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Abstract

Background: and aim Glyceryl trinitrate (GTN) infusion is a reliable method to provoke migraine-like headaches in humans. Previous studies have simulated this human model in anaesthetized or in awake rodents using GTN doses 10,000 times higher than used in humans. The relevance of such toxicological doses to migraine is not certain. Anaesthesia and low blood pressure caused by high GTN doses both can affect the expression of nociceptive marker c-fos. Therefore, our aim was to simulate the human GTN migraine model in awake rats using a clinically relevant dose.

Methods: Awake rats were infused with GTN (4 µg/kg/min, for 20 min, i.v.), a dose just 8 times higher than in humans. mRNA and protein expression for c-fos were analysed in the trigeminal vascular system at various time points using RT-PCR and immunohistochemistry, respectively.

Results: A significant upregulation of c-fos mRNA was observed in the trigeminal nucleus caudalis at 30 min and 2 h that was followed by an upregulation of Fos protein in the trigeminal nucleus caudalis at 2 h and 4 h after GTN infusion. Pre-treatment with sumatriptan attenuated the activation of Fos at 4 h, demonstrating the specificity of this model for migraine.

Conclusion: We present a validated naturalistic rat model suitable for screening of acute anti-migraine drugs.

Keywords

Glyceryl trinitrate sumatriptan awake rat model migraine nociception c-fos

Introduction

Glyceryl trinitrate (GTN) infusion in humans is a valid and reliable model to provoke migraine attacks (1–4). In contrast to clinical research there is no consensus on a single animal model for pre-clinical migraine research. Therefore, better and more naturalistic animal models for migraine research are required. NO liberated from GTN is thought to be the main cause of migraine provocation in humans (5). Previous studies have therefore used GTN in animal models analysing the expression of Fos as a measure of nociception, but in a dose that is 1000–10,000 times higher than the dose given in humans (6,7). Such enormous doses are not only beyond clinical and pharmacological relevance but also induce Fos expression (8) via non-specific mechanisms. Other studies using a more realistic dose of GTN have studied it in anaesthetized rats under acute surgical interventions (9,10). Anaesthesia and surgery by themselves affect expression of c-fos, an extensively used marker to study nociception in animals (11,12). In anaesthetized rats GTN-induced hypotension also upregulated c-fos (8). Therefore, in the earlier studies the experimental readout, Fos expression, has been confounded by number of factors, albeit to a varying extent. Increase in Fos expression within the caudal region of the trigeminal nucleus caudalis (TNC), particularly in layers I and II, may indicate the activation of the trigeminal vascular system (TVS) (13,14). Meningeal deafferentation reduced Fos expression induced by injection of noxious chemicals into the cisterna magna (15). This explains the involvement of trigeminal vascular fibres in detecting and transmitting the nociceptive information. The headache phase of migraine is very likely a manifestation of the increased nociceptive traffic in the TVS (16). This neuro-vascular circuit comprises of dural and large cerebral arteries and their sensory nerve supply, the trigeminal ganglion (TG) and the TNC. Higher pain centres are eventually activated all the way to the sensory cortex, where headache is finally perceived.

In the present study we developed a new model in unanaesthetized freely moving rats. Under these circumstances, unlike previous models, a clinically relevant dose of GTN caused a robust expression of Fos in TNC probably because confounders like anaesthesia, acute surgical stress and hypotension were eliminated.

Methods

Animals

Seventy-seven male Sprague-Dawley rats weighing 320–340 g (Taconic M&B, Denmark) were used for the study. The rats were maintained in cages with a 12 h light/dark cycle and free access to food and water. All the experimental protocols were approved by the Danish committee for experiments with animals (2009/561-1664).

