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Aqueous Oxygen Attenuation of Reperfusion Microvascular Ischemia in a Canine Model of Myocardial Infarction

Richard Spears, J.*; Prcevski, Petar*; Xu, Rui*; Li, Li*; Brereton, Giles; DiCarli, Marcello*; Spanta, Ali*; Crilly, Richard*; Lavine, Steven*; vander Heide, Richard*

doi: 10.1097/01.MAT.0000094665.72503.3C
ORIGINAL ARTICLES
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Uncorrected microvascular ischemia may contribute to left ventricular impairment during reperfusion after prolonged coronary artery occlusion. Attenuation of such ischemia in microvessels with impaired erythrocyte flow may require delivery of oxygen at high levels in plasma. Intraarterial infusion of aqueous oxygen (AO) can be used in a site specific manner to achieve hyperoxemic levels of oxygenation in the perfusate. With this new approach, the hypothesis was tested that reperfusion microvascular ischemia can be attenuated.

After a 90 min coronary balloon occlusion in a canine model, AO hyperoxemic intracoronary perfusion was performed for 90 min after a 30 min period of autoreperfusion. Control groups consisted of normoxemic reperfusion, both passive (autoreperfusion) and active (roller pump). A significant improvement in left ventricular ejection fraction (p < 0.05) at 2 hr of reperfusion was noted only in the AO hyperoxemia group (17 ± 6% by two dimensional echo), without a significant reduction in the improvement 1 hr after termination of treatment. During AO hyperoxemic perfusion, ECG ST segment isoelectric deviation normalized, and frequency of ventricular premature contractions was significantly reduced, in contrast to the autoreperfusion control group (p < 0.05). Microvascular blood flow, measured as the ischemic/normal left ventricular segment ratio by radiolabeled microspheres immediately after AO hyperoxemic perfusion, was double the value of the autoreperfusion control group at 2 hr of reperfusion (p < 0.05).

We conclude that reperfusion microvascular ischemia is attenuated by intracoronary AO hyperoxemic perfusion and acutely improves left ventricular function in this model.

From the *Cardiovascular Research Laboratory, Wayne State University, Departments of Medicine and Pathology, Detroit, Michigan; and †Michigan State University, Lansing, Michigan.

Received for consideration January 2003;

Accepted for publication May 2003.

Dr. Spears owns stock in TherOx, Inc.

Address correspondence to: J. Richard Spears, MD, Professor of Medicine, Wayne State University, Louis M. Elliman Research Bldg., Rm 1107, 421 E. Canfield Rd., Detroit, MI 48201.

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.

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Methods

Animal Model

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.

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Experimental Groups

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.

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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.

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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.

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Data Analysis

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.

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Results

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).

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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).

Figure 1

Figure 1

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).

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Electrocardiographic Changes

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).

Figure 2

Figure 2

Figure 3

Figure 3

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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).

Figure 4

Figure 4

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Discussion

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).

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Conclusions

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.

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Acknowledgment

This work was supported by grants from TherOx, Inc., and the National Institutes of Health (HL56436).

