Effect of Delay in Retroperfusion Therapy on Infarct Size Reduction Christopher M. Feindel, MD, Reena Sandhu, MSc, Jorge Cruz, MD, and Gregory J. Wilson, MSc, MD Cardiovascular Surgical Research Laboratory, Department of Cardiovascular Surgery, Toronto Western Division, Toronto Hospital Corporation, Toronto, Ontario, Canada
Retroperfusion of arterial blood through the coronary sinus reduces infarct size if therapy starts immediately after coronary artery occlusion. To determine if a new system of non-electrocardiogram-synchronized retroperfusion is able to reduce infarct size after delays consistent with clinical use, anesthetized pigs were subjected to 4 hours of left anterior descending coronary artery occlusion followed by 1 hour of reperfusion. Retroperfusion of arterial blood commenced immediately after occlusion of the left anterior descending coronary artery in the no-delay group (n = 10) and after a 1-hour (n = 10) and a 2-hour (n = 8) delay in two other groups. In the control group (n = lo), no therapy was used. In all groups, retroperfusion of arterial blood was terminated after 4 hours of occlusion of the left anterior descending coronary artery. Infarct size, expressed as a percentage of
the in vivo area at risk (k the standard deviation), was smaller in the no-delay group (44.1 f 12.9) and marginally smaller in the 1-hour delay group (71.0 f 9.8) compared with controls (86.3 2 7.5) ( p < 0.05). Infarct size in the 2-hour delay group (75.0 2 10.7) was not significantly different from controls. Mean coronary sinus pressure (f the standard deviation) was 56 -C 25 mm Hg, 39 2 9 mm Hg, and 47 k 9 mm Hg in the no-delay, 1-hour delay and 2-hour delay groups, respectively. Thus, this new retroperfusion system limits infarct size by 50% if it is started immediately after coronary occlusion. However, if institution of retroperfusion is delayed, only marginal benefit is achieved in a model of minimal intercoronary collateralization.
I
creating a temporary obstruction in the coronary venous system. This results in the development of a pressure gradient, which forces the actively retroperfused arterial (ie, well-oxygenated) blood into the ischemic zone where it can serve as nutritional support. The present study was conducted in the pig, an animal with few intercoronary collaterals [ 10-121, because there is a subset of patients who have few, if any, angiographically demonstrated collaterals [13]. As the amount of collateral flow is a critical determinant of myocardial infarct size [lo, 14-16], it is assumed that in the pig model with minimal collateralization, we are defining a lower time limit for delaying the implementation of ICSR, a limit still effective in limiting infarct size.
n the last 15 years, numerous experimental studies have demonstrated that retrograde administration of arterial blood through the coronary sinus is effective in reducing myocardial infarct size [l-31 and improving cardiac function [l, 4-91 when therapy is applied at the time of experimental coronary artery occlusion. Although application of therapy at the time of occlusion represents a situation where optimal benefit can be demonstrated, it does not represent the realistic delays that can occur between the onset of symptoms and the administration of therapy in patients with acute myocardial infarction. The purpose of this study was to define the time window within which arterial retroperfusion must be applied after a coronary occlusion to salvage ischemic myocardium. The intermittent coronary sinus retroperfusion (ICSR) system we tested differs from the electrocardiogram-synchronized system developed by Meerbaum and co-workers [4] and previously investigated in that retroperfusion is not limited to diastole but occurs over a period of several cardiac cycles, typically during inflation of a coronary sinus balloon catheter, followed recurrently by a period of no retroperfusion when the coronary sinus balloon catheter is deflated. Presumably, coronary venous retroperfusion works by Accepted for publication March 23, 1992 Address reprint requests to Dr Feindel, Department of Cardiovascular Surgery, Rm 14-222, Eaton Wing, The Toronto General Hospital, 200 Elizabeth St, Toronto, Ont, Canada M5G 2C4.
0 1992 by The Society of Thoracic Surgeons
(Ann Tkorac Surg 1992;54:1120-5)
Material and Methods The ZCSR System The ICSR device (supplied by Rocky Mountain Research Inc, Salt Lake City, UT) used in this study has two major components: a controller and a delivery device that is connected to a 16F triple-lumen balloon-tipped coronary sinus catheter (Fig 1). The delivery system uses a miniature roller pump driven by a direct-current stepping motor to provide perfusion to the central lumen of the catheter. A solenoid-actuated syringe is used to inflate and deflate the balloon on the tip of the coronary sinus catheter. The third lumen of the catheter is connected to a pressure transducer and is used to monitor coronary sinus 0003-4975/92/$5.00
FEINDEL ET AL RETROPERFUSION AND INFARCT SIZE
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CONTROLLED
ROLLER PUMP
b
\\
PRESSUREh
ARTERIAL
BLOOD
Fig 1. Intermittent coronary sinus retroperfusion system (generously supplied by Rocky Mountain Research Inc, Salt Lake City, UTJ.
pressure. The controller allows both the inflation and deflation times of the balloon to be adjusted independently between 0 and 99 seconds and the roller pump speed to be set to deliver blood at rates ranging from 0 to 600 mL/min. Perfusion can be set to run continuously or only in the inflation part of the cycle, as desired.
Experimenfal Preparation Yorkshire pigs weighing 24 to 34 kg were initially sedated with ketamine hydrochloride (30 mg/kg). After endotracheal intubation, they were anesthetized and ventilated by a Narco Air-Shield ventilator on a Boyle anesthetic machine delivering a mixture of oxygen (SO%), nitrous oxide (20%), and isoflurane (0.75% to 1.5%).The left ear vein was catheterized for administration of intravenous fluids. A midline sternotomy and pericardiotomy were performed to expose the heart and great vessels of the neck. A catheter inserted into the left carotid artery was used for monitoring arterial pressure and for sampling blood. A catheter placed in the innominate artery was connected through the roller pump of the retroperfusion device to the central lumen of the coronary sinus catheter to serve as the source of arterial blood. A triple-lumen thermodilution catheter (American Edwards Laboratories) was positioned in the pulmonary artery through the left jugular vein to determine cardiac output. A triple-lumen balloon-tipped Foley catheter (16F 50-mL balloon; Reich of Canada) was manually advanced into the orifice of the coronary sinus through the superior vena cava. The catheter was positioned such that the balloon was approximately 0.5 cm inside the coronary sinus. Correct positioning of the catheter was confirmed periodically throughout the experiment by manual palpation and direct visualization. Because the left hemiazygos vein empties into the proximal coronary sinus in the pig, it was necessary to ligate this vein to prevent the retroperfusate from escaping through this route. The left anterior descending coronary artery (LAD)was dissected at the level of the second diagonal branch with
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great care taken to prevent damage to the accompanying coronary veins. After a period of stabilization, lidocaine hydrochloride (50 mg) and heparin sulfate (10,000 U) were administered intravenously. The LAD was occluded for 4 hours with a silk ligature, and then the ligature was released to allow 1 hour of reperfusion. Hemodynamic measurements (heart rate, arterial pressure, coronary sinus pressure, and cardiac output) were measured before occlusion, and these measurements were repeated 120, 230, and 290 minutes after occlusion. The animals were treated humanely in compliance with the "Guide to the Care and Use of Experimental Animals," volume 1 (1980) and volume 2 (1984), Canadian Council on Animal Care.
