reduction of myocardial infarct size by a fluorocarbon-oxygenated reperfusate

Reduction of Myocardial infarct Size by a Fluorocarbon-Oxygenated Reperfusate

PHILIPPE MENASCHE, MD, MICHEL FAUCHET, MD, ANNE LAVERGNE, MD,

PIERRE COMMIN, MD, CHRISTIANE MASQUET, MD, PAGO LORENTE, MD,

PIERRE BIRKUI, MD, ROBERT P. GEYER, PhD, and ARMAND PIWNICA, MD

This investigation assesses whether the size of an acutely revascularized myocardial infarct (MI) could be reduced by altering the compositlon of the initial reperfusate. Nlneteen open-chest dogs underwent 4-hour occlusion of the. left anterior descending coronary artery and were then assigned to a treatment group: 12 dogs to selective intracoronary infusion of the modified reperfusate over 30 minutes before resumption of blood flow for 60 minutes and 7 to a control group (90 minutes of unmodified blood reperfusion). The modified reperfusate consisted of 566 ml of a fluorocarbon-oxygenated crystaloid solution (POP 650 mm Hg; total O2 content 5.5 ~01% ) whose composition was adjusted by decreasing Ca++ (0.25 mM), increasing pH (7.60) and adding glucose (1.8 g/liter). Four hours after occlusion, technetium-99mplabeled microspheres were in- jected into the left atrium. After 90 minutes of reperfusion, the heart was removed and sliced transversely. Areas not perfused by microspheres (areas at risk) were traced, planimetered and compared with the areas of necrosis after incubation

in triphenyltetrazolium chloride. Areas were then converted into weights. In control dogs, the weight of necrotic myocardium was not significantly different from the weight at risk (5.0 f 0.7 vs 7.0 f 0.6 g, respectively [mean f standard error of the mean]), whereas it was markedly reduced in treated dogs (5.9 f 0.5 vs 9.4 f 0.7 g, respectively, p <O.OOl). The weight of salvaged myocardium was 3.4 f 0.5 g in treated dogs vs 1.9 f 0.4 g in the control group (p <0.02). When the dogs were classified according to the amount of left ventricle at risk, the protective effects of the modlfied reperfusate were apparent mainly in the largest areas at risk (>40% of the left ventricle). We conclude that a modlfied initial reperfusate can significantly reduce MI size, possibly by preventing some reperfusion-related damage in the reversibly injured myocardial areas, and fluorocarbons are effective in oxygenating such crystalloid reperfusion solutions that might enhance the beneficial effects of intracoronary fibrinoiysis.

(Am J Cardiol 1964;53:608-613)

Early intracoronary infusion of streptokinase is increasing in importance as a mode of therapy in patients with acute myocardial infarction (MI).iJ However, 1 potential limitation of the effectiveness of early thrombolysis may be that under certain circumstances, reperfusion might extend myocardial damage.3

From the Service de Chirurgie Cardio-Vasculaire, Service de Cardiol- ogie, Service Central de Siophysique et de Wcine Nucleaire. Service d’Anatomie Pathologique et INSERM U-141, Hbpital Lariboisiire, Paris, France, and School of Public Health, Harvard University, Boston, Massachuuset. This work was support&i by a grant from the F(rdtuatian de Cardiologie. the UER Lariboisiere-Saint-Louis and Grant HL 15520 and Contract l-Hb-2912 from the National Heart, Lung, and Blood In- stitute, National Institutes of Health, U.S. Public Health Service, Be- thesda, Maryland. Manuscript received May 12, 1983; revised manuscript received October 11, 1983, accepted October 14. 1983.

Address for reprints: Philippe Menasche, MD, Service de Chirurgie Cardio-Vasculaire, Hopital Larlboisiere, 2 Rue Ambroise Pare, 75475 Paris Cedex 10, France.

