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Comparison of three cardioplegic solutions during hypothermic ischemic arrest in neonatal blood-perfused rabbit hearts
J THoRAc CARDIOV ASC SURG 1989;98: 1132-7
Comparison of three cardioplegic solutions during hypothermic ischemic arrest in neonatal blood-perfused rabbit hearts Inadequate myocardial preservation continues to be an important cause of postoperative morbidity and mortality after pediatric cardiac operations. To investigate methods of improving preservation in neonatal myocardium, we compared three cardioplegic solutlons with topical hypothermia during 120 minutes of ischemic arrest in isolated, blood-perfused, neonatal rabbit hearts. Topical hypothermia (150 C) without cardioplegia resulted in 71 % ± 5 % recovery of preischemic contractile function. A high potassium (30 mEqjL) cardioplegic solution resulted in it 76 % ± 6 % recovery of function, not significantly different from that obtained with hypothermia alone. In contrast, the St. Thomas' Hospital and Hopital Lariboisiere cardioplegic soluti~ resulted in recoveries of 89 % 1:- 6 % and 88% ± 7%, respectively, both of which were significantly greater (p < 0.001) than recoveries obtained with the high potassium solution or hypothermia alone. Thus the cardioplegic solutio~ used at St. Thomas' Hospital and Hopital Lariboisiere provided exceUent protection during 2 hours of hypothermic ischemic arrest jn neonatal rababit hearts and resulted in functional recovery superior to that achieved with hypothermia alone or with the high potassium cardioplegic solution.
Takashi Konishi, MD, and Carl S. Apstein, MD, Boston, Mass.
Inadequate preservation of neonatal myocardium during cardiac operations remains a significant clinical problem, especially with the advent of aggressive surgical strategies for correcting certain congenital cardiac defects soon after birth. For example, Bull, Cooper, and Stark' recently concluded that 50% of pediatric cardiac surgical deaths were attributable to inadequate myocardial preservation despite cardioplegia, especially when the ischemic period exceeded 85 minutes. Despite these obser-
From Cardiac Muscle Research Laboratory, Cardiovascular Institute, Boston University School of Medicine, and the Cardiology Section, Thorndike Memorial Laboratory, Boston City Hospital. Boston, Mass. Dr. Konishi was supported by a Research Fellowship from the Massachusetts Affiliate of the American Heart Association, No. 13-437856; this research was supported by U.S. Public Health Service Grant HL-35675. Received for publication Dec. 19, 1988. Accepted for publication March 21, 1989. Address for reprints: Carl S. Apstein, MD, Director, Cardiac Muscle Research Laboratory, Boston University School of Medicine, 80 East Concord St. (R-217), Boston, MA 02118.
12/1/13096
1 1 32
vations, neonatal myocardium had generally been reported to be more resistant to hypoxic and ischemic injury than adult myocardium.e? though some workers have reached the opposite conclusion.f We reasoned that protection of neonatal myocardium during a prolonged period of ischemic arrest may depend on the composition of the cardioplegic solution that is used. Although the composition of cardioplegic solutions has been studied extensively in adult hearts, relatively few comparative studies have been performed in neonatal hearts. Therefore, in the current study, we used an experimental protocol designed to closely simulate clinical surgical conditions and we compared three widely used cardioplegic solutions: (1) a high potassium solution, (2) St. Thomas' Hospital solution No.2, and (3) Hopital Lariboisiere solution. Groups of neonatal (4- to 6-day-old) isolated blood-perfused rabbit hearts were subjected to 120 minutes of hypothermic (150 C) ischemic arrest, either without cardioplegic protection (controls) or with one of three types of cardioplegic solutions (Table I). Methods Isolated heart preparation. We used an isolated isovolumic (balloon in the left ventricle) rabbit heart model perfused with fresh whole blood, a system that we2• 9 have recently developed
Volume 98 Number 6
Comparison of cardioplegic solutions
December 1989
I I33
Blood Line
D=~==~
Pressure Regulator
Saline
Filter Oxygenator Syringe
t
Pulmonary Artery Cannula
~~~~
Pacer
Intra-LV Balloon Apical Drain Pump
Fig. 1. Isolated blood-perfused rabbit heart preparation. As previously reported.! heparinized blood from a donor rabbit is pumped through an oxygenator and filter to the coronary arteries through a cannula in the aortic stump. Coronary perfusion pressure (CPP) is controlled by a pressure regulator, and coronary flow regulation is determined by coronary vascular autoregulation. A thin-walled latex balloon fills the left ventricular cavity and is attached to a pressure transducer (Statham P23Db) for measurement of left ventricular pressure (LVP). Left ventricular balloon volume determines left ventricular volume and is held constant throughout each experiment so that changes in left ventricular end-diastolic pressure reflect changes in left ventricular diastolic distensibility. Left ventricular thebesian drainage is vented by an apical drain, and all coronary venous efflux is collected by a cannula in the ligated pulmonary artery. Heart rate is controlled by a right ventricular pacer, and temperature is maintained at 3r C and monitored with an intraventricular temperature probe (not shown).
