INTRODUCTION

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BRIEF PERIODS OF MYOCARDIAL ISCHEMIA are known to induce both an early (16) and a delayed (13) preconditioning against the deleterious consequences of subsequent myocardial ischemia. Many substances, such as opioids (9), nitric oxide (NO) donors (25), monosphosphoryl lipid A (27), and adenosine A₁- and A₃-receptor agonists (2, 4, 21) have been used to pharmacologically mimic ischemic delayed preconditioning (I-DPC). Among them, adenosine A₁-receptor stimulation-induced delayed preconditioning (A₁-DPC) has been one of the most extensively investigated and has been reported to afford protection against myocardial infarction (2-4, 8, 12, 21, 29). In most of these studies, infarct size after A₁-DPC was measured after short durations of reperfusion (i.e., not exceeding 3 h) (2-4, 8, 29), but one group reported a similar beneficial effect after 3 days of reperfusion (12, 21). Interestingly, Miki et al. (15) demonstrated in conscious rabbits that I-DPC also decreases infarct size when assessed after 3 h of reperfusion, but this cardioprotective effect was no longer observed after 72 h of reperfusion. Such differential effects, depending on the duration of reperfusion, have also been reported with another cardioprotective intervention, i.e., administration of N-2-mercaptopropionyl glycine (14). Therefore, it may be hypothetized that the duration of postischemic coronary artery reperfusion could also be critical for the infarct-limiting effect of A₁-DPC.

Accordingly, the aim of this study was to compare the cardioprotective effects of A₁-DPC after short (3 h) and long (72 h) durations of reperfusion. We also performed a similar investigation with I-DPC. To avoid the confounding effects of numerous factors associated with the open-chest state, such as anesthesia, hypothermia, trauma, and elevated catecholamines, we performed this study in a model of conscious chronically instrumented rabbits.

	METHODS

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The animal instrumentation and the ensuing experiments were performed in accordance with the official regulations edicted by the French Ministry of Agriculture (Agreement no. A94-043-12).

Animal surgery. Male New Zealand White rabbits (2-2.5 kg) were anesthetized with a mixture of tiletamine (25 mg/kg iv) and zolazepam (25 mg/kg iv) and intubated and mechanically ventilated with 100% oxygen via a positive pressure respirator. The ventilation rate was 25 breaths/min, and the tidal volume was ~25 ml. Subsequent anesthesia was maintained with pentobarbital sodium (20-30 mg/kg iv). An external electrocardiogram (ECG) was recorded during the surgery. A left thoracotomy was performed at the fourth intercostal space under sterile conditions. A pneumatic occluder fashioned from 18-gauge Tygon tubing was implanted around a major branch of the left coronary artery according to a technique previously described (6). Proper functioning of the occluder was confirmed by observing cyanosis of the distal myocardium and ST segment deviation of the ECG after a brief inflation of the occluder. Conversely, hyperemia and normalization of the ECG were verified after its deflation. The chest was closed in layers, and a small tube was left in the thorax to evacuate air and fluids after surgery. Internal ECG leads were attached to intercostal muscles. The occluder and internal ECG wires were exteriorized between the scapulae. During the postoperative period, rabbits were treated for 3 days with buprenorphine (0.02 mg/kg sc) and flunixine meglumate (1 mg/kg im) for analgesia. Gentamycin (0.5 mg/kg im) was also administered during 5 consecutive days. Rabbits were allowed to recover for a minimum of 10 days after surgery.

Experimental protocol. Throughout the experiment, rabbits were conscious and kept in a box in a quiet, dimly lit room. The internal ECG wires were connected to an amplifier (Gould Instruments; Cleveland, OH). An intra-arterial catheter was introduced into the ear artery, and arterial pressure was measured using a Statham P23 ID strain gauge transducer (Statham Instruments; Oxnard, CA). ECG and arterial pressure were recorded on a multichannel oscillograph (DMS 1000, Graphtec, Vanderbilt; Irvine, CA).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Experimental protocol. I-DPC, ischemia-induced delayed preconditioning; A1-DPC100, N6-cyclopentyladenosine (CPA)-induced delayed preconditioning, 100 µg/kg; A1-DPC400, CPA-induced delayed preconditioning, 400 µg/kg; CAO, coronary artery occlusion.

