Techniques of preservation and storage of the donor

Transcription

1 Continuous Perfusion Improves Preservation of Donor Rat Hearts: Importance of the Implantation Phase David K. Nickless, MB, BS, Marc Rabinov, MB, BS, PhD, Stephen M. Richards, PhD, Robert A. J. Conyers, FRCPA, and Franklin L. Rosenfeldt, FRACS Baker Medical Research Institute and The Alfred Hospital, Melbourne, Australia Background. Continuous hypothermic perfusion of donor hearts may provide extra protection for long ischemic times and suboptimal donors. The aim of three separate studies was to assess the effect of continuous hypothermic perfusion during simulated donor heart storage and implantation. Methods. In study 1 twelve isolated rat hearts underwent 10 minutes of normothermic ischemia to simulate the effect of brain death on the heart and 5 hours of cardioplegic arrest, using University of Wisconsin solution. Six hearts were statically stored in University of Wisconsin solution at 2 C, and six were perfused with University of Wisconsin solution. To assess the effect of simulated implantation, in study 2 an additional 12 hearts were statically stored for 5.5 hours in University of Wisconsin solution, six of which were rewarmed to a mean of 16 C over the last 30 minutes of arrest. To assess the effect of simulated perfusion, in study 3 during implantation 12 hearts were rewarmed to a mean of 16 C over the last 30 minutes of arrest, during which time six were perfused with 2 C solution. Results. Hearts perfused during storage demonstrated greater recovery of prearrest power, 85.8% 1.8%, than hearts preserved by static storage, 72.7% 3.0% (p < 0.01). The simulated warm implantation period reduced recovery of power from 68.3% 5.1% to 40.2% 2.0% (p < 0.001). Perfusion during warm implantation improved recovery to 61.8% 3.9% (p < 0.01). In all experiments improved function was accompanied by improved metabolic energy status. Conclusions. During the implantation period of heart transplantation the donor heart sustains injury that could amount to 50% of total ischemic injury. Continuous perfusion during the cold storage phase and during simulated implantation improves recovery of the donor heart. (Ann Thorac Surg 1998;65: ) 1998 by The Society of Thoracic Surgeons Techniques of preservation and storage of the donor heart have changed very little since the first human transplants, despite almost 30 years of research. Arrest by a potassium cardioplegic solution and hypothermic storage is the technique used in the majority of cases. This simple technique provides acceptable myocardial protection for ischemic times up to 6 hours. To provide protection for the average donor heart during even longer ischemic times, or to provide extra protection for suboptimal donor hearts, many investigators have recommended the use of continuous hypothermic perfusion [1 3]. However, continuous perfusion has not gained acceptance in clinical transplantation because it is more complicated than conventional static storage and because perfusion with an asanguineous fluid may cause myocardial edema [1, 2]. Our experience of the prolonged ischemic times required to procure donor organs from remote locations [4] and the development of simpler techniques of perfusion such as microperfusion [5] led us Accepted for publication Dec 8, Address reprint requests to Dr Rosenfeldt, Baker Medical Research Institute, PO Box 6492, Melbourne, Victoria 8008, Australia ( frank. rosenfeldt@baker.edu.au). to believe that continuous perfusion of the donor heart during transport and implantation may have a role in future transplant practice and merited further study. It is well recognized that brain death causes a severe and variable degree of myocardial depression [6]. This depression reduces myocardial recovery after a subsequent period of cardioplegic arrest [3]. It has been shown that a period of unprotected global normothermic ischemia also reduces myocardial recovery after a subsequent period of cardioplegic arrest [7]. Therefore, in the present study, to model donor heart status, we used normothermic ischemia to produce reproducible myocardial depression comparable with that produced by brain death. We believe that the implantation period poses a particular hazard to the donor heart. During this final stage of ischemia, while the anastomoses are completed, the donor heart is removed from ice storage and placed in the pericardial cavity where it rewarms. Additional ischemic injury occurring during the implantation phase could constitute a significant proportion of the total ischemic injury sustained by the donor heart during transplantation. Therefore, in the present study, on the basis of our clinical experience we used a simulated 1998 by The Society of Thoracic Surgeons /98/$19.00 Published by Elsevier Science Inc PII S (98)