Study Protocol The animals were divided into four groups based on when retroperfusion of arterial blood was begun. In all four groups, the LAD was occluded for 4 hours and then reperfused for 1 hour. In the no-delay group, ICSR commenced immediately after coronary occlusion and continued until the end of the 4-hour occlusion period. In the 1-hour and 2-hour delay groups, ICSR commenced 1 hour and 2 hours after coronary occlusion, respectively, and also continued until the end of the 4-hour occlusion period. In the control group, no therapy was given (no coronary sinus catheter), but the left hemiazygos vein was ligated. Intermittent coronary sinus retroperfusion consisted of 5 seconds of balloon inflation followed by 5 seconds of balloon deflation with retroperfusion of arterial blood at 60 mWmin during the inflation part of the cycle.
Determination of Area at Risk and Infarct Size The area at risk of infarction was delineated at the end of the reperfusion period by again occluding the LAD at the previous ligation site and injecting 1 million radioactive microspheres, 15 f 3 pm in diameter and labeled with cerium 141 (Dupont-NEN, Montreal, Que), into the left atrium over a 1-minute period. The microspheres were suspended in 10 mL of 0.9% sterile saline solution containing 0.01% Tween 80. One hour after reperfusion, the animals were killed with an overdose of isoflurane and potassium chloride. The hearts were excised, sliced transversely into sections 1 cm thick, weighed, and assessed for necrosis by incubation in tetrazolium chloride according to the method of Lie and colleagues [17]. Tetrazoliumpositive tissue (dark red) was considered viable, whereas areas that failed to stain with tetrazolium (pale) were considered necrotic. Each slice was then sandwiched gently between transparent plates to hold it in a fixed orientation. Care was taken to prevent tissue distortion when placing the heart slices into the device. The heart slices were photographed on both sides using 10 x 15-cm Kodak Ektachrome 64T tungsten film. This film produces color transparencies showing the actual size of heart slices. These transparencies were used for later planimetric assessment of infarct size. Autoradiographs were then made by placing Kodak X-Omatic x-ray film on each side of the slice and exposing the film for 48 hours at 4°C. After it was developed, the
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FEINDEL ET AL RETROPERFUSION AND INFARCT SIZE
Ann Thorac Surg 1992;54:112O-5
exposed film was carefully aligned with the original full-format heart photograph. Correct alignment of the heart slices was facilitated by the placement of small metallic markers on the transparent plates. As these markers were visible on both the photographs and the autoradiographs, they served for the alignment of the two sheets. Once developed, the x-ray film revealed the extent of trapping of the 14'Ce-tagged microspheres and thus delineated the area at risk at the time of microsphere injection (ie, 1 hour after reperfusion of the LAD). The outlines of the area at risk, the area infarcted, and the area occupied by the left and right ventricles were analyzed by computerized planimetry. By using the mass of each slice, the percentage of necrotic myocardium within the area at risk was calculated for each heart as described previously [MI.
Statistical Analysis All values are expressed as the mean & one standard deviation. All data first were analyzed to determine if the assumptions for parametric testing (normal distributions and equal variances) were met. Because they were met, a one-way analysis of variance was used to compare area at risk and infarct sizes between the four groups. The Scheffe F test was employed to determine which differences were significant. Hemodynamic data were analyzed using an analysis of variance for repeated measures. Deaths caused by ventricular fibrillation were compared between the four groups using Fisher's exact test. Significance was defined as a p value of less than 0.05.
Results There were no significant differences in the mean values for body weight and left ventricular weight between the four experimental groups.
Exclusions Of the 52 pigs initially entered into the study, 13 did not complete the protocol. Two animals died during the surgical procedure owing to technical difficulties associ-
m Y Gj
Z Y
i
30
CONTROL 2
H DELAY 1
INFARCT SIZE AREA AT RISK SIZE
H DELAY
NO DELAY
GROUP Fig 2. Comparison of infarct and area-at-risk sizes expressed as percentage of the left ventricle between the four groups. (" p < 0.05 versus control group by Schefft! test.)
Y
120 100
1
* Y ' i
80
El
A
8
60
Y
"y A
40
A A
I LU
*
CONTROL
2 H
1 H
DELAY
DELAY
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GROUP Fig 3 . Comparison of percentage of area at risk infarcted between the four groups. (* p < 0.05 versus control group by Schefft! test; t p < 0.05 versus no-delay group by Schefft! test.)
ated with LAD dissection. Two pigs in the 2-hour delay group were excluded from the study because of technical complicationswith the autoradiograms. Two animals died of ventricular fibrillation within the first few minutes of coronary occlusion before they were assigned to a group. A further 6 pigs (2 in the no-delay group and 2 each in the 1-hour and 2-hour delay groups) died of ventricular fibrillation within the first 30 minutes of retroperfusion therapy. One pig in the 2-hour delay group died minutes after reperfusion had begun. There were no deaths caused by ventricular fibrillation in the control group. Thus, 2, 2, 3, and 0 deaths were due to ventricular fibrillation in the no-delay, 1-hour delay, 2-hour delay, and control groups, respectively. The number of deaths due to ventricular fibrillation was not significantly different between the four groups ( p = 0.44, Fisher's exact test).
