Download Reperfusion of ischemic myocardium: Ultrastructural and

J AM COLL CARDIOL
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1983;1(4) 1037-46
Reperfusion Of Ischemic Myocardium: Ultrastructural and
Histochemical Aspects
JUTTA SCHAPER, MD, WOLFGANG SCHAPER, MD
Bad Nauheim, West Germany
The effects of reperfusion on ischemic myocardium generally depend OQ the severity of the preceding ischemic
injury. Reperfusion of myocardium, irreversibly injured
by ischemia, produces further progression of myocardial
necrosis that is accompanied by simultaneously occurring stimulation of interstitial cell proliferation resulting
in scar formation. Reperfusion of reversibly injured
myocardium leads to structural improvement and reorganization. Thus, it may be stated from the ultrastructural part of this study that reperfusion of ischemic myocardium induces 1) slow structural recuperation after
reversible injury, and 2) accelerated cellular destruction
and symptoms of scar formation after irreversible ischemic injury. We observed that the reduced tissue content
of nicotinamide adenine dinucleotide (NAD), rather than
reduced dehydrogenase activity, is the basis of histochemical reactions employing tetrazolium salts. Directly
measured NAD tissue content in ischemic tissue correlated well with the degree of ultrastructural injury and
Emergency coronary bypass surgery and aggressive thrombolytic therapy are gaining widespread interest and are currently advocated and used in treatment of unstable angina
and impending myocardial infarction (1-8). Reduction or
complete prevention of myocardial infarction is the rationale
for the proposed beneficial effects of reperfusion (9-12),
which are still controversial. Jennings and Reimer (13) and
Schaper (14), in recent reviews on reperfusion of ischemic
myocardium, showed that reflow is able to limit infarct size
within 3 hours and may still be successful up to 5 to 6 hours
after onset of the ischemic event. Others (15-18) claim that
the reperfusion itself has harmful effects on ischemic cardiac
tissue. This conviction may be partially a matter of the
experimental model of ischemia used, that is, the globally
From the Department of Experimental Cardiology, Max-Planck-Institute, Bad Nauheim, West Germany.
Address for reprints: Jutta Schaper, MD, Max-Planck-Institute, Department of Experimental Cardiology, Benekestrasse 2, 0-6350 Bad Nauheim, West Germany.
© 1983 by the Amencan College of Cardiology
with macroscopic differential staining.
Occlusion of two small coronary arteries in the same
heart followed by reperfusion of only one artery (identical occlusion times for both arteries) showed identical
infarct sizes for reperfused and nonreperfused myocardium for occlusion times of 3 and 6 hours. When the
effects of occlusion times of less than 3 hours are studied
with tetrazoliuni salts, a difficulttechnical problem arises:
during that time, tissue-NAD concentrations have not
decreased enough to enable differential staining. Reperfusion leads to washout of NAD, thus producing differential staining; this may be a harmful effect of reperfusion. However, because early reperfusion leads to significant structural and functional recovery and to small
infarcts, reperfusion injury is unlikely to occur. Both
ultrastructural and histochemical evidence suggest that
reperfusion is beneficial for reversibly injured tissue but
accelerates the decay of irreversibly injured tissue.
ischemic heart (18) or the model of the oxygen or calcium
(Ca 2 +) paradox (19).
Bulkley and Hutchins (20,21) reported the occurrence of
necrotic foci in the presence of patent grafts in human myocardium, which they ascribed to the harmful effect of reflow
after the intraoperative nonperfusion period. Montoya et al.
(22) described hemorrhage into the myocardium of patients
undergoing revascularization for unstable angina. Deleterious effects of myocardial reperfusion due to hemorrhage
have been described in the canine heart by Breshnahan et
al. (23), who also observed an extension of the origional
infarct size. Several investigators (24-26) showed that infarct size extension due to reperfusion is only present after
longer periods of coronary artery occlusion, and Corday et
al. (27) suggested that the enlargement of infarct size was
merely caused by hemorrhage into necrotic myocardium but
not gy damage to salvageable tissue. Mclvamara et al. (26)
also claimed that hemorrhage into the infarct is probably
not automatically deleterious. Muller et al. (28) stated that
reperfusion with blood seems to be the optimal method to
salvage ischemic myocardium in human beings, but in their
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opinion, a definite confirmation of the beneficial effects of
reperfusion was still needed.