Determination of myocardial area at risk and infarct size. After reperfusion was completed, animals received an injection of heparin (1,000 IU iv) and were reanesthetized with pentobarbital sodium (50 mg/kg iv). Potassium chloride was administered intravenously to induce cardiac arrest. The hearts were excised. The ascending aorta was cannulated and perfused (120 mmHg) retrogradely with saline followed by Evans blue (1%). The right ventricle was then removed, and the left ventricle was cut into five to six slices. These slices were weighed and incubated in 1% triphenyltetrazolium chloride (TTC, Sigma; Poole, UK) in a pH 7.4 buffer during 15 min at 37°C to identify the infarcted myocardium. Slices were overnight fixed in 10% formaldehyde and then photographed with a digital camera. With the use of a computerized planimetric program (Scion Image, Scion; Frederick, MD), the area at risk and the infarcted zones were quantified. The area at risk was identified as the nonblue region and was expressed as a percentage of the left ventricle weight. Infarcted area was identified as the TTC-negative zone and was expressed either as a percentage of the area at risk or as a percentage of the left ventricle weight.

Histological analysis. Formalin-fixed slices were further embedded in paraffin for histological analysis. Two 5-µm-thick sections were cut from each paraffin block using a microtome. One was stained with hematoxylin-eosin-safranin (HES) and the other with Masson's trichrome. For morphometry, all HES-stained sections obtained from rabbits subjected to 72 h of CAR were observed in a microscope at a ×2 magnification. Successive digital photographs of the adjacent microscopic fields encompassing the complete cardiac circumference were recorded. A computerized reconstruction (Photoline, Computerinsel; Bad Gögging, Germany) of the complete cardiac slice at a ×2 magnification was made by adequate juxtaposition of the different and complementary digital photographs. Infarct was delimited from this photographic reconstitution by drawing its contour with a computer mouse. Simultaneous observation of the histologic section on a microscope at ×4 or ×10 magnifications allowed an accurate detection of the infarcted area. Myocardial infarction was considered as a central region of coagulation necrosis with a border of myocytolysis and inflammatory infiltration. Finally, planimetry was performed as previously described for the TTC-technique, and infarct sizes were calculated.

Data analysis. Data are reported as means ± SE. The effects of saline, CPA (100 or 400 µg/kg), and I-DPC on heart rate and mean arterial pressure were analyzed on day 1 by a paired Student t-test. On day 2, comparisons were made only between the four groups using ANOVA for repeated measurements. Comparisons were performed among control, I-DPC, A₁-DPC₁₀₀, and A₁DPC₄₀₀ groups at each duration of CAR using a one-way ANOVA and post hoc Fisher's prottected least-significant difference test if necessary. Significant differences were determined as P < 0.05.

	RESULTS

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Sixty-one rabbits successfully underwent the CAO and subsequent reperfusion protocol and were used in this study: 10 in control, 7 in I-DPC, 6 in A₁-DPC₁₀₀, 6 in A₁-DPC₄₀₀ with 3 h of CAR and 12 in control, 6 in I-DPC, 8 in A₁-DPC₁₀₀, and 6 in A₁-DPC₄₀₀ with 72 h of CAR.

Hemodynamic. At day 1, baseline values of heart rate and mean arterial pressure were not significantly different among groups (heart rate: 214 ± 9, 205 ± 9, 202 ± 8, and 212 ± 10 beats/min; mean arterial pressure: 72 ± 3, 69 ± 3, 74 ± 3, and 70 ± 4 mmHg for control, I-DPC, A₁-DPC₁₀₀, and A₁-DPC₄₀₀, respectively). Intravenous injection of saline did not affect heart rate and mean arterial pressure in control (data not shown). In I-DPC, the ischemic preconditioning protocol induced a significant increase in heart rate during each CAO compared with baseline (e.g., +12 ± 4% during the last CAO), but mean arterial pressure did not change. In A₁-DPC₁₀₀ and A₁-DPC₄₀₀, CPA decreased both heart rate (16 ± 3% and 20 ± 4%, respectively) and mean arterial pressure (33 ± 3% and 39 ± 4%, respectively) compared with baseline. On day 2 (Table 1), heart rate and mean arterial pressure were not significantly different among control, I-DPC, A₁-DPC₁₀₀, and A₁-DPC₄₀₀ at baseline and during CAO and CAR.

