Oxygen radicals
in cerebral ischemia
CHARLES
W. NELSON,
ENOCH
P. WEI, JOHN T. POVLISHOCK,
HERMES
A. KONTOS,
AND MICHAEL
A. MOSKOWITZ
Departments
of Medicine and Anatomy, Medical College of Virginia, Virginia Commonwealth University,
Richmond, Virginia 23298; and Neurosurgery and Neurology Services, Massachusetts General Hospital,
Harvard Medical School, Boston, Massachusetts 02114
OF univalent reduction of oxygen, superoxide, hydrogen peroxide, and hydroxyl radical are very
reactive and capable of inducing tissue injury (6). They
have been suggested as possible mediators of the tissue
injury in ischemia-reperfusion in many tissues, including the brain. The generation of reactive oxygen species
and their role in tissue injury in ischemia-reperfusion of
the brain have received experimental support from a
variety of sources. These include direct demonstration
of superoxide production by showing the presence of
superoxide dismutase (SOD) -inhibitable reduction of
nitro blue tetrazolium (NBT) in the early part of reperfusion after ischemia in newborn pigs (1) and the finding
of increased hydrogen peroxide production in gerbils
subjected to brain ischemia (17). The latter authors also
showed that after inactivation of xanthine oxidase the
increased hydrogen peroxide production was eliminated
and mortality and brain edema were reduced, suggesting
that xanthine oxidase was the source of hydrogen peroxide. A number of attempts to inhibit xanthine oxidase
in the brain have met with mixed results. Some investigators found that inhibition of xanthine oxidase reduced infarct size and mortality, whereas others found
THE PRODUCTS
H1356
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$2.00
Copyright
no effect (2). Liu et al. (13) found that pretreatment
with polyethylene glycol-conjugated SOD and polyethylene glycol-conjugated catalase reduced infarct size in
rats subjected to middle cerebral artery occlusion. Attempts to demonstrate products of interaction of reactive oxygen species with tissue components have met
with irregular and limited success. For example, products of lipid peroxidation have not been found with uniformity after ischemia-reperfusion of the brain (21).
In the present research, we investigated the time
course of superoxide production after ischemia-reperfusion in cats, and we examined the role of superoxide and
its derivatives in the cerebral vascular changes that occur after ischemia of the brain.
METHODS
Animal preparation.
Experiments
were conducted on cats
anesthetized
with pentobarbital
sodium (30 mg/kg iv). After
tracheostomy,
each animal was ventilated with a positive pressure respirator. End-tidal CO, was measured continuously
with
an infrared CO, analyzer and was maintained
at a constant level
of -30 mmHg. After operative procedures were completed, the
animals received 5 mg/kg gallamine triethiodide
intravenously
for skeletal muscle paralysis. The left subclavian artery was
ligated, and an occluder was placed around the brachiocephalic
artery. Large-bore (PE-240) tubing was placed in the abdominal
aorta for subsequent phlebotomy.
Arterial blood pressure was
measured with a Statham transducer connected to a cannula
introduced into the aorta via the femoral artery. Arterial blood
samples were taken periodically
for arterial blood gas and pH
measurements
using a Corning blood gas analyzer. Hematocrit
was measured with a micromethod.
Single or double cranial windows were implanted
over the
parietal cortex as described in detail previously
(12). When
double windows were used, rubber 0 rings were placed between
the dura and skull to prevent mixing of the contents between
window chambers. The space under the window was filled with
artificial cerebrospinal
fluid (CSF) identical in composition
to
endogenous CSF of cats. Each window had ports for superfusion
of reagents and for monitoring
intracranial
pressure. In experiments in which vessel caliber was measured, we observed several small and large arterioles (smaller or larger than 100 pm,
respectively). Arteriolar
diameter was measured with a Vickers
image-splitting
device attached to a Wild dissecting microscope.
Superoxide measurements. Superoxide production
was measured as the SOD-inhibitable
rate of NBT reduction
as described in detail previously (8). During the period of interest, a
2.4 mM solution
of NBT was placed under both cranial windows. SOD (60 U/ml; 3,000 U/mg protein from bovine blood)
was placed under one window during the same period. At the
end of the assay period, the reagents were flushed from the
window with fresh CSF. The brain then was perfused via both
carotid arteries, first with 500 ml of heparinized
0.9% sodium
chloride solution and then by 500 ml of a freshly prepared
mixture of 2.5% glutaraldehyde
and 2% paraformaldehyde
in 0.1
M phosphate buffer.
