Cardiopulmonary bypass (CPB) induces complex changes in blood, leading to a systemic inflammatory response. Although technical improvements achieved over the past years have contributed to the reduction of operative and postoperative mortality and morbidity, post-CPB systemic inflammatory response worsens the prognoses for high-risk patients. Indeed, enlarging patterns of surgical indications increased concomitantly the number of older and/or high-risk patients, who are more prone to develop postoperative single or multiple organ dysfunction syndromes (MODS).
Fifteen years ago, Kirklin et al1 hypothesized that these deleterious effects were linked to the contact of blood components with CPB circuit's walls. These mechanisms involve both humoral inflammatory mediators (Factor XII, kallikrein-kinin, fibrinolytic and complement systems, cytokines)2,3 and cellular activation (neutrophils and endothelial cells).4 Evidence suggests that activated leukocytes release cytotoxic proteases and large amounts of oxygen-derived free radicals (OFR)5 that could precipitate to the bypass-induced injury and impairment of myocardial recovery. Clermont et al6 showed the production of systemic, and not only coronary, alkyl and alkoxyl radicals during CPB to be a consequence of systemic inflammation syndrome (SIRS). Several approaches to reduce the negative consequences of CPB have been tested, including the administration of drugs such as aprotinin or the modification of CPB procedures such as the use of pulsatile blood flow.
The present study was designed to observe the potentially beneficial consequences of reducing the inflammatory process in patients undergoing cardiac surgery. We hypothesized that modulating the inflammatory response could decrease OFR production and reduce postoperative complications. The first aim of this study was to assess inflammatory and oxidative stress status in a high-risk population for cardiac surgery (Parsonnet score7 more than 20) undergoing a classic CPB or a protocol of CPB designed to reduce the inflammatory response (ie, heparin-coated circuits, aprotinin, and pre-CPB hemofiltration). In a second window, we looked into the clinical complications and the patient's management in the intensive care unit (ICU) throughout the investigation period (first postoperative month).8,9
Study Population and Study Design
Our study complies with the Declaration of Helsinki. After approval of the local research ethics committee and the patient's informed written consent, 27 patients (Parsonnet. score7 more than 20) undergoing coronary artery revascularization or valvular replacement (alone or combined) under CPB were included in this prospective study. Patients were randomized to 1 of the 2 study groups, based on a table of random numbers. The study groups were defined as follows: the normal procedure (NP) group (n = 14) underwent conventional CPB, and the antiinflammatory procedure (AIP) group (n = 13) underwent conventional CPB associated with Duraflo II heparin-coated circuits (Baxter Healthcare Corp, Bentley Laboratories Division, Irvine, California) plus high doses of aprotinin (complete Royston dose) and preoperative low-volume ultra-filtration (10 milliliters per kg−1/min−1). The same surgeon operated on all the patients.
The following criteria led to the exclusion of the patient from the protocol: surgical emergencies, past medical history of heparin or aprotinin allergy, intercurrent inflammatory chronic pathologies, coagulation troubles (except heparin or aspirin treatment), and aneurysmectomy-associated surgery. All routine cardiac medication was continued until the morning of surgery, except for angiotensin-converting enzyme inhibitors, which were stopped the day before surgery so that no effect on systemic vascular resistance was carried forward during the study. Anticoagulation therapy was stopped 10 days before surgery. Patients were premedicated with midazolam (0.1 mg/kg−1) orally plus hydroxyzine (1 mg/kg−1) 90 minutes before receiving anesthesia.
Before the induction of anesthesia, a complete hemodynamic monitoring was performed in the operating room. Monitoring consisted of a pulmonary artery catheter for the continuous measurement of cardiac output and SvO2 through the right internal jugular vein, a peripheral 14 gauge venous catheter, a 20 gauge arterial catheter, a pulse oxymetry, and 5-leads electrocardiogram (ECG). The hemodynamic profile consisted of heart rate, mean pulmonary artery pressure, pulmonary capillary wedge pressure, cardiac index, and systemic vascular resistance using a Baxter Vigilance monitor.
