Preservation of myocyte structure and mitochondrial integrity in subzero cryopreservation of mammalian hearts for transplantation using antifreeze proteins--an electron microscopy study.

OBJECTIVE: Freeze tolerant fish and insects in nature are able to survive subzero temperatures by noncolligatively lowering the freezing temperature of their body fluids using a family of thermal hysteresis proteins (antifreeze proteins, AFPs) specific for each species. Past efforts to cryopreserve mammalian hearts using these proteins were unsuccessful. We report the first successful subzero cryopreservation of rat hearts using fish derived antifreeze proteins with preservation of myocyte structure. METHODS: Heterotopic heart transplantations were performed in isoimmunic Sprague Dawley rats. Donors' hearts were arrested using University of Wisconsin (UW) solution and preserved in UW solution containing AFP I (six experiments) or AFP III (six experiments) at concentrations of 15-20 mg/cc for 2-6 h at subzero temperatures ranging from -1.1 to -1.3 degrees C. Four control experiments were performed by preserving harvested hearts in UW solution alone at -1.3 degrees C for 6 h. In all experiments ice was added in the solution for crystallization. Heterotopic transplantations were performed in the abdomen of the recipient rats. Viability was visually assessed and graded on a scale of 1 (poor contraction) to 6 (excellent contraction). The hearts were then fixed in vivo and processed for electron microscopy study. RESULTS: All hearts preserved at subzero temperatures using AFP I or AFP III survived displaying viability scores of 4-6 1 h after transplantation. Three of the four control hearts that were preserved at -1.3 degrees C without the protective effect of AFP froze and died upon reperfusion. Electron microscopy study of hearts preserved with AFP demonstrated preservation of myocyte structure and mitochondrial integrity.CONCLUSION: Subzero cryopreservation of mammalian hearts for transplantation using AFP I or AFP III is feasible with preservation of myocyte structure and mitochondrial integrity.     

Successful nonfreezing, subzero preservation of rat liver with 2,3-butanediol and type I antifreeze protein

BACKGROUND: Organ cryopreservation is hindered by ice inflicted damage. Nonfreezing preservation of livers at subzero temperatures might offer advantages over current preservation. METHODS: Sprague-Dawley rats were divided into three groups. UW livers (n = 6) were stored in University of Wisconsin (UW) solution at +4 degrees C. UWB livers (n = 6) were perfused ex vivo with UW + 10% 2,3-butanediol at < or =7 degrees C and stored at -4 degrees C. AFP livers (n = 4) were preserved identical to UWB livers, except for addition of 1 mg/ml of type I antifreeze protein. After 24 h livers were perfused with Krebs-Henseleit buffer (37 degrees C) for 60 min. Bile production, O(2) consumption (O(2)C), taurocholate extraction, and lactate dehydrogenase (LDH) release during perfusion and liver adenine nucleotide content and energy charge at the end of perfusion were measured. Cell membrane integrity was determined by trypan blue infusion. RESULTS: Ice formation was prevented in all livers stored at -4 degrees C. Bile production, O(2)C, and taurocholate extraction were similar among three groups. Livers stored at -4 degrees C contained significantly more adenine nucleotides than livers stored at +4 degrees C but the energy charge was similar. LDH release was significantly greater (P < 0.05) in the AFP group vs UWB and UW (63 vs 28 and 21 mU/min/g liver, respectively). Hepatocyte and sinusoidal cell trypan blue uptake was similar in all three groups. CONCLUSIONS: Butanediol with or without AFP was effective in preventing ice formation up to 24 h in rat livers stored at -4 degrees C. Although as effective as current +4 degrees C protocols, subzero preservation for longer periods needs to be achieved prior to clinical application.     

