Ann Thorac Surg 1997;63:459-464
© 1997 The Society of Thoracic Surgeons
Original Article: Cardiovascular
Beneficial Effects of Fluosol–Polyethylene Glycol Cardioplegia on Cold, Preserved Rabbit Heart
Joginder N. Bhayana, MD,
Zhong T. Tan, MD, PhD,
Jacob Bergsland, MD,
Daya Balu, MD,
Jaswant K. Singh, MD,
Eddie L. Hoover, MD
Departments of Surgery and Pathology, State University of New York at Buffalo and Buffalo General Hospital, Buffalo, and Department of Veterans Affairs, Samuel S. Stratton Medical Center, Albany, New York
Accepted for publication September 9, 1996.
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Abstract
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Background. Because of its high oxygen-carrying capacity, especially at low temperatures, fluosol may enhance heart preservation.
Methods. Hearts of male New Zealand white rabbits (1.5–2.0 kg) were excised and flushed through the aorta with 0°C St. Thomas' Hospital solution, fluosol, or polyethylene glycol or fluosol–polyethylene glycol cardioplegic solution. Hearts were then stored for 12 hours at 0°C and reperfused with Krebs-Henseleit buffer at 36.5°C for 60 minutes using a Langendorff system.
Results. Myocardial contractile function was significantly greater in the fluosol–polyethylene glycol cardioplegia–preserved group (p < 0.01) and polyethylene glycol–cardioplegia preserved group (p < 0.05) than in the St. Thomas' Hospital solution–preserved group. The myocardial high-energy phosphate content was significantly higher in the fluosol–polyethylene glycol–cardioplegia–preserved group (p < 0.01), with reduced release of lactate dehydrogenase (p < 0.01) in comparison with the St. Thomas' Hospital solution–preserved group.
Conclusions. The addition of fluosol and polyethylene glycol to the cardioplegic solution may enhance long-term cold heart preservation.
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Introduction
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Safe heart preservation is limited clinically to a few hours of cold storage [1], because hypothermia inhibits oxidative phosphorylation, resulting in the depletion of myocardial high-energy phosphates and the accumulation of toxic reductive metabolites [2, 3]. In addition, neutrophils, platelets, and complement are activated by ischemia. Further, at reperfusion, oxygen produces radicals, thereby enhancing lipid peroxidation [4]. The resulting cell adhesion, aggregation, and chemotaxis may in turn damage endothelial and myocardial cells [5–9]. Reperfusion is also associated with incomplete restoration of the microcirculation [10, 11].
Perfluorochemicals are blood substitutes with a high oxygen-carrying capacity, low viscosity, and small particle size [12]. These solutions are stable, sterile, and physiologic and are capable of dissolving oxygen and carbon dioxide. Because they are also acellular, the endothelial and myocardial injury mediated by neutrophils and platelets may be ameliorated. Because of its various properties, fluosol emulsion may penetrate collateral capillaries of the myocardium [13, 14], clear blood-borne elements, and improve postischemic microcirculation. Fluosol is also superior to blood in transporting oxygen at low temperatures [13]. The addition of fluosol to the cardioplegic solution may therefore be beneficial during cardiac preservation. University of Wisconsin cardioplegic solution modified by the addition of polyethylene glycol (PEG) has been reported to have superior protective effects [15]. On the basis of this, we hypothesized that the addition of fluosol to the PEG cardioplegic solution might have a further protective effect. In this study, fluosol and PEG were added to the cardioplegic solution used to preserve rabbit hearts for 12 hours at 0°C.
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Material and Methods
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Fifty male rabbits (1.5–2.0 kg) were purchased from Becker Farm (Lockport, NY). Fluosol was obtained from Alpha Therapeutical Corporation (Los Angeles, CA), and PEG from Union Carbide Chemicals & Plastics Company Inc (Danbury, CT). Other chemicals were purchased from Sigma Chemical Company (St. Louis, MO).
