Ann Thorac Surg 1997;63:1664-1668
© 1997 The Society of Thoracic Surgeons
Original Article: Cardiovascular
Intravenous Phenylephrine Preconditioning of Cardiac Grafts From Non–Heart-Beating Donors
Jeffrey T. Cope, MD,
Michael C. Mauney, MD,
David Banks, BS,
Oliver A. R. Binns, MD,
Christopher L. Moore, BS,
Jeffrey J. Rentz, BS,
Kimberly S. Shockey, MS,
R. Christoper King, MD,
Irving L. Kron, MD,
Curtis G. Tribble, MD
Division of Thoracic and Cardiovascular Surgery, Department of Surgery, University of Virginia Health Sciences Center, Charlottesville, Virginia
Accepted for publication December 14, 1996.
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Abstract
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Background. Hypoxia and warm ischemia produce severe injury to cardiac grafts harvested from non–heart-beating donors. To potentially improve recovery of such grafts, we studied the effects of intravenous phenylephrine preconditioning.
Methods. Thirty-seven blood-perfused rabbit hearts were studied. Three groups of non–heart-beating donors underwent intravenous treatment with phenylephrine at 12.5 (n 8), 25 (n 7), or 50 µg/kg (n 7) before initiation of apnea. Non–heart-beating controls (n 8) received saline vehicle. Hypoxic cardiac arrest occurred after 6 to 12 minutes of apnea, followed by 20 minutes of warm in vivo ischemia. A 45-minute period of ex vivo reperfusion ensued. Nonischemic controls (n 7) were perfused without antecedent hypoxia or ischemia.
Results. Phenylephrine 25 µg/kg significantly delayed the onset of hypoxic cardiac arrest compared with saline controls (9.6 0.5 versus 7.7 0.4 minutes; p 0.00001), yet improved recovery of left ventricular developed pressure compared with saline controls (57.1 5.3 versus 41.0 3.4 mm Hg; p 0.04). Phenylephrine 25 µg/kg also yielded a trend toward less myocardial edema than saline vehicle (p 0.09).
Conclusions. Functional recovery of nonbeating cardiac grafts is improved by preconditioning. We provide evidence that the myocardium can be preconditioned with phenylephrine against hypoxic cardiac arrest.
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Introduction
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Cardiac transplantation in the United States is plagued by a serious crisis of organ shortage. Although the number of patients undergoing heart transplantation annually in this country has remained at a plateau over the last several years, the number of individuals in need of a transplant has risen steadily [1]. As a result, waiting-list deaths have risen as high as 40% at some centers [2, 3]. To supplement the critical shortage of organs available from conventional brain-dead donors, there has been renewed interest in the potential procurement of cardiac grafts from non–heart-beating donors (NHBDs). Non–heart-beating donors are defined as patients who become eligible for organ donation after declaration of cardiopulmonary death rather than brain death. A potential NHBD includes an apneic individual in a persistent vegetative state who does not fulfill the strict criteria for brain death, but from whom the patient's family and physicians have elected to terminate life-sustaining measures. After withdrawal of mechanical ventilatory support, hypoxic cardiac arrest would ensue. The patient would be declared dead by cardiopulmonary criteria and thus become an eligible NHBD. The onset of hypoxic cardiac arrest in such patients heralds the inception of a period of global, warm, in vivo ischemia, during which the organs are explanted as rapidly as possible to minimize ischemic injury.
As demonstrated by recent studies from our laboratory, the sequential hypoxic and warm ischemic intervals inherent to an NHBD harvest combine to produce a unique and severe myocardial injury [4, 5]. The profound graft dysfunction that results from this injury has dissuaded clinicians from using NHBDs for human cardiac transplantation, although a few reports have appeared in the recent literature claiming successful transplantation of non–heart-beating cardiac grafts in the experimental setting [6–9]. As such, the development of cardioprotective interventions applied at specific times before, during, or after the actual NHBD procurement procedure will be necessary to preserve the viability of these hearts for transplantation. The period just before withdrawal of ventilation presents an opportunity to pretreat NHBDs in an effort to render the hearts more resistant to the deleterious consequences of the ensuing hypoxic and ischemic periods.
