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Volume 17, Number 11,
Issue of June 1, 1997
pp. 4180-4189
Copyright ©1997 Society for Neuroscience
Reduction of CuZn-Superoxide Dismutase Activity Exacerbates
Neuronal Cell Injury and Edema Formation after Transient Focal Cerebral
Ischemia
Takeo Kondo1, 2,
Andrew
G. Reaume4,
Ting-Ting Huang3,
Elaine Carlson3,
Kensuke Murakami1, 2,
Sylvia F. Chen1, 2,
Eric K. Hoffman4,
Richard W. Scott4,
Charles J. Epstein3, and
Pak H. Chan1, 2
Departments of 1 Neurological Surgery,
2 Neurology, and 3 Pediatrics, University of
California School of Medicine, San Francisco, California 94143, and
4 Department of Molecular Biology, Cephalon, Incorporated,
West Chester, Pennsylvania 19380
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Apoptotic neuronal cell death has recently been associated with the
development of infarction after cerebral ischemia. In a variety of
studies, CuZn-superoxide dismutase (CuZn-SOD) has been shown to protect
the brain from ischemic injury. A possible role for CuZn-SOD-related
modulation of neuronal viability is suggested by the finding that
CuZn-SOD inhibits apoptotic neuronal cell death in response to some
forms of cellular damage. We evaluated this possibility in the model of
transient focal cerebral ischemia in mice bearing a disruption of the
CuZn-SOD gene (Sod1). Homozygous mutant (Sod1
 /) mice had no detectable CuZn-SOD activity, and heterozygous mutants (Sod1 +/) showed a 50% decrease
compared with wild-type mice. Sod1 / mice showed a
high level of blood-brain barrier disruption soon after 1 hr of middle
cerebral artery occlusion and 100% mortality at 24 hr after ischemia.
Sod1 +/ mice showed 30% mortality at 24 hr after
ischemia, and neurological deficits were exacerbated compared with
wild-type controls. The Sod1 +/ animals also had
increased infarct volume and brain swelling, accompanied by increased
apoptotic neuronal cell death as indicated by the in situ
nick-end labeling technique to detect DNA fragmentation and
morphological criteria. These results suggest that oxygen-free radicals, especially superoxide anions, are an important factor for
the development of infarction by brain edema formation and apoptotic
neuronal cell death after focal cerebral ischemia and reperfusion.
Key words:
CuZn-superoxide dismutase;
focal cerebral ischemia;
blood-brain barrier;
Evans blue extravasation;
neuronal apoptosis;
TUNEL;
oxidative stress
INTRODUCTION
Oxygen-free radicals are believed to be
involved in the pathogenesis after cerebral ischemia and reperfusion.
During cerebral ischemia, a number of events that predispose the brain
to the formation of oxygen-free radicals may occur (Siesjö, 1984 ;
McCord, 1985). After reperfusion, these events can set off a cascade of other biochemical and molecular sequelae such as the xanthine-xanthine oxidase reaction and phospholipase activation, leading to free-radical production (Gaudet and Levine, 1979; Chan et al., 1984; Siesjö, 1984; McCord, 1985). Among these oxygen-free radicals, superoxide anion
(O2 ), being directly toxic to neurons
(Fridovich, 1986; Patel et al., 1996), may initiate a free
radical-mediated chain reaction causing additional CNS damage (Saugstad
and Aasen, 1980; Chan, 1994).
One of the manifestations of CNS damage after cerebral ischemia is the
formation of brain edema caused by the breakdown of the blood-brain
barrier (BBB). CuZn-superoxide dismutase (CuZn-SOD), a cytosolic
protein, prevents vasogenic brain edema after several kinds of injuries
(Chan et al., 1991; Kinouchi et al., 1991; Shukla et al., 1993),
suggesting that O2 is an important factor for
BBB disruption. Another manifestation of CNS damage is the direct
injury of neuronal cells, including excitatory events that are induced
by glutamate release after cerebral ischemia. Glutamate elevates
cytosolic free calcium (Ca2+) (Choi, 1988), which activates
Ca2+-dependent enzymes and leads to free radical production
(Orrenius et al., 1992; Pazdernik et al., 1992). Recent studies suggest that excitotoxic injury causes apoptotic neuronal cell death in some
neuronal subpopulations (Kure et al., 1991; Ankarcrona et al., 1995).
Both CuZn-SOD (Greenlund et al., 1995) and BCL-2, which recently has
been shown to have an antioxidant property (Kane et al., 1993), inhibit
apoptotic neuronal cell death, suggesting the possibility that
oxygen-free radicals may modulate neuronal apoptosis. Apoptotic
neuronal cell death may also play an important role in focal ischemic
brain injury (Linnik et al., 1993; Tominaga et al., 1993; MacManus et
al., 1994; Li et al., 1995b).
The development of mice deficient in the mouse CuZn-SOD gene
(Sod1) has provided a model for assessing the role of
CuZn-SOD in nervous system injuries (Reaume et al., 1996) (T. Huang, M. Yasunami, E. Carlson, A. Gillespie, A. Reaume, E. Hoffman, P. Chan, R. Scott, C. Epstein, unpublished data). These mutant mice with a decrease
in or complete absence of endogenous CuZn-SOD activity do not show any
phenotypic abnormalities under normal physiological conditions.
However, we hypothesized that the mutant mice might be vulnerable to
oxidative stress because of additional oxygen-free radical formation
after ischemia and reperfusion. Using an in vivo model of
focal cerebral ischemia and reperfusion, we now show early BBB
disruption and high mortality in the mutant mice with reduced CuZn-SOD
activity. In addition, using an in situ technique to detect
the DNA free 3 -OH ends, we have found that apoptotic neuronal cell
death is increased in the mutant mice after ischemia and reperfusion.