2 1266 NICKLESS ET AL Ann Thorac Surg DONOR HEART PERFUSION 1998;65: implantation phase of 30 minutes at a mean myocardial temperature of 16 C. The aims of the present study were: (1) to compare the effect on myocardial recovery of continuous hypothermic perfusion during simulated donor heart storage; (2) to assess the magnitude of ischemic injury occurring in the donor heart during a simulated implantation phase; and (3) to ascertain whether perfusion of the donor heart during the simulated implantation phase improved recovery. Material and Methods Animals Hearts were from male Munich Wistar rats (250 to 350 g body weight). Animals were treated in accordance with the Code of Practice of the National Health and Medical Research Council of Australia and protocols were approved by the institutional ethics committee. Experimental Preparation Hearts were perfused on an isolated, working rat heart apparatus [8]. The perfusion medium was modified Krebs-Henseleit buffer containing (in mmol/l): NaCl, 118.0; KCl, 4.7; CaCl 2, 2.5; MgSO 4, 1.2; KH 2 PO 4, 1.2; NaHCO 3, 25.0, glucose 11.0, ph 7.4 gassed with 95% O 2 and 5% CO 2. Animals were anesthetised with halothane in oxygen. After heparinization the heart was rapidly excised and immersed in ice-cold buffer for 1 minute. The aorta was cannulated and the heart was perfused in the nonworking state for 15 minutes at a pressure of 100 cm H 2 O to allow the heart to stabilize, during which time the left atrial appendage was cannulated. For measurements of function, a left atrial pressure of 15 cm H 2 O and an aortic pressure of 100 cm H 2 O were used. CALCULATIONS. The rate of performing external cardiac work (power) was calculated as follows: Power (mj s 1 g 1 dry weight) [(P LAP) CO ]/W, where P peak aortic pressure (mm Hg), LAP left atrial pressure (11 mm Hg); CO cardiac output (ml/min), and W heart dry weight (g). Myocardial oxygen consumption (MVO 2 )in L O 2 min 1 g 1 dry weight was calculated as follows: MVO 2 [(Pao 2 Pvo 2 ) CF ]/760 W, where Pao 2 and Pvo 2 (mm Hg) are arterial and venous partial pressures of oxygen measured across the coronary circulation, 760 is the barometric pressure (mm Hg), and CF is coronary flow (ml/min). The mechanical efficiency of the heart was calculated by dividing the cardiac power by MVO 2 and expressing the result as a percentage of the expected energy equivalent of complete oxygen combustion, which is J/mL of O 2 [9]: Myocardial efficiency [Cardiac power 6000 (%)]/MVO , where power is expressed as mj s 1 g 1, and MVO 2 as L O 2 min 1 g 1. CARDIOPLEGIC SOLUTIONS. Hearts were arrested and stored in or perfused with the University of Wisconsin (UW) solution, the composition of which has been described Fig 1. Protocols: Study 1: perfusion during storage; study 2: effect of warm implantation; study 3: effect of perfusion during implantation. (UW University of Wisconsin solution.) elsewhere [10]. Dexamethasone (sodium phosphate) was added (final concentration 8 mg/l) immediately before use. In study 3 hearts were stored in or perfused with the St. Thomas Hospital No. 1 solution during the warm implantation phase, the composition of which has been described elsewhere [11]. CONTINUOUS PERFUSION APPARATUS. The hearts underwent continuous hypothermic perfusion using the following system. The perfusate was delivered to the heart from a syringe pump (Perfusor VI; B. Braun, Melsungen, Germany). A simple membrane oxygenator, consisting of a 30-cm coil of narrow diameter silicone tubing within a sealed glass bottle containing 100% oxygen, raised the oxygen tension of the perfusate to 600 to 700 mm Hg. The perfusate passed through a m porosity filter and bubble trap before entering the aortic cannula. The lower half of the perfused heart was immersed in the perfusate to keep the epicardium moist. The whole system was maintained in a cold room at 2 C. Simulated Effect of Brain Death In pilot studies, to simulate the myocardial depressant effect of brain death, we subjected rat hearts to varying periods of global ischemia, then selected a period that was sufficient to depress myocardial function to 70% to 80% of control. To quantitate the depression in functional and metabolic terms we subjected six hearts to 10 minutes of global ischemia without active cooling by clamping the aortic cannula. Function was measured before ischemia, and after ischemia with 10 minutes reperfusion (Fig 1). The hearts were then freeze-clamped for measurement of high energy phosphates. Control (preischemic) levels of adenine nucleotides were measured in another six normal hearts without ischemia, freezeclamped on the apparatus. FUNCTION. Freshly excised hearts achieved an aortic flow rate of ml min 1 g 1, generated a power of mj s 1 g 1, and had a myocardial oxygen consumption of L O 2 g 1 min 1. After 10

