Sage Journals: Discover world-class research

Abstract

Objective

To evaluate whether intraganglionic calcitonin gene-related peptide induced differential migraine-like responses in male and female rats.

Methods

Calcitonin gene-related peptide was injected in the trigeminal ganglion of male and female rats followed by assessment of periorbital mechanical allodynia with von Frey hairs. The influence of systemic treatment with sumatriptan or intraganglionic treatment with minocycline and propentofylline was determined on the calcitonin gene-related peptide-induced mechanical allodynia in male and female rats. One additional group was exposed to an aversive light 24 h after calcitonin gene-related peptide priming, followed by evaluation of periorbital mechanical threshold, and another group was tested in the elevated-plus maze.

Results

Intraganglionar calcitonin gene-related peptide-induced periorbital mechanical allodynia in female (0.5 to 6 h) and male rats (0.5 to 4 h). Systemic sumatriptan briefly attenuated the mechanical allodynia, but intraganglionar minocycline or propentofylline injection was effective only in male rats. Calcitonin gene-related peptide induced photic sensitivity in female and male rats (lasting 4 h and 1 h, respectively), as well as anxiety-like behavior.

Conclusions

Intraganglionar calcitonin gene-related peptide may play a major role in migraine-like responses, including periorbital mechanical allodynia, light sensitivity and anxiety like-behavior. Female rats are likely to be more susceptible to calcitonin gene-related peptide effects and a better understanding of the sexual dimorphism in calcitonin gene-related peptide signaling may help to improve migraine therapy.

Keywords

Trigeminal ganglion mechanical allodynia light sensitivity anxiety-like behavior

Introduction

Migraine is a severe and disabling brain condition characterized by throbbing head pain, with sensitivity to movement, visual, auditory, and other afferents inputs, as well as nausea and vomiting (for review see (1)). It is considered the top cause of years lived with disability between the ages of 15–49, being two to three times more common in women than in men (2,3). Moreover, there is increasing evidence that migraine often coexists with psychiatric disorders such as anxiety and depression (4).

Soon after its discovery in 1982, calcitonin gene-related peptide (CGRP) was implicated in the physiopathology of migraine. Recently, the first antibody toward the CGRP receptor (erenumab) was approved by the FDA for episodic and chronic migraine prophylactic therapy, and it is expected to be marketed worldwide soon (5). As already described, other migraine therapies, including certain triptans and CGRP-related antibodies, have little or no ability to cross the blood-brain barrier (6,7). Therefore, peripheral structures of the trigeminovascular system, such as the trigeminal ganglion (TG) and dura mater, are likely to be the targets of CGRP-related therapies in migraine treatment.

It has been demonstrated that about 50% of the neurons of human and rat TG contain CGRP (8,9). This structure is devoid of barriers to the peripheral circulation and is suggested to represent a source of CGRP during a migraine attack. The CGRP receptor is expressed by approximately one third of neurons, mainly large sized neurons, and glial cells in the TG (8,9). Thus, it has been suggested that blocking CGRP transmission within the TG may be sufficient to abort or prevent a migraine crisis (for review see (5)).

Epidural injection of CGRP induced a variety of migraine like-responses in rodents (10,11) and it was recently suggested that females are more sensitive to peripherally administered CGRP (12). Likewise, intraganglionic injection of CGRP was recently shown to induce long-lasting facial heat hyperalgesia in rats so as to induce glial-dependent up-regulation of pro-inflammatory cytokines and Nav 1.7 sodium channels on TG neurons (13). This paper investigates whether intraganglionic CGRP induced differential migraine-like responses (i.e facial mechanical allodynia; light sensitivity and anxiety-like behavior) in male and female rats and the sex influence in the blockade of nociceptive response by sumatriptan or glial cells inhibition.

Methodology

Animals

Male and female Wistar rats 2 months old (220–280 g) provided by The Federal University of Paraná colony were maintained in a climate-controlled room (22 ± 2℃) on a 12-hour light/dark cycle with food and water ad libitum. All animals were housed together for at least 1 week before testing (males and females were housed separately), in groups of four in plastic boxes with wood shaving bedding, under controlled conditions of light (12:12 hour light: dark cycle) and temperature (22 ± 1℃), and with chow and water ad libitum. Experiments were performed during the light cycle, between 8 am and 5 pm. The animals were allowed to acclimate to the environment for at least 48 h before any experiment. All studies were performed by experimenters (EIA and JMT) who were blinded to the treatment conditions, with a different experimenter (ARB) randomly assigning each animal by sortition (i.e. a simple randomization method) to different groups and dosing. All experimental procedures were performed in accordance with the ARRIVE guidelines, and in accordance with the guidelines of the Federal University of Paraná and Brazilian regulations on animal welfare and were approved by the University Ethics Committee (CEUA/BIO-UFPR; #1273). Determining the sample size is a requirement of the Committee on the Ethical Use of Animals of our institution before approval of the experimental protocols. Based on an a priori power analysis using the GPower 3.1 software (14), which, with a large standardized effect size of F = 0.5, power of 0.8, and α = 0.05, estimated eight rats per group. Based on this observation, in previous studies that have used the same animal models (12,15,16) and considering replacement, reduction, and refinement in optimizing animal use, it was determined a sample size of 6–9 animals per group.

