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Ann Thorac Surg 1999;67:1689-1695
© 1999 The Society of Thoracic Surgeons

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Original Articles

Ischemic preconditioning reduces neutrophil accumulation and myocardial apoptosis

^a Departments of Department of Cardiothoracic Surgery, Emory University School of Medicine, Atlanta, Georgia, USA
^b Department of Hematology/Oncology, Emory University School of Medicine, Atlanta, Georgia, USA
^c Department of Pathology, Emory University School of Medicine, Atlanta, Georgia, USA

Accepted for publication December 18, 1998.

Address reprint requests to Dr Vinten-Johansen, Division of Cardiothoracic Surgery, Department of Surgery, Carlyle Fraser Heart Center, Emory University School of Medicine, 550 Peachtree St NE, Atlanta, GA 30365-2225

	Abstract

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Background. This study tested the hypothesis that ischemic preconditioning (IP) inhibits myocardial apoptosis after a short period of ischemia and reperfusion.

Methods. In 9 anesthetized dogs, the left anterior descending (LAD) coronary artery was occluded for 30 min and reperfused for 3 h (control), while in 9 others, LAD occlusion was preceded by 5 min of occlusion and 5 min of reperfusion (IP). DNA from frozen myocardial tissue samples was extracted, and apoptosis were identified as "ladders" by agarose gel electrophoresis or confirmed histologically using the terminal transferase UTP nick end-labeling (TUNEL) assay. Neutrophil accumulation was detected by measuring cardiac myeloperoxidase activity.

Results. Thirty minutes of LAD occlusion caused a significant decrease in blood flow (colored microspheres), which was comparable between groups. In the control group, DNA ladders occurred in the area at risk (AAR) in six out nine experiments. In contrast, DNA laddering in the AAR was not observed in any of the IP group. AAR in the control group showed a greater percentage of apoptotic cells than IP (6.7 ± 0.9% vs 1.2 ± 0.2%; p < 0.01). Cardiac myeloperoxidase activity (U/g tissue) was significantly reduced from 0.07 ± 0.004 in control to 0.04 ± 0.01 in IP group (p < 0.05).

Conclusions. We conclude that ischemic preconditioning attenuates apoptosis and neutrophil accumulation in the AAR in a model of nonlethal acute ischemia and reperfusion.

	Introduction

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Transient ischemia is often performed on a beating and regionally ischemic heart during minimally invasive and off-pump procedures, and requires temporary interruption of coronary blood flow in the recipient artery during anastomosis of the vascular graft to the target vessel. To reduce coronary artery motion and protect the heart from the risk of myocardial ischemic injury during short episodes of coronary artery occlusion, some approaches have emerged, including the use of pharmacologic therapy to reduce heart rate, such as adenosine [1], the applications of electrical stimulation of vague nerve and mechanical epicardial stabilization [2], and limitation of myocardial injury by ischemic preconditioning (IP) [3–6]. IP, as a highly effective method of protecting the heart from infarction and vascular injury in models of coronary artery occlusion, was first reported by Murry and associates in 1986 [3]. Since then, many studies have shown that IP attenuates the incidence and severity of postischemic arrhythmias, preserves coronary endothelial function, and reduces myocardial infarct size after a subsequent prolonged period of myocardial ischemia followed by reperfusion [3, 7]. IP has now been used during minimally invasive and off-pump procedures [8].

Apoptosis or programmed cell death is a genetically controlled biochemical and morphological response for cells to commit suicide. The features of this type of cell death are characterized by DNA fragmentation or laddering. In contrast to necrosis, apoptosis requires energy and protein synthesis, maintains cell membrane integrity, and avoids an inflammatory response. Recently, two studies have shown that IP protects myocardium from irreversible ischemia-reperfusion injury in an in vitro rabbit and in vivo rat model by preventing apoptotic cell death [9, 10]. It is not known, however, whether a short duration of reversible ischemia in vivo induces programmed cell death, or whether IP can also protect the myocardium by inhibiting apoptosis. Therefore, we tested the hypothesis that a single cycle of IP (5 min of ischemia and 5 min of reperfusion) before 30 min of coronary occlusion reduces myocardial apoptosis. The effect of IP on myocardial blood flow and neutrophil accumulation in myocardium were determined, and the presence of myocardial apoptosis was evaluated using DNA laddering by agarose gel electrophoresis and the terminal transferase UTP nick end-labeling (TUNEL) assay.

