Proc. Nati. Acad. Sci. USAVol. 80, pp. 5784-5788, September 1983Physiological Sciences

A flow- and time-dependent index of ischemic injury afterexperimental coronary occlusion and reperfusion

(myocardial ischemia/myocardial infarction)

LAURENCE W. V. DEBOER*, ROBERT E. RUDEt, ROBERT A. KLONERt, JOANNE S. INGWALLtPETER R. MAROKO§, MICHAEL A. DAVISt, AND EUGENE BRAUNWALDtIItDepartment of Medicine and Pathology, Harvard Medical School and Brigham and Women's Hospital, Boston, Massachusetts 02115; *Massachusetts GeneralHospital, Boston, Massachusetts 02114; tDepartment of Internal Medicine, University of Texas Health Science Center, Dallas, Texas, 75235; §Deborah HeartInstitute, Browns Mills, New Jersey 08015; and lUniversity of Massachusetts Medical Center, Worcester, Massachusetts 01605

Contributed by Eugene Braunwald, June 13, 1983

ABSTRACT The purpose of the present study was to deter-mine whether an ischemic index-expressed as the product of flowdeprivation (FD) and the duration of occlusion (T), FD x T-cor-related with biochemical and early morphologic alterations of thesubendocardial myocardium and could predict ultimate devel-opment of irreversible injury after coronary reperfusion. Myo-cardial biopsy specimens for measurement of ATP and other pu-rines and for ultrastructure studies were obtained in vivo duringcoronary occlusion in a canine model and were considered relativeto development of necrosis after coronary reperfusion. FD X Tcorrelated negatively with ATP content [ATP, nmol/mg of cardiacprotein = 23.6 - 0.24(FD X T) + 0.0007(FD X T)2; r = -0.81]and with a semiquantitative early histologic index of damage (r =0.70). Values of (FD x T) < 18 were associated with reversibleinjury-i.e., complete salvage after coronary reperfusion. (FD xT) > 18 was associated with varying degrees of necrosis; necrosiswas severe (78 ± 12% of subendocardial biopsy specimens) whenATP < 10 nmol/mg of protein and total purine pool was de-creased by 50%. FD X T correlated with the eventual percentageof subendocardial necrosis (r = 0.85). Accordingly, as an index ofischemic injury, FD x T may be useful in assessing whether isch-emic myocardial tissue will benefit from early restoration of bloodflow to the ischemic area.

Acute myocardial infarction can be treated by reperfusion (1),accomplished either by thrombolysis (streptokinase) or by cor-onary artery bypass surgery. The exact factors that determinewhether myocardial cells will be salvaged by reperfusion re-main to be clarified. The duration of ischemia is one factor thatdetermines the fate of injured cells, but other factors such ascollateral blood flow may be important. We tested whether asingle index derived from consideration of the duration of isch-emia and from the collateral blood flow to the ischemic myo-cardium could account for the biochemical and ultrastructuralchanges occurring during ischemic injury and ultimately for theamount of myocardium that was salvageable by reperfusion.We focused our attention on the subendocardium where severeischemic injury is anticipated during the first hours of coronaryocclusion.

MATERIALS AND METHODSAnimal Preparation. Seventy-four adult mongrel dogs (15-

25 kg body weight) were anesthetized, maintained on respi-ratory support, and treated as described (2). A thoracotomy wasperformed in the fifth left intercostal space and the heart wassuspended in a pericardial cradle. The left anterior descending

coronary artery was occluded with an atraumatic Schwartz ar-terial clamp for 10-180 min. Five minutes after coronary arteryocclusion, regional myocardial blood flow (RMBF) was deter-mined by using the plastic microsphere ("13Sn, 57Co, 141Ce, 46Sc)technique (3, 4) as described.

Myocardial biopsy specimens were obtained from the centralischemic zone (the area with obvious epicardial cyanosis) of eachheart at the end of the occlusion period and immediately there-after from normally perfused myocardium for analysis of high-energy phosphates. Biopsy specimens for ultrastructural anal-ysis were taken from immediately adjacent normal and isch-emic areas. In some animals not reperfused, specimens weretaken within 1 min of sacrifice at the end of the occlusion pe-riod and were fixed immediately in Karnovsky's fixative.

