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The Journal of Neuroscience, January 1, 1998, 18(1):205-213
Mitochondrial Susceptibility to Oxidative Stress Exacerbates
Cerebral Infarction That Follows Permanent Focal Cerebral Ischemia in
Mutant Mice with Manganese Superoxide Dismutase Deficiency
Kensuke
Murakami1,
Takeo
Kondo1,
Makoto
Kawase1, 2,
Yibing
Li1,
Shuzo
Sato1,
Sylvia F.
Chen1, and
Pak H.
Chan1, 2
1 CNS Injury and Edema Research Center, Departments of
Neurological Surgery and Neurology, University of California, School of
Medicine, San Francisco, California 94143-0651, and
2 Departments of Neurosurgery, Neurology, and Neurological
Sciences, Stanford University Medical Center, Stanford, California
94305-5784
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ABSTRACT |
Mitochondrial injury has been implicated in ischemic neuronal
injury. Mitochondria, producing adenosine triphosphate by virtue of
electron flow, have been shown to be both the sites of superoxide anion
(O2 ) production and the target of free
radical attacks. We evaluated these mechanisms in an in
vivo cerebral ischemia model, using mutant mice with a
heterozygous knock-out gene (Sod2  /+) encoding mitochondrial manganese superoxide dismutase (Mn-SOD).
Sod2 /+ mice demonstrated a prominent increase in
O2 production under normal
physiological conditions and in ischemia, as evidenced by specific
oxidation of a fluorescent probe, hydroethidine, reflecting decreased
activity of Mn-SOD. A mitochondrial viability assay that used rhodamine
123, which is accumulated by transmembrane potential of viable
mitochondria, demonstrated accelerated development of mitochondrial
injury. This rapid progress of ischemic injury resulted in exacerbation
of infarct size and hemisphere enlargement, causing advanced
neurological deficits but without altering DNA fragmentation induction.
The present study suggests that O2
overproduced in a mitochondrial compartment, when uncoupled from antioxidant defenses, induces impairment of mitochondrial function and
causes exacerbation of cerebral infarction after ischemia.
Key words:
cerebral ischemia; oxidative stress; manganese superoxide
dismutase; superoxide anion; mitochondrial injury; DNA
fragmentation
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INTRODUCTION |
Oxygen radicals have been implicated
in the pathogenesis of ischemic neuronal injury (Smith et al., 1984 ;
Kontos, 1985). These oxygen radicals induce lipid peroxidation, protein
oxidation, and DNA damage, causing both acute and chronic neuronal
injury. Among oxygen radicals, superoxide anion
(O2) plays a key role in the oxidative
chain reaction, producing a highly reactive oxidant. These free
radicals are processed by antioxidant enzymes and scavengers, including
superoxide dismutases (SODs), glutathione peroxidase, and catalase.
With the use of transgenic mice overexpressing copper-zinc SOD
(CuZn-SOD), it has been demonstrated that CuZn-SOD (SOD1), a cytosolic
antioxidant isoenzyme, plays a protective role in transient focal
ischemia-reperfusion and in traumatic brain injury (Kinouchi et al.,
1991; Yang et al., 1994; Mikawa et al., 1996), whereas cerebral
infarction after permanent focal ischemia is not reduced (Chan et al.,
1993). These results suggest that CuZn-SOD plays a protective role
mainly during reperfusion after ischemia. Furthermore, recent evidence
indicates that CuZn-SOD, as well as Bcl-2, has an inhibitory property
in the apoptotic process via an antioxidant pathway (Kane et al., 1993;
Greenlund et al., 1995; Kondo et al., 1997b).
Manganese SOD (Mn-SOD, SOD2) is an isoenzyme of SOD localized to the
mitochondria. The mitochondria provide ATP for cellular homeostasis,
function by means of oxidative phosphorylation and electron transport,
and are also the sites of O2
production in ischemia as well as under normal physiological conditions. In NMDA receptor-mediated excitotoxicity, which is an
important process of ischemic neuronal injury, mitochondrial production
of oxygen radicals has been demonstrated to be a key event, as well as
arachidonic cascade and the nitric oxide pathway (Dugan et al., 1995).
Despite the mitochondrial detoxification, overproduced oxygen radicals,
once uncoupled from this defense, induce oxidative stress. Mitochondria
also are suggested to be the targets of free radical attacks, which
impair ATP generation that causes energy depletion and increases free
radicals (Hillered and Chan, 1989; Coyle and Puttfarcken, 1993).
Knock-out mutant mice with target-disrupted Sod2 genes
encoding Mn-SOD have been established (Li et al., 1995b). We have
demonstrated that infarct size is greater in the knock-out mutants than
in the wild-type mice after mild focal ischemia-reperfusion but is not
different after moderate ischemia-reperfusion, despite early progression of ischemic injury (Mikawa et al., 1995; Chan et al., 1996;
Kondo et al., 1997a). We also have demonstrated that deficiency of
Mn-SOD triggers neuronal cell death via both necrosis and apoptosis after ischemia-reperfusion in the mutant knock-outs (Kondo et al.,
1997a). Although mitochondria are being recognized as the targets for
oxidative stress and their mode of dysfunction can affect the outcome
of cell death either by apoptosis or necrosis (Ankarcrona et al.,
1995), it is still unknown whether Mn-SOD is neuroprotective in
permanent ischemia without restoration of cerebral blood flow supplying
oxygen as a substrate for oxygen radicals.
