(Stroke. 1998;29:2600-2606.)
© 1998 American Heart Association, Inc.
Original Contributions |
Expression and Vascular Effects of Cyclooxygenase-2 in Brain
From the Departments of Anesthesia (J.E.B.), Pathology (S.A.M.), and Internal Medicine and Pharmacology, Cardiovascular Center (F.M.F.), University of Iowa College of Medicine, Iowa City.
Correspondence to J.E. Brian, Jr, MD, Department of Anesthesia 6 JCP, University of Iowa College of Medicine, Iowa City, IA 52242. E-mail eddie-brian{at}uiowa.edu
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Background and Purpose—Cyclooxygenase-2 (COX-2) is an inducible isoform of cyclooxygenase. Several types of brain cells in culture can express COX-2 when treated with lipopolysaccharide (LPS) or some cytokines. LPS produces dilatation of cerebral arterioles in vivo through a mechanism that is partially inhibited by indomethacin. In the present study we examined the hypothesis that LPS causes increased expression of COX-2 in brain as well as COX-2–dependent dilatation of cerebral arterioles.
Methods—Cranial windows were implanted in anesthetized rats and used to measure diameter of cerebral arterioles under control conditions and during topical application of various agonists and antagonists. Windows were flushed every 30 minutes for 4 hours with vehicle (artificial cerebrospinal fluid; n=5), LPS (100 ng/mL; n=8), LPS and NS-398 (100 µmol/L; n=8), a selective inhibitor of COX-2, or LPS and dexamethasone (1 µmol/L; n=5), which attenuates expression of COX-2. To examine expression of COX-2 protein in vivo, other animals were injected intracisternally with artificial cerebrospinal fluid (n=3) or LPS (40 ng; n=4). Four hours after injection, the leptomeninges were harvested and analyzed by Western blot for expression of COX-2 protein. In a third group of experiments, COX-2 expression and prostaglandin E2 (PGE2) production were determined in leptomeningeal tissue treated for 4 hours ex vivo with vehicle (n=4), LPS (100 ng/mL; n=4), LPS and NS-398 (100 µmol/L; n=4), or LPS and dexamethasone (1 µmol/L; n=4).
Results—LPS caused marked, progressive dilatation of cerebral arterioles, with a maximum increase in diameter of 55±9% (mean±SEM) at 4 hours. Coapplication of either NS-398 or dexamethasone with LPS reduced dilatation of cerebral arterioles at hours 2 through 4 (P<0.05). In contrast, NS-398 did not inhibit dilatation of cerebral arterioles in response to bradykinin or ADP. In animals injected intracisternally with vehicle, COX-2 protein was expressed at a very low level in leptomeningeal tissue. Intracisternal injection of LPS increased COX-2 protein expression by approximately 20-fold (P<0.05). In leptomeningeal tissue treated ex vivo with LPS, there was also expression of COX-2. Both dexamethasone and NS-398 markedly reduced COX-2 protein expression in ex vivo LPS-treated tissue. PGE2 production was detectable under control conditions in leptomeningeal tissue incubated in vehicle ex vivo for 4 hours (6.5±1.1 pmol/mg protein). LPS treatment significantly increased PGE2 production to 12.8±1.1 pmol/mg protein (P<0.05). Both dexamethasone and NS-398 significantly attenuated LPS-induced PGE2 production (P<0.05).
Conclusions—LPS increased expression of COX-2 protein in leptomeningeal tissue and caused COX-2–dependent dilatation of cerebral arterioles in vivo. Ex vivo, both NS-398 and dexamethasone suppressed LPS-induced PGE2 production and COX-2 expression in leptomeningeal tissue. Inhibition of LPS-induced dilatation of cerebral arterioles in vivo by NS-398 and dexamethasone suggests that the dilatation was dependent on expression and activity of COX-2. These findings support the concept that exposure of brain to LPS causes cerebral vasodilatation that is dependent in part on expression and activity of COX-2.
