Reduced susceptibility to ischemic brain injury and N-methyl-D-aspartate-mediated neurotoxicity in cyclooxygenase-2-deficient mice

Abstract

Cyclooxygenase-2 (COX-2), a prostanoid-synthesizing enzyme that
contributes to the toxicity associated with inflammation, has recently
emerged as a promising therapeutic target for several illnesses,
ranging from osteoarthritis to Alzheimer's disease. Although COX-2 has
also been linked to ischemic stroke, its role in the mechanisms of
ischemic brain injury remains controversial. We demonstrate that
COX-2-deficient mice have a significant reduction in the brain injury
produced by occlusion of the middle cerebral artery. The protection can
be attributed to attenuation of glutamate neurotoxicity, a
critical factor in the initiation of ischemic brain injury, and to
abrogation of the deleterious effects of postischemic inflammation, a
process contributing to the secondary progression of the damage. Thus,
COX-2 is involved in pathogenic events occurring in both the early and
late stages of cerebral ischemia and may be a valuable therapeutic
target for treatment of human stroke.

Full text

Reduced susceptibility to ischemic brain injury and
N
-methyl-D-aspartate-mediated neurotoxicity in
cyclooxygenase-2-deficient mice
Costantino Iadecola*
†
, Kiyoshi Niwa*, Shigeru Nogawa*, Xueren Zhao*, Masao Nagayama*, Eiichi Araki*,
Scott Morham
‡
, and M. Elizabeth Ross*
*Center for Clinical and Molecular Neurobiology, Department of Neurology, University of Minnesota Medical School, Minneapolis, MN 55455;
and ‡Myriad Genetics, Salt Lake City, UT 84108
Communicated by Philip Needleman, Monsanto Company, St. Louis, MO, November 21, 2000 (received for review July 27, 2000)
Cyclooxygenase-2 (COX-2), a prostanoid-synthesizing enzyme that
contributes to the toxicity associated with inflammation, has
recently emerged as a promising therapeutic target for several
illnesses, ranging from osteoarthritis to Alzheimer’s disease. Al-
though COX-2 has also been linked to ischemic stroke, its role in the
mechanisms of ischemic brain injury remains controversial. We
demonstrate that COX-2-deficient mice have a significant reduc-
tion in the brain injury produced by occlusion of the middle
cerebral artery. The protection can be attributed to attenuation
of glutamate neurotoxicity, a critical factor in the initiation of
ischemic brain injury, and to abrogation of the deleterious effects
of postischemic inflammation, a process contributing to the sec-
ondary progression of the damage. Thus, COX-2 is involved in
pathogenic events occurring in both the early and late stages of
cerebral ischemia and may be a valuable therapeutic target for
treatment of human stroke.
middle cerebral artery occlusion 兩prostanoids 兩cerebral blood
flow 兩NS398 兩stroke
Stroke remains a major cause of death and disability world-
wide (1– 4). After many years of setbacks, effective treat-
ments for stroke, based on thrombolysis and restoration of flow,
have been developed (5, 6). However, these therapies can be
safely administered only in those patients who reach medical
attention during the first few hours after the onset of the stroke
(7, 8). Therefore, it would be important to develop treatments
that target downstream events in the ischemic cascade. Further-
more, it would be desirable to combine thrombolytic therapy
with other therapeutic strategies aimed at protecting the brain
from the residual ischemia (9). Immediately after induction of
ischemia, activation of glutamate receptors initiates the ischemic
cascade and contributes to the damage that occurs in the early
stages of cerebral ischemia (refs. 10 and 11; see ref. 12 for a
review). At later times after ischemia, the tissue damage con-
tinues to evolve (13), and inflammation and programmed cell
death are thought to be major factors in the progression of the
injury (refs. 14 –17; see ref. 18 for a review).
Cyclooxygenase (COX)-1 and COX-2 are enzymes involved in
the first step of the synthesis of prostanoids (see ref. 19 for a
review). COX-1 is expressed constitutively in many organs and
contributes to the synthesis of prostanoids involved in normal
cellular functions. COX-2 is thought to be an inducible enzyme
whose expression is up-regulated in pathological states, most
notably those associated with inf lammation (see ref. 20 for a
review). There is substantial evidence that COX-2 reaction
products are responsible for cytotoxicity in models of inflam-
mation (see ref. 21 for a review). Thus, COX-2 inhibitors are
potent antiinflammatory agents and have recently been intro-
duced in clinical practice with notable success (22, 23).
In brain, COX-2 is present in selected neurons (24–26), and
its expression is up-regulated in several neurological diseases,
including stroke, Alzheimer’s dementia, and seizures (24, 27–
29). In rodents as in humans, cerebral ischemia up-regulates
COX-2 in neurons, blood vessels, and inflammator y cells infil-
trating the injured brain (27, 28, 30). However, experimental
evidence linking COX-2 to the mechanisms of brain injury
associated with these conditions is lacking. For example, al-
though it is well established that cerebral ischemia increases
COX-2 expression in the damaged brain, studies using COX
inhibitors in models of focal cerebral ischemia have yielded
conflicting results (28, 31, 32). Furthermore, studies using
COX-2 inhibitors cannot exclude effects unrelated to COX-2,
such as modification of gene expression or activation of perox-
isome proliferator-activated receptors (33, 34). In view of the
increasing use of COX-2 inhibitors as antiinflammatory agents
in the elderly—a population with an increased risk for stroke—it
would be of great interest to define the role of COX-2 in ischemic
brain injury.
