Ann Thorac Surg 1999;67:1345-1349
© 1999 The Society of Thoracic Surgeons
Original Articles
Effects of cardioplegic flushing, storage, and reperfusion on coronary circulation in the pig
Algimantas Budrikis, MDa,
Qiuming Liao, MDa,
Ramunas Bolys, MDa,
Boris Westerlaken, MDa,
Stig Steen, MD, PhDa
a Department of Cardiothoracic Surgery, University Hospital, Lund, Sweden
Accepted for publication November 18, 1998.
Address reprint requests to Dr Steen, Dept of Cardiothoracic Surgery, University Hospital of Lund, SE-221 85 Lund, Sweden
e-mail: stig.steen{at}thorax.lu.se
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Abstract
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Background. The aim of the study was to investigate how flush-perfusion of the heart with cold cardioplegic solution, 2 or 12 hours of cold ischemic storage, and 24 hours of reperfusion affect coronary endothelial function and coronary vascular resistance.
Methods. Porcine coronary arterial endothelial and smooth muscle function was studied in organ baths. An adult porcine working heart model was used to investigate coronary vascular resistance after 24 hours of reperfusion.
Results. Flushing the heart with 1 L of St. Thomas’ cardioplegic solution, using a perfusion pressure of 60 to 65 mm Hg, significantly reduced endothelium-dependent relaxation. Flushing followed by 12 hours of storage gravely impaired endothelium-dependent relaxation, and 24 hours of reperfusion worsened it still more.
Conclusions. Flushing the heart with cold cardioplegic solution impairs endothelium-dependent relaxation, as does prolonged cold ischemic storage. Reperfusion of injured coronary endothelium may injure it still more. A correlation was found (p < 0.001) between high coronary vascular resistance and low endothelium-dependent relaxation.
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Introduction
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Heart preservation for clinical transplantation is achieved in most centers by flushing the heart with a cold cardioplegic solution followed by cold ischemic storage. Regardless of the type of cardioplegic solution used, most centers do not accept more than 4 to 5 hours of ischemic time before reperfusion with oxygenated blood is initiated. Longer ischemic times are correlated directly to an impaired postimplantation myocardial function. There is no general global agreement about which cardioplegic solution is best. In the Scandinavian countries, St. Thomas’ cardioplegic solution is still the most frequently used solution for clinical heart preservation. In our institution we use St. Thomas’ solution enriched with 3.5% dextran-40. The dextran-40 gives the solution an oncotic pressure besides supplying it with other potentially beneficial properties [1]. Recently, we published a study on adult pig hearts preserved for 2 or 12 hours with this solution [2]; the hearts preserved for 12 hours started to deteriorate after 4 to 5 hours of reperfusion, whereas the coronary vascular resistance (CVR) increased steadily during the 12-hour observation period.
Impairment of the endothelial function of pig coronary arteries or even loss of coronary endothelial cells has been observed after flush perfusion of the heart [3, 4]. The length of the cold ischemic storage time affects the function of the endothelial cells [5, 6] and the restoration of blood flow after acute ischemia may cause additional reperfusion injury to the endothelium [7].
The present study is designed to address these questions (ie, to sort out the effects of cardioplegic flushing, cold ischemic storage, and reperfusion on hearts preserved as for transplantation).
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Material and methods
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Animals
Sixteen domestic pigs with a mean weight of 20 kg (range, 18 to 22 kg) were used for investigation of the effects of the storage per se on the coronary arteries (eight animals served as fresh controls and eight were used for 2- and 12-hour storage of the vessels). Fifteen 60-kg pigs (range, 55 to 64 kg) were used to obtain coronary arteries for fresh controls (n = 9), and for flush-perfusion and subsequent storage for 2 and 12 hours (n = 6 in each group). Another 24 pigs with a mean weight of 60 kg (range, 56 to 63 kg) were used as heart donors and support animals (12 in each group) for investigation of the effects of reperfusion after flush-perfusion and storage. All the animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals," published by the National Institutes of Health (NIH publication no. 85-23, revised 1985).
