Ann Thorac Surg 1999;68:499-504
© 1999 The Society of Thoracic Surgeons
Original Articles
Effects of cyanosis and hypothermic circulatory arrest on lung function in neonatal lambs
Mitsugi Nagashima, MDa,
Ulrich Stock, MDa,
Georg Nollert, MDa,
Jason Sperling, MDa,
Dominique Shum-Tim, MDa,
Shinichi Hatsuoka, MDa,
John E. Mayer, Jr, MDa
a Department of Cardiovascular Surgery, Children’s Hospital and Harvard Medical School, Boston, Massachusetts, USA
Address reprint requests to Dr Mayer, Department of Cardiovascular Surgery, Children’s Hospital, 300 Longwood Ave, Boston, MA 02115
Presented at the Thirty-fifth Annual Meeting for The Society of Thoracic Surgeons, San Antonio, TX, Jan 25–27, 1999.
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Abstract
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Background. Lung function is often impaired after cardiac surgery and cardiopulmonary bypass (CPB), particularly in chronically cyanotic patients. This study aimed to evaluate lung function in a surgically created chronic cyanotic neonatal lamb model after CPB and deep hypothermic circulatory arrest (DHCA) and to assess the role of nitric oxide (NO) in the pathogenesis of increased pulmonary vascular resistance.
Methods. A chronic cyanosis model was surgically created in 7 lambs (4.7 ± 0.8 days old) by anastomosing the pulmonary artery (PA) to the left atrium (LA). Another 7 lambs underwent a sham operation (control). One week later, the animals underwent shunt takedown and CPB with 90 minutes of DHCA at 18°C. Cardiac index (CI), pulmonary vascular resistance index (PVRI), lung dynamic compliance (Cdyn), alveolar-arterial oxygen difference (AaDO2), left atrial plasma nitrate/nitrite (NO metabolites) levels, and pulmonary cGMP production (concentration difference between LA and PA) were measured before CPB and at 1 and 2 hours after reperfusion.
Results. The cyanosis model consistently produced significantly lower arterial oxygen tension (34.8 ± 2.3 vs 93.1 ± 8.8 torr in control, p < 0.001) and Qp/Qs (0.6 ± 0.1 vs 1.0 ± 0.0 in control, p < 0.001) than controls. Postoperative PVRI was significantly lower in the cyanosis group than in controls, although CPB with DHCA significantly elevated PVR in both cyanotic and control animals. There were no significant differences in AaDO2 and Cdyn after CPB between groups. The level of NO metabolites did not change before or after CPB in either cyanotic or acyanotic animals. NO metabolite levels tended to be higher in the cyanotic animals (p = 0.08). There was no significant difference in pulmonary cGMP production between both groups.
Conclusions. These findings suggest that CPB with DHCA, per se, does not affect NO production in cyanotic or acyanotic neonatal lambs but causes increased PVR in both groups. Chronic cyanosis does not result in reduced pulmonary function after CPB with DHCA, and is associated with lower PVR. The mechanism may involve an increased NO production in cyanotic animals.
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Introduction
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Cardiopulmonary bypass (CPB) causes increased postoperative pulmonary vascular resistance (PVR) [1–3]. Impaired production of nitric oxide (NO), a key mediator of vasodilation, may be involved in increased PVR postoperatively. NO metabolites significantly decrease after 120 min of CPB, and the reduction of NO was associated with pulmonary vasoconstriction in a piglet model [4]. However, Bando and associates [5] reported that NO metabolites actually increased after CPB in children who underwent cardiac surgery. Thus, the relationship between CPB and NO production is still controversial. Moreover, although cyanosis is quite common in pediatric cardiac surgery, the influence of existing cyanosis on lung function after CPB is still unknown. Therefore, the current study examined how CPB affects PVR and NO production, and investigated the effect of chronic cyanosis on NO production and lung function after CPB with deep hyothermic circulatory arrest (DHCA) in a neonatal lamb model [6]. We also measured cyclic guanosine 3,5-monophosphate (cGMP), which is a second messenger of NO, as an indicator of NO production [7].