Induction of anesthesia was performed with intravenous midazolam (0.02 mg/kg−1), sufentanil (0.2 μg/kg−1), and hypnomidate (0.3 mg/kg−1). After verifying correct manual ventilation, cisatracurium dibesylate (0.15 mg/kg−1) was injected. Patients were orally intubated and ventilated with FiO2: 0.4. In both groups, anesthesia was maintained with sufentanil and cisatracurium as required and inhaled isoflurane. After CPB weaning, isoflurane inhalation was stopped and intravenous propofol relay was established (1.5 milligrams per kg−1/h−1).
After intravenous injection of heparin (300 IU/kg−1), CPB was achieved in a standard fashion. A nonpulsatile pump was primed with Ringer's lactate solution (1000 mL) and hydroxy-ethyl starch (500 mL). CPB flow rate was maintained between 2.4 L.min−1/m−2 (37°C) and 1.7 liters per min−1/m−2 (28°C). Moderate body hypothermia (32°C) was used. An anterograde cardioplegia catheter was inserted in the ascending aorta, and a retrograde cardioplegia canula was placed in the coronary sinus. After applying aortic cross-clamping, topical slush was applied on the heart and 500 milliliters of St Thomas' Hospital cardioplegic solution were infused. Infusions were repeated every 30 minutes of clamping, resulting in 1500 to 2000 milliliters of total cardioplegia. Rewarming began 10 minutes prior to aortic unclamping, and 100 milliliters of mannitol solution (20%) was infused. Reperfusion on CPB was continued for 25-40 minutes after clamp removal. Finally, CPB was discontinued and protamine was given for heparin reversal at the dose of 450 IU/kg−1 for patients in the NP group and at the dose of 225 IU/kg−1 for patients in the AIP group. In both groups, the patient would benefit from a blood infusion in the case of severe anemia (blood hematocrit <22% during CPB or <26% after CPB).
Use of sympathomimetic drugs or intraaortic balloon pumping (IABP) during CPB weaning was left to the discretion of clinicians according to the hemodynamic constants. The patients were transferred to the post operative intensive care unit (ICU), then to semi-intensive care service, and finally to surgery service. Patients did not receive nonsteroidal antiinflammatory drugs, if required, until postoperative day 3. Clinical and biological parameters were chronologically recorded (Figure 1).
Hemodynamic profile was noted before and after CPB, during the 3rd and the 6th postoperative hour (POH3 and POH6), and during the first and second postoperative day (POD1 and POD2). Complications such as arrhythmia, blood loss, infection, and organ failure were registered. ICU length of stay was noted. The criteria for discharging the patients from ICU were clinical (no ECG abnormalities, blood pressure stability, absence of fever [T < 38°C], absence of rhythm disturbances or pace dependence, efficient prophylactic antiarrhythmic and anticoagulation treatments, no major organ dysfunction [respiratory, renal, or hepatic failure], no use of inotropic drugs, correct management of postoperative pain) and biological (Hb > 105 g/L, normalization of cardiac enzymes, PO2 > 90 mm Hg, normal hepatic enzymes).
Blood sampling for biochemical measurements is depicted in Figure 1. Standard biology (blood formula, platelet count, renal function, blood gases, pH, and lactic acid) was performed before, during, and after CPB (POH3 and POH6) and at POD1 and POD2. Arterial concentrations of C3a and IL-6 were evaluated using enzyme-linked immunosorbent assays (ELISA) just before CPB, during CPB, and immediately after aortic unclamping (for cost reasons, the evaluation was not performed further on time). Arterial concentrations of C3, C4, B factor, and C-reactive protein (CRP, immunonephelometric assay and monoclonal antibody, Dade Baehring BNII) were evaluated before, during, and after CPB and at POD1 and POD2. I-troponin and creatine kinase-MB (CK-MB) were measured before, during, and after CPB and at POD1. Protein plasma levels were evaluated by the standard method.
