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INTRODUCTION. Fhe pathogenic role of polymorphonuclear leukocytes. (neu- rophils) during the development of ..... 0.5-jim-thick, plastic-embedded sec- tions.
Quantification of neutrophil migration following ischemia and reperfusion in cats and dogs Kurt H. Albertine, Andrew and Allan M. Lefer Departments Cardiology,

of Physiology Johns

and

Hopkins

S. Weyrich, Medicine,

University,

Xin-liang

Jefferson

Medical

Baltimore,

of

infiltration reperfusion,

of we

neutrophils during morphometrically

the early quantified

the tissue distribution of neutrophils in cats and dogs. At the end of the reperfusion period, the base of the heart was cross-clamped to preserve neutrophil location at the moment of death. Point-counting methods were used to determine the distribution of neutrophils inside and outside coronary arterioles and venules ( 100 m in diameter) as well as coronary capillaries 5-10 tm in diameter in O.5-jm-thick, plastic-embedded sections. Ischemia-reperfusion resulted in a threefold increase in neutrophil number in the lumen of arterioles and venules at 60 mm of reperfusion and up to a sevenfold increase at 270 mm of reperfusion (P < .05) compared to timematched control nonischemic hearts. The ratio of intravascular neutrophils in venules to arterioles was 2:1. Intracapillary neutrophils increased, but not significantly, at 60 mm of reperfusion. At 270 mm of reperfusion, intracapillary neutrophils increased 1 1-fold (P < .05). The percentage of total neutrophils that accumulated outside arterioles and venules in cat hearts was 8% at 60 mm of reperfusion (not significant, NS) and 28% at 270 mm of reperfusion (P < .05). In dog hearts, the percentages were 26% (NS) and 44% (P < .05), respectively. The percentage of total neutrophils that accumulated outside capillaries was < 6% in both cat and dog hearts (NS). The combination of rapid intravascular sequestration, delayed extravascular infiltration, and low incidence of neutrophil-cardiac myocyte contact in situ in these two species suggests that neutrophil-mediated cardiac myocyte injury during early reperfusion may initially depend )n diffusion of inflammatory mediators and subsequently require direct contact between neutrophils and cardiac rnyocytes.J. Leukoc. Biol. 55: 557-566; 1994.

F(cy hil

Words: margination

endothelium creatine

polymorphonuclear kinase activity

leukocytes

neutro-

INTRODUCTION Fhe pathogenic role of polymorphonuclear rophils) during the development schemia-reperfusion injury has been )articular, accumulation of neutrophils vith arrhythmias, coronary endothelial to-reflow phenomenon, and cardiac

College,

David

J. Lefer,

Philadelphia,

Lewis

Pennsylvania,

C. Becker, and

Division

of

Maryland

Abstract: Endothelial cell dysfunction and cardiac myocyte injury resulting from ischemia and reperfusion have been associated with accumulation of neutrophils in the myocardium. To determine whether the accumulation is related primarily to intravascular sequestration or extravascular period

Ma,

myocardial

Neutrophils function

are thought to contribute release of inflammatory

oxygen-derived free physical obstruction ment of neutrophils

to endothelial mediators,

cell dyssuch as

radicals and hydrolytic enzymes, of microvessels. Fu rthermore, to cardiac myocytes has been

and by attachproposed

as

a mechanism contributing to myocyte injury [9, 10]. The association between neutrophils and cell injury in the ischemic-reperfused heart has been supported by histopathologic studies showing that neutrophils accumulate in the injured region [11-13]. These studies have shown that the location of the accumulated neutrophils shifts from primarily intravascular to significantly extravascular. For example, in the dog heart, which has high collateral blood flow, neutrophil migration into the ischemic and reperfused myocardium became evident at 3 h of reperfusion and was extensive at 5 h ofreperfusion following 1 h ofcoronary artery occlusion [11]. In the rat heart, which has low collateral blood flow, the time course of neutrophil accumulation has been shown to be early or late depending on the duration of the ischemic period. When rat hearts were made ischemic for only 30 mm, the majority of the accumulated neutrophils re-

mained

intravascular

during

the

first

6 h of reperfusion

[12].

