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J Thorac Cardiovasc Surg 2001;121:570-579
© 2001 The American Association for Thoracic Surgery
Cardiopulmonary Support and Physiology |
From the Division of Cardiothoracic Surgery, Carlyle Fraser Heart Center, Emory University School of Medicine, Atlanta, Ga.
Supported by the Carlyle Fraser Heart Center of Crawford Long Hospital, Emory University, and a Scientific Development Award (Z.-Q.Z.) and a Grant-in-Aid (J.V.-J.) from the National American Heart Association.
Received for publication July 28, 2000. Revisions requested Sept 7, 2000; revisions received Sept 27, 2000. Accepted for publication Sept 29, 2000. Address for reprints: Jakob Vinten-Johansen, MD, Cardiothoracic Research Laboratory, 550 Peachtree St, NE, Atlanta, GA 30365-2225 (E-mail: jvinten{at}emory.edu).
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Abstract |
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AbsUnits · min–1 · g–1 tissue). In isolated postischemic left anterior descending coronary artery rings, the maximal relaxation response to the endothelium-dependent vasodilator acetylcholine was significantly greater in the adenosine group than in the vehicle group (97.9% ± 5.6% vs 64.7% ± 6.5%, P < .05). |
Introduction |
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1,2 To avoid prolonged ischemia and possible reperfusion injury, we recently introduced perfusion-assisted direct coronary artery bypass (PADCAB), 3 a novel technique by which to perfuse the grafted vessels by using a computer-controlled blood delivery system during multivessel OPCABG. This technique allows rapid perfusion of grafted segments before completion of the proximal anastomosis.
Although many studies have identified effective cardioprotective agents to attenuate ischemia-reperfusion (I-R) injury, 1,2 a clinically useful strategy to protect the ischemic myocardium has not been developed for OPCABG when extracorporeal circulation and cardioplegia are not options. Among the various cardioprotective drugs, adenosine has been extensively investigated both in clinical and experimental studies. 4,5 Potent cardioprotective effects of adenosine on I-R injury are derived from reversal of microvascular vasoconstriction, inhibition of neutrophil functions, reduction of platelet aggregation, and replenishment of high-energy stores in endothelial and myocardial cells. 5,6 A1-receptor–, 7 A2-receptor–, 8 and possibly A3-receptor–mediated 9 mechanisms are involved in adenosine cardioprotection.
Accordingly, the present study tested the hypothesis that the selective delivery of adenosine to a target vessel with a computer-controlled perfusion delivery pump system during simulated off-pump revascularization would attenuate reperfusion injury, reduce coronary artery endothelial dysfunction, and salvage myocardium.
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Methods |
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A pair of 5-MHz piezoelectric ultrasonic crystals 2.5 to 3.0 mm in diameter was placed in the subendocardium of the myocardium perfused by the LAD (ischemic-reperfused segment) to measure instantaneous segmental dimensions by means of a sonomicrometer (model 120; Triton Technology, San Diego, Calif). A model SPC-320 2F solid-state pressure transducer (Millar Instruments) was inserted into a small right ventricular branch of the LAD artery distal to the site of ligation to measure instantaneous LAD intracoronary pressure(Fig 1
, A). The position of the transducer catheter did not interfere with LAD blood flow. The dogs were systemically heparinized with an initial 300-U/kg dose of heparin sodium supplemented with 300 U/kg every 90 minutes.
Experimental protocol
The protocol is schematically presented in fig1, B. Hemodynamic and segmental function data were obtained, and colored microspheres were injected at baseline. Each dog then received a bolus injection of lidocaine (1.2 mg · kg–1) followed by continuous infusion (0.3 mg · kg–1 · min–1), after which the LAD was ligated just distal to the first diagonal branch for 75 minutes. After LAD ligation, a 2-mm diameter cannula (DLPlegia Highflow Vessel Cannulae; Medtronic DLP, Grand Rapids, Mich) was inserted into the LAD distal to the site of ligation. Ventricular fibrillation, when encountered, was converted by direct-current countershock of 10 W/s. When more than 4 countershocks were required, ventricular fibrillation was termed intractable, and the experiment was terminated. At the end of 75 minutes of LAD occlusion, all data were again collected. The LAD cannula was then attached to a delivery line from the Myocardial Protection System (MPS) cardioplegia and perfusion delivery system (Quest Medical Inc, Allen, Tex), and reperfusion was established by continuous withdrawal of femoral arterial blood. Mean LAD pressure was immediately matched to mean systemic arterial pressure by varying flow rate during the entire reperfusion period. The dogs were randomly given saline solution vehicle (vehicle group, n = 7) or 10 µmol/L blood adenosine (adenosine group, n = 7) at a rate of 10 mL/L blood into the LAD during the first 30 minutes of reperfusion, followed by unmodified reperfusion for 90 minutes. Adenosine was administered through side ports situated just proximal to the heart in the perfusion line to avoid rapid deamination during transit. After reperfusion was established with the MPS system, the perfusion line was used for injection of colored microspheres and withdrawal of reference samples to determine myocardial blood flow specifically in the area at risk (AAR). Coronary blood flow rate was increased to compensate for reference sample withdrawal from the catheter.
