Ann Thorac Surg 2006;81:2167-2171
© 2006 The Society of Thoracic Surgeons
Original article: Cardiovascular
Resuscitation of Non-Beating Donor Hearts Using Continuous Myocardial Perfusion: The Importance of Controlled Initial Reperfusion
Satoru Osaki, MD,
Kozo Ishino, MD
*
,
Yasuhiro Kotani, MD,
Osami Honjo, MD,
Takanori Suezawa, MD,
Kazushige Kanki, MD,
Shunji Sano, MD
Department of Cardiovascular Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan
Accepted for publication January 18, 2006.
* Address correspondence to Dr Ishino, Department of Cardiovascular Surgery, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama City, 700–8558, Japan. (Email: ishino{at}tb3.so-net.ne.jp).
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Abstract
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BACKGROUND: Warm ischemia is a major cause of cardiac allograft failure in transplants from non–heart-beating donors. To minimize myocardial ischemia, we used a continuous myocardial perfusion technique for resuscitation of donor hearts. The purpose of the present study was to investigate an optimal duration of controlled initial reperfusion.
METHODS: Cardiac arrest was induced by asphyxia in 18 donor pigs. The hearts were harvested 30 minutes after global warm ischemia. Continuous myocardial reperfusion was immediately commenced from the aortic root with blood cardioplegic solution (20°C, 40 mm Hg) and then with oxygenated blood (20° to 37°C, 40 to 60 mm Hg). Animals were divided into three groups according to the duration of the initial reperfusion: group I = 5 minutes, group II = 20 minutes, and group III = 60 minutes. Orthotopic transplantation was performed while keeping the heart beating by continuous myocardial perfusion. Cardiac function was evaluated before anoxia and after transplantation. Lactate extractions were determined during reperfusion. Myocardial edema was assessed by heart weight and posterior wall thickness of the left ventricle.
RESULTS: Recovery rates of cardiac function in group II hearts after transplantation were better than in groups I and III (cardiac output, 61% ± 9% versus 41% ± 5% versus 44% ± 4%, respectively; p < 0.05; left ventricular end-systolic pressure–volume ratio, 64% ± 8% versus 36% ± 9% versus 42% ± 6%, respectively; p < 0.05). Lactate extractions in groups II and III returned to 0 within 20 minutes of reperfusion. Myocardial edema after transplantation in group II hearts was less than in groups I and III.
CONCLUSIONS: The best recovery was observed in the non-beating donor hearts resuscitated by continuous myocardial perfusion when the initial controlled reperfusion with lukewarm blood cardioplegic solution at 40 mm Hg lasted for 20 minutes.
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Introduction
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Non-beating donor hearts (NHBDs) have been proposed as a means to expand the donor pool for heart transplantation. Because severe myocardial damage as a result of hypoxic perfusion during the agonal period and an indeterminate period of warm ischemia is unavoidable, this strategy has not yet been used beyond the laboratory. Previous studies have clearly demonstrated that death owing to respiratory arrest in a possible cardiac allograft NHBD results in more severe depletion of myocardial energy stores than death owing to bleeding [1]. Although Shirakura and associates [2] reported satisfactory functional recovery of canine hearts after a 24-hour period of preservation in an agonally arrested transplantation model and Gundry and colleagues [3] achieved long-term survival of baboons receiving transplantation of hearts harvested from asphyxiated NHBDs, the successful outcomes of these experiments are largely attributable to the application of ethically unacceptable multiple pretreatments.
Investigators in the 1960s attempted to resuscitate canine and human cadaver hearts by resumption of coronary perfusion with normothermic oxygenated blood using either the host animal for canine hearts [4] or heart-lung machines in the case of human donors [5]. When cardiomyocytes are reoxygenated after a prolonged period of energy depletion, severe cytosolic calcium overload and reactivation of energy production cause deleterious hypercontracture ("oxygen paradox" [6]). Therefore, the initial reperfusion should maintain cardiac arrest and prevent this hypercontracture, allowing a brief interval during which ionic balance can recover more normally during immediate reoxygenation.
To reduce ischemia we resuscitated and preserved cadaver hearts by a continuous myocardial perfusion technique, but without use of any cardioprotective pretreatments. The present study was undertaken to determine an optimal duration of controlled initial reperfusion with leukocyte-depleted hyperkalemic blood cardioplegia for hearts harvested from asphyxiated NHBDs in a pig transplantation model as a first step toward clinical use of NHBD cardiac allografts.