Animal preparation
Anesthesia was induced with intramuscular ketamine (50 mg/mL; Ketalar; Morris Plains, NJ) at a dose of 30 mg/kg body weight. Thiopental sodium (Pentotal; Abbott Laboratories, North Chicago, IL) at a dose of 5 to 8 mg/kg body weight, was given intravenously before tracheostomy. Anesthesia and muscular relaxation were maintained with a continuous infusion of 10 mL or 30 mL per hour (for 20-kg and 60-kg pigs, respectively) of a mixture of 8 g of ketamine, 300 mg of pancuronium bromide (2 mg/mL; Pavulon; Organon Teknika, Boxtel, the Netherlands), and 30 mg of midazolam (5 mg/mL; Dormicum; Roche, Basel, Switzerland) dissolved to 500 mL in 10% glucose. The animals were ventilated with a Siemens Servoventilator 300 (Siemens; Elma AB, Solna, Sweden). A volume-controlled, pressure-regulated ventilation with a volume of 5 L/min and a frequency of 30 breaths/minute was used for the 20-kg pigs. The 60-kg pigs were ventilated with 10 L/min and 20 breaths/min. Positive end-expiratory pressure was adjusted to 5 cm H2O and the inspired oxygen fraction was 0.5 in all animals.
A median sternotomy was performed, and heparin (4 mg/kg) was given through a central venous catheter. The heart was excised and placed in cold (4°C) St. Thomas’ Hospital Cardioplegic Solution (Cardioplegi; Kabi Pharmacia, Uppsala, Sweden). The distal part of the left anterior descending coronary artery was immediately excised from the myocardium and blood was removed by dripping oxygenated Krebs solution at 4°C through the lumen of the vessel. A dissecting microscope (LEIKA WILD M 691; Wild Leitz Ltd, Heerbrugg, Switzerland) was used for visualization. The coronary artery was dissected free from adherent connective tissue, cut into 1-mm long segments, and either immediately or after 2 or 12 hours of storage in St. Thomas’ solution at 4°C, transferred to organ baths.
Another six 60-kg pigs were used to investigate the effects of cardioplegic flushing and subsequent storage. The hearts were harvested after being arrested with 1,000 mL of St. Thomas’ solution at 4°C, infused at a pressure of 60 mm Hg to 65 mm Hg into the aortic root, and the hearts were then stored in this solution at the same temperature. Segments of coronary arteries from these hearts were dissected out immediately and transferred to organ baths. After 2 and 12 hours of storage, other segments from the same hearts were harvested and investigated. During the dissection procedure the hearts were fully immersed in St. Thomas’ solution at 4°C.
Reperfusion of the heart after flush-perfusion and cold storage
After flush-perfusion and 2 or 12 hours of storage in St. Thomas’ solution at 4°C, 12 hearts obtained from 60-kg pigs (6 in each storage group) were reperfused for 24 hours with arterial blood from support animals. The working heart blood perfusion model (Fig 1) is described in detail in another study [2]. Briefly, a latex balloon connected to a Y-shaped valve apparatus was inserted into the left ventricle. One branch of the apparatus was connected to the bottom of the reservoir and another branch to the top. The reservoir was filled with saline. The artificial valves allowed only unidirectional flow in each branch when the heart started to beat. The heart pumped saline in a closed circle during the entire reperfusion period. The perfusion pressure in the aortic root of the isolated heart was maintained at 90 mm Hg (range, 80 to 95 mm Hg) by adjustment of the pump speed. The left ventricular-developed pressure, cardiac output, coronary flow, and coronary vascular resistance were recorded at the end of the reperfusion, just before harvesting the coronary arteries for organ bath studies. The oxygen consumption (MVO2) of the isolated heart, in milliliters per minute for 100 grams of heart muscle, was calculated by means of the following equation: 
, where SaO2 and SvO2 are the oxygen saturation (in %) in the blood in the aorta and coronary sinus, respectively; Hb (grams per liter) is the hemoglobin content; PaO2 and PvO2 (kPa) are oxygen tensions of the aortic and coronary sinus blood, respectively; W (grams) is the heart weight after the experiment; and CBF (milliliters per minute) is the coronary blood flow. The hemodynamic parameters of all support animals in both the 2-hour and 12-hour group were stable throughout the 24-hour experimental period, with no need for inotropic support.