Surgery

Rats were first anaesthetized by injecting ketamine and xylazine (100 mg/kg and 7.5 mg/kg) intraperitoneally (i.p.). Before surgery the animals were injected with enrofloxacin (commercial name Baytril, antimicrobial; 10 mg/kg s.c.) and carprofen (commercial name Rimadyl, NSAID; 5 mg/kg subcutaneously). Surgery was performed aseptically. The incision was first made in the left inguinal area and the femoral vein was isolated. The catheter was then placed subcutaneously by inserting it from the neck into the trocar and pulled out at the other end of the trocar facing the femoral vein. The vein was cannulated using the polythene tubing (0.40 mm internal diameter, 0.80 mm external diameter, Portex®, Smiths medical ASD, USA), and checked for patency. The catheter was then sealed using a blocking solution (Haemaccel-Heparin, 2 : 1). The animal was monitored until fully recovered from anaesthesia as indicated by the ability to ambulate, and core body temperature was maintained using an automatic regulated heating plate (Letica HB101, Panlab, Barcelona, Spain). Enrofloxacin and carprofen treatment was repeated for 72 h after surgery, while treatment with buprenorphine (analgesic, commercial name Temgesic, Schering Plough Europe, Belgium) was repeated 48 h after surgery with an interval of 24 h. Rats were then allowed to recover for a period of ten days. The indwelling catheter was flushed once in a week with heparinized saline (100 IU/ml) and sealed again with blocking solution. After a recovery period of 10 days the rats were moved to accusampler cages (Dilab, Lund AB, Sweden) and were acclimatized for 2 days. The cannula was connected through a tether at the nape of the neck and the rats were allowed to move freely in the cage. These rats ambulated normally without any signs of discomfort. On the third day, GTN (4 µg/kg/min or 30 µg/kg/min for 20 min) was administered intravenously (i.v.) and the rats were observed and finally killed by perfusion at the required time points (30 min, 1 h, 2 h, 4 h and 6 h) after anaesthetizing with pentobarbital. This dose was chosen after a standardization procedure. Saline- or vehicle (0.18% ethanol)-treated rats were perfused after 2 h. Sumatriptan (0.3–0.6 mg/kg/min for 3 min) was infused i.v. 5 min before the GTN infusion.

Drugs

GTN stock solution (5 mg/ml in 95% ethanol) was obtained from Nycomed (Roskilde, Denmark). Sumatriptan was obtained from Glostrup hospital pharmacy, (Glostrup, Denmark). Enrofloxacin stock solution (50 mg/ml) an antimicrobial and xylazine (20 mg/ml) were from Rompun® (Bayer Inc., Germany), Heparin solution (1000 IU/ml) was obtained from Glostrup hospital pharmacy, Haemaccel (500 IU/ml) were from Voluven®, carprofen stock solution (50 mg/ml) was from Pfizer Inc (NY, USA), buprenorphine stock solution (0.3 mg/ml), analgesic, was purchased from Schering Plough (Europe, Belgium), Ketamine (50 mg/ml) was obtained from Intervet (Skovlunde, Denmark) and lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (Steinhelm, Germany).

For in vivo use all stock solutions were administered without dilution or further diluted in saline to their final concentrations just before administration. All stock solutions were stored either at −20°C or at 4°C and the dilutions required for the studies were freshly prepared.

Blood pressure measurements

For assessment of mean arterial blood pressure (MABP) in awake rats, four rats underwent left femoral artery and vein cannulation and were allowed to recover for 10 days as described above. These rats showed signs of discomfort while moving. However, these rats were used only for the blood pressure studies. Rats were then infused with GTN through the femoral vein and the MABP was measured through the femoral artery throughout the duration of infusion. Finally we infused a bolus of CGRP to check the intravenous patency, and these rats were used only for the blood pressure studies. For MABP studies in anaesthetized rats, five rats were anaesthetized with 65 mg/kg pentobarbital and their femoral artery and vein was cannulated. The body temperature was maintained at 37.0 ± 0.5°C throughout the experiment using the heating plate. Following intubation the animal was mechanically ventilated by a respirator (SAR-830/AP, CWE, Ardmore, PA, USA) with 30/70% air mixture of O₂/N₂O, a stroke volume of 3.5–4.0 ml and a stroke rate of 55–65 per minute. Rats were subsequently infused with GTN (4 µg/kg for 20 min), as mentioned above.

mRNA expression studies

Rats were anaesthetized with pentobarbital at various different time points (30 min, 1 h, 2 h, 4 h and 6 h) after the termination of the GTN infusion and perfused transcardially with 400 ml phosphate buffered saline (PBS). The TG and TNC were carefully dissected and divided into small chunks. Tissue chunks were immediately submerged in a RNA stabilization solution (RNAlater™, Ambion, Naerum, Denmark) and kept at 4°C overnight to be either further processed or stored at −80°C for later use. The RNA stabilized tissue was disrupted and homogenized thoroughly with a rotor–stator homogenizer. Total RNA was purified using RNeasy Mini Kit (Qiagen® GmbH, Hilden, Germany). Yield and purity of the extracted RNA was assessed spectrophotometrically by measuring the absorbance at 260 nm and by determining the 260 nm/280 nm ratio.

