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Abstract

Nitric oxide (NO) directly activates trigeminal afferents innervating the dura mater and up-regulates inflammatory mediators. We evaluated NO-mediated up-regulation of cyclooxygenase-2 (COX-2), tumour necrosis factor-alpha (TNF-α) and matrix metalloproteinase-9 (MMP-9), and the effect of glucocorticoid administration in an experimental animal model of migraine. COX-2 and TNF-α expression and MMP-9 activity were increased after continuous intravenous infusion of glyceryl trinitrate (GTN), a NO donor. Immunofluorescence staining demonstrated strong expression of these inflammatory mediators in the meningeal blood vessels. Methylprednisolone (MP) down-regulated MMP-9, which was reversed by RU486, a glucocorticoid receptor antagonist. COX-2 and TNF-α expression was not affected by MP or RU486 administration. These results suggest proinflammatory mediators are involved in the NO-mediated cascade of migraine pathogenesis. Further understanding of the activation of these inflammatory mediators at the transcriptional level may have therapeutic implications for future migraine treatments.

Keywords

Neurogenic inflammation meninges transcription factor blood-brain barrier corticosteroids

Introduction

Inflammation within the dura mater correlates with provoked migraine headache (1). Trigeminal fibres innervating the meningeal layer of the dura mater function as the main path for headaches and mediate pain sensation. Headaches are maintained under an aseptic inflammatory condition induced by increased vessel permeability and vasodilation, oedema and mast cell degranulation following neuropeptide release from the perivascular axons of trigeminal nerves (2–4).

Nitric oxide (NO) may play a pivotal role in migraine, as workers exposed to glyceryl trinitrate (GTN, an exogenous nitric oxide donor) in explosives factories had headaches (5). Intravenous infusions of GTN produced a delayed headache in migraineurs, which was indistinguishable from a spontaneous migraine attack (6). NO is generated from NO synthase (NOS), and NOS inhibitors can be used as a treatment for spontaneous migraine attacks (7).

A GTN challenge produced a delayed migraine response 4–6 h following up-regulation of new genes and cytokines important to the pathogenesis of an attack (8). Moreover, since GTN activates nuclear factor (NF)-κB, its blockade provides a novel target as a treatment for migraine headache (9). NF-κB induces iNOS (10) and correlates with up-regulation of cyclooxygenase-2 (COX-2) and matrix metalloproteinase-9 (MMP-9) (11).

Methylprednisolone (MP) is an anti-inflammatory agent that works via the glucocorticoid receptor (GR). Glucocorticoids (GC) inhibit two major proinflammatory transcription factors, NF-κB and AP-1. Suppression by GC does not affect NF-κB translocation, nor binding to DNA, which suggests interference in transcriptional activation of NF-κB (12). GC repress the expression of proinflammatory cytokines and also affect the production of inflammatory mediators, including prostaglandins and NO (13).

Based on the data implicating GTN and NO in migraine and inflammation, we investigated the effects of GTN infusion on the expression of inflammatory factors such as COX-2 and MMP-9, as well as tumour necrosis factor-α (TNF-α) in the rat dura mater, and studied the effects of MP on COX-2, TNF-α and MMP-9 expression by the suppression of NF-κB activation. Furthermore, we used RU486, a GR antagonist, to block the effects of MP.

Methods

Administration of drugs

Adult male Sprague-Dawley rats weighing 270–300 g were used for these experiments. All procedures were performed in accordance with the guidelines for the care and use of laboratory animals approved by the Institutional Animal Care and Use Committee, and in research facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.

Rats were anaesthetized with an intraperitoneal (i.p.) injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). GTN (Pohl-Boskamp Co. GmbH, Hohenlockstedt, Germany) or vehicle (GTN, 10 µg/kg/min for 30 min; vehicle, 0.9% NaCl) were intravenously infused at a rate of 1 ml/h after anaesthesia. The infusion rate of GTN in our experiment was higher than that generally used in humans. At first we tested a GTN dosage of 2 µg/kg/min, but it resulted in a weak signal intensity on Western blot for both COX-2 and TNF-α. A dosage of 10 µg/kg/min was found appropriate to demonstrate increased signals on both Western blot and immunohistochemistry. MP (30 mg/kg) was administered via the tail vein immediately after GTN infusion. RU486 [15 mg/kg mixed in dimethylsulphoxide (DMSO) 10% intraperitoneally] or a vehicle (DMSO 10% intraperitoneally) was administered 1 min before GTN infusion. All rats were sacrificed with an overdose of an anaesthetic agent at 1, 3, 6 or 18 h after GTN infusion. Sham-operated control animals were sacrificed immediately after vehicle infusion. Dura mater and brain were isolated and used for further experiments.

