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Ann Thorac Surg 2006;82:131-137
© 2006 The Society of Thoracic Surgeons
Department of Cardiovascular and Thoracic Surgery, Xinhua Hospital Shanghai Children's Medical Center, Shanghai Second Medical University, Shanghai, People's Republic of China
Accepted for publication February 27, 2006.
* Address correspondence to Dr Yang, Biosystems, Institute for Biodiagnostics, National Research Council of Canada, 435 Ellice Avenue, Winnipeg, Manitoba, Canada R3B 1Y6 (Email: victor.yang{at}nrc-cnrc.gc.ca).
Presented at the Forty-second Annual Meeting of The Society of Thoracic Surgeons, Chicago, IL, Jan 30–Feb 1, 2006.
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Abstract |
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 Top Abstract IntroductionMaterial and MethodsResultsCommentSouthern Thoracic Surgical...DiscussionFootnotesAcknowledgmentsReferences |
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METHODS: Sixteen piglets underwent 90-minute deep hypothermia, either with circulatory arrest or low-flow perfusion (30 mL · kg–1 · min–1) at 18°C. Disaturated phosphatidylcholine, total phospholipids, and total proteins from tracheal aspirates were measured serially until the end of cardiopulmonary bypass. Lung static compliance, airway resistance, and arterial blood oxygen partial pressure to inspired oxygen fraction were also measured.
RESULTS: The DHLF caused more significant decrement of pulmonary static compliance than DHCA (3 ± 0.4 mL · cmH2O–1 vs 3.5 ± 0.3 mL · cmH2O–1 at 90 minutes of deep hypothermia). Arterial blood oxygen partial pressure to inspired oxygen fraction decreased more significantly after cardiopulmonary bypass in the DHLF group than the DHCA group (205 ± 84 mm Hg vs 302 ± 96 mm Hg). The DHLF caused more severe decrement of disaturated phosphatidylcholine total phospholipids (50% ± 21% vs 67% ± 23% of baseline at 90 minutes of deep hypothermia) and disaturated phosphatidylcholine total proteins (58% ± 23% vs 73% ± 23% of baseline at 90 minutes of deep hypothermia) than DHCA. More significant water retention developed in the lung in the DHLF group than in the DHCA group. The extent of surfactant depletion was statistically correlated with the extent of pulmonary functional deterioration in either group.
CONCLUSIONS: The DHCA induces less injury on pulmonary surfactant metabolism and pulmonary function than DHLF.
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Introduction |
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TopAbstractIntroductionMaterial and MethodsResultsCommentSouthern Thoracic Surgical...DiscussionFootnotesAcknowledgmentsReferences |
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Pulmonary surfactant lines the alveolar surface and reduces its tension at the air-liquid interface, thereby preventing alveolar collapse. This complex lipoprotein material is synthesized by alveolar type II cells and is mainly composed of phospholipids (80%) and surfactant-associated proteins (SP-A, SP-B, SP-C, and SP-D) [5]. The phospholipid moiety, chiefly involving disaturated phosphatidylcholine (DSPC; 
50%), is a critical determinant for surfactant surface activity [6]. Data on surfactant metabolism after deep hypothermic CPB with either strategy in neonates are insufficient. The currently available information regarding surfactant metabolism after cardiac surgery is derived mainly from older children and does not involve either DHCA or DHLF [7–9]. Therefore, this study was carried out to determine the effects of DHCA and DHLF on pulmonary surfactant integrity and pulmonary function with a piglet model.
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Material and Methods |
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TopAbstractIntroductionMaterial and MethodsResultsCommentSouthern Thoracic Surgical...DiscussionFootnotesAcknowledgmentsReferences |
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Surgical Preparations
All piglets were anesthetized with ketamine (22 mg/kg, intramuscular) and acepromazine (1.1 mg/kg, intramuscular) then intubated, and mechanically ventilated. Continuous pentobarbital infusion (20 mg/kg per hour), intermittent fentanyl citrate (10 µg/kg per hour), and pancuronium bromide (0.1 mg/kg per hour) were used throughout the experimental protocol to maintain deep general anesthesia and muscular relaxation.
