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Ann Thorac Surg 1997;64:1354-1359
© 1997 The Society of Thoracic Surgeons
Division of Cardiac Surgery, Department of Cardiology, Department of Pharmacology, and Department of Anesthesiology, Catholic University, Rome, Italy
Accepted for publication May 30, 1997.
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Abstract |
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Methods. Three cardioplegic technique were tested in porcine hearts arrested for 32 to 36 minutes and reperfused for 30 minutes: IAWBC, antegrade cold blood cardioplegia (ACBC), and antegrade cold crystalloid cardioplegia (ACCC). In the hearts arrested with IAWBC, three different intervals of ischemia were used: three 10-minute intervals (IAWBC1), two 15-minute intervals (IAWBC2), and one 30-minute interval (IAWBC3). Rings from the coronary arteries were used to evaluate in vitro the contractile responses to U46619 and the relaxant responses to bradykinin, A23187, and sodium nitroprusside.
Results. All six groups (treatment groups and control group) displayed similar responses to U46619 (30 nmol/L) and nitroprusside. In the IAWBC1, IAWBC2, and ACBC groups, endothelium-dependent relaxations to bradykinin and A23187 were preserved compared with controls, whereas those of the ACCC and IAWBC3 groups were significantly impaired (bradykinin: control, 8.72 ± 0.07; IAWBC1, 8.73 ± 0.03; IAWBC2, 8.65 ± 0.05; IAWBC3, 8.30 ± 0.07 [p < 0.05]; ACBC, 8.50 ± 0.03; ACCC, 8.25 ± 0.09 [p < 0.05]; A23187: control, 7.07 ± 0.08; IAWBC1, 7.07 ± 0.06; IAWBC2, 7.04 ± 0.03; IAWBC3, 6.64 ± 0.01 [p < 0.05]; ACBC, 6.80 ± 0.05; ACCC, 6.60 ± 0.08 [p < 0.05]; nitroprusside: control, 6.19 ± 0.1; IAWBC1, 6.19 ± 0.07; IAWBC2, 6.03 ± 0.03; IAWBC3, 6.08 ± 0.05; ACBC, 6.04 ± 0.2; ACCC, 6.05 ± 0.03; all values are expressed as the negative logarithm of the concentration producing 50% of the maximal response).
Conclusions. Myocardial preservation with IAWBC with ischemic intervals of 15 minutes or shorter does not alter the endothelium-dependent relaxation to bradykinin or A23187 in porcine coronary arteries, but these responses are significantly impaired by ACCC and IAWBC with an ischemic interval of 30 minutes.
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Introduction |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentReferences |
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As initially proposed [2], warm blood cardioplegia involves continuous antegrade and retrograde infusion. In clinical settings, however, the infusion is rarely continuous because interruptions are necessary for construction of distal anastomoses. The heart is thus exposed to normothermic ischemia, prolonged periods of which have been shown to cause endothelial cell dysfunction in experimental models [3]. For this reason, warm blood cardioplegia has not gained wide acceptance: in the national survey [1] just mentioned, only 10% of respondents used this technique on a routine basis. The more recently introduced technique of intermittent antegrade warm blood cardioplegia (IAWBC) [4] is, to some extent, a recognition of the noncontinuous nature of warm blood cardioplegic techniques already being used. It provides a specific protocol based on consecutive intervals of normothermic ischemia, each lasting a maximum of 10 minutes, an interval that is sometimes difficult to respect.
The present study was designed to assess the effects of progressively longer ischemic intervals during infusion of IAWBC on the endothelium-dependent vasodilation and to compare the effects of this technique with those of the conventional antegrade cold crystalloid cardioplegia (ACCC) and antegrade cold blood cardioplegia (ACBC) using an animal model simulating the clinical conditions.
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Material and Methods |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentReferences |
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After induction of anesthesia with ketamine hydrochloride (20 mg/kg) and sodium pentobarbital (10 mg/kg given intravenously), female Landrace pigs weighing 25 to 28 kg were intubated and ventilated with 100% oxygen. Pancuronium bromide was used at regular intervals to maintain muscle relaxation. The femoral artery was exposed and cannulated to monitor systemic arterial pressure, and a median sternotomy was performed.
Study Groups
CONTROL GROUP.
The caval veins were clamped, and after a few beats to empty the heart, the aorta was clamped. The heart was rapidly excised and submerged in a cold (4°C), oxygenated Krebs solution containing the following (in millimoles per liter): NaCl, 118.5; KCl, 4.8; MgSO4, 1.2; KH2PO4, 1.2; CaCl2, 1.9; NaHCO3, 25.0; and glucose, 10.1. The same solution was injected gently into the coronary ostia. The right coronary artery was then dissected and maintained in the cold, oxygenated Krebs solution prior to the in vitro studies described later.
