Intracoronary adenosine administration during reperfusion following 3 hours of ischemia: Effects on infarct size, ventricular function, and regional myocardial blood flow

lntracoronary adenosine administration during reperfusion following 3 hours of ischemia: Effects on infarct size, ventricular function, and regional myocardial blood flow Previous studies have demonstrated that adenosine significantly enhances myocardial salvage after 90 minutes of regional ischemia. To determine its effect after prolonged ischemia, closed-chest dogs underwent 3 hours of left anterior descending artery occlusion followed by 72 hours of reperfusion. lntracoronary adenosine (3.75 mg/min; at 1.5 ml/min:total volume = 90 ml; n = 10) or an equivalent volume of saline (1.5 ml/min: total volume = 90 ml; n = 9) was infused into the left main coronary artery during the first 60 minutes of reperfusion. Regional myocardial blood flow was assessed serially with microspheres and regional ventricular function was assessed by contrast ventriculography. Infarct size was determined histologically. Light and electron microscopy were utilized to assess neutrophil infiltration and microvascular injury. Adenostne failed to reduce infarct size expressed as a percentage of the area at risk (36.0 + 4.9% versus 34.6 + 4.6%; p = NS) or to improve regional ventricular function as measured by the radial shortening method (3.2 + 1.6% versus 2.2 * 3.1%; p = NS) at 72 hours after reperfusion. Vasodilatory effects were not observed in the endo- and midmyocardial regions of the ischemic zone during adenosine administration. This was associated with a similar extent of capillary endothelial changes and neutrophil Infiltration in both adenosine-treated and saline control groups. These results suggest that severe functional abnormalities are present in the vasculature after 3 hours of ischemia and that adenosine therapy is ineffective in enhancing myocardial salvage. (AM HEART J 1990;120:808.)

David G. Babbitt, MD, Renu Virmani, MD,a Harry D. Vildibill Jr, BS, Elizabeth Daughtry Norton, DVM, and Mervyn B. Forman, MD, PhD. Nashville, Tenn., and Washington, D.C.

The rationale for reperfusion therapy was provided by the observation that irreversible myocardial injury occurs as a “wave front” phenomenon progressing from the subendocardium to subepicardium with increasing periods of occlusion.ls 2 We37 4 have previously demonstrated that the administration of the potent coronary arteriolar vasodilator adenosine during reperfusion significantly enhances myocardial salvage after 90 and 120 minutes of proximal left anterior descending coronary occlusion. Infarct size reFrom the Department of Medicine, Division of Cardiology, Vanderbilt versity School of Medicine; and *the Department of Cardiovascular ogy, Armed Forces Institute of Pathology.

UniPathol-

Supported in part by National Institutes of Health grant ROl HL 40829-01. Dr. Forman is a recipient of a First Award from the National Institutes of Health. Received

for publication

Reprint requests: Mervyn bilt University Medical Nashville, TN 37232. 4/l/22828

808

March

26, 1990;

accepted

B. Forman, MD, Division Center, Room CC-2218

May

21, 1990.

of Cardiology, Medical Center

VanderNorth,

duction was associated with relative preservation of structural and functional changes in the microvasculature.3, 5 Although reperfusion can rarely be accomplished in man within 3 hours of the onset of acute myocardial infarction, numerous clinical studie#lo have demonstrated a reduction in mortality associated with reduced infarct size and improved ventricular function if successful reperfusion was achieved within 4 to 5 hours of ischemia. We therefore investigated the effect of intracoronary adenosine after prolonged coronary occlusion on infarct size and ventricular function. in the canine closed-chest model of reperfusion. The effects of adenosine on regional myocardial blood flow, neutrophil infiltration, and ultrastructural changes were also determined. METHODS Experimental preparation. Thirty-seven mongrel dogs of both sexes weighing 20 to 30 kg were quarantined for 2 weeks prior to study to ensure that they were free of canine diseases. Seven days prior to the experiment, the animals

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1. hemodynamic Measurements ( B, O-l, O-2,0-3, R-l, R-2, R-3, R-72) 2. Regional Myocordial Blood Flow ( B, O-1, R, R-l, R-2, R-3 1 3. CVG ( B, O-l,