Preservation and Reperfusion of Rabbit Hearts
All animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" (NIH publication 85-23, revised 1985). Rabbits were anesthetized with sodium pentobarbital (50 mg/kg) through an ear vein injection. The chest was opened, and 50 mL of St. Thomas' Hospital solution was injected into the inferior vena cava. The hearts were immediately excised and the root of the aorta flushed with 25 mL of ice-cold St. Thomas' Hospital solution. Hearts were further flushed with 25 mL of ice-cold (0°C) St. Thomas' Hospital solution, fluosol, PEG cardioplegic solution [15], or fluosol-PEG cardioplegic solution and preserved for 12 hours in ice-cold Krebs-Henseleit bicarbonate buffer (mmol/L: Na+, 139; K+, 5.9, Ca2+, 1.2; Mg2+, 1.2; Cl-, 125.1; HCO3-, 21; SO42-, 1.2; H2PO4-, 1.2; glucose, 11, in double-distilled water). To assess the recovery of myocardial function, after preservation the hearts were reperfused with oxygenated Krebs-Henseleit buffer using a Langendorff system, and this was done according to the method of Apstein and associates [16].
Measurements of systolic and diastolic function were recorded on a Gould TA11 physiologic recorder (Gould Instrumentation Systems, Inc., Valley View, OH). The left ventricular developed pressure and the maximum rate of increase (+dp/dt) and decrease (-dp/dt) of left ventricular pressure were also recorded.
Preservation Solutions
The composition of the preservation solutions is as follows:
St. Thomas' Hospital solution (mmol/L): Na+, 120.0; K+, 16.0; Mg2+, 16.0; Ca2+, 1.2; CL-, 160.4; HCO3- 10.0. pH: 7.3–7.4.
Fluosol (mmol/L): Na+, 127.6; K+, 5.5; Ca2+, 2.4; Mg2+, 2.1; Cl-, 116.2; HCO3- 25.0. W/V: 20% perfluorodecalin and perfluorotripropylamine, 2.72% poloxamer 188, 0.80% glycerin, 0.18% glucose, 0.40% egg yolk phospholipids, oxygenated. pH: 7.3–7.4.
PEG cardioplegic solution (mmol/L): Na+, 140.0; K+, 125.0; Mg2+, 5.0; CL-, 140.0; SO42-, 5.0; H2PO4-, 25.0; lactobionate, 100.0; raffinose, 30.0; glutathione, 3.0. PEG: 20 mol/L 100 g/L. pH: 7.3–7.4.
Fluosol-PEG cardioplegic solution: PEG added with fluosol components (W/V): 10% perfluorodecalin and perfluorotripropylamine oxygenated, 1.36% poloxamer 188, 0.40% glycerin, 0.20% egg yolk phospholipids. pH: 7.3–7.4.
Experimental Groups
Group I (control group) hearts were excised and immediately perfused without preservation. Group II (St. Thomas' group) hearts were flushed with 50 mL of ice-cold St. Thomas' Hospital solution and preserved at 0°C for 12 hours. Group III (fluosol group) hearts were flushed with 25 mL of ice-cold St. Thomas' Hospital solution and 25 mL of ice-cold oxygenated fluosol and preserved at 0°C for 12 hours. Group IV (PEG group PEG) hearts were flushed with 25 mL of ice-cold St. Thomas' Hospital solution and 25 mL of ice-cold PEG cardioplegic solution and preserved at 0°C for 12 hours. Group V (fluosol-PEG group) hearts were flushed with 25 mL of ice-cold St. Thomas' Hospital solution and 25 mL of ice-cold fluosol-PEG cardioplegic solution and preserved at 0°C for 12 hours.
Measurement of Myocardial Function
The left ventricular developed pressure, +dp/dt, -dp/dt, coronary flow rate, and heart rate were measured after 60 minutes of reperfusion. Measurements were performed at varying balloon volumes in increments of 0.05 mL.
Analysis of Myocardial High-Energy Phosphates
After the functional assessment, hearts were freeze-clamped and stored in liquid nitrogen. Left ventricular muscle (0.1–0.2 g) was homogenized with ice-cold 7.1% perchloric acid and centrifuged at 1,000 g for 10 minutes at 0°C. Adenosine triphosphate and creatinine phosphate were analyzed in the supernatant by enzymatic determination using a spectrophotometer (Stasar, Gilford Instrument Laboratories, Oberlin, OH) at an ultraviolet light wavelength of 340 nm, according to the method of Yoshikawa and associates [17].
