Myeloperoxidase activity as a quantitative assessment of neutrophil infiltration into ischemie myocardium

Myeloperoxidase Activity as a Quantitative Assessment of Neutrophil Infiltration into lschemic Myocardium

KEVIN M. MULLANE, ROSEMARYKRAEMER,AND BRUCE SMITH

The infiltration of neutrophils into ischemic myocardium exacerbates myocardial damage upon reperfusion, whereas drugs that inhibit neutrophil activity or function reduce infarct size. Consequently, it is important to accurately assess the myocardial neutrophil content. Histologic sections and radiolabeled cells have been used, but have major limitations. We have developed a method to measure the neutrophils present in cardiac tissue by utilizing a spectrophotometric assay for the neutrophil-specific myeloperoxidase enzyme (MPO) (Bradley et al., 1982a). Coronary artery occlusion and reperfusion in the anesthetized dog induces neutrophil accumulation into the ischemic heart, which shows a linear relationship with time. An increase in activity from 0.014 + 0.001 units (u) MPO/lOO mg tissue to 0.091 -t 0.02 u MPO/lOO mg is already apparent at the end of the 90-min occlusion period. This activity increases over 5 hr reperfusion to 0.32 ‘_ 0.018 u MPO/lOO mg tissue. Histologic analyses confirmed the temporal association of neutrophil accumulation. Moreover, there is a correlation between infarct size and tissue MP0 activity. Measuring the MP0 content in preparations of canine neutrophils, which is directly correlated with cell number, allows units of MP0 activity to be converted into a tissue neutrophil content. This assay is simple, sensitive, and provides a quantitative index of myocardial neutrophil accumulation that can be used to study the relationship between leukocyte infiltration and myocardial

injury.

Key Words:

Myeloperoxidase;

Neutrophils;

Myocardial ischemia; Dog

INTRODUCTION Mortality

resulting

from

myocardial

infarction

is directly

related

to infarct

size

and has led to the development of drugs aimed at reducing the amount of tissue that becomes irreversibly damaged (Maroko et al., 1971; Hillis and Braunwald, 1977). Early thrombolytic therapy with streptokinase can restore coronary blood flow to an ischemic region (Goldberg et al., 1982; Khaja et al., 1983) and has become an important agent in the coronary care unit. However, reperfusion of ischemic myocardium

induced

perimental

by thrombolytic

studies

of reperfused

agents

From the Department of Pharmacology, New Address

reprint

lege, Valhalla, Received

requests

to Dr.

could

myocardium

K. M. Mullane,

York

exacerbate

show extensive

Medical

Department

College,

myocardial morphologic

Valhalla,

of Pharmacology,

injury;

ex-

and en-

New York. New York

Medical

Col-

NY 10595.

January

7, 1985;

revised

and accepted March

28, 1985. 157

Journalof Pharmacological Methods 14, 157-167 (1985) 0 1985 Elsevier Science Publishing

Co., Inc., 52 Vanderbilt

Avenue, New York, NY 10017

158

K. M. Mullane et al. zymic

alterations

Meerbaum, Recent

(Hearse,

1977;

Meerbaum

and

Corday,

1975;

Corday

and

1983). studies

have implicated

perfusion-induced

myocardial

invading

damage

neutrophils

(Mullane

in the exacerbation

et al., 1984;

Romson

of re-

et al., 1983;

Engler et al., 1983; Bednar et al., 1985a). Polymorphonuclear leukocytes (PMNs) accumulate rapidly in the ischemic myocardium and the ensuing response has all the hallmarks of an acute inflammatory event (Mullane et al., 1984). The role of PMNs and the possible contribution of PMN-derived mediators to myocardial injury have recently venom

been

factor

reviewed

(Maroko

(Mullane

et al., 1978),

Lucchesi,

1983), nafazatrom

or agents

which

(Bednar

deplete

circulating

When

studying with

the relationship

the effects

(Mullane

neutrophils

such as cobra 1982; jolly and

(Romson

between

PMN infiltration

etal.,

antisera

et al., 19841, which are directed reduce infarct size.

of drugs that inhibit

cells (Romson

Drugs

such as specific

function,

1982),

(Romson

against

and myocardial

neutrophil

to have a quantitative assessment of cell infiltration studies have relied on either histologic assessment of radiolabeled

1983).

and Moncada,

et al., 1985a), or ibuprofen

et al., 19831, or hydroxyurea (Mullane to prevent their activation/infiltration, together

and Moncada,

BW755C

PMNs

damage,

it is important

into the myocardium. Previous (Mullane et al., 1984) or the use

et al., 1982; Thakur

et al., 1979).

