Ann Thorac Surg 2000;69:1121-1126
© 2000 The Society of Thoracic Surgeons
ORIGINAL ARTICLES: CARDIOVASCULAR
Ulinastatin attenuates reperfusion injury in the isolated blood-perfused rabbit heart
Zhi-Li Cao, MDa,
Yukio Okazaki, MDa,
Kozo Naito, MDa,
Tetsuya Ueno, MDa,
Masafumi Natsuaki, MDa,
Tsuyoshi Itoh, MDa
a Department of Thoracic and Cardiovascular Surgery, Saga Medical School, Saga, Japan
Address reprint requests to Dr Itoh, Department of Thoracic and Cardiovascular Surgery, Saga Medical School, 5-1-1 Nabeshima, Saga City, Saga 849-8501, Japan
e-mail: itoht2{at}post.saga-med.ac.jp
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Abstract
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Background. Ventricular dysfunction after long cardioplegic arrest has been observed in cardiac operations. Urinary trypsin inhibitor, also called ulinastatin, may attenuate myocardial ischemia-reperfusion injury. The present study was designed to determine the protective efficacy of ulinastatin in blood-perfused parabiotic isolated rabbit hearts as a surgically relevant model with long (4-hour) cardioplegic arrest.
Methods. Each isolated rabbit heart, with a latex balloon inserted in the left ventricle, was parabiotically blood-perfused using a modified Langendorff column. The left ventricular developed pressure, rate of pressure development, and coronary flow with a left ventricular end-diastolic pressure of 10 mmHg were measured before ischemia and 15, 30, 45, and 60 minutes after reperfusion began (control, n = 10). Ulinastatin (15,000 U/kg) was administered to the support animal just before reperfusion began (group U-1, n = 10) or at the beginning of the extracorporeal circulation and readministered before reperfusion (group U-2, n = 10). The endothelium of the coronary artery was observed by scanning electron microscopy to evaluate the extent of endothelial ischemia-reperfusion injury.
Results. Ulinastatin enhanced the recovery of developed pressure in both the U-1 (p < 0.05) and U-2 (p < 0.01) groups compared with the control group. Although ulinastatin given just before reperfusion (group U-1) did not enhance the recovery of the rate of pressure development or the coronary flow compared with the control, earlier administration did improve the recovery of the rate of pressure development compared with the control (U-2, p < 0.05), and there was improvement of the recovery of coronary flow after 60 minutes of reperfusion (U-2, p < 0.05). Scanning electron microscopy showed that ulinastatin had ameliorated coronary endothelial damage.
Conclusions. Ulinastatin improved functional recovery after long cardioplegic arrest and reduced coronary endothelial injury. Administration of ulinastatin at the beginning of cardiopulmonary bypass and just before reperfusion may be useful clinically in cases requiring prolonged aortic cross-clamping.
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Introduction
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Despite great advances in myocardial protection during cardiac operations, myocardial injury associated with prolonged aortic cross-clamping can cause postcardiotomy cardiogenic shock. Myocardial contractile dysfunction after ischemia-reperfusion has been thought to be caused by many interrelated factors. Leukocytes, especially polymorphonuclear neutrophils (PMNs), have been shown to have a significant effect on the development of reperfusion injury [1–4]. Conversely, cardiopulmonary bypass (CPB) causes the activation of PMNs and circulating adhesion molecules [5,6]. Activated PMNs can adhere to myocytes as well as to coronary endothelium, and the PMNs release a variety of cell-activating and cytotoxic substances, such as PMN elastase, complement products, cytokines, and free radicals, which can cause both endothelial and myocardial injuries. Therefore, methods that reduce ischemia-reperfusion injury of the coronary endothelium and myocytes that is caused by PMNs are needed to make cardiac procedures safer.