Infarct Size Figure 2 shows the infarct and area-at-risk sizes expressed as a percentage of the left ventricle. The microspheredetermined area at risk, as measured at the end of the experiment, was nearly identical in the four groups. Infarct size, when considered as a percentage of the left ventricle, was significantly smaller only in the no-delay group compared with controls ( p < 0.01). The percentage of the area at risk that was infarcted was 44.1% k 12.9%in the no-delay group, 71.0% & 9.8% in the 1-hour delay group, 75.0% 2 10.7% in the 2-hour delay group, and 86.3% 5 7.5% in the control group, which received no retroperfusion therapy (Fig 3). When comparisons were made with the control group, application of retroperfusion reduced infarct size as expressed as a percentage of the area at risk in the no-delay group ( p < 0.001) and the 1-hour delay group ( p = 0.03). The administration of ICSR therapy after a 2-hour delay did not significantly reduce infarct size ( p = 0.06). Immediate commencement of retroperfusion (no delay) achieved significantly greater
FEINDEL ET AL RETROPERFUSION AND INFARCT SIZE
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1123
Hemodynamics
Y
v,
The hemodynamic data for the four experimental groups are summarized in Table 1. Two-way analysis of variance for repeated measures revealed a significant time effect for each of the hemodynamic variables ( p < 0.0001). However, there was no significant group effect for any of these variables, indicating that the changes across time in the control group were not different from the corresponding changes across time in the retroperfused groups. Mean coronary sinus pressure was 56.1 25.4 mm Hg, 38.0 8.5 mm Hg, and 47.3 8.9 mm Hg in the no delay, 1-hour delay, and 2-hour delay groups, respectively. Throughout the entire 4-hour occlusion period, coronary sinus pressures promptly fell to a right atrial pressure level immediately on balloon deflation in all three groups receiving retroperfusion, indicating that the coronary sinus had not thrombosed while the catheter was in position. Mean coronary sinus pressure was higher in the no-delay group compared with the 1-hour delay group, but this did not quite reach significance ( p = 0.052).
U
IQ:
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I I-
*
*
s TIME DELAY IN ICSR ADMINISTRATION
Fig 4. Relationship between delay in administration of intermittent coronary sinus retroperfusion (ICSR) and percentage of the area at risk salvaged.
*
Comment
salvage than that achieved after a 1-hour or 2-hour delay ( p < 0.001). Immediate application of ICSR resulted in a 50% reduction in infarct size; however, the amount of myocardium salvaged by ICSR falls to 17% and 11%after delays in administration of 1 hour and 2 hours, respectively (Fig 4). Macroscopically, infarcts in the control, 1-hour, and 2-hour delay groups appeared as confluent areas, whereas in the no-delay group, they appeared as small, patchy areas interspersed among salvaged tissue. A few animals in each of the retroperfused groups exhibited small hemorrhagic areas within the myocardium. In all cases, these areas were confined to the borders of the infarct.
This study demonstrates that retroperfusion with arterial blood is able to salvage ischemic myocardium in animals with minimal collateralization. However, time of initiation of therapy was of critical importance in obtaining a beneficial effect, as initiation of retroperfusion immediately after coronary occlusion produced substantially more salvage than did administration delayed for 1 hour or 2 hours after occlusion. The amount of myocardium available for salvage by the application of retroperfusion after a 1-hour or 2-hour delay depends on (1)the effectiveness of retroperfusion in salvaging ischemic myocardium and (2) how much of the myocardium within the area at risk has already under-
Table I. Hernudunarnic Data fur the Four Experimental Groupsa Variable Mean arterial pressure (mm Hg) Before occlusion 120 minutes after occlusion 230 minutes after occlusion 290 minutes after occlusion Heart rate (beatsimin) Before occlusion 120 minutes after occlusion 230 minutes after occlusion 290 minutes after occlusion Rate-pressure product (beats x mm Hg/min) Before occlusion 120 minutes after occlusion 230 minutes after occlusion 290 minutes after occlusion
Control 83.7 4 10.4 67.7 2 10.7 65.8 4 8.4 61.5 4 6.6 111.4 4 100.9 4 99.8 4 99.1 k 11,492.8 4 9,057.6 4 8,672.1 4 8,422.0 ?
8.6 8.7 7.4 5.7 1,440.1 1,631.6 1,435.7 1,170.8
No Delay
81.7 4 7.3 69.7 ? 11.0 68.1 2 12.1 63.4 4 14.7 107.0 ? 101.7 4 100.2 ? 100.3 4 11,222.5 ? 9,327.6 ? 8,949.7 4 8,593.4 4
9.2 7.2 6.4 4.3 1,502.2 1,097.1 956.2 1,198.6
1-Hour Delay 92.5 4 10.0 72.2 2 18.8 57.7 2 13.2 57.9 4 13.5 108.7 t 14.2 95.3 8.8 98.0 4 19.0 104.0 t 17.3
*
12,088.0 ? 8,552.3 ? 7,292.7 4 7,928.0 4
1,973.8 1,859.9 1,216.2 1,172.2
2-Hour Delay 87.4 67.6 69.0 56.8
?
10.4 11.6 16.0 16.6
106.5 ? 99.0 ? 92.6 +90.5 4
10.2 7.0 14.2 15.0
4
? 4
11,492.8 ? 8,840.9 ? 8,453.6 4 7,197.0 rt
1,440.2 1,636.5 2,576.2 2,174.2
For each of the hemodynamic variables, repeated-measures analysis of variable indicated a significant time effect ( p < 0,001); however, there were no significant group effects ( p < 0.05) for any of the hemodynamic variables measured.
a
1124
FEINDEL ET AL RETROPERFUSION AND INFARCT SIZE
gone necrosis by the time retroperfusion is applied. Schaper and co-workers [14] documented that the rate of infarct development in the pig is rapid: more than 90% of the area at risk is necrotic after 45 minutes of coronary occlusion, and maximal infarct size is achieved after 90 minutes of occlusion. These results are similar to those of Fujiwara and associates [16] and suggest that the time window in which any therapy can potentially demonstrate a beneficial effect in the pig model is very small, perhaps less than 45 minutes in a situation of minimal collateralization. However, Horneffer and co-authors [19] observed that only 43% and 71% of the area at risk was infarcted in the pig after 45 and 90 minutes of occlusion, respectively. They noted that infarction was still occurring up to 180 minutes of occlusion, which suggests that the time window for therapeutic intervention in this species may be larger than that proposed by Schaper [14], Fujiwara [16], and their associates. Other investigators [lo, 2&22] also observed a slower rate of infarct progression with approximately 60% or less of the area at risk being infarcted after periods of occlusion of 60 minutes or longer. The difference between the rates of infarct progression in these studies may be attributed to several factors including differences in myocardial oxygen consumption, collateral flow, length of the reperfusion period, and differences in the size of the area at risk. In the current investigation, the observation that retroperfusion was able to salvage myocardium after a 1-hour delay suggests that noninfarcted myocardium was still present at this time in this model of minimal intercoronary collateralization, and that consequently, the rate of infarct progression was slower in this study compared with that of Fujiwara and co-workers [16] or Schaper and colleagues [14]. This may have been due to the lower myocardial oxygen consumption in the present study, as the product of rate times pressure (an indicator of myocardial oxygen demand) fell significantly in all experimental groups during the occlusion and reperfusion periods. The importance of myocardial oxygen consumption and hernodynamics on ultimate infarct size has been previously examined in the pig [14, 201 and is known to play a role in governing the rate at which an infarct evolves. The reason for the declines in the measured hemodynamic variables cannot be determined from these experiments. However, there was no significant group effect for any of the hemodynamic variables, indicating that all of the groups were similar in this regard.