Table I. Components of cardioplegic solutions (mmoljL) Solutions
K+
\' + .a
Ca++
Mg++
0-
HCOj--
Glucose
Mannitol
Glutamate
Lidocaine
pH
High potassium 51. Thomas' Hospital No.2 Hopital Lariboisicre
30 16 15
84 110 100
0 1.2 0.25
0 16 16
107 160 148
7 10 0
139 0 0
0 0 68
0 0 20
0 0.9 0
7.4 7.8 7.4
(Fig. 1). In this system. heparinized blood is pumped from a venous reservoir. through an oxygenator, into a pressurized ar'terial reservoir, and through a 40 /-lm filter (SQ40, Pall Biomedical Products Corporation, Glen Cove, N.Y.) before entering an aortic cannula. Coronary venous blood collects in the venous reservoir and is recirculated through the system. A heparinized (500 units/kg) and anesthetized (sodium pentobarbital, 50 rug/kg) 2 to 3 kg male New Zealand white rabbit served as a blood donor, contributing 70 to 80 ml of fresh whole blood via the carotid artery. Gentamicin (6 mg/100 ml) was added to the blood. The hematocrit value of the blood perfusate was 29% to 33%, blood glucose was maintained between 80 and
120 mg/dl, oxygen tension was 70 to 150 mm Hg, and pH was 7.35 to 7.45. A neonatal albino New Zealand rabbit (4 to 6 days old, male or female) was heparinized (200 units) and anesthetized with sodium pentobarbital (50 mg). The thorax was rapidly opened, and the heart and proximal great vessels were excised and placed in iced saline. The aortic stump was perfused with the isolated heart system described earlier. The time from thoracotomy to initiation of coronary perfusion on the isolated heart apparatus was approximately I to 2 minutes. Coronary perfusion pressure was adjusted to 60 mm Hg and coronary flow was determined by coronary autoregulation. A coronary perfusion pressure of 60
1134
The Journal of Thoracic and Cardiovascular
Konishi and Apstein
Surgery
Table II. Ventricular function before and after hypothermic ischemic arrest End-ischemia
Pre-ischemia DP Group Topical cooling High potassium St. Thomas Hospital H6pital Lariboisicrc
N (mm Hgj
EDP
CF/G
(mm Hgj
(mlfm)
9
92 ± 13
10 ± 1
2.1 ± 0.8
9
97 ± 7
!I ± !
3.3 ± 2.0
9
99 ± 31
9±!
1.9 ± 0.6
8
91 ± 16
J1 ± I
2.5 ± 0.8
Contracture (mm
Hgt
::::)1: •
Reperfusion
DP (mm Hgi 65 ± II
%REC 71 ± 5
73 ± 10
t
:j: 76 ± 6}:j:
CF/G
WeI weigh:
tDP imm Hgi
tmlfmin,
img)
Wetfdrv
9 ± 3
2.0 ± U
282 :': 5f>
4.9 ± 0.7
II ± 2
2.8 ± 1.3
244 ± 35
4.f> ± 0.3
26 ± 8
88 ± 27
89 ± 6
+12 ::!: 4
1.8:,: 0.7
331 ± 74
5.0 ± 1.2
24 ± 6
80 ± 13
88 ± 7
11 ± 3
2.4 ± 0.8
2f>6 ± 35
4.8 ± 1.4
Values arc mean ± standard deviation. :\. ~ umber of animals: DP. developed pressure: EDP. cnd-diastolic pressure: C Fi/G. coronary 110\'. , left vcntricula r weight in grams: '7, REC. percent recovery of developed pressure: wet/dry. ratio of wet to dry weight of the left ventricle.
"n < 0.05. tp
mm Hg was maintained during the preischemic and recovery periods, since we? have previously shown that this coronary perfusion pressure results in optimal performance of the neonatal heart. Coronary perfusion pressure was monitored from a side arm of the aortic perfusion cannula connected to a pressure transducer (Statham P23Db, Spectramed Inc., Critical Care Division, Oxnard, Calif.). A polyethylene cannula was passed across the left ventricular apex to decompress the left ventricle of thebesian drainage. Another cannula was inserted into the right ventricle via the pulmonary artery for complete collection of coronary sinus effluent and to keep the right ventricle decompressed throughout the experiment. A pacing wire (7F, Cordis Corp., Miami, Fla.) was introduced into the right ventricle through the right atrium. Left ventricular pressures were measured with a collapsed latex balloon placed in the left ventricle via the left atrium. The left ventricular balloon was filled with saline to achieve a physiologic left ventricular end-diastolic pressure (7 to IS mm Hg). Left ventricular pressure was monitored through a short, rigid, fluid-filled polyethylene tubing (Intra-Medic, PE240; Clay Adams, Division of Becton Dickinson and Co., Parsippany, N J.) attached toa pressure transducer (Statham P23Db). The heart was then suspended in warm saline at 37 8 C. Bath temperature was monitored throughout the experiment and maintained by adjusting the temperature of the water circulating through the water-jacketed heart chamber. During a 15- to 20-minute preischemic period, the heart was allowed to stabilize at a temperature of 37° C, a paced heart rate of 240 beats/min, a coronary perfusion pressure of 60 mm Hg, and a left ventricular balloon volume that produced a left ventricular end-diastolic pressure of 7 to 15 mm Hg. Hearts were discarded if a left ventricular developed pressure of at least 60 mm Hg was not reached during the preischemic stabilization period; approximately 5% of experimental preparations were rejected because of this criterion. Coronary flow rate was measured by collecting timed samples of the coronary sinus effluent via the pulmonary artery cannula. All animals used in this study received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and
the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes Of Health (NIH Publication No. 80-23, revised 1978). Cardioplegic solutions. We compared three cardioplegic solutions that are currently used in major medical centers (see Table I). The high potassium solution had a potassium concentration of 30 mmol /L: it contained neither calcium nor magnesium, and the glucose content was 2Slr (139 rnmol/L). The St. Thomas' Hospital solution 1\0. 2 had a potassium concentration of 16 mrnol/L, a calcium content of 1.2 rnrnol/L, a high magnesium level, was alkalotic, and contained lidocainc.!" The Hopital Lariboisiere solution had a potassium concentration of 15 mmol/L, was hypocalcemic at 0.25 mmol/L, had a high magnesium level, mannitol, glutamate as substrate, and a pH of 7.4. 11 Experimental protocol. Four groups of hearts were compared during a 120-minute period of hypothermic ischemic arrest and 30 minute of recovery. Each group consisted of eight or nine hearts. The choice of perfusate (i.c., control or one of three types of cardioplegic solution) was alternated among groups so that the experiments among all four groups were performed concurrently. The control group was made hypothermic but received no coronary infusions. The high potassium group, St. Thomas' Hospital group, and Hopital Lariboisiere group received cardioplegic solutions as described earlier and in Table 1. All hearts underwent a 15- to 20-minute baseline prcischcmic period as described above. Hypothermic ischemic arrest was initiated in this manner: The pacemaker was turned off, the 37' C saline in the heart chamber of the perfusion apparatus was rapidly exchanged for saline at 4 0 C, the left ventricular balloon was deflated. and then coronary perfusion was stopped. In the three groups that received cardioplegic protection, the cardioplegic solution was then immediately infused into the aortic cannula at a pressure of 60 mm Hg. Myocardial perfusion by the cardioplegic solution was confirmed by visual observation of clear cardioplegic solution flow from the pulmonary artery cannula. The three cardioplegia groups received an infusion of 1.5 ml of cardioplegic solution at 4° C at the start of the ischemia period and every