ACCC GROUP.
After sternotomy and full heparinization, the animals were placed on conventional cardiopulmonary bypass, which was monitored and conducted with standard technique maintaining an aortic pressure of 50 to 80 mm Hg throughout the bypass procedure. The mean hematocrit during bypass was 26%.
After the condition of the animal had stabilized, the aorta was cross-clamped, and 500 mL of cold (4°C) St. Thomas' II cardioplegic solution was infused into the aortic root at a controlled pressure of 70 mm Hg. Ice slush was placed inside the pericardium and over the entire heart. The animal was cooled to a temperature of 28°C. Ten minutes before the aortic clamp was removed, the animal was rewarmed. The total period of cross-clamping was 36 minutes (Fig 1
). After the clamp was released, the heart was defibrillated if necessary, and the animal was left on the pump (to avoid pressure variations that might affect endothelial function in the coronary arteries) [5] for a 30-minute reperfusion period at 37°C. At the end of reperfusion, the animal was weaned from cardiopulmonary bypass, and the heart was removed and stored as described for the control group.
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IAWBC1 GROUP.
Bypass was initiated as already described, but the body temperature was maintained at 37°C throughout the procedure. Table 1 shows the IAWBC protocol (see Fig 1
). After the aorta had been cross-clamped, IAWBC was infused into the ascending aorta for 2 minutes at a controlled pressure of 70 mm Hg, after which the infusion was interrupted for 10 minutes. This procedure was repeated three times, so that the heart was subjected to a total of 30 minutes of ischemia and 6 minutes of cardioplegia infusion. At the end of the third ischemic interval, the aortic clamp was removed and the animal reperfused as in the previous two groups.
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IAWBC3 GROUP.
The procedure was identical to that of the IAWBC1 group except the duration of ischemia was prolonged to a single period of 30 minutes. The total cross-clamp time was 32 minutes (see Fig 1).
In Vitro Studies
Three rings 3 mm in length were obtained from the right coronary artery of each animal. The rings were placed in 5-mL organ baths containing the Krebs solution already described. All the experiments were performed in the presence of fenoprofen (20 µmol/L) to prevent synthesis of endogenous prostanoids. The bath solution was maintained at 37°C and bubbled with a mixture of 95% oxygen and 5% carbon dioxide; under these conditions, the solution pH was 7.4. The segments were suspended in the organ baths and connected to an isotonic force transducer (Harvard or Ugo Basile Biological Research Apparatus model 7006). In preliminary experiments, the arterial segments were contracted with U46619 (0.1 µmol/L), which mimics the action of thromboxane A2, under increasing loads (1 to 8 g, increased in 1-g steps). Maximal contraction was achieved with a 6-g load, which was used in all subsequent experiments. The mechanical responses of the segments, magnified 200 to 400 times, were visualized on a Rikadenki R-01 or R-02 recorder.
The rings were allowed to equilibrate for 60 minutes. Cumulative concentration–response curves for U46619 (1 nmol/L to 0.3 µmol/L) were plotted for all six groups. A bath concentration that produced submaximal concentration in the control segments (30 nmol/L) was chosen to precontract segments used in studies of relaxant responses. The test drugs in these latter experiments were bradykinin (0.1 nmol/L to 0.1 µmol/L), the calcium ionophore A23187 (10 nmol/L to 3 µmol/L), and sodium nitroprusside (30 nmol/L to 30 µmol/L). The drugs were added when peak contraction had been achieved with U46619 (30 nmol/L), and cumulative concentration–response curves were plotted for each drug. In the treatment groups, only one concentration–response curve was obtained from each ring to eliminate any effects of time (eg, possible recovery from damage inflicted by the treatment) on the relaxant responses.
The following drugs were used: bradykinin acetate salt; the calcium ionophore A23187; 9,11-dideoxy-11
-9-epoxy-methanoprostaglandin F2 (U46619) (Sigma, St. Louis, MO); fenoprofen sodium salt dihydrate (Eli Lilly and Co, Indianapolis, IN); and sodium nitroprusside (Malesci, Florence, Italy). U46619 and A23187 were dissolved in methanol (concentration, 1 mmol/L) and dimethyl sulfoxide (concentration, 3 mmol/L), respectively. All other drugs were dissolved in bidistilled water. Stock solutions were stored at -30°C. The drugs were diluted with Krebs solution on the day of each experiment and stored on ice until used.