Comparison of Retroperfusion With Reperfusion Of all the therapies tested for salvage of ischemic myocardium, only early reperfusion has been adopted for widespread clinical use [23]. Therefore, if reperfusion is used as the gold standard by which to compare the efficacy of retroperfusion, studies initiating retroperfusion and reperfusion at comparable intervals after coronary occlusion would yield important information. Geary and associates [3] compared the degree of myocardial salvage obtained with antegrade arterial reperfusion and electrocardiogram-synchronized retroperfusion in baboons subjected to LAD occlusion and found that retroperfusion
Ann Thorac Surg 1992;541120-5
after 1 hour of coronary occlusion resulted in salvage similar to that obtained with antegrade reperfusion after 2 hours of occlusion. The mechanisms by which retroperfusion exerts a beneficial effect on the ischemic myocardium are uncertain but are presumably related to the provision of arterial blood to the ischemic myocardium and the washout of toxic metabolites. However, it is unclear what proportion of the arterial retroperfusate actually serves as nutritional support for the ischemic myocardium and how much is simply shunted through venous anastomoses [24].
Validity of Tetrazolium Staining to Quantify Necrosis In this investigation, myocardial infarct size was quantified using tetrazolium staining. Previous investigations in our laboratory in pigs subjected to a protocol similar to that in the no-delay group have checked tetrazolium staining against electron microscopy, as it has been suggested that tetrazolium nonstaining may not be an adequate predictor of myocardial necrosis [25]. Our studies demonstrated that in the model we have used, electron microscopy (using either conventional or a low-denaturation [26] tissue processing technique) validated the tetrazolium chloride staining technique and that areas appearing unstained using tetrazolium were clearly irreversibly damaged as exemplified by the presence of sarcolemmal disruptions and the presence of mitochondria containing multiple, large electron-dense deposits. On the other hand, areas that were stained by tetrazolium (tetrazoliumpositive-red) appeared either normal or only mildly ischemic. We are confident, therefore, that the tetrazolium staining used in this study provided an adequate end point for the identification of areas of necrosis.
Clinical Implications In a clinical setting, it is important to know when the application of retroperfusion therapy is no longer likely to be effective in limiting infarct size. We have demonstrated that in an animal with minimal collateralization, ICSR results in only modest salvage of ischemic myocardium if it is instituted after delays of an hour or more. However, patients with acute myocardial infarction show a great deal of variability in the extent of preformed collaterals and, as a result, a great deal of variability in the rates at which an infarct will evolve. Thus, it is possible that patients with extensive preformed collaterals may benefit from the effects of retroperfusion for longer periods after coronary occlusion. The finding that the ICSR system reduced infarct size substantially in the no-delay group supports the potential use of this system in an emergency “bail-out” procedure for abrupt coronary closure during percutaneous transluminal coronary angioplasty. Because a coronary sinus catheter can be placed within minutes and with little difficulty, the possible use of the ICSR system in this situation needs to be investigated. The ICSR system offers some advantages over the electrocardiogram-synchronized system, which has been previously reported [l-91. First, because the system does not synchronize to the electrocardiogram, it is compact
Ann Thorac Surg 1992;54:1120-5
and inexpensive and can be easily transported with a patient. Second, difficulties in tracking the heart beat, which are particularly great in the presence of arrhythmias, are avoided. A potential disadvantage of the ICSR system is the presence of high coronary sinus pressures that are maintained over several cardiac cycles unlike in the synchronized system, in which the elevated coronary sinus pressures are relieved during each systole. Additional studies are needed to optimize the ICSR system. They should determine the coronary sinus pressures and the occlusion/release cycles needed for myocardial protection. This work was generously supported by grant AN429 from the Ontario Heart and Stroke Foundation. We gratefully acknowledge Ms Patty Boylen for technical assistance.
References 1. Farcot JC, Meerbaum S, Lang T, Kaplan L, Corday E.
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Synchronized retroperfusion of coronary veins for circulatory support of jeopardized ischemic myocardium. Am J Cardiol 1978;41:1191-201. Smith GT, Geary GG, Blanchard W, McNamara JJ. Reduction in infarct size by synchronized selective coronary venous retroperfusion of arterialized blood. Am J Cardiol 1981;48: 1064-70. Geary GG, Smith GT, Suehiro GT, Zeman C, Siu 6, McNamara JJ. Quantitative assessment of infarct size reduction by coronary venous retroperfusion in baboons. Am J Cardiol 1982;50:142&30. Meerbaum S, Lang TW, Osher JV, et al. Diastolic retroperfusion of acutely ischemic myocardium. Am J Cardiol 1976; 3758S98. Yamazaki S, Drury JK, Meerbaum S, Corday E. Effects of synchronized retroperfusion on left ventricular function measured by two dimension echocardiography. In: Mohl W, Wolner E, Glogar D, eds. The coronary sinus. Darmstadt: Steinkopff Verlag, 1984:375-9. Gundry SR. Modification of myocardial ischemia in normal and hypertrophied hearts utilizing diastolic retroperfusion of the coronary veins. J Thorac Cardiovasc Surg 1982;83:659-69. Yamazaki S, Drury JK, Meerbaum S, Corday E. Synchronized coronary venous retroperfusion: prompt improvement of left ventricular function in experimental myocardial ischemia. J Am Coll Cardiol 1985;5:655-63. Farcot JC, Berdeaux A, Guidicelli JF, Vilaine JP, Bourdarias JP. Diastolic synchronized retroperfusion versus reperfusion: effects on regional left ventricular function and myocardial blood flow during acute coronary occlusion in dogs. Am J Cardiol 1983;51:141&21. Morkov AK, Lehan PH, Hellems HK. Reversal of acute myocardial ischemia in closed chested animals by retrograde perfusion of the coronary sinus with arterial blood. Acta Cardiol (Brux) 1976;185-99. Savage RM, Guth 6, White FC, Hagan AD, Bloor CM. Correlation of regional myocardial blood flow and function with myocardial infarct size during acute myocardial ischemia in the conscious pig. Circulation 1981;64:699-707.
