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Stroke. 1998;29:2600-2606

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(Stroke. 1998;29:2600-2606.)
© 1998 American Heart Association, Inc.

Original Contributions

Expression and Vascular Effects of Cyclooxygenase-2 in Brain

Johnny E. Brian, Jr, MD; Steven A. Moore, MD, PhD Frank M. Faraci, PhD

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


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 down arrowIntroduction 
 down arrowReferences 
 
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


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 down arrowIntroduction 
 down arrowReferences 
 
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


*    Materials and Methods
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 up arrowIntroduction
 *Materials and Methods
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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 {approx}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 ({approx}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 ({approx}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.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 down arrowIntroduction 
 down arrowReferences 
 
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 1ADown). In contrast, LPS (n=8; 100 ng/mL) caused time-dependent dilatation of cerebral arterioles, reaching 55±9% at 4 hours (Figure 1ADown). 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 1BDown; P<0.05). Coapplication of dexamethasone (n=5; 1 µmol/L) with LPS also attenuated LPS-induced vasodilatation at hours 2 to 4 (Figure 1BDown; 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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  		Figure 1. A, Treatment of cranial windows with aCSF ({bullet}) every 30 minutes for 4 hours did not change cerebral arteriolar diameter (P>0.05). LPS ({blacksquare}; 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 ({diamondsuit}; 100 µmol/L) or dexamethasone (DEX) ({blacktriangledown}; 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 2ADown). 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 2BDown).



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  		Figure 2. A, Bradykinin caused dose-dependent dilatation (10-6 mol/L [filled bars], 10-5 mol/L [open bars]) under control conditions. After treatment of cranial windows every 30 minutes for 4 hours with NS-398 (100 µmol/L), dilatation to bradykinin was not different (P>0.05). B, ADP produced dose-dependent dilatation (10-5 mol/L [filled bars], 10-4 mol/L [open bars]) under control conditions. After treatment of cranial windows every 30 minutes for 4 hours with NS-398 (100 µmol/L), dilatation to ADP was not different (P>0.05). Data are mean±SEM.

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 3Down). Intracisternal injection of LPS markedly increased COX-2 protein expression in leptomeningeal tissue ({approx}20-fold; n=4; Figure 3Down).



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  		Figure 3. COX-2 Western blots were performed on leptomeningeal tissue from rats 4 hours after intracisternal injection of aCSF (control) or 40 ng of LPS. Cytokine-activated, murine RAW 264.7 macrophages were used as a source of COX-2 standard. All 3 animals injected with aCSF had detectable bands for COX-2. However, COX-2 bands are markedly increased in the 4 animals injected with LPS. In the lanes labeled experiment 2, the COX-2 bands are darker because of a longer film exposure to better demonstrate COX-2 expression in tissue from the animal injected with aCSF. Doublets in the less intensely exposed lanes represent the different glycosylation states of COX-2 protein.

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 4ADown). Incubation of leptomeningeal tissue ex vivo with media for 4 hours induced expression of COX-2 (n=4; Figure 4ADown). LPS treatment (100 ng/mL; n=4) of leptomeningeal tissue caused an additional increase in COX-2 protein expression (n=4; Figure 4ADown), 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 4BDown).



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  		Figure 4. Analysis of leptomeningeal tissue treated ex vivo. All portions of this figure represent data from the same experimental preparations. A, Western blot of leptomeningeal tissue immediately after removal from the brain (no incubation) or after incubation ex vivo for 4 hours in DMEM with or without LPS (100 ng/mL), dexamethasone (Dex; 1 µmol/L), or NS-398 (100 µmol/L). This blot is representative of 4 separate sets of samples. B, Densitometry of Western blots of leptomeningeal tissue incubated ex vivo for 4 hours in DMEM with or without LPS, dexamethasone, or NS-398. LPS significantly increased COX-2 protein expression, and both dexamethasone and NS-398 reduced LPS-induced COX-2 expression. C, PGE2 accumulation in supernatant during 4 hours of leptomeningeal tissue incubation ex vivo. LPS significantly increased PGE2 production, which was reduced by both dexamethasone and NS-398. *P<0.05 compared with control. **P<0.05 compared with LPS. Data are mean±SEM.

In leptomeningeal tissue incubated ex vivo with LPS, PGE2 production approximately doubled compared with incubation with media (P<0.05; Figure 4CUp). Dexamethasone and NS-398 both prevented LPS-induced increase in PGE2 (Figure 4CUp).


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 down arrowIntroduction 
 down arrowReferences 
 
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 3Up) and in freshly harvested leptomeningeal tissue (Figure 4AUp). 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-{kappa}B.35 36 Both activator protein-1 and nuclear factor-{kappa}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 {approx}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


*    Acknowledgments
 
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.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
down arrowIntroduction 
 down arrowReferences 
 

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Editorial Comment

Hermes A. Kontos, MD, PhD

Associate Editor for Basic Science, Virginia Commonwealth University, Medical College of Virginia Campus, Richmond, Virginia


*    Introduction 
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 up arrowReferences
 *Introduction 
down arrowReferences 
 
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.


*    References 
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 up arrowReferences
 up arrowIntroduction 
 *References 
 

  1. Gu C, Castellino A, Chan JY-H, Chao MV. BRE: a modulator of TNF action. FASEB J.. 1998;12:1101–1108.[Abstract/Free Full Text]
  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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