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The Journal of Neuroscience, December 1, 1998, 18(23):9727-9732
Attenuation of Focal Ischemic Brain Injury in Mice Deficient in
the  1 (NR2A) Subunit of NMDA Receptor
Eiharu
Morikawa1, 3, 4,
Hisashi
Mori2,
Yuji
Kiyama2,
Masayoshi
Mishina2, 4,
Takao
Asano3, and
Takaaki
Kirino1, 4
Departments of 1 Neurosurgery and
2 Molecular Neurobiology and Pharmacology, University of
Tokyo Faculty of Medicine, Bunkyo-ku, Tokyo 113-8655, Japan,
3 Department of Neurosurgery, Saitama Medical
School/Center, Kawagoe, Saitama 350-8550, Japan, and 4 Core
Research for Evolution and Technology, Japan Science and Technology
Corporation, Kawaguchi, 332-0012, Japan
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ABSTRACT |
The role of glutamate neurotoxicity in cerebral ischemia has long
been advocated but still remains controversial, because various
glutamate receptor (GluR) antagonists showed inconsistent protective
efficacy in brain ischemia models. To address this central issue of
ischemic brain damage more directly, we used mutant mice deficient in
the GluR 1 (NR2A) subunit of NMDA receptor with or without
additional heterozygous mutation in the GluR2 (NR2B) subunit. Those
mutant mice, as well as their littermates, were subjected to focal
cerebral ischemia by introducing a 6-0 nylon suture from left
common carotid artery. Brain injury volumes after 2 hr of suture
insertion, as evaluated by 2,3,5-triphenyltetrazolium chloride staining
at 24 hr after ischemia, revealed significantly smaller injury size in
GluR1 subunit knock-out mice compared with their wild-type
littermates. The reduction in injury volume was not attributable
to differences in body temperature or in blood flow during ischemia.
Additional heterozygous GluR2 subunit disruption did not result in
further reduction in injury volume. These data directly demonstrate
relevance of NMDA receptor-mediated tissue injury after brain ischemia
and provide evidence that GluR1 subunit is involved in these
injurious mechanisms.
Key words:
NMDA receptor; GluR1 subunit; neurotoxicity; glutamate; brain ischemia; cerebral infarction
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INTRODUCTION |
Brain ischemia causes massive
elevation of extracellular glutamate concentration. An excess amount of
glutamate is postulated to induce glutamate receptor (GluR)-mediated
cascade reactions, which eventually lead to brain damage (Choi, 1988 ;
Lipton and Rosenberg, 1994). Molecular cloning studies have identified
five NMDA receptor subunits called GluR 1 (NR1) and
GluR1-GluR4 (NR2A-NR2D) (Nakanishi, 1992; Seeburg, 1993, Mori
and Mishina, 1995). Among these five subunits, GluR1, GluR1
(NR2A), and GluR2 (NR2B) are the major components of the
hetero-oligomeric NMDA receptor channel complex distributed in the
mature forebrain (Watanabe et al., 1992). The GluR1 and GluR2
subunits are the constituents of the receptor molecule during stages of
embryonic development. Mice with a null mutation in the GluR1 or the
GluR2 subunit gene die shortly after birth and have a defect in the
formation of neuronal pattern in the brainstem (Forrest et al., 1994;
Li et al., 1994; Kutsuwada et al., 1996). In contrast, GluR1 subunit is expressed only in the postnatal brain, and mice lacking the gene for
this subunit (1 /) develop and mate normally.
Detailed electrophysiological and behavioral evaluation, however, has
revealed that the GluR1 subunit knock-out (1KO) mice have reduced
hippocampal long-term potentiation (LTP) and impaired spatial and
contextual learning, which confirms that disruption of this gene
disturbs normal NMDA receptor functions (Sakimura et al., 1995; Kiyama
et al., 1998). We subjected these mice to focal cerebral ischemia to
investigate the significance of the GluR1 subunit of NMDA receptor
in the development of tissue injury after brain ischemia. Furthermore,
we mated these 1KO mice with other genetically engineered mice
harboring a heterozygous GluR2 mutation
(1+/+2+/, Kutsuwada et
al., 1996) to create mice with both a null mutation in the GluR1
gene and a heterozygous mutation in the GluR2 gene (1/2+/, 1KO2H).
