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J Thorac Cardiovasc Surg 1994;107:1512-1518
© 1994 Mosby, Inc.
CARDIOPULMONARY BYPASS, |
Syracuse, N.Y.
Supported in part by the American Heart Association–New York Affiliate and the Hendricks Foundation, State University of New York.
Received for publication July 30, 1993. Accepted for publication Oct. 25, 1993. Address for reprints: Leslie J. Kohman, MD, Department of Surgery, SUNY Health Science Center, 750 East Adams St., Syracuse NY 13210.
Abstract
We designed an experiment to compare single-dose versus multidose cardioplegia (calcium 0.3 mmol/ L) in neonatal rabbit hearts 1, 4 and 6 weeks of age at 25° C and 32° C. Isolated hearts had a stabilization period of retrograde perfusion, a working period, a period of ischemia with single or multidose cardioplegia, reperfusion, and a final working period. We measured hemodynamic recovery, creatine kinase during reperfusion, and coronary vascular resistance during administration of the cardioplegic solution. One-week and 4-week-old hearts exhibited better recovery with single-dose than with multidose cardioplegia. Six-week-old hearts, on the other hand, showed better recovery with multidose cardioplegia. Four-week-old hearts at 25° C showed increased creatine kinase release with multidose cardioplegia. The 6-week-old hearts tended toward lower creatine kinase release with multidose cardioplegia. Coronary vascular resistance rose with subsequent administrations in 1-week and 4-week-old hearts at 25° C but did not rise in 1- and 4-week-old hearts at 32° C or in 6-week-old hearts at either temperature. On the basis of hemodynamic recovery, single-dose cardioplegia appears to provide better protection than multidose cardioplegia to 1- and 4-week-old isolated rabbit hearts. Once the rabbit has reached 6 weeks of age, multidose cardioplegia has some advantage over single-dose cardioplegia, similar to the findings in adult hearts. Creatine kinase release and coronary vascular resistance data corroborate the hemodynamic findings. (J T HORAC C ARDIOVASC S URG 1994;107:1512-8)
Despite clinical limitations,
1 a vast majority of surgeons treating infants with congenital heart disease use cardioplegia during their cases. 2 Research now focuses on refinement of the composition and administration schedule to best optimize cardioplegic protection of the infant heart during operative correction of congenital defects. The superiority of multidose over single-dose cardioplegia in the adult does not extend to the neonate. 3-5 Temperature and timing of delivery of the cardioplegic solution also affect outcome. We designed an experiment to compare single-dose versus multidose cardioplegia in neonatal rabbit hearts of various ages at two temperatures, both in the mild hypothermia range.
MATERIALS AND METHODS
Hearts from New Zealand White rabbits, aged 1 week (7 to 10 days), 4 weeks (22 to 28 days), and 6 weeks (36 to 42 days), were evaluated in an isolated working heart apparatus. All animals received care according to the "Guide for the Care and Use of Laboratory Animals" (NIH Publication No. 86-23, revised 1985). The experiment had the approval of the Committee for the Humane Use of Animals of the State University of New York Health Science Center at Syracuse.
Animals were anesthetized with intraperitoneal ketamine hydrochloride (5 mg/kg) and xylazine (10 mg/kg). Heparin (150 units/kg or 1.5 mg/kg) was given intraperitoneally. The lungs of all animals were ventilated with room air via a tracheostomy with a small-animal respirator. Each heart was then excised, cannulated, and mounted on our left-heart perfusion circuit as previously described. 6 The perfusate, a Krebs-Henseleit buffer solution, contained NaCl, 120 mmol/L; NaHCO3, 25 mmol/L; KH2PO4, 1.2 mmol/L; KCl, 4.7 mmol/L; MgSO4, 1.2 mmol/L; CaCl2, 1.5 mmol/L; and glucose, 11.1 mmol/L. The solution was oxygenated with 95% oxygen and 5% carbon dioxide (323 mOsm/L, pH 7.4 at 37° C). The perfusate was double filtered, first through a qualitative filter, then through an inline 5 µm filter. The cardioplegic solution (324 mOsm/L, pH 7.8) contained NaCl, 110 mmol/L; KCl, 16.0 mmol/L; MgCl2, 16.0 mmol/L; NaHCO3, 10.0 mmol/L; and CaCl2, 0.3 mmol/ L.
Fig. 1 illustrates the time course of the experimental protocol. We perfused each heart for 15 minutes in a retrograde fashion (Langendorff mode) at 55 cm H2O at 39° C. We rejected hearts that had an aortic flow of less than 10 ml/min or a coronary flow of less than 2 ml/min. We replaced these hearts until we had at least six hearts in each group. After 15 minutes of retrograde perfusion, the hearts were converted to the working mode by perfusion through the left atrium. The height of the perfusion column was adjusted individually for each heart to obtain a left atrial pressure of 3 to 8 mm Hg and then kept at this height for the remainder of the experiment. After a 30-minute working heart period, we recorded hemodynamic function and transferred the hearts to a hypothermic (25° C or 32° C) chamber and rendered them ischemic for 120 minutes. Cardioplegic solution (4° C) was given for 3 minutes through the aortic root at 40 cm H2O. Hearts were randomly assigned to receive either a single dose of cardioplegic solution or multiple doses given every 30 minutes. After 120 minutes of ischemia the hearts were moved back to the 39° C chamber and received retrograde reperfusion for 15 minutes. During this time all coronary effluent was collected on ice to be analyzed for creatine kinase content. After 15 minutes, the hearts were converted to the working mode for an additional 30 minutes and hemodynamic parameters were remeasured at 30 minutes after reprefusion.
