); 90 minutes of normothermic cardiopulmonary bypass (bypass group); 90 minutes of cardiopulmonary bypass and treated with prostacyclin during cardiopulmonary bypass (continuous nebulization with continuous positive airway pressure until the end of the cardiopulmonary bypass; prostacyclin group). After 60 minutes of reperfusion, swine were put to death and pulmonary arteries harvested. After contraction to phenylephrine, endothelium-dependent relaxation to bradykinin and acetylcholine was studied in standard organ chamber experiments. The pulmonary artery intravascular cyclic adenosine monophosphate content was compared between the 3 groups (post–cardiopulmonary bypass).
RESULTS: There was a statistically significant improvement of the endothelium-dependent relaxation to bradykinin in the prostacyclin group when compared with the bypass group (P < .05). There was no statistically significant difference for endothelium-dependent relaxation to acetylcholine (P > .05) between the prostacyclin and the bypass groups. There was a statistically significant decrease in the cyclic adenosine monophosphate content and a statistically significant increase of the mean pulmonary artery pressure in the bypass group only (P < .05).
CONCLUSION: Prophylactic use of inhaled prostacyclin has a favorable impact on the pulmonary endothelial dysfunction induced by cardiopulmonary bypass associated with preservation of pulmonary intravascular cyclic adenosine monophosphate content and the pulmonary vascular tone.
Pulmonary endothelium, presenting a total surface area of 200 m2 in adults, is in constant interaction with the blood elements.5 Beyond its roles as selective barrier, the endothelium allows inhibition of platelet aggregation and neutrophil adhesion and elaborates powerful vasoactive substances that control blood flow and vascular remodeling. Appropriate endothelial function results from the equilibrium between endothelium-dependent relaxing factors (EDRF), such as nitric oxide (NO) and prostacyclin, and endothelium-dependent contracting factors (EDCF), such as endothelin. Endothelial dysfunction can be defined as an imbalance between relaxing factors and contracting factors and results in the loss of the normal protective role of the endothelium in the homeostasis of the vascular wall.6 In vascular reactivity studies, only the vasomotor control function of the endothelium is studied.
Prostacyclin (PGI2) is an endogenous prostaglandin derived from arachidonic acid metabolism through the cyclooxygenase pathway in the vascular endothelium. PGI2 binds to a Gs-protein–related receptor, which when activated increases cyclic adenosine monophosphate (cAMP) concentration, activating a protein kinase A to decrease free intracellular calcium concentration (Figure 1). The physiological effects are vascular dilatation (predominantly in resistance vessels), inhibition of endothelin secretion, inhibition of platelet aggregation, and inhibition of leukocyte adhesion to the endothelium.7
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A selective endothelial dysfunction of the pulmonary arterial tree involving relaxation mediated by muscarinic receptors to acetylcholine (Gi-coupled) following CPB in a porcine model was demonstrated in our laboratory.10 The aim of this experimental study in a porcine model was first to determine the effect of prophylactic use of inhaled PGI2 before CPB on the pulmonary endothelial function and second to examine its effect on hemodynamic data, on the A-aDO2, and on pulmonary artery pressures.
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Materials and methods |
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Experimental groups
Group 1: control (n = 6)
The mediastinum was exposed via a median sternotomy. Heparin (300 UI/kg; Leo Pharma, Inc, Ajax, Ontario, Canada) was given intravenously. After 1 hour of general anesthesia with 1% isoflurane, the animal was exsanguinated and the heart and lung harvested.
To study the effect of these drugs on pulmonary vascular reactivity, a preliminary experiment was conducted. Four animals underwent median sternotomy. After 1 hour of general anesthesia with 1% isoflurane, anesthesia was changed to propofol for 1.5 hours (0.1-0.2 mg/kg/min, same dosage as in the CPB groups), followed by 1 hour with 1% isoflurane to match the time and drugs used in the other groups. Arterial pressure was maintained with crystalloid (Ringer's lactate). The animal was then exsanguinated and the lungs harvested. Endothelium-dependent relaxation to bradykinin (BK) and acetylcholine (ACh) was comparable to the control group used in the study (data not shown). These organ chamber experiments confirmed that normal animals could be used as parallel control animals.