In numerous animal experiments, the possible beneficial
or harmful effects of reperfusion on ischemic myocardium
were investigated and many abnormalities were noted. The
common denominator observed in ischemic reperfused cardiac tissue was that functional recovery occurred only very
slowly and often was absent (29-35). Many different pathophysiologic factors have been implicated In causing delayed functional recovery. Impaired mitochondrial function
(29,31,36,37), possibly associated with mitochondrial Ca2+
loading (30,31,38), but also Ca 2 + loading alone (39-42)
have been discussed as possible mechanisms. Kotaka et al.
(43) showed that mitochondrial dysfunction during coronary
reperfusion was partially caused by acyl coenzyme A accumulation. Wood et al. (29) reported loss of glycogen and
decreased phosphorylase activity, and Hess et al. (44) described decreased Ca2 + uptake by the sarcoplasmic reticulum and decreased activity of myofibrillar adenosine triphosphatase (ATPase).
Delayed resynthesis of the depleted adenine nucleotide
pool (30-32,45,46) has also been strongly associated with
delayed recovery. Reimer et al. (46) showed that the adenine
nucleotide pool was markedly dcreased after 15 minutes of
reversible ischemia and that it was still slightly suppressed
after 4 days of reflow. Microvascular defects causing persistence of reduced blood flow to the ischemic region (34,4750) and edema formation (51-53) combined with Ca2 + loading
(53) have also been associated with slow recovery during
reperfusion after an ischemic event.
Only a few studies have described the effects of reperfusion on ischemic myocardium from an ultrastructural point
of view. Jennings and his co-workers (54-59) reported most
of the data on myocardial ultrastructure in ischemia and
reperfusion and showed very clearly that tissue reversibly
injured by ischemia shows structural recovery after reperfusion, whereas irreversibly injured tissue deteriorates further during reflow. Our own studies (60-62) on the ultrastructural changes in regional ischemia in the dog heart
agreed with those of Jennings et al. From their work and
ours, it becomes evident that it is nearly impossible to estimate the effects of coronary reperfusion on previously
ischemic myocardium when the differentiation of reversible
versus irreversible ischemic injury is lacking.
Duration of coronary artery occlusion has been used in
many studies (17 ,37,45,63) as an indicator of the degree of
injury produced. However, because time is only one of
several factors determining the development of myocardial
ischemia and infarction (14), a more direct method needs
to be used to investigate the effects of reperfusion. The present study, therefore, was undertaken to investigate the effects of ischemia of varying duration and of reperfusion in
the same heart by electron microscopy. Ultrastructural evaluation of tissue obtained by small needle biopsy offers the
unique opportunity to determine the degree of sichemic injury and the phase of recorvery during reperfusion with high
accuracy and in a continuous manner using very small tissue
samples.
Methods
Experimental design. In IS anesthetized open chest dogs, the
anterior descending branch of the left coronary artery was occluded
for 45, 90 or 180 minutes. At the end of the ischemic interval,
transmural needle biopsy specimens from the center of the ischemic
area were taken; thereafter, the arterial ligature was opened and
the reperfusion period started. The chest was closed and the animals were sacrificed after 48 hours of reperfusion after transmural
biopsy samples were again taken from the same previously ischemic area of the still beating heart.
From the large number of dogs used in this experimental series,
only three for each ischemic interval (each interval being defined
as one group) will be described here. The experiments presented
were deliberately selected from each group for their special and
very typical features representative of the entire population.
Preparation of tissue for electron microscopy. All tissue
samples were subdivided into a subendocardial and a subepicardial
part. Both samples were separately numbered and treated during
the embedding, cutting and evaluation procedures. All biopsy material was immediately fixed in 3% cold glutaraldehyde, prepared
with 0.1 M cacodylate buffer at pH 7.4. Immersion fixation for 2
to 6 hours was followed by frequent rinsing in cold cacodylate
buffer during 3 consecutive days; thereafter the tissue was embedded in Epon following fixation in 2% osmic acid anhydride and
dehydration in a series of ethanol plus substitution by propylenoxide using a Wakura automatic tissue processor.
Semithin (1 to 2 ~m) sections were prepared and stained with
toluidine blue from all samples. Artifact-free areas were selected
from these for the preparation of thin sections (500 to 600 A) for
electron microscopy. These sections were attached to uncoated
copper grids, stained with uranyl acetate and lead citrate and viewed
in a Philips EM 300 electron microscope. All micrographs, 30 to
40 per sample, were evaluated using our standardized system (62,64).
Results and Discussion
The ultrastructural results for each group are shown in
Table 1 and the typical symptoms of cellular recovery or
degradation are described and illustrated in the text.