3 Ann Thorac Surg NICKLESS ET AL 1998;65: DONOR HEART PERFUSION 1267 Table 1. Biochemical Data Variable Before Brain Death Controls Study 1 Study 2 Study 3 After Brain Death Static Perfused Static Warm Implant Warm Implant Warm Implant Perfused ATP ( mol/g dry wt) a b b ADP ( mol/g dry wt) b a AMP ( mol/g dry wt) a TAN ( mol/g dry wt) b b a Energy charge a % Water a c Significantly different from corresponding values: a p 0.05, b p 0.01, c p ADP adenosine diphosphate; AMP adenosine monophosphate; ATP adenosine triphosphate; TAN total adenine nucleotides. minutes of global ischemia followed by reperfusion, aortic flow declined to 70% (p ), cardiac power to 76% (p ), and oxygen consumption to 91% (p 0.05) of the corresponding preischemic values. Efficiency decreased from 21.4% 0.6% to 17.7% 0.6% (p 0.01). BIOCHEMISTRY. The normothermic ischemic period had no effect on myocardial adenine nucleotide content or energy charge; however, the tissue water content of hearts after global ischemia was higher than hearts in the preischemic group (76.1% versus 73.5%, p 0.05) (Table 1). Study 1: Perfusion During After simulating the effect of brain death on the heart by 10 minutes of normothermic global ischemia and 10 minutes of reperfusion, prestorage function was measured during 15 minutes in the working mode (see Fig 1). The hearts were then returned to the nonworking mode and were infused for 2 minutes with 2 C UW solution at a pressure of 60 cm H 2 O. The heart and the cannula were then detached from the isolated working heart apparatus, the heart submerged in UW solution at 2 C. They were then randomly allocated to one of two different preservation methods that simulated preservation of the donor heart during transport. In the static storage group, hearts (n 6) were stored in 2 C UW solution for 5 hours without perfusion. In the continuous perfusion group (n 6) the demountable cannula system was connected to the continuous perfusion apparatus and retrograde aortic perfusion with oxygenated 2 C UW solution was performed at a flow-rate of 3 ml/h for 5 hours. This flow rate was approximately twice that necessary to satisfy the calculated myocardial oxygen demand at this temperature. At the end of the storage or perfusion period, all hearts were reconnected to the isolated working heart apparatus and reperfused in the nonworking mode for 30 minutes. They were then converted to the working mode for a period of 15 minutes for measurement of functional recovery. Finally a sample of myocardium was excised for determination of water content and the remainder was freeze-clamped and stored in liquid nitrogen for analysis of adenine nucleotide content. Study 2: The Effect of Implantation The protocol was the same as in study 1 except that a 30-minute period of rewarming completed the 5.5-hour preservation period to simulate the phase of implantation. Hearts were allocated to one of two groups (see Fig 1). Hearts in the warm implantation group (n 6) were removed from the ice-cold UW solution and partly immersed in 25 C UW solution for 30 minutes (mean myocardial temperature 16 C), whereas hearts in the static storage group (n 6) continued to be preserved in ice-cold UW solution. At the end of the implantation phase, all hearts were returned to the isolated working heart apparatus for reperfusion; measurement of functional recovery and collection of tissue samples were the same as in study 1. Study 3: Perfusion During Simulated Implantation The protocol was the same as in study 2 except that 5 minutes before the implantation phase all hearts received a 5-mL infusion of St. Thomas Hospital solution at a flow-rate of 1 ml/min to remove the UW solution from the coronary circulation. A thermocouple temperature probe (Shiley Instruments, Irvine, CA) was inserted into the right ventricular cavity through the preexisting incision in the pulmonary artery for measurement of cardiac temperature. Hearts were allocated to one of two groups (see Fig 1). In the warm implantation group (n 6), hearts were partly immersed in 25 C St. Thomas Hospital solution for 30 minutes. In the implantation perfused group (n 6) the hearts were partly immersed in 25 C St. Thomas Hospital solution and were perfused with 2 C oxygenated St. Thomas Hospital solution at 12 ml/h. At the end of the implantation phase all hearts were returned to the isolated working heart apparatus for reperfusion, measurement of functional recovery, and collection of tissue samples as described for study 1. Determination of Cardiac Tissue Water and Adenine Nucleotide Content Samples of cardiac tissue removed for analysis of water content were dried to constant weight. The percentage tissue water was calculated according to the formula: [(1 Dry weight)/wet weight] 100%.

