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Abstract

Neuropeptide release and the expression of c-fos like immunoreactivity (c-fos LI) within trigeminal nucleus caudalis neurons (TNC) are activation markers of the trigeminal nerve system. Glyceryltrinitrate (GTN) is believed to stimulate the trigeminal nerve system, thereby causing headache. We examined the effects of a 30 min NO-donor infusion on CGRP release in jugular vein blood and c-fos LI within TNC of the rat. GTN (2 and 50 μg/kg/min) or NONOate infusion (25 nmol/kg/min) did not cause any CGRP release during and shortly after infusion, whereas administration of capsaicin resulted in strongly increased CGRP levels. GTN infusion (2 μg/kg/min for 30 min) did not lead to enhanced c-fos LI after 2 h and 4 h, whereas capsaicin infusion caused a time- and dose-dependent expression of c-fos LI within laminae I and II of the TNC. Surprisingly, GTN attenuated capsaicin-induced c-fos expression by 64%. The nitric oxide synthase (NOS) inhibitor L-NAME (5 and 50 mg/kg) reduced capsaicin-induced c-fos LI dose dependently (reduction by 13% and 59%). We conclude that GTN may lead to headaches by mechanisms independent of CGRP release from trigeminal nerve fibres. GTN doses comparable to those used in humans did not activate or sensitize the trigeminal nerve system. Both GTN and L-NAME reduced capsaicin-induced c-fos LI. This is most likely due to a feedback inhibition of nitric oxide synthases, which indicates that the c-fos response to capsaicin within TNC is mediated by NO dependent mechanisms.

Keywords

CGRP release c-fos expression trigeminal nucleus caudalis migraine nitric oxide rat

Introduction

Nitric oxide (NO) has been implicated in primary headache disorders, e.g. migraine. Infusion of the NO donor glyceryltrinitrate (GTN 0.5–2 µg/kg/min) leads to an immediate short lasting migraine-like headache in migraineurs and a less intense headache in control subjects (1). After a pain free interval a delayed (4–5 h) migraine headache follows only in migraineurs (2, 3). Moreover, the nonselective NOS inhibitor N^G-methyl-L-Arginine (L-NMMA) attenuated spontaneous migraine attacks in a small study (4). Based on these studies and a host of other clinical observations, NO, a powerful endogenous vasodilator with a multitude of biological actions (5), is believed to play a key role in migraine pathogenesis (6–8).

It remains unclear why only migraineurs develop a delayed headache after GTN whereas control subjects do not. One possibility may be sensitization of the trigeminal nerve system as demonstrated in migraineurs during and between the attack (9–11). Thus, migraineurs may be more susceptible to NO than control subjects due to a sensitized state of the trigeminal nerve system.

In experimental animals bolus administration of GTN (10 mg/kg i.p.) led to c-fos expression within the trigeminal nucleus caudalis (TNC) (12). C-fos is an immediate early gene and widely used as a histological marker of trigeminal nerve activation (13). High dose GTN i.a./i.p. activates central and peripheral trigeminal neurons as demonstrated by electrophysiological recordings and induced hyperalgesia in rats 2 h and 4 h after administration (14, 15). In contrast, GTN in doses comparable to those used in human subjects did not result in c-fos expression within the rat trigeminal brain stem nucleus (16).

Based on human and experimental animal evidence we hypothesized that sensitization of the trigeminal nerve system, e.g. by low dose capsaicin, results in increased susceptibility to NO. This may lead to the expression of c-fos.

In the brain and meninges large cerebral vessels and the venous sinuses are able to generate nociceptive signals due to their innervation by sensory nerve fibres mainly originating from cell bodies within the trigeminal ganglion (17). Upon stimulation these perivascular nerves release neuropeptides, e.g. calcitonin gene-related peptide (CGRP) (18–20). CGRP levels are elevated in the external jugular vein (which drains extracranial tissues including the dura) during a migraine attack, indicating activation of the trigeminovascular system (21). CGRP release can be attenuated by the antimigraine drug sumatriptan, which also aborts spontaneous migraine attacks (22).

It has been proposed that NO induces headache due to the activation of perivascular meningeal sensory afferent nerve fibres (possibly via 5-HT_2B receptors), thereby causing the release of CGRP (23–25). In line with this hypothesis we have recently demonstrated in the rat that a 30 min intravenous infusion of GTN causes a delayed inflammatory response (after 4 h) in the dura mater (26).

