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Ann Thorac Surg 1997;64:1099-1107
© 1997 The Society of Thoracic Surgeons

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Original Article: Cardiovascular

Gradual Reperfusion Reduces Infarct Size and Endothelial Injury but Augments Neutrophil Accumulation

Department of Cardiothoracic Surgery, Bowman Gray School of Medicine, Winston-Salem, North Carolina

Accepted for publication April 22, 1997.

	Abstract

Top Footnotes Abstract Introduction Material and Methods Results Coronary Blood Flow to... Segmental Function Segmental Stiffness Infarct Size Plasma Creatine Kinase Activity Tissue Myocardial... In Vitro Coronary Artery... Comment Acknowledgments References

 
Background. Reperfusion causes injury to the coronary artery endothelium primarily by neutrophil-mediated mechanisms. However, factors other than neutrophils may govern the extent of myocardial necrosis. This study tests the hypothesis that gradual initiation of reflow will reduce reperfusion injury and preserve postischemic endothelial function.

Methods. In 16 anesthetized dogs, the left anterior descending artery was ligated for 60 minutes. In one group, reperfusion was initiated abruptly (abrupt, n = 8), whereas in the gradual reperfusion group (ramp, n = 8), flow was slowly initiated during the first 30 minutes of reperfusion. After reperfusion, coronary artery segments were isolated to assess postischemic endothelial function.

Results. Infarct size (area of necrosis/area at risk) was significantly reduced in the ramp group (28.2% ± 2.0%) compared with abrupt (41.6% ± 1.4%). Neutrophil accumulation (myeloperoxidase) in the area at risk was significantly greater in the ramp group compared with abrupt (8.0 ± 1.3 versus 3.5 ± 0.8 U/g tissue). In isolated postischemic left anterior descending arterial rings, the concentration of acetylcholine that elicited a response 50% of the maximum possible response was significantly greater in abrupt (-6.88 ± 0.04 log [mol/L]) than ramp (-7.62 ± 0.04 log [mol/L]) and control (-7.68 ± 0.003 log [mol/L]), suggesting endothelial dysfunction. The concentration of A23187 that elicited a response 50% of the maximum possible response was similarly greater in abrupt (-7.24 ± 0.03 log [mol/L]) versus ramp (-7.62 ± 0.03 log [mol/L]) and control (-7.8 ± 0.04 log [mol/L]). Smooth muscle dysfunction (response to sodium nitrite) also occurred in the abrupt rings.

Conclusions. Gradual reperfusion of an ischemic area reduces infarct size and preserves endothelial function but paradoxically increases neutrophil accumulation within the area at risk.

	Introduction

Top Footnotes Abstract Introduction Material and Methods Results Coronary Blood Flow to... Segmental Function Segmental Stiffness Infarct Size Plasma Creatine Kinase Activity Tissue Myocardial... In Vitro Coronary Artery... Comment Acknowledgments References

 
Reperfusion salvages myocardium that would otherwise succumb to necrosis with delayed or failed reperfusion. However, numerous studies have shown that reperfusion imposes deleterious effects that either exaggerate or manifest injury not present at the end of ischemia [1–3]. This "reperfusion injury" may lead to more extensive necrosis and contractile or diastolic dysfunction that may be treated with target-specific therapy aimed at various contributors to the inflammatory-like responses after reperfusion [1]. The actions of neutrophils have been particularly emphasized in the pathophysiology of postischemic injury [4, 5]. Neutrophils and neutrophil-derived products are key in injuring the vascular endothelium and in the pathogenesis of necrosis. In many studies, a reduction in postischemic injury to specific interventions has been attributed to reduction of neutrophil accumulation and inhibition of superoxide production [6, 7].

Recent studies using surgical models of ischemia, involving cardioplegic arrest and subsequent reperfusion, have developed a strategy in which reperfusion injury can be significantly reduced by modifying the conditions of reperfusion, as well as the composition of the initial reperfusate [2, 8]. In this regard, "gentle reperfusion" at a low intracoronary pressure has been an effective modification of the conditions of reperfusion that have reduced postischemic injury [9, 10]. This concept of gentle reperfusion has been adopted not only in delivery of cardioplegic solutions, but also for the initiation of reperfusion after unclamping the aorta [11].

Recently, we reported that gradual restoration of coronary blood flow during the initial 30 minutes of reperfusion to achieve a "gentle reperfusion" reduced infarct size and postischemic myocardial blood flow defects in a nonsurgical model of ischemia and reperfusion [12]. In the current study, we tested the hypothesis that controlled hydrodynamics of reperfusion reduces postischemic coronary artery endothelial dysfunction and inhibits neutrophil accumulation in the area at risk. We found that gradual reperfusion (1) reduced infarct size, (2) attenuated coronary artery endothelial dysfunction to stimulators of nitric oxide, but (3) paradoxically increased neutrophil accumulation in the area at risk.

