CGRP infusion in unanesthetized rats increases expression of c-Fos in the nucleus tractus solitarius and caudal ventrolateral medulla,but not in the trigeminal nucleus caudalis

Abstract

Background and aims

Calcitonin gene-related peptide (CGRP) and glyceryl trinitrate (GTN) infusion in migraineurs provokes headache resembling spontaneous migraine, and CGRP receptor antagonists are effective in the treatment of acute migraine. We hypothesized that CGRP infusion would increase molecular markers of neuronal activation in migraine-relevant tissues of the rat.

Methods

CGRP was infused intravenously (i.v.) in freely moving rats to circumvent factors like anesthesia, acute surgery and severe hypotension, the three confounding factors for c-Fos expression. The trigeminal nucleus caudalis (TNC) was isolated at different time points after CGRP infusion. The level of c-Fos mRNA and protein expression in TNC were analyzed by qPCR and immunohistochemistry. c-Fos-stained nuclei were also counted in the nucleus tractus solitarius (NTS) and caudal ventrolateral medulla (CVLM), integrative sites in the brain stem for processing cardiovascular signals. We also investigated Zif268 protein expression (another immediate early gene) in TNC. The protein expression of p-ERK, p-CREB and c-Fos was analyzed in dura mater, trigeminal ganglion (TG) and TNC samples using Western blot.

Results

CGRP infusion caused a significant dose-dependent fall in mean arterial blood pressure. No significant activation of c-Fos in the TNC at mRNA and protein levels was observed after CGRP infusion. A significant increase in c-Fos protein was observed in the NTS and CVLM in the brain stem. Zif268 expression in the TNC was also not changed after CGRP infusion. p-ERK was increased in the dura mater 30 minutes after CGRP infusion.

Conclusion

CGRP infusion increased the early expression of p-ERK in the dura mater but did not increase c-Fos and Zif268 expression in the TNC. The rats may, thus, differ from migraine patients, in whom infusion of CGRP caused headache and a delayed migraine attack. The rat CGRP infusion model with c-Fos or Zif268 as neuronal pain markers in TNC is unsuitable for antimigraine drug testing.

Keywords

Migraine c-Fos CGRP neuronal activation zif268 vasodilation

Introduction

Calcitonin gene-related peptide (CGRP) is a potent vasodilator (1). CGRP receptor antagonists olcegepant (2) and telcagepant (3,4) are effective against acute migraine, demonstrating a key role of CGRP in migraine pathogenesis. CGRP infusion in migraine patients caused an immediate headache during and shortly after the infusion, and a delayed headache resembling spontaneous migraine attacks (5,6). As CGRP has a half-life of approximately 10 minutes, the direct effects of CGRP cannot account for the delayed headaches. CGRP therefore seems to trigger a cascade of events probably involving gene activation that results in delayed headache in humans. CGRP and CGRP receptor components are present in the dura mater, trigeminal ganglion (TG), trigeminal nucleus caudalis (TNC) and other pain-processing structures inside the rat brain (7 –9).

The immediate early genes (IEGs) c-fos and zif268 have been used extensively to study pain pathways in different parts of the brain in a number of disease models (10 –12). In the few studies conducted in animal models of migraine using other migraine-provoking agents, the increase in c-Fos expression has been ambiguous (13,14). These studies were performed in anesthetized rats subjected to acute surgery, which is known to up-regulate c-Fos expression (15). These interfering factors can be minimized in unanesthetized freely moving rats, as shown recently in a glyceryl trinitrate (GTN) infusion model from our lab (16,17). We therefore hypothesize that CGRP infusion in unanesthetized freely moving rats will induce migraine-like pain reflected by increase in c-Fos and Zif268 in the TNC. We also studied expression of p-extracellular signal-regulated kinase (p-ERK), p-cyclic AMP-response element binding protein (p-CREB) and c-Fos using Western blot in the dura mater, TG and TNC samples.

Methods

One hundred and four male Sprague-Dawley (SD) rats weighing 300–360 g (Taconic M&B, Denmark) were used in this study. The rats were maintained in cages with a 12-hour 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). Rats were weighed pre-surgery (331 ± 5 g, n = 76) and after the recovery period of eight days (354 ± 3 g, n = 76). Rats recovered well after surgery as indicated by body gain and there were no visible signs of distress or infection.

Drugs

CGRP (NeoMPS, Strasbourg, France) stock solution 0.5 mg ml⁻¹ was prepared in water and aliquots were stored at −20℃. CGRP stock solution was further diluted in saline to their final concentrations just prior to administration. All the dilutions required for the study were freshly prepared.

Measurement of mean arterial blood pressure (MABP)

For assessment of MABP in unanesthetized rats, six rats underwent carotid artery and femoral vein cannulation. Rats were allowed to recover for one to three days and infused with CGRP (0.1, 0.25, 0.5 and 1 µg kg⁻¹ minute⁻¹ for 20 minutes at 30-minute intervals between two doses) through the femoral vein, and the MABP was measured via the carotid artery throughout the infusion.