We have performed two sets of experiments based on the evidence above. We examined whether NO-donor infusion in doses comparable to those used in human studies causes the release of CGRP into jugular vein blood. In addition, we studied whether prestimulation with capsaicin alters the susceptibility of the trigeminal nerve system to subsequent GTN infusion as determined by c-fos LI within the TNC.

Materials and methods

One hundred and nine rats (CGRP n = 26, c-fos n = 83;) were used for this study. All experiments were approved by the Landesamt für Gesundheitsschutz und Tierschutz (no. G0265/01), which is the responsible body for animal experimentation in Berlin.

CGRP study

Male Sprague-Dawley rats (250–350 g; Charles River, Germany) were anaesthetized with sodium thiopentone (60 mg/kg i.p.; Trapanal, Byk Gulden, Denmark). Supplemental doses were administered during the experiments. The rats were placed on a heating blanket and the body temperature was maintained at 37 ± 0.5°C using a rectal probe. Animals were tracheotomized and mechanically ventilated using supplemental oxygen. Endexpiratory CO₂ was continuously monitored (EGM 1, Heyer, Bad Ems, Germany).

One femoral artery was cannulated (Portex Polythene Tubing PE 50, neoLab GmbH, Heidelberg, Germany) for the monitoring of arterial blood gases and arterial blood pressure. One catheter was inserted into the femoral vein for fluid substitution (NaCl 0.9%; Berlin Chemie, Berlin, Germany).

For the collection of blood samples and determination of CGRP levels another catheter (PE, 0.86 mm ID) was placed in the jugular vein. One carotid artery was canulated (PE 50) for drug administration.

Drug administration and blood sampling

Animals were divided into four groups. Rats in group 1 (n = 7) received GTN at a dose of 2 µg/kg/min (Nitrolingual, Pohl-Boskamp, Hohenlockstedt, Germany) dissolved in NaCl 0.9% (1 ml). Animals in group 2 received GTN 50 µg/kg/min (n = 7). Rats in group 3 (n = 6) were administered 25 nmol/kg/min diethylamine NONOate (Calbiochem, Bad Soden, Germany) and rats in the fourth group (control; n = 6) received vehicle (1 ml). All drugs were administered over a time period of 30 min into the left internal carotid artery.

For positive control, 8-methyl-N-vanillyl-6-nonenamide (capsaicin 4 µmol/kg Sigma, Steinheim, Germany) was administered at the end of the experiments into the left internal carotid artery to a subset of rats (n = 3 or 4/group) in each group. Capsaicin is known to induce CGRP release from trigeminal nerve fibres (27). Immediately after capsaicin administration, a blood sample was taken from the jugular vein to determine CGRP release.

Blood samples of 1 ml each were drawn from the jugular vein and collected in prechilled Eppendorf tubes containing EDTA (1 mg/ml blood) and the protease inhibitor Aprotinin (0.55 TIU/ml blood). All samples were immediately cold centrifuged (6000 g at 3°C) and plasma samples were stored at −80°C until further analysis.

For baseline CGRP determination one sample was collected 15 min before drug infusion. Further blood samples were taken 2, 15 and 35 min after the start of the infusion. To minimize the effect of the blood volume loss, saline was injected (1 ml over 5 min) into the femoral vein after the baseline and the 15 min samples were taken.

CGRP determination

Samples were acidified with 1% trifluoroacetic acid and extracted using Sep – Pak C18 cartridges (Waters, Eschborn, Germany). The eluates were concentrated by freeze drying overnight and dissolved in radioimmunoassay buffer on the following day. CGRP concentrations were determined using a [¹²⁵I]-CGRP radioimmunoassay (Bachem, Meyerside, UK) according to the manufacturer's instructions. The concentration of CGRP was determined in duplicates and above the detection limit in all plasma samples.

Statistical analysis

All data are expressed as mean ± SEM. Differences among groups were evaluated by One-way anova with Bonferroni's multiple comparison as post hoc test. Statistical significance was assumed when P < 0.05.

C-fos study

Male Sprague-Dawley rats were anaesthetized and equipped with catheters in the femoral vein and artery as described in the experiments above. Mean arterial blood pressure (MABP) and arterial blood gases were monitored during drug infusion and for 20 min afterwards in randomly selected animals of all groups (n = 3/group). After drug administration the wounds were sutured and anaesthesia (Xylocain 5%) was locally applied to the wounds. The rats were kept in a quiet place on a heating blanket and body temperature was monitored in all rats until the end of the experiment.

