Postischemic myocardial reperfusion is commonly associated with focal regions of microvascular impairment, even when global coronary flow is normal. The problem ranges in severity from microvascular stunning to no reflow, 1–3 depending primarily on the duration and severity of ischemia. Such impairment may progress over several days after reperfusion 4 and, when severe, portends a poor prognosis, 5 even when infarct size is relatively small. 6 The etiology of microvascular impairment progression is unknown, but has been attributed primarily to metabolic abnormalities 7–9 associated with reperfusion at flows presumed to be initially adequate to correct ischemia. However, experimental perfusion imaging studies with novel tracers suggest that microvascular ischemia, rather than simply hypoperfusion to nonviable tissue, persists during reperfusion. 10,11 Recently, we demonstrated that aqueous oxygen (AO) hyperoxemic reperfusion in a porcine model reduced indices of acute injury, including infarct size. 12 Attenuation of a self-perpetuating cycle of microvascular ischemia during reperfusion may have been responsible for the observed benefits. In the present study, we present additional experimental evidence that microvascular ischemia is present during reperfusion and can be ameliorated by intracoronary aqueous oxygen.
The animal protocol was approved by the Wayne State University School of Medicine Institutional Review Board before initiation of studies. All animals were housed and studied in a facility approved by the American Association for Accreditation of Laboratory Animal Care.
Under intravenous pentobarbital and morphine sulfate anesthesia in adult mongrel dogs weighing 21 ± 4 kg (n = 36), a proximal circumflex coronary occlusion was performed for 90 min with an angioplasty balloon that was 10% larger at nominal pressure compared with coronary lumen diameter. Injection of contrast medium (loversal) was performed at the beginning and end of the occlusion period to confirm the absence of flow distal to the balloon. Additional injections were performed during reperfusion to assess Thrombolysis In Myocardial Infarction (TIMI) flow grade. Mechanical ventilation with air was provided throughout each study. Heparin boluses were given to maintain an activated clotting time >300 sec. Lidocaine boluses (1 mg/kg) were given only before and during balloon occlusion. When ventricular fibrillation occurred, these animals were excluded from the study (n = 10). Arterial blood perfusion after removal of the balloon was performed at 100 ml/min through a no. 7 French guide catheter positioned in the left main coronary. The distal end of the guide was adjusted fluoroscopically, with test injections of contrast medium (5 ml at 15 min intervals) through a sidearm into the proximal end of the catheter during blood perfusion, to provide roughly equal flow to the circumflex and left anterior descending coronary arteries. AO containing 0.8 to 1.0 ml O2/g of lactated Ringer’s solution (2.8–3.4 MPa O2 pressure) was infused at 2.5 to 3.5 ml/min into arterial blood withdrawn with a roller pump at 100 ml/min via a no. 4 French catheter in a femoral artery via a small (20 ml priming volume) extracorporeal circuit to produce hyperoxemia. Transthoracic two dimensional echo was used to confirm the absence of microbubbles. All animals received an intravenous normal saline infusion of approximately 8 ml/kg/hour, or approximately 600 ml over a 3.5 hour study period. During the 1.5 hour period of AO infusion in the treatment group, saline was given at a similar rate (mean = 8.6 ml/kg/hour from AO infusion) as the only source of saline infusion.
Animals were randomly assigned to one of three groups: 1) autoreperfusion (physiologic, passive reperfusion with normoxemic blood) alone, 2) normoxemic reperfusion (normoxemic, “active,” arterial blood perfusion at 100 ml/min via a roller pump into the left coronary artery through the guide catheter) and the initial 30 min period of autoreperfusion, and 3) AO hyperoxemic reperfusion after 30 min of autoreperfusion. Excluding animals with ventricular fibrillation that occurred during coronary balloon occlusion, 11 animals were treated with AO, 7 with autoreperfusion alone, and 8 with normoxemic reperfusion.