Heart preservation in HTK solution: role of coronary vasculature in recovery of cardiac function

BACKGROUND: Poor myocardial tolerance to prolonged cold ischemia remains a major concern in heart transplantation. In this study, we estimated superiority of Histidine-Tryptophan-Ketoglutarate (HTK) over University of Wisconsin (UW) as a cardiac preservation solution. METHODS: Isolated rat hearts were mounted on a Langendorff apparatus to estimate the baseline cardiac function. The hearts were arrested and stored at 4 degrees C in UW and HTK solution for 8 hours, and then reperfused. The aortic flow, coronary flow, cardiac output, rate pressure product, and left ventricular dp/dt in the HTK group recovered significantly more than the UW group. The values of myocardial total adenine nucleotides and the adenosine triphosphate to adenosine diphosphate ratio were higher in the HTK than in the UW group. We also examined coronary vascular responsiveness using left coronary arteries dissected from the rat hearts before flushing, before storage, after storage, and after reperfusion. RESULTS: The maximal relaxation response to acetylcholine was significantly higher in the HTK than in the UW group after reperfusion, although there were no significant differences at each stage before reperfusion. In addition, the endothelium-independent relaxation response to sodium nitroprusside in the HTK group was also well preserved after reperfusion. CONCLUSIONS: These results indicate that HTK is superior to UW solution for cardiac preservation. HTK protects coronary vasculature during preservation, which together with reperfusion might lead to improved functional cardiac recovery following preservation.     

UW is superior to Celsior and HTK in the protection of human liver endothelial cells against preservation injury.

Celsior solution (CS), a new preservation solution in thoracic organ transplantation, was evaluated for its efficacy in cold preservation of human liver endothelial cells (HLEC) and was compared to University of Wisconsin solution (UW) and histidine-tryptophan-ketoglutarate solution (HTK, Custodiol). HLEC cultures were preserved at 4 degrees C in CS, UW, and HTK, for 2, 6, 12, 24, and 48 hours, with 6 hours of reperfusion. Levels of lactate dehydrogenase (LDH), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and adenosine 5'-triphosphate (ATP) were measured after each interval of ischemia and the respective phase of reperfusion. Preservation injury of HLEC as measured by LDH release, intracellular ATP level, and MTT reduction were overall significantly (P CS > HTK.     

Comparison of intermittent injection of nondepolarizing solution with a single flush of UW solution for donor heart preservation

Isolated canine hearts were preserved for 6 h at 5°C followed by normothermic reperfusion for 2 h. The dogs were divided into two groups of nine hearts each; group 1 received a nondepolarizing preservation solution in multidose, and group 2 received a single flush of University of Wisconsin (UW) solution. Serum MB-CK and mitochondrial aspartate aminotransferase (m-AAT) concentrations and calcium overload during reperfusion were lower in group 1 than in group 2. At the end of reperfusion, myocardial ATP and total adenine nucleotide concentrations were higher and mitochondrial morphology appeared more intact in group 1 than in group 2. Left ventricular diatolic function was preserved better in group 1 than in group 2. These results suggest that in 6-h heart preservation, a nondepolarizing solution applied in multidose fashion protects the myocardium from the deleterious effects of hypothermia and cardioplegia better than a single flush of UW solution.     

A novel bisindolylmaleimide derivative enhances functional recovery of heart after long-term hypothermic heart preservation

BACKGROUND: Functional recovery following heart transplantation mainly depends on the ability of preservative solution in providing the physical and biochemical environment so as to maintain the viability of the tissue during preservation and in reperfusion. Here we demonstrate the protective effects of a novel bisindolylmaleimide derivative, MS1, on enhancing the functional recovery of the heart following long-term hypothermic preservation when added to the preservative solution. METHODS: After anesthesia and artificial ventilation, the hearts were rapidly isolated and perfused with Kreb's Henseleit buffer at 37 degrees C in working mode. After 30 minutes of perfusion, the hearts were arrested with cardioplegic solution and preserved in University of Wisconsin solution with (UW-MS1 group) or without MS1 (UW-Vehicle group) for 12 h at 4 degrees C. After 12 hours, the hearts were reperfused for 60 minutes. RESULTS: MS1 treated hearts showed: a) significant recovery of cardiac functions (P<0.001), b) well-preserved myocardial ATP levels (P<0.001), c) less myocardial water content (P<0.01), d) reduced oxidative stress (P<0.001), e) less intracellular swelling and well-preserved mitochondria, and g) activation of cell survival cascades compared to the control hearts preserved in UW solution without MS1. In contrast, these protective effects of MS1 were abolished on opening the permeability transition pore before MS1 treatment. CONCLUSION: These results altogether indicate the efficacy of this compound in protecting the myocardium against reperfusion injury and thus making this drug a clinically useful tool in patients undergoing reperfusion after cardiac surgeries.     

