Recently, it has been shown that early reperfusion after acute myocardial infarction improves survival rate and preserves left ventricular function. However, the extent of the improvement in left ventricular function has been found to be less than expected, particularly in late reperfusion. Reperfusion in itself may induce myocardial tissue damage in terms of reperfusion injury. Although the underlying mechanisms are not well known, several may be involved in this phenomenon. In these mechanisms, oxygen free radical generation has been proposed to be a major mechanism in the pathogenesis of reperfusion injury.1-3
Thus, a free radical scavenger has been developed to interfere with the generation of free radicals and to salvage jeopardized myocardium in the reperfusion.4,5 Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) has potent effects as a free radical scavenger and helps provide protection from brain damage in patients with acute cerebral infarction.6,7
Research over the past decade has analyzed the characteristics of left ventricle contraction using the LV end-systolic pressure-volume relationship (ESPVR), which has recently been recognized as useful for studying hemodynamics of the heart. The end-systolic volume elastance (Ees), which is the slope of the ESPVR, is relatively independent of the preload and afterload and is a reliable index of LV systolic function (contractility) within the physiologically normal range of LV pressures.8-11 This index may be suitable for judgment of the recovery of left ventricular function after reperfusion. The difference in this index between controls and those under treatment could serve as a measure of the quantity of protection from reperfusion injury.
The aim of the current study was to evaluate whether edaravone acting as a free radical scavenger works against reperfusion injury in an acute myocardial ischemia-reperfusion rat model.
All experimental procedures and protocols used in the study conformed to the guiding principles of Dokkyo University School of Medicine with regard to the NIH guidelines for the care and use of laboratory animals published by the US National Institutes of Health (NIH publication No 85-23, revised 1985).
Male Sprague-Dawley rats (Oriental Bioservice Kanto Inc, Ibaraki, Japan) 7 weeks of age weighing 200-300 g were given ad libitum access to standard rat chow and water. Under anesthesia with an intraperitoneal injection of sodium pentobarbital (20 mg/kg), the trachea was intubated with PE-240 tubing connected to a small animal ventilator (Harvard respirator) to permit controlled ventilation. A midsternal incision was made, and the positive end-expiratory pressure was held constant by submerging the outflow tube tip of the respirator in water at a depth of 10 mm to prevent pulmonary atelectasis. The chest was opened, and coronary occlusion was achieved by ligating the proximal left main coronary artery, confirmed by regional cyanosis of the myocardial surface. Edaravone (EDA, 3 mg/kg, n = 11) and saline (CON, n = 18) were administered intravenously from 2 to 3 minutes after coronary artery ligation. The dose was determined according to the previously demonstrated effects in the rat ischemic brain and the hindlimb amputation models.12,13 Five minutes after coronary occlusion, the exteriorized end of the ligature was pulled free, and the ischemic myocardium was reperfused. Contractility (Ees), LVEDP, LVSP, and HR were measured by pressure-volume relationships at every 5 minutes before ligation to 25 minutes after reperfusion (Fig. 1).
Contractility (End-Systolic Elastance, Ees)
The LV end-systolic pressure-volume relationship (ESPVR) was determined by the previously reported conductance catheter technique.14-16 Briefly, the conductance catheter was inserted into the left ventricle through the apex and was pushed until the distal tip was withdrawn to create the normal loop, and segmental volume was measured by each sensing electrode. Then, the total conductance volume was calculated by VPR 1002 (Unique Medical, Tokyo, Japan). Another 2.5-Fr catheter-tip micromanometer (SPR-524, Millar Instruments, Houston, TX) was inserted into the left ventricle through the apex to permit continuous measurement of LV pressure. To change the preload, a snare was placed around the inferior vena cava. We recorded the conductance volume and LV pressure simultaneously during gradual inferior vena cava occlusion. Electrical signals were digitized through an analog-to-digital converter (AD12-8, Contec, Tokyo, Japan) at a sampling frequency of 1000 Hz with 12-bit resolution and stored on a personal computer (Dynabook SS 330, Toshiba, Tokyo, Japan). The points of the ESPVR were determined by a previously reported iterative technique.14,15,17 In several consecutive pressure-volume loops, the points of each cardiac cycle with maximum pressure-to-volume ratio were first determined. Linear regression of these points with the expression
yielded estimates for the slope, or the end-systolic elastance (Ees), and the volume-axis intercept (V0), where Ped and Ved are the end-systolic pressure and volume, respectively. This initial estimate for V0 was used to determine the points of maximal P/(V − V0) for each cardiac cycle, and these new points were again fitted by linear regression, leading to new estimates for Ees and V0. These procedures were repeated using Integra 3 software (Unique Medical, Tokyo, Japan) until convergence (2%) was achieved. All data were obtained during a 10-second period when the respirator was turned off in the expiration phase to avoid respiratory drift. Finally, a parallel conductance volume was measured by the hypertonic saline-dilution method in which 0.02 mL of saturated NaCl solution was injected into the pulmonary artery. The conductance catheter was placed into the left ventricle in situ. The conductance volume measured by the conductance catheter was actually the sum of the LV blood conductance volume and the parallel conductance volume resulting from other structures extrinsic to the LV blood volume. Therefore, the true LV conductance volume was predicted by subtracting the parallel conductance volume from the actual measured conductance volume.