Twenty-five dogs were sacrificed immediately after the bi-opsy specimens were obtained. In 49 other dogs, the coronaryclamp was released after the specimens were obtained so thateventual necrosis after reperfusion could be assessed. The chestwas then closed, and the dogs were allowed to recover. After72 hours, each dog was reanesthetized and sacrificed by intra-venous injection of KC1. The heart was rapidly removed andplaced on ice. Samples of myocardium were removed for de-termination of RMBF and determination of infarction by lightmicroscopy or triphenyltetrazolium chloride staining (see be-low).

Biochemical Techniques. Transmural biopsy specimens (30-50 mg) were divided and the subendocardial half was analyzedfor total purines (ATP, ADP, AMP, adenosine, adenine, inosine,and hypoxanthine) by HPLC as described (2). Data are ex-pressed as nmol/mg of cardiac protein determined by the Lowrytechnique (5).

Morphologic Analysis. Biopsy specimens were obtained byusing a commercially available 16-gauge Travenol biopsy needleor biopsy drill. One-micrometer sections were cut on a Porter-Blum microtome (model MT2B) and stained for light micros-copy with toluidine blue. Morphologic analysis of ischemic in-jury from the central ischemic zone was then carried out byusing a published grading system (6); 0 = normal; 1 + = nuclearchromatin clumping either alone or in combination with rarevacuoles, myocardial relaxation as manifest by wide I bands; 2+= 1+ and intermyofibrillar edema and more vacuoles; 3+ =2+ and numerous vacuoles or sarcolemmal membrane liftedoff the myofibrils; 4+ = severe swelling and architectural dis-ruption. The mean grade of approximately 60 cells was used toobtain a mean ischemic score which reflects a semiquantitative

Abbreviation: RMBF, regional myocardial blood flow.1ITo whom reprint requests should be addressed at: Brigham and Wom-en's Hospital, 75 Francis St., Boston, MA 02115.

5784

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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assessment of the severity of cellular injury (6). Thin sections(600 A) were cut from some specimens on a Porter-Blum mi-crotome (MT2B), stained with uranyl acetate and lead citrate,and examined in a Philips 201 electron microscope for the pres-ence or absence of glycogen, intermyofibrillar edema, nuclearchanges, wide I bands, mitochondrial swelling, intramitochon-drial amorphous dense bodies, and breaks within the sarcolem-mal membrane.

In reperfused hearts, presence of necrosis was determinedby light microscopy (n = 26) or incubation of heart slices in tri-phenyltetrazolium chloride (n = 7) (7-9). A 1-cm-square sec-tion of the subendocardial half of the myocardium was cutproximal to and immediately adjacent to the sites of samplingsfor biochemical and ultrastructural studies, with care taken toavoid the needle tracts. For histologic analysis, this tissue wasfixed in 10% buffered formalin and embedded in paraffin. Sec-tions 5-7 ,um thick were cut and stained with hematoxylin/eosinand periodic acid-Schiff stains and examined by light micros-copy. Images of the sections were photo-optically enlarged andpercentage necrosis of the subendocardium was determined byplanimetry.The severity of ischemic injury was expressed as a function

of the duration (time of occlusion) of ischemia (T, in min) andthe severity of ischemia. The latter was calculated in terms offlow deprivation (FD)-i.e., blood flow in the ischemic zoneas a fraction of flow in the normal zone: FD = 1.0 - (RMBFischemic/RMBF nonischemic). The product FD X T is in unitsof minutes and theoretically represents the severity of ischemicinsult in terms of an equivalent number of minutes of total isch-emia-i.e., absence of blood flow.Data Analysis. All biochemical, electron microscopic, and

histologic analyses were made on coded specimens by an in-vestigator not aware of the origin of the sample. All group dataare presented as mean ± SEM. Analysis of variance and Tu-key's test for multiple comparison were used to compare bio-chemical and morphologic data among groups of dogs with low,high, and intermediate values for FD x T. Paired and unpairedtests were used as appropriate. Regression by least squares fitwas performed on a Hewlett-Packard 9815 calculator.