Mice with a null mutation of the COX-2 gene have been a
useful model for investigating the role of COX-2 in systemic
inflammation, thermoregulation, and cerebrovascular regula-
tion (refs. 35 and 36; see ref. 37 for a review). In the present
study, therefore, we used COX-2-deficient mice to gain further
insight into the role of COX-2 in ischemic brain injury.
Methods
Animals. COX-2-null mice were obtained from a colony estab-
lished at the University of Minnesota (35, 36). Mice (SV129 ⫻
C57BL
兾
6J) were back-crossed to C57BL
兾
6J mice five or six
times and were studied at age 2–3 months. Experiments were
performed in age-matched littermates [wild-type (⫹
兾
⫹), het-
erozygous (⫹
兾
⫺), and homozygous (⫺
兾
⫺)] to minimize con-
founding effects deriving from the genetic background of the
mice. The genotype of all COX-2 mice was determined by PCR
with the use of primers and methods described previously (35).
No alterations in the anatomy of large cerebral vessels were
noticed in COX-2-null mice. C57BL
兾
6J mice were obtained
from The Jackson Laboratory.
Induction of Focal Cerebral Ischemia. Focal cerebral ischemia was
produced by occlusion of the middle cerebral artery (MCA) (38).
Mice were anesthetized with 2% halothane
兾
100% oxygen. Body
temperature was maintained at 37 ⫾0.5°C by a thermostatically
Abbreviations: NMDA, N-methyl-D-aspartate; COX, cyclooxygenase; MCA, middle cerebral
artery; RT-PCR, reverse transcription–PCR; PBD, porphobilinogen deaminase; CBF, cerebral
blood flow; PGE2, prostaglandin E2.
†To whom reprint requests should be addressed at: Department of Neurology, University
of Minnesota Medical School, Box 295, UMHC, 516 Delaware Street SE, Minneapolis, MN
55455. E-mail: iadec001@tc.umn.edu.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
1294–1299
兩
PNAS
兩
January 30, 2001
兩
vol. 98
兩
no. 3

controlled infrared lamp. A 2-mm hole was drilled in the inferior
portion of the temporal bone to expose the left MCA. The MCA
was elevated and cauterized distal to the origin of the lenticu-
lostriate branches. Mice in which the MCA was exposed but not
occluded served as sham-operated controls. Wounds were su-
tured and mice were allowed to recover and were returned to
their cages. Rectal temperature was controlled until mice re-
gained full consciousness. Thereafter, rectal temperature was
measured daily until the time of sacrifice. There were no major
differences in rectal temperature among COX-2 ⫹
兾
⫹,⫹
兾
⫺,
and ⫺
兾
⫺mice before MCA occlusion or after ischemia. For
example, 48 h after MCA occlusion rectal temperature was
36.2 ⫾0.1°C in COX-2 ⫹
兾
⫹, 36.6 ⫾0.1 in ⫹
兾
⫺, and 36.0 ⫾0.2
in ⫺
兾
⫺mice (P⬎0.05, analysis of variance).
Reverse Transcription–PCR (RT-PCR). mRNA for COX-2 was de-
tected by RT-PCR as previously described (28). Mice were killed
24 h after ischemia (n⫽4 per time point), and their brains were
removed. This time point was chosen because COX-2 mRNA
expression reaches a plateau 24 h after MCA occlusion in mice
(39). A 4-mm-thick coronal brain slice was cut at the level of the
optic chiasm, and samples including the infarcted cortex and the
corresponding area of the contralateral cortex were collected
and frozen in liquid nitrogen. Total RNA was extracted from the
tissue according to the method of Chomczynski and Sacchi (40).
RNA integrity was determined on denaturing formaldehyde
gels. Aliquots of total RNA (0.25
␮
g) were used in the RT
reaction mixed with 0.5
␮
g of oligo(dT) primer as directed
(18-mer; New England Biolabs). First-strand cDNA synthesis
was then carried out with the use of Moloney murine leukemia
virus reverse transcriptase (New England Biolabs) according to
the manufacturer’s instructions. After heating at 95°C for 10 min,
5
␮
l from each RT reaction mixture was used for PCR ampli-
fication. Primers (0.2
␮
M each) for the sequence of interest and
for porphobilinogen deaminase (PBD), a ubiquitously expressed
sequence, were used in a final volume of 50
␮
l. COX-2 primers
were as follows: forward, 5⬘-CCAGATGCTATCTTTGGG-
GAGAC-3⬘; reverse, 5⬘-GCTTGCATTGATGGTGGCTG-3⬘,
which result in a PCR product of 249 bp. COX-1 primers were
as follows: forward, 5⬘-GAATACCGAAAGAGGT TTG-
GCTTG-3⬘; reverse, 5⬘-TCATCTCCAGGGTAATCTGGCAC-
3⬘, which yields a 374-bp product. PBD primers were as follows:
forward, 5⬘-GCCACCACAGTCTCGGTCTGTATGCGAGC-
3⬘; reverse, 5⬘-TGTCCGGTAACGGCGGCGCGGCCA-
CAAC-3⬘. The ‘‘hot start’’ method was used with the following
cycle parameters: 94°C, 15 s; 68°C, 30 s; 73°C, 20 s, for five cycles,
then 94°C, 15 s; 64°C, 30 s; 73°C, 20 s, for 35 cycles; and 73°C,
15 min. Reaction products were then separated on an 8%
polyacrylamide gel, stained with ethidium bromide, and photo-
graphed. Each set of PCRs included control samples run without
RNA or in which the RT step was omitted to ensure that PCR
products resulted from amplification from the COX-2 mRNA
rather than genomic DNA. The OD of the bands was determined
by a gel image analysis system (Molecular Analyst; Bio-Rad). In
some studies, measurements were normalized to the OD of the
PBD band used as an internal standard (28, 41).