Two or four hours after the beginning of capsaicin/drug infusion the animals received an additional sublethal dose of thiopentone (100 mg/kg i.p.) for euthanasia. We chose this paradigm because c-fos LI within TNC was maximal in other studies at these time points (12, 28). The thorax was opened and the rats were perfused via the ascending aorta with saline 200 ml followed by 400 ml of ice cold paraformaldehyde 4% (PFA) in 0.1 m PBS (pH 7.4). The brain and cervical spinal cord were removed and stored in 4% PFA in 0.1 m PBS overnight followed by 48 h in 20% sucrose at 4°C. The brainstem and attached cervical spinal cord were cut serially in 50 µm coronal sections using a freezing microtome (HM 500 OM, Microm, Heidelberg, Germany) beginning at the level of the obex through the C2 segment of the cervical cord, covering a distance of 6 mm. Every fourth section was processed for immunohistochemistry and cell counting (30 sections in total per animal) as described by Mitsikostas et al. (28).

C-fos immunohistochemistry

Cryostat sections from control and experimental animals were processed simultaneously free floating. PBS (0.1 m, pH 7.0) was used for antibody dilutions and washes. Sections were placed in 0.03% hydrogen peroxide (Sigma-Aldrich GmbH) for 30 min followed by 2 h blocking with 3% normal goat serum in PBS and 0.25% Triton X-100. Sections were then incubated with the primary polyclonal rabbit anti-rat c-fos antibody 1 : 8000 overnight (Ab-5, Oncogene Research Products, USA) and 0.25% Triton X-100 (Sigma- Aldrich). C-fos LI was visualized using standard avidin-biotin peroxidase techniques. After incubation with the secondary antibody (biotinylated goat anti-rabbit IgG serum; Alexis; Switzerland; 1 : 600, 2 h at room temperature) the sections were washed again and placed in avidin-biotin peroxidase complex (Vector Laboratories Inc., Burlingame, CA, USA) and finally in a solution of 40% 3,3′-diaminobenzidine tetrahydrochloride (DAB; Sigma-Aldrich) and 0.003% hydrogen peroxide. To terminate the chromogenic reaction the sections were rinsed in PBS two times and hereafter mounted serially on slides, air-dried, dehydrated and cover-slipped.

Cell counting

C-fos LI nuclei in lamina I and II of TNC were identified under bright field microscopy by their black nickel enhanced nuclei from the background and only considered positive if typical staining pattern within the nucleus was visible through a wide range of magnification (between × 20 and × 4) (29, 30);. C-fos LI nuclei were counted by an observer naive to the treatment (K.S.) and confirmed in randomly selected sections by a second investigator (N.O.) also blinded to the treatment protocol. Distributions of c-fos LI in the TNC were quantified by counting positive nuclei over six consecutive levels (from obex 6 mm to spinal cord, each level 1 mm). Five sections (50 µm) per mm were assessed (30 sections in total per animal) and averaged from each individual as cells/section per level to investigate the distribution pattern or over all six levels as cells/section within the entire TNC (30 sections) to compare different treatment conditions.

Study design and drug administration

Capsaicin-induced c-fos LI

The first experiments (n = 30) were performed to show the ability of systemic intravenously (i.v.) administered capsaicin to activate the trigeminal nerve system as demonstrated by the induction of c-fos LI in the trigeminal nucleus caudalis. The distribution and time pattern of c-fos LI induction in TNC was investigated using capsaicin doses of 1.5, 3 and 4 µmol/kg i.v. for 30 min dissolved in vehicle (saline/ethanol/Tween-80 in a ratio 8 : 1 : 1) and diluted in NaCl 0.9% to a final delivery volume of 1 ml (n = 4 per dose and time point). In controls, an identical amount of vehicle in NaCl 0.9% was infused over 30 min (n = 3 per time point). Animals were sacrificed 2 h or 4 h after infusion.

In all further experiments a dose of 3 µmol/kg capsaicin was used for sensitization/prestimulation since this induced a sufficient, but submaximal c-fos expression (as determined by c-fos LI) at the 2 h and 4 h time point.

To determine specificity of the response to capsaicin the 5-HT_1B/1D-receptor agonist sumatriptan (300 µg/kg i.p.,dissolved in NaCl 0.9%; n = 4) or NaCl 0.9% (n = 4) was administered 10 min prior to 3 µmol/kg capsaicin infusion.