Volume 98 Number 6 December 1989
20 minutesduring ischemia. The temperature of the heart was maintained at IS ° C during the ischemic period. At the end of the 120-minute ischemic period, the left ventricular balloon was filled to the same volume present during the preischemic baseline period to measure the "contracture" pressure in the arrested heart before reperfusion. Reperfusion was initiated at 60 mm Hg with arterial bloodat 26° C. The arterial perfusate and heart were gradually rewarmed to 37'" C over a 5-minute period. When myocardial temperature became 37° C, the pacemaker was turned on at a rate of 240 beats/min. Left ventricular function was monitored during 30 minutesof reperfusion. At the end of the reperfusionperiod,the heart was dried to constant weight to determine the dry/wet weight ratio. Data analysis. Data are reported as the mean ± standard deviation and were analyzed by analysis of variance followed by analysis of protected least significant difference when the analysis of variance demonstrated significant intergroup differences (i.e., F < 0.05). Significant differences for the protected least significant difference test were considered to be p < 0.05.12
Results The results are summarized in Table II. During the preischemic baseline period, all four groups had comparable ventricular function and coronary flow rates. Developed pressure was greater than 90 mm Hg in all groups, which indicated good myocardial function. At the end of the 12Q-minute ischemic period, significant contracture had occurred in all of the groups. The high potassium solution group had significantly less contracture than the other three groups, among which the extent of contracture was similar. During 30 minutes of reperfusion, the contracture that had developed during the ischemic period was completely reversed in all groups, such that left ventricular end-diastolic pressures were similar among all groups at the end of the reperfusion period. However, there were significant differences with regard to contractile performance. The St. Thomas' Hospital group and the Hopital Lariboisiere group each recovered significantly better developed pressure than either of the two other groups. There was no significant difference in recovery of developed pressure between the St. Thomas' Hospital and Hopital Lariboisiere groups (89% versus 88%), nor was there a significant difference between the high potassium cardioplegia group and the group that received no cardioplegic protection (76% versus 71%). Recovery of coronary vasomotor function was assessed indirectly by return of coronary flow values. At the end of reperfusion, coronary flow values were comparable to the preischemic values and there were no significant differences among groups. There was no significant difference in edema (wet/dry weight ratio) among the four groups at the end of the reperfusion period.
Comparison of cardioplegic solutions
1 135
Discussion Our results demonstrate that neonatal myocardial preservation during prolonged (120 minutes) hypothermic ischemic arrest was significantly improved by the St. Thomas' Hospital solution No.2 and the Hopital Lariboisiere cardioplegic solutions, but not by the high potassium glucose-containing cardioplegic solution. Our goal was to compare three cardioplegic solutions under experimental conditions that simulated those of clinical cardiac surgery. We used a blood-perfused model and relatively long hypothermic ischemic arrest period. To assess any potential protective effect of cardioplegia, we selected a relatively long arrest period to ensure that the control group would have incomplete preservation and be able to serve as a reference group against which a protective cardioplegic effect might be compared. We maintained hypothermia at 15° C during the ischemic arrest to simulate clinical conditions. Most comparative studies of different cardioplegic solutions have used adult hearts. Immature, neonatal myocardium differs from mature myocardium in terms of sarcoplasmic reticular function, ion pumps, and contractile proteins. 13-1 5 Therefore we reasoned that the immature myocardium may have unique, specific requirements regarding composition of the cardioplegic solution. Despite such a theoretical possibility, our results indicate that two cardioplegic solutions that have been shown to confer good protection against ischemia in adult myocardium (the St. Thomas' Hospital and Hopital Lariboisiere solutions) also confer excellent protection in the neonatal myocardium. Components common to these two cardioplegic solutions are a high magnesium level, low calcium content, and absence of glucose (Table I). Since the St. Thomas' Hospital and Hopital Lariboisiere solutions resulted in similar functional recovery, it is reasonable to conclude that their differences in calcium content, mannitol, glutamate, lidocaine, and pH had no significant effect on myocardial preservation in this model. This result appears to be different from that in the neonatal pig heart, where the cardioplegic calcium level significantly influenced subsequent recovery.l" The specific component responsible for the poorer recovery of the glucose-high potassium solution cannot be determined from these experiments. A potassium concentration of 30 mfiq/L may be higher than optimal.'? Infusion of a high glucose concentration before the onset of zero-flow normothermic ischemia appeared to increase tissue injuryl''; however, if adequate washout of tissue lactate is maintained, then a high level of perfusate glucose appears to reduce ischemic injury." Also, the complete absence of calcium in the high potassium solution may cause some degree of "calcium paradox" injury.I"
The Journal of
1136
Thoracic and Cardiovascular Surgery
Konishi and Apstein