Successful cardiac preservation for 12 hours using nondepolarizing cold cardioplegia. A canine model

Abstract Isolated canine hearts were preserved for 12 h at 5 °C followed by normothermic reperfusion for 2 h. Dogs were divided into two groups: group 1 ( n = 7) received a nondepolarizing preservation solution in multidose, and group 2 ( n = 6) received single-flushed University of Wisconsin (UW) solution, both administered in multidose fashion. At the end o reperfusion, the myocardial adenosine triphosphate concentration and left ventricular systolic and diastolic function were preserved better in group 1 than in group 2. Myocardial mitochondrial ultra-tructural integrity was identical in the two groups. These results suggested that in a 124 heart preservation, nondepolarizing solution administered in multidose fashion protects the myocardium from the deleterious effects of hypothermia and cardioplegia better than UW solution.     

Effect of initial reperfusion temperature on myocardial preservation

The effect of initial postischemic reperfusion temperature on myocardial preservation was studied in the isolated working rat heart model. After baseline measurement of aortic flow rate, coronary flow rate, and heart rate, 40 hearts were subjected to 60 minutes of ischemic arrest at 15 degrees C induced with a single dose of cold potassium cardioplegic solution. Hearts were then revived with a 10 minute period of nonworking reperfusion at 28 degrees, 31 degrees, 34 degrees, or 37 degrees C (10 hearts each), followed by 5 minutes of nonworking reperfusion at normothermia, followed by 30 minutes of working perfusion. Repeat measurements of function were obtained and postischemic release of creatine kinase into coronary effluent was determined. Recovery of aortic flow was significantly reduced at lower initial reperfusion temperatures (75% at 28 degrees C versus 88% at 37 degrees C) and the effect was approximately linear throughout the range studied (p less than 0.05). Release of creatine kinase into coronary effluent was greater at lower initial reperfusion temperatures (421 ImU/min/gm wet weight at 28 degrees C versus 115 ImU/min/gm wet weight at 37 degrees C), also in a linear manner (p less than 0.05). In this model, initial postischemic hypothermic reperfusion is deleterious to cellular integrity and functional recovery of the preserved myocardium. Studies in higher animals and humans are warranted to further evaluate the effect of initial reperfusion temperature on myocardial preservation.     

A comparative study of Celsior and University of Wisconsin solutions based on 12-hr preservation followed by transplantation in canine models

BACKGROUND: Celsior is a recently developed extracellular-type preservation solution that is effective in organ preservation. This experimental study was designed to compare the effects of Celsior and University of Wisconsin (UW) solutions in myocardial protection, using 12-hour preservation followed by orthotopic transplantation. METHODS: Fourteen pairs of adult mongrel dogs were divided into 2 groups. In the UW group (n = 7), UW solution at 4 degrees C was used for coronary vascular washout and storage following cardiac arrest with glucose-insulin-potassium (GIK) solution. In the Celsior group (n = 7), Celsior solution was used to produce cardiac arrest, for coronary vascular washout, and for storage. After 12-hour cold preservation, orthotopic transplantation was performed under cardiopulmonary bypass (CPB). The rate of recovery (%) of cardiac function of donor hearts was compared 1 and 2 hours after weaning from CPB, and then the transplanted hearts were harvested for histological study. RESULTS: Hemodynamic parameters including cardiac output, left ventricular pressure (LVP), and the maximum rates of positive and negative increase of LVP after transplantation were significantly (p < 0.05) higher in the Celsior group than in the UW group 2 hours after weaning from CPB. The transmission electron microscopic study found that degeneration of the mitochondria in the Celsior group was less extensive than in the UW group. CONCLUSION: Celsior solution enhanced the cardiac function of hearts preserved for 12 hours prior to transplantation compared to UW solution. Our results indicate that Celsior solution is equivalent or superior to UW solution for cardiac preservation.     