The present study was designed to investigate the role of Mn-SOD and
mitochondrial injury in ischemia to clarify whether mitochondria with
decreased Mn-SOD activity could be vulnerable targets of O2 relative to normal
mitochondria.
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MATERIALS AND METHODS |
Permanent focal ischemia. The Sod2 gene
knock-out mutants (heterozygous) with CD1/SV129 background were
backcrossed with CD1 for at least five generations (F5), and littermate
wild-type mice with the identical genetic background as Sod2
heterozygous mice (3-month-old males, 35-40 gm) were subjected to
permanent focal cerebral ischemia. Focal ischemia was induced by
intraluminal middle cerebral artery (MCA) occlusion with a nylon
monofilament suture (Yang et al., 1994).
Animals were anesthetized with 2.0% isoflurane in 30%
O2/70% N2O. The rectal temperature was
controlled at 37 ± 0.5°C with a homeothermic blanket
(Homeothermic Blanket System, Harvard Apparatus, South Natick, MA) and
a heating pad. A skin incision was made on the midline of the ventral
neck. The left external carotid artery was exposed, and its branches
were electrocoagulated. An 11 mm 5-0 monofilament nylon suture
(Dermaron 1756-41, Devis & Geck, Manati, Puerto Rico) was introduced
from the left external carotid artery stump into the left internal
carotid artery. The skin incision was sutured. These surgical
procedures were performed within 10 min. After surgery, the animals
were taken care of in individual cages at 20°C.
To determine both the anatomical and physiological background of the
ischemia, we evaluated cerebral vasculature and change of regional
cerebral blood flow (rCBF) in both groups of animals. The vasculature
was evaluated by carbon black injection (K. Murakami T. Kondo, and P.H.
Chan, unpublished observations). After anesthesia with ketamine (200 mg/kg) and xylazine (10 mg/kg), the mice were killed by transcardial
perfusion with 200 ml of 10 U/ml heparin in saline and 200 ml of 3.7%
formaldehyde in PBS. Carbon black in an equal volume of 20% gelatin in
H2O was injected from the ascending aorta. The brain was
removed and fixed in 3.7% formaldehyde in PBS for 24 hr. The cerebral
vasculature was observed with a dissecting microscope (Stemi 2000C,
Zeiss, Oberkochen, Germany).
Change in rCBF was evaluated in both groups of animals with a laser
Doppler flowmeter (LASERFLO BPM2, Vasomedic, St.
Paul, MN). The laser Doppler probe was placed on the cranial window
(made by using a dental drill) above the MCA territory cortex (0.5 mm
posterior and 4 mm lateral from the bregma). The rCBF was monitored
continuously from 10 min before until 30 min after the ischemia
induction. Residual rCBF during MCA occlusion was calculated as
(ischemia rCBF/ pre-ischemia rCBF) × 100% and compared between
knock-out mutants and wild-type animals.
In situ detection of O2
production. The spatial production of
O2 in cerebral ischemia was
investigated by the method of in situ detection of oxidized
hydroethidine (HEt) (Bindokas et al., 1996; Kondo et al., 1997a). HEt
is oxidized to ethidium (Et) selectively by
O2, but not by other reactive oxygen
species such as hydrogen peroxide, hydroxyl radical, or peroxynitrite
(Bindokas et al., 1996). HEt solution (200 µl; stock solution of HEt,
100 mg/ml in dimethylsulfoxide, diluted to 1 mg/ml with PBS) was
administered intravenously 15 min before ischemia induction. In a
preliminary kinetic study after the fluorescence of HEt and Et, we have
determined that HEt rapidly disappeared in the serum and penetrated
into the brain shortly after intravenous injection, whereas Et remained
mainly in the serum. In the brain of intravenously HEt-injected
animals, fluorescence was assessed microscopically at Ex = 355 nm
and Em > 415 nm for HEt detection, or at Ex = 510-550 nm
and Em > 580 nm for Et detection. Animals were killed at 1 or 4 hr after the ischemia induction by transcardial perfusion as described
above. After post-fixation in 3.7% formaldehyde for 2 hr, brain
sections 50 µm in thickness at the level of the anterior commissure
were placed on a glass slide, using a vibratome. These sections were observed with a microscope (Axioplan, Zeiss) under fluorescent light
(HBO W/2, Zeiss). Photomicrographs of the fluorescent microscopy were
taken in both the ischemic and nonischemic hemispheres, and the
intensity and expression patterns of the oxidized HEt were observed and
compared between wild-type and knock-out mutant mice.
Evaluation of mitochondrial injury. To assess mitochondrial
viability in ischemic and nonischemic brain tissue, we used rhodamine 123 (Rh 123), a cell-permeant cationic fluorescent probe selectively accumulated by transmembrane potential in viable mitochondria. Animals
were perfused with ice-cold PBS and were killed at 4 and 24 hr after
ischemia. The brains were removed immediately after decapitation and
frozen in 2-methyl butane at 20°C. Frozen brain sections at the
level of the anterior commissure (20 µm in thickness) were used
quantitatively to evaluate and compare mitochondrial injury between the
wild-type and knock-out mutant mice. The frozen sections were dried at
room temperature and were incubated with 2.0 × 103 µM Rh 123 (Molecular Probes,
Eugene, OR) and with 2.5 × 103 µg/ml
Hoechst 33258 (Molecular Probes) in PBS for 15 min. After being fixed
in 3.7% formaldehyde in PBS for 15 min, the sections were rinsed in
ddH2O and mounted with glycerol. Furthermore, to confirm
that the mitochondrial accumulation of Rh 123 in fact is caused by the
mitochondrial transmembrane potential, we tested agents that have been
shown to disrupt the mitochondrial membrane potential, valinomycin
(potassium ionophore; Sigma, St. Louis, MO) and 2,4-dinitrophenol [DNP
(proton ionophore); Sigma], for their effects on the
probe-mitochondrial interaction (Johnson et al., 1981). The brain
sections were pretreated with 5 ng/ml valinomycin or 1.0 × 103 M DNP for 60 min at room
temperature before the incubation with Rh 123. The control sections
were incubated with PBS. Then the effects of these inhibitors were
determined.