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Material and methods
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Creation of cyanosis
Fourteen neonatal lambs were sedated with an intramuscular injection of ketamine hydrochloride (50 mg/kg). After intubation, the animals were maintained by inhalation anesthesia with isoflurane. A left thoracotomy was performed through the third intercostal space. The ductus arteriosus was ligated after the pericardium was entered. A chronic cyanosis model was created in 7 lambs by an anastomosis between the pulmonary artery (PA) and the left atrium (LA). Two partial side-biting clamps were placed on the left PA and on the LA. The length of the longitudinal incision on the PA was matched to the estimated diameter of the left pulmonary artery (5–7 mm). Then, the LA was anastomosed to the PA using a continuous 7-0 monofilament suture. Another 7 lambs (control) underwent a sham operation that did not include the PA to LA anastomosis but did include 20 minutes of side clamp of the PA and the LA as well as PDA ligation. The animals were given intramuscular injection of antibiotics during the procedure and at 12 hours after the procedure, and buprenorpine hydrochrolide (0.01 mg/kg) was used to minimize discomfort and pain at 8-hour intervals for 48 hours after the procedure. All animals in both groups survived for 7 days and underwent the second surgical procedure.
Deep hypothermic circulatory arrest study
Seven days after the first surgery, the lambs were sedated with an intamuscular injection of ketamine hydrochloride (50 mg/kg). After intubation, the animals were artificially ventilated by a volume-controlled ventilator (Servo Ventilator 900C; Siemens-Elema, Danvers, MA) with a tidal volume of 20 mL/kg and 4 cm H2O PEEP (positive end-expiratory pressure) under 100% of inspired oxygen fraction (FiO2) during the experiment. After a bolus intravenous infusion of 300 µg/kg of fentanyl, anesthesia was maintained by continuous intravenous infusion of ketamine (5 mg/kg/h), midazolam (0.2 mg/kg/h), pancronium (0.1 mg/kg/h), and fentanyl (50 µg/kg/h) throughout the experiment. A 19-gauge catheter was inserted via the left femoral artery and passed into the thoracic aorta for measurements of systemic arterial pressure and for blood gas sampling. Through a median sternotomy, the heart was exposed. A 5 F side-holed catheter (Berman angiographic catheter; Arrow International Inc, Reading, PA) was inserted to measure pulmonary arterial pressure. After systemic heparinization, a 5 F polyurethane sheath was inserted into the LA for pressure measurement and blood sampling. A micromanometer catheter (SPC-350; Millar Instruments Inc, Houston, TX) was placed into the left ventricular (LV) cavity through the apex to measure LV pressure. Two pairs of ultrasonic transducers (Sonometrics, London, Canada) were implanted into the LV midmyocardium to measure the long- and short-axis dimensions of the LV.
Cardiopulmonary bypass and post-CPB management
The circuit for CPB consisted of a roller pump and a membrane oxygenator (VPCML; COBE Laboratories, Arvada, CO). The pump prime consisted of 200 mL of Normosol R (Abbott Laboratories, North Chicago, IL) and 500 mL of homologous, heparinized fresh donor blood to achieve a hematocrit of 20%. An 8 F arterial cannula and a 24 F venous cannula were placed into the right femoral artery and right atrium, respectively. Immediately after CPB was started, the PA-LA shunt was clamped in the cyanosis group. The animals were cooled to 18°C over 30 minutes using a pH stat strategy. CPB flow was maintained at 150 mL/kg/min. The esophageal temperature was kept at 18°C using a temperature-controlled blanket during the 90 minutes of DHCA. The patent foramen ovale was closed through a right atriotomy during DHCA. The animals were rewarmed on bypass to achieve a rectal temperature of 38°C over 40 minutes. The heart was defibrillated as necessary at a temperature of 32°C. The animals were then weaned from bypass and observed for 2 hours after reperfusion. The esophageal temperature was kept at 37°C after the termination of CPB. Protamine sulfate was added for neutralization of heparin. Sodium bicarbonate was added when the base deficit exceeded -3.0 mmol/L, and the hematocrit was kept at 20% during CPB and between 23% and 25% after CPB. No inotropic agents or vasodilators (including NO donors) were used. Normosol-R containing 5 g/L of glucose and homologous blood were used for volume repletion.