The amounts of reduced NBT were determined after pyridine
0 1992 the American
Physiological
Society
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Nelson,
Charles
W., Enoch
P. Wei, John T. Povlishock,
Hermes A. Kontos,
and Michael
A. Moskowitz.
Oxygen radicals in cerebral ischemia. Am. J. Physiol. 263 (Heart
Circ. Physiol. 32): H1356-H1362,
1992.-Superoxide
production was measured as the superoxide dismutase (SOD)-inhibitable portion of nitro blue tetrazolium
(NBT) reduction after
cerebral ischemia-reperfusion
in anesthetized
cats equipped
with cranial windows.
Significant
superoxide production
was
found in the early reperfusion period and continued for more
than 1 h after ischemia. Superoxide was not detected in control
animals not subjected to ischemia, during ischemia, and at 120
min of reperfusion. After ischemia, the vasoconstrictor
response
to arterial hypocapnia was reduced. This effect was prevented
by pretreatment
with SOD plus catalase or by deferoxamine.
The response to topical acetylcholine was converted to vasoconstriction after ischemia. The normal vasodilator response reappeared spontaneously at 120 min of reperfusion. The vasodilator
response to acetylcholine
was preserved in animals pretreated
with SOD plus catalase. Blood-brain
barrier permeability
to
labeled albumin and horseradish peroxidase was increased after
ischemia. These effects were minimized
by pretreatment
with
SOD and catalase. We conclude that superoxide generation occurs during reperfusion after cerebral ischemia for a fairly long
period and that superoxide and its derivatives are responsible at
least in part for the vasodilation and the abnormal reactivity as
well as for the increase in blood-brain
barrier permeability
to
macromolecules
seen after ischemia. Furthermore,
the findings
suggest that the agent responsible for the vascular abnormalities
is hydroxyl radical generated via the iron-catalyzed
HaberWeiss reaction.
cerebral microcirculation;
hydroxyl radical; blood-brain
barrier;
endothelium-dependent
vasodilation
OXYGEN
RADICALS
IN CEREBRAL
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determined,
15 min of global complete ischemia was induced.
Vessel responses to acetylcholine
were measured again 30, 45,
60, 75, 90, and 120 min after the onset of reperfusion.
The effect of oxygen radical scavengers on arteriolar
responses to acetylcholine
after ischemia-reperfusion
was studied
in five cats equipped with double cranial windows. In seven cats,
one window was pretreated with SOD (60 U/ml) plus catalase
(40 U/ml) while the other window was left untreated. Responses
to topical acetylcholine
( 10e7 M) were determined before and 60
min after the onset of reperfusion
after a 15-min period of
complete global ischemia.
The effect of oxygen radical scavenging agents on arteriolar
responses to hypocapnia after ischemia-reperfusion
was studied
in 12 cats equipped with double cranial windows. One window
was pretreated with SOD (60 U/ml) plus catalase (40 U/ml).
Responses to hypocapnia were measured before and at 60 min of
reperfusion after complete global ischemia. Hypocapnia was induced by hyperventilation
via increasing the volume and rate of
the respirator.
In five cats we studied the same responses to
hypocapnia except that, instead of pretreatment
with SOD and
catalase, we pretreated one window with deferoxamine
(1 mM)
to scavenge iron and thereby inhibit the generation of hydroxyl
radical via the iron-catalyzed
Haber-Weiss
reaction.
We also studied the effect of ischemia-reperfusion
on the
permeability
of the blood-brain
barrier to proteins by using two
methods. The first method determined the permeability
of the
blood-brain
barrier to human plasma albumin (mol wt 70,000)
labeled with lZr,I . Ten minutes before testing, the animal received 50 &i of labeled albumin. At the end of the experiment,
the blood containing the radioactive albumin was removed from
the cerebral vessels by perfusion of the brain transcardially,
first
with 0.9% sodium chloride solution and then with fixative consisting of 2.5% glutaraldehyde,
2% paraformaldehyde,
and 0.1
M phosphate buffer as described above. This technique reliably
eliminated
all blood from the blood vessels. Fixation permitted
cutting the brain samples in precise fashion to measure radioactivity in the desired thickness of the cerebral cortex. Appropriate samples of the brain then were cut and weighed, and their
radioactivity
was determined
in an LKB 1282 gamma counter.
Blood samples were drawn immediately before the onset of reperfusion and 1 h later. A permeability
index was calculated by
expressing the concentration
of labeled albumin per unit weight
of brain as a percent of the mean concentration
of radioactive
albumin in blood. The effectiveness of perfusion in eliminating
blood from the cerebral vessels was verified by examination
of
the brain surface. In a properly perfused preparation,
the vessels
were no longer visible because of the absence of red blood cells.