R-3, R-72)

4. CAG 1 B, O-I, R, R-72) 5. ADENOSINE

Levels (B,O-l,O-3,

6. Light and Electron

Microscopy

R-l, R-3) (9

1. Summary of experimental protocol. Intracoronary adenosinewas administered during the first hour of reperfusion. CAG, Coronary angiogram; CVG, contrast ventriculogram; O-1, O-2, O-3 = 1,2, or 3 hours of occlusion;R-l = 10 to 15 minutes after reperfusion. Fig.

were anesthetized with intravenous pentobarbital sodium (30mg/kg) and ventilated with a Harvard positive-pressure respirator (Harvard Apparatus Inc., S. Natick, Mass.). The heart wasexposedvia a left thoacotomy and the proximal left anterior descendingartery was isolated. A snare (surgical monofilament) enclosedin a polyethylene tube (model 601-325, Dow Corning Corp., Medical Materials, Midland, Mich.) wasimplanted on the proximal vesseland was securedto the epicardium with two sutures.The snarewas placed proximal to the first large diagonal artery when technically possible.An incision wasmadein the left atria1 appendageand a heparinized medical-gradetubing (model 602-285 Dow Corning Corp.) was secured utilizing a purse-string suture. The pericardium and chest were subsequently closedand the lines were buried in a subcutaneous pocket in the subscapularregion. All animalsreceived prophylactic antibiotics (600,000units Cornbiotic [penicillin and dihydrostreptomycin], Pfizer Inc., New York, N.Y.) for 48 hoursafter the operative procedureand wereallowed 5 to 7 days to recover before instrumentation. Experimental protocol (Fig. 1). On the day of the experiment, the dogs were randomized to receivg either intracoronary adenosineor intracoronary saline (control group). The animalswere anesthetized with pentobarbital sodium(30 mg/kg intravenously), intubated, and placedon a Harvard positive-pressureventilator (Harvard Apparatus Inc.) to maintain an arterial pH of 7.4 f 0.5. Anesthesia was maintained throughout the experiment with morphine sulfate (meandose,10mg/dog) and diazepam (mean dose, 15 mg/dog). Heart rate and electrocardiographic changeswere monitored continuously utilizing leads 1, aVF, and a%. Under sterile conditions, bilateral femoral

artery and vein cut-downs were performed for subsequent placement of 7F Cordis sheaths (Cordis Corp., Miami, Fla.). A 7F pigtail catheter was used to obtain measurements of phasic and meanarterial blood pressureand left ventricular end-diastolic pressure.Selective coronary angiography was performed utilizing a modified 7F right Cordis guiding catheter. A summary of the experimental protocol is shownin Fig. 1. After baselinehemodynamic parameterswere recorded, a contrast ventriculogram was obtained in the 30-degree right anterior oblique projection using 10 to 12 cc of meglumine diatrizoate (Renografin-76, E. R. Squibb & Sons, New Brunswick, N.J.) injected through a power injector. All subsequent ventriculograms were performed utilizing the samedegreeof rotation. Regional myocardial blood flow wasassessed serially utilizing 15 pm radioactive microsphereslabelled with lz51,51Cr,141C!e, s5Sr and 85Nb and 46Sc,respectively (3M Company, St. Paul, Minn.), injected at z 2 X lo6 microspheres/injection into the left atrial catheter followed by a 6 ml bolus of normal saline. Femoral arterial blood sampleswerewithdrawn at a rate of 7.5 ml/min for subsequentcalculation of myocardial blood flow. Patency of the left anterior descending(LAD) artery was confirmed by selective coronary angiography. The snarewasthen retrieved from the subcutaneouspocket and wastightened in two stagesover approximately 10minutes. All animalsreceived lidocaine at occlusionand at reperfusion (3 mg/kg bolus), followed by an infusion of 0.12 mg/kg/min that was maintained for 1 hour into occlusion and for the first 30 minutes of reperfusion. Hemodynamic measurements,regional myocardial blood flow, and a contrast ventriculogram were performed at 60 minutes into