Key Words: cerebral arteries • cyclooxygenase • dexamethasone • lipopolysaccharides • vasodilation
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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Cyclooxygenase is the rate-limiting enzyme in the production of prostaglandins, converting arachidonic acid to prostaglandin H2 (PGH2).1 Many tissues express a constitutive isoform of cyclooxygenase (COX-1) under normal conditions.1 A second isoform of cyclooxygenase (COX-2) has been described.2 In most cultured cells, COX-2 is not expressed under normal conditions, but inflammatory stimuli, including lipopolysaccharide (LPS), reactive oxygen species, and some cytokines and growth factors, can upregulate COX-2 expression.3 4 5 6 Expression of COX-2 leads to increased production of prostanoids.3 6 In brain, prostanoids are potent vasoactive7 and inflammatory substances.8
In brain of adult animals, a subpopulation of neurons normally expresses COX-2.9 Other brain cells, including microglia, astrocytes, and vascular cells, do not normally express significant levels of COX-2 but upregulate COX-2 expression after inflammatory stimuli.10 11 12 In addition, certain pathophysiological conditions, including ischemia and hypoxia, are associated with increased expression of COX-2 in brain.13 14 15
We and others have shown that indomethacin can partially inhibit LPS-induced dilatation of cerebral arterioles and increased cerebral blood flow.16 17 Because indomethacin inhibits activity of both COX-1 and COX-2,18 it is not known which isoform of COX is responsible for LPS-induced cerebral vasodilatation. In the present study we examined the hypothesis that exposure of the cerebral cortex to LPS would cause expression of COX-2 and dilatation of cerebral arterioles. We further hypothesized that LPS-induced dilatation of cerebral arterioles would be inhibited by dexamethasone, which prevents expression of COX-2 and NS-398, a selective inhibitor of COX-2 enzymatic activity.2 19 20 21
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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Cranial Windows
Male Sprague-Dawley rats (n=41; weight, 341±3 g) were anesthetized with pentobarbital (50 mg/kg IP). A tracheostomy was performed, and ventilation was maintained with a small-animal ventilator. PaCO2 was adjusted to
40 mm Hg by altering minute ventilation, and
PaO2 was maintained at >100
mm Hg by supplementing room air with oxygen. Anesthesia
was supplemented by administration of additional pentobarbital (5 to 15
mg/kg per hour) through the femoral vein. Rectal temperature was
measured and maintained at 37±0.5°C with a heating pad.
A closed cranial window was prepared in a manner similar to that
described in rabbits.22 The scalp, muscle, and
periostium overlying the parietal area of the skull were reflected, and
bleeding was controlled with ferric chloride solution. A
craniotomy (3x4 mm) was made in the parietal
bone with the use of an air-cooled drill, and bone bleeding was
controlled with bone wax. The dura overlying an arteriole was incised.
Two blunt needles were affixed to a dam of bone wax surrounding the
craniotomy, and a circular glass coverslip (12 mm)
was fused to the wax. The window was reinforced with dental acrylic. An
outlet tube was affixed to one needle and set to maintain intracranial
pressure at 10 cm H2O. A stopcock was attached to
the other needle, and the window was filled with artificial
cerebrospinal fluid (aCSF) warmed to 37°C and equilibrated with 90%
N2/5% O2/5%
CO2 (pH 7.34±0.003;
PO2 72±1 mm Hg;
PCO2 41±0.3 mm Hg). Cerebral
arterioles were observed with a microscope equipped with a video
camera, and images were recorded on videotape. Arteriolar diameter
was measured with a calibrated video micrometer. The
preparation was allowed to equilibrate for 30 minutes, during which
time the window was flushed with 2 mL of aCSF every 15 minutes.
Flushing the window with aCSF did not alter diameter of cerebral
arterioles.
After the equilibration period, arteriolar diameter was measured under control conditions and in response to topical ADP (10-5 and 10-4 mol/L), an activator of endothelial nitric oxide (NO) synthase.23 Responses to ADP were examined to test responsiveness of the preparation. The window was then flushed with aCSF several times, and the preparation was allowed to recover for 30 minutes. After a second measurement of baseline vessel diameter, animals were randomly allocated to receive (1) aCSF alone (n=5); (2) aCSF containing LPS (Sigma; 100 ng/mL; n=8); (3) aCSF containing LPS (100 ng/mL) and an inhibitor of COX-2, NS-398 (Calbiochem; 100 µmol/L; n=8); or (4) aCSF containing LPS (100 ng/mL) and dexamethasone (Sigma; 1 µmol/L; n=5).
The diameter of cerebral arterioles was recorded at baseline and compared between groups. After baseline measurements, the cranial windows were flushed every 30 minutes for 4 hours with 2 mL of aCSF or aCSF containing LPS with or without inhibitors. Diameter of arterioles was measured at 30 minutes and 1, 2, 3, and 4 hours before flushing of the window. Changes in arteriolar diameter are expressed as percent change in diameter compared with baseline. Arterial blood pressure was continuously monitored, and arterial blood gases were measured at regular intervals.