Data collection and analysis
Hemodynamic data, including left ventricular, systemic arterial (femoral arterial), and LAD pressures and segmental length data were acquired during the 10-second period of respiratory apnea. The data from each channel were digitized and processed by computer algorithms by means of an interactive videographics program developed in our laboratory (SPECTRUM; Wake Forest University, Winston-Salem, NC), as described previously. 10 Percentage of segmental shortening was calculated as follows:
100 x (EDL – ESL)/EDL,
where EDL and ESL are end-diastolic length and end-systolic length, respectively.
Determination of AAR and infarct size
After postexperimental excision of the heart, unisperse blue dye was injected under 100 mm Hg perfusion pressure to stain the normally perfused region blue and demarcate the AAR. The left ventricle (LV) was cut into 4- to 5-mm thick transverse slices. The AAR was separated from the nonischemic zone and incubated for 15 minutes in 1% solution of triphenyltetrazolium chloride (Sigma Chemical, St Louis, Mo) at 37°C to differentiate the necrotic zone (pale) from the ischemic nonnecrotic zone (red). The AAR was calculated as the sum of the weights of the nonnecrotic and necrotic tissue within the AAR divided by the weight of the LV and expressed as a percentage (AAR/LV). The infarct size was calculated as the weight of necrotic tissue divided by the weight of the AAR (An/AAR) and expressed as a percentage.
Regional myocardial blood flow
Regional myocardial blood flow (RMBF) with 1 to 2 million 15-µm color-labeled microspheres (Triton Technology, San Diego, Calif) was calculated by the reference sampling method, as previously described. 11 Reference samples were withdrawn from the femoral artery or the perfusion line at a constant rate for 3 minutes. Postexperimental myocardial tissue and reference blood samples were analyzed with a spectrophotometer (DU7400; Beckman, Fullerton, Calif). Blood flow was calculated in the nonischemic epicardium, nonischemic endocardium, AAR epicardium, and the AAR endocardium, respectively, as follows:
RMBF = (CT x FR/CR)/WT,
where CT and CR are the absorbances from dispersed microspheres in the tissue and reference blood samples, respectively; FR is the flow rate; and WT is the total weight of the tissue sample in grams. Results are expressed as milliliters per minute per gram of tissue.
Plasma creatine kinase activity
Arterial blood samples (3 mL) were centrifuged at 2500g and 4°C for 10 minutes. The plasma was drawn off and analyzed spectrophotometrically for creatine kinase (CK) activity and for protein concentration (CK-10 kit; Sigma Diagnostics, St Louis, Mo). Plasma CK activity is expressed as international units per gram of protein.
Determination of myocardial edema
Postexperimental transmural myocardial tissue samples were taken from the nonischemic zone and from the AAR. Each sample was further subdivided into subepicardial and subendocardial regions, blotted of surface moisture, weighed, and desiccated for 48 hours. The percentage of myocardial water content is defined as follows:
[(wet weight – dry weight)/wet weight] x 100
Cardiac myeloperoxidase activity
Postexperimental transmural myocardial tissue samples 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 in myocardium, as described previously. 12 MPO activity is described as the rate of hydrogen peroxide degradation–induced color change per minute per 100 mg of tissue and expressed as the change in absorbance units per minutes per gram of myocardial tissue.
Postexperimental coronary artery endothelial function
Both the ischemic-reperfused LAD and nonischemic circumflex coronary arteries (LCx) were carefully dissected from the heart and cut into rings of approximately 2 mm in length. The rings were mounted on stainless steel hooks and placed in organ chambers filled with 37°C Krebs-Henseleit solution oxygenated with 95% oxygen and 5% carbon dioxide, having the following composition: NaCl, 118.1 mmol/L; KCl, 4.7 mmol/L; KH2PO4, 1.2 mmol/L; MgSO4, 1.2 mmol/L; CaCl2 · 2H2O, 2.5 mmol/L; NaHCO3, 25.0 mmol/L; and glucose, 11.1 mmol/L. Changes in isometric force were measured by isometric force transducers (model TR001; Radnoti, Monrovia, Calif), as described previously, 10 after 60 minutes of equilibration with 10 µmol/L indomethacin (INN: indometacin) to prevent vascular responses to endogenous prostacyclin. The coronary rings were precontracted with the 10 nmol/L thromboxane A2 mimetic agent U46619 (Upjohn, Kalamazoo, Mich). Once a stable contraction was observed, cumulative concentration responses to acetylcholine (Sigma Chemical), a muscarinic receptor-mediated endothelium-dependent stimulator of nitric oxide synthase, were obtained. After the rings were washed with fresh Krebs-Henseleit solution and equilibrated to baseline, the procedure was repeated for A23187 (Sigma Chemical), a receptor-independent stimulator of nitric oxide synthase, and for sodium nitroprusside (Sigma Chemical) to test endothelium-independent vascular smooth muscle relaxation. Relaxation is expressed as a percentage of U46619-induced constriction. Drug concentrations are expressed as the final concentration in the organ chamber.
