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Ann Thorac Surg 1996;62:702-709
© 1996 The Society of Thoracic Surgeons
Department of Cardiovascular Surgery, University of Kiel, Kiel, and Department of Pathology, University Hospital, Heidelberg, Germany
Accepted for publication April 13, 1996.
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Abstract |
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 TopFootnotes Abstract IntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Methods. We assessed the effects of (1) rapid cooling by topical hypothermia alone, (2) slow prearrest cooling by coronary perfusion hypothermia, and (3) cardioplegic cardiac arrest with St. Thomas' Hospital solution no. 2 for protection of isolated immature rat hearts (age, 28 days) during 8 hours of global ischemia at 10°C. Myocardial hypertrophy was induced noninvasively by lifelong feeding of a low iron diet. Recovery of left ventricular function, metabolism, and myocardial fine structure were assessed.
Results. In hypertrophied hearts, protection by topical hypothermia alone resulted in significantly improved postischemic recoveries of maximum left ventricular pressure and rate of pressure rise compared with the method of slow cooling or application of cardioplegia (40.6% ± 5.0% and 38.1% ± 5.9%, mean ± standard error of the mean; p < 0.05). The same pattern of recovery was observed among nonhypertrophied control hearts. Regardless of the method of protection, hypertrophied hearts revealed a significantly larger interstitial space at the end of reperfusion than control hearts. In hypertrophied hearts, postischemic adenosine triphosphate concentrations were higher with topical hypothermia alone for protection than with the other methods.
Conclusions. Rapid cooling by topical hypothermia alone provides superior protection of hypertrophied immature rat hearts as compared with slow prearrest cooling. Application of St. Thomas' Hospital cardioplegic solution no. 2 does not improve protection and even hinders postischemic functional recovery.
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Introduction |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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The explanation for inadequate protection in young hearts may relate in part to the differences in metabolic profiles of adult and pediatric hearts. Other common factors of congenital cardiac defects, however, such as chronic cyanosis and myocardial hypertrophy, also may contribute to inadequate protection in this age group [4]. With regard to this aspect, previous reports emphasized the importance of using experimental models that simulate clinically relevant conditions before drawing conclusions about the safety of cardioprotective strategies [5]. One common feature of many congenital cardiac defects with an intracardiac shunt is the rapid development of postnatal myocardial hypertrophy as a sequel of chronic volume overload. We therefore simulated this pathway to early postnatal myocardial hypertrophy by using an experimental model of nutritional anemia, which was induced in neonatal rats by feeding them a diet low in iron. This measure has been described as leading to rapid development of cardiac hypertrophy by compensatory hypercirculation [6]. This experimental model produced the sequelae of chronic volume overload noninvasively.
At the age of 28 days, hearts of rats were subjected to isolated perfusion to determine the effects of (1) rapid cooling to 10°C by topical hypothermia alone, (2) slow prearrest cooling by coronary perfusion hypothermia, and (3) cardioplegic cardiac arrest using St. Thomas' Hospital cardioplegic solution no. 2 (STS 2) plus topical hypothermia for myocardial protection during 8 hours of global ischemia at 10°C. Indices of left ventricular function, metabolism, and myocardial fine structure were assessed.
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Material and Methods |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Induction of Myocardial Hypertrophy
Since mating, nutritional anemia was induced in mother animals by feeding a diet low in iron (Diet C 1038; Altromin, Lage, Germany; iron content, 5.1 mg/kg) and distilled water. After birth, litters were reduced to 8 male members and the mother animal. After weaning on the 18th day, littermates received a low iron diet for 10 days before the perfusion experiments, performed on the 28th day. Rats were allocated randomly to study groups.
Pilot Experiments
The first set of pilot perfusion experiments was performed to evaluate the duration of ischemia at hypothermia of 10°C that was followed by 50% or less recovery of maximum developed left ventricular pressure. Nonhypertrophied control hearts (four experimental groups, n = 6 in each) were subjected to in vitro perfusion according to the same protocol as described later for the subsequent perfusion experiments. Hearts were protected by topical hypothermia alone, as described later for "protocol 1." Global ischemia times of 4, 6, 7, and 8 hours at a hypothermia level of 10°C were assessed.
