| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Ann Thorac Surg 1995;59:699-706
© 1995 The Society of Thoracic Surgeons
Department of Cardiovascular Surgery, University of Kiel, Kiel, and Department of Thoracic and Cardiovascular Surgery, Hannover Medical School, Hannover, Germany
Accepted for publication November 26, 1994.
 |
Abstract |
|---|
 TopFootnotes Abstract IntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
|---|
|
Introduction |
|---|
|
TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
|---|
In the present study, we therefore used the isolated nonworking immature rat heart to determine the efficacy of topical hypothermia alone, additional cardioplegic arrest with St. Thomas' Hospital cardioplegic solution II (STS II), and slow-perfusion hypothermia for myocardial protection during 8 hours of global ischemia at 10°C in a clinically relevant experimental model of chronic cyanosis. Unlike in previous studies, the present model allowed for the sequelae of chronic cyanosis to be produced noninvasively, because neonatal rats were exposed for 25 days to low air pressure (54 kPa), which is equivalent to a high altitude of 5,000 m above sea level. Four indices of myocardial injury and protection-myocardial function, endothelial function, creatine kinase (CK) leakage, and myocardial energy-rich phosphates-were assessed.
|
Material and Methods |
|---|
|
TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
|---|
Preliminary Experiments
To assess the degree of hypoxia induced by the simulated high-altitude conditions in our experimental model, two sets of preliminary experiments were conducted, as follows: Twenty-eight-day–old rats were anesthetized with diethyl ether and placed supine. The right carotid artery was exposed after incision and cannulated with a flexible catheter, which was brought through the skin of the animal's neck before closure of the wound. The rats were then transferred into a decompression chamber, allowing the maintenance of a constant low air pressure. The arterial catheter was guided outside the chamber through an airtight drill hole in the chamber wall to enable sampling of arterial blood for blood gas analysis under the simulated high-altitude conditions. The animals were then progressively subjected within 60 minutes to a chamber pressure simulating an altitude of 5,000 m above sea level. For the next 60 minutes, the animals were kept hypoxic at the simulated 5,000-m altitude conditions (54-kPa air pressure) before the pressure was progressively increased during 1 hour back to the normal local air pressure of 60 m above sea level. Arterial blood samples were taken for blood gas analysis (Corning 158; Ciba-Corning, Medfield, MA) before, during (at 5000 m and 3000 m), and after simulation of the high-altitude conditions.
Arterial blood from 6 animals, adapted to hypoxia in the same way and using the same time course as used for the perfusion experiments, was assessed. Blood from another group of rats not exposed to high-altitude conditions was also evaluated.
Perfusion Experiments
INDUCTION OF CHRONIC HYPOXIA.
Litters of 8 animals, including the mother, were placed into the decompression chamber at 3 days of age. There were no nutritional restrictions throughout the exposure time. Over the course of 10 days, animals were progressively subjected to hypoxic conditions equivalent to 5,000 m above sea level, which was then maintained until the end of exposure on day 28, except for short periods necessary for cleaning and feeding. At the end of the exposure period, animals were brought back to normal local air pressure within 1 hour and were then ready for the next experimental step.
PERFUSION METHODOLOGY.
Rats were injected with sodium heparin (1 mg per rat intraperitoneally). After 30 minutes, the animals were anesthetized by the inhalation of diethyl ether, and, after laparotomy, 1 mL of blood was drawn by puncture of the inferior caval vein for measurement of the hemoglobin level and for the hematocrit determination. The chest was then opened and the thoracic cavity filled with cold (4°C) Krebs-Henseleit buffer solution. With the heart thus immersed in the cold buffer solution, the pulmonary artery was incised near its origin and the aorta was cannulated with a blunted 16-gauge needle (diameter, 1 mm). The heart was then excised and mounted onto the perfusion apparatus by means of the aortic cannula. Perfusion of the coronary arteries was performed according to the Langendorff technique [5] at a perfusion pressure of 60 mm Hg (80 cm H2O) using oxygenated modified Krebs-Henseleit buffer solution with the following composition (in millimoles per liter): NaCl, 118; KH2PO4, 1.2; KCl, 4.9; CaCl2, 2.5; MgSO4, 1.2; NaHCO3, 25, and glucose, 11.1. The perfusate was filtered before use (5-µm pore size), aerated with a mixture of 95% oxygen and 5% carbon dioxide to maintain the pH at 7.4, and kept at 37°C. Each heart was housed in a thermostatically controlled heart chamber maintained at 37°C during the preischemic and postischemic periods. During a 15-minute washout period after cannulation, an intraventricular balloon was inserted into the left ventricle through the mitral valve. The ultrathin balloons were designed so that they matched the ventricular dimensions of the heart. The balloon was filled with fluid and attached to a pressure transducer through a fluid-filled tube. The volume of the balloon was adjusted by means of a water-tight microsyringe attached to the sidearm of the transducer. The left ventricular (LV) pressure signal was recorded and processed on-line by an Analog-Digital Converter (Plugsys, Type 663; Sachs Electronic, March, Germany). Data processing was performed using an IBM-compatible personal computer equipped with standard laboratory software (Hemodyn; Sachs Electronic).