Effects of GTN on c-fos expression

To determine whether GTN infusion causes c-fos expression (after 2 h and 4 h) within TNC we infused GTN 2 µg/kg/min for 30 min to a subset of animals (n = 4). Because we could not detect increased c-fos LI, we then tried to enhance the response to GTN by capsaicin. GTN (2 µg/kg/min, for 30 min; n = 4) or GTN-vehicle (n = 4) were infused prior to capsaicin treatment (3 µmol/kg, i.v.). For control one further group received GTN vehicle followed by capsaicin vehicle (n = 3). Animals were sacrificed after 4 h.

Effects of GTN on capsaicin-induced c-fos LI

In order to study whether GTN enhances the c-fos response to a certain stimulus (n = 11), capsaicin infusion (3 µmol/kg) was followed by either GTN 0.5 µg/kg/min (3 µmol/kg capsaicin + 0.5 µg/kg/min GTN; n = 3) or GTN 2 µg/kg/min (3 µmol/kg capsaicin + 2 µg/kg/min GTN; n = 4) (for 30 min) or vehicle (n = 4).

Effects of NOS – inhibitors on capsaicin-induced c-fos LI

Because GTN reduced the number of capsaicin-induced c-fos LI cells we hypothesized that this is possibly mediated via NOS. To determine the significance of NOS for capsaicin-induced enhanced c-fos LI, we therefore studied the effects of NOS inhibition on capsaicin-induced c-fos LI (n = 15). Rats were treated with 3 µmol/kg capsaicin infusion and subsequently either N(G)-nitro-L-arginine methyl ester i.v. (L-NAME, 50 mg/kg (n = 4) or 5 mg/kg (n = 5) dissolved in 0.3 ml saline) or 50 mg/kg of the inactive stereoisomer N(G)-nitro-D-arginine methyl ester (D-NAME; n = 3) or 0.3 ml saline (n = 3) were administered.

Statistical analysis

Statistical analysis was performed in cooperation with the Institut für Statistik, Charité, Humboldt University of Berlin, Germany. From each animal the mean number of c-fos LI cells/section either for each level below obex or for all levels of the entire TNC were used for further statistical analysis. Data are presented as the average number of c-fos LI cells/section/animal per treatment group as mean ± SEM.

One-way anova with Dunnett's test for multiple comparisons between groups was used. Statistical analysis was performed for both, differences between corresponding levels and between the means of cells/section within the entire TNC. Student's two tailed t-test was used when appropriate for comparisons between means of two groups (P ≤ 0.05 was considered significant).

Results

CGRP release

Physiological variables

MABP was significantly reduced during and shortly after infusion in the GTN 50 µg/kg/min (by 18 mmHg) and NONOate group (by 13.5 mmHg). Arterial blood gases and body temperature were maintained within physiological limits throughout all experiments and did not differ between groups (anova with Bonferroni post hoc analysis).

CGRP release

Intracarotid infusion of GTN (2 or 50 µg/kg/min for 30 min) did not cause any enhanced CGRP release in jugular vein blood after 2 min, 15 min and 35 min as compared to baseline CGRP levels (Fig. 1). The synthetic NO donor diethylamine NONOate (25 nmol/kg/min) did not alter CGRP concentrations in the jugular vein neither did vehicle. In contrast, capsaicin injection in the carotid artery at the end of some experiments led to significantly elevated CGRP concentrations in external jugular vein blood (P < 0.05; Fig. 1). There were no statistically significant differences between the capsaicin treatment groups.

Figure 1

shows CGRP levels measured in jugular vein blood samples of the rat collected 15 min prior (baseline), 2 min, 15 min and 35 min after intra arterial infusion of GTN (2 (□) and 50 () µg/kg/min for 30 min in 1 ml NaCl; n = 7 per group) or DEA-NONOate(25 nmol/kg/min () for 30 min; n = 6) or vehicle infusion (▪) (n = 6). CGRP levels did not change during the observation period. In contrast, capsaicin infusion(4 µmol/kg) at the end of a subset of experiments caused strong CGRP release (P < 0.05) in all groups. Data are expressed as mean ± S.E.M.

We could also not detect any CGRP increase in a subset of experiments after 4 h using both GTN doses (2 and 50 µg/kg/min) or after infusion of GTN in the jugular or femoral vein (data not shown).

C-fos LI within TNC

Physiological variables

Arterial blood gases and body temperature were not different between groups. A transient increase of MABP could be observed during capsaicin infusion in all groups.