	Material and Methods

Top Footnotes Abstract Introduction Material and Methods Results Coronary Blood Flow to... Segmental Function Segmental Stiffness Infarct Size Plasma Creatine Kinase Activity Tissue Myocardial... In Vitro Coronary Artery... Comment Acknowledgments References

 
All animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH 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 microfilaria-free mongrel dogs of either sex were initially anesthetized with intravenous 20 mg/kg sodium thiamylal. After endotracheal intubation and cannulation of the left femoral vein, 350 µg fentanyl citrate and 5 mg diazepam were infused for deep anesthesia followed by an infusion of fentanyl (0.3 µg • kg^-1 • min^-1) and diazepam (0.03 mg • kg^-1 • min^-1). The dog was ventilated with oxygen-enriched room air using a Harvard volume-cycled respirator to maintain arterial oxygen tension greater than 100 mm Hg. Arterial carbon dioxide tension was maintained between 35 and 45 mm Hg by adjusting respiratory volume, rate, or both. Metabolic acidosis was counteracted with intravenous sodium bicarbonate as necessary to maintain pH between 7.35 and 7.45. The chest was opened by median sternotomy and a pericardial cradle was formed. Millar MPC-500 (Millar Instruments, Inc, Houston, TX) solid-state pressure transducers were inserted into the aortic root through the right internal mammary artery and into the left ventricle (LV) through an apical stab wound to measure instantaneous aortic and left ventricular blood pressures, respectively. Umbilical tape snares were placed loosely around the inferior and superior venae cavae for later transient bicaval occlusion to measure regional diastolic function. A proximal portion of the left anterior descending (LAD) coronary artery was dissected free and loosely encircled with a 3-0 suture. A pair of 2.5-mm diameter, 5-MHz piezoelectric ultrasonic crystals were implanted in the subendocardium of the myocardium perfused by the LAD to measure segmental systolic contractile function and diastolic characteristics using a model 120 sonomicrometer (Triton Technology, Inc, San Diego, CA). The left carotid artery was cannulated with a silicone elastomer catheter. The LAD was cannulated with a 12-gauge Angiocath and immediately perfused by the carotid catheter. The cannulation procedure interrupted LAD blood flow for an average of 41 ± 5 seconds. Left anterior descending arterial blood flow was measured by a cannulating flow probe interposed in the circuit from the carotid to the LAD and connected to an ultrasonic blood flow meter (Transonics Systems, Inc, Ithaca, NY).

Experimental Protocol
Hemodynamic and segmental function data were collected in the baseline state. The carotid–LAD fistula was clamped to occlude blood flow for 60 minutes of ischemia. The LAD catheter was disconnected to vent collateral blood flow, thereby creating a model of coronary collateral flow-diverted ischemia. Previous studies have shown that diversion of collateral blood flow in this manner produces a consistent infarct size without the necessity of correlating infarct size with collateral blood flow by microspheres [13]. After 60 minutes of LAD occlusion, the extracorporeal circuit was reconnected and reperfusion was reinstituted in one of two ways: (1) the clamp on the carotid–LAD shunt was immediately released, and blood flow was abruptly restored (abrupt group); or (2) reperfusion was gradually restored by slowing releasing an occlusive clamp on the carotid–LAD shunt, and gradually increasing blood flow during the next 30 minutes following an exponential trajectory in blood flow [12]. In both groups, reperfusion was continued for 3 hours. The perfusion circuit could supply approximately 100 mL/min blood at a perfusion pressure of 100 mm Hg, and therefore was not an impairment to reactive hyperemic blood flow.

Data Collection and Analysis
Hemodynamic data, including instantaneous left ventricular, aortic, and systemic arterial blood pressures, myocardial segment length data from the area at risk, and mean LAD blood flow were acquired in triplicate during 12-second periods of respiratory apnea at baseline, at the end of ischemia, and at 15, 60, 120, and 180 minutes of reperfusion using computerized acquisition as described previously [14]. The data were analyzed by computer using an interactive video graphics program developed in our laboratory (SPECTRUM Cardiovascular Data Acquisition and Analysis System; Bowman Gray School of Medicine and Triton Technology, San Diego, CA). Percent segmental shortening was calculated as 100 x [(EDL - ESL)/EDL], where EDL and ESL are end-diastolic length and end-systolic length, respectively. Segmental work per beat was calculated using point-by-point integration of the pressure-segment length loop during the entire cardiac cycle. The characteristics of segment stiffness were determined by fitting the end-diastolic pressure-segment length data of the variably loaded pressure-segment length loops obtained during bicaval occlusion to the exponential relationship P_ed = (e^ßLed), where P_ed is the end-diastolic pressure, (mm Hg) and ß (unitless) are coefficients that measure the end-diastolic pressure axis intercept and the degree of curvature, respectively, and L_ed is the end-diastolic segment length.