Statistical Analysis
Data are reported as the group mean ± the standard error of the mean; n indicates the number of animals used for a given experiment. In cumulative concentration–response curves, contractions to U46619 were expressed as percentages of the maximum, ie, that produced by the highest concentration used (0.3 µmol/L); responses to relaxant agonists were expressed as percentages of inhibition of the precontraction induced by U46619 (30 nmol/L). The maximal U46619-induced contraction as well as contractions produced by U46619 (30 nmol/L) prior to the administration of relaxant agonists was also measured as an absolute value (ie, reduction in ring diameter in millimeters). Concentrations producing 50% of the maximal response (EC50) were calculated for individual concentration–response curves using nonlinear regression analysis; negative logarithms of the EC50 (-log EC50) are also shown.
Mean values between treatment groups were compared using one-way analysis of variance, followed by Dunnett's test for multiple comparisons with a control group whenever a significant F value emerged. Significance was set at a p value of less than 0.05.
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Results |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentReferences |
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Precontractions induced by U46619 (30 nmol/L) in experiments with the relaxant agonists were essentially similar in all six groups (control, 1.16 ± 0.04 mm; IAWBC1, 1.23 ± 0.05 mm; IAWBC2, 1.22 ± 0.06 mm; IAWBC3, 1.21 ± 0.05 mm; ACBC, 1.31 ± 0.04; ACCC, 1.22 ± 0.06 mm).
Bradykinin produced concentration-dependent inhibition of the U46619-induced contraction in all groups (Table 2). The drug displayed similar potency in the control, ACBC, IAWBC1, and IAWBC2 groups. Compared with the control group, however, the concentration–response curves for the ACCC and IAWBC3 groups were shifted significantly to the right (p < 0.05) (ACCC, -log EC50: 8.25 ± 0.09 nmol/L; IAWBC3, 8.30 ± 0.07 nmol/L) (Fig 2).
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Comment |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentReferences |
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. Like all new techniques, IAWBC has raised a number of questions regarding both its safety and its efficacy. One of the potential advantages of this technique has emerged from recent studies [6] using phosphorus 31 nuclear magnetic resonance imaging, which indicate that IAWBC can preserve high-energy phosphates in the myocardium. Nonetheless, a number of studies [7, 8] have shown that normothermic ischemia causes prolonged, but reversible endothelial dysfunction that becomes evident during the subsequent period of reperfusion. In a canine model of global ischemia [3], aortic cross-clamping for 60 minutes followed by a similar period of reperfusion reduced the relaxant responses of the artery to aggregating platelets, adenosine diphosphate, and 5-hydroxytryptamine with respect to controls. However, damage is evident only after short periods of reperfusion. In fact, Tsao and co-workers [9] showed that coronary artery segments precontracted with U46619 relaxed normally in response to acetylcholine after exposure to ischemia alone, whereas no response was observed in those subjected to postischemic reperfusion for 2.5 minutes. The resulting reduction in release of the endothelium-derived relaxing factor facilitates vasospastic responses to platelet aggregation [7] and can be responsible for the no-reflow phenomenon [10].
These findings have raised concern that excessive prolongation of the ischemic interval during infusion of normothermic blood cardioplegia might impair the endothelial response to vasoactive and thrombogenic substances in coronary arteries. The goals of the present study were to assess the effects of progressively longer ischemic intervals during infusion of IAWBC on the endothelium-dependent vasodilation and to compare the effects of this technique with those of the conventional methods—ACCC and ACBC.
In the IAWBC1, ACBC, and ACCC treatment groups, the aortas remained clamped for a total of 36 minutes. With hypothermic techniques such as ACCC and ACBC, the cardioplegic solution is generally infused in a few minutes, after which the infusion is interrupted for up to 30 consecutive minutes; the cycle can be repeated depending on the duration of the surgical procedure. In the IAWBC1 group, the 10-minute interruption, preceded by a 2-minute interval of blood cardioplegia infusion, is the interval thought to be safe during infusion of standard continuous warm blood cardioplegia as originally described by Salerno and coauthors [2]. This interval was prolonged to 15 minutes twice in the IAWBC2 group and to 30 minutes for a single period of ischemia in the IAWBC3 group (see Fig 1).
We have chosen not to exceed a period of 36 minutes of ischemia for the following reasons:
In our model, the consecutive periods of normothermic ischemia (10 minutes each and 15 minutes each) and reperfusion (2 minutes each) used in the IAWBC1 and IAWBC2 groups, respectively, had no cumulative effect on endothelial function. In contrast, significant impairment was noted in the ACCC and IAWBC3 segments, which had been exposed to hypothermic ischemia for 30 consecutive minutes.