	Material and methods

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Surgical preparation
Eighteen dogs of either gender weighing 20 to 35 kg were initially premedicated with 4 mg/kg morphine sulfate. Endotracheal intubation was then performed after administration of fentanyl (20 µg/kg) and diazepan (0.25 mg/kg). Subsequent anesthesia was maintained by continuous infusion of 0.3 µg/kg/min of fentanyl and 0.03 mg/kg/min of diazepan during the experiment. The dogs were ventilated with tidal volume of 10 mL/kg and rate of 12 breaths per minute adjusted to maintain pH 7.35–7.45, pO₂ > 100 mm Hg, pCO₂ within 35–45 mm Hg. Catheters were placed in the left femoral artery for reference blood sampling of regional blood flow measurement. Limb leads and a precordial lead were placed for electrocardiographic monitoring. The chest was opened by median sternotomy, and the pericardium was incised anteriorly to expose the heart. A high-fidelity transducer-tipped catheter (Model MPC-500; Millar Instruments, Houston, TX) was secured in the ascending aorta for systemic blood pressure measurement, and a second catheter was inserted into the left ventricular cavity via a small puncture wound in the apex. A 14-gauge catheter was placed in the left atrium for injection of colored microspheres. A 2-0 silk suture was passed just below the first diagonal branch of the left anterior descending artery (LAD), and the ends of the tie were threaded through a small plastic (PE120) tube (Clay Adams, Parsippany, NJ) to form a snare for later coronary occlusion. All dogs were then systemically heparinized with 300 U/kg sodium heparin before starting the experiment. A bolus of lidocaine (2 mg/kg) was intravenously given and a drip maintained (1 mg/min).

The experimental procedures complied with the Guiding Principles in the Use and Care of Animals approved by the Council of the American Physiological Society, as well as with state and federal regulations. The experimental protocol was approved by the Institutional Animal Care and Use Committee.

Experimental protocol
After a 20- to 30-minute postsurgical stabilization period, steady-state baseline hemodynamic measurements were acquired in duplicate. The LAD was reversibly occluded by pulling up on the snare to produce a zone of regional ischemia in the left ventricle. After 30 minutes of ischemia, the ligature was loosened, and the ischemic myocardium was reperfused for 3 hours. All animals were randomized to two groups: 1) control (n = 9): dogs were not preconditioned before ischemia; and 2) ischemic preconditioning (IP, n = 9) animals underwent 5 minutes of LAD occlusion followed by 5 minutes of reperfusion before 30 minutes of LAD occlusion. At the end of the 3 hours of the reperfusion period, tissue samples from the heart were used to evaluate tissue myeloperoxidase (MPO) activity, myocardial blood flow, myocardial apoptosis, and infarct size (see below).

Hemodynamic measurements
Heart rate, mean aortic pressure, and left ventricular systolic and diastolic pressures were acquired and processed using a videographics program developed in our laboratory (Spectrum, Wake Forest University, Winston-Salem, NC) [11]. Measurements were taken before coronary artery occlusion (control), at the end of 30 minutes of ischemia, and at 30, 90, and 180 minutes of reperfusion. Peak left ventricle (LV) pressure, dp/dt max, LV end-diastolic pressure, and mean aortic pressure were averaged from no less than 10 beats.

Regional myocardial blood flow
Colored microspheres including red, yellow, and blue (15 µM diameter; Triton Technology, San Diego, CA) were separately injected at baseline, the end of ischemia, and 180 min of reperfusion to quantify collateral blood flow in the area at risk during ischemia, and postischemic myocardial blood flow using the reference sampling method as previously described [12]. Myocardial tissue samples from nonischemic and ischemic zones were isolated before triphenyltetrazolium chloride (TTC) staining. Regional myocardial blood flow (RMBF) was calculated as RMBF = (CT x FR/CF) x WT, where CT and CF are the absorbance from dispersed microspheres in the tissue and reference blood samples, respectively, FR is reference flow rate (3 mL/min), and WT is total weight of the tissue sample in grams. Results are expressed as mL/min/g tissue.