Determination of Area at Risk and Infarct Size
At the end of each experiment, the LAD perfusion circuit was again occluded, and 5 mL of Unisperse blue dye (Ciba-Geigy, Newport, DE) was injected into the left atrium and allowed to circulate for at least 10 seconds to demarcate the in vivo area at risk (AAR). The heart was then rapidly arrested with a bolus injection of 300 mg sodium pentobarbital and excised, and the necrotic tissue within the AAR was identified using a 1% solution of 37°C triphenyltetrazolium chloride in phosphate buffer (pH 7.4). The AAR and area of necrosis (AN) were determined gravimetrically as described previously [12, 14]. The AAR was calculated as a percent of the left ventricular mass as [(weight of nonnecrotic + necrotic area at risk)/(total weight of LV) x 100]. The AN as a percent of the left ventricular mass (AN/LV) was calculated as [(weight of necrotic tissue in area at risk)/(weight of LV) x 100]. The AN as a percent of the AAR (AN/AAR) was calculated as [(weight of necrotic tissue in area at risk/total AAR) x 100].

Plasma Creatine Kinase Activity
Blood samples for measuring creatine kinase (CK) activity were withdrawn from the femoral artery at the same time points as contractile function was assessed. The blood was centrifuged, and the plasma was analyzed spectrophotometrically for CK activity (CK-10 Kit; Sigma Diagnostic, St. Louis, MO) and protein concentration (Sigma Diagnostic). Creatine kinase activity was expressed as international U/g of protein.

Cardiac Myeloperoxidase Activity
Tissue samples weighing approximately 0.4 g were taken from the nonischemic zone and from the nonnecrotic and necrotic areas of the AAR for spectrophotometric analysis of myeloperoxidase (MPO) activity as an assessment of neutrophil accumulation (resident, adherent, embolized) in myocardium as described in detail previously [15]. Previous experiments have shown that Unisperse blue dye and triphenyltetrazolium chloride staining do not interfere with the MPO assay.

In Vitro Coronary Artery Ring Studies
Both the ischemic-reperfused LAD and nonischemic circumflex artery were carefully dissected from the heart after excision, placed in cold Krebs-Henseleit solution, and cleaned of adipose and connective tissue. The isolated coronary artery segments from the LAD and circumflex artery were each cut into four rings of approximately 2 mm in length. The rings were mounted on stainless steel hooks, placed in organ chambers that were filled with Krebs-Henseleit solution (37°C, gassed with 95% O₂–5% CO₂), and connected to isometric force transducers (model TR001; Radnoti, Monrovia, CA). Changes in isometric force were digitized at 3 Hz using an analog to digital converter (DT2827; Data Translation, Marlboro, MA) and IBM PC computer. After 60 minutes of equilibration, the rings were placed at the optimal point of their length-tension relationship and incubated with 10 µmol/L indomethacin to prevent vascular responses to endogenous prostacyclin. Dose-response curves to the thromboxane A₂ mimetic agent, U46619 (Upjohn), were performed and cumulative concentration-response curves to acetylcholine (concentrations from 0.01 to 1 mmol/L; Sigma), a muscarinic receptor-dependent, endothelium-dependent stimulator of nitric oxide synthase, were obtained. The rings were washed several times with KH solution and equilibrated to baseline levels of passive tension. Dose-dependent vascular responses were also determined for the calcium ionophore A23187, a non–receptor-mediated, endothelium-dependent stimulator of nitric oxide synthase, and for acidified (pH 2.0) sodium nitrite (NaNO₂), an endothelium-independent smooth muscle relaxing agent and donor of nitric oxide. Relaxation is expressed as a percentage of U46619-induced constriction. The concentration of the drug that elicts a response 50% of the maximal possible response (EC₅₀, log [mol/L]) was calculated as the dose of the drug required to cause 50% relaxation from preconstricted levels. Neither saline solution at pH 2.0 nor NaNO₂ at pH 7.4 elicited relaxation responses. Drug concentrations are expressed as the final concentration in the organ chamber. Left anterior descending arterial segments were also taken from four hearts not subjected to coronary ischemia and reperfusion to serve as controls.