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Material and Methods
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Animal Preparation
All experimental animals were cared for in accordance with institutional guidelines and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication 86-23, revised 1985). The experimental protocol was approved by the Okayama University School of Medicine Experimental Animals Committee. Eighteen weight-matched pairs of Yorkshire pigs (male or female, 20 ± 2 kg) were used. After premedication with an intramuscular injection of 10 mg/kg ketamine hydrochloride, an ear vein was cannulated and anesthesia was induced with 50 mg of thiamylal sodium and 0.5 mg of atropine sulfate and maintained with isoflurane inhalation (0.5% to 2.0%) and 0.2 mg/kg pancuronium. An endotracheal tube was inserted by means of a tracheostomy, and mechanical ventilation was begun with a tidal volume of 10 mL/kg.
Surgical Procedure
After median sternotomy, 500 U/kg heparin was given intravenously. Catheters were inserted into the carotid artery, the internal jugular vein, and the left atrium for pressure monitoring. A 5F Swan-Ganz catheter was inserted into the main pulmonary artery for the measurement of cardiac output. A 3F pressure-tip catheter (Miller Instruments, Houston, TX) and a conductance catheter (2S-RH6DA-116, Alpha Medical Instruments, Mission Viejo, CA) were inserted from the apex into the left ventricle. The left azygos vein, which drains into the coronary sinus, was ligated. A 3F catheter was placed in the coronary sinus to obtain blood samples. A cardiopulmonary bypass circuit incorporating a pediatric oxygenator (D902 Lilliput 2, Dideco, Mirandola, Italy) was filled with 500 mL of donated blood, 10,000 U of heparin, and 500 mg of methylprednisolone. Cardiopulmonary bypass was established by bicaval and aortic cannulation and was maintained with a flow rate of 75 mL/min per kilogram. At the end of each experiment, the animal was killed by an intravenous injection of potassium chloride.
Experimental Protocol
Asphyxiation was induced by turning the ventilator off. After cardiac arrest was achieved with standstill or ventricular fibrillation, the animals were left for 30 minutes in room temperature. The heart, including both atria, ventricles, and the aortic arch, was excised and weighed. After insertion of a perfusion cannula into the aortic root, the ascending aorta was clamped and then isolated continuous myocardial perfusion was initiated with blood cardioplegia at 20°C with a perfusion pressure of 40 mm Hg (Fig 1). The blood cardioplegia was composed of leukocyte-depleted oxygenated blood mixed with a modified St. Thomas's Hospital solution (20% D-mannitol 100 mL, sodium bicarbonate 19 mEq, potassium chloride 18.5 mEq, magnesium sulfate 37 mEq in 1 L of 5% glucose) in a 4:1 ratio. Animals were divided into three groups (n = 6 each) according to the duration of the controlled initial reperfusion: group I = 5 minutes, group II = 20 minutes, and group III = 60 minutes. After the initial reperfusion, myocardial perfusion was stopped for 30 seconds to weigh the heart. Myocardial perfusion was then resumed with leukocyte-depleted oxygenated blood at 20°C with a perfusion pressure of 40 mm Hg. After 20 minutes the perfusion pressure was elevated to 60 mm Hg and the temperature was increased stepwise to 37°C during a period of 30 minutes. At this stage the orthotopic heart transplantation was performed, with the heart beat being maintained by continuous myocardial perfusion. Two hours after the aorta was released from the clamp, animals were weaned from cardiopulmonary bypass with an infusion of 0.1 µg/min per kilogram epinephrine.
Cardiac Function, Lactate Level, and Myocardial Edema
Cardiac function was assessed before induction of anoxia as control value. Because respiratory function after transplantation deteriorated with time, probably owing to left ventricular dysfunction, measurements were repeated 1 hour after weaning from cardiopulmonary bypass. Cardiac output was measured by the thermodilution method. The maximal rate of increase of left ventricular pressure was obtained from a continuous pressure curve recorded using a high-fidelity micromanometer catheter. Left ventricular (LV) end-systolic pressure–volume ratio (LV Emax) was calculated from pressure–volume relation curves during transit volume unloading induced by the occlusion of both venae cavae. Calibration for blood conductivity was performed just before each measurement. Blood samples were obtained from the aorta and the coronary sinus to measure lactate concentrations before anoxia (control value), at 1 minute, every 10 minutes to the end of the controlled initial reperfusion, and subsequently at 30-minute intervals. Lactate extraction (E) was calculated by the following equation: E (%) = (A – V)/A x 100, where A is the arterial substrate concentration and V is the coronary sinus substrate concentration. Myocardial edema was assessed by the wet weight of the heart and posterior wall thickness of the LV. The wet weight including the aortic arch was measured at three time points: after harvesting (control value), at the end of the controlled initial reperfusion, and at the end of the experiment. The posterior wall thickness was measured by surface echocardiography at short-axis imaging of the LV at the level of the papillary muscles before anoxia (control value) and after transplantation.
Statistical Analysis
Wilcoxon's signed-rank test was used for comparison of pretransplant and posttransplant values. Statistically significant differences among groups were determined by one-way analysis of variance, followed by Scheffe's test for multiple comparisons. A p value less than 0.05 was considered to be statistically significant. Results are given as mean ± standard deviations.