Data analysis and measurements
Qp/Qs was estimated using the following formula:
where Sat O2 is the oxygen saturation at various positions. It was assumed that O2 saturation in the pulmonary vein (PV) was 98% under 21% oxygen and 100% under 100% oxygen. LV volume was calculated by the ellipsoidal model:
where DL is long-axis length and DS is short-axis length obtained by ultrasonic transducers. Cardiac index (CI) was calculated by:
where, Ved is end-diastolic LV volume and Ves is end-systolic LV volume. Body surface area (BSA) was calculated by this formula [8]:
Lung function: Pulmonary vascular resistance index (PVRI) was calculated using the formula: PVRI (woods x units) = [mean PA pressure (mm Hg) - mean LA pressure (mm Hg)]/Op (L/min/m2)
Before bypass, Qp in the cyanosis group was calculated by Qp = Qp/Qs x C.I. Lung dynamic compliance (Cdyn) was calculated with the lung mechanics calculator (Servo 940; Siemens-Elema) according to the formula: Cdyn (mL/cm H2O) = expiratory tidal volume (mL)/pause pressure (cm H2O) - end expiratory lung pressure (cm H2O)]
Cdyn was measured at respiratory rate of 25/min to eliminate influences by the respiratory rate. Alveolar - arterial oxygen tension difference (AaDO2) was calculated using the following equation: AaDO2 (mm Hg) = [1 x (760 - 47) - PACO2/R] - measured PaO2
where PACO2 is alveolar carbon dioxide tension, R is the respiratory exchange ratio, and PaO2 is arterial oxygen tension. It was assumed that R = 0.85 and PACO2 was the same as arterial carbon dioxide.
Serial blood samples were taken from the LA and the PA line before CPB and at 60 and 120 minutes of reperfusion. Total plasma concentrations of NO2- and NO3- nitric oxide metabolites, were measured by modified Griess reaction (nitrate/nitrite colorimetric assay kit; Cayman Chemical, Ann Arbor, MI). Cyclic GMP was measured by competitive enzyme immunoassay (cGMP immunoassay kit; Cayman Chemical).
Animals in this study received humane care in compliance with "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication no. 86-23, revised in 1985).
Statistics
All values are expressed as mean ± standard error (SEM). Data were compared using the two-tailed unpaired Student’s t test or analysis of variance (ANOVA). A p value less than 0.05 was considered significant.
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Results
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Validation of cyanosis
One week after the surgical creation of cyanosis, the cyanosis model consistently produced significantly lower arterial oxygen tension, arterial oxygen saturation, and Qp/Qs than control at room air (Table 1).
Baseline measurements
There was no significant difference in cardiac index and lung function between groups with regard to baseline data before CPB (Table 2).
Cardiac index
There was no significant difference in cardiac index between groups after CPB (Fig 1).
Lung function
PVRI after CPB was significantly increased in both groups compared with pre-CPB (p < 0.05). PVRI was less in the cyanosis group at 60 and 120 minutes of reperfusion than in controls (Fig 2). There was no significant difference in AaDO2 and Cdyn after CPB between the two groups, although Cdyn in the cyanosis group had a tendency towards a worse recovery (Figs 3 and 4).
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Fig 2. The change in pulmonary vascular resistance index before and after CPB. Data are expressed as mean ± standard error. *p < 0.05 compared with control.
