- Email: [email protected]
Low-Flow Hypothermic Crystalloid Perfusion Is Superior to Cold Storage During Prolonged Heart Preservation
Low-Flow Hypothermic Crystalloid Perfusion Is Superior to Cold Storage During Prolonged Heart Preservation R. Oua, Y.W. Lima, J.W. Choonga, D.S. Esmoreb, R.F. Salamonsenc, C. McLeand, J. Forbese, M. Baileyf, and F.L. Rosenfeldta,* a
Cardiac Surgical Research, Department of Cardiothoracic Surgery, Alfred Hospital, and the Department of Surgery, Monash University, Melbourne, Australia; bDepartment of Cardiothoracic Surgery, Alfred Hospital, and the Department of Surgery, Monash University, Melbourne, Australia; cDepartment of Intensive Care, Alfred Hospital, Melbourne, Australia; dDepartment of Pathology, Alfred Hospital, Melbourne, Australia; eGlycation and Diabetes, Baker IDI Heart and Diabetes Research Institute, Melbourne, Australia; and fDepartment of Epidemiology and Preventive Medicine, School of Public Health & Preventive Medicine, Monash University, Melbourne, Australia
ABSTRACT Background. Preservation of donor hearts for transplantation has traditionally been performed with the use of static cold storage. We have developed and tested a novel gravitypowered system of cold crystalloid perfusion for prolonged donor heart preservation. Methods. Greyhounds were anesthetized; their hearts were arrested with cold cardioplegic solution and excised. Hearts were allocated to 12 hours of perfusion preservation (n ¼ 6) or cold storage in ice (n ¼ 5). Non-preserved hearts (n ¼ 5) served as a normal reference group. Perfusion hearts were perfused (20 mL/min, 8e12 C) with a novel oxygenated nutrient-containing preservation solution. After preservation, the recovery of the hearts was assessed in a blood-perfused working heart rig over 2 hours in terms of function, blood lactate level, myocardial adenosine triphosphate, and histology. Results. After 2 hours of reperfusion, in comparison with cold storage hearts, perfused heart function curves showed superior recovery of cardiac output (P ¼ .001), power (P ¼ .001), and efficiency (0.046 0.01 vs 0.004 0.003 joules/mL O2, P ¼ .034). Myocardial adenosine triphosphate content (mmol/mg protein) was reduced significantly from the normal level of 26.5 (15.9, 55.8) to 5.08 (0.50, 10.4) (P ¼ .049) in cold storage hearts but not in perfused hearts. Over a period of 2 hours, lactate levels in the blood perfusate were significantly lower in the perfusion group than in the cold storage group (P < .05). Conclusions. Continuous hypothermic crystalloid perfusion provides myocardial preservation superior to cold storage for long-term heart preservation, with potential applicability to marginal and donation after circulatory death hearts.
T
HE SHORTAGE of donor hearts limits the number of transplants worldwide. Surgeons are increasingly accepting for transplantation older donors and allografts with prolonged ischemic time [1,2]. The increasing utilization of donor kidneys, lungs, and livers from donation after circulatory death (DCD) donors raises the possibility of using hearts from deceased donors if techniques of myocardial preservation could be improved. After more than 40 years of clinical transplantation, static cold storage is still the standard method for preserving the donor heart [3]. Cold storage limits the safe storage time to 4 to 6 hours [4]. Improving methods of donor heart preservation such as
the use of continuous perfusion may allow more distant procurement of donor hearts, alleviate the detrimental effects of storage on marginal donor hearts, and may even resuscitate damaged hearts such as those from DCD donors. Numerous experimental studies have shown the superiority of perfusion preservation over static cold storage [5,6]. Compared with cold storage, continuous perfusion of donor *Address correspondence to Franklin L. Rosenfeldt, Cardiac Surgical Research, Department of Cardiothoracic Surgery, Alfred Hospital, PO Box 315, Prahran, VIC 3181, Australia. E-mail: [email protected]
ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/14 http://dx.doi.org/10.1016/j.transproceed.2014.09.149
Transplantation Proceedings, 46, 3309e3313 (2014)
3309
3310
OU, LIM, CHOONG ET AL
hearts has been shown to allow a longer safe preservation interval with superior functional recovery and maintenance of aerobic metabolism [7,8]. In a previous study, we demonstrated that perfusion with warm oxygenated blood provided preservation superior to cold storage in DCD hearts from greyhound dogs [9]. However, in clinical practice, the logistic difficulties and excessive costs associated with continuous warm blood perfusion technology limit its use [10]. A simple, portable, and reliable perfusion system for clinical usage in donor hearts is needed. We have formulated an oxygenated, nutrient-containing perfusion solution for use in a gravity-feed low-flow hypothermic perfusion device. The object of this study was to assess the efficacy of this novel perfusion technique in comparison with conventional cold storage for preserving normal hearts during a 12-hour period. METHODS This study was approved by the institutional ethics committee, in accordance with the relevant Australian code of practice. In 16 greyhound dogs, anesthesia was induced by use of propofol, and the animals were intubated and mechanically ventilated with isofluorane and oxygen. The internal jugular vein and femoral vein and artery were cannulated for fluid infusion and hemodynamic monitoring. After sternotomy, the pericardium was opened and the heart was exposed. Before explantation, heparin (10,000 U) was administered intravenously to allow the collection of blood (600e900 mL) to prime a working heart rig. Collected blood was replaced with intravenous Ringer’s solution. Cardioplegia was induced antegrade through the ascending aorta with 1000 mL of St Thomas’ Hospital II cardioplegic solution at 4 C, saturated with oxygen, and the heart explanted.