Incomplete
perfusion resulted in retention of red blood cells,
which rendered the vessels visible by virtue of the presence of
hemoglobin.
The concentration
of labeled albumin was measured in a 2-mm surface layer of brain in each of two cranial
windows. One window was pretreated with SOD (60 U/ml) plus
catalase (40 U/ml) before the induction
of a 15-min period of
complete global ischemia while the other window was left untreated. We also measured the extravasation
of labeled albumin
in animals that underwent
the same operative procedures but
were not subjected to ischemia and therefore served as controls.
A second technique was used to measure the permeability
of
the blood-brain
barrier to horseradish
peroxidase
(mol wt
40,000). The animals received 50 mg/kg of horseradish peroxidase intravenously
(Sigma type VI) 5 min before the experimental interventions
were begun. This type of peroxidase
causes no significant change in the animal’s arterial blood pressure and no alteration
in the caliber of pial vessels. After the
experimental
interventions
were performed, the animal’s head
was perfused with fixative as described above to eliminate the
blood from the cerebral vessels and to fix the brain. The brain
was removed and the cortex was serially sectioned at a thickness
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extraction of brain homogenates or by surface spectrophotometry using a two-wavelength
technique described in detail previously (8). The surface spectrophotometric
measurements were
calibrated by comparing the results with direct chemical determination
of the reduced NBT after extraction
with pyridine.
The two methods yielded identical results.
Pyridine extraction involved first removing a cylindrical core
of tissue from under each cranial window and from under another part of each fixed brain. A 2-mm-thick
surface section
from each sample then was homogenized
in 0.1% sodium hydroxide and 1% sodium dodecyl sulfate (SDS). The homogenate
then was centrifuged for 20 min at 20,000 g, and the pellet was
removed and resuspended
in 4 ml of pyridine.
After 1 h of
heating at 85”C, the samples were recentrifuged
for 10 min at
10,000 g. The absorbance at 515 nm then was measured with a
Beckman spectrophotometer
to analyze samples from under
each window and control areas. We used an extinction
coefficient of 17.9 x 1O’j M-l cm-’ to calculate the amount of reduced NBT. By subtracting the amount of reduced NBT found
in the presence of SOD from the amount reduced in the absence
of SOD, the rate of SOD-inhibitable
NBT reduction was calculated. The results are expressed in nanomoles per minute per
liter assuming a constant rate of NBT deposition
throughout
the assay period.
Comparisons
between groups were made using analysis of
variance followed by t tests modified for multiple comparisons.
Ischemia-reperfusion
protocol. Before the induction
of complete global cerebral ischemia, the animals were given a constant
intravenous
infusion of 1 mM ATP solution titrated to lower
mean arterial blood pressure to 70 mmHg. The cannula in the
abdominal
aorta was connected to a reservoir that could be
raised or lowered after ischemia induction to keep arterial blood
pressure at 70 mmHg while the ATP infusion was continued as
above until controlled bleeding was sufficient by itself to control
arterial blood pressure. When the ischemic period was completed, the animal’s blood was reinfused at rates regulated to
keep mean arterial blood pressure below 130 mmHg. Ischemia
was induced by completely occluding the brachiocephalic
artery.
This maneuver typically resulted in reflex hypertension
that
was attenuated with controlled bleeding and ATP infusion. The
completeness of ischemia was verified visually by noting that
blood flow in pial vessels ceased completely or that vessels emptied completely
of blood and the brain surface became pale
without visible blood vessels. Ischemic time was measured after
complete ischemia was established. This occurred usually -5
min after arterial occlusion. In four cats we measured blood flow
with radioactive microspheres
and verified that blood flow in
the brain under the cranial window was zero during the period
of ischemia.
Experimental
design. We measured superoxide production
in
the following groups of cats: 1) during complete ischemia; 2)
during reperfusion at O-15 min after complete ischemia; 3) during reperfusion at 60-75 min after complete ischemia; 4) during
reperfusion at 120-135 min after complete ischemia; 5) during
reperfusion at O-15 min after incomplete
ischemia. This was
accomplished using the same protocol as for complete ischemia,
except that no attempt was made to control the blood pressure.
Under these conditions,
there was intermittent
or continuous
sluggish flow through the vessels under the cranial window.
Superoxide production
also was measured in control animals
subjected to the same operative procedures but without
the
induction
of ischemia.
We conducted the following experiments
to determine
the
effects of oxygen radicals on postischemic arteriolar
reactivity.
