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 Previous Article | Next Article 
The Journal of Neuroscience, October 15, 1998, 18(20):8292-8299
Overexpression of SOD1 in Transgenic Rats Protects Vulnerable
Neurons Against Ischemic Damage After Global Cerebral Ischemia and
Reperfusion
Pak H.
Chan1, 3,
Makoto
Kawase1, 3,
Kensuke
Murakami1,
Sylvia F.
Chen1, 3,
Yibing
Li1,
Bernard
Calagui1, 3,
Liza
Reola1, 3,
Elaine
Carlson2, and
Charles J.
Epstein2
CNS Injury and Edema Research Center, Departments of
1 Neurological Surgery and Neurology and
2 Department of Pediatrics, University of California,
School of Medicine, San Francisco, California 94143, and
3 Departments of Neurosurgery, Neurology and Neurological
Sciences, Stanford University School of Medicine, Palo Alto, California
94304
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ABSTRACT |
Transient global cerebral ischemia resulting from cardiac arrest is
known to cause selective death in vulnerable neurons, including
hippocampal CA1 pyramidal neurons. It is postulated that
oxygen radicals, superoxide in particular, are involved in cell death
processes. To test this hypothesis, we first used in situ imaging of superoxide radical distribution by
hydroethidine oxidation in vulnerable neurons. We then generated SOD1
transgenic (Tg) rats with a five-fold increase in copper zinc
superoxide dismutase activity. The Tg rats and their non-Tg wild-type
littermates were subjected to 10 min of global ischemia followed by 1 and 3 d of reperfusion. Neuronal damage, as assessed by cresyl
violet staining and DNA fragmentation analysis, was significantly
reduced in the hippocampal CA1 region, cortex, striatum,
and thalamus in SOD1 Tg rats at 3 d, as compared with the non-Tg
littermates. There were no changes in the hippocampal CA3
subregion and dentate gyrus, resistant areas in both SOD1 Tg and non-Tg
rats. Quantitative analysis of the damaged CA1 subregion
showed marked neuroprotection against transient global cerebral
ischemia in SOD1 Tg rats. These results suggest that superoxide
radicals play a role in the delayed ischemic death of hippocampal
CA1 neurons. Our data also indicate that SOD1 Tg rats are
useful tools for studying the role of oxygen radicals in the
pathogenesis of neuronal death after transient global cerebral
ischemia.
Key words:
superoxide dismutase; transgenic rat; superoxide
radicals; transient global cerebral ischemia; delayed neuronal
degeneration; DNA fragmentation
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INTRODUCTION |
Free radicals with unpaired
electrons are generally highly reactive molecules that initiate radical
chain reactions and damage cellular macromolecules, including proteins,
DNA, and lipids, ultimately leading to cell death. Superoxide radicals
and other oxygen radicals have been implicated in neuronal cell death
in acute CNS injury and in chronic neurodegenerative diseases (Kontos, 1985 ; Coyle and Puttfarcken, 1993; Chan, 1994). One particular role of
oxygen free radicals in brain injury appears to involve reperfusion
after cerebral ischemia (Chan, 1996). Reperfusion supplies oxygen to
the ischemic region of the brain in which oxygen is being used in
mitochondria to generate adenosine triphosphate, and superoxide
radicals and H2O2 are produced as a by-product (Boveris and Chance, 1973). Mitochondria are known to be the site for
the production of reactive oxygen species in cultured cortical neurons
after exposure to NMDA (Dugan et al., 1995). Although several
antioxidant enzymes (including superoxide dismutases [SODs], glutathione peroxidases, and catalase) process these oxygen radicals, when they are overproduced, they generally exceed the capacity of the
endogenous antioxidant enzymes, causing oxidative stress or injury of
brain cells during reperfusion after an ischemic insult. We have
demonstrated that overexpression of cytosolic copper zinc (CuZn) SOD in
transgenic (Tg) mice plays a protective role in several types of brain
injury, including cold-injury-induced brain edema (Chan et al., 1991),
transient focal cerebral ischemia (Kondo et al., 1997b), traumatic
brain injury (Mikawa et al., 1996), and hypoxic and excitotoxic
neuronal injury in cultures (Chan et al., 1990; Copin et al., 1996).
Although reperfusion after transient global cerebral ischemia resulting
from cardiac arrest is known to produce oxygen radicals that damage
selective vulnerable neurons, it is not clear whether increased CuZn
SOD activity will provide neuroprotection against this ischemic insult. Mice that overexpress SOD1 would be a perfect choice to address this
issue. However, because of the plasticity of the arteries in most mouse
strains, SOD1 Tg mice are not well suited for transient global cerebral
ischemia, which requires the hypoplasticity of the posterior
communicating arteries (Murakami et al., 1997, 1998a).