Experimental Design The explanted hearts were randomly allocated to perfusion preservation, cold storage, or a normal reference group. In the perfusion group (n ¼ 6), after cardioplegia, the heart was excised, a cannula was inserted into the aorta, and the heart was transferred to the perfusion device and perfused for 12 hours. The perfusion device comprised an insulated container in which bags of perfusion solution were attached to a manifold and drip chamber (Fig 1). The perfusion solution [Organ Perfusion Solution (OPS), patent WO 2102/027787 A1] comprised an extracellular electrolyte solution containing nutrients, oncotic agents, a vasodilator, and an antioxidant (Table 1). Before use, the perfusate was saturated with 100% oxygen by means of direct bubbling (PO2 ¼ 600 mm Hg). The box was kept cold with ice packs. The aortic cannula was attached to the circuit and the heart received gravity-feed perfusion at a flow rate of 20 mL/min and a pressure of 8 to 10 cm H2O. Myocardial temperature was monitored and maintained at 8 to 12 C. Perfused hearts were assessed for oxygen consumption during preservation by measurement of coronary perfusate flow and the oxygen content. In the cold storage group (n ¼ 5), after the administration of the cardioplegic solution, the heart was immersed in cold Ringer’s solution in a plastic bag and placed in ice in an insulated cooler for 12 hours. Myocardial temperature was kept at 4 C. In the normal reference group (n ¼ 5), after cardioplegia and explantation, the hearts were immediately transferred to the blood-perfused working heart rig for reperfusion with blood followed by functional and metabolic assessment.
Fig 1. Perfusion device for heart preservation.
Reperfusion Procedures After a 40-minute simulated ischemic implantation period and before reperfusion, the hearts from the perfusion and cold storage groups were infused with a protective 2-part modified acidic St Thomas’ Hospital II cardioplegia containing cyclosporine (mitochondrial protectant) and cariporide (sodium-hydrogen exchange inhibitor) [9]. The hearts were then reperfused on a working heart rig in which blood could be pumped into the aorta (resting mode) or into the left atrium (working mode). The hearts were electrically defibrillated, reperfused in resting mode for 1 hour, 45 minutes, and then were switched to working mode for functional and metabolic studies over a period of 2 hours.
Functional and Metabolic Assessment The pump flow was initially set to achieve a left atrial pressure of 10 mm Hg and was then gradually increased to generate cardiac function curves of cardiac power versus left atrial pressure. Cardiac power was derived from developed pressure (mean aortic pressure Table 1. Composition of Organ Perfusion Solution Composition
Function
Concentration
Potassium chloride Calcium chloride Magnesium chloride Trizma HCl Sodium bicarbonate Adenosine Gluthathione (reduced) Sodium lactobionate Sodium L-aspartate Fructose-1,6 bisphosphate D-Glucose Insulin Oxygen Sodium hydroxide Osmolarity
Cardioplegic Cardioplegic Cardioplegic Buffer Buffer Vasodilatation Anti-oxidant Oncotic agent Energy preservation Energy preservation Energy preservation Energy preservation Aerobic metabolism Adjust pH Prevent edema
15 (mmol/L) 0.5 (mmol/L) 7.5 (mmol/L) 20 (mmol/L) 20 (mmol/L) 5 (mmol/L) 3 (mmol/L) 70 (mmol/L) 20 (mmol/L) 5 (mmol/L) 14 (mmol/L) 6 (units) 600 (mm Hg) pH 7.3 380 (mOsm)
LOW-FLOW HYPOTHERMIC CRYSTALLOID PERFUSION
3311
minus left atrial pressure) and cardiac output. In working mode, myocardial oxygen consumption was calculated from hemoglobin, oxygen saturation, and partial pressure of oxygen in arterial and coronary venous blood samples. Blood samples were taken to determine the baseline lactate level immediately before hearts were attached to the rig and then hourly in the working mode over a period of 2 hours. Intact mitochondria from left ventricular biopsies were isolated by means of differential centrifugation. The pellets from the heart were re-suspended in buffer and the freshly isolated mitochondria were used immediately for the analysis of adenosine triphosphate (ATP) levels with the use of chemoluminescence [11].
Water Content and Histological Assessment Samples of left ventricular myocardium were excised and then were dried to a constant weight to assess myocardial water content. Finally, the heart was perfused with 10% formalin at physiological pressure and placed in formalin. Transmural sections of the left ventricle were examined microscopically by a pathologist (CMcL) blinded to the experimental groups for the presence and extent of edema, ischemic changes, and infarction.
Statistical Methods Groupwise comparisons were performed with the use of analysis of variance (ANOVA) for normally distributed data and KruskalWallis tests otherwise with specific post hoc pairwise comparisons adjusted by means of a Bonferroni correction for multiple comparisons. Results are reported as mean (standard errors) or as median (interquartile range). A value of P < .05 was considered significant.