Both methods

have

major drawbacks that limit their usefulness. Neutrophil infiltration is not homogenous. Consequently, a large number of histologic sections need to be examined for each

heart

(Mullane

et al., 1984)-a

procedure

which

is time-consuming

and

provides only semiquantitative data. The use of leukocytes labeled with radioisotopes such as indium-111 is a more quantitative method to assess cell infiltration into a tissue, (Romson

but requires

the removal,

et al., 1982; Thakur

the activity

and characteristics

activity

when

Thakur

et al., 1979), which

isolation,

of neutrophils

the cells are reintroduced

the bloodstream. In order to circumvent

and labeling

et al., 1979). These

indicates these

procedures

as demonstrated

into the circulation

that “damaged”

problems,

of neutrophils can dramatically

alter

by the loss of radio(Romson

cells are being

we have utilized

in vitro

et al., 1982;

removed

from

a spectrophotometric

assay for the neutrophil-specific myeloperoxidase enzyme (Bradley et al., 1982a) to measure neutrophil accumulation in ischemic myocardium. The results indicate that the myeloperoxidase enzyme serves as a useful quantitative indicator of PMN infiltration

because

temporal

changes

in activity

correlate

with

histologic

analyses

of

cell invasion. MATERIALS Induction

AND

METHODS

of Myocardial

Male dogs weighing

lschemia

9.0-19.2

in Dogs

kg were fasted overnight,

then anesthetized

initially

with Biotal (thiamylated sodium) and maintained with chloralose (80 mg/kg) supplemented as required. The dogs were intubated and artificially ventilated with room air. Catheters were inserted into the femoral artery and vein. The heart was exposed through a left thoracotomy at the fifth intercostal space and suspended in a peri-

MP0

Activity in lschemic Myocardium

cardial cradle. A segment of the left anterior descending (LAD) coronary artery was cleared just below the first major diagonal branch. A screw clamp and an occlusive snare ligature were placed around the coronary artery. LAD coronary blood flow was measured by placing an electromagnetic flow probe (Carolina Medical Electronics) distal to the screw clamp. The screw clamp was then adjusted to effect a critical stenosis so that basal coronary flow was not altered, but the hyperemic response following a IO-set occlusion of the vessel was reduced by more than 70%. Reperfusion into a critical stenosis reduces the incidence of ventricular fibrillation and hemorrhagic infarction. Myocardial ischemia was induced by occlusion of the LAD coronary artery for 90 min and followed by reperfusion for 0 (n = S), 3 (n = S), or 5 hr (n = 6) in order to assess the time course of neutrophil accumulation. Demarcation

of the Area at Risk and Infarct

Zones

Those portions of the myocardium at risk of becoming infarcted, and that are infarcted, are delineated by a dual staining technique, which we have described previously (Mullane et al., 1984; Mullane and Moncada, 1982). At the end of the reperfusion period, 50 ml of whole blood was collected into a tube containing heparin (IO units/ml final concentration). The previously occluded segment of the LAD was cannulated and the heparinized blood reinfused at mean systemic blood pressure. Simultaneously, 20 ml of Evan’s blue dye (Sigma Chemical Co.) was injected into the femoral vein, rendering all parts of the heart blue except that area which was perfused with the undyed blood. The dog was sacrificed and the heart was removed and sliced (0.5 cm thick) in a breadloaf fashion from apex to base. The area free of the blue dye indicates that portion of the left ventricle served by the occluded segment of the LAD, called the risk area. The slices of myocardium were then incubated with triphenyltetrazolium (TPT) in phosphate-buffered saline, pH 7.4 (Sigma Chemical Co.) for 20 min (Lie et al., 1975). While the viable tissue stains brick red, the infarcted area remains colorless.