Grading of Ultrastructural Alterations in Ischemic
and Reperjused Cardiac Tissue
Electron microscopic investigations of ischemic and reperfused myocardium allow exact definition of the stage of
progression of cellular damage and degree of recovery during reperfusion when all ultrastructural changes are evaluated in a standardized way. On the basis of earlier studies
in ischemia (64), normal myocardium (Fig. 1) was differ-
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1983;1(4):1037-46
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1
Figure l. Normal myocardium . Magnification 30.000 x .
Figure 2. Reversible slight Ischemic injury . Magnification 13.800 x ,
mset rnagmfication 48.000 x ,
Figure 3. Reversible moderate Ischemic mJury. Magnification 13.800 x ;
mset magnification 30.000 x .
Figure 4. Reversible severe Ischemic mjury. Magnificanon 13,800 x :
inset magnification 20,400 x ,
Figure 5. Irreversible ischerruc mjury. Magruficauon 13,800 x ; inset
magnification 48.000 x .
entiated from myocardium undergoing slight (Fig. 2), moderate (Fig. 3) and severe (Fig. 4) reversible ischemic injury
and irreversible injury (Fig. 5).
The estimation of ultrastructural changes in mitochondria, nuclei and myofilaments provides the most reliable and
reproducible identification of the degree of ischemic injury;
the systematic assessment of all other subcellular cornpo-
nents , including blood vessels and extravascular space of
the myocardium, contributes substantial information on
pathogenetic mechanisms operative in the progression of
ischemia and infarction, According to our experience with
ultrastructural changes in ischemic myocardium obtained in
approximately 120 dogs (62), it appears that regional ischemia may provoke a cascade of events on the cellular
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.
~
SCHAPER AND SCHAPER
. ,..
."
Figure 7. Endothelial prohferation and presence of increased numbers of
intravascular neutrophils during reperfusion. Magnificanon 9,900 x.
Figure 6. Early stage of structural recovery during coronary reperfusion.
Magnification 9,900 x . Inset shows incomplete recovery of mitochondria;
magnification 24,000 x.
level characterized by a close interaction among myocytes,
all interstitial and vascular cells and cells from the circulating blood.
Structural recovery during reperfusion after reversible ischemic injury. The early stage of structural recuperation is shown in Figure 6. Myocardial mitochondria
were still electron lucent; they contained only a few matrix
granules and a reduced number of cristae, and the cells exhibited numerous lipid droplets. Many microvessels showed
endothelial proliferative activity and contained a disproportionately high number of neutrophils (Fig. 7) and platelets.
Intermediate stages were characterized by slight mitochondrial clearing (Fig. 8), while complete recovery of cardiac cells was indicated by mitrochondria exhibiting a dense
matrix and numerous tortuous cristae, a typical symptom
of high metabolic activity (Fig. 9). At all stages of cellular
recovery, these cells showed a significantly increased number of primary and secondary lysosomes and many very
active parts of the Golgi apparatus (Fig. 8 and 9).
Progression of lesions during reperfusion after irreversible injury. Different stages of cellular degradation
after irreversible ischemic injury were observed after 48
hours of reperfusion. Destruction of myocytes depended on
the duration of the preceding ischemic interval and on myocardial localization; that is, the subendocardial samples
showed further developed necrosis and repair processes than
did the subepicardial tissue. After short-term ischemia, destroyed myocytes were found next to intact recovered cells
(Fig. 10), but after longer ischemic periods, all cells from
one particular sample were irreversibly injured. Typical ultrastructural findings in necrotizing cardiac muscle cells were
disappearance of nuclei, dissolution or condensation with
phospholipid of mitochondria and disappearance of myofilaments, especially Z band and I band material or persisting
contracture bands (Fig. 10 to 12). The following cell types
were involved in cellular degradation and simultaneous repair processes during reperfusion: neutrophil granulocytes,
blood platelets and monocytes, the latter transforming into
tissue macrophages, proliferating fibroblasts and endothelial
cells. These cells were numerous and contained a large
number of primary and secondary lysosomes (Fig. 10 to
12).
Importance of Cardiac Lysosomes in Ischemia
and Reperfusion
Lysosomal changes were especially obvious in the tissue
studied and, therefore, they will be described in a more
detailed manner. With increasing severity of ischemia, the
number of primary lysosomes gradually decreased within
myocardial cells, whereas the amount of secondary lysosomal structures, that is, phagosomes, autophagic vacuoles
and phospholipid-lipofuscin complexes, remained unaltered. After irreversible injury occurred, intact lysosomes
were increasingly less evident and virtually absent at late
stages of ischemia. Neither intact primary lysosomes nor an
increased amount of secondary lysosomal structures was
observed in myocytes during the development of necrosis.