Western blot analysis

Rats were perfused transcardially with cold 0.9% NaCl at 1, 3, 6 or 18 h after GTN infusion. Samples were homogenized with Teflon homogenizer (800 rpm, 30 strokes) in seven volumes of ice-cold lysate buffer [20 mm HEPES (pH 7.6), 250 mm sucrose, 1 mm dithiothreitol, 1.5 mm MgCl₂, 10 mm KCl, 1 mm ethylenediamine tetraaceticacid, 1 mm ethylene glycol tetraaceticacid, 1 mm phenylmethylsulphonyl fluoride, 2 µm leupeptin, 1.5 µm pepstatin, 0.7 µm aprotinin]. Insoluble materials were removed by centrifugation at 13 000 rpm for 5 min at 4°C. The supernatant was collected and the protein concentration of each sample determined. Aliquots containing 20 µg of protein were separated by 12% sodium dodecyl sulphate (SDS)–polyacrylamide gels and transferred to polyvinylidenefluoride membranes. The membranes were blocked for 1 h with 5% non-fat milk in phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBS–T) and then incubated overnight at 4°C in the presence of the following antibodies: rabbit polyclonal anti-COX-2 (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), monoclonal anti-rat TNF-α/TNFSF1A antibody (1:200; R&D Systems, Inc., Minneapolis, MN, USA), and monoclonal anti-β-actin (1:5000; Sigma, St Louis, MO, USA). The membranes were washed with PBS–T and incubated with horseradish peroxidase-conjugated, anti-rabbit immunoglobulin G (IgG) antibodies or antimouse IgG for 1 h. After washing with PBS–T, immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Life Sciences, Piscataway, NJ, USA). Optical density measurements were performed using the Gelpro software (Media Cybernetics, Bethesda, MD, USA).

Gelatin zymography

Dissected dura mater was homogenized in cold lysate buffer. Samples were separated by electrophoresis on a 10% SDS–polyacrylamide gel containing 0.1% gelatin as substrate under non-reducing conditions at 4°C. Gels were washed twice with 2.5% Tritox X-100 for 15 min each to remove the SDS. Gels were incubated with the developing buffer [50 mmol/l Tris–HCl buffer (pH 7.5) containing 0.15 mol/l NaCl, 10 mmol/l CaCl₂ and 0.2 mg/ml NaN₃] at 37 °C for 16 h and stained with 0.1% Coomassie Brilliant Blue R250, followed by destaining with a solution containing methanol/acetic acid/water (20:10:70).

Immunohistochemistry

Rats were transcardially perfused with ice-cold 0.9% saline solution 18 h after GTN infusion. The dura was dissected rapidly and mounted onto poly-L-lysine-coated glass slides. Tissues were dried completely and post-fixed in absolute ethanol at −20°C for 10 min. After several washes in 0.05 m PBS, the tissue was incubated with 5% normal goat serum (NGS; Vector Laboratories, Inc., Burlingame, CA, USA) in PBS containing 0.1% Triton X-100 (PBS–Tx) for 1 h at room temperature. Immunohistochemistry for COX-2, MMP-9 or TNF-α was performed using the following: monoclonal anti-MMP-9 antibody (Oncogene^TM Research Products, San Diego, CA, USA), rabbit polyclonal anti-COX-2 (Santa Cruz Biotechnology, Inc.) or monoclonal antirat TNF-α/TNFSF1A antibody (R&D Systems, Inc.). Antibodies were diluted with PBS–Tx containing 2% NGS (1:300) and then incubated overnight at 4°C. After three rinses in PBS–Tx, the tissue for staining against COX-2 or TNF-α was incubated for 1 h at room temperature with Alexa Fluor 488 goat antirabbit IgG or goat antimouse IgG (1:200; Molecular Probes, Carlsbad, CA, USA) in PBS–Tx containing 2% NGS; for MMP-9, it was incubated with Alexa Fluor 568 goat antimouse IgG.