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occlusion. Occlusionof the LAD wasvisualized with coronary angiography. The snare was releasedafter 180 minutes of LAD occlusionand patency wasconfirmed by arteriography. Animals randomized to adenosinetherapy received a 3.75 mg/min intracoronary infusion through the distal tip of the guiding catheter positioned in the left main coronary ostium. The volume administeredwas1.5ml/min over a go-minute period for a total volume of 90 ml (225mg of adenosine).Control animalsreceivedthe samevolume of saline. Regional myocardial blood flow was subsequently obtained at 10 to 15 minutes after initiation of adenosine or salineinfusion, at 1 hour after reperfusion just prior to discontinuing adenosine,and at 2 and 3 hours after reperfusion. Hemodynamic measurementswere obtained serially at these time points. After obtaining a contrast ventriculogram at 3 hours after reperfusion, the animals wereweanedfrom the respirator, given antibiotics, and allowed to recover. At 72 hours the animals were reanesthetized with 25 mg/kg intravenous pentobarbital sodium and hemodynamicmeasurementsandventriculography wereperformed utilizing the sameprojection. After obtaining a coronary angiogram to confirm vesselpatency, the left thorax was opened and the snare was ligated under direct vision. Monastral blue dye (E. I. Du Pont de Nemours & Co., Wilmington, Del.) wasinjected through a pigtail catheter positioned in the ascendingaorta in a doseof 1 ml/kg approximately 2 minutes after ligating the snareto define the area at risk. This method provides for a more physiologic risk region, becausewhen infarction occursthe bed at risk is not being perfused and therefore has a low flow state. While in vitro techniquesinject colored dyes at fixed pressure, in vivo methodsallow administration of a known volumeand vicosity of dye in a situation more analogousto the conditions at the time of previous myocardial ischemia. Animals werethen put to death with pentobarbital sodium and potassiumchloride and the heartswererapidly excised and washed.The hearts were cut into 1 cm tranverse sections parallel to the atrioventricular groove and were numbered 1 through 5 from apex to base. Analysis of area at risk and infarction. Slices1,2,4, and 5 were fixed for 3 days in 10% phosphate-bufferedformaldehyde and were then photographed to define the area at risk (AR), unstained by Monastral blue. The sectionswere then dehydrated and embedded in paraffin. Microscopic sections,7 pm thick, were cut and stained with hematoxylin-eosin and Mallory’s trichrome stain. The areaof necrosis(including hemorrhagewithin the area of necrosis)was traced from the projection of histologic slicesstained with Mallory’s stain (which stains the area of necrosispurplish blue). Each slide wasmagnified X7 and the outlines of the infarct and risk regionwere drawn and werethen measured by computerized planimetry by an observer blinded to the treatment group. AN representsthe total area of necrosis including hemorrhagewithin the infarct. The valuesof AN and AR werre corrected for tissue weights. Light microscopy. The secondand fourth slice sections from 10 control and 8 adenosine-treated animals were stained with hematoxylin and eosinand were examinedby