Compounds

Full length rat calcitonin gene-related peptide (CGRP) (Sigma-Aldrich, St. Louis, MO, USA) was diluted in saline and administered to animals at 0.1 nmol or 380 ng/10 µL, sumatriptan (Libbs, Embu das Artes, SP, BR) was diluted in water for injection (1 mg/kg, i.p.), minocycline (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in water for injection (100 µg/10 µL) and propentofylline (Santa Cruz Biotech, Dallas, TX, USA) was dissolved in saline (25 µg/10 µL). Doses were selected based on previous studies (13,17 –20).

Intraganglionic administration of drugs

The intraganglionar injection of CGRP, minocycline and propentofylline was performed according to (21), with minor modifications. After brief inhalatory anesthesia with a halothane (4%) O₂ mixture, the heads of the animals were restrained using one hand, and with the other hand a sterile long 27-G needle was introduced into the zygomatic process through the infraorbital foramen. The needle was connected to a Hamilton 0.5-mL syringe and positioned at an ∼ 10° angle relative to the midline of the head. Consequently, the needle was inserted through the infraorbital canal until its tip passed the foramen rotundum and reached the right TG. The injection volume of each drug was 10 µL.

Periorbital mechanical allodynia

Animals were placed individually into acrylic observation cages of 30 cm³ for at least 2 h for habituation. Animals that did not respond to the 8 g filament in the baseline assessment were included in the experiments. Mechanical thresholds of the periorbital region were assessed with von Frey filaments (Semmes-Weinstein monofilaments, Stoelting, Wood Dale, IL, USA) by applying them in the midline of the forehead near the eyes. Each filament was applied three times, ranging from 0.04 to 8.0 g, until each animal presented two nociceptive responses consisting of a sharp withdrawal of the head (22).

CGRP-induced photic sensitivity

Twenty-four hours after CGRP injection, the rats were exposed to 5000–6000 lux for a period of 1 h in their home cage, by exposure to a lamp of 180 W. The illumination of the room during the test was 0 to 1 lux. This protocol was based on previous studies, with minor modification (23 –25). Briefly, rats were placed in acrylic boxes for mechanical allodynia assessment.

Elevated plus maze

The elevated plus maze test was performed to evaluate anxiety-like behavior, according to previous studies (26). The apparatus used consisted of two open arms (50 cm length × 10 cm width) without sidewalls, two closed arms (50 cm length × 10 cm width × 30 height) with sidewalls and a central area (10 × 10 cm) that connects the arms. The maze was elevated 60 cm above the floor and placed in a moderately lit room (40 lux). In this test the animals were placed in the center of the apparatus facing the open arm for 5 min and the time and entries spent in both arms were recorded. The EPM was cleaned between each test with a 1% ethanol solution to eliminate odors left by other rats. The parameters evaluated were time and entries in open and closed arms. Animals avoid open spaces due to fear or anxiety. Therefore, rats that spend less time and enter less into open arms compared to control groups are considered to display anxiety-like behavior.

Experimental design

CGRP or saline was directly injected into the right TG after mechanical threshold baseline assessment. Mechanical allodynia in the periorbital area was evaluated 30 min after the injection, in male and female rats, and hourly up to 6 h. An independent group of male and female rats received Sumatriptan (1 mg/kg i.p., administered 15 min before CGRP injection), which was used as a positive control (Figure 1(a)). The paw mechanical threshold was also assessed in an independent group of female rats in 1 h intervals after intraganglionar CGRP injection (Supplementary method).

Figure 1.

Effect of intraganglionar CGRP injection and sumatriptan on periorbital allodynia in male and female rats. Experimental protocol (a). Mechanical threshold time-course in male (b) and female (c) rats after pretreatment with sumatriptan (Suma; 1.0 mg/Kg, i.p.) or vehicle (Veh; same volume; i.p.) followed by intraganglionar CGRP (CGRP; 0.1 nmol/10 µL; i.g.) or saline (Sal; 10 µL; i.g.) injection. *p < 0.05, compared to Veh-Sal, #p < 0.05, compared to Veh-CGRP. Two-way ANOVA with repeated measures followed by the Bonferroni post hoc test. (d) Area under the curve (AUC) of the mechanical threshold of male and female rats. *p < 0.05, compared to the male group. One-way ANOVA followed by the Bonferroni post hoc test. Data are expressed as mean ± SEM (n = 6–7).