Pre-validated gene-specific primers from Qiagen's QuantiTect Primers Assay source were used to detect each mRNA transcript. The cDNA synthesis and qPCR analysis were done as described previously (17). A non-regulated reference gene (β-actin) was used to calculate the relative abundance of mRNA transcripts in each experimental sample. A calibrator (cDNA from tissues known to express target gene) was included in every PCR run to provide a constant calibration point. Rats were administered with lipopolysaccharides (LPS) (5 mg/kg, i.p.) and perfused after 5 h. The liver was used as a calibrator for c-fos mRNA. Expected end product sizes were evaluated by analyzing the PCR products by agarose gel separation

Fos immunohistochemistry and cell counting

The primary antibody used for Fos expression in the present study was purchased from Calbiochem, Darmstadt, Germany (Cat. No. PC38). Unless otherwise stated, the description of the antibody is from the manufacturer’s published data. For the primary antibody used, omission eliminated all staining from the corresponding secondary antibody. The antibody against Fos was raised in rabbit. The rabbit anti-Fos antibody was characterized by preabsorption immunohistochemistry (IHC) (18). The rats were anaesthetized with pentobarbitol (65 mg/kg) 2 h and 4 h after the GTN or saline infusion and transcardially perfused with isotonic saline for 2 min, followed by 4% paraformaldehyde (PFA) for 10 min. TNC were removed and post-fixed overnight in 4% PFA. The tissue was then stored in PBS at 4°C. Four days before cryosectioning, the PBS was exchanged for a 30% sucrose solution. Cross-sections of TNC were cut, with each section having a thickness of 40 µm. Every 10th section was subjected to Fos immunostaining (with a total of 11 stained sections per rat). The immunolabelling procedures were performed according to the Avidin–Biotin–peroxidase Complex (ABC) protocol. Sections were incubated overnight with polyclonal rabbit anti-Fos antibody. The sections were then incubated with biotinylated goat anti-rabbit IgG (Jackson Immunoresearch, Suffolk, UK) for 1 h. Fos protein visualization was completed by using diaminobenzidine (DAB). Buffer controls omitted either primary or secondary antibodies. The sections were subsequently mounted onto the glass slides and covered with coverslips, and were observed with a Leica DMR HCS microscope using a 40X objective lens. Counting of the Fos-positive cells in the region of interest was performed by an observer blinded to the treatment. For comparison between the control and the treated cells, Fos-positive cells were counted in the TNC starting from 0.8 mm to 5.12 mm caudally to the obex, which includes C1 and C2. The total number of Fos-positive cells were counted in lamina I–IV of the TNC, including the medullary and upper cervical dorsal horn. Representative images of the treated and control sections were captured using a Leica DC 300F digital camera attached to the microscope.

Data analysis

All values are given as mean ± SEM. Experiments were compared using ANOVA (Kruskal-Wallis test), which was followed by Dunn’s post hoc test to determine the significant difference from the saline-treated group. Two-way ANOVA was used followed by Bonferroni post hoc test for the comparisons of Fos-positive cells between the treatment groups in different level of brain stem sections. The Wilcoxon paired t-test was used for non-parametric analysis of paired data and the Mann-Whitney t-test was used for analysis of non-paired data. Differences were considered significant at p < 0.05. GraphPad Prism (GraphPad Prism software, San Diego, CA, USA) was used for statistical analysis.

