JOURNAL
OF SURGICAL
RESEARCH
51,133-137
Neonatal
(19%)
Myocardium LESLIE J. KOHMAN,
Department
of Surgery,
SUNY Submitted
Resists Reperfusion M.D.,’
AND
Health
Science
for publication
The response of neonatal myocardium to ischemia and reperfusion was observed in an isolated working heart model using neonatal rabbits and compared to that of the adult rabbit heart. Lipid peroxidation occurring during ischemia and that occurring during reperfusion were evaluated separately. Malondialdehyde (MDA) in heart tissue was measured as an index of lipid peroxidation, and the occurrence of oxygen free radical damage was assessed by the effects of the scavengers, superoxide dismutase and catalase, on MDA production. Baseline MDA levels were similar in neonatal and adult hearts, were changed little by treatment with normoxic cardioplegia, and were elevated in both groups by treatment with hyperoxic cardioplegia. Thus, the degree of lipid peroxidation during ischemia is similar in neonatal and adult hearts. After 10 min of retrograde reperfusion subsequent to treatment with anoxic cardioplegia, the MDA content of adult hearts was significantly greater than that of similarly treated neonatal hearts. Addition of free radical scavengers to the reperfusion medium lowered the MDA content of adult hearts significantly, but not to the level of neonatal hearts. After 60 min of reperfusion subsequent to hyperoxic cardioplegia, adult hearts had higher MDA than neonates; addition of scavengers to the cardioplegia did not lower the MDA significantly in either group. Only 5 of 12 adult hearts recovered function after hyperoxic cardioplegia, while all 12 neonatal hearts recovered. Our results indicate that neonatal myocardium suffers less damage from oxygen-centered free radicals during reperfusion than does adult myocardium. 0 1991 Academic Press, Inc.
INTRODUCTION
Myocardial injury resulting from cardiac surgery has many causes, including numerous operative and patient variables. Most of these variables are not reproducible in a laboratory model. Common to all procedures, however, are the stressful periods of ischemia and reperfusion. ’ To whom reprint requests should Surgery, 750 E. Adams St., Syracuse,
be addressed NY 13210.
at Department
of
LINDA Center, June
J. VEIT, Syracuse,
Injury
B.S. New
York
13210
8, 1990
“Reperfusion injury” has been attributed in large part to the rapid reintroduction of oxygen and subsequent damage from oxygen-centered free radicals acting on already compromised myocytes [l]. Free radical damage has been demonstrated in the myocardium of neonatal animals [2] and clinically in pediatric patients undergoing correction of congenital heart defects [3]. However, the unique characteristics of neonatal myocardium in its response to oxygen damage and reperfusion injury have not been fully assessed. Oxygen free radicals exert one of their major damaging effects on lipid membranes, where the unsaturated bonds of fatty acids can undergo peroxidation. The consequences of lipid peroxidation include increased membrane permeability, decreased calcium transport in the sarcoplasmic reticulum, altered mitochondrial function, and formation of toxic metabolites which impair cardiac function [4]. These cellular membranes have different lipid composition in neonatal and adult myocardium [5]. This fact has stimulated our interest in age-related differences of lipid peroxidation in response to hyperoxic injury, and how it may be related to differences in the tolerance of neonatal and adult myocardium to various stresses. In a previous study in our laboratory, we measured malondialdehyde (MDA) in heart tissue as a marker of lipid peroxidation in neonatal and adult rabbit hearts undergoing ischemia in a laboratory model of cardiopulmonary bypass and cardioplegic arrest [6]. Adult hearts not protected by cardioplegia had a significantly higher MDA content at the end of ischemia than did neonatal hearts. When the hearts were protected with standard cardioplegia and evaluated after reperfusion, adult and neonatal hearts had equal and low MDA levels. Hyperoxygenated cardioplegia, however, was associated with a significantly elevated postreperfusion MDA content in the adult hearts but not in the neonatal hearts. These data suggest that some lipid peroxidation occurs during ischemia, and that neonatal myocardium may be less susceptible to this injury. In order to further elucidate the differences between neonatal and adult myocardium with regard to lipid peroxidation during such a stress, an experiment was designed to separate the lipid peroxidation occurring dur-
133 All
c opyrlght 0 1991 rights of reproduction
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JOURNAL
OF
SURGICAL
RESEARCH:
VOL.