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11. Maxwell MP, Hearse DJ, Yellon DM. Species variation in the coronary collateral circulation during regional myocardial ischemia: a critical determinant of the rate of evolution and extent of myocardial infarction. Cardiovasc Res 1987;21: 737-46. 12. Hearse DJ, Muller CA, Fukanami M, Kudoh Y, Opie LH, Yellon DM. Regional myocardial ischemia: characterization of temporal, transmural and lateral flow interfaces in the porcine heart. Can J Cardiol 1986;2:4%61. 13. Dewood MA, Heit J, Spores J, et al. Anterior transmural myocardial infarction: effects of surgical coronary reperfusion on global and regional left ventricular function. J Am Coll Cardiol 1983;1:1223. 14. Schaper W, Binz K, Sass S, Winkler 6. Influence of collateral blood flow and of variations in MV02 on tissue-ATP content in ischemic and infarcted myocardium. J Mol Cell Cardiol 1987;19:19-37. 15. Schaper W. The collateral circulation of the heart. Prog Cardiovasc Dis 1988;31:57-77. 16. Fujiwara H, Matsuda M, Fujiwara Y, et al. Infarct size and the protection of ischemic myocardium in pig, dog and human. Jpn Circ J 1989;53:1092-7. 17. Lie JT, Pairolero PC, Holley KE, Titus JL. Macroscopic enzyme mapping verification of large, homogenous experimental myocardial infarcts of predictable size and location in dogs. J Thorac Cardiovasc Surg 1975;69:599-605. 18. Feindel CM, Tait GA, Wilson GJ, Klement P, MacGregor DC. Multidose blood versus crystalloid cardioplegia. Comparison by quantitative assessment of irreversible myocardial injury. J Thorac Cardiovasc Surg 1984;87585-95. 19. Horneffer PJ, Healy B, Gott VL, Gardner TJ. The rapid evolution of a myocardial infarction in an end-artery coronary preparation. Circulation 1987;76(Suppl5):3942. 20. Garcia-Dorado D, Theroux P, Elizaga J, Fernandez F, Alonso J, Solares J. Influence of tachycardia and arterial hypertension on infarct size in the pig. Cardiovasc Res 1988;22:6204. 21. Klein HH, Schubothe M, Nebandahl K, Kreuzer H. Temporal and spatial development of infarcts in porcine hearts. Basic Res Cardiol 1983;79:440-7. 22. Warltier DC, Zyvoloski MG, Gross GJ, Brooks HL. Subendocardial versus transmural myocardial infarction: relationship to the collateral circulation in the canine and porcine hearts. Can J Physiol Pharmacol 1982;60:1700-6. 23. Reimer KA, Jennings RB. Experimental models and endpoints for evaluating interventions in the acute phase of myocardial infarction: an anatomic approach. In: Morganroth J, Moore EN, eds. Interventions in the acute phase of myocardial infarction. New York: Martinus Nijhoff, 1984: 1-11. 24. Cohen MV, Matsuki T, Downey JM. Pressure-flow characteristics and nutritional capacity of coronary veins in dogs. Am J Physiol 1988;255:H834-46. 25. Barnard RJ, Okamoto F, Buckberg GD, et al. Studies of controlled reperfusion after ischemia. 111. Histochemical studies: inability of triphenyltetrazolium chloride nonstaining to define tissue necrosis. J Thorac Cardiovasc Surg 1986;92:502-12. 26. Sjostrand F, Allen BS, Buckberg GD, et al. Studies of controlled reperfusion after ischemia. IV. Electron microscopic studies: importance of embedding techniques in quantitative evaluation of cardiac mitochondria1 structure during regional ischemia and reperfusion. J Thorac Cardiovasc Surg 1986;92: 51S24.
Retroperfusion of arterial blood through the coronary sinus reduces infarct size if therapy starts immediately after coronary artery occlusion. To determine if a new system of non-electrocardiogram-synchronized retroperfusion is able to reduce infarct size after delays consistent with clinical use, anesthetized pigs were subjected to 4 hours of left anterior descending coronary artery occlusion followed by 1 hour of reperfusion. Retroperfusion of arterial blood commenced immediately after occlusion of the left anterior descending coronary artery in the no-delay group (n = 10) and after a 1-hour (n = 10) and a 2-hour (n = 8) delay in two other groups. In the control group (n = lo), no therapy was used. In all groups, retroperfusion of arterial blood was terminated after 4 hours of occlusion of the left anterior descending coronary artery. Infarct size, expressed as a percentage of
the in vivo area at risk (k the standard deviation), was smaller in the no-delay group (44.1 f 12.9) and marginally smaller in the 1-hour delay group (71.0 f 9.8) compared with controls (86.3 2 7.5) ( p < 0.05). Infarct size in the 2-hour delay group (75.0 2 10.7) was not significantly different from controls. Mean coronary sinus pressure (f the standard deviation) was 56 -C 25 mm Hg, 39 2 9 mm Hg, and 47 k 9 mm Hg in the no-delay, 1-hour delay and 2-hour delay groups, respectively. Thus, this new retroperfusion system limits infarct size by 50% if it is started immediately after coronary occlusion. However, if institution of retroperfusion is delayed, only marginal benefit is achieved in a model of minimal intercoronary collateralization.
I
creating a temporary obstruction in the coronary venous system. This results in the development of a pressure gradient, which forces the actively retroperfused arterial (ie, well-oxygenated) blood into the ischemic zone where it can serve as nutritional support. The present study was conducted in the pig, an animal with few intercoronary collaterals [ 10-121, because there is a subset of patients who have few, if any, angiographically demonstrated collaterals [13]. As the amount of collateral flow is a critical determinant of myocardial infarct size [lo, 14-16], it is assumed that in the pig model with minimal collateralization, we are defining a lower time limit for delaying the implementation of ICSR, a limit still effective in limiting infarct size.
n the last 15 years, numerous experimental studies have demonstrated that retrograde administration of arterial blood through the coronary sinus is effective in reducing myocardial infarct size [l-31 and improving cardiac function [l, 4-91 when therapy is applied at the time of experimental coronary artery occlusion. Although application of therapy at the time of occlusion represents a situation where optimal benefit can be demonstrated, it does not represent the realistic delays that can occur between the onset of symptoms and the administration of therapy in patients with acute myocardial infarction. The purpose of this study was to define the time window within which arterial retroperfusion must be applied after a coronary occlusion to salvage ischemic myocardium. The intermittent coronary sinus retroperfusion (ICSR) system we tested differs from the electrocardiogram-synchronized system developed by Meerbaum and co-workers [4] and previously investigated in that retroperfusion is not limited to diastole but occurs over a period of several cardiac cycles, typically during inflation of a coronary sinus balloon catheter, followed recurrently by a period of no retroperfusion when the coronary sinus balloon catheter is deflated. Presumably, coronary venous retroperfusion works by Accepted for publication March 23, 1992 Address reprint requests to Dr Feindel, Department of Cardiovascular Surgery, Rm 14-222, Eaton Wing, The Toronto General Hospital, 200 Elizabeth St, Toronto, Ont, Canada M5G 2C4.
