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Texas Heart Institute Journal. Effect of Myocardial .... A 6-0 Prolene stitch was placed around the left an- ... the stitch that had been placed previously. The area at.
Laboratory Investigation

Effect of Myocardial Reperfusion on the Release of Nitric Oxide after Regional Ischemia An Experimental Model of Beating-Heart Surgery

Koki Nakamura, MD Off-pump coronary artery bypass surgery is increasing in popularity worldwide. HowSharif Al-Ruzzeh, PhD, FRCS ever, very little is known about the effect of regional myocardial ischemia–reperfusion Caroline Gray, PhD on nitric oxide release. Magdi Yacoub, FRCS In an animal model mimicking off-pump bypass, male Sprague-Dawley rats (350–450 Mohamed Amrani, PhD, FRCS

g) were mechanically ventilated under general anesthesia. After left lateral thoracotomy, the animals underwent occlusion of either the left anterior descending artery (for 3, 5, 7.5, 10, 12.5, 15, or 20 minutes) or the circumflex artery (for 5, 10, or 15 minutes). Twentyfour hours after reperfusion, heart tissue was stained for determination of the area at risk and the infarcted area. Blood samples obtained before ischemia, 10 minutes after reperfusion, and 24 hours after reperfusion were analyzed for plasma concentrations of nitric oxide. After occlusion of the left anterior descending artery, the size of the infarcted area increased dramatically as the duration of occlusion increased, and was significantly larger after 12.5, 15, or 20 minutes of occlusion than after 3 minutes. After occlusion of the circumflex artery, the size of the infarcted area increased steadily and was significantly larger after 15 minutes of occlusion than after 5 minutes. There was no significant correlation between the duration of coronary occlusion and the plasma concentration of nitric oxide: 10 minutes after reperfusion, this concentration was significantly lower than that before ischemia, but it was twice the baseline level 24 hours after reperfusion. We concluded that the duration of regional ischemia did not affect the plasma concentration of nitric oxide in the systemic circulation. (Tex Heart Inst J 2006;33:35-9) Key words: Animal model; coronary artery bypass, off-pump; coronary artery occlusion, temporary; myocardial ischemia, regional reversible; nitric oxide; rats, SpragueDawley; reperfusion injury From: The National Heart and Lung Institute, Imperial College of Science, Technology and Medicine, University of London, Harefield Hospital, Middlesex, United Kingdom Address for reprints: Sharif Al-Ruzzeh, PhD, FRCS, FRCSEd, Leeds General Infirmary, 18 Fielding Way, Leeds LS27 9AB, United Kingdom

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ince its revival during the past decade, off-pump coronary artery bypass surgery (OPCAB) has been increasing in popularity worldwide.1 Growing evidence regarding the hazards of using cardiopulmonary bypass (CPB) for multivessel coronary artery bypass grafting has prompted many cardiac surgeons in the United Kingdom to convert to OPCAB.2 However, very little is known about the correlation between nitric oxide (NO) release and regional ischemia–reperfusion. We and others3 - 5 have shown that endothelial dysfunction is common after ischemia–reperfusion injury and that postischemic impairment of the endothelium results from a reduction in the release of NO. Nitric oxide has numerous beneficial effects, including vasodilatory properties, an inhibitory action on platelet aggregation, and antioxidant effects.6 -12 Consequently, interventions aimed at reversing the impairment of NO synthesis could have important implications for cardiac function. However, the relationship between the ischemic period and the release of NO has not been completely ascertained, nor has the time course of NO release before and after ischemia–reperfusion. Therefore, we performed this study to investigate the effect of temporary coronary artery occlusion on the plasma concentration of NO and its changes over time.

Materials and Methods

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Animals © 2006 by the Texas Heart ® Institute, Houston

Texas Heart Institute Journal

Male Sprague-Dawley rats (350–450 g) were used in all experiments. In all studies, animals received humane care in compliance with the “Principles of Laboratory Effect of Myocardial Reperfusion on Nitric Oxide Release

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Animal Care” formulated by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH publication 8523, revised 1996). Coronary Artery Occlusion