Despite recent success with the use of Tg and knock-out mutant mice in
elucidating the oxidative mechanisms of brain injury after stroke,
there are many obvious advantages to using the rat species for
construction of Tg animals. The development of a Tg rat to be applied
specifically toward gaining understanding of the underlying mechanisms
of oxidative stress in stroke would provide many advantages over the
use of Tg or knock-out mice. One of these advantages is the fact that
rat global cerebral ischemia models are well established (Pulsinelli et
al., 1982; Smith et al., 1984), whereas, application of these models is
lacking or scarce in mice. In addition, the cerebrovascular structure,
brain anatomy, various physiological parameters, blood hemodynamics, and many stroke risk factors have been characterized in rats. The
larger size of the animal would permit additional studies, including
but not limited to, electrophysiology, microdialysis, studies with
systemic cardiovascular variables, regional cerebral blood flow and
metabolism, multiorgan physiology, and interaction in both anesthetized
and awake animals. For these reasons, as well as for ease of
manipulation and performance of physiological measurements, Tg rats
that overexpress SOD1 have been made. Using these SOD1 Tg rats, we now
provide evidence that the death of vulnerable hippocampal
CA1 neurons is significantly reduced after transient global
cerebral ischemia, suggesting a role that superoxide radicals play in
delayed ischemic neuronal death.
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MATERIALS AND METHODS |
Transgene purification and preparation. A 14 kb
BamHI-EcoRI genomic fragment of the SOD1 gene
that contains five exons, and its own promoter, was used for generating
Tg rats. The SOD1 genomic DNA was purified from low-melting agarose gel
after electrophoresis using EluTip (S & S). The eluted DNA was diluted
to 2 ng/µl in 10 mM Tris, pH 7.4, 2 mM EDTA
for injection.
Production of Tg rats. Female Sprague Dawley rats (60-90
gm; Charles River, Holister, CA) were superovulated with 15 U
gonadotropin from pregnant mare serum (G4877; Sigma, St. Louis, MO) at
12 P.M. to 2 P.M. followed 48 hr later with 5-15 U human chorionic
gonadotropin (CG-5; Sigma), mated to fertile Sprague Dawley males, and
checked for "plugs" the following morning. Fertilized eggs were
flushed from oviducts using M2 media (M7167; Sigma) at ~2 P.M. the
following day, incubated in hylauronidase (U/ml in M2 media) for 5 min
at room temperature, washed through several changes of M2, placed in a
20 µl drop of M16 (M7292; Sigma) overlaid with mineral oil, and
incubated at 37°C in 7% CO2.
Fertilized eggs with visible pronuclei were placed in an injection well
prepared on a LabTek two-chamber slide with M2 media (50 µl M2
media spread to a diameter of ~1 cm) and overlaid with mineral oil.
The slide was secured to the stage of a Leica DMIRB inverted
light microscope equipped with Nomarski DIC optics and two Narishige
(Greenvale, NY) three-axis hanging joystick micromanipulators. The DNA
was injected using an Eppendorf (Fremont, CA) transjector 5246 and
needles fabricated from microcapillaries (TW100F-4; World Precision
Instruments, Sarasota, FL) pulled using a Sutter Instruments (Novato,
CA) model P-77 micropipette puller. DNA was injected at a concentration
of 2 ng/µl.
Injected zygotes were transferred the same day (8-10 P.M.) to the
right oviduct of 24 hr postcoitus (with vasectomized Sprague Dawley
male) "pseudopregnant" female Sprague Dawley rats. Pups were born
24 d from the day of transfer.
Fluorescence in situ hybridization. The site of
transgene integration was determined by fluorescence in
situ hybridization (FISH) on metaphase chromosome
spreads prepared from blood lymphocytes, as described (Shi et al.,
1994). The 12 kb SOD1 transgene fragment was labeled with
digoxigenin-11-dUTP (Boehringer Mannheim, Indianapolis, IN) by nick
translation (Enzo Biochem Inc., New York, NY) and detected using
fluorescein isothiocyanate-conjugated sheep anti-digoxigenin Fab
fragment (Boehringer Mannheim) (Shi et al., 1994). The slides were
counterstained with propidium iodide and 4'6-diamidino-2-phenylindole in antifade solution (Oncor, Inc., Gaithersburg, MD) and photographed using ASA 400 Kodak Gold (Eastman Kodak, Rochester, NY) and a Zeiss
Photoscope III.
Isoelectric focusing gel electrophoresis. Transgenic animals
were identified by expression of the human SOD1 enzyme from red blood
cell lysates using horizontal isoelectric focusing gel electrophoresis (pH 4.5-6.0; Multiphor; Pharmacia Biotech) and staining for SOD1 activity with nitroblue tetrazolium (Sigma) (Epstein et al., 1987). Five microliters of whole blood was lysed in 500 µl 2 mM
EDTA and 0.5% NP-40. One microliter of lysate was mixed with 10 µl 1% glycine, loaded onto the gel, and electrophoresed for 2 hr at 2000 V and 12 A, limited with 0.1 M  -alanine cathode and 0.1 M glutamic acid in 0.5 M
H3PO4 anode. After electrophoresis, the gel was
soaked with gentle mixing in the dark for 10 min in 100 ml of 0.036 M phosphate buffer, pH 7.8, with added 16.1 mg nitroblue tetrazolium, 175 µl
N,N,N',N'-tetramethylethylenediamine
(Sigma), and 20 µl 0.5 M EDTA, pH 8.0, and then removed
from the solution, placed on a glass plate, and transferred to a light
box to develop for 5-10 min. For analysis of transgene expression in
tissues, 2-4 µg of tissue homogenates was electrophoresed as
above.
