Methods

All animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (National Institutes of Health Publication 85–23, revised 1985). The protocol was also approved by the Animal Care and Use Committee of the Bowman Gray School of Medicine of Wake Forest University.

Twenty-two mongrel dogs of either gender were initially anesthetized with sodium thiamylal (20 mg/kg i.v.). After endotracheal intubation and cannulation of the right femoral vein, deep anesthesia was maintained by continuous infusion of 0.3 μg/kg/min fentanyl citrate and 0.03 mg/kg/min diazepam. The dog was ventilated with oxygen-enriched room air using a Harvard volume-cycled respirator to maintain arterial pO₂ greater than 100 mm Hg. Normal blood gas levels and acid-base status were maintained by adjustment of the rate and volume of respiration or by i.v. administration of sodium bicarbonate, as appropriate. A left thoracotomy was performed at the fifth intercostal space, and the heart was suspended in a pericardial cradle. Millar MPC-500 solid-state pressure transducers (Millar Instruments, Houston, TX) were inserted into the aortic rootvia the right internal mammary artery and into the LV through a puncture in the apex of the heart, to measure MAP and instantaneous LV blood pressure, respectively. An umbilical snare was placed around the inferior vena cava for transient preload reduction. A pair of piezoelectric crystals were placed into the subendocardium of the area of the LV perfused by the LAD in the direction of circumferential fiber shortening, to measure segmental systolic and diastolic length. Anticoagulation was achieved with bolus injections of 300 U of sodium heparin given every 90 min, to maintain activated clotting times of >400 sec. The left carotid artery was cannulated and connected to a large-bore silastic catheter in which a Transonic ultrasonic cannulating-type flow probe (Transonic Systems, Ithaca, NY) was interposed. This circuit was capable of accommodating up to 95-ml/min flow at 100 mm Hg mean pressure. A proximal segment of the LAD was isolated and cannulated with a 14-gauge angiocatheter and immediately perfused via the left carotid artery. Ischemia encountered during the cannulation process lasted no longer than 40 sec and did not alter segmental function, relative to the precannulation period.

Experimental protocol.

Hemodynamic and segmental function data were collected before ischemia for baseline, at the end of ischemia and at 15, 60, 120 and 180 min of reperfusion. After base-line measurements were recorded, 60 min of collateral-deficient ischemia was created by clamping the carotid-LAD perfusion circuit, disconnecting the perfusion circuit from the coronary catheter and venting the collateral blood flow into a beaker. Arteriotomy and collateral diversion reduce the variability in infarction size observed in the canine model of ischemia-reperfusion by reducing the nutritive collateral blood flow (Eng et al., 1987). After 55 min of collateral diverted ischemia, an intracoronary infusion of either CGS-21680 at 0.2 μg/kg/min (n = 8) or vehicle (n = 8) was begun by reconnecting the extracorporeal circuit to the cannula in the LAD, while the circuit remained clamped, and inserting a 25-gauge needle into the silastic tubing just proximal to the cannula and distal to the clamp. At the end of 60 min of ischemia, reperfusion was initiated by removing the clamp, and the intracoronary infusion of CGS-21680 or vehicle was continued for the next 55 min. The AAR was reperfused for a total of 3 hr.

Drug preparation.

CGS-21680 was prepared from a dry powder and dissolved in 0.5 ml of dimethylsulfoxide. When completely dissolved, the drug was diluted with normal saline to achieve a dose of 0.2 μg/kg/min, to be delivered by 60-min intracoronary infusion. Fresh drug was prepared for each experiment. The saline vehicle was prepared with dimethylsulfoxide and saline in the same concentration as used in the CGS-21680-treated group. The dose of drug was calculated to deliver a concentration of drug of about 180 nM, which was a compromise between maximal neutrophil inhibition determined by in vitrotests and systemic vasodilation.

Data collection and analysis.

LV and aortic pressures, segment length data and LAD blood flow measurements were collected in duplicate or triplicate during 12-sec intervals of respiratory apnea. Data were digitized at 200 Hz using a 12-bit analog-to-digital converter (model DT2821; Data Translation Devices, Marlboro, MA) and an IBM-PC computer. Data were stored both on removable media and on the hard disk for archival and subsequent analysis. Two measurements were taken during steady state and three measurements were taken during caval occlusion (for determination of segmental diastolic stiffness). The data were processed using SPECTRUM (Triton Technology, San Diego, CA), a video-graphical interface computer algorithm program developed in our laboratory. The beginning of systole was marked when instantaneous LV dP/dt exceeded 250 mm Hg/sec, and the end-systolic point was marked at peak negativedP/dt. These points were checked visually and adjusted manually as necessary. Segmental shortening was calculated by the formula [(EDSL − ESSL)/EDSL] × 100 and expressed as a percentage. Segmental work was calculated using a point-by-point integration of the pressure-segment length loop over the entire cardiac cycle. Segmental stiffness was calculated by fitting the end-diastolic pressure-segment length data of the variably preloaded pressure-segment length loops obtained during caval occlusion to the equationP _ED = α(e ^{β L}), whereP _ED is the end-diastolic pressure, α and β coefficients are the end-diastolic pressure axis intercept and the degree of curvature, respectively, and L is the EDSL. The β coefficient is a unitless measure of the curvature of the end-diastolic relation (modulus of stiffness).

Determination of AAR and infarction size.

