Extracorporeal life support (ECLS) devices, such as the cardiopulmonary bypass (CPB), preserve life by providing adequate oxygen supply and blood flow to vital organs.1 However, cardiac surgery using CPB is often accompanied by a systemic inflammatory response, which significantly influences morbidity and mortality after CPB.2
The inflammatory response arises during CPB because of endotoxemia and interactions between blood and large artificial surfaces.3 Protein adsorption initiates interactions between blood and artificial surfaces. A series of chain reactions causes the formation and release of numerous powerful inflammatory mediators, including hormones and autacoids.4
The current study compares inflammatory responses in a rat model of CPB using a new small circuit and a relatively conventional large circuit.5–7 We tested the hypothesis that the small CPB circuit with reduced PV and blood contact with surfaces would attenuate the systemic inflammatory response and reduce levels of inflammatory cytokines as well as organ tissue damage. We therefore compared levels of serum CD11b, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10) and the biochemical markers lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) between groups of rats given CPB with the new small or the conventional large circuit. The lung wet-to-dry weight (W/D) ratio was also measured as an index of pulmonary edema.
Materials and Methods
The Animal Care and Use Committee at the National Cerebral and Cardiovascular Center Research Institute approved this study, and all procedures met the National Institutes of Health guidelines for animal care.
Sprague-Dawley rats (male 400–450 g) were housed (n = 3 per cage) under a 12 h light–dark cycle with food and water available ad libitum.
Anesthesia, Surgical Preparation, and Cardiopulmonary Bypass
The animals were anesthetized with isoflurane (5%) in oxygen-enriched air, placed in the supine position and then a rectal thermocouple was inserted. The rats were orotracheally intubated using a 14G cannula (Insyte BD Medical, Sandy, UT) and ventilated with a Model 687 respirator (Harvard Apparatus Ltd., Edenbridge, Kent, UK). Ventilation was volume-controlled at a frequency of 70/min, a tidal volume of 8–10 ml/kg body weight and 40% inspired oxygen fraction. Anesthesia was maintained with 2.0–2.5% isoflurane and the rectal temperature was maintained at 36°C throughout the study. Arterial blood pressure was monitored using a Model 870 PowerLab system (AD Instruments, Castle Hill, NSW, Australia) via the femoral artery, which was cannulated with SP-31 polyethylene tubing (Natsume Seisakusho Co. Ltd, Tokyo, Japan). The left common carotid artery was cannulated with SP-55 polyethylene tubing (Natsume Seisakusho) to serve as the arterial return cannula for the CPB circuit. Heparin sodium (500 IU/kg) was then administered through this cannula. A 16 G cannula (Togomedkit Co. Ltd, Tokyo, Japan) was advanced through the right external jugular vein into the right atrium to serve as a conduit for venous uptake.
The CPB circuit comprised a membranous oxygenator (Senko Medical Co. Ltd., Osaka, Japan), tubing (Senko Medical) and a Micro tube MP-3 roller pump (Tokyo Rikakikai Co. Ltd., Tokyo, Japan). The total priming volumes of the conventional large and new small circuits were 15 and 7 ml, respectively, and surface areas of 0.044 and 0.034 m2, respectively, were in contact with blood (Figure 1). The large and small CPB circuits were primed with 14 and 6 ml, respectively, of acetate Ringer’s solution and 1 ml (1,000 IU) of heparin each.
The rats were assigned to groups that underwent a SHAM operation, or CPB with high and low PV (n = 7 each). The SHAM group underwent surgical preparation without CPB. The rats were systemically anticoagulated using 500 IU/kg heparin. The target activated coagulation time was 250–300 seconds and CPB was initiated and maintained at 70 ml/kg/min. The arterial pressure of carbon dioxide (PaCO2) and of oxygen (PaO2) was maintained at 35–45 and 300–400 mm Hg respectively, in all groups. Blood samples were collected before (pre-CPB), and at 60 and 120 min after starting CPB (end-CPB).
The CD11b positive activation ratio was measured using CD11b FITC flow cytometry kits (BD Pharmingen, Franklin Lakes, NJ). Levels of TNF-α, IL-6, and IL-10 were measured using Quantikine enzyme-linked immunosorbent assay (ELISA) kits (R&D systems, Minneapolis, MN) to evaluate inflammatory responses.8 Plasma concentrations of LDH, AST, and ALT served as biochemical markers of organ damage9 and were measured by automated colorimetry using a DRI-CHEM 7000 Analyzer (FUJIFILM, Kanagawa, Japan).