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We assayed creatine kinase (CK) in the coronary sinus effluent with a Sigma Diagnostics, Inc., Kit CK47-UV (St. Louis, Mo.). We calculated coronary vascular resistance (CVR) during each administration of cardioplegic solution by the following formula:
Statistical analysis
Mean percent recovery of baseline preischemic values was calculated for each group and each measured parameter. Results are expressed as means ± standard error of the mean (SEM). Analysis of variance and Student-Neuman-Keuls test were used to determine if significant differences existed between groups. In hearts that did not recover to the time of postischemic evaluation, we recorded values of zero for hemodynamic parameters. Significance was accepted at the p < 0.05 level.
RESULTS
Approximately 19% of the original hearts were eliminated because of insufficient preischemic function. Table I shows baseline preischemic function of acceptable hearts. Tables II to IV show percent recovery of hemodynamic parameters at the end of the 30-minute postischemic working heart period. One-week-old hearts exhibited better recovery with single than multidose cardioplegia, significant for cardiac output and left ventricular pressure at 25° C. Four-week-old hearts exhibited a similar pattern of improved recovery with single compared with multidose cardioplegia, significant for aortic flow, cardiac output, and stroke volume at 25° C. Six-week-old hearts, on the other hand, showed better recovery with multidose cardioplegia.
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Table V) showed no significant difference between single and multidose cardioplegia in 1- and 4-week-old hearts, except that 4-week-old hearts at 25° C showed increased CK release with multidose cardioplegia. On the contrary, the 6-week-old hearts tended toward lower CK release with multidose than with single-dose cardioplegia.
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Table VI), rose with subsequent administrations in 1-week-old and 4-week-old hearts at 25° C but did not rise in 1-week-old and 4-week-old hearts at 32° C or in 6-week-old hearts at either temperature.
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Our studies show that, on the basis of hemodynamic recovery, single-dose cardioplegia appears to provide better protection than multidose cardioplegia to 1- and 4-week-old isolated rabbit hearts, with statistical significance at 25° C but not at 32° C. By 6 weeks, multidose cardioplegia has some advantage over single-dose cardioplegia, similar to the findings in adult hearts. 7
Numerous laboratory investigations have proved the superiority of cardioplegia over hypothermia alone in the neonatal heart. 8-12 The ideal composition and method of administration remain fruitful fields for inquiry. A number of articles have compared single-dose and multidose cardioplegia in the experimental model of the neonatal heart. 3-5,7,13-15 The calcium content in the cardioplegic solutions in these experiments ranged from 1.2 to 2.5 mmol/L. Baker and associates 16 recently demonstrated that optimal myocardial protection in 1-week-old rabbit hearts occurred at a calcium content of 0.3 mmol/L. We therefore modified our cardioplegic solution to this calcium concentration.
Several of the previous studies 5,7,13,14 showed that single-dose cardioplegia results in a better outcome than multidose cardioplegia in hearts from rabbits ranging in age from birth to 2 days 5 up to 3 to 4 weeks. 13,14 These studies all kept the ischemic heart at 10° to 15° C. Kempsford and Hearse 15 confirmed that multidose cardioplegia is deleterious in the neonatal heart at 10° C, but they found that it improved recovery at 20° C. Our study expands these investigations to cover conditions of higher temperatures and greater age range, as well as optimal calcium concentration. In contrast to Kempsford and Hearse, 15 we found that single-dose cardioplegia continues to maintain its superiority in 1- and 4-week-old hearts at 25° C but has less advantage at 32° C. Murashita and Hearse 3 speculated that there was a transition temperature between 32° and 34.5° C at which multidose cardioplegia loses its advantage. They quoted theoretical reasons why multidose should be superior to single-dose cardioplegia at 37° C and stated that with hypothermia the differences between the two modes are less predictable and more vulnerable to influencing factors. We believe that age is one of these factors. The 6-week-old hearts resemble those of adults; multidose cardioplegia exhibited some advantage at 25° C.
CK release, indicative of global myocardial injury, corroborates the hemodynamic findings: at 25° C, 4-week-old hearts subjected to multidose cardioplegia released more CK than did those subjected to single-dose cardioplegia, whereas at 6 weeks hearts subjected to multidose cardioplegia released less CK than hearts subjected to single-dose cardioplegia at both 25° and 32° C. CVR results also support the findings. An increase of CVR during ischemia represents a tendency toward diminished recovery. 15 In our study, CVR rose with subsequent administrations of multidose cardioplegia in 1- and 4-week-old hearts at 25° C, indicating progressive damage; this rise did not occur in 1- and 4-week-old hearts at 32° C or in 6-week-old hearts at either temperature.