0 1992 by The Society of Thoracic Surgeons
(Ann Tkorac Surg 1992;54:1120-5)
Material and Methods The ZCSR System The ICSR device (supplied by Rocky Mountain Research Inc, Salt Lake City, UT) used in this study has two major components: a controller and a delivery device that is connected to a 16F triple-lumen balloon-tipped coronary sinus catheter (Fig 1). The delivery system uses a miniature roller pump driven by a direct-current stepping motor to provide perfusion to the central lumen of the catheter. A solenoid-actuated syringe is used to inflate and deflate the balloon on the tip of the coronary sinus catheter. The third lumen of the catheter is connected to a pressure transducer and is used to monitor coronary sinus 0003-4975/92/$5.00
FEINDEL ET AL RETROPERFUSION AND INFARCT SIZE
Ann Thorac Surg 1992;54:1120-5
CONTROLLED
ROLLER PUMP
b
\\
PRESSUREh
ARTERIAL
BLOOD
Fig 1. Intermittent coronary sinus retroperfusion system (generously supplied by Rocky Mountain Research Inc, Salt Lake City, UTJ.
pressure. The controller allows both the inflation and deflation times of the balloon to be adjusted independently between 0 and 99 seconds and the roller pump speed to be set to deliver blood at rates ranging from 0 to 600 mL/min. Perfusion can be set to run continuously or only in the inflation part of the cycle, as desired.
Experimenfal Preparation Yorkshire pigs weighing 24 to 34 kg were initially sedated with ketamine hydrochloride (30 mg/kg). After endotracheal intubation, they were anesthetized and ventilated by a Narco Air-Shield ventilator on a Boyle anesthetic machine delivering a mixture of oxygen (SO%), nitrous oxide (20%), and isoflurane (0.75% to 1.5%).The left ear vein was catheterized for administration of intravenous fluids. A midline sternotomy and pericardiotomy were performed to expose the heart and great vessels of the neck. A catheter inserted into the left carotid artery was used for monitoring arterial pressure and for sampling blood. A catheter placed in the innominate artery was connected through the roller pump of the retroperfusion device to the central lumen of the coronary sinus catheter to serve as the source of arterial blood. A triple-lumen thermodilution catheter (American Edwards Laboratories) was positioned in the pulmonary artery through the left jugular vein to determine cardiac output. A triple-lumen balloon-tipped Foley catheter (16F 50-mL balloon; Reich of Canada) was manually advanced into the orifice of the coronary sinus through the superior vena cava. The catheter was positioned such that the balloon was approximately 0.5 cm inside the coronary sinus. Correct positioning of the catheter was confirmed periodically throughout the experiment by manual palpation and direct visualization. Because the left hemiazygos vein empties into the proximal coronary sinus in the pig, it was necessary to ligate this vein to prevent the retroperfusate from escaping through this route. The left anterior descending coronary artery (LAD)was dissected at the level of the second diagonal branch with
1121
great care taken to prevent damage to the accompanying coronary veins. After a period of stabilization, lidocaine hydrochloride (50 mg) and heparin sulfate (10,000 U) were administered intravenously. The LAD was occluded for 4 hours with a silk ligature, and then the ligature was released to allow 1 hour of reperfusion. Hemodynamic measurements (heart rate, arterial pressure, coronary sinus pressure, and cardiac output) were measured before occlusion, and these measurements were repeated 120, 230, and 290 minutes after occlusion. The animals were treated humanely in compliance with the "Guide to the Care and Use of Experimental Animals," volume 1 (1980) and volume 2 (1984), Canadian Council on Animal Care.
Study Protocol The animals were divided into four groups based on when retroperfusion of arterial blood was begun. In all four groups, the LAD was occluded for 4 hours and then reperfused for 1 hour. In the no-delay group, ICSR commenced immediately after coronary occlusion and continued until the end of the 4-hour occlusion period. In the 1-hour and 2-hour delay groups, ICSR commenced 1 hour and 2 hours after coronary occlusion, respectively, and also continued until the end of the 4-hour occlusion period. In the control group, no therapy was given (no coronary sinus catheter), but the left hemiazygos vein was ligated. Intermittent coronary sinus retroperfusion consisted of 5 seconds of balloon inflation followed by 5 seconds of balloon deflation with retroperfusion of arterial blood at 60 mWmin during the inflation part of the cycle.
Determination of Area at Risk and Infarct Size The area at risk of infarction was delineated at the end of the reperfusion period by again occluding the LAD at the previous ligation site and injecting 1 million radioactive microspheres, 15 f 3 pm in diameter and labeled with cerium 141 (Dupont-NEN, Montreal, Que), into the left atrium over a 1-minute period. The microspheres were suspended in 10 mL of 0.9% sterile saline solution containing 0.01% Tween 80. One hour after reperfusion, the animals were killed with an overdose of isoflurane and potassium chloride. The hearts were excised, sliced transversely into sections 1 cm thick, weighed, and assessed for necrosis by incubation in tetrazolium chloride according to the method of Lie and colleagues [17]. Tetrazoliumpositive tissue (dark red) was considered viable, whereas areas that failed to stain with tetrazolium (pale) were considered necrotic. Each slice was then sandwiched gently between transparent plates to hold it in a fixed orientation. Care was taken to prevent tissue distortion when placing the heart slices into the device. The heart slices were photographed on both sides using 10 x 15-cm Kodak Ektachrome 64T tungsten film. This film produces color transparencies showing the actual size of heart slices. These transparencies were used for later planimetric assessment of infarct size. Autoradiographs were then made by placing Kodak X-Omatic x-ray film on each side of the slice and exposing the film for 48 hours at 4°C. After it was developed, the
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FEINDEL ET AL RETROPERFUSION AND INFARCT SIZE
Ann Thorac Surg 1992;54:112O-5
exposed film was carefully aligned with the original full-format heart photograph. Correct alignment of the heart slices was facilitated by the placement of small metallic markers on the transparent plates. As these markers were visible on both the photographs and the autoradiographs, they served for the alignment of the two sheets. Once developed, the x-ray film revealed the extent of trapping of the 14'Ce-tagged microspheres and thus delineated the area at risk at the time of microsphere injection (ie, 1 hour after reperfusion of the LAD). The outlines of the area at risk, the area infarcted, and the area occupied by the left and right ventricles were analyzed by computerized planimetry. By using the mass of each slice, the percentage of necrotic myocardium within the area at risk was calculated for each heart as described previously [MI.
Statistical Analysis All values are expressed as the mean & one standard deviation. All data first were analyzed to determine if the assumptions for parametric testing (normal distributions and equal variances) were met. Because they were met, a one-way analysis of variance was used to compare area at risk and infarct sizes between the four groups. The Scheffe F test was employed to determine which differences were significant. Hemodynamic data were analyzed using an analysis of variance for repeated measures. Deaths caused by ventricular fibrillation were compared between the four groups using Fisher's exact test. Significance was defined as a p value of less than 0.05.