However, this situation was entirely different in moderate
to severe reversible ischemic injury followed by cellular
recovery during myocardial reperfusion. After 48 hours of
reperfusion, many new intact primary lysosomes were present at the nuclear poles and in the periphery of the myocardial cells, and the number of secondary Iysosomes had
REPERFUSION OF ISCHEMIC MYOCARDIUM
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1983,1(4).1037-46
Figure 9. Complete structural recovery after reversible ischemia. Note
numerous lysosomal figures and active Golgi apparatus. Magnification
30,000 x. Inset shows part of a mitochondrion with prominent tortuous
cristae; magnification 90,000 x.
Figure 8. Intermediate state of cellular recovery after reversible ischemic
injury, Note the increased number of various lysosomal structures. Magnification 24,600 x. Inset also shows different stages of lysosomes close
to parts of a Golgi apparatus; magnification 54,000 x.
significantly increased compared with that in the nonischemic tissue of the same heart. This finding implicates the
very important role of lysosomal enzyme activity in removing, most probably by autophagocytosis, those intracellular structures that were injured by the earlier ischemic
event. Leakage of proteolytic enzymes from lysosomes
causing degradation at the myofibrillar level is structurally
indicated by the disarrangement of sarcomeres in reperfused
tissue, but apparently this process must have ended at a
certain state during reperfusion, perhaps when normal cellular pH was reached again.
This view is reinforced by our own ultrastructural observations on human myocardium obtained as intraoperative
needle biopsy specimens from patients undergoing aortocoronary bypass surgery (65). The myocardial cells of patients with chronic coronary heart disease exhibited a very
typical morphologic pattern: most of the myocytes obtained
from the poststenotic area showed a decrease in size, distinct
disarray of myofibrils, normal-appearing mitochondria and
nuclei, but an increased number of intact primary lysosomes
and a large amount of secondary lysosomal structures. Apparently, these myocytes, when underperfused during an
attack of angina pectoris, were stimulated to release and,
thereafter, to reproduce lysosomal enzymes to permit removal of cellular components injured by ischemia.
Lysosomes in reversible versus irreversible ischemia.
In summary, in irreversible cellular damage leading to
myocardial infarction, lysosomes and their enzymes apparently gradually disappear from myocardial cells after contributing to the destruction of the tissue. In contrast, in
reversible ischemic injury followed by long-term reperfusion, lysosomal enzyme activity may act as protective mechanism by scavenging intracellular debris, thereby contributing to the structural reorganization and recovery of
ischemically injured myocytes. The cellular recovery from
ischemic injury (during reperfusion) is characterized by a
massive induction of lysosomes that clear the cells of debris
accumulated during ischemia. The result of this endocytotic
"clear up" is a live myocardial cell. Irreversibly damaged
myocytes are the target of scavenger cells; their own lysosomal system apparently does not contribute to
autophagocytosis.
Reperfusion After Regional Ischemia:
Beneficial or Harmful?
From the data presented in this study (Table I), it may
be concluded that the degree of injury may vary from slight
to severe reversible damage in all groups. After 45 minutes,
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one sample showed many irreversibly injured cells, whereas
after 90 and 180 minutes, irreversible injury, when present,
was ubiquitous. The subendocardial samples showed more
severe damage than did those from the subepicardium. The
great range of severity of .ischemi~ in al~ g~oups supports
the finding (14) that the time of Ischemia IS only One of
several factors determining whether myocardial infarction
occurs .
Types of cellular reaction to reperfusion. The effects
of reperfusion on ischemic cardiac tissue appeared to be
very different, again independent of the time of ischemia.
Three different types of cellular reaction to reperfusion were
observed. I) Structural recovery after reversible ischemic
injury was observed in dogs from all three groups. Improvement of cellular structures was usually present after
moderate and severe reversible ischemic injury. 2) The situation did not change from that observed after the ischemic
interval that usually occurred after slight to moderate reversible injury. This tissue reaction may be similar to that
observed frequently in well perfused tissue during or after
an ischemic insult (own observations). 3) Marked structural
deterioration of myocardium that was already irreversibly
injured by ischemia occurred, indicative of further progression of necrosis and the beginning of scar formation .