For examining the increased inflammatory factors in dural vessels, double-staining with von Willebrand factor (vWF, Dako rabbit antihuman Von Willebrand Factor; Carpinteria, CA, USA) as an endothelial cell marker was performed. The tissue stained by vWF was visualized after incubation with Alexa Fluor 568 or Alexa Flour488 goat antirabbit IgG for 1 h at room temperature. After washing with PBS, nuclei were stained by Hoechst 33482 (Molecular Probes) and coverslipped with mounting medium (Vectasheild, Vector Laboratories, Inc.). Immunostained sections were examined with a Nikon Eclipse E600 microscope (Nikon, Tokyo, Japan), and images were captured and processed with Laser Sharp2000 software (BioRad, Hercules, CA, USA).

Statistical analysis

Data are expressed as mean ± s.d. Differences among groups were analysed by one-way analysis of variance (anova) followed by Tukey's post hoc test. All analysis was performed with SPSS for Windows 10.0 (SPSS Inc., Chicago, IL, USA). P-values < 0.05 were considered to be significant.

Results

Effects of GTN on COX-2 and TNF-α expression

We tested whether NO induction increased COX-2, another inflammatory factor, or TNF-α, a proinflammatory cytokine. On Western blot analysis, basal expression of COX-2 and TNF-α protein was observed in vehicle-treated animals. COX-2 protein expression was increased at 3 h after GTN infusion and continuously up-regulated to 18 h (136 ± 39% of control, n = 3 for each time point, Fig. 1a). TNF-α protein was expressed 1 h after GTN infusion (150 ± 30% of control), rapidly decreased at 3 h, and then reappeared at 18 h (163 ± 36% of control, n = 3, Fig. 1b).

Figure 1

Western blot of COX-2 and TNF-α protein after GTN infusion. (a) COX-2 protein expression was increased 3 h after GTN infusion (10 µg/kg/min), and continuously up to 18 h. (b) TNF-α protein was expressed at 1 h after GTN infusion, with rapid reduction around 3 h and reappearance at 18 h.

On immunohistochemistry, both COX-2 and TNF-α protein were strongly expressed in meningeal blood vessels after GTN infusion (Fig. 2). Vehicle-treated animals showed low basal levels of COX-2 and TNF-α in the meningeal blood vessels.

Figure 2

Double immunofluorescence staining of COX-2 (A–H) and TNF-α (I–P) with the endothelial cell marker, vWF. Both COX-2 and TNF-α were also strongly expressed in dilated vessels after GTN infusion. The intensities of COX-2 (E–H) and TNF-α (M–P) immunoreactivity were lower in vehicle-treated animals than GTN-treated animals. Scale bar = 100 µm.

Enzyme activity of MMP-9 following NO induction

We measured MMP-9 enzyme activity, which is induced by proinflammatory mediators such as NO or cytokines, using zymography and immunohistochemistry (14–16). GTN infusion induced MMP-9 expression at either low (2 µg/kg/min intravenously, data not shown) or high doses (10 µg/kg/min). MMP-9 activity increased twofold as early as 1 h after GTN infusion (10 µg/kg/min) and decreased gradually until 18 h (Fig. 3a). Fluorescence microscopy revealed an increase in MMP-9 immunoreactivity in vWF+ blood vessels. MMP-9 expression appeared scattered throughout the remainder of the dura mater (Fig. 3b).

Figure 3

Zymographic analysis of MMP-9 and double immunofluorescence staining of MMP-9 and vWF. (a) MMP-9 activity was upregulated as early as 1 h and decreased gradually up to 18 h after GTN infusion. (b) MMP-9 double immunohistochemistry with vWF as a marker of vascular endothelial cells. Dura mater of GTN-infused rats show increased MMP-9 immunoactivity in vascular structures. Moreover, the vessels were dilated by GTN infusion. Scale bar = 100 µm.