RESULTS
Oxygen was consumed throughout the entire perfusion preservation period. At the standard flow rate of 20 mL/ min, myocardial oxygen consumption was approximately 0.09 mL O2 per 100 g heart weight per minute. The perfusion group showed that with increasing left atrial pressure, cardiac output and cardiac power equated to levels attained by normal, non-ischemic hearts (P ¼ 1.0 for both) and was significantly higher for levels of cardiac output (P ¼ .001) and cardiac power (P ¼ .001) than in cold storage hearts, which showed no increase with increasing left atrial pressure (2-way ANOVA for all) (Fig 2A). The myocardial ATP content (mmol/mg protein) was reduced significantly from the normal level of median 26.5 (15.9, 55.8) to 5.1 (0.5, 10.4) in cold storage hearts, (P ¼ .049) but not in the perfusion group 16.80 (8.74, 21.43) (P > .05) (Fig 2B). Myocardial oxygen efficiency in perfused hearts was significantly higher than that in the cold storage group (0.046 0.01 vs 0.004 0.003 joules/mL O2, P ¼ .034) and was not significantly different from the normal group (0.034 0.010 joules/mL O2, P ¼ 1.00) (Fig 2B). The cold storage hearts had an efficiency of only 14% of normal (P ¼ .043). In both groups, there was an increase in perfusate lactate after initial reperfusion and a decrease thereafter. However, the lactate levels over time were significantly lower in the perfusion group than in the cold storage group (P < .05) (Fig 2C). Water content of the cold storage group was 79.6% 0.70% and in the perfusion group was 80.6% 0.71%. The
Fig 2. (A) Cardiac function curves of cardiac output and power versus left atrial pressure (LAP) after 2 hours of reperfusion (P values given are for perfusion vs cold storage); (B) myocardial adenosine triphosphate (ATP) content (median, range, and interquartile range), oxygen efficiency (mean SEM) after reperfusion; and (C) lactate change after reperfusion (mean SEM).
3312
myocardial water content in the normal group was 79.1% 0.73%. There were no significant differences among all 3 groups (P ¼ .17). On microscopic examination, the normal hearts showed the appearance of healthy myocardium. The appearance of the perfusion hearts was similar to the normal hearts, myofiber striations were evident, and there were no obvious signs of ischemia. In the cold storage group, signs of ischemic damage were evident, the myofiber striations of the hearts were obliterated, contraction bands were present, and the cytoplasm appeared hypereosinophilic, in keeping with early stage ischemic change. DISCUSSION
In the current study, we have shown the superiority of the technique of low-flow hypothermic crystalloid perfusion over cold storage for long-term preservation. The perfusate (OPS) is an extracellular cardioplegic solution containing an oncotic agent (sodium lactobionate), buffers, nutrients (glucose, aspartate, and fructose bisphosphate), a vasodilator (adenosine), and antioxidants (reduced glutathione and sodium lactobionate) (Table 1). Sodium lactobionate, a common component of organ preservation solutions, provides oncotic support and prevents cell swelling [12]. Aspartate acts in the tricarboxylic acid cycle as a salvageable amino-acid for regeneration of oxaloacetate, thus increasing pyruvate and ATP generation in the tricarboxylic acid cycle [13]. Adenosine has a cardioprotective action through several mechanisms. By activating the A1 adrenergic receptors, it causes coronary vasodilation and improves myocardial blood flow and functional recovery [14]. Adenosine acts on mitochondrial KATP channels to simulate ischemic pre-conditioning. Adenosine can also act as an ATP precursor for restoring post ischemic ATP levels [15]. Reduced glutathione is an antioxidant that improves preservation of coronary endothelial function [16]. Fructose 1-6 bisphosphate (FDP) is formed from fructose 6-PO4 in glucose metabolism. This process is an energy-requiring process catalyzed by phosphofructokinase. The goal of providing FDP in a perfusate solution is to provide extra substrate economically for the glycolytic pathway. It has been shown that the administration of FDP results in significantly improved myocardial function after ischemia [17]. FDP also lowers blood viscosity by inhibiting platelet aggregation [18] and chelating extracellular calcium [19] and acts as an antioxidant [20]. At 8 to 12 C in the potassium-arrested canine heart, myocardial oxygen consumption is reduced to 0.14 mL/100g/ min, which is only 3% of the level in a normothermic beating non-working heart [21]. Oxygenation of our crystalloid perfusion solution before use can maintain a PO2 of around 300 mm Hg for 6 hours or more in plastic bags of solution stored in ice (our laboratory data). The oxygen saturated perfusate at 4 C contains 4.2 mL of oxygen/100 mL [22]. Thus, theoretically, at the flow rate of 20 mL/min, the oxygenated perfusate can provide up to 0.84 mL oxygen/ min, which is similar to the 0.63 mL/min calculated oxygen
OU, LIM, CHOONG ET AL
requirement of the 450-gram hearts in our study. During cold perfusion, the measured oxygen consumption of the hearts was 0.4 mL/min, which is just below theoretical requirements. We chose a low flow rate sometimes called “microperfusion” [23] as a compromise between satisfying demands for oxygen on the one hand and causing myocardial edema on the other [24]. With the use of the single-pass gravity system, we aimed to reduce the flow rate to the minimum necessary to preserve myocardial integrity while avoiding the necessity of frequent replenishment of the bags of perfusate in the perfusion box. At the perfusate flow rate of 20 mL/min, perfusion pressure remained consistently low. Low perfusion pressures (8e10 mm Hg) might cause inadequacies in flow distribution across the myocardial wall. Although we did not monitor flow distribution, regional flow insufficiencies could not have been a major problem on account of the excellent function recovery, lack of lactate production during reperfusion, and the maintenance of trans-myocardial mitochondrial ATP content at near normal levels. The protective action of the continuous perfusion technique can be ascribed to a combination of the cardioprotective action of the cardioplegic base solution, the nutrients, and the antioxidants as well as the provision of oxygen and the washout of metabolites. This study used normal hearts followed over an ischemic period far greater than used clinically. The rationale was that improvements in myocardial protection showing up under these circumstances would also apply to marginal and DCD donor heart situations. Study Limitations
