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a Department of Clinical Research, University of Bern, Bern, Switzerland
b Department of Cardiovascular Surgery, University Hospital, Bern, Switzerland
c Department of Anesthesiology, University Hospital, Bern, Switzerland
d Department of Cardiology, University Hospital, Bern, Switzerland
Received 5 December 2007; received in revised form 24 April 2008; accepted 5 May 2008.
* Corresponding author. Address: University of Bern, Department of Clinical Research, Murtenstrasse 31, 3010 Bern, Switzerland. Tel.: +41 31 632 9669; fax: +41 31 632 8837. (Email: robert.rieben{at}dkf.unibe.ch).
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Abstract |
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 Top Abstract 1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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Key Words: Ischemia • Reperfusion • Complement inhibition • Cytoprotection • Endothelium
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1. Introduction |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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Activation of the coagulation and complement system, leukocytes, endothelial cells and other pro-inflammatory mediators, contribute to organ damage [2]. Complement inhibition [3] and technical modifications such as ultrafiltration devices to remove excess water and pro-inflammatory mediators [4], may improve clinical outcome to a certain extent. However, the problem of CPB-associated inflammation and in particular IR-injury is not entirely prevented by their use. Furthermore, cardiac arrest induced with cardioplegic solution for the purpose of myocardial preservation during CPB does not specifically protect the endothelium. IR-induced endothelial cell activation with shedding of its natural anticoagulant and anti-inflammatory surface glycocalyx layer [5] may, in part, contribute to CPB-associated tissue damage. Restoration of an anti-inflammatory and anti-coagulant environment through functional replacement of the shed heparan sulfate proteoglycan (HSPG) may therefore attenuate cardio-pulmonary dysfunction. To this purpose, low molecular weight dextran sulfate (DXS, MW 5000), a sulfated glycosaminoglycan analog and potent inhibitor of complement [6,7] and coagulation [8] that has been shown to bind to HSPG-denudated endothelium [7] and to ameliorate acute myocardial IR-injury in vivo [9], was tested in a porcine model of CPB. We hypothesized that DXS would preserve cardiovascular and pulmonary function by complement inhibition and endothelial cell protection in CPB mediated inflammation and IR-injury.
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2. Materials and methods |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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2.1 Animals and anesthesia
Eighteen large white pigs (60 ± 3 kg) were premedicated with intramuscular ketamine (20 mg/kg) and xylazine (2 mg/kg), followed by intravenous administration of midazolam (0.5 mg/kg) and atropine (0.05 mg/kg), and were intubated and mechanically ventilated with a volume-controlled ventilator (Servo 900, Siemens AG, Solna, Sweden) with 5 cm H2O end-expiratory pressure. Anesthesia was maintained with continuous intravenous infusion of pentobarbital (8 mg/kg/h), fentanyl (0.03 mg/kg/h), and pancuronium chloride (1 mg/kg/h) for muscle relaxation.
Arterial, central venous and Swan-Ganz catheters were introduced into the internal and external jugular veins and carotid artery respectively. The urinary bladder was catheterized through a midline incision, and urinary output measured regularly. Rectal and central venous blood temperatures (thermistor in pulmonary artery catheter) were measured continuously. A heating pad was used to maintain the body temperature between 37.5–38.5 °C, except during CPB-induced hypothermia.
2.2 Surgical procedure
A full midline sternotomy and pericardiotomy was performed. Methylprednisolone (Solu-Medrol, Pfizer, Canada, 1 g) was administered intravenously and the pigs were heparinized with 400 IU/kg of heparin. Activated clotting time (ACT) was controlled regularly using high-range ACT cartridges (Medtronic, Minneapolis, MN) and an automatized ACT analyzer (Medtronic HemoTec, Parker, CO). If necessary additional heparin was administered to keep ACT above 250 s. A 5-F high-fidelity pressure-sensitive tip transducer (Millar Instruments; Houston, TX) was introduced through the base of the brachiocephalic trunk into the left ventricle. Data were recorded with the PowerLab system (Chart 5.2, PowerLab, ADInstruments Castle Hill, Australia).