Extending myocardial viability during heart preservation with cyclosporine A.

BACKGROUND AND AIM OF STUDY: Hypothermic preservation (PRES) of donor hearts is limited to 12-14 hours for complete functional recovery after reperfusion. In a canine heterotopic heart transplant model, 50% to 60% functional recovery returned after 18 hours of PRES with University of Wisconsin (UW) solution. Concomitant with functional changes were marked increases in apoptotic cells at 2 (2.69%) and 6 (5.98%) hours of reperfusion with a concomitant decrease in lamin B1 (2% and 7.6%, respectively) with no evidence of necrotic cells. These results suggested that blockade of apoptosis may prolong myocardial viability during PRES and reperfusion. METHODS: Donor hearts were subjected to 18 and 24 hours of PRES (2 degrees C to 4 degrees C) with and without cyclosporine A (CyS) treatment (apoptosis blocker). CyS was given to the donor animal (10 mg/kg), in the PRES solution (10(-5) mol/L), slowly infused during the PRES period (1 mL/min), and also to the recipient animal (2.5 mg/kg). RESULTS: After 18 hours of PRES with CyS, function returned to 100% within 1 hour and stayed at this level throughout a 6-hour recovery period. Apoptotic myocytes were reduced (55%) after 18 hours PRES with CyS treatment, and 6-hour reperfusion lamin B1 was reduced to only 3.7%. Twenty-four hour PRES in UW resulted in no functional recovery. However, after CyS treatment, functional recovery returned to 100% after 4 hours of reperfusion. Adenosine triphosphate (ATP) and creatine phosphate (CP) concentrations were surprisingly the same with or without CyS treatment at 18 hours and lower with 24 hours. CONCLUSIONS: Use of CyS in the PRES solution prolongs myocardial viability during donor heart PRES. The mechanism of action may be associated with the mitochondrial permeability transition (MPT) pore via cyclophilin D binding.     

Prolonged preservation of the blood-perfused canine heart with glycolysis-promoting solution.

BACKGROUND: Prolonged ischemia and inadequate myocardial preservation remain significant perioperative risk factors in cardiac transplantation. Long-term preservation techniques that have been effective in small rodent hearts have not been as effective in larger animal models or in clinical studies. We developed a cardioplegia solution formulated to promote high-energy phosphate production from glycolysis and determined its efficacy in a blood perfused canine heart model subjected to 24 hours of ischemia. METHODS: Hearts harvested from adult dogs (n = 6 per group) were flushed with a histidine-buffered cardioplegia solution containing glucose or University of Wisconsin solution. The hearts were maintained at 4 degrees C for 24 hours then reperfused with autologous blood. After reperfusion, left ventricular pressures were measured with an intracavitary balloon at varying balloon volumes and compared with control nonischemic hearts. Predicted stroke volume and ejection fraction were calculated at an end-systolic pressure of 70 mm Hg and end-diastolic pressure of 15 mm Hg. RESULTS: Developed pressure was better preserved in the hearts that received histidine-buffered solution (93+/-9 versus 38+/-7 mm Hg, p<0.05), along with a higher end-diastolic volume at 15 mm Hg (31+/-3 versus 22+/-2 mL histidine-buffered versus University of Wisconsin solutions, respectively, p<0.05). Stroke volume and ejection fraction were also higher in the histidine group (17+/-2.5 versus 2.3+/-1.2 mL and 50%+/-3.5% versus 9% +/-4.5%, respectively) in the presence of dobutamine. CONCLUSIONS: The highly buffered glycolysis-promoting cardioplegia solution provided effective preservation of the blood perfused canine heart with superior recovery of pump performance after 24 hours of hypothermic ischemia compared with University of Wisconsin solution in this model.     