4 1268 NICKLESS ET AL Ann Thorac Surg DONOR HEART PERFUSION 1998;65: ADP AMP] and Adenylate energy charge [0.5ADP ATP]/[ATP ADP AMP]. Statistical Analysis Data pertaining to cardiac function, tissue water, and adenine nucleotide content between groups within each study were compared using the Student s unpaired t test. In study 3 cardiac temperature data were analyzed using repeated measures analysis of variance. Comparisons of coronary flow before versus after preservation were made using the Student s paired t test. A probability value of less than 0.05 was considered significant. All results were expressed as mean standard error of the mean. Fig 2. Study 1: recovery of cardiac power in perfused hearts versus static storage hearts. (**p 0.01.) The adenine nucleotide content of freeze-clamped hearts was determined by HPLC analysis of neutralized perchlorate extracts [12]. Adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) were separated isocratically and were quantified by peak area at 254 nm. The values obtained were used to calculate the following indices of myocardial energy status: Total adenine nucleotides [ATP Results Study 1: Perfusion During After arrest, storage perfused hearts demonstrated significantly higher recovery of prearrest aortic flow (p 0.05) and power (p 0.01) (Fig 2), than static storage hearts (Table 2). There was near complete recovery of prearrest coronary flow, oxygen consumption, and efficiency in both groups following reperfusion with no between-group differences. Hearts in the storage perfused group had higher levels of ATP (p 0.05), ADP (p 0.01), AMP (p 0.05), and Table 2. Physiological Parameters (g dry wt) Study 1 Study 2 Study 3 Parameter Static Perfused Static Warm Implant Warm Implant Warm Implant Perfused Aortic flow (ml min 1 g 1 ) Initial Final c a % Recovery a b a Power (mj s 1 g 1 ) Initial Final a c b % Recovery b c b Coronary flow (ml min 1 g 1 ) Initial Final b % Recovery a MVO 2 ( L O 2 min 1 g 1 ) Initial Final a a % Recovery a Efficiency (%) Initial Final c b % Recovery c a Significantly different: a p 0.05, b p 0.01, c p

5 Ann Thorac Surg NICKLESS ET AL 1998;65: DONOR HEART PERFUSION 1269 total adenine nucleotides (p 0.01) than hearts in the static storage group (see Table 1). At the end of the experiment, hearts preserved using continuous perfusion contained 7% more water than nonperfused hearts (p ). Study 2: Effect of the Implantation Phase Hearts in the warm implantation group had markedly lower recovery of prearrest aortic flow (p 0.01) and cardiac power (p 0.001; Fig 3) than hearts in the static storage group (see Table 2). There was a reduction in coronary flow in the static storage group (p 0.05) and the warm implantation group (p 0.01) but no betweengroup difference in recovery. Despite the deterioration in cardiac function in the warm implantation group there was no decline in oxygen consumption. This reduced the recovery of efficiency in these hearts to half that of the static storage group (p ) (see Table 2). The ATP content of the warm implantation group was significantly lower than that of the static storage group (p 0.01) as was the total adenine nucleotides content (p 0.05) (see Table 1). At the end of the experiment the tissue water content of hearts in both groups was identical. Study 3: Continuous Perfusion During the Implantation Phase Hearts in the implantation perfused group demonstrated a higher recovery of aortic flow (p 0.05) and produced