Urinary trypsin inhibitor, also called ulinastatin (Mochida Pharmaceutical Co, Ltd, Tokyo, Japan), is a protease inhibitor that is purified from the fresh urine of healthy men [7]. Ulinastatin decreases elastase release from PMNs [8] and suppresses the activity of PMN elastase [9]. It also stabilizes lysosomal membranes and suppresses the release of lysosomal enzymes [10]. Although several investigators have shown beneficial effects of ulinastatin on ischemia-reperfusion injury of the lung [11], liver [12], kidney [13], and heart [14], its cardioprotective effect after prolonged cardioplegic arrest in the blood-perfused heart has not been investigated yet. The aims of this study were to investigate the effects of ulinastatin on myocardial ischemia-reperfusion injury after 4 hours of cardioplegic arrest and to examine the optimal timing for its administration. We tested ulinastatin in an isolated parabiotic blood-perfused Langendorff system by measuring the left ventricular pressure-volume relationships and coronary flow rate. We also observed the damaged endothelium of the coronary artery by scanning electron microscopy.
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Material and methods
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Preparation of the support animals
Adult male Japanese white rabbits weighing 3.0 to 3.4 kg were used as support animals. The support animal was anesthetized intramuscularly with ketamine (50 mg/kg). A vascular access catheter was established by introducing an indwelling catheter (24G; Terumo, Tokyo, Japan) into the left ear vein for giving pharmacologic agents and lactated Ringer’s solution. The animal was anesthetized with pentobarbital sodium (5 to 7 mg/kg, intravenously) and pancuronium bromide (0.08 mg/kg, intravenously), and tracheostomy was done for mechanical ventilation support. After heparinization (500 U/kg) the right femoral artery was cannulated (18G, Terumo) for arterial blood pressure monitoring using a pressure transducer (model RM-6000; Nihon Kohden, Tokyo, Japan). The right carotid artery (18G, Terumo) and the left jugular vein (16G, Terumo) were cannulated to establish extracorporeal circulation. The support rabbit was ventilated (model SN-480-5, Shinano Co, Tokyo, Japan) with 100% oxygen throughout the experiment. Anesthesia was maintained by intravenous administration of ketamine (25 mg/kg).
Preparation of the donor animals
Adult male Japanese white rabbits weighing 2.7 to 3.0 kg were used as heart and blood donors. The heart donor animal was anesthetized and ventilated in the same way as the support rabbit. After heparinization (500 U/kg) the right carotid artery was cannulated for blood retrieval to prime the extracorporeal circuit. To save the blood donor animal, 45 mL of blood was retrieved from the heart donor animal after the infusions of lactated Ringer’s solution. Cardiectomy was done by median sternotomy.
Establishment of the blood-perfused, parabiotic, isolated heart Langendorff model
A modified Langendorff column (Fig 1) was primed with blood from the heart donor rabbit and heparinized (15 mg/column) lactated Ringer’s solution. The carotid artery cannula was connected to the pump (model MP-3; Rikakikai Co Ltd, Tokyo, Japan). Arterial blood was drained actively using this pump and was fed into a modified Langendorff apparatus. The height of the column was set at 80 cm H2O. The venous flow from the Langendorff column was returned to the jugular vein of the support animal using the same type of pump. The systolic arterial pressure of the support animal was maintained above 70 mm Hg, and the central venous pressure was maintained between 0 and 2 cm H2O. The perfusion temperature was maintained at 37°C.
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Fig 1. Modified Langendorff column used. Arterial blood from the support animal was used to perfuse the isolated heart. The height of the column was set at 80 cm H2O.
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The aorta of the isolated heart was cannulated, and coronary perfusion was instituted through the Langendorff column immediately after the cardiectomy. The chambers of the extracorporeal circuit were filled with the remaining blood from the heart donor animal. Left atriotomy was done, and a vent (20G, Terumo) was inserted into the left ventricle from the apex. A fluid-filled latex balloon was placed in the left ventricle through the mitral valve and secured with a purse-string suture around the mitral annulus. The balloon was connected to a pressure transducer (model RM-6000, Nihon Kohden) for measurement of the left ventricular pressure-volume relationships. The zero-pressure reference was set at the level of the aortic valve. The heart was paced (model EDP 30/s; Biotronik Inc, Berlin, Germany) at a constant rate (150 to 200 beats per minute) throughout the experiment. The coronary blood flow rate was directly measured by timed collection.