Beneficial effect of additional cardioplegia flush during hypothermic static cardiac preservation

BACKGROUND: The effects of various preservative solutions and methods have been studied to prolong the safety period of cardiac preservation. In this study, we used cardioplegic solution (CS) during cardiac preservation and investigated how flush CS yields good preservation of isolated hearts compared with only cold immersion. METHODS: Male Wistar rat hearts were arrested with 4 degrees C St. Thomas crystalloid CS. All hearts were immersed for 6 hr in a 4 degrees C Euro-Collins solution. Hearts were classified into seven groups by period and number of infusions of CS (20 ml/kg) during simple immersion of hearts. Infusion of CS during preservation was not used for group I. Infusion was performed at two hours after starting immersion for group II, at 3 hr for group III, at 4 hr for group IV, at 5 hr for group V, every hour for group VI, and every 2 hr for group VII. After preservation, the hearts were reperfused with blood using a support rat. Myocardial adenosine triphosphate was measured immediately after immersion of hearts. Biochemical examination of coronary effluents was performed at 15 min after reperfusion, and cardiac function was evaluated at 40 min after reperfusion. Myocardial specimens were subsequently taken for measurement of water content. RESULTS: Percentage recovery of left ventricular developed pressure and dp/dt in groups III, VI, and VII were higher than those in group I at each balloon volume, and left ventricular end-diastolic pressure in these groups was also significantly lower than that in group I. Levels of creatine kinase-MB and lactate in groups VI and VII after reperfusion were significantly lower than those in group I. Myocardial adenosine triphosphate was significantly better preserved in groups III, IV, VI, and VII than in group I. However, no significant difference in cardiac function or myocardial adenosine triphosphate was found among groups III, IV, VI, and VII. CONCLUSIONS: The use of CS during cardiac preservation is effective in preserving cardiac function and myocardial enzymes, and infusion may be sufficient if performed once-only at 3 or 4 hr from starting immersion in 6 hr storage of isolated rat hearts.     

In a canine model, lung preservation at 10 degrees C is superior to that at 4 degrees C. A comparison of two preservation temperatures on lung function and on adenosine triphosphate level measured by phosphorus 31-nuclear magnetic resonance.

Techniques for organ preservation generally use hypothermia to retard metabolic requirements. However, excessive hypothermia may also produce injury. Using a canine left lung allotransplantation procedure, we compared two preservation temperatures (4 degrees and 10 degrees C) in terms of subsequent lung function measured by temporary occlusion of the right pulmonary artery after implantation of the preserved left donor lung. The lungs were flushed with low-potassium dextran electrolyte solution, inflated with 100% oxygen, and preserved for 18 hours. To investigate possible changes of energy stores at different temperatures, we performed phosphorus 31-nuclear magnetic resonance analyses of lung samples. Sequential determinations of adenosine triphosphate levels in lung tissue preserved at 4 degrees, 10 degrees, and 22 degrees C were studied. After transplantation, lungs preserved at 10 degrees C (n = 6) provided significantly better arterial oxygen tension than those preserved at 4 degrees C (n = 6), 451 +/- 46 mm Hg versus 243 +/- 86 mm Hg (p less than 0.05), and lower pulmonary vascular resistance, 581 +/- 68 dynes.sec.cm-5 versus 1006 +/- 157 dynes.sec.cm-5 (p less than 0.05). Adenosine triphosphate levels at 4 degrees and 10 degrees C were stable and did not differ from each other at the end of the 18-hour preservation period: 0.86 +/- 0.04 mumol/gm wet weight for control versus 0.86 +/- 0.07 mumol/gm wet weight for 4 degrees C and 0.93 +/- 0.06 mumol/gm wet weight for 10 degrees C after 18 hours of preservation. Preservation at 22 degrees C caused a 28% depression of adenosine triphosphate after 18 hours of preservation. These results lead us to conclude the following: (1) Optimal temperature for lung preservation is in the vicinity of 10 degrees C, and (2) lung dysfunction caused by excessive hypothermia is not due to a failure to maintain adenosine triphosphate levels. We suspect that adenosine triphosphate is generated by oxidative phosphorylation during lung preservation.     

Improved myocardial ischemic tolerance by contractile inhibition with 2,3-butanedione monoxime.