Intraganglionar minocycline and propentofylline were used to inhibit TG glial cells and were administered 30 min before CGRP (minocycline 100 µg/10 µL i.g.; propentofylline 25 µg/10 µL i.g.; CGRP 0.1 nmol/10 µL, i.g.). Control rats received intraganglionar injection of saline (10 µL) followed by a second injection of saline in the TG (10 µL). Periorbital mechanical allodynia was evaluated 30 min after the last injection and hourly up to 6 h (Figures 2(a) and 3(a)).

Figure 2.

Effect of intraganglionar minocycline on periorbital allodynia induced by CGRP. Experimental protocol (a). Mechanical threshold time-course in male (b) and female (c) rats after pre-treatment with minocycline (Mino; 100 µg/10 µL, i.g.) or vehicle (Veh; 10 µL) followed by intraganglionar CGRP (CGRP; 0.1 nmol/10 µL; i.g.) or saline (Sal; 10 µL; i.g.) injection. *p < 0.05, compared to Veh-Sal, #p < 0.05, compared to Veh-CGRP. Two-way ANOVA with repeated measures followed by the Bonferroni post hoc test. (d) Area under the curve (AUC) of the mechanical threshold of male and female rats. *p < 0.05, compared to the male group. One-way ANOVA followed by the Bonferroni post hoc test. Data are expressed as mean ± SEM (n = 7–9).

Figure 3.

Effect of intraganglionar propentofylline on periorbital allodynia induced by CGRP. Experimental protocol (a). Mechanical threshold time-course in male (b) and female (c) rats after pre-treatment with propentofylline (PPF; 25 µg/10 µL, i.g.) or vehicle (Veh; 10 µL) followed by intraganglionar CGRP (CGRP; 0.1 nmol/10 µL; i.g.) or saline (Sal; 10 µL; i.g.) injection. *p < 0.05, compared to Veh-Sal, #p < 0.05, compared to Veh-CGRP. Two-way ANOVA with repeated measures followed by the Bonferroni post hoc test. (d) Area under the curve (AUC) of the mechanical threshold of male and female rats. *p < 0.05, compared to the male group. One-way ANOVA followed by the Bonferroni post hoc test. Data are expressed as mean ± SEM (n = 7–9).

To investigate the possibility of CGRP to induce light sensitization, animals were exposed to an aversive bright light 24 h after receiving CGRP (0.1 nmol/10 µL) or saline (10 µL) intraganglionar injection. Mechanical allodynia was evaluated at 30 min after light exposure and hourly up to 4 or 6 h in male and female rats, respectively (Figure 4(a)). Additionally, anxiety-like behavior was assessed using the Elevated Plus Maze (EPM) test 1 h after CGRP (0.1 nmol/10 µL) or saline (10 µL) intraganglionar administration in independent groups of male and female rats (Figure 5(a)).

Figure 4.

Effect of intraganglionar CGRP injection on photic sensitivity. Experimental protocol (a) Mechanical threshold time-course in male (b) and female (c) rats 24 h after intraganglionar CGRP (CGRP; 0.1 nmol/10 µL; i.g.) or saline (Sal; 10 µL; i.g.). All animals were exposed during 60 min to an aversive light after baseline measure of the mechanical threshold (BL). *p < 0.05, compared to Veh-Sal. Two-way ANOVA with repeated measures followed by the Bonferroni post hoc test. (d) Area under the curve (AUC) of the mechanical threshold of male and female rats. *p < 0.05, compared to the male group. One-way ANOVA followed by the Bonferroni post hoc test. Data are expressed as mean ± SEM (n = 7–8).

Figure 5.

Effect of intraganglionar CGRP injection on anxiety-like behavior. Experimental protocol (a). Number of entries displayed by male (b) and female (d) rats and time spending in the open and closed arms of the elevated plus maze (EPM) by male (c) and female (e) rats. The test was performed 1 h after intraganglionar administration of CGRP (CGRP; 0.1 nmol/10 µL; i.g.) or saline (Sal; 10 µL; i.g.). *p < 0.05, compared to Sal. Data are expressed as mean ± SEM (n = 8–9). T-test.

Statistical analysis

All data were expressed as mean ± standard error of the mean (S.E.M.) of 7–10 animals per group. Two-way analysis of variance (ANOVA) with repeated measures followed by Bonferroni post-hoc test was used to analyze the time-course of mechanical allodynia with treatment and time as the independent factors. Area under the curve (AUC) data was analyzed by one-way ANOVA followed by Bonferroni post-hoc test. Student’s t-test was performed to analyze the anxiety-like behavior. Results were considered statistically significant if p < 0.05. GraphPad Prism version 6 for Windows was used for statistical data (GraphPad Software, San Diego, CA, USA).