MEASUREMENTS.
Preischemic baseline measurements of systolic LV function and coronary flow were performed 15 minutes after the onset of in vitro perfusion during isovolumic contractions by inflating the intraventricular balloon to an LV end-diastolic pressure of 10 mm Hg. The volume necessary to produce this pressure was registered. The LV maximum developed pressure (LVP) and its first derivative (dp/dt) were recorded. Coronary endothelial function was assessed by the change in the coronary flow in response to the endothelium-dependent vasodilator acetylcholine (10-5 mol/L), which was administered by means of a separate perfusion column during 5 minutes as a lement to the perfusate.
The recovery in systolic function and coronary flow was measured 60 minutes after the onset of reperfusion at an LV end-diastolic pressure of 10 mm Hg. The volume necessary to produce this pressure was recorded and-in relation to the preischemic volume-judged as an estimate for the recovery of diastolic function. Recovery of endothelial function was estimated by the coronary flow response to acetylcholine at 40 minutes of reperfusion in relation to the preischemic response.
The blood hemoglobin concentration was measured with an automated analyzer using a standard test kit (Merckotest Haemoglobin 3317; E. Merck, Darmstadt, Germany). The hematocrit was assessed by centrifugation of blood in heparinized hematocrit capillaries.
Samples for measurement of the CK leakage were obtained from the coronary effluent at 2, 10, 20, and 40 minutes during reperfusion. Each sample was stored at 4°C until it could be assayed using a standard enzymatic test (Granutest 2.5; E. Merck).
At the end of reperfusion, hearts were rapidly frozen with Wollenberger tongs precooled in liquid nitrogen. After perchloric acid extracts were prepared from tissue samples according to the method of Lowry and Passonneau [6], tissue was dried for 48 hours at 80°C before determining the myocardial dry weight. Adenosine triphosphate (ATP) and creatine phosphate levels (in micromoles per gram, dry weight) were assessed using a standard enzymatic assay [7].
PRESERVATION TECHNIQUES AND EXPERIMENTAL GROUPS.
The perfusion experiments were carried out in six experimental groups (13 animals in each group). Animals in groups 1H to 3H were exposed to chronic hypoxia and animals in groups 1C to 3C served as age-matched controls not exposed to hypoxia. At the end of the control perfusion period, three different preservation protocols were applied for the induction of global myocardial ischemia during 8 hours at 10°C.
In protocol 1 (rapid cooling by topical hypothermia alone), preischemic perfusion was discontinued in the hearts of animals in groups 1H and 1C and cardiac arrest was induced simultaneously by rapid topical cooling with cold Krebs-Henseleit perfusate.
Protocol 2 consisted of slow cooling by perfusion hypothermia and was carried out in the hearts of groups 2H and 2C. Hypothermia was induced slowly during 10 minutes, from normothermia to 10°C, by perfusion with perfusate at gradually decreasing temperatures. During this period, the temperature inside the water-jacketed perfusion chamber of the heart was adjusted thermostatically to the temperature of the perfusate.
Protocol 3 consisted of single-dose cardioplegia plus topical hypothermia. The hearts of animals in groups 3H and 3C were arrested with STS II, which was administered at a temperature of 10°C for 3 minutes through a sidearm of the aortic cannula from a reservoir located 60 cm above the heart. The composition of STS II was as follows (in millimoles per liter): NaCl, 110; KCl, 16; MgCl2, 16; CaCl2, 1.2; and NaHCO3, 10 (pH adjusted to 7.8; osmolarity, 324 mOsm/L). Topical hypothermia was applied additionally as done in protocol 1.
After initial preservation, all hearts were disconnected from the perfusion apparatus and stored for 8 hours at 10°C in a beaker containing Krebs-Henseleit perfusate.
STATISTICAL ANALYSIS.