Left Ventricular Function
Analysis of transthoracic echocardiographic % left ventricular ejection fraction (LVEF) and regional wall motion (% fractional shortening of the posterior wall) was performed from end diastolic and end systolic endocardial outlines in video recordings (Dextra D-200 offline analysis system, Lakewood, CA) obtained in the canine model. A short axis view, at the level of the papillary muscles, was used for all analyses. One technician performed all analyses, which were performed with masked assignment. Intraobserver variability was found to be 1.6 ± 1.2% for frames analyzed 2 weeks apart.
Myocardial Blood Flow
At 5 min before balloon deflation, 30 min of autoreperfusion, and 120 min after balloon deflation, 10 mCi of either Sc46-, Tin113-, or Co57-labeled microspheres (15 μm, Dupont NEN), respectively, were injected into the left ventricle in dogs in the first 8 animals in the AO reperfusion group and in all 7 animals of the autoreperfusion group. In the AO group, the last injection of microspheres was performed 2 min after termination of AO hyperoxemic perfusion. From each heart, 8 transmural samples weighing 0.8–1.0 g were removed from each of the normally perfused and postischemic regions (n = 32 samples) for measurement of radioactivity. Regional myocardial blood flow/g of tissue was determined from a reference blood flow.
Masked assignment was used for all analyses. Two way repeated unbalanced analysis of variance (ANOVA) (SPSS 9.0) was used for comparisons of mean values over time to baseline values. ANOVA was used for comparison of mean values between groups at equivalent time periods. For comparisons of more than two groups, a Bonferroni type correction was applied to p values, which were considered significant at <0.05.
Arterial Oxygen Tension
Animals subjected to normoxemic reperfusion had a mean arterial PO2 = 14 ± 1 kPa (108 ± 8 mm Hg). Animals subjected to AO reperfusion had a mean coronary perfusate PO2 = 70 ± 20 kPa (530 ± 150 mm Hg).
Left Ventricular Function
Acutely after balloon coronary occlusion, a significant mean fall in LVEF was noted (mean absolute 18% compared to baseline values; p < 0.05, Figure 1A). At 30 min of autoreperfusion after balloon deflation and catheter removal, a nonsignificant recovery in LVEF was observed in all three groups (p > 0.05). In the autoreperfusion control group, no significant improvement in LVEF was observed during the 3 hr period of reperfusion compared to values noted during coronary occlusion (p > 0.05).
AO hyperoxemic reperfusion, however, was associated with a significant mean improvement in LVEF (at 90 min AO treatment, mean absolute increase = 17 ± 6%) compared to both the mean value noted during the initial autoreperfusion period in the same animals and the mean value of the autoreperfusion control group at the same time period (Figure 1A, p < 0.05). Mean LVEF was not significantly different (p > 0.05), 1 hr after termination of hyperoxemic perfusion, from the values during hyperoxemic perfusion. Regional wall motion data paralleled the LVEF results (Figure 1B). A similar impairment of regional wall motion between the 3 groups was noted during ischemia. No significant improvement in regional wall motion was noted during autoreperfusion. AO hyperoxemic reperfusion was associated with a significant improvement in regional wall motion compared with the baseline autoreperfusion period in the same animals as well as compared with the autoreperfusion control group at comparable intervals (p < 0.05).
No significant difference in mean values between groups was noted in heart rate or arterial pressure at equivalent time periods (p > 0.05).
The mean absolute deviation of the ST segment from the isoelectric point did not improve significantly in the control group subjected to autoreperfusion alone for 3 hours (Figure 2). In contrast, the ST segment became isoelectric in the treatment group by the end of the 90 min period of AO hyperoxemic perfusion. Relative frequency of ventricular extrasystoles peaked at 45 min reperfusion in the autoreperfusion control group (Figure 3). At this period, the relative frequency of ventricular extrasystoles was significantly lower in the AO perfusion group (15 min of treatment) compared to the autoreperfusion control group (p < 0.05).