2.3 Cardiopulmonary bypass (CPB)
A minimal extracorporeal circulation (MECC) system comprising a membrane oxygenator (Quadrox, Jostra Inc., Hirrlingen, Germany) and a centrifugal pump (Jostra Inc.) with a priming volume of 600–800 ml was used. Standard aortic and bicaval cannulation was performed. A 7-F catheter was inserted across the right atrium into the coronary sinus for blood sampling throughout the experiment. Extracorporeal circulation was performed with moderate systemic hypothermia (32 °C) and a non-pulsatile mean flow rate of 3.3 (2.6–3.6) l/min/m2. Ringer solution or hydroxyethyl starch was used for volume substitution. Antegrade cold modified Buckberg blood cardioplegia (BCP, using Cardioplegin as the crystalloid cardioplegic solution) was administered after aortic cross-clamping, initiating a period of 60 min of cardiac arrest. Cold BCP (8 °C) was repeated after 30 min, and warm BCP (35 °C) 30 min thereafter. To the two repeat-cardioplegia solutions a total of either 10 ml phosphate buffered saline (PBS, n
= 8) or 10 ml of low molecular dextran sulfate in PBS (DXS, MW 5000, Fluka Chemie, Buchs, Switzerland, 300 mg, equivalent to a total of 5 mg/kg body weight, n
= 10) were added. The total volume of cardioplegia solution used was weight-adapted and similar in both groups (1420 ± 520 ml and 1330 ± 330 ml). After aortic declamping a reperfusion phase of 30 min was initiated followed by weaning off CPB and a post-CPB observation phase of 120 min. The animals were then sacrificed (intravenous bolus of potassium chloride) and the heart and lungs excised for further analysis.
2.4 Experimental groups
All experimenters were blinded with regard to treatment regimen. Randomization of the animals into the two groups was done prior to the experimental work, using a randomization code (DXS = 0, PBS = 1), created by a random number generator (SAS, version 9.1.2, SAS Institute Inc., Cary, NC, USA). The samples (DXS solution or PBS) were prepared according to the randomization output and stored at –20 °C until use. Randomization and sample preparation was performed by an independent laboratory technician. Prior to premedication of the animals, the corresponding vial was allocated to the pig (sequential number of vial = sequential number of the pig). All 18 consecutively enlisted pigs were treated according to this protocol. No animal was initially excluded. Two experiments (DXS = 1, PBS = 1) were terminated 1 h prematurely due to hemodynamic deterioration after sudden, intractable arrhythmia and electromechanical dissociation, respectively.
2.5 Hemodynamic monitoring and management
Heart rate, carotid and pulmonary arterial blood and central venous pressures were recorded continuously. Right ventricular and left atrial pressures were measured invasively. Left ventricular pressure as well as dp/dt
max and dp/dt
min were recorded with a 5-F high-fidelity pressure-sensitive tip transducer (Millar Instruments). Unless recorded continuously, all functional data were measured at following time points: baseline, 30 min after cessation of CPB and every 30 min thereafter. Mean arterial pressure was kept at a minimum of 50 mmHg throughout the procedure, by adjusting fluid balance (with hydroxyethyl starch) and noradrenalin, 5 µg boli, followed by continuous intravenous infusion if pressure could not be maintained.
2.6 Echocardiography
Epicardial echocardiography was obtained using a transesophageal (TEE) probe (ACUSON ultrasound system, Siemens, Malvern, PA, USA) placed retrocardially to obtain following views according to the standard TEE views: two and four chamber views, transgastric midpapillary short axis view and transgastric two chamber view. Left ventricular ejection fraction was measured using the biplane method (or modified Simpson method). Measurements were obtained at baseline and simultaneously with other hemodynamic measurements.