We studied the time course of endothelium-dependent
vasodilation from acetylcholine
after ischemia-reperfusion
in eight
cats equipped with single cranial windows. After control responses to topical application
of acetylcholine
(low7 M) were
ISCHEMIA
H1358
OXYGEN
RADICALS
IN CEREBRAL
ISCHEMIA
of 40 pm on a Vibratome.
The sections were reacted for light
microscopic
visualization
of the protein
reaction
product
through the use of the cobalt glucose oxidase technique
(8).
Next the sections were mounted on glass slides, dehydrated, and
cleared for microscopic analysis. Corresponding
anatomic areas
from oxygen radical scavenger-treated
and untreated windows
were compared by a blinded neuroanatomist
to assess qualitatively the intensity and localization
of the peroxidase reaction
product.
RESULTS
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ISCHEMIA
REPERFUSION
o-15
60-75
(MN)
120-135
Fig. 1. Superoxide
production,
measured
as superoxide
dismutase
(SOD)-inhibitable
reduction
of nitro blue tetrazolium
(NBT),
in control
animals not subjected to complete
ischemia
(n. = 4), during ischemia
(n
= 4), and at O-15 (n = 6), 60-75 (n = 7), and 120-135
(n = 6) min after
complete
ischemia.
Columns,
means k SE of NBT reduction
with and
without
SOD and difference
between
them. Difference,
SOD-inhibitable NBT reduction
and a measure
of superoxide
production.
No
superoxide
was detected under control conditions,
during ischemia,
and
late in reperfusion
period
(120-135
min). Significant
superoxide
production (P < 0.05) was found at O-15 and 60-75 min during reperfusion
after ischemia.
o0
30
TIME AFTER
Fig. 2. Arteriolar
diameter
after complete
ischemia.
chemic diameters
(means
significantly
dilated (P <
line diameters
from which
60
REPERFUSION
120
90
(min)
changes at various times during reperfusion
Values are %change
compared
with preis75 vessels). Vessels are
t SE, n = 11 animals,
0.001) throughout
reperfusion
period. Base%changes were calculated
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Fig. 3. Effect of treatment
with SOD + catalase or with deferoxamine
on
change in arteriolar
diameter
after complete
ischemia.
Results are
%change
measured
60 min after ischemia
compared
with preischemic
values (means + SE; SOD + catalase: n = 7 animals,
42 vessels; deferoxamine:
n = 5 animals,
36 vessels). Treatment
with SOD + catalase or
with deferoxamine
resulted in significantly
reduced vasodilation
(P <
0.001) after ischemia
compared
with untreated
vessels. Baseline diameters from which %changes were calculated
are displayed
above columns
(means k SE).
Figure 4 shows the response to topical acetylcholine
before and during the period of reperfusion after complete
ischemia. The normal vasodilator response to acetylcholine seen before ischemia was converted to a vasoconstriction during the early phase of reperfusion. This abnormal
vasoconstrictor response persisted until 60 min of reperfusion when the response to acetylcholine reverted back
to vasodilation. At the end of the observation period of 2
h the response was vasodilator, but it was significantly
less than that seen before ischemia.
Figure 5 shows that at 60 min of reperfusion after complete ischemia, the response to acetylcholine was vasoconstrictor in the untreated window, whereas pretreatment with SOD and catalase resulted in a retained
vasodilator response to acetylcholine.
Figure 6 shows that the vasoconstrictor responses to
hypocapnia were significantly reduced at 60 min of reperfusion after complete ischemia and that this effect was
inhibited by pretreatment
with SOD plus catalase or by
deferoxamine.
Figure 7 shows that the permeability
index to albumin
increased considerably in animals subjected to ischemiareperfusion without pretreatment.
Pretreatment
with
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Superoxide production. Figure 1 shows that superoxide
generation was not seen in control animals not subjected
to ischemia, during the period of complete ischemia, and
120-135 min of reperfusion
after complete ischemia.
There was significant superoxide production at O-15 and
60-75 min of reperfusion after complete ischemia. Superoxide production was much greater in the early part of
reperfusion than later on. Thus superoxide production
after ischemia was time dependent. NBT reduction rates
in nanomoles per minute per liter during O-15 min of
reperfusion after incomplete
ischemia were as follows:
NBT alone, 5.42 t 0.86; NBT plus SOD, 3.80 t 0.61;
giving a rate of SOD-inhibitable
NBT reduction of 1.62 t
0.75. The latter was significantly
lower than that seen
during the corresponding period after complete ischemia.