Intravenous (i.v.) CGRP administration

Rats were first anesthetized by intraperitonial injection of a premixed solution of ketamine (Ketaminol®Vet, Intervet, Skovlunde, Denmark) (100 mg kg⁻¹) and xylazine (Rompun®Vet, Bayer Inc, Germany) (7.5 mg kg⁻¹). Prior to surgery the animals were injected with enrofloxacin (Baytril®Vet, Bayer Inc, Germany) (10 mg kg⁻¹subcutaneously (s.c.)), an antimicrobial, and caprofen (Rimadyl®Vet, Pfizer Oy Animal Health, Helsinki, Finland) (5 mg kg⁻¹ s.c.), a nonsteroidal anti-inflammatory drug (NSAID). Surgery was performed aseptically. The incision was first made in the left inguinal area and the femoral vein was isolated. The catheter was then inserted 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 2 cm polythene tubing (0.40 mm, ID, 0.80 mm OD, Portex®, Smiths Medical ASD, USA), attached with polyurethane tubing and checked for patency. The catheter was then sealed using a blocking solution (Haemaccel, Heparin 500 IU ml⁻¹, Voluven®). The animal was monitored until fully recovered from anesthesia as indicated by the ability to ambulate. The core body temperature was maintained using an automatic regulated heating plate. Enrofloxacin and caprofen treatments were repeated once in a day for the next two days, while buprenorphin (Temgesic®, RB Pharmaceuticals Limited, Berkshire, UK) (0.03 mg kg⁻¹), an analgesic, treatment was repeated once a day for the next day. After a recovery period of eight days, the patency of the cannula was checked by infusing 100 µl of five-times diluted ketamine and xylazine solution. Afterwards, the cannula was connected to external polyurethane tubing coming out from a tether at the nape of the neck and the rats were allowed to move freely in AccuSampler cages (Dilab, Lund AB, Sweden) and were acclimatized for two days. On the third day, CGRP (0.25 µg kg⁻¹ min⁻¹ or 1 µg kg⁻¹ min⁻¹, for 20 minutes) was administered i.v. and the rats were sacrificed by pentobarbitone (50 mg kg⁻¹, i.v.) at the required time points. Control rats were sacrificed and perfused after two hours of saline infusion.

Effect of anesthesia on c-Fos expression

To assess the effect of anesthesia on c-Fos expression, six rats (femoral vein cannulated rats, on day 10) were infused i.v. with 200 µl of diluted ketamine and xylazine anesthesia. This gives 15 to 25 minutes of anesthesia to rats; during that time period saline was infused in these rats, after which the rats recovered from anesthesia in the cage. These rats were deeply anesthetized with pentobarbitone and perfused two hours after the saline infusion.

mRNA expression studies

The rats were anesthetized with pentobarbital and perfused transcardially with cold 400 ml phosphate-buffered saline (PBS). TNCs were carefully dissected out and stored at −80℃ for later use. The tissues were homogenized thoroughly in RLT buffer (Qiagen® GmbH, Hilden, Germany) with a rotor-stator homogenizer. Total RNA was purified using an RNeasy Mini Kit (Qiagen® GmbH, Hilden, Germany) and its yield and purity were assessed spectrophotometrically (Nanodrop 200C, Thermo Fisher Scientific Inc). The cDNA synthesis and quantitative polymerase chain reaction (qPCR) analysis were performed as described previously (9). Briefly, cDNA was synthesized using the QuantiTect Reverse Transcription Kit (Qiagen), and all samples were subjected to reverse transcription-PCR (RT-PCR). qPCR was performed with LightCycler technology (Roche) and QuantiFast SYBR Green I dye (Qiagen). mRNA was quantified using a calibrator-normalized relative quantification approach. The detection ratios between each target gene and a non-regulated reference gene (β-actin) were used to calculate the relative abundance of mRNA transcripts in each experimental sample. A calibrator was included in every PCR run to provide a constant calibration point for all samples both within a LightCycler run and between LightCycler runs. Consequently, all final expression data were expressed as a target/reference ratio in experimental samples, normalized by the target/reference ratio of the calibrator. The TNC of the capsaicin-treated rat was used as the calibrator for all the target genes.

Pre-validated gene-specific primers (Table 1) from Qiagen’s QuantiTect Primers Assay source were used to detect each mRNA transcript. A master mix of SYBR Green and appropriate primers were applied to LightCycler glass capillaries together with 1 µl of the cDNA samples according to the protocol by Qiagen. All prepared PCR samples were run using the real-time cycler program. At the end of each run, a melting-curve analysis was performed to verify single-product detection of primers. All fluorescence data were processed by a post-PCR data analysis software program (LightCycler Relative Quantification Software version 1.0 (Roche)). The quantification data were graphically visualized. 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 analyzing the amplicons generated from all the Qiagen primers. The expression of different mRNA in the tissues was detected using qPCR. To test for the contamination of the mRNA template with genomic DNA, negative controls were tested in which the RT 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 mRNA from the TNC of capsaicin-treated rats (3 µg kg⁻¹ minute⁻¹ i.v. for 20 minutes, in anesthetized rats) was extracted, converted to cDNA and subsequently served as a calibrator for all target mRNA in the qPCR experiments.

Table 1.

The primers used in the experiment were designed by Qiagen from NCBI GenBank extracted sequences (acc. no.).

Transcript	NCBI GenBank acc. no	Calibrator (rat tissue cDNA)	Amplicon length (base pairs)
Fos: FBJ osteosarcoma oncogene-rat	NM_022197	TNC	73
Actb: actin, beta-rat	NM-031144	TNC	145

NCBI: National Center for Biotechnology Information; TNC: trigeminal nucleus caudalis. Amplicon length denotes the expected size of amplified PCR products, as provided by Qiagen.

Antibody characterization

The details of antibodies used in this study are compiled in Table 2. Unless otherwise stated, the descriptions of the antibodies are from the manufacturer’s published data. Omission of primary antibody eliminated all staining. The c-Fos and Zif268 antibodies were previously characterized by pre-absorption immunohistochemistry (IHC) (18,19). The antibody for p-extracellular signal-regulated kinase (ERK) recognizes only the phosphorylated form of ERK1/2 and it has been characterized previously by a pre-absorption protocol (20). The polyclonal rabbit p-cyclic AMP-response element binding protein (CREB) antibody detects a band of 43 kb. The c-Fos antibody used for Western blot was previously characterized by pre-absorption IHC (21). A 62 kDa band was reported as c-Fos protein. This antibody gives other bands of Fos-related antigens at 35 and 47 kDa. Only the 62 kDa band was reported to be changed by stimulus (21). We also used lysate of mouse c-Fos transfected 293T cells as a positive control (SC-118888, Santa Cruz Biotechnology).

Table 2.

A list of primary and secondary antibodies used for Western blot (WB) and immunohistochemistry (IHC).