The heart was exposed by median sternotomy. The CPB circuit consisted of a Sarns 8000 nonpulsatile roller pump (Sarns, Terumo CVS, Ann Arbor, MI) and a Medtronic Minimax Plus infant membrane oxygenator (Medtronic, Minneapolis, MN) with venous reservoir forming the CPB circuit. Circuit blood gases were monitored using a CDI-300 (CDI; 3M Healthcare, Irvine, CA) continuous in-line blood gas analyzer. A pediatric arterial filter (COBE Cardiovascular, Arvada, CO) was incorporated in the bypass circuit. The circuit was primed with fresh heparin-treated donor pig whole blood to maintain a hematocrit of approximately 25%. After the animals were given heparin (500 U/kg), an 8F arterial cannula and a 14F venous cannula were inserted in the ascending aorta and right atrium, respectively, for initiation of CPB. Pharmacologic agents for blood pressure controlling were not administered during CPB. Sodium bicarbonate was given, as necessary, to maintain the base excess between –3 and 3 mmol/L. Alpha-stat blood gas management was used throughout the experiment.
Experimental Groups and Protocol
Sixteen piglets were randomly assigned to two groups: the DHCA group (n = 8), hypothermic CPB followed by 90-minute circulatory arrest at 18°C and rewarming; and the DHLF group (n = 8), hypothermic CPB followed by 90-minute low-flow perfusion with 30 mL · kg–1· min–1 at 18°C and rewarming. The experimental protocol is shown in Table 1.
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Measurement of lung function
Measurement of lung static compliance (Cstat), airway resistance (Raw), and arterial oxygen partial pressure to inspiratory oxygen fraction (PaO
2/FIO
2) were detailed in our previous study [4]. Briefly, a Servo 900C ventilator (Siemens-Elema AB, Solna, Sweden) was used to assess Cstat and Raw. The measurement was carried out at the beginning of CPB, during hypothermic CPB at 25°C and 18°C, every 15 minutes during either DHCA or DHLF, during rewarming at 25°C and 37°C, as well as every 15 minutes during normothermic CPB. The peak inspiratory pressure and positive end-expiratory pressure were kept constant at 25 cm H2O and 2 cm H2O, respectively, while Cstat was assessed. Static compliance was measured as a tidal volume divided by the applied pressure (end-inspiratory plateau pressure minus end-expiratory pressure) and expressed in mL · cm H2O–1. A mean value of Cstat was calculated for a minimum of six breaths at each stage. Airway resistance is the inversion of Cstat divided by inspiration time and expressed in cm H2O · mL–1 · S–1. A blood sample from the left atrium was aspirated to determine the level of PaO
2. The PaO
2/FIO
2 was used to reflect pulmonary oxygenation capacity before and after DHCA or DHLF.
Chemical analysis of pulmonary surfactant
Tracheal aspirates containing pulmonary surfactant were suctioned using 10 mL normal saline at room temperature. The timing set for aspiration is at the beginning of CPB, at the end of hypothermic CPB, every 30 minutes during DHCA or DHLF, and at the end of normothermic CPB. Aliquots of aspirates were extracted with threefold volumes of chloroform-methanol (2:1, vol/vol) to isolate the phospholipids in the chloroform phase. Disaturated phosphatidylcholine (DSPC), the major surface-tension lowering component of lung surfactant, was separated from other phospholipids as described by Mason and colleagues [10]. Briefly, samples from the chloroform phase were dried under nitrogen gas, oxidized with a small volume of osmium tetroxide in carbon tetrachloride for 15 minutes, and dried again under nitrogen, dissolved in chloroform-methanol (20:1, vol/vol), and passed through a neutral aluminum column. The DSPC fraction was collected by adding a mobile phase of chloroform-methanol-7M ammonium hydroxide (70:30:2, vol/vol/vol) to the column. Amounts of DSPC and total phospholipids (TPL) were determined according to the methods described by Bartlett [11] and corrected by the total volume of the aspirates. Values for TPL are presented as mg/kg, and those for DSPC as a percentage of TPL (DSPC/TPL), a parameter reflecting the extent of surfactant change due to injurious factors. Total proteins (TP) in the aspirates were measured according to the method of Lowry and colleagues [12], using bovine serum albumin as the standard, and corrected by the total volume of the aspirated fluid; these values are presented as milligrams per kilogram (mg/kg). The DSPC and TP ratio (DSPC/TP) was expressed as micrograms per milligram (µg/mg). This ratio has been indicated to mainly reflect the balance between alveolar surfactant and plasma proteins leaking into the airspaces as a consequence of vascular injurious factor including CPB [13–16].