Vascular caliber and reactivity. Figure 2 shows the
changes in arteriolar caliber observed during the period of
reperfusion after complete ischemia. Marked vasodilation, seen in the early period of reperfusion, became progressively less pronounced. However, at the end of the 2-h
observation, the cerebral arterioles were still significantly
dilated when compared with their caliber before ischemia.
Figure 3 shows that the dilation of cerebral arterioles at
60 min of reperfusion after complete ischemia was significantly reduced in the windows pretreated with SOD plus
catalase or with deferoxamine when compared with the
untreated side.
OXYGEN
RADICALS
IN CEREBRAL
H1359
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deferoxamine
Fig. 6. Effect of treatment
with SOD + catalase or with deferoxamine
on
change in arteriolar
diameter
in response to hyperventilation
before and
after ischemia.
Values are %change
from diameters
immediately
before
hyperventilation
(means t SE; SOD + catalase:
n = 7 animals,
42
vessels; deferoxamine:
n = 5 animals,
36 vessels). Treatment
has no
effect on vasoconstrictor
response to hyperventilation
before ischemia.
After ischemia,
treated vessels responded
normally
to hyperventilation,
whereas untreated
vessels had a significantly
(P < 0.001) attenuated
response. Baseline diameters
from which %changes
were calculated
are
displayed
above columns
(means t SE).
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untreated
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+
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untreated
sod
+
catalase
Fig. 5. Effect of pretreatment
with SOD + catalase on change in arteriolar diameter
in response to topical
10ey M acetylcholine
application
at 60 min of reperfusion
after complete
ischemia.
Results are %change
compared
with diameter
immediately
before acetylcholine
application
(means k SE; n = 5 animals,
28 vessels). SOD + catalase had no effect
on acetylcholine
responses
before ischemia.
Untreated
vessels constricted in response to acetylcholine
application
after ischemia.
Treated
vessels dilated in response to acetylcholine
after ischemia,
but response
was attenuated
when compared
with preischemic
responses
(see also
Fig. 4). Baseline
diameters
from which %changes
were calculated
are
displayed
above columns
(means 2 SE).
SOD and catalase reduced this effect but did not eliminate it.
Extravasation of horseradish peroxidase was observed
consistently within the brain parenchyma underlying the
cranial windows in all animals subjected to ischemiareperfusion. The altered cerebral vascular permeability
was seen in relation to the crest of the gyri as well as in
the deeper brain structures. Oxygen radical scavengers
did not completely prevent the permeability change
induced by ischemia-reperfusion but reduced it significantly (Fig. 8). The brain parenchyma exposed to topically applied SOD plus catalase showed easily recognizable reduction in permeability when compared with the
ISCHEMIA
+
SOD +
CATALASE
ISCHEMIA
UNTREATED
Fig. 7. Effect of treatment
with SOD + catalase on changes in permeability
to albumin
after ischemia
(n = 5 per each group).
Values are
means +: SE of permeability
index, which is ratio of radiolabeled
albumin content
in fixed brain to that in whole blood (x100)
measured
60
min after ischemia or in control period. Treatment
with SOD + catalase
reduced but did not completely
prevent
increase in permeability
index
seen after ischemia.
untreated side. SOD alone (60 U/ml) had a consistent but
small effect on the increased permeability induced by
ischemia-reperfusion. SOD in a concentration of 120
U/ml had a more pronounced effect. Catalase (80 U/ml)
alone had no detectable effect on the increased permeability from ischemia-reperfusion.
DISCUSSION
The important findings of these experiments are as
follows. 1) Superoxide generation occurs during the period of reperfusion after either complete or incomplete
cerebral ischemia. There is no superoxide production under resting conditions in the absence of ischemia or during the period of complete cerebral ischemia. Superoxide
generation is much greater after complete rather than
incomplete ischemia, and superoxide generation in the
former condition lasts for >l h. 2) The cerebral arteriolar
changes induced by ischemia-reperfusion are characterized by sustained dilation, reduced responsiveness to the
vasoconstrictor effects of arterial hypocapnia, reversal of
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Fig. 4. Change in arteriolar
diameter
caused by topical
lop7 M acetylcholine application
before ischemia
and at various times during reperfusion after complete
ischemia.
Results are %change
compared
with
diameters
immediately
before acetylcholine
application
(means t SE; n
= 8 animals,
45 vessels). Acetylcholine-induced
vasoconstriction
occurred during first 60 min of reperfusion.
Significant
vasodilation
occurred with acetylcholine
after 120 min of reperfusion
but was reduced
when compared
with response seen before ischemia
(P c 0.005). Baseline diameters
from which
%changes
were calculated
are displayed
above or beside columns
(means t SE).