After the experiment, blood samples were obtained from the jugular vein, and the ischemic area was measured using perfusion of black dye from the jugular vein. Hearts were immediately excised, and the left ventricle carefully separated from the atria and the right ventricle; then ischemic and nonischemic areas were weighed. The percentage of the ischemic area was calculated by the ischemic area weight/total LV weight.
Measurement of Blood Malondialdehyde Levels
Lipid peroxidation estimated the levels of malondialdehyde (MDA) precursors, including hydroperoxide, epiperoxide, polyperoxide, and endoperoxide. Plasma was isolated from fresh blood samples by centrifugation at 3000 rpm for 10 minutes at 4°C. Then 1/12N H2SO4O and 10% phosphotungstic acid were added to the plasma, and the sample was vortexed and centrifuged at 3000 rpm for 10 minutes at 4°C. The organic layer was separated and suspended in distilled water. Thiobarbituric acid was added to the suspension. The sample was heated in a 100°C water bath for 1 hour after vortexing for 10 seconds. The reaction was stopped by cooling the product on ice for 5 minutes. The n-butanol was added, and the TBA-MDA product was extracted after mixing for 20 seconds. The organic layer was separated from the aqueous layer via centrifugation at 2000 rpm for 10 minutes at 4°C. The synchronous fluorescence spectrum of the supernatant was measured (Hitachi F-2500, Tokyo, Japan). The net fluorescent intensity was determined at 553 nm and quantified by comparison with a standard curve prepared with a standardized sample. The value was expressed as nanomoles MDA per liter in plasma.
All data are expressed as the mean ± SD. Because all continuous data were normally distributed, unpaired Student t test was used to determine the statistical significance of differences. Discrete variables were compared with the χ2 for independence test. Statistical significance was accepted at P < 0.05 level.
Blood Pressure, Heart Rate, Area at Risk Size
Table 1 shows that systolic left ventricular pressure and heart rate at all measuring points did not differ between EDA and CON rats. Figure 2 shows that the ischemic area ratio between EDA and CON rats also did not differ. However, end-diastolic pressure was significantly lower at 5, 15, and 20 minutes after reperfusion in EDA compared with CON rats (Fig. 3, P < 0.01, respectively).
Five of the 18 CON rats died with lethal reperfusion ventricular tachyarrhythmia confirmed by ECG signals. In contrast, none of the EDA-treated rats died even when reperfusion ventricular tachyarrhythmia was observed. Thus, the survival ratio was 100% in EDA-treated rats and 72% in CON rats 25 minutes after reperfusion, showing that the survival rate improved in the EDA-treated rats compared with CON rats (Table 2, P < 0.05).
Contractility (End-Systolic Elastance, Ees)
The pressure-volume loops recorded in an EDA-treated rat and a CON rat are shown in Figure 4. Ees in EDA treated and CON rats just before and 25 minutes after reperfusion are shown Figure 5. Ees were not different before coronary ligation between EDA-treated and CON rats. In contrast, Ees were significantly greater from reperfusion to 25 minutes afterward in EDA-treated rats compared with CON rats (P < 0.01, respectively). These data suggested that with EDA treatment just before reperfusion, contractility of the left ventricle was preserved after ischemic reperfusion. Furthermore, the degree of improvement of Ees from reperfusion to 25 minutes was greater in EDA-treated rats (1029.8 ± 710.3 mm Hg/mL) than in CON rats (696.1 ± 450.6 mm Hg/mL).
Blood Malondialdehyde Levels
Blood MDA levels were significantly higher in CON rats than in normal sham-operated and EDA-treated rats. These levels did not differ between normal sham-operated and EDA-treated rats. Thus, the treatment of EDA just before reperfusion perfectly inhibited the increases of MDA level (Fig. 6).
The present study demonstrated that edaravone, a newly synthesized free radical scavenger, not only inhibited deterioration of cardiac function but also prevented increasing serum malondialdehyde levels after 5 minutes of ischemia-reperfusion in rats. The 3 mg/kg dose of edaravone was chosen because this has been shown to be the dose effective in the rat ischemic forebrain and hindlimb amputation models.12,13
It was reported that the pathophysiology of the reperfusion injury may be involved in calcium overload, osmotic pressure overload, and particularly in free radicals that were demonstrated to be produced in myocardial ischemic-reperfusion.2 Thus, free radicals have been proposed as one of the major factors in reperfusion injury.