The second set of pilot experiments was performed to assess the degree of myocardial hypertrophy that occurs as a sequel of long-term low iron nutrition. Hearts from anemic and nonanemic control animals (3 per group) were perfused at the age of 4 weeks for 10 minutes at normothermia for washout of the blood components. Then, perfusion was discontinued and the hearts were fixed in formaldehyde (10%) before embedding in Histoform-plastic (Technovit 7100; Haeraeus-Kulzer, Werheim, Germany). Transverse histologic sections were cut at the widest ventricular diameter, stained with hematoxylin and eosin, and assessed by light microscopy.
Perfusion Experiments
The perfusion experiments were performed in six experimental groups (n = 17 to 19 in each group). Hearts of groups 1H, 2H, and 3H were hypertrophied because of nutritional anemia; hearts of groups 1C, 2C, and 3C served as age-matched, nonhypertrophied controls. At the end of the preischemic perfusion period, we applied three different protocols for protection during global myocardial ischemia for 8 hours at 10°C.
Protocol 1 consisted of rapid cooling by topical hypothermia alone. In the hearts of groups 1H and 1C, preischemic perfusion was discontinued and cardiac arrest was induced simultaneously by rapid topical cooling with cold (4° to 6°C) Krebs-Henseleit solution.
Protocol 2 involved slow prearrest cooling by coronary perfusion hypothermia. In the hearts of groups 2H and 2C, hypothermia was induced slowly over 10 minutes from normothermia to 10°C by perfusion with oxygenated perfusate at gradually decreasing temperature. During this period, the temperature inside the water-jacketed perfusion chamber of the heart was adjusted to the temperature of the perfusate thermostatically.
Protocol 3 was cardioplegic cardiac arrest with STS 2 plus topical hypothermia. Hearts of groups 3H and 3C were arrested with STS 2, which was administered at a temperature of 10°C through a sidearm of the aortic cannula from a reservoir located 60 cm above the heart, for 3 minutes as a single dose. The composition of STS 2 was as follows: NaCl 110 mmol/L, KCl 16 mmol/L, MgCl2 16 mmol/L, CaCl2 1.2 mmol/L, and NaHCO3 10 mmol/L (pH adjusted to 7.8; osmolarity, 324 mOsm/L). Topical hypothermia was applied additionally as described in protocol 1.
After initial protection, all the hearts were disconnected from the perfusion apparatus, placed in a beaker containing Krebs-Henseleit solution, and stored in a refrigerator for 8 hours at 10°C.
Perfusion Methodology
Rats, randomly selected from litters, were injected with sodium heparin (1 mg per rat intraperitoneally). After 30 minutes, the animals were anesthetized by inhalation of diethyl ether, and 1 mL of blood was withdrawn by puncture of the inferior caval vein for measurement of hemoglobin and hematocrit after laparotomy. The chest was then opened, and the thoracic cavity was filled with cold (4°C) Krebs-Henseleit solution. With the heart thus immersed in cold solution, the pulmonary artery was incised near its origin and the aorta was cannulated with a blunted 16-gauge needle (1 mm outer diameter). The heart was then excised and mounted on the perfusion apparatus through the aortic cannula. Perfusion of the coronary arteries was performed according to the Langendorff technique [7], at a perfusion pressure of 60 mm Hg (80 cm H2O), using oxygenated modified Krebs-Henseleit solution with the following composition (in mmol/L): 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, and kept at 37°C. Each heart was housed in a thermostatically controlled heart chamber and maintained at 37°C during the preischemic and postischemic periods. During a 15-minute washout period after cannulation, an intraventricular balloon was inserted in the left ventricle through the mitral valve. The ultrathin balloon was designed to match the ventricular dimensions of the heart. The balloon was filled with distilled water and attached to a pressure transducer through a fluid-filled tube. The volume of the balloon was adjusted by means of a watertight microsyringe attached to the sidearm of the transducer. The left ventricular pressure signal was recorded and processed on-line using 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).
Functional and Hematologic Measurements
Preischemic and postischemic functional and hematologic indices were obtained from all hearts of the six experimental groups. Preischemic baseline measurements of systolic left ventricular function, coronary flow, and heart rate were performed 15 minutes after the onset of in vitro perfusion during isovolumic contractions by inflating the intraventricular balloon to a left ventricular end-diastolic pressure of 10 mm Hg. The volume necessary to produce this pressure (V10) was registered. The maximum developed left ventricular pressure (LVDP) and its first derivative (maximum peak dP/dt) were recorded.