For the measurement of radical species, spin-trapping experiments were performed with α-phenyl-N-tert-butylnitrone (PBN, Aldrich Chemical Co, St Quentin, France) highly purified through a double sublimation. PBN-saline solutions (120 mM, ice-cold, N2 gassed) were prepared just prior to use (10 minutes of stirring) and protected from light throughout the experiment. Blood samples (5 mL), collected at various times (before and during CPB, 3 and 10 minutes after unclamping, and at POH3), were immediately mixed with 2.5 milliliters of PBN-saline solution by gentle inversion and then centrifuged at 3500 rpm for 5 minutes. The resulting plasma/spin trap supernatants were extracted immediately with 1 milliliter of ice-cold, N2-gassed high-grade toluene (20 seconds of stirring, 10 minutes of centrifugation at 4500 rpm). The toluene extracts were stored in liquid N2 for less than 24 hours, then thawed and transferred into a quartz Electron Spin Resonance (ESR) cell for analysis. All ESR spectra were recorded at room temperature on a Bruker (Wissembourg, France) ESP 300 EX-band spectrometer, using a TM110 cavity. PBN spin-trap preparation, extraction, storage, and analysis were performed sheltered from light to prevent any photolytic degradation of the trap. The concentrations of PBN adducts were determined through a double integration of the spectra using TEMPO nitroxide radical as a standard.
Student's t test and F test for paired sample and unpaired samples were used for statistical analysis of the difference between pre-bypass values and different time points within 1 group or of differences between groups at the same time points. Data not normally distributed were analyzed using Mann-Whitney or Wilcoxon tests. Categorical variables were analyzed using χ2 test. Significance levels for acceptance were P < 0.05. Results in tables and text are presented as mean ± standard error (SEM).
Clinical Characteristics and Operative Data
There were no significant initial differences between the 2 groups in preoperative patient's demographic criteria, comorbidity, surgery, aortic cross-clamping, CPB, or ventilation times (Table 1). There was also no statistical difference between the 2 groups concerning the administration of sympathomimetic drugs for CPB weaning or the amounts of blood transfusion or losses. No increased mortality was noted during the first postoperative month.
In return, there was a statistically significant difference for ICU stay length, with a shorter period in patients in the AIP group (control: 6.0 ± 6.6 days; treated: 3.4 ± 2.2 days; P < 0.05) (Table 1).
We did not observe any significant difference in functional parameters: heart rate, systolic and diastolic blood pressures, pulmonary artery wedge pressure, cardiac index, and systemic vascular resistance index. No ECG modifications were observed. In the AIP patient group, SvO2 was significantly higher at POH3 (Table 2).
No difference was noted between the 2 groups for blood formula, platelet, and white cell counts before surgery and at POD2. CK-MB and I-troponin increased significantly in the 2 groups throughout the entire study period (Figure 2). CK-MB plasma levels started to increase after protamine injection, then reached a peak at the 6th hour of the postoperative period and remained significantly elevated at POD1. Plasma levels of CK-MB and I-troponin presented a similar postoperative pattern of evolution during and after CPB time. However, AIP-treated patients released lower amounts of I-troponin in the plasma from the 3rd hour following operation (3.72 ± 2.53 vs 10.77 ± 14.2 μg/L, P < 0.01) until the end of the observation period (6.37 ± 2.80 vs 12.72 ± 16.13 μg/L, P < 0.05). At the 6th hour of the post operative period, this significant difference observed for I-troponin (6.29 ± 3.86 vs 13.35 ± 15.1 μg/L, P < 0.01) was confirmed by CK-MB data, which were lower in patients in the AIP group as compared with the NP group (12.72 ± 7.47 vs 38.25 ± 16.86 UI/L, P < 0.05).
Lactic acid levels had a similar evolution in both patient groups, except at POH 6, where they were 2 times higher in patients in the NP group, showing a peak at this particular time point (Table 2). Starting from normal values at baseline, CRP increased in both groups, showing a significantly higher increase at the end of the study (POD2) in the NP group (NP: 200.42 ± 63.92 mg/L, AIP: 153.09 ± 80.75 mg/L, P < 0.05). Similar results were obtained for the evolution of C3 and C4 levels, which were lower in the AIP group. In the 2 groups, the C3a plasma level started to increase during pre-ACD, but the variations were not significantly different between the groups along CPB.