Primary antibody	Company and catalogue number	Concentration	Secondary antibody	Concentration
c-Fos-Rb (IHC)	Calbiochem (PC 38)	1:4000	Donkey anti-Rb IgG, F(ab')2 fragment specific	1:2000
c-Fos-Rb (WB)	Santa Cruz Biotechnology (SC-52)	1:500	Goat-anti Rb HRP conjugate	1:10000
Egr-1-(C-19)-Rb (IHC)	Santa Cruz Biotechnology (SC-189)	1:1000	Donkey anti-Rb IgG, F(ab')2 fragment specific	1:2000
p-ERK-Rb (WB)	Cell Signaling (#4376)	1:2000	Goat-anti Rb HRP conjugate	1:15000
p-CREB-Rb (WB)	Cell Signaling (#9191)	1:1000	Goat-anti Rb HRP conjugate	1:15000
GAPDH-Rb (WB)	Cell Signaling (#5174)	1:20000	Goat-anti Rb HRP conjugate	1:15000

Ms: mouse; Rb: rabbit; HRP: horseradish peroxidase; Ig: immunoglobulin; p-ERK: p-extracellular signal-regulated kinase; p-CREB: p-cyclic AMP-response element binding protein; GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

c-Fos and Zif268 IHC

At two and four hours after the end of the infusion, the animals were sacrificed with a high dose of pentobarbitone injection and perfused with PBS (7.4 pH) and paraformaldehyde (PFA) (4% in 7.4 pH PBS). TNCs were removed and post-fixed overnight in 4% PFA. The tissues were then stored in 30% sucrose solution in PBS for four days. The brain stems and upper cervical spinal cords were sectioned serially with a freezing microtome (Leica CM 3050S, Leica Biosystems Nussloch GmbH, Germany) from 0.5 mm rostral to obex to the C2 level (150 ± 5 sections), and every seventh 40-µm section (23 selected sections) was saved and processed for IHC. The immunolabeling procedures were performed according to the Avidin-Biotin-peroxidase Complex (ABC) method as described elsewhere (22). Sections were incubated overnight with polyclonal rabbit anti-c-Fos or anti-Zif268 antibody. Biotinylated donkey anti-rabbit immunoglobulin (Ig)G used as the secondary antibody. Buffer controls omitted either primary or secondary antibodies. The sections were subsequently mounted onto the glass slides and coverslipped, and observed through a Leica DMR HCS microscope using a 20× objective lens. Counting of c-Fos- and Zif268-positive cells in the region of interest was performed by an observer (c-Fos counting by ST and Zif268 counting by DKB) blinded to the treatment. The total numbers of c-Fos-positive cells were counted in lamina I–II of the TNC including the medullary and upper cervical dorsal horn. The distribution was counted from 0.5 mm rostral to obex to the C2 level. c-Fos counting was also performed in the nucleus tractus solitarius (NTS) and caudal ventrolateral medulla (CVLM), integrative sites for processing cardiovascular signals in the brain stem. The representative images of the treated and control sections were captured using a Nikon DS-Fi2 digital camera attached to a Nikon Eclipse (Ni) microscope.

Western blot

The rats were anesthetized with pentobarbital at 30 minutes and two hours post-infusion and perfused transcardially with ice-cold PBS. Dura mater, TG and TNC were isolated and immediately frozen in dry ice. Samples were grounded using a mortar and pestle kept in dry ice and transferred to a micro-centrifuge tube containing 300 µl lysis buffer. Tubes were left on ice for a minimum of half an hour. Afterwards, samples were briefly sonicated and were centrifuged at 12,000 g for 20 minutes at 4℃. Supernatant were collected and stored at −80℃ until further processing. Bio-Rad DC protein assay kit (Bio-Rad, CA, USA) was used to measure protein content. Protein samples were diluted with sample buffer and heated at 95℃ for five minutes, immediately kept on ice and centrifuged. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel (12%) electrophoresis; 25 µg protein was loaded into the wells. The separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane. The PVDF blots were blocked in 5% bovine serum albumin (BSA) in tris-buffered saline containing 1% Tween-20 (TBS-T) for one hour at room temperature. The primary antibodies were made in 5% BSA dissolved in 1% TBS-T. PVDF blots were kept in different primary antibodies overnight at 4℃. The next day, PVDF blots were washed three times with TBS-T for 10 minutes each wash. The respective horseradish peroxidase-conjugated secondary antibodies were diluted in TBS-T and PVDF blots were incubated in this for one hour at room temperature. The PVDF blots were washed three times with PBS-T for 10 minutes each wash and detection was performed using the Amersham™ ECL™ Select Western Blotting Detection Reagent kit. The PVDF blots were scanned by an electronically cooled CCD camera system (Fujifilm LAS-1000) and chemifluorescent images were captured and stored digitally. Densitometric analysis was conducted for each band by Multi Gauge V3.2 and expressed as ratio to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) signal (loading control). Densitometric data were graphically visualized. For the expression of different proteins of interest in the same PVDF blot, the blots were stripped using stripping buffer followed by several washes and treated as mentioned above.

Data analysis

All values are presented as mean ± SEM. For blood pressure experiments, one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test was used to determine the significant difference from the saline-treated group. For comparing c-fos mRNA and c-Fos-immunopositive cell count per section between different time points and treatments, Kruskal-Wallis test followed by Dunnett’s post hoc test were used. Mann-Whitney test or unpaired t-test was used for analysis of non-paired data. Differences were considered significant at p < 0.05. GraphPad Prism 5 (GraphPad Prism Software Inc, San Diego, CA, USA) was used for statistical analysis.

Results

Effect of CGRP infusion on MABP in unanesthetized rats

The 0.25, 0.5 and 1.0 µg kg⁻¹ minute⁻¹ infusion of CGRP for 20 minutes caused a significant fall in MABP of awake rats (N = 6) (Figure 1), whereas the infusion of 0.1 µg kg⁻¹ minute⁻¹ of CGRP for 20 minutes did not change the MABP significantly. For further studies of c-Fos expression, we chose to infuse 0.25 and 1 µg kg⁻¹ minute⁻¹ (for 20 minutes) of CGRP.

Figure 1.

