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Ann Thorac Surg 1998;66:73-78
© 1998 The Society of Thoracic Surgeons

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Original articles: cardiovascular

Function of adult pig hearts after 2 and 12 hours of cold cardioplegic preservation

^a Department of Cardiothoracic Surgery, University Hospital of Lund, Lund, Sweden

Accepted for publication February 6, 1998.

Address reprint requests to Dr Steen, Department of Cardiothoracic Surgery, University Hospital of Lund, S-221 85 Lund, Sweden

	Abstract

Top Abstract Introduction Material and methods Results Comment References

 
Background. Most cardioplegic solutions have been developed using the classic Langendorf heart perfusion model, which only allows a short experimental follow-up. Our aim was to investigate hearts after prolonged storage by using a physiologic model including prolonged perfusion with normal, fresh blood.

Methods. Sixteen hearts from 60-kg pigs were preserved with dextran-enriched (dextran-40, 35 g/L) St. Thomas’ solution for 2 or 12 hours after which they were continuously reperfused for 12 hours with normal blood, supplied by a support pig. A flexible balloon, fixed to an artificial valve apparatus connected to a circuit system, was inserted in the left ventricle for obtaining measurements of hemodynamic performance.

Results. During the first 3 to 4 hours of reperfusion there was no significant difference in left ventricular developed pressure, cardiac output, minute work output, or oxygen consumption between the two groups. After this time left ventricular developed pressure (p < 0.001), cardiac output (p < 0.01), minute work output (p < 0.01), and oxygen consumption were significantly lower in the 12-hour group. Coronary flow was higher (p < 0.01) and coronary vascular resistance lower (p < 0.01) during the first 5 to 6 hours of reperfusion in the 12-hour group. After 12 hours of reperfusion coronary vascular resistance was significantly higher (p < 0.01) in the 12-hour group.

Conclusions. High-degree and long-lasting coronary hyperemia at the beginning of reperfusion can be a sign of unsatisfactory preservation of the heart. This investigation shows the importance of reperfusion with normal blood and a long follow-up period after postischemic reperfusion when studying the effect of cardioplegic solutions.

	Introduction

Top Abstract Introduction Material and methods Results Comment References

 
It has been shown that the dysfunction of hearts exposed to cold cardioplegic arrest is at its maximum 2 to 6 hours after the start of reperfusion [1–3]. The most commonly used experimental method for evaluating heart preservation has been the classic Langendorf rat heart model. The gold standard of crystalloid cardioplegia, St. Thomas’ Hospital solution, was developed using that method (for review see [4]) in which the rat heart is perfused with an extracellular buffer solution aerated with 95% oxygen and 5% carbon dioxide at 37°C. No proteins or colloids can be added to the aerated solution because of foam created by the aeration; ie, no oncotic pressure is present to counteract interstitial edema, which therefore necessarily develops over time, and makes the method unstable within 1 hour of reperfusion. Tsao and coworkers [5] have shown that significant myocardial injury occurs within 3 hours of postischemic heart reperfusion and that it is related to the activation of neutrophil leukocytes and the accumulation of these cells in the myocardium. Thus for a proper evaluation of hearts preserved for prolonged periods of time, it is mandatory to use a stable, working heart model that includes at least 6 hours of reperfusion of the coronary vasculature with normal blood.

The aim of the present study was to present such a model and to test it on short-term and long-term preservation of adult porcine hearts, which are being considered for use as xenografts in clinical heart transplantation in the near future.

	Material and methods

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Thirty-two Swedish pigs of native breed with a mean weight of 60 kg (range, 57 to 63 kg) were used (16 as donors and 16 as support pigs). Eight donor hearts were preserved for 2 hours (control group) and 8 for 12 hours in dextran-enriched (dextran-40, 35 g/L) St. Thomas’ Hospital solution (Cardioplegi; Kabi Pharmacia, Uppsala, Sweden). The animals were treated in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication 85-23, revised 1985).