Capsaicin i.v. causes a dose dependent increase of c-fos LI within TNC

Capsaicin caused a dose and time dependent expression of c-fos LI in laminae I,II of rat TNC. Capsaicin at a dose of 1.5 µmol/kg/min for 30 min led to 12.3 ± 2.7 c-fos LI cells/section after 2 h and 6.8 ± 3.5 positive cells/section after 4 h (n.s. P > 0.05); The infusion of 3 µmol/kg capsaicin resulted in 38.1 ± 5.8 c-fos LI cells/section after 2 h and 18 ± 2.5 c-fos LI cells after 4 h (P < 0.05); 4 µmol/kg capsaicin led 83.9 ± 7.7 c-fos LI cells (2 h) and 35.5 ± 2.6 cells/section (4 h) to express c-fos LI (4 h P < 0.05; Fig. 2a). In contrast, vehicle infusion only led to minor expression of c-fos in 1.8 ± 0.5 cells/section (2 h) and 1.9 ± 0.7 cells/section (4 h).

Figure 2

(a) shows the number of c-fos LI cells within lamina I and II of the trigeminal nucleus caudalis 2 h (▴) and 4 h () after infusion of 1.5, 3, and 4 µmol/kg capsaicin. C-fos LI was significantly higher in rats after 2 h at 3 and 4 µmol/kg capsaicin; ∗P < 0.05. Infusion of capsaicin vehicle did result in a non significant expression of c-fos LI (less than 2 cells/section). Data are demonstrated as mean values ± S.E.M. per section for the entire TNC. (b) demonstrates the distribution of c-fos LI within TNC 2 h after infusion of capsaicin vehicle (□; water: ethanol: tween 80 ratio 8 : 1 : 1), capsaicin (▪; Cps 3 µmol/kg), or GTN (; 2 µg/kg/min for 30 min). Cps but not GTN infusion caused a significant increase in c-fos LI at all of the TNC levels examined after 2 h and 4 h (4 h data not shown). Data are shown as mean values ± S.E.M.

The distribution of c-fos-LI within the TNC after capsaicin i.v. was bilateral and maximal staining was detected at obex −2 till − 3.00 mm in accordance with data from the literature (Fig. 2b)(28).

To demonstrate that capsaicin-induced c-fos LI was mediated via the trigeminal nerve system sumatriptan (300 µg/kg) was administered i.p. 15 min prior to capsaicin. Sumatriptan, but not vehicle reduced c-fos LI significantly (sumatriptan 8.2 ± 1.3 vs. vehicle 23.4 ± 5.2 cells/section P < 0.05) when determined 2 h after capsaicin administration (Fig. 3).

Figure 3

Sumatriptan (300 µg/kg i.p.; n = 4) administered 10 min prior to infusion reduced the number of capsaicin (Cps 3 µmol/kg i.v.; n = 4)) induced c-fos LI within laminae I,II of the trigeminal nucleus caudalis after 2 h significantly (P < 0.05). Data are shown as mean values ± S.E.M. ∗P < 0.05.

In all further experiments capsaicin was used at a dose of 3 µmol/kg i.v. since this dose caused stable but submaximal c-fos LI within TNC. C-fos LI staining in representative TNC sections are illustrated in Figs 4 + 5.

Figure 4

(a, b) illustrate representative images of c-fos LI in the TNC (coronal section) −3 mm caudal from obex after treatment with 3 µmol/kg capsaicin i.v. (a: magnification 10×; b: 20x). (c, d) representative sections show markedly decreased c-fos LI in laminae I,II of TNC after i.v. infusion of 3 µmol/kg^–capsaicin followed by 2 µg/kg/min GTN for 30 min (a, c: magnification 10×; c, d: 20×).

Figure 5

Schematic drawings of c-fos LI in coronal hemisections (Obex −2.0 mm) from rat brainstem. The drawings show the distribution of c-fos LI within TNC following: (a) capsaicin 3 µmol/kg i.v.; (b) sumatriptan 300 µg/kg i.p. followed by capsaicin 3 µmol/kg i.v.; (c) vehicle infusion i.v.; (d) capsaicin 3 µmol/kg i.v followed by L-NAME 50 mg/kg i.v.; (e) GTN 2 µg/kg/min i.v. plus capsaicin 3 µmol/kg i.v. (f) GTN 2 µg/kg/min i.v.; (g) capsaicin 3 µmol/kg i.v. followed by GTN 2 µg/kg/min i.v.