Lactate Dehydrogenase Activity
Coronary effluent was collected continuously during reperfusion, and the lactate dehydrogenase activity in the effluent was analyzed by an enzymatic assay at an ultraviolet light wavelength of 340 nm, according to the method of Schmid and colleagues [18].
Histologic Study
A full-thickness piece of left ventricle wall was fixed at the end of the experiment with ice-cold electron microscopy fixative (1% glutaraldehyde, 4% formaldehyde) and another piece with light microscopy fixative (10% formalin), and the resulting samples were studied by electron microscopy and light microscopy, respectively. The integrity of the cellular and nuclear membranes and of the ultrastructure were scored and the findings among groups compared.
Statistical Analysis
Variable measures were expressed as the mean ± standard error. Statistical analysis was performed using multiple or one-way analysis of variance to compare means between groups. A p value of less than 0.05 was considered significant.
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Results
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Myocardial Function
The myocardial function in the five groups of hearts after 60 minutes of reperfusion is summarized in Table 1
. All groups showed a decreased +dp/dt, -dp/dt, and left ventricular developed pressure (group II, p < 0.001; groups III and IV, p < 0.005; group V, p < 0.01) in comparison with the control group. The coronary flow rate (p < 0.01) and heart rate (p < 0.05) in group II were also decreased. In comparison with group II, group III showed an increase in the +dp/dt, -dp/dt, and left ventricular developed pressure, but this did not reach statistical significance. Group IV showed a slight increase (p < 0.05) and group V more of an increase (p < 0.01).
Myocardial Compliance
Figure 1 shows the left ventricular end-diastolic pressure-volume relationships at 60 minutes of reperfusion. All four preserved groups showed a leftward shift of the regression line in comparison with group I. Group II showed the worst myocardial compliance (p < 0.01). Group III showed no significant improvement in comparison with Group II. However, groups IV and V showed improved myocardial compliance versus group II (p < 0.05) as well as versus the control group (p < 0.05).
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Fig 1. . Left ventricular compliance at the end of reperfusion after preservation. In comparison with group I, *p < 0.05 and **p < 0.01 at 0 or 25 mm Hg left ventricular end-diastolic pressure (LVEDP). In comparison with group II, *p < 0.05. Group IV versus group V = p > 0.05.
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Myocardial High-Energy Phosphates
The left ventricular high energy-phosphate contents for the five groups are shown in Figure 2. All groups showed a decrease in the adenosine triphosphate and creatinine phosphate contents in comparison with group I (groups II, III, and IV: p < 0.001; group V, p < 0.01). Groups III and IV showed no significant increase in comparison with group II (p > 0.05), but group V showed a significant increase (p < 0.01). In comparison with group III, group IV showed no significant difference (p > 0.05) but group V showed an increase (p < 0.01).
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Fig 2. . Myocardial high-energy phosphate contents at the end of reperfusion after preservation. In comparison with group I, *p < 0.01 and **p < 0.001. Group V versus groups II, III, and IV = p < 0.01. (ATP = adenosine triphosphate; CP = creatinine phosphate.)
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Release of Myocardial Lactate Dehydrogenase
Figure 3 shows the activities of the lactate dehydrogenase released from the myocardium during reperfusion. All groups showed a higher release than group I (groups II, III, and IV: p < 0.001; group V, p < 0.01). Groups III and IV showed no significant difference in comparison with group II, but group V showed a significant decrease (p < 0.01).
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Fig 3. . Activities of lactate dehydrogenase (LDH) released from reperfused heart after preservation. In comparison with group I, *p < 0.01 and **p < 0.001. Group V versus groups II, III, and IV = p < 0.001.
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Histologic Study-Light Microscopy
Subtle changes, such as pink, wavy myocardial fibers, interstitial edema, and contraction bands, were observed in all groups. However, the band changes were not prominent in any of the groups. There was no evidence of acute myocardial necrosis.
Ultrastructural Analysis
Electron microscopy findings indicated that all subcellular organelles undergo some changes during hypothermia and reperfusion. The control group showed only minimal alterations. Group II showed irreversibly injured myocytes, whereas groups III, IV, and V showed minimal nonspecific changes similar to those seen in the control group. The electron microscopy findings are summarized in Table 2 and shown in Figures 4 and 5.