Myeloperoxidase activity
The activity of myeloperoxidase, an enzyme located almost exclusively in neutrophils, was measured in lung tissue as a representative indicating the extent of neutrophil infiltration. After homogenization of the frozen lung tissue samples (50 mg) in 0.5% hexadecyltrimethylammonium bromide dissolved in 10 mmol/L 3-[N-morpholino] propane sulfonic acid, centrifugation was set at 21,000g for 20 minutes at 4°C. The supernatant was mixed with sodium phosphate (80 mmol/L, pH 5.5) and tetramethyl benimide (16 mmol/L) and incubated at 25°C for 5 minutes. After hydrogen peroxide (1 mmol/L) was added, the samples were incubated exactly 3 minutes at 25°C. A blank without hydrogen peroxide was also analyzed for each tissue. The reaction was stopped by adding aliquots of cold acetic acid (2M). The optical density was measured at 650 nm on a spectrophotometer. Myeloperoxidase activity was the quantity of enzyme degrading 1 µmol hydrogen peroxide per minute at 37°C.
Wet-to-dry lung weight ratio
A piece of lung tissue (about 1 g) from the posterior part of the left lower lobe was cut and its wet weight determined in an automatic electric balance (AP250D, Ohaus, Florham, NJ). The piece of lung tissue was then dried in an oven at 80°C for 48 hours and weighed again to obtain its dry weight for calculation of the wet-to-dry weight ratio.
Statistical Analysis
Statistical analysis was performed using the statistical analysis system (SAS Institute, Cary, NC). All data are presented as mean ± standard deviation. Two-way repeated measures analysis of variance (ANOVA) was used to test for significant differences in the effect of (1) bypass modes, (2) time, and (3) interaction of bypass modes and time. The Bonferroni multiple comparison post-test was used to assess significant differences in the effect of bypass modes at different time points. The Pearson test was used for correlation study between changes of pulmonary surfactant and lung function. A p value less than 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterial and MethodsResultsCommentSouthern Thoracic Surgical...DiscussionFootnotesAcknowledgmentsReferences |
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Wet-to-Dry Lung Weight Ratio
Averaged ratios of the wet-to-dry lung weight ratio for each of the two groups were 5.9 ± 1.9 in the DHCA group and 7.4 ± 2.1 in the DHLF group. The differences were statistically significant (p = 0.04).
Relationship of Pulmonary Surfactant to Lung Function
The correlation coefficients between pulmonary surfactant in the tracheal aspirates and lung function were determined separately for the two groups (Table 2). In the DHCA group, significant correlations were noted between DSPC/TPL or DSPL/TP and Cstat or PaO
2/FIO
2. Similarly, significant correlations were also obvious between pulmonary surfactant and Cstat or PaO
2/FIO
2 in the DHLF group.
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Comment |
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TopAbstractIntroductionMaterial and MethodsResultsCommentSouthern Thoracic Surgical...DiscussionFootnotesAcknowledgmentsReferences |
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Recent studies have indicated that CPB could alter the pulmonary surfactant system including changes in the DSPC, TPL, and its protein fractions and may result in impaired surface tension properties of the surfactant isolates [7–9]. Consistent with these studies, we noted a decrease in both DSPC/TPL and DSPC/TP from the tracheal aspirates during either DHCA or DHLF.
Also, compared with DHCA, DHLF caused more intense deterioration of pulmonary surfactant. In this study, the mean exposure time of the lung to perfusate was approximately 120 minutes for piglets in the DHCA group and approximately 210 minutes for piglets in the DHLF group. We believe that this mainly was the result of the much longer duration of exposure of the lung tissue to the perfusate during DHLF. Although the main objective of DHLF is to provide blood to the brain, it has been proven that the flow toward the brain is limited [21]. Other organs, including the lung, remain perfused during the period of low-flow perfusion under deep hypothermia. It has been speculated that the maintenance of more or less blood perfusion to respective important organs, including the lung, could reduce the extent of ischemic injury to these organs and, consequently, serve as a rationale for global low-flow perfusion rather than regional cerebral low-flow perfusion. However, our results indicated that this might not be the case during deep hypothermia in terms of its influence on the lung tissue per se.