Experiments were performed in a total of 74 dogs. Of the25 dogs with permanent occlusion, 11 developed ventricularfibrillation and were excluded from the study. Of 49 dogs sub-jected to coronary occlusion followed by reperfusion, 13 de-veloped ventricular fibrillation during occlusion and 3 devel-oped it upon release of the occlusion; these 16 dogs were excludedfrom further study. Thus, of the original 74 dogs entered intothe study, there were 47 survivors, 14 without reperfusion and33 with reperfusion. Biochemical analysis of biopsy specimenswas always performed at the end of the occlusion period and

With necrosis

*%̂, I*F ..........

No necrosis

0 10203040 60 80 100 120 140 160 180 200 220FD x T, min

FIG. 1. Relation between FD x T during occlusion and subsequentdevelopment of necrosis in subendocardial tissue.

was successful in 34 dogs; ultrastructural analysis was carriedout in 33 dogs. The extent of necrosis was determined in all 33surviving dogs subjected to coronary reperfusion.

RESULTSOf the 33 dogs successfully reperfused, none developed ne-crosis when FD X T during occlusion was < 18 min (Table 1;Figs. 1 and 2A). When (FD X T) > 18 min, varying degreesof necrosis developed (Figs. 1 and 2). There was a significantcorrelation between FD X T and % necrosis that was best de-scribed by a parabolic model [Fig 3; % necrosis = -14.9 +1.45(FD X T) - 0.005(FD X T)2; r = 0.85; P < 0.001]. Therewas no significant correlation between % necrosis and FD alone(r = 0.22) and only a moderate correlation between % necrosisand T alone [% necrosis = 5.66 + 0.31(T); r = 0.60].

Biochemical values were determined at the end of the oc-clusion period in 12 animals without reperfusion and in 22 an-imals subjected to reperfusion after the specimens had beenobtained (Table 1). For convenience we analyzed the resultsaccording to FD X T: (FD x T) < 18 min, no necrosis; be-tween 18 and 80 min, intermediate necrosis, 33 ± 6% of thespecimen (Fig. 2B); > 80 min, severe necrosis, 78 ± 12% ofthe specimen (Fig. 2C).(FD x T) < 18 Min. In nonischemic tissue the mean ATP

value was 33.2 ± 1.4 nmol/mg of protein (n = 34). ATP con-centration in the ischemic zone when (FD x T) < 18 min (re-versible injury; i.e., not associated with histologic or histo-chemical evidence of necrosis) was 19.9 ± 1.7 nmol/mg ofprotein (P < 0.001 vs. nonischemic). Total purines were slightlybut not significantly decreased from 48.2 ± 1.8 nmol/mg innonischemic tissue to 42.2 ± 3.3 nmol/mg in ischemic tissue.There was a significant increase in inosine but not in hypoxan-thine in the ischemic zone. The average ischemic score in I-,msections was 0.69 ± 0.08 and it never exceeded 1.00. Features

Table 1. FD X T index and biochemical and morphologic correlates of ischemia

Duration nmol/mg of protein % subendocardialof occlusion, min FD x T ATP ADP AMP Ino Hypo I purinest MIS necrosis*

Nonischemic (n = 34) - 33.2 ± 1.4 8.4 ± 0.6 3.5 ± 0.4 1.9 ± 0.8 1.6 ± 0.3 48.2 ± 1.8<18 (n = 12) 13.1 ± 0.6 19.9 ± 1.7t0 9.9 ± 0.9 4.1 ± 0.9 6.3 ± 1.3 2.2 ± 0.5 42.2 ± 3.3 0.69 ± 0.08§ 0I§>18, <80 (n = 14) 45.3 ± 5.3 14.8 ± 2.0t§ 7.3 ± 1.0 3.8 ± 0.6 5.7 ± 1.3** 4.2 ± 1.4 36.2 ± 3.9 1.01 ± 0.12 32.9 ± 6.3§>80 (n = 8) 141.9 ± 14.8 3.3 ± 1.2t 5.9 ± 1.6 5.3 ± 1.8 4.3 ± 1.1** 6.0 ± 1.1** 24.8 ± 4.0** 1.89 ± 0.85 77.8 ± 12.3

Ino, inosine; Hypo, hypoxanthine; MIS, mean ischemic score.* From animals with reperfusion only (n = 33).t No detectable adenine or adenosine.t For difference from nonischemic, P < 0.001.§For difference from >80 min, P < 0.01.For difference from >80 min, P < 0.05.OFor difference from >18, <80, P < 0.01.** For difference from normal, P < 0.05.