Blood gases, pH, hemoglobin (Hb) concentrations, and electrolytes were measured using an ABL800 FLEX system (Radiometer, Copenhagen, Denmark). Rats in which the Hb level declined to <7 g/dl at any point were excluded from the study. All rats were sacrificed at the end of CPB by an intracardiac injection of potassium chloride and then the left lung was harvested from each rat and divided into three parts. The W/D ratio was calculated in the superior third. The lung block was weighed before and after desiccation for 72 h in a dry oven at 70°C.
All data are expressed as means ± standard error (SE). Groups were compared using the analysis of variance (ANOVA). Groups at the same time points were subsequently compared using the Fisher PLSD posthoc test. All data were statistically analyzed using Stat View 5.0 (Abacus Concepts, Berkeley, CA). Significance was set at p < 0.05.
Table 1 compares changes in hemodynamic variables, Hb concentrations, PaO2, PaCO2, and levels of electrolytes in the SHAM, high and low PV groups. Mean arterial pressure (MAP) and Hb significantly decreased during CPB in both the high and low PV groups compared with the SHAM group. Furthermore, MAP and Hb significantly decreased during CPB in the high, compared with the low PV group. However, hemoglobin levels never fell below 7 g/dl in any of the rats so none were excluded from any of the groups. Levels of PaO2 were higher in the two CPB groups than in the SHAM group, whereas PaCO2 levels and pH did not significantly differ among these groups. Levels of potassium tended to increase in both CPB groups. Hemolysis was not among the macroscopic findings of blood plasma from all groups.
The CD11b positive activation ratio as well as levels of inflammatory and biochemical markers in plasma did not significantly differ among the three groups before CPB.
The CD11b positive activation ratio significantly increased in the high and low PV groups compared with the SHAM group during CPB. The ratio was lower in the low, than in the high PV group during CPB but the difference did not reach statistical significance (high vs. low PV: 75.6 ± 4.8 vs. 68.7 ± 6.7% at 60 min; 79.7 ± 5.2 vs. 74.6 ± 7.2% at 120 min; Figure 2).
Inflammatory and biochemical markers remained constant during the experimental periods in the SHAM group, whereas TNF-α significantly increased in the high PV group (high vs. low PV: 856 ± 65 vs. 444 ± 34 pg/ml) at 60 min after starting CPB. However, TNF-α values did not significantly differ between these groups at the end of CPB (high vs. low PV: 1,237 ± 61 vs. 1,129 ± 137 pg/ml; Figure 3A). Levels of IL-6 were significantly increased in the high PV group compared with the low PV group (695 ± 62 vs.: 292 ± 85 pg/ml) at 60 min, but did not significantly differ at the end of CPB (high vs. low PV: 1,226 ± 132 vs. 1158 ± 150 pg/ml; Figure 3B). Levels of IL-10 significantly increased in the high PV group compared with the low PV group at the end of CPB (632 ± 40 vs. 385 ± 55 pg/ml; Figure 3C).
Levels of biochemical markers significantly increased in the high PV group compared with the low PV group at 120 min after starting CPB (LDH, 882 ± 62 vs. 707 ± 126 U/L; AST, 233 ± 20 vs. 159 ± 14 U/L; ALT, 91 ± 11 vs. 76 ± 7 U/L; Figure 3D–F).
The W/D ratio was significantly higher in the high and low PV groups than in the SHAM group (6.01 ± 0.10 and 5.46 ± 0.09 vs. 4.68 ± 0.08; Figure 4). The increase in the W/D ratio was significantly suppressed in the low PV group compared with the high PV group.
We discovered that levels of proinflammatory cytokines and biochemical markers of organ damage were significantly more increased in groups of rats after CPB than in SHAM rats. These findings were similar to those of our previous studies.5–7
Levels of the proinflammatory cytokines TNF-α, IL-6, and the biochemical markers LDH, AST, and ALT were significantly more elevated in the high PV CPB group than in the low PV CPB group at 60 min, whereas none of the markers significantly differed between them at 120 min.
The W/D ratio in the lungs at the end of CPB was higher in the high PV group than in the low PV group, indicating more water accumulation in the high PV group. Therefore, lung edema was reduced and the W/D ratio was significantly suppressed in the CPB group with low PV.