The ex vivo rig we used in the current study assessed functional recovery of the left ventricle and not the right ventricle. Also, we studied only the early stage of recovery of the hearts. It is unknown how these results would have changed had hearts been allowed to recover for hours or days. However, in clinical transplantation, good early function generally predicts good later function [3]. This model of clinical transplantation included two distinct protective modalities: the mitochondrial-protective reperfusate designed to reduce reperfusion injury, followed by oxygenated hypothermic perfusion [9]. The experimental design did not allow us to separate the relative benefit of these modalities. However, there is good evidence in the literature of the efficacy of each of the components of these modalities in limiting reperfusion injury and preserving myocardial integrity [12e20]. CONCLUSIONS
We conclude that hypothermic perfusion preservation provides excellent preservation of donor hearts for a 12-hour period, with functional and aerobic metabolic recovery approaching normal levels. By contrast, the conventional technique of cold storage results in poor recovery with
LOW-FLOW HYPOTHERMIC CRYSTALLOID PERFUSION
minimal contractile function and persistent anaerobic metabolism. We believe that cold low-flow crystalloid perfusion shows promise for improving prolonged preservation of brain-dead and DCD donor hearts. We have now successfully applied this technique of hypothermic gravity perfusion in human DCD hearts evaluated in the laboratory [25]. REFERENCES [1] Marasco SF, Esmore DS, Richardson M, et al. Prolonged cardiac allograft ischemic time: no impact on long-term survival but at what cost? Clin Transplant 2007;21:321e9. [2] Wittwer T, Wahlers T. Marginal donor grafts in heart transplantation: lessons learned from 25 years of experience. Transpl Int 2008;21:113e25. [3] Iyer A, Kumarasinghe G, Hicks M, et al. Primary graft failure after heart transplantation. J Transplant 2011. Article ID 175768, Page: 9. [4] Ozeki T, Kwon MH, Gu J, et al. Heart preservation using continuous ex vivo perfusion improves viability and functional recovery. Circ J 2007;71:153e9. [5] Mownah OA, Khurram MA, Ray C, et al. Development of an ex vivo technique to achieve reanimation of hearts sourced from a porcine donation after circulatory death model. J Surg Res 2014;189:326e34. [6] Collins MJ, Moainie SL, Griffith BP, Poston RS. Preserving and evaluating hearts with ex vivo machine perfusion: an avenue to improve early graft performance and expand the donor pool. Eur J Cardiothorac Surg 2008;34:318e25. [7] Rosenbaum DH, Peltz M, DiMaio JM, et al. Perfusion preservation versus static preservation for cardiac transplantation: effects on myocardial function and metabolism. J Heart Lung Transplant 2008;27:93e9. [8] Peltz M, He TT, Adams 4th GA, et al. Perfusion preservation maintains myocardial ATP levels and reduces apoptosis in an ex vivo rat heart transplantation model. Surgery 2005;138:795e805. [9] Repse S, Pepe S, Anderson J, et al. Cardiac reanimation for donor heart transplantation after cardiocirculatory death. J Heart Lung Transplant 2010;29:747e55. [10] Wagner FM. Donor heart preservation and perfusion. Appl Cardiopulmon Pathophysiol 2011;15:198e206. [11] Drew B, Phaneuf S, Dirks A, et al. Effects of aging and caloric restriction on mitochondrial energy production in gastrocnemius muscle and heart. Am J Physiol Regul Integr Comp Physiol 2003;284:R474e80.
3313 [12] Cobert ML, Peltz M, West L, Jessen ME. Importance of organ preservation solution composition in reducing myocardial edema during machine perfusion for heart transplantation. Transplant Proc 2010;42:1591e4. [13] Choong YS, Gavin JB, Buckman J. Long-term preservation of explanted hearts perfused with L-aspartate enriched cardioplegic solution: improved function, metabolism, and ultrastructure. J Thorac Cardiovasc Surg 1992;103:210e8. [14] Petsikas D, Ricci MA, Baffour R, et al. Enhanced 24-hour in vitro heart preservation with adenosine and adenosine monophosphate. J Heart Transplant 1990;9:114e8. [15] Bolling SF, Bove EL, Gallagher KP. ATP precursor depletion and postischemic myocardial recovery. J Surg Res 1991;50: 629e33. [16] Kevelaitis E, Nyborg NC, Menasche P. Protective effect of reduced glutathione on endothelial function of coronary arteries subjected to prolonged cold storage. Transplantation 1997;64: 660e3. [17] Cohen JE, Atluri P, Taylor MD, et al. Fructose 1,6diphosphate administration attenuates post-ischemic ventricular dysfunction. Heart Lung Circ 2006;15:119e23. [18] Cavallini L, Deana R, Francesconi MA, Alexandre A. Fructose-1,6-diphosphate inhibits platelet activation. Biochem Pharmacol 1992;43:1539e44. [19] Rizzoli V, Battilana G, Galzigna L. Fructose-1,6diphosphate counteracts ethanol-stimulated calcium uptake in isolated BHK cells. Mol Cell Biochem 1990;95:89e93. [20] Tavazzi B, Cerroni L, Di Pierro D, et al. Oxygen radical injury and loss of high-energy compounds in anoxic and reperfused rat heart: prevention by exogenous fructose-1,6-bisphosphate. Free Radic Res Commun 1990;10:167e76. [21] Buckberg GD. Left ventricular subendocardial necrosis. Ann Thorac Surg 1977;24:379e93. [22] Graham M. The solubility of oxygen in physiological salines. Fish Physiol Biochem 1987;4:1e4. [23] Okada K, Yamashita C, Okada M, Okada M. Successful 24-hour rabbit heart preservation by hypothermic continuous coronary microperfusion with oxygenated University of Wisconsin Solution. Ann Thorac Surg 1995;60: 1723e8. [24] Cobert ML, West LM, Jessen ME. Machine perfusion for cardiac allograft preservation. Curr Opin Organ Transplant 2008;13:526e30. [25] Rosenfeldt F, Ou R, Woodard J, et al. Twelve-hour reanimation of a human heart following donation after circulatory death. Heart Lung Circ 2014;23:88e90.