Our observations may have clinical relevance but are subject to the inherent limitations of the experimental conditions. First, there may be important species differences. For example, Como and associates" reported that St. Thomas' Hospital solution No.2 did not improve the functional recovery of the neonatal pig heart relative to topical cooling without cardioplegia. However, Bove, Stammers, and Gallagher" showed that the St. Thomas' Hospital solution No.2 protected the buffer-perfused neonatal rabbit heart against ischemia at 28° C. Our results in blood-perfused hearts at 15° C are consistent with those of Bove, Stammers, and Gallagher. Our results should be compared with the recent report of Baker, Boerboom, and Olinger," in which St. Thomas' Hospital solution No.2 appeared to be harmful to buffer-perfused immature rabbit myocardium, especially when multidose cardioplegia was used. Differences between Baker's and our studies include the buffer versus blood-perfusion methodology, as well as the age of the rabbits that were used. Baker used 7- to lO-day-old rabbit pups, whereas ours were 4 to 6 days old. Relatively small differences in degree of immaturity appear to have a profound influence on myocardial cation accumulation during ischemia and reperfusion. Pridjian and associates'? have shown that newborn rabbit pups from 0 to 7 days old have a relatively high susceptibility to calcium accumulation after ischemia, whereas hearts of 14to 21-day-old rabbits are relatively resistant. Thus our experiments were performed on immature hearts still in their "calcium sensitive" phase of development, and Baker's experiments were done on older hearts that were no longer as susceptible to calcium overload. This difference in age may account for our observed beneficial effects of the St. Thomas' Hospital and Hopital Lariboisiere solutions, both of which are "anticalcium" in design, by virtue of their low-calcium high-magnesium composition. Our experiments also differed from the clinical situation because we studied normal neonatal myocardium, as have most others. However, neonatal cardiac operations are usually performed to repair a defect that has resulted in hypertrophied myocardium which also may have been exposed to chronic hypoxia. Thus caution must be exercised in extrapolating our results obtained in normal myocardium to hypertrophied or hypoxic myocardium. In summary, our results indicate that multidose cardioplegia with both the St. Thomas' Hospital solution No. 2 and the Hopital Lariboisiere solution confers excellent protection during 2 hours of hypothermic ischemic arrest in neonatal rabbit hearts. Both of these solutions provided protection superior to that of hypothermia alone or cardioplegia with a glucose-high potassium solution. We are grateful for the helpful advice of Dr. Joanne S. Ingwall, Dr. John Mayer, Dr. Paul Hickey, William N. Grice, and
for the expert manuscript preparation and editorial assistance of Maryanne Mills. REFERENCES 1. Bull C, Cooper J, Stark J. Cardioplegic protection of the child's heart. J THORAC CARDIOVASC SURG 1984;88:28793. 2. Grice WN, Konishi T, Apstein CS. Resistance of neonatal myocardium to injury during normothermic and hypothermic ischemic arrest and reperfusion. Circulation I987;76('Pt 2):VI50-5. 3. Nishioka K, Jarmakani JM. Effect of ischemia on mechanical function and high-energy phosphates in rabbit myocardium. Am J Physiol 1982;242:H 1077-83. 4. Coles JG, Watanabe T; Wilson GJ, et al. Age-related differences in the response to myocardial ischemic stress. J THORAC CARDIOVASC SUR9 1987;94:526-34. 5. Yano Y, Braimbridge MV, Hearse DJ. Protection of the pediatric myocardium. J THORAC CARDIOVASC SURG 1987;94:887-96. 6. Baker JE, Boerboom LE, Olinger G N. Age-related changes in the ability of hypothermia and cardioplegia to protect ischemic rabbit myocardium. J THORAC CARDIOVASC SURG 1988;96:717-24. 7. Bove EL, Stammers AH. Recovery of left ventricular function after hypothermic global ischemia: age-related differences in the isolated working rabbit heart. J THoRAe CARDIOVASC SURG 1986;91:115-22. 8. Parrish M, Payne A, Fixler DE. Global myocardial ischemia in the newborn, juvenile, and adult isolated isovolumic rabbit heart. Circ Res 1987;61:609-15. 9. IsoyamaS, Apstein CS, Wexler LF, Grice WN, Lorell BH. Acute decrease in left ventricular diastolic chamber distensibility during simulated angina in isolated hearts. Circ Res 1987;61:925-33. 10. Hearse DJ, Braimbridge MV, Jynge P. Protection of the ischemic myocardium: Cardioplegia. New York: Raven Press, 1981:347. 11. Menasche P, Grousset C, Apstein CS, Marotte F, Mouas C, Piwnica A. Increased injury of hypertrophied myocardium with ischemic arrest: preservation with hypothermia and cardioplegia. Am Heart J 1985;110:1204-9. 12. Snedecor G, Cochran W. Statistical methods. 7th ed. Ames, Iowa: Iowa State University Press, 1980:233-7. 13. Nassar R, Reedy MC, Anderson PAW. Developmental changes in the ultrastructure and sarcomere shortening of the isolated rabbit ventricular myocyte. Circ Res 1987;611:465-83. 14. Downing SE, Talner NS, Gardner TH. Ventricular function in the newborn lamb. Am J Physiol 1965;208: 931-7. 15. Friedman WF. The intrinsic physiologic properties of the developing heart. Prog Cardiovasc Dis 1972;15:87-110. 16. Como AF, Bethencourt OM, Laks H, et al. Myocardial protection in the neonatal heart: a comparison of topical hypothermia and crystalloid and blood cardioplegic solutions. J THORAC CARDIOVASC SURG 1987;93:163-72. 17. Hearse DJ, Braimbridge MV, Jynge P. Protection of the
Volume 98 Number 6 December 1989
ischemic myocardium: Cardioplegia. New York: Raven Press, 1981:231-5. 18. Hearse OJ, Stewart A, Braimbridge MY. Myocardial protection during ischemic cardiac arrest: possible deleterious effects of glucose and mannitol in coronary infusates. J THORAC CARDIOVASC SURG 1978;76:16-23. 19. Apstein CS, Gravino FN, Haudenschild Cc. Determinants of a protective effect of glucose and insulin on the ischemic myocardium. Circ Res 1983;52:515-26. 20. Hearse OJ, Braimbridge MY, Jynge P. Protection of the ischemic myocardium: Cardioplegia. New York: Raven Press, 1981:219-24.
Comparison of cardioplegic solutions
I I 37
21. Bove EL, Stammers AH, Gallagher KP. Protection of the neonatal myocardium during hypothermic ischemia: effect of cardioplegia on left ventricular function in the rabbit. J THORAC CARDIOVASC SURG 1987;94:115-23. 22. Pridjian AK, Levitsky S, Krukenkamp I, Silverman NA, Feinberg H. Developmental changes in reperfusion injury: a comparison of intracellular cation accumulation in the newborn, neonatal, and adult heart. J THORAC CARDlOVASC SURG 1987;93:428-33.