Determination of necrotic myocardium
At the end of reperfusion, the LAD ligature was retied, and Unisperse blue (Ciba Geigy, Newport, DE) was injected into the aortic root to stain the normally perfused region blue and outline the area at risk. After excision, the left ventricle was cut into transverse slices. The area at risk was separated from the nonischemic zone for identification of necrosis either by histology (see Tissue Preparation) or by 1.0% triphenyl tetrazolium chloride (TTC) staining.

Determination of tissue myeloperoxidase (MPO) activity
After TTC staining, tissue samples weighing approximately 0.3 g were taken from the nonischemic and ischemic zones for analysis of MPO activity. The samples were frozen and stored at -70°C until assayed. The samples were processed and analysed as previously described [11]. The activity of MPO was measured spectrophotometrically at 460 nM (SPECTRAmax; Molecular Devices, Sunnyvale, CA) and expressed as U/g tissue.

Tissue preparation
Myocardial tissue samples from nonischemic and ischemic zones determined by Unisperse blue dye staining were isolated after harvesting heart. The fresh tissues for identifying apoptosis by gel electrophoresis were then placed in tubes for DNA isolation. Samples for analysis of apoptosis by the TUNEL assay were placed immediately in molds oriented appropriately for sectioning and embedded in optimal cutting temperature compound (OCT; Miles Laboratories, Torrance, CA), frozen in liquid nitrogen, and stored at -70°C in airtight bags for detection of apoptotic cells using TUNEL staining (see below). The tissues for identifying necrosis by histology were fixed in 4% paraformaldehyde buffered with 0.1 M Na₂PO₄ (pH 7.4) for 1–3 hours at 4°C, cryoprotected in 15% sucrose-phosphate-buffered serum overnight, and were then embedded in OCT. Cryosections (7 µM) of all these samples were obtained using a Hacker-Bright cryostat and thaw-mounted onto Vectabond (Vector Laboratories, Burlingame, CA) -coated slides or Fisher-Plus (Fisher Scientific, Pittsburgh, PA) slides, refrozen, and stored at -70°C until use. Histological slides were stained with hematoxylin and eosin and evaluated for the presence of myocardial necrosis.

DNA isolation and gel electrophoresis
Freshly frozen nonischemic and ischemic myocardium (30–50 mg) were minced in 600 µL of lysis buffer (Puregene DNA Isolation Kit; Gentra Systems Inc, Minneapolis, MN) and were quickly homogenized using 30–50 strokes with a tube pestle. The tissue was digested with 100 µg/mL of proteinase K (Sigma, St. Louis, MO) at 56°C for 3–4 hours and incubated with RNase at 37°C for 1 hour. After incubation, tissues were precipitated and centrifuged at 16,000g for 5 minutes. Supernatants containing DNA were precipitated with isopropanol. After centrifugation at 16,000g for 5 minutes, the resulting DNA pellets were washed with 75% ethanol, and dissolved in DNA hydration solution, and analyzed at 260 nm by spectrophotometry. DNA (10 µg) was loaded into 1.5% agarose gel containing 0.5 µg/mL ethidum bromide. DNA electrophoresis was carried out at 80 V for 1.5 to 2 hours. DNA electrophoretic patterns were visualized under ultraviolet light.