Statistical Analysis
Two-way analysis of variance for repeated measures was used to determine if time- and group-related differences existed in hemodynamic, functional, and CK data. If a significant difference was found, Duncan's multiple range test was applied to locate the source of differences. Time and group–time multiple comparisons were corrected for appropriate interactions. A one-way analysis of variance was used to analyze discreet variables such as AAR, infarct size, MPO data. Means and standard errors of the mean are reported. Significance was assigned at less than or equal to 5% probability (p 0.05).

	Results

Top Footnotes Abstract Introduction Material and Methods Results Coronary Blood Flow to... Segmental Function Segmental Stiffness Infarct Size Plasma Creatine Kinase Activity Tissue Myocardial... In Vitro Coronary Artery... Comment Acknowledgments References

 
Hemodynamic Data
Hemodynamic data during the course of the experiment are shown in Table 1. There were no significant differences between the two groups in any of the hemodynamic variables at baseline. During ischemia, heart rate increased significantly in each group, but with no significant differences between groups. There was a tendency for mean aortic pressure to be higher in the ramp group compared with the abrupt group, but this was statistically significant only at 60 minutes of reperfusion. Neither maximum rate of increase of LV pressure (dP/dt_max) nor the pressure-rate product were different between either group at any time.

	Coronary Blood Flow to the Area at Risk

Top Footnotes Abstract Introduction Material and Methods Results Coronary Blood Flow to... Segmental Function Segmental Stiffness Infarct Size Plasma Creatine Kinase Activity Tissue Myocardial... In Vitro Coronary Artery... Comment Acknowledgments References

View larger version (21K): [in this window] [in a new window]   Fig 1. . Mean left anterior descending (LAD) coronary artery blood flow (mL/min) through the carotid–LAD shunt during the course of the experiment. (Cntl = control; Isch = ischemia; open boxes = abrupt group; filled circles = ramped reperfusion group; *p < 0.05 versus abrupt group.)

	Segmental Function

Top Footnotes Abstract Introduction Material and Methods Results Coronary Blood Flow to... Segmental Function Segmental Stiffness Infarct Size Plasma Creatine Kinase Activity Tissue Myocardial... In Vitro Coronary Artery... Comment Acknowledgments References

View larger version (25K): [in this window] [in a new window]   Fig 2. . (A) Segmental work in the area at risk during baseline (cntl), ischemia (Isch), and 15, 60, 120, and 180 minutes of reperfusion. All data at the ischemia and reperfusion time points were different from their respective group baseline value. (B) The modulus of segmental diastolic stiffness determined as the curvature of the exponential end-diastolic pressure-length relationship during transient bicaval occlusion. The modulus of stiffness (ß coefficient) is a unitless number. There were no group differences.

	Segmental Stiffness

Top Footnotes Abstract Introduction Material and Methods Results Coronary Blood Flow to... Segmental Function Segmental Stiffness Infarct Size Plasma Creatine Kinase Activity Tissue Myocardial... In Vitro Coronary Artery... Comment Acknowledgments References

	Infarct Size

Top Footnotes Abstract Introduction Material and Methods Results Coronary Blood Flow to... Segmental Function Segmental Stiffness Infarct Size Plasma Creatine Kinase Activity Tissue Myocardial... In Vitro Coronary Artery... Comment Acknowledgments References

 
The mass of the LV was comparable between the two groups, averaging 117.2 ± 8.4 g in the abrupt group and 107.8 ± 6.6 g in the ramp group. The AAR as a percentage of the left ventricular mass was also comparable between groups (p = 0.52) (Fig 3A). The AN as a percentage of the left ventricular mass was significantly reduced in the ramp group compared with the abrupt group (p = 0.02) (see Fig 3A). The AN, normalized to the AAR, was reduced by 32.7% in the ramp group compared with the abrupt group.

View larger version (19K): [in this window] [in a new window]   Fig 3. . (A) Size of the area at risk (AAR) relative to the left ventricular mass and infarct size represented as area of necrosis (AN) relative to the AAR (AN/AAR) and left ventricle (AN/LV). (B) Total plasma creatine kinase (CK) levels expressed as international units (IU) per gram of protein during the course of the experiment. (*p < 0.05 versus abrupt group; abbreviations are as in Figure 1.)

