Cloricromene (8-monochloro-3-beta-diethylaminoethyl-4-methyl-7-ethoxycarbonyl methoxycoumarin) is a coumarin derivative currently marketed in Europe for symptoms of claudication in patients with atherosclerotic peripheral vascular disease . In animal models, cloricromene reduces injury in skeletal muscle after an ischemic-reperfusion insult , improves survival in hemorrhagic and endotoxic shock models [3,4], and decreases myocardial infarct size after an ischemic-reperfusion injury [5-8]. These varying pathophysiologic states all share a component of microcirculatory dysfunction in which cloricromene may exert its action.
Effective early reperfusion of the myocardium reduces myocardial damage in an acute myocardial infarction. Although cloricromene is thought to enhance myocardial reperfusion, only one study examines myocardial infarct size when cloricromene administration is limited to the reperfusion period. Squadrito et al.  administered a single dose of cloricromene five minutes after reperfusion in a rat model and demonstrated a marked decrease in infarction size. No other work has examined this time-related effect.
The purpose of this study was to evaluate the effect of time of administration of cloricromene in an in vivo model of myocardial infarction in rabbits.
Fifty-seven male New Zealand White rabbits weighing between 2.9 and 4.7 kg were used for the experiment. The experimental protocol was approved by our Animal Care and Use Committee and complied with the Guiding Principles in the Use and Care of Animals approved by the Counsel of American Physiological Society.
Anesthesia was initiated with intramuscular ketamine (35 mg/kg) and xylazine (6 mg/kg). The left femoral vein was cannulated, and ketamine (25 mg/h) and xylazine (20 mg/h) were administered by a microprocessor-controlled infusion system. Femoral artery cannulation allowed continuous mean arterial pressure (MAP) monitoring and intermittent blood sampling. A tracheostomy was performed, and supplemental oxygen was administered to the spontaneously breathing rabbits. Arterial blood gases were maintained within the physiologic range by adjusting the amount of inspired oxygen or by the institution of mechanical ventilation. Heart rate (HR) was continuously monitored.
The chest was opened by median sternotomy, and the heart was exposed. Blood volume loss was equivalently replaced with hetastarch. The rabbits were systemically heparinized with a standard dose of 1500 U of heparin (beef lung). The left atrial appendage was cannulated with a 16-gauge catheter and secured with a purse string suture. The catheter provided immediate access to the left-sided circulation for staining the area at risk (AAR). The descending branch of the left coronary artery was identified, and a 5-0 proline suture with a removable snare was placed around this vessel.
After a 20-min stabilization period, the rabbits were randomly assigned to one of five groups: VEH received an infusion of saline vehicle starting at the onset of ischemia; IR received an infusion of cloricromene starting at the onset of ischemia at 8 micro g/kg-1/min-1; R(-5) and R(+30) received an infusion of cloricromene at 8 micro g [center dot] kg-1 [center dot] min-1 starting 5 min before reperfusion and 30 min after reperfusion, respectively; and RB(-5) received 300 micro g/kg of cloricromene 5 min before reperfusion followed by an infusion of 8 micro g [center dot] kg-1 [center dot] min-1. All infusions were continued to the end of the reperfusion period.
In all groups, the descending branch of the left coronary artery was occluded for 30 min by securing the snare to produce a zone of regional ischemia in the left ventricular free wall. This ischemic zone was confirmed visually by cyanosis and dyskinesis. After 30 min, the snare was released, and a 120-min reperfusion period started.
At the end of reperfusion, the snare was tightened, and 5 mL of Unisperse blue dye (Ciba-Geigy, Summit, NJ) were injected into the left atrium to stain the perfused myocardium. The AAR was demarcated by the lack of blue stain. The rabbits were then killed with pentobarbital (100 mg/kg), and the heart was excised. The atria, right ventricle, and the great vessels were removed, and the left ventricle (LV) was sectioned parallel to the atrioventricular sulcus into 2-mm thick slices from apex to base. The unstained region was separated from the blue-stained nonischemic zone and incubated for 10 min in a 1% solution of triphenyltetrazolium chloride (TTC) in phosphate buffer (pH 7.4) warmed to 37 degrees C. In the presence of intact dehydrogenase enzyme systems or the oxidized form of nicotinamide adenine dinucleotide/reduced nicotinamide adenine dinucleotide, triphenyltetrazolium dye forms a red precipitate demarcating nonnecrotic tissue. Areas of necrosis lack activity of these enzyme components and therefore remain a pale color. At the end of the TTC incubation, tissue samples were placed in a 10% solution of cold buffered formalin for fixation. The AAR was then subdivided into nonnecrotic (TTC positive, red color) and necrotic (TTC negative, pale color) regions by an assistant who remained blinded to the study group identity. The AAR and the area of necrosis (AN) were then determined gravimetrically as described elsewhere [10,11].
MAP and HR were continuously monitored. After the stabilization period, baseline hemodynamic measurements were obtained. Additional hemodynamic data were acquired and recorded at the end of ischemia and at 15, 60, and 120 min after reperfusion (R15, R60, and R120). The modified pressure-rate product (PRP) was calculated from the data recorded (MAP x HR).
Standard exclusion criteria were: 1) unclear demarcation of the AAR after coronary occlusion by Unisperse blue staining, 2) failure to complete the entire protocol, 3) an area at risk/left ventricular area (AAR/LV%) greater than 45%, and 4) no evidence of reperfusion after snare release (i.e., no hyperemia in the ischemic zone immediately after snare release).
Triplicate acquisitions of hemodynamic data were averaged at each time point. An analysis of variance for repeated measures was used to determine whether time- and group-related differences existed. Duncan's multiple range test was used to correct for pairwise multiple comparisons.