SITE OF OCCLUSION

FIGURE 1. Schematic diagram of the model of myocardial ischemia indicating the site of coronary occlusion. Following a dual staining technique at the end of the experiment. three regions of the left ventricle are demarcated (the right ventricle is discarded). These regions are the normal myocardium (white area), the risk area (hatched region), and, within that, the infarcted zone (stippled area). Also indicated are the four sites of tissue sampling for the MP0 assay (see Methods).

159

160

K. M. Mullane et al. Sections of tissue of approximately 250 mg net weight were taken from the central infarct zone, the risk area, and the “interface” region overlapping the infarct and risk areas in 2-3 separate rings of myocardium (see Figure 1) to obtain an overall picture for each heart. The mean values of each individual heart were used for statistical analysis. Because the Evan’s blue dye interfered with the spectrophotometric assay, samples of control myocardium were obtained from dogs that fibrillated upon coronary occlusion and thus did not receive the dye, or that were obtained from other experiments not involving the heart. Neutrophil

Isolation

Canine neutrophils were harvested from the peritoneum following the instillation of 400 ml 0.1% glycogen into the peritoneal cavity for 3 hr in a separate group of dogs (n = 10). Contaminant red blood cells in the aspirated peritoneal fluid were destroyed by hypotonic lysis. The leukocytes were washed in normal saline and resuspended in Hank’s balanced salt solution. Wright-Giemsa staining showed that the leukocyte suspension contained 85-95% neutrophils. Aliquots of cells were diluted to equal volumes containing 105-IO6 cells/ml. Platelet Isolation Platelets were prepared and washed according to the technique of Radomski and Moncada (1983). Briefly, 25 ml of blood was collected into a tube containing 2.7 ml of 3.15% sodium citrate and 2 pg/ml (final concentration) prostacyclin (PGI, in IM Tris buffer, pH 8.4). The blood was centrifuged at 220g for 12 min to obtain plateletrich plasma. The platelets were isolated by centrifugation and washed with Tyrode’s buffer containing 0.3 kg/ml PG12 before being resuspended in PGL-free buffer at IO’ or IO8 platelets/ml. Myeloperoxidase

Assay

Activity of the neutrophil-specific myeloperoxidase enzyme (MPO) was measured by an adaptation of the method described by Bradley et al. (1982a). MPO, contained within the azurophilic granules of the neutrophils, was liberated as follows: myocardial tissue or canine neutrophils were frozen in liquid nitrogen and pulverized. The thawed tissue was then suspended in 6 ml 50 mM potassium phosphate buffer containing 0.5% hexadecyltrimethylammonium bromide (HTAB) to solubilize the enzyme. The homogenate was blended for 1.5 min in a Waring blender and further disrupted with four vertical strokes with a mechanical mortar and pestle (Oxwell Model 3000). The samples were then sonicated for 10 set (Fisher Sonic Dismembrator, Model 300), freeze-thawed three times with liquid nitrogen, and sonicated for a further 10 sec. The released enzyme was separated from the cell debris by centrifugation at 40,OOOg for 15 min on a Beckman x 575B ultracentrifuge. The volume of supernatant (which contains MPO) was measured. The pellet contains virtually no MP0 activity (Bradley et al., 1982a). Platelet suspensions were handled in a similar manner. MP0 activity was assayed in duplicate by mixing 0.1 ml of the supernatant with 2.9 ml of 50 mM phosphate buffer (pH 6.0) containing 0.167 mg/ml 0-dianisidine

MP0

Activity in lschemic Myocardium

hydrochloride (Sigma Chemical Co.) and 0.0005% hydrogen peroxide (J.T. Baker Co.). The change in absorbance at 460 nm was measured every 30 set for 2 min using a Beckman Model 26 spectrophotometer. The change in absorbance between 30 and 90 set was used for comparison, and the results were expressed as units (u) of MPO/lOO mg tissue (wet weight), where 1 u of MP0 activity is defined as that degrading 1 Fmol peroxide/min at 25°C (Bradley et al., 1982a). The intraassay variation was 9 + 3% for duplicated samples. The average for the duplicates was used for analysis. Histologic

Analyses

Sections of myocardium were prepared as described previously (Mullane et al., 1984). The tissue was placed in neutral-buffered formalin for at least 72 hr, and then routinely processed for paraffin histology. The tissue blocks were orientated so that when sections were cut the vertical axis of the block extended from the epicardium to the endocardium. The blocks were cut at 6 TVand the sections were stained with hematoxylin and eosin. RESULTS Neutrophil

MP0

Activity

MP0 activity measured in preparations of canine neutrophils elicited from the peritoneal cavity contained 0.6 + 0.09 u MPO/I06 cells (mean + S.E.M., n = IO). Activity was linear from 105-IO6 cells (Figure 2), which represents the major range of MP0 activity obtained in myocardial tissue, and thus was used as a standard curve to assess actual numbers of neutrophils contained within the myocardium.