Bound volumes available to subscribers Bound volumes ofTHE JOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY are available to subscribers (only) forthe 1989 issues from the Publisher, at a costof $55.00 ($72.00 international) for Yol. 97 (January-June) and Yol. 98 (July-December). Shipping charges are included. Each bound volume contains a subject and author index and all advertising is removed. Copies are shipped within 60 days after publication of the last issue of the volume. The binding is durable buckram with the JOURNAL name, volume number, and year stamped in goldon the spine. Payment must accompany all orders. ContactThe c.y. Mosby Company, Circulation Department, 11830 Westline Industrial Drive, St. Louis, Missouri 63146-3318, USA; phone (800) 325-4177, ext. 351. Subscriptions must be in force to qualify. Bound volumes are not available in place of a regular JOURNAL subscription.
Comparison of three cardioplegic solutions during hypothermic ischemic arrest in neonatal blood-perfused rabbit hearts Inadequate myocardial preservation continues to be an important cause of postoperative morbidity and mortality after pediatric cardiac operations. To investigate methods of improving preservation in neonatal myocardium, we compared three cardioplegic solutlons with topical hypothermia during 120 minutes of ischemic arrest in isolated, blood-perfused, neonatal rabbit hearts. Topical hypothermia (150 C) without cardioplegia resulted in 71 % ± 5 % recovery of preischemic contractile function. A high potassium (30 mEqjL) cardioplegic solution resulted in it 76 % ± 6 % recovery of function, not significantly different from that obtained with hypothermia alone. In contrast, the St. Thomas' Hospital and Hopital Lariboisiere cardioplegic soluti~ resulted in recoveries of 89 % 1:- 6 % and 88% ± 7%, respectively, both of which were significantly greater (p < 0.001) than recoveries obtained with the high potassium solution or hypothermia alone. Thus the cardioplegic solutio~ used at St. Thomas' Hospital and Hopital Lariboisiere provided exceUent protection during 2 hours of hypothermic ischemic arrest jn neonatal rababit hearts and resulted in functional recovery superior to that achieved with hypothermia alone or with the high potassium cardioplegic solution.
Takashi Konishi, MD, and Carl S. Apstein, MD, Boston, Mass.
Inadequate preservation of neonatal myocardium during cardiac operations remains a significant clinical problem, especially with the advent of aggressive surgical strategies for correcting certain congenital cardiac defects soon after birth. For example, Bull, Cooper, and Stark' recently concluded that 50% of pediatric cardiac surgical deaths were attributable to inadequate myocardial preservation despite cardioplegia, especially when the ischemic period exceeded 85 minutes. Despite these obser-
From Cardiac Muscle Research Laboratory, Cardiovascular Institute, Boston University School of Medicine, and the Cardiology Section, Thorndike Memorial Laboratory, Boston City Hospital. Boston, Mass. Dr. Konishi was supported by a Research Fellowship from the Massachusetts Affiliate of the American Heart Association, No. 13-437856; this research was supported by U.S. Public Health Service Grant HL-35675. Received for publication Dec. 19, 1988. Accepted for publication March 21, 1989. Address for reprints: Carl S. Apstein, MD, Director, Cardiac Muscle Research Laboratory, Boston University School of Medicine, 80 East Concord St. (R-217), Boston, MA 02118.
12/1/13096
1 1 32
vations, neonatal myocardium had generally been reported to be more resistant to hypoxic and ischemic injury than adult myocardium.e? though some workers have reached the opposite conclusion.f We reasoned that protection of neonatal myocardium during a prolonged period of ischemic arrest may depend on the composition of the cardioplegic solution that is used. Although the composition of cardioplegic solutions has been studied extensively in adult hearts, relatively few comparative studies have been performed in neonatal hearts. Therefore, in the current study, we used an experimental protocol designed to closely simulate clinical surgical conditions and we compared three widely used cardioplegic solutions: (1) a high potassium solution, (2) St. Thomas' Hospital solution No.2, and (3) Hopital Lariboisiere solution. Groups of neonatal (4- to 6-day-old) isolated blood-perfused rabbit hearts were subjected to 120 minutes of hypothermic (150 C) ischemic arrest, either without cardioplegic protection (controls) or with one of three types of cardioplegic solutions (Table I). Methods Isolated heart preparation. We used an isolated isovolumic (balloon in the left ventricle) rabbit heart model perfused with fresh whole blood, a system that we2• 9 have recently developed
Volume 98 Number 6
Comparison of cardioplegic solutions
December 1989
I I33
Blood Line
D=~==~
Pressure Regulator
Saline
Filter Oxygenator Syringe
t
Pulmonary Artery Cannula
~~~~
Pacer
Intra-LV Balloon Apical Drain Pump
Fig. 1. Isolated blood-perfused rabbit heart preparation. As previously reported.! heparinized blood from a donor rabbit is pumped through an oxygenator and filter to the coronary arteries through a cannula in the aortic stump. Coronary perfusion pressure (CPP) is controlled by a pressure regulator, and coronary flow regulation is determined by coronary vascular autoregulation. A thin-walled latex balloon fills the left ventricular cavity and is attached to a pressure transducer (Statham P23Db) for measurement of left ventricular pressure (LVP). Left ventricular balloon volume determines left ventricular volume and is held constant throughout each experiment so that changes in left ventricular end-diastolic pressure reflect changes in left ventricular diastolic distensibility. Left ventricular thebesian drainage is vented by an apical drain, and all coronary venous efflux is collected by a cannula in the ligated pulmonary artery. Heart rate is controlled by a right ventricular pacer, and temperature is maintained at 3r C and monitored with an intraventricular temperature probe (not shown).