Experimental protocol
After a 30-minute equilibration period, the left ventricular pressure-volume relationships and coronary flow rate were measured as baseline data. Saline solution was infused into the intraventricular latex balloon to generate end-diastolic pressures (EDPs) of 5, 10, 15, 20, and 25 mm Hg. The left ventricular developed pressure (DP) and the rate of positive pressure development (dp/dt) at an EDP of 10 mm Hg were selected as representative data in this series. If the baseline DP at an EDP of 10 mm Hg was less than 70 mm Hg the heart was excluded from the study. After acquisition of the baseline data, the fluid in the latex balloon was adjusted to obtain an EDP of 0 mm Hg.
To achieve cardioplegic arrest similar to that of a clinical procedure 50 mL of St. Thomas’ Hospital solution (in mmol/L: NaCl, 110.0; NaHCO3, 10.0; KCl, 16.0; MgCl2, 16.0; and CaCl2, 1.2) at 4°C was infused from a height of 80 cm H2O through a separate column. The effluent from the coronary sinus was collected and discarded from the circuit. After cardiac arrest the hearts were immersed in saline solution at 20°C for 4 hours. The heart was given additional cardioplegic solution (25 mL) every 30 minutes during the 4 hours of global ischemia. Blood was recirculated continuously between the support rabbit and the circuit of Langendorff apparatus at a flow rate of 10 mL/minute to allow the potential activation of PMNs as in a clinical CPB operation.
Heparin (15 mg/column) was added after 4 hours of cardioplegic arrest. Then the heart was reperfused with blood for 60 minutes. The left ventricular pressure-volume relationships and coronary flow rate were measured 15, 30, 45, and 60 minutes after reperfusion began. Blood gas analysis was repeated to ensure the stability of the support animals throughout the experiments. At the conclusion of the experiment the cardioplegic solution was perfused into the isolated heart. Then the tissue was fixed by perfusing 2.5% glutaraldehyde in 0.1 M cacodylate buffer with 3% sucrose at a perfusion pressure of 80 cm H2O.
Experimental design
No ulinastatin was administered to the control group (n = 10). In the first test group, ulinastatin (15,000 U/kg) was administered to the support animal just before reperfusion began (group U-1, n = 10). In the second test group, ulinastatin (15,000 U/kg) was administered to the support animal at the beginning of the extracorporeal circulation (before ischemia) and also just before reperfusion began (group U-2, n = 10).
Observation of the coronary endothelium by scanning electron microscopy
The heart was stored in the same fixative until it was studied. After the heart was rinsed with 0.1 M cacodylate buffer it was dehydrated through an ethanol series and freeze dried. The tissue specimen was coated with gold (IB-3 ion coater; Eiko Ltd, Mito, Japan) and observed by scanning electron microscopy (JSM-5200LV; JEOL Ltd, Tokyo, Japan).
Statistical analysis
Results are expressed as the mean ± standard deviation. Statistical analyses were performed by analysis of variance (factorial ANOVA); when a significant F value was obtained, comparisons between groups were done by Scheffé test as a post-hoc test. Differences were considered significant at the level of p less than 0.05.
Animal care
All of the animals involved in this study received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication no. 86-23, revised 1985). All procedures were approved by the Animal Research Committee of the Saga Medical School.
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Results
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The conditions of the support animals were stable throughout the experiments. There were no significant differences among the three groups in baseline systolic blood pressures, blood gas data, and hemoglobin concentrations of the support animals (Table 1). During reperfusion, no significant differences were found in those values (Table 2).
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Table 2. Systolic Blood Pressure, Blood Gas Data, and Hemoglobin Concentrations of the Support Animals After 30 Minutes of Reperfusiona
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No hearts were excluded because of baseline DP lower than 70 mm Hg at an EDP of 10 mm Hg. No significant differences in the preischemia baseline data of the coronary flow rate, DP, and dp/dt at an EDP of 10 mm Hg were observed in the three groups (Table 3).
Recovery of developed pressure
The percentage recoveries of DP during reperfusion after 4 hours of cardioplegic arrest are shown in Figure 2. The control hearts without ulinastatin showed approximately 50% recovery compared with preischemia baseline data. Treatment with ulinastatin just before reperfusion (group U-1) enhanced the recovery of DP compared with the controls throughout the reperfusion period (p < 0.05). Treatment with ulinastatin at the beginning of the extracorporeal circulation and also just before reperfusion (group U-2) substantially improved the recovery of DP compared with the controls throughout the reperfusion period (p < 0.001). No significant differences were found between the DP recoveries of the U-1 and U-2 groups except at 30 minutes after reperfusion began.