In a previous study from our laboratory4 dealing with a globally ischemic heart subjected to 2 hours of ischemia under cardioplegic protection, we showed that the recovery of cardiac function could be significantly improved by modifying the chemical composition of the initial reperfusate. Thus, the present study assesses whether the concept of a modified reperfusate could successfully be applied to a model of regional ischemia. Our hypothesis is that limitation of reperfusion damage in the ischemic border zone may enhance recovery and, thus, ultimately reduce infarct size.5

Methods

Operative procedure: Nineteen mongrel dogs of either sex weighing 20 to 30 kg were anesthetized with sodium pento- barbital (1 cg/kg) and endotracheally intubated and ventilated with a Harvard respirator at 100% FiOz. The common femoral

606

February 1, 1984 THE AMERICAN JOURNAL OF CARDIOLOGY Volume 53 609

4-hour L.A.D. occlusion

Control (n:7) treated (n: 12)

1

30 mn delivery of

I the reporfuslon

solution

1 90 mn-wblood roperfusIon+ 60mn

FIGURE 1. The experimental protocol. LAD = left anterior descending coronary artery.

artery was cannulated with a No. 7F Command catheter. Ar- terial pressure was monitored with a Statham P 23Db pressure transducer. Electrocardiographic standard limb leads and arterial pressure were recorded on a multichannel recorder (Hellige multiscriptor, 9400/6). Arterial blood gases and pH were intermittently monitored and maintained within the normal range.

The chest was opened at the fifth left intercostal space and the heart suspended in a pericardial cradle. The left anterior descending coronary artery (LAD) was dissected free from the adjacent tissues and occluded just beyond the first major di- agonal branch with a vascular clamp. All dogs received an i.v. bolus of xylocaine, 1 mg/kg, immediately before occlusion. Occlusion was maintained 4 hours, after which the myocardium at risk of infarction was evaluated by injecting 10 mCi of technetium-99m-labeled human albumin microspheres, 15 f 5 pm in diameter (CEA) into the left appendage. After 2 minutes the coronary clamp was released.

Experimental groups: The dogs were randomly assigned to either the control (n = 7) or the treated (n = 12) group (Fig. 1). The control dogs were reperfused with their own unmodified blood for 90 minutes. In the treated group, the initial reperfusate consisted of 500 ml of the reperfusion solution being studied. In this group, a No. 8F coronary angiographic catheter was inserted through the left carotid artery and in- troduced under fluoroscopic control into the LAD proximal to the site of occlusion. After release of the clamp, the catheter was used to deliver the reperfusion solution selectively into the previously ischemic area. The catheter was connected to a roller pump which allowed infusion of the solution at a controlled low flow rate (17 ml/min) to avoid myocardial damage related to the perfusion itself. The selectiveness of the fluorocarbon reperfusion was confirmed by a coronary an- giogram taken just before the start of the infusion that showed that the catheter almost totally occluded the vessel so that a minimal amount of blood passed around the catheter, and the progressively white staining of the reperfused area while the solution was being delivered, indicating that the reopened LAD and its tributaries were predominantly filled with the milky fluorocarbon emulsion.

Assessment of results: Five and a half hours after coronary occlusion, the dogs were killed with an overdose of barbiturate and the heart was excised. The patency of the reperfused LAD was assessed by gross examination. The left ventricle was excised and sectioned transversely from the atrioventricular groove to the apex into l-cm-thick transverse slices. The slices were incubated for 20 min in triphenyltetrazolium chloride at 37°C. Triphenyltetrazolium chloride is a reliable method

TABLE I Composition of the Fluorocarbon-Oxygenated Repertuslon Solution

Solution

Perfluorotributylamine (FC-43) Pluronic F-68 Na+ K+ Ca++ NaHC03 Glucose PO2 PCO* Total O2 content PH Osmolaritv