A one-way analysis of variance was used to analyze group differences in LV, AAR, and AN. Results are reported as means and standard errors of the mean. Significance was assigned when P < 0.05. The AAR/LV, AN/LV, and AN/AAR percentages were each analyzed using an analysis of variance to determine whether differences between treatment groups existed.
Based on the exclusion criteria, eight rabbits were excluded: 3 from Group VEH, 1 from Group IR, 3 from Group R(-5), and 1 from Group R(+30). Data from 49 rabbits were included in the final analysis: 11 from Group VEH, 9 from Group IR, 9 from Group R(-5), 11 from Group RB(-5), and 9 from Group R(+30).
Baseline hemodynamic variables were similar in all groups (Table 1). There was a significant time effect for HR, MAP, and PRP, with a tendency toward increasing HR during coronary occlusion and decreasing MAP and PRP during the experimental protocol. There were no differences among the groups at each time point. There was no difference between groups for the mass of the LV or the AAR (Table 2). The AN was significantly reduced from that of Group VEH in Groups IR and RB(-5).
The AAR/LV% was similar among all groups (Figure 1A). The AN/LV% is presented in Figure 1B. These data show a tendency toward infarct reduction in all treatment groups compared with VEH, and this reduction was significant in Groups IR and RB(-5). When the AN was normalized for the AAR (AN/AAR%), infarct size was reduced when cloricromene was given during ischemia and reperfusion. However, there was a comparable reduction in infarct size when cloricromene was given just before reperfusion either as an infusion or as a bolus plus infusion [Groups R(-5) and RB(-5)]. These data are presented in Figure 1C.
Our results suggest that cloricromene exerts a time-dependent cardioprotective benefit in a rabbit model of ischemic-reperfusion injury. Cloricromene's cardioprotective benefit is exerted as effectively when given just prior to reperfusion as when given during both ischemia and reperfusion. This suggests that the mechanisms of action are exerted during the reperfusion period. Further, we show that when cloricromene administration is delayed until 30 minutes into the reperfusion period, all effective cardioprotection is lost. These data clearly indicate that cloricromene's time of action is in the early reperfusion period.
We selected our groups based on the previous literature using cloricromene in rabbit models [5-8]. The rabbit provides a consistent model for infarction study because there are few, if any, coronary collateral blood vessels. Furthermore, previous work has demonstrated significant infarct reduction with cloricromene in the rabbit when the drug is given during ischemia and reperfusion at a dose of 7.1 micro g [center dot] kg-1 [center dot] min-1. Thus, our study included both a negative and a positive control group-VEH and IR, respectively. The unique aspect of this study is that the time of administration was varied to evaluate the influence of cloricromene during the reperfusion period only. We achieved this by administering the drug five minutes before and 30 minutes after the onset of reperfusion. Previous studies [11,12] have shown that infarct development during reperfusion is largely complete after 30 minutes of reflow. As predicted, the VEH group had the largest infarct size, and cardioprotection was seen when cloricromene was given during both ischemia and reperfusion. The data demonstrated injury reduction when the drug was given five minutes before reperfusion but not when administration was delayed for 30 minutes into reperfusion when the acute phase of infarct development was complete. When comparing Groups IR and R(-5), there was a tendency for greater cardioprotection in the IR group. This observation raises the question as to whether there is cardioprotective action during ischemia or whether there is simply more drug present in the IR group at the time of reperfusion. Because it is impossible to give the drug only during ischemia (cloricromene will be present during reperfusion even if stopped at the end of ischemia), we included another study group to examine this possible loading effect. In group RB(-5), a bolus dose of cloricromene was administered, followed by an infusion. We found that groups IR and RB(-5) were nearly identical in their cardioprotective profiles. In fact, these two groups had the greatest cardioprotection seen in this study. These data support the hypothesis that cloricromene acts during reperfusion and that the infusion during ischemia only serves as a time of pharmacologic drug loading. Although there may be some cardioprotection exerted during ischemia, the predominate effect occurs during reperfusion.
Squadrito and associates  found a 2 mg/kg bolus of cloricromene to be cardioprotective in rats when given five minutes after the onset of reperfusion. These investigators reported a 50% reduction in infarct size (68% +/- 4% AN/AAR% for placebo; 30% +/- 1.2% AN/AAR% for cloricromene group). This infarct size reduction is similar to ours and demonstrates the effectiveness of cloricromene in a rat model when the drug is given during the reperfusion period. The major differences between the study by Squadrito et al.  and the present study remain the choice of animal model and drug administration regimen. Our study is the first to evaluate the time effect in a rabbit model. Additionally, we have further defined the critical time of this cardioprotection by demonstrating that cardioprotection is lost after 30 minutes of reperfusion. Therefore, our work and that of Squadrito et al.  collectively support the hypothesis that cloricromene interferes with the deleterious side effects present during the early reperfusion period.
Although the mechanisms of cloricromene's cardioprotective effects were not evaluated in this study, we can conclude that the cardioprotection occurs via some pathway other than vasodilation during ischemia or via an alteration in the myocardial oxygen supply/demand ratio. The rabbit offers a collateraldeficient model, and therefore increased collateral flow by vasodilation cannot be responsible for the results. In addition, hemodynamics were equal across groups, which eliminates oxygen supply/demand alterations as a mechanism. What role, if any, cloricromene has on neutrophil activation or endothelial injury in the reperfusion period has yet to be determined.
In conclusion, we found that cloricromene reduces myocardial infarct size after an ischemic-reperfusion injury in rabbit myocardium. This benefit is achieved either when the drug is given during ischemia and reperfusion or when it is administered just prior to reperfusion. Cardioprotection is lost when the drug is initiated 30 minutes into the reperfusion period. These data suggest that the critical period of action for this compound is during the early reperfusion period.
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© 1997 International Anesthesia Research Society
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