These data indicate that oxygen-free radicals, especially
O2, are important mediators of brain edema
and apoptotic neuronal cell death in focal ischemic brain injury.
MATERIALS AND METHODS
Sod1-deficient mice. In the present study, we used
two independent sources of mutant mice. One strain of mutant mice
designated 129/CD1-Sod1<tm1 Cep> was produced at Cephalon
(West Chester, PA) (Reaume et al., 1996). This mutant was made by
targeted deletion of the Sod1 gene in embryonic stem (ES)
cells using a positive-negative selection scheme that replaced all
Sod1 coding sequences (7.6 kb) with the neomycin resistance
gene. The mutant ES cells were in turn used to create mice that were
either heterozygous (Sod1 +/ cep) or
homozygous (Sod1 / cep) mutants. Another
strain of mutant mice designated CD1-Sod1<tm1 Cje> was
produced in the Department of Pediatrics, University of California, San
Francisco (T.-T. Huang et al., unpublished data). This mutant was made
by targeted deletion of the Sod1 gene in ES cells using an
alternative positive-negative selection scheme that replaced a portion
of exon 3 and the entire exon 4 of the Sod1 gene (1.9 kb)
with the neomycin resistance gene. The mutant ES cells were in turn
used to create mice that were heterozygous (Sod1 +/
uc) mutants. All these mutant mice were bred with the CD1
strain of mice. In the present study, age-matched wild-type (Wt)
129/CD1 mice were used as controls.
Two methods were used for assessing of CuZn-SOD activity in the mutant
mice. For the gel electrophoresis assay, previously described methods
(Epstein et al., 1987) were used. For the measurement of
CuZn-SOD-specific activity, supernatant fluids from red blood cell
lysates or brain homogenates were assayed by a previously described
method based on cytochrome C reduction (McCord and Fridovich, 1969).
CuZn-SOD activity, which reduces the cytochrome C deoxidative capacity
by half, was taken as one unit. Tissue protein was quantified using BCA
protein assay reagent (Pierce, Rockford, IL), and CuZn-SOD activity was
calculated in relation to the mass of protein.
Focal cerebral ischemia and neurological assessment. Focal
cerebral ischemia was achieved with suture monofilament blockade of the
middle cerebral artery (MCA) using the previously described method of
Yang et al. (1994), with some modification. Male 2- to 3-month-old Wt
or mutant mice (35-40 gm) were anesthetized with 1.5% isoflurane in a
30% O2/70% N2O mixture under spontaneous breathing. The rectal temperature was controlled at 37.0 ± 0.5°C during surgery with a feedback regulated heating pad. After
exposing the left carotid artery, a 5-0 Dermalon suture (1756-41, Devis and Geck, Manati, Puerto Rico) was advanced into the internal carotid artery 12 mm from the lumen of the external carotid artery. The
ipsilateral common carotid artery was occluded with a small surgical
clip immediately after suture blockade. The animals underwent MCA
occlusion for 1 hr and were then subjected to reperfusion by removing
the suture and the clip. Mean arterial blood pressure and cerebral
blood flow (CBF) were monitored as physiological parameters during
ischemia, and arterial blood gas was analyzed after reperfusion. After
recovering from anesthesia, the animals were maintained in an air
conditioned room at 20°C during the reperfusion periods (2-96
hr).
At 24 hr after 1 hr of MCA occlusion, surviving mice were evaluated by
a blinded observer for their neurological deficits. The neurological
deficit score assignment of 0 to 4 was based on the methods described
previously by Yang et al. (1994). After evaluating the scores, the
brains were removed and rapidly frozen and then were analyzed
histologically with cresyl violet staining. Animals that had massive
hematomas in the brain or no infarction in the brain were omitted from
the neurological analysis.
Measurement of Evans blue extravasation. A quantitative
assay of Evans blue was based on the method described previously by Chan et al. (1991). Just after reperfusion, 0.1 ml of 4% Evans blue
(Sigma, St. Louis, MO) in 0.9% saline was intravenously injected. At
2, 8, or 24 hr after the injection, the animals were killed by
transcardiac perfusion using 200 ml of heparinized saline (10 U/ml
heparin in 0.9% of saline), and the brains were removed. After
removing the cerebellum and brainstem, the cerebral hemispheres were
separated into the ischemic hemispheres and nonischemic hemispheres. Each hemisphere was well homogenized in 1 ml of 0.1 M PBS.
After 1000 × g spin for 5 min, 0.7 ml of supernatant
was taken, and 0.7 ml of 100% (w/v) trichloroacetic acid was
added. The mixture was incubated at 4°C for 18 hr, then centrifuged
at 1000 × g for 30 min. The Evans blue concentration
of the supernatant was measured at 610 nm by a spectrophotometer.
Results are presented as milligrams of Evans blue/hemisphere by
comparing it with the standard solution.
Histological analysis. At 8, 24, 48, or 96 hr after 1 hr of MCA occlusion, the brains were removed and frozen rapidly. The brains were sectioned with a cryostat into a 20 µm thickness from the
anterior side to the posterior side at 500 µm intervals. The sections
were stained with cresyl violet using standard histological criteria.
The staining image was scanned by Color One Scanner 600/27 (Apple
Computer, Cupertino, CA) at 1200 dpi resolution. The infarct area and
the bilateral hemispheric area were measured by the National Institutes
of Health system in 14 sections from the anterior tip of the brain. The
total infarct volume and total hemispheric volume were calculated
according to the method of Liu et al. (1989).