In conclusion, the ultrastructural data presented in this study
provide evidence that reperfusion of ischemic myocardium
induces : I) structural recuperation after reversible injury ,
Figure 10. Heterogeneity of myocardial ultrastructure after a short period
of ischemia plus reperfusion. Irreversible injury at left an~ top of the
micrograph. but recovering almost intact myo~ardial cell at bottom and
right. A very active interstitial cell is present m the center of the micrograph. Magnification 3,000 x.
and 2) accelerated cellular destruction and symptoms of scar
formation after irreversible ischemic injury. Reperfusion,
therefore, is beneficial for reversibly injured tissue.
Histochemistry
Dehydrogenase-stain (tetrazolium salts). One of the
most precise measurements of infarct size utilizes tetrazolium salts . The compounds act as electron acceptors in reactions catalyzed by dehydrogenases . Colorless soluble tetrazolium salts become intensely colored (production of
formazan dye), and because these dyes are insoluble in
water, they precipitate precisely at places where the reaction
occurred. These methods were developed to diagnose human myocardial infarcts postmortem (66-68). The method
is usually called the "dehydrogenase-stain" because of the
mechanism involved. Dehydrogenases exhibit reduced activity in human myocardial infarction, that is, only normal
muscle stains; infarcted muscle does not stain . The reaction
can be carried out in ultrathin sections for electron microscopy, on thin sections for light microscopy and on myocardial rings for inspection and macroscopic size determination (planimetry and point counting).
Addition of succinate or NAD. In recent years , the
method has gained great popularity because true anatomic
infarct size can be obtained more easily (compared with
conventional light microscopy) and at much greater speed
(69). However. most worker s did not recognize that the
proposed mechanism of action (dehydrogenase activity and
the lack thereof) is not operative in short-term (up to 6 hours)
occlusions, because dehydrogenase activity does not appreciably decrease within 24 hours after coronary occlusion,
at least not to a degree that affects the staining reaction (70).
Yet, if an experimental occlusion lasts for 3 hours or longer,
Figure II. Cellular degradation after irreversible ischemic injury. Myofilaments and mitochondna (*) are dissolving (top) . Adherence of a neutrophilic (right) and mfiltration by a phagocyt ic (bottom left) cell. Magnification 13,800 x
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differential staining with tetrazolium is obtained. Furthermore, we observed that in the presence of differential staining (normal tissue stained, infarcted tissue not stained), the
addition of either succinate or nicotinamide adenine dinucleotide (NAD) caused a uniform dye precipitate, that is,
previously nonstained infarcted tissue became stained. It
was found that differential staining after relatively shortterm occlusions is caused not by the lack of dehydrogenases,
but rather by the lack of their coenzymes, that is, NAD
(70,71). In a series of experiments, we showed that the sum
of NAD plus NADH remained constant in regionally ischemic tissue as long as the degree of damage was reversible
by ultrastructural criteria. Total NAD decreased when the
point of no return was reached. When the tissue concentration of total NAD fell below 200 pmol/g wwt (normal =
700), the tissue lost its ability to produce the formazan dye.
This ability is quickly restored in vitro by the addition of
NAD to the incubation medium. Succinate dehydrogenase,
which becomes active when its substrate is added to the
incubation medium, produces the insoluble formazan dye
in infarcted muscle because this enzyme does not need NAD
as a coenzyme, The reaction that takes place on the cut
surface of myocardial cells is interpreted as an enzymatic
cycling (70).
Quantifying myocardial damage after short occlusion
times. The question whether reperfusion is good or bad for
previously ischemic heart muscle assumes practical significance when trying to quantitatively define the amount of
damage inflicted by relatively short occlusion times (between 20 minutes and 3 hours). Within that time, it is not
possible to measure infarct size with tetrazolium salts. It is,
of course, possible to diagnose irreversible ischemic damage
at the end of a short occlusion time, but without reperfusion,
a size determination is difficult if not impossible. This, in
essence, means that a subendocardial infarction that was
caused by an occlusion of 2 hours' duration cannot be diagnosed quantitatively with tetrazolium salts unless the tissue is reperfused. Reperfusion aids identification of tissue
that has undergone irreversible ischemic damage within these
2 hours of occlusion. Reperfusion removed (washed out)
NAD from severely ischemic tissue and thus permitted differential staining. At this point, it may be necessary again
to question whether reperfusion might have caused (in part)
the effect that only indicates like a litmus test. It is also
necessary to discuss the modes of disappearance of NAD
from ischemic myocardium. NAD can disappear from ischemic myocardium by one or more of the following routes:
1) by washout through leaky membranes; 2) by enzymatic
degradation through activation of glycohydrolase (72); and
3) by depressed de novo synthesis in the presence of accelerated degradation (73).