51, NO.
2, AUGUST
1991
ing ischemia from that occurring during reperfusion. To investigate the relationship of lipid peroxidation to free radical damage, free radical scavengers (superoxide dismutase (SOD) and catalase) were introduced during these two periods separately. If scavengers reduced lipid peroxidation, the lipid peroxidation could be assumed to be caused by oxygen free radicals.
iao iio Time Sequence wIluteB)
MATERIAL
AND
METHODS
Experimental model. Hearts were obtained from New Zealand white rabbits. Neonates were between 7 and 10 days of age (weights ranged from 120 to 250 g) and adults were older than 5 months (2.0-3.7 kg). All animals received care according to the Guide for the Care and Use of Laboratory Animal-s (NIH Publication No. 85-23, revised 1985). Animals were anesthetized and hearts excised as previously reported [6, 71. For analysis of baseline MDA, six neonatal and four adult hearts were immediately clamp-frozen and placed in liquid nitrogen. In all other groups, consisting of six hearts each, the aorta was cannulated and perfusion of the coronary arteries established by retrograde (Langendorff) perfusion of the aortic root. The perfusion pressure was 100 cm water for adults and 55 cm water for neonates. After stabilization, the heart was converted to the working mode by perfusing through the left atrium instead of the aorta. The perfusion pressure was adjusted to maintain a physiologic left atria1 pressure of 6-10 cm H,O, monitored by a fluid-filled transducer, and held constant throughout the remainder of the experiment. The hearts ejected against a column height equivalent to the Langendorff perfusion pressure. The apparatus has been previously described [7]. Perfusates. The perfusate during the baseline period and during reperfusion was a Krebs-Henseleit buffer containing NaCI, 120 miW; NaHCO,, 25 mM; KH,PO,, 1.2 m&f; KCl, 4.7 mM; MgSO,, 1.2 mM; CaCl,, 1.5 mM; and glucose, 11.1 miV, bubbled with oxygen 95%/CO, 5%. The cardioplegia was a standard St. Thomas’ Hospital solution containing NaCl, 110 mM; KCl, 16.0 mA4; MgCl,, 16.0 mA4; CaCl,, 1.2 n-&f; and NaHCO,, 10.0 mM. The oxygen partial pressure in the cardioplegia was t5 mm Hg (anoxic) achieved by bubbling nitrogen 95%/ CO, 5% through the reservoir, 100-200 mm Hg (normoxie) achieved by equilibration with room air, or >650 mm Hg (hyperoxic) achieved by bubbling with oxygen 95%/CO, 5%. The free radical scavengers were superoxide dismutase and catalase (Sigma Scientific, St. Louis, MO), added to the cardioplegia or to the Krebs-Henseleit buffer to yield a final concentration of 150,000 units of each enzyme per liter of solution. Assessment of Lipid Peroxidation. Hearts were frozen with tongs cooled to the temperature of liquid nitrogen and assayed for MDA by the thiobarbituric acid reaction [8]. The values are expressed as nanomoles per mil-
FIG.
1.
Experimental
protocol.
ligram protein. Protein was assayed by the Lowry method [9]. Assessment of function. Hemodynamic measurements included aortic flow, coronary flow, and heart rate. Aortic pressure was measured by a fluid-filled pressure transducer on a side arm of the aortic ejection line. Left ventricular pressure was measured by a Millar ultraminiature pressure transducer (SPR-249, Millar Instruments, Inc., Houston, TX) inserted through the left atria1 line, and left atria1 pressure was confirmed by the same Millar transducer. The first derivatives of ventricular pressure during the ascending and descending phase, +dP/dt and -dP/dt, were recorded via 8805D and 8805B carrier amplifiers. The degree of hemodynamic recovery for each group of hearts was determined by postischemic values of the above parameters, expressed as percentage of preischemic values. Protocol. The experimental protocol is shown in Fig. 1. Perfused hearts underwent 10 min of retrograde (Langendorff ) perfusion (37” C) followed by conversion to the working mode, also at 37°C. After 30 min, baseline hemodynamic measurements were made. The hearts were then rendered globally ischemic for 90 min by cessation of perfusion and transfer to a hypothermic (30°C) chamber. Cardioplegia at 30°C was given via the aortic root and coronary arteries for 3 min at the start of ischemia, and for 3 min every 25 min thereafter. This temperature, rather than a cooler one, was chosen because we wished to induce measurable damage to allow demonstration of therapeutic effects, if any, from our interventions. The cardioplegia was anoxic, normoxic, or hyperoxic, as described under Perfusates above, and thus the hearts in each group were categorized as being anoxic, normoxie, or hyperoxic, respectively. Retrograde reperfusion with buffer was then carried out for 10 min at 37°C. Hearts that failed to generate pressure or flow after the retrograde reperfusion period were immediately clampfrozen for MDA analysis. Hearts that recovered were perfused for 60 min in the working mode. Hemodynamic measurements were then repeated and the hearts clampfrozen. To isolate the lipid peroxidation occurring during ischemia, hyperoxic or normoxic cardioplegia was administered to different groups of neonatal and adult hearts,
KOHMAN
.-z al ;;
a
F A
6
d z
VEIT:
NEONATAL
REPERFUSION
IO
k
E E.