At the end of the experiment, the heart was rapidly arrested with a bolus injection of sodium pentobarbital and excised. The proximal aorta was cannulated, and the heart was stained using a gentian violet infusion deliveredvia the ascending aorta (infusion pressure was maintained at 100 mm Hg). The atria and the right ventricle were removed, and the LV was cut into 3- to 4-mm-thick sections. The unstained region (AAR) was then separated from the blue-stained nonischemic area and incubated for 10 min in a 1% solution of triphenyltetrazolium chloride solution in phosphate buffer, warmed to 37°C. After incubation, all tissues were placed in a 10% formalin solution overnight. The AAR was then subdivided into pale necrotic and brick-red nonnecrotic areas. The AAR and area of necrosis were determined gravimetrically and expressed as a percentage of the total LV mass. The AAR was calculated as 100 × [(mass of necrotic tissue + mass of nonnecrotic ischemic-reperfused tissue)/total mass of LV]. This gravimetric method of infarction sizing compares favorably with the planimetric method (Vinten-Johansenet al., 1992).

Plasma CK activity.

Blood samples for measuring plasma CK activity were taken from the left femoral artery at the same time points at which hemodynamic data were collected. The plasma was analyzed spectrophotometrically for total CK activity (CK-10 kit; Sigma Diagnostics, St. Louis, MO) and protein content. CK levels were expressed as international units per gram of protein.

Cardiac MPO activity.

Tissue samples were taken from the nonischemic zone and the nonnecrotic and necrotic zones of the AAR for spectrophotometric analysis of MPO activity as a measure of neutrophil accumulation in the myocardium, using a method described previously (Mullane et al., 1985; Sato et al., 1995). MPO activity (units per gram of tissue) was calculated as micromoles per minute = ΔA ₄₆₀/11.3 μmol/(CM × milliliters of reactant ×D)/sample weight, where ΔA ₄₆₀ is equal to the absorbance change over time at 460 nm, 11.3 is the molar extinction coefficient for H₂O₂ at 460 nm,CM represents the cuvette path length and D is the volume of supernatant used. One unit of MPO activity is defined as the enzyme activity degrading 1 mmol of H₂O₂/min at 25°C.

In vitro PMN adherence assay.

Adherence of canine neutrophils to the endothelial cell surface of normal, homologous, coronary arteries (from naive animals not subjected to ischemia and reperfusion) was assessed using neutrophils labeled with Zynaxis PKH26 (Zynaxis Cell Science, Malvern, PA) vital fluorescent dye. One milliliter each of diluent and PKH26 dye (4 μM) was added to a neutrophil suspension of 5 × 10⁶ cells/ml and incubated for 5 min. Two milliliters of PBS containing 10% plasma were subsequently added to stop the labeling reaction, and another 5 ml of PBS were added to the cell suspension. The cells were then centrifuged at 600 × g for 10 min at 4°C. The pellet was resuspended in PBS, and the cells were counted by an automated Coulter counter to determine the population per milliliter of cell suspension. This labeling procedure yields cells with normal function and morphology. In validation studies performed with the labeling procedure, unlabeled and labeled neutrophils demonstrated 95% and 97% viability, respectively, using trypan blue exclusion.

Canine coronary arteries were carefully isolated in cold oxygenated Krebs-Henseleit buffer, so as not to disturb the endothelium. After removal of the fat and connective tissue, the arteries were cut into segments 2 to 3 mm in length. These segments were carefully opened and placed in plastic dishes containing 3 ml of Krebs-Henseleit buffer at 37°C. Labeled neutrophils (4 × 10⁶ cells) were added to the bath alone or in combination with different concentrations of CGS-21680 (1–50 μM). PAF (100 nM) was added to the dishes 5 min after incubation with CGS-21680, and the incubation was continued for an additional 15 min. After incubation, the segments were removed, washed three times with Krebs-Henseleit buffer, placed on glass slides (endothelial side up) and covered with immersion oil and a glass coverslip. Adherence was measured by counting the number of neutrophils adhering to the endothelial surface in six separate microscopic fields under epifluorescence microscopy (490-nm excitation, 504-nm emission).

In vitro superoxide production by PMNs.

Adherence-independent superoxide radical production by activated neutrophils was determined by measuring the SOD-inhibitable reduction of ferricytochrome c to ferrocytochrome c. Neutrophils (5 × 10⁶ cells/ml) were prewarmed to 37°C in the presence of 160 μM cytochrome c and incubated with cytochalasin B (5 μg/ml) and CGS-21680 (1–50 μM) for 5 min. The neutrophils were then stimulated with PAF (100 nM) in a final reaction volume of 0.5 ml. The tests were run in two groups, with and without SOD, to correct for nonspecific activity or color generation. The tubes were centrifuged at 500 × g for 10 min at 4°C. Cytochrome c reduction was measured spectrophotometrically by determining the optical density of the supernatant at 500 nm, using a V_max kinetic microtiter plate reader (Molecular Devices, Palo Alto, CA). Superoxide radical production was calculated using an extinction coefficient of 21 mM⁻¹ cm⁻¹ for cytochrome c. Results are reported as nanomoles of SOD-inhibitable O₂ ⁻ produced by a suspension of 5 × 10⁶ neutrophils during the 5-min measurement period.

Statistical analysis.

Data are expressed as mean ± S.E.M. Differences were considered significant when P < .05. Differences in end-point variables between groups were analyzed by Student’s t test. Time-related differences within groups and group differences were analyzed by two-way analysis of variance for repeated measures, with post hoc t tests to determine the points that differed at each time. In vitro data were analyzed with one-way analysis of variance followed by Duncan’s multiple comparisons analysis.