On the other hand, when the ischemic period was 1 h, about 75% ofthe neutrophils were intravascular after 1 h of reperfusion, whereas only about 30% were intravascular after 2 h of reperfusion [131. Some of the variability among the results of previous histopathological studies may be due, at least partly, to differences in species and ischemia-reperfusion protocols. Therefore, the temporal relationship between neutrophil intravascular sequestration and extravascular infiltration has not been well defined for the early period of reperfusion following myocardial ischemia. Moreover, the precise distribution of intravascular neutrophils (i.e., arterioles, capillaries, or venules) and extravascular neutrophils (i.e., adventitial or among myocytes) has not been reported. In an effort to clarify these issues, we performed experiments in two species employing the same ischemia and reperfusion protocol. Two species were selected to evaluate the role of low collateral blood flow (cat heart) versus high collateral blood flow (dog heart) on the time course of neutrophil accumulation in the ischemic and reperfused myocardium. Precise morphometnc methods were used to determine neutrophil sequestration and extravasation in hearts subjected to 90 mm of ischemia and

leukocytes (neuof acute myocardial well documented. In has been associated cell dysfunction, the myocyte injury [1-8].

by

either

60

Abbrcvtattons:

PM N, Reprint Medical Received

Journal

or

270

CK,

creatine

polymorphonuchear requests: College, October

mm

Allan 1020 6,

of Leukocyte

of

reperfusion.

kirtase;

LAD,

leukocyte;

PRI,

M.

pressu

Lefer,

Department

Locust

Street,

Philadelphia,

1993;

accepted

December

Biology

Volume

left

anterior

re-rate of

Physiology, PA

29,

55,

descending; index. Jefferson

19107. 1993.

May

1994

557

METHODS

myocardium

in the

Surgical

preparation

from

Two separate animal species were used to quantify the tissue distribution ofpolymorphonuclear leukocytes (PMNs) in the heart during ischemia-reperfusion injury. Fifteen adult male cats (3.6 ± 0.2 kg body weight) and 15 adult male dogs (23.5 ± 0.7 kg body weight) were anesthetized with pentobarbital sodium (35 mg/kg body weight, intravenously). The anesthetized animals were animal respirators (Harvard Polyethylene catheters were

jugular into

vein, the

blood

for

right pressure

injection

femoral using

mechanically

Apparatus, inserted

ventilated

South into the

of supplemental artery Statham

for

using

Natick, MA). right external

anesthetic,

measurement P23 pressure

and

of arterial transducers.

Mean arterial blood pressure was continuously recorded on an oscillographic recorder (Gould, Cleveland, OH). Heart rate and ST segment elevations were recorded every 20 mm from lead II of a scalar electrocardiogram. Pressure-rate indcx (PRI) was calculated as the product of mean arterial blood pressure and heart rate divided by 1000. PR! was used as an approximation of myocardial oxygen demand [14]. A midline sternotomy was made in the cats, and a left lateral thoracotomy was performed in the dogs to expose the heart. Once the pericardial sac was opened, a silk ligature (2-0) was placed around the left anterior descending (LAD) coronary artery, 10-12 mm from its origin.

Experimental

protocol

The 15 cats and 15 dogs were divided into three groups: (1) three sham-operated controls (360 mm), called “sham ischemia”; (2) five to six 90-mm mschemia+60-min reperfusion; and (3) five to six 90-mm ischemia+270-min reperfusion. The ischemmc period was initiated 30 mm after the surgical procedures were completed to allow stabilization of hemo-

dynamic variables. Myocardial totally occluding the LAD with

ischemia

was

produced

by

the silk ligature. Reperfusion was initiated by releasing the silk ligature. Prior approval for these experiments was obtained from the Animal Care Cornmittee ofThornasJefferson University for the use ofcats and of Johns Hopkins University for the use of dogs. One dog and one cat were discarded because of poor fixation of tissues.