To test selectivity of NS-398 on LPS-induced dilatation of cerebral arterioles, 3 other groups of animals were studied. In 1 group (n=6), vasodilatation to ADP (10-5, 10-4 mol/L) was tested. After recovery, cranial windows were flushed with aCSF containing NS-398 (100 µmol/L) every 30 minutes for 4 hours. Dilatation to ADP (10-6 and 10-5 mol/L) was then retested in the presence of NS-398 (100 µmol/L). In the second group of animals (n=6), dilatation to bradykinin (10-6, 10-5 mol/L) was tested, and cranial windows were then flushed with NS-398 (100 µmol/L) every 30 minutes for 4 hours. Dilatation to bradykinin was then retested in the presence of NS-398 (100 µmol/L). Bradykinin causes an immediate, reversible dilatation in cerebral arterioles that is dependent on cyclooxygenase24 25 (presumably COX-1, since the time course and reversibility of the bradykinin-dependent dilatation are not consistent with COX-2 expression and activity). In a third group of animals (n=3), dilatation to bradykinin (10-6, 10-5 mol/L) was tested, cranial windows were flushed with aCSF every 30 minutes for 4 hours, and dilatation to bradykinin was then retested.
Intracisternal Injection
In a separate group of rats (n=7), expression of COX-2 protein
was documented by Western blot analysis. Preliminary studies
demonstrated that harvesting brain directly beneath the cranial window
did not yield sufficient tissue to allow adequate protein for Western
blot analysis. To circumvent this problem, intracisternal
injection of LPS was performed to expose the entire brain surface to
LPS. The amount of LPS injected (40 ng) was calculated on the basis of
the estimated cerebrospinal fluid (CSF) volume in an adult rat (300 to
400 µL) to yield a concentration of LPS equivalent to that used in
the cranial window experiments (100 ng/mL). The concentration of LPS in
CSF produced with this procedure is probably less than the total dose
delivered in cranial windows because the windows were flushed with
LPS-containing CSF every 30 minutes for 4 hours.
For intracisternal injections, rats were anesthetized with pentobarbital (50 mg/kg IP) and atropine (15 µg/kg IP) to inhibit respiratory secretions. During the course of the procedure, the pentobarbital was supplemented as needed (5 to 15 mg/kg per hour) to maintain an adequate level of anesthesia. Animals were placed in a stereotaxic head frame, and the atlanto-occipital membrane was exposed through a small incision. The atlanto-occipital membrane was punctured with a 27-gauge needle in a stereotaxic arm and confirmed by aspiration of CSF. After aspiration of 100 µL of CSF, 100 µL of aCSF (with or without LPS, 40 ng) was injected over 15 minutes. The needle was left in place for 30 minutes to allow the injected CSF time to diffuse away from the site of injection. The incision was closed with sutures after removal of the needle. The animals were then maintained under anesthesia for 4 hours.
Western Blot Analysis
Animals were killed with an overdose of pentobarbital, the brain
was rapidly removed, and the leptomeninges containing
pial blood vessels were separated from the cortex. While the surface
was kept moist with ice-cold phosphate-buffered saline, the meninges
were incised and peeled from the surface with fine-tipped forceps under
a dissecting microscope. Portions of meninges (20 to 40
mm2) were removed as intact sheets of tissue. The
isolated leptomeninges were homogenized by
sonication in ice-cold lysis buffer (50 mmol/L Tris, pH 7.5,
150 mmol/L NaCl, 100 µg/mL phenylmethylsulfonyl
fluoride, 1 µg/mL aprotinin, 1 µg/mL leupeptin, 1
mmol/L diethyldithiocarbamic acid, 1% Nonidet P-40, and 1% sodium
deoxycholate), and protein content was
determined.26 Equal amounts of protein per lane
(100 µg) were loaded onto a 7.5% polyacrylamide gel and
separated by electrophoresis at 200 V for 45 minutes. Proteins were
then transferred to nitrocellulose at 100 V for 1 hour, and the
membrane was blocked with 5% nonfat dry milk/0.5% Tween-20 in
Tris-buffered saline. The nitrocellulose was then incubated with a
rabbit polyclonal antibody specific for COX-2 (Cayman Chemical, catalog
No. 160106; 1:1000) overnight at 25°C followed by horseradish
peroxidase–conjugated secondary antibodies (donkey anti-rabbit,
Amersham) for 1 hour at 25°C. Antibody labeling was detected by
chemiluminescence (Pierce). To verify antibody specificity and the size
of the COX-2 bands, protein extracts from cultured,
cytokine-activated RAW 264.7 macrophages were
used as controls for COX-2. Color molecular weight standards were also
run on each gel. Western blot results were quantified by
densitometry.