Similar to adults undergoing cardiac surgery, CPB will also incur strong systemic inflammatory response in children [22, 23]. The intensity of inflammation is, to some extent, related to the degree of lung injury during CPB, as evidenced by reduction of postoperative pulmonary dysfunction by the strategies aimed at inhibiting systemic inflammatory response, especially neutrophils sequestration in the lung tissue [24–27].
Moreover, Tassani and colleagues [28] demonstrated that DHLF, in contrast to DHCA, would cause more significant systematic inflammatory response in newborns. In that clinical research, they found that after rewarming, the concentration of complement 3A in the perfusate was significantly lower in the DHCA group than in the DHLF group. The interleukin-8 level was significantly lower, and the interleukin-6 level had a tendency to be lower in the DHCA group compared with levels in the DHLF group. There was less weight gain on the first postoperative day in the DHCA group due to reduced edema formation in the DHCA group. Our results indicated that, at the end of rewarming, sequestration of neutrophils in the lung tissue tended to be more significant in the DHLF group than in the DHCA group, as reflected by the myeloperoxidase activity in the lung. In addition, significantly more water retention was found in the DHLF group.
In the current study we demonstrated that DHLF, compared with DHCA, resulted in more intense pulmonary injury characterized by more significant depletion of pulmonary surfactant and deterioration of lung function. More significant systemic inflammatory activation, as reflected by more neutrophil sequestration in the lung in the DHLF group, is likely the major reason for this discrepancy.
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Southern Thoracic Surgical Association: Fifty-Third Annual Meeting |
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TopAbstractIntroductionMaterial and MethodsResultsCommentSouthern Thoracic Surgical...DiscussionFootnotesAcknowledgmentsReferences |
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Manuscripts accepted for the Resident Competition must be submitted to the STSA headquarters office no later than September 15, 2006. The Resident Award will be based on abstract, presentation, and manuscript.
Applications for membership should be completed be September 15, 2006, and forwarded to Chairman of Membership Committee, Southern Thoracic Surgical Association, 633 N Saint Clair St, Suite 2320, Chicago, IL 60611-3658.
| Please visit the STSA (http://www.stsa.org) or CTSNet (http://www.ctsnet.org) websites for detailed information on submitting abstracts. All abstracts must be submitted electronically for consideration.
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Discussion |
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TopAbstractIntroductionMaterial and MethodsResultsCommentSouthern Thoracic Surgical...DiscussionFootnotesAcknowledgmentsReferences |
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DR YANG: Oh, we inserted a left ventricle vent.
DR PIGULA: And I guess that brings up the question in my mind, is whether or not this phenomena is related to a washout effect or if there is any kind of washout of the surfactant or the surfactant-related analogs into the vent and whether or not that can be measured?
DR YANG: Do you mean the drainage?
DR PIGULA: Whether the venting and the removal of that blood.
DR YANG: We also considered this problem. As a result, every time we injected 10 mL normal saline into the trachea, and then we tried to aspirate the same amount out. Also, we compared the ratio of the disaturated phosphatidylcholine to the total phospholipid in the aspirates. Although we diluted it, the ratio should remain the same, that's the reason why we used the ratio.
In addition, to further reduce the within-subject variation among different time points, we set the initial results measured as 100 percent and normalized all the later measurements to it.
DR PIGULA: I understand. I'm just wondering, with hypothermic circulatory arrest the circulation is stopped, there is no drainage of the heart, there is no pulmonary venous return. I think even with low flow bypass, there is still some pulmonary blood flow even if it's based on the bronchial circulation and there will still be pulmonary venous return.
DR YANG: Yes.
DR PIGULA: So my question is whether that low level of perfusion has any impact on either surfactant generation or degradation. Because I think if you have a difference, it's accounted by either decreased generation or increased degradation of the surfactant.
DR YANG: At first we also anticipated that the low-flow perfusion during deep hypothermia might maintain better surfactant integrity as there was more or less perfusate into the lung. However, contrary to this expectation, we did find that low-flow perfusion caused even worse surfactant integrity. As a result, it seemed that the negative influence outweighed the beneficial effects provided by the incessant low-level circulation.