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light microscopyin a blinded manner. The extent of patchy myocardial necrosis,the degreeof hemorrhage,and the extent of contraction band necrosis were evaluated. The acute inflammatory infiltrate within vesselsand in the surrounding myocardium (interstitium) and macrophageinfiltration were assessedin the ischemic and nonischemic zones.An averageof 20high-power fields (HPF) (X400) per slide was evaluated. The degree of neutrophil and macrophage infiltration and the patchinessof the infarct were assessedsemiquantitatively according to the method of Romsonet al.,” with a score of 4+ being assignedto the most severeinfiltrate and a scoreof 0 assignedwhen rare or no changesare seen.The extent of contraction band necrosiswas quantitated by the method of Tazelar et a1.,12 with a rare presencegiven a scoreof 1+ and a diffuse presencea scoreof 4+. Electron microscopy. Myocardial biopsy sampleswere taken within 60 secondsof death from the central ischemic zone (anterior wall) and the nonischemiczone (posterior wall) and were divided into endocardial and epicardial halves. The tissue obtained at biopsy was cut in 1 mm3 piecesand fixed in 3 % buffered glutaraldehyde for transmission electron microscopy. Thirty-six specimenswere examined from nine (five adenosine-treated;four control) randomly selectedanimals.Tissue wasallowed to fix for 1 to 6 hours and wasthen transferred to 1% osmiumtetroxide in 0.1 mol/L cacodylate buffer, dehydrated, and embedded in Epon (E.F. Fullam Inc., Latham, N.Y.). Semithin sectionswere cut, stained with toluidine blue, and examined by light microscopy. The artifact-free areaswith the most capillarieswereselectedfor ultrathin sectioncutting, stainedwith uranyl acetate lead citrate, and examined with a Zeiss109 IGF electron microscope(Carl ZeissInc., Thornwood, N.Y.). Ultrastructural changeswere descriptively evaluated as mild, moderate, and severe. Calculations of regional myocardial blood flow. The third transverse slice was sectionedinto three zones:central ischemic,lateral ischemic,and nonischemicposterior wall. Each zone was further subdivided into epicardial, midmyocardial, and endocardial sectionsweighing 0.3 to 1.0 gm. Myocardial sectionsand reference blood samples were counted for 5 minutes in a multichannel analyzer (Model 5986,Packard Instrument Co., DownersGrove, Ill.) with background correction and the overlapping radioactivity betweenisotopeswascorrected using a matrix correction method (CompusphereSoftware, Packard Instrument Co.).13 Analysis of ventricular function. Regional left venticular function wasmeasuredby digitization of end-diastolic and end-systolic cineangiographicframes. Regional function wasassessed in the ischemiczone (segmentsthat were akinetic or dyskinetic at 1 hour of occlusion)at baseline,at occlusion,at 3 hours and then at 72 hours after reperfusion usinga radial shortening method with customizedsoftware validated in this laboratory. l3 Briefly, a longitudinal axis was constructed that connected the middle of the aortic valve plane to the apex of the heart for both the end-diastolic and end-systolic silhouettes.The midpoint of this axis was determined and 36 radii were constructed from this

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point at lo-degree intervals. Radial axesthat involved the mitral and aortic valves were excluded from analysis.Percent shortening of each radius was determined with the following formula: percentage shortening = (end-diastolic length - systolic length)/(end-diastolic length x 100). Adenosine levels. Blood samplesfor determination of plasmaadenosineconcentrations were collected from the left atria1 line in four adenosine-treated and five control dogs.Sampleswere obtained at the following time points: at baseline, 1 and 3 hours into occlusion, and at 1 and 3 hours into reperfusion. A procedure for measuringadenosinein rat plasmathrough ultraviolet microbore high-performance liquid chromatography, originally described by Jackson and Ohnishi,14was utilized. Statistical analysis. Repeated measurestwo-way analysis of variance wasutilized to analyze serial results in the groups (hemodynamics,myocardial blood flow). If a statistically significant difference was found between paired results, further analysis was performed by two-tailed t test (NCSS Software, Dr. Jelly Hintze, Kaysville, Utah). Comparisons between two groups (i.e., infarct size, histologic variables) were analyzed by a nonpaired Student’s t test with a two-tailed discriminant score.All data are expressed as mean -+ standard error of the mean. RESULTS

Thirty-seven dogs were randomized to receive either intracoronary adenosine or intracoronary saline

during reperfusion. Twelve animals were excluded due to inadequate myocardial ischemia that was prospectively defined as area at risk <20 % of the left ventricle, minimal left ventricular wall motion abnormality at occlusion, and epicardial blood flow in the central ischemic region of more than 0.6 ml/gm/ min. Five dogs died with occlusion-precipitated ventricular fibrillation, one died during anesthesia induction, and one animal failed to reperfuse. Seven animals (four adenosine-treated, three controls) were successfully resuscitated from occlusion-precipitated ventricular fibrillation and were alive 72 hours after reperfusion. Therefore a total of 19 animals (9 control and 10 adenosine-treated) completed the experimental protocol and form the basis of this analysis. In all animals successfully defibillated no more than two cardioversions (300 W/set) were employed and cardiopulmonary resuscitation was not required. Hemodynamic variables (Fig. 2). No significant differences were observed in heart rate or systolic blood pressure, mean blood pressure (data not shown), rate-pressure product, or left ventricular end-diastolic pressure. Paoz values were comparable in both groups (data not shown). infarct size (Fig. 3). Infarct size parameters were obtained from tissue weight. The area at risk expressed as a percentage of the total left ventricle was similar in both experimental groups (42.3 + 3.8%

