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Interact CardioVasc Thorac Surg 2008;7:785-788. doi:10.1510/icvts.2008.177576 © 2008 European Association of Cardio-Thoracic Surgery
The effect of sivelestat sodium on post-cardiopulmonary bypass acute lung injury in a neonatal piglet model
a Department of Pediatric Cardiac Surgery, Sakakibara Heart Institute, 3-16-1 Asahi-cho, Fuchu-si, Tokyo, 183-0003, Japan Received 11 February 2008; received in revised form 29 May 2008; accepted 9 June 2008
*Corresponding author. Tel.: +81-42-314-3111; fax: +81-42-314-3150.
Cardiopulmonary bypass may cause acute lung injury and can seriously affect postoperative outcome, especially in younger patients. A synthesized neutrophil elastase inhibitor, sivelestat sodium, may be most effective when used during cardiopulmonary bypass. After anesthesia induction, sivelestat (2 mg/kg/h) was given to the SS group (n=7), and 0.9% saline solution to the placebo group (n=7). Piglets were placed on hypothermic cardiopulmonary bypass and subjected to myocardial ischemia (2 h) induced by cold crystalloid cardioplegia. At 24 h after surgery, PaO2/FiO2 ratio and alveolar-arterial oxygen difference were significantly better in the SS group (379.1±93.9 mmHg and 250.5±89.3 mmHg) than the placebo group (232.4±105.3 mmHg, and 378.3±90.8 mmHg, P<0.05). Interleukin-8 level in the epithelial lining fluid was above the lowest standard in 6 out of 7 (4.5, 12.9, 24.6, 27.7, 37.7, and 159.8; mean=44.5±57.6 g/l) in the placebo group, and in 2 out of 7 (36.1 and 67.8 g/l) in the SS group (P<0.05). The median histological score of acute lung injury in the harvested lung was 3 (2–5) in the placebo group and 1 (1–5) in the SS group (P<0.05). Intraoperative administration of sivelestat effectively reduced neutrophil induction and activation in the lung and improved oxygenation after cardiopulmonary bypass in a piglet model.
Key Words: Sivelestat sodium; Neutrophil activation; Systemic inflammatory response; Cardiopulmonary bypass
Acute lung injury (ALI) is a core symptom of the systemic inflammatory response following cardiopulmonary bypass (CPB) and can seriously affect postoperative outcome. Younger patients are more susceptible to this response. Neutrophil induction and activation are thought to be the prime event of ALI. The degree of neutrophil induction is reported to predict postoperative oxygenation in pediatric patients undergoing heart surgery [1]. Neutrophil elastase is a cytotoxic enzyme that destroys components of connective tissue, and is considered to be a key enzyme in development of systemic inflammation and ALI [2]. A synthesized neutrophil elastase inhibitor, sivelestat sodium (SS), has recently become commercially available in Japan (Elaspol®, Ono Pharmaceutical Co., Osaka, Japan). This low-molecular weight antiprotease competitively inhibits the activity of neutrophil elastase [3]. However, little is known regarding its effect on post CPB inflammatory response and ALI, and no in vivo study using an infant lung model has been published. Since the primary pulmonary insult can be generated at the time of lung reperfusion, SS may be most effective when the maximal blood level is achieved during CPB, especially at the time of myocardial reperfusion [4]. The paper examines the effect of SS on ALI following CPB in a neonatal piglet model.
Fourteen neonatal piglets were randomly assigned to receive either SS or placebo using a computer-generated permutation table. Ages and body weight ranged from 7 to 14 (median=10) days and from 7.4 to 11.3 (mean= 9.7±1.3) kg, respectively. The piglet was anesthetized and intubated. The central venous and arterial monitoring catheters were inserted into the femoral vein and artery. Simultaneous with anesthesia induction, SS (2 mg/kg/h) was given to the SS group, and 0.9% saline solution to the placebo group. The statistician (M.A.) and the pathologist (T.M.) were blinded to the injection protocol. The operation was done through a median sternotomy. CPB was established with ascending aortic and right atrial cannulations, with left ventricular venting. Heparinized homologous blood, harvested from an adult pig on the same day, was added to the prime to achieve a hematocrit of about 25%. Once the predetermined flow (150 ml/kg/min) was achieved, the ascending aorta was clamped and cardioplegic arrest was induced by infusion of 20 ml/kg of crystalloid cardioplegic solution (Miotecter®, Mochida Pharmaceutical Co., Ltd., Tokyo, Japan). Piglets were cooled to 28 °C, and the recirculated blood was ultrafiltered to keep the hematocrit at the pre-CPB level. Cardioplegia (10 ml/kg) was given every 30 min. After 2 h of myocardial ischemia, the aortic clamp was removed. Dopamine (5 µg·kg–1·min–1) was then initiated, and was adjusted at the discretion of the anesthesiologist. Piglets were rewarmed to 35 °C and the CPB was discontinued.