Statistical analysis
The data were analyzed by 1-way or 2-way analysis of variance for repeated measures to identify group, time, and group-time interactions. If significant interactions were found, then further pairwise analysis was performed by using post hoc analysis to locate the source of differences. Comparisons between the 2 groups in the AAR, infarct size, MPO activity, myocardial edema, and percentage of relaxation of coronary rings at each concentration were analyzed by the nonpaired Student t test. Mean ± SEM values are reported.
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Results |
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Hemodynamic data and segmental function
Hemodynamic data and segmental shortening during the course of the experiment are shown inTable I. There were no significant differences between the 2 groups in any of the hemodynamic variables and segmental shortening in the AAR at baseline. Furthermore, there were no changes in mean arterial pressure during the course of the experiment in either group. Note that delivery of adenosine to the AAR did not produce hypotension (see R15 inTable I). At ischemia and 15 minutes of reperfusion, left ventricular end-diastolic pressure increased significantly in the vehicle group but subsequently returned to the baseline level, and no significant differences were found between groups. Ischemia caused paradoxical systolic expansion of the ischemic segment. During reperfusion, akinesis or dyskinesis of systolic shortening persisted in both groups, with no group differences.
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). During the remainder of reperfusion, LAD blood flow remained comparable between the 2 groups. At the end of 2 hours of reperfusion, LAD blood flow averaged 24.0 ± 4.0 mL/min in the adenosine group and 24.2 ± 5.1 mL/min in the vehicle group.
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RMBF |
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).
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, B). In the adenosine group there was a significant reduction in myocardial edema in the ischemic subendocardium (81.9% ± 1.1%) compared with that in the vehicle group. Myocardial edema in the AAR subepicardium tended to be lower in the adenosine group but with no significant difference between the 2 groups. In the normal zone myocardial edema was comparable between the 2 groups.
Cardiac MPO activity
MPO activity in the nonischemic zone was low and comparable between the 2 groups(Fig 5). In the triphenyltetrazolium chloride– negative necrotic tissue within the AAR, MPO activity was 36% less in the adenosine group than in the vehicle group, although there was no statistical difference. MPO activity in the AAR nonnecrotic tissue was comparable between the 2 groups.
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, B) relaxed comparably by all 3 vasodilators with no group differences at any concentration.
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Discussion |
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13,14 the technique limits the application of cardioprotective strategies that have been extensively investigated over the past 20 years (ie, cardioplegia and hypothermia). During OPCABG, reperfusion after a period of coronary artery occlusion is established without any active cardioprotection. For the moment, strategies such as proximal first technique or the use of intraluminal coronary shunts have been applied to minimizing the effects of ischemia during elective coronary occlusion, especially in multivessel OPCABG. 15,16 Measures such as ischemic preconditioning of target vascular beds have also been advocated, but its effects are not clear, and clinical efficacy for regional ischemia is not known. 17,18 In this regard the novel PADCAB technique for off-pump operations allows not only rapid reperfusion but also the delivery of cardioprotective agents directly into the ischemic-reperfused target area. 3 The PADCAB technique of coronary perfusion was originally applied to graft conduits after the completion of the distal anastomosis. However, with the aid of an intraluminal device, the target coronary artery can be perfused directly, thereby avoiding ischemia induced by vascular snares. This intraluminal perfusion closely simulates the experimental conditions used in the present study.
In the present study the myocardium at risk underwent 75 minutes of ischemia at the time of reperfusion. This duration of ischemia simulates acute myocardial infarction rather than routine OPCABG revascularization in an elective procedure. However, even during OPCABG operations, a relatively long ischemic period may be required, and full perfusion of the target area may not be achieved until the proximal anastomosis is completed, thereby involving a longer duration of compromised blood supply. In a patient with a low-grade stenosis, the ischemia may not extend beyond removal of the ligature. However, the presence of a severe stenosis may impair adequate blood flow until the proximal anastomosis is completed. Additionally, even the short ischemic period (usually <15 minutes) necessary for routine OPCABG may produce subtle injury (metabolic disarrangement or reversible myocardial dysfunction), 19,20 although it may not produce severe injury. In any event the indications for OPCABG may extend to patients with acute myocardial infarctions in the future, for which strategies to attenuate I-R injury will be important for postoperative cardiac function and improvement of mortality.