NO. OF NEUTROPHILS FIGURE 2. Relationship between units of MP0 activity and the number of canine peritoneal neutrophils. Each point represents the mean of ten separate experiments performed in duplicate.

161

162

K. M. Mullane et al.

Control

n=19

90’ n=5

90’+3h n=5

90’+5h n=6

FIGURE 3. Time-related increase in MPO activity in ischemic myocardium following 90 min occlusion and 0,3, or 5 hr reperfusion. Results of the MPO assay are compared to histologic sections of the ischemic heart, which demonstrate the degree of cellular infiltration (H and E, x 46).

In contrast,

two separate

preparations

of canine

platelets

contained

MPO activity (< 0.0002 u MPO/I08 platelets). Control myocardium taken from dogs that underwent

ventricular

coronary

where

occlusion

(n = 5) or from other

experiments

no detectable fibrillation

upon

the myocardium

was

not stained (n = 7) showed very low MPO activity of 0.014 + 0.001/100 mg tissue. This activity was less than that which could be accurately measured in preparations of neutrophils. Extrapolating from the neutrophil standard curve, this level of activity represents

approximately

1.5

x IO4 neutrophils/lOO

may be endogenous to the cardiac ments. Ninety minutes of coronary

mg tissue.

tissue or it may represent occlusion increased MPO

This MPO trapped activity

activity

blood eleto 0.091 t

0.02 u/l00 mg in the ischemic tissue (infarct and risk areas), which represents 1.4 & 0.4 x IO’ neutrophils (n = 5). MPO activity increased in a time-related manner upon reperfusion equivalent to 5.3 temporal changes

(Figure 3) to a peak level of 0.32 + 0.018 u MPO/lOO mg tissue, f 0.3 x IO5 neutrophils (n = 6), after 5 hr reperfusion. The in myocardial MPO activity in this ischemic region matched the

histologic analyses (Figure 3). Light microscopy revealed an absence of neutrophils in the control sections of myocardium. After 3 and 5 hr reperfusion, when MPO activity was high, large numbers of leukocytes could be observed in the histologic sections. The results were very similar to those we have reported previously (Mul-

MP0

Activity in lschemic Myocardium

163

lane et al., 1984), where using a semiquantitative O-4 scoring system for neutrophil infiltration control cardiac tissue had a score of 0, while after 3 hr reperfusion the score was 2, and after 5 hr it was 3-4. Transmural

Distribution

of MP0

Activity

Because neutrophil infiltration is heterogeneous, the transmural distribution of MP0 activity across the ischemic region was studied in segments of tissue taken from the infarct, risk, and interface zones. In dogs subjected to 90 min of coronary occlusion and 5 hr reperfusion, the infarct size was 55 2 3% of the risk area, the latter being 36 ? 2% of the total left ventricle. Highest MP0 activity was found at the interface-that region overlapping the junction between the infarcted and risk areas-where activity at 5 hr was 0.377 * 0.02 u MPO/IOO mg tissue (= 6.4 ? 0.4 x IO5 neutrophils; Figure 4). Activity in the central infarct zone was 0.30 ? 0.01 u MPO/IOO mg tissue (= 4.9 ? 0.2 x IO5 neutrophils), whereas lower activity was observed in the central risk area at 0.24 -t 10.02 u MPOIIOO mg, equivalent to 3.8 t 0.35 x IO5 PMNs. In dogs subjected to 90 min and 5 hr of myocardial ischemia, but not given Evan’s blue dye (n = 6), MP0 levels in regions of the normal myocardium perfused via the unoccluded circumflex coronary artery were 0.0165 * 0.005 u MPO/IOO mg (= 1.7 ? 0.4 x IO4 PMNs). This value was not significantly different from that obtained in normal cardiac tissue of control animals.