Segments of the coronary arteries from the hearts that had been reperfused for 24 hours were obtained using the same technique as for the fresh control group and immediately transferred to the organ baths.
Recording of contractility and endothelium-dependent relaxation
Isometric tension was measured using a myograph consisting of a chamber with a volume of 5 mL, water-mantled to keep the temperature of the bath solution constant (37°C). Krebs solution, the medium used in all experiments, was bubbled with 95% oxygen and 5% carbon dioxide, giving a pH of approximately 7.4. The composition of the Krebs solution was (in mmol/L) NaCl, 119; NaHCO3, 15; KCl, 4.6; NaH2PO4, 1.2; MgCl2, 1.2; CaCL2, 1.5; and glucose, 11. Each ring segment wassuspended between two metal holders (0.2 mm in diameter). One holder was attached to a Grass FT 03 transducer (Grass Instrument Co, Quincy, MA), connected to a Grass polygraph for continuous recording of isometric tension. The other metal holder was fixed to an adjustable unit, by means of which the vessel segments were stretched repeatedly until a basal tension of about 10 mN was reached. In separate experiments it was found that maximum response was obtained at this tension. A stable contraction was then induced with the thromboxane A2 analog U-46619 (The Upjohn Company, Kalamazoo, MI) added at a concentration of 3 x 10-7 mol/L. In separate experiments concentration–response curves with U-46619 showed that a concentration of 3 x 10-7 mol/L induces a contraction that is 95% to 100% of the maximum, regardless of the presence or absence of endothelium. After repeated washes, resulting in the restoration of basal tension, a second contraction was induced with the same concentration of U-46619 and when it had reached a stable plateau, increasing concentrations (10-14 to 10-6 mol/L) of substance P (ICN Biomedicals Inc, Aurora, OH) were cumulatively added to the baths. Substance P stimulates the release of endothelium-derived relaxing factor by stimulating receptors in the endothelium. In eight fresh coronary segments obtained from different pigs the endothelium was destroyed by flushing carbogen gas (95% O2, 5% CO2) through the lumen. The flushing pressure was 20 cm H2O and the duration of the procedure, 15 minutes. In all vessel segments with destroyed endothelium, substance P elicited no relaxation (Fig 2). The response to the different concentrations of substance P was expressed as a percentage of the U-46619-induced contraction. If the relaxation induced by substance P was impaired compared to fresh controls, the endothelium-independent vasodilator papaverine (10-4 mol/L) was added to the bath to ascertain whether complete relaxation could be obtained.
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Fig 2. Endothelium-dependent relaxation of the coronary arteries. Data are shown as the mean ± standard error of the mean, *p < 0.05, **p < 0.01, ***p < 0.001. When error bars are not visible they are hidden by the symbols. (endothelium (-) = endothelium destroyed.)
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Data analysis
The maximum relaxation produced by substance P was determined. The pEC50 value was determined from each curve as the negative logarithm of the concentration giving half maximal relaxation. Results were expressed as the mean ± the standard error of the mean, n being the number of animals used in each group. Student’s t test with the post hoc test of Bonferroni was used for calculation of statistical differences between the groups. Regression analysis was performed with the least-squares method to check for correlation between endothelial dysfunction and high CVR. A p value of less than 0.05 was considered statistically significant.
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Results
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There was no significant difference in endothelium-dependent relaxation (EDR) or in contractile capacity when the segments of fresh coronary arteries obtained from 20-kg pigs were compared to segments from 60-kg pigs. Cardioplegic flushing parameters are shown in Table 1.
Endothelium-dependent relaxation after cold storage without flush-perfusion
The EDR after 2 hours of storage in St. Thomas’ solution at 4°C was not significantly different from that in fresh coronary arteries. After 12 hours of storage the coronary arteries lost 8% of their EDR (p < 0.05) and the sensitivity of the vessels to substance P, expressed as the pEC50 value, became significantly lower (p < 0.001) (Fig 2).