Contracture of the arrested myocardium during prolonged storage of the heart results in both systolic and diastolic dysfunction, and is a major limitation to extended preservation. We studied the effects of a reversible contractile inhibitor, 2,3-butanedione monoxime (BDM), on myocardial ischemic tolerance. Isolated rabbit hearts were flushed with University of Wisconsin (UW) solution with and without 30 mmol/L BDM and 1 mmol/L CaCl, stored at 4 degrees C for 24 hours, and subsequently reperfused for 60 minutes. Left ventricular pressure-volume relationships and adenine nucleotide content were determined before reperfusion. Left ventricular systolic pressure, diastolic volume, and adenine nucleotide content were measured after reperfusion. Hearts stored in UW solution underwent contracture and adenosine triphosphate (ATP) depletion during storage, and exhibited systolic dysfunction, impaired diastolic relaxation, and poor ATP regeneration upon reperfusion. The addition of calcium worsened contracture and ATP depletion (p < 0.005) and slightly improved function and ATP regeneration (p = not significant). Hearts stored in the presence of BDM experience no contracture during storage; ATP was preserved (10.7 versus 15.7 nmol/mg; p < 0.05), and left ventricular systolic pressure and ATP content recovered to 74% and 93% of control on reperfusion, respectively (p < 0.005). Left ventricular diastolic volume remained depressed, however, although less than with UW solution (0.87 versus 0.45 mL; p < 0.001). When both BDM and calcium were included in the UW solution, calcium-stimulated ATP hydrolysis and contracture were prevented, left ventricular systolic pressure returned to 87% of control, and left ventricular diastolic volume and ATP content returned to control levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-term heart preservation using a new portable hypothermic perfusion apparatus.

OBJECTIVE: Perfusion storage is not often used clinically compared with simple immersion because of complicated circuits and demanding management. We developed a new apparatus for preservation combined with simple immersion and continuous coronary perfusion. METHODS: The main characteristics of this apparatus are as follows: (1) hypothermic storage, (2) does not require any energy source, (3) variable perfusion pressure, and (4) portability. The perfusion apparatus is composed of a storage chamber, a cooling chamber, and metal bars from which a perfusate bag is suspended. Adult mongrel dogs were divided into two groups: the coronary perfusion group (CP, n = 6) and the simple immersion group (SI, n = 6). Coronary vascular beds of the dog were washed out with a University of Wisconsin (UW) solution following cardiac arrest obtained using a GIK solution. The hearts were then excised. In the CP group, the heart graft, which was immersed in a 4 degrees C UW solution, was perfused with the same solution at a flow rate of 35 approximately 50 ml/hr. In the SI group, the heart graft was immersed in a 4 degrees C UW solution only. The heart graft was preserved for 12 hours in both groups. Beta-adenosine triphosphate (beta-ATP), phosphocreatine (Pcr), and inorganic phosphate (Pi) levels were measured immediately after excision of the heart, and at 3, 6, and 12 hours after preservation. Beta-ATP, Pcr, and Pi values were expressed as a percentage of control values, which had been obtained immediately after excision of the heart. Water content of the myocardium was measured prior to and after 12-hour preservation. The preserved graft was then evaluated through orthotopic transplantation. RESULTS: Beta-ATP/Pi levels at 6 and 12 hours after preservation were significantly higher in the CP group than in the SI group (62 +/- 5 versus 39 +/- 7%, 48 +/- 5 versus 22 +/- 8%, respectively, p < 0.05). Pcr/Pi levels at 6 and 12 hours after preservation were 30 +/- 9% and 22 +/- 8%, respectively in the CP group, while Pcr/Pi levels in the SI group were detected in only one case. There was no significant difference in water content either prior to or after 12-hour preservation between the two groups. Histopathologically, irregular expansion and/or contraction of myocardial fibers were more severe in the SI group than in the CP group. The recovery rate of hemodynamic parameters 2 hours after heart transplantation was significantly (p < 0.05) higher in the CP group than in the SI group. CONCLUSION: Stable and safe long-term canine heart preservation with continuous coronary perfusion associated with immersion is possible using this new apparatus, and may have broad clinical application.     