Myocardial Blood Flow
Mean blood flow in the ischemic segment, examined in the dog model, was similar between the control (autoreperfusion alone) and treatment (AO perfusion) groups during both balloon occlusion and at the initial (30 min) autoreperfusion period (p > 0.05) (Figure 4). However, after AO perfusion, the mean blood flow in the treatment group was double that of the control group (p < 0.01), whether expressed as absolute blood flow (AO mean = 0.92 ± 0.35 ml/g/min; autoreperfusion = 0.43 ± 0.19 ml/g/min) or the ratio of blood flow of the ischemic segment to the normal segment in the same animals. The improvement in flow after AO hyperoxemic reperfusion was similar in subepicardial and subendocardial tissues (p > 0.05).
The clinical importance of microvascular integrity during postinfarction myocardial reperfusion is now well recognized. The presence of a “no reflow” region portends a relatively high clinical event rate. 5,6,13,14 The fact that such regions often exist despite TIMI III angiographic flow after angioplasty or thrombolytic therapy has been emphasized only recently. 5 However, Hori et al.15 demonstrated more than a decade ago that resting blood flow was reduced experimentally by microsphere embolization only when >70% of total embolization of a myocardial region was performed, despite significant ischemic changes in function and lactate production at even 10% embolization.
In the present study, TIMI III angiographic flow in the infarct vessel was observed during all periods of reperfusion in all animals. Nevertheless, it is likely that microvascular ischemia was present during reflow. LVEF and electrocardiographic abnormalities (both ST segment deviation and premature ventricular contraction frequency) were significantly improved with AO hyperoxemic reperfusion but not with either autoreperfusion or active normoxemic reperfusion. The most likely explanation for these effects is that oxygen delivery during reflow is inadequate to correct the residual ischemia in all segments of the infarct zone.
Experimental studies have demonstrated that hypoxia has profound effects on endothelial morphology and function. 16–23 An increase in endothelial permeability and loss of barrier function results in edema of both endothelial cells and adjacent tissue, and the associated transformation to a globular shape as well as expression of adhesion molecules would be expected to impede blood flow. Low velocity flow 24,25 and hypoxia 26 each can result in leukocyte activation which may further amplify problems with local tissue flow. The results of the current study demonstrated that microvascular blood flow, immediately after AO reperfusion, was twice that observed with ordinary reperfusion (autoreperfusion).
The level of improvement in microvascular blood flow noted in our study is similar to that noted in other studies of the effects of enhanced oxygenation of reperfused tissues. Capillary density was doubled after hyperbaric oxygen (HBO) treatment of reperfused striated skeletal muscle, 27 and tissue blood flow was markedly increased by HBO during reperfusion of skin flaps. 28
AO hyperoxemic perfusion has been shown to prevent low flow myocardial ischemia. 29 In reperfused regions of myocardium with relatively low blood flow, it is possible that delivery of oxygen at high partial pressures in plasma may attenuate endothelial cell hypoxia and reverse a potentially self-propagating cycle (ischemia and responses to ischemia) that compromises microvascular flow. The possibility exists that diffusion of oxygen at high partial pressures between perfused and occluded capillaries 30 may also provide some benefit. This proposed effect may help explain the reduction in infarct size associated with hyperoxemic reperfusion 12,31,32 and in the sustained improvement in LVEF noted 1 hr after termination of AO treatment in the present study and in the AO porcine reperfusion model. 12 The results of a recent clinical trial of AO reperfusion immediately after angioplasty and stenting of the infarct coronary artery on left ventricular function 33 are consistent with this hypothesis. Echocardiographic regional wall motion score index improved at each time period examined (1 day, as well as 1 and 3 months after reperfusion) and was sufficiently great to explain the chronic improvement in LVEF at 3 months (7% mean absolute increase compared to postangioplasty, before AO reperfusion).
Our results support the hypothesis that inadequate reperfusion at the tissue level commonly occurs despite TIMI III reflow. AO hyperoxemia improved left ventricular function and electrocardiographic evidence of ischemia, very likely as a result of augmentation of oxygen delivery in plasma. Marked improvement in myocardial blood flow after AO infusion was found, which may explain the improvement in LV function after treatment.
This work was supported by grants from TherOx, Inc., and the National Institutes of Health (HL56436).
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