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Fig 2. Percentage recoveries of developed pressure during reperfusion after 4 hours of cardioplegic arrest. *p < 0.05 versus control; **p < 0.001 versus control; ***p < 0.05 versus group U-1. (Control = no ulinastatin; U-1 = single dose of ulinastatin (15,000 U/kg) administered just before reperfusion began; U-2 = ulinastatin (15,000 U/kg) administered twice, once at the beginning of the extracorporeal circulation (before ischemia) and once just before reperfusion began.)
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Recovery of rate of developed pressure
The control hearts showed percent recoveries of dp/dt during reperfusion after 4 hours of cardioplegic arrest of approximately 50% compared with preischemia baseline dp/dt data (Fig 3). Treatment with ulinastatin at the beginning of the extracorporeal circulation and just before reperfusion (group U-2) significantly improved the recovery of dp/dt compared with the controls throughout the reperfusion period (p < 0.05 at 15 minutes, p < 0.01 at 30, 45, and 60 minutes). However, a single dose of ulinastatin just before reperfusion (group U-1) did not enhance the recovery of dp/dt compared with the controls throughout the reperfusion period. No significant differences were detected between the U-1 and U-2 groups except at 60 minutes after reperfusion began.
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Fig 3. Percentage recovery of rate of developed pressure (dp/dt) during reperfusion after 4 hours of cardioplegic arrest. *p < 0.05 versus control; **p < 0.01 versus control; ***p < 0.05 versus group U-1. Abbreviations as in Figure 2.
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Recovery of coronary flow
The percentage recoveries of coronary flow during reperfusion after 4 hours of cardioplegic arrest are shown in Figure 4. There were no significant differences between the U-1 and control hearts throughout the reperfusion period. In contrast, treatment with ulinastatin at the beginning of the extracorporeal circulation and just before reperfusion (group U-2) significantly improved the recovery of coronary flow compared with the control hearts only after 60 minutes of reperfusion (p < 0.05). No significant differences were detected between the U-1 and U-2 groups throughout the reperfusion period.
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Fig 4. Percentage recovery of coronary flow during reperfusion after 4 hours of cardioplegic arrest. *p < 0.05 versus control. Abbreviations as in Figure 2.
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Scanning electron microscopy of the coronary endothelium
After 4 hours of cardioplegic arrest followed by 60 minutes of reperfusion with blood, scanning electron microscopy showed that the coronary endothelial cells of the control hearts without ulinastatin were damaged (Fig 5). Many endothelial cells were detached and collagen fibers under the endothelial cells were exposed. Blood cells were deposited on the exposed collagen fibers. In comparison, the coronary endothelium of rabbits treated with ulinastatin at the beginning of the extracorporeal circulation and before reperfusion (group U-2) showed less damage and fewer delaminated endothelial cells (Fig 6). Although the damage seen in the scanning electron micrographs was not quantified in this series, the scanning electron microscopy findings suggest that treatment with ulinastatin reduces coronary endothelial damage.
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Fig 5. Scanning electron micrograph of the coronary endothelium after reperfusion with blood for 60 minutes after 4 hours of cardioplegic arrest, without ulinastatin (control). Many endothelial cells were delaminated. Blood cells were deposited not only in the delaminated area but also on the damaged endothelial cells. (A) original magnification x350. (B) original magnification x1,500.
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Fig. 6. Scanning electron micrograph of the coronary endothelium after reperfusion with blood for 60 minutes after 4 hours of cardioplegic arrest with ulinastatin administration (group U-2). Endothelial delamination was reduced compared with controls. Fewer platelets were deposited than in the controls, and platelets were only seen in the delaminated area. (A) original magnification x350. (B) original magnification x1,500.