Results There were no significant differences in the mean values for body weight and left ventricular weight between the four experimental groups.
Exclusions Of the 52 pigs initially entered into the study, 13 did not complete the protocol. Two animals died during the surgical procedure owing to technical difficulties associ-
m Y Gj
Z Y
i
30
CONTROL 2
H DELAY 1
INFARCT SIZE AREA AT RISK SIZE
H DELAY
NO DELAY
GROUP Fig 2. Comparison of infarct and area-at-risk sizes expressed as percentage of the left ventricle between the four groups. (" p < 0.05 versus control group by Schefft! test.)
Y
120 100
1
* Y ' i
80
El
A
8
60
Y
"y A
40
A A
I LU
*
CONTROL
2 H
1 H
DELAY
DELAY
NO DELAY
GROUP Fig 3 . Comparison of percentage of area at risk infarcted between the four groups. (* p < 0.05 versus control group by Schefft! test; t p < 0.05 versus no-delay group by Schefft! test.)
ated with LAD dissection. Two pigs in the 2-hour delay group were excluded from the study because of technical complicationswith the autoradiograms. Two animals died of ventricular fibrillation within the first few minutes of coronary occlusion before they were assigned to a group. A further 6 pigs (2 in the no-delay group and 2 each in the 1-hour and 2-hour delay groups) died of ventricular fibrillation within the first 30 minutes of retroperfusion therapy. One pig in the 2-hour delay group died minutes after reperfusion had begun. There were no deaths caused by ventricular fibrillation in the control group. Thus, 2, 2, 3, and 0 deaths were due to ventricular fibrillation in the no-delay, 1-hour delay, 2-hour delay, and control groups, respectively. The number of deaths due to ventricular fibrillation was not significantly different between the four groups ( p = 0.44, Fisher's exact test).
Infarct Size Figure 2 shows the infarct and area-at-risk sizes expressed as a percentage of the left ventricle. The microspheredetermined area at risk, as measured at the end of the experiment, was nearly identical in the four groups. Infarct size, when considered as a percentage of the left ventricle, was significantly smaller only in the no-delay group compared with controls ( p < 0.01). The percentage of the area at risk that was infarcted was 44.1% k 12.9%in the no-delay group, 71.0% & 9.8% in the 1-hour delay group, 75.0% 2 10.7% in the 2-hour delay group, and 86.3% 5 7.5% in the control group, which received no retroperfusion therapy (Fig 3). When comparisons were made with the control group, application of retroperfusion reduced infarct size as expressed as a percentage of the area at risk in the no-delay group ( p < 0.001) and the 1-hour delay group ( p = 0.03). The administration of ICSR therapy after a 2-hour delay did not significantly reduce infarct size ( p = 0.06). Immediate commencement of retroperfusion (no delay) achieved significantly greater
FEINDEL ET AL RETROPERFUSION AND INFARCT SIZE
Ann Thorac Surg 1992;54:112&5
1123
Hemodynamics
Y
v,
The hemodynamic data for the four experimental groups are summarized in Table 1. Two-way analysis of variance for repeated measures revealed a significant time effect for each of the hemodynamic variables ( p < 0.0001). However, there was no significant group effect for any of these variables, indicating that the changes across time in the control group were not different from the corresponding changes across time in the retroperfused groups. Mean coronary sinus pressure was 56.1 25.4 mm Hg, 38.0 8.5 mm Hg, and 47.3 8.9 mm Hg in the no delay, 1-hour delay, and 2-hour delay groups, respectively. Throughout the entire 4-hour occlusion period, coronary sinus pressures promptly fell to a right atrial pressure level immediately on balloon deflation in all three groups receiving retroperfusion, indicating that the coronary sinus had not thrombosed while the catheter was in position. Mean coronary sinus pressure was higher in the no-delay group compared with the 1-hour delay group, but this did not quite reach significance ( p = 0.052).
U
IQ:
Q: W
I I-
*
*
s TIME DELAY IN ICSR ADMINISTRATION
Fig 4. Relationship between delay in administration of intermittent coronary sinus retroperfusion (ICSR) and percentage of the area at risk salvaged.
*
Comment
salvage than that achieved after a 1-hour or 2-hour delay ( p < 0.001). Immediate application of ICSR resulted in a 50% reduction in infarct size; however, the amount of myocardium salvaged by ICSR falls to 17% and 11%after delays in administration of 1 hour and 2 hours, respectively (Fig 4). Macroscopically, infarcts in the control, 1-hour, and 2-hour delay groups appeared as confluent areas, whereas in the no-delay group, they appeared as small, patchy areas interspersed among salvaged tissue. A few animals in each of the retroperfused groups exhibited small hemorrhagic areas within the myocardium. In all cases, these areas were confined to the borders of the infarct.
This study demonstrates that retroperfusion with arterial blood is able to salvage ischemic myocardium in animals with minimal collateralization. However, time of initiation of therapy was of critical importance in obtaining a beneficial effect, as initiation of retroperfusion immediately after coronary occlusion produced substantially more salvage than did administration delayed for 1 hour or 2 hours after occlusion. The amount of myocardium available for salvage by the application of retroperfusion after a 1-hour or 2-hour delay depends on (1)the effectiveness of retroperfusion in salvaging ischemic myocardium and (2) how much of the myocardium within the area at risk has already under-
Table I. Hernudunarnic Data fur the Four Experimental Groupsa Variable Mean arterial pressure (mm Hg) Before occlusion 120 minutes after occlusion 230 minutes after occlusion 290 minutes after occlusion Heart rate (beatsimin) Before occlusion 120 minutes after occlusion 230 minutes after occlusion 290 minutes after occlusion Rate-pressure product (beats x mm Hg/min) Before occlusion 120 minutes after occlusion 230 minutes after occlusion 290 minutes after occlusion
Control 83.7 4 10.4 67.7 2 10.7 65.8 4 8.4 61.5 4 6.6 111.4 4 100.9 4 99.8 4 99.1 k 11,492.8 4 9,057.6 4 8,672.1 4 8,422.0 ?
8.6 8.7 7.4 5.7 1,440.1 1,631.6 1,435.7 1,170.8
No Delay
81.7 4 7.3 69.7 ? 11.0 68.1 2 12.1 63.4 4 14.7 107.0 ? 101.7 4 100.2 ? 100.3 4 11,222.5 ? 9,327.6 ? 8,949.7 4 8,593.4 4
9.2 7.2 6.4 4.3 1,502.2 1,097.1 956.2 1,198.6
1-Hour Delay 92.5 4 10.0 72.2 2 18.8 57.7 2 13.2 57.9 4 13.5 108.7 t 14.2 95.3 8.8 98.0 4 19.0 104.0 t 17.3
*
12,088.0 ? 8,552.3 ? 7,292.7 4 7,928.0 4
1,973.8 1,859.9 1,216.2 1,172.2
2-Hour Delay 87.4 67.6 69.0 56.8
?