Results

Assessment of periorbital mechanical allodynia induced by intraganglionar CGRP in male and female rats

Intraganglionar CGRP induced a reduction in the mechanical threshold in the periorbital area compared with saline injected group from 0.5 to 4 h in male rats (time factor: F (7, 112) = 26.07, p < 0.0001; treatment factor: F (2, 16) = 31.00, p < 0.0001; interaction factor: F (14, 112) = 11.59, p < 0.0001; Figure 1(b)). Additionally, sumatriptan administered 30 min before intraganglionar CGRP reduced periorbital allodynia during 30 min (Figure 1(b)). Otherwise, female rats presented longer-lasting periorbital allodynia (from 0.5 to 6 h) compared with the corresponding control group; that is, vehicle-injected female rats (time factor: F (8, 120) =19.95, p < 0.0001; treatment factor: F (2, 15) = 30.73, p < 0.0001; interaction factor: F (16, 120) = 8.670, p < 0.0001; Figure 1(c)). However, the effect of sumatriptan pre-treatment was slightly more prolonged, reducing the allodynia from 0.5 to 1 h (Figure 1(c)). The integrated AUC calculated from 0 to 6 h post-CGRP injection revealed that female rats presented a significant additional decrease in the mechanical threshold compared to male rats (interaction factor: F (5,31) = 31.53, p < 0.0001; Figure 1(d)). However, there was no change in the effect of sumatriptan between male and female rats (interaction factor: F (5,31) = 31.53, p > 0.999, Figure 1(d)). Control animals that received intraganglionar saline injection did not present a significant variation of the mechanical threshold (Figure 1(b)–(d)). Moreover, female rats showed a reduction in the paw mechanical threshold during 6 h after injection of CGRP in the TG compared with saline-treated rats (time factor: F (7, 112) = 3.994, p = 0.0006; treatment factor: F (1, 16) =33.03, p < 0.0001; interaction factor: F (7, 112) = 3.225, p = 0.0038; Supplemental material, Figure 1).

Effect of intraganglionar minocycline on the periorbital mechanical allodynia induced by intraganglionar CGRP in male and female rats

Intraganglionar CGRP promoted periorbital mechanical allodynia in male rats during 4 h (Figure 2(b)), which was significantly reduced (up to 1 h after treatment) by previous intraganglionar injection of minocycline in the TG (time factor: F (7, 189) = 18.40, p < 0.0001; treatment factor: F (3, 27) = 17.56, p < 0.0001; interaction factor: F (21, 189) = 8.802, p < 0.0001; Figure 2(b)). In sharp contrast, female rats showed longer-lasting periorbital mechanical allodynia after CGRP injection in the TG, which was not affected by intraganglionar minocycline pre-treatment (time factor: F (7, 203) = 25.13, p < 0.0001; treatment factor: F (3, 29) = 107.9, p < 0.0001; interaction factor: F (21, 203) = 8.731, p < 0.0001; Figure 2(c)). The integrated AUC calculated from 0 to 6 h post-CGRP injection confirm the previous finding that female rats are more sensitive to CGRP than male rats (interaction factor: F (7,54) = 42.39, p = 0.0083, Figure 2(d)). Additionally, there is a significant difference in the minocycline effect, which increased the mechanical threshold of male, but not of female rats (interaction factor: F (7,54) = 42.39, p < 0.0001, Figure 2(d)). The control groups, which received two intraganglionar saline injections 30 min apart, or one intraganglionar minocycline injection followed by saline, did not show significant changes in the mechanical threshold during the testing period (Figure 2(b)–(d)).

Effect of intraganglionar propentofylline on the periorbital mechanical allodynia induced by intraganglionar CGRP in male and female rats

Intraganglionar CGRP promoted periorbital mechanical allodynia in male rats during 4 h (Figure 3(b)), which was significantly reduced (up to 1 h after treatment) by previous intraganglionar injection of propentofylline in the TG (time factor: F (7,189) = 27.49, p < 0.0001; treatment factor: F (3,27) = 18.45, p < 0.0001; interaction factor: F (21,189) = 11.66, p < 0.0001, Figure 3(b)). In sharp contrast, female rats showed longer lasting periorbital mechanical allodynia after CGRP injection in the TG (up to 6 hours), which was not affected by intraganglionar propentofylline pre-treatment (time factor: F (7,154) = 63.39, p < 0.0001; treatment factor: F (3,22) = 127.1, p < 0.0001; interaction factor: F (21,154) = 21.18, p < 0.0001; Figure 3(c)). The integrated AUC calculated from 0 to 6 h post CGRP injection confirms the previous finding that female rats are more sensitive to CGRP than male rats (interaction factor: F (7,48) =41.51, p = 0.0324, Figure 3(d)). As observed with minocycline, propentofylline also increased the mechanical threshold of male, but not of female rats (interaction factor: F (7,48) = 41.51, p < 0.0001; Figure 3(d)). The control groups, which received two intraganglionar saline injections 30 min apart, or one intraganglionar propentofylline injection followed by saline, did not show significant changes in the mechanical threshold during the testing period (Figure 3(b)–(d)).