To study the significance of the remaining GluR2 subunit in 1KO
mice, we also evaluated the development of ischemic brain injury in
these double-mutant mice.
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MATERIALS AND METHODS |
Production of mice with GluR1 and/or GluR2 subunit
gene mutation. Generation and characterization of the GluR1
subunit knock-out (Sakimura et al., 1995) and GluR2 subunit
knock-out (Kutsuwada et al., 1996) in TT2 embryonic stem cells,
followed by back-crossing with C57BL/6 mice, have been described
elsewhere. Mice with GluR1 homozygous and GluR2 heterozygous
mutation were produced by mating the homozygous GluR1 mutant mice
(1/2+/+) with GluR2
heterozygous mutant mice
(1+/+2+/). The resultant
heterozygous mutants for both GluR1 and GluR2 (1+/2+/) were crossed
with GluR1 homozygous mutants
(1/2+/+) to generate
GluR1 homozygous and GluR2 heterozygous mutant mice
(1/2+/). The
offspring mice
(1/2+/) were mated
again with GluR1 homozygous mutants
(1/2+/+) to expand the
colony of GluR1 homozygous and GluR2 heterozygous mutant mice
(1/2+/) and
homozygous mutant mice for GluR1
(1/2+/+).
Western blot analysis of GluR1 and GluR2 subunit proteins
in the forebrain. Western blot analysis was done essentially as described previously (Sakimura et al., 1995; Takahashi et al., 1996;
Watanabe et al., 1998). In brief, the wild-type (WT) and mutant mice
were decapitated under anethesia, and each forebrain was removed
rapidly and homogenized. The proteins (100 mg) prepared from each
forebrain homogenate were fractionated by SDS-PAGE and electroblotted
onto a nitrocellulose membrane. The blots were immunoreacted with
anti-GluR1, anti-GluR2, or anti-neuron-specific enolase (NSE)
antibody, and the protein bands were visualized by
chemiluminescence (ECL detection system; Amersham, Arlington Heights, IL). The rabbit anti-GluR1, anti-GluR2, and
anti-NSE antibodies were prepared previously (Sakimura et al., 1980,
1995; Watanabe et al., 1998). For quantitative analysis, the
immunoreactive bands were scanned using a computing densitometry and
analyzed with NIH Image software (version 1.58). The quantity of the
immunoreactive bands of GluR1 and GluR2 proteins were corrected
by comparing them with the band of NSE protein as an internal standard
for the amount of protein loaded onto the gels.
Production of focal cerebral ischemia. Nonfasted mice of
either sex were subjected to cerebral ischemia by an investigator (E.M.) blinded to the group identity. Anesthesia was induced and maintained by halothane (3 and 1%, respectively) plus 50% nitrous oxide and the balance of oxygen under spontaneous ventilation. Because
repeated blood withdrawal from mice was not compatible with further
ischemia experiments, the samples for physiological data (arterial
pO2, pCO2, pH, and blood glucose)
were taken from a separate set of animals (n = 5 each).
These physiological parameters were maintained within normal range
under the setting of ischemia induction, and there were no differences
between the groups. Focal cerebral ischemia was produced as described
previously (Huang et al., 1994). After ligation of the left proximal
common carotid artery, a 6-0 nylon monofilament (Ethilon;
Ethicon, Inc., Somerville, NJ) with a heat-modified head was introduced
into the distal internal carotid artery and was advanced ~11 mm
distal to the carotid bifurcation. The filament was wedged into the
circle of Willis, producing focal cerebral ischemia in the left middle
cerebral artery territory. The wound was closed, and the animal was
returned to its cage. In transient focal cerebral ischemia, the nylon
filament was withdrawn from the carotid artery 2 hr after insertion
under halothane anesthesia. During the surgical procedure, the rectal
temperature was kept normothermic (36.5-37.5°C) by a heating pad and
a heating lamp (ATB-1100; Nihon Koden, Tokyo, Japan).