Regulation of COX-2 and TNF-a protein expression by methylprednisolone

Since oxidative stress promotes NF-κB activity and NO raises oxidative stress, this mechanism probably triggers NF-κB activity in the dura mater following GTN infusion (17, 18). Steroids affect the action of NF-κB and cytokine production (19–21). Thus, we tested whether MP or RU486 could regulate GTN-induced changes in NF-κB, COX-2 or TNF-α. Western blot analysis of COX-2 was performed at 3 h after GTN infusion; MP and RU486 did not change COX-2 expression (Fig. 4a). MP did not suppress TNF-α activation (138 ± 39% vs. 118 ± 29% of vehicle-treated group) at 3 h after GTN infusion, and the administration of RU486 did not affect TNF-α expression (118 ± 29% vs. 114 ± 53% of vehicle-treated group, n = 4 for each group, Fig. 4b). Additional study at different time points (1 and 18 h after GTN infusion) failed to reveal MP or RU486 effects on TNF-α expression (data not shown).

Figure 4

Effect of MP on COX-2 and TNF-α expression after GTN infusion. (a) COX-2 expression was not significantly changed after MP or RU486 treatment. (b) TNF-α level was not altered by MP or RU486 administration.

MMP-9 enzyme activity is affected by glucocorticoids

We examined whether MP could also suppress MMP-9 activity. MMP-9 activity was evaluated by gelatin zymography at 3 h after GTN infusion. MP decreased GTN-induced increases in MMP-9 activity (181 ± 42% vs. 111 ± 23% of vehicle-treated group, P < 0.05), which could be reversed by RU486 (111 ± 23% vs. 168 ± 29% of vehicle-treated group, P < 0.05, n = 5 for each group; Fig. 5).

Figure 5

Zymographic analysis of the effects of MP on MMP-9 activation after GTN infusion. MP reduced GTN-induced MMP-9 expression (P < 0.05), and RU486 reversed the effects of MP (P < 0.05).

Discussion

Our study has demonstrated that infusion of GTN up-regulated COX-2, TNF-α and MMP-9 within meningeal blood vessels. Meningeal vessels showed increased COX-2, TNF-α and MMP-9 immunoreactivity among endothelial cells after GTN infusion. GC down-regulated TNF-α expression and MMP-9 activity, and these changes were reversed by the administration of GR antagonist.

NO induces inflammatory gene expression via NF-κB activation (22). Nuclear translocation of NF-κB and expression of iNOS and interleukin (IL)-6 regulates immunity and inflammation in meningeal blood vessels in GTN-infused rats (9). Here, NO production after GTN infusion up-regulated COX-2, MMP-9 and TNF-α, which are downstream of NF-κB.

COX-2 regulation is important in the pathogenesis and treatment of migraine. Non-selective COX inhibitors, such as acetylsalicylic acid, have been used in migraine therapy for decades (23). A multicentre study of migraine patients has demonstrated that rofecoxib, a selective COX-2 inhibitor, was effective and generally well-tolerated in patients experiencing migraine attacks with or without aura (24). In addition, administration of parecoxib significantly reduces plasma protein extravasation in experimentally induced neurogenic inflammation of the rat dura mater, and it significantly attenuates c-fos expression in lamina I or II of the trigeminal nucleus caudalis in a model of electrically stimulated rat trigeminal ganglion (25).

Our data have shown a dual peak of TNF-α expression after GTN infusion. There are transitory increases in levels of the proinflammatory cytokines IL-1β, IL-6 and TNF-α in the jugular blood of migraine patients in the early hours of migraine attacks, as well as increases in soluble intercellular adhesion molecule-1 (26). NO production could be responsible for the early activation of NF-κB and subsequent TNF-α production, as suggested by our group (9).

Neurogenic inflammation is elicited by the activation of unmyelinated sensory neurons through noxious stimuli and subsequent release of neuropeptides such as substance P and calcitonin gene-related peptide (CGRP) from peripheral nerve endings (7). CGRP stimulates the production and release of proinflammatory cytokines such as TNF-α, IL-1β and IL-6 from human lymphocytes in vitro through specific receptors on these cells (27). In addition, CGRP, whose jugular plasma levels are increased in the first hours of migraine attacks, is associated with neurogenic inflammation and plasma protein leakage in animal models of migraine (28, 29). The first peak of TNF-α expression might be caused by NO-mediated transactivation of NF-κB, whereas the secondary peak could be induced by other inflammatory stimuli such as other cytokines or CGRP release.