In situ labeling of DNA fragmentation. At 8, 24, 48, or 96 hr after 1 hr of MCA occlusion, the brain sections at the level of the
caudate putamen that show typical infarction were stained using an
in situ technique to detect the DNA free 3-OH ends (TUNEL) reaction (Gavrieli et al., 1992). Frozen brain sections were fixed for
30 min in 4% formaldehyde in 0.1 M PBS, pH 7.4. The slides were placed in 1 × terminal deoxynucleotidyl transferase buffer (Life Technologies, Gaithersburg, MD) for 15 min, followed by 10 µl/ml of terminal deoxynucleotidyl transferase enzyme (Life Technologies) with 40 µl/ml biotinylated 16-dUTP (Boehringer
Mannheim, Indianapolis, IN) at 37°C for 60 min. The slides were then
washed in 2× SSC (150 mM sodium chloride, 15 mM sodium citrate, pH 7.4) for 15 min, followed by PBS two
times for 15 min. Avidin-biotin horseradish peroxidase solution (ABC
kit, Vector Laboratories, Burlingame, CA) was applied to the sections
for 30 min, then washed with PBS two times for 15 min. Staining was
visualized using 0.025% diaminobenzidine and 0.075%
H2O2 in PBS. After staining, the slides were
rinsed with water. Slides were then stained with methyl green for 10 min and dehydrated and mounted. Slides were observed with either a
bright-field or a dark-field phase-contrast microscopy (Diaphot-TMD,
Nicon, Tokyo, Japan). In the latter, a yellow filter, which is used for
detection of fluorescent isothiocyanate, was used without excitation.
Consecutive sections were stained with hematoxylin and eosin (H & E) to
observe standard morphological criteria.
Cell counts. A blinded observer counted the number of
TUNEL-positive cells in 625 µm2 pixels in 400×
magnification of light-field microgram. Positive cells were determined
carefully by the criteria described by Charriaut-Marlangue and Ben-Ari
(1995) and counted in 200 pixels (0.125 mm2) with random
movement under blinded conditions to avoid intentional selection.
Individual regions were determined as follows: inner boundary of the
caudate putamen from the medial edge of the infarction to 200 µm
toward infarct area; center of the caudate putamen from external
capsule to 500 µm toward midline; cortical penumbra from the edge of
the infarction to 1 mm toward infarct area; the piriform cortex inside
of the piriform cortex neuronal cell layer. The total positive cell
number in 200 pixels was expressed as cells/mm2.
Statistical analysis. All measurements, except the
neurological deficit score, were statistically analyzed using one-way
ANOVA followed by Fisher's protected least-significant difference
(PLSD) test. The neurological deficit score was analyzed statistically using a nonparametric test (Kruskal-Wallis test for overall effects, followed by the Mann-Whitney U test for individual group
comparisons). Spearman rank correlation followed by linear regression
analysis was used for correlation study.
RESULTS
CuZn-SOD activity and physiological parameters in mutant mice
Nondenatured gel electrophoresis followed by nitroblue tetrazolium
staining confirms that CuZn-SOD activity is reduced in both
Sod1 +/uc and Sod1
+/cep and completely absent in Sod1
/cep (Fig. 1). A quantitative assay of
CuZn-SOD activity shows ~50% reduction in both Sod1
+/uc and Sod1 +/cep and only a
trace of activity in Sod1 /cep (Table
1). Although the gene-targeted locus is different in the
two independent lines of heterozygous mutant mice (Sod1
+/uc and Sod1 +/cep), these mice
show the same level of CuZn-SOD activity. The minor level of activity
in Sod1 /cep likely represents background
from the assay, although it also could reflect an endogenous SOD
activity contributed by the extracellular form of CuZn-SOD that exists
in the brain and plasma. Physiological studies show no significant
differences in arterial blood gas analysis, middle arterial blood
pressure, or CBF between Wt and all mutant mice (Table
2).
Fig. 1.
CuZn-SOD activity in mutant mice. CuZn-SOD
activity analysis of blood (lanes
1-4) and brain tissue homogenate
(lanes 5-8). Nondenatured gel
electrophoresis followed by nitroblue tetrazolium staining shows a
white band (arrowhead), corresponding to CuZn-SOD activity. There are also visible bands, corresponding to manganese (Mn)-SOD and hemoglobin (Hb).
Lanes 1 and 5, Wt; 2 and
6, Sod1 +/uc;
3 and 7, Sod1
+/cep; 4 and 8,
Sod1 /cep.
[View Larger Version of this Image (64K GIF file)]
Mortality and neurological deficits are exacerbated in
mutant mice
The mortality of Wt and mutant animals during 24 hr after 1 hr of MCA occlusion is shown in Table 3. All
Sod1 /cep mice were dead within several
hours after ischemia, and the rate of mortality was significantly
higher than that of Wt mice (p < 0.01) (Table
3). Although the rate of mortality was moderately higher in both
Sod1 +/uc and Sod1
+/cep than in Wt mice at 24 hr, no significance was seen
(Table 3). However, the mortality was significantly higher for
Sod1 +/ animals at 48 and 96 hr after ischemia, compared
with Wt mice. In surviving animals, neurological deficits were
exacerbated significantly in Sod1 +/uc and
Sod1 +/cep mice, compared with Wt mice at 24 hr (p < 0.05-0.01) (Table
4).
Table 4.