All data are expressed as the mean ± standard deviation. Nonparametric methods were used for data evaluation. Comparison of group means was carried out by the Kruskal Wallis test [8]. When significant differences were found, further analysis by the Mann-Whitney U test was performed. Statistical significance was set at p < 0.05.
|
Results |
|---|
|
TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
|---|
).
|
|
. The body weight of chronic hypoxic animals was lower than that of the control animals (p < 0.05). Hypoxia resulted in a significant increase in the serum hemoglobin concentration and hematocrit (p < 0.05). Among hearts exposed to hypoxia, the myocardial dry weight to body weight ratio was significantly greater than that in controls (p < 0.01). No significant differences were observed among the three subgroups of hypoxia and control animals.
|
. The LVP was higher in the hypoxia groups than in the control groups (p < 0.05). Except for group 2H (hypoxia plus slow cooling), the mean preischemic dp/dt and coronary flow were also significantly higher in the hypoxia groups than in the control groups (p < 0.025). No differences between the hypoxic and control groups were observed in the preischemic heart rate, increase in coronary flow brought about by acetylcholine infusion, and the balloon volume needed to produce an LV end-diastolic pressure of 10 mm Hg. No significant differences were observed among the three subgroups of hypoxia and control animals.
|
. In the chronic hypoxic hearts, recovery of LVP and dp/dt was greatest after topical cooling alone. Among the hypoxia groups, the difference in the recovery of LVP was significant (70.6% ± 18% of the preischemic value). In addition, the mean values of dp/dt and coronary flow were highest after topical cooling alone, although the numerical differences were not consistently significant. In hypoxic hearts, the balloon volume needed to produce an LV end-diastolic pressure of 10 mm Hg was significantly higher (p < 0.01) in group 1H hearts (hypoxia plus rapid cooling) than it was in group 2H hearts (hypoxia plus slow cooling) and group 3H hearts (STS II).
|
The same pattern of recovery as that observed in hypoxic hearts was seen in control hearts. The greatest recovery in LVP and dp/dt was seen in the hearts subjected to topical cooling alone (44.4% ± 19.4% and 41.4% ± 20.1%, respectively). However, except for the recovery in dp/dt in comparison with group 2C (slow cooling), the differences failed to achieve statistical significance. The response to acetylcholine was measured to be 62% ± 37% of the preischemic value after topical cooling, but was not recordable with the other protection methods tested.
Myocardial High-Energy Phosphates
As shown in Figure 3, the mean postischemic myocardial concentrations of ATP and creatine phosphate ranged from 3.6 to 6.5 µmol/g dry weight and from 8.7 to 14.5 µmol/g dry weight, respectively. In comparison with preischemic control values, the postischemic ATP concentrations were reduced by 70% to 80%, whereas the creatine phosphate concentrations were decreased by 60% to 70%. No statistically significant differences were observed among the six subgroups of animals.
|
|
Comment |
|---|
|
TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
|---|
Results from several experimental studies indicate a greater inherent tolerance to ischemia in immature hearts than in adult hearts [9, 10]. In contrast, a strong correlation between early postoperative death after pediatric cardiac operations and the duration of myocardial ischemia has been described [1]. This discrepancy may be explained by the fact that most of the studies evaluating cardioprotective strategies in immature hearts have been conducted in healthy animals. Little is known about the possible impact of cyanosis and hypertrophy, as occur with many congenital heart defects, on the response of the heart to measures for myocardial protection.
Because the surgical measures alone that are necessary to create chronic cyanosis experimentally might disturb postischemic myocardial function, this might increase the sampling error. Thus, we employed an experimental model that allowed for the development of sequelae of chronic hypoxia noninvasively: Neonatal rats were exposed to a simulated high altitude of up to 5,000 m above sea level for approximately 4 weeks. This procedure is well characterized for studies that examine the metabolic and morphologic effects of chronic hypoxia on the heart [11]. Because it has not previously been used in connection with myocardial protection, however, we measured several variables to test its clinical relevance to our intended study before beginning the actual study. The changes that occurred at a simulated high altitude in the serum hemoglobin level, hematocrit, body weight, the relative myocardial weight, and particularly the arterial oxygen tension and oxygen saturation recommended this model for the purpose of our study (see Figs 1, 2
; Table 1).
Several factors must be considered when comparing our results with those of other studies.