Table I. Components of cardioplegic solutions (mmoljL) Solutions
K+
\' + .a
Ca++
Mg++
0-
HCOj--
Glucose
Mannitol
Glutamate
Lidocaine
pH
High potassium 51. Thomas' Hospital No.2 Hopital Lariboisicre
30 16 15
84 110 100
0 1.2 0.25
0 16 16
107 160 148
7 10 0
139 0 0
0 0 68
0 0 20
0 0.9 0
7.4 7.8 7.4
(Fig. 1). In this system. heparinized blood is pumped from a venous reservoir. through an oxygenator, into a pressurized ar'terial reservoir, and through a 40 /-lm filter (SQ40, Pall Biomedical Products Corporation, Glen Cove, N.Y.) before entering an aortic cannula. Coronary venous blood collects in the venous reservoir and is recirculated through the system. A heparinized (500 units/kg) and anesthetized (sodium pentobarbital, 50 rug/kg) 2 to 3 kg male New Zealand white rabbit served as a blood donor, contributing 70 to 80 ml of fresh whole blood via the carotid artery. Gentamicin (6 mg/100 ml) was added to the blood. The hematocrit value of the blood perfusate was 29% to 33%, blood glucose was maintained between 80 and
120 mg/dl, oxygen tension was 70 to 150 mm Hg, and pH was 7.35 to 7.45. A neonatal albino New Zealand rabbit (4 to 6 days old, male or female) was heparinized (200 units) and anesthetized with sodium pentobarbital (50 mg). The thorax was rapidly opened, and the heart and proximal great vessels were excised and placed in iced saline. The aortic stump was perfused with the isolated heart system described earlier. The time from thoracotomy to initiation of coronary perfusion on the isolated heart apparatus was approximately I to 2 minutes. Coronary perfusion pressure was adjusted to 60 mm Hg and coronary flow was determined by coronary autoregulation. A coronary perfusion pressure of 60
1134
The Journal of Thoracic and Cardiovascular
Konishi and Apstein
Surgery
Table II. Ventricular function before and after hypothermic ischemic arrest End-ischemia
Pre-ischemia DP Group Topical cooling High potassium St. Thomas Hospital H6pital Lariboisicrc
N (mm Hgj
EDP
CF/G
(mm Hgj
(mlfm)
9
92 ± 13
10 ± 1
2.1 ± 0.8
9
97 ± 7
!I ± !
3.3 ± 2.0
9
99 ± 31
9±!
1.9 ± 0.6
8
91 ± 16
J1 ± I
2.5 ± 0.8
Contracture (mm
Hgt
::::)1: •
Reperfusion
DP (mm Hgi 65 ± II
%REC 71 ± 5
73 ± 10
t
:j: 76 ± 6}:j:
CF/G
WeI weigh:
tDP imm Hgi
tmlfmin,
img)
Wetfdrv
9 ± 3
2.0 ± U
282 :': 5f>
4.9 ± 0.7
II ± 2
2.8 ± 1.3
244 ± 35
4.f> ± 0.3
26 ± 8
88 ± 27
89 ± 6
+12 ::!: 4
1.8:,: 0.7
331 ± 74
5.0 ± 1.2
24 ± 6
80 ± 13
88 ± 7
11 ± 3
2.4 ± 0.8
2f>6 ± 35
4.8 ± 1.4
Values arc mean ± standard deviation. :\. ~ umber of animals: DP. developed pressure: EDP. cnd-diastolic pressure: C Fi/G. coronary 110\'. , left vcntricula r weight in grams: '7, REC. percent recovery of developed pressure: wet/dry. ratio of wet to dry weight of the left ventricle.
"n < 0.05. tp
mm Hg was maintained during the preischemic and recovery periods, since we? have previously shown that this coronary perfusion pressure results in optimal performance of the neonatal heart. Coronary perfusion pressure was monitored from a side arm of the aortic perfusion cannula connected to a pressure transducer (Statham P23Db, Spectramed Inc., Critical Care Division, Oxnard, Calif.). A polyethylene cannula was passed across the left ventricular apex to decompress the left ventricle of thebesian drainage. Another cannula was inserted into the right ventricle via the pulmonary artery for complete collection of coronary sinus effluent and to keep the right ventricle decompressed throughout the experiment. A pacing wire (7F, Cordis Corp., Miami, Fla.) was introduced into the right ventricle through the right atrium. Left ventricular pressures were measured with a collapsed latex balloon placed in the left ventricle via the left atrium. The left ventricular balloon was filled with saline to achieve a physiologic left ventricular end-diastolic pressure (7 to IS mm Hg). Left ventricular pressure was monitored through a short, rigid, fluid-filled polyethylene tubing (Intra-Medic, PE240; Clay Adams, Division of Becton Dickinson and Co., Parsippany, N J.) attached toa pressure transducer (Statham P23Db). The heart was then suspended in warm saline at 37 8 C. Bath temperature was monitored throughout the experiment and maintained by adjusting the temperature of the water circulating through the water-jacketed heart chamber. During a 15- to 20-minute preischemic period, the heart was allowed to stabilize at a temperature of 37° C, a paced heart rate of 240 beats/min, a coronary perfusion pressure of 60 mm Hg, and a left ventricular balloon volume that produced a left ventricular end-diastolic pressure of 7 to 15 mm Hg. Hearts were discarded if a left ventricular developed pressure of at least 60 mm Hg was not reached during the preischemic stabilization period; approximately 5% of experimental preparations were rejected because of this criterion. Coronary flow rate was measured by collecting timed samples of the coronary sinus effluent via the pulmonary artery cannula. All animals used in this study received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and
the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes Of Health (NIH Publication No. 80-23, revised 1978). Cardioplegic solutions. We compared three cardioplegic solutions that are currently used in major medical centers (see Table I). The high potassium solution had a potassium concentration of 30 mmol /L: it contained neither calcium nor magnesium, and the glucose content was 2Slr (139 rnmol/L). The St. Thomas' Hospital solution 1\0. 2 had a potassium concentration of 16 mrnol/L, a calcium content of 1.2 rnrnol/L, a high magnesium level, was alkalotic, and contained lidocainc.!" The Hopital Lariboisiere solution had a potassium concentration of 15 mmol/L, was hypocalcemic at 0.25 mmol/L, had a high magnesium level, mannitol, glutamate as substrate, and a pH of 7.4. 11 Experimental protocol. Four groups of hearts were compared during a 120-minute period of hypothermic ischemic arrest and 30 minute of recovery. Each group consisted of eight or nine hearts. The choice of perfusate (i.c., control or one of three types of cardioplegic solution) was alternated among groups so that the experiments among all four groups were performed concurrently. The control group was made hypothermic but received no coronary infusions. The high potassium group, St. Thomas' Hospital group, and Hopital Lariboisiere group received cardioplegic solutions as described earlier and in Table 1. All hearts underwent a 15- to 20-minute baseline prcischcmic period as described above. Hypothermic ischemic arrest was initiated in this manner: The pacemaker was turned off, the 37' C saline in the heart chamber of the perfusion apparatus was rapidly exchanged for saline at 4 0 C, the left ventricular balloon was deflated. and then coronary perfusion was stopped. In the three groups that received cardioplegic protection, the cardioplegic solution was then immediately infused into the aortic cannula at a pressure of 60 mm Hg. Myocardial perfusion by the cardioplegic solution was confirmed by visual observation of clear cardioplegic solution flow from the pulmonary artery cannula. The three cardioplegia groups received an infusion of 1.5 ml of cardioplegic solution at 4° C at the start of the ischemia period and every