Group 2: cardiopulmonary bypass (n = 6)
After sternotomy, the aorta and right atrium were cannulated following heparin administration (300 UI/kg), when activated coagulation time (ACT, assessed with Hemochron 801; Technidyne, Edison, NJ) was superior to 300 seconds. CPB was initiated when ACT was superior to 400 seconds. Ventilation and isoflurane were stopped but a continuous positive airway pressure (CPAP) of 3 cm H2O was maintained throughout the CPB period. Anesthesia was maintained with a continuous infusion of propofol (0.1-0.2 mg/kg/min). The pump flow was adjusted to obtain an index of 2.4 L/min/m2 and assessed by venous gases to maintain mixed venous saturation over 60%. FIO2 was kept constant at a level of 68%. Mean systemic arterial pressure was maintained between 50 and 70 mm Hg with crystalloid (Ringer's lactate). The temperature was allowed to drift to 36°C. The heart was left beating, empty. No aortic crossclamping or cardioplegic solution was used. Before CPB weaning, swine were rewarmed to 38°C (normal porcine temperature). After 90 minutes of CPB, mechanical ventilation and isoflurane anesthesia were reinstituted and CPB was weaned. No protamine was given due to its potential cardiovascular effects such as complement activation and NO production.11 Normal circulation was restored for 60 minutes, at which time the animal was exsanguinated into the cardiotomy reservoir. The beating heart and the lungs were excised "en bloc" and immediately immersed in a cold modified Krebs–bicarbonate solution (composition in mmol/L: NaCl 118.3, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, glucose 11.1, CaCl2 2.5, NaHCO3 25, and ethylenediaminotetraacetic acid 0.026).
Group 3: cardiopulmonary bypass and inhaled prostacyclin (n = 5)
The same procedure was followed as in the CPB group (group 2). The only difference was that a bolus of 2 to 2.5 µg/kg of PGI2 was given via the endotracheal tube through a nebulizer during the 30 minutes preceding the initiation of CPB (Figure 1). Ventilation was then stopped but a continuous nebulization of PGI2 at a rate of 0.1 µg/kg/min with CPAP of 3 cm H2O was then instituted until the end of the CPB. Before weaning of CPB, nebulization was stopped and ventilation reinstituted. The dosages were based on review of the literature7 and our experience with this medication.8,12
PGI2 (Flolan, Glaxo Wellcome, Mississauga, Ontario, Canada) was given as epoprostenol salt 1.5 mg dissolved in 100 mL of sterile glycine buffer diluent for a concentration of 15 µg/mL. The drug was administered through a conventional in-line nebulizer kit (Salter Labs, Arvin, Calif) connected to the inspiratory limb of the ventilator.
Hemodynamic and biochemical data
Arterial and venous blood gases were measured at regular intervals during the experiment (baseline, during CPB, immediately after weaning from CPB, and at death) Pulmonary artery pressures were measured with a Swan-Ganz catheter at different intervals during the procedure: after induction, before CPB, and after weaning of CPB (0 minute, 10 minutes, 15 minutes, 45 minutes, and 60 minutes).
Vascular reactivity studies
Less than 10 minutes after en bloc excision, the heart was removed and the primary pulmonary artery was dissected. Branches of second-degree pulmonary arteries were isolated and dissected free of connective and adventitial tissue and divided into rings (4 mm wide; 16 rings per animal). All rings were placed in organ chambers (Emka Technologies Inc, Paris, France) filled with 20 mL modified Krebs–bicarbonate solution continuously heated at 37°C and oxygenated with a carbogen mixture (95% O2 and 5% CO2). The rings were suspended between 2 metal stirrups with the upper 1 connected to an isometric force transducer connected to a signal amplifier and then allowed to stabilize for 30 minutes. Data were collected with a biological signal data acquisition software (IOX 1.203; Emka Technologies Inc). Each arterial ring was stretched to the optimal point of its active length-tension curve (3.5 g) as determined by measuring the contraction to potassium chloride (KCl) 60 mmol/L at different levels of stretch (data not shown). The maximal contraction of rings was then obtained with addition of potassium chloride (KCl 60 mmol/L). After obtainment of a plateau, all baths were washed twice with modified Krebs–bicarbonate solution and indomethacin (10–5 mmol/L; to exclude production of endogenous prostanoids) was added in each bath. After 60 minutes of stabilization, phenylephrine (PE; range 2 x 10–7 mol/L to 3 x 10–6 mol/L) was added to obtain a contraction averaging 50% of the maximal contraction to KCl.