Aderwsine and reperfusion injury

8 11

versus 42.2 f 4.0%). Intracoronary adenosine infusion given during the first hour of reperfusion failed to reduce infarct size, either when expressed as a percentage of the area at risk (adenosine: 38.0 f 4.9 % versus control: 34.8 + 4.6 % ; p = NS) or as a percentage of the total left ventricle (adenosine: 17.4 + 3.2 % versus control: 15.5 + 3.5%; p = NS). Subset analysis of six adenosine-treated animals and six control animals that were not cardioverted also showed no difference in infarct size as a percent of the area at risk (adenosine: 33.8 f 10% versus control: 29.4 f 6%;p=NS). Regional ventricular function (Fig. 4). Serial ventricular function is shown in Fig. 4. No significant differences were noted in baseline contractile function (22.3 + 3.2% versus 20.3 f 2.8%) and the number of radii in the ischemic zone (14.6 -+ 1.0 versus 14.1 + 0.2) in adenosine-treated and control animals, respectively. Both groups developed similar degrees of dyskinesis after 1 hour of oclusion. At 72 hours after reperfusion, a small and similar improvement in contractile function was noted in both groups (3.2 f 1.8% versus 2.2 -t 3.1%; p = NS). Regional myocardial blood flow (Fig. 5). Results of regional myocardial blood flow are depicted in Fig. 5. There was no significant difference in baseline blood flow between adenosine-treated and control animals. Both groups exhibited a >90% decrease in subendocardial flow in the central and lateral zones (central zone: 0.08 + 0.04 ml/min/gm versus 0.03 + 0.01 ml/min/gm) suggestive of comparable ischemia. Selective administration of intracoronary adenosine during the first hour of reperfusion failed to increase blood flow in the endocardial and midmyocardial sections of the central and lateral ischemic zones. A vasodilatory effect was noted in the epicardial segments of the ischemic and nonischemic zone (posterior wall) in the adenosine-treated group. The latter is most likely related to blood recirculation through the pulmonary vascular bed. A progressive decline in regional blood flow in the inner two thirds of the myocardium in both adenosine-treated and control animals was observed, compatible with the “noreflow”phenomenon. Relationship

between

infarct

size and collateral

blood

flow (Fig. 6). Since collateral blood flow is an important determinant of infarct size in this model, these two variables were assessed using linear regression analysis. A significant correlation between collateral blood flow determined 1 hour after occlusion and infarct size, expressed as a percentage of the area at risk, was noted in both experimental groups (adenosine-treated: F = -0.66, p < 0.05; control: r = -0.67, p < 0.05). The y-intercepts (49.2 versus 46.8; p = NS)

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Fig. 2. Hemodynamic changesin adenosine-treatedand control animalsduring the experimental protocol. No significant differences were noted in heart rate (HR), systolic blood pressure@BP), rate-pressure product (RPP), or left ventricular end-diastolic pressure(LVEDP). Abbreviations as in Fig. 1.

and slopes (-97.2 versus -50.6; p = NS) of the linear regression lines were not statistically different. Therefore adenosine failed to reduce infarct size for any degree of collateral flow. Adenosine levels (Fig. 7). Left atrial concentrations of adenosine were measured in four adenosine-treated and five control animals. A continual increase in levels was observed during the occlusion period in both groups. Just prior to discontinuation of adenosine after one hour of reperfusion, levels were higher in the adenosine-treated group. Light and electron microscopy (Fig. 8 and Table I). Light microscopy failed to reveal any differences in interstitial and intravascular neutrophil accumula-

tion within the ischemic myocardium between the two groups. The degrees of patchy infarction, hemorrhage, and contraction band necrosis were also similar. Electron microscopy of the endocardial ischemic regions in both control and adenosine-treated animals showed predominant changes of irreversible injury. Irreversible changes included peripheral margination of nuclear chromatin and shrunken nuclei; disruption of sarcolemmal membranes and bleb formation; and mitochondrial changes of clearing of matrix, fragmentation of cristae, and the presence of amorphous and granular matrix densities. Reversible ischemic injury was noted in occasional myocytes and