Percentage change in mean arterial blood pressure after increasing doses of CGRP infusion in conscious normotensive rats. One-way ANOVA followed by Dunnett’s multiple comparison test was used to determine the significant difference from the saline-treated group. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001 (n = 5–8).

c-fos mRNA expression

We studied the c-fos expression in the TNC as a marker of neuronal activation. After CGRP infusion (0.25 µg kg⁻¹ minute⁻¹ for 20 minutes), c-fos mRNA levels were unchanged at all time points (20 minutes, 30 minutes, one hour, two hours, four hours, six hours) in the TNC, compared to the saline-treated group (Figure 2(a)). However, at the higher (1 µg kg⁻¹ minute⁻¹ for 20 minutes) dose of CGRP, a non-significant increase in c-fos mRNA was observed in the TNC samples at the two-hour time point (Figure 2(b)).

Figure 2.

Normalized ratio of c-fos mRNA in TNC at different time points after CGRP (a) (0.25 µg kg⁻¹ minutes⁻¹, 20 minutes) and (b) (1.0 µg kg⁻¹ minutes⁻¹, 20 minutes) intravenous infusion in unanesthetized rats. Kruskal-Wallis test followed by Dunnett’s post hoc test were used to determine the significant difference from the saline-treated group.

c-Fos and Zif268 protein expression

Representative images of c-Fos-immunoreactivity in the NTS and CVLM two hours after CGRP infusion is shown in Figure 3. Representative images of c-Fos and Zif268 immunoreactivity in the spinal trigeminal nucleus is shown in Figure 4. There was no significant difference observed in c-Fos-immunopositive cells per section in the lamina I–II of the TNC comparing CGRP (1 µg kg⁻¹ minute⁻¹, 20-minute infusion, two hours and four hours post-infusion) and saline-treated rats (Figure 5(a)). c-Fos expression in the NTS (Figure 5(c)) and CVLM (Figure 5(e)) was significantly increased two hours after the CGRP infusion. Four hours after the CGRP infusion, c-Fos expression in the NTS (Figure 5(c)) and in the CVLM (Figure 5(e)) was not significantly different from saline-infused rats. A lower dose of CGRP (0.25 µg kg⁻¹ minute⁻¹, 20-minute infusion, two hours post-infusion) also increased c-Fos expression in NTS (4 ± 1.7 (saline treated) to 10 ± 1.5, p = 0.004, n = 7) and in CVLM (2 ± 0.3 (saline treated) to 10 ± 1.3, p < 0.0001, n = 7) significantly, but it did not increase c-Fos count in TNC (8 ± 1.5 (saline treated) to 4 ± 0.8, p = 0.08, n = 7).

Figure 3.

Representative image of c-Fos-immunoreactivity in the nucleus tractus solitarius (NTS) and caudal ventrolateral medulla (CVLM). Cross-sections through the medulla with the NTS and CVLM stained for the activation marker c-Fos in neuronal nuclei. Representative specimen fixed two hours after a 20-minute intravenous (i.v.) CGRP (1 µg kg⁻¹ minute⁻¹) infusion in unanesthetized rats. Magnification bars 100 µm.

Figure 4.

Representative images of c-Fos (a) and Zif268 (b) immunoreactivity in the spinal trigeminal nucleus. Cross-sections through the medulla with the trigeminal nucleus caudalis stained for the activation marker c-Fos and Zif268 in the neuronal nuclei. Representative specimen fixed two hours after a 20-minute intravenous (i.v.) CGRP (1.0 µg kg⁻¹ minute⁻¹) infusion in unanesthetized rats and stained for c-Fos and Zif268. (a)1 and (b)1 are areas showing mediolateral trigeminal nucleus caudalis at higher magnification. Magnification bars 100 µm.

Figure 5.

Changes in c-Fos-immunoreactive cell count per section at two hours and four hours after intravenous (i.v.) CGRP (1 µg kg⁻¹ minute⁻¹, 20 minute) infusion in unanesthetized rats, compared to the saline-infused rats. (a) TNC; (c) NTS and (e) CVLM. ^***p < 0.001 and ^**p < 0.01 (Kruskal-Wallis test followed by Dunnett’s post hoc test). Changes in c-Fos-immunoreactive cell count per section after saline (20 minutes) infusion in anesthetized rats, compared to the saline infusion (20 minutes) in unanesthetized rats. (b) TNC; (d) NTS, and (f) CVLM. ^***p < 0.001 and ^**p < 0.01 (Mann-Whitney test, compared to control rats).

Two hours after saline infusion in the anesthetized rats, the c-Fos expression (per section) in the lamina I and II of the TNC (23 ± 6) (Figure 5(b)), in the NTS (63 ± 12) (Figure 5(d)) and in the CVLM (29 ± 5) (Figure 5(f)) was significantly increased, compared to c-Fos expression in lamina I and II of the TNC (8 ± 1), in the NTS (4 ± 0.4) and in the CVLM (4 ± 1) of saline-treated unanesthetized rats. There was no difference observed in Zif268-immunopositive cells per section in the lamina I–II of the TNC between CGRP- (176 ± 18, n = 12, two hours post-infusion) and saline- (150 ± 27, n = 12, two hours post-infusion) treated rats.

Western blot

A characteristic band of the p-ERK protein was identified at 42 and 44 KDa. A significant increase in the intensity of the p-ERK band was observed in the dura mater samples 30 minutes after CGRP (1 µg kg⁻¹ minute⁻¹, 20 minutes) infusion, compared to vehicle control (Figure 6(a)). Phospho-CREB (Ser133) antibody detects endogenous levels of CREB only when phosphorylated at serine 133 and shows a band at 43 KDa. Thirty minutes after the CGRP infusion, p-CREB expression did not change in the dura mater (Figure 6(a)). Two hours after the CGRP infusion, no significant changes in the levels of p-ERK and p-CREB were observed in the dura mater (Figure 6(b)). Thirty minutes (Figure 7(a)) and two hours (Figure 7(b)) after the CGRP infusion, p-ERK levels were not changed in the TG. Furthermore, there was no significant change in the expression of p-ERK (p = 0.06), p-CREB and c-Fos levels observed in the TNC at 30 minutes (Figure 8(a)) and two hours (Figure 8(b)) after the CGRP infusion.

Figure 6.