Ex Vivo Leptomeningeal Treatment
Rats (n=8) were anesthetized with ether and decapitated.
Leptomeningeal tissue was removed as described above. In each animal,
tissue from each hemisphere was used as a single sample. Each treatment
group contained leptomeningeal tissue from 4 different rats. Control
leptomeningeal tissue was placed immediately after dissection in
ice-cold lysis buffer for protein determination and analysis of
COX-2 expression by Western blot. Experimental samples were placed into
serum-free Dulbecco's modified Eagle's medium (DMEM) with or without
LPS (100 ng/mL), LPS and dexamethasone (1 µmol/L),
or LPS and NS-398 (100 µmol/L). When meninges were harvested for
NS-398 or dexamethasone treatment, the buffer used to
moisten brains during harvesting contained appropriate
concentrations of NS-398 or dexamethasone.
Tissue was incubated at 37°C for 4 hours, and the media was removed
and analyzed for prostaglandin
E2 (PGE2) concentration.
Tissue was homogenized for protein determination and
Western blot analysis. PGE2 was
quantified by radioimmunoassay with the use of an antibody specific for
PGE2 (Seragen). Cross-reactivity of this antibody
with other eicosanoids is <0.1%. Prostaglandin
production was normalized to protein content of each
sample.
Statistical Analysis
Data are expressed as mean±SEM. Data between groups were
compared by ANOVA and Duncan's post hoc test. Data within groups were
analyzed by repeated-measures ANOVA and post hoc comparison by
means contrast. P<0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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Effect of NS-398 on LPS-Induced Dilatation of Cerebral Arterioles
Baseline arteriolar diameter was not different between groups and averaged 53±1 µm. Dilatation of cerebral arterioles in response to ADP (10-5, 10-4 mol/L) performed at the beginning of each study averaged 8±1% and 17±1%, respectively.
In control animals treated with aCSF (n=5), there was no change
in arteriolar diameter over the 4 hours of study (P>0.05;
Figure 1A
). In contrast, LPS (n=8; 100
ng/mL) caused time-dependent dilatation of cerebral arterioles,
reaching 55±9% at 4 hours (Figure 1A). LPS-induced changes in
arteriolar diameter were significantly different than those in the aCSF
group at hours 2 to 4 (P<0.05). When NS-398 (n=8; 100
µmol/L) was coapplied with LPS, vasodilatation was significantly
reduced at hours 2 to 4 (Figure 1B; P<0.05). Coapplication
of dexamethasone (n=5; 1 µmol/L) with LPS also
attenuated LPS-induced vasodilatation at hours 2 to 4 (Figure 1B;
P<0.05). There were no differences either across time or
between groups in mean arterial pressure or
arterial blood gas values, which averaged 125±1
mm Hg, pH 7.39±0.003, PaCO2
40±0.3 mm Hg, and PaO2
205±2 mm Hg, respectively (P>0.05).
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)
every 30 minutes for 4 hours did not change cerebral arteriolar
diameter (P>0.05). LPS (
; 100 ng/mL) caused a
marked, time-dependent dilatation that reached 55±9% by 4 hours. The
LPS-induced dilatation was significant compared with aCSF at hours 2
through 4 (P<0.05). *P<0.05 compared
with aCSF. B, Coapplication of either NS-398 (
; 100 µmol/L)
or dexamethasone (DEX) (
; 1 µmol/L) with LPS (100
ng/mL) significantly reduced LPS-induced dilatation at hours 2 through
4 (P<0.05). +P<0.05 compared with
LPS. Data are mean±SEM.