We also determined whether brain slices under freezing conditions would
reduce the Rh 123 uptake. The animals were anesthetized deeply with
methoxyflurane and were perfused with ice-cold saline. The
animals (n = 6) were killed, and the brains were
removed and immersed in ice-cold PBS. Brain sections of 200 µM were cut in ice-cold PBS on glass slides and were
dried at room temperature for 5 min. The brain sections of three
animals were frozen at 80°C for 1 hr, and the slices from the other
one-half of the animals were kept in ice-cold PBS for the same amount
of time. Both the frozen and control sections were incubated with Rh
123 and Hoechst at the same concentrations as just described. Sections were fixed with 3.7% formaldehyde in PBS for 30 min, followed by a 5 min wash with ddH2O. The sections were mounted on slides with aqueous mounting medium.
The sections were observed with a microscope under fluorescent light.
Photomicrographs were taken with high-powered magnification (400×) in
the lateral caudoputamen of both the ischemic and nonischemic hemispheres. These photomicrographs were taken by double exposure to Rh
123 and Hoechst 33258. Total cells and Rh 123-positive cells were
quantified on the photomicrographs by a blinded investigator. The ratio
of Rh 123-positive cells to total cells was calculated and compared
between wild-type and knock-out mutant mice.
Evaluation of neurological deficits and histological outcome.
Neurological deficits of the experimental animals were graded on a
scale of 0-5 as described by Yang et al. (1994), with minor modification, before death at 24 hr. It also was confirmed whether the
dead mice died of cerebral ischemia or surgical complications such as
subarachnoid hemorrhage caused by the mechanical damage of blood
vessels by the inserted suture.
Frozen brain sections taken at 500 µm intervals by a cryostat were
stained with cresyl violet. The unstained area was measured on each
section by an image analysis system; then the infarct volume was
calculated by multiplying the unstained area by the distance (Swanson
et al., 1990; Swanson and Sharp, 1994). Hemisphere enlargement was also
calculated and expressed as ipsilateral hemisphere volume/contralateral
hemisphere volume × 100%.
In situ detection of DNA-fragmented cells. DNA
fragmentation after permanent focal cerebral ischemia was determined by
the TUNEL (terminal deoxynucleotidyl transferase-mediated uridine 5 -triphosphate-biotin nick end labeling) method in both groups of
animals. Frozen brain sections at the anterior commissure were dried at
room temperature and fixed with 3.7% formaldehyde for 45 min.
Endogenous peroxidase was inactivated with 60 mM hydrogen peroxide and 100 mM sodium azide for 30 min. After the
slides were washed with PBS, they were immersed in terminal
deoxynucleotidyl transferase buffer (Life Technologies, Gaithersburg,
MD) at room temperature for 15 min and incubated with terminal
deoxynucleotidyl transferase (Life Technologies) and
biotin-16-uridine-5-triphosphate (Boehringer Mannheim, Indianapolis,
IN) at 37°C for 60 min. The reaction was stopped by washing with 6 mM sodium citrate and 60 mM sodium chloride for
30 min. Then the slides were incubated with 2% bovine serum albumin in
PBS. After the slides were washed with PBS, the sections were incubated
with avidin-biotin-horseradish peroxidase (ABC kit, Vector
Laboratories, Burlingame, CA) for 30 min at room temperature, and
staining was visualized with 3 mM 3,3-diaminobenzidine
tetrahydrochloride and 18 mM hydrogen peroxide in PBS.
After being rinsed in ddH2O, the sections were stained with
methyl green for 10 min and then were dehydrated and mounted.
TUNEL-labeled cells were quantified with a light microscope. These
cells, displaying morphological features of apoptosis, were counted in
the regions of interest, including the medial caudoputamen, piriform
cortex, MCA territory cortex, border zone cortex, and lateral
caudoputamen, by a blinded investigator who used high-powered
magnification (400×). The number of TUNEL-labeled cells was expressed
as per millimeter squared in each region and compared between the
animals at 4 and 24 hr ischemia.
Statistical analysis. The grading score of neurological
deficit outcome was analyzed with nonparametric statistics of the Mann-Whitney U test. The statistical significance of
difference was evaluated by Student's t test in infarct
volume and hemisphere enlargement between wild-type and knock-out
mutant mice and by ANOVA in the evaluation of mitochondrial injury and
DNA fragmented cells between each time point and between mouse
groups.
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RESULTS |
Cerebral vasculature and reduction of rCBF in focal
cerebral ischemia
Because mice with different genotypes were used in the present
study, although both groups originated from the same CD-1 strain, the
cerebral vasculature and change of rCBF were analyzed to confirm that
both anatomic and physiological backgrounds were the same in this
study. Cerebral vasculature was determined by carbon black injection
(Fig. 1A). The method
used herein allowed us to determine the structure of major blood
vessels in the brain. There was no remarkable difference between
wild-type mice and knock-out mutants, not only in the structure of
Willis' circle but also in that of the MCA. The distribution of the
MCA trunk and branch appeared to be anatomically identical.