Cardiac Surgical Research, Department of Cardiothoracic Surgery, Alfred Hospital, and the Department of Surgery, Monash University, Melbourne, Australia; bDepartment of Cardiothoracic Surgery, Alfred Hospital, and the Department of Surgery, Monash University, Melbourne, Australia; cDepartment of Intensive Care, Alfred Hospital, Melbourne, Australia; dDepartment of Pathology, Alfred Hospital, Melbourne, Australia; eGlycation and Diabetes, Baker IDI Heart and Diabetes Research Institute, Melbourne, Australia; and fDepartment of Epidemiology and Preventive Medicine, School of Public Health & Preventive Medicine, Monash University, Melbourne, Australia
ABSTRACT Background. Preservation of donor hearts for transplantation has traditionally been performed with the use of static cold storage. We have developed and tested a novel gravitypowered system of cold crystalloid perfusion for prolonged donor heart preservation. Methods. Greyhounds were anesthetized; their hearts were arrested with cold cardioplegic solution and excised. Hearts were allocated to 12 hours of perfusion preservation (n ¼ 6) or cold storage in ice (n ¼ 5). Non-preserved hearts (n ¼ 5) served as a normal reference group. Perfusion hearts were perfused (20 mL/min, 8e12 C) with a novel oxygenated nutrient-containing preservation solution. After preservation, the recovery of the hearts was assessed in a blood-perfused working heart rig over 2 hours in terms of function, blood lactate level, myocardial adenosine triphosphate, and histology. Results. After 2 hours of reperfusion, in comparison with cold storage hearts, perfused heart function curves showed superior recovery of cardiac output (P ¼ .001), power (P ¼ .001), and efficiency (0.046 0.01 vs 0.004 0.003 joules/mL O2, P ¼ .034). Myocardial adenosine triphosphate content (mmol/mg protein) was reduced significantly from the normal level of 26.5 (15.9, 55.8) to 5.08 (0.50, 10.4) (P ¼ .049) in cold storage hearts but not in perfused hearts. Over a period of 2 hours, lactate levels in the blood perfusate were significantly lower in the perfusion group than in the cold storage group (P < .05). Conclusions. Continuous hypothermic crystalloid perfusion provides myocardial preservation superior to cold storage for long-term heart preservation, with potential applicability to marginal and donation after circulatory death hearts.
T
HE SHORTAGE of donor hearts limits the number of transplants worldwide. Surgeons are increasingly accepting for transplantation older donors and allografts with prolonged ischemic time [1,2]. The increasing utilization of donor kidneys, lungs, and livers from donation after circulatory death (DCD) donors raises the possibility of using hearts from deceased donors if techniques of myocardial preservation could be improved. After more than 40 years of clinical transplantation, static cold storage is still the standard method for preserving the donor heart [3]. Cold storage limits the safe storage time to 4 to 6 hours [4]. Improving methods of donor heart preservation such as
the use of continuous perfusion may allow more distant procurement of donor hearts, alleviate the detrimental effects of storage on marginal donor hearts, and may even resuscitate damaged hearts such as those from DCD donors. Numerous experimental studies have shown the superiority of perfusion preservation over static cold storage [5,6]. Compared with cold storage, continuous perfusion of donor *Address correspondence to Franklin L. Rosenfeldt, Cardiac Surgical Research, Department of Cardiothoracic Surgery, Alfred Hospital, PO Box 315, Prahran, VIC 3181, Australia. E-mail: [email protected]
ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/14 http://dx.doi.org/10.1016/j.transproceed.2014.09.149
Transplantation Proceedings, 46, 3309e3313 (2014)
3309
3310
OU, LIM, CHOONG ET AL
hearts has been shown to allow a longer safe preservation interval with superior functional recovery and maintenance of aerobic metabolism [7,8]. In a previous study, we demonstrated that perfusion with warm oxygenated blood provided preservation superior to cold storage in DCD hearts from greyhound dogs [9]. However, in clinical practice, the logistic difficulties and excessive costs associated with continuous warm blood perfusion technology limit its use [10]. A simple, portable, and reliable perfusion system for clinical usage in donor hearts is needed. We have formulated an oxygenated, nutrient-containing perfusion solution for use in a gravity-feed low-flow hypothermic perfusion device. The object of this study was to assess the efficacy of this novel perfusion technique in comparison with conventional cold storage for preserving normal hearts during a 12-hour period. METHODS This study was approved by the institutional ethics committee, in accordance with the relevant Australian code of practice. In 16 greyhound dogs, anesthesia was induced by use of propofol, and the animals were intubated and mechanically ventilated with isofluorane and oxygen. The internal jugular vein and femoral vein and artery were cannulated for fluid infusion and hemodynamic monitoring. After sternotomy, the pericardium was opened and the heart was exposed. Before explantation, heparin (10,000 U) was administered intravenously to allow the collection of blood (600e900 mL) to prime a working heart rig. Collected blood was replaced with intravenous Ringer’s solution. Cardioplegia was induced antegrade through the ascending aorta with 1000 mL of St Thomas’ Hospital II cardioplegic solution at 4 C, saturated with oxygen, and the heart explanted.