Donor procedure
Anesthesia was induced with intramuscular ketamine hydrochloride (Ketalar; Parke-Davis, Morris Plains, NJ) at a dosage of 25 mg/kg of body weight. Atropine sulphate (Atropin; Kabi Pharmacia, Uppsala, Sweden) 0.5 mg and sodium thiopental (Pentothal; Abbot Laboratories, North Chicago, IL) 5 to 8 mg/kg were given intravenously before tracheostomy (tube no. 7). Anesthesia and muscular relaxation were maintained with a continuous, 33 mL/hour infusion of a mixture of 600 mg of pancuronium bromide (Pavulon; Organon Teknika, Boxtel, the Netherlands) and 16 g of ketamine hydrochloride dissolved to 1,000 mL with 10% glucose. The animals were ventilated with a Servo Ventilator 300 (Siemens; Elema AB, Solna, Sweden) using volume-controlled ventilation, (10 L/min, 20 breaths/min, positive end expiratory pressure = 5 cm H₂O, inspired oxygen fraction = 0.5). Central venous and aortic pressure catheters were established through the neck vessels. The pressures were displayed on flouroscopes (HP78353B and HP78342; Hewlett Packard, Andover, MA).

The heart and aortic arch were exposed through a median sternotomy. After systemic heparinization (500 IU/kg) the ascending aorta was cannulated with a 9-gauge aortic root cannula (Medtronic, Inc, Grand Rapids, MI) providing a separate pressure catheter through which the perfusion pressure was continuously measured. The left azygos vein was ligated and the superior and inferior caval veins were clamped. Subsequently the ascending aorta was cross-clamped, 1,000 mL of cold (4°C), dextran-enriched St. Thomas’ solution was infused in the aortic root at a pressure of 60 mm Hg. The appendices of the left and right atrium were opened to keep the heart decompressed, and saline ice-slush (0.5°C) was placed around the heart. After the cardioplegic solution had been infused, the heart was excised. The heart was promptly immersed in St. Thomas’ solution at 4°C and stored at this temperature.

The support animal
The support pigs were anesthetized and ventilated in the same way as the donor pigs. After systemic heparinization (500 IU/kg) the left femoral artery and vein were cannulated with 18F and 24F venous cannulas, respectively (Research Medical Inc, Midvale, UT). These cannulas were connected to a tubing system (-inch internal diameter) with two roller pumps included (Stöckert Instrumente, Munich, Germany) (Fig 1). Blood gases in the support pig were measured repeatedly, and adjustments in ventilation or correction of metabolic acidosis with sodium bicarbonate were made accordingly.

View larger version (32K): [in this window] [in a new window]   Fig 1. Isolated heart model. Measured variables are denoted in the middle of the figure. (ECG = echocardiogram; SaO2 = oxygen saturation in arterial blood; SvO2 = oxygen saturation in coronary sinus blood.)

Reperfusion of the isolated heart
One roller pump (pump A in Figure 1) was used to supply the coronary arteries of the isolated heart with arterial blood from the support pig. The perfusion pressure of the isolated heart was maintained at 90 mm Hg (range, 85 to 95 mm Hg) by adjustment of the pump speed. All hearts were weighed before and after reperfusion, which continued for 12 hours. To avoid temperature reduction, the tube was encased in a water jacket where the temperature was maintained near 38°C using a heat exchanger (Jostra, Hirlingen, Germany). Blood flow was measured with a clamp-on probe (12 mm in diameter) connected to a Transonic flow-meter (T201D; Transonic Systems Inc, Ithaca, NY). The blood from the coronary sinus was collected into a water-jacketed reservoir, where the heart was submerged in the blood up to the atrial level and then returned to the femoral vein of the support pig by means of the second roller pump (pump B in Figure 1). Temperatures were measured in the aortic root and inside the right ventricle. The temperature in the aortic root was adjusted to keep the heart temperature at 37°C. All cannulas and tubes used for blood flow were coated with covalently bound heparin (Carmeda, Täby, Sweden). Heparin was administered continuously to the support pig to keep the Hemochron time near 600 seconds.

Measurements of function of the isolated heart
Intraventricular pressure was measured through a catheter (1 mm in diameter) placed in the middle of the balloon and passing through the stiff wall of the tube just above the velour patch on the ventricular branch of the artificial valve apparatus. A plastic tube, connected to the aortic branch of the apparatus, was elevated 60 cm above the heart before entering an open (atrial) reservoir. With a flow of 5 L/min through this part of the tube system the resistance was calculated to be 1,300 dynes · s · cm^-5, which is comparable with the systemic vascular resistance measured in the donor pigs before harvesting of the heart (Table 1). Another tube connected the bottom of the atrial reservoir with the atrial branch of the valve apparatus. Balloon, tubes, and reservoir were filled with 0.9% NaCl at 37°C. The reservoir was placed at the level necessary to create a filling pressure of 20 mm Hg in the balloon inserted into the left ventricle of the nonbeating heart. A second flow probe (8 mm in diameter; Transonic Systems Inc) was implanted to measure the flow through the aortic branch, ie, cardiac output (CO).

