Heart transplantation is a well-established treatment for patients in end-stage heart failure. One of the primary causes of early death after transplantation is cardiac graft dysfunction, suggesting that early survival is influenced by preservation and implantation injury.1 A prolonged ischemic period is regarded as a major factor in postischemic reperfusion injuries leading to cardiac dysfunction. Many techniques have been used to protect myocardium from reperfusion injury, including the use of intraoperative free radical scavengers2,3 and the optimization of the oxygen supply/demand balance during reperfusion.4,5 Currently, one major problem in heart transplantation is the shortage of donors. However, the donor heart is occasionally not utilized because an appropriate recipient cannot be found.6 Usually, the ischemic time is limited to 4 hours. Improved cardiac preservation techniques may allow better donor allocation and maximize the donor usage.
Perfluorochemicals (PFCs), one type of artificial blood substitute, are hydrocarbons in which most or all of the hydrogen atoms have been replaced with fluorine.7 They have been investigated as adjuncts in cardioplegia solutions,8 in preserving myocardium during transient ischemia during angioplasty,9,10 and in improving cardiac function after heart transplantation.11,12 These results suggest that PFCs may have protective effects on cardiac function after cardiac ischemia and heart transplantation.
Conversely, Tabayashi and associates13 reported that there was no difference between oxygenated crystalloid solution and oxygenated fluorocarbon emulsion cardioplegic solution in myocardial protection after 2-hour global ischemia. Thus, the effect of PFC emulsion on cardiac protection remains controversial.
Perfluoro-octyl bromide (PFOB) emulsion, a PFC, was recently developed by a new base and emulsification technique from second-generation PFCs.14 We introduced a new emulsifying technology and improved the stability and oxygen transport capacity.15 We hypothesized that PFOB emulsion added to cardiac storage solution may have an advantage for cardiac protection and lead to prolonging storage time. The purpose of this study was to evaluate the cardioprotective effects of PFOB emulsion in storage solution after 6-hour cold storage.
Materials and Methods
Male Hartely guinea pigs (n = 24) weighing from 300 to 400 g (Sankyo Lab, Japan) were used in this study. All animal care was in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources published by the National Institute of Health (NIH publication No. 86-23, revised 1985).
The animals were randomly divided into the three groups: nonoxygenated St. Thomas Hospital II (STS) group, oxygenated STS (O2 STS) group, and oxygenated STS with 30% PFOB emulsion (O2 STS + PFOB) group. STS had the following composition (mM): sodium, 120.0; potassium, 16.0; calcium, 1.2; magnesium, 16.0; chloride, 160.4; bicarbonate, 10.0.
Artificial Blood Substitute
The PFOB emulsion (100 ml) used in this study was 28% PFOB having about 210 nm of average particle size as a base, 12% of an perfluoroalchol esters with oleic acid (FO - 982), 2.4% of yolk lecithin, and 0.12% of polyethyleneglycol (PEG), 1, 2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-PEG (DSPE-50H). The composition of electrolyte in the O2 STS + PFOB group was modified to be the same as that of the other experimental groups. In addition, 5% bovine serum albumin was added to the O2 STS + PFOB group to modulate osmolarity.
The experimental design is summarized in Figure 1; 30 mg ketamine and 1,000 U heparin were injected into peritoneum 15 minutes before animals were killed. After death, the hearts were immediately perfused at 30 mm Hg aortic pressure and 10 mm Hg left ventricular end-diastolic pressure in the Langendorff apparatus through the aortic root with 37°C Krebs-Henseleit solution (KHS) equilibrated with 95% O2 and 5% CO2. KHS (pH 7.4 ± 0.1, Po 2 557.55 ± 22.2) composition was as follows (mM): NaCl, 118.2; NaHCO3, 25.0; KH2PO4 1.0; KCl, 4.83; MgSO4, 2.37; CaCl2, 2.5; glucose, 11.1. In each group, hearts were stabilized for 30 minutes, followed by baseline for 30 minutes. Hearts were cooled from 37°C to 16.8 ± 1.1°C in < 1 minute, after which perfusion was switched from KHS to STS. Perfused STS was stopped at 12.1 ± 0.7°C (10 minutes after initiating cooling). Hearts were then stored (no flow, no bubbling) in each experimental group (STS, O2 STS, or O2 STS + PFOB) for 6 hours at 4°C. The storage solution was oxygenated for 20 seconds before 6-hour cold storage. On rewarming to 23.4 ± 0.3°C in < 1 minute, perfusion with STS was continued for 10 minutes and at 25.1 ± 0.4°C, and then perfusion solution was switched back to KHS, and KHS was perfused for 60 minutes.