Competitive RT-PCR was used to determine more accurately
the magnitude of mRNA induction (28). A deletion construct
was synthesized that consisted of the same sequence amplified
from the endogenous COX-2 message but missing an internal
79-nucleotide fragment. To generate the construct, a pair of
COX-2 primers was prepared: forward: 5⬘-CCAGATGC-
TATCTTTGGGGAGAC-3⬘; reverse: 5⬘-GCTTGCATTGAT-
GGTGGCTG-3⬘, which result in a 249-bp PCR product. The
PCR product was then digested with a restriction enzyme,
Sau3AI (New England Biolabs). The sample was then religated
at 14°C overnight with T4 DNA ligase (New England Biolabs),
and the ligase was inactivated at 65°C for 15 min. A 1-
␮
l aliquot
of the religated sample was then amplified with the use of the
COX-2 forward and reverse primers described above. Products
were separated on a polyacrylamide gel, and a main product of
170 bp was excised from the gel. The construct was eluted from
the crushed gel with T.E. buffer (10 mM Tris
兾
1 mM EDTA, pH
8.0) at 55°C for 4 h and purified for use in the competition assay.
The RT reaction mixtures (5
␮
l each) of the animals killed 24 h
after ischemia (n⫽4 per group) were coamplified with known
amounts of deletion construct (0.25–25 fg). The PCR products
were then separated on a gel, and the gel was stained with
ethidium bromide and photographed. The OD of the bands was
determined by image analysis. For data analysis, the log of the
OD ratio (COX-2
兾
construct) was plotted as a function of the log
of the concentration of the construct and fitted by linear
regression analysis (28, 42). The 0 value of the log of the ratio
(COX-2
兾
construct) (yaxis) represents the point at which the
COX-2 PCR product and the construct are present in equal
amounts. Therefore, the amount of the construct corresponding
to the 0 ratio (xaxis) represents the amount of the COX-2 PCR
product before PCR amplification (42, 43).
Prostaglandin E
2
(PGE
2
) Enzyme Immunoassay. Tissue concentration
of PGE
2
, a COX reaction product, was measured 24 h after MCA
occlusion or3hafterN-methyl-D-aspartate (NMDA) injection.
Fig. 1. COX-2 mRNA expression in the brain of COX-2-null mice 24 h after
MCA occlusion. (A) Representative gel illustrating COX-2 mRNA expression in
the ischemic cortex (s) and contralateral cortex (n) assessed by RT-PCR. The
ubiquitous sequence PBD was also studied and used as an internal control. std,
standards; bl, sample without the reverse transcriptase step. (B) COX-2 mRNA
expression assessed by competitive PCR. COX-2 expression is reduced in COX-2
⫹
兾
⫺mice and is absent in COX-2 ⫺
兾
⫺mice (n⫽4 per group; *,P⬍0.05 from
contralateral; #, P⬍0.05 from ⫹
兾
⫹stroke).
Iadecola et al. PNAS
兩
January 30, 2001
兩
vol. 98
兩
no. 3
兩
1295
PHARMACOLOGY

PGE
2
concentration was determined in the cerebral cortex
ipsilateral and contralateral to the lesion with the use of an
enzyme immunoassay kit (Cayman Chemicals, Ann Arbor, MI)
as previously described (28). Prostanoids were extracted with
100% methanol according to the method of Powell (see ref. 28),
and the PGE
2
concentration was determined spectrophotometri-
cally according to the instructions provided with the kit.
NMDA Microinjection in Neocortex. In halothane-anesthetized mice
the dura overlying the parietal cortex was exposed, and NMDA
(20 nmol in 200–300 nl of sterile 0.1 M PBS, pH 7.4) was injected
with a glass micropipette (tip 40 –50
␮
m) connected to a micro-
injection device. The micropipette was inserted into the parietal
cortex at a site 1.5 mm caudal to bregma, 4.0 mm from the
midline, and 0.8 mm below the dural surface. The micropipette
was left in place for 10 min, to minimize back-f lux of NMDA, and
then removed. Mice were returned to their cages and allowed to
survive for 3 h for PGE
2
measurement and for 24 h for
determination of lesion volume.
Determination of Lesion Volume. Mice were killed 1 and 4 days
after MCA occlusion or 1 day after NMDA injection. Brains
were removed and frozen in cooled isopentane (⫺30°C). Coro-
nal forebrain sections (thickness ⫽30
␮
m) were serially cut in
a cryostat, collected at 90-
␮
m intervals, and stained with thionin
for determination of lesion volume by an image analyzer (MCID;
Imaging Research, St. Catherine’s, ON, Canada; ref. 38). In
studies of cerebral ischemia, infarct volume in cerebral cortex
was corrected for swelling to factor out the contribution of
ischemic edema to the total volume of the lesion (38, 44).
Monitoring of Cerebral Blood Flow (CBF). Techniques used for
monitoring CBF after MCA occlusion have been published (38).