In situ detection of cell death by TUNEL assay
Freshly frozen nonischemic and ischemic myocardial samples were cut at 6–7 mm thickness, fixed in 4% paraformaldehyde in PBS for 20 minutes at room temperature, and incubated with proteinase K (1 µg/mL) in PBS for 30 minutes. DNA fragments in the tissue sections were determined using an in situ cell death detection kit (Boehringer Mannheim, Ridgefield, CT). Briefly, the enzyme terminal deoxynucleotidyl transferase (TdT) was used to incorporate digoxigenin-conjugated dUTP to the ends of DNA fragments. The TdT-mediated dUTP nick end-labeling sites were then detected by an anti-fluorescein antibody conjugated with alkaline phosphatase, a reporter enzyme that catalytically generates a red-colored product from Vector Red substrate. The slides were dehydrated in graded alcohols and coverslipped with hematoxylin counterstaining. The slides were washed, dried, and mounted in Permount medium. For each slide, color video images of 280–360-µM fields were captured and digitized by use of a x25 objective with a Sony DXC-760MD video camera, a RasterOps 24 XLTV video card, and Media Grabber software on a Macintosh Quadra 950 computer. The cells with clear nuclear labeling were defined as TUNEL-positive cells. The apoptotic cells were calculated as percentage of TUNEL-positive cells using the following formula: number of TUNEL-positive cell nuclei/(number of TUNEL-positive cell nuclei + number of total cell nuclei) x 100.

Statistical analysis
A one-way analysis of variance followed by Duncan’s post-test was used to analyze group differences such as myeloperoxidase and apoptotic myocyte data. Hemodynamic and blood flow data were analyzed by repeated measures analysis of variance. A p < 0.05 was considered significant. Results are reported as mean and standard error of the mean.

	Results

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Hemodynamics
Hemodynamic data for heart rate (HR), mean aortic pressure (MAP), dP/dt_max, and left ventricular end-diastolic pressure (LVEDP) in the two groups are shown in Figure 1. There were no significant differences in any measured parameters between the two groups at baseline. There was no group difference during the course of the experiment in HR and MAP. Coronary occlusion was associated with an increase in LVEDP that was comparable in both groups. Although coronary occlusion caused a depression in maximal dp/dt in both groups, IP was associated with a significant improvement in the dP/dt_max during 90 to 180 minutes of reperfusion.

View larger version (29K): [in this window] [in a new window]   Fig 1. Graphs showing hemodynamic data at baseline, during ischemia, and after 90 and 180 minutes of reperfusion. HR = heart rate; MAP = mean aortic pressure; dp/dt = positive dp/dt; LVEDP = left ventricular end-diastolic pressure. Ischemia = 30 minutes of coronary occlusion; R90 and R180MIN = 90 and 180 minutes after reperfusion. IP = one cycle of ischemic preconditioning (5-minutes coronary occlusion plus 5 minutes of reperfusion). Error bars = SEM. *p < 0.05 vs control group.

View larger version (96K): [in this window] [in a new window]   Fig 2. Detection of DNA fragmentation using agarose gel electrophoresis. Numbers at bottom of figure indicate: lane 1 = nonischemic zone and lane 2 = ischemic zone in the control group; lane 3 = nonischemic zone and lane 4 = ischemic zone in the IP group. IP significantly attenuated the presence of DNA ladders in ischemic myocardium.

View larger version (125K): [in this window] [in a new window]   Fig 3. Detection of apoptotic myocytes using the terminal transferase UTP nick end-labeling (TUNEL) technique in nonischemic zones (A) in the control group and (B) in the IP group, and ischemic zones (C) in the control group and (D) in the IP group. Red staining indicates apoptotic myocytes in ischemic myocardium (x100). Apoptotic myocytes were not detected in nonischemic zone after ischemia and reperfusion.

	Comment

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The role of neutrophils in the development of myocardial infarction after irreversible ischemia and reperfusion has been well documented [17–19]. However, the role of neutrophils in apoptosis is not understood. Neutrophils adherent to the vascular endothelium during the early phase of reperfusion not only directly induce endothelial damage, but also elicit myocyte injury by inflammatory mediators released from activated neutrophils. Although the factors that may induce apoptosis in the ischemic/reperfused myocardium are not fully known, mediators such as reactive oxygen species and various cytokines released from accumulated neutrophils in ischemic/reperfused myocardium have been suggested as triggers in the development of apoptosis [13, 14, 20]. As shown in the present study, IP significantly decreases neutrophil accumulation in area at risk myocardium. This observation is in agreement with Gross and Auchampach [21], in which inhibition of neutrophil accumulation in the nonlethal coronary artery occlusion may be of significance for the reduction of myocardial apoptosis. Furthermore, to explore the protective mechanisms of IP for the reduction in infarct size, myoprotective agents released during IP such as endogenous adenosine and endothelium-derived relaxing factor (nitric oxide) have been reported to be involved in IP-mediated cardioprotection [22, 23]. It is unclear, however, whether these processes are also involved in the IP-reduced apoptosis seen in the present study.