Plasma cytokine levels (TNF-α, IL-6, and IL-10) and biochemical markers (LDH, AST, ALT) were significantly elevated in the CPB groups compared with the SHAM group, indicating a systemic inflammatory response and organ damage in our rat CPB model. However, systemic blood pressure and Hb were maintained at around 80 mm Hg and 10 g/dl, respectively, during CPB. These data indicated that our rat model of CPB is equivalent to the established human CPB procedure, which is often associated with systemic inflammation and organ damage.10 Possible factors responsible for the inflammatory response during CPB include contact between blood and the surface of the extracorporeal circulation unit, endotoxemia, surgical trauma, ischemic reperfusion injury, and blood loss.3 Many studies have shown that blood contacting the surface of the CPB circuit activates white blood cells, platelets, and the complement system. An increase in cytokines, such as interleukins and necrosis factor,11 aggravates the inflammatory response and complex interactions during CPB lead to further inflammation.12
Schnoering et al.13,14 studied a new miniaturized heart-lung machine (MiniHLM) in animals and found that reducing the priming volume does not reduce the inflammatory response. Their study did not focus on the blood contact area and combinations of various factors, such as hypothermia, cardiac arrest, and reperfusion.13,14 With respect to this, the CD11b positive activation ratio did not significantly differ between the high and low PV groups in the current study. CD11b expression indicates leukocyte (monocyte and granulocyte) activation. The results might differ if leukocyte (monocyte and granulocyte) activity is suppressed during long-term CPB.
We postulated that using a smaller CPB circuit with a reduced priming volume and a smaller surface area would attenuate the systemic inflammatory response with a reduction of inflammatory cytokine levels and organ tissue damage during CPB.
Our data indicated that decreasing the surface area by about 20% and the priming volume by about 50% reduced the significantly more elevated proinflammatory markers TNF-α and IL-6 in the high compared with the low PV and SHAM groups at 60 min after starting CPB. However, these values did not significantly differ among the groups at 120 min after starting CPB.
These findings suggested that in addition to the blood contact surface area, the duration of exposure to CPB is also involved in evoking the systemic inflammatory response.
The systemic inflammatory response could be suppressed by reducing levels of inflammatory cytokines and organ tissue damage during brief CPB for procedures, such as coronary artery bypass grafting or single-valve replacement. However, such suppression tends to be masked during a longer duration of CPB.
According to our findings, CPB is the fundamental cause of an initial increase in cytokines derived from early reactions by leukocytes (monocytes, neutrophils) and then further increases are derived from vascular endothelial injury. Although the values of proinflammatory cytokines did not differ, the W/D ratio was higher in the high PV group. We could not confirm detailed mechanisms of the increase in the cytokine levels during CPB and this will require further study. We believe that the PV is a major cause of edema, but the relationship between inflammation and edema remains unclear. Future studies should investigate the roles of factors involved in vascular permeability, such as leukotrienes and leukocyte protease. In addition, recent findings indicate the importance of maintaining normal colloid osmotic pressure during CPB.15,16 Colloid osmotic pressure was not assessed in the current study, but will be the focus of a future investigation. In addition, we would like to consider using hydroxyethyl starch formulations and albumin preparations in future studies. Because the Hb level decreased significantly more during CPB in the high, than in the low PV group, we also intend to focus a future investigation on relationships among the inflammatory response, oxygen delivery, and consumption.
The short CPB circuit system has become increasingly popular.17–20 Minimal extracorporeal circulation technology includes shorter tubes, which decreases PV. The additional benefits of such technology include a decrease in the coagulation cascade and a reduction in the need for blood transfusions in patients undergoing cardiac surgery.17–20 This technology should become more important and become even more popular in the future. However, our data suggested that the duration of CPB exposure is an important factor in eliciting the systemic inflammatory response in addition to blood contacting artificial surfaces. The duration of CPB should be minimal to ensure the safe application of minimal extracorporeal circulation technology.
Further studies are needed to clarify the effects of minimal extracorporeal circulation and the mechanisms of the pathophysiological changes that arise during artificial perfusion.
This study has several limitations. We evaluated parameters during CPB, but not after CPB. Further studies are needed to assess the systemic inflammatory response and organ damage after CPB. All CPB circuits are generally coated and the coating materials should be evaluated. Although open CPB circuits with an open reservoir and operative field suction tubes are commonly used during cardiac surgery, only closed CPB circuits were used in the current study. Future studies should analyze the impact of blood in contact with air during open-circuit CPB. Potassium levels tended to increase during CPB in the high and low PV groups, indicating hemolysis during CPB group. In addition, hemolysis is linked to inflammation and thrombosis during CPB, but we did not assess these topics in the current study. Damage to blood cells will be analyzed in the future.
We consider that our rat model of CPB is equivalent to the established human CPB procedure. However, the effects on inflammatory responses during and after CPB should be assessed in larger animal models before our findings could be clinically applied.
Systemic inflammatory response and organ damage including pulmonary edema were induced in rat models of CPB.