Level of Hypothermia
Improved myocardial protection with STS II has been reported for immature rats, rabbits, and piglets. In these studies, however, the myocardial temperature was kept normothermic or mildly hypothermic during the period of ischemia, whereas our experiments were conducted at 10°C during myocardial ischemia, a temperature more closely related to clinical conditions. The reason for the improved protection that we observed in association with the sole use of topical hypothermia may be twofold. First, the efficacy of protection produced by topical hypothermia at this temperature will be even more pronounced in rat hearts than in rabbit or piglet hearts, because homogeneous hypothermia is induced more rapidly in small hearts than in bigger hearts. Second, the unequivocal benefit of cardioplegia may be offset by detrimental effects of coronary perfusion using crystalloid solutions; such effects have been observed at lower temperatures [4]. Thus, it remains controversial whether the application of both cardioplegia and hypothermia at 10°C is beneficial. Magovern and associates [12] conjectured that there were damaging effects from STS II in immature rabbit hearts at 10°C that are not seen at 28°C. Kempsford and colleagues' [4] findings differed from those of Magovern's group, in that a trend toward improved protection from the use of STS II at this temperature was observed in neonatal rabbit hearts subjected to a global ischemia time of 18 hours. Interestingly, multidose cardioplegia conferred better protection at 20°C in this study but was deleterious at 10°C. The authors concluded that multiple infusions might bring about detrimental effects by providing fluid for the development of cellular edema at 10°C rather than at 20°C, which may be of particular relevance in view of the greater capillary permeability of the immature heart [4, 13]. Our own results confirm, on the one hand, that cardioplegia at 10°C may be detrimental in the immature heart, even when applied as a single dose. On the other hand, slow cooling by means of coronary perfusion with Krebs-Henseleit solution and subsequent topical hypothermia also failed to improve protection. Results were consistent in both normal hearts and hearts that were hypertrophied after exposure to a simulated high altitude. As discussed later, there is some evidence that coronary perfusion with asanguineous solutions at lower temperatures per se, regardless of the perfusate or cardioplegia used, has a detrimental effect on the immature heart.
Role of Calcium
It has been reported that slow cooling by coronary perfusion with asanguineous perfusate before arrest may accelerate the ATP depletion rate [14]. Because the maintenance of intracellular homeostasis, and hence a low cytosolic calcium level, requires ATP, depletion of myocardial energy-rich phosphates may finally result in partial cell membrane damage and the sudden influx of calcium into the cell, with further deleterious effects. Thus, it is conceivable that detrimental sequelae of cytosolic calcium loading as a result of preischemic hypothermic perfusion with Krebs-Henseleit solution contributed to the reduced postischemic recovery seen in our experiments after slow cooling.
The same final pathway may account for the relatively poor protection that we observed with STS II. This solution was used in our studies as an example of a crystalloid cardioplegic solution that has been well characterized and is in current clinical use. It was developed specifically to protect the ischemic adult heart [15]. The efficacy of this solution in protecting the ischemic child's heart, however, has been questioned, mainly for its calcium concentration of 1.2 mmol/L [16]. This concentration was reported to be optimal only in the setting of normothermic ischemia in immature rabbit hearts [17]. Results of further studies performed under hypothermic conditions have indicated, however, that the calcium dose–response curve is shifted downward with cold, such that 0.3 mmol/L is the optimal calcium concentration in St. Thomas' Hospital cardioplegic solution [18]. The reason for this phenomenon is currently unknown. Lipid phase transitions of sarcolemmal membranes that occur below 20°C, together with age-related changes in the susceptibility to shifts in transmembrane calcium gradients, might be involved in this process [13].
Myocardial Protection in Hypertrophied Immature Hearts
One aspect of this study was to assess whether disease states such as hypertrophy and cyanosis alter the response of the immature heart to measures for myocardial protection. Our results indicate that this is not the case, in that, neither in normal nor hypertrophied hearts does cardioplegia or slow cooling afford greater protection than does topical hypothermia alone. However, the difference between the topical hypothermia group and both other groups in terms of the postischemic recovery of LVP and dp/dt tended to be more pronounced in hypertrophied hearts (see Table 3). In this context, whether hypertrophy may render the myocardium more susceptible to the development of edema when perfused with asanguineous solutions, regardless of whether the potassium concentration is high (35 mmol/L) or low (5 mmol/L), warrants consideration [19]. This phenomenon, together with the greater capillary permeability of the immature heart, might act synergistically and contribute to the more profound myocardial edema formation that occurs in hypertrophied rat hearts after asanguineous hypothermic perfusion.