2.7 Complement and coagulation
Blood samples were collected (EDTA plasma and serum) at baseline, after going on CPB, after 30 and 60 min on CPB, after 30 min weaning, and every 30 min thereafter. Samples were kept on ice until centrifugation and stored at –80 °C until further analysis.
Classical pathway complement activity (venous samples) was determined by standard CH50 assay [10].
Activated partial thromboplastin time (aPTT, venous samples) was measured using Dade Actin FS reagent in a standard coagulation assay.
Thrombin–anti-thrombin III (TAT) complexes (coronary sinus) were measured by micro-enzyme immunoassay (Enzygnost Micro, Behringwerke AG, Marburg, Germany).
2.8 Ischemic and inflammatory markers
Circulating troponin I and creatine kinase (CK-MB fraction) were determined by enzyme immunoassays (AxSYM microparticle enzyme immunoassay platform, Abbott laboratories, Abbott Park, IL, USA) in coronary sinus blood samples. Both assays were tested for cross-reactivity to porcine troponin I and CK-MB. The effect of DXS in concentrations <1 mg/ml (in the fluid phase) was tested and found to be negligible in the multiplex suspension array setting described below in detail (data not shown).
TNFalpha, IL-1beta, IL-6 and Il-8 were measured by sandwich immunoassay using the Luminex fluorescent bead technology. Matching antibody pairs (‘DuoSet’) specific for porcine antigens were purchased from R&D Systems, Minneapolis, MN, USA, and included anti-TNFalpha, anti-IL-1beta, anti-IL-6 and anti-IL-8. The capture antibodies were coupled to carboxylated beads using the Bio-Plex Amine Coupling Kit (Bio-Rad Laboratories, Hercules, CA). The biotinylated counterparts were used as the detection antibodies. Analysis was done with the Bio-Plex system (Bio-Rad Laboratories). Data analysis was done with Bio-Plex Manager version 4.0 software (Bio-Rad Laboratories) with five-parametric curve fitting.
2.9 Endothelin-1 (ET-1)
Plasma and tissue ET-1 levels (coronary sinus samples) were determined by specific radioimmunoassay after solid phase extraction on C18 reverse phase columns as previously described by Shaw et al. [11].
2.10 Histology and immunostaining
Representative samples from the heart and lungs were fixed in 4% buffered formaldehyde, paraffin-embedded, and 3 µm sections stained with hematoxylin-eosin. Neutrophil tissue numbers were determined by counting neutrophils in 10 randomly selected high power viewing fields from various samples of each experiment. Five µm sections were cut from all snap frozen tissue samples, air-dried, acetone fixed, hydrated and labeled using a two/three-step indirect immunofluorescence technique. The following antibodies were used: rabbit anti-human C1q, C3b/c and C4b/c (Dako); goat anti-human C6 (Quidel) cross-reactivity with the respective porcine antigens was verified. Secondary antibodies were goat anti-rabbit IgG(H + L)-FITC (Southern Biotechnology Associated), rabbit anti-mouse Ig-FITC (Dako) and biotinylated goat anti-rat IgG (Southern Biotechnology) followed by streptavidin-FITC (Amersham Biosciences). Samples from all experiments were stained and graded for complement deposition: 0: no staining, 1: minimal focal or diffuse staining, 2: moderately strong staining, 3: extensive staining.
2.11 Tissue water content
Water content of the lungs was determined through desiccation of the lungs at 100 °C for 48 h. Tissue water content was calculated using the formula [(wet weight – dry weight)/wet weight] x 100.
2.12 Statistics
Non-parametric tests were used for data analyses because of the relatively low number of data points rendering assumption for Gaussian distribution of the data rather speculative despite the fact that common tests did not reject the hypothesis of normal distribution. Differences between the two groups were compared by Mann–Whitney U-test. For parameters measured over time, baseline values were subtracted from the end-point measurements, to account for random differences between the two groups at baseline. All analyses were two-sided and differences were considered statistically significant with a p value of <0.05. Analyses involved baseline and end point measurements only, as significant between-group differences (in biochemical markers) were expected at these time points. Repeated, multiple testing was not performed to avoid corresponding problems with type I errors.