Experimental Design The explanted hearts were randomly allocated to perfusion preservation, cold storage, or a normal reference group. In the perfusion group (n ¼ 6), after cardioplegia, the heart was excised, a cannula was inserted into the aorta, and the heart was transferred to the perfusion device and perfused for 12 hours. The perfusion device comprised an insulated container in which bags of perfusion solution were attached to a manifold and drip chamber (Fig 1). The perfusion solution [Organ Perfusion Solution (OPS), patent WO 2102/027787 A1] comprised an extracellular electrolyte solution containing nutrients, oncotic agents, a vasodilator, and an antioxidant (Table 1). Before use, the perfusate was saturated with 100% oxygen by means of direct bubbling (PO2 ¼ 600 mm Hg). The box was kept cold with ice packs. The aortic cannula was attached to the circuit and the heart received gravity-feed perfusion at a flow rate of 20 mL/min and a pressure of 8 to 10 cm H2O. Myocardial temperature was monitored and maintained at 8 to 12 C. Perfused hearts were assessed for oxygen consumption during preservation by measurement of coronary perfusate flow and the oxygen content. In the cold storage group (n ¼ 5), after the administration of the cardioplegic solution, the heart was immersed in cold Ringer’s solution in a plastic bag and placed in ice in an insulated cooler for 12 hours. Myocardial temperature was kept at 4 C. In the normal reference group (n ¼ 5), after cardioplegia and explantation, the hearts were immediately transferred to the blood-perfused working heart rig for reperfusion with blood followed by functional and metabolic assessment.
Fig 1. Perfusion device for heart preservation.
Reperfusion Procedures After a 40-minute simulated ischemic implantation period and before reperfusion, the hearts from the perfusion and cold storage groups were infused with a protective 2-part modified acidic St Thomas’ Hospital II cardioplegia containing cyclosporine (mitochondrial protectant) and cariporide (sodium-hydrogen exchange inhibitor) [9]. The hearts were then reperfused on a working heart rig in which blood could be pumped into the aorta (resting mode) or into the left atrium (working mode). The hearts were electrically defibrillated, reperfused in resting mode for 1 hour, 45 minutes, and then were switched to working mode for functional and metabolic studies over a period of 2 hours.
Functional and Metabolic Assessment The pump flow was initially set to achieve a left atrial pressure of 10 mm Hg and was then gradually increased to generate cardiac function curves of cardiac power versus left atrial pressure. Cardiac power was derived from developed pressure (mean aortic pressure Table 1. Composition of Organ Perfusion Solution Composition
Function
Concentration
Potassium chloride Calcium chloride Magnesium chloride Trizma HCl Sodium bicarbonate Adenosine Gluthathione (reduced) Sodium lactobionate Sodium L-aspartate Fructose-1,6 bisphosphate D-Glucose Insulin Oxygen Sodium hydroxide Osmolarity
Cardioplegic Cardioplegic Cardioplegic Buffer Buffer Vasodilatation Anti-oxidant Oncotic agent Energy preservation Energy preservation Energy preservation Energy preservation Aerobic metabolism Adjust pH Prevent edema
15 (mmol/L) 0.5 (mmol/L) 7.5 (mmol/L) 20 (mmol/L) 20 (mmol/L) 5 (mmol/L) 3 (mmol/L) 70 (mmol/L) 20 (mmol/L) 5 (mmol/L) 14 (mmol/L) 6 (units) 600 (mm Hg) pH 7.3 380 (mOsm)
LOW-FLOW HYPOTHERMIC CRYSTALLOID PERFUSION
3311
minus left atrial pressure) and cardiac output. In working mode, myocardial oxygen consumption was calculated from hemoglobin, oxygen saturation, and partial pressure of oxygen in arterial and coronary venous blood samples. Blood samples were taken to determine the baseline lactate level immediately before hearts were attached to the rig and then hourly in the working mode over a period of 2 hours. Intact mitochondria from left ventricular biopsies were isolated by means of differential centrifugation. The pellets from the heart were re-suspended in buffer and the freshly isolated mitochondria were used immediately for the analysis of adenosine triphosphate (ATP) levels with the use of chemoluminescence [11].
Water Content and Histological Assessment Samples of left ventricular myocardium were excised and then were dried to a constant weight to assess myocardial water content. Finally, the heart was perfused with 10% formalin at physiological pressure and placed in formalin. Transmural sections of the left ventricle were examined microscopically by a pathologist (CMcL) blinded to the experimental groups for the presence and extent of edema, ischemic changes, and infarction.
Statistical Methods Groupwise comparisons were performed with the use of analysis of variance (ANOVA) for normally distributed data and KruskalWallis tests otherwise with specific post hoc pairwise comparisons adjusted by means of a Bonferroni correction for multiple comparisons. Results are reported as mean (standard errors) or as median (interquartile range). A value of P < .05 was considered significant.
RESULTS
Oxygen was consumed throughout the entire perfusion preservation period. At the standard flow rate of 20 mL/ min, myocardial oxygen consumption was approximately 0.09 mL O2 per 100 g heart weight per minute. The perfusion group showed that with increasing left atrial pressure, cardiac output and cardiac power equated to levels attained by normal, non-ischemic hearts (P ¼ 1.0 for both) and was significantly higher for levels of cardiac output (P ¼ .001) and cardiac power (P ¼ .001) than in cold storage hearts, which showed no increase with increasing left atrial pressure (2-way ANOVA for all) (Fig 2A). The myocardial ATP content (mmol/mg protein) was reduced significantly from the normal level of median 26.5 (15.9, 55.8) to 5.1 (0.5, 10.4) in cold storage hearts, (P ¼ .049) but not in the perfusion group 16.80 (8.74, 21.43) (P > .05) (Fig 2B). Myocardial oxygen efficiency in perfused hearts was significantly higher than that in the cold storage group (0.046 0.01 vs 0.004 0.003 joules/mL O2, P ¼ .034) and was not significantly different from the normal group (0.034 0.010 joules/mL O2, P ¼ 1.00) (Fig 2B). The cold storage hearts had an efficiency of only 14% of normal (P ¼ .043). In both groups, there was an increase in perfusate lactate after initial reperfusion and a decrease thereafter. However, the lactate levels over time were significantly lower in the perfusion group than in the cold storage group (P < .05) (Fig 2C). Water content of the cold storage group was 79.6% 0.70% and in the perfusion group was 80.6% 0.71%. The
Fig 2. (A) Cardiac function curves of cardiac output and power versus left atrial pressure (LAP) after 2 hours of reperfusion (P values given are for perfusion vs cold storage); (B) myocardial adenosine triphosphate (ATP) content (median, range, and interquartile range), oxygen efficiency (mean SEM) after reperfusion; and (C) lactate change after reperfusion (mean SEM).