Evaluation of tissue injury after ischemia. Twenty-four
hours after arterial occlusion, the mice were anesthetized with 3% halothane and decapitated. Brains were removed immediately and placed
in ice-cold saline for 10 min and sectioned coronally into 5 slices
(2-mm-thick) in a rodent brain matrix (Mouse Brain Slicer; Medical
Agent Co. Ltd., Tokyo, Japan). Brain slices were placed in 2%
2,3,5-triphenyltetrazolium chloride monohydrate (TTC) (Sigma, St.
Louis, MO) at room temperature for 30 min (Bederson et al., 1986),
followed by 10% formalin overnight. Brain slices were directly scanned
on an image scanner (JX-320M; Sharp, Tokyo, Japan). The area of
ischemic injury was measured (NIH Image software) on the posterior surface of each section. The injury volume was calculated by
numeric integration of the sequential areas. Eight mice could not
survive for 24 hr after ischemia. Because exclusion of these mice could
affect overall effects of the gene mutation on the ischemic injury, the
brains of those animals were removed as soon as the animal's death was
confirmed, and the size of the brain injury was evaluated by the same
method as above.
Genotyping of mice by Southern blot hybridization analysis.
Genotyping of the mice that were used for the brain injury analysis was
done in a completely blind manner by another investigator (H.M.).
Genotype of each mouse was identified by Southern blot hybridization
analysis of the genomic DNA extracted from tail specimen taken at the
time of death as described above. In brief, Southern blot
hybridization analysis of the GluR1 locus was done using
BamHI-digested tail DNA hybridized with a 1.2 kb
SmaI-SmaI fragment of the GluR1 gene (Sakimura
et al., 1995). Southern blot hybridization analysis of the GluR2
locus was performed using EcoRI-digested tail DNA hybridized
with a 0.4 kb HindIII-XbaI fragment of the
GluR2 gene (Kutsuwada et al., 1996).
Evaluation of regional cerebral blood flow by laser-Doppler
flowmetry. Under halothane anesthesia with spontaneous ventilation as described above, regional cerebral blood flow (CBF) at
dorsolateral cerebral cortex was determined by laser-Doppler flowmetry
(LBF-IIIF; Biomedical Science, Tokyo, Japan) as demonstrated previously
(Morikawa et al., 1996). After exposing and thinning the skull at 1 mm
posterior and 4 mm lateral to the bregma, the head of the mouse was
fixed in a stereotaxic frame (Narishige, Tokyo, Japan) in supine
position, and the tip of the flowmetry probe (0.8 mm in diameter) was
secured perpendicular to the skull surface. After baseline measurements were obtained, the left common carotid artery was ligated, and the
nylon suture was inserted. Regional CBF was measured for 30 min after
suture insertion.
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RESULTS |
Expression of the GluR1 and GluR2 subunit proteins in
mutant mice
We first examined the expression levels of GluR1 and GluR2
subunit proteins in the forebrain of the WT and mutant mice using Western blot analysis. The intensities of immunoreactive bands of
GluR1 and GluR2 proteins were quantitatively analyzed and compared with those of NSE, a marker protein of neurons (Schmechel et
al., 1978), as an internal standard. The amounts of GluR1 and
GluR2 subunit proteins were comparable between male and female WT
mice and between male and female 1KO mice, indicating that there was
no appreciable gender difference in the expression of GluR1 and
GluR2 proteins (Fig.
1A). These analyses
also confirmed the absence of GluR1 protein in 1KO and
1KO2H mice (Fig. 1A,B). The
expression of GluR2 protein in 1KO mice was not appreciably affected by the GluR1 gene disruption, whereas the amount of GluR2 protein in 1KO2H mice was decreased to approximately half of that in WT littermates as reported previously (Fig.
1B) (Sakimura et al., 1995; Kutsuwada et al.,
1996).
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Figure 1.
A, Western blot analysis of
forebrain proteins from WT and 1KO mice with anti-GluR1
(top), anti-GluR2 (middle), and
anti-NSE (bottom) antibodies. Samples from two males
(M) and two females
(F) of each genotype were presented.
B, Western blot analysis of forebrain proteins from WT,
1KO, and 1KO2H male mice with anti-GluR1
(top) and anti-GluR2 antibodies
(bottom).