Infarct sizes after 3 h of CAR. Left ventricular weights and area at risk sizes were similar among control, I-DPC, A₁-DPC₁₀₀, and A₁-DPC₄₀₀ with 3 h of CAR (Table 2). As illustrated in Fig. 2A, infarct sizes determined by TTC staining were similarly and significantly decreased in I-DPC, A₁-DPC₁₀₀, and A₁-DPC₄₀₀ (36 ± 5%, 41 ± 4%, and 38 ± 5% of the area at risk, respectively) compared with control (55 ± 3% of the area at risk). Because infarct size can be influenced by the size of the area at risk (28), the effects of I-DPC and A₁-DPC were also investigated by plotting these two parameters expressed as percentages of the left ventricle. As shown in Fig. 2B, the infarct size/area at risk regression line was shifted downward by I-DPC, A₁-DPC₁₀₀, and A₁-DPC₄₀₀ compared with control. The overall histologic pattern of the heart sections obtained from rabbits subjected to 3 h of CAR was similar among the four groups. Infarcts consisted of myocyte necrosis with contraction bands and mainly of hemorrhages and edema. However, no clear-cut delimitation of the infarcted area was possible, and hence no accurate histologic determination of infarct size was possible after such a short CAR duration.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   A: infarct sizes (expressed as percentage of area at risk) measured after 3 h of CAR. Infarction was detected after triphenyltetrazolium chloride (TTC) staining. Circles indicate individual and average values, respectively. B: scatterplots of the relationship between the sizes of the infarct and area at risk (expressed as percentage of left ventricular weight). Regression lines are represented for each group: control (r = 0.76), I-DPC (r = 0.79), A1-DPC100 (r = 0.79), and A1-DPC400 (r = 0.95). *P < 0.05 vs. control.

Infarct sizes after 72 h of CAR. Left ventricular weights and area at risk sizes were similar among control, I-DPC, A₁-DPC₁₀₀, and A₁-DPC₄₀₀ with 72 h of CAR (Table 2). As illustrated in Fig. 3A, infarct sizes determined by TTC staining were not significantly different among control, I-DPC, A₁-DPC₁₀₀, and A₁-DPC₄₀₀ (41 ± 3, 42 ± 3, 40 ± 4, and 42 ± 3% of the area at risk, respectively). Furthermore, the infarct size/area at risk regression lines were not different among the four groups (Fig. 3B).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   A: infarct sizes (expressed as percentage of area at risk) measured after 72 h of CAR by the TTC technique. Circles indicate individual and average values, respectively. B: scatterplots of the relationship between the sizes of the infarct and area at risk (expressed as percentage of left ventricular weight). Regression lines are represented for each group: control (r = 0.89), I-DPC (r = 0.89), A1-DPC100 (r = 0.79), A1-DPC400 (r = 0.81).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   A: infarct sizes (expressed as percentage of area at risk) measured after 72 h of CAR by histology. Circles indicate individual and average values, respectively. B: scatterplots of relationship between the sizes of the infarct and area at risk (expressed as percentage of left ventricular weight). Regression lines are represented for each group: control (r = 0.88), I-DPC (r = 0.90), A1-DPC100 (r = 0.81), A1-DPC400 (r = 0.81).

	DISCUSSION

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In this study, we investigated the infarct-limiting effect of a pharmacological delayed preconditioning induced by two different doses of an adenosine A₁-receptor agonist (CPA) in chronically instrumented conscious rabbits. In agreement with previous reports (2, 7, 8, 29), our results demonstrate that after 3 h of CAR, A₁-DPC decreases infarct size. Similarly, I-DPC significantly decreased infarct size when assessed after 3 h of CAR, as also previously described in anesthetized (13) and conscious rabbits (14, 17). Surprisingly, both A₁-DPC₁₀₀ and A₁-DPC₄₀₀ failed to limit infarct size when measurements were performed after 72 h of CAR with both the TTC technique and the histologic analysis. Similarly, I-DPC failed to demonstrate any cardioprotective effect against myocardial infarction with 72 h of CAR.