	Plasma Creatine Kinase Activity

Top Footnotes Abstract Introduction Material and Methods Results Coronary Blood Flow to... Segmental Function Segmental Stiffness Infarct Size Plasma Creatine Kinase Activity Tissue Myocardial... In Vitro Coronary Artery... Comment Acknowledgments References

	Tissue Myocardial Myeloperoxidase Activity

Top Footnotes Abstract Introduction Material and Methods Results Coronary Blood Flow to... Segmental Function Segmental Stiffness Infarct Size Plasma Creatine Kinase Activity Tissue Myocardial... In Vitro Coronary Artery... Comment Acknowledgments References

 
Myeloperoxidase activity in ischemic and nonischemic myocardium is shown in Figure 4. In the nonischemic zone, MPO activity was low and was comparable between the two groups. In the triphenyl tetrazolium chloride-positive (nonnecrotic) area at risk, MPO activity was 127% greater in the ramp group than in the abrupt group (p = 0.004). A similar pattern was demonstrated in the necrotic tissue within the AAR, with MPO activity in the ramp group being 118% greater than that in the abrupt group (p = 0.0001). Therefore, a reduction in infarct size in the ramp group was associated paradoxically with an increase in neutrophil accumulation.

View larger version (14K): [in this window] [in a new window]   Fig 4. . Myeloperoxidase (MPO) activity in units per gram tissue in the nonischemic zone (NIZ), the nonnecrotic ischemic zone (IZ), and the necrotic ischemic zone (NEC). (*p < 0.05 versus abrupt group.)

	In Vitro Coronary Artery Vascular Function

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ENDOTHELIUM-DEPENDENT RELAXATIONS.
Figure 5A shows vasodilator responses to the endothelium-dependent and muscarinic (M₂) receptor-mediated vasodilator acetylcholine in the LAD rings. The concentration response curves in LAD rings from the ramp and abrupt groups were shifted markedly to the right compared with that of the control group, without a reduction in the maximum relaxation. However, the responses in the ramp group were significantly (p < 0.05) greater at a given concentration of acetylcholine than those in the abrupt group. The EC₅₀ also increased in the ramp (-7.04 ± 0.04 log [mol/L]) and abrupt (-6.88 ± 0.04 log [mol/L]) groups compared with the control (-7.68 ± 0.03 log [mol/L]) group, but the increase in EC₅₀ of the ramp group was significantly (p < 0.05) less compared with the abrupt group (Table 3).

View larger version (24K): [in this window] [in a new window]   Fig 5. . Concentration-response curves of left anterior descending coronary artery segments to acetylcholine (A), the calcium ionophore A23187 (B), and the smooth muscle relaxing agent, acidified NaNO2 (C). (*p < 0.05 abrupt or ramp group versus control; **p < 0.05 abrupt group versus ramp group; p < 0.05 versus previous time point.)

ENDOTHELIUM-INDEPENDENT RELAXATIONS.
There were no differences between the control and the ramp groups (Figure 5C) in response to NaNO₂. However, the curves in the abrupt group were modestly shifted to the right compared with the control and the ramp groups. The EC₅₀ also was greater in the abrupt (-5.07 ± 0.05 log [mol/L]) group compared with the control (-5.46 ± 0.05 log [mol/L]) and the ramp (-5.34 ± 0.04 log [mol/L]) groups (Table 3). These data indicate that gradually restoring the reperfusion flow (ramp) protects not only the coronary receptor-mediated and nonmediated endothelial function but also smooth muscle function from ischemia and reperfusion damage.

	Comment

Top Footnotes Abstract Introduction Material and Methods Results Coronary Blood Flow to... Segmental Function Segmental Stiffness Infarct Size Plasma Creatine Kinase Activity Tissue Myocardial... In Vitro Coronary Artery... Comment Acknowledgments References

 
During reperfusion of ischemic areas of the heart, the myocardium is susceptible to damage manifest as microvascular injury, endothelial cell dysfunction, and infarct extension secondary to no-reflow, edema, and necrosis. Neutrophils play an important role in the inflammatory-like component of reperfusion injury pathophysiology [1, 16]. In the current study, we found that gentle coronary reperfusion following an incremental, or "ramped," protocol for increase in flow for the first 30 minutes of reflow reduced postischemic infarct size by 33% compared with an abruptly reperfused group, a result consistent with previous studies [12, 17]. This reduction in infarct size was associated with a significant decrease in plasma CK activity. In addition, gentle reperfusion attenuated endothelial injury. This preservation of endothelial function and reduction in infarction was not, however, associated with better postischemic systolic or diastolic function. Paradoxically, gentle reperfusion increased the accumulation of neutrophils in both the nonnecrotic and necrotic areas at risk.