Enzyme activity. The total activity of CuZn SOD was
determined as previously described (Epstein et al., 1987).
Evaluation of anatomical background. To determine the
anatomical background of the ischemia, we evaluated cerebral
vasculature in both groups of animals using carbon black injection
(Murakami et al., 1998a) with minor modifications. After anesthesia
with ketamine (80 mg/kg) and xylazine (12 mg/kg), the rats were killed by transcardial perfusion with 200 ml of 10 U/ml heparin in saline and
300 ml of 3.7% formaldehyde in 0.1 M 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).
Transient global cerebral ischemia. Transient global
ischemia was induced by bilateral common carotid artery (CCA) occlusion and bleeding to lower the mean arterial blood pressure to 35-40 mmHg,
using the previously described method of Smith et al. (1984) with some
modifications. Male SOD1 Tg rats (325-540 gm) and control non-Tg
littermates were anesthetized with 5% isoflurane in 70% N2O and 30% O2, and maintained during
surgery at a level of 1.5-2% isoflurane in 70% N2O and
30% O2 under spontaneous breathing. The rectal temperature
was controlled at 37.0 ± 0.5°C during surgery with a
feedback-regulated heating pad. The femoral artery was exposed and
catheterized with a PE-50 catheter to allow continuous recording of the
arterial blood pressure and removal of blood samples for blood gas
analysis. The right jugular vein was isolated and cannulated with a 23 gauge butterfly needle for intravenous injection of 500 U/kg
heparin dissolved to 100 U/ml with 0.9% saline. After exposing the
bilateral CCAs, blood was quickly withdrawn from the jugular vein
cannula to achieve a reduction of the mean arterial blood pressure.
When the blood pressure reached 40 mmHg, the bilateral CCAs were
temporarily occluded with metal clips. The blood pressure was
maintained at 35-40 mmHg by additional blood withdrawal and
reinfusion. After 10 min, ischemia was terminated by removal of the CCA
clips, and the blood was reinfused. Blood flow in the forebrain,
measured by laser Doppler flowmetry, dropped to 10% of the basal level
during ischemia and rapidly returned to 100% after reperfusion. After
recovery of the arterial blood pressure, the arterial blood was
collected for blood gas analysis. The animals regained consciousness
and were maintained in an air-conditioned room at 20°C for 1 and
3 d after reperfusion.
At the end of the recovery period, the rats were deeply anesthetized
with methoxyflurane, and the brains were removed and rapidly frozen.
Coronal sections of the brains (20 µm) were cut with a cryostat and
mounted onto slides. The sections were stained with cresyl violet for
histological assessment of neuronal cell damage. The brain regions
examined were the hippocampus, cortex, thalamus, and striatum. The
histopathological damage was measured using a neuropathological scoring
system of 0-6, in which 0 = no damage, 1 = 0-10%, 2 = 10-25%, 3 = 25-50%, 4 = 50-75%, 5 = 75-100%, and
6 = complete neuronal death (McBean et al., 1995). Scores for SOD1
Tg rats were compared with those for the non-Tg littermates by means of
a nonparametric analysis using the Mann-Whitney U test. The
lesion areas of the hippocampal CA1 subregion were quantified using the image analysis system of Swanson et al. (1990) with some modification. The present method was created to evaluate infarct size in focal ischemia by measuring areas of the stained sections that had optical densities exceeding a threshold value and was
applied to measure the neuronal damage in the CA1 subregion after global ischemia without sampling error and observer bias. Fresh
frozen brains were sectioned with a cryostat into a 20 µm thickness
from the anterior side to the posterior side at 1 mm intervals,
consecutively. The sections were mounted on slide glass and stained
with cresyl violet using standard histological criteria. The stained
images were scanned by a Color One scanner (Apple Computer, Cupertino,
CA). In each hippocampal section the lengths of the unstained and total
CA1 subregions were measured by the NIH image program. The
area of the damaged and total CA1 pyramidal cell layer was
calculated by integrating the length of the damaged and total
CA1 pyramidal cell layers by the distance. The ratio of
CA1 damage was calculated as (area of CA1
damage/area of total CA1 subregion) × 100%. The
hemisphere area at the posterior commissure level in the coronal
sections was also measured using the same method to evaluate brain
swelling and edema. Results were expressed as mean ± SE. The
statistical significance of differences between the SOD1 Tg rat and
non-Tg littermates was evaluated by Fisher's protected least
significant difference test followed by the nonparametric t
test. Significance between groups was assigned at a level of <5%
probability.
In situ detection of superoxide anion (O2 )
production. The spatial production of O2 during
cerebral ischemia was investigated by the in situ detection of oxidized hydroethidine (HEt) method as previously described (Kondo
et al., 1997a; Murakami et al., 1998b) with minor modifications. HEt
(Molecular Probes, Eugene, OR) is taken up by living cells and oxidized
to a red fluorescent dye, ethidium, specifically by
O2, but not by other reactive oxygen species in the
cells (Bindokas et al., 1996). The rats were anesthetized with 2%
isoflurane in 30% O2 and 70% N2O. The HEt
solution (1 ml; 1 mg/ml in 1% dimethylsulfoxide with PBS) was
administered intravenously 1 hr before killing. The rats were
killed at 1 hr and 1 and 3 d after ischemia by transcardial perfusion with 10 U/ml heparin in saline and 3.7% formaldehyde. After
post-fixation, the brains were cut into slices of 50 µm thickness at
the level of the anterior commissure and the hippocampus using a
vibratome and placed on glass slides. These sections were analyzed
under fluorescent light (HBO 100 W/2, Zeiss), and fluorescence was assessed at excitation = 510-550 nm and emission > 580 nm for detection of ethidium. Photomicrographs of the hippocampus and the cortex were taken, and the intensity and expression patterns of
the oxidized HEt were compared with control nonischemic brains and
between each period after 10 min of global ischemia.