Minimal amounts of hyaluronidase in HTK or UW solution substantially improve the recovery of preserved hearts

Abstract  Rat hearts were preserved by simple storage for 18 h at 0–1 °C and reperfused parabiotically with whole blood from a host rat. The preservation solutions used for flush perfusion and storage were the commercial solutions EuroCollins, HTK, or UW with or without adding 40 mg/l hyaluronidase or Euro-Flush-Glutathione (EFG) solution, especially designed for prolonged heart storage. All solutions were filtered (0.45 pm) before use. The functional recovery was measured using a latex balloon in the left ventricle for LVP, dp/dt, and isotonic stroke volume. The metabolic recovery as well as the edema formation was determined from freeze-clamped myocardium at the end of reperfusion. In hearts preserved with hyaluronidase-containing solutions, the edema formation during reperfusion was reduced combined with an improvement in the coronary flow. Functional and metabolic recovery were improved in these hearts with significant increase in the stroke volume and ECP in all groups versus hearts preserved in the hyaluronidase-free basic solutions. The effectiveness of HTK preservation was significantly improved by hyaluronidase in all parameters measured in our study. The best functional and metabolic recovery was found in hearts preserved by HTK + H- or EFG-solu-tion. Thus, preservation solutions containing hyaluronidase, especially HTK + H and EFG, seem best suited for the prolonged storage preservation of the heart.     

Effects of nucleoside transport inhibition on long-term ex vivo preservation of canine hearts.

The effect of nucleoside transport inhibition on 24-hour preservation of canine hearts was studied in 36 hearts arrested either with a cold hyperkalemic cardioplegic solution without (group I) or with supplementation of a specific nucleoside transport inhibitor (R75231, 1 mg/L) (groups II and III). The hearts were excised and stored for 24 hours at 0.5 degrees C. Then they were reperfused for 3 hours with use of a closed perfusion system primed with normal blood (groups I and II) or with blood supplemented with the same nucleoside transport inhibitor (0.32 mg/L) (group III). Serial biopsy specimens for determination of myocardial purines were taken. Creatine kinase and heat-stable lactate dehydrogenase release from the myocardium were examined during reperfusion. Recovery of function was studied during reperfusion by measurement of isometric contraction in a fluid-filled intraventricular balloon. After 24 hours of preservation, without the use of the drug, myocardial inosine and hypoxanthine accumulated to, respectively, 4.05 +/- 1.18 and 0.28 +/- 0.08 mumol/gm dry weight. In the drug-treated groups (II and III pooled), significantly less inosine and hypoxanthine accumulated (1.68 +/- 0.33 and 0.05 +/- 0.02 mumol/gm dry weight, respectively) (p < 0.05 versus group I). Upon reperfusion, intramyocardial adenosine was lost in the control hearts and maintained in the drug-treated hearts. Hypoxanthine accumulated significantly (p < 0.05) during reperfusion in group I (1.08 +/- 0.43 versus 0.16 +/- 0.13 in group II and 0.03 +/- 0.03 mumol/gm dry weight in group III). The rate of creatine kinase and heat-stable lactate dehydrogenase release was significantly lower (p < 0.05) in group III (that is, pretreatment and posttreatment with the drug) than in the control group. Functional recovery of hearts in group III was superior to that in group II (p < 0.05), while hearts in group I showed no recovery at all. We conclude that nucleoside transport inhibition improves long-term preservation of the heart and that the mechanism of this protection may be related to an increase in endogenous adenosine and reduction of myocardial hypoxanthine content.     

A non-heart-beating donor model to evaluate functional and morphologic outcomes in resuscitated pig hearts.

A non-heart-beating donor model was considered to examine whether pig hearts from the abattoir could be resuscitated by whole blood reperfusion. For preservation, machine perfusion using University of Wisconsin (UW) solution was compared with storage on ice. Nineteen hearts from abattoir pigs, harvested 25 +/- 3 min after exsanguination, were harvested and transported to the laboratory. Controls (n = 7) were immediately reperfused with homologous whole pig blood in an isolated heart model for 60 min with monitoring of left ventricular developed pressure (LVDP), contractility, and coronary flow. UW solution hearts (UW, n = 6) were perfused for 4 h with 10 degrees C cold UW solution before blood reperfusion. In the cold storage group (CS, n = 6), the organs were stored for an additional 4 h on ice before blood reperfusion. In all hearts, histology was performed after 60 min of blood reperfusion to evaluate myocardial reperfusion injury. All three groups showed significant increases in LVDP (p <.001), although this functional recovery was earliest in the control group and latest in the UW group. Significant declines were observed for both LVDP and contractility from the peak values in each group to the end of blood reperfusion. Coronary flow increased steadily over the time course for the UW group, whereas in the control and CS groups flow increased during the first 15 min of blood reperfusion and then decreased. In the UW and CS groups, there were significant positive correlations between coronary flow and LVDP (p <.001). Microscopic examination revealed no differences between the three groups. Thus, hearts from an abattoir with 25 min of warm ischemic time can be resuscitated. For storage of these organs, continuous machine perfusion with UW solution is superior to cold storage on ice.     