The explanation for the superiority of single-dose cardioplegia in neonatal hearts at all but the warmest temperatures must relate to either the content of the solution or the effect of repeated administration. Several components (calcium, mannitol, glutamate, lidocaine, and pH) have been discredited as a cause of these effects. 10 Simple repeated administration alone cannot explain it: multidose Krebs buffer had no detrimental effect when compared with single-dose Krebs buffer. 7
Magovern, Pae, and Waldhausen 14 speculated that the explanation for the reduced benefit of multidose cardioplegia must result from the effects of reinfusion of cardioplegic solution into hypothermic hearts or the total dose of cardioplegic solution. Murashita and Hearse 3 maintain that these effects cannot be explained on the basis of the increased volume of cardioplegic solution. The depression in function does not appear to be due to depletion of myocardial energy stores. 7,13,14
The source of the decrement in functional recovery after multidose cardioplegia must be age related. Proposals for the mechanism of this age-related difference include effects on the glycolytic capacity of the immature myocardium, direct membrane toxicity, or alterations in calcium metabolism 14; edema promoted by repeated resupply of calcium, sodium, and water 3; and tolerance of the neonatal myocardium to increased lactate production and its more efficient anaerobic metabolism. 5
By using a reduced dose of calcium in the cardioplegic solution, we have shown that this effect is not due to simple calcium concentration: our results with reduced-calcium cardioplegic solution are similar to those reported with standard amounts of calcium. Murashita and Hearse 3 support the concept that in the neonatal heart calcium concentration in the cardioplegic solution has little direct effect. However, even at low calcium concentrations, the repeated administration may still cause damage: rabbits from birth to 7 days of age have higher susceptibility to calcium accumulation after ischemia than hearts of 14 to 21-day-old rabbits. 17
Edema as a cause of these age-related differences might be confirmed by ultrastructural studies. Sawa and colleagues 5 demonstrated significant intracellular edema and mitochondrial damage, more prominent with multidose cardioplegia. They proposed that repeated administration of cardioplegic solution may damage vulnerable endothelial cells of the more permeable neonatal microvasculature.
Some of our results indicate that hearts made ischemic at 32° C recovered better than those at 25° C. This observation seems counter to general belief, but hypothermia itself increases the vulnerability of cells to ischemia. 3 Neonatal hearts may be as small as 1 gm; these hearts cool extremely rapidly when moved to a hypothermic chamber. There is some evidence that such rapid cooling, much faster than the clinical situation, may have a deleterious effect on ischemic tolerance in immature myocardium. 18 This effect is based on intracellular calcium distribution; our lower-calcium cardioplegic solution may contribute to these observations.
We have confirmed by three types of measurements (hemodynamic recovery, CK release, and CVR) that single-dose low-calcium cardioplegia provides superior benefit to multidose cardioplegia in neonatal rabbit hearts aged 1 and 4 weeks challenged with an ischemic period of mild hypothermia (25° C). This advantage does not extend to older hearts (6 weeks of age) and becomes less significant at warmer temperature (32° C). We agree with Konishi and Apstein 10 that relatively small differences in degree of maturity have a profound influence on myocardial cation accumulation during ischemia and reperfusion.
We realize the inherent weaknesses in a laboratory model using asanguineous perfusates in normal hearts. The myocardium of patients with congenital heart defects has often bs in normal hearts. The myocardium of patients with congenital heart defects has often been exposed to cyanosis, acidosis, and hypertrophy. However, the vast majority of laboratory work on this topic has been done in just such models, and crystalloid cardioplegia has proved effective in mature hearts in both experimental and clinical settings. The expense of large animals and the technical difficulties of blood perfusion in small hearts preclude these options. Additionally, the model must allow for myocardial damage during ischemia to show differences in various treatments; we know that the ischemic heart of the rabbit is damaged by St. Thomas' Hospital cardioplegic solution. 16 The isolated working heart model provides stability and allows rapid alterations of temperature, pressure, perfusate, and other conditions necessary for the performance of these studies. Historically, many similar laboratory investigations have led to improvements in clinical management. We hope that this additional evidence from the laboratory will help rationalize the design of cardioplegic maneuvers in clinical practice.
References
This article has been cited by other articles:
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  K. G. Warner, M. G. Sheahan, S. M. Arebi, A. Banerjee, J. M. Deiss-Shrem, and K. R. Khabbaz Proper timing of blood cardioplegia in infant lambs: superiority of a multiple-dose regimen Ann. Thorac. Surg., March 1, 2001; 71(3): 872 - 876. [Abstract] [Full Text] [PDF]
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P. J. del Nido Myocardial Protection and Cardiopulmonary Bypass in Neonates and Infants Ann. Thorac. Surg., September 1, 1997; 64(3): 878 - 879. [Full Text]
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