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Results
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Arrest time (time from turning the ventilator off to cardiac arrest), warm ischemic time, isolated myocardial perfusion time, transplantation time, and cardiopulmonary bypass time did not differ among the groups (Table 1). Control values of each variable for cardiac function before induction of anoxia also did not differ among the groups (Table 2). All transplanted animals survived for 1 hour after weaning from cardiopulmonary bypass. Recovery rates of cardiac output, maximal rate of increase of left ventricular pressure, minimal rate of increase of left ventricular pressure, and LV Emax after transplantation in group II were significantly better than in the other two groups, although they remained at approximately 60%.
Lactate extraction is shown in Figure 2. A massive release of lactate was observed at the beginning of the controlled initial reperfusion in all groups. Transmyocardial lactate difference decreased gradually and reached insignificant levels by 20 minutes in groups II and III. The percentage change in heart weight after the initial controlled reperfusion and after transplantation as compared with after 30 minutes of global ischemia is shown in Figure 3. The heart weight increased according to the duration of the initial controlled reperfusion. Although the weight further increased after transplantation in all groups, the rate of weight gain in group II hearts was significantly less than those in the other groups (p < 0.05). In addition, the percentage increase in LV posterior wall thickness after transplantation in group II hearts was significantly less than those in the other groups (group I = 35% ± 4%, II = 20% ± 4%, III = 34% ± 6%; p < 0.05).
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Fig 2. Lactate extraction before anoxia, at 1 and 5 minutes, every 10 minutes to the end of the controlled initial reperfusion, and subsequently at 30-minute intervals. *p < 0.05 versus control value. (Group I, open bars; group II, shaded bars; group III, solid bars.)
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Fig 3. Percent change in heart weight after controlled initial reperfusion (open bars) and after transplantation (solid bars). *p < 0.05 versus groups I and III.
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Comment
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Although a number of approaches to procurement of cadaver hearts have been studied in the laboratory, understanding of the outcomes is confused by several experimental factors, including species of animals used, mode of death (exsanguination or asphyxia) and varied period of postmortem warm ischemia. Tam and coworkers [5] showed that human cadaver hearts were more amenable to ischemic injury than canine hearts, when exposed to 60 minutes of ischemia after arrest. Lundsgaard-Hansen and colleagues [1] found that exsanguination maintained better myocardial metabolic state than asphyxia in cadaver rabbit hearts. Regarding postmortem ischemic time, Casavilla and associates [7] reported important information from their clinical experiences with liver and kidney transplantation from NHBDs whose families requested that life support be withdrawn or who experienced cardiac arrest after brain death (categories 3 and 4: The Maastricht International Workshop [8]). In the 24 patients in that study, the mean interval between cardiac arrest and thoracic aorta cross-clamping was 24 minutes in category 3 and 37 minutes in category 4. Therefore, we used pigs in this study because of similarity of the myocardium and the coronary anatomy to that of humans, and, to mimic category 3 NHBD, we used the asphyxiation model and set the duration of postmortem ischemia at 30 minutes.
The NHBD would have inevitably suffered a profound myocardial injury during hypoxic perfusion before arrest as well as indeterminate period of warm ischemia after arrest. Preservation of NHBD cardiac graft with simple cardioplegic flush and hypothermic nonperfused storage has allowed functional recovery only when multiple cardioprotective pretreatments are used [2, 3]. Before clinical application of this treatment strategy, therefore, establishment of legally defensible and effective techniques for resuscitation of the cardiac allograft and reliable methods for evaluating its transplantability is essential. In the present study, we induced cardiac arrest by asphyxiation without the use of any pretreatment except anticoagulation and, to avoid additional myocardial ischemia, resuscitated the hearts by continuous myocardial perfusion with blood cardioplegia and subsequently with oxygenated blood. The controlled initial reperfusion is critically important to prevent reoxygenation-induced hypercontracture after global warm ischemia and to protect the myocardium against ischemia–reperfusion injury. Although at the cellular level reoxygenation-induced hypercontracture can be prevented if the contractile apparatus is blocked for the first 15 minutes after global ischemia [9], the optimal duration of the controlled reperfusion in whole ischemic heart has not been investigated. Our results demonstrated that a 20-minute initial reperfusion with blood cardioplegia led to the best posttransplant recovery of NHBD hearts, when compared with 5-minute and 60-minute perfusions.