GTN infusion did not cause c-fos LI within TNC

GTN at a dose of 2 µg/kg/min for 30 min did not show any difference in the total number of c-fos LI cells in TNC compared to vehicle (2 h) treated animals. (GTN 2.4 ± 0.9 and vehicle 1.8 ± 0.5 cells/section P > 0.05; Fig. 2b). Subanalysis for each level did also not reveal any significant differences.

GTN (2 µg/kg/min for 30 min) application prior to capsaicin infusion (23.9 ± 1.3 cells/section) did not increase the number of c-fos LI cells compared to capsaicin treatment alone (27.2 ± 2.2 cells/section) within the entire TNC (Fig. 6a). In both groups the number of positively stained cells was significantly increased compared to vehicle treated animals (7.0 ± 1.6 cells/section, P < 0.05; Fig. 6a).

Figure 6

(a) illustrates the number of c-fos LI cells after capsaicin infusion (Cps 3 µmol/kg, 27 ± 2 cells). GTN (2 µg/kg/min i.v. for 30 min) administration prior to capsaicin infusion did not increase the number of c-fos LI cells (24 ± 1 cells). Cps vehicle infusion resulted in very low c-fos LI, which was different from both, Cps and GTN + Cps administration (∗P < 0.05). Data are shown as mean values ± S.E.M. (b) capsaicin (Cps 3 µmol/kg) was used as a prestimulator of the trigeminal nerve system. Subsequent GTN administration did not enhance c-fos LI within TNC. In contrast, the number of c-fos LI was attenuated by 65% (2 h after capsaicin administration; ∗P < 0.05) by subsequent GTN 0,5 or 2 µg/kg/min i.v. for 30 min. Data are shown as mean values ± S.E.M.

Capsaicin-induced c-fos LI was attenuated by GTN

GTN infusion after capsaicin(3 µmol/kg) administration significantly attenuated c-fos LI within TNC (Fig. 6b). In the subanalysis GTN at a dose of 0.5 µg/kg and 2 µg/kg reduced the number of c-fos LI cells/section within the entire TNC by 65.4% (P < 0.05) and 64.3% (P < 0.05), respectively.

Comparing corresponding TNC levels (cells/section per level) the 3 µmol/kg capsaicin + 2 µg/kg/min GTN group showed a significant reduction of c-fos LI at all levels, 3 µmol/kg capsaicin + 0.5 µg/kg/min GTN reduced c-fos LI compared to 3 µmol/kg capsaicin at all except one (4–5 mm caudal to obex) levels (data not shown).

NOS inhibition attenuates capsaicin-induced c-fos LI in TNC

Administration of the NOS-Inhibitor L-NAME (50 mg/kg) following capsaicin (3 µmol/kg) infusion also decreased the number of c-fos LI cells/section within TNC by 59% (P < 0.05; Fig. 7). L-NAME (5 mg/kg) was without a significant effect (13.9% reduction). Distribution analysis revealed a significant reduction of c-fos LI cells/section at all levels in the L-NAME 50 mg/kg treated group. To determine specificity the inactive enantiomer D-NAME (50 mg/kg) was administered. D-NAME did not change capsaicin-induced c-fos LI (Fig. 7).

Figure 7

Capsaicin (Cps 3 µmol/kg) induced c-fos LI is significantly attenuated after 2 h by subsequent administration of the NOS inhibitor L-NAME (50 mg/kg i.v.; n = 4; P < 0.05) but not 5 mg/kg L-NAME (i.v.; n = 5). ∗P < 0.05. The inactive enantiomer D-NAME (50 mg/kg i.v.; n = 3) was without effect. Data are shown as mean values ± S.E.M.

Discussion

We have shown here that the infusion of the NO-donors glyceryltrinitrate and diethylamine NONOate does not cause CGRP release in an experimental animal model of migraine. In contrast, administration of capsaicin resulted in strongly increased CGRP levels within jugular vein blood. GTN did not lead to enhanced c-fos LI within the trigeminal nucleus caudalis and did not sensitize the trigeminal nerve system to subsequent capsaicin stimulation.

A preactivated state of the trigeminal nerve system was generated with capsaicin. In contrast to our expectations the subsequent GTN infusion reduced the number of c-fos LI cells. Inhibition of NOS by L-NAME led to similar findings indicating that capsaicin causes c-fos expression via NO dependent mechanisms.

Taken together, these data suggest that GTN may cause headaches by mechanisms independent of neuropeptide release. In addition, capsaicin-induced c-fos expression is at least in part mediated by nitric oxide.