Two hours after the myocardial reperfusion, bronchial epithelial lining fluid (ELF) was collected using a polyester probe (Micro Sampling Probe®, Olympus Medical Systems, Inc., Tokyo, Japan). This probe is attached to a stainless-steel guidewire, contained in a plastic sheath. It was advanced through the endotracheal tube into the terminal bronchus under fluoroscopic guidance. The inner probe was first advanced until it was outside of its sheath, remaining in this position for 10 s, and then withdrawn into the sheath. Both were removed from the endotracheal tube to avoid contamination. The polyester probe was cut from the tip of the guidewire, weighed (wet weight, unit=µg) using a precision laboratory scale (DRAGON 204, 4001®, Mettler-Toledo Co., Ltd., Shanghai, China), and then placed in 3 ml of saline. This was repeated three times and the absorbed ELF was released from the probe to saline by agitation using a vortex mixer. The probe was then taken out, dried for seven days, and weighed. The dilution ratio was calculated using the formula: [3000+(wet weight)–(dry weight)]÷[(wet weight–(dry weight)]. The same procedure was repeated at 6 h after myocardial reperfusion, and the piglet was extubated. Finally, the piglet was euthanized after performing blood gas analysis and hemodynamic measurements at 24 h. Both lungs were harvested; the left lung was dried for seven days and the wet-to-dry weight ratio was calculated. The right lung was fixed in 10% buffered formaldehyde. The entire fixed lung was cut into six sections with equal thickness, enabling gross examination of the lung parenchyma. One tissue block was taken from each segment, including grossly abnormal areas whenever present. It was stained with hematoxylin and eosin, and sent for microscopic evaluation by a blinded pathologist. Acute lung injury was graded from 0 (almost none) to 5 (severe infiltration) on the basis of neutrophil infiltration of lung alveoli (Fig. 1). Arterial blood gas analysis included arterial partial oxygen tension (PaO2), arterial partial carbon dioxide tension (PCO2), HCO3 partial pressure, pH, base excess, and hematocrit. The measurement was done after anesthesia induction, during CPB, 6 h after myocardial reperfusion, and 24 h after myocardial reperfusion. The arterio-alveolar oxygen difference (a-A DO2) was calculated as FiO2x(PB–PH2O)–PaCO2/R–PaO2, where FiO2=fraction of inspired oxygen, PB=barometric pressure (=760), PH2O= water vapor pressure (=47), and R=respiratory quotient (=0.8). The PaO2/FiO2 ratio and a-A DO2 were calculated by assuming FiO2=0.9. Blood samples were transferred to a sterile vacuum flask containing acid ethylenediaminotetraacetic, and immediately centrifuged. The separated plasma and the ELF dilution were frozen at
Piglets were treated in compliance with the Guide for the Care and Use of Laboratory Animals (NIH publication 85-23, revised 1985), and the protocol was approved by our Institutional Review Board.
SPSS statistical software for Windows (version 11.0; SPSS Inc., Chicago, IL, USA) was used for data analysis. Values are expressed as mean±standard deviation or median. Differences between groups were examined by Student's t-test (continuous variables) or
Results are summarized in Table 1. The operative parameters were all similar between the SS and placebo groups. There was no between-group difference in PO2 including PO2 measured 6 h after myocardial reperfusion. The only significant difference was observed 24 h after myocardial reperfusion, with the PO2 significantly better for the SS group than the placebo group. Throughout the protocol, PCO2 was similar between groups. Hemodynamic parameters including mean arterial pressure, mean central venous pressure, and heart rate were all similar between groups. These parameters were successfully maintained by using dopamine (  5 µg·kg–1·min–1) in all but one piglet in each group. Throughout the protocol, the hematocrit was kept at the pre-CPB level, and no significant between-group difference developed. The net water balance during the operation and the change in body weight were similar between groups. PaO2/FiO2 ratio was 512.0±214.5 at anesthesia induction and 274.4±70.7 at 6 h after myocardial reperfusion for the SS group and 424.9±104.5 and 96.6±44.5, respectively, for the placebo group. Alveolar-arterial oxygen difference was 105.0±13.0 at anesthesia induction and 274.4±70.7 at 6 h after myocardial reperfusion for the SS group and 96.6±44.5 and 327.6±140.5, respectively, for the placebo group. These differences were not significant. However, at 24 h after surgery, PaO2/FiO2 ratio and alveolar-arterial oxygen difference were significantly better in the SS group (379.1±93.9 mmHg and 250.5±89.3 mmHg) than the placebo group (232.4± 105.3 mmHg, and 378.3±90.8 mmHg, P<0.05) (Fig. 2). Interleukin-8 level in the ELF was above the lowest standard (62.5 pg/ml at measuring the diluted specimen in the saline) in 6 out of 7 (4.5, 12.9, 24.6, 27.7, 37.7, and 159.8; mean=44.5±57.6 g/l) in the placebo group, and in 2 out of 7 (36.1 and 67.8 g/l) in the SS group (P=0.0308;  2-test). Serum interleukin-8 level, however, did not exceed the lowest standard in any of the specimens. The median histological score of ALI was 3 (2–5) in the placebo group and 1 (1–5) in the SS group (P=0.0404; 2-test).