Numerous studies have demonstrated that a reduction in neutrophil activity and accumulation is associated with a concomitant reduction in infarct size [7, 14]. However, this was clearly not the mechanism operative in this study as neutrophils accumulated to a greater extent in the ramped reperfusion group. A likely mechanism for the observed reduction in infarction is that gentle reperfusion, with lower intracoronary pressure, reduced the transmigration of fluid from the vascular space to the interstitial space (ie, reduced interstitial edema). Coronary hydrostatic pressure is a major coefficient in the Starling forces relating the movement of fluids between the intravascular and interstitial compartments. Under normal conditions, the intravascular–interstitial hydrostatic pressure differential driving the movement of water into the interstitial spaces is partially counterbalanced by forces applied in the opposite direction (ie, tissue pressure and oncotic pressure). The slight overdrive toward movement of water into the interstitium is drained by the lymphatic system, thereby maintaining fluid balance in the tissue. In ischemia-reperfusion, disruptions in the tight junctions and increases in permeability of the endothelial cells by humoral factors (histamine, leukotriene B₄) increase the facility with which water moves interstitially at any given driving pressure. The resulting interstitial edema enhances extravascular compressive forces and hence may ablate microvascular patency and impede the distribution of blood flow to the subserved region, producing areas of no-reflow. A reduction in the intracoronary pressure during the period of ramping, measured in a previous study [12], may reduce the hydrostatically driven component of water migration toward the interstitium and, hence, may reduce defects in regional blood flow distribution. Consistent with this, the distribution of blood flow to the ischemic-reperfused myocardium was improved by ramped reperfusion in our previous study [12] with the greatest improvement being shown in the subendocardium. In the abruptly reperfused group, there was a pronounced defect in blood flow distribution to the subendocardium [12].

In most studies of ischemia-reperfusion in which neutrophil events are measured, including studies from our laboratory [14, 15, 18], a reduction in vascular injury and in the extent of necrosis has been associated with a reduction in neutrophil accumulation [4]. The process of accumulation within the AAR begins immediately after the onset of reperfusion [4, 19], with P-selectin-mediated loose attachment to the coronary vascular endothelium, and progresses for several hours with firm adherence and diapedesis into the parenchyma mediated by ß₂-integrins [20]. The period of ramping, with its low flow status, would coincide with these early events of neutrophil–endothelial cell interactions. The initial P-selectin-mediated loose attachment of neutrophils to the coronary endothelium is, in part, dependent on shear forces at the endothelial–vascular interface. High shear forces generated during high-flow states (ie, postischemic reactive hyperemia) mechanically inhibit the tenuous interaction of the P-selectin glycoprotein molecule on the endothelium with its counter ligand, sialyl Lewis^x, on the neutrophil. In addition, high shear forces promote the release of nitric oxide [21], which inhibits neutrophil adherence to endothelial cells [22]. However, the initial cell–cell interactions may be enhanced during low-flow states when intravascular shear forces are reduced, leading to increased adherence and, presumably, to increased accumulation in the arterioles [23] and venules [24, 25] of the reperfused AAR. Bienvenu and associates [25] showed that low flow rates in normal as well as postischemic tissue are associated with increased neutrophil adherence. Additionally, activated neutrophils become less deformable, and thus, more likely to plug the capillaries [26]. This effect is magnified under conditions of low flow, and can result in the trapping of neutrophils even in the absence of adhesion molecule expression [26]. These phenomena may be responsible for the greater accumulation of neutrophils observed in the ramped group compared with the abruptly reperfused group. Although our results do not challenge the basic understanding of the role of neutrophils in ischemia-reperfusion, greater inhibition of postischemic injury may be achieved by combining gentle reperfusion with antineutrophil therapy. However, the inhibition of neutrophil accumulation within the AAR during controlled reperfusion with antiadhesion therapy, such as L-arginine or monoclonal antibodies directed at the selectins or ß₂ integrins, has not been tested.

The greater accumulation of neutrophils in the gently reperfused group appears inconsistent with better coronary (macrovascular) function. However, the apparent discrepancy can be explained by either the distribution (large epicardial versus microvessels) or the disposition (adherent versus embolized) of neutrophils, either of which would be measured as accumulation by the tissue MPO activity technique. This accumulation would have created injury at the level of the microvessel, without similar concomitant injury in the conduit arteries, as observed by Quillen and colleagues [27]. Alternatively, this adherence may have been at the level of the coronary veins, rather than in arteries, where adherence and subsequent emigration into the parenchyma may predominate. Finally, neutrophils may have embolized in the small arterioles of the microvasculature, rather than adhering to vessels directly. The microembolization of neutrophils would be consistent with the lower LAD blood flows previously observed in the ramp group [12], as embolized neutrophils would contribute to increases in postischemic vascular resistance and blood flow defects [28]. We did not examine the conduit coronary arteries for adherence of neutrophils, nor did we test microvessel function or neutrophil adherence to the coronary microvasculature, so we cannot determine the distribution of accumulated neutrophils between larger vessels and the distal microvessels. Our results, however, agree with those of Sawatari and associates [29], who demonstrated in lambs that a low initial reperfusion pressure after cardiopulmonary bypass resulted in an increased preservation of the endothelium in vivo.