In situ detection of cells with fragmented DNA. DNA
fragmentation after global ischemia was determined by the terminal
deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin
nick end labeling (TUNEL) method in the brains of Tg and control rats.
Frozen hippocampal brain sections were stained as previously described
(Murakami et al., 1998b) with minor modifications. In brief, frozen
brain sections were fixed with 3.7% formaldehyde. After endogenous
peroxidase was inactivated with 0.3% H2O2 for
30 min, the sections were immersed in terminal deoxynucleotidyl
transferase (TdT) buffer (Life Technologies, Gaithersburg, MD) and
incubated with TdT and biotin-16-uridine-5'-triphosphate (Boehringer
Mannheim). After blocking with 2% bovine serum albumin in PBS, the
sections were incubated with avidin-biotin-horseradish peroxidase (ABC
kit; Vector Laboratories, Burlingame, CA) and visualized with 3 mM 3,3'-diaminobenzidine tetrahydrochloride and 18 mM hydrogen peroxide in PBS. The slides were counterstained with methyl green and mounted.
TUNEL-positive cells were quantified with a light microscope by a
blinded investigator. The total number of cells and the number of
TUNEL-positive cells within a grid were counted using high-powered
magnification (400×). The ratio of the number of TUNEL-positive
neurons to the total injured neurons was calculated and expressed as
percent of the TUNEL-positive cells in each group.
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RESULTS |
Characterization of SOD1 Tg rats
SOD1 Tg rats [number 66, University of California San Francisco
(UCSF)] have been successfully produced. In this strain, the human
SOD1 transgene was incorporated into a single chromosome as shown by
FISH (Fig. 1). Because the human and rat
SOD proteins comigrate in a nondenaturing gel system (Epstein et al.,
1987), we developed a new nondenaturing isoelectric focusing gel
electrophoresis (IFGE) method to separate these two proteins, thereby
permitting semiquantitative assay of these two enzymes. The human
homodimer and the heterodimer composed of human and rat segments were
clearly demonstrated in various brain regions, including the
hippocampus, brainstem, cerebellum, spinal cord, pituitary gland,
cerebral cortex, and striatum, as well as in heart tissue in the IFGE
gel (Fig. 2). Because of the high
expression of the human transgene relative to the endogenous rat genes,
the rat CuZn SOD homodimer was rarely visualized. Total CuZn SOD
activity ranged from 8.2-27.2 U/mg in various brain regions as
compared with the range of 2.5-5.3 U/mg in non-Tg rat counterparts
(Table 1). The CuZn SOD activity, therefore, increased ~5.2-6.2-fold in the Tg rats.
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Figure 1.
Incorporation of human CuZn SOD (SOD1) transgene
into a single rat chromosome detected by fluorescence in
situ hybridization. Note the spots of bright fluorescence
located in one of the rat chromosomes.
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Figure 2.
Expression of human CuZn SOD in various brain
regions and heart tissue in SOD1 Tg rat. The tissue homogenates from
various brain regions and from heart tissue were subjected to
isoelectric focusing gel electrophoresis. The short
arrow indicates the homodimer of rat CuZn SOD. The long
arrow indicates the homodimer of the human CuZn SOD. The band
between the rat and human CuZn SOD homodimers is the rat/human
heterodimer.
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The cerebral vasculature was analyzed to confirm that both anatomical
backgrounds were the same in SOD1 Tg rats and non-Tg littermates.
Cerebral vasculature was determined by carbon black injection (Fig.
3). The method used herein allowed us to
determine the structure of major blood vessels (Fig. 3A,B)
in the brain and the posterior communicating artery (Fig.
3C,D) that influence the ischemic status in global ischemia.
There was no remarkable difference between SOD1 Tg rats and non-Tg
littermates.
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Figure 3.
Photographs showing similarity in cerebral
vasculature in non-Tg littermates (left) and SOD1 Tg
rats (right). The major blood vessels involved in the
circle of Willis were almost the same in both animals (A,
B). The plasticity of the posterior communicating artery
(arrows) that influences the outcome of hippocampal
injury after global ischemia was similar between the two animals
(C, D). ACA, Anterior cerebral artery; MCA, middle
cerebral artery; PCA, posterior cerebral artery; SCA, superior
cerebellar artery; BA, basilar artery.