The basic component of the blood–brain barrier, endothelial basal lamina, can be degraded by a group of proteolytic zinc-dependent enzymes known as MMPs. Migrainous patients show higher MMP-9 plasma levels during headache attacks than in asymptomatic periods, in migraines both with and without aura (30). The direct link between MMP-9 activation and the development of migraine has not been clearly demonstrated. However, in an experimental animal model, Gursoy-Ozdemir et al. have shown that cortical spreading depression activates and up-regulates MMPs and promotes sustained changes in vascular permeability (31). Here, MMP-9 was also rapidly activated by GTN, supporting the involvement of MMP-9 in NO-mediated migraine pathogenesis.

Steroids are occasionally recommended for patients with intractable migraine or status migrainosus (32, 33). Because we intended to test the effect of glucocorticoids in a more clinically relevant condition, MP was scheduled to be given immediately after the GTN infusion. Our data have demonstrated that MP down-regulated MMP-9, an effect that was blocked by RU486, which inhibits MP binding to GR. MP treatment also suppressed MMP-9 expression after spinal cord injury in rats, which was reversed by a GR antagonist (34). The anti-inflammatory action of GC requires GC binding to cytosolic GR to form aGR, which translocates into the nucleus to serve as a transcription factor. Thus, inhibiting transcription factors such as NF-κB or AP-1 by blocking GR may control inflammation-related factors. Blood–brain barrier and clinical symptoms can be reduced with different inhibitors of MMPs, including activators of tissue inhibitor of metalloproteinases-1 (TIMP-1), the cognate tissue inhibitor of MMP-9. Dexamethasone induces TIMP-1 expression in the murine cerebral vascular endothelial cell line (35). The inhibition of MMP-9 by GC in our models might be mediated by transcriptional activation of the TIMP-1 gene, in addition to the suppression of MMP-9 transcription. Although both COX-2 and TNF-α are downstream of NF-κB, our results suggest that they may be controlled by divergent pathways. These observations suggest that COX-2 and TNF-α exerts inflammatory activity by regulating other transcription factors, presumably by a different pathway than MMP-9 (36, 37). Our assumption is that the blockade of NF-κB transcription by MP could enhance other transcription factor activities, such as epithelium-specific Ets (ESE)-1 or CAAT/enhancer-binding protein (C/EBP) β, which may also transactivate COX-2 expression.

GTN-induced iNOS expression occurs within dural resident macrophages that reside particularly in perivascular regions (8). The different patterns of COX-2 and MMP-9 expression in our experiment may be caused by different dosages of GTN and different procedures for tissue processing. Because it is already known that GTN infusion activates NF-κB as reflected in a reduction in the inhibitory protein-κ-Bα (IκBα) and increased DNA binding activity (9), we did not repeat the experiments for NF-κB DNA binding activity.

In conclusion, proinflammatory mediators are involved in the NO-mediated cascade of migraine pathogenesis. Further understanding of these inflammatory mediators in the diverse pathways of transcription factors may have therapeutic implications for future migraine treatments.

Acknowledgement

This work was supported by a grant from the Ministry of Korean Health, Project 02-PJ1-PG1-CH05-0003.

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Differential Effects of Corticosteroids on The Expression of Cyclooxygenase-2,Tumour Necrosis Factor-Alpha and Matrix Metalloproteinase-9 in An Animal Model of Migraine

Abstract

Keywords

Introduction

Methods

Administration of drugs

Western blot analysis

Gelatin zymography

Immunohistochemistry

Statistical analysis

Results

Effects of GTN on COX-2 and TNF-α expression

Enzyme activity of MMP-9 following NO induction

Regulation of COX-2 and TNF-a protein expression by methylprednisolone

MMP-9 enzyme activity is affected by glucocorticoids

Discussion

Acknowledgement

References