Stages of sarcolemmal damage during ischemia.
Results from our previous experiments suggest that, with
regard to NAD deprivation of ischemic tissue, ischemia causes a two-stage damage to sarcolemma and prob-
Table 1. Ultrastructural Results of Coronary Occlusion and
Reperfusion in Nine Dogs
Ischemia
Dog
Min
EPI
Endo
h
Epi
Endo
I
2
3
4
5
45
45
45
90
90
90
180
180
180
Slight
Mod
Mod
Mod
Slight
Mod
Mod
Mod
Irre
Sev
Sev
Mod
Mod
Irre
Irre
Irre
Mod
Irre
48
48
48
48
48
48
48
48
48
NI
Slight
Slight
Nl
Mod
Slight
Irre
Nl
Irre
Slight
Mod
Mod
Mod
Irre
Irre
Irre
Slight
Irre
6
Figure p. Myocardial cell destruction after irreversible ischemic injury,
dissolutionof mitochondria (*) but persistmg contracture of myofilaments
and presence of an active macrophage with numerous lysosomes. Magnification 39,000 x ,
Reperfusion
7
8
9
Endo ~ endocardial layer: Epi = epicardial layer: h = hours: Irre = irreverstble:
MIn = minutes: Mod = moderate. NI = normal. Sev = severe
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ably other membranes. At a first stage (very probably early
irreversible injury), the membranes become leaky and on
reperfusion, NAD is washed out. At a later stage (advanced
necrosis), the membrane-bound enzyme glycohydrolase is
activated and cleaves NAD (70-72). The presence and activity of glycohydrolase in heart, brain and kidney were
demonstrated by us (70,71) with the following experiment.
Freshly obtained tissue was rapidly homogenized in the
presence of oxygen, and aliquots of the homogenates were
taken at 1 minute intervals and assayed for NAD and NADH.
The pyridine nucleotides decreased within minutes through
the action of activated glycohydrolase (homogenization),
and the rate of decline was very fast in brain, followed by
that in heart and kidney (70,71). This is also the order of
susceptibility to ischemic damage. The decrease of the pyridine nucleotides in the tissue homogenates could be completely prevented when nicotinamide (the end product) was
added to the incubation medium.
It is unresolved which mechanism contributes most to the
observed loss of total NAD in ischemic tissue. The observation of differential staining without reflow in the presence
of infarcts at time intervals longer than 3 hours after coronary occlusion would suggest activation of glycohydrolase,
but tissues exposed longer to ischemia were also perfused
longer by collateral flow; that is, a washout effect, even in
nonreperfused myocardium, cannot be ruled out.
Does reperfusion irreversibly damage ischemic tissue? Because pyridine nucleotides playa key role in energy
metabolism, their loss through reperfusion at a time when
the diagnosis of irreversible ischemic injury by means other
than NAD histochemistry is difficult, leaves doubt whether
reperfusion is so beneficial. However, the observation (made
using tetrazolium salts) that up to 80% of the myocardium
at risk can be salvaged if reperfusion is established within
45 minutes after occlusion (69,74) suggests that a deleterious effect of reperfusion must be very small especially in
comparison with its beneficial effects. The question whether
already existing infarcts can become larger by reperfusion
was addressed with the following experiment (74): in the
same heart (canine, open chest), two medium size coronary
arteries were prepared and occluded for a period of 3 hours;
thereafter one artery was reperfused but not the other. This
was possible by occluding one artery 1 hour before the other
to allow for a 1 hour reperfusion period. Rings of left ventricular myocardium were then incubated in p-nitro blue
tetrazolium and infarct sizes were compared in each heart.
In another experiment, the duration of occlusion was extended to 6 hours. The purpose of the experiment was to
test the hypothesis that reperfusion may irreversibly damage
ischemic tissue that may have been, at the moment of reperfusion, only reversibly injured. If this were true, the
infarct at the reperfused side should have been larger than
at the nonreperfused side of the same heart. This was not
the case at 3 or 6 hours of occlusion. The incidence of
hemorrhagic infarcts was one of eight reperfused 3 hour
occlusions and eight of eight in occlusions lasting 6 hours.
It should be noted that Althaus et al. (75) found that reperfused hemorrhagic infarcts in the pig produced tougher
scars than did nonreperfused, nonhemorrhagic infarcts.
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