As is seen in Figure 3, there was a significant increase in the release of IL-6 during CBP, especially after protamine injection. At this particular time point, the peak of IL-6 was significantly lower in patients in the AIP group as compared with the NP group (NP: 853 ± 250 pg/mL, AIP: 553 ± 192 pg/mL, P < 0.05).
Time course of PBN-adduct concentration during the different periods of the study is shown in Figure 4. The ESR spectra detected in all samples were characteristic of alkyl and alkoxyl PBN adducts (N = 13.5 G, aH = 2.1 G, g = 2.012).5 The PBN-adduct plasma concentration did not differ significantly between the 2 groups at pre-CPB and per-CPB. During preaortic cross-unclamping the concentration of PBN-adducts increased in the blood and stayed at a high level during post-ACU; however, this increase was significantly lower in the AIP group of patients (P < 0.05).
The main result of our study is that pre-CPB therapeutic optimization can modulate the inflammatory response and reduce oxidative stress in high-risk patients undergoing valvular replacement or coronary surgery. These results may have clinical consequences in reducing postoperative complications and reducing ICU length of stay.
Several therapeutics have been tested for their antiinflammatory properties. Prophylactic use of aprotinin in cardiac surgery has been shown in many studies to decrease bleeding and to reduce transfusion.10-12 High doses of aprotinin also reduce the inflammatory response to CPB, probably through kallikrein inhibition.13 However, the potential harmful effects of aprotinin during cardiac surgery are a matter of heated debate, especially the risk of serious end-organ damage,14 but the results are contradictory. In our study we have not observed any increased renal failure, stroke, or encephalopathy from the use of aprotinin, but the number of patients is probably too low to exclude a probable deleterious effect of aprotinin. To date, neither the U.S. Food and Drug Administration nor the French Drug Agency has modified the authorization to use this drug in its specific indications. Initial interest in heparin-coated or uncoated CPB circuits was focused on postoperative bleeding and improvement of biocompatibility.15 However, heparin, as a C3-convertase inhibitor, reduces in vitro protease liberation and activation after leukocytes' activation. Studies.16,17 indicated that heparin-coated circuits reduce the production of both proinflammatory and antiinflammatory cytokines (IL-6, -8, and -10). Removal of excess fluid with ultra-filtration has been proposed as a method for removing proinflammatory mediators during cardiac surgery. Journois et al showed that ultra- filtration could lower plasma levels of tumor necrosis factor-alpha (TNF-α), IL-1, IL-10, myeloperoxidases, and C3a in cardiac pediatric patients.18 Patients also had less fever, reduced postoperative blood losses, and reduced time to extubation, suggesting, without proving, a causal relationship between cytokinemia and clinical end points. Whereas these 3 therapeutics seemed to have individually beneficial effects on the inflammatory response to CPB, they never had been associated before.
Recently the efficacy of a biocompatible surface alone and in combination with methylprednisolone during CPB has been studied.19 It was suggested as a potential beneficial effect for combined strategies to minimize inflammation after CPB in elderly patients. In patients undergoing elective coronary artery bypass grafting procedures, low doses of methylprednisolone induced a reduction of inflammatory reaction during and after CPB by inhibition of proinflammatory cytokine release and of free radical generation after the aortic cross-clamp reverse.20
The myocardial ischemia-reperfusion sequence that occurs during CPB can explain the cardiac dysfunction.5 The cellular injury is clearly linked to an oxidative stress, but the physiopathology remains multifactorial and involves the production of cytokines such as IL-6, IL-8, IL-1, and TNF-α8 Correlation between OFR production, CK-MB, and I-troponin increase during CPB and in the first postoperative hour has already been highlighted, confirming that myocardial lesions can also be caused by reperfusion of the ischemic heart with blood modified and inflammatory-activated by CPB.6 Clermont et al6 reported no significant difference between peripheral and coronary sinus OFR concentrations, leading to the conclusion that the majority of oxidative stress seems to originate from a systemic activation.