Effect of NS-398 on Bradykinin- and ADP-Induced Dilatation of
Cerebral Arterioles
In separate groups of animals, there were no significant
differences (P>0.05) in arteriolar diameter, mean
arterial pressure, or arterial blood gases,
which averaged 52±2 µm, 128±1 mm Hg, pH 7.40±0.003,
PaCO2 40±0.3 mm Hg, and
PaO2 206±3 mm Hg,
respectively. In 1 group of animals (n=6), bradykinin
(10-6, 10-5
mol/L) produced vasodilatation that was not significantly
affected (P>0.05) by treatment with NS-398 (100
µmol/L) every 30 minutes for 4 hours (Figure 2A). In a second group of animals (n=3),
vasodilatation to bradykinin (10-6,
10-5 mol/L) was similar (P>0.05)
before (18±7%; 40±8%) or after (21±9%; 38±9%) flushing windows
with aCSF every 30 minutes for 4 hours. In a third group of animals
(n=6), vasodilatation to ADP (10-5,
10-4 mol/L) was not significantly
different (P>0.05) before or after treatment with NS-398
(100 µmol/L) every 30 minutes for 4 hours (Figure 2B).
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LPS-Induced COX-2 Expression In Vivo
In animals injected intracisternally with vehicle (n=3), COX-2
expression in leptomeningeal tissue was slight but detectable by
Western blot analysis (Figure 3).
Intracisternal injection of LPS markedly increased COX-2 protein
expression in leptomeningeal tissue (20-fold; n=4; Figure 3).
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LPS-Induced COX-2 Expression and PGE2Production Ex Vivo
In freshly harvested leptomeningeal tissue, there was no
detectable COX-2 protein expression by Western blot (n=4; Figure 4A). Incubation of leptomeningeal tissue
ex vivo with media for 4 hours induced expression of COX-2 (n=4; Figure 4A). LPS treatment (100 ng/mL; n=4) of leptomeningeal tissue caused an
additional increase in COX-2 protein expression (n=4; Figure 4A), which
was significantly reduced (P<0.05) by
dexamethasone (1 µmol/L/ml; n=4) or NS-398 (100
µmol/L/ml; n=4; Figure 4B).
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In leptomeningeal tissue incubated ex vivo with LPS,
PGE2 production approximately doubled
compared with incubation with media (P<0.05;
Figure 4C
). Dexamethasone and NS-398 both prevented
LPS-induced increase in PGE2 (Figure 4C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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There are several new findings in this study. First, LPS produced time-dependent dilatation of cerebral arterioles in vivo that was inhibited by NS-398 and dexamethasone. Second, NS-398 appears to be a relatively selective inhibitor of COX-2 in brain in vivo. Third, direct application of LPS to brain cortical surface caused significant upregulation of COX-2 protein expression in leptomeningeal tissue. Fourth, exposure of ex vivo leptomeningeal tissue to LPS upregulated COX-2 protein expression and PGE2 production. Both dexamethasone and NS-398 suppressed COX-2 expression and PGE2 production ex vivo. Data from ex vivo experiments are consistent with in vivo data demonstrating COX-2–dependent dilatation of cerebral arterioles and expression of COX-2 protein after treatment with LPS. Because LPS-induced dilatation in brain was significantly reduced by both NS-398 and dexamethasone, these findings support the concept that LPS-induced cerebral vasodilatation is dependent in part on expression and activity of COX-2.
COX-2 Expression in Brain
COX-2 is an inducible isoform of
cyclooxygenase, distinct from the constitutive
isoform, COX-1.1 In most tissues, COX-2 is not
expressed under basal conditions but is upregulated by certain factors,
including inflammatory stimuli.3 4 5 6 Once
expressed, COX-2 increases production of
prostaglandins.3 The principal
prostaglandin produced during inflammation is
PGE2,1 and
PGE2 is a potent dilator of cerebral blood
vessels.7 Cyclooxygenase can
also produce reactive oxygen species, which are vasodilators in
brain.24
In brain, a number of cell types can express COX-2 during inflammatory conditions in vitro. Cultured astrocytes, microglia, and microvascular cells (both endothelium and smooth muscle) express COX-2 and increase prostanoid production after stimulation with LPS or cytokines.11 27 28 29 In vivo, intravenous LPS or cytokines cause expression of COX-2 in the cerebral endothelium, vascular, perivascular, and leptomeningeal cells.12 30 31 In the present study we did not identify the specific cells expressing COX-2 but rather documented LPS-induced expression of COX-2 protein in leptomeningeal tissue by Western blot.