The strategy of gentle reperfusion is often applied in cardiac surgery when systemic blood pressure is lowered at the time of aortic unclamping. The rationale behind this reperfusion strategy is a reduction of edema and mechanically induced microvascular injury. The results from the present study suggest that ischemic myocardium is sensitive to the modality (conditions) of reperfusion as suggested by previous studies [2, 10], and suggests that potentially deleterious mechanisms, such as neutrophil accumulation, may be inadvertently evoked by this strategy. Although the net effect of a gentle reperfusion strategy may be beneficial, the full benefit may not be realized by these opposing mechanisms. The increased accumulation of neutrophils in the ramped reperfusion group may have introduced an additional component of injury, thereby potentially underrepresenting the amount of tissue that was salvaged by the ramping process itself.

	Acknowledgments

Top Footnotes Abstract Introduction Material and Methods Results Coronary Blood Flow to... Segmental Function Segmental Stiffness Infarct Size Plasma Creatine Kinase Activity Tissue Myocardial... In Vitro Coronary Artery... Comment Acknowledgments References

 
We gratefully acknowledge the assistance of Mrs Martha Oldland for preparation of the manuscript. These studies were supported in part by grant HL46179 from the National Heart, Lung, and Blood Institute of the National Institutes of Health (Dr Vinten-Johansen) and by a grant from the American Heart Association—North Carolina Affiliate (Dr Zhao).

	Footnotes

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Address reprint requests to Dr Vinten-Johansen, Cardiothoracic Research Laboratory, Carlyle Fraser Heart Center, Crawford Long Hospital of Emory University, 550 Peachtree St, NE, Atlanta, GA 30365-2225.