The recovery of systolic function, coronary flow, and heart rate was measured 60 minutes after the onset of reperfusion at a left ventricular end-diastolic pressure of 10 mm Hg. V10 was recorded and judged, in relation to the preischemic volume, as an estimate for the recovery of diastolic function.
Blood hemoglobin concentration was measured with an automated analyzer using a standard test kit (Merckotest Haemoglobin 3317; E. Merck, Darmstadt, Germany). Hematocrit was assessed by centrifugation of blood in heparin-treated hematocrit capillaries.
Determination of Myocardial High-Energy Phosphates
At the end of reperfusion, 13 hearts per group were frozen rapidly with Wollenberger tongs precooled in liquid nitrogen. After perchloric acid extracts had been prepared from tissue samples according to the method of Lowry and Passonneau [8], the tissue was dried for 48 hours at 80°C before measuring the myocardial dry weight. Creatine phosphate and adenosine triphosphate (ATP) levels (in micromoles per gram dry weight) were assessed using a standard enzymatic assay [9].
Light and Electron Microscopic Assessment
At the end of reperfusion, 4 to 6 hearts per experimental group were fixed by aortic perfusion with ice-cold 2.5% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer (pH 7.4). Transmural slices (up to 3 mm thick) of the left ventricular wall were then cut and immersed in the same fixative. After frequent rinsing in buffer, tissue blocks were postfixed for 90 minutes in 2% osmium tetroxide. After dehydration in graded series of ethanol, samples were embedded in epoxy resin. After polymerization, 6 tissue blocks per heart were prepared. Semithin sections (0.5 to 1 µm) were cut and prepared for light microscopy by staining with fuchsin and methylene blue. Artifact elimination was done for every kind of embedding or mechanical alteration, eg, cutting edge lesions. Then, the ratio of the interstitial space and myocytes as a relative measure of interstitial edema was assessed morphometrically by the "point counting" method according to Weibel [10]. Three hundred points per semithin section, ie, 1,800 points per heart, were evaluated using a light microscope (Standard 25; Zeiss, Oberkochen, Germany) with an ocular integrated test system (Integrationsplatte II; Zeiss).
Additionally, the degree of preservation of the myocardial bundle structure [11] was assessed on six semithin sections per heart by light microscopy (magnification, x100) and classified into four groups as follows: group 1, myocardial bundle structure detectable in nearly no areas of the section (0% to 10%); group 2, myocardial bundle structure detectable in less than half of the section (10% to 50%); group 3, myocardial bundle structure detectable in more than half of the section (50% to 90%); and group 4, myocardial bundle structure detectable in nearly all or every area of the section (90% to 100%).
Ultrathin sections (50 to 80 nm) were prepared using an ultramicrotome (Ultracut E; Reichert-Jung, Vienna, Austria) and were contrasted with uranyl acetate and lead citrate. Cellular alterations were identified by electron microscope (EM 10; Zeiss). Light and electron microscopic specimens were assessed in a blinded fashion.
Nine additional hearts (4 hypertrophied, 5 nonhypertrophied) were prepared accordingly at the end of the preischemic perfusion period to assess the impact of ischemia and reperfusion on the myocardial fine structure.
Statistical Analysis
All data are expressed as mean ± standard error of the mean. All multiple comparisons, except for evaluation of the myocardial bundle structure, were performed by analysis of variance. When significant F values were obtained, intergroup comparisons were performed with Duncan's test. Data for myocardial bundle structure were analyzed by the Kruskall-Wallis test and subsequent Mann-Whitney U test for intergroup comparisons. A probability of less than 5% (p < 0.05) that a difference between groups occurred by chance was accepted as statistically significant.
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Results |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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. After 4 hours of global ischemia at 10°C, recovery measured more than 80% of the preischemic values. Increase in ischemia time resulted in a decrease of postischemic recovery of LVDP, such that approximately 50% recovery was measured after 8 hours of ischemia. Hearts in all subsequent perfusion experiments were therefore subjected to 8 hours of global ischemia at 10°C.