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5786 Physiological Sciences: DeBoer et al.

A

100

0 10203040 60 80 100 120 140 160 180 200 220FD x T, min

FIG. 3. Myocardial necrosis in the subendocardium as a function ofFD X T [% necrosis = -14.9 + 1.45(FD X T) - 0.005(FD x T)2].

of this reversibly injured tissue prior to reperfusion includedglycogen loss, nuclear chromatin clumping and margination,intermyofibrillar edema, wide I bands suggesting myofibril re-laxation, occasionally swollen mitochondria with decrease inmatrix density, and separation of cristae (Fig. 4). This tissuevirtually never demonstrated intramitochondrial amorphousdense bodies or breaks in the sarcolemmal membrane.(FD x T) > 80 min. In these dogs (with severe necrosis),

ATP values were severely depressed (3.3 ± 1.2 nmol/mg ofprotein; P < 0.01 vs. nonischemic; P < 0.01 vs. (FD X T) =18-80). ATP values in this group were always <10 nmol/mgof protein. Total purine levels were depleted by approximately50% [P < 0.01 vs. nonischemic; P < 0.05 vs. (FD X T) < 18min]. There was a significant increase in inosine levels com-pared to nonischemic tissue and there was a trend toward lowerinosine values compared to (FD X T) < 18 min. There was anearly 4-fold increase in hypoxanthine levels compared to non-

FIG. 2. (A) Heart with reversible injury (complete salvage = no ne-crosis) in the subendocardium after injury. FD X T = 15 min. (B) In-complete salvage of the subendocardium after an injury (FD x T = 60min), showing a mixture of necrotic cells (bottom) and non-necrotic cells(top left). (C) Heart subjected to severe injury (FD x T = 136 min). Theentire subendocardium is necrotic (irreversible injury).

FIG. 4. Electron micrograph of ischemic myocardium which showedsalvage by reperfusion [(FD x T) = 15 min]. Note depletion ofglycogengranules, mild edema (e), and wide I bands (I). Nucleus (n) shows chro-matin clumping and margination.

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010203040 60 80 100 120 140 160 180 200 220FD x T, min

FIG. 6. Relationship between ATP concentration andFD x T- ATP= 23.6 - 0.24(FD x T) + 0.0007(FD x T)2; r = -0.81.

FIG. 5. Electron micrograph of the sarcolemmal membrane andmitochondria from an animal with (FD x T) = 136 min, in which ne-

crosis developed upon reperfusion. Sarcolemmal breaks are seen (ar-rows) as well as amorphous dense bodies (arrowhead) in the mitochon-dria (i).

ischemic tissue (P < 0.005) and a trend toward higher levelscompared to tissue with (FD X T) < 18 min.

Morphologic analysis revealed an average ischemic score of1.89 ± 0.85 [P < 0.01 vs. (FD X T) < 18 min; P < 0.05 vs.

(FD x T) = 18-80 min]. Many, but not all, cells containedamorphous dense bodies within the mitochondria and well-de-lineated breaks in the sarcolemmal membrane (Fig. 5), findingsnot present when (FD X T) < 18 min.(FD x T) = 18-80 min. In dogs with intermediate values

of FD x T, histologic, biochemical, and ultrastructural valueswere also intermediate. ATP was 14.8 ± 2.0 nmol/mg [P <0.001 vs. nonischemic; P NS vs. (FD X T) < 18 min; P < 0.01vs. (FD x T) > 80 min]. Total purines were decreased fromnormal by approximately 25%. Ultrastructural analysis revealedheterogeneous populations of cells, some appearing reversiblyinjured and some showing severe ultrastructural abnormalitiesincluding amorphous dense bodies within the mitochondria.