Endothelium-dependent relaxation
The NO-mediated relaxation pathway was studied by constructing concentration-response curves to ACh (10–10 to 10–5 mol/L; an agonist of M2 receptors coupled to Gi-proteins) and to (10–12 to 10–6 mol/L; an agonist of B2 receptors coupled to Gq-proteins).
Endothelium-independent relaxation
At the end of the experiment, endothelium-independent relaxation was studied with the use of 10–5 mol/L sodium nitroprusside (SNP), a nitric oxide donor.
Drugs
All drugs were prepared daily. ACh, BK, indomethacin, and sodium SNP were obtained from Sigma Chemical Co (Oakville, Ontario, Canada). Phenylephrine was obtained from Cayman Chemical Company (Ann Arbor, Mich).
Determination of pulmonary artery intravascular cAMP content
To determine the vascular content of porcine pulmonary arteries, rings from the 3 groups were collected after death, frozen in liquid nitrogen, and stored at –70°C. At the time of analysis, all segments were pulverized in a liquid nitrogen–cooled stainless steel mortar, and then transferred in trichloracetic acid solution (6.25% w/v). The acid extracts were then centrifuged at 4°C for 15 minutes at 12,000g (3000 revolutions per minute) to precipitate cell debris and proteins. The pellets were used for total protein determination using the Bradford microassay technique (Bio-Rad, Mississauga, Ontario, Canada). To remove trichloracetic acid, the supernatants were extracted 4 times with water-saturated diethyl ether. Any residual diethyl ether was removed by heating the samples to 90°C for 3 to 5 minutes. cAMP quantification was done using an enzyme immunoassay system with acetylation based on rabbit anti-cAMP antibody (Amersham Pharmacia Biotech, Baie d'Urfé, Quebec, Canada). The amount of cAMP in each blood vessel ring was standardized to pmol cAMP · mg–1 protein.
Total antioxidant plasma status
The assay antioxidant levels in plasma samples rely on the ability of antioxidants in the sample to inhibit the oxidation of ABTS (2,2'-Azino-di-[3-ethylbenzthiazoline sulphonate]) to ABTS+ by metmyoglobin (a peroxidase). The amount of ABTS+ produced can be monitored by reading the absorbance at 600 nm. Under the reaction conditions used, the antioxidants in the sample cause suppression of the absorbance at 600 nm to a degree that is proportional to their concentration. Blood samples from groups were drawn from right atrium before and after CPB in ethylenediaminetetraacetic acid to prevent coagulation and subsequently were centrifuged. The isolated plasma was then frozen and kept at –70°C until analysis. The plasma samples were analyzed with Total Antioxidant status activity Assay Kit (Calbiochem, San Diego, Calif). The concentration of ABTS+ were measured with a spectrophotometer set at 600 nm.
Statistical analysis
All values are expressed as the mean ± standard error of the mean (SEM). Contractions to PE are expressed as a percentage of the maximal contraction to KCl (60 mmol/L). Relaxations are expressed as the percentage of the maximal contraction to PE for each ring. Two-way analyses of variance (ANOVA) were performed with 1 repeated measure (ACh and BK doses) to compare the concentration-response curves between the 3 groups, followed by pairwise comparisons when the global ANOVA was significant. One-way ANOVA was performed to compare groups for contractions and cAMP. Two-way ANOVA was performed with 1 repeated measure (time) to compare the hemodynamic data (mean arterial pressure [mAP], mean pulmonary artery pressure [mPAP], pulmonary vascular resistance index [PVRI], SvO2, A-aDO2, lactic acid levels), followed by pairwise comparisons when the global ANOVA was significant. Statistical analysis was performed with the computer software SAS (SAS Institute, Inc, Cary, NC).