Results

Effect of GTN infusion on MABP in anaesthetized and unanaesthetized rats

Rats were weighed pre-surgery, one day after surgery and after the recovery period of 10 days. Rats recovered well after surgery and there was a gain in weight after the recovery period as compared with one day after surgery. In addition, there were no visible signs of distress or infection, and food and water intake was normal. To compare the awake rats with anaesthetized ones, the MABP response was studied. Infusion of GTN (4 µg/kg/min) over 20 min did not cause a drop in MABP of awake rats (N = 4), whereas the same dose of GTN in anaesthetized rats caused a significant drop in MABP, as shown in Figure 1. The anaesthetized rats showed higher baseline values for MABP, which could be due to the effect of barbiturates on autonomic reflexes (19,20). Therefore we also measured the MABP during the infusion with saline and did not observe any drop in blood pressure. Hence the drop in blood pressure during the infusion was the effect of GTN. In the rest of the series for Fos expression studies only the femoral vein was cannulated for the infusion of GTN to reduce the surgical stress to the animals.

Figure 1.

A representative trace showing the effect of glyceryl trinitrate (GTN) (4 µg/kg/min for 20 min i.v.) infusion on mean arterial blood pressure (MABP) in (A) anaesthetized rat (not used in the rest of the study) (B) awake rat (used in the rest of the study). Results are shown in bar diagrams in (C), paired t-test (Wilcoxon test): **p < 0.01 (N = 4–5). Note that there is no change in MABP in unanaesthetized rats.

GTN dose response on Fos protein expression

During the pilot experiments, the total number of Fos-positive cells both in the vehicle (0.18% ethanol) and saline-treated rats in the entire TNC were measured. No significant difference were seen in the number of Fos-positive nuclei between the groups treated with saline (22.2 ± 4.1) or the vehicle, 0.18% ethanol (21.3 ± 1.4) (Figure 3A), hence for the subsequent experimental analysis saline treatment was used as the standard control. For dose selection of GTN, Fos protein expression studies were performed by infusing two different doses, 4 µg/kg/min and 30 µg/kg/min, for 20 min. Total Fos expression was measured in the brainstem throughout the TNC. Fos-positive cells were stronger between 2 mm to 4 mm (rostral to caudal) from the obex, in the regions of TNC, where caudal regions C1 and C2 of the spinal cord were also included. More Fos-positive cells were seen in the lamina I and II and few in lamina III/IV. The average number of Fos-positive cells in the TNC per section was compared between the saline- and GTN-treated groups. Both the GTN-treated groups showed a significant increase in Fos protein expression at 2 h. However, we did not observe any significant difference in the Fos expression between the two doses (Figures 2 and 3B) and hence for further experiments the dose 4 µg/kg/min was used.

Figure 2.

Effect of two different i.v. doses of GTN (4 µg/kg/min and 30 µg/kg/min for 20 min) on Fos protein expression at 2 h in TNC by immunohistochemistry. Representative images of the saline control (A, A1, A2), 4 µg/kg/min GTN (B, B1, B2) and 30 µg/kg/min GTN (C, C1, C2). Scale bars represent 100 µm (A, B and C) and 50 µm (A1, A2, B1, B2, C1 and C2). N = 6–7.

Figure 3.

Summary of total count of Fos-positive cells in the TNC. Saline vs. vehicle control (A). Saline vs. GTN 4 µg/kg/min and 30 µg/kg/min (B). *p < 0.05, paired t-test (Wilcoxon test) (N = 6–7).

Effect of GTN infusion on c-fos mRNA and Fos protein expression

Investigation of mRNA expression by quantitative real-time PCR (qPCR) studies

Primers designed by Qiagen were first tested on cDNA transcribed from specific tissue RNA. Melting curve analysis showed a single product with specific melting temperature peaks when analysing the amplicons generated from all the Qiagen primers. The expression of c-fos mRNA in the tissues was detected using qPCR, and an amplified PCR product of c-fos was seen at 73 bp in the ethidium bromide stained agarose gel. To test for the contamination of the mRNA template with genomic DNA, negative controls were tested in which the reverse transcriptase was omitted from the runs. No product was amplified under these conditions, indicating that genomic DNA was completely cleared by the mRNA isolation procedure. The liver of LPS-treated rats was used to extract mRNA, which was converted to cDNA and which subsequently served as a calibrator for c-fos in the qPCR experiments.