3312
myocardial water content in the normal group was 79.1% 0.73%. There were no significant differences among all 3 groups (P ¼ .17). On microscopic examination, the normal hearts showed the appearance of healthy myocardium. The appearance of the perfusion hearts was similar to the normal hearts, myofiber striations were evident, and there were no obvious signs of ischemia. In the cold storage group, signs of ischemic damage were evident, the myofiber striations of the hearts were obliterated, contraction bands were present, and the cytoplasm appeared hypereosinophilic, in keeping with early stage ischemic change. DISCUSSION
In the current study, we have shown the superiority of the technique of low-flow hypothermic crystalloid perfusion over cold storage for long-term preservation. The perfusate (OPS) is an extracellular cardioplegic solution containing an oncotic agent (sodium lactobionate), buffers, nutrients (glucose, aspartate, and fructose bisphosphate), a vasodilator (adenosine), and antioxidants (reduced glutathione and sodium lactobionate) (Table 1). Sodium lactobionate, a common component of organ preservation solutions, provides oncotic support and prevents cell swelling [12]. Aspartate acts in the tricarboxylic acid cycle as a salvageable amino-acid for regeneration of oxaloacetate, thus increasing pyruvate and ATP generation in the tricarboxylic acid cycle [13]. Adenosine has a cardioprotective action through several mechanisms. By activating the A1 adrenergic receptors, it causes coronary vasodilation and improves myocardial blood flow and functional recovery [14]. Adenosine acts on mitochondrial KATP channels to simulate ischemic pre-conditioning. Adenosine can also act as an ATP precursor for restoring post ischemic ATP levels [15]. Reduced glutathione is an antioxidant that improves preservation of coronary endothelial function [16]. Fructose 1-6 bisphosphate (FDP) is formed from fructose 6-PO4 in glucose metabolism. This process is an energy-requiring process catalyzed by phosphofructokinase. The goal of providing FDP in a perfusate solution is to provide extra substrate economically for the glycolytic pathway. It has been shown that the administration of FDP results in significantly improved myocardial function after ischemia [17]. FDP also lowers blood viscosity by inhibiting platelet aggregation [18] and chelating extracellular calcium [19] and acts as an antioxidant [20]. At 8 to 12 C in the potassium-arrested canine heart, myocardial oxygen consumption is reduced to 0.14 mL/100g/ min, which is only 3% of the level in a normothermic beating non-working heart [21]. Oxygenation of our crystalloid perfusion solution before use can maintain a PO2 of around 300 mm Hg for 6 hours or more in plastic bags of solution stored in ice (our laboratory data). The oxygen saturated perfusate at 4 C contains 4.2 mL of oxygen/100 mL [22]. Thus, theoretically, at the flow rate of 20 mL/min, the oxygenated perfusate can provide up to 0.84 mL oxygen/ min, which is similar to the 0.63 mL/min calculated oxygen
OU, LIM, CHOONG ET AL
requirement of the 450-gram hearts in our study. During cold perfusion, the measured oxygen consumption of the hearts was 0.4 mL/min, which is just below theoretical requirements. We chose a low flow rate sometimes called “microperfusion” [23] as a compromise between satisfying demands for oxygen on the one hand and causing myocardial edema on the other [24]. With the use of the single-pass gravity system, we aimed to reduce the flow rate to the minimum necessary to preserve myocardial integrity while avoiding the necessity of frequent replenishment of the bags of perfusate in the perfusion box. At the perfusate flow rate of 20 mL/min, perfusion pressure remained consistently low. Low perfusion pressures (8e10 mm Hg) might cause inadequacies in flow distribution across the myocardial wall. Although we did not monitor flow distribution, regional flow insufficiencies could not have been a major problem on account of the excellent function recovery, lack of lactate production during reperfusion, and the maintenance of trans-myocardial mitochondrial ATP content at near normal levels. The protective action of the continuous perfusion technique can be ascribed to a combination of the cardioprotective action of the cardioplegic base solution, the nutrients, and the antioxidants as well as the provision of oxygen and the washout of metabolites. This study used normal hearts followed over an ischemic period far greater than used clinically. The rationale was that improvements in myocardial protection showing up under these circumstances would also apply to marginal and DCD donor heart situations. Study Limitations
The ex vivo rig we used in the current study assessed functional recovery of the left ventricle and not the right ventricle. Also, we studied only the early stage of recovery of the hearts. It is unknown how these results would have changed had hearts been allowed to recover for hours or days. However, in clinical transplantation, good early function generally predicts good later function [3]. This model of clinical transplantation included two distinct protective modalities: the mitochondrial-protective reperfusate designed to reduce reperfusion injury, followed by oxygenated hypothermic perfusion [9]. The experimental design did not allow us to separate the relative benefit of these modalities. However, there is good evidence in the literature of the efficacy of each of the components of these modalities in limiting reperfusion injury and preserving myocardial integrity [12e20]. CONCLUSIONS