Mice were anesthetized with halothane (maintenance 1%), and
the femoral artery and trachea were cannulated. Mice were
artificially ventilated with an oxygen–nitrogen mixture by a
mechanical ventilator (SAR-830; CWE, Ardmore, PA). The
oxygen concentration in the mixture was adjusted to maintain
arterial partial O
2
pressure between 120 and 140 mmHg. End-
tidal CO
2
was continuously monitored with a CO
2
analyzer
(Capstar-100; CWE) (38). CBF was monitored with two laser-
Doppler flow probes (Vasamedic, Minneapolis, MN) placed
through burr holes drilled in the center (3.5 mm lateral to the
midline and 1 mm caudal to bregma) and the periphery (1.5 mm
lateral to the midline and 1.7 mm rostral to lambda) of the
ischemic territory (38). The location of the probe was selected in
preliminary experiments to correspond to the region of brain
that is spared from infarction in COX-2-deficient mice. After
placement of the probes, the MCA was occluded and CBF was
monitored for 90 min. CBF data are expressed as a percentage
of the preocclusion value. Arterial pressure and blood gases did
not differ between COX-2 ⫹
兾
⫹and ⫺
兾
⫺mice (10 min after
MCA occlusion: COX-2 ⫹
兾
⫹: arterial pressure: 83 ⫾3 mmHg;
paCO
2
: 34.6 ⫾0.6 mmHg; arterial partial O
2
pressure: 125 ⫾6
mmHg; pH: 7.35 ⫾0.02; COX-2 ⫺
兾
⫺: arterial pressure: 84 ⫾
2; paCO
2
: 33.1 ⫾0.8; arterial partial O
2
pressure: 126 ⫾5; pH:
7.33 ⫾0.02).
Renal Function. Because COX-2-null mice develop renal disease
(35), plasma creatinine was measured by standard colorimetric
methods. Furthermore, histological analysis of the kidneys was
performed in paraffin-embedded sections stained with hema-
toxylin and eosin. Creatinine did not differ between COX-2
⫹
兾
⫹(0.18 ⫾0.07 mg
兾
dl; n⫽6) and ⫹
兾
⫺mice (0.18 ⫾0.05;
n⫽6) (P⬎0.05 from COX-2 ⫹
兾
⫹). In COX-2 ⫺
兾
⫺mice (n⫽
6), creatinine was elevated (0.43 ⫾0.08; P⬍0.05 from COX-2
⫹
兾
⫹) but still in the normal range (45). COX-2 ⫺
兾
⫺mice had
hypertrophy of the juxtaglomerular apparatus and macula densa
but no alterations in the glomeruli or tubules.
Data Analysis. Data in the text and figures are expressed as
means ⫾SEM. Multiple comparisons were evaluated statisti-
cally by the analysis of variance and Tukey’s test. Two-group
comparisons were analyzed by the two-tailed Student’s ttest for
independent samples. For all procedures, probability values of
less than 0.05 were considered statistically significant.
Results
Postischemic COX-2 Expression Is Attenuated in COX-2-Null Mice.
First, we sought to establish whether the increase in COX-2
expression that occurs after cerebral ischemia is reduced in
COX-2-null mice. In COX-2 ⫹
兾
⫹mice, COX-2 mRNA was
increased in the ischemic cortex 24 h after MCA occlusion by 2-
to 3-fold (Fig. 1A). The up-regulation was reduced in COX-2
⫹
兾
⫺(P⬍0.05, analysis of variance) (Fig. 1B). COX-2 mRNA
was not detected in either the ischemic or the nonischemic cortex
in COX-2 ⫺
兾
⫺mice. In contrast, expression of COX-1 mRNA
was comparable in COX-2 ⫹
兾
⫹,⫹
兾
⫺, and ⫺
兾
⫺mice (Fig.
2A). To determine whether the reduction in COX-2 mRNA
expression is associated with a reduction in COX-2 enzymatic
Fig. 2. COX-1 mRNA expression and PGE2concentration in the brain of
COX-2-null mice 24 h after MCA occlusion. (A) Representative gel illustrating
COX-1 mRNA expression assessed by RT-PCR 24 h after MCA occlusion. Similar
results were obtained in four separate groups of COX-2 ⫹
兾
⫹,⫹
兾
⫺, and ⫺
兾
⫺
mice. COX-1
兾
PBD OD ratios in the ischemic cortex of COX-2 ⫹
兾
⫹,⫹
兾
⫺, and
⫺
兾
⫺mice were 1.7 ⫾0.4, 1.6 ⫾0.2, and 1.8 ⫾0.5, respectively (n⫽4 per
group; P⬎0.05). Abbreviations are as in Fig. 1. (B) Effect of MCA occlusion on
PGE2concentration in the ischemic cortex (Stroke) and contralateral cortex.
The PGE2elevation in the ischemic cortex in COX-2 ⫹
兾
⫹(n⫽8) was attenu-
ated in COX-2 ⫹
兾
⫺(n⫽7) and abolished in COX-2 ⫺
兾
⫺mice (n⫽7) (*,P⬍
0.05 from contralateral; #, P⬍0.05 from ⫹
兾
⫹stroke).
1296
兩
www.pnas.org Iadecola et al.

activity, the concentration of the COX reaction product PGE
2
was measured in the ischemic and nonischemic cortex 24 h after
MCA occlusion. PGE
2
concentration was significantly elevated
in the ischemic cortex of COX-2 ⫹
兾
⫹mice (Fig. 2B). The
elevation was attenuated in COX-2 ⫹
兾
⫺mice and absent in
COX-2 ⫺
兾
⫺mice (Fig. 2B).