Quantitative PCR was performed to obtain an overview of the c-fos mRNA transcript level in the TG and TNC of the rats subjected to different treatments. We studied the pain relaying areas of the TVS, TG and TNC for neuronal activation using c-fos as a marker for nociception. After GTN infusion, we observed a 4-fold and 3-fold increase in c-fos mRNA levels at 30 min and 2 h in TNC, respectively, when compared with the saline-treated group (Figure 4A). There was a similar trend indicating higher expression of c-fos mRNA in TG, especially at the 2 h interval, but the values did not reach significance (Figure 4B). The c-fos mRNA levels in TNC were examined at 2 h after the pre-treatment with sumatriptan (0.3 mg/kg) before GTN infusion and compared with the c-fos mRNA levels at 2 h with the GTN-treated group. Sumatriptan-induced inhibition of c-fos mRNA did not reach significance (Figure 4C).

Figure 4.

Effect of GTN infusion on c-fos mRNA expression in (A) the TNC, and (B) the TG at five different time points (30 min, 1 h, 2 h, 4 h and 6 h). (C) Effect of GTN infusion on c-fos mRNA expression with sumatriptan (SUMA) pre-treatment in the TNC at 2 h. Data are presented as mean ± SEM. **p < 0.01, ***p < 0.001 (ANOVA (Kruskal Wallis) followed by Dunn's post hoc test) compared with saline, (N = 5–6).

Fos protein expression

The density of the Fos-immunopositive cells was counted in the TNC, including medullary and upper cervical dorsal horn, in both control and treated groups. The distribution was counted rostrocaudally, every 0.72 mm starting from 0.8 mm and ending at 5.12 mm. Fos-positive cells were counted in the superficial laminae (I/II) and deeper laminae (III/IV) of the entire TNC. We did not observe any labelling of Fos-positive neurons in lamina V. In three sets of control and GTN-treated rats we observed a significant increase of Fos expression in lamina I/II at 4 h after GTN infusion when compared with control (Figure 7A), but no difference was seen in the lamina III/IV between the groups (Figure 7B). Furthermore, only the superficial laminae (I/II) were examined rostrocaudally in all the groups. A significant increase in the Fos-positive cells were observed in the superficial laminae between 2.24 mm to 3.68 mm at 2 h after the GTN infusion when compared with the saline-treated group (Figure 5 and 8). In the group treated with GTN and killed after 4 h, a significant increase was seen between 0.80 mm to 4.40 mm from the obex as compared to the group treated with saline (Figure 5 and 8). Our result suggests that more Fos-positive cells were located between 2.24 mm to 4.40 mm from the obex. No differences were detected between the left and the right TNC in any of the groups. Furthermore, the Fos-positive cells were distributed along the dorsolateral, midlateral and ventrolateral axis, with a slightly higher concentration in the latter. Control experiments were performed to assure the specificity of the antibodies with omission of the primary antibody or incubation of sections with buffer only; no staining was observed in either case.

Figure 5.

Effect of GTN on expression of Fos protein at 2 h and 4 h TNC after the infusion. Representative image of the saline control (A, A1, A2), GTN 2 h (B, B1, B2) and GTN 4 h (C, C1, C2). Scale bars represent 100 µm (A, B and C) and 50 µm (A1, A2, B1, B2, C1 and C2). N = 6–7.

Figure 6.

Effect of GTN (4 µg/kg/min, i.v.) infusion with or without the pre-treatment of sumatriptan (SUMA) on expression of Fos protein. Representative images of the GTN 4 h (A, A1, A2) and SUMA + GTN 4 h (B, B1, B2). Scale bars represent 100 µm (A and B) and 50 µm (A1, A2, B1 and B2). N = 4–6.

Figure 7.

Total Fos-positive cells along the rostrocaudal axis from the obex (per 0.72 mm). Fos-positive cells in superficial laminae (I/II) (A) and deeper laminae (III/IV) (B) of saline-treated compared with GTN (4 h)-treated rats, *p < 0.05. No differences were seen between the groups in the deeper laminae (III/IV), (N = 3).

Figure 8.

Fos-positive cells in the superficial lamiae (I/II) of rats treated with saline, GTN for 2 h, GTN for 4 h and SUMA (sumatriptan) plus GTN for 4 h. Data are presented as mean ± SEM. p < 0.05, two-way ANOVA. This was followed by Bonferroni’s post hoc test, *p < 0.05 when compared with saline, ^#p < 0.05 when compared with saline treatment, ^$p < 0.05 when compared with GTN treatment for 4 h. N = 6–8.