CPB can trigger a systemic inflammatory response. Younger patients are more susceptible to this response for several reasons including higher metabolic demands, reactive pulmonary vasculature, and immature organ systems [5]. The lung is both a source and a target of the inflammatory response to CPB. The inflammatory injury related to CPB results in increased pulmonary vascular resistance, decreased compliance, decreased functional residual capacity, increased ventilation–perfusion mismatch with intrapulmonary shunting, leakage of fluid into the interstitial space, and reduced surfactant activity [6]. Potential therapeutic options in the ICU for ALI include administration of corticosteroid, conservative fluid management, use of end-expiratory positive pressure and low tidal volume ventilation, nitric oxide, surfactant, prostaglandins, albuterol, acetylcysteine, and prone positioning [7]. Although these strategies reduce the symptoms of ALI, their effect is indirect and limited [8].
There is an increasing awareness that the human neutrophil is a key mediator of tissue destruction in post-CPB inflammatory responses, including ALI [9]. Neutrophils secrete oxygen species and proteolytic enzymes. Neutrophil elastase is considered to be one of the most potent proteolytic enzymes released by neutrophils, and it degrades elastin and other tissue components. Some proteases, including SS competitively inhibits neutrophil elastase, and this inhibition is refractory to inactivation by superoxide [3]. SS is usually administered at a dose of 0.2 mg·kg–1·h–1 for several days beginning on arrival in the intensive care unit. A study showed that 4-day administration of SS resulted in a significantly lower serum neutrophil elastase and interleukin-8, significantly lower respiratory index, and significantly higher PaO2/FiO2 after 24 h of treatment compared with the control group [11]. SS has been used in the clinical setting to treat adult patients with acute lung injury due to various reasons, including infection [12] and insult of major surgery [13]. On the other hand, it can be assumed that SS is especially effective in preventing development of ALI if the maximal blood concentration is achieved when the inflammatory cascade is initiated during CPB [14]. Interestingly, PO2/FiO2, and a-A DO2 were similar at 6 h but significantly better in the SS group at 24 h. We found that SS effectively reduced the sequel (depressed oxygenation 24 h postsurgically) of ALI, as is sometimes seen clinically. The histological findings were negligible in more than half of the specimens in the SS group. In the placebo group, lung neutrophil infiltration had increased to varying degree in all specimens. Bronchial ELF was harvested using a polyester (microsampling) probe. The probe is shielded in a plastic tube, and can be delivered to the terminal bronchus under fluoroscopic guidance without contamination. It allows collection of ELF without the need for saline administration, which is mandatory for bronchoalveolar lavage and, therefore, allows collection of the local ELF, which contains a high concentration of biochemical markers [15].
IL-8 chemo attracts neutrophils, and induces neutrophil degranulation and respiratory burst. The Quantikine porcine IL-8 immunoassay is enzyme-based and uses E. coli-expressed, recombinant porcine IL-8 and antibodies raised against it to measure porcine IL-8 in cell culture supernates and serum. Using this test, we found IL-8 was below the lowest standard in five piglets in the SS group and in one in the placebo. Thus, Overall, intraoperative administration of SS effectively reduced neutrophil induction/activation in the lung and improved oxygenation after CPB in a piglet model. Our encouraging results suggest that SS may be effective for small infants undergoing cardiac surgery and, therefore, further evaluation of its effectiveness is mandatory.
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Abstract
1. Introduction
1-proteinase, normally control elastase-mediated tissue damage by forming enzyme-inhibitor complexes. However, triggered neutrophils may create an environment that enables elastase to simultaneously oxidize and inactivate these inhibitory proteases 
nik J, Siniewicz K, Moll JA, Moll J, Baj Z, Sysa A, Zeman K. Effect of cardiopulmonary bypass on neutrophil activity in pediatric open-heart surgery. Arch Immunol Ther Exp (Warsz) 2005;53:272–277.