Off-pump coronary artery bypass surgery is increasing in popularity worldwide. However, very little is known about the effect of regional myocardial ischemia-reperfusion on nitric oxide release.

In an animal model mimicking off-pump bypass, male Sprague-Dawley rats (350 --450 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). Twenty-four 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.

Key words:: Animal model, coronary artery bypass, off-pump, coronary artery occlusion, temporary, myocardial ischemia, regional reversible, nitric oxide, rats, Sprague-Dawley, reperfusion injury

Since 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 others^{3 --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.

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 circumflex 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.

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 blue-stained and the unstained (pink-to-red) tissue. So that ischemic and infarcted tissue could be differentiated, the AR was incubated in 0.1% nitro-blue tetrazolium (NBT) in a phosphate buffer (pH 7.4) at 37[deg]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).

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 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.

We found no significant correlation between the duration of coronary artery occlusion and the plasma concentration of NO. In the time-course study, the plasma NO had decreased significantly at the beginning of reperfusion but was twice the baseline level 24 hours after reperfusion.

Endothelium-derived NO not only modulates the tone of the underlying vascular smooth muscle but also inhibits several proatherogenic processes, including smooth muscle proliferation and migration, platelet aggregation, oxidation of low-density lipoproteins, monocyte and platelet adhesion, and synthesis of inflammatory cytokines.¹² Consequently, interventions aimed at reversing impaired NO synthesis could have important implications for cardiac function.

In the present study, reperfusion affected NO production independent of the duration of coronary occlusion. Many studies have shown that an important feature of ischemia --reperfusion injury is the postischemic impairment of the endothelium, which is partially due to a reduction in NO release.^{3 --5} Indeed, our study also showed that beginning the reperfusion process decreased the plasma concentration of NO. The decrease in NO production at 10 minutes after reperfusion was likely due to the endothelial dysfunction caused by reperfusion.

A few reports have shown that the release of NO increases because of the inflammatory response associated with the use of CPB in cardiac surgery.^{13 --15} In those circumstances, the production of such a large amount of NO might be inhibited by treating CPB patients with glucocorticoids, aprotinin, and hemofiltration.¹³ On the other hand, in human beings it has also been shown that, even during CPB, NO production in the coronary sinus decreases significantly after grafting with an internal thoracic artery.¹⁶

L-arginine, the physiologic substrate of NO, reverses low coronary reflow and improves postischemic recovery of cardiac mechanical function.³ Therefore, supplementation with L-arginine may be effective in increasing NO production after coronary surgery ---particularly if OPCAB is used (avoiding CPB).

The increase in the NO concentrations detected 24 hours after reperfusion in our study may have been related to the systemic inflammatory reaction induced by the surgical procedures, or caused by an alternative mechanism that counteracted the reduction in NO concentration. Ferreiro and colleagues¹⁷ reported that hypoxia down-regulated endothelial nitric oxide synthase (NOS) activity in the cardiac tissue of cyanotic children; in contrast, inducible NOS activity was up-regulated as a counteraction.

We speculate that, in our study, the plasma NO concentration was independent of the degree of ischemia because of the change in NO release, which may have been diluted in the systemic circulation. One study limitation was that the blood samples were obtained from the femoral vein. Obtaining samples from the coronary sinus would have better reflected the changes in NO release in the heart. However, accessing the coronary sinus in rats is technically difficult, and we considered that the change in NO concentrations in the systemic circulation would indirectly reflect such changes in the heart. Future studies in larger animals may allow collection of blood samples from the coronary sinus so that minor changes in NO concentrations in the heart can be investigated. Another limitation of this study was that the rat heart model involved healthy coronary arteries. Although this limitation would make any extrapolation difficult, we believe that this model could assist in the investigation of several aspects of regional ischemia in OPCAB.

In conclusion, the duration of regional ischemia did not affect the plasma concentration of NO in the systemic circulation. This concentration decreased at the beginning of reperfusion but was twice as high as baseline 24 hours after reperfusion.