Western blot analysis of p-ERK and p-CREB in the dura mater 30 minutes (a) and two hours (b) after a 20-minute intravenous (i.v.) CGRP (1.0 µg kg⁻¹ minute⁻¹) infusion in unanesthetized rats. ^*p < 0.05, unpaired t-test.

Figure 7.

Western blot analysis of p-ERK and p-CREB in the TG 30 minutes (a) and two hours (b) after a 20-minute intravenous (i.v.) CGRP (1.0 µg kg⁻¹ minute⁻¹) infusion in unanesthetized rats.

Figure 8.

Western blot analysis of p-ERK, p-CREB and c-Fos in the TNC 30 minutes (a) and two hours (b) after a 20-minute intravenous (i.v.) CGRP (1.0 µg kg⁻¹ minute⁻¹) infusion in unanesthetized rats. ^*p < 0.05, unpaired t-test.

Discussion

Effect of CGRP infusion on MABP

CGRP infusion decreased blood pressure dose dependently, which is in line with a study showing a blood pressure-lowering effect (–24 ± 4 mmHg) after i.v. bolus injection of CGRP (0.1–5 µg kg⁻¹) in unanesthetized rats (23). As we could not detect an increased expression of c-Fos-positive nuclei in the TNC after infusion of the lower dose (0.25 µg kg⁻¹ minute⁻¹, 20 minutes) of CGRP, we decided to infuse a higher dose (1.0 µg kg⁻¹ minute⁻¹, 20 minutes) of CGRP. At this concentration the fall in MABP was distinct; therefore we also investigated c-Fos expression in the NTS and in the CVLM, which together with the rostral ventrolateral medulla (RVLM) are integrative sites for processing cardiovascular signals in the brain stem (24,25). Two hours after infusion of CGRP, both at the lower dose and the higher dose, c-Fos-positive cells in NTS and CVLM were significantly upregulated compared to saline-infused rats. Infusion of CGRP causes a decrease in MABP and an increase in c-Fos-positive cells in NTS and CVLM, which confirms the involvement of the baroreceptor reflex after CGRP infusion.

Effect of CGRP infusion on c-Fos expression in the TNC

CGRP infusion in migraineurs caused an immediate headache during infusion and shortly after a delayed headache resembling spontaneous migraine attacks (6). We assumed that changes in c-Fos protein expression at the two-hour and four-hour time points will match the immediate and delayed phase of human migraine provocation studies, respectively. We have used a validated model in which infusion of GTN in unanesthetized freely moving rats caused an increase of c-Fos in TNC, and pretreatment with sumatriptan (16) and olcegepant (17) attenuated c-Fos expression. Mechanical stimulation of dural blood vessels produced c-Fos-positive labeling primarily in lamina I, whereas facial stimulation produced c-Fos-positive labeling both in lamina I and in the outer part of lamina II (26). Capsaicin and GTN infusion in rats also increased c-Fos-positive labeling only in lamina I and the outer part of lamina II (16,27). The number of c-Fos-positive nuclei were not increased significantly in lamina III–IV in all three previous studies. In the present study, we have counted c-Fos-positive nuclei only in lamina I and the outer part of lamina II. Neither a lower nor higher dose of CGRP activated c-Fos in TNC both at the mRNA level and at the protein level. This finding was supported by two studies: In the first study, both topical (on dura surface) and systemic infusion of CGRP in rats were not able to activate or sensitize mechanosensitive meningeal nociceptors in the TG (28). More recently in the second study, it has been shown that the activity of the spinal trigeminal neurons with meningeal afferent input is normally not controlled by CGRP receptor activation or inhibition in the TG (29), emphasizing that CGRP-induced vasodilation and CGRP application on TG may not be an effective stimulus for the activation of pain. Both studies suggested a more central site of action to explain the facilitatory effect of CGRP on central neuron discharge. In contrast to the previous two studies, it has been reported that a bolus injection of CGRP produced facilitation of responses to innocuous vibrissal stimulation in non-nociceptive brain stem trigeminal neurons (30).

Our findings are in agreement with a number of studies that have failed to detect a hyperalgesic effect of peripherally administered CGRP (31,32). Anti-nociceptive effects of CGRP administered intracerebroventricularly (i.c.v.) and into the nucleus raphe magnus were reported in rats by tail-flick and hot plate tests, respectively (33,34). Furthermore, it has been shown that CGRP receptors are not present on sensory axons in cranial dura mater (7). Activation of meningeal nociceptors resulted in activation of trigeminal neurons and i.v. administration of the CGRP receptor antagonist olcegepant at a very high (0.9 mg kg⁻¹) dose inhibited neuronal activity, whereas the local meningeal application of olcegepant had no effect (35). Pre-treatment with olcegepant (0.9 mg kg⁻¹) inhibited capsaicin-induced c-Fos expression throughout the spinal trigeminal nucleus (18). These results suggest a more central site of action of olcegepant in inhibiting migraine. However, a peripheral mode of action of CGRP cannot be excluded as human positron-emission tomography (PET) studies revealed that central antagonism of CGRP receptors is not necessary for therapeutic efficacy in migraine pain relief (36,37).

Effect of CGRP infusion on Zif268 expression in the TNC

We also tested another IEG, Zif268, which follows a similar expression time profile as c-Fos. IHC for Zif268 was conducted only in limited samples to verify c-Fos data. It has been shown that Zif268 (early growth response protein 1 (EGR-1)) and c-Fos levels were increased in TNC after formalin injection in the whisker pad of the rat (12). Compared to the c-Fos-positive cell, we have seen more Zif268-positive cells in saline-infused rats, which is in accordance with previous studies (12,38). Zif268-positive cells per section were also not increased significantly in TNC two hours after CGRP (1.0 µg kg⁻¹ min⁻¹, 20 minutes) infusion. Both IEGs gave similar results, which further confirmed that CGRP infusion did not activate second-order neurons in TNC.