25
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Fig. 8. These light micrographs demonstrate altered cerebral vascular permeability to horseradish peroxidase occurring
after ischemia with reperfusion in presence of various concentrations of SOD and/or catalase. In A, note that combined
use of 120 U/ml SOD + 80 U/ml catalase reduced overall cortical extravasation of peroxidase (*) compared with
untreated controls (B, arrows). Note that in both control and treated hemispheres, permeability change deep within
brain parenchyma (arrowheads) is unaffected by SOD-catalase treatment. This most likely reflects inability of employed
agents to diffuse rapidly to deep cortical sites. With use of 120 U/ml SOD, a marked reduction in altered cerebrovascular
permeability to horseradish peroxidase (C, *) is seen compared with control hemisphere (D, arrows). Note again that use
of this superoxide anion scavenger did not affect permeability change deep within cortex.
OXYGEN
RADICALS
IN CEREBRAL
H1361
polypeptides from sensory fibers is involved (19). It is also
likely that adenosine may contribute because its concentration during ischemia rises markedly (23).
The time course of the abnormal response to acetylcholine correlates with that of the superoxide production,
suggesting that the two are causally related. This is further supported by the inhibition of the abnormality
after
pretreatment
with SOD and catalase. It is well known
that oxygen radicals attack directly and inactivate the
endothelium-derived
relaxing factor from acetylcholine
(3, 9, 18). We believe this is the mechanism involved in
view of the spontaneous
return of the vasodilator
response to acetylcholine
2 h after reperfusion.
Had the
mechanism been irreversible
damage of the endothelium,
we would have expected that this would not have occurred. From the practical standpoint,
the response to
acetylcholine
may be used to gauge the time course of
superoxide production during reperfusion after ischemia.
It is likely that the increase in baseline vascular caliber
caused by oxygen radicals also may have contributed
to
the reduction in the vasodilator
responses to acetylcholine by nonspecific
mechanisms.
However,
this factor
cannot fully explain the findings because it cannot explain the reversal of the response from vasodilator
to
vasoconstrictor.
Other investigators
have noted inconsistent
responses
to acetylcholine
application after cerebral ischemia-reperfusion in cats (15). They started measuring responses
after 60 min of reperfusion
and saw both dilation and
constriction.
Our data show that 60 min is approximately
the time that responses return toward normal; therefore
heterogeneous
findings would not be unexpected. Their
period of ischemia was shorter than in our studies, which
would affect the time course to recovery, because the
amount of reperfusion injury is related to the intensity of
initial ischemia.
The response to hypocapnia in large cerebral vessels is
not endothelium dependent (20). It would appear, therefore, that the inhibition
of the vasoconstrictor
response
to hypocapnia observed in the present experiments
after
ischemia is due to effects of oxygen radicals directly on
vascular smooth muscle. Because this effect was inhibited
by both SOD plus catalase and by deferoxamine,
we attributed it to the direct effects of hydroxyl radical. This
radical is very reactive and short-lived.
Consequently,
it
does not survive more than a few molecular diameters
from its site of formation.
Because it is accessible to
deferoxamine,
SOD and catalase, which are unlikely to
enter into the interior of cells easily, it is likely that its
action is on the cell membrane.
It has been reported by others that oxygen radicals can
damage membrane enzyme systems and affect ion fluxes
leading to permeability
changes after different types of
cerebral injury (14, 16). Our findings show that the increased permeability
of the blood-brain
barrier to proteins during reperfusion
after ischemia is mediated by
oxygen radicals. The precise mechanism
by which this
occurs was not identified. It appears from the use of different scavengers that superoxide is the agent immediately responsible for the increased permeability
because
SOD, but not catalase alone, was effective in minimizing
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the endothelium-dependent
vasodilator
response to acetylcholine, and disruption
of the blood-brain
barrier to
proteins. These changes are induced by oxygen radicals
because they are inhibited by pretreatment
with specific
scavengers. In the case of the sustained vasodilation
and
the abnormal response to hypocapnia after ischemia-reperfusion, we identified hydroxyl radical as the agent responsible for the abnormalities.
Unlike what occurs in hearts in which the production
of oxygen radicals during reperfusion
after ischemia is
brief (lo), in the in vivo blood-perfused
brain superoxide
production is prolonged, lasting for >l h. This is similar
to what is seen in other experimental
pathophysiological
conditions
such as acute hypertension
and fluid-percussion brain injury, in which superoxide production
lasts
for at least 1 h after the insult (7, 22). The prolonged
generation of superoxide
in ischemia-reperfusion
may
have practical significance because it affords a better opportunity
for therapeutic
intervention.