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Figure 1.
A, Photomicrographs showing
similarity in cerebral vasculature in wild-type (Wt) and
knock-out mutant mice (Sod2 /+), respectively. The
origins of the MCA and the other major blood vessels involved in the
Willis' circle were almost the same in both animals. The distribution
of the MCA territory was also similar between the two animals.
B, Residual rCBF of the MCA territory cortex measured by
laser Doppler flowmetry during MCA occlusion. The ischemia was induced
at the same level in both wild-type and knock-out mutant
mice.
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Change of rCBF was measured indirectly by laser Doppler flowmetry,
which showed a prominent reduction in rCBF in the ischemic cortex of
both animal groups. As shown in Figure 1B, the rCBF was reduced to 9.7 ± 2.6% and 8.0 ± 1.0% of the baseline
in wild-type and knock-out mutant mice, respectively, by MCA occlusion.
There was no statistically significant difference in residual rCBF
during ischemia between the two groups.
Production of O2 in
cerebral ischemia
Production of O2 was determined
by using HEt, a fluorescent dye oxidized to Et selectively by
O2 at 1 and 4 hr after ischemia
induction. Because the MCA was occluded permanently in the present
study, HEt was administered 15 min before MCA occlusion. This
fluorescent probe was distributed sufficiently, even to the brain
tissue of the ischemic area, where the blood vessels were occluded. As
previously observed by Kondo et al. (1997a),
O2 production was shown by oxidized
HEt signals as small particles in the cytosol, suggesting mitochondrial
production of O2. These signals were
increased in knock-out mutants as compared with wild-type mice in the
nonischemic area (Fig.
2A,B). Some cells that
are within the microvessel area showing the morphological characteristic of endothelial cells displayed strong signals under normal physiological conditions as well as in ischemia (Fig.
2C,D). At 1 hr after ischemia induction, however, although
the ischemic area showed a slightly increased mitochondrial expression
of oxidized HEt signals relative to the nonischemic hemisphere, the
difference was not marked between wild-type and knock-out mutant mice
at this time point (Fig. 2E,F). The cellular
and subcellular patterns of oxidized HEt signals appeared to be changed
at 4 hr after ischemia. Some brain cells exhibited diffuse cytosolic
expression of HEt fluorescent signals in the ischemic area (Fig.
2G,H). The cytosol was filled with the diffuse HEt
signals in these cells, and the shape of the cytosolic space, including
dendrites, was represented by the fluorescent signal. This cytosolic
pattern fluorescence of oxidized HEt was more intense and more
frequently identified in knock-out mutants than in wild-type mice.
Furthermore, these cells were observed more frequently in the cortex,
the cortical penumbra in particular, than in the caudoputamen.
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Figure 2.
Representative photomicrographs showing increased
production of O2 by the expression of
oxidized HEt in Sod2 /+ mouse brains. The basal level
of O2 was increased even under normal
physiological conditions in the knock-out mutant mice
(B) as compared with wild-type animals
(A), reflecting decreased activity of Mn-SOD.
Under normal physiological conditions, HEt signals were detected as
small particles in the cytosol, indicating mitochondrial production of
O2. These small particles were likely
to be increased and intense in knock-out mutants. Endothelial cells
demonstrated strong fluorescent signals rather than other cell
populations under both normal (C) and ischemic
conditions (D). E, F, Ischemic
area showing increased production of
O2 at 1 hr after ischemia induction.
In the ischemic brain tissue, including the cortex and caudoputamen,
these small red particles were increased slightly in both wild-type
animals (E) and knock-out mice
(F) at 1 hr. However, these HEt signals
were likely to indicate not a cytosolic, but a mitochondrial,
expression of O2, and the difference
was not remarkable between wild-type mice and knock-out mutants.
G, H, At 4 hr, although a great deal of HEt was supposed
to have been oxidized to Et, the intense and different subcellular
pattern of the HEt signal was observed. Some cells in the ischemic area
exhibited cytosolic expression of O2.
This fluorescent signal, which filled up the cytosolic space, displayed
the shape of those cells. Furthermore, these cells with a cytosolic
expression pattern of HEt were observed more frequently in the
Sod2 knock-out mutants (G) than in
the wild-type mice (H), particularly in
the penumbra area. Scale bars: 25 µm in A,
B; 100 µm in E-H; 10 µm in
C, D and the high magnification in
A, B, E-H.
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Mitochondrial injury was exacerbated in knock-out mutants
We evaluated the temporal change of Rh 123-accumulated cells to
determine whether decreased activity of Mn-SOD causes mitochondrial susceptibility in knock-out mutants. Examination of the mitochondrial viability assay revealed vesicular accumulation of the cell-permeant cationic compound, Rh 123, in the brain cells. To confirm that Rh 123 accumulated in the mitochondria by the remaining transmembrane potential, we tested two agents for their effect on the
probe-mitochondrial interaction. Pretreatment with these ionophores,
which decrease the transmembrane potential of mitochondria, diminished
this accumulation of Rh 123 as compared with the control that was
treated with PBS (Fig. 3A).