Our results were not supported by a recent study showing an increase in c-Fos expression in TNC 60 minutes after a single i.v. injection of CGRP (0.6 µg kg⁻¹); however, this study was performed in pentobarbitone-anesthetized rats. Increase in transient receptor potential cation channel subfamily V member 1 (TRPV1), CGRP, phosphorylated protein kinase C and phosphorylated CREB protein was also reported in TG (39). In our study, infusion of saline in ketamine- and xylazine-anesthetized rats caused a significant increase in c-Fos expression in the lamina I–II of the TNC, in the NTS and in the CVLM, compared to saline-infused rats in unanesthetized rats. It is already known that anesthesia and acute surgery by themselves affect expression of c-Fos (15,40,41). The study by Chatchaisak and colleagues (39) raised a few points. First, the authors have shown that after 45 minutes and 60 minutes of CGRP infusion TRPV1 content is increased in TG; even in the chronic pain models a change in TRPV1 expression takes days (42). Second, they have shown an increase in CGRP content in TG after 60 minutes of CGRP infusion; even in TG culture it takes a minimum of four-hours’ incubation of CGRP to promote CGRP mRNA synthesis (43). Furthermore, in isoflurane-anesthetized rats four hours after i.v. infusion of GTN (250 µg kg⁻¹ over a period of two hours), an increase in CGRP and neuronal nitric oxide synthase (nNOS) immunoreactivity was reported in the TG (44). After one short bolus infusion of CGRP in rats, the 60-minute time window is very short to expect increased TRPV1 and CGRP protein expression. In the rat closed-cranial window (CCW) model, a 20-minute i.v. infusion of CGRP caused a robust increase in middle meningeal artery (MMA) diameter and decrease in blood pressure; these effects returned to normal baseline within 10 minutes after the infusion was stopped (45). We have used the same cannulas (polyurethane) for CCW and infusion studies. A robust increase in MMA diameter by CGRP infusion excludes the possibility of any absorption of CGRP by the polyurethane cannula.

Western blot

We have seen a significant activation of p-ERK in the dura mater 30 minutes after CGRP infusion. The mitogen-activated protein kinase (MAPK) cascades represent major downstream effectors of CGRP (46). CGRP receptor activation produced protein kinase A (PKA) and protein kinase C-dependent mechanical hyperalgesia and central sensitization (47). ERK signaling pathway plays an important role in peripheral sensitization after noxious stimulation to the peripheral tissues (48). Both c-Fos and p-ERK are used as a marker of neuronal activation and central sensitization after noxious stimulation tissue injury (49). Compared to c-Fos, pERK can be induced within a minute, which can be correlated well with the induction of immediate pain after noxious stimulus. c-Fos is localized in the nuclei of neurons but pERK can be induced in different subcellular structures of neurons such as nuclei, cytoplasm, axons and dendrites (49). Recently, it was reported that infusion of GTN evoked a delayed meningeal nociceptor sensitization that was associated with a robust ERK phosphorylation in the meningeal arteries (50).

We did not see any delayed activation of p-ERK two hours after CGRP infusion in the dura mater and in the TNC. Capsaicin-stimulated p-ERK activation was reported in the TG within 10 minutes, and the p-ERK activation was not affected by pre-treatment with the CGRP receptor antagonist, olcegepant (27). ERK is the principal mediator of Ca²⁺-dependent c-Fos induction via two different modules, (i) ERK/Elk-1 (on serum response element, SRE site) and (ii) ERK/? (probably via an indirect mechanism involving the family of ribosomal S6 kinase, RSKs)/CREB (on cAMP-response element, CRE site), a pathway positively controlled by calcium-calmodulin kinases (CaM-Ks) (20,51). Previously, CREB expression was reported to be upregulated by CGRP in cultured spinal neurons and in a primary culture of TG enriched for glia (52,53). However, we did not see activation of p-ERK and p-CREB in TG 30 minutes and two hours after CGRP infusion. Compared to GTN, CGRP has much smaller blood-brain-barrier permeability. The increase in c-Fos expression in TNC after GTN infusion might not directly correlate to the dural artery vasodilation induced by released nitric oxide (NO). In migraine patients, it has been reported that migraine pain was not accompanied by MMA dilation, and only slight intracerebral artery dilation was reported (54). The low frequency of delayed headaches after infusion of CGRP in control subjects as compared to migraineurs (5) suggests that sensitization might be required before migraine can be induced by CGRP. CGRP infusion in spontaneous trigeminal allodynia rats (SD) induced acute trigeminal sensitivity, marked by decreased von Frey head-withdrawal threshold, compared to normal SD rats (55). Neurogenic inflammation is supposed to be induced by a co-release of CGRP, substance P (SP) and neurokinin A (NKA) (56). Injection of CGRP together with SP or NKA in the human temporal muscle elicited a significant pain sensation (57). Thus, co-administration of CGRP with SP and/or NKA might be needed to obtain a measurable pain response in rats. Recently, we have shown that i.c.v. injection of CGRP did not increase c-Fos in the TNC (9). It might be due to the limited permeability of i.c.v. injected CGRP in the rat brain. Taken together; the rat CGRP infusion model with c-Fos or Zif268 as neuronal pain markers in TNC is unsuitable for antimigraine drug testing.

Clinical implications

Calcitonin gene-related peptide (CGRP) infusion activates blood pressure-regulating centers in the brain stem.

CGRP infusion increased the early expression of p-extracellular signal-regulated kinase (p-ERK) in the dura mater.

CGRP-induced vasodilation of dural arteries is not enough to activate neurons in the trigeminal nucleus caudalis.

The rat CGRP infusion model with c-Fos or Zif268 as neuronal pain markers in TNC is unsuitable for antimigraine drug testing.

Footnotes

Funding

This work was supported by an International Mobility Grant (Faculty of Health and Medical Sciences, University of Copenhagen), Candy’s Foundation, Lundbeck Foundation and the A.P. Møller Foundation for the Advancement of Medical Science.

Conflict of interest

None declared.

References

Brain

Williams

Tippins

. Calcitonin gene-related peptide is a potent vasodilator. Nature 1985; 313: 54–56.

Olesen

Diener

Husstedt

. Calcitonin gene-related peptide receptor antagonist BIBN 4096 BS for the acute treatment of migraine. N Engl J Med 2004; 350: 1104–1110.