Blood

formed

on

blood

samples

expressed

the

outside

inside

and

out.

stored

The

heart

for 4-5

immersed

was

days

at 4#{176}C to fix it

in.

Ischemic tissue specimens were cut from the portion of the left ventricular wall that was supplied by the LAD coronary artery. These tissue specimens were full mural thickness (i.e., epicardium to endocardium). Non-ischemic control tissue

specimens

from

the

same

heart

were

excised

from

the

wall

area supplied by the left circumflex coronary artery [17]. Since the cat heart has been shown to have a consistently low collateral blood flow to the left ventricle [18, 19], we took one tissue specimen from the feline left ventricle. The dog heart exhibits a variable degree of collateral blood flow to the left ventricle. Therefore, we took three tissue blocks spaced at 2-cm intervals from the LAD field ofthe canine left ventricle. One tissue slice, which spanned the full thickness of the yentricular wall, was cut from each specimen. The fixed slices were dehydrated in a graded series ofacetone (i.e., 50, 75, 90, and 100%), infiltrated in methacrylate (Immunobed; Polysciences, Warrington, PA), and embedded in the same medium. Thick sections (0.5 tm) were cut with the aid of glass knives struments, counterstain

mounted on a Historange Ijamsville, MD). Toluidine the tissue sections.

microtome blue

(LKB used

was

Into

Neutrophil distribution was determined using quantitative morphological (i.e., morphometric) methods [20]. Neutrophil distribution in the ischemic-reperfused and nonischemic subepicardium, midmyocardium, and subendocardium was categorized as either inside arterioles (15-100 j.tm in diameter), capillaries (5-10 am), and venules (15-100 m in diameter) or outside each vessel type. Neutrophil location was identified by using 0.5-jim-thick, plastic-embedded sections which provide sufficient resolution to identify endothelial cells, including those lining coronary capillaries. The ischemic-reperfused myocardial tissue was analyzed twice, first to quantify neutrophil distribution throughout the injured myocardial tissue and second to determine whether the accumulated neutrophils were preferentially located around the border of injured cardiac myocytes or situated among the myocytes. These analyses were accomplished by observing video-projected, calibrated images of the original histological sections using a Dage video camera (Michigan City, IN) mounted on a Zeiss Axioplan light microscope (Thornwood,

in

tubes before at the end were per-

cat

and dog blood using a hemocytorneter. The were then centrifuged at 2000g and 4#{176}Cfor The plasma was analyzed spectrophotornetrically for kinase (CK) activity [15]. Plasma CK activity was

as international

Plasma

protein

method

[16].

Determination

concentration

units

per

was

microgram

determined

of protein. by the

biuret

of PMN distribution

base of the heart was double-clamped to arrest blood flow and to trap blood in the coronary vessels in situ. The heart was excised with one clamp still attached. Freshly prepared fixative (2.5% glutaraldehyde, 1% paraformaldehyde in Millonig’s phosphate buffer, pH 7.45, 320 mOsm/kg H20) was injected into the lumen of the left ventricle to fix the

Sham 360’

The

358

the

fixative

sampling

Samples of arterial blood (2 ml) were withdrawn containing 200 IU heparin sodium immediately ischemia, at the end of the ischemic period, and of the reperfusion period. Total leukocyte counts

20 mm. creatine

from

same

Journal

of Leukocyte

Biology

Volume

55,

May

1994

90’I

(3) Fig.

1. Bar

mia

(I)

kinase

ber

graph

and

either

(CK) of

cats

activity in

each

+

60’R

90’I

(5) (mean 60

± SEM) or

in cats. group.

270 The

P