a significantly greater recovery of cardiac power (p 0.01) (Fig 4), than warm implantation (nonperfused) hearts (see Table 2). There was a reduction in coronary flow in the warm implantation group (p 0.01) and the implantation perfused group (p 0.01). Recovery of coronary flow in the implantation perfused group was greater than the warm implantation group (p 0.05) (see Table 2). The oxygen consumption of both groups after arrest declined similarly compared with values obtained before cardioplegic arrest. However, the recovery of efficiency of implantation perfused hearts was significantly improved compared with the warm implantation group (p 0.05) (see Table 2). The implantation perfused group demonstrated a significantly higher ATP content (p 0.01), a higher total adenine nucleotides content (p 0.05) (see Table 1) and a higher energy charge (p 0.05) than the warm implantation group. At the end of the experiment the tissue water content of both groups of hearts was similar. Before the implantation period, the cardiac temperature in both groups was identical at 2.0 C. However after 5 minutes the temperature increased rapidly to C in perfused hearts and C in nonperfused hearts. Beyond this point the cardiac temperature in the implantation perfused group remained in a range 13.6 to 14.0 C, but in the warm implantation group it continued to rise to a final value of 20.9 C (p ). Comment In these three studies in an experimental model of preservation of donor hearts subjected to simulated brain death, we separated and quantified the depressive effect on myocardial function of the transport and implantation stages of transplantation. We then measured the effect of continuous hypothermic perfusion in each of these stages. Study 1 showed that continuous hypothermic perfusion significantly improved myocardial preservation during a simulated organ storage period of 5.5 hours at 2 C. Study 2 suggested that the injury sustained by the donor heart during implantation can be of similar magnitude to that sustained during storage and transport. Study 3 showed that continuous hypothermic perfusion can substantially reduce the amount of injury occurring during implantation. Effects of Brain Death on the Donor Heart Experimental studies of heart preservation should take into account the suboptimal nature of the donor heart at the time of procurement. Studies conducted on freshly excised hearts may overestimate the ability of the heart from a brain-dead animal to withstand the preservation technique being studied. Although the effects of brain death on the circulation have been well studied, the effects of brain death on the donor heart itself remain controversial. Wicomb and associates [3] reported that compared to freshly excised pig hearts, hearts excised from brain-dead animals demonstrated a 40% reduction in cardiac output without any change in ATP content. Galinanes and coworkers [13] observed a 50% decline in function in brain-dead rats. In the present study we used a 10-minute period of normothermic global ischemia to produce a functional depression of 25%, without affecting myocardial adenine nucleotide levels. The 10-minute period of normothermic ischemia was, in the rat, sufficiently prolonged to outweigh any protective effect of ischemic preconditioning, which is usually induced by only 3 to 5 minutes of ischemia. Fig 3. Study 2: recovery of cardiac power in warm implantation hearts versus static storage hearts. (**p 0.01.)