Neurological deficits of Wt and mutant mice at 24 hr after
ischemia
| Genotype |
Neurological deficit
scoresa
|
| 0 |
1 |
2 |
3 |
4 |
Mean ± SEMb |
|
| Wt |
3 |
7 |
5 |
0 |
1 |
1.31
± 0.25 |
| Sod1+/uc |
0 |
1 |
2 |
3 |
3 |
2.89
± 0.35** |
| Sod1+/cep |
0 |
4 |
9 |
2 |
1 |
2.00
± 0.20* |
|
|
a
Neurological deficit scores of 0-4: 0, no neurological deficit; 1, failed to extend right forepaw; 2, circled
to the right; 3, fell to the right; 4, unable to walk spontaneously.
Each value represents the number of animals in the respective scores.
b
Each value represents mean ± SEM of
neurological deficit scores;
*
P < 0.05,
**
P < 0.01, significantly different from Wt mice (Mann-Whitney U
test).
|
|
Evans blue extravasation indicates the early BBB disruption in
mutant mice
At 2 hr after 1 hr of MCA occlusion, no Evans blue leakage
occurred in the entire brain of Wt mice, including the ischemic region
(Fig. 2A). In contrast,
Sod1 +/ mice showed a minor level of Evans blue
extravasation around the center of the ischemic region (Fig.
2A). The Evans blue extravasation was much more
intensified and extended to the entire ischemic region in the
Sod1 / mice (Fig. 2A).
Fig. 2.
Evans blue extravasation in mutant mice after
ischemia. A, Representative photographs of Evans blue
extravasation in the brains of Wt mice, heterozygous
(Sod1 +/) and homozygous (Sod1 /) mutants 2 hr after ischemia. The dark stained area in the left hemisphere indicates Evans blue extravasation. B,
Quantitative assay of Evans blue at 2, 8, or 24 hr after ischemia.
Values are mean ± SEM of the amount of Evans blue in the
hemisphere. Ctrl., Basal value of Evans blue in the
nonischemic hemisphere of Wt (n = 16),
Sod1 +/ (n = 19), or
Sod1 / (n = 5) mice. Data in respective time points indicate the amount of Evans blue in the ischemic hemisphere of Wt, Sod1 +/, or
Sod1 / mice (n = 4-8). Asterisks indicate a significant increase of Evans blue
leakage compared with Wt mice (p < 0.01),
and NS indicates no significance between Wt and
Sod1 +/ (Fisher's PLSD test).
[View Larger Version of this Image (33K GIF file)]
As illustrated in Figure 2B, quantitative assay of
Evans blue revealed a low background basal level of Evans blue
extravasation in the nonischemic hemisphere: Wt, 0.23 ± 0.02;
Sod1 +/, 0.22 ± 0.01; Sod1 /,
0.24 ± 0.05 (mean ± SEM of µg/hemisphere; no significant
difference in any of the groups, by ANOVA). At 2 hr after 1 hr of MCA
occlusion, the amount of Evans blue leakage was not increased in the
ischemic hemisphere of the Wt mice (0.26 ± 0.02 µg/hemisphere),
although it was moderately increased in the ischemic hemisphere of
Sod1 +/ (0.81 ± 0.15 µg/hemisphere; p < 0.01) (Fig. 2B) and was highly
increased in the ischemic hemisphere of Sod1 /
(2.21 ± 0.49 µg/hemisphere; p < 0.01) (Fig.
2B). Evans blue leakage increased in Wt mice at 8 hr
after ischemia and gradually increased up to 24 hr (Fig.
2B). Also, Sod1 +/ mice showed an increasing amount of Evans blue extravasation up to 24 hr, although no
significant difference was seen, compared with Wt at 8 and 24 hr after
ischemia (Fig. 2B). No Evans blue leakage studies were performed in Sod 1 / mice at 8 and 24 hr because of
100% animal mortality. These results suggest that a decreased level of
CuZn-SOD activity in the mutant mice mediates early BBB breakdown after
cerebral ischemia.
Infarct volume and brain swelling are exacerbated in
mutant mice
To investigate the correlation between the degree of
ischemic infarction and CuZn-SOD activity, we compared the infarct size between Wt and heterozygous mutant mice after 1 hr of MCA occlusion. Hemisphere enlargement was also measured to evaluate the brain swelling
contributed by brain edema. As shown in Figure
3A, the infarct area and the normal area can
be clearly defined by cresyl violet staining. Distribution of the
infarction and brain swelling at 24 hr after ischemia are shown in
Figure 3B. Distribution of the infarction is similar in the
two different strains of mutants (Sod1 +/uc
and Sod1 +/cep), which is remarkably extended
to the posterior side of the brain, compared with that of Wt mice (Fig.
3B1). The hippocampal CA1 pyramidal neurons,
especially, which were spared in Wt mice, are involved in the
infarction in the heterozygous mutant mice (Fig. 3A). In
contrast, the percentage of hemisphere enlargement was continuously
greater in all of the brain sections in both of the heterozygous
mutants (Sod1 +/uc and Sod1
+/cep) than in the Wt mice (Fig.
3B2).
Fig. 3.
Histological analysis of Wt and
Sod1 +/ mice after ischemia. A,
Representative low-magnification photograph of cresyl violet staining
at 24 hr after 1 hr of MCA occlusion. The caudate putamen level at 3 mm
from the anterior tip (top) and the hippocampal level at
6 mm from the anterior tip (bottom) are represented. Infarction is represented in the left hemisphere as a pale, stained area. The ischemic hemisphere is much more enlarged in
Sod1 +/ than in Wt mice. Note that hippocampal CA1
pyramidal neurons are also involved in the infarction in
Sod1 +/ mice. Scale bar, 2 mm. B,
Distribution of infarction and hemisphere enlargement in the brain.
B1, Sequential changes of percentage of infarct
area expressed as infarct area/ischemic hemisphere area × 100%.