It is interesting that although the ischemic and reperfusion periods in the ACBC group were identical to those of the ACCC and IAWBC3 groups, the first of these did not show any endothelial impairment. The absence of damage in the IAWBC1, IAWBC2, and ACBC groups is probably due to various and partially different factors. The fact that ischemic endothelial impairment has been demonstrated only after reperfusion [7, 9, 10] indicates the possible involvement of an oxidative mechanism. Therefore it is reasonable to assume that the presence of antioxidant factors in the blood solutions is at least partially responsible for the preservation of endothelial function observed in these groups. Similar protective effects have, in fact, been reported not only with blood cardioplegic solutions but also crystalloid-albumin solutions [13]. Another possible explanation is the greater buffering capacity of blood cardioplegic solutions [14] compared with that of the crystalloid cardioplegias.
In the IAWBC1 and IAWBC2 groups, the absence of endothelial damage might also be a result of the duration of the ischemic insult. It is unlikely that each single ischemic interval (maximum, 15 minutes) was short enough to allow complete recovery of the endothelial function during the following 2-minute period of infusion of blood cardioplegia. In fact, in 1992, it was reported that even brief periods of ischemia lasting 5 minutes followed by reperfusion result in sustained endothelial dysfunction [11]. More recently, Tian and co-workers [6] found that an initial 10-minute interruption of warm blood cardioplegia in a porcine model produced a 45% reduction in myocardial phosphate levels with a 0.12-unit decrease in intracellular pH. There were no further changes in either of these variables during five subsequent 10-minute consecutive ischemic periods. During reperfusion, no differences could be detected between animals subjected to intermittent or continuous warm blood cardioplegia.
This adaptive response to the first ischemic insult observed in the myocardial muscle cells is typical of the phenomenon of preconditioning [15]. Thus, a similar protective mechanism can explain the preserved endothelial responses in the IAWBC1 and IAWBC2 groups. This protective mechanism is probably overcome by the duration of the ischemic interval in the IAWBC3 group. These results are consistent with the metabolic studies of Ikonomidis and associates [16]. These researchers could not detect any change in the production of lactate or in the consumption of oxygen with the use of IAWBC and intervals of 15 minutes.
Other possible causes of endothelial damage related to cardioplegic solutions are the potassium concentrations and hypothermia. Potassium, which is a standard component of cardioplegic solutions, has been shown to cause transient endothelial dysfunction when infused at high concentrations [17, 18]. It is unlikely that potassium was responsible for the sustained impairment of endothelial function observed in the ACCC group. First, normal responses to bradykinin and A23187 were observed in the other two groups, which were exposed to identical potassium ion concentrations (20 mEq/L). Moreover, the potassium-induced endothelial damage observed in one of the studies cited [17] occurred with concentrations of 25 mEq/L or more. The more recent study [18] demonstrated that potassium concentrations of 20 mEq/L produce transient impairment of relaxation mediated by endothelium-derived hyperpolarizing factor. Kilpatrick and Cocks [19], however, found that the endothelium-derived hyperpolarizing factor–mediated component of porcine coronary artery relaxant responses to bradykinin or A23187 is significant only when nitric oxide synthesis is blocked, as was true in the study of He and Yang [18]. Under normal conditions, the relaxant response to these agonists is almost totally mediated by nitric oxide, and this relaxation was not affected at all by exposure to potassium ion concentrations ranging from 25 to 75 mEq/L.
Hypothermia is considered by some authors to be another possible source of endothelial damage. This is a controversial issue [20, 21], however, as it has been demonstrated that endothelium-dependent relaxation in rat infrarenal aortic transplants is best preserved at storage temperatures of 4° to 8°C [22]. In any case, the absence of damage in the ACBC group tends to exclude a role for hypothermia in the impairment noted in the ACCC segments because the temperatures used in these two groups were identical.
Results similar to ours have been reported by Murphy and colleagues [23], who found that the endothelial responses of left ventricular coronary microvessels were not impaired in dogs subjected to continuous antegrade warm blood cardioplegia, which is difficult to use in clinical settings.
The present study does not address the possible myocardial alterations caused by the different cardioplegic solutions used because it is clear from the literature that the endothelial damage precedes much of the myocardial alteration [9, 11].
As a last consideration, the available ischemic interval of 15 minutes might appear relatively short for the construction of delicate anastomoses in the case of complex arterial revascularizations. In a group of patients undergoing operation with the use of IAWBC, Isomura and colleagues [24] reported the implantation of at least two arterial conduits in 45% of patients with a mean ischemic interval of 13.9 minutes.
In summary, IAWBC is a new technique that simplifies the complex delivery of the blood cardioplegic solution required by the continuous retrograde technique provided the coronary perfusion is interrupted for periods not longer than 15 minutes.
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Footnotes |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentReferences |
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References |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentReferences |
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This article has been cited by other articles:
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  Q. Yang and G.-W. He Effect of Cardioplegic and Organ Preservation Solutions and Their Components on Coronary Endothelium-Derived Relaxing Factors Ann. Thorac. Surg., August 1, 2005; 80(2): 757 - 767. [Abstract] [Full Text] [PDF]
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