According to Little's test, missing values (max 15%) in the datasets were all missing completely at random (MCAR). The method of the last observation carried forward (LOCF) was used for imputation of missing data.
SAS Version 9.1.2 (SAS Institute Inc., Cary, NC, USA) and SPSS Version 12.0.1 (SPSS Inc., Chicago, IL, USA) software were used for all analyses. Data (corrected for hematocrit, where appropriate) are presented as mean ± standard deviation.
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3. Results |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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Systolic function, measured as dp/dt max was slightly, though not significantly, increased in both groups 30 min off CPB and remained relatively stable throughout the remaining post-CPB period (Fig. 1C, p = 0.989).
Diastolic function, measured as dp/dt min, was impaired in PBS controls during the post-CPB period as compared to baseline (p = 0.057 baseline vs end of experiment) and did not fully recover until the end of the experiment, whereas diastolic function remained relatively unchanged in the DXS group (Fig. 1D, p = 0.782).
RV (Fig. 2A) and pulmonary artery pressures (Fig. 2B) post-CPB were increased in both groups as compared to baseline, and were significantly higher in the PBS controls as compared to the DXS group at the end of the experiment (p = 0.021 and p = 0.002, respectively).
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3.2 Echocardiography
LV ejection fraction remained stable and within normal values during the whole procedure. No significant between-group differences were noted at the end of the observation period (68 ± 11 mmHg for DXS vs 70 ± 4 mmHg for PBS; p
= 0.341; p
= 0.341, results not shown). LV regional wall motion was preserved and similar in both groups.
3.3 Histology
Myocardial samples from both groups very focally revealed changes consistent with IR damage including focal wavy fibers and contraction bands, and were accompanied by neutrophil infiltration (not shown). The severity of the changes was more pronounced in samples from the PBS group.
Samples of lung tissue revealed comparable non-specific atelectatic changes in the lower lobes in both groups. Neutrophil accumulation was observed in edematous septa as well as in lung alveoli, particularly at sites of focal hemorrhage. The extent of infiltration and edema was more pronounced in PBS controls as compared to DXS treated animals (Fig. 2C).
3.4 Lung water content
Water content of the lungs was significantly higher in the PBS controls as compared to the DXS treated animals (81 ± 3% vs 78 ± 3%, p
= 0.047) (Fig. 2C). This finding correlated with increased histological signs of tissue edema in samples from the PBS experiments (Fig. 2D).
3.5 Soluble coagulation and complement parameters
Baseline aPTT was comparable in both groups (DXS 32.8 ± 4.4 s vs PBS 39.4 ± 16.5 s; p
= 0.317). Values remained above 300 s in both groups following heparin administration (not shown).
Thrombin anti-thrombin (TAT) levels markedly increased in the PBS group during CPB and values were slow to recover, whereas levels remained largely unchanged throughout in the DXS group, with significantly less TAT complexes than in the PBS group at the end of the observation period (12.8 ± 4.1 µg/ml for DXS vs 20.7 ± 1.0 µg/ml for PBS; p = 0.043, Fig. 3 ).
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3.6 Markers of ischemia
Plasma troponin I values increased in both groups throughout the experiment, but differences were not statistically significant at the end of the observation period (48.6 ± 21.0 µg/ml for DXS vs 56.8 ± 18.70 µg/ml for PBS; p
= 0.175, Fig. 4A). CK-MB values continuously increased in both groups, with significantly increased values in the PBS as compared to the DXS group at the end of the experiment (peak levels 35.9 ± 11.1 ng/ml for DXS vs 43.4 ± 14.8 ng/ml for PBS; p
= 0.042, Fig. 4B).