Preconditioning is a term originally used to describe the ability of a brief (5 minute) ischemic episode to induce an endogenous adaptive response in the myocardium, allowing it to better withstand an ensuing ischemic interval of longer duration []. Numerous pharmacologic agents, particularly 
1-adrenoceptor agonists, when given a few minutes before extended ischemia mimic the effects of ischemic preconditioning by delaying the onset of myocardial necrosis and improving the recovery of postischemic cardiac function [11–17]. This so-called pharmacologic preconditioning has been proposed as a potential cardioprotective intervention to use before clinical events in which there is a planned ischemic episode, such as elective cardiac operations, percutaneous transluminal coronary angioplasty, or transplantation. Because of the scheduled nature of an NHBD harvest, pharmacologic preconditioning is an attractive pretreatment option for preserving postischemic graft function. We hypothesized that intravenous pretreatment of NHBDs with the 1-adrenoceptor agonist phenylephrine before termination of mechanical ventilation would protect these cardiac grafts against injury incurred during the incipient period of global, warm in vivo ischemia. To test our hypothesis, we used a rabbit model of NHBDs and studied graft function on an ex vivo blood-perfused isolated heart apparatus.
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Material and Methods
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Adult New Zealand white rabbits were used for all protocols. The Animal Review Committee of the University of Virginia reviewed and approved the protocols for this study. All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH publication 86-23, revised 1985).
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Preparation of Isolated Heart Donors
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Intramuscular xylazine and ketamine were administered to adult (2.8 to 3.0 kg) New Zealand white rabbits of either sex. All animals underwent tracheostomy and volume ventilation (12 mL/kg) with 100% oxygen, followed by placement of a femoral artery catheter (18-gauge) for monitoring of blood pressure and heart rate. Intravenous metocurine (0.2 mg/kg) was given to achieve pharmacologic paralysis.
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Experimental Design
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An isolated heart apparatus was assembled in a manner identical to that described recently [4], using a support rabbit system for the continuous provision of fresh arterial blood perfusate. Five groups of rabbits were studied. A nonischemic control group (n = 7) underwent paralysis, systemic heparin administration (2,000 U intravenously), cannulation of the ascending aorta, and immediate perfusion on the isolated heart circuit without any antecedent hypoxia or ischemia. Four groups of NHBDs were prepared as described in the previous section, except that before termination of ventilation, the animals received a predetermined intravenous dose of phenylephrine HCl (PE) or 0.9% saline solution. Phenylephrine 1% (10 mg/mL; American Regent Laboratories, Shirley, NY) was diluted in the appropriate volume of 0.9% saline solution to yield final concentrations of 250, 500, and 1,000 µg/mL. Fifteen minutes before the planned induction of pharmacologic paralysis and immediate ventilatory withdrawal, three NHBD groups received intravenous pretreatment with a consistent volume (0.5 mL/kg) of one of the three PE solutions. These solutions were administered as a rapid bolus into the marginal ear vein at final doses of 12.5 µg/kg (n = 8), 25 µg/kg (n = 7), or 50 µg/kg (n = 8). These dosages and the 15-minute "washout" period before termination of ventilation were based on a recent investigation in which intravenous PE preconditioning was applied to an open-chest rabbit model of regional ischemia [17]. A fourth group of NHBD saline-treated controls (SC, n = 7) was pretreated with 0.9% saline vehicle only. Systolic blood pressure, diastolic blood pressure, and heart rate were recorded immediately before both pretreatment and ventilatory withdrawal. Also recorded were the peak blood pressures and the lowest heart rate attained by each animal after treatment with PE or 0.9% saline solution.
Cessation of mechanical ventilation initiated a period of systemic hypoxia, which ended in cardiac arrest within 6 to 12 minutes. The onset of hypoxic cardiac arrest was heralded by loss of the femoral arterial pressure waveform. Heparin sodium (2,000 U intravenously) was administered at the time of cardiac arrest and was circulated with 2 minutes of chest compressions at a consistent rate and amplitude, as determined by the femoral arterial pressure waveform. This practice was based on the policy of our institution's medical ethics committee, which forbids prearrest heparin treatment in the clinical setting because it could be argued that systemic anticoagulation in a vegetative patient could induce intracranial hemorrhage and thus convert a sublethal brain injury into brain death.