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Production of O2 during global
cerebral ischemia
Before the use of SOD1 Tg rats for transient global cerebral
ischemia studies, we first wanted to verify the increased production of
O2 in the brains of animals after transient cerebral
ischemia, especially in the areas with vulnerable neurons (i.e.,
hippocampal CA1 subregion). Therefore, production of
O2 was determined using HEt in the cortex and
hippocampal CA1 and CA3 subregions at 1 hr and
1 and 3 d after 10 min global ischemia. As previously observed by
Kondo et al. (1997a), O2 production was shown by
oxidized HEt signals as small particles in the cytosol, suggesting
possible mitochondrial production of O2 under normal
physiological conditions (Fig.
4A-C).
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Figure 4.
Superoxide radical imaging in ischemic brain.
Representative photographs showing the production of
O2 by the presence of oxidized HEt in cortex
(left), hippocampal CA1
(middle), and CA3 (right)
subregions. As previously observed by Kondo et al. (1997a),
O2 production was demonstrated under normal
physiological conditions by oxidized HEt signals appearing as small
particles in the cytosol, suggesting possible mitochondrial production
of O2 (A-C). At 1 hr
after ischemia and reperfusion, the diffuse cytosolic oxidized HEt
signal was observed in cortical cells (D).
Although a high background could be observed, the cytosolic signals
were not increased in the hippocampal CA1
(E) and CA3 (F)
subregions. At 1 d after ischemia, the diffuse cytosolic signals
attenuated to preischemic levels in cortical cells except for
endothelial cells (G). However, the marked
diffuse cytosolic signal remained in the hippocampal CA1
subregion (H) but not in the
CA3 subregion (I). The diffuse
signal was not found at 3 d after ischemia
(J-L). Scale bar, 200 µm (lower
magnification); 20 µm (higher magnification).
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At 1 hr after ischemia and reperfusion, the increase in the diffuse
cytosolic signal of oxidized HEt was markedly observed in cortical
cells (Fig. 4D). In the hippocampus the diffuse
cytosolic signal of oxidized HEt was also observed, but the intensity
was similar to that of the preischemic level (Fig.
4E,F). At 1 d after ischemia, the diffuse
cytosolic signals in cortical cells returned to the preischemic level,
and, except for the endothelial cells (Fig. 4G), the marked
expression of the signal was present in the hippocampal CA1
subregion (Fig. 4H). The cytosol was filled with
diffuse oxidized HEt fluorescence in these cells. However, signals were
not observed in the CA3 subregion (Fig.
4I). The oxidized HEt signals decreased at 3 d
after ischemia (Fig. 4J-L). The
observation of increased production of O2 in
vulnerable brain regions prompted us to proceed to the use of SOD1 Tg
rats for the transient global cerebral ischemia studies.
Delayed neuronal injury after transient global
cerebral ischemia
There were no marked differences in mortality between SOD1 Tg rats
(10%) and non-Tg littermates (7.7%) within 72 hr after global
ischemia and reperfusion. No significant difference was observed in any
physiological parameters between the two groups (Table
2). As shown in Figure
5, the ischemic damage can be defined by
cresyl violet staining. Ischemic change and severe brain swelling at
3 d after global ischemia were seen in the non-Tg littermates (Fig. 5A). In contrast, this damage was reduced in the SOD1
Tg rats. Additionally, the quantitative analysis of the area of
hemisphere at the level of the posterior commissure clearly showed that
the swelling was markedly reduced in SOD1 Tg rats (57.3 ± 0.8 mm2) compared with non-Tg littermates (62.2 ± 1.2; p < 0.01). The pyramidal neurons were not clearly
stained in the non-Tg littermates, especially in the hippocampal
CA1 subregion (Fig. 5C,E). However, many CA1 neurons in the SOD1 Tg rats were clearly observed
as being without degeneration (Fig.
5D,F). The grading data of
the neuronal damage were significantly reduced in the cortex of SOD1 Tg
rats compared with non-Tg littermates, striatum, thalamus, and in the
hippocampal CA1 subregion, whereas no significant changes were seen after ischemia and reperfusion in the dentate gyrus (Fig.
6A). There was no
significant reduction of injuries in the CA3 subregion,
although a trend toward significance was observed. This was because
damage to the CA3 subregion did occur in the SOD1 Tg rats.
The quantitative analysis of the damaged area of the CA1
subregion showed a markedly protective effect against global ischemia
in SOD1 Tg rats compared with non-Tg littermates 3 d after
ischemia (Fig. 6B). However, no significant
differences were detected 1 d after reperfusion.
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Figure 5.
Microscopic photographs showing neuronal damage
stained with cresyl violet in SOD1 Tg rats and non-Tg littermates
3 d after transient global ischemia. More severe edema was
observed in non-Tg littermates (A) than in Tg
rats (B). The CA1 pyramidal cell
layer in non-Tg littermates was weakly stained by cresyl violet
(C, arrowheads), and the nuclei of
CA1 neurons were shrunken (E,
arrowheads) compared with those of SOD1 Tg rats
(D, F, arrows). Scale
bars: A, B, 1 mm; C,
D, 500 µm; E, F, 10 µm.
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Figure 6.
Neuronal injury after transient global cerebral
ischemia in rats. A, Data showing the scatterplot of the
neuropathological scores for a variety of brain areas for SOD1 Tg rats
(closed circles, n = 8) and non-Tg
littermates (open circles, n = 8)
after global ischemia of 10 min and reperfusion of 3 d. Each
column shows the mean score of injury of each group.