6 1270 NICKLESS ET AL Ann Thorac Surg DONOR HEART PERFUSION 1998;65: the static storage group. Although this edema could have produced an undetected impairment in diastolic function, more importantly systolic function was substantially improved by perfusion. Furthermore, coronary flow was not reduced after the 5-hour perfusion period in study 1 and was not significantly different from the static storage group (Table 2). Although significant, this degree of edema is considerably lower than that reported in most other continuous perfusion studies [1, 2] because of the shorter duration of preservation in our study (5 hours) and the use of UW solution, which contains colloid and high molecular weight impermeants, which reduce tissue edema. Fig 4. Study 3: recovery of cardiac power in warm implantation perfused hearts versus warm implantation hearts. (**p 0.01.) Continuous Hypothermic Perfusion The total number of heart transplants performed around the world is restricted by the limited availability of donor organs [14]. Improving donor heart preservation would permit the use of hearts that would otherwise be rendered unusable by depressed function or geographic remoteness, thus expanding the donor organ pool. Continuous hypothermic perfusion has been previously shown experimentally to improve preservation of isolated hearts during prolonged ischemia [1 3]. During donor organ transport and implantation, the heart is dependent on limited cellular energy reserves to maintain viability. Ischemia and anerobic metabolism result in the rapid accumulation of metabolites, including lactate, which decrease myocardial ph and inhibit enzymes necessary for cellular viability [15]. Continuous perfusion removes these metabolites and limits the decline in ph. In addition, oxygen and substrates can be delivered so that the energy requirements of the hypothermic potassium-arrested heart can be fully met, particularly during implantation, when energy requirements are greater because of the warmer myocardial temperature. The improvements in myocardial energy status we found using continuous perfusion during both the simulated storage (study 1) and implantation (study 3) support this concept. Myocardial edema is a well-recognized consequence of continuous hypothermic perfusion [1, 2]. Bethencourt and Laks [16] examined the effect of edema on myocardial compliance during hypothermic perfusion using a modified Krebs-Henseleit solution with added potassium (20 meq/l). After 24 hours of preservation, heart water content increased by nearly 30%, passive ventricular compliance decreased significantly and coronary artery resistance increased. In their study ultrastructural analysis revealed the edema was largely confined to the interstitial space without disruption to intracellular organelles. In our study the tissue water content of the storage perfused group was only 7% higher than that of Effect of Myocardial Warming During Implantation Phase At the beginning of the implant procedure the donor heart is removed from hypothermic storage, placed in the warm thoracic cavity of the recipient, and exposed to the ambient temperature of the operating room. Until the aortic anastomosis is completed and the cross-clamp removed, the heart remains ischemic. There is little information in the literature to assist in quantifying the amount of warm ischemic damage occurring during the implantation phase of transplantation. In clinical practice, during implantation, effective topical cooling of the donor heart is difficult until the atrial anastomoses have been completed and the pericardial cavity can be flooded with cold saline. We have recorded myocardial temperatures in several human donor hearts during implantation and found that left ventricular temperatures increased steadily from 2 to 4 C after removal of the donor heart from the ice box, to 19 to 23 C just before crossclamp removal, averaging 14 C over the 40- to 70-minute period. In the model of implantation used in the present study, the heart was partly immersed in fluid at 25 C, which warmed it from 2 to 21 C (average, 16.4 C), which would be a reasonable approximation of the warm ischemia that normally occurs during implantation. Although the larger surface area to volume ratio of the rat heart causes it to rewarm more rapidly than a human heart in an equivalent environment, we shortened the simulated implantation period in the rat model to adjust for this difference in organ size. Study 2 showed that there was only 40% recovery of cardiac power in the warm implantation group, where 30 minutes of rewarming followed 5 hours of static storage, compared with 68% recovery in the static storage group. Thus, approximately half the reduction in recovery due to cold storage plus implantation was attributable to the implantation phase alone. The lower adenine nucleotide content of hearts subjected to the simulated implantation process probably reflects the additional ischemic injury occurring at the warmer temperature. Thus, within the limitations of an experimental study such as this (rats versus humans and laboratory environment versus operating room conditions), our results suggest that the amount of ischemic injury occurring during the implantation period can be of a similar magnitude to the amount occurring during the storage and transport period. This