Ischemic Brain Injury Is Reduced in COX-2-Null Mice. We then studied
the brain injury produced by MCA occlusion in COX-2-null
mice. Mice were killed 96 h after ischemia, and the infarct
volume (mm
3
) was measured in brain sections stained with
thionin (38). The 96-h time point was selected on the basis of the
fact that, at this time, the damage resulting from postischemic
inflammation is fully expressed (38). Infarct volume was signif-
icantly smaller in COX-2-null mice than in wild-type littermates
(Fig. 3A). The reduction was greater in COX-2 ⫺
兾
⫺(⫺38 ⫾
4%) than in ⫹
兾
⫺mice (⫺20 ⫾3%) (Fig. 3A). These data
suggest that COX-2 contributes to ischemic brain injury.
To determine whether COX-2 is also involved in pathogenic
processes that occur in the initial stages of cerebral ischemia, we
studied infarct volume in mice killed 24 h after ischemia. At this
time the damage resulting from postischemic inflammation does
not contribute to tissue injury (38). For example, in mice lacking
inducible nitric oxide synthase, an enzyme that plays a critical
role in inflammatory responses, infarct volume is not reduced
24 h after ischemia, but only at 96 h (38). We found that in
COX-2-null mice infarct volume is also reduced 24 h after
ischemia (⫺34 ⫾3%; Fig. 3B). These data suggest that COX-2
is also involved in pathogenic events occurring in the early stages
of cerebral ischemia.
Effect of MCA Occlusion on CBF in COX-2-Null Mice. The intensity of
the ischemic insult has a profound impact on the ensuing brain
damage (see ref. 46 for a review). We therefore studied the effect
of MCA occlusion on neocortical CBF in COX-2 ⫹
兾
⫹and ⫺
兾
⫺
mice. CBF was monitored both in the center of the ischemic
territory and in the peripheral region that is spared from
infarction in COX-2 ⫺
兾
⫺mice. As illustrated in Fig. 3 Cand D,
the reduction in CBF in the center or periphery of the ischemic
territory did not differ between COX-2 ⫹
兾
⫹and ⫺
兾
⫺mice
(P⬎0.05). These data demonstrate that the reduction in CBF
in areas that are spared from infarction in COX-2 ⫺
兾
⫺mice is
comparable to that observed in COX-2 ⫹
兾
⫹mice. Therefore,
differences in the intensity of the ischemic insult cannot account
for the neuroprotection observed in COX-2-null mice.
NMDA-Mediated Injury
in Vivo
Is Attenuated in COX-2-Null Mice.
Glutamate receptors play a critical role in the initiation of
ischemic brain injury (12). Therefore, we sought to determine
whether the reduction in ischemic damage in COX-2-null mice
could be attributed to reduced susceptibility to glutamate re-
ceptor-mediated damage. The glutamate receptor agonist
NMDA was microinjected directly into the cerebral cortex, and
injury volume was determined in thionin-stained brain sections
24 h later. At this time after NMDA microinjection, there is no
histological evidence of inf lammation in the area of the lesion
(data not shown). In normal mice, NMDA microinjection in-
creased the local concentration of PGE
2
and produced a well-
defined neocortical lesion (Fig. 4 Aand B). The PGE
2
elevation
was attenuated by treatment with the COX-2 inhibitor NS398 (20
mg
兾
kg i.p.; 1 h before NMDA), demonstrating that COX-2 was
responsible for the increase in this prostaglandin (Fig. 4A).
NS398 administration (20 mg
兾
kg i.p.; 1 h before and1hafter
NMDA) also reduced the volume of the lesion (Fig. 4 Cand D).
In addition, the NMDA-induced damage was markedly attenu-
ated in COX-2 ⫺
兾
⫺mice (Fig. 5). Administration of NS398
reduced injury volume in COX-2 ⫹
兾
⫹but not in COX-2 ⫺
兾
⫺
mice (Fig. 5C), attesting to the specificity of the effect of NS398
on COX-2. Thus the brain damage produced by the activation of
glutamate receptors is attenuated by COX-2 inhibition and in
COX-2-null mice.
Fig. 3. (A) Infarct size in COX-2 ⫹
兾
⫹(n⫽6), ⫹
兾
⫺(n⫽7), and ⫺
兾
⫺(n⫽8) null mice 96 h after MCA occlusion (MCAO). Ctx (E.C.), cerebral cortical infarct
corrected for swelling. (*,P⬍0.05 from COX-2 ⫹
兾
⫹and ⫹
兾
⫺mice; #, P⬍0.05 from COX-2 ⫺
兾
⫺and ⫹
兾
⫹mice). (B) Infarct size in COX-2-null mice 24 h after
MCAO (n⫽6 per group; *,P⬍0.05 from COX-2 ⫹
兾
⫹mice). (Cand D) CBF reduction in the center (C) and periphery (D) of the ischemic region in COX-2 ⫹
兾
⫹
and ⫺
兾
⫺mice after MCAO (n⫽6 per group).