Endothelium-dependent relaxation after cold flush-perfusion
Flush-perfusion alone did not affect substance P-induced maximal EDR, but the sensitivity of the coronary arteries to this drug was significantly decreased (p < 0.05) (Fig 2).
Endothelium-dependent relaxation after flush-perfusion and cold storage
There was no significant difference in EDR between flush-perfused vessels with subsequent 2-hour storage and fresh controls, but EDR was reduced by 6% after flush-perfusion and 12 hours of subsequent cold storage (p < 0.01), (Fig 2). The sensitivity of the coronary arteries to substance P decreased after flush-perfusion with subsequent 2-hour (p < 0.05) and 12-hour storage (p < 0.05) compared to that in fresh controls.
Endothelium-independent relaxation
In cases where full relaxation (compared with fresh controls) was not obtained with substance P, 10-4 mol/L papaverine, an endothelium-independent vasodilator, was added to the baths; complete relaxation was then elicited in all cases.
Contractile capacity of the coronary arteries
The contractile capacity of the coronary arteries to U-46619 was not significantly different from that found in fresh controls (Fig 3).
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Fig 3. Contractile response of pig coronary arteries to thromboxane A2 analog U-46619. Each bar represents the mean ± standard error of the mean. There were six animals in each group except the fresh control group (n = 9).
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Heart function, coronary vascular resistance, and endothelium-dependent relaxation after cardioplegic flushing, cold storage, and 24 hours of reperfusion
The left ventricular-developed pressure, cardiac output, coronary flow, and oxygen consumption were significantly decreased and the coronary vascular resistance significantly increased in hearts preserved for 12 hours, compared to the 2-hour storage group (Table 2).
Substance P-induced EDR was 98% ± 1% in the fresh coronary arteries. It was reduced by 6% (p < 0.01) in flush-perfused vessels stored for 2 hours and reperfused for 24 hours; and even more reduced (by 58%, p < 0.001) after flushing, 12 hours of storage, and 24 hours of subsequent reperfusion (Fig 2). The difference between these two groups was significant (p < 0.01).
The regression analysis showed that a high CVR correlated with a low EDR (p < 0.001) and also with a low pEC50 value (p < 0.01) (Fig 4).
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Fig 4. Correlation between maximal endothelium-dependent relaxation, pEC50, and CVR after 24 hours of reperfusion of the isolated hearts; n = 6 in both groups.
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Comment
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Acetylcholine is the drug classically used in both human and animal vascular preparations to elicit EDR. However, in porcine coronary arteries acetylcholine does not induce EDR and therefore, cannot be used. We found substance P to be the best alternative because it is the drug to which the normal porcine coronary arterial endothelium is most sensitive; substance P does not affect the smooth vascular muscle cells directly as is demonstrated when the endothelium is destroyed (Fig 2, top).
The present study indicates that cold cardioplegic flushing of the heart impairs the coronary endothelial function, as a significant reduction in sensitivity to substance P was seen in the coronary arteries after flushing. Cold ischemic storage for 2 hours did not affect endothelial function, but after 12 hours of cold ischemic storage, it was significantly impaired. The study also indicates that if the endothelium is impaired before the start of reperfusion, the reperfusion may add further damage.
In an earlier study where we measured the coronary flow and coronary vascular resistance continuously during the first 12 hours of reperfusion [2], both coronary flow and resistance remained constant in the hearts preserved for 2 hours, but in those preserved for 12 hours coronary vascular resistance increased linearly with time, with the consequence that less and less blood passed through the coronary circulation.
It is known that reperfusion of ischemic vessels might activate leukocyte adhesion molecules, procoagulant factors, and vasoconstrictive agents and may lead to additional damage to the endothelium [7]. The dysfunction seen in the hearts after 12 hours of preservation and 24 hours of reperfusion (Table 2) may have been caused mainly by an insufficient blood supply to the myocardium, attributable to inadequate preservation of the coronary vascular endothelium. As seen in Figure 4, there was a significant negative correlation between endothelial function and coronary vascular resistance (ie, the more the endothelial function was impaired, the higher the coronary vascular resistance), supporting the hypothesis that impaired heart function after heart transplantation may be caused by bad preservation of the coronary endothelium.
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References
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Abstract
Introduction