200 g/liter 25.6 g/liter 100 mhflliter 4.5 mhflliter

0.25 mAMiter 30 m/d/liter 1.8 g/liter

650 mm Hg 45 mm Hg

5.5 vol% 7.60 (at 37’C) 290 mosm/liter

of quantifying necrosis.6 The area of necrosis (AN) appears as a pale gray area, whereas the normal myocardium stains dark red. Subsequently, the slices were counted in a gamma camera (Opticamera, CGR) for visualization of the nonperfused myocardium at risk of developing infarction before reperfusion. The window for technetium-99m was 140 f 14 keV. The gamma camera was linked to a digital computer (Cine 200, Intertechnique) and the images were recorded on a magnetic disk. The digital computer was programmed to recall and display images recorded on an oscilloscope screen within a 64 X 64 matrix of squares. Areas at risk (AR) were constructed on the visual display unit with an electronic light pen. The edge of the area of interest was placed where radioactivity decreased by 50% of the maximal activity.7 Specific AR, assessed by microspheres, and AN, assessed by triphenyltetrazolium chloride, were planimetered and expressed as a percentage of the total area of the corresponding slice. The weights of the AR and AN were then calculated using the weight of the left ventricular slice and multiplying it by the AR and AN, respectively.

Thus, by analyzing the volume of infarcted myocardium as a function of the volume of myocardium at risk of necrosis, variability among dogs resulting from differences in collateral flow and size of native coronary arterial beds was greatly di- minished because each dog served as its own control.6

Reperfusion solution: The composition of the reperfusion solution is listed in Table I. Despite the high solubility of carbon dioxide in perfluorochemicals, bicarbonate buffer was considered essential for maintaining the proper pH and PCOZ. Emulsions were prepared by sonification in the presence of carbon dioxide to prevent fluoride formation. Centrifugation was used to ensure removal of large particles so that the average particle size was O.l/*, with no particles larger than 0.4~. The preparation was stored at 4’C before use.

Statistical evaluation: Within each group, relations between variables (weights at risk being the independent variables) were tested by linear regression. The null hypothesis (equality of regression coefficients and elevations in the 2 populations) was tested against the alternate l-tailed hypothesis, which states that regression coefficients or elevations, or both, were lower in the treated than in the control group. In addition, a 2-way replicated analysis of variance (model 1) was used to determine the presence of significant differences in the group mean values.s When analysis of variance demonstrated a statistically significant difference (p <0.05), the resultant simultaneous multiple comparisons between experimental groups were carried out using Bon- ferroni’s method to correct for the set of comparisons.* Fur- thermore, in each group, the values of the areas at risk, expressed as a percentage of the total area of the corresponding left ventricular slice, were distributed in 2 classes from the cutoff point of 40%. The corresponding values of the areas of

610 FLUOROCARBONS IN REPERFUSED INFARCTS

lSCHEMlC INFARCTED

I 0 CONTROL

m FC-REPERFUSED

FIGURE 2. The weight of the left ventricle (LV) at risk and the weight of the left ventricle that became infarcted for control and fluorocarbon (FC)-reperfused dogs. There was no significant difference in the weight of myocardium at risk between the 2 groups. However, infarct weight, expressed as a percent of the weight at risk, was significantly smaller in treated than in control dogs (p <O.OOl). Results are presented as the mean f standard deviation.

necrosis in each class were then compared using the Mann- Whitney test. Data are given as the mean f standard error of the mean.

Results

No significant differences between the 2 groups were found in pre- and postocclusion mean arterial pressure (105 f 10 and 112 f 9 mm Hg, respectively, in control dogs and 127 f 6 and 118 f 6 mm Hg, respectively, in reperfused dogs) or pre- and postocclusion heart rate (174 f 19 and 172 f 13 beats/min, respectively, in control dogs, and 171 f 10 and 162 f 10 beats/min, respectively, in reperfused dogs). Thus, differences in ultimate infarct size between the 2 groups cannot be ascribed to differences in these variables. No dog had ventricular fibrillation during the study or episodes of prolonged ventricular tachycardia.