CGRP is known to be released during spontaneous migraine attacks, which can be aborted by so called ‘triptans’ (22). A new CGRP receptor antagonist also attenuates spontaneous migraine attacks (31). Among others, these findings led to the proposal that CGRP is a causal factor for migraine headaches. Human data show that the infusion of GTN up to 0.5 µg/kg/min for 20 min to non-migraineurs causes an immediate headache, but does not increase the release of CGRP up to 60 min after infusion (32). Moreover, CGRP administration leads to the development of migraine headache after 4–6 h in a subset of migraineurs similar to the effects of GTN infusion (33). If CGRP is a key mediator of GTN-induced headaches, one would expect an immediate release of CGRP once the GTN infusion is started. This would indicate trigeminal nerve activation.

Our findings differ from results of previous studies which show that the effects of NO-donor application is mediated by CGRP (23, 25). Wei et al. (23) demonstrated that cortical blood flow changes in the guinea pig after GTN can be attenuated by topical application of a peptidergic CGRP receptor antagonist thereby indicating a CGRP dependent mechanism. Strecker et al. (25) confirmed these findings in the rat by administering diethylamine-NONOate and they also demonstrated CGRP release in an in vitro preparation of the skull due to NO-donor application. One may speculate that the failure to detect increased CGRP release in our hands may be due to the mode of NO administration. In this study the NO-donors entered the cranial circulation intraluminally while in the two aforementioned studies the NO-donors were applicated extraluminally. However, the administration route is unlikely to account for the different findings since GTN diffuses freely across biological membranes.

Alternatively, the differences could be explained by the low NO-donor concentrations we have used. However, GTN infusion at a dose of 50 µg/kg/min for 30 min reduced mean arterial blood pressure significantly, but did not cause any CGRP release as compared to the 2 µg/kg/min dose. Higher i.v. GTN doses are not practicable in the rat mainly due to induced hypotension. Moreover, the GTN dose we have used has also been successfully and repeatedly applied by other groups to study GTN related headache mechanism. Among others, cerebral blood flow changes and neurogenic inflammation have been demonstrated (26, 34). A significant increase in CGRP levels after capsaicin infusion also proves that the CGRP assay has been used correctly. Indeed, we found it is possible to measure the release of endogenous CGRP from jugular vein blood in the rat as previously demonstrated, e.g. after unilateral electrical stimulation of the trigeminal ganglion (35).

Our findings indicate a CGRP independent mode of action of GTN to induce pathophysiolgical events related to headaches in experimental animals. However, we cannot entirely exclude that excessive GTN causes CGRP release. Moreover, our results in the rat are in agreement with data from human studies (32) and may indicate a different mode of action of GTN to generate headaches.

We have demonstrated that systemic capsaicin infusion causes a dose dependent increase in c-fos LI within trigeminal nucleus caudalis with a distribution pattern similar to that of intracisternal capsaicin injection and dural blood vessel stimulation (28, 36). We intentionally used an i.v. infusion mode in order to avoid central trigeminal nervous system activation caused by trauma. Because capsaicin does not penetrate the blood brain barrier (37) we conclude that activation of peripheral trigeminal sensory neurons, e.g. from the meninges, leads to c-fos expression within TNC (13). In line with previous studies capsaicin-induced c-fos LI is attenuated by the 5-HT_1B/D receptor agonist sumatriptan, which furthermore demonstrates specificity of the response to capsaicin (38).

Low dose GTN did not lead to increased c-fos LI within trigeminal nucleus caudalis after 2 h and 4 h in accordance with data published by Jones et al. (39). These results vary from observations by Tassorelli & Joseph (12). This discrepancy could be explained by the use of a lower GTN dose in our study. Observation periods did not differ between the studies. We did not observe that GTN enhances the c-fos expression pattern to capsaicin, which is in contrast to one previously published manuscript (39). Drug application modes (i.v. vs. local capsaicin) or the use of different anaesthetics (barbiturates vs. urethane) may account for these differences. Various anaesthetics act as stimulants of neurons, thereby leading to enhanced c-fos expression. In light of our findings it is worthwhile to mention that urethane, in contrast to barbiturate anaesthesia (which was used in our study), strongly promotes c-fos expression (40).