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Brain injury volumes after ischemia
A total of 105 mice were subjected to focal cerebral
ischemia by occluding the left middle cerebral artery using nylon
filament. The brain injury volume after permanent focal cerebral
ischemia was not significantly different between 1KO and WT mice
[91 ± 14 (n = 13) and 108 ± 8 (n = 14) mm3, respectively].
Although there was a trend toward smaller injury volumes in female
1KO mice, there was not a statistically significant difference
between males and females (Fig. 2). Two
male WT mice died at 19.5 and 16.5 hr of ischemia, and their injury
volumes were 149 and 141 mm3, respectively. There
was not a premature mortality in 1KO mice.
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Figure 2.
Cerebral injury volumes after permanent focal
cerebral ischemia. There were no statistically significant
differences in injury volumes between WT and 1KO mice in either
males (n = 8 and 4 for WT and 1KO, respectively)
or females (n = 6 and 9 for WT and 1KO,
respectively). Values are expressed as mean ± SEM.
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After 2 hr of transient focal ischemia, 1KO mice developed
significantly smaller brain injury volumes compared with WT mice [41 ± 7 (n = 19) and 87 ± 8 (n = 28) mm3 for 1KO and WT,
respectively; p = 0.0004; unpaired t test]. When the data were analyzed separately with regard to gender, the
difference was more prominent in females [30 ± 9 (n = 7) and 94 ± 12 (n = 14)
mm3, respectively; p = 0.0039;
unpaired t test] than in males [48 ± 10 (n = 12) and 80 ± 10 (n = 14)
mm3, respectively; p = 0.044;
unpaired t test] (Fig. 3).
There was, however, no statistically significant difference in injury
volumes between males and female in either WT or 1KO mice. Five WT
mice (one male and four females) died between 18 and 22 hr of ischemia and had large injury volumes (between 135 and 187 mm3). On the other hand, there was no premature
mortality in 1KO mice. The difference in mortality rate reached
statistical significance when the data of the above two studies were
combined (p < 0.05;  2
test).
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Figure 3.
Cerebral injury volumes after 2 hr of transient
focal cerebral ischemia. 1KO mice had significantly smaller injury
volume compared with WT littermate mice in both males
(n = 14 and 12 for WT and 1KO, respectively;
*p = 0.044) and females (n = 14 and 7 for WT and 1KO, respectively; **p = 0.0039). Values are expressed as mean ± SEM.
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The injury volume in 1KO2H mice after 2 hr of transient focal
ischemia was similar to those of 1KO mice, and there was no
additional reduction of injury volume by further disrupting one copy of
the GluR2 subunit gene [34 ± 8 (n = 17) and
43 ± 9 (n = 14) mm3 for
1KO2H and 1KO, respectively] (Fig.
4). One male 1KO2H mouse died at 22 hr of ischemia and had an injury volume of 28 mm3.
There was no premature mortality in 1KO mice.
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Figure 4.
Cerebral injury volumes after 2 hr of transient
focal cerebral ischemia compared with 1KO2H mice and their
littermates 1KO. There were no significant differences between these
two groups in either males (n = 6 each) or females
(n = 8 and 11 for 1KO and 1KO2H,
respectively). Values are expressed as mean ± SEM.
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When the injury size was examined in 2-mm-thick consecutive slices, the
reduction of ischemic injury in mutant mice compared with WT mice was
evident in all five slices (p < 0.01; one-way ANOVA, followed by Fisher's PLSD test) (Fig.
5A). Notably, the attenuation
of injury was more evident in the dorsolateral cerebral cortex, which
is the area of milder ischemia called "ischemic penumbra" (Fig.
5B,C).
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Figure 5.
A, Areas of cerebral injury on each
brain slice after 2 hr of transient focal cerebral ischemia. Data from
two experiments are combined [1KO (n = 33),
1KO2H (n = 17), and WT (n = 28)]. Reduction of injury area was observed on all the brain slices
in 1KO and 1KO2H mice compared with WT
(p < 0.01; one-way ANOVA, followed by
Fisher's PLSD test). Values are expressed as mean ± SEM.