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Plasma nitrate/nitrite and cGMP level
Total concentrations of nitrate/nitrite were not significantly different before and after CPB in both groups, and PA and LA concentrations were not significantly different (data not shown). Repeated measure ANOVA revealed a tendency to higher NO metabolite levels in the cyanotic animals (p = 0.08) than acyanotic animals throughout the experiments. LA plasma NO metabolites in the cyanosis group were significantly higher than in controls at 1 hour after reperfusion (Fig 5). The cGMP levels decreased in both LA and PA samples at 1 hour of reperfusion and increased in the LA, but further decreased in the PA at 2 hours of reperfusion. Consequently, the pulmonary cGMP production (concentration difference from LA to PA) significantly increased at 2 hours of reperfusion compared with the pre-CPB value or at 1 hour of reperfusion (Fig 6). There was a weak but significant (p < 0.05) correlation between PVRI and NO metabolite levels but no correlation between PVRI and cGMP production (Fig 7).
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Fig 6. Pulmonary cGMP production (concentration difference from the LA to the PA) before and after CPB.
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Fig 7. (A) Plot chart showing the significantly inverse relationship between pulmonary vascular resistance index and NO metabolite levels (R = 0.36, p <0.05). (B) Plot chart showing no significant correlation between pulmonary vascular resistance index and cGMP production.
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Comment
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The current study revealed that CPB with DHCA did not significantly affect NO production in either cyanotic or acyanotic neonatal lambs. PVRI increased in both groups after CPB compared with pre-CPB values. However, cyanotic animals had a lower PVR associated with higher NO production than acyanotic animals.
CPB causes pulmonary vasoconstriction, but its precise mechanism has not been clarified. Increased postoperative PVR may result from an imbalance between vasoconstrictors and vasodilators. Several researchers suggested that an increase of endothelin-1 and an inadequate NO production after CPB could play an important role in an increased PVR [5, 9]. Impaired production of NO, which is a key vasodilator, may be connected with an increased postoperative PVR. Morita and associates [4] showed that CPB caused pulmonary hypertension and reduced pulmonary NO production by 70% after 120 minutes of CPB in a piglet model. They inferred that an impairment of NO production after CPB was due to endothelial cell dysfunction or inactivation of NO by free radicals. However, it still remains controversial whether CPB causes decreased NO production, especially in a clinical setting. Seghaye and associates [10] demonstrated that NO metabolite concentration of peripheral arterial plasma was not significantly different between pre-CPB values and post-CPB values in children who underwent open heart surgery. Bando and associates [5] also found that peripheral arterial plasma NO metabolites increased significantly for at least 6 hours after CPB in children with both pulmonary low flow and low pressure (eg, tetralogy of Fallot) and pulmonary high flow and high pressure (eg, ventricular septal defect and truncus arteriosus). Our current study revealed that the level of left atrial NO metabolites did not significantly change after CPB in either cyanotic or acyanotic neonatal animals. These discrepancies in findings from studies of NO production after CPB may depend on a variety of factors including the duration of CPB, temperature, age, the presence of preoperative heart failure (which has been reported to increase plasma NO [11, 12], the site and the timing of blood collection, and especially the use of exogenous NO donors or other vasodilators. The process of NO production is not simple, particularly in a case where inflammation is involved. NO is produced by the endothelium through constitutive NO synthase (cNOS) under physiologically normal conditions and contributes to basal vasoregulation [13]. However, with inflammation, NO is produced not only by endothelium but also myocardium [14] and inflammatory cells like neutrophils and macrophages through inducible NO synthase (iNOS) [15]. CPB activates inflammatory reactions and probably causes increased NO production due to over-expression of iNOS. We did not define the site or isoforms of NOS responsible for the NO production in this study.