	References

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1. Forman MB, Virmani R, Puett DW. Mechanisms and therapy of myocardial reperfusion injury. Circulation 1990;81(Suppl 4):69–78.
2. Buckberg GD. Strategies and logic of cardioplegic delivery to prevent, avoid, and reverse ischemic and reperfusion damage. J Thorac Cardiovasc Surg 1987;93:127–39.[Medline]
3. Becker LC, Ambrosio G. Myocardial consequences of reperfusion. Prog Cardiovasc Dis 1987;30:23–44.[Medline]
4. Mehta JL, Nichols WW, Mehta P. Neutrophils as potential participants in acute myocardial ischemia: relevance to reperfusion. J Am Coll Cardiol 1988;11:1309–16.[Abstract]
5. Lefer AM, Tsao PS, Lefer DJ, Ma X. Role of endothelial dysfunction in the pathogenesis of reperfusion injury after myocardial ischemia. FASEB J 1991;5:2029–34.[Abstract]
6. Nakanishi K, Vinten-Johansen J, Lefer DJ, et al. Intracoronary L-arginine during reperfusion improves endothelial function and reduces infarct size. Am J Physiol 1992;263:H1650–8.[Medline]
7. Romson JL, Hook BG, Kunkel SL, Abrams GD, Schork MA, Lucchesi BR. Reduction of the extent of ischemic myocardial injury by neutrophil depletion in the dog. Circulation 1983;67:1016–23.[Abstract/Free Full Text]
8. Buckberg GD. Myocardial protection: an overview. Semin Thorac Cardiovasc Surg 1993;5:98–106.[Medline]
9. Vinten-Johansen J, Buckberg GD, Okamoto F, Rosenkranz ER, Bugyi H, Leaf J. Studies of controlled reperfusion after ischemia. V. Superiority of surgical versus medical reperfusion after regional ischemia. J Thorac Cardiovasc Surg 1986;92:524–34.
10. Okamoto F, Allen BS, Buckberg GD, Bugyi H, Leaf J. Studies of controlled reperfusion after ischemia. XIV. Reperfusion conditions: importance of ensuring gentle versus sudden reperfusion during relief of coronary occlusion. J Thorac Cardiovasc Surg 1986;92:613–20.[Abstract]
11. Vinten-Johansen J, Hammon JW Jr. Myocardial protection during cardiac surgery. In: Utley J, Gravlee GP, eds. Cardiopulmonary bypass: principles and practice. Baltimore: Williams & Wilkins, 1993:155–206.
12. Vinten-Johansen J, Lefer DJ, Nakanishi K, Johnston WE, Brian CA, Cordell AR. Controlled coronary hydrodynamics at the time of reperfusion reduces postischemic injury. Cor Art Dis 1992;3:1081–93.
13. Eng C, Sangho C, Factor S, Kirk ES. A nonflow basis for the vulnerability of the subendocardium. J Am Coll Cardiol 1987;9:374–9.[Abstract]
14. Jordan JE, Zhao Z-Q, Sato H, Vinten-Johansen J. Adenosine A₂ activation attenuates reperfusion injury by inhibiting neutrophil accumulation, superoxide generation and coronary endothelial adherence. J Pharmacol Exp Therapeutics 1997;280:301–9.[Abstract/Free Full Text]
15. Nakanishi K, Zhao Z-Q, Vinten-Johansen J, Hudspeth DA, McGee DS, Hammon JW Jr. Blood cardioplegia enhanced with nitric oxide donor SPM-5185 counteracts postischemic endothelial and ventricular dysfunction. J Thorac Cardiovasc Surg 1995;109:1146–54.[Abstract/Free Full Text]
16. Entman ML, Michael L, Rossen RD, et al. Inflammation in the course of early myocardial ischemia. FASEB J 1991;5:2429–37.
17. Hori M, Kitakaze M, Sato H, et al. Staged reperfusion attenuates myocardial stunning in dogs. Role of transient acidosis during early reperfusion. Circulation 1991;84:2135–45.[Abstract/Free Full Text]
18. Sato H, Zhao Z-Q, Vinten-Johansen J. L-arginine inhibits neutrophil adherence and coronary artery dysfunction. Cardiovas Res 1996;31:63–72.[Medline]
19. Dreyer WJ, Michael LH, West MW, et al. Neutrophil accumulation in ischemic canine myocardium: insights into time course, distribution, and mechanism of localization during early reperfusion. Circulation 1991;84:400–11.[Abstract/Free Full Text]
20. Sluiter W, Pietersma A, Lamers JMJ, Koster JF. Leukocyte adhesion molecules on the vascular endothelium: their role in the pathogenesis of cardiovascular disease and the mechanisms underlying their expression. J Cardiovasc Pharmacol 1993;22(Suppl 4):S37–44.
21. Nerem RM, Harrison DG, Taylor WR, Alexander RW. Hemodynamics and vascular endothelial biology [Review]. J Cardiovasc Pharmacol 1993;21(Suppl 1):S6–10.[Medline]
22. Lefer DJ, Nakanishi K, Johnston WE, Vinten-Johansen J. Antineutrophil and myocardial protection actions of a novel nitric oxide donor after acute myocardial ischemia and reperfusion in dogs. Circulation 1993;88:2337–50.[Abstract/Free Full Text]
23. Ritter LS, Wilson DS, Davis-Gorman G, Williams SK, Copeland JG, McDonagh PF. During reperfusion after myocardial ischemia, reduced blood flow enhances leukostasis in the coronary microcirculation [Abstract]. Circulation 1996;92(Suppl 1):713.
24. Yuan Y, Mier RA, Chilian WM, Zawieja DC, Granger HJ. Interaction of neutrophils and endothelium in isolated coronary venules and arterioles. Am J Physiol 1995;268:H490–8.[Medline]
25. Bienvenu K, Granger DN. Molecular determinants of shear rate-dependent leukocyte adhesion in postcapillary venules. Am J Physiol 1993;264:H1504–8.[Medline]
26. Mazzoni MC, Schmid-Schönbein GW. Mechanisms and consequences of cell activation in the microcirculation. Cardiovasc Res 1996;32:709–19.[Medline]
27. Quillen JE, Sellke FW, Brooks LA, Harrison DG. Ischemia-reperfusion impairs endothelium-dependent relaxation of coronary microvessels but does not affect large arteries. Circulation 1990;82:586–94.[Abstract/Free Full Text]
28. Engler RL, Dahlgren MD, Morris D, Peterson MA, Schmid-Schönbein G. Role of leukocytes in response to acute myocardial ischemia and reflow in dogs. Am J Physiol 1986;251:H314–23.[Medline]
29. Sawatari K, Kadoba K, Bergner KA, Mayer JA Jr. Influence of initial reperfusion pressure after hypothermic cardioplegic ischemia on endothelial modulation of coronary tone in neonatal lambs: impaired coronary vasodilator response to acetylcholine. J Thorac Cardiovasc Surg 1991;101:777–82.[Abstract]

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