B, Spatial distribution of injury areas on five brain
slices. Black areas indicate regions of brain injury
observed in most of the animals (>70%) in the group. Dark
gray and light gray areas demonstrate
regions of injury observed less frequently (40-70 and 20-40%,
respectively). C, Representative scanned images of
TTC-stained brain slices analyzed for injury volume.
White areas indicate regions of tissue injury.
Calculated injury volumes for these particular animals were 100, 39, and 35 mm3 for WT, 1KO, and 1KO2H,
respectively.
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Regional CBF after brain ischemia
To exclude the possibility that gene disruptions could cause
different CBF after middle cerebral artery occlusion, which could influence the results, we measured regional CBF before and after the
occlusion in a separate set of animals (n = 5 in each
group). Regional CBF at dorsolateral cerebral cortex, as measured
continuously by laser-Doppler flowmetry, demonstrated reduction of CBF
by ~35% when common carotid artery was occluded. The CBF further
decreased to ~17% of baseline after insertion of the nylon suture,
which recovered and remained at a level of ~22% of baseline during
the first 30 min without any significant difference between the two groups (Fig. 6). Thus, the reduction in
ischemic injury volumes was not attributable to the regional CBF
differences in the dorsolateral cerebral cortex, or ischemic penumbra,
in the acute stage.
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Figure 6.
Regional CBF profiles measured at dorsolateral
cerebral cortex using laser-Doppler flowmetry after vessel occlusion
in WT (n = 5) and 1KO (n = 5) mice. Values are expressed as mean ± SEM. Regional CBF was
reduced by ~35% of baseline after common carotid artery occlusion.
Insertion of the nylon suture resulted in further reduction of the CBF
to ~20% of baseline for 30 min. No significant differences were
detected in CBF between 1KO and WT mice.
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DISCUSSION |
The present investigation clearly demonstrates for the first time
that mice lacking the GluR1 subunit of NMDA receptor channel are
less prone to cerebral injury after focal brain ischemia. Functional
NMDA receptor channel complexes contain both GluR and GluR
subunits. In the forebrain of mature mice, GluR constituents making
up the hetero-oligomeric receptors are primarily the GluR1 and
GluR2 subunits (Watanabe et al., 1992). Western blot analyses in
this study confirmed that the targeted disruption of the GluR1 subunit gene resulted in complete absence of GluR1 subunit protein in the brain homogenate and that there was no compensatory increase in
the content of GluR2 subunit protein. In fact, the NMDA receptor channel activity in the hippocampal CA1 region of 1KO mice is decreased to about half of that of WT mice, and correspondingly, thresholds for hippocampal LTP induction and contextual learning are
increased (Sakimura et al., 1995; Kiyama et al., 1998). Thus, a
straightforward explanation of our results will be that the decrease in
the number of functional NMDA receptor channels on the cell membrane of
forebrain neurons in 1KO mice has made cells more resistant to
glutamate-induced neurotoxicity. Consistent with this, it was reported
that the suppression of GluR1 subunit synthesis by administrating
antisense oligodeoxynucleotides reduced infarction volumes produced by
focal brain ischemia (Wahlestedt et al., 1993).
This explanation implies that the another constituent of NMDA
receptor channels, the GluR2 subunit, would also be involved in
glutamate-induced neurotoxicity. In agreement with this, it is
demonstrated that selective GluR2 subunit antagonist provides brain
protection in feline brain ischemia (Di et al., 1997). In our study,
however, further disruption of one copy of the GluR2 subunit gene in
1KO mice (1KO2H mice) appeared to have no additional protective effects against ischemia. It is possible that the extent of
reduction in GluR2 subunit protein may not be significant enough to
bring additional protective effects already bestowed by complete
deletion of the GluR1 gene.
Alternatively, the GluR1 subunit may indeed play a more pivotal role
in glutamate neurotoxicity than the GluR2 subunit. Interestingly, in
this regard, it is demonstrated that vulnerability of CA1 neurons to
glutamate is intrinsically less in newborns and more in 3-week-old
rats, suggesting that the expression of the GluR1 subunit, whose
expression occurs postnatally, increases the glutamate neurotoxicity
(Marks et al., 1996).