Iadecola et al. PNAS
兩
January 30, 2001
兩
vol. 98
兩
no. 3
兩
1297
PHARMACOLOGY

Discussion
We used COX-2-null mice to investigate the role of COX-2 in the
mechanisms of cerebral ischemic injury. We found that, after
MCA occlusion, COX-2-null mice do not express COX-2 mRNA
and have a reduction in the elevation in PGE
2
produced by
cerebral ischemia. Furthermore, the volume of brain damage
produced by MCA occlusion is markedly reduced in COX-2-null
mice. The reduction in injury volume, like the elevation in
COX-2 mRNA and PGE
2
, is more marked in homozygous than
in heterozygous null mice. These findings provide strong evi-
dence that COX-2 reaction products are involved in ischemic
brain damage. Furthermore, the observation that, in COX-2-null
mice, infarct volume is also reduced 24 h after MCA occlusion,
supports the hypothesis that COX-2 is also involved in patho-
genic events that take place in the early stages of cerebral
ischemia.
The neuroprotection observed in COX-2-null mice cannot be
attributed to differences in the intensity of the ischemic insult,
because the reduction in CBF produced by MCA occlusion is
comparable in COX-2 ⫹
兾
⫹and ⫺
兾
⫺mice. However, COX-
2-null mice were found to be more resistant to the damage
produced by the glutamate receptor antagonist NMDA. The
reduction in excitotoxicity cannot be a consequence of alter-
ations in the NMDA receptors in COX-2-null mice, because
acute administration of the COX-2 inhibitor NS398 produced
comparable attenuation in NMDA-induced injury. Conversely,
the neuroprotection exerted by NS398 is unlikely to be due to
effects of the drug unrelated to COX-2 inhibition, because
NS398 did not confer protection in COX-2-null mice. These
observations, collectively, provide evidence that COX-2 reaction
products contribute to NMDA-mediated cytotoxicity. This con-
clusion is also supported by the observations that NS398 protects
neuronal cultures from NMDA (47) and that neuronal cultures
from transgenic mice overexpressing COX-2 are more suscep-
tible to glutamate excitotoxicity (48). Considering the critical
role that NMDA receptors play in ischemic brain injury (see ref.
12 for a review), the data suggest that attenuation of glutamate
receptor-dependent ischemic damage contributes to the reduc-
tion in ischemic injury observed in COX-2-null mice.
The mechanisms by which COX-2 contributes to neurotoxicity
remain to be defined. Superoxide radicals, a COX-2 reaction
product (49, 50), are well known to participate in ischemic brain
injury and could mediate tissue damage either directly or by
reacting with nitric oxide to form the strong oxidant peroxyni-
trite (see ref. 51 for a review). Furthermore, COX-2 reaction
products could activate poly(ADP-ribose) polymerase, an en-
zyme involved in DNA repair that contributes to neurotoxicity
(52, 53). In addition, PGE
2
, a major reaction product of COX-2
(54), could mediate toxicity by facilitating glutamate release
from astrocytes (55). However, the role of PGE
2
in neurotoxicity
remains controversial because this prostaglandin has also been
reported to counter the cytotoxic effects of glutamate (56).
However, attenuation of glutamate neurotoxicity is not the
sole mechanism of the protection from ischemic injury observed
in COX-2-null mice. Cerebral ischemia is associated with an
inflammatory reaction that contributes to tissue damage (57).
COX-2 is a critical factor in the cytotoxicity associated with
inflammation (21). It is therefore likely that COX-2 plays a role
also in the mechanisms by which inflammation contributes to
ischemic damage. This hypothesis is supported by the observa-
tion that the COX-2 inhibitor NS398 reduces ischemic injury
even when administered6hafterMCAocclusion (28). At this
time after focal ischemia, activation of NMDA receptors does
not contribute to the damage, as evidenced by the fact that
NMDA receptor antagonists are no longer protective (58).
Therefore, the evidence suggests that COX-2 also plays a role in
the late stages of ischemic brain injury.
Fig. 4. Effect of NS398 on the lesion produced by direct microinjection of
NMDA into the cerebral cortex of C57BL
兾
6J mice. (A) PGE2concentration in
the injured cortex, 3 h after NMDA injection, in mice receiving vehicle (n⫽5)
or NS398 (n⫽5; *,P⬍0.05 from vehicle). (Band C) Thionin-stained brain
sections showing the lesion produced by NMDA (arrow) in mice receiving
vehicle or NS398. (D) Effect of NS398 (n⫽6) or vehicle (n⫽7) on the volume
of the lesion produced by NMDA (*,P⬍0.05 from vehicle). (E) Effect of
NS398 on the lesion area at different rostrocaudal levels from the anterior
commissure.
Fig. 5. Lesion produced by microinjection of NMDA into the cerebral cortex
of COX-2 ⫹
兾
⫹and ⫺
兾
⫺mice. (Aand B) Thionin-stained brain sections
showing the lesion produced by NMDA injection (arrow) in COX-2 ⫹
兾
⫹(A)
and ⫺
兾
⫺(B) mice. (C) Effect of NS398 on the volume of the lesion produced
by NMDA microinjection in COX-2 ⫹
兾
⫹and ⫺
兾
⫺mice (n⫽6 per group; *,P⬍
0.05 from vehicle). (D) Lesion area at different rostrocaudal levels relative to
the anterior commissure in COX-2 ⫹
兾
⫹and ⫺
兾
⫺mice (n⫽6 per group).
1298
兩
www.pnas.org Iadecola et al.

The findings of the present study suggest that COX-2 is a
promising pharmacological target for the treatment of ischemic
stroke. COX-2 inhibition offers several advantages over other
prospective neuroprotective strategies. First, by targeting both
‘‘early’’ and ‘‘late’’ components of ischemic injury, COX-2
inhibitors can act as a bimodal neuroprotective strategy, with a
likelihood of success greater than that of unimodal therapies.