Rats were anesthetized with 50 mg/kg of sodium pentobarbital administered intraperitoneally. Rats underwent oral intubation with an 18-G soft venous cannula and artificial ventilation with room air. During the experiments, the room temperature was maintained at 23 to 25 °C. In addition, a heating pad was placed beneath the animals to maintain a body temperature of 36.0 to 37.0 °C as determined by rectal temperature probe. A left lateral thoracotomy was performed at the level of the 4th or 5th intercostal space. The pericardium was opened, and the heart was exposed. A 6-0 Prolene stitch was placed around the left anterior descending coronary artery (LAD), 2 mm distal to the left atrial appendage and around the circumf lex artery (Cx), beneath the left atrial appendage along the atrioventricular groove. A short length of polyethylene tubing was placed over the suture as an occluder. The suture was tightened and clamped to produce coronary occlusion and was released to produce reperfusion. The LAD was occluded for 3, 5, 7.5, 10, 12.5, 15, or 20 minutes; the Cx was occluded for 5, 10, or 15 minutes. Reperfusion followed. Approximately 10 minutes later, the chest was closed, and the rats were allowed to awake spontaneously (1– 2 hours later). Control rats underwent thoracotomy followed by closure of the chest without occlusion of a coronary artery. Myocardial Tissue Analysis

Twenty-four hours after reperfusion, the rats were again anesthetized in the manner described above and were intubated via tracheostomy. The chest was opened while the animals were artificially ventilated with room air. The coronary artery was re-occluded with the stitch that had been placed previously. The area at risk (AR) was stained with Evans blue dye (2 mL of 2% w/v) injected via the femoral vein. (The properties of Evans blue dye are such that the dye solution stains the perfused myocardium, but the occluded nonperfused myocardium remains uncolored.) The heart was excised, the atrial and right ventricular walls were removed, and the left ventricle (LV) was cut into 4 or 5 horizontal slices. The AR, which included infarcted and ischemic myocardium, was separated from the nonischemic myocardium by following the line of demarcation between the bluestained and the unstained (pink-to-red) tissue. So that ischemic and infarcted tissue could be differentiated, 36

Effect of Myocardial Reperfusion on Nitric Oxide Release

the AR was incubated in 0.1% nitro-blue tetrazolium (NBT) in a phosphate buffer (pH 7.4) at 37 °C for 15 minutes. (The NBT dye forms a blue formazan complex in the presence of coenzymes and dehydrogenases.) The infarcted myocardium was then separated from the ischemic myocardium that did not reach the degree of necrosis. The 3 portions of the LV myocardium (nonischemic, ischemic, and infarcted) were weighed: AR was the sum of the ischemic and infarcted areas (IF). The following percentages were calculated and the results analyzed: the portion of the left ventricle that was ischemic or infarcted (AR/LV), and the portion of AR that was infarcted (IF/AR). Analysis of the Plasma Concentrations of Nitric Oxide

The plasma concentrations of NO were analyzed using 0.6-mL blood samples collected before thoracotomy and ischemia, 10 minutes after reperfusion, and 24 hours after reperfusion. The samples were centrifuged, and the plasma was stored at –70 °C until the analyses were performed. Total NO production was determined with chemiluminescence by an NO analyzer (NOA 270, Sievers Instruments, Inc.; Boulder, Colo) to assay the amount of nitrite (the NO breakdown product) that was present, as previously described.3 Levels of NO were expressed in µmol/L (µM). Nitrite is measured as an index of total NO production, because NO2– is the principal oxidation product in an aqueous solution devoid of any biological contaminants. Statistical Analysis

All values are expressed as mean ± SEM. The 2 groups (LAD and Cx occlusion) were compared by using Student’s t-test after confirmation that the data were normally distributed. Paired t-tests were used for timecourse studies of NO levels. The relationship between the plasma NO level and the duration of coronary artery occlusion was analyzed by use of Spearman’s rank correlation test. For multiple comparisons, 1-way analysis of variance (ANOVA) was performed, followed by Fisher’s post hoc test to determine which relationships were statistically significant. A P-value of less than 0.05 was considered statistically significant.

Results Sixty-nine rats were used for the study: 40 in the LAD group, 25 in the Cx group, and 4 as a control group (no occlusion). Forty-six rats (27 in the LAD group, 16 in the Cx group, and 3 in the control group) survived for 24 hours after reperfusion. The survival rate for the LAD group was 67.5%, and that for the Cx group was 64.0% (difference not statistically significant). Among the surviving animals, staining failed in Volume 33, Number 1, 2006

6 rats, and occlusion was not properly performed for 3 rats (as confirmed by staining). Consequently, the final analysis included 37 rats: 23 in the LAD group, 11 in the Cx group, and 3 in the control group. Myocardial Ischemia Induced by Coronary Artery Occlusion

The AR weighed significantly more in the LAD group (0.305 ± 0.014 g) than in the Cx group (0.206 ± 0.026 g; P