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, respectively. Massive right and left ventricular hypertrophy developed under conditions of nutritional anemia by feeding a diet low in iron.
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). The mean myocardial dry weight was higher in the hypertrophied hearts than in corresponding controls. Among the hypertrophied hearts of anemic animals, the myocardial dry weight to body weight ratio was significantly greater than in the controls. Feeding a low iron diet resulted in marked reductions in serum hemoglobin concentration and hematocrit.
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. Both LVDP and maximum peak dP/dt were significantly higher in hypertrophied hearts than in corresponding control hearts. The numeric values of V10 were higher in hypertrophied hearts than in corresponding controls. Coronary flow was increased in hypertrophied hearts (p < 0.05 versus corresponding controls). No differences were observed between hypertrophied hearts and control hearts in preischemic heart rates. Within the hypertrophy groups and control groups, no significant differences were observed during preischemic perfusion.
POSTISCHEMIC MYOCARDIAL FUNCTION.
Recovery rates of myocardial function after 1 hour of reperfusion are shown in Table 2. In hypertrophied hearts, the recovery rates of LVDP and maximum peak dP/dt were greatest after protection with the use of topical hypothermia alone (40.6% ± 5.0% and 38.1% ± 5.9%, respectively). The numeric differences in the recoveries of LVDP and of maximum peak dP/dt were statistically significant compared with group 2H (slow cooling) and 3H (STS 2). The recovery of V10 was highest in group 1H (topical hypothermia alone). However, this numeric difference was not significant in comparison with group 2H or group 3H. No differences among the hypertrophy groups were observed with regard to recovery of coronary flow and heart rate.
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MYOCARDIAL HIGH-ENERGY PHOSPHATES.
The highest numeric concentrations of ATP after 60 minutes of normothermic reperfusion were measured in hearts protected by topical hypothermia alone (group 1H, 6.1 ± 0.7 µmol/g dry weight; group 1C, 6.3 ± 0.8 µmol/g dry weight) (see Table 2). Among the hypertrophy groups, ATP values were significantly increased in group 1H compared with groups 2H (slow cooling) and 3H (STS 2). The same pattern was observed with respect to the creatine phosphate concentrations. Again, the numerically highest values were measured in group 1H (topical hypothermia alone). Among the control groups, no statistically significant differences were observed in the postischemic concentrations of myocardial high-energy phosphates.
LIGHT AND ELECTRON MICROSCOPIC FINDINGS.
Histologically, the interstitial space was larger in the hypertrophied hearts after ischemia and subsequent reperfusion than in the corresponding control hearts or the preischemic controls. Analysis of variance revealed a significant increase of the morphometrically determined interstitium to myocyte ratio in reperfused hypertrophied hearts compared with corresponding control hearts (Table 3). No such difference was observed among preischemic hearts of hypertrophied and control animals. In regard to the method of protection, all comparisons among corresponding groups indicated a significant increase of this ratio in hypertrophied hearts except for group 3H versus group 3C (protection by STS 2), which narrowly failed to reach statistical significance.
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) than in the hypertrophied hearts of anemic animals regardless of the method used for myocardial protection (Fig 3B; see Table 3). Subsequent group-by-group comparisons with respect to each method of myocardial protection revealed that the bundle structure of hearts in group 1C was significantly better preserved than in group 1H (protection by topical hypothermia alone).
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). However, these features of irreversible myocardial damage were often found close to reversibly altered or even morphologically intact myocytes, as observed more frequently in nonhypertrophied hearts of control groups (see Fig 3C). |
Comment |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Several studies have indicated a greater inherent tolerance to ischemia of immature hearts than of the adult heart [12, 13]. On the other hand, a strong correlation of early postoperative death with the duration of myocardial ischemia in pediatric cardiac operations has been described [1]. A reason for this discrepancy may be that most studies evaluating cardioprotective strategies in immature hearts were conducted in healthy animals. Little is known about the impact of cyanosis or hypertrophy, as observed with many congenital heart defects, on the response of the heart to measures for myocardial protection.