Oxygen consumption (MVO₂) expressed per 100 g of heart muscle was calculated every 4 hours by means of the following equation: , where W is the weight of the heart (in g), SaO₂ and SvO₂ are the oxygen saturation (in %) in the aortic and coronary sinus blood, respectively, Hb is hemoglobin content (in g/L), and PaO₂ and PvO₂ are the oxygen tension (in kPa) of the aortic and coronary sinus blood, respectively. Minute work output was calculated by multiplying cardiac output by left ventricular mean pressure and subtracting mean left ventricular inflow pressure. Arterial blood gases and oxygen saturation of the blood going to and from the isolated heart were analyzed every 4 hours by means of blood analyzers (ABL505 and OSM3; Radiometer, Copenhagen, Denmark).

Data analysis
The results are given as mean ± standard error of the mean. For comparison between groups, analysis of variance with repeated measurements was used. To determine at which time interval differences occurred, linear contrasts were used. Student’s unpaired two-tailed t test with the post-hoc test of Bonferroni was used when statistical differences in minute work output and oxygen consumption were calculated. A p value of less than 0.05 was considered significant. Professional statisticians were consulted (Clinical Data Care AB, Lund, Sweden).

	Results

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Support animal
The hemodynamic condition of all animals in both groups was stable throughout the 12-hour experimental period, with no need for inotropic support. No blood transfusions were given. Urine production was around 100 mL/hour without any sign of hemolysis. The PaO₂ level was approximately 30 kPa (inspired oxygen fraction = 0.5) (Fig 2, upper panel). There was no statistically significant difference at any time between the groups regarding PaO₂ and hematocrit (Fig 2).

View larger version (22K): [in this window] [in a new window]   Fig 2. Arterial oxygen tension (paO2) (upper panel) and hematocrit (lower panel) of the support animals during the 12-hour reperfusion period. Data are shown as the mean ± standard error of the mean (n = 8 in each group).

The hemodynamic performance of the isolated hearts is shown in Figs 3 to 5. During the first 3 hours no statistically significant difference was seen between the groups in left ventricular developed pressure and CO, but then these measurements slowly decreased in the 12-hour group and continued to do so until the end of the experiment so that eventually there was a significant difference between the two groups (p < 0.001 and p < 0.01, respectively) (Fig 3). Heart rate was slightly higher in the 12-hour group, but the difference was not statistically significant (Fig 3).

View larger version (30K): [in this window] [in a new window]   Fig 3. Pressure that developed in the left ventricle (upper panel), cardiac output (middle panel), and heart rate (lower panel) of the isolated pig heart after 2 and 12 hours of cold storage in St. Thomas’ solution. Data are shown as the mean ± standard error of the mean (n = 8 in each group). (**p < 0.01; ***p < 0.001.)

View larger version (32K): [in this window] [in a new window]   Fig 4. Coronary flow (upper panel) and coronary vascular resistance (CVR) (lower panel) of the isolated pig heart after 2 and 12 hours of cold storage in St. Thomas’ solution. Data are shown as the mean ± standard error of the mean (n = 8 in each group). (**p < 0.01.)

View larger version (25K): [in this window] [in a new window]   Fig 5. Minute work output (upper panel) and O2 consumption (lower panel) of the isolated pig heart after 2 and 12 hours of storage in St. Thomas’ solution. Data are shown as the mean ± standard error of the mean (n = 8 in each group). (*p < 0.05; **p < 0.01; ***p < 0.001.)

The minute work output and oxygen consumption were lower in the 12-hour group, but a statistically significant difference was seen only at 8 hours (p < 0.001 and p < 0.05, respectively) and 12 hours (p < 0.01 and p < 0.05, respectively) of reperfusion (Fig 5). No significant gain in weight occurred during the 12 hours of reperfusion in any of the groups (Table 2).

	Comment

Top Abstract Introduction Material and methods Results Comment References

Many heart preservation studies have been performed using the Langendorf model: the heart is perfused with a cell-free buffer solution, eg, Krebs-Henseleit solution, and in most cases the perfusion period is shorter than 3 hours. Heart function is evaluated isovolumetrically by means of a balloon implanted in the left ventricle, a situation which is far from the normal working condition of the heart. The results obtained from such studies must therefore be interpreted with great caution.