After the onset of hypoxic cardiac arrest, all NHBD groups were subjected to a 20-minute period of warm in vivo global myocardial ischemia to simulate the amount of time needed in the clinical setting of a non–heart-beating harvest to perform a median sternotomy and explant the donor heart. Just before the end of the 20-minute ischemic period, a sternotomy was performed, a left atrial blood gas sample was collected, and a saline-filled glass cannula was inserted into the ascending aorta of the donor heart. At 20 minutes of ischemia, NHBD hearts were excised and immediately reperfused ex vivo on the blood-perfused isolated heart apparatus described earlier. After 45 minutes of reperfusion, left ventricular developed pressure, percentage myocardial water content, and myocardial oxygen consumption were determined as described previously [4]. The experimental design for the NHBD groups is depicted in Figure 1
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Statistical Analyses
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All results are expressed as mean ± standard error of the mean. Data were analyzed for between-group differences using analysis of variance. The post hoc test of Tukey's multiple comparisons or planned comparison test of hypothesis was used where appropriate. Significant differences were identified with a confidence level of p less than 0.05. All statistical analyses were performed using Statistica software (Statsoft, Tulsa, OK).
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Results
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Systolic and diastolic blood pressure data for the four groups of NHBDs at baseline (just before pretreatment with PE or saline), shortly after treatment, and at the time of ventilatory withdrawal are outlined in Table 1. Baseline mean systolic and diastolic arterial pressures were similar among the groups. The administration of 0.5 mL/kg saline to the SC group did not change arterial pressure from baseline, whereas PE treatment at 12.5, 25, and 50 µg/kg yielded increases above baseline of 35%, 53%, and 73% in systolic pressure and 52%, 61%, and 82% in diastolic pressure, respectively. However, by the end of the 15-minute washout period (ventilatory withdrawal), systolic and diastolic pressures in all groups had returned to approximately baseline values, and none were significantly elevated above those of SC.
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Table 1. . Mean Systolic and Diastolic Arterial Pressures at Baseline, Just After Treatment with Phenylephrine or Saline Solution, and at Ventilatory Withdrawal
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The time required to produce hypoxic cardiac arrest after terminating ventilation in NHBDs is presented in Figure 2. As illustrated, treatment with PE 25 µg/kg significantly prolonged the time to hypoxic cardiac arrest when compared with SC (9.6 ± 0.5 versus 7.7 ± 0.4 minutes; p = 0.005 by analysis of variance). However, despite this difference in hypoxic intervals, all NHBD groups sustained equivalent degrees of hypercarbia, acidemia, and hypoxemia, as demonstrated by analysis of left atrial blood gases collected at the time of explantation (Table 2).
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Fig 2. . Time (minutes) required to produce hypoxic cardiac arrest after withdrawal of ventilation from non–heart-beating donors. Phenylephrine (PE) 25 µg/kg versus saline controls (SC), p = 0.005 by analysis of variance.
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Early in the reperfusion period, 1 of the SC hearts sustained severe ischemic contracture, precluding any attempts at intraventricular balloon placement. As a result, this heart was excluded from reperfusion data analysis, leaving a total of 7 hearts in the SC group for final analysis of left ventricular developed pressure, myocardial water content, and myocardial oxygen consumption. Figure 3 depicts mean left ventricular developed pressure for all five groups after 45 minutes of reperfusion. Although all NHBD groups had a significantly lower mean left ventricular developed pressure than nonischemic controls (p = 0.00001 by analysis of variance), treatment with PE 25 µg/kg yielded significantly improved left ventricular functional recovery when compared with SC (57.1 ± 5.3 versus 41.0 ± 3.4 mm Hg; p = 0.04 by planned comparison test of hypothesis). Similarly, Table 3 reveals that each of the four NHBD groups had a significantly higher myocardial water content than nonischemic controls (p = 0.00002 by analysis of variance). However, PE25 exhibited an obvious trend toward less myocardial edema formation than SC (p = 0.09 by planned comparison test of hypothesis). Examination of myocardial oxygen consumption data revealed no significant differences or identifiable trends among groups.
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Fig 3. . Mean left ventricular developed pressures in all groups after 45 minutes of reperfusion. Nonischemic controls (NC) versus all others, p = 0.00001 by analysis of variance. Phenylephrine (PE) 25 µg/kg versus saline controls (SC), p = 0.04 by planned comparison test of hypothesis.