Evaluation of periorbital mechanical allodynia induced by aversive light after intraganglionar injection of CGRP in male and female rats

Twenty-four hours after CGRP injection, male and female rats do not present significant periorbital mechanical allodynia. At this time point, exposure to a bright light promoted periorbital mechanical allodynia during 1 h in male rats (time factor: F (6, 84) =4.582, p = 0.0005; treatment factor: F (1, 14) = 4.347, p < 0.0559; interaction factor: F (6, 84) = 3.620, p = 0.0030; Figure 4(b)). Female rats presented a more prolonged reduction in the mechanical threshold when compared with male rats, from 1 to 4 h, after exposure to light (time factor: F (8, 128) = 6.806, p < 0.0001; treatment factor: F (1, 16) = 12.72, p =0.0026; interaction factor: F (8, 128) = 4.969, p < 0.0001; Figure 4(c)). The integrated AUC calculated from 0 to 4 h post-light exposure revealed a more pronounced reduction in the mechanical threshold in female than in male rats (interaction factor: F (3,26) = 9.551, p = 0.0002). Exposure to the bright light did not induce a significant alteration of the mechanical threshold in rats that received intraganglionar saline injections (Figure 4(b) and (d)).

Analysis of the anxiety-like behavior induced by intraganglionar CGRP in male and female rats

Intraganglionar CGRP induced a reduction in the number of entries into the open arm of the EPM apparatus 1 h after the injection in male (t = 2.267, p = 0.0398; Figure 5(b)), and female rats (t = 2.925, p = 0.0099; Figure 5(d)). The number of entries into the closed arms was not different when male and female rats were compared to their respective control groups. Furthermore, the time spent in the open arms was reduced in male (t = 2.403, p = 0.0307; Figure 5(c)) and female rats (t = 3.156, p = 0.0061, Figure 5(e)) compared with their respective control groups. Male rats did not show statistical difference from control groups in the time spent in the closed arms, but female rats injected with CGRP spent more time in the closed arms compared to their respective control group (t = 2.415, p = 0.0281; Figure 5(e)).

Discussion

There is mounting evidence that CGRP contributes to migraine pain through peripheral mechanisms. This idea has been reinforced by the efficacy of peripherally restricted anti-CGRP agents in migraine sufferers. The present study provided evidence that CGRP injected directly into the TG induced long-lasting periorbital mechanical allodynia in male and female rats, as well as other migraine-related responses such as light sensitivity and anxiety-like behavior. In addition, our results corroborate recent demonstrations of a sexual dichotomy in CGRP-induced nociceptive responses.

It has been recently demonstrated that intraganglionar CGRP injection in male rats evokes heat hyperalgesia parallel to an activation of glial cells and release of several pro-inflammatory cytokines, including IL-1β, IL-6 and TNFα (13). In line with these observations, herein it was shown that intraganglionar CGRP also evokes long-lasting periorbital mechanical allodynia, in addition to hindpaw allodynia, which may reflect cutaneous allodynia seen in patients with migraine as well as the development of central sensitization (22). Interestingly, female rats were more sensitive to CGRP effect, showing prolonged mechanical allodynia when compared to male rats. Sexual dichotomy has been demonstrated in response to CGRP. Dural CGRP injection has been shown to evoke nociceptive responses in female, but not in male rats (12). Female rats seem to have significantly lower levels of CGRP receptor components in the TG, compared to male counterparts, but no difference in CGRP mRNA was detected (27). On the other hand, it has been suggested that estrogen fluctuation modulated CGRP levels in the trigeminal system of females (28) and that female rats present significantly higher levels of one component of the CGRP receptor (i.e. RCP) in the upper cervical spinal cord (29).

Sexual dichotomy has also been demonstrated in the effect of anti-migraine drugs. It has been suggested that females have a stronger CGRP response than males and hence would be less susceptible to sumatriptan blockade (11). Herein, this difference in the effect of sumatriptan was not detected. This discrepancy may be related to the different species used in the present study compared to the study of Rea et al. (i.e. rats vs. mice), as well as by the fact that we injected sumatriptan 30 min before CGRP, while they co-administered CGRP and sumatriptan (11). Sumatriptan is considered an abortive migraine treatment, but there is mounting clinical evidence that it is far more likely to provide complete pain relief if administered before rather than after the establishment of cutaneous allodynia (30 –32). In addition, several pre-clinical studies using different migraine models have shown the effectiveness of sumatriptan when administered before the induction of migraine (33 –35). In fact, a previous study reported that periorbital allodynia induced by dural administration of an inflammatory soup was abolished by systemic pre-treatment with sumatriptan and significantly reduced by the post-treatment (i.e. 30 min later). However, when the treatment was performed 1.5 or 2.5 h after the stimulus, sumatriptan failed to affect the mechanical allodynia (22). These data are corroborated by in vitro observations that early, but not late, sumatriptan administration in rats prevented the development of central sensitization; that is, prevented neuronal firing increase to innocuous periorbital skin stimulation (30). Moreover, sumatriptan pre-treatment has been shown to almost completely inhibit the release of CGRP evoked by electrical stimulation of the TG (36).