The evaluation method used in the present study, where normal arterial blood perfused the isolated heart, allowed us to continue with the perfusion throughout the planned 12-hour period, during which the heart could work with high end-diastolic and low end-systolic volumes (not isovolumetrically). The artificial valve apparatus with the connected tube system was designed to give values for left ventricular developed pressure and a systemic vascular resistance in the control group (2-hour group) that were close to the values obtained in the donor pigs before harvesting. Conversely, hemodynamic instability in the support animals may affect assessments of myocardial performance in the isolated heart. An accurate monitoring of the support animal and correction of the ventilation and fluid balance allowed us to avoid that influence.

Bolli and coworkers [6] analyzed the recovery of left ventricle function by measuring systolic wall thickening in patients after cardiac operations. They found that the maximal deterioration in heart function occurred between 2 and 6 hours after aortic declamping and that recovery came within 24 to 48 hours. Other investigators have obtained similar results [2, 3]. Bolli [7] suggests that myocardial stunning may be masked in the immediate postoperative period by the use of inotropic drugs, afterload-reducing therapy, or both. In the present study, afterload was constant and no inotropic drugs were used. The myocardial dysfunction still appeared only after 3 hours of reperfusion, suggesting that it takes a few hours of reperfusion before the dysfunction is manifested. The study by Tsao and coworkers [5] showed that 3 hours of reperfusion were needed before a significant invasion of leukocytes into a postischemic myocardium appeared.

The deterioration in myocardial function seen in the present study was not caused by a coronary flow that was too low, because hearts that had been preserved for 12 hours had a much higher coronary flow during the first 6 hours than those preserved for 2 hours. One reason for the high initial coronary flow is probably coronary reactive hyperemia, which is a phenomenon observed at the beginning of myocardial reperfusion after ischemia [8]. The initial increase in coronary flow plays an important role in the recovery of the postischemic myocardium [9]. The underlying mechanisms that may be involved include release of adenosine [8], activation of K⁺_ATP channels [10], and release of endothelium-derived relaxing factor [11]. Changes in interstitial osmolarity and levels of potassium, hydrogen ions, carbon dioxide, catecholamines, and prostaglandins may also be involved in mediating the reactive hyperemia [12]. There seems to be a relationship between the duration of ischemia and of postischemic reactive hyperemia: the longer the ischemic period, the longer the vasodilatation of the coronary arteries [8].

Another reason for the high initial flow seen in the present study can be arteriovenous shunting in the myocardium. Extensive studies of the coronary vasculature of the pig have been published by Kassab and coworkers [13, 14]; there appears to be no structural evidence for significant arteriovenous shunting in the myocardium [15]. Thus the high initial coronary flow seen in our study can not be explained by direct shunting of the arterial blood to the veins. However, others have suggested that not all myocardial capillaries are functional at all times [16]. They can be regulated by precapillary sphincters [17] or pericytes that surround the capillary wall [18]. These pericytes contain microfilaments, and it has been suggested that these microfilaments are able to contract, thereby narrowing the capillary lumen. The storage time may have influenced the number of functional capillaries, thereby explaining the difference in flow between the groups at the beginning of reperfusion.

Coronary vascular resistance increased in an almost-linear fashion in the 12-hour group during the reperfusion period, and at 12 hours it was significantly greater than in the 2-hour group. A similar increase in vascular resistance in lungs preserved for 24 hours has been reported and attributed to endothelial dysfunction [19]. In the present study, we suggest that the endothelium was better preserved in the 2-hour group than in the 12-hour group [20, 21]; however, initially, because of the hyperemia, this finding was masked. When all toxic, vasodilating metabolites had been washed from the myocardium, the vascular tone would increase, and it would have increased more in the 12-hour group if the vascular endothelium in that group had been more injured.

In conclusion, evaluation of heart preservation needs a stable model including prolonged reperfusion with normal, fresh blood with intact blood cells, circulating in a body where chemotactic substances from the preserved heart can reach organs delivering leukocytes and other inflammatory cells.

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Algimantas Budrikis Richard Ingemansson Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Budrikis, A. Articles by Steen, S. Search for Related Content PubMed PubMed Citation Articles by Budrikis, A. Articles by Steen, S.