Intracoronary administration of adenosine (10 µmol/L) during the first 30 minutes of reperfusion, as an additive to the blood perfusing the ischemic myocardial segment, reduced infarct size by 60% of that observed in the saline vehicle group. This reduction in infarct size was consistent with previous studies 8,21 and was associated with a significant decrease in plasma CK activity and myocardial edema. The reduction in myocardial edema is likely related to preservation of the microvascular endothelium, consistent with our data in epicardial vessels. In addition, brief adenosine treatment significantly attenuated endothelial injury and tended to reduce the accumulation of neutrophils in the necrotic AAR. Moreover, the selective intracoronary administration of adenosine did not alter systemic blood pressure. Hence, transient intracoronary adenosine could be safely delivered to the target vessel myocardium to attenuate reperfusion injury, although this reduction of I-R injury was not associated with recovery of postischemic segmental function at the end of 2 hours of reperfusion.
Previous studies have demonstrated the potent cardioprotective effects of adenosine, including direct inhibition of neutrophils (superoxide anion generation and expression of adhesion molecules) and inhibition of endothelial activation, which attenuated neutrophil-endothelial interactions. 21-23 These effects are largely mediated by A2a receptor interactions, 5,24 although interaction with the newly identified A3 receptor has been reported to attenuate neutrophil adherence to endothelium. 9,25 These mechanisms, rather than increased blood flow, have been suggested to play major roles in the reduction of infarction after myocardial ischemia and subsequent reperfusion when adenosine was administered at reperfusion. 21
The strategy of administering exogenous adenosine to reduce reperfusion injury has been described recently, 6,21,26,27 and numerous studies have demonstrated that adenosine can restore various biochemical alterations with or without functional recovery. 5,28,29 Consistent with previous studies in which adenosine was administered by intracoronary infusion, 6,30,31 the present study demonstrated a reduction of infarct size and preservation of endothelial function, although postischemic segmental function showed no recovery, presumably because of our relatively short reperfusion duration. As another possible reason, it has been demonstrated that neutrophils are not involved in the pathogenesis of regional contractile dysfunction, 32 although this is still controversial. Therefore, inhibition of neutrophil actions with adenosine may not be associated with improvement of regional segmental function in infarct models. Considering the short half-life (<15 seconds) of adenosine in whole blood, 33 the most effective method of adenosine administration is likely to be selective intracoronary treatment introduced into the blood just proximal to the coronary catheter.
In the present study 10 µmol/L adenosine was infused directly into the LAD during the first 30 minutes for a total volume of 40 to 60 mL (10-15 mg adenosine). Although the concentration is fairly low and the infusion period is short compared with that found in previous studies, 6,30,31 the findings from our study suggest that our delivery method of adenosine is effective in salvaging myocardium and attenuating endothelial injury in the PADCAB setting without hypotension. The short infusion period is clinically relevant, especially when PADCAB is used for multiple grafts, although it is not known whether 30 minutes of intracoronary adenosine is optimal. In addition, the delivery of adenosine can be precisely controlled by the computerized pump system with an accuracy of ±1% over a wide range of flow rates. 34 In the present study coronary perfusion pressure was matched to systemic arterial pressure by varying flow rate during the entire reperfusion period. It is important that coronary pressure is measured directly with an intraluminal catheter or estimated from transcatheter pressure differentials because several previous studies have demonstrated that there is a safe limit of pressure for the vulnerable myocardium at the time of reperfusion, beyond which myocardial damage (interstitial edema) is observed. 35 In this regard the perfusion pressure used in our study was comparable with that of passive flow systems, although the pump flow was not pulsatile.
In summary, the present study determined that intracoronary perfusion of at-risk myocardium with adenosine during the first 30 minutes of reperfusion reduced the extent of myocardial infarction in a simulated PADCAB model. In addition, endothelial function was highly preserved with adenosine treatment. This novel reperfusion strategy for OPCABG can be used in cases requiring single, as well as multiple, grafting and prolonged ischemia and for patients with acute evolving myocardial infarction.
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J. Vinten-Johansen Does adenosine have cerebro-protective effect during cardiopulmonary bypass?: Reply Ann. Thorac. Surg., October 1, 2001; 72(4): 1448 - 1448. [Full Text] [PDF]
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