*:: . .-: 1 ..‘... . ,*. I.:... . . . >:.:.* . . : . . . . . . . -::.. . . . . . . l::: *::.* -.*:: . . ._. .:.I... ._._. . .*.*.I. *::.* *::.* 1.1.1.:

RISK -+

INFARCT

3h reperfusion

RISK-+

INFARCT

5h reperfusion

FIGURE 4. Transmural distribution of MP0 activity in ischemic myocardium after 3 and 5 hr reperfusion. The interface region is denoted by the white bars. After 3 hr reperfusion (n = 5) the infarct and “interface” regions contained significantly @ < 0.05) higher MP0 activity than the risk area. At 5 hr reperfusion (n = 6), the “Interface” region had the highest activity @ < 0.05 versus infarct zone, p < 0.02 versus risk area), while the infarct zone demonstrated greater MP0 activity than the risk area @ < 0.05).

164

K. M. Mullane et al.

60-

0 units

0.1

MP0 /lOOmg

0.2 infarcted

0.3 tissue

FIGURE 5. Relationship between infarct size and units of MP0 activity in segments of infarcted myocardium. A, basal (unoccluded), n = 4; 0, control infarct, n = 3; 0, nafazatromtreated infarct, n = 4.

MP0

Activity and Infarct Size

The relationship between MP0 activity and infarct size was studied in 7 dogs subjected to 90 min occlusion and 5 hr reperfusion. Four of the infarcted dogs were pretreated with nafazatrom (IO mg/kg p.0.) prior to coronary occlusion to reduce infarct size (Bednar et al., 1985a), while three were untreated. MP0 activity in control segments of normal cardiac tissue from four dogs was also included. Thus, a range of infarct sizes and MP0 activities could be compared. There was a significant correlation (r = 0.88, p < 0.001) between the area of myocardial injury and the extent of neutrophil infiltration after 5 hr of reperfusion. DISCUSSION By use of the neutrophil-specific myeloperoxidase enzyme, we have followed the infiltration of neutrophils into myocardium rendered ischemic by coronary artery occlusion and reperfusion. The MP0 assay is a sensitive, quantitative index of myocardial neutrophil content, and it can reproducibly detect as little as approximately 8 x 104 neutrophils. MP0 activity in preparations of canine peritoneal neutrophils is linear over the range 105-IO6 cells, which covers the major range of enzyme activity observed in the cardiac tissue. This enables MP0 activity to be converted to actual numbers of neutrophils. Other leukocyte populations contain minimal amounts of this enzyme (Bradley et al., 1982b), and we could not detect MP0 activity in preparations of platelets. The use of MP0 activity as an index of neutrophil infiltration avoids the problems and limitations of histologic (Mullane et al., 1984; Fishbein et al., 1978) and radiolabeled techniques (Romson et al., 1982; Thakur et al., 1979). Histologic analyses