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4. Discussion |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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With respect to cardiac function, DXS improved diastolic function in comparison to PBS controls. Diastolic dysfunction is frequently observed following open-heart surgery, may have serious clinical implications and be difficult to treat. Systolic function however, as measured by conductance catheter and echocardiography, was unaffected. This may be because systolic function remained essentially unaffected throughout the experiment. Furthermore, echocardiography may not be sensitive enough here to assess differences and changes in ejection fraction of less than 5–10%. It therefore remains speculative what, if any, effects DXS may have on hearts with obviously impaired systolic LV function. Whilst the reduction in inflammation (discussed below) may not always correlate with improvement in clinical parameters and outcome, a few such possible correlations are highlighted in the following paragraphs.
DXS significantly reduced episodes of atrial fibrillation in this short-term post-CPB follow-up. From a clinical standpoint, atrial fibrillation is still an important source of postoperative morbidity and prolonged hospital stay, occurring in up to 50% of patients after CPB [12]. The prospect of reducing this rate is therefore desirable. Diminished local cardiac inflammation following DXS use may partly be responsible for the observed reduction of atrial fibrillation. Indeed, the incidence of atrial fibrillation correlates with inflammation [13]. Nevertheless, an important proportion of hemodynamically relevant episodes of atrial fibrillation manifest themselves 48–72 h postoperatively. In the short post-CBP follow-up in the current study, no predictions can be made as to the possibly continued, indirect positive anti-arrhythmic effect DXS may have had in a later post-CBP phase. DXS administration is associated with significant attenuation of myocardial damage and complement deposition and evidently reduced CK-MB levels. Presently, although a trend towards less troponin I in the DXS group was observed, the differences did not reach statistical significance; possibly because of a too short observation period, the peak for troponin release being slightly delayed as compared to CK-MB.
With respect to the lungs, DXS treated animals revealed significantly less neutrophil infiltration, complement deposition and edema than controls, indicative of reduced inflammation and possibly endothelial damage. As a positive consequence thereof, pulmonary artery as well as right ventricular (RV) pressures were significantly less elevated after CPB in the DXS group compared to PBS controls.
Lung injury following CPB is multifactorial and includes sequelae associated with IR-injury, changes in the integrity of the bronchoalveolar architecture and infiltration by inflammatory cells [14]. Such injury represents an important cause of postoperative morbidity with high mortality, and may lead to respiratory failure. Indirect attenuation of pulmonary artery pressures, either by reducing the cuffing effect on the arterioles provoked by accumulated lung water, and/or preservation of diastolic LV function by DXS may therefore prove critical in a clinical setting. Furthermore, preservation of perioperative RV function by DXS may be central to ensure optimal postoperative recovery. Indeed, the use of DXS (MW 8000) during CPB in a pediatric population has been shown to ameliorate immediate post-CPB respiratory index [15].
A minimal extracorporeal circulation (MECC) system was used in our experiments. This system has proven to be safe and may also reduce morbidity as well as CPB-associated inflammation in humans [16]. Whilst MECC may not yet be as established as conventional ECC, precisely because of its reduced pro-inflammatory potential it was utilized in this study. Although differences in reduction of the anti-inflammatory potential of a substance may be more difficult to elucidate when the level of inflammation itself is possibly lower than in a conventional ECC system, any differences that are observed may be of even greater value. Indeed, DXS modulated several components of inflammation and coagulation.
Thrombin levels, measured as thrombin anti-thrombin (TAT) complexes, rapidly increase after CPB [17]. Reduction of thrombin generation by anti-thrombin III ameliorates IR-injury by attenuating leukocyte recruitment [18]. Inhibition of overall total thrombin production by DXS correlated with reduced cardiac and pulmonary neutrophil recruitment and may provide one mechanism by which damage was ameliorated.