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Results |
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Vascular reactivity study
Contractions
The amplitude of the contraction to KCl (60 mmol/L) and to PE and the concentration of PE used were quantified for both groups in Table 2. The amplitude of contraction to KCl (endothelium-independent agent) was significantly lower in the control group and higher in the CPB group without PGI2. Conversely, there was no difference in contraction to PE (endothelium-dependent agent) between the 2 CPB groups, but the CPB group without PGI2 required a significantly higher PE concentration to achieve the same level of contraction (P < .05).
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Endothelium-independent relaxation
No statistically significant difference in relaxation to the SNP was observed between groups, with all rings achieving 100% relaxation (data not shown).
Influence of indomethacin
In some baths, indomethacin was not added in order to eliminate its influence on PGI2. No statistically significant difference in contraction to KCl or PE or relaxation to ACh or BK was demonstrated in baths with and without indomethacin between the 3 groups (data not shown).
Pulmonary artery intravascular cAMP content
There was a statistically significant lower cAMP content in the vascular rings of the CPB group without PGI2 when compared with the controls and with the CPB group with PGI2 (P < .05; Figure 4).
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Discussion |
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Congenital and adult heart surgery such as mitral valve replacement are commonly followed by pulmonary hypertension in the postoperative period.13 CPB can directly cause pulmonary vascular injury. Endothelial dysfunction may leave the pulmonary adrenergic system unopposed, with an increase in vascular tone, which can result in RV failure and low output syndrome.13 Morita and colleagues14 demonstrated in a porcine model that CPB causes a significant increase in pulmonary vascular resistance and depresses the RV function by more than 50%. Several studies have demonstrated that RV dysfunction is a negative prognostic factor after cardiac surgery. Perioperative mortality ranges from 44% to 86% in patients with RV dysfunction.15 Furthermore, RV systolic dysfunction has been shown to be associated with poor outcome in patients undergoing myocardial revascularization with reduced left ventricular function.16 After CPB, pulmonary hypertension can be prevented by avoidance of hypoxia, hypercarbia, acidosis, and hypothermia as well as optimization of airway pressures and depth of anesthesia.13
Numerous experimental and human studies have demonstrated elevation of PGI2 levels during CPB with return to the baseline level immediately following CPB.17 As the relative decrease of PGI2 following CPB may explain the rise in pulmonary artery pressure, our main hypothesis was that PGI2 loading before CPB could prevent the pulmonary artery pressure elevation and secondary RV dysfunction.
In humans, pulmonary hypertension is defined as an mPAP of more than 25 mm Hg but no such definition exists in swine. The results of the present study demonstrate that administration of inhaled PGI2 before CPB effectively attenuates pulmonary artery pressure elevation secondary to CPB. Also, the adverse systemic effects of inhaled PGI2 on oxygen exchange are minimal due to its pulmonary selectivity,8 as shown by the absence of hypotension and preservation of the A-aDO2 in the PGI2 group compared with the CPB only group. The inhaled route of delivery is most likely responsible for the improvement in A-a gradient in this model.