We compared the effects of high and low PV during CPB and found that low PV reduced lung edema. Levels of the proinflammatory cytokines TNF-α and IL-6 were significantly elevated in the high PV CPB group compared with the low PV CPB group at 60 min, but none of the examined markers significantly differed between these groups at 120 min. These findings suggest that in addition to the amount of surface area in contact with blood, the duration of CPB exposure is also an important trigger of the systemic inflammatory response.
1. Walker G, Liddell M, Davis C: Extracorporeal life support - state of the art. Paediatr Respir Rev 2003.4: 147–152.
2. Gao D, Grunwald GK, Rumsfeld JS, et al: Variation in mortality risk factors with time after coronary artery bypass graft operation. Ann Thorac Surg 2003.75: 74–81.
3. Butler J, Rocker GM, Westaby S: Inflammatory response to cardiopulmonary bypass. Ann Thorac Surg 1993.55: 552–559.
4. Li S, Price R, Phiroz D, Swan K, Crane TA: Systemic inflammatory response during cardiopulmonary bypass and strategies. J Extra Corpor Technol 2005.37: 180–188.
5. Fujii Y, Shirai M, Tsuchimochi H, et al: Hyperoxic condition promotes an inflammatory response during cardiopulmonary bypass in a rat model. Artif Organs 2013.37: 1034–1040.
6. Fujii Y, Shirai M, Inamori S, et al. Insufflation of hydrogen gas restrains the inflammatory response of cardiopulmonary bypass in a rat model. Artif Organs 2013.37:136–141.
7. Fujii Y, Shirai M, Inamori S, Takewa Y, Tatsumi E: A novel small animal extracorporeal circulation model for studying pathophysiology of cardiopulmonary bypass. J Artif Organs 2015.18: 35–39.
8. Pasquale MD, Cipolle MD, Monaco J, Simon N: Early inflammatory response correlates with the severity of injury. Crit Care Med 1996.24: 1238–1242.
9. Hongchi J, Fanqiang M, Wei L, Liquan T, Haiquan Q, Xueying S. Splenectomy ameliorates acute multiple organ damage induced by liver warm ischemia reperfusion in rats. Surgery 2007.141:32–40.
10. Boyle EM, Pohlman TH, Johnson MC, Verrier ED. Endothelial cell injury in cardiovascular surgery: The systemic inflammatory response. Ann Thorac Surg 1997.63: 277–84.
11. Engelman RM, Rousou JA, Flack JE 3rd, Deaton DW, Kalfin R, Das DK: Influence of steroids on complement and cytokine generation after cardiopulmonary bypass. Ann Thorac Surg 1995.60: 801–804.
12. Cremer J, Martin M, Redl H, et al: Systemic inflammatory response syndrome after cardiac operations. Ann Thorac Surg 1996.61: 1714–1720.
13. Schnoering H, Arens J, Detering SM, et al: Expression of inflammation in myocardial tissue of rabbits: Comparison of two miniaturized heart-lung machines. Artif Organs 2013.37: 541–548.
14. Schnoering H, Arens J, Detering SM, et al: Development of a rabbit animal model for miniaturized heart-lung machines. ASAIO J 2013.59: 152–156.
15. Golab HD, Scohy TV, de Jong PL, Kissler J, Takkenberg JJ, Bogers AJ: Relevance of colloid oncotic pressure regulation during neonatal and infant cardiopulmonary bypass: A prospective randomized study. Eur J Cardiothorac Surg 2011.39: 886–891.
16. Loeffelbein F, Zirell U, Benk C, Schlensak C, Dittrich S: High colloid oncotic pressure priming of cardiopulmonary bypass in neonates and infants: Implications on haemofiltration, weight gain and renal function. Eur J Cardiothorac Surg 2008.34: 648–652.
17. Immer FF, Pirovino C, Gygax E, Englberger L, Tevaearai H, Carrel TP: Minimal versus conventional cardiopulmonary bypass: Assessment of intraoperative myocardial damage in coronary bypass surgery. Eur J Cardiothorac Surg 2005.28: 701–704.
18. Skrabal CA, Steinhoff G, Liebold A: Minimizing cardiopulmonary bypass attenuates myocardial damage after cardiac surgery. ASAIO J 2007.53: 32–35.
19. Fromes Y, Gaillard D, Ponzio O, et al: Reduction of the inflammatory response following coronary bypass grafting with total minimal extracorporeal circulation. Eur J Cardiothorac Surg 2002.22: 527–533.
20. Vohra HA, Whistance R, Modi A, Ohri SK: The inflammatory response to miniaturised extracorporeal circulation: A review of the literature. Mediators Inflamm 2009.2009: 707042.