MP0

Activity in lschemic Myocardium

require the tedious inspection of multiple tissue samples-because neutrophil infiltration is heterogenous-to arrive at an arbitrary semiquantitative index of cell invasion. The MP0 assay on tissue samples of 100 mg or more allows the problems of heterogeneity to be avoided, as these samples contain mixed areas of leukocyte infiltration. The radiolabeling of cells mandates their prior removal from the circulation, isolation, and labeling in vitro-procedures that can drastically alter the integrity and viability of these leukocytes, and which are unnecessary in this assay. There is a linear increase in MP0 activity with time in cardiac tissue from ischemic regions of the heart. The increase observed after 90 min occlusion probably reflects the margination and accumulation of cells onto the endothelium within the blood vessels before they migrate into the tissue (Mullane et al., 1984). Upon reperfusion, there is a large increase in MP0 activity over 5 hr. Histologic analyses paralleled the MP0 changes, adding further credence to the use of this enzyme as a quantitative marker of myocardial neutrophil content. This time course of cell invasion is very similar to that we have described previously, using histologic techniques (Mullane et al., 1984), and has been shown, by use of radiolabeled cells, to peak at 24-72 hr (Thakur et al., 1979). In addition to a time-related increase in MP0 activity after myocardial ischemia, there is a transmural distribution of enzyme activity across the wall of the ventricle. The “interface” between the risk area and infarcted zone contains the highest levels, with the central infarct zonecontaining intermediate levels and the risk area showing slightly lower activity. This transmural distribution is the same as that which we have found for platelet accumulation (Bednar et al., 1985b). Neutrophils can release up to 50% of their MP0 during inflammation induced by staphylococci (Bradley et al., 1982b). It is not clear what proportion of the cardiac MP0 is free or still contained within the leukocyte. Moreover, in the preparations elicited of peritoneal neutrophils, some MP0 may be released before the cells are harvested so that the measured activity per neutrophil is correspondingly lower. However, the levels of canine neutrophil MP0 content are in very close agreement with those reported in rat cells (Bradley et al., 1982a), while the same neutrophil MP0 levels are used for comparison between different preparations of myocardium, so that any errors will not invalidate the quantitative comparison between tissue specimens. The time course, transmural distribution, and histology confirm the accuracy of the MP0 determinations. The contribution of neutrophils to ischemia-induced myocardial injury is becoming increasingly apparent. Myocardial ischemia induces complement activation (Pinckard et al., 1981), which in turn can stimulate neutrophils, causing them to aggregate (Jacob et al., 1980). Indeed, neutrophil aggregation in vitro may reflect a mechanism of tissue injury that is readily amenable to the study of drugs directed against neutrophil-mediated damage (Jacob et al., 1980; Bednar et al., 1985a). Drugs that prevent neutrophil activation and infiltration (Mullane et al., 1984; Maroko et al., 1978; Mullane and Moncada, 1982; Jolly and Lucchesi, 1983; Romson et al., 1982) or agents that deplete circulating neutrophils (Mullane et al., 1984; Romson et al., 1983) reduce infarct size. Consequently, the development of a simple enzymic assay as a quantitative index of neutrophil infiltration into the myocardium will be useful

165

166

K. M. Mullane

et al.

to demonstrate in vivo that the ability of some drugs to influence leukocyte behavior correlates with their myocardial protective effects. To this effect, we have recently found that nafazatrom (Bay g 6575), which inhibits leukocyte function in vitro, salvages ischemic myocardium in vivo commensurate with a reduction in cardiac MP0 activity (Bednar et al., 1985a). It remains to be determined if drugs that reduce infarct size by a mechanism independent of the neutrophil (Hillis and Braunwald, 1977; Goldberg et al., 1982) also alter the degree of cell invasion (MP0 activity). However, the MP0 assay provides a useful, quantitative index of tissue neutrophil content. This

study

was supported

by a Grant-in-Aid

from

uted in part by the American

Heart Association,

recipient

Manufacturers

of a Pharmaceutical

The authors for typing

wish

this

to thank

the American

Association

Dr. J.C. McGiff

for helpful

Faculty

Hillis

M, Smith

trom-induced

Bednar

KM,

M, Smith

belled

platelet

Bradley

B, Pinto

depletion

cardium.

function

myocardium.

accumulation

PP, Priebat

Circ

enzyme

marker.

of neutrophil

PP, Christensen

LD, Braunwald

RD,

inflammation.

ischemic

status

RD, Rothstein inflam-

content with an

Rothstein

C (1982b)

myeloperoxidase

Blood

Corday E, Meerbaum the present

Forestieri

and Pam Blank

in py-

60:618-622.

of reperfusion

Jacob HS,

Craddock

ischemia.

aggregation.

An unsuspected

ease. N Engl]

Med

L, O’Neill

infarction.

N En@/

Cardiol

therapy

Med

Cardiovasc

in an

ischemic

my-

JF, Lo E, Osterberger

HT, Weiss

R, Lee T, Kurian S (1983) Intra-

in acute myocardial

308:1305-1311.

PC, Holley

KE, Titus

enzyme-mapping

of predictable

of

Pitt B, Goldstein

fibrinolytic

of the acutely

Co//

Colfer

coronary

homogeneous,

of dis-

1 106:8-13.

JA, Brymer AD,

Lie JT, Pairolero

model

Am Heart

WW,

T, Goldberg

mechanism

BR (1983) Effect of BW755C

injury.