7 Ann Thorac Surg NICKLESS ET AL 1998;65: DONOR HEART PERFUSION 1271 suggests that more attention should be given to this phase of donor heart preservation. We found that continuous hypothermic perfusion during the simulated implantation phase enhanced the recovery of function and improved the efficiency of oxygen utilization compared with nonperfused hearts. The higher adenine nucleotide content showed that perfused hearts sustained less ischemic injury. The beneficial effect of using intermittent antegrade cold blood cardioplegia during clinical donor heart implantation has been described [17]. Carrier and coworkers [18] recently described a randomized clinical trial of continuous, retrograde perfusion by the coronary sinus using warm blood cardioplegia during donor heart implantation, compared with topical cooling with cold saline and ice slush. The results showed a higher rate of primary graft failure in the topical cooling group (25%) than in the perfusion group (0%). Currently our own clinical preference is to use continuous or semicontinuous antegrade perfusion with cold blood cardioplegia during implantation of the donor heart, as this should provide better protection of the right ventricle than retrograde perfusion [19]. Clinical Aspects of Continuous Hypothermic Perfusion Over the past 30 years many investigators have demonstrated in the laboratory the ability of continuous hypothermic perfusion to preserve the donor heart for periods of up to 48 hours [1 3]. Extracellular edema due to the hydrostatic perfusion pressure has been observed but can be minimized by using a perfusate that exerts an adequate osmotic and oncotic pressure. Blood-based perfusates would probably be best but there are difficulties in obtaining an adequate volume of blood to perfuse a donor heart continuously during a storage and transport period of several hours. With only one report of clinical transplantation (heterotopic) after continuous perfusion during donor heart transport [20], it is clear that the technique has not gained widespread acceptance. One good reason is that perfusion is inherently more complicated than conventional static storage. To enable perfusion to be used during the transport, requires simple techniques such as a low-flow, nonrecirculating, gravity feed system using dilute blood cardioplegia. During implantation, the warmer myocardial temperatures mandate use of a blood-based perfusate with its superior oxygen-carrying capacity, substrate composition, and oncotic properties. Clearly further work is necessary to select optimal solutions and to construct perfusion systems that are mechanically simple, reliable, and portable. Critique of Model Used In these three studies we sought to simulate in the rat the clinical process of cardiac transplantation. The basic implications of the model may be transferrable to larger animals as previous study of hypothermic cardioplegia has shown close similarity between the response of the isolated rat heart and the dog heart on bypass [21]. In the present study a 5-hour period of simulated organ transport was chosen to produce a 60% depression of functional recovery in these deliberately damaged hearts. This depression is of a similar order of magnitude to that observed after ischemic times of 7 hours or more, which occur not infrequently in countries like Australia due to the remote geographic location of donor organs [4]. A 5-hour period of simulated organ transport in the rat would be equivalent to a longer ischemic time for a human heart, due to the greater metabolic rate in the rat. The 3 ml/h continuous perfusion flow rate used during simulated donor organ transport would easily satisfy calculated myocardial oxygen demand and represents a flow rate equivalent to 1 L/h for a human heart, which would be practicable for clinical donor runs. This rate was increased to 12 ml/h during the simulated implantation period in study 3, in agreement with the Van t Hoff rule that for every 10 C increase in temperature, the velocity of organic reactions increases by a factor of approximately two. University of Wisconsin solution was used as the preservation solution during the cold static storage period in all three study protocols because of its superiority at lower temperatures [22]. There is evidence that UW solution may exert an adverse effect on endothelial function in the heart at temperatures above 20 C [23]. It is likely that this effect is mediated by the high potassium content of UW solution. To avoid any confounding effect of UW solution, in study 3 St. Thomas Hospital solution was used during the simulated warm implantation phase with and without perfusion because of its lower potassium concentration. However, in the event, the results showed no evidence of an adverse effect of UW in the rewarming period, that is the recovery of the warm implantation groups in study 2 (UW present) and in study 3 (UW washed out by St. Thomas solution) were the same. Probably the myocardial temperature, 16 C, was not high enough to elicit the adverse effects of UW solution. In conclusion, during the implantation phase of donor heart preservation, due to a progressive increase in myocardial temperature, the donor heart sustains injury that can be of a similar magnitude to ischemic injury occurring during the longer cold storage period. Continuous perfusion can minimize the myocardial injury occurring during implantation as well as during the cold storage phase of donor heart preservation, which is evident in improved recovery of function and energy status. We acknowledge the assistance and advice of Dr Donald Esmore, Head of the Alfred Hospital Cardiac Transplant Unit. References 1. Copeland JG, Jones M, Spragg R, Stinson EB. In vitro preservation of canine hearts for 24 to 28 hours followed by successful orthotopic transplantation. Ann Surg 1973;178: Cooper DKC, Wicomb WN, Rose AG, Barnard CN. Orthotopic allotransplantation and autotransplantation of the baboon heart following 24-hr storage by a portable hypothermic perfusion system. Cryobiology 1983;20:

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