In the TG, satellite glial cells that contain CGRP receptors are often organized around a CGRP-expressing neuron, indicating neuron–glia communication (5). In fact, CGRP has been shown to activate the release of inflammatory cytokines and nitric oxide from ganglionic glial cells that in turn enhance CGRP release. This mechanism has been proposed to contribute to peripheral and central sensitization of trigeminal neurons (37,38). Herein, we have shown that blockade of glial cells with minocycline and propentofylline significantly reduced periorbital mechanical allodynia in male rats, reinforcing the idea that glial cells contribute to CGRP effect. However, it is important to point out that both minocycline and propentofylline are non-selective glial inhibitors (19,20). Although we have injected these drugs directly into the TG, at a volume that has been shown not to cause solution spreading to adjacent structures (21,39) and at a dose that has been shown to cause glial cell inhibition (17,19), it is possible that they are modulating different targets. Minocycline has been shown to inhibit satellite glial cells of the TG, resulting in attenuation of CGRP-induced thermal nociception and down-regulation of several pro-nociceptive cytokines (13). Moreover, minocycline has several inhibitory effects on microglial cells, including inhibition of microglial proliferation and hypertrophic morphology and reduction in the production of proinflammatory factors (40 –42). In this regard, there is evidence for the existence of resident microglial cells in the normal rat TG (43), as demonstration of microglia activation in different orofacial pain models (44,45). Finally, minocycline is suggested to decrease the excitability of different neuronal populations by decreasing glutamatergic transmission (46 –48). On the other hand, propentofylline is a phosphodiesterase inhibitor that has a broad inhibitory action on both microglia and astrocytes, by reducing adenosine uptake and the expression of adenosine receptors (49,50). In contrast to minocycline, it does not affect the expression or hypertrophic morphology of glial cells and seems to exert less influence on release of cytokines (20). Thus, the present data, obtained with two mechanistically different glial inhibitors, does not indicate a conclusive target for these drugs, but allows the suggestion that cooperative interactions between TG glial cells and neurons may contribute to CGRP allodynic actions.

The current findings also corroborate previous studies showing sex differences in the processing of orofacial pain. There are several reports indicating that variations in hormone levels and hormone receptor expression play a crucial role in orofacial pain perception (for review see (51)). Although the majority of these studies have not used migraine models (52 –55), the influence of hormonal fluctuations in migraine frequency and severity is well established in the clinical setting (56 –60). Moreover, in a rat migraine model induced by nitroglycerin, distinct cerebral areas were activated in female compared to male and neutered female rats (61), reinforcing the idea that different hormonal levels may contribute to sexual differences in migraine. Other studies using inflammatory and neuropathic trigeminal pain models have suggested that changes in genes and proteins expression in the TG differ between male and female rats (62,63). In this regard, it is important to mention the study of Kuzawińska et al., which demonstrated that induction of peripheral inflammation in trigeminal innervated tissues caused a significant increase in CGRP expression in female compared to male mice, but the increase in expression of different cytokines (e.g. IL-1 beta, TNF alpha, IL-6) and in BDNF levels in the TG was found to be more prominent in males (62). In the TG, glial cells are the main source of cytokine release, and according to our data, glial cells blockade inhibit CGRP allodynic responses in male, but not female rats. This observation corroborates previous demonstrations of a male-dominant microglial signaling in some pain conditions (19,64,65). It has been proposed that while spinal inhibition of microglia inhibits allodynia in male mice, it has no effect in female mice, seeming to trigger a T cell-dependent response to maintain pain sensitization (19). However, it has been suggested that this sex dependence is restricted to the spinal cord, and to some microglia markers that contribute to spinal cord plasticity and central sensitization (65). Thus, it remains to be investigated which mechanisms contribute to increased sensitivity of CGRP detected in female rats, as well as the lack of response of female rats to glial inhibitors under our conditions.

Photophobia is one of the key symptoms of migraine, which is present in about 80% of migraineurs (66,67). The precise mechanism of photophobia remains to be elucidated; but it has been suggested that CGRP actions on peripheral and central sites contribute to this effect (68). Our results demonstrated that CGRP priming causes sensitization to light in male and female rats, but females present longer lasting periorbital mechanical allodynia. In line with our observation, it has been reported that female mice show a trend of greater CGRP-induced light aversion compared to males (11,68). Interestingly, exposure to bright light enhances excitability of TG neurons, which show increased expression of CGRP and nitric oxide synthase, as soon as 5 min after photic stimulation and persisting for up to 12 h. Additionally, glial cell activation was also demonstrated by a gradual increase in pERK1/2 expression from 2 to 12 h after photic stimulation (25). Thus, the fact that we have injected CGRP directly into the TG may have favored the observation of a sexual difference in this effect.