Small particles displayed by the accumulated Rh 123 were decreased by
preincubation with both inhibitors. Furthermore, the intensity of
vesicular fluorescent signals also was weakened by these agents,
although background fluorescence was increased slightly. These results
were observed both in the caudoputamen and in the cortex, suggesting
that the transmembrane potential is likely to remain even in frozen
brain tissue and that the small particles shown by the accumulated Rh
123 under fluorescence indicate the existence of viable mitochondria.
We also tested the effects of freezing on Rh 123 staining. The Rh
123-positive cells were 75 ± 10% and 69 ± 3%
(p > 0.05) for controls (without freezing) and
for frozen slices, respectively.
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Figure 3.
Evaluation of mitochondrial injury after
ischemia. A, Representative microphotographs showing
mitochondrial accumulation of the fluorescent probe, Rh 123, and the
effects of mitochondrial membrane ionophores DNP and valinomycin.
Top, middle, and bottom rows are photographs singly exposed to Rh 123, both doubly exposed to
Rh 123 and counterstained by Hoechst 33258, and with high-powered magnification, respectively. Small red particles represent the fluorescent signals of Rh 123 accumulated by the transmembrane potential of
mitochondria, suggesting the existence of viable mitochondria. This
accumulation of Rh 123 was diminished by pretreatment with ionophores
DNP and valinomycin. The vesicular fluorescent signals were observed to
be weakened and decreased by the preincubation of the ionophores. Scale
bars: 25 µm, top and middle; 10 µm,
bottom. B, Fluorescent photomicrographs
in both nonischemic (top) and ischemic hemispheres
(bottom) of wild-type (Wt;
left) and Sod2 knock-out mutant mice
(right). Note that, in both wild-type and knock-out
mutant mice, some cells were Rh 123-negative in the nonischemic area;
Rh 123-positive cells also were observed in the ischemic brain tissue.
Thus, not all of the cells without the small Rh 123 fluorescent
particles necessarily indicate a mitochondrial injury. Scale bar, 10 µm. C, Ratio of the number of Rh 123-positive cells to
that of total cells in the lateral caudoputamen of the ischemic
hemisphere, a possible ischemic core, in the present ischemia model at
4 and 24 hr ischemia. Values are mean ± SE;
*p < 0.05, **p < 0.01, and
***p < 0.001 versus control, ANOVA.
 p < 0.05 versus wild-type, ANOVA. At 4 hr
ischemia, the ratio of Rh 123-positive cells was inclined to decrease
as compared with the control value, but no significant difference was
observed between wild-type and knock-out mutant mice. However, these
cells decreased in both groups at 24 hr ischemia and were significantly less in knock-out mutants than in wild-type mice.
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Using the present method, we determined and compared mitochondrial
viability between wild-type and Sod2 knock-out mutant mice after ischemia. Ischemic cells demonstrated shrunken nuclei without an
intense fluorescent signal of Rh 123, showing ischemic change lacking
the mitochondrial membrane potential (Fig. 3B). These findings were not remarkably different between the animal groups. Figure 3C demonstrates the temporal pattern of mitochondrial
injury development after ischemia in wild-type and knock-out mice. The ratio of Rh 123-positive cells in the nonischemic hemisphere was 53.1 ± 11.7% in wild-type mice and 58.9 ± 11.3% in
Sod2 knock-out mice. There was no significant difference
between wild-type and knock-out mice in the nonischemic hemisphere. At
4 hr ischemia, a slight decrease of Rh 123-positive cells was observed;
the ratio was 45.3 ± 12.6% (n = 4) in wild-type
animals and 41.9 ± 9.8% (n = 4) in knock-out
mice. The decrease of Rh 123-positive cells was significant in
knock-out mice as compared with the control value
(p < 0.05, ANOVA). This ratio was decreased
remarkably at 24 hr: 35.5 ± 4.4 (n = 7) in
wild-type mice and 17.8 ± 5.2% (n = 8) in
knock-out mice. The ratio of Rh 123-positive cells to total cells was
decreased significantly in both wild-type and knock-out mice relative
to the control value (p < 0.01 in wild-type mice and p < 0.001 in knock-out mice, ANOVA), and
mitochondrial injury was exacerbated significantly more in the
knock-out mice than in the wild-type mice (p < 0.05, ANOVA).
Neurological deficit and histological damage were advanced in
knock-out mutants
Among the surviving wild-type and knock-out mutant mice, a
significant difference was not seen in neurological outcome at 24 hr
ischemia. Higher mortality was seen in the knock-out mice as compared
with the wild-type mice (12.5% in wild-type mice and 50.0% in
knock-out mice). Although it was not statistically significant, there
is a possibility that severely injured mice were already dead and thus
have been excluded from the neurological evaluation. Therefore,
autopsies were conducted to determine the cause of death; then the dead
mice that were confirmed to have no surgical complications such as
subarachnoid hemorrhage were assigned a score of 5 in the evaluation of
neurological deficits. Dead mice showed severe brain swelling, and
these findings are consistent with our previous data demonstrating that
correlations existed between hemispheric enlargement and neurological
deficits (Kondo et al., 1997a). The mean scores of wild-type and
knock-out mice were 2.4 (n = 8) and 3.8 (n = 16), respectively, and neurological outcome was
significantly exacerbated in the knock-out mice
(p < 0.05, Mann-Whitney U test;
Fig. 4).
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Figure 4.