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Comment
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Polymorphonuclear neutrophils have important effects in myocardial ischemia-reperfusion injury [1–4], and CPB activates PMNs and enhances circulating adhesion molecules [5,6]. Consequently it is important to protect the myocardium from activated PMNs during open heart operations. Although the biocompatibility of CPB with heparin-coated surfaces reduces complement activation [15], optimal biocompatibility of CPB has not been achieved yet. New strategies to reduce myocardial ischemia-reperfusion injury, especially with long cardioplegic arrest during CPB, should be investigated to reduce postoperative morbidity and mortality.
In the present study we found that ulinastatin attenuated ischemia-reperfusion injury in parabiotic blood-perfused isolated rabbit hearts; myocardial contractility improved and coronary endothelial damage was ameliorated. Ulinastatin decreases elastase release from PMNs [8] and suppresses the activity of PMN elastase [9]. It also decreases the blood level of PMN elastase after CPB [16]. In contrast, Shibata and colleagues [17] reported that ulinastatin had no effect on functional recovery or enzyme leakage when added to a cardioplegic or reperfusion solution. However, in that study, the isolated hearts were not reperfused with blood. It is likely, therefore, that ulinastatin cannot improve postischemia-reperfusion cardiac function unless PMNs are present during reperfusion. In the present study, the baseline data showed that ulinastatin did not affect cardiac contractility before ischemia. Ulinastatin may, therefore, specifically improve ischemia-reperfusion injury associated with PMNs.
Ulinastatin was administered just before reperfusion began in group U-1, whereas it was given before perfusion with the modified Langendorff column (extracorporeal perfusion), as well as just before reperfusion in group U-2. The administration of ulinastatin just before reperfusion appeared to be effective when the U-1 and control groups were compared; however, the U-2 group showed more improvement compared with the U-1 group, which suggests that activation of the PMNs during the 4 hours of circulation through the modified Langendorff apparatus was also attenuated by ulinastatin.
Because we could not measure the levels of PMN elastase, cytokines, or circulating adhesion molecules in the perfused blood because of the unavailability of antibodies for those substances in rabbit, the inflammatory response associated with extracorporeal circulation, which was simulated using a modified Langendorff column, was not evaluated in this study. It is likely that the PMNs were less activated in the modified Langendorff column than they would be in a CPB circuit because PMNs are highly activated by passage through the membranous oxygenator in the CPB circuit.
The coronary endothelial cells appeared to be severely damaged in the control hearts after 4 hours of cardioplegic arrest and 1 hour of blood reperfusion. In contrast, Tsao and colleagues [18] found only functional damage after occlusion of the left anterior descending artery for 90 minutes and reperfusion for up to 270 minutes without morphologic damage to the surface of the endothelium, observed by scanning electron microscopy, in cat. In the present study, the longer period of ischemia and extracorporeal circulation resulted in morphologic damage to the surface of the endothelium. Endothelial cells were delaminated and became detached where blood cells were deposited. These changes appeared to be compatible with the no-reflow phenomenon that occurs after ischemia and reperfusion, in which coronary flow was reduced after 60 minutes of reperfusion. Ulinastatin attenuated the morphologic ischemia-reperfusion injury of the endothelium, but this effect was not quantified in this study. It is important in cardiac operations to prevent ischemia-reperfusion injury of the coronary endothelium because it is related to the no-reflow phenomenon.
It is obvious that PMNs and related substances have important effects in ischemia-reperfusion injury. Practical methods of attenuating ischemia-reperfusion injury associated with PMNs in cardiac operations should be established to prevent postcardiotomy cardiogenic shock. Long periods of cardioplegic arrest cause not only longer myocardial ischemia but also heavy activation of PMNs, which are associated with longer extracorporeal circulation times. Ulinastatin may clinically attenuate ischemia-reperfusion injury especially in cases of prolonged cardioplegic arrest.
In conclusion, ulinastatin improved myocardial contraction after 4 hours of cardioplegic arrest followed by 60 minutes of reperfusion with blood. Damage to the endothelium was reduced by the administration of ulinastatin. The administration of ulinastatin before extracorporeal circulation as well as just before reperfusion may lead to less damage to the myocardium and coronary endothelium in the clinical setting.
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Accepted for publication September 28, 1999.
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Abstract
Introduction