The relationship between CPB and plasma cGMP level is also poorly described. NO activates guanylate cyclase in vascular smooth muscle, increasing intracellular levels of cGMP, which induces vascular relaxation. Increased intracellular cGMP also causes a release of cGMP into blood. Therefore, the level of plasma cGMP has been used as an indicator of NO production [7] because it is very difficult to measure NO itself directly due to its short half-life. This study demonstrated that pulmonary cGMP production did not change at 1 hour of reperfusion, and significantly increased at 2 hours of reperfusion compared with pre-CPB values in both cyanotic and acyanotic animals. However, the level of NO metabolites was comparable with pre-CPB values in both cyanotic and acyanotic animals. The level of NO metabolites in the LA plasma did not change before or after CPB. Moreover, there was a weak but significant correlation between PVR and plasma NO metabolite levels, but no significant correlation between PVR and pulmonary cGMP production. These findings may suggest that cGMP is not a specific indicator for NO production in an in vivo setting and especially after CPB. The dissociation of cGMP level from the NO metabolite concentration during reperfusion period may be accounted for by the release of atrial natriuretic peptide (ANP). ANP, which is released from atrial myocardium by stimulation of a stretch, also has been reported to increase plasma cGMP level [10]. It has been demonstrated that the plasma ANP level increases a few hours after CPB [10]. In our model, as RA and LA pressure significantly increased after CPB, ANP may have been released from both atria and caused an increase of the pulmonary cGMP production in the late reperfusion period.
The present study revealed that in cyanotic neonatal lambs, a lower PVR was found after CPB with DHCA than in acyanotic animals. Somewhat higher levels of NO metabolites were observed in the cyanosis group compared with controls. Although the underlying mechanism still remains unknown, a possible explanation for our finding of higher NO metabolites in the chronic cyanosis group is that chronic hypoxia may activate cNOS in endothelium in the lung. Scarborough and associates [16] reported that alveolar hypoxia increased cNOS expression in the lungs in a piglet model. Le Cras and associates [17] also found that alveolar chronic hypoxia caused an increase of expression of cNOS and iNOS in rat lungs, although it is unclear whether alveolar hypoxia is relevant to cyanosis due to an intracardiac right to left shunt. We speculate that an increased amount of endothelium could result from collateral vessel development or that increased cNOS or iNOS expression in the pulmonary endothelium may lead to higher NO production, although it is unclear how much angiogenesis is induced by 1 week of cyanosis.
Cardiopulmonary bypass, which temporarily interrupts and reestablishes pulmonary artery flow, can produce ischemia-reperfusion injury in the lung, characterized by endothelial cell injury. The higher NO production in the lung vasculature could cause worse recovery of alveolar gas exchange and compliance in cyanotic animals because NO can react with super oxide anion (O2-) to generate the cytotoxic species, peroxynitrite (OONO-) [18]. Peroxynitrite may cause lung dysfunction, but AaDO2 and lung compliance were only slightly worse in cyanotic animals than acyanotic animals in our study, and the differences did not reach statistical significance. The hypothesis that reoxygenation of cyanotic animals would produce severe organ dysfunction by formation of peroxynitrite was not substantiated in this model.
In summary, a chronically cyanotic neonatal model was established by a surgical creation of a right to left shunt through an anastomosis of the pulmonary artery to the left atrium. This surgical cyanotic model is consistent and useful for future research to investigate the effect of CPB or ischemia/reperfusion in conditions of chronic cyanosis. Although the cyanosis was maintained for 1 week, it may be argued that the model is not truly a "chronic" cyanosis model. However, the establishment of cyanosis in the first few days of life during the transition from a fetal to a normoxic circulation is similar to that encountered clinically in neonates who then undergo neonatal repair. CPB with DHCA, per se, did not affect pulmonary NO production in either chronically cyanotic or acyanotic animals but did cause increased PVR in both groups. In addition, cyanotic animals had a lower PVR that was associated with a tendency to higher NO metabolite levels. To clarify the precise underlying mechanism by which chronic cyanosis has a tendency to cause higher NO production after CPB than acyanotic animals, further investigation, especially the site or isoform responsible for NO production, will be required. However, the role of NO in the changes in PVR associated with CPB/DHCA does not seem to be dominant in this model.
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Abstract
Introduction