We found that 30 minutes of LAD coronary occlusion produced a sustained depression in maximal dp/dt that persisted throughout the reperfusion period. In the absence of observed changes in heart rate and arterial pressure, this decrease in maximal dp/dt may suggest a concomitant decrease in left ventricular contractile dysfunction. IP significantly improved the maximal dp/dt during 90 to 180 minutes of reperfusion, suggesting a better recovery of contractile function under the hemodynamic conditions observed. Although we did not measure regional or global contractile function directly using ultrasonic dimension transducers or impedance catheter techniques, respectively, conflicting results regarding effects of IP on postischemic regional contractile dysfunction have been reported. Cohen and associates [24] demonstrated that in an infarct rabbit model IP improved wall motion after 30 minutes of coronary occlusion. In contrast, Ovize and associates [25] found that in the noninfarct dog model, IP did not attenuate myocardial stunning after 15 minutes of coronary occlusion. In the present study, although we can not conclude the protective effect of IP on regional contractile function from this noninfarct model, several explanations for the increased tolerance to ischemia induced by IP have been proposed. IP slows down the rate of ATP depletion during subsequent initial minutes of coronary occlusion [5], stimulates cardiac A₁-receptors by adenosine released during preconditioning ischemia [22], and opens ATP-sensitive potassium channels [21].

Postischemic perfusion defects are common consequences of ischemia/reperfusion injury. In the present study, myocardial blood flow to the area at risk in the control group showed a progressive decrease in perfusion over the 3 hour reperfusion period. Although improvement in postischemic blood flow by IP at the end of reperfusion did not reach significance compared with the control group, there was a trend toward a better recovery. It has been proposed that neutrophils play an important role in myocardial perfusion defects after ischemia and reperfusion [17–19, 26]. Accumulated neutrophils at the site of reperfused myocardium may mechanically occlude the capillary, and also may effect endothelial function by releasing neutrophil-derived superoxide anion and soluble proinflammatory mediators. In addition, interstitial edema, increased vascular permeability, endothelial cell swelling, as well as impaired release of vasodilator substances (ie, nitric oxide, adenosine), and enhanced release of vasoconstrictor substances (ie, endothelin, superoxide anion) secondary to damaged endothelium may all participate in microvascular perfusion defects after reperfusion. IP significantly attenuated neutrophil accumulation in the area at risk, as indicated by a reduction of tissue myeloperoxidase activity in the present study, it can not eliminate, however, all ischemia/reperfusion-induced damage in the microvasculature. This may partly explain why IP only had modest protective effects on postmyocardial blood flow.

In conclusion, the present study provides evidence that a nonlethal duration of coronary occlusion increases neutrophil accumulation and triggers both endothelial dysfunction and myocardial apoptosis. IP, as one of the most potent protective methods to attenuate cell death and endothelial dysfunction secondary to ischemia and reperfusion, modestly improved postischemic myocardial blood flow, decreased neutrophil accumulation, and inhibited myocardial apoptosis in the area at risk. These results suggest that IP may be applied to protect the heart from other manifestations of ischemia/reperfusion injury (endothelial dysfunction, apoptosis), although its role in attenuating regional contractile dysfunction is still controversial. The application of IP in off-pump cardiac surgery may attenuate apoptosis and vascular defects related to obligatory short-term coronary occlusion.

	Acknowledgments

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This work was supported by grants from National American Heart Association (Scientist Development Award, Z.-Q. Zhao) and (Grant-In-Aid, J. Vinton-Johansen) and the Carlyle Fraser Heart Center Foundation of Crawford Long Hospital and Emory University School of Medicine.

We thank L. Susan Schmarkey, Sara L. Katzmark, and Jill Robinson for their technical assistance.

	References

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