B2, Sequential changes of percentage of
hemisphere enlargement expressed as (ischemic hemisphere area nonischemic hemisphere area)/nonischemic hemisphere area × 100%.
X-axis shows the distance of the sections from the
anterior tip of the brain. Data show mean ± SEM
(n = 5-8). C, Temporal profile of
infarct volume and hemisphere enlargement in the whole brain of Wt and
Sod1 +/cep mice. The percentages of total
infarct volume (C1) and percentages of total
hemisphere enlargement (C2) were calculated by
the accumulation in consecutive brain sections and expressed in the
same manner as in Figure 3B. Data show mean ± SEM
(n = 4-8). Asterisks indicate a
significant increase, compared with Wt mice (*p < 0.05, **p < 0.01), and NS indicates
no significance between Wt and Sod1 +/cep
(Fisher's PLSD test).
[View Larger Version of this Image (51K GIF file)]
Next we assessed the total infarct volume and hemisphere enlargement in
the whole brain by calculating the cumulative areas in consecutive
brain sections at 8, 24, 48, and 96 hr after ischemia. As illustrated
in Figure 3C1, infarction was not obvious at 8 hr after ischemia, except in the lateral caudate putamen (i.e., core of
the ischemia), both in Wt and Sod1 +/cep mice
(4.2 ± 0.3% vs 7.1 ± 0.5%; p = 0.68). The
infarct volume was increased at 24 hr after ischemia and was
significantly greater in Sod1 +/cep (53.0 ± 5.5%) than in Wt mice (28.4 ± 3.4%; p < 0.01) (Fig. 3C1). The infarct volume remained at
48 hr after ischemia both in Wt and in Sod1
+/cep mice (33.1 ± 3.3% vs 55.0 ± 6.1)
(p < 0.01) (Fig. 3C1).
Although the infarct volume was reduced slightly at 96 hr after
ischemia in Sod1 +/cep mice (47.7 ± 3.3%), it was still significantly greater than in Wt mice (32.2 ± 3.5%; p < 0.05) (Fig. 3C1).
One possibility to explain the unlikely phenomenon of the reduction of
infarct volume in temporal resolution is the high mortality in
Sod1 +/cep mice, as demonstrated in Table 3.
The mortality rates of the Sod1 +/ animals were 50 and
63.6% for 48 and 96 hr, whereas the mortality rates of Wt mice were
28.6 and 55.6%, respectively. Thus, the animals with severe infarction
could not survive for longer periods after ischemia, which might result
in the reduction of infarct volume in Sod1
+/cep animals at 96 hr after ischemia.
Hemisphere enlargement showed similar results as the infarct volume
(Fig. 3C2). The percentage of hemisphere
enlargement was slightly increased at 8 hr after ischemia both in Wt
and in Sod1 +/cep mice (4.4 ± 1.1% vs
6.9 ± 0.9; p = 0.63) (Fig.
3C2). The percentage of hemisphere enlargement
highly increased in Sod1 +/cep mice (22.7 ± 4.6%) and was significantly greater than that in Wt mice (8.9 ± 1.6; p < 0.01) (Fig. 3C2).
It gradually decreased in Sod1 +/cep mice up
to 96 hr after ischemia (48 hr, 18.8 ± 3.0%; 96 hr, 16.6 ± 4.3%), and no significant difference was seen, compared with Wt mice
(48 hr, 10.4 ± 1.3%; 96 hr, 11.8 ± 1.9%) (Fig.
3C2).
Severity of infarction and brain swelling correlates the
exacerbation of neurological deficits
We analyzed the correlation between infarct volume and
hemisphere enlargement in individual animals at 24 hr after ischemia. Linear regression analysis (Fig. 4A)
shows a positive correlation between the percentage of infarct volume
and the percentage of hemisphere enlargement in Wt and Sod1
+/ mice (r = 0.80). Individual data plots also
demonstrate the differences in the subpopulation of infarct volume and
hemisphere enlargement between Wt and Sod1 +/ mice. These
results indicate that a decrease in the level of CuZn-SOD in
Sod1 +/ mice exacerbates both infarction and brain edema
at 24 hr after 1 hr of MCA occlusion.
Fig. 4.
Correlation between histological findings and
neurological deficits at 24 hr after ischemia. The results in
individual Wt (open circles, n = 8)
and Sod1 +/ (solid circles,
n = 13) mice are shown. Lines are
linear regression that fit through the data points. A,
Linear regression of percentage of infarct volume and percentage of
hemisphere enlargement in Wt and Sod1 +/ mice
(r = 0.80). B, Linear regression of
percentage of infarct volume on neurological deficit score in Wt and
Sod1 +/ mice (r = 0.79). C, Linear regression of percentage of hemisphere
enlargement on neurological deficit score in Wt and Sod1
+/ mice (r = 0.70).
[View Larger Version of this Image (19K GIF file)]
To examine the effects of the histological findings on the neurological
deficits, next we analyzed them by plotting variations of the
percentage of infarct volume (Fig. 4B) or the
percentage of hemisphere enlargement (Fig. 4C) versus
neurological deficit scores in individual animals at 24 hr after
ischemia. Linear regression analysis indicates that neurological
deficits in individual animals were positively correlated with the
percentage of infarct volume (r = 0.79) and the
percentage of hemisphere enlargement (r = 0.70). These
findings indicate that increased formation of infarction and brain
edema causes exacerbation of neurological deficits in Sod1
+/ mice.
Reduction of CuZn-SOD enhances neuronal apoptosis
To evaluate the role of oxygen-free radicals in the induction of
apoptotic neuronal cell death in focal ischemic brain injury, we
examined DNA fragmentation in the infarcted brain sections from Wt and
Sod1 +/ mice. As shown in Figure
5B, TUNEL staining does not label the normal
neuronal cells in the noninfarct area. In contrast, two different
patterns of staining were observed in the neuronal cells in the
infarcted area (Fig. 5D,F).