AND
135
INJURY
10 ‘~~05
vs. Neonate
“N”
2.al i; ii
a
E”
6
f ‘~~05
vs.
Adult
A+FRS
5
4
E 5
4
2 2 0 N H B Neonate
B
N
H
0
FIG. 2.
MDA levels at baseline and after normoxic and hyperoxic cardioplegia. B, baseline (no intervention); N, normoxic cardioplegia, no reperfusion; H, hyperoxic cardioplegia, no reperfusion.
and MDA was evaluated at the end of the ischemic period (no reperfusion). To isolate the lipid peroxidation of reperfusion, other groups of hearts were given anoxic cardioplegia to minimize any oxygen effect during ischemia and were reperfused retrograde for 10 min with or without free radical scavengers in the reperfusion medium. A third experiment was designed to allow both functional and biochemical assessment. Hyperoxic cardioplegia during ischemia, with or without addition of free radical scavengers, was administered and the hearts were then perfused in the working mode for 60 min after initial retrograde reperfusion. All groups consisted of six hearts. Statistical analysis. Analysis of variance (ANOVA) and the Student Newman-Keuls test were used to compare measurements among groups. The results are presented as means f SEM, and P values of less than 0.05 were considered significant.
A+FRS
A
Adult
A
A+FRS
’
Adult
Neonate
FIG. 3. fusion. anoxic
MDA levels after anoxic cardioplegia and 10 min of reperA, anoxic cardioplegia, Krebs-Henseleit reperfusion; A + FRS, cardioplegia, SOD and catalase added to reperfusate.
In the hearts treated with anoxic cardioplegia, adult hearts had significantly higher MDA levels after 10 min of retrograde reperfusion than did neonatal hearts: neonate 2.18 + 0.42 nmol/mg protein rt SEM vs adult 7.76 f 1.41, P < 0.05 (Fig. 3). The addition of free radical scavengers to the reperfusate had little effect on the MDA content of neonatal hearts (reduced to 0.99 f 0.22), but the MDA content of adult hearts was significantly lowered by the addition of scavengers to the reperfusate: 4.04 +- 0.72, P < 0.05. Of the hearts given anoxic cardioplegia during the ischemic period, none in either age group recovered. The addition of free radical scavengers had no effect on function in these hearts. In the third experiment, receiving hyperoxic cardioplegia with or without free radical scavengers and performing work after reperfusion (Fig. 4), adult hearts in both groups exhibited significantly higher MDA levels
RESULTS 10
Baseline values of MDA, measured without any intervention, were similar in neonatal and adult myocardium: neonates (n = 6) 1.05 + 0.29 nmol/mg protein + SEM vs adult (n = 4) 1.21 + 0.51 (Fig. 2). When either normoxic or hyperoxic cardioplegia was infused, MDA content of neonatal hearts, measured after ischemia and without reperfusion, was not significantly different from that of adult hearts: neonate normoxie 1.21 f 0.23 (nmol/mgprotein + SEM), adult normoxie 2.77 + 0.52, neonate hyperoxic 6.18 + 2.05, and adult hyperoxic 5.62 f 1.05 (Fig. 2). Within each age group, hyperoxic cardioplegia resulted in higher MDA levels than normoxic cardioplegia, supporting the proposition that MDA actually measures an effect of oxygen damage. This elevation in MDA reached statistical significance in the neonatal hearts (Fig. 2).
8
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