Other Correlations. There was a significant inverse corre-

lation between FD x T and ATP levels which was best fit bya parabolic function; ATP = 23.6 - 0.24(FD X T) + 0.0007(FDX T)2; r = -0.81; P < 0.001 (Fig. 6). There were moderatecorrelations between FD x T and total purines [1P = 44.6 -

0.16(FD X T); r = -0. 62], between FD X T and mean isch-emic score [MIS = 0.60 + 0.01(FD X T); r = 0.70], and be-tween ATP and % necrosis [% necrosis = 83.5 - 6.7(ATP) +0.14(ATP)2; r = -0.70].

DISCUSSIONThe present study developed an index of ischemic injury, FDx T (the product of the flow deprivation and the duration ofischemia), and indicates that the extent of irreversible ischemic

damage to the subendocardium after coronary artery occlusionmay be expressed as a function of this index. Appropriatenessand usefulness of the index have been verified experimentallyby the findings that FD X T correlates with biochemical andultrastructural measurements of ischemic injury determinedduring coronary occlusion, and ultimately FD x T correlateswith the percentage of necrosis in the subendocardium afterreperfusion. FD x T accounts for 72% (r2) of the variabilityseen in the subendocardial necrosis despite variability in heartrates, hemodynamics, and presumably MVO2 among these dif-ferent, randomly selected animals.

The usefulness of focusing on the subendocardium in study-ing myocardial ischemic injury has been demonstrated by ex-

amination of the posterior papillary muscle (3, 10-12). Thepresent investigation differs, however, from these studies on

the posterior papillary muscle model in that we obtained sam-

ples in vivo, allowing comparison of the biochemical and ul-trastructural findings existing at the end of the ischemic periodwith the ultimate development of necrosis in tissue immedi-ately adjacent to the biopsy site in the same animal after a pe-riod of reperfusion. In addition, the current study focused on

changes in ATP in relationship to flow deprivation as well as

duration of occlusion.The findings of this investigation indicate that specific con-

stellations of biochemical and ultrastructural abnormalities thatoccur during myocardial ischemia are associated with reversibleand irreversible injury. Decrease in ATP to a mean of 19.91.7 nmol/mg was associated with reversible injury-i.e., no

necrosis was present after reperfusion. In our study, tissue thatbecame severely necrotic (78 ± 12% of the subendocardialspecimen) always demonstrated ATP content < 10 nmol/mg ofcardiac protein. This finding is also consistent with the obser-vations of other investigators (13, 14). Less information is avail-able about the catabolites of ATP during reversible and irre-versible ischemic injuries. The total purine pool as calculatedin this study remained relatively intact in completely salvagedtissue because a large increase in inosine concentration and a

smaller increase in hypoxanthine concentration compensatedfor the reduction in ATP. In contrast, total purines fell by 50%in irreversibly injured tissue, despite a nearly 4-fold increasein hypoxanthine concentration. Irreversible injury was also as-

sociated with a mean ischemic score exceeding 1.0 and, in manycells, amorphous intramitochondrial dense bodies and breaksin the sarcolemmal membrane (11, 13) (Fig. 5).

It is tempting to attribute irreversible myocardial injury tothe marked decrease in ATP concentration or to the anatomicalabnormalities seen in this study; however, there was no efforton our part to dissociate these factors or to consider other fac-tors including lysosome or fatty acid-induced injury. Similarly,we do not know whether the effects of pharmacological agentswill alter the relationship between FD x T and ischemic injury.

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5788- PhysiologicaliSciences DeBoer et al.

We express our gratitude to Nancy Carlson; Carol Hare, Ann Di-nello, Susan St. John, Alice Carmel, and John Tumas for their technicalassistance and to Nancy Watterson for-preparation of this manuscript.This work was supported by Research Grants HL 23140 and HL 26215(Specialized Center of Research) and Training Grant T32HL-70749-03from the National Institutes of Health. L. W.V. DeB. is the recipient ofa National Institutes of Health Service Award; J.S.I. and R.A. K. areEstablished Investigators of the American Heart Association. Animalsused in this study were maintained in accordance with the guidelinesof the Committee on Animals of. the Harvard Medical School and ofthe Committee on Care and Use-of Laboratory Animals of the Instituteof Laboratory Animals Resources, National Research Council [DHEWpublication no. (NIH) 78-23, revised 1978].

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