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Comment
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The obligatory periods of in vivo hypoxia and warm ischemia that characterize the procurement of organs from NHBDs represent a profound insult to cardiac graft function, as demonstrated by recent work from our laboratory [4, 5]. In the current investigation, we endeavored to determine whether it is possible to decrease the severity of this graft dysfunction by harnessing the cardioprotective effects of pharmacologic preconditioning and thus enhancing the ischemic tolerance of the donor heart. Our results suggest that preconditioning NHBDs with the 1-adrenoceptor agonist PE at an intravenous dose of 25 µg/kg partially protects cardiac grafts from subsequent in vivo ischemic injury. These conclusions are based on a significant improvement in postischemic left ventricular functional recovery and a trend toward less myocardial edema formation in NHBDs pretreated with PE 25 µg/kg compared with saline-pretreated donors. These results are in accord with previous studies in other models of myocardial ischemia in which PE preconditioning was found to be protective [11–17].
An unexpected conclusion from the present study is that preconditioning may also afford myocardial protection against the deleterious consequences of hypoxia, as evidenced by a modest, yet significant, prolongation in the time required for hearts to sustain hypoxic arrest when pretreated with PE 25 µg/kg. However, this is contradictory to the conclusion of a previous study, which failed to demonstrate any preconditioning-mediated protection against hypoxic injury [18]. Possible explanations for the discordant conclusions of our study and that of Cave and associates [18] are the use of more physiologic blood perfusate in this study as opposed to crystalloid in the latter, or a difference in the preconditioning stimulus, model of hypoxia, or end points of each study. We do not believe that prolongation of the hypoxic period would preclude the clinical application of PE preconditioning to non–heart-beating cardiac donation, as this period was only modestly lengthened in the PE 25 µg/kg group, and the postischemic ventricular functional recovery of these hearts was still significantly better than in saline-treated hearts. If future studies confirm our findings, the concept of intravenous pharmacologic preconditioning against the deleterious effects of myocardial hypoxia may have even more far-reaching clinical applications, such as preventing cardiac arrest in hypoxic patients.
The exact mechanism mediating preconditioning remains elusive, although a host of theories have been proposed. Based on the results of this study, the most appealing candidate for a preconditioning mechanism is a reduction in intracellular acidosis. Preconditioning has been shown to reduce the severity of myocardial acidosis during prolonged ischemia [19, 20]. It is possible that in the present study, preconditioned hearts sustained a slower accumulation of H+ ions during the hypoxic period, thus delaying the negative inotropy and eventual onset of cardiac arrest from severe myocardial acidosis.
The range of doses and timing of administration of PE were based on a previous study in open-chest rabbits, which documented a significant reduction in myocardial infarct size by an intravenous bolus of PE 50 µg/kg given 15 minutes before extended regional ischemia [17]. However, we failed to detect any preconditioning effect of the same PE dose in our NHBD model, in terms of either a delay in the time to onset of hypoxic arrest or an improvement in postischemic cardiac functional indices. The posttreatment hypertension after a dose of 50 µg/kg was of markedly greater magnitude in our study than in the previous study; Hale and Kloner [17] reported only a 44% increase in systolic pressure and a 57% increase in diastolic pressure. We believe that the disparities in the hemodynamic responses to an identical dose of PE could be attributed to the presence of an open chest in the regional ischemia study or a difference in anesthetic levels. The former could conceivably impair systemic venous return and blunt the hemodynamic response to PE. Failure of PE 50 µg/kg to elicit a preconditioning effect in our study might be explained simply by the magnitude of the resultant blood pressure increase; mechanical injury from left ventricular strain may have counterbalanced any functional benefits conferred by preconditioning. Given the favorable response to a PE dose of 25 µg/kg, 12.5 µg/kg is probably below the threshold dose for eliciting a preconditioning response.
In summary, the results of this study demonstrate that the function of cardiac grafts procured from NHBDs can be significantly improved by intravenous preconditioning with the 1-adrenoceptor agonist PE. This pretreatment strategy may prove invaluable in ensuring the clinical feasibility of non–heart-beating cardiac donation. An additional important finding of the current study is that the myocardium can be preconditioned against the deleterious effects of hypoxia. The clinical applicability of this latter finding should be the focus of future investigations.
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Acknowledgments
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This work was supported by a National Research Service Award (Fellowship 1 F32 HL09065-01A2), National Heart, Lung, and Blood Institute, National Institutes of Health.
We express our sincere appreciation for the technical assistance of Anthony J. Herring and Taylor N. Cope.
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Footnotes
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Address reprint requests to Dr Tribble, Department of Surgery, University of Virginia Health Sciences Center, Box 181-95, Charlottesville, VA 22908.
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Abstract
Introduction