Neurological deficit scores in wild-type
(Wt) and knock-out mutant mice (Sod2
/+) at 24 hr ischemia. Small circles and
bars are the scores of each animal and mean scores,
respectively. *p < 0.05, Mann-Whitney
U test. 0 = No observable neurological deficit, 1 = failed to extend right forepaw, 2 = circled to the right, 3 = fell to the right, 4 = could not walk spontaneously, and
5 = dead. Mean scores of knock-out mice were significantly higher than those of wild-type mice.
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We next evaluated both the infarct size and hemisphere enlargement at
24 hr ischemia. Figure 5A
shows the typical findings of histology in wild-type and knock-out mice
at 24 hr. The infarct area extended throughout the entire MCA
territory, including both the caudoputamen and cortex in wild-type and
knock-out mice. Infarct volume was 74.1 ± 27.4 mm3 (n = 7) in wild-type animals and
121.0 ± 31.2 mm3 (n = 8) in
knock-out mice and was significantly greater in knock-out than in
wild-type mice (p < 0.01, Student's
t test; Fig. 5B). Furthermore, as shown in Figure
5C, brain swelling, a possible index of ischemic brain
edema, was also more severe in knock-out than in wild-type mice
(p < 0.001, Student's t test).
Hemisphere enlargement was 17.9 ± 5.4% (n = 7)
in wild-type and 34.3 ± 7.8% (n = 8) in
knock-out mice.
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Figure 5.
Histological analysis after 24 hr of MCA
occlusion. A, Photomicrograph showing the histological
changes after 24 hr of MCA occlusion in wild-type (Wt)
and knock-out mutant mice (Sod2 /+). The infarct area
was localized in the caudoputamen and MCA territory cortex in both mice
groups. However, cortical infarction extended to the boundary zone of
the anterior cerebral artery territory, and brain swelling was
extremely severe in the knock-out mutant mice. Also shown are infarct
volume (B) and hemisphere enlargement (C) in wild-type and knock-out mutant mice at 24 hr ischemia. Values are mean ± SE; **p < 0.01 and ***p < 0.001, Student's t test. Cerebral infarction and hemisphere enlargement were significantly more severe in knock-out than in wild-type mice. Scale bar, 1 mm.
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DNA fragmentation is not altered by reduction of Mn-SOD in
permanent focal cerebral ischemia
To determine the role of Mn-SOD in DNA fragmentation after
ischemia, we evaluated the contribution of DNA fragmentation to ischemic neuronal injury with in situ TUNEL staining.
TUNEL-labeled cells were distributed mainly in the inner boundary
zones, such as the medial caudoputamen, at 24 hr ischemia in both
wild-type and knock-out mutant mice (Fig.
6A,B). These cells were
densely labeled in the nuclei and showed characteristic features of
morphology, nuclei condensation, cell shrinkage, and, occasionally,
fragmentation of nuclei, suggesting apoptotic cells (Fig.
6C,D). No morphological differences were observed between
the cells of knock-out mutants and wild-type mice. These TUNEL-labeled
cells were not observed in the nonischemic area. To determine the
temporal pattern and anatomical distribution of DNA fragmented cells in
ischemia, we counted TUNEL-labeled cells with the morphological
features mentioned above in the five regions at 4 and 24 hr (Fig.
6E). Quantitative analysis demonstrated that the DNA
fragmentation was induced as early as 4 hr and greatly increased at 24 hr and that the contribution of DNA-fragmented cells to the ischemic
injury was statistically the same level between wild-type and knock-out
mutant mice. At 4 hr ischemia, a few cells were labeled in most of the
regions, but not in the MCA area cortex of wild-type mice nor in the
lateral caudoputamen of knock-out mutant mice. These
TUNEL-labeled cells were dispersed throughout the entire ischemic
lesion at 24 hr but frequently were observed at the inner boundary
zone. These observations are consistent with previous reports (Li et
al., 1995a; Murakami et al., 1997). However, no significant difference was seen in any regions between wild-type and knock-out mutant mice at
24 hr.
View larger version (96K):
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|
Figure 6.
In situ detection of DNA
fragmentation after ischemia by TUNEL staining. Photomicrographs show
the distribution of DNA-fragmented cells labeled by TUNEL staining in
the inner boundary zone in wild-type (Wt)
(A) and knock-out mutant mice (Sod2
/+) (B). TUNEL-labeled DNA fragmented cells
were distributed mainly in the medial caudoputamen adjacent to the
inner boundary. C, D, High-magnification
photomicrographs showing morphological features of TUNEL-labeled cells.
These cells represent cell shrinkage, nuclei condensation, or
fragmentation (arrowhead). E, Temporal
profile of DNA fragmentation in each region of ischemic area, the
medial caudoputamen, piriform cortex, MCA territory cortex, penumbra
area cortex, and lateral caudoputamen. DNA fragmentation was induced as
early as 4 hr in both the wild-type and knock-out mutant mice. The
TUNEL-labeled cells were dispersed throughout the entire ischemic area
at 24 hr, particularly in the inner boundary zone. Quantitative
analysis demonstrated no statistically significant difference in the
induction of DNA fragmentation between wild-type and knock-out mice.
CC, Corpus callosum; LV, lateral
ventricle.
|
|
| |
DISCUSSION |
The brain is one of the most vulnerable organs to ischemia because
of its high energy demand for cellular function and its relatively
modest defense against secondary oxidative stress. Under normal
physiological conditions, brain mitochondria generate ATP by virtue of
effective electron transport in their respiratory chain for cellular
maintenance and integrity. The biochemical function of mitochondria
strongly depends on the membrane, which is one of the oxygen radical
target sites. Disturbance of mitochondrial membrane integrity causes
mitochondrial dysfunction, leading brain cells to energy failure.