*p < 0.03, **p < 0.01, significantly different from non-Tg littermates (Mann-Whitney
U test). B, The quantitative analysis of
the damaged area of the CA1 subregion at 1 and 3 d
after 10 min of global ischemia, and reperfusion showed a marked
increase in the size of the damaged area between 1 and 3 d in both
groups. The protective effect of CuZn SOD against global ischemia was
significant in SOD1 Tg rats (48.4 ± 11.7%; closed
bar) compared with non-Tg littermates (87.3 ± 1.7%;
open bar; n = 6, p < 0.01) at 3 d after ischemia. However, no
significant differences were observed 1 d (n = 4) after ischemia. Values are mean ± SE (Fisher's protected
least significant difference test followed by nonparametric
t test).
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DNA fragmentation
To determine the role of CuZn SOD in DNA fragmentation in delayed
neuronal damage, we used in situ TUNEL staining to evaluate the contribution of DNA fragmentation to neuronal damage after global
ischemia. We did not observe DNA-fragmented nuclei labeled by TUNEL
staining at 1 d after global ischemia (Fig.
7A,B). At 3 d after
global ischemia, the TUNEL-positive cells were restricted to the
hippocampal CA1 subregion (Fig. 7C,D), and the
number of TUNEL-positive cells observed in the CA1
subregion of the non-Tg littermates (Fig. 7C) was much
greater than in the SOD1 Tg rats (Fig. 7D).
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Figure 7.
DNA fragmentation. Representative photographs
showing DNA fragmentation by TUNEL staining at 1 d (A,
B) and 3 d (C, D) after 10 min of global
ischemia and reperfusion. The DNA-fragmented cells labeled by TUNEL
staining were restricted to the hippocampal CA1 subregion
at 3 d after global ischemia, but not at 1 d after global
ischemia. A marked number of TUNEL-positive cells was observed in the
CA1 subregion in non-Tg littermates
(C), compared with SOD1 Tg rats
(D). Scale bar, 500 µm. Quantitative analysis
showed the ratio of TUNEL-positive neurons in the hippocampal
CA1 subregion after global ischemia and reperfusion. The
ratio was significantly ameliorated in Tg rats (closed
bar) compared with non-Tg littermates (open bar)
at 3 d (n = 6; p < 0.03).
However, the ratio was the same between Tg and non-Tg rats at 1 d
(n = 4). Values are mean ± SE (Fisher's
protected least significant difference test followed by nonparametric
t test). E, Quantitative analysis of
TUNEL-positive cells in the hippocampal CA1 subregion in
SOD1 Tg rats and non-Tg littermates after transient global cerebral
ischemia. TUNEL-positive cells in the hippocampal CA1
subregion were counted by imaging analysis. There was a significant
reduction in these DNA-fragmented cells in SOD1 Tg rats as compared
with non-Tg littermates at 3 d after 10 min of ischemia.
*p < 0.03, Fisher's protected least significant
difference test followed by nonparametric t test.
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To determine the temporal pattern of DNA fragmentation after global
ischemia, we counted TUNEL-positive cells in the hippocampal CA1 subregion at 1 and 3 d (Fig. 7E).
Quantitative analysis showed that the TUNEL-positive neurons in the
hippocampal CA1 subregion did not appear 1 d after
global ischemia and reperfusion. Although DNA-fragmented cells were
observed 3 d after global ischemia in both groups, DNA
fragmentation damage was significantly ameliorated in the Tg rats
compared with the non-Tg littermates (n = 6, p < 0.03, Fisher's protected least significant
difference test, followed by the nonparametric t test).
However, the same level of TUNEL-stained cells was observed between the
Tg and non-Tg rats 1 d after reperfusion (n = 4).
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DISCUSSION |
Early superoxide radical formation in hippocampal CA1
subregion after transient global cerebral ischemia in rats
It is well established that transient global cerebral ischemia
manifested by cardiac arrest causes selective neuronal death in
vulnerable regions, such as hippocampal CA1 pyramidal
cells, Purkinje cells of the cerebellum, and neurons in the third to fifth layers of the cerebral cortex (Kirino, 1982; Pulsinelli et al.,
1982). The cell death mechanisms of these vulnerable neurons after
transient cerebral ischemia have been extensively studied and are
attributed to many factors, including glutamate neurotoxicity, calcium,
expression of various genes, mitochondrial dysfunction, and oxygen free
radicals (Abe et al., 1995; Ito et al., 1997). The oxygen free radical
hypothesis is especially attractive because of the phenomenon of oxygen
radical production, superoxide radicals in particular, that is
associated with reperfusion injury (McCord, 1985; Chan, 1996). However,
it is technically difficult to prove or disprove this hypothesis
because of the lack of a quantitative method for oxygen radical
measurement in the ischemic brain and the lack of a direct correlation
between the increased antioxidant levels and their neuroprotection in
experimental animal models of transient global cerebral ischemia (Chan
et al., 1996).