In the present study we detected minimal expression of
COX-2 protein in leptomeningeal tissue after intracisternal injection
of aCSF (Figure 3) and in freshly harvested leptomeningeal tissue
(Figure 4A). The leptomeningeal tissue consists principally of the
arachnoid and pial layers of the meninges and the accompanying vascular
structures. There may be slight contamination with underlying
superficial brain tissue, which contains primarily glial cells. We
believe that it is unlikely that the leptomeningeal preparation
included neurons that constitutively express COX-2 because
these neurons are found in deeper brain
structures.9 Furthermore, when we
analyzed freshly harvested leptomeningeal tissue from animals
that had not undergone intracisternal injection, we did not detect
COX-2 protein. It is likely that a mild inflammatory reaction occurred
after intracisternal injection of aCSF. We prepared the aCSF under
sterile conditions and used sterile, disposable supplies when
performing intracisternal injections. Low levels of LPS could be
present in the aCSF, which could account for the minimal COX-2
expression in animals injected with aCSF. However, intracisternal
injection of LPS (40 ng) increased COX-2 expression in leptomeningeal
tissue in vivo by 20-fold.
Leptomeningeal tissue from animals that had not undergone intracisternal injection did not express COX-2. However, incubation of leptomeningeal tissue in DMEM for 4 hours caused expression of COX-2. We used sterile supplies and endotoxin-free reagents for the incubation but cannot completely rule out LPS contamination as a causative factor for COX-2 expression. It is also possible that the process of stripping the leptomeningeal tissue from the brain could activate mechanisms, resulting in expression of COX-2. As in vivo, LPS treatment of leptomeningeal tissue ex vivo caused an additional increase in COX-2 protein expression, which was attenuated by both dexamethasone and NS-398. In cultured microglia, PGE2 and cAMP have a positive effect on COX-2 protein expression.28 32 33 Consistent with this, the promoter region of the COX-2 gene contains a cAMP response element.32 34 Because NS-398 inhibits prostanoid production from COX-2, it is possible that NS-398 attenuated COX-2 expression in ex vivo leptomeningeal tissue by reducing prostanoid and cAMP production.
Dexamethasone suppresses LPS or
cytokine-induced expression of COX-2 in cultured
astrocytes, microglia, and cerebrovascular
cells.1 11 27 29 The COX-2 gene lacks a
glucocorticoid response element by which dexamethasone
could suppress COX-2 expression.34 However,
dexamethasone may suppress expression of COX-2 by other
mechanisms, including direct inhibition of the transcription factors
activator protein-1 and nuclear
factor-
B.35 36 Both activator
protein-1 and nuclear factor-B bind to the promoter region of COX-2,
activating COX-2 transcription.11 37 Although we
did not study the mechanism by which dexamethasone
prevented LPS-induced dilatation, there is good evidence that
dexamethasone suppresses expression of COX-2.
Dexamethasone appears to have minimal, if any, direct vascular effects. We have previously reported that prolonged exposure of cerebral arterioles in vivo to dexamethasone does not alter resting diameter.38 Furthermore, dexamethasone does not inhibit ADP-mediated dilatation of cerebral arterioles (which is NO dependent).38 Others have reported that dexamethasone does not alter constrictor or dilator responses of cerebral and extracerebral vessels.39 40 Dexamethasone does not affect the activity of COX-1 in vivo.41 Thus, it is unlikely that dexamethasone reduced LPS-induced dilatation by a nonspecific effect on vascular tone.
We have previously reported that dexamethasone and indomethacin inhibited LPS-induced dilatation of cerebral arterioles in rabbits.16 In both the previous and present studies, dexamethasone tended to produce more suppression of LPS-induced dilatation than indomethacin or NS-398, although this was not statistically significant. LPS can also cause expression of inducible NO synthase, which is inhibited by dexamethasone.16 42 Thus, dexamethasone could produce a greater reduction of LPS-induced dilatation because of suppression of both inducible NO synthase and COX-2 expression. On the basis of the present data, we cannot comment on the relative contribution of COX-2 versus inducible NO synthase to the observed dilatation. Our findings also do not exclude potential interaction of inducible NO synthase and COX-2. We used NS-398 and dexamethasone as pharmacological tools to demonstrate involvement of the specific systems under study.