The sites of generation of superoxide and its enzymatic
sources were not investigated
in this study. In another
investigation,
we used a histochemical
technique to localize superoxide in the same model used in the present
experiments
(5). We found that the production
of superoxide occurred in endothelial and smooth muscle cells of
the cerebral arterioles, and we also localized superoxide in
the extracellular
space in proximity
to the blood vessels.
We showed earlier that superoxide escapes into the extracellular space from the ceils of the vessel wall via the
anion channel (8). In this histochemical
investigation
(5)
we found no superoxide production
in the brain parenchyma, but its absence there may be due to incomplete
penetration of the reagents that were applied topically on
the brain surface.
The enzymatic sources of superoxide during reperfusion after brain ischemia have not been identified. As
noted at the outset of this article, the available evidence
with respect to xanthine oxidase is conflicting. Armstead
et al. (1) found that indomethacin
inhibited superoxide
production during the early part of reperfusion after ischemia in newborn pigs. Therefore they concluded that cyclooxygenase
was the source of superoxide.
We have
shown previously that cyclooxygenase
generates superoxide in vitro (8, 11). It should be noted, however, that the
rate of SOD-inhibitable
reduction of NBT reported by
Armstead et al. (1) is -100 times greater than what we
found.
The microvascular
abnormalities
seen during reperfusion after complete ischemia, including sustained arteriolar dilation, abnormal reactivity to hypocapnia, reversal
of the endothelium-dependent
vasodilation
response to
acetylcholine
and increased permeability
of the bloodbrain barrier to proteins, are clearly due to the generation
of oxygen radicals because they were inhibited strongly by
pretreatment
with oxygen radical scavengers. We did not
investigate the role of radicals in the more pronounced
reactive dilation that occurs in the earlier phases of reperfusion. Although the time course of the dilation is similar to the time course of generation of superoxide, suggesting a causal relationship,
it is likely that this earlier
dilation is multifactorial.
We found earlier that release of
ISCHEMIA
H1362
OXYGEN
RADICALS
IN CEREBRAL
the response. The increased permeability
to proteins coupled with the sustained arteriolar dilation, which should
lead to increased capillary pressure, would be expected to
contribute to the induction of edema with secondary adverse effects on brain parenchymal
function.
This research was supported
by National
Institutes
of Health Grants
HL-21851,
NS-19316,
NS-26361,
and HL-07537.
Address for reprint
requests: H. A. Kontos,
Box 662, MCV Station,
Medical
College of Virginia,
Richmond,
VA 23298.
Received
31 March
1992; accepted
in final
form
16 June
1992.
11. Kukreja,
R., H. A. Kontos,
M. L. Hess, and E. F. Ellis.
PGH
synthase
and lipoxygenase
generate
superoxide
in the presence of
NADH
or NADPH.
Circ. Res. 59: 612-619,
1986.
112. Levasseur,
J. E., E. P. Wei, A. J. Raper,
H. A. Kontos,
and
J. L. Patterson,
Jr. Detailed
description
of a cranial window
technique
for acute and chronic
experiments.
Stroke 6: 308-317,
1975.
13. Liu, T. A., J. S. Beckman,
B. A. Freeman,
E. L. Hogan,
and
C. Y. Hsu. Polyethylene
glycol-conjugated
superoxide
dismutase
and catalase
reduce ischemic
brain injury.
Am. J. Physiol.
256
(Heart
Circ. Physiol.
25): H589-H593,
1989.
14. Lo, W. D., and A. L. Betz.
Oxygen
free-radical
reduction
of
brain capillary
rubidium
uptake. J. Neurochem.
46: 394-397,
1986.
W. G., S. M. Amundsen,
F. M. Faraci,
and D. D.
15. Mayhan,
Heistad.
Responses
of cerebral
arteries
after ischemia
and reperfusion in cats. Am. J. Physiol. 255 (Heart
Circ. Physiol. 24): H879H884, 1988.
16. Palmer,
G. C. Free radicals
generated
by xanthine
oxidase-hypoxanthine
damage adenylate
cyclase and ATPase
in gerbil cerebral cortex.
Metab.
Brain Dis. 2: 243-257,
1987.
17. Patt,
A., A. H. Harken,
L. K. Burton,
T. C. Rodell,
D.
Piermattei,
W. J. Schorr,
N. B. Parker,
E. M. Berger,
I. R.
Horesh,
L. S. Terada,
S. L. Linas,
J. C. Cheronis,
and J. E.
Repine.
Xanthine
oxidase-derived
hydrogen
peroxide
contributes
to ischemia
reperfusion-induced
edema in gerbil brains. J. Clin.