To address the first issue, we have developed an in situ
imaging method for superoxide radical measurement (Kondo et al., 1997a;
Murakami et al., 1998b). This method is based on the selective oxidation of HEt by superoxide radicals (Bindokas et al., 1996). At 1 hr after 10 min of ischemia and reperfusion, we observed diffuse cytosolic expression of oxidized HEt signals in
cortical cells (Fig. 4D). These oxidized HEt signals
were significantly increased in vulnerable hippocampal CA1
neurons, whereas they were not observed in the ischemia-resistant
CA3 neurons. The increased level of superoxide radicals
precedes the occurrence of the majority of neuronal cell deaths and DNA
fragmentation detected 3 d after reperfusion in rats (Figs. 5, 7).
These data prompted our further investigation of the role of SOD1 in
CA1 neuroprotection after transient global cerebral
ischemia.
The making of SOD1 Tg rats for transient global
cerebral ischemia
CuZn SOD has been extensively used in attempts to reduce brain
injury induced by ischemia and reperfusion. Various degrees of success
and failure were obtained in neuroprotection when exogenous CuZn SOD
was used (Chan et al., 1993). However, the neuroprotective role of CuZn
SOD in transient focal cerebral ischemia was found in Tg mice
overexpressing SOD1 activity (Kinouchi et al., 1991; Yang et al.,
1994). Conversely, increased neuronal injury was observed in knock-out
mutant mice deficient in SOD1 activity (Kondo et al., 1997b). However,
the extremely high mortality of mice that were subjected to global
cerebral ischemia and the variability in the anatomy of the
cerebrovasculature and in the genetic background of the Tg and
knock-out mutant mice prompted us to produce SOD1 Tg rats. Using
standard pronuclear DNA injection in rat embryos, we have succeeded in
making several strains of transgenic rats that overexpress human CuZn
SOD activity in the brain and in other systemic organs (E. Carlson, C. Epstein, and P. H. Chan, unpublished data). A heterozygous Tg strain
(number 66 UCSF) that overexpresses CuZn SOD has been fully
characterized by FISH (Fig. 1), IFGE (Fig. 2), and by direct enzymatic
assay (Table 1). Additional Southern blots and reverse transcriptase
PCRs have been used during the breeding to confirm both the SOD1
gene and the mRNA, respectively (data not shown). There were no
observable phenotypic differences between the SOD1 Tg rats and the
littermates.
Reduction of neuronal death and DNA fragmentation in vulnerable
hippocampal CA1 neurons after transient global ischemia
When non-Tg rats were subjected to 10 min ischemia they developed
selective neuronal damage at 3 d in vulnerable regions, including
the hippocampal CA1 subregion, cerebral cortex, thalamus, and striatum, whereas the neurons in the dentate gyrus were not affected (Fig. 6A). The hippocampal CA1
subregion was the most vulnerable region, and 90% of the
CA1 neurons were lost (Fig. 6A,B). In
contrast, neuronal damage in various brain regions was significantly
reduced in SOD1 Tg rats (Fig. 7A,B)
at 3 d after reperfusion. The reduction of cell damage in the
CA1 region was ~50% (Fig. 6B) in Tg
rats. Although there was no significant cell damage in the
CA1 subregion at 1 d after reperfusion in either SOD1
Tg rats or their littermates (Fig. 6B), the
significant increase at 3 d in the non-Tg animals suggests that a
delayed neurodegeneration occurs in the vulnerable CA1
neurons and that increased endogenous SOD1 activity can significantly
prevent cell death. Although a neuroprotective effect has been observed
in vulnerable neurons of SOD1 Tg rats after transient global cerebral
ischemia, it is not clear whether this neuroprotection is permanent.
Such issues can be addressed by future studies of long-delayed cell
injury and recovery after ischemia (i.e., 6 months).
The mechanisms of oxidative stress-induced delayed death of hippocampal
CA1 pyramidal cells after transient global cerebral ischemia are unclear. Recent studies, although still somewhat controversial, have identified some apoptotic features by biochemical and morphological evidence such as TUNEL (MacManus et al., 1993; Sei et al., 1994; Petito et al., 1997) and internucleosomal DNA fragmentation as indicated by the DNA laddering pattern (Héron et
al., 1993). Because TUNEL staining indicates DNA damage, and its
specificity for apoptosis is questionable, we have used TUNEL staining
only as an indication of DNA damage in cells. Whereas only a small
fraction of cells (<5%) are TUNEL-positive 1 d after reperfusion, we have demonstrated a tremendously delayed increase in
TUNEL-positive cells in the hippocampal CA1 subregion in
wild-type animals 3 d after reperfusion (Fig. 7E) with
up to 40% of the total cells being TUNEL-positive. The TUNEL-positive
cells are likely caused by the increased level of superoxide radicals
during reperfusion because <20% of the hippocampal CA1
cells are TUNEL-positive in SOD1 Tg rats at 3 d after reperfusion.
Our data also suggest that if TUNEL-positive cells are mainly apoptotic
in nature, the damage to hippocampal CA1 neurons would
involve both necrosis (60%) and apoptosis. However, much more
stringent criteria for apoptosis (i.e., DNA laddering, caspases
induction, cytochrome c release, and ultrastructural features) will be
needed in future studies so that the apoptosis process can be
confirmed. Whatever cell death processes are involved in the delayed
death of hippocampal CA1 neurons, it is mediated by
superoxide radicals. Because only a 50% reduction in cell death is
achieved in SOD1 Tg rats after transient global cerebral ischemia,
mechanisms or factors other than superoxide radicals are likely to be
involved. It is also noteworthy that superoxide production in the
hippocampal CA1 subregion is well developed 24 hr after
ischemia, at a time when neuronal damage is not yet observed. This
might suggest that superoxide radicals do not immediately damage these
neurons but that an interval of time is required for the full
expression of the injury. This delayed cell injury might provide a
window of opportunity for therapeutic interventions using antioxidants.