Second, potent and selective COX-2 inhibitors have already
proved to be relatively safe and well tolerated (22). Therefore,
examining their efficacy in stroke patients would be easier than
testing potential neuroprotective agents with an unknown safety
profile. Third, a large number of patients already take COX-2
inhibitors for treatment of osteoarthritis or pain (22). Epidemi-
ological studies could provide important clues to the usefulness
of COX-2 inhibitors in stroke prevention and improvement of
outcome. On the basis of these considerations, COX-2 inhibition
seems an attractive therapeutic strategy for stroke and other
diseases associated with glutamate excitotoxicity.
We thank Dr. Dale Cooper for help with the measurement of blood urea
nitrogen and creatinine, Dr. Carlos Manivel for pathological examina-
tion of mouse kidneys, Ms. Tracy Aber for assistance in experiments
involving RT-PCR, and Mr. Tim Murphy and Ms. Andrea Hyde for
editorial assistance. This work was supported by National Institutes of
Health Grant NS35806.
1. Jorgensen, H. S., Nakayama, H., Pedersen, P. M., Kammersgaard, L.,
Raaschou, H. O. & Olsen, T. S. (1999) Clin. Geriatr. Med. 15, 785–799.
2. Johansson, B., Norrving, B. & Lindgren, A. (2000) Stroke (Dallas) 31, 481– 486.
3. Kita, Y., Okayama, A., Ueshima, H., Wada, M., Nozaki, A., Choudhury, S. R.,
Bonita, R., Inamoto, Y. & Kasamatsu, T. (1999) Int. J. Epidemiol. 28,
1059–1065.
4. Warlow, C. P. (1998) Lancet 352, Suppl. 3, SIII1– 4.
5. Lyden, P. D., Grotta, J. C., Levine, S. R., Marler, J. R., Frankel, M. R. & Brott,
T. G. (1997) Neurology 49, 14 –20.
6. Kwiatk owski, T. G., Libman, R. B., Frankel, M., Tilley, B. C., Morgenstern,
L. B., Lu, M., Broderick, J. P., Lewandowski, C. A., Marler, J. R., Levine, S. R.
& Brott, T. (1999) N. Engl. J. Med. 340, 1781–1787.
7. Hamann, G. F., del Zoppo, G. J. & von Kummer, R. (1999) Thromb. Haemost.
82, Suppl. 1, 92–94.
8. Lyden, P. D. (1999) Prog. Cardiovasc. Dis. 42, 175–183.
9. Steiner, T. & Hacke, W. (1998) Eur. Neurol. 40, 1–8.
10. Benveniste, H., Drejer, J., Schousboe, A. & Diemer, N. H. (1984) J. Neurochem.
43, 1369–1374.
11. Butcher, S. P., Bullock, R., Graham, D. I. & McCulloch, J. (1990) Stroke
(Dallas) 21, 1727–1733.
12. Lee, J. M., Zipfel, G. J. & Choi, D. W. (1999) Nature (London) 399, A7–A14.
13. Dereski, M. O., Chopp, M., Knight, R. A., Rodolosi, L. C. & Garcia, J. H.
(1993) Acta. Neuropathol. 85, 327–333.
14. Du, C., Hu, R., Csernansky, C. A., Hsu, C. Y. & Choi, D. W. (1996) J. Cereb.
Blood Flow Metab. 16, 195–201.
15. Endres, M., Namura, S., Shimizu-Sasamata, M., Waeber, C., Zhang, L.,
Gomez-Isla, T., Hyman, B. T. & Moskowitz, M. A. (1998) J. Cereb. Blood Flow
Metab. 18, 238–247.
16. Chopp, M., Li, Y., Jiang, N., Zhang, R. L. & Prostak, J. (1996) J. Cereb. Blood
Flow Metab. 16, 578–584.
17. Connolly, E. S., Jr., Winfree, C. J., Springer, T. A., Naka, Y., Liao, H., Yan,
S. D., Stern, D. M., Solomon, R. A., Gutierrez-Ramos, J. C. & Pinsky, D. J.
(1996) J. Clin. Invest. 97, 209–216.
18. Dirnagl, U., Iadecola, C. & Moskowitz, M. A. (1999) Trends Neurosci. 22,
391–397.
19. Vane, J. R., Bakhle, Y. S. & Botting, R. M. (1998) Annu. Rev. Pharmacol.
Toxicol. 38, 97–120.
20. Seibert, K., Zhang, Y., Leahy, K., Hauser, S., Masferrer, J. & Isakson, P. (1997)
Adv. Exp. Med. Biol. 400A, 167–170.
21. Seibert, K., Masferrer, J., Zhang, Y., Gregory, S., Olson, G., Hauser, S., Leahy,
K., Perkins, W. & Isakson, P. (1995) Agents Actions Suppl. 46, 41–50.
22. Hawkey, C. J. (1999) Lancet 353, 307–314.
23. Willoughby, D. A., Moore, A. R. & Colville-Nash, P. R. (2000) Lancet 355,
646– 648.
24. Yamagata, K., Andreasson, K. I., Kaufmann, W. E., Barnes, C. A. & Worley,
P. F. (1993) Neuron 11, 371–386.
25. Kaufmann, W. E., Worley, P. F., Pegg, J., Bremer, M. & Isakson, P. (1996) Proc.
Natl. Acad. Sci. USA 93, 2317–2321.