Invest.
81: 1556-1562,
1988.
G. M., and P. M. Vanhoutte.
Superoxide
anions and
18. Rubanyi,
hyperoxia
inactivate
endothelium-derived
relaxing
factor. Am. J.
Physiol.
250 (Heart
Circ. Physiol.
19): H822-H827,
1986.
19. Sakas,
D. E., M. A. Moskowitz,
E. P. Wei, H. A. Kontos,
M.
Kano,
and C. S. Ogilvy.
Trigeminovascular
fibers increase
blood flow in cortical
gray matter by axon reflex-like
mechanisms
during acute severe hypertension
or seizures.
Neurobiology
CPH.
86: 1401-1405,
1989.
20. Toda,
N., Y. Hatano,
and K. Mori.
Mechanisms
underlying
response
to hypercapnia
and bicarbonate
of isolated
dog cerebral
arteries. Am. J. Physiol. 257 (Heart Circ. Physiol. 26): H141-H146,
1989.
21. Watson,
B. D., R. Busto,
W. J. Goldberg,
M. Santiso,
S.
Yoshida,
and M. D. Ginsberg.
Lipid
peroxidation
in vivo induced by reversible
global ischemia
in rat brain. J. Neurochem.
42:
268-274,
1984.
22. Wei, E. P., H. A. Kontos,
C. W. Christman,
D. S. Dewitt,
and J. T. Povlishock.
Superoxide
generation
and reversal
of
acetylcholine-induced
cerebral
arteriolar
dilation
after acute hypertension.
Circ. Res. 57: 781-787,
1985.
23. Winn,
H. R., R. Rubio,
and R. M. Berne.
Brain
adenosine
production
in the rat during 60 seconds of ischemia.
Circ. Res. 45:
486-492,
1979.
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.1 on July 31, 2017
REFERENCES
1. Armstead,
W. M., R. Mirro,
D. W. Busija,
and C. W. Leffler. Postischemic
generation
of superoxide
anion by newborn
pig
brain. Am. J. Physiol.
255 (Heart
Circ. Physiol.
24): H401-H403,
1988.
2. Betz,
A. L., J. Randall,
and D. Martz.
Xanthine
oxidase is not
a major source of free radicals
in focal cerebral
ischemia.
Am. J.
Physiol.
260 (Heart
Circ. Physiol.
29): H563-H568,
1991.
3. Gryglewski,
R. J., R. M. J. Palmer,
and S. Moncada.
Superoxide
anion is involved
in the breakdown
of endothelium-derived vascular
relaxing
factor. Nature
Lond. 320: 454-456,
1986.
4. Itoh, K., A. Konishi,
S. Nomura,
N. Mizuno,
Y. Nakamura,
and T. Sugimoto.
Application
of coupled oxidation
reaction
to
electron
microscopic
demonstration
of horseradish
peroxidase:
cobalt-glucose
oxidase method.
Brain Res. 175: 341-346,
1979.
5. Kontos,
C. D., E. P. Wei, J. I. Williams,
H. A. Kontos,
and
J. T. Povlishock.
Cytochemical
detection
of superoxide
in cerebral inflammation
and ischemia.
Am. J. Physiol.
263 (Heart
Circ.
Physiol.
32): H1234-H1242,
1992.
6. Kontos,
H. A. Oxygen
radicals
in CNS damage.
Chem. Biol.
Interact.
72: 229-255,
1989.
7. Kontos,
H. A., and E. P. Wei. Superoxide
production
in experimental
brain injury.
J. Neurosurg.
64: 803-807,
1986.
8. Kontos,
H. A., E. P. Wei, E. F. Ellis,
L. W. Jenkins,
J. T.
Povlishock,
G. T. Rowe,
and M. L. Hess. Appearance
of superoxide
anion radical
in cerebral
extracellular
space during
increased prostaglandin
synthesis
in cats. Circ. Res. 57: 142-151,
1985.
9. Kontos,
H. A., E. P. Wei, J. T. Povlishock,
R. C. Kukreja,
and M. L. Hess. Inhibition
by arachidonate
of cerebral arteriolar
dilation
from acetylcholine.
Am. J. Physiol. 256 (Heart
Circ. Physiol. 25): H665-H671,
1989.
10. Kramer,
J. H., C. M. Arroyo,
B. F. Dickens,
and W. B.
Weglicki.
Spin-trapping
evidence
that graded myocardial
ischemia alters post-ischemic
superoxide
production.
Free Radical
Biol. Med. 3: 153-159,
1987.
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