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Fig 4. . Group II. (A) Marked edema with loss of myofibrils (long black arrows). Edema and swelling of mitochondria (black arrowheads). (x7000 before 47% reduction.) (B) Mitochondria showed amorphous and granular densities (long black arrows). Swelling of cristae and increase in matrix space with edema of mitochondria (black arrowheads). (x21,000 before 43% reduction.)
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Fig 5. . Group IV. (A) Normal myofibrillar arrangement (long white arrows) and closely packed, normal-appearing mitochondria (long black arrows). (x8,000 before 37% reduction.) (B) Normal-appearing mitochondria (long black arrows) in association with glycogen (white arrowheads). (x24,000 before 37% reduction.)
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Comment
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Rabbit hearts preserved with fluosol-PEG cardioplegic solution for 12 hours showed improved recovery of left ventricular function, myocardial compliance, and high-energy phosphate contents and less loss of myocardial lactate dehydrogenase than all other groups (except the control group). The integrity of the ultrastructure of the myocytes in the fluosol-PEG cardioplegic solution group was well maintained. It has been reported that the addition of either PEG or fluosol to the cardioplegic solution during long-term preservation enhances the protective effects [13–15]. This study confirms the beneficial effects of PEG cardioplegic solution containing fluosol as well.
The mechanism responsible for enhancing protection is not clearly understood. In the current study, fluosol alone showed the same protective effects as St. Thomas' Hospital solution, but the addition of fluosol to the PEG enhanced the protective effects. It is speculated that these enhanced protective effects are related to either a better oxygen supply to remote areas of preserved hearts or to the small particle size of perfluorochemicals. The mean particle size of fluosol is less than 270 nm, allowing for the solution to penetrate to collateral capillaries of the myocardium. Perfluorochemicals may also clear blood-borne elements and transport oxygen to remote myocytes [14]. Fluosol has a greater capacity to bind to and release oxygen under hypothermic conditions than blood does [14]. As oxidative phosphorylation ceases during heart preservation, reductive metabolites, such as lactic acid, reduced nicotinamide adenine nucleotide, reduced nicotinamide adenine dinucleotide phosphate, acetyl coenzyme A, acetyl carnitine, and hydrogen ions accumulate. These reductive metabolites are toxic to the lipid structures of the cell and to the subcellular organelles [2–4]. This high capacity of fluosol to bind to and release oxygen under hypothermic conditions may cause reductive toxic metabolites to be oxidized and their levels reduced during ischemia. Reduced release of LDH, observed in the fluosol-PEG cardioplegic solution–preserved group, may be attributed to the protective effects of the cardioplegic solution on cellular membranes.
A limitation to this study was that there was no direct determination of the differences in oxygen uptake between the group receiving fluosol-PEG cardioplegic solution and the control group or the group receiving St. Thomas' Hospital solution. The addition of fluosol to PEG cardioplegic solution may have led to the improved intracellular penetration of any agent. If so, the changes in morphology may have been related to fluosol entry into the cell, which had the effect of improving nitric oxide production with its subsequent antineutrophil adhesion effects. This, of course, requires measurement of the myeloperoxidase activity, which we are planning to do. The most dramatic conclusion yielded by this pilot study is that adding PEG and a high oxygen containing solution, such as fluosol, to the cardioplegic solution causes a significant improvement in myocardial contractile function and in the cellular biochemical variables. Additional measurements of conjugated "dienes" will be done in subsequent studies to assess the limitation of the oxygen free radical injury. The current study underscores the importance of site-related injury in determining the degree of preservation. Because of the clinical relevance of PEG to a cardioprotective solution, new metabolic studies have been started to clarify these effects. In this regard, we are now testing the addition of PEG and fluosol to a cardioplegic solution to determine its effectiveness in limiting oxygen free radical injury and cellular oxygen uptake.
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Acknowledgments
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We appreciate the support of the Buffalo Heart Surgical Associates. This study was supported by the Heart Transplant Fund and the Buffalo General Hospital Foundation.
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Footnotes
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Address reprint requests to Dr Bhayana, Department of Surgery, Buffalo General Hospital, 100 High St, Buffalo, NY 14203.
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Abstract
Introduction