The weight of the left ventricle at risk of developing necrosis was found to be similar in the control and fluorocarbon-reperfused dogs (7.0 f 0.8 and 9.4 f 0.7 g, respectively; difference not significant). Analysis of variance showed that in the control group the weight of necrotic myocardium (5.0 f 0.7 g) was not significantly different from the weight at risk, whereas in the treated group the weight of necrosis (5.9 f 0.5 g) was markedly smaller than the weight at risk (t = 4.100, p X0.001) (Fig. 2). The protective effects of the reperfusion solution were further confirmed by comparative analysis of the weight of salvaged myocardium, which was calculated for each individual slice as the difference between the weight of necrosis and the weight at risk. Thus, a significantly greater amount of myocardial mass was found to be salvaged in the treated group (3.4 f 0.5 vs 1.9 f 0.4 g in the control dogs, p <0.02). A similar pattern was observed when the extent of myocardial damage was expressed as a percentage of the mass of myocardium at risk that developed necrosis, that is, weight of necrosis/weight at risk X 100 (Fig. 3). Only 63.3 f 4.5% of the weight at risk went on to necrosis in fluorocarbon-reperfused dogs, compared with 72.3 f

CONTROL

FC-REPERFUSED

00 p-zo.02

7

1 1 ‘2 2

-

00

T

FIGURE 3. Percentage of infarction expressed as a percentage or the ischemic myocardium at risk of necrosis in control and fluorocarbon (FQreperfused dogs. Results are presented as the mean f standard deviation.

5.7% in the control dogs reperfused with unmodified blood (p <0.02).

Individual analysis of myocardial tissue slices showed that in 4 of 27 slices in the control group and in 5 of 51 slices in the reperfused group, the weight of necrosis was slightly greater than the weight at risk. However, the difference was consistently small (<lo% increase in tissue damage), so that this pattern is unlikely to be of physiologic relevance.

Furthermore, a difference that was at the limit of significance (p = 0.05) was found among the slopes of the regression lines plotting the weight of necrotic myocardium as a function of the weight of myocardium at risk (Fig. 4). This finding suggested that the larger the An, the larger the salvaged zone in the treated group. This hypothesis tended to be confirmed by analysis of the ultimate infarct size as a function of the extent of the initial area of hypoperfusion. The AN was similar in magnitude in both control and treated dogs when the size of the An was less than 40% of the area of a left ventricular slice (Fig. 5). Conversely, when the AR was more than 40% a marked discrepancy emerged between the 2 groups insofar as the initial reperfusion with the fluorocarbon-containing solution resulted in significantly smaller infarcts (p <0.03) than did immediate reperfusion with unmodified blood.

Discussion

Concept of reperfusion solution: A major concern raised by intracoronary thrombolysis in acute MI is that reperfusion of an ischemic area might cause further damage.3 Although the areas of hemorrhage9 and cell swelling10 related to reperfusion are both consistently smaller and contained within the borders of necrosis, the possibility that some damage related to restoration

February 1, 1964 THE AMERICAN JOURNAL OF CARDIOLOGY Volume 53 611

4t

- CONTROL (r=0.671 ) --- FC REPERFUSED (r=0.720)

MYOCARDIUM AT RISK (GM )

FIGURE 4. Relation between the weight of myocardium at risk, the percent of left ventricle and the weight of infarcted myocardium in control and fluorocarbon (FC)-reperfused dogs. The equations of the regression lines are as follows: control: y = 0.791x -0.481, r = 0.871; FC-reperfused: y = 0.571x +0.580, r = 0.720. These regression lines were constructed from an analysis of 27 slices of left ventricle in the control group (mean 3.81dog) and 5 1 slices in the treated group (mean 4.2/dog). The average values of the weight at risk (mean f standard deviation) were 7.0 f 4 g in the control group (n = 7) and 9.4 f 5 g in the reperfused group (n = 12) whereas the weight of necrosis was, respectively, 5.0 f 3.7 and 5.9 f 3.9 g. For both regression lines, the confidence interval of the slopes is 95%. The difference between the slopes of the 2 regression lines is at the limit of significance (p = 0.05).

of blood flow also affecting the ischemic border zone has not been ruled out. Long et all1 related the delayed appearance of positive pyrophosphate scans in human myocardial infarction to reperfusion necrosis associated with the return of blood flow in predominantly epicar- dial areas.