NS-398 and COX-2
NS-398 has been reported to be a selective inhibitor
of COX-2 enzymatic activity. In vitro, NS-398 in concentrations up to
100 µmol/L does not inhibit activity of COX-1 in isolated enzyme
preparations.19 20 21 In vivo, NS-398 does not
reduce COX-1 activity in gastric tissue, even when there is complete
suppression of COX-2 activity in inflammatory
exudates.43 In the present study bradykinin
caused dose-dependent dilatation of cerebral arterioles, which was not
significantly inhibited by NS-398. The specific isoform of COX
activated by bradykinin has not been definitely identified but
is most likely COX-1, since acute application of bradykinin caused
immediate, reversible dilatation of cerebral arterioles, which can be
blocked with indomethacin.24 25
This time course is not consistent with bradykinin causing
expression of COX-2 with subsequent dilatation. In addition,
bradykinin-induced dilatation of cerebral arterioles is
endothelium dependent, and it appears unlikely that
COX-2 is expressed in cerebral vessels of adult animals under normal
conditions.9 24 25 NS-398 also did not affect
dilatation of cerebral arterioles due to activation of
endothelial NO synthase with ADP. Our data suggest that
NS-398 is selective for COX-2 in brain in vivo and that the
inhibitory effect of NS-398 on LPS-induced dilatation is
due to inhibition of COX-2 activity. We have previously reported that
in rabbit cerebral arterioles in vivo, indomethacin
reduced LPS-induced dilatation by 50%,16
which is consistent with the findings of the present
study.
In summary, we have demonstrated that topical application of LPS caused marked, time-dependent dilatation of cerebral arterioles that was inhibited by NS-398, a selective inhibitor of COX-2, as well as dexamethasone. We documented LPS-mediated increased expression of COX-2 protein in leptomeningeal tissue in vivo and ex vivo by Western blot analysis, consistent with our pharmacological data that COX-2 contributes to LPS-induced dilatation of cerebral arterioles in vivo. We also demonstrated COX-2–dependent PGE2 production in LPS-treated leptomeningeal tissue ex vivo. These data suggest that expression and activity of COX-2 are important components of the vascular response to inflammation in brain in vivo. While this report was under revision, another study was published which also suggests that expression of COX-2 contributes to cerebral vasodilatation after treatment with LPS.44
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Acknowledgments |
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This study was supported by National Institutes of Health grants NS-24621 and HL-38901, by American Heart Association Grant-in-Aid 96 50661N, and by research funds from the Department of Anesthesia, University of Iowa College of Medicine. Dr Faraci is an Established Investigator of the American Heart Association. The authors wish to express thanks to Paula Ludwig and Elizabeth Yoder for technical assistance.
Received March 9, 1998; revision received July 20, 1998; accepted August 24, 1998.
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Editorial Comment
Associate Editor for Basic Science, Virginia Commonwealth University, Medical College of Virginia Campus, Richmond, Virginia
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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Prostaglandins are involved in the regulation of a wide variety of normal and pathophysiological biological functions, including inflammation.1 It has recently been recognized that the inducible form of cyclooxygenase (COX-2) plays a central role in the induction and amplification of inflammation.2
The above article as well as that of Okamoto et al,3 published earlier in Stroke, confirm the crucial role of COX-2 in the induction of vasodilation, one of the cardinal manifestations of inflammation in the brain. In both studies the inflammatory reaction was induced by the administration of lipolysaccharide, an endotoxin product. It was demonstrated that COX-2 was induced, its activity increased with resulting increased production of prostaglandins, and the inhibition of the induction of the enzyme or inhibition of its activity resulted in a reduction in vasodilation.
There are significant practical benefits from the use of agents that inhibit either the induction or the activity of COX-2 in preventing the adverse consequences of inflammation of the brain. The inflammatory process in the brain causes, in addition to vasodilation, edema and, ultimately, neuronal dysfunction. Inhibition of the process is very likely to minimize or prevent the consequent brain dysfunction. The confirmation, therefore, that COX-2 plays an important role in the inflammation of the brain constitutes a significant advance.
Received March 9, 1998; revision received July 20, 1998; accepted August 24, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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1. Gu C, Castellino A, Chan JY-H, Chao MV. BRE: a modulator of TNF action. FASEB J.. 1998;12:1101–1108.
2. Needleman P, Isakson PC. The discovery and function of COX-2. J Rheumatol.. 1997;24:6–8.
3. Okamoto H, Osamu I, Roman RJ, Hudetz AG. Role of inducible nitric oxide synthase and cyclooxygenase-2 in endotoxin-induced cerebral hyperemia. Stroke.. 1998;29:1209–1218.
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