However, no direct cause-and-effect relationship has ever been demonstrated between high proinflammatory cytokine plasma levels and poor outcome after cardiac surgery. Akira and Maruo reported an initiating and promoting role of IL-6 in the leucocyte activation.21,22 Analyses during dog experimental myocardial necrosis pointed out that IL-6 intervenes as an essential mediator of interactions between activated endothelium and leucocytes. Finkel et al23 showed that IL-6 could be implicated in the clinical nevus that causes human reversible myocardial depression. Kawamura already described a peak of IL-6 and IL-8 after aortic unclamping that suggests the participation of these 2 cytokines in reperfusion injuries resulting from neutrophil activation.24 Our results agree with these data and show an IL-6 secretion peak occurring between unclamping and protamine injection following CPB. Fransen et al showed that the increase in IL-6 plasma levels could be linked with blood transfusion.25 Our blood samples were collected apart from any blood transfusion that could interfere and blood protein levels do not differ significantly between the 2 groups, excluding any skew from hemodilution.
CRP, which originates from the liver during immunoinflammatory pathologies, does not vary during the CPB but, in return, increases dramatically at POD 1 and 2. In treated patients, this increase in CRP was lower 2 days after surgery, a result that is concordant with the reduction of IL-6 activity, 1 of the proinflammatory cytokines with IL-1 and TNF-α that stimulates its hepatic production.26
C3a anaphylatoxin is produced during the cleavage of C3 by C3 convertase. It could play a role in postoperative complications attributable to complement activation.2 Indeed, C3a is responsible for the activation of neutrophils, monocytes, and platelets in experimental CPB models. Several studies brought back an increase in the C3 fraction during cardiac surgery,2,3,8 but no relation between postoperative prognosis and C3a rates could be highlighted. Our results agree with data from the literature indicating an increase of C3a after CPB, but in our clinical conditions the variations were not statistically significant.
At the same time, we observed an increase in the serum rates of CK-MB and I-troponin. Lasocki et al showed a direct correlation between high I-troponin plasma levels and higher in-hospital mortality.27 In our work, the concentrations of IL-6 seemed to correlate with CK-MB levels, but no significant relationship that would have confirmed the contribution of IL-6 to myocardial reperfusion lesions was noted.
During general anesthesia, a reduction of cardiac index was observed as a result of the various drugs used (morphinics, intravenous hypnotics, and inhaled halogens). Nevertheless, no significant difference among hemodynamic parameters during the perioperational observational period was noted between the 2 groups, although SvO2 remained higher in the treated group at POH3, expressing a better reoxygenation in these patients. Paradoxically, at the 6th postoperative hour, a lactate peak was observed in the control group only, which could not be explained by a reduction of their cardiac index. Therefore, the increase in lactic acid levels in the control group was more likely to originate from a large whole-body inflammatory phenomena and tissue suffering than solely to the reperfusion of the ischemic myocardium, explaining the discrepancies between the 2 groups concerning cardiac index.
Measurement of OFR in patients is difficult because of the transient nature of these species. The spin trapping technique associated with ESR is an elegant approach to evaluate oxidative stress. In this technique, the unstable radicals react with the spin trap to form more stable adducts. In our study, radical species trapped by PBN were consistent with alkyl-alkoxyl radicals. We demonstrated that the production of free radicals was persistent and that pre-CPB therapeutic optimization induced a reduction of oxidative stress. Recently it has been reported that, in patients undergoing coronary artery bypass grafting for the first time, the infusion of the free radical scavenger deferoxamine, for 8 hours starting immediately after the induction of anesthesia, improved the postischemic recovery of the left-ventricular function and that the benefit remained for the 12 months of follow-up.28
In conclusion, a therapeutic modulation of inflammation was useful in high-risk patients undergoing cardiac surgery under CPB, resulting in a reduction of markers of inflammation and cardiac injury, a reduction of oxidative stress plasma levels, and a decrease in their hospital length of stay in the ICU department. These beneficial effects of a therapeutic modulation of inflammation could be related to a better tissue perfusion through the reduction of IL-6 and OFR production. Our investigation suggests that a patient's outcome after cardiac surgery is linked to the degree of the inflammatory response during CPB.
This work was supported in part by CHU Dijon, the Region Bourgogne, Faculté de Médecine de Dijon, Fondation de France, and Association de Cardiologie de Bourgogne.
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