Based on the data for functional myocardial recovery, it is tempting to conclude that the hearts exposed to a simulated high altitude performed better than do normal hearts, irrespective of the measure used for myocardial protection. However, comparison of postischemic functional data between normal and cyanotic hearts is not valid, because the preischemic LVP and dp/dt were higher in the cyanotic than in the normal hearts. This increase in hemodynamic function is apparently, at least in part, a result of adaptive mechanisms triggered in response to the chronic hypoxia produced by the high-altitude conditions; these mechanisms include an increase in the rate of oxidative energy production, in the glycolytic fluxes, and in the accumulation of respiratory enzymes [20].
The recovery of endothelial function was assessed by the coronary flow response to the endothelium-dependent vasodilator acetylcholine [21]. Unlike what occurs with topical hypothermia, there was no measurable recovery of endothelial function with cardioplegia or perfusion hypothermia. Aoki and colleagues [22] have suggested that only excessively cold cardioplegia (<4°C) might cause endothelial dysfunction in neonatal lamb hearts, whereas cardioplegia at 10°C is not harmful. The discrepancy with our results is probably due to differences in the experimental protocols of the two studies. Thus, it is very conceivable that the detrimental effects of a long ischemic interval such as 8 hours, together with those of cardioplegia, lead to suppression of postischemic endothelial function that might not be seen when coronary perfusion with crystalloid solution is completely omitted or when ischemia is of shorter duration, as in Aoki and associates' study. We also cannot rule out that the relatively low infusion pressure of 60 cm H2O produced endothelial dysfunction by causing shear stress and barotrauma, as even control of the infusion pressure does not guarantee normal shear stress in epicardial coronary arteries [23]. Although the design of this study did not allow for isolation of the effects of shear stress and pressure during the infusion of cold asanguineous perfusates, it is evident that impairment of postischemic vascular function may limit the recovery of mechanical function [21].
The postischemic myocardial concentrations of ATP and creatine phosphate were reduced in all groups by 70% to 80% and 60% to 70%, respectively, versus preischemic control values. This marked reduction indicates a severe metabolic derangement after 8 hours of global ischemia, but there was no correlation between the level of myocardial high-energy phosphates and functional recovery. Because of the postischemic variance in the myocardial ATP and creatine phosphate concentrations, no significant differences were observed among the six subgroups. This result supports previously reported results, in that a postischemic myocardial concentration of high-energy phosphates does not necessarily correlate with postischemic functional recovery [24, 25].
The wide variability in the CK leakage during postischemic reperfusion leads us to question the value of this variable as an indicator of ischemic injury in this experimental system. Because CK leakage is a flow-dependent phenomenon, it is very conceivable that small changes in coronary vascular resistance would considerably alter the enzyme washout [4]. This may be even more relevant in relatively small rat hearts with low postischemic coronary flow rates.
Limitations of the Study
The interpretation of our results in the context of the clinical situation in pediatric cardiac surgery patients must be performed with caution. The volume of cardioplegic solution delivered per gram of heart weight was probably much greater than that used clinically in the human heart. Thus, a volume-related detrimental mechanism of cardioplegia cannot be discounted. In addition, there are many obvious differences between the isolated rat heart and the human heart in situ. Noncoronary collateral flow could alter the balance between beneficial and detrimental effects of cardioplegia. The prevention of sequelae of noncoronary collateral flow, such as rewarming and the return of electrical activity, would require reinfusion of hypothermic cardioplegia. This measure would be expected to be more beneficial under such conditions.
Conclusions
The major point to emerge from this study is that the use of a crystalloid perfusate in the immature heart, particularly at deep hypothermia, is not well tolerated. The adverse consequences of crystalloid perfusion at hypothermic temperatures could well account for the improved function that we observed after protection by topical hypothermia alone. This method of protection appears even more beneficial for chronic hypoxic and hypertrophied immature hearts than it does for noncyanotic, nonhypertrophied control hearts. The mechanism involved remains to be elucidated, but it is likely that perfusion with cold asanguineous solutions triggers detrimental effects that are more pronounced in hypertrophied than in normal immature myocardium. The data favor the use of topical hypothermia alone in the surgical treatment of congenital cardiac defects in the neonatal period. How much other methods such as blood cardioplegia improve myocardial protection in the immature heart must be further investigated.
|
Acknowledgments |
|---|
|
TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
|---|
|
Footnotes |
|---|
|
TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
|---|
|
References |
|---|
|
TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
|---|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ANN THORAC SURG | ASIAN CARDIOVASC THORAC ANN | EUR J CARDIOTHORAC SURG |
| J THORAC CARDIOVASC SURG | ICVTS | ALL CTSNet JOURNALS |