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was similar in both treatment groups. These included mitochondrial swelling, loss of normal dense mitochondrial matrix, and absence of amorphous and granular flocculent densities. The capillary changes in the ischemic region in control and adenosine-treated dogs were moderate to severe. Endothelial swelling with a decrease in pinocytotic vesicles was frequently observed. Capillary lumina were totally or partially obstructed with swollen endothelial protrusions and membranebound vesicles, interdigitating with white and red cells and platelets. Although endothelial disruption was seen, it was not prominent in either group. Neutrophils and red cells were infrequently observed in the interstitium. Capillaries in the nonischemic myocardium of control and adenosine-treated animals were intact with occasional endothelial folds and swelling, and the myocardium showed no changes of irreversible ischemia.

%

DISCUSSION Present study. In this study the effect of intracoro-

Fig.

nary adenosine was assessed after prolonged (3 hours) regional ischemia in the canine model. Treatment did not reduce infarct size or improve regional ventricular function 72 hours after reperfusion. Physiologic reactive hyperemia was seen upon reperfusion in both groups. Adenosine, a potent arteriolar vasodilator, failed to increase regional myocardial blood flow in the inner two thirds of the ischemic zone soon after reperfusion. Adenosine therapy also did not prevent the progressive decrease in blood flow occurring during the first 3 hours of reperfusion. However, a vasodilatory effect was observed in the epicardial region of the ischemic zone where the myocytes remained viable. These observations suggest that the microvasculature was functionally unresponsive due to prolonged ischemia. Electron microscopy of the capillaries in the ischemic subendocardium showed severe changes in both groups, with endothelial swelling and protrusions, vascular plugging by red and white cells, and occasional endothelial disruption. Mechanical obstruction of capillaries may be responsible for the progressive decrease in blood flow observed during the first 3 hours of reperfusion. These results are contrary to those of previous observations, which demonstrated marked structural and functional preservation of the microvasculature with adenosine therapy after 90 or 120 minutes of regional myocardial ischemia.3, 5 Therefore the beneficial pharmacologic effects of adenosine therapy in enhancing myocardial salvage in the canine model

8 13

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AR/LV

AN/AR

ANILV

3. Myocardial infact size (AN) expressedas a percentage of the area at risk (AR) and of the total left ventricle (LV) is shown. Adenosine treatment failed to enhance myocardial salvage72 hours after reperfusion.

are possible only when the duration chemia is less than 180 minutes.

of myocardial

is-

Comparison with previous studies of prolonged ischemia. Only a few studies have reported the effects

of therapeutic intervention on myocardial preservation after prolonged (3 hours) periods of regional myocardial ischemia followed by reperfusion.15, l6 Gallager et al. l5 failed to show infarct size reduction 24 hours after reperfusion with superoxide dismutase and catalase in conscious dogs subjected to 3 hours of coronary occlusion. Similarly, the administration of verapamil and ibuprofen to unconscious open-chest animals did not enhance myocardial salvage.16 The time course of irreversible injury was less in this study compared with the observations of Reimer and Jennings.2 Previously reported studies of permanent occlusion of the LAD artery in our laboratory17 have shown infarct size of 70 f 3% expressed as area at risk, which is markedly greater than the 38% observed in the present study. Therefore significant myocardial salvage was still obtainable (approximately 45% ) in the closed-chest model of LAD occlusion with blood reperfusion. These findings are in contrast to the open-chest left circumflex occlusion model, where only 11% to 33 % was salvageable after 3 hours’ occlusion and reperfusion when compared

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Fig. 4. Serial changes in radial shortening in the ischemic zone at baseline (B), occlusion(OCC), 3 hours (R-3HR) and 72 hours after reperfusion (R-72HR). Adenosine failed to improve regional contractile function compared with that of control animals.