Previous experimental studies showed that the reperfusion injury reported after a period of global ischemia could largely be avoided by modifying the composition on the initial blood12 or crystalloid4 reperfusate with respect to electrolytes, pH and substrates. Consequently, in the present work, we tested the hypothesis that an appropriately modified reperfusate might also be effective after regional ischemia by preventing a potential reperfusion injury in the reversibly damaged areas, thereby contributing to a reduction in the ultimate size of the infarction.

Use of fluorocarbons: A major characteristic of the present study was the use of a crystalloid reperfusate oxygenated with fluorocarbons. Perfluorochemicals are synthetic compounds that can dissolve large amounts of various gases including oxygen, nitrogen and carbon dioxide and have thus been proposed for use in artificial blood substitutes.13 However, fluorocarbons cannot be used in biologic systems without emulsification, which requires that the emulsion be stable to avoid embolic reaction. The preparation used in our study consisted of FC-43 and Pluronic F-68, a nonionic surfactant and detergent.14 The resulting emulsion can dissolve 41~01% of oxygen at 37”C,r4 and was selected because of its stability. The potential disadvantage of long-term tissue retention was not considered a significant problem in an acute infarction model. The particle size of the emulsion is a critical factor that determines the surface

!O -

15_

lo_

5,

1 CONTROL

a FC-RERERFUSED

eee P c 0.03

(40% >40%

AREA AT RISK ( % LV)

FIGURE 5. Amount of salvaged myocardium, percent of left ventricle (LV), as a function of the extent of the area at risk. The percentage of salvaged myocardium was determined, for each individual slice of left ventricle, as the difference between the areas of necrosis and the area at risk both expressed as a percentage of the left ventricular slice area. For graphic reasons, results are expressed as the group means f the standard error of the mean (instead of the standard deviation). FC = fluorocarbon.

available for gaseous exchange, viscosity and, to a large extent, toxicity, l3 In the present study, the mean particle size was O.lp, which is in keeping with the values now established as imperative.14 Because the volume of 02 dissolved in FC changes linearly along with POs, a 100% 02 atmosphere was provided throughout the experiments. However, “bloodless” animals exchange-transfused with fluorocarbons have survived in 60% 0540% N2,13 a ratio that is more relevant to clinical use.

Asanguineous fluorocarbon-oxygenated reperfusion solutions have advantages over autologous modified blood solutions: (1) improved delivery of the reperfusate at the microvascular level, resulting from the low viscosity of the emulsion14; (2) a higher oxygen extrac- tion,15 largely due to the small size of the particles which offer more surface area for gas exchange than red blood cells in the same volume14; (3) the possibility of con- trolling PO2 and total 02 content, a consideration that may be clinically relevant in view of the possible 02.. related toxicity during the early phase of reflowls; and (4) the high level of quality control, clinical availability and ease of storage of asanguineous perfusates.

Other components of the reperfusate: A consistent feature of irreversible myocardial injury is increased tissue calcium content during early reflowI with sub- sequent precipitation of calcium phosphate crystals within mitochondria and myocardial fibers.18 Thus, in the present study, the concentration of Ca++ was initially decreased to 0.25 mM to reduce the extracellular Ca++ load available for cellular influx. This concept is consistent with the data of Shine et alI9 documenting the protective effects of low Ca++ reperfusion. For the same purpose, the reperfusate Na+ concentration was

612 FLUOROCARBONS IN REPERFUSED INFARCTS

adjusted to a high level (100 mM) because any decrease in extracellular Na+ would result in increased intracellular Ca++ contentzO

Intracellular acidosis develops during ischemia and may be exacerbated during reflow as a consequence of an efflux of protons from mitochondria to cytosol in response to a mitochondrial uptake of Ca++.21 In view of the negative inotropic effect of intracellular acidosis, these findings provide a rationale for increasing the reperfusate pH. In the present study, pH was increased to 7.60 (at 37°C) during early reperfusion. This value was selected after studies from our laboratory4 and others12 on globally ischemic hearts and is consistent with that successfully reported by Regan et a122 in a canine model of LAD occlusion.