Khaja F, Walton

DE, Molgranulocyte

302:789-794.

occlusion-reperfusion

on

Part 1. / Am

PR, Hammerschmidt

dow CF (1980) Complement-induced

roscopic

S (Eds) (1983) Symposium

myocardium,

E (1977) Myocardial

Med296:971-978,1034-1041,1003-1096.

ocardial

Dermato/78:206-209.

and extracellular

ogenic

is the

Award.

and Ginny

Jolly SR, Lucchesi

myo-

(in press).

of cutaneous

/ invest

(1985b3

in infarcted

DA, Christensen

estimation

Cellular

KM,

indium-lll-la-

Pharmacol

Measurement

mation: Bradley

A, Mullane

suppresses

/ Cardiovasc

(1982a)

contrib-

M. Mullane

(1985a) Nafaza-

of neutrophil

salvage of ischemic

Neutrophil

C

6, Mullane, inhibition

(in press).

Res

with funds

Kevin

Development

discussions

N Englj

mediates

Chapter.

manuscript.

REFERENCES Bednar

Heart Association,

Westchester/Putnam

JL (1975) Mac-

verification

experimental

of large,

myocardial

size and location

infarcts

in dogs. / Thorac

Surg 69:599-605.

1:1031-1080. Engler

RL,

Schmid-Schonbein

(1983) Leukocyte ischemia

GW,

capillary plugging

and reperfusion

Pavalec

RS

in myocardial

in the dog. Am)

Pathol

3:98-111. Fishbein

MC,

Maclean

histopathologic tion.

Chest

S, Maroko

ronary

thrombolysis

tion.

Ann

infarc-

lntem

Med

Corell

PR,

PR, Engel

TR

(1982)

Intraco-

for acute myocardial

infarc-

96:115-117. of the ischemic

influencing coronary Maroko

/ MO/ Cell Cardio/9:605-616.

JK, Sobel

crosis

BE, Watanabe

J Jr, Braunwald

infarct

size

PR, Carpenter

experimental

Circulation

CB, Chiariello

by cobra venom after coronary

T,

E (1971) Factors

following

artery occlusions.

MC, Radvany P, Krostman

43:67-82.

M, Fishbein

JD, Hale SL (1978) Refactor of myocardial

artery

occlusion.

ne-

/ C/in In-

vest 61:661-670. Meerbaum

myo-

Kjekshus

JW, Ross

duction

DJ (1977) Reperfusion

cardium.

PR (1978) The

of myocardial

73:843-849.

Goldberg

Hearse

D, Maroko

evolution

Maroko

perfusion

S, Corday during

Parts 1 and 2. Am

E (1975) acute

Symposium

myocardial

/ Cardio/36:211-261,

on re-

infarction,

368-406.

MP0

Activity in lschemic Myocardium

Mullane KM, Moncada S (1982) The salvage of ischaemic myocardium by BW755C in anaesthetised dogs. frostag/andins 24:255-266.

Radomski M, Moncada S (1983) An improved method for washing human platelets with prostacyclin. Thromb Res 30:383-389.

Mullane KM, Moncada S (1983) Cytoprotection with prostacyclin and with dual enzyme inhibitors. In Mechanism of Drug Action. Eds. TP Singer, TE Mansow, RN Ondarza. Academic Press. pp. 229-242.

Romson JL, Hook BG, Kunkel SL, Abrams CD, Schork MA, Lucchesi BR (1983) Reduction of the extent of ischemic myocardial injury by neutrophi1 depletion in the dog. Circulation 67:10161023.

Multane KM, Read N, Salmon JA, Moncada S (1984) Role of leukocytes in acute myocardial infarction in anesthetized dogs: relationship to myocardial salvage by anti-inflammato~ drugs. j Pharmacof Exp Ther 228:510-522.

Romson IL, Hook BG, Rigot VH, Schork MA, Swanson DP, Lucchesi BR (1982) The effect of ibuprofen on accumulation of indium-Ill-labeled platelets and leukocytes in experimental myocardial infarction. Circulation 66:1002-1011.

Pinckard RN, O’Rourke RA, Crawford MH, Grover FS, McManus LM, Chidoni JJ, Storrs SB, Olson MS (1980) Complement localization and mediation of ischemic injury in baboon myocardium. J C/in Invest 66:1050-1056.

Thakur ML, Gottschalk A, Zaret BL (1979) Imaging experimental myocardial infarction with indiumIll-labeled autologous leukocytes: effects of infarct age and residual regional myocardial blood flow. Circulation 60:297-305.

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