Interestingly, we found that the capsaicin-induced c-fos LI within TNC is nitric oxide dependent. Inhibition of nitric oxide synthases by L-NAME reduced capsaicin-induced c-fos LI dose dependently. The inactive enantiomer D-NAME was without effect, demonstrating specificity of the L-NAME response. In line with our results it has been shown, e.g. in hypothalamic nuclei and the amygdala, that neuronal NOS inhibition by L-NAME attenuates capsaicin-induced neuronal activation and c-fos expression (41). Moreover, in the headache model of sagittal sinus stimulation L-NAME attenuated c-fos expression within trigeminocervical complex of the cat. This also indicates NO dependency of electrically induced trigeminal nerve mediated c-fos expression (42).

Capsaicin-induced c-fos LI is attenuated by subsequent GTN infusion, which may be due to a negative feedback mechanism on NOS. In line with this hypothesis, NO derived from cortical neurons has been shown to autoregulate its own formation through feedback inhibition of the NO synthesizing enzyme. In this study, application of exogenous NO-donors such as GTN or sodium nitroprusside also inhibited neuronal NOS activity in the NOS activity assay (43). A similar mechanism has been demonstrated for endothelial NOS (44) and in inflammatory conditions. In cultured macrophages IFNγ/lipopolysaccharide stimulated NOS activity and NO release was significantly attenuated by subsequent administration of exogenous NO donors, e.g. sodium nitroprusside (45). In the same study, sodium nitroprusside also inhibited enhanced neuronal NOS activity (due to proinflammatory stimuli) from brain tissue.

In summary, our data support a negative feedback mechanism of exogenous nitric oxide derived from NO-donors such as GTN on NOS activity. Although we did not measure NOS activity in our assay, we have clearly shown NOS dependency of capsaicin-induced c-fos expression by adding the NOS-inhibitor L-NAME.

Our findings point to a crucial role of NOS for c-fos expression within TNC after capsaicin. Others have shown the importance of NOS for electrically induced c-fos expression after sagittal sinus stimulation (42). Together, these results point to a general role of NOS and subsequent NO formation for c-fos expression. In light of these results it would be interesting to know whether excessive bolus GTN-induced c-fos LI (12) within brain tissues, e.g. trigeminal nucleus caudalis, is specific for trigeminal afferent nerve pathways and whether it is attenuated by a selective 5-HT_1B/D receptor agonist. To our knowledge this question has not been addressed yet. Alternatively, c-fos LI after bolus GTN might be a stereotypical response caused by excessive NO formation in several neuronal tissues.

Migraineurs appear to be susceptible to NO as demonstrated by the development of delayed headaches after GTN infusion. Our purpose, to generate a sensitized state in the rat mimicking human conditions, failed due to complex interactions between capsaicin, c-fos expression and NOS. Our data do not exclude the possibility that GTN administration may lead to enhanced activation of trigeminal neurons in an otherwise NO-independent systemically sensitized animal.

While we demonstrate here the inhibitory properties of GTN on stimulus induced NOS, others have clearly demonstrated the feed forward activation of GTN on nNOS in TNC and iNOS in meningeal macrophages (46, 47). These findings are in accordance with the often described dual role of NO and nitric oxide synthases as a pro- and anti- inflammatory agent (48).

In summary, our data reveal that capsaicin-induced c-fos expression within TNC is NO dependent. Moreover, our findings indicate that GTN causes pathophysiological events consistent with headaches by a mechanism independent of CGRP release.

Footnotes

Acknowledgements

UR and GA are supported by the Kopfschmerz BMBF, Project A1 and the GSK fellowship for clinical migraine research 2001 (UR). The authors are deeply indebted to Ms. Sonja Blumenau for excellent technical assistance.

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CGRP Release and c-fos Expression within Trigeminal Nucleus Caudalis of the Rat following Glyceryltrinitrate Infusion

Abstract

Keywords

Introduction

Materials and methods

CGRP study

Drug administration and blood sampling

CGRP determination

Statistical analysis

C-fos study

C-fos immunohistochemistry

Cell counting

Study design and drug administration

Capsaicin-induced c-fos LI

Effects of GTN on c-fos expression

Effects of GTN on capsaicin-induced c-fos LI

Effects of NOS – inhibitors on capsaicin-induced c-fos LI

Statistical analysis

Results

CGRP release

Physiological variables

CGRP release

C-fos LI within TNC

Physiological variables

Capsaicin i.v. causes a dose dependent increase of c-fos LI within TNC

GTN infusion did not cause c-fos LI within TNC

Capsaicin-induced c-fos LI was attenuated by GTN

NOS inhibition attenuates capsaicin-induced c-fos LI in TNC

Discussion

Footnotes

Acknowledgements

References