CONTROL

ZONE

CENTRAL

ZONE

LATERAL

ZONE

~~~~~~~

di$jJ!! /A /J& B

0

B=BASELINE,

R

lHR2HR3HR O=OCCLUSION,

B

0

R lHR2HR3HR

B

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R=REPERFUSION

Fig. 5. Serial changes in regional myocardial blood flow in the control zone (posterior wall), central, and lateral ischemic zones. A vasodilatory response to adenosine was noted only in the control zone and in the epicardial regions of central and lateral ischemic zones. Note that there was a progressive decrease in subendocardial and midmyocardial blood flow in both adenosine-treated and control animals during the first 3 hours of reperfusion, consistent with the “no-reflow” phenomenon. Abbreviations as in Fig. 1.

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ADENOSINE r =-0.66

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p < 0.05

p< 0.05

I

I

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I

. 0.1 0.2 0.3 0.4 0.5 0.6 TRANSMURAL COLLATERAL FLOW (mVmin/g) (CENTRAL ISCHEMIC ZONE) Fig. 6. Relationship between infarct size expressedas a percentage of the area at risk (AN/AR) and transmural collateral blood flow in the central ischemiczone measured1 hour after occlusion.Note similarity in slopesbetween control and adenosine-treatedanimals, indicating that adenosinedid not reduce infarct size for any degreeof ischemia.Asterisks refer to animalsthat were successfullycardioverted.

with permanent occlusion.lp 2 This difference may be related to the open-chest’s model having higher myocardial oxygen consumption compared with the closed-chest preparation and also may be due to mechanical effects on the heart while it rests in a pericardial cradle. A thoracotomy may also result in mobilization and activation of neutrophils, which could result in enhanced tissue injury during the perireperfusion period. Finally, the use of the circumflex as opposed to the LAD artery and the marked variability in collateral blood flow inherent in the canine model may also have contributed to this discrepancy. This study is in agreement with other studies and demonstrates that closed-chest preparations have smaller infarcts with comparable occlusion times and that this preparation may be a more appropriate model to assess the efficacy of various therapeutic interventions in enhancing myocardial salvage after reperfusion.159 l6 Comparison

with previous

studies

utilizing

adenosine.

A number of methodologic differences exist between this study and prior studies from our laboratory37 4

that have shown that adenosine reduces infarct size after 90 and 120 minutes of regional ischemia. These differences include the administration of thrombolytic agents and the route of adenosine administration. Thirty thousand units of streptokinase was given in our initial study of 90 minutes of coronary occlusion.3 The lack of streptokinase administration is unlikely to account for the negative results in this study. Kloner et al. I* demonstrated that both small and large doses of tissue plasminogen activator did not enhance myocardial salvage or improve the no-reflow phenomenon compared with animals given saline after reperfusion. In addition, intravenous adenosine without streptokinase significantly reduced infarct size after 72 hours of reperfusion, and this was associated with amelioration of the noreflow phenomenon. lg Although adenosine was infused subselectively into the proximal LAD in a previous study, later observations showed beneficial effects of adenosine infusion into the left main artery after 120 minutes of ischemia.39 4 Furthermore, the intravenous administration of adenosine in a dose of

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October 1990 Heart Journal

Fig. 7. Serial measurementsof left atria1 adenosineconcentrations were determined at baseline,at 1 and 3 hours of occlusion (OCC.lHR, OCC.3HR), and at 1 and 3 hours after reperfusion (REP). The data were similar prior to reperfusionin the two groups(four adenosine-treated,five control) and werepooled.Treated animals demonstrated increasedlevels during the early reperfusion period.

I. Semiquantitative assessment of histologic parameters by light microscopy Table

Parameter

Hemorrhagic infarct Patchiness of infarct Contraction band necrosis Acute inflammation Interstitial Intravascular Macrophages

Control (n = 10)

Adenosine (n = 8)

p Value

2.0 i 0.5 1.9 + 0.3 1.4 i 0.3

1.5 t 0.5 2.0 AI 0.3 2.2 +- 0.3

0.50 0.82 0.07

0.7 dz 0.2 0.8 k 0.2 1.9 * 0.3

1.0 + 0.3 0.9 f 0.3 2.1 * 0.4

0.43 0.83 0.66

shown that neutrophil activation occurs after reper-