Effect of pre-treatment with sumatriptan on Fos protein expression

Sumatriptan-induced inhibition of c-fos mRNA with 0.3 mg/kg did not reach significance (Figure 3C). Therefore, for the protein expression studies we doubled the dose of sumatriptan to 0.6 mg/kg. The rostrocaudal distribution of the Fos-positive nuclei in lamina I/II were analysed in the sumatriptan pre-treated group at 4 h after GTN infusion. Rats pre-treated with 0.6 mg/kg of sumatriptan before GTN infusion showed a significant decrease in Fos-positive cells in the superficial laminae between 2.96 mm to 3.36 mm compared with the GTN-treated rats (Figure 6 and 8).

Discussion

This study introduces a novel model of migraine in awake freely moving rats, simulating GTN-induced migraine provocation in humans. GTN infusion in this model caused an upregulation of mRNA and protein expression of the nociceptive marker c-fos in TNC. These changes were reversed by a specific anti-migraine drug sumatriptan at the protein level.

Acute surgery is known to upregulate Fos (11). In our model of freely moving rats the experiments were carried out only when animals had fully recovered, 10 days after the surgery. Migraine-provoking experiments in humans are conducted in the conscious state and in our model rats were similarly also unanaesthetized and freely moving. Some previous studies have used anaesthetized rats (21,22). Anaesthesia is warranted for the surgical interventions for invasive investigations such as in vivo electrophysiology but is still a significant departure from the human model. Anaesthesia suppresses cortical pain experience and cross-talk between cortex and lower centres and more importantly, anaesthesia can itself modulate c-fos expression (11,12). This may be the reason why previous studies had to use doses of GTN that were beyond pharmacological and clinical relevance. The GTN dose (4 µg/kg/min for 20 min) chosen for our study was only 8 times the dose given in humans (23). In anaesthetized rats this dose caused a significant drop in MABP, but in the unanaesthetized rats it did not. A similar difference in haemodynamic effects in anaesthetized rats and unanaesthetized rats has been described for other compounds such as anandamide (24). Rats normally need 5–10 times human doses of drugs, based on the body surface area to dose ratio (25), as given in the present study. In contrast, most previous studies with GTN have used 10 mg/kg, which is 10,000 times the human dose (6,7). The high dose caused a drastic (37%) and prolonged (75 min) drop in blood pressure in previous studies in anaesthetized rats (26). Decreased blood pressure is thus a major side effect associated with the administration of high doses of GTN, and is known to increase c-fos expression (8). The dose used in our study in awake rats avoids this confounder. Low doses of GTN have shown to potentiate the response of central neurons to dural and facial stimulation in anaesthetized rats, but GTN per se did not show any activation in the TNC of these rats (27). Moreover, another study shows that 100 µg/kg dose, i.p., which is slightly higher than the dose used i.v. in our model, causes signs of pain and allodynia even in naïve rats (28). This observation supports our contention that even the clinically relevant doses of GTN may elicit migraine-relevant behaviour/effects in animals. Previous animal models based on GTN infusion are attractive in their simplicity, but are confounded by the factors mentioned above. Other brain disorders like stroke, Alzheimer’s disease and Parkinson’s disease are characterized by specific changes in biomarkers or morphology, which can be easily monitored in animal models. In contrast preclinical migraine research lacks such biomarkers or morphological changes. As migraine is relatively subtle, episodic and non-progressive disorder, the stimulus used for modelling migraine should be in accordance. Furthermore, while simulating a human model in animals, it is imperative to keep experimental conditions as close to original conditions as possible. This was achieved in our unanaesthetized freely moving rats.

Rats cannot verbalize the pain experience, and pain is therefore estimated using bio-markers such as c-fos (29,30). This is an immediate early gene of which the mRNA reaches its peak 30–40 min after nociceptive stimulation followed by an increase in the Fos protein level within approximately 2 h. Noxious cranial stimuli are known to consistently induce c-fos in nociresponsive neurons located in the caudal TNC, especially in lamina I and II (31,32). Earlier studies have also used c-fos as an indicator of nociception in animal migraine models (18,33). In the present study, two different doses of GTN, 4 µg/kg/min and 30 µg/kg/min, were used to analyse the GTN dose response effect on Fos expression. There was no difference in the expression of Fos protein between the two doses, in keeping with previous human data showing a ceiling effect of 0.5 µg/kg/min for headache induction (2). Hence the lower dose was used in the further studies.