Ferrari

Dodick

. Efficacy and tolerability of MK-0974 (telcagepant), a new oral antagonist of calcitonin gene-related peptide receptor, compared with zolmitriptan for acute migraine: A randomised, placebo-controlled, parallel-treatment trial. Lancet 2008; 372: 2115–2123.

Olesen

Dodick

. Antimigraine efficacy of telcagepant based on patient's historical triptan response. Headache 2011; 51: 64–72.

Hansen

Hauge

Olesen

. Calcitonin gene-related peptide triggers migraine-like attacks in patients with migraine with aura. Cephalalgia 2010; 30: 1179–1186.

Lassen

Haderslev

Jacobsen

. CGRP may play a causative role in migraine. Cephalalgia 2002; 22: 54–61.

Lennerz

Ruhle

Ceppa

. Calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), and calcitonin gene-related peptide (CGRP) immunoreactivity in the rat trigeminovascular system: Differences between peripheral and central CGRP receptor distribution. J Comp Neurol 2008; 507: 1277–1299.

Eftekhari

Edvinsson

. Calcitonin gene-related peptide (CGRP) and its receptor components in human and rat spinal trigeminal nucleus and spinal cord at C1-level. BMC Neurosci 2011; 12: 112–112.

Bhatt

Gupta

Ploug

. mRNA distribution of CGRP and its receptor components in the trigeminovascular system and other pain related structures in rat brain, and effect of intracerebroventricular administration of CGRP on Fos expression in the TNC. Neurosci Lett 2014; 559: 99–104.

10.

Mitsikostas

Sanchez del Rio

. Receptor systems mediating c-fos expression within trigeminal nucleus caudalis in animal models of migraine. Brain Res Brain Res Rev 2001; 35: 20–35.

11.

Cutrer

Mitsikostas

Ayata

. Attenuation by butalbital of capsaicin-induced c-Fos-like immunoreactivity in trigeminal nucleus caudalis. Headache 1999; 39: 697–704.

12.

Otahara

Ikeda

Sakoda

. Involvement of NMDA receptors in Zif/268 expression in the trigeminal nucleus caudalis following formalin injection into the rat whisker pad. Brain Res Bull 2003; 62: 63–70.

13.

Offenhauser

Zinck

Hoffmann

. CGRP release and c-Fos expression within trigeminal nucleus caudalis of the rat following glyceryltrinitrate infusion. Cephalalgia 2005; 25: 225–236.

14.

Jones

Lever

Bingham

. Nitric oxide potentiates response of trigeminal neurones to dural or facial stimulation in the rat. Cephalalgia 2001; 21: 643–655.

15.

Takayama

Suzuki

Miura

. The comparison of effects of various anesthetics on expression of Fos protein in the rat brain. Neurosci Lett 1994; 176: 59–62.

16.

Ramachandran

Bhatt

Ploug

. A naturalistic glyceryl trinitrate infusion migraine model in the rat. Cephalalgia 2012; 32: 73–84.

17.

Ramachandran

Bhatt

Ploug

. Nitric oxide synthase, calcitonin gene-related peptide and NK-1 receptor mechanisms are involved in GTN-induced neuronal activation. Cephalalgia 2014; 34: 136–147.

18.

Sixt

Messlinger

Fischer

. Calcitonin gene-related peptide receptor antagonist olcegepant acts in the spinal trigeminal nucleus. Brain 2009; 132: 3134–3141.

19.

Slade

Knight

Carter

. Nocturnal light pulses selectively induce Egr-1/NGFI-A protein in periventricular hypophysiotrophic somatostatinergic neurons. J Mol Neurosci 2001; 17: 271–277.

20.

Papadeas

Blake

Knapp

. Sustained extracellular signal-regulated kinase 1/2 phosphorylation in neonate 6-hydroxydopamine-lesioned rats after repeated D1-dopamine receptor agonist administration: Implications for NMDA receptor involvement. J Neurosci 2004; 24: 5863–5876.

21.

Basheer

Sherin

Saper

. Effects of sleep on wake-induced c-Fos expression. J Neurosci 1997; 17: 9746–9750.

22.

Hundahl

Hannibal

Fahrenkrug

. Neuroglobin expression in the rat suprachiasmatic nucleus: Colocalization, innervation, and response to light. J Comp Neurol 2010; 518: 1556–1569.

23.

Lappe

Slivjak

Todt

. Hemodynamic effects of calcitonin gene-related peptide in conscious rats. Regul Pept 1987; 19: 307–312.

24.

Minson

Llewellyn-Smith

Arnolda

. C-fos expression in central neurons mediating the arterial baroreceptor reflex. Clin Exp Hypertens 1997; 19: 631–643.

25.

Murphy

Ennis

Shipley

. Directionally specific changes in arterial pressure induce differential patterns of fos expression in discrete areas of the rat brainstem: A double-labeling study for Fos and catecholamines. J Comp Neurol 1994; 349: 36–50.

26.

Strassman

Mineta

Vos

. Distribution of fos-like immunoreactivity in the medullary and upper cervical dorsal horn produced by stimulation of dural blood vessels in the rat. J Neurosci 1994; 14: 3725–3735.

27.

Sixt

Messlinger

Fischer

. Calcitonin gene-related peptide receptor antagonist olcegepant acts in the spinal trigeminal nucleus. Brain 2009; 132: 3134–3141.

28.

Levy

Burstein

Strassman

. Calcitonin gene-related peptide does not excite or sensitize meningeal nociceptors: Implications for the pathophysiology of migraine. Ann Neurol 2005; 58: 698–705.

29.

Covasala

Stirn

Albrecht

. Calcitonin gene-related peptide receptors in rat trigeminal ganglion do not control spinal trigeminal activity. J Neurophysiol 2012; 108: 431–440.

30.

Cumberbatch

Williamson

Mason

. Dural vasodilation causes a sensitization of rat caudal trigeminal neurones in vivo that is blocked by a 5-HT1B/1D agonist. Br J Pharmacol 1999; 126: 1478–1486.

31.