Therefore, energy failure because of mitochondrial dysfunction is
likely to be an important factor in ischemic brain injury.
In the present study it was shown that the increased HEt oxidation, a
possible index of O2 production, was
increased in nonischemic areas as well as in ischemic areas in
Sod2 gene knock-out mutants, as compared with wild-type
mice, reflecting decreased activity of mitochondrial Mn-SOD. This
increased production of O2 was
remarkable in the penumbra cortex, where blood flow is reduced to a
critical level for neuronal cells to survive, but oxygen, as a
substrate for reactive oxygen radicals, continued to be supplied. Furthermore, the mitochondrial injury progressed, as evidenced by the
lack of transmembrane potential, and thus cerebral infarction and
neurological deficit outcome were exacerbated in knock-out mutant mice.
Therefore, these results suggest that mitochondria are the important
sites at which oxygen radicals are produced under both normal and
pathological conditions and that mitochondrial Mn-SOD plays a
protective role as an antioxidant enzyme of mitochondrially produced
O2 in cerebral ischemia.
However, caution must be taken when the results are interpreted.
Although HEt has been shown to be a highly selective compound for
superoxide radical reaction, it is unlikely to compete with Mn-SOD for
superoxide, because the latter reaction is extremely fast. The only
exception is that nitric oxide reacts with superoxide faster than
Mn-SOD, but it is unlikely that HEt, an artificial exogenous agent,
would react at the same rate as nitric oxide. However, it will be of
special interest and importance to determine the rate constant of the
HEt oxidation. On the other hand, HEt could be oxidized by oxidatively
damaged mitochondrial electron transport proteins such as succinate
dehydrogenase, the activity of which is known to be reduced in
Sod2 knock-out mutant mice (Li et al., 1995b).
In an in vivo study of cerebral ischemia, both the
anatomical and physiological backgrounds of ischemia are likely to be
important factors affecting the condition of ischemia. Recently,
variations of the plasticity of Willis' circle have been demonstrated
to affect the infarct size after focal ischemia among some strains of
mice (Barone et al., 1993). The analysis of cerebral vasculature showed
similar structures of Willis' circle and the MCA between the two
animal groups. However, because the anatomical analysis is neither
quantitative nor functional, the change in rCBF was investigated also.
No significant difference was seen between wild-type and knock-out
mutant mice, indicating that the ischemic condition induced by the
present method was, in fact, the same level and did not contribute to
the exacerbation of ischemic brain injury in Sod2 knock-out
mutant mice. On the basis that the ischemic condition was the same
level in both groups of animals, O2
production, mitochondrial injury, and the outcome of neurology and
histology, including the involvement of DNA fragmentation, were
evaluated to elucidate the role of Mn-SOD in permanent focal ischemia.
Because HEt is oxidized continuously to Et by both normally and
physiologically produced O2 and the
oxidization of HEt was likely to reach the maximum within several hours
after the injection, O2 production
could be evaluated only in the early phase of ischemia. Oxidized HEt
signals were seen as a small vesicular pattern under normal
physiological conditions, and these vesicular signals were increased in
knock-out mice even under normal physiological conditions. This finding
may indicate that the mitochondria are the important sites of
O2 production and that Mn-SOD plays an
important role as an antioxidant, even under normal
physiological conditions. However, considering that there was no
evidence of oxidative injury in the brains of heterozygous knock-out
mutants (Sod2 /+) such as that seen in the hearts of
homozygous knock-out mutants (Sod2 /) that died within
several postnatal days (Li et al., 1995b), this increase in
O2 production in knock-out mutant mice
may not exceed the toxic threshold for neuronal injury. An intense and
cytosolic signal of oxidized HEt was observed in some brain cells of
the ischemic cortex, particularly in the cortical penumbra. These
signals were observed more strongly and frequently in knock-out mutants
than in wild-type mice. We first applied this fluorescent probe to detect O2 production in in
vivo ischemia-reperfusion brain and observed that
O2 production greatly progresses in
the reperfusion state several hours after transient ischemia in mice
(Kondo et al., 1997a). In consideration of these findings in both
transient and permanent ischemia, O2
is likely to contribute to ischemic brain injury, but this might be
more prominent in the penumbra during both permanent ischemia and the
reperfusion state after transient ischemia when oxygen is effectively
supplied as a substrate for O2
production. The present findings suggest that relatively increased O2, which might be uncoupled from the
antioxidant defense mechanism of mitochondria in knock-out mutant mice
with decreased Mn-SOD, may exacerbate ischemic brain injury and result
in the extension of the infarct area, especially in the penumbra.
Mitochondrial viability assay, which was evaluated by the
integrity of mitochondrial membrane potential, demonstrated that the
decreased level of Mn-SOD causes mitochondrial susceptibility in
knock-out mutant mice. Rh 123 visualizes the mitochondria with normally
inner negative potential at the membrane, which is generated by the
electrochemical gradient of the proton (Johnson et al., 1981). Thus, Rh
123-negative cells are likely to be those that have lost mitochondrial
viability. Accumulated Rh 123 exhibited red fluorescence as small
vesicles around the nuclei, which were diminished by ionophores
disrupting the potential. Interestingly, these vesicular patterns in Rh
123 fluorescent signals were similar to those observed in mitochondrial
HEt expression. In ischemic brain tissue Rh 123-positive cells were
decreased more prominently in knock-out than in wild-type animals.