26. Breder, C. D., Dewitt, D. & Kraig, R. P. (1995) J. Comp. Neurol. 355, 296–315.
27. Miettinen, S., Fusco, F. R., Yrjanheikki, J., Keinanen, R., Hirvonen, T.,
Roivainen, R., Narhi, M., Hokfelt, T. & Koistinaho, J. (1997) Proc. Natl. Acad.
Sci. USA 94, 6500–6505.
28. Nogawa, S., Zhang, F., Ross, M. E. & Iadecola, C. (1997) J. Neurosci. 17,
2746–2755.
29. Pasinetti, G. M. (1998) J. Neurosci. Res. 54, 1–6.
30. Iadecola, C., Forster, C., Nogawa, S., Clark, H. B. & Ross, M. E. (1999) Acta
Neuropathol. 98, 9–14.
31. Hara, K., Kong, D. L., Sharp, F. R. & Weinstein, P. R. (1998) Neurosci. Lett.
256, 53–56.
32. Cole, D. J., Patel, P. M., Reynolds, L., Drummond, J. C. & Marcanton io, S.
(1993) J. Pharmacol. Exp. Ther. 266, 1713–1717.
33. Lehmann, J. M., Lenhard, J. M., Oliver, B. B., Ringold, G. M. & Kliewer, S. A.
(1997) J. Biol. Chem. 272, 3406–3410.
34. Grilli, M., Pizzi, M., Memo, M. & Spano, P. (1996) Science 274, 1383–1385.
35. Morham, S. G., Langenbach, R., Loftin, C. D., Tiano, H. F., Vouloumanos, N.,
Jennette, J. C., Mahler, J. F., Kluckman, K. D., Ledford, A., Lee, C. A., et al.
(1995) Cell 83, 473–482.
36. Niwa, K., Araki, E., Morham, S. G., Ross, M. E. & Iadecola, C. (2000)
J. Neurosci. 20, 763–770.
37. Langenbach, R., Loftin, C. D., Lee, C. & Tiano, H. (1999) Ann. N.Y. Acad. Sci.
889, 52–61.
38. Iadecola, C., Zhang, F., Casey, R., Nagayama, M. & Ross, M. E. (1997)
J. Neurosci. 17, 9157–9164.
39. Nogawa, S., Forster, C., Zhang, F., Nagayama, M., Ross, M. E. & Iadecola, C.
(1998) Proc. Natl. Acad. Sci. USA 95, 10966–10971.
40. Chomczynski, P. & Sacchi, N. (1987) Anal. Biochem. 162, 156 –159.
41. Iadecola, C., Zhang, F., Casey, R., Clark, H. B. & Ross, M. E. (1996) Stroke
(Dallas) 27, 1373–1380.
42. Galea, E. & Feinstein, D. L. (1992) PCR Methods Appl. 2, 66– 69.
43. Diviacco, S., Norio, P., Sentilin, L., Menzo, S., Clementi, M., Biamonti, G.,
Riva, S., Falaschi, A. & Giacca, M. (1992) Gene 122, 313–320.
44. Lin, T.-N., He, Y. Y., Wu, G., K han, M. & Hsu, C. Y. (1993) Stroke (Dallas)
24, 117–121.
45. Loeb, W. F. & Quimby, F. W. (1989) The Clinical Chemistry of Laborator y
Animals (Pergamon, New York).
46. Hossmann, K.-A. (1994) Ann. Neurol. 36, 557–565.
47. Hewett, S. J., Uliasz, T. F., Vidwans, A. S. & Hewett, J. A. (2000) J. Pharmacol.
Exp. Ther. 293, 417–425.
48. Kelley, K. A., Ho, L., Winger, D., Freire-Moar, J., Borelli, C. B., Aisen, P. S.
& Pasinetti, G. M. (1999) Am. J. Pathol. 155, 995–1004.
49. Kontos, H. A., Wei, E. P., Povlishock, J. T., Dietrich, W. D., Magiera, C. J. &
Ellis, E. F. (1980) Science 209, 1242–1245.
50. Chan, P. H. & Fishman, R. A. (1980) J. Neurochem. 35, 1004 –1007.
51. Chan, P. H. (1996) Stroke (Dallas) 27, 1124 –1129.
52. Eliasson, M. J., Sampei, K., Mandir, A. S., Hurn, P. D., Traystman, R. J., Bao,
J., Pieper, A., Wang, Z. Q., Dawson, T. M., Snyder, S. H. & Dawson, V. L.
(1997) Nat. Med. 3, 1089 –1095.
53. Endres, M., Scott, G., Namura, S., Salzman, A. L., Huang, P. L., Moskowitz,
M. A. & Szabo, C. (1998) Neurosci. Lett. 248, 41– 44.
54. Brock, T. G., McNish, R. W. & Peters-Golden, M. (1999) J. Biol. Chem. 274,
11660–11666.
55. Bezzi, P., Carmignoto, G., Pasti, L., Vesce, S., Rossi, D., Rizzini, B. L., Pozzan,
T. & Volterra, A. (1998) Nature (London) 391, 281–285.
56. Cazevieille, C., Muller, A., Meynier, F., Dutrait, N. & Bonne, C. (1994)
Neurochem. Int. 24, 395–398.
57. Barone, F. C. & Feuerstein, G. Z. (1999) J. Cereb. Blood Flow Metab. 19,
819– 834.
58. Hossmann, K.-A. (1994) Brain Pathol. 4, 23–36.
Iadecola et al. PNAS
兩
January 30, 2001
兩
vol. 98
兩
no. 3
兩
1299
PHARMACOLOGY