Some neuronal cells in the infarcted area are densely labeled in their nuclei using TUNEL staining, accompanied by small particles around the
nuclei that resemble apoptotic bodies (Fig. 5D). These
neuronal cells show cellular shrinkage and chromatin condensation in
the nuclei using H & E staining (Fig. 5C). This
cellular morphology is consistent with the apoptotic cell death
process. Besides these typical apoptotic neuronal cells, slightly
TUNEL-stained cells are also observed (Fig. 5F).
These cells show diffuse nuclear staining by TUNEL and cellular
swelling and nuclear lysis by H & E staining (Fig.
5F,E). This cellular morphology is
consistent with necrosis. These results are consistent with those of
previous reports, in which the coexistence of cells with either
necrotic or apoptotic features is observed after focal cerebral
ischemia and in which necrotic neurons are slightly labeled using TUNEL staining (Charriaut-Marlangue and Ben-Ari, 1995; Li et al., 1995b). The
present study also confirms that apoptotic and necrotic features can be
distinguished by the different patterns in TUNEL staining and that
these differences in TUNEL staining are readily visualized by
dark-field phase-contrast microscopy (Fig. 5G,H).
Fig. 5.
Representative findings of TUNEL staining in Wt
mice at 24 hr after ischemia. High magnification of neuronal cells in
the caudate putamen with H & E staining (A,
C, E) and TUNEL staining with methyl
green counter staining (B, D,
F). A, B, The
nuclear and cellular morphology of the neurons in the nonischemic
caudate putamen is typical of normal neurons with smooth nuclear
membrane and uniform chromatin formation (A). TUNEL
staining does not label normal neuronal cells (B).
C, D, Representative morphology of the
cells in the marginal zone of the infarction, which shows apoptotic
features such as cell shrinkage and chromatin condensation in the
nuclei (C). Apoptotic neuronal cells in the marginal
zone are recognized with TUNEL staining with markedly labeled small particles, called "apoptotic bodies," around the nuclei
(D, arrow). E,
F, Representative morphology of the cells in the
infarction, which shows necrotic features such as cellular swelling
(pale color on eosinophilic staining) and irregularly shaped nuclei (E). These neuronal cells are slightly labeled with
TUNEL staining in the nuclei (F, open arrow).
G, H, Lower magnification of TUNEL staining by bright-field (G) and dark-field
(H) phase-contrast microscopy. Two patterns of
the staining quality are emphasized in the dark-field phase-contrast
photomicrographs: apoptotic neurons with dense TUNEL labeling as
bright yellow spots (H, rotated
arrow) and necrotic neurons with slight TUNEL labeling as
faint, small, yellow spots (H,
arrowhead). Scale bars:
A-F, 20 µm; G,
H, 200 µm.
[View Larger Version of this Image (85K GIF file)]
No differences were observed in the morphological features of apoptotic
or necrotic neurons between Wt and Sod1 +/ mice. However,
the distribution and number of apoptotic neurons showed a notable
difference between these groups. In Wt mice, apoptotic neurons were
observed primarily at the margin of the inner boundary of the caudate
putamen (Fig, 6A). In other lesions of the infarction (i.e., center of the caudate putamen, the cortical penumbra, and the
piriform cortex), the main population of the cells showed necrotic
features (Fig, 6A,C,E). In contrast, in
Sod1 +/ mice, an increased number of apoptotic neurons
were observed in the caudate putamen (Fig.
6B), and the main population of the
cells showed apoptotic features in the cortical penumbra (Fig.
6D) and the piriform cortex (Fig.
6F).
Fig. 6.
Distribution of TUNEL-positive neurons in
Wt and Sod1 +/ mice. Representative dark-field
phase-contrast photographs of TUNEL staining in Wt (A,
C, E) and heterozygous mutant
(B, D, F) mice at
24 hr after 1 hr of MCA occlusion. Normal, noninfarcted area gives
yellow reticular background, and infarct area is defined as dark area
(A, C, E,
arrowheads). In the infarcted area, bright yellow spots representing apoptotic neurons with dense TUNEL
labeling and faint yellow spots representing necrotic
neurons with slight TUNEL labeling are observed. In the caudate
putamen, the apoptotic neurons are located primarily at the margin of
the inner boundary, and the center of the caudate putamen was occupied
with necrotic neurons in Wt mice (A). In contrast,
apoptotic neurons were increased in the margin of the inner boundary
and extend to the center of the caudate putamen in Sod1
+/ mice (B). In the cortical penumbra, an increased
number of apoptotic neurons is observed in Sod1 +/ mice (D), compared with Wt mice (C). In
the piriform cortex, apoptotic neurons are scarce in Wt mice
(E), whereas almost all neurons show apoptotic features
in Sod1 +/ mice (F).
LV, Lateral ventricle; CP, caudate
putamen; EC, external capsule; SN, septal
nucleus; AC, territory of anterior cerebral artery;
MC, territory of middle cerebral artery;
Pi, piriform cortex. Scale bar, 500 µm.