In the first 10 minutes of reperfusion, sample tubes for coronary effluent were replaced every 2 minutes; tubes were replaced every 5 minutes during the last 50 minutes. The volume of coronary effluent collected for each period was measured and subsequently analyzed for creatinine kinase (CK) and lactate dehydrogenase (LDH).
Measurement of Cardiac Mechanical and Metabolic Variances
The heart rate (beats per minute) was analyzed from the electrocardiogram (average three R – R intervals). Left ventricular developed pressure (LVDP) was measured as left ventricular end-systolic pressure minus left ventricular end-diastolic pressure. Coronary flow rate (milliliters per minute per gram) was calculated from the coronary effluent (milliliters), sampling time (minutes) and cardiac wet weight (grams).
At the point of just before cooling as the baseline and at the time of just after reperfusion, 10 and 60 minutes after reperfusion, we measured percent O2 extraction, myocardial O2 consumption (MVO2), and cardiac efficiency. Oxygen concentration of perfusate was used as arterial Po2 and oxygen concentration of coronary effluent was used as venous Po2. MVO2 and cardiac efficiency were calculated as coronary flow volume /g × (perfusate Po2N coronary effluent Po2) × 24 μl O2/ml at 760 mm Hg and as LVDP × heart rate/MVO2, respectively.
Measurement of Creatinine Kinase and Lactate Dehydrogenate
Creatine kinase and LDH were analyzed by CX-7 (Beckmann Coulter Inc.). Measurement detection limits were 5–1,200 IU/l for CK and 20–1,200 IU/l for LDH, respectively.
Measurement of Myocardial Water Content
After mechanical and metabolic variances were collected, the aorta, pulmonary artery, and right and left atria were removed and cardiac wet weight was measured. The hearts were then dried at room temperature for 2 weeks and reweighed to obtain cardiac dry weight. Myocardial water content (MWC, %) was calculated as follows: (1 – cardiac dry weight/cardiac wet weight) × 100.
All experimental values were expressed as mean and standard error of the mean (SEM) in each group. In mechanical and metabolic variances, comparisons were analyzed by repeated measurement analysis of variance (ANOVA) and/or one-way ANOVA, using Bonferroni t test for post hoc analysis. P value < 0.05 was deemed significant in the experimental data.
There were no significant difference among the three groups in heart rate and coronary flow rate (Figures 2A and 2B). However, the value of developed pressure in the O2 STS + PFOB group was markedly higher than those of the STS and O2 STS groups at 60 minutes reperfusion (p < 0.01) (Figure 2C). There was no difference among the three groups in the time to spontaneous heart rhythm and return to sinus rhythm (data not shown).
As shown in Table 1, there was no difference among the three groups in O2 extraction and cardiac efficiency; however, O2 STS + PFOB group significantly improved MVO2 during reperfusion (p < 0.01).
Creatinine Kinase and Lactate Dehydrogenate
CK release during 60 minutes reperfusion in O2 STS + PFOB group was markedly attenuated compared to STS group and O2 STS groups (p < 0.01) (Figure 3). There was no significant difference among the three groups in the baseline values.
The LDH release during 60-minute reperfusion in the O2 STS + PFOB group was markedly attenuated compared with the STS group and O2 STS groups (p < 0.01) (Figure 4). There was no significant difference among the three groups in values at baseline.
Myocardial Water Content
The MWC in the O2 STS + PFOB group was significantly decreased compared with the STS group (p < 0.01, (Figure 5).
Primary graft dysfunction is the main cause (approximately 50%) of early mortality after heart transplantation reported from the International Society for Heart and Lung Transplantation.1 Perioperative cardiac ischemia/reperfusion injuries may augment the late coronary vasculopathy, leading late to death after heart transplantation.1,16 These reports suggest that the improvements in cardiac preservation may decrease both early and late mortality after heart transplantation. In this study, we have demonstrated that PFOB emulsion added to cardiac storage solution may improve graft dysfunction by preserving cardiac function and inhibiting CK and LDH release after 6-hour cardiac cold storage.
In general, artificial blood substitutes are divided into two groups as modified hemoglobin and PFCs, including PFOB. Hemoglobin has a sigmoid-shaped oxygen dissociation curve that shifts leftward, thus attenuating its ability to release oxygen at low temperatures.17,18 Blood also potentially contains other elements considered important in cellular injury, such as leukocytes, complement, and cytokines. In contrast, PFCs have no injurious factors, have a high affinity for oxygen, and release oxygen in a linear fashion unaffected by temperature.19 Therefore, PFOB may be more suited than hemoglobin for supplying oxygen during cardiac cold storage.