Neurological and histological analyses also demonstrated
significant exacerbation in knock-out mutants. Mortality was markedly high in knock-out mutant mice. Neurological deficits were exacerbated significantly in knock-out mutant mice relative to wild-type. Further,
decreased activity of Mn-SOD also exacerbated histological outcome.
Infarct volume and hemisphere enlargement were significantly larger in
knock-out than in wild-type mice. In both mice groups the infarct area
was observed in the entire MCA territory, including the caudoputamen
and the frontoparietal cortex in the MCA area, after 24 hr of permanent
MCA occlusion. However, because almost the whole caudoputamen was
involved in the infarct area in both mice groups, enlargement of the
infarct area might have resulted from the expansion of the infarct area
to the cortical penumbra in knock-out mutants.
A recent study has demonstrated that mitochondrial function is required
in the apoptotic process of neuronal death and that succession of the
two distinct pathways to neuronal death, apoptosis or necrosis, is
dependent on the preservation of mitochondrial function (Ankarcrona et
al., 1995). We have demonstrated that ischemia-reperfusion injury was
accelerated and that both necrosis and apoptosis contribute to
the early progression of ischemia-reperfusion injury in
Sod2 knock-out mutant mice (T. Kondo, K. Murakami, and P.H.
Chan, unpublished observations). Therefore, we determined whether
occurrence of apoptotic cell death in ischemia alone was altered in
knock-out mutant mice. DNA-fragmented cells labeled by TUNEL staining
were quantified cautiously in five regions of interest, as described by
Charriaut-Marlangue and Ben-Ari (1995), although whether TUNEL-labeled
cells are exactly apoptotic cells is still controversial. However,
although the distribution of DNA fragmentation was consistent with
previous demonstrations (Li et al., 1995a; Murakami et al., 1997), no
significant difference was seen between wild-type and knock-out mice in
any regions. This insignificance could be attributable to the transient
nature of apoptosis, which we might have missed during the course of the studies. This issue has been discussed in our previous studies involving CuZn-SOD knock-out mutant mice (Kondo et al., 1997b). We have
demonstrated that DNA fragmentation was induced less in permanent focal
ischemia than in transient focal ischemia followed by reperfusion and
that severe ischemia, such as that induced in permanent MCA occlusion,
might cause relatively increased necrotic cell death (Murakami et al.,
1997). Recent evidence has shown that relatively intense insults,
including a combination of hypoxia and substrate deprivation and
exposure to high concentrations of NMDA, incline neuronal cell death to
necrosis (Bonfoco et al., 1995; Copin et al., 1996). In the present
study permanent ischemia induces such an intense ischemic insult that a
significant difference could not be seen in the alternative apoptotic
pathways to neuronal cell death between wild-type and knock-out mutant
mice.
We have demonstrated previously that overexpressed CuZn-SOD plays
a protective role in transient focal ischemia followed by reperfusion,
but not in permanent focal ischemia (Chan et al., 1993; Yang et al.,
1994). In permanent focal ischemia, cerebral blood flow is reduced
severely to the level at which the ischemic brain yields to energy
depletion; thus, neurons in the ischemic core rapidly die, whereas
distant neurons in the penumbra remain viable. Therefore, an increased
or decreased level of SOD is likely to play a protective or critical
role, respectively, in the penumbra, but not in the ischemic region.
However, considering the results obtained from the permanent focal
ischemia studies conducted in both CuZn-SOD overexpressed transgenic
mice and in Sod2 knock-out mutant mice herein, brain tissue
in the penumbra might be exposed to very mild ischemia as compared with
the ischemic core in permanent focal ischemia. Thus, knock-out mice
brains might sustain oxidative injury by mitochondrially produced
O2, which would be detoxified
instantly in wild-type brains, causing exacerbation of cerebral
infarction after permanent focal ischemia. Another possibility for this
discrepancy between the CuZn overexpresser and the Mn-SOD knock-out
mutant mice after permanent focal cerebral ischemia may be attributable
to the compartmentalizing role of this enzyme when oxygen delivery is
cut off completely. It is likely that the mitochondrial compartment and
the level of Mn-SOD are relatively sensitive to the lack of oxygen
availability, which may lead to the increased ischemic injury.
Finally, we demonstrated that decreased Mn-SOD activity, in fact,
causes increased oxidative stress and/or injury in cerebral ischemia
and that mitochondria are both the sites of
O2 production and the targets of free
radical attacks. We conclude that mitochondrial Mn-SOD plays a
protective role as an antioxidant defense in ischemic neuronal
injury.
| |
FOOTNOTES |
Received June 2, 1997; revised Sept. 30, 1997; accepted Oct. 21, 1997.
The present study was supported by National Institutes of Health Grants
NS14543, NS25372, NO1 NS 5-2334, NS36147, and AG08938. We thank Dr.
Charles J. Epstein, Dr. Ting-Ting Huang, and Elaine Carlson for their
collaborative efforts in generating the mutant mice; Liza Reola and
Bernard Calagui for their technical assistance; and Cheryl Christensen
for her editorial assistance.
Correspondence should be addressed to Pak H. Chan, PhD, Neurosurgical
Laboratories, Stanford University, 701B Welch Road, #148, Palo Alto, CA
94304.
| |
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Top
Abstract
Introduction