DR CHRISTOPHER A. CALDARONE (Toronto, Ontario, Canada): Frank, are you alluding to the potential for multiple insufflations of saline into the airway in one group to cause an artifact due to washing out an important factor in one group and not washing it out in the other group?
DR PIGULA: Well, no. Really, my point was whether or not if there is active venting in the heart, whether or not the removal of the pulmonary venous return, or there is blood flow through the lungs, whether that has any effect on either removal of surfactant or any impact on surfactant levels in the lung. Because, really, that's a fundamental difference here in your model. In one there is circulation, even though it's low level circulation, and in the other there is no circulation. And whether the fact that there is that ongoing circulation impacts the surfactant levels that you will measure in the lung.
DR YANG: From what I could understand, the low-level circulation in the lung during the whole body low-flow perfusion did lead to worse surfactant integrity, which outweighed the beneficial effects of nutrients and oxygen provision. The potential explanation, we believed, is the more significant inflammation found inside the lung in this group after perfusion.
DR WILLIAM M. DECAMPLI (Orlando, FL): I think we all are concerned, and should be concerned about pulmonary function during the variety of ways that we conduct both cardiopulmonary bypass as well as ischemic arrest. The emphasis now, and certainly today and in during's talks, have been placed on brain protection. We know that we've all thought for decades now about cardiac protection. But the lung in the infant and neonate is equally important in our ability to get a good postoperative recovery. You spoke a little bit about the mechanisms by which you thought surfactant is depleted. I'm just wondering if you have given consideration, perhaps in the next step in your studies, to actually look at the alveolar histology, maybe do some cellular functional studies, to determine exactly what it is that may be going on here.
My second question relates to whether you think that since a lot of the dysfunction is a mechanical dysfunction here, that whether intermittent ventilation of the lungs during cardiopulmonary bypass and(or) circulatory arrest might improve the situation? I know that there have been a couple papers, and in particular one paper in the past 18 months, I can't remember the source, but looked at tetralogy of Fallot repairs and compared groups that had had ventilation during the repair versus no ventilation. There was a significant improvement in early recovery of lung function with intermittent ventilation. So I thought if you could comment on that.
DR YANG: We have begun our histologic study to view the difference between the two perfusion modes, esp., the extent of the inflammation. We also wanted to know the integrity of the whole alveolar membrane. Histological results have revealed that low flow perfusion caused more severe damage in the integrity of the alveolar epithelial layer. As a result, depletion of surfactant may be the result of injured alveolar epithelial layer.
In addition, we also paid special attention to recent achievements in this field of research, including effect of the ventilatory modes. Our pilot results indicated that continuous ventilation using low frequency and tidal volume might be beneficial for patients, but more detailed study is required.
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Acknowledgments |
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TopAbstractIntroductionMaterial and MethodsResultsCommentSouthern Thoracic Surgical...DiscussionFootnotesAcknowledgmentsReferences |
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Footnotes |
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TopAbstractIntroductionMaterial and MethodsResultsCommentSouthern Thoracic Surgical...DiscussionFootnotesAcknowledgmentsReferences |
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References |
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TopAbstractIntroductionMaterial and MethodsResultsCommentSouthern Thoracic Surgical...DiscussionFootnotesAcknowledgmentsReferences |
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  Y. Yang, Z. Su, J. Cai, S. Wang, J. Liu, Z. Xu, and W. Ding Continuous Pulmonary Infusion of L-Arginine During Deep Hypothermia and Circulatory Arrest Improves Pulmonary Surfactant Integrity in Piglets Ann. Thorac. Surg., August 1, 2008; 86(2): 429 - 435. [Abstract] [Full Text] [PDF]
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) and DHLF (
) groups. Data are means ± SD of 8 piglets in each group. (A) Static pulmonary compliance (Cstat) levels. (B) Gas exchange (Pa
O
2
/F
IO
2
) levels (white bars = DHCA; vertically lined bars = DHLF). (* p < 0.05, ** p < 0.01, two-way repeated measures ANOVA with Bonferroni multiple comparison post-test. CA = circulatory arrest; CPB-b = baseline levels obtained during initial cardiopulmonary bypass (CPB); DHCA = deep hypothermia with circulatory arrest; DHLF = deep hypothermia with low-flow; LF = low-flow perfusion; NS = not significant; Temp. = temperature.