Recently, the harmful effects of cardiac cold storage have been reviewed and include: 1) cellular swelling, 2) extracellular edema, 3) cellular acidosis leading to decreased oxygen delivery by hemoglobin, 4) depletion of metabolic substrate, 5) reperfusion injury, 6) calcium overload, and 7) vascular endothelial cell injury.20 The use of PFOB emulsion during cardiac cold storage may have some advantages for delivering oxygen under the condition of low temperature, improving cardiac contractile function, and attenuating extracellular edema.
However, for oxygen delivery during cardiac cold storage, there was no significant difference in O2 content in the storage solution between the O2 STS group and the O2 STS + PFOB before and just after 6-hour cardiac cold storage (data not shown). This finding suggests that PFOB emulsion may have a cardioprotective effect by itself. In vitro studies have shown that PFOB reduces cytokines release by human alveolar macrophage21 and attenuates oxidative injury in rat pulmonary artery endothelial cells.22 Furthermore, neutrophil adhesion to lung epithelial cells and subsequent cytolysis are inhibited by PFOB during the activation with a proinflammatory stimulus,23 and PFOB attenuates neutrophil adhesion to activated endothelial cells.24 These phenomena may be related, at least in part, to the cardioprotective effect of PFOB. However, the mechanism of these PFOB effects has never been clearly elucidated.
There have been many reports about the cardioprotective effects of PFCs. The amount of myocardial ATP and mortality after heart transplantation were improved in oxygenated University of Wisconsin solution supplemented with PFCs in a unique heart preservation technique, but not in nonoxygenated PFCs solution.25 Furthermore, PFCs improved the cardiac function of transplanted hearts11,12 and the survival time (hyperacute rejection) of guinea pig heart xenografts by inhibiting thrombus formation in the xenografted heart.26 Other investigators have shown that PFCs impair the function of the postischemic heart.9,10 It has also been demonstrated that there was no difference in cardioprotective effects between an oxygenated crystalloid and oxygenated fluorocarbon solution after 2-hour ischemic arrest.13 Thus, the cardioprotective effect of PFCs has not been conclusively demonstrated.
The results of this study support the cardioprotective effect of PFCs found in previous studies.9–11,25,26 However, the discrepancy between a previous study13 and our study may be explained by 1) the use of a different base (fluorocarbon vs. PFOB), and 2) the presence or absence of emulsion. In this study, PFOB was modified by yolk lecithin and PEG. However, the protective effects of this emulsion have not been known until now.
There are a few reports of the cardioprotective effect of PEG. Storage solution (University of Wisconsin) containing PEG improved cardiac functions after cold storage.27 In one comparison study among STS, fluosol, PEG, or fluosol-PEG storage solution for cardiac preservation, only fluosol-PEG markedly improved cardiac contractile function, preserved myocardial high-energy phosphate content, and decreased LDH release after cold storage.28 PEG was suggested to be an important contributor in the cardioprotective effect in this study. However, there has never been a report about this effect by using PFOB emulsion surrounded by PEG, and the cardioprotective effect of PEG has not been clarified.
In this study, we did not perfuse the coronary circulation with PFOB emulsion before 6-hour cardiac cold storage, because there was no difference when it was added to cold storage solution alone or to cold storage solution and preinfused with PFOB emulsion in our preliminary experiment (data not shown). Our findings in this study cannot explain this result. However, previous studies have demonstrated that PEG conjugates may enhance the permeability and the retention effect.29,30 It is possible that PFOB emulsion may penetrate via the cardiac wall or extramural coronary vessels during cardiac cold storage.
In conclusion, our study suggests that PFOB emulsion added to storage solution may enhance myocardial preservation. It can improve cardiac function, inhibit cardiac damage, and attenuate myocardial water content after 6-hour cold storage. PFOB emulsion may be used to prolong the cold ischemic time of donor heart. However, it is necessary to evaluate the benefit of PFOB emulsion for cardioprotective effect both in large experimental animals (canine or porcine heart) and in more prolonged cardiac cold storage times.
This study was supported in part by grants from the Ministry of Education, Science and Culture (No.90312363, 11557112 and 11694229), and by Health Sciences Research Grant of Ministry of Health, Welfare and Labor of Japan for Research on Advanced Medical Technology. The authors thank Dr. Eiji Uchida, associate professor at Rakunogekuen University, for teaching the methods of CK and LDH assay, and Dr. S. Ullah of Arkansas Children's Hospital for his help with the manuscript.
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