Subnormothermic machine perfusion protects against rat liver preservation injury: a comparative evaluation with conventional cold storage

Hypothermic machine perfusion (MP) of the liver has been reported to improve graft function reclaiming marginal livers, such as those from non-heart-beating donors. Livers from obese donors often have fatty infiltrates and are more susceptible to hypothermic conditions. No data exist about MP at temperatures >4 degrees C. This study evaluated liver function after organ preservation by comparing MP at 20 degrees C with conventional cold storage. METHODS: For MP, rat livers were perfused for 6 hours using an oxygenated Krebs-Henseleit (KH) solution at 20 degrees C (pH 7.4). For cold storage, livers were perfused in situ and preserved with Celsior solution at 4 degrees C for 6 hours. The reperfusion period with KH (2 hours at 37 degrees C) was performed under the same conditions both among livers preserved by MP or cold storage. Hepatic enzyme release (aspartate aminotransferase [AST], alanine aminotransferase [ALT], lactate dehydrogenase [LDH], and gamma-glutamyl transferase [GGT]), bile production, and ATP levels were measured during MP and reperfusion. RESULTS: At the end of reperfusion, livers preserved by MP showed significantly decreased liver damage compared with cold storage: AST, 18 +/- 4 vs. 45 +/- 6 mU/mL (P < .01); ALT, 1.5 +/- .07 vs. 6 +/- 0.5 mU/mL (P < .01); and LDH, 82 +/- 2 vs. 135 +/- 29 mU/mL (P < .05). No difference was observed between bile production between MP and cold storage. High levels of biliary GGT and LDH were found in cold preserved livers. ATP levels were higher in livers preserved with MP compared with those preserved by cold storage. CONCLUSIONS: MP at 20 degrees C resulted in a better quality of liver preservation, improving hepatocyte survival, compared with conventional cold storage. This may provide a new method for successful utilization of marginal livers, in particular fatty livers.     

Celsior solution compared with University of Wisconsin solution (UW) and histidine–tryptophan–ketoglutarate solution (HTK) in the protection of human hepatocytes against ischemia–reperfusion injury

Celsior, a new preservation solution in thoracic organ transplantation was evaluated for efficacy in cold preservation of human hepatocytes and compared with University of Wisconsin solution (UW) and histidine-tryptophan-ketoglutarate solution (HTK, Custodiol). Human hepatocyte cultures were preserved at 4 degrees C in Celsior, UW and HTK for 2, 6, 12, 24 and 48 h with 6 h of reperfusion. Levels of lactate dehydrogenase (LDH; cell necrosis), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; mitochondrial function), and adenosine 5'-triphosphate (ATP; loss of intracellular energy) were measured. Cell necrosis, mitochondrial dysfunction, and loss of ATP were significantly ( P<0.001, P<0.001, P<0.002, respectively) lower in Celsior than in HTK. The amount of cell necrosis and mitochondrial dysfunction in Celsior solution (CS) and UW was equal ( P=n.s.) up to 24 h and significantly lower in UW after 48 h ( P<0.001). Additionally, the intracellular level of ATP was significantly higher after ischemia ( P<0.001) and reperfusion from long-term ischemia (24, 48 h) ( P<0.002). We can conclude that Celsior was superior to HTK and equal to UW in the protection of human hepatocytes against cold preservation injury from ischemia and reperfusion. Furthermore, Celsior was effective in long-term preservation of human hepatocytes.