We conclude that hypothermic perfusion preservation provides excellent preservation of donor hearts for a 12-hour period, with functional and aerobic metabolic recovery approaching normal levels. By contrast, the conventional technique of cold storage results in poor recovery with
LOW-FLOW HYPOTHERMIC CRYSTALLOID PERFUSION
minimal contractile function and persistent anaerobic metabolism. We believe that cold low-flow crystalloid perfusion shows promise for improving prolonged preservation of brain-dead and DCD donor hearts. We have now successfully applied this technique of hypothermic gravity perfusion in human DCD hearts evaluated in the laboratory [25]. REFERENCES [1] Marasco SF, Esmore DS, Richardson M, et al. Prolonged cardiac allograft ischemic time: no impact on long-term survival but at what cost? Clin Transplant 2007;21:321e9. [2] Wittwer T, Wahlers T. Marginal donor grafts in heart transplantation: lessons learned from 25 years of experience. Transpl Int 2008;21:113e25. [3] Iyer A, Kumarasinghe G, Hicks M, et al. Primary graft failure after heart transplantation. J Transplant 2011. Article ID 175768, Page: 9. [4] Ozeki T, Kwon MH, Gu J, et al. Heart preservation using continuous ex vivo perfusion improves viability and functional recovery. Circ J 2007;71:153e9. [5] Mownah OA, Khurram MA, Ray C, et al. Development of an ex vivo technique to achieve reanimation of hearts sourced from a porcine donation after circulatory death model. J Surg Res 2014;189:326e34. [6] Collins MJ, Moainie SL, Griffith BP, Poston RS. Preserving and evaluating hearts with ex vivo machine perfusion: an avenue to improve early graft performance and expand the donor pool. Eur J Cardiothorac Surg 2008;34:318e25. [7] Rosenbaum DH, Peltz M, DiMaio JM, et al. Perfusion preservation versus static preservation for cardiac transplantation: effects on myocardial function and metabolism. J Heart Lung Transplant 2008;27:93e9. [8] Peltz M, He TT, Adams 4th GA, et al. Perfusion preservation maintains myocardial ATP levels and reduces apoptosis in an ex vivo rat heart transplantation model. Surgery 2005;138:795e805. [9] Repse S, Pepe S, Anderson J, et al. Cardiac reanimation for donor heart transplantation after cardiocirculatory death. J Heart Lung Transplant 2010;29:747e55. [10] Wagner FM. Donor heart preservation and perfusion. Appl Cardiopulmon Pathophysiol 2011;15:198e206. [11] Drew B, Phaneuf S, Dirks A, et al. Effects of aging and caloric restriction on mitochondrial energy production in gastrocnemius muscle and heart. Am J Physiol Regul Integr Comp Physiol 2003;284:R474e80.
3313 [12] Cobert ML, Peltz M, West L, Jessen ME. Importance of organ preservation solution composition in reducing myocardial edema during machine perfusion for heart transplantation. Transplant Proc 2010;42:1591e4. [13] Choong YS, Gavin JB, Buckman J. Long-term preservation of explanted hearts perfused with L-aspartate enriched cardioplegic solution: improved function, metabolism, and ultrastructure. J Thorac Cardiovasc Surg 1992;103:210e8. [14] Petsikas D, Ricci MA, Baffour R, et al. Enhanced 24-hour in vitro heart preservation with adenosine and adenosine monophosphate. J Heart Transplant 1990;9:114e8. [15] Bolling SF, Bove EL, Gallagher KP. ATP precursor depletion and postischemic myocardial recovery. J Surg Res 1991;50: 629e33. [16] Kevelaitis E, Nyborg NC, Menasche P. Protective effect of reduced glutathione on endothelial function of coronary arteries subjected to prolonged cold storage. Transplantation 1997;64: 660e3. [17] Cohen JE, Atluri P, Taylor MD, et al. Fructose 1,6diphosphate administration attenuates post-ischemic ventricular dysfunction. Heart Lung Circ 2006;15:119e23. [18] Cavallini L, Deana R, Francesconi MA, Alexandre A. Fructose-1,6-diphosphate inhibits platelet activation. Biochem Pharmacol 1992;43:1539e44. [19] Rizzoli V, Battilana G, Galzigna L. Fructose-1,6diphosphate counteracts ethanol-stimulated calcium uptake in isolated BHK cells. Mol Cell Biochem 1990;95:89e93. [20] Tavazzi B, Cerroni L, Di Pierro D, et al. Oxygen radical injury and loss of high-energy compounds in anoxic and reperfused rat heart: prevention by exogenous fructose-1,6-bisphosphate. Free Radic Res Commun 1990;10:167e76. [21] Buckberg GD. Left ventricular subendocardial necrosis. Ann Thorac Surg 1977;24:379e93. [22] Graham M. The solubility of oxygen in physiological salines. Fish Physiol Biochem 1987;4:1e4. [23] Okada K, Yamashita C, Okada M, Okada M. Successful 24-hour rabbit heart preservation by hypothermic continuous coronary microperfusion with oxygenated University of Wisconsin Solution. Ann Thorac Surg 1995;60: 1723e8. [24] Cobert ML, West LM, Jessen ME. Machine perfusion for cardiac allograft preservation. Curr Opin Organ Transplant 2008;13:526e30. [25] Rosenfeldt F, Ou R, Woodard J, et al. Twelve-hour reanimation of a human heart following donation after circulatory death. Heart Lung Circ 2014;23:88e90.