Infusion of 4 µg/kg/min of GTN resulted in the activation of neurons in TNC, including the medullary and upper cervical dorsal horn at 2 h and 4 h, as shown by upregulation of Fos protein in superficial laminae (I/II). In rats, localized mechanical dural stimulation has been shown to activate the nociresponsive neurons only in the superficial laminae I (34) whereas chemical stimulation or induction of cortical spreading depression strongly induced Fos-positive neurons in both lamina I and II (15,35,36). Destruction of unmyelinated C-fibres or chronic sectioning of the trigeminal nerve innervating the meninges resulted in the reduction of Fos expression in lamina I and II in the TNC (15,36). This shows the involvement of the TVS in the activation of second order neurons. Studies have also shown that facial stimulation can activate both lamina I and II (37). Moreover, in our model the distribution of the Fos-positive cells mainly in the ventrolateral axis of the dorsal horn represents most of the dorsal half of the head and the face, which can be compared with the pain referral regions induced by dural stimulation in humans (38). In humans we do not observe any facial pain after GTN infusion, although there are reports of decrease in pain thresholds in regions interposed with myofacial tissue (39). However, in our animal model Fos-positive cells were also seen in the dorsolateral axis of the dorsal horn, which mainly refer to the afferents terminating from the V2 (maxillary) and V3 (mandibular) regions of TG. This could reflect either the facial pain or an expansion in the receptive field due to the weak or silent synapses that has been demonstrated in dorsal horn neurons in anatomical and electrophysiological studies (40,41). These results suggest that NO released by GTN may cause increased nociception. GTN, like NO, is neutral in charge and hydrophobic and therefore diffuses freely across the blood-brain barrier. Hence activation of Fos can be a direct CNS effect of NO as well as being caused by amplified afferent nociceptive signals.

In humans the headache induced by GTN is decreased by the administration of the anti-migraine drug sumatriptan (42). Sumatriptan is a specific and clinically effective anti-migraine agent that exerts a part of its anti-migraine effect by constriction of meningeal arteries (43), via a constricting effect on vascular smooth muscle cells and by inhibiting the release of vasoactive neuropeptides from trigeminal nerve terminals (44). Sumatriptan has also shown to decrease the evoked potential and firing of the central trigeminal vascular neurons (45). Sumatriptan significantly inhibited the GTN induced Fos expression at 4 h throughout the rostrocaudal axis of TNC. This shows that sumatriptan inhibits the activation of central neurons of the TVS and that this model has a predictive validity.

Although this model most closely approximates the human model, there are a few limitations of this study. First we have used naïve rats in this study, and GTN usually causes delayed headache in migraineurs. It is possible that 8 times higher dose of GTN in rats may have sensitized the naïve rats. We observe an increase in Fos expression both at 2 h and 4 h, so 4 h may represent a delayed phase (22). However, we are not certain whether this represents an immediate or a delayed phase.

In conclusion, the present study presents and validates a migraine model in the freely moving unanaesthetized rat. The specificity of this model for migraine was confirmed by effect of sumatriptan on Fos expression. The model will be useful in understanding the pathophysiological mechanisms of migraine and for the screening of acute migraine drugs.

Footnotes

Funding

This work was supported by Candy’s Foundation, Lundbeck foundation and Danish Agency of Science, Technology and Innovation (271-07-0773).

Conflict of interest

We declare that there is no conflict of financial interest with regard to our manuscript.

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A naturalistic glyceryl trinitrate infusion migraine model in the rat

Abstract

Keywords

Introduction

Methods

Animals

Surgery

Drugs

Blood pressure measurements

mRNA expression studies

Fos immunohistochemistry and cell counting

Data analysis

Results

Effect of GTN infusion on MABP in anaesthetized and unanaesthetized rats

GTN dose response on Fos protein expression

Effect of GTN infusion on c-fos mRNA and Fos protein expression

Investigation of mRNA expression by quantitative real-time PCR (qPCR) studies

Fos protein expression

Effect of pre-treatment with sumatriptan on Fos protein expression

Discussion

Footnotes

Funding

Conflict of interest

References