fusion in spite of therapeutic concentrations of lidocaine as utilized in this study. Adenosine inhibition of neutrophil adhesion to cultured endothelial cells is not potentiated by the addition of pharmacologic concentrations of lidocaine. Further studies will be required to determine if an interaction occurs between adenosine and lidocaine in vivo. Role of microvascular injury in myocardial tion. Kloner et a1.22 have shown that

preserva-

structural changes in the endothelium progress at a slower rate compared with changes in the myocytes after comparable durations of ischemia, suggesting that the former cells are more resistant to ischemic injury. In

2.8 mg/min reduced

infarct size and augmented regional blood flow. lg A significant increase in blood flow was noted in the epicardial zone of the ischemic bed and in the nonischemic zone in the current study. Therefore the route of administration is unlikely to have influenced the results of this study. A preliminary observation by Hoffmeister et a1.20 suggests that the beneficial effects of adenosine of myocardial reperfusion injury are dependent on lidocaine administration. We21 have previously

contrast to myocytes, only 20% of endothelial cells show structural evidence of irreversible injury after 60 minutes of permanent ischemia.22 A marked disparity is noted histologically in the microcirculation between models of permanent occlusion and reperfusion.22-24 Reperfusion after 90 minutes of ischemia results in acceleration of endothelial injury in association with rapid influx of cellular elements, particularly neutrophils. 21124-26These changes may contribute to the progressive decline in capillary

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Fig. 8. Electron photomicrographs of the ischemic subendocardial region from adenosine-treated (A and B) and control animals (C and D). Note marked endothelial swelling in both treated and control animals with decrease in pinocytotic vesicles. The lumen contains either white (IV) cells or red (R) cells and memAdenosine-treated and control animals also showed luminal obstrucbrane-bound vesicles (arrowheads). tion either by endothelial protrusions (arrowheads in B and C) with entrapped red cells (R). Control animal in D shows luminal obstruction with neutrophil (IV) and red (R) cells. Note endothelial disruption focally (arrowheads) with intact basement membrane in D.

blood flow after reperfusion to areas of potentially viable myocytes. 27 We and others28-30 have observed that reperfusion results in abnormalities in endotheM-independent and dependent vascular reactivity in areas of prior ischemia. We3*5 have previously demonstrated that intracoronary adenosine administered in the reperfusion period after 90 and 120 minutes of ischemia increased myocardial blood flow, resulting in attenuation in both structural and functional abnormalities in the reperfused bed. This was associated with enhanced myocardial salvage and improved ventricular function.3s 5 In the present study adenosine administration failed to increase regional myocardial blood flow and prevent the progressive decline in flow in the reperfused bed after 180 minutes of ischemia. Ultrastruc-

tural changes were severe and were similar in both groups. These consisted of endothelial swelling, cytoplasmic projections, presence of membrane-bound vesicles, and focal disruption. Cellular elements were seen occasionally plugging capillary lumens and in the interstitium. These findings suggest that microvascular injury may be irreversible in the inner two thirds of the myocardium after 180 minutes of ischemia. This is supported by the observation that adenosine failed to induce vasodilatation after 180 minutes of coronary occlusion in these segments. Implications. This study demonstrates that the administration of intracoronary adenosine after 180 minutes of ischemia failed to reduce infarct size and improve regional ventricular function. These findings are contrary to previous observations in our lab-

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oratory, where significant myocardial salvage was observed with 90 and 120 minutes of occlusion. The failure of adenosine to augment myocardial blood flow, in association with histological observations of severe endothelial changes, would suggest that enhanced myocardial salvage cannot be achieved with intracoronary adenosine after 180 minutes of coronary occlusion in the closed-chest canine model. We thank Carolyn Coffey and Linda Hawkins for expert secretarial assistance. REFERENCES

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