10.4 11.6 16.0 16.6
106.5 ? 99.0 ? 92.6 +90.5 4
10.2 7.0 14.2 15.0
4
? 4
11,492.8 ? 8,840.9 ? 8,453.6 4 7,197.0 rt
1,440.2 1,636.5 2,576.2 2,174.2
For each of the hemodynamic variables, repeated-measures analysis of variable indicated a significant time effect ( p < 0,001); however, there were no significant group effects ( p < 0.05) for any of the hemodynamic variables measured.
a
1124
FEINDEL ET AL RETROPERFUSION AND INFARCT SIZE
gone necrosis by the time retroperfusion is applied. Schaper and co-workers [14] documented that the rate of infarct development in the pig is rapid: more than 90% of the area at risk is necrotic after 45 minutes of coronary occlusion, and maximal infarct size is achieved after 90 minutes of occlusion. These results are similar to those of Fujiwara and associates [16] and suggest that the time window in which any therapy can potentially demonstrate a beneficial effect in the pig model is very small, perhaps less than 45 minutes in a situation of minimal collateralization. However, Horneffer and co-authors [19] observed that only 43% and 71% of the area at risk was infarcted in the pig after 45 and 90 minutes of occlusion, respectively. They noted that infarction was still occurring up to 180 minutes of occlusion, which suggests that the time window for therapeutic intervention in this species may be larger than that proposed by Schaper [14], Fujiwara [16], and their associates. Other investigators [lo, 2&22] also observed a slower rate of infarct progression with approximately 60% or less of the area at risk being infarcted after periods of occlusion of 60 minutes or longer. The difference between the rates of infarct progression in these studies may be attributed to several factors including differences in myocardial oxygen consumption, collateral flow, length of the reperfusion period, and differences in the size of the area at risk. In the current investigation, the observation that retroperfusion was able to salvage myocardium after a 1-hour delay suggests that noninfarcted myocardium was still present at this time in this model of minimal intercoronary collateralization, and that consequently, the rate of infarct progression was slower in this study compared with that of Fujiwara and co-workers [16] or Schaper and colleagues [14]. This may have been due to the lower myocardial oxygen consumption in the present study, as the product of rate times pressure (an indicator of myocardial oxygen demand) fell significantly in all experimental groups during the occlusion and reperfusion periods. The importance of myocardial oxygen consumption and hernodynamics on ultimate infarct size has been previously examined in the pig [14, 201 and is known to play a role in governing the rate at which an infarct evolves. The reason for the declines in the measured hemodynamic variables cannot be determined from these experiments. However, there was no significant group effect for any of the hemodynamic variables, indicating that all of the groups were similar in this regard.
Comparison of Retroperfusion With Reperfusion Of all the therapies tested for salvage of ischemic myocardium, only early reperfusion has been adopted for widespread clinical use [23]. Therefore, if reperfusion is used as the gold standard by which to compare the efficacy of retroperfusion, studies initiating retroperfusion and reperfusion at comparable intervals after coronary occlusion would yield important information. Geary and associates [3] compared the degree of myocardial salvage obtained with antegrade arterial reperfusion and electrocardiogram-synchronized retroperfusion in baboons subjected to LAD occlusion and found that retroperfusion
Ann Thorac Surg 1992;541120-5
after 1 hour of coronary occlusion resulted in salvage similar to that obtained with antegrade reperfusion after 2 hours of occlusion. The mechanisms by which retroperfusion exerts a beneficial effect on the ischemic myocardium are uncertain but are presumably related to the provision of arterial blood to the ischemic myocardium and the washout of toxic metabolites. However, it is unclear what proportion of the arterial retroperfusate actually serves as nutritional support for the ischemic myocardium and how much is simply shunted through venous anastomoses [24].
Validity of Tetrazolium Staining to Quantify Necrosis In this investigation, myocardial infarct size was quantified using tetrazolium staining. Previous investigations in our laboratory in pigs subjected to a protocol similar to that in the no-delay group have checked tetrazolium staining against electron microscopy, as it has been suggested that tetrazolium nonstaining may not be an adequate predictor of myocardial necrosis [25]. Our studies demonstrated that in the model we have used, electron microscopy (using either conventional or a low-denaturation [26] tissue processing technique) validated the tetrazolium chloride staining technique and that areas appearing unstained using tetrazolium were clearly irreversibly damaged as exemplified by the presence of sarcolemmal disruptions and the presence of mitochondria containing multiple, large electron-dense deposits. On the other hand, areas that were stained by tetrazolium (tetrazoliumpositive-red) appeared either normal or only mildly ischemic. We are confident, therefore, that the tetrazolium staining used in this study provided an adequate end point for the identification of areas of necrosis.
Clinical Implications In a clinical setting, it is important to know when the application of retroperfusion therapy is no longer likely to be effective in limiting infarct size. We have demonstrated that in an animal with minimal collateralization, ICSR results in only modest salvage of ischemic myocardium if it is instituted after delays of an hour or more. However, patients with acute myocardial infarction show a great deal of variability in the extent of preformed collaterals and, as a result, a great deal of variability in the rates at which an infarct will evolve. Thus, it is possible that patients with extensive preformed collaterals may benefit from the effects of retroperfusion for longer periods after coronary occlusion. The finding that the ICSR system reduced infarct size substantially in the no-delay group supports the potential use of this system in an emergency “bail-out” procedure for abrupt coronary closure during percutaneous transluminal coronary angioplasty. Because a coronary sinus catheter can be placed within minutes and with little difficulty, the possible use of the ICSR system in this situation needs to be investigated. The ICSR system offers some advantages over the electrocardiogram-synchronized system, which has been previously reported [l-91. First, because the system does not synchronize to the electrocardiogram, it is compact
Ann Thorac Surg 1992;54:1120-5
and inexpensive and can be easily transported with a patient. Second, difficulties in tracking the heart beat, which are particularly great in the presence of arrhythmias, are avoided. A potential disadvantage of the ICSR system is the presence of high coronary sinus pressures that are maintained over several cardiac cycles unlike in the synchronized system, in which the elevated coronary sinus pressures are relieved during each systole. Additional studies are needed to optimize the ICSR system. They should determine the coronary sinus pressures and the occlusion/release cycles needed for myocardial protection. This work was generously supported by grant AN429 from the Ontario Heart and Stroke Foundation. We gratefully acknowledge Ms Patty Boylen for technical assistance.
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