Additional therapeutic or pharmacological regimens in SOD1 Tg rats will
be useful to further dissect the mechanisms involved in delayed
vulnerable cell death after transient global cerebral ischemia.
Our success in making SOD1 Tg rats also provides an impetus for stroke
researchers and neuroscientists to develop and to employ these animals
for studying the oxidative mechanisms in acute brain injuries and
chronic neurodegenerative diseases. Some of these studies are currently
being undertaken in our laboratory.
| |
FOOTNOTES |
Received June 8, 1998; revised July 20, 1998; accepted July 30, 1998.
This work was supported by National Institutes of Health contract,
"Transgenic Rat for Stroke Research", NO1-NS-5-2334 and NO1-NS-8-2386 (P.H.C.), and National Institutes of Health Grants NS
14543 (P.H.C.), NS 25372 (P.H.C.), NS 36147 (P.H.C.), and AG 08938 (C.J.E., P.H.C.). P.H.C. is a recipient of the Jacob Javits Neuroscience Investigator Award. We thank Cheryl Christensen for editorial assistance.
Correspondence should be addressed to Dr. Pak H. Chan, Neurosurgical
Laboratories, Stanford University, 701B Welch Road, #148, Palo Alto, CA
94304.
| |
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Overexpression of Copper-Zinc Superoxide Dismutase Attenuates Acute Activation of Activator Protein-1 After Transient Focal Cerebral Ischemia in Mice
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K. Sampei, A. S. Mandir, Y. Asano, P. C. Wong, R. J. Traystman, V. L. Dawson, T. M. Dawson, P. D. Hurn, and C. Y. Hsu
Stroke Outcome in Double-Mutant Antioxidant Transgenic Mice Editorial Comment
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N. Derugin, M. Wendland, K. Muramatsu, T. P. L. Roberts, G. Gregory, D. M. Ferriero, Z. S. Vexler, and W. D. Dietrich
Evolution of Brain Injury After Transient Middle Cerebral Artery Occlusion in Neonatal Rats Editorial Comment
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M. Fujimura, Y. Morita-Fujimura, N. Noshita, T. Sugawara, M. Kawase, and P. H. Chan
The Cytosolic Antioxidant Copper/Zinc-Superoxide Dismutase Prevents the Early Release of Mitochondrial Cytochrome c in Ischemic Brain after Transient Focal Cerebral Ischemia in Mice
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M. Fujimura, Y. Morita-Fujimura, P. Narasimhan, J.-C. Copin, M. Kawase, P. H. Chan, and C. Y. Hsu
Copper-Zinc Superoxide Dismutase Prevents the Early Decrease of Apurinic/Apyrimidinic Endonuclease and Subsequent DNA Fragmentation After Transient Focal Cerebral Ischemia in Mice • Editorial Comment
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M. Kawase, K. Murakami, M. Fujimura, Y. Morita-Fujimura, Y. Gasche, T. Kondo, R. W. Scott, P. H. Chan, and M. S. Wolin
Exacerbation of Delayed Cell Injury After Transient Global Ischemia in Mutant Mice With CuZn Superoxide Dismutase Deficiency • Editorial Comment
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H. Sheng, D. T. Laskowitz, G. B. Mackensen, M. Kudo, R. D. Pearlstein, D. S. Warner, and C. Iadecola
Apolipoprotein E Deficiency Worsens Outcome From Global Cerebral Ischemia in the Mouse • Editorial Comment
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M. Kawase, M. Fujimura, Y. Morita-Fujimura, P. H. Chan, and C. Iadecola
Reduction of Apurinic/Apyrimidinic Endonuclease Expression After Transient Global Cerebral Ischemia in Rats : Implication of the Failure of DNA Repair in Neuronal Apoptosis • Editorial Comment: Implication of the Failure of DNA Repair in Neuronal Apoptosis
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K. Kitagawa, M. Matsumoto, Y. Tsujimoto, T. Ohtsuki, K. Kuwabara, K. Matsushita, G. Yang, H. Tanabe, J.-C. Martinou, M. Hori, et al.
Amelioration of Hippocampal Neuronal Damage After Global Ischemia by Neuronal Overexpression of BCL-2 in Transgenic Mice • Editorial Comment
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T. Sugawara, M. Fujimura, Y. Morita-Fujimura, M. Kawase, and P. H. Chan
Mitochondrial Release of Cytochrome c Corresponds to the Selective Vulnerability of Hippocampal CA1 Neurons in Rats after Transient Global Cerebral Ischemia
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K.-M. Noh and J.-Y. Koh
Induction and Activation by Zinc of NADPH Oxidase in Cultured Cortical Neurons and Astrocytes
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Top
Abstract
Introduction
Compound via MeSH