Pre-clinical and clinical reports on CGRP-inducing anxiety are scarce. Additionally, some studies that used CGRP as a model of migraine evaluate anxiety-like behavior in the open field test using high levels of illumination (69 –71). However, it has been reported that high illumination conditions cause diminished locomotor behavior (72), in addition to potential bias, as CGRP is able to induce photophobia. Therefore, it is difficult to differentiate the light aversion from the anxiety behavior using these parameters. On the other hand, both pre-clinical and clinical studies have shown the association between migraine and anxiety. Herein, the elevated plus maze test was used in low light conditions, which is a more validated method to evaluate the anxiety-like behavior (26). Some mechanisms have been proposed to explain the contribution of CGRP to anxiety related to migraine. Injection of CGRP was shown to potentiate anxiety-like behaviors and increase neural activity in the bed nucleus of the stria terminalis, which is a relay site within the hypothalamic-pituitary-adrenal axis to regulate its activity in response to acute stress caused by real or perceived threats (72 –74). Although we have injected CGRP in the TG in the current study, it has been demonstrated that it can act as a neurohormone, contributing to multiple aspects of migraine by acting in locations distant from its release (75 –77). Another possibility is that peripheral CGRP is able to activate trigeminovascular projections from the medullary dorsal horn to selective areas in the midbrain, hypothalamus and amygdala, which contribute to an altered emotional state during a migraine crisis (78). In line with this hypothesis, it has been suggested that peripherally restricted CGRP blockers are effective in patients with migraine comorbidities such as depression and anxiety (79). According to our data, male and female rats present anxiety-like behavior after intraganglionic CGRP, but no sex difference was detected. This fact may be related to technical limitations, since the test used evaluate an ethological variable and control female rats show a trend of higher anxiety levels compared to the male counterpart. Clinical studies show that anxiety is more prevalent in women and men with migraine compared with the general population, but data regarding the prevalence in one or other sex are discrepant (2,80 –82).

In conclusion, the present data indicate that intraganglionar CGRP is able to evoke migraine-like responses, including periorbital mechanical allodynia, light sensitivity and anxiety like-behavior. In general, female rats are likely to be more susceptible to CGRP effect, and differently from male, its action seems not to be dependent on glial cells (Supplemental material, Figure 2). The findings of the present study raise several questions regarding sex-related mechanistic differences that contribute to CGRP effects. Although anti-CGRP therapies have been efficacious in both sexes (80 –82), a better understanding of the sexual dimorphism in CGRP signaling may help to improve migraine therapy.

Article highlights

Intraganglionar CGRP induces mechanical allodynia, light sensitivity and anxiety-like behavior in male and female rats.

Female rats are likely to be more susceptible to CGRP effects.

Different mechanisms contribute to CGRP-induced allodynia in male and female rats.

Supplemental Material

CEP896539 Supplemental Material - Supplemental material for Contribution of intraganglionic CGRP to migraine-like responses in male and female rats

Supplemental material, CEP896539 Supplemental Material for Contribution of intraganglionic CGRP to migraine-like responses in male and female rats by Erika Ivanna Araya, Joelle de Melo Turnes, Amanda Ribeiro Barroso and Juliana Geremias Chichorro in Cephalalgia

Footnotes

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES, Finance Code 001) and National Council for Scientific and Technological Development (CNPq).

ORCID iD

Erika Ivanna Araya

References

Goadsby

Holland

Martins-Oliveira

, et al. Pathophysiology of migraine: A disorder of sensory processing. Physiol Rev 2017; 97: 553–622.

Victor

Campbell

, et al. Migraine prevalence by age and sex in the United States: A life-span study. Cephalalgia 2010; 30: 1065–1072.

Steiner

Stovner

Vos

, et al.

Migraine is first cause of disability in under 50s: Will health politicians now take notice?

J Headache Pain 2018; 19: 17–20.

Ligthart

Gerrits

MMJG

Boomsma

, et al. Anxiety and depression are associated with migraine and pain in general: An investigation of the interrelationships. J Pain 2013; 14: 363–370.

Edvinsson

Haanes

Warfvinge

, et al. CGRP as the target of new migraine therapies – successful translation from bench to clinic. Nat Rev Neurol 2018; 14: 338–350.

Edvinsson

. CGRP blockers in migraine therapy: Where do they act? Br J Pharmacol 2008; 155: 967–969.

Edvinsson

. Novel migraine therapy with calcitonin gene-regulated peptide receptor antagonists. Expert Opin Ther Targets 2007; 11: 1179–1188.

Eftekhari

Salvatore

Calamari

, et al. Differential distribution of calcitonin gene-related peptide and its receptor components in the human trigeminal ganglion. Neuroscience 2010; 169: 683–696.

Lennerz

Rühle

Ceppa

, et al. 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.

10.

Yao

Huang

Wang

, et al. Behavioral study of a rat model of migraine induced by CGRP. Neurosci Lett 2017; 651: 134–139.

11.