On the other hand, the hypocontractility state without myocardial necrosis continued from a few hours to a few days after a short period (5 or 10 minutes) of ischemia-reperfusion and then slowly recovered. This phenomenon was defined as myocardial stunning; calcium overload and free radicals may be involved in this mechanism.
In this study, contractility (Ees) was already greater in edaravone-treated rats than in controls just before reperfusion after 5 minutes ischemia. Thus, it is suggested that edaravone partially inhibited ischemic myocardial damage and maintained contractility. Moreover, contractility was better maintained in edaravone-treated rats than in controls after reperfusion to 25 minutes, and the degree of the improvement of contractility after reperfusion was also greater in edaravone-treated rats than in controls, suggesting a reduction of reperfusion injury and protection of myocardial stunning. Left ventricular end-diastolic pressure, which is a good index for estimation of cardiac function, was lower in edaravone-treated rats than in controls at 5, 15, and 20 minutes after reperfusion. The findings also supported the idea that edaravone was useful for maintenance of cardiac function after ischemia-reperfusion.
Edaravone has been reported to delete hydrogen oxide radicals that have higher reactivity to trigger the lipid peroxidation and so inhibit the initiation and propagation of lipid peroxidation by hydrogen peroxide radicals.18
Oxygen free radical damage can be monitored by measuring malondialdehyde. In the present study, the low levels of malondialdehyde in edaravone-treated rats as compared with controls suggest that edaravone has a free radical scavenging property after ischemic myocardial reperfusion in rats. Numerous studies have reported that edaravone protects against detrimental phenomena of cerebral ischemia and reperfusion. Because it has not been demonstrated that edaravone has any actions other than scavenging of free radicals, it is conceivable that all favorable cardiac protective effects are caused by the free radical scavenger action of edaravone. Thus, the results of our study might indicate that injection of edaravone just before reperfusion suppressed myocardial stunning and reperfusion injury after myocardial ischemia-reperfusion via the scavenging of free radicals.
The main productive sources of free radicals in myocardial tissue in ischemia-reperfusion are suggested to involve the xanthine oxidase-hypoxanthine system,19 the electron transmission system in mitochondria,20 the arachidonic cascade,21 and the NADPH oxidase system in polynuclear white blood cells.22,23 Free radicals react with lipid so that lipid peroxidation progresses.24 Arroyo et al25 reported that free radicals such as hydrogen oxide were produced in myocardial ischemia-reperfusion.
The present study demonstrated that 5 of 18 (27.8%) controls and none of 11 (0%) edaravone-treated rats died of life-threatening ventricular tachyarrhythmia. Treatment with edaravone just before 5 minutes of ischemia-reperfusion suppressed life-threatening ventricular tachyarrhythmia and ventricular fibrillation and increased the survival rate at 25 minutes after reperfusion. Moreover, malondialdehyde in the blood 25 minutes after reperfusion was significantly lower in edaravone-treated rats than in controls. Therefore, it is suggested that the augmentation of free radicals might be involved in the occurrence of the life-threatening reperfusion ventricular arrhythmia. Matsuura and Shattock26 reported that single ventricular cells exposed to an oxidant exhibited spontaneous membrane potential fluctuations at plateau potentials and at the level of the resting membrane potential. These voltage fluctuations in the resting potential occasionally induced triggered activity. It was also reported that oxidant stress inhibited the current of the Na+-K+ pump and increased Ca2+ release from the sarcoplasmic reticulum. Thus, Ca2+ overload may have led to the triggered activity.27 These electrophysiological changes producing oxidants might be involved in the occurrence of lethal reperfusion ventricular arrhythmia.
Manning et al28 reported that the occurrence of reperfusion ventricular fibrillation was significantly controlled, and even the death rate decreased by allopurinol treatment after 5 minutes of ischemia-reperfusion in rat heart. They suggested that the production of free radicals by the xanthine and xanthine oxidase system is related to vulnerability in ischemia-reperfusion. Bernier et al29 also demonstrated that the occurrence of ventricular fibrillation was increased by FeCl3 or adenosine diphosphate, a free radical-generating system, administration in contrast to a decrease by a radical scavenger such as SOD or catalase administration. Further, Zweier et al30 confirmed that free radicals could be directly detected by electron spin resonance spectroscopy 10 seconds after reperfusion in rat heart. These data all support the concept that free radicals are concerned with the occurrence of life-threatening reperfusion ventricular arrhythmias.
In conclusion, this study suggested that edaravone protected the deterioration of cardiac function and suppressed occurrence of lethal ventricular tachyarrhythmia after ischemia-reperfusion in rat heart. The action of edaravone as a free radical scavenger controlled myocardial ischemic injury as well as reperfusion injury, so it is conceivable that it is also effective in the protection of cardiac function and life-threatening ventricular tachyarrhythmias after myocardial ischemia-reperfusion.
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