One might argue that our negative results after 72 h of CAR are due to insufficient doses of CPA. However, this is unlikely because these doses were chosen on the basis of a dose-response curve as being the ED₅₀ and the ED₈₀ at decreasing mean arterial pressure, suggesting that the level of adenosine A₁-receptor stimulation was high enough. Furthermore, the dose of 400 µg/kg was the highest hemodynamically well tolerated (26). Nevertheless and after 3 h of CAR, both doses of CPA elicited a significant reduction in infarct size and of the same magnitude, suggesting that the dose of 100 µg/kg was already sufficient to achieve a maximal cardioprotective effect. Importantly, the critical effect of the duration of reperfusion (i.e., 3 h vs. 72 h of CAR) was also observed with I-DPC in agreement with the data of Downey's group (15). It is important to consider that the absence of cardioprotection afforded by A₁-DPC and I-DPC after 72 h of CAR was observed regardless of the method used to measure infarct size (TTC technique and histologic analysis).

In apparent contradiction with the present results, Bolli's group reported with similar experimental conditions (chronically instrumented conscious rabbits, TTC-technique), a significant limitation of infarct size after 72 h of CAR with I-DPC (17, 19, 22, 23) and with A₁-DPC induced by 2-chloro-N⁶-cyclopentyladenosine (12, 21). This group further obtained a TTC-independent evidence for an infarct-limiting effect of I-DPC, i.e., an enhancement of the recovery of postinfarction myocardial function (24). Nevertheless, we also confirmed our results by a TTC-independent technique, i.e., histologic analysis. This discrepancy remains to be elucidated, but rabbits' strain specificity might, at least in part, provide an explanation, as previously illustrated for I-DPC in mice hearts (1). Finally, we cannot rule out favorable remodeling effects of A₁-DPC or I-DPC, which may enhance recovery of regional myocardial function in our experimental conditions.

The factors explaining the critical role of the duration of reperfusion on the cardioprotective effect of I-DPC and A₁-DPC have not been investigated in the present study, but a number of hypotheses may be raised. According to most of the studies investigating infarct-limiting procedures, we defined myocardial infarction as being the TTC-unstained zone. TTC is a chemical that is converted to formazan dye by dehydrogenase enzymes and cofactors retained in the viable tissue (11). Tissue that is not stained by TTC, i.e., TTC-negative zone, is consequently considered as being infarcted. However, it is important to consider that tissue converting TTC into formazan could also be a dead tissue from which dehydrogenase enzymes have not yet been washed. Indeed, some interventions (e.g., intravenous superoxide dismutase administration) that could preserve capillary permeability and delay the dehydrogenases wash out might lead to a transient decrease of the TTC-negative zone without actual and prolonged cardioprotective effect (10, 20). Consequently, we cannot exclude that A₁-DPC or I-DPC-induced infarct limitation after 3 h of CAR could be related to a delay in the loss of dehydrogenase enzymes in necrotic tissue secondary to a protection of the vasculature. Such a hypothesis, considered as unlikely by Downey's group regarding I-DPC (15), has also been advocated to explain the inability of the free radical scavenger N-2-mercaptopropioglycine to induce a prolonged limitation of infarct size (14). Finally, because tissue edema, hemorrhage, and acute inflammation are known to affect infarct size (18), one might suggest that the protection observed with A₁-DPC and I-DPC after 3 h of CAR could be related to an attenuation of these phenomenons. Indeed, tissue edema and hemorrhage, which may artificially increase infarct size after a short period of reperfusion, could be inhibited by delayed preconditioning, resulting in a decrease in infarct weight despite minor and no actual cardioprotective effect. Such a hypothesis is not further supported according to our qualitative analysis of infarction showing that huge hemorrhage and edema observed after 3 h of CAR are of similar extend in all groups of rabbits.

According to Birnbaum et al. (5), infarct size assessed by TTC following 30 min of myocardial ischemia, is smaller when measured after 2 h instead of after 4 h of CAR. It is reasonable to speculate that I-DPC and A₁-DPC may delay the very early evolution of infarct size assessed by the TTC technique. This could explain the only transient protection observed with 3 h of CAR, whereas infarct size measured with 72 h of CAR is not significantly different from nonpreconditioned animals.

In conclusion, this study is the first to investigate in conscious rabbits the infarct-limiting effect of A₁-DPC after both 3 and 72 h of CAR. A₁-DPC, at the two CPA investigated doses, as well as I-DPC, significantly decreased infarct size after 3 h of CAR, but this effect was no longer observed after 72 h of CAR with both the TTC technique and histology.