Differences in gating and pharmacological properties, as well as in
various modulations, are noted between GluR1/1 and GluR2/1 channels (Seeburg, 1993; Mori and Mishina, 1995). Calcium-dependent inactivation of NMDA receptor channels, observed in the cultured hippocampal neurons (Legendre et al., 1993), is shown to be
subunit-specific, requires the presence of the GluR1 subunit, and is
less pronounced in GluR2 subunit-containing heteromers (Krupp et
al., 1996). The offset decay time constant of the GluR1/1 channel
is ~120 msec and that of the GluR2/1 channel is ~400 msec
(Monyer et al., 1994). The duration of NMDA receptor channel-mediated
EPSCs in neurons of the visual cortex layer IV and superior
colliculus is longer at early developmental stages and becomes
progressively shorter (Hestrin, 1992; Carmignoto and Vicini, 1992).
Neurons of postnatal neocortex expressing the GluR1 subunit have
faster EPSCs, and the proportion of cells expressing this subunit
increases developmentally (Flint et al., 1997). The absence of the
GluR1 subunit, therefore, would lead to qualitative changes of NMDA receptor channel functions in the brain. In fact, alterations in NMDA
receptor channel properties have been demonstrated in 1KO mice. In
cerebellar granule cells, the decay time course of NMDA
receptor-mediated EPSCs was slower in the mutants compared with WT
(Takahashi et al., 1996). In hippocampal CA3 pyramidal neurons,
reduction in the NMDA receptor-mediated EPSCs was observed selectively
at the synapses formed by commissural-associational inputs (Ito
et al., 1997). These differences between GluR1 and GluR2
heteromers in receptor channel functions, however, do not necessarily
support the hypothesis that the GluR1 subunit is more involved in
neurotoxicity than the GluR2 subunit.
Although glutamate neurotoxicity has been implicated as a major
mechanism of ischemic brain injury (Choi, 1988; Lipton and Rosenberg,
1994), the effects of antagonists directed against NMDA receptors have
been unsatisfactory in clinical trials (Lipton, 1993; Schehr, 1996), as
well as in experimental animals (Buchan and Pulsinelli, 1990). In the
current study, significant reduction in injury volume was observed in
the GluR1 subunit-deficient mice in transient brain ischemia but not
in permanent ischemia in which only a trend toward smaller injury
volume was observed in females. It is also noted that the attenuation
of tissue injury was predominantly in the so-called ischemic penumbra
and was less in the core region. Thus, the resistance provided by the
GluR1 subunit ablation was prominent in mild to moderate ischemia
but less so in severe ischemic insult. This is in accordance with the
notion that mechanisms other than the NMDA receptor-mediated type may
become dominant in severe brain ischemia (Siesjo, 1992). It is
hypothesized that brain ischemia induces downregulation of the GluR2
subunit of AMPA receptors, which renders AMPA receptor channels
permeable to calcium and, consequently, increases glutamate-induced neurotoxicity after ischemia (Pellegrini-Giampietro et al., 1992; Gorter et al., 1997). Our results, however, indicate significant involvement of the NMDA receptor channel-mediated neurotoxicity in
focal cerebral ischemia and reperfusion.
Although it has been demonstrated that estrogens attenuate excitotoxic
injury (Goodman et al., 1996) and also upregulate NMDA receptors
(Woolley et al., 1997), which may exacerbate glutamate-induced neurotoxicity, statistically significant gender difference was not
observed in the current study in terms of ischemic injury. There was
not a significant gender difference in the amounts of GluR1 and
GluR2 subunit proteins in brain homogenate taken from WT or 1KO
mice, either. In any case, our data demonstrate direct evidence that
NMDA receptor channels, specifically the GluR1 subunit, play a major
role in ischemic brain injury in both males and females.
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FOOTNOTES |
Received May 7, 1998; revised Sept. 21, 1998; accepted Sept. 22, 1998.
This work was supported in part by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Science, and Culture of Japan.
We thank Dr. Keisuke Ueki for helpful comments and advice and Dr.
Nobuto Saito for technical assistance.
Correspondence should be addressed to Dr. Takaaki Kirino, Department of
Neurosurgery, University of Tokyo Faculty of Medicine, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-8655, Japan.
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Abstract
Introduction