Systemic complement inhibition in CPB, including soluble complement receptor type 1 [19] has proven beneficial in ameliorating post-CPB recovery. C5b-9 directly contributes to IR injury and lower levels are associated with reduced complications and better recovery post-CPB [20]. Furthermore, C5b-9 levels correlate with lung water accumulation and recovery of LV wall motion [19]. DXS reduced tissue deposition of all complement components, including C5b-9 in heart and lungs.
IL-6, an acute-phase inflammatory cytokine, is secreted by activated endothelial cells, amongst others. Plasma IL-6 is markedly increased following CPB and its production is stimulated by thrombin [21]. Reduction of plasma IL-6, possibly also in relation with a partial reduction in thrombin production by DXS, may therefore play an important role in curbing post-CPB inflammation. Furthermore, DXS also significantly reduced TNFalpha, IL-1beta and IL-8 levels, cytokines in part known to be activated and injurious during CPB and cardiac surgery in general.
Not only high plasma ET-1, but also elevated tissue ET-1 levels have been shown to predispose to alterations in pulmonary function and myocardial contractility [22]. Focally reduced tissue ET-1 levels after DXS treatment may therefore have contributed to partly improved cardio-pulmonary function post-CPB.
Of note, methylprednisolone (equivalent to 30 mg/kg) was administered to all animals prior to the experiment to prevent vasoplegia and hypotension as a reaction to CPB. Whilst various clinical studies [23], have shown a positive effect of methylprednisolone on reduction of inflammatory responses, the effect on clinical outcome is not so clear and certain patient populations may not necessarily benefit from its use [24]. Whilst a certain overall reduction in inflammation may be attributable to the use of methylprednisolone in this model, any methodological bias would be present in all experiments, including the controls. Whether DXS, in the applied dose, would suffice to reduce the inflammatory response in an equivalent manner in a protocol lacking methylprednisolone would have to be tested in further experiments.
4.1 Study limitations
As with all experimental models, the animals used in this CPB study were healthy. Furthermore, with limited numbers in each group, the power of this study is reduced. Additionally, the observation period post-CPB may have been too short to elucidate potentially significant, clinically relevant differences between the two groups. The use of hydroxyethyl starch may influence inflammation. However, comparable amounts were used in both groups, whereby observed differences between the groups are unlikely to be due to its use. With the aim to mimic a clinically realistic situation, we chose to adjust the use of vasoactives and volume to maintain a mean pressure of 50 mmHg, in effect however taking into account the limited expressiveness of these pressure-based functional measurements and the possibility of masking subtle influences of DXS upon function. Indeed load independent determination of cardiac function using a pressure-volume platform would have been able to provide more consistent and possibly more reliable data. However, despite these limitations, we made several reliable observations in a double-blinded pilot study with settings closely mimicking clinical reality. Indeed, in the complex setting of CBP-associated injury and inflammation, even small advances in ameliorating aspects of post-CBP damage may be of relevance for optimizing future treatment strategies.
In summary, the addition of low molecular weight dextran sulfate to standard blood cardioplegia significantly reduces local complement activation in the heart and lungs as well as the release of pro-inflammatory mediators. Furthermore, DXS in part improved the hemodynamic situation by ameliorating diastolic LV function and RV– and pulmonary artery pressures post-CPB. Whilst DXS has been used in a few patient trials [25], including CPB in children, [15] larger randomized clinical trials would be needed before judging the possible importance of the use of DXS in altering current clinical practice. However, in light of the fact that DXS at the current dose was not associated with any adverse events and was well tolerated, it remains to be substantiated whether a more aggressive treatment regimen, evaluated in a longer post-CBP follow-up and ultimately analyzed in human trials, may more markedly and sustainably improve post-CPB cardiac function and outcome.
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Acknowledgments |
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Footnotes |
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Yara Banz is a recipient of an MD-PhD grant of the Swiss Academy of Medical Sciences. This study was subsidized in part by the Swiss National Science Foundation, grant no. 3200B0-104228. |
References |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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