Myocardial metabolic products play a role in the development of ischemic damage. Increased lactate and the associated rise in cytosolic reduced nicotinamide adenine dinucleotide were shown to inhibit glycolysis and reduce anaerobic adenosine triphosphate production [10]. Neely and colleagues [11] demonstrated that the decrease in contractile force of ischemic hearts was associated with increased tissue lactate and that the onset of irreversible damage was also related to the continued presence of high lactate levels. Although a cause-and-effect relationship between tissue lactate and irreversible myocardial damage has not been established, the harmful effects of lactate accumulation depend on the exposure of the heart to high lactate levels not only during ischemia but also during reperfusion [12]. Thus, adequate washout of increased tissue lactate during reperfusion may lead to better functional recovery. In the present study, a massive release of lactate was observed at the initiation of reperfusion in all hearts, but the transmyocardial lactate difference was normalized within 20 minutes of the initial controlled reperfusion. It is likely that the hearts undergoing a 5-minute controlled reperfusion had insufficient washout of lactate, and therefore, their posttransplant recovery rate was worse than those undergoing a 20-minute reperfusion.
Several investigators have demonstrated that myocardial edema is associated with cardiac dysfunction [13, 14]. As myocardial edema accumulates within the interstitial spaces, interstitial pressure rises, thus increasing the stiffness and decreasing the compliance of both ventricles. We evaluated myocardial edema as the percentage increase in weight of the explanted heart and also by the percentage increase in LV posterior wall thickness. Although heart weight did not increase when the initial controlled reperfusion was performed for 5 or 20 minutes, the hearts undergoing 60 minutes of reperfusion showed significant weight gain. Mehlhorn and coworkers [15] previously demonstrated that when continuous blood cardioplegia was delivered for 60 minutes in arrested canine hearts, blood cardioplegia itself caused cardiac dysfunction owing to myocardial edema caused by the combination of increased myocardial fluid filtration and decreased cardiac lymph drainage in the absence of rhythmic cardiac contraction. After transplantation, the hearts undergoing 5-minute and 60-minute controlled reperfusions developed severe myocardial edema, probably caused by ischemia–reperfusion injury as a result of inadequate myocardial protection during the controlled reperfusion. Our results indicate that a 20-minute controlled reperfusion with blood cardioplegia leads to better preservation of cardiac function, at least in part, by preventing myocardial edema.
Teoh and associates [16] showed in 1986 that, in cardioplegic-arrested hearts, terminal warm-blood cardioplegia accelerated myocardial metabolic recovery and preserved high-energy phosphate content. In case of NHBD hearts undergoing global warm ischemia, however, an optimal temperature of blood cardioplegia for the initial reperfusion has not been determined. Suehiro and colleagues [17] resuscitated exsanguinated canine NHBD hearts after 60 minutes of ischemia by warm-blood cardioplegia before cold storage and again before aortic unclamping at transplantation. Martin and coworkers [18] used an exsanguinated pig NHBD model in which they clearly demonstrated that controlled reperfusion with cold-blood cardioplegia (4°C) immediately after a 30-minute warm ischemia and a second controlled reperfusion with lukewarm-blood cardioplegia (20°C) at transplantation preserved the cardiac function better than two reperfusions with warm-blood cardioplegia. Because of the excellent cardioprotective property of lukewarm-blood cardioplegia [19] together with the experimental results of Martin and colleagues [18], we used a temperature of 20°C for the controlled initial reperfusion with blood cardioplegia in our pig NHBD model.
The ideal pressure of initial reperfusion that minimizes microvascular fluid filtration and ensures homogeneous myocardial perfusion to prevent ischemia has not been determined. Okamoto and coworkers [20] showed that after 4 hours of left descending coronary artery ligation of canine beating hearts, gentle coronary reperfusion (40 to 50 mm Hg) with normal blood provided better recovery of contractility and less edema than sudden reperfusion (80 mm Hg). Li and associates [21] demonstrated that in isolated pig hearts arrested by cardioplegia and preserved by continuous myocardial perfusion with 10°C blood cardioplegia for 6 hours, perfusion at 40 cm H2O provided better functional recovery, more coronary flow, less coronary vascular resistance, and lower magnitude of lactate release than perfusion at 80 cm H2O. In the present study, therefore, we set the pressure of the initial reperfusion and subsequent oxygenated blood perfusion at 40 mm Hg.
In conclusion, without the use of any cardioprotective pretreatment, resuscitation of pig NHBD hearts harvested after 30 minutes of global warm ischemia is feasible by means of continuous myocardial perfusion, although the recovery rate of cardiac function is at best approximately 60%. Our results indicate that the controlled initial reperfusion with leukocyte-depleted lukewarm-blood cardioplegia at 40 mm Hg for 20 minutes provides better recovery of cardiac function, less myocardial edema, and lower magnitude of lactate release than if the initial reperfusion lasted for 5 or 60 minutes. Adequate recovery of the NHBD hearts for successful transplantation might be possible by supplementation of the reperfusate with some cardioprotective agents such as a free radical scavenger and also by prolongation of resuscitation by continuous myocardial perfusion.
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Abstract
Introduction