Nakamura-Craig

Gill

. Effect of neurokinin A, substance P and calcitonin gene related peptide in peripheral hyperalgesia in the rat paw. Neurosci Lett 1991; 124: 49–51.

32.

Chu

Choy

Foster

. A comparative study of the ability of calcitonin gene-related peptide and adrenomedullin_13–52 to modulate microvascular but not thermal hyperalgesia responses. Br J Pharmacol 2000; 130: 1589–1596.

33.

Huang

Brodda-Jansen

Lundeberg

. Anti-nociceptive effects of calcitonin gene-related peptide in nucleus raphe magnus of rats: An effect attenuated by naloxone. Brain Res 2000; 873: 54–59.

34.

Pecile

Guidobono

Netti

. Calcitonin gene-related peptide: Antinociceptive activity in rats, comparison with calcitonin. Regul Pept 1987; 18: 189–199.

35.

Fischer

Koulchitsky

Messlinger

. The nonpeptide calcitonin gene-related peptide receptor antagonist BIBN4096BS lowers the activity of neurons with meningeal input in the rat spinal trigeminal nucleus. J Neurosci 2005; 25: 5877–5883.

36.

Vermeersch

SGG

Hoon

J de

De Saint-Hubert

. PET imaging in healthy subjects and migraineurs suggests CGRP receptor antagonists do not have to act centrally to achieve clinical efficacy. J Headache Pain 2013; 14 (Suppl 1): P224–P224.

37.

Hostetler

Joshi

Sanabria-Bohorquez

. In vivo quantification of calcitonin gene-related peptide receptor occupancy by telcagepant in rhesus monkey and human brain using the positron emission tomography tracer [11C]MK-4232. J Pharmacol Exp Ther 2013; 347: 478–486.

38.

Clark

Bhattacharya

Miller

. Induction of c-Fos, Zif268, and Arc from acute bouts of voluntary wheel running in new and pre-existing adult mouse hippocampal granule neurons. Neuroscience 2011; 184: 16–27.

39.

Chatchaisak

Srikiatkhachorn

Maneesri-le Grand

. The role of calcitonin gene-related peptide on the increase in transient receptor potential vanilloid-1 levels in trigeminal ganglion and trigeminal nucleus caudalis activation of rat. J Chem Neuroanat 2013; 47: 50–56.

40.

Stenberg

Ovlisen

Svendsen

. Effect of local anaesthesia on neuronal c-Fos expression in the spinal dorsal horn and hypothalamic paraventricular nucleus after surgery in rats. Basic Clin Pharmacol Toxicol 2005; 96: 381–386.

41.

Sommers

Nguyen

Veening

. Suppression of noxious-induced c-Fos expression in the rat lumbar spinal cord by isoflurane alone or combined with fentanyl. Anesth Analg 2008; 106: 1303–1308. table.

42.

Kim

Park

Cho

. Differential changes in TRPV1 expression after trigeminal sensory nerve injury. J Pain 2008; 9: 280–288.

43.

Zhang

Winborn

Marquez de Prado

. Sensitization of calcitonin gene-related peptide receptors by receptor activity-modifying protein-1 in the trigeminal ganglion. J Neurosci 2007; 27: 2693–2703.

44.

Dieterle

Fischer

Link

. Increase in CGRP- and nNOS-immunoreactive neurons in the rat trigeminal ganglion after infusion of an NO donor. Cephalalgia 2011; 31: 31–42.

45.

Bhatt DK, Gupta S, Olesen J, et al. PACAP-38 infusion causes sustained vasodilation of the middle meningeal artery in the rat: Possible involvement of mast cells. Cephalalgia. Epub ahead of print 21 February 2014. DOI:10.1177/0333102414523846.

46.

Walker

Conner

Poyner

. Regulation of signal transduction by calcitonin gene-related peptide receptors. Trends Pharmacol Sci 2010; 31: 476–483.

47.

Sun

Lawand

. Calcitonin gene-related peptide receptor activation produces PKA- and PKC-dependent mechanical hyperalgesia and central sensitization. J Neurophysiol 2004; 92: 2859–2866.

48.

Dai

Iwata

Fukuoka

. Phosphorylation of extracellular signal-regulated kinase in primary afferent neurons by noxious stimuli and its involvement in peripheral sensitization. J Neurosci 2002; 22: 7737–7745.

49.

Gao

RR.

c-Fos and pERK, which is a better marker for neuronal activation and central sensitization after noxious stimulation and tissue injury?

Open Pain J 2009; 2: 11–17.

50.

Zhang

Kainz

Zhao

. Vascular extracellular signal-regulated kinase mediates migraine-related sensitization of meningeal nociceptors. Ann Neurol 2013; 73: 741–750.

51.

Zanassi

Paolillo

Feliciello

. cAMP-dependent protein kinase induces cAMP-response element-binding protein phosphorylation via an intracellular calcium release/ERK-dependent pathway in striatal neurons. J Biol Chem 2001; 276: 11487–11495.

52.

Vause

Durham

. Calcitonin gene-related peptide differentially regulates gene and protein expression in trigeminal glia cells: Findings from array analysis. Neurosci Lett 2010; 473: 163–167.

53.

Seybold

McCarson

Mermelstein

. Calcitonin gene-related peptide regulates expression of neurokinin1 receptors by rat spinal neurons. J Neurosci 2003; 23: 1816–1824.

54.

Amin

Asghar

Hougaard

. Magnetic resonance angiography of intracranial and extracranial arteries in patients with spontaneous migraine without aura: A cross-sectional study. Lancet Neurol 2013; 12: 454–461.

55.

Oshinsky

Sanghvi

Maxwell

. Spontaneous trigeminal allodynia in rats: A model of primary headache. Headache 2012; 52: 1336–1349.

56.

Williamson

Hargreaves

. Neurogenic inflammation in the context of migraine. Microsc Res Tech 2001; 53: 167–178.

57.

Pedersen-Bjergaard

Nielsen

. Calcitonin gene-related peptide, neurokinin A and substance P: Effects on nociception and neurogenic inflammation in human skin and temporal muscle. Peptides 1991; 12: 333–337.