[View Larger Version of this Image (165K GIF file)]
To quantify the apoptotic neurons, we assessed temporal resolution of
TUNEL-positive cells between Wt and Sod1 +/ mice. The number of apoptotic neurons was counted at 8, 24, 48, and 96 hr after 1 hr of MCA occlusion in the inner boundary of the caudate putamen, the
center of the caudate putamen, the cortical penumbra, and the piriform
cortex, respectively. As illustrated in Figure 7,
apoptotic neurons were scarce at 8 hr after ischemia. They were
remarkably increased at 24-48 hr and decreased at 96 hr after ischemia, both in Wt and in Sod1 +/ mice. These results
are consistent with those of previous reports for the temporal profile
of apoptotic neurons after focal cerebral ischemia in the rat (Li et
al., 1995a). Although there are no differences in the pattern of
temporal profile in the apoptotic neurons between Wt and
Sod1 +/ mice, apoptotic cell numbers are increased
significantly in Sod1 +/ than in Wt mice at 24 and 48 hr
after ischemia (p < 0.05-0.01) (Fig. 7). These
experiments demonstrate that in ischemic-reperfusion injury, apoptotic
neuronal cell death is increased in Sod1 +/ mice. These findings are consistent with the idea that oxidative stress mediates apoptotic neuronal cell death (Kane et al., 1993; Troy and Shelanski, 1994; Greenlund et al., 1995). Here we have extended this concept to
the role of oxygen-free radicals in apoptotic neuronal cell death in
focal ischemic brain injury.
Fig. 7.
Temporal profile of TUNEL-positive neurons in Wt
and Sod1 +/ mice. Apoptotic neuronal cells were
counted at 8, 24, 48, and 96 hr after 1 hr of MCA occlusion in the
inner boundary of the caudate putamen (A), the center of
the caudate putamen (B), the cortical penumbra
(C), and the piriform cortex (D). Data
are expressed as cells/mm2 (for details, see Results). Data
represent mean ± SEM (n = 4). Asterisks indicate a significant increase, compared with
Wt mice (*p < 0.05, **p < 0.01; Fisher's PLSD test).
[View Larger Version of this Image (39K GIF file)]
DISCUSSION
Targeted disruption of the Sod1 gene results in the
partial or complete loss of CuZn-SOD activity in mutant mice. In this study, we present data that show that the formation of brain
edema and neuronal cell injury, including apoptotic neuronal cell
death, is exacerbated in mutant mice after focal cerebral ischemia.
Because phenotypic abnormalities are not seen in the mutant mice under normal physiological conditions, these findings are consistent with the
hypothesis that an increased level of oxygen-free radicals, especially
O2, mediates these pathologies in the mutant
mice after focal cerebral ischemia.
The present study demonstrated high mortality and exacerbated
neurological deficits in mutant mice after focal cerebral ischemia. The
mechanisms underlying exacerbated mortality and neurological deficits
in mutant mice are unclear at present. However, in ischemic brain
injury, brain edema is known to be an important factor for the acute
phase of mortality because of the development of severe brain swelling
and herniation. Our results indicate that BBB disruption occurs
unusually early in mutant mice, with the amount of Evans blue leakage
being dependent on the degree of decreased CuZn-SOD activity. In
addition, at later time points, mutant mice showed severe brain
swelling that was correlated to neurological deficits. This severe
brain edema is suggested to be the primary factor causing the
exacerbation of mortality and neurological deficits in the mutant
animals. The BBB, which is composed of endothelial cells, is intact
several hours after ischemia (Menzies et al., 1993), suggesting that
endothelial cells are relatively resistant to ischemic injury
(Brightman, 1992). Because the endothelial cells are a major cellular
source of O2 through high levels of xanthine
oxidase (Betz, 1985; Terada et al., 1991), our findings suggest that
O2 may contribute to the ischemic
vulnerability of endothelial cells in mutant mice.
O2 is known to produce highly toxic hydroxyl
radicals through two distinct pathways; the first is reaction with
H2O2 through the Haber-Weiss reaction; the
second is peroxynitrite self-decomposition through a reaction with
nitric oxide. However, the former pathway appears unlikely in the
mutant mice, because O2 is hardly converted
to H2O2 in cytosol, especially in the
Sod1 / mutants, because of the lack of CuZn-SOD
activity. The latter pathway more likely explains the phenomenon in the
mutants, because endothelial cells are an abundant source of nitric
oxide from their constitutive nitric oxide synthase. Consistent with
this inference is recent evidence suggesting that the constitutive endothelial nitric oxide synthase is activated after focal cerebral ischemia and reperfusion (Nagafuji et al., 1995).
It is known that exogenously supplied SOD improves the histological
outcome in ischemic animals by increasing the CBF (Cerchiari et al.,
1987). However, our previous reports demonstrated that endogenously
increased CuZn-SOD activity in transgenic mice has no effect on CBF
during and after ischemia (Chan et al., 1993; Yang et al., 1994; Kondo
et al., 1996). The present study also confirms that a decreased level
of CuZn-SOD in mutant mice does not affect the residual CBF during
ischemia. Recently, Barone et al. (1993) reported a significant
difference between mouse strains in their sensitivity to focal ischemia
that was related to the structural and functional differences in the
potency of the circle of Willis. Although the functional vascular
anatomy was not examined in the present study, a similar level of CBF decreases during ischemia in Wt and mutant animals clearly suggests that functional vascular anatomy does not play a role in the outcomes after focal cerebral ischemia. In addition, two different mutant mouse
strains (Sod1 +/uc and Sod1
+/cep) with different genetic backgrounds showed almost
the same distribution and volume of infarction, which were remarkably
different from those in Wt mice. These results again suggest that
because of the decreased level of CuZn-SOD activity, the severity of
the infarction in the mutant mice is related to the biochemical
reaction of oxygen-free radicals.
