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