Because the operative measures alone that are necessary to create chronic volume overload experimentally might disturb postischemic myocardial function, this might increase the sampling error. Thus, we used an experimental model that produced the sequelae of chronic volume overload noninvasively. Neonatal rats were subjected to a diet low in iron for approximately 4 weeks. This measure is known to cause rapid development of cardiac hypertrophy by compensatory hypercirculation [6]. Measurements of hemoglobin, hematocrit, myocardial weight, and gross histologic examination of hearts from anemic rats indicated (1) that severe iron deficiency anemia occurred under our experimental conditions and (2) that this was followed by rapid development of myocardial hypertrophy. These observations recommended this model for the purposes of our study (see Figs 2A, 2B; Table 1).
Improved protection with STS 2 has been reported in immature rats and rabbits [14, 15]. In these studies, however, the myocardial temperature was maintained at normothermia or mildly hypothermic during ischemia, whereas the experiments of this study were conducted at 10°C during myocardial ischemia, a temperature more closely related to clinical conditions. The excellent recoveries that we observed in the pilot perfusion experiments with noncardioplegic protection after shorter ischemia times required extension of the ischemia interval, such that the recoveries of contractile function were 50% or less, thereby leaving enough scope for improved protection. However, cardioplegic protection did not afford improved protection in this study. The reason for the superior results that we observed with topical hypothermia alone 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 than in bigger hearts. Second, it is possible that the combination of topical cooling at 4° to 6°C and additional hypothermic cardioplegic arrest induces extremely rapid arrest in the immature heart with subsequent myocardial injury caused by rapid cooling contracture [3].
Our unfavorable results with perfusion hypothermia match the findings of Hosseinzadeh and colleagues [16], who reported that slow cooling by coronary perfusion with asanguinous perfusate may accelerate ATP depletion. Because the maintenance of intracellular homeostasis and hence a low cytosolic calcium level requires ATP, the depletion of myocardial high-energy phosphates may result in cell membrane damage and sudden influx of calcium into the cell. Thus, it is conceivable that the 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.
One major aspect of this study was to assess whether hypertrophy alters the functional response of the immature heart to methods for myocardial protection. Our results indicate that this is not the case; in neither normal nor hypertrophied hearts did cardioplegia or slow cooling afford greater protection than topical hypothermia alone. However, the difference between the topical hypothermia group and both other groups in terms of postischemic recovery of LVDP and maximum peak dP/dt tended to be more pronounced in hypertrophied hearts (see Table 3). In this context, the report of Mavroudis and Ebert [17] warrants consideration. They found that hypertrophy renders the adult myocardium more susceptible to the development of interstitial edema during postischemic reperfusion. Our results indicate that the same applies for the hypertrophied immature heart: morphometric assessment of the interstitium to myocyte ratio [10, 11] revealed greater interstitial edema in reperfused hypertrophied hearts than in the nonhypertrophied control hearts. Consequently, the myocardial bundle structure was found to be less well preserved in hypertrophied hearts than in controls. However, unlike evaluation of postischemic myocardial function, the electron microscopic assessment of alterations of subcellular structures did not reveal a constant pattern depending on one of the three methods for myocardial protection [18].
Measurements of postischemic myocardial concentrations of ATP and creatine phosphate support the functional results, as the highest numeric concentrations of both phosphates were observed after protection using topical hypothermia alone. However, the numeric differences were statistically significant only among hypertrophied hearts after exposure to a low iron diet (see Table 2).
The interpretation of our results in relation to the clinical situation in pediatric cardiac operations must be made with caution because of the obvious differences between the isolated perfused rat heart and the human heart in situ. The limitations of this type of perfusion system are well known. However, the major points to emerge from this study are as follows. (1) The model of biventricular hypertrophy as established noninvasively by nutritional iron restriction may provide insight into cardioprotective strategies for relatively immature hypertrophied hearts. (2) Neither STS 2 nor extended hypothermic prearrest cooling improves myocardial protection of the immature heart. The adverse consequences of crystalloid perfusion at low temperatures could well account for the equal or even improved function observed after protection by topical hypothermia alone. (3) Hypertrophied immature myocardium develops a more severe interstitial edema during postischemic reperfusion than do nonhypertrophied hearts. This observation supports the clinical concept of timely correction of congenital cardiac defects before marked hypertrophy has occurred.
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Acknowledgments |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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We express our gratitude to Dr Wolfgang Caliebe, Department of Biostatistics, University of Kiel, for his help in the statistical analysis of the data.
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Footnotes |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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References |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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