Volume 98 Number 6 December 1989
20 minutesduring ischemia. The temperature of the heart was maintained at IS ° C during the ischemic period. At the end of the 120-minute ischemic period, the left ventricular balloon was filled to the same volume present during the preischemic baseline period to measure the "contracture" pressure in the arrested heart before reperfusion. Reperfusion was initiated at 60 mm Hg with arterial bloodat 26° C. The arterial perfusate and heart were gradually rewarmed to 37'" C over a 5-minute period. When myocardial temperature became 37° C, the pacemaker was turned on at a rate of 240 beats/min. Left ventricular function was monitored during 30 minutesof reperfusion. At the end of the reperfusionperiod,the heart was dried to constant weight to determine the dry/wet weight ratio. Data analysis. Data are reported as the mean ± standard deviation and were analyzed by analysis of variance followed by analysis of protected least significant difference when the analysis of variance demonstrated significant intergroup differences (i.e., F < 0.05). Significant differences for the protected least significant difference test were considered to be p < 0.05.12
Results The results are summarized in Table II. During the preischemic baseline period, all four groups had comparable ventricular function and coronary flow rates. Developed pressure was greater than 90 mm Hg in all groups, which indicated good myocardial function. At the end of the 12Q-minute ischemic period, significant contracture had occurred in all of the groups. The high potassium solution group had significantly less contracture than the other three groups, among which the extent of contracture was similar. During 30 minutes of reperfusion, the contracture that had developed during the ischemic period was completely reversed in all groups, such that left ventricular end-diastolic pressures were similar among all groups at the end of the reperfusion period. However, there were significant differences with regard to contractile performance. The St. Thomas' Hospital group and the Hopital Lariboisiere group each recovered significantly better developed pressure than either of the two other groups. There was no significant difference in recovery of developed pressure between the St. Thomas' Hospital and Hopital Lariboisiere groups (89% versus 88%), nor was there a significant difference between the high potassium cardioplegia group and the group that received no cardioplegic protection (76% versus 71%). Recovery of coronary vasomotor function was assessed indirectly by return of coronary flow values. At the end of reperfusion, coronary flow values were comparable to the preischemic values and there were no significant differences among groups. There was no significant difference in edema (wet/dry weight ratio) among the four groups at the end of the reperfusion period.
Comparison of cardioplegic solutions
1 135
Discussion Our results demonstrate that neonatal myocardial preservation during prolonged (120 minutes) hypothermic ischemic arrest was significantly improved by the St. Thomas' Hospital solution No.2 and the Hopital Lariboisiere cardioplegic solutions, but not by the high potassium glucose-containing cardioplegic solution. Our goal was to compare three cardioplegic solutions under experimental conditions that simulated those of clinical cardiac surgery. We used a blood-perfused model and relatively long hypothermic ischemic arrest period. To assess any potential protective effect of cardioplegia, we selected a relatively long arrest period to ensure that the control group would have incomplete preservation and be able to serve as a reference group against which a protective cardioplegic effect might be compared. We maintained hypothermia at 15° C during the ischemic arrest to simulate clinical conditions. Most comparative studies of different cardioplegic solutions have used adult hearts. Immature, neonatal myocardium differs from mature myocardium in terms of sarcoplasmic reticular function, ion pumps, and contractile proteins. 13-1 5 Therefore we reasoned that the immature myocardium may have unique, specific requirements regarding composition of the cardioplegic solution. Despite such a theoretical possibility, our results indicate that two cardioplegic solutions that have been shown to confer good protection against ischemia in adult myocardium (the St. Thomas' Hospital and Hopital Lariboisiere solutions) also confer excellent protection in the neonatal myocardium. Components common to these two cardioplegic solutions are a high magnesium level, low calcium content, and absence of glucose (Table I). Since the St. Thomas' Hospital and Hopital Lariboisiere solutions resulted in similar functional recovery, it is reasonable to conclude that their differences in calcium content, mannitol, glutamate, lidocaine, and pH had no significant effect on myocardial preservation in this model. This result appears to be different from that in the neonatal pig heart, where the cardioplegic calcium level significantly influenced subsequent recovery.l" The specific component responsible for the poorer recovery of the glucose-high potassium solution cannot be determined from these experiments. A potassium concentration of 30 mfiq/L may be higher than optimal.'? Infusion of a high glucose concentration before the onset of zero-flow normothermic ischemia appeared to increase tissue injuryl''; however, if adequate washout of tissue lactate is maintained, then a high level of perfusate glucose appears to reduce ischemic injury." Also, the complete absence of calcium in the high potassium solution may cause some degree of "calcium paradox" injury.I"
The Journal of
1136
Thoracic and Cardiovascular Surgery
Konishi and Apstein