The formulation of our reperfusion solution also ad- dressed the principle of providing substrate in the form of glucose. The choice of glucose was based on the assumption that the metabolic patterns of the border zone remain predominantly oxydative, with glucose ac- counting for more of the residual oxygen uptake than free fatty acids. 23 The addition of glucose also contrib- uted to the osmolarity of the solution. There is evidence that cellular swelling may be a major cause of reperfusion damage.24 Although the osmolarity of the present reperfusate was not increased above 280 mosm/liter to avoid increasing the viscosity of the fluorocarbon emulsion, care was taken to deliver the solution at a low flow rate in an attempt to limit postischemic myocardial edema.25

Effectiveness of reperfusion solutions: Fluoro- carbon-oxygenated cardioplegic and reperfusion solutions have been successfully used in vitro4p26 and in vivo27 in globally ischemic hearts. In regional ischemia, fluorocarbons reduce infarct size in dogs subjected to 6-hour permanent coronary artery ligation.28 The present study shows that the dogs receiving a fluorocarbon-oxygenated (PO2 650 mm Hg, 02 content 5.5 vol%), hypocalcemic, alkalotic solution as an initial reperfusate after 4 hours of coronary occlusion had smaller infarcts than did those immediately reperfused with their own unmodified blood (PO2 448 f 43 mm Hg, 02 content 16 f 0.9 ~01%).

The weight of myocardium at risk was greater, although not significantly, in the reperfused group. This difference between the 2 groups may be related to the variability in the distribution of collateral vessels among dogs and, hence, to the variability in the amount of blood flow still suppling the jeopardized myocardium beyond the site of occlusion. This assumption is based on the observations that assignment of the dogs to 1 of the 2 groups was done on a random basis, all procedures were performed consecutively by the same investigator, using a standardized operative technique, especially with respect to the site of occlusion, and determinations of the An were carried out without knowledge of the technique of reperfusion used. This fortuitous difference in the weights at risk between the 2 groups is unlikely to have biased the results in favor of reperfusion, because comparison of data between the 2 groups showed that although the treated dogs had initially larger regions at risk than the control animals, they did

not develop larger infarcts, comparison of data within each group demonstrated that the percent of salvage of jeopardized myocardium was significantly greater in the reperfused group, and the extent of necrosis was also assessed after the values of An were pooled into classes of similar size. With regard to the latter point, it is clinically relevant that the protective effects of the fluorocarbon reperfusate were mainly apparent when the AR involved more than 40% of a left ventricular slice because the evolution of such large ischemic areas towards necrosis carries a poor prognosis.2e

Furthermore, the endpoint of this study clearly was to assess the overall effects on infarct size of a complex reperfusate whose principles of formulation and mode of delivery have previously been investigated separately. Consequently, the design of the study does not allow to determine whether the reduction in infarct mass was predominantly related to improved oxygen delivery by the fluorocarbon particles to the border zone or, more generally, to a beneficial effect of enhanced oxygen supply, limitation of Ca++ overload, counteraction of acidosis and low-flow initial2 reperfusion with reduced oxygen content. However, our pilot studies on the isolated working rat heart models0 suggest that each of these mechanisms has a role.

Although preliminary clinical trials with fluorocarbons have been encouraging,31 their use as an adjunct to intracoronary thrombolysis must await additional experimental data. These data should be aimed at de- fining more clearly the basic protective mechanisms of these modified reperfusates, the comparative effects of chemically different fluorocarbon particles, the inter- actions between the oxygen-carrying compounds and the other components of the reperfusion solution, and the long-term effects of fluorocarbons on tissue via- bility.

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reduction of myocardial infarct size by a fluorocarbon-oxygenated reperfusate

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