Contraction to KCl is a receptor-independent response of vascular smooth cells. Contractions with KCl were greater in the CPB group without PGI2. However, a higher concentration of PE was necessary to achieve the same level of contraction in the aforementioned group than in the CPB group with PGI2, which can be explained by a decreased number of pulmonary 
-receptors or reduced sensitivity of these receptors. In humans, CPB is associated with an impairment of -adrenergic responsiveness, apparently due to desensitization of -receptors.18 This may be due to the influence of hemodilution, hypothermia, alteration of drug pharmacokinetics, or increased level of plasmatic catecholamines.18 One explanation for the better -adrenergic responsiveness in the PGI2 group comes from the findings from Wachtogel and colleagues.19 The loss of platelet -adrenergic receptors during extracorporeal circulation is prevented with prostaglandin E1 (PGE1),19 a vasodilator prostaglandin similar in structure and effects to PGI2.17
A decreased pulmonary production of NO after CPB has been demonstrated in numerous studies.14,20 ACh induces a dose-dependent vasomotor effect dependent on the preexisting vascular tone. Under resting conditions, ACh induces vasoconstriction and vasodilatation under conditions of elevated tone.21 ACh-related NO pathway appears to be preferentially damaged.10,22 Indeed, Gagnon and colleagues10 have shown in a porcine model that CPB with reperfusion of the pulmonary tree induces a decrease in endothelium-dependent relaxation mediated by muscarinic receptors, which was also demonstrated in a dog model by Zanaboni and colleagues.22 Although ACh induces endothelium-dependent relaxation through both the NO and cAMP pathways,23 more specific signal transduction pathways alterations may explain the lack of benefit of inhaled PGI2 in ACh-induced dysfunction in this study.
BK is an agonist that binds to B2 receptors coupled to Gq-proteins, which causes release of NO in pulmonary endothelium but also stimulates prostacyclin release,21 resulting in vasodilatation independently of the preexisting vascular tone. This study demonstrates complete relaxation of the pulmonary rings with BK after PGI2 administration, which can be attributed to this prostacyclin stimulation. This interaction between cAMP and NO has been recently studied in canine coronary arteries in which activation of the cAMP signal transduction increases endothelial NO production.24
PGI2 binds to Gs-protein receptors and induces an increase in cAMP concentration. In this study, CPB causes a significant decrease in cAMP content. However, administration of inhaled PGI2 before CPB increases these levels and prevents its subsequent decrease. These data indicate that cAMP signal transduction may be an important potential compensatory pathway to the dysfunctional NO pathway.
The CPB-induced systemic inflammatory response contributes to the release of reactive oxygen species (ROS), which may in part explain the vasoconstriction following weaning of CPB due to scavenging of NO by ROS.7 PGI2 has cytoprotective effects due to its ability to scavenge the ROS and reduces the synthesis of proinflammatory cytokines such as tumor necrosis factor .7 In the current study, the antioxidant activity was measured from the right atrium instead of lung tissue or left atrium plasma, which may explain in part the lack of difference between the CPB and the PGI2 groups.
Clinical implications
Congenital heart disease and pretransplantation status are frequently associated with a preexisting pulmonary hypertension. Inhaled PGI2 has been used to evaluate the reversibility of pulmonary hypertension of potential candidates for heart transplantation.25 To our knowledge, no study has examined the role of pre-CPB use of vasodilators such as PGI2 and NO to prevent post-CPB pulmonary hypertension. The inhaled form is especially effective due to pulmonary selectivity and prevention of hypotension, which could be deleterious in high-risk patients.
Limitations
The amount of inhaled PGI2 reaching the alveoli cannot be precisely determined due to drug deposition in the ventilator and orotracheal tubing. Data have demonstrated that there is less than 10% of alveolar deposition of drug during mechanical ventilation.26 However, the PGI2 concentration use in our study was comparable to other published studies12,26 with evident hemodynamic results. There was no inhalation of a placebo in the CPB group, so we cannot exclude a possible interference of CPAP on our results and the inhaled route of delivery may be responsible for the improvement in the A-a gradient. However, it has been previously demonstrated that a CPAP of 5 cm H2O does not prevent the post-CPB lung function impairment.27 Also, the PGI2 group has not been compared with an inhaled glycine group, the buffer vehicle used to transport PGI2 molecules. However, glycine has no hemodynamic and respiratory parameter influence and is not associated with major signs of acute pulmonary toxicity.28,29 Use of healthy swine in this study instead of a model with pre-CPB pulmonary hypertension could have minimized our results. Finally, a species specificity of swine intravascular macrophages cannot be excluded, which could limit the clinical relevance of the present study.
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Acknowledgments |
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