Our observations may have clinical relevance but are subject to the inherent limitations of the experimental conditions. First, there may be important species differences. For example, Como and associates" reported that St. Thomas' Hospital solution No.2 did not improve the functional recovery of the neonatal pig heart relative to topical cooling without cardioplegia. However, Bove, Stammers, and Gallagher" showed that the St. Thomas' Hospital solution No.2 protected the buffer-perfused neonatal rabbit heart against ischemia at 28° C. Our results in blood-perfused hearts at 15° C are consistent with those of Bove, Stammers, and Gallagher. Our results should be compared with the recent report of Baker, Boerboom, and Olinger," in which St. Thomas' Hospital solution No.2 appeared to be harmful to buffer-perfused immature rabbit myocardium, especially when multidose cardioplegia was used. Differences between Baker's and our studies include the buffer versus blood-perfusion methodology, as well as the age of the rabbits that were used. Baker used 7- to lO-day-old rabbit pups, whereas ours were 4 to 6 days old. Relatively small differences in degree of immaturity appear to have a profound influence on myocardial cation accumulation during ischemia and reperfusion. Pridjian and associates'? have shown that newborn rabbit pups from 0 to 7 days old have a relatively high susceptibility to calcium accumulation after ischemia, whereas hearts of 14to 21-day-old rabbits are relatively resistant. Thus our experiments were performed on immature hearts still in their "calcium sensitive" phase of development, and Baker's experiments were done on older hearts that were no longer as susceptible to calcium overload. This difference in age may account for our observed beneficial effects of the St. Thomas' Hospital and Hopital Lariboisiere solutions, both of which are "anticalcium" in design, by virtue of their low-calcium high-magnesium composition. Our experiments also differed from the clinical situation because we studied normal neonatal myocardium, as have most others. However, neonatal cardiac operations are usually performed to repair a defect that has resulted in hypertrophied myocardium which also may have been exposed to chronic hypoxia. Thus caution must be exercised in extrapolating our results obtained in normal myocardium to hypertrophied or hypoxic myocardium. In summary, our results indicate that multidose cardioplegia with both the St. Thomas' Hospital solution No. 2 and the Hopital Lariboisiere solution confers excellent protection during 2 hours of hypothermic ischemic arrest in neonatal rabbit hearts. Both of these solutions provided protection superior to that of hypothermia alone or cardioplegia with a glucose-high potassium solution. We are grateful for the helpful advice of Dr. Joanne S. Ingwall, Dr. John Mayer, Dr. Paul Hickey, William N. Grice, and
for the expert manuscript preparation and editorial assistance of Maryanne Mills. REFERENCES 1. Bull C, Cooper J, Stark J. Cardioplegic protection of the child's heart. J THORAC CARDIOVASC SURG 1984;88:28793. 2. Grice WN, Konishi T, Apstein CS. Resistance of neonatal myocardium to injury during normothermic and hypothermic ischemic arrest and reperfusion. Circulation I987;76('Pt 2):VI50-5. 3. Nishioka K, Jarmakani JM. Effect of ischemia on mechanical function and high-energy phosphates in rabbit myocardium. Am J Physiol 1982;242:H 1077-83. 4. Coles JG, Watanabe T; Wilson GJ, et al. Age-related differences in the response to myocardial ischemic stress. J THORAC CARDIOVASC SUR9 1987;94:526-34. 5. Yano Y, Braimbridge MV, Hearse DJ. Protection of the pediatric myocardium. J THORAC CARDIOVASC SURG 1987;94:887-96. 6. Baker JE, Boerboom LE, Olinger G N. Age-related changes in the ability of hypothermia and cardioplegia to protect ischemic rabbit myocardium. J THORAC CARDIOVASC SURG 1988;96:717-24. 7. Bove EL, Stammers AH. Recovery of left ventricular function after hypothermic global ischemia: age-related differences in the isolated working rabbit heart. J THoRAe CARDIOVASC SURG 1986;91:115-22. 8. Parrish M, Payne A, Fixler DE. Global myocardial ischemia in the newborn, juvenile, and adult isolated isovolumic rabbit heart. Circ Res 1987;61:609-15. 9. IsoyamaS, Apstein CS, Wexler LF, Grice WN, Lorell BH. Acute decrease in left ventricular diastolic chamber distensibility during simulated angina in isolated hearts. Circ Res 1987;61:925-33. 10. Hearse DJ, Braimbridge MV, Jynge P. Protection of the ischemic myocardium: Cardioplegia. New York: Raven Press, 1981:347. 11. Menasche P, Grousset C, Apstein CS, Marotte F, Mouas C, Piwnica A. Increased injury of hypertrophied myocardium with ischemic arrest: preservation with hypothermia and cardioplegia. Am Heart J 1985;110:1204-9. 12. Snedecor G, Cochran W. Statistical methods. 7th ed. Ames, Iowa: Iowa State University Press, 1980:233-7. 13. Nassar R, Reedy MC, Anderson PAW. Developmental changes in the ultrastructure and sarcomere shortening of the isolated rabbit ventricular myocyte. Circ Res 1987;611:465-83. 14. Downing SE, Talner NS, Gardner TH. Ventricular function in the newborn lamb. Am J Physiol 1965;208: 931-7. 15. Friedman WF. The intrinsic physiologic properties of the developing heart. Prog Cardiovasc Dis 1972;15:87-110. 16. Como AF, Bethencourt OM, Laks H, et al. Myocardial protection in the neonatal heart: a comparison of topical hypothermia and crystalloid and blood cardioplegic solutions. J THORAC CARDIOVASC SURG 1987;93:163-72. 17. Hearse DJ, Braimbridge MV, Jynge P. Protection of the
Volume 98 Number 6 December 1989
ischemic myocardium: Cardioplegia. New York: Raven Press, 1981:231-5. 18. Hearse OJ, Stewart A, Braimbridge MY. Myocardial protection during ischemic cardiac arrest: possible deleterious effects of glucose and mannitol in coronary infusates. J THORAC CARDIOVASC SURG 1978;76:16-23. 19. Apstein CS, Gravino FN, Haudenschild Cc. Determinants of a protective effect of glucose and insulin on the ischemic myocardium. Circ Res 1983;52:515-26. 20. Hearse OJ, Braimbridge MY, Jynge P. Protection of the ischemic myocardium: Cardioplegia. New York: Raven Press, 1981:219-24.
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21. Bove EL, Stammers AH, Gallagher KP. Protection of the neonatal myocardium during hypothermic ischemia: effect of cardioplegia on left ventricular function in the rabbit. J THORAC CARDIOVASC SURG 1987;94:115-23. 22. Pridjian AK, Levitsky S, Krukenkamp I, Silverman NA, Feinberg H. Developmental changes in reperfusion injury: a comparison of intracellular cation accumulation in the newborn, neonatal, and adult heart. J THORAC CARDlOVASC SURG 1987;93:428-33.
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