The effects of oxidative stress on the biochemical mechanisms in focal
ischemic brain injury have been shown in a variety of experimental
studies. In the present study, sequential changes in the infarct area
demonstrated that infarction was extended to the posterior side of the
cortex in heterozygous mutant mice. This finding suggests that the
decreased level of CuZn-SOD is related to or accelerates the damage of
the neuronal cells located in the cortical penumbra area. One important
metabolic event in the penumbra area is the depolarization that causes
Ca2+ overload by excitatory amino acids (Simon and
Shiraishi, 1989). A recent study provides direct evidence that
excitotoxic neuronal cell death requires O2
generation in cultured cortical neurons (Patel et al., 1996), suggesting that the excitotoxic events may contribute to the formation of infarction through a high level of O2 in
the penumbra area in the mutant mice. The present study also demonstrates that hippocampal CA1 neurons are involved in the infarction in mutant mice. A variety of experimental models of global
cerebral ischemia have demonstrated that CA1 hippocampal pyramidal
neurons are the cells most vulnerable to ischemic injury (Pulsinelli
and Brierley, 1979; Kirino, 1982), in which abundant NMDA receptors are
expressed (Monaghan and Cotman, 1985), suggesting that activation of
NMDA receptors contributes to CA1 vulnerability. These findings again
support the possibility that excitotoxic events may contribute to
neuronal cell injury in the mutant mice.
The appearance of apoptotic neuronal cell death in focal ischemic brain
injury has recently been observed. DNA laddering (a biochemical
hallmark of apoptosis) after MCA occlusion in the rat is associated
with increased intranucleosomal endonuclease activity (Tominaga et al.,
1993). Recent morphological studies with TUNEL staining demonstrate
that the inner boundary zone of the caudate putamen is vulnerable to
apoptotic neuronal cell death after focal cerebral ischemia (Li et al.,
1995b; Charriaut-Marlangue et al., 1996), and the number of apoptotic
neurons was maximized at 24-48 hr after ischemia (Li et al., 1995a).
Our results in Wt mice are consistent with these previous reports. The
present study provides a new perspective that whereas a small number of neurons in the core of the ischemia show apoptotic neuronal cell death
in Wt mice, the number is increased significantly in the mutant mice.
Because neurons in the core of the ischemic region rapidly die, it is
difficult to explain this phenomenon. Recent studies have demonstrated
that a neurotoxic dose of glutamate induced acute necrosis in a
subpopulation of cerebellar granule cells during and immediately after
exposure, whereas the remaining neurons died from delayed-onset
apoptosis (Ankarcrona et al., 1995). Furthermore, excitotoxic animal
models suggest that apoptotic and necrotic mechanisms of neuronal death
may occur simultaneously within individual dying cells in the injured
brain (Portera-Cailliau et al., 1995). These findings are likely to
explain tissue injury such as in focal ischemic brain injury, in which
cells with either necrotic or apoptotic features coexist. Thus, even in
the core of the infarction, some neuronal subpopulations die rapidly
via the necrotic process, and the remaining neuronal cells may die gradually by the apoptotic process, which could be enhanced by a
decreased level of CuZn-SOD in the mutant mice.
More importantly, heterozygous mutant mice demonstrate
significantly increased apoptotic neuronal cell death in the cortical penumbra and piriform cortex, suggesting that oxygen-free radicals, in
particular O2, are important factors for
inducing apoptotic neuronal cell death in focal ischemic brain injury.
These results are consistent with recent studies in which CuZn-SOD, as
well as BCL-2 (recently shown to have antioxidant properties), has been
found to delay or prevent neuronal apoptosis by growth factor
deprivation in culture (Hockenbery et al., 1993; Kane et al., 1993). In
addition, Araki et al. (1992) demonstrated that SOD attenuated
increases in intracellular Ca2+ concentration after MCA
occlusion, suggesting that a decreased level of CuZn-SOD in the mutant
mice induces Ca2+ overload, subsequently leading to
apoptosis. A recent study has demonstrated that mild focal ischemia
causes delayed infarction, related to the appearance of apoptotic
neurons (Du et al., 1996), suggesting the important role of apoptotic
neuronal cell death in the development of ischemic brain injury. This
finding supports our hypothesis that CuZn-SOD-related modulation of
neuronal viability is related to the inhibition of apoptotic neuronal
cell death after focal cerebral ischemia and reperfusion.
We have presented data showing that mutant mice exhibit exacerbation of
ischemic brain injury through early, severe BBB disruption and
subsequent infarction with increased apoptotic neuronal cell death. The
early BBB breakdown suggests that the vulnerability of the endothelial
cells makes them a target for early oxygen-free radical damage in the
mutant mice. Differences in cellular distribution of necrosis and
apoptosis in heterozygous mutants suggest that excitotoxic events play
a critical role in brain injury induced by oxidative stress after focal
cerebral ischemia. Current data are consistent with the hypothesis that
a series of biochemical events involving the generation of
O2, perhaps peroxynitrite formation and
Ca2+ overload, is involved. This schema, although it is
still incomplete, provides a framework for additional mechanistic
studies.
FOOTNOTES
Received Jan. 30, 1997; revised March 17, 1997; accepted March 21, 1997.
This work was supported by National Institutes of Health Grants NS14543
and NS25372 (P.H.C.) and AG08938 (C.J.E., P.H.C.), and by the Lucille
Markey Foundation to the University of California, San Francisco,
Program in Biological Sciences. We thank L. F. Reola and B. E. Calagui
for their expert technical assistance and C. Christensen for her
editorial assistance. We particularly thank F. R. Sharp, J. Honkaniemi,
K. Lamborn, and H. Kinouchi for their suggestions.
Correspondence should be addressed to Dr. P.H. Chan, Departments of
Neurological Surgery and Neurology, University of California, Box 0651, San Francisco, CA 94143.
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