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References

1. Bolli R, Triana JF, Jeroudi MO. Prolonged impairment of coronary vasodilation after reversible ischemia: evidence for microvascular “stunning.” Circ Res 67: 332–343, 1990.
2. Maxwell L, Gavin JB. The contribution of ischemia to the development of microvascular incompetence in the myocardium. Cardiovasc Res 25: 491–495, 1991.
3. Kloner RA, Ganote CE, Jennings RB. The “no reflow” phenomenon after temporary coronary occlusion in the dog. J Clin Invest 54: 1496–1508, 1974.
4. Rochitte CD, Lima JA, Bluemke DA, et al: Magnitude and time course of microvascular obstruction and tissue injury after acute myocardial infarction. Circulation 98: 1006–1014, 1998.
5. Gibson CM, Cannon CP, Murphy SA, et al: Relationship of TIMI myocardial perfusion grade to mortality after administration of thrombolytic drugs. Circulation 101: 125–130, 2000.
6. Wu KC, Zerhouni EA, Judd RM, et al: Prognostic significance of microvascular obstruction by magnetic resonance imaging in patients with acute myocardial infarction. Circulation 97: 765–772, 1998.
7. McCord JM. Oxygen-derived radicals: a link between reperfusion injury and inflammation. Fed Proc 46: 2402–2406, 1987.
8. Bagchi D, Wetscher GJ, Bagchi M, et al: Interrelationship between cellular calcium homeostasis and free radical generation in myocardial reperfusion injury. Chem Biol Interact 104: 65–85, 1997.
9. Halestrap AP, Kerr PM, Javadov S, et al: Elucidating the molecular mechanism of the permeability transition pore and its role in reperfusion injury of the heart. Biochem Biophys Acta 1366: 79–94, 1998.
10. Ince C, Vink H, Wieringa PA, et al: Heterogeneous NADH fluorescence during post-anoxic reactive hyperemia in saline perfused rat heart. Adv Exp Med Biol 277: 477–482, 1990.
11. Weinstein H, Reinhardt CP, Leppo JA. Direct detection of regional myocardial ischemia with technetium-99m nitroimidazole in rabbits. J Nucl Med 39: 598–607, 1998.
12. Spears JR, Henney C, Prcevski P, et al. Aqueous oxygen hyperbaric reperfusion in a porcine model of myocardial infarction. J Invasive Cardiol 14: 160–166, 2002.
13. Ito H, Tomooka T, Sakai N, et al: Lack of myocardial perfusion immediately after successful thrombolysis: a predictor of poor recovery of left ventricular function in anterior myocardial infarction. Circulation 85: 1699–1705, 1992.
14. Claeys MJ, Bosmans J, Veenstra L, et al: Determinants and prognostic implications of persistent ST-segment elevation after primary angioplasty for acute myocardial infarction: importance of microvascular reperfusion injury on clinical outcome. Circulation 99: 1972–1977, 1999.
15. Hori M, Inoue M, Kitakaze M, et al: Role of adenosine in hyperemic response of coronary blood flow in microembolization. Am J Physiol 250: H509–H518, 1986.
16. Pinsky DJ, Yan SF, Lawson C, et al: Hypoxia and modification of the endothelium: implications for regulation of vascular homeostatic properties. Semin Cell Biol 6: 283–294, 1995.
17. Ogawa S, Gerlach H, Esposito C, et al: Hypoxia modulates the barrier and coagulant function of cultured bovine endothelium: increased monolayer permeability and induction of procoagulant properties. J Clin Invest 85: 1090–1098, 1990.
18. Al-Haboubi HA, Tomlinson DR, Ward BJ. The influence of hypoxia on transvascular leakage in the isolated rat heart: quantitative and ultrastructural studies. J Physiol (Lond) 482: 157–166, 1995.
19. Dauber IM, VanBenthuysen KM, McMurtry IF, et al: Functional coronary microvascular injury evident as increased permeability due to brief ischemia and reperfusion. Circ Res 66: 986–998, 1990.
20. Mazzoni MC, Borgstrom P, Warnke KC, et al: Mechanisms and implications of capillary endothelial swelling and luminal narrowing in low-flow ischemias. Int J Microcirc Clin Exp 15: 265–270, 1995.
21. Zund G, Nelson DP, Neufeld EJ, et al: Hypoxia enhances stimulus-dependent induction of E-selectin on aortic endothelial cells. Proc Natl Acad Sci USA 93: 7075–7080, 1996.
22. Ginis I, Mentzer SJ, Li X, Faller DV: Characterization of a hypoxia-responsive adhesion molecule for leukocytes on human endothelial cells. J Immunol 155: 802–810, 1995.
23. Arnould T, Michiels C, Janssens D, et al: Hypoxia induces PMN adherence to umbilical vein endothelium. Cardiovasc Res 30: 1009–1016, 1995.
24. Ritter LS, McDonagh PF. Low-flow reperfusion after myocardial ischemia enhances leukocyte accumulation in coronary microcirculation. Am J Physiol 273: H1154–H1165, 1997.
25. Kubes P. The role of shear forces in ischemia/reperfusion-induced neutrophil rolling and adhesion. J Leukoc Biol 62: 458–464, 1997.
26. Baudry N, Danialou G, Boczkowski J, et al: In vivo study of the effect of systemic hypoxia on leukocyte-endothelium interactions. Am J Respir Crit Care Med 158: 477–483, 1998.
27. Sirsjo A, Lehr HA, Nolte D, et al: Hyperbaric oxygen treatment enhances the recovery of blood flow and functional capillary density in postischemic striated muscle. Circ Shock 40: 9–13, 1993.
28. Zamboni WA, Roth AC, Russell RC, Smoot EC. The effect of hyperbaric oxygen on reperfusion of ischemic axial skin flaps: a laser Doppler analysis. Ann Plast Surg 28: 339–341, 1992.
29. Spears JR, Wang B, Wu X, et al: Aqueous oxygen: a highly O2-supersaturated infusate for regional correction of hypoxemia and production of hyperoxemia. Circulation 96: 4385–4391, 1997.
30. Goresky CA, Goldsmith HL. Capillary-tissue exchange kinetics: diffusional interactions between adjacent capillaries. Adv Exp Med Biol 37: 773–781, 1973.
31. Thomas MP, Brown LA, Sponseller DR, et al: Myocardial infarct size reduction by the synergistic effect of hyperbaric oxygen and recombinant tissue plasminogen activator. Am Heart J 120: 791–800, 1990.
32. Sterling DL, Thornton JD, Swafford A, et al: Hyperbaric oxygen limits infarct size in ischemic rabbit myocardium in vivo. Circulation 88: 1931–1936, 1993.
33. Dixon SR, Bartorelli AL, Marcovitz PA, et al: Initial experience with hyperoxemic reperfusion after primary angioplasty for acute myocardial infarction: results of a pilot study utilizing intracoronary aqueous oxygen therapy. J Am Coll Cardiol 39: 387–392, 2002.
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