Rea

Wattiez

Waite

, et al. Peripherally administered calcitonin gene-related peptide induces spontaneous pain in mice: Implications for migraine. Pain 2018; 159: 2306–2317.

12.

Avona A, Burgos-vega C, Burton MD, et al. Dural calcitonin gene-related peptide produces female-specific responses in rodent migraine models. J Neurosci 2019; 39: 4323–4331.

13.

Afroz

Arakaki

Iwasa

, et al. CGRP induces differential regulation of cytokines from satellite glial cells in trigeminal ganglia and orofacial nociception. Int J Mol Sci 2019; 20: 711–711.

14.

Faul F, Erdfelder E, Lang AG, et al. G*Power 3: A flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behavior Research Methods 2007; 39: 175–191.

15.

Valdivieso

Baughan

Canavati

, et al. Effects of pregabalin on neurobehavior in an adult male rat model of PTSD. PLoS One 2018; 13: e0209494–e0209494.

16.

Gambeta

Kopruszinski

dos Reis

, et al. Facial pain and anxiety-like behavior are reduced by pregabalin in a model of facial carcinoma in rats. Neuropharmacology 2017; 125: 263–271.

17.

Ledeboer

Sloane

Milligan

, et al. Minocycline attenuates mechanical allodynia and proinflammatory cytokine expression in rat models of pain facilitation. Pain 2005; 115: 71–83.

18.

Oshinsky

Sanghvi

Maxwell

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

19.

Sorge

Mapplebeck

JCS

Rosen

, et al. Different immune cells mediate mechanical pain hypersensitivity in male and female mice. Nat Neurosci 2015; 18: 1081–1083.

20.

Fujita

Tamano

Yoneda

, et al. Ibudilast produces anti-allodynic effects at the persistent phase of peripheral or central neuropathic pain in rats: Different inhibitory mechanism on spinal microglia from minocycline and propentofylline. Eur J Pharmacol 2018; 833: 263–274.

21.

Neubert

Mannes

Keller

, et al. Peripheral targeting of the trigeminal ganglion via the infraorbital foramen as a therapeutic strategy. Brain Res Brain Res Protoc 2005; 15: 119–126.

22.

Edelmayer

Vanderah

Majuta

, et al. Medullary pain facilitating neurons mediate allodynia in headache-related pain. Ann Neurol 2009; 65: 184–193.

23.

De Felice

Ossipov

Wang

, et al. Triptan-induced latent sensitization a possible basis for medication overuse headache. Ann Neurol 2010; 67: 325–337.

24.

Kopruszinski

Xie

Eyde

, et al. Prevention of stress- or nitric oxide donor-induced medication overuse headache by a calcitonin gene-related peptide antibody in rodents. Cephalalgia 2017; 37: 560–570.

25.

Okada

Saito

Matsuura

, et al. Upregulation of calcitonin gene-related peptide, neuronal nitric oxide synthase, and phosphorylated extracellular signal-regulated kinase 1/2 in the trigeminal ganglion after bright light stimulation of the eye in rats. J Oral Sci 2019; 61: 146–155.

26.

Pellow

Chopin

File

, et al. Validation of open: closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J Neurosci Methods 1985; 14: 149–167.

27.

Stucky

Gregory

Winter

, et al. Sex differences in behavior and expression of CGRP-related genes in a rodent model of chronic migraine. Headache 2011; 51: 674–692.

28.

Labastida-Ramírez

Rubio-Beltrán

Villalón

, et al. Gender aspects of CGRP in migraine. Cephalalgia 2019; 39: 435–444.

29.

Rizk

Voulalas

, et al. Sex differences in the expression of calcitonin gene-related peptide receptor components in the spinal trigeminal nucleus. Neurobiol Pain 2019; 6: e100031–e100031.

30.

Burstein

Jakubowski

. Analgesic triptan action in an animal model of intracranial pain: A race against the development of central sensitization. Ann Neurol 2004; 55: 27–36.

31.

Landy

McGinnis

McDonald

. Clarification of developing and established clinical allodynia and pain-free outcomes. Headache 2007; 47: 247–252.

32.

Aurora

Barrodale

McDonald

, et al. Revisiting the efficacy of sumatriptan therapy during the aura phase of migraine: Research submission. Headache 2009; 49: 1001–1004.

33.

Ferrari

Levine

Green

. Mechanisms mediating nitroglycerin-induced delayed-onset hyperalgesia in the rat. Neuroscience 2016; 317: 121–129.

34.

Huang

Ren

Qiu

C-S

, et al. Characterization of a mouse model of headache. Pain 2016; 157: 1744–1760.

35.

Dallel

Descheemaeker

Luccarini

. Recurrent administration of the nitric oxide donor, isosorbide dinitrate, induces a persistent cephalic cutaneous hypersensitivity: A model for migraine progression. Cephalalgia 2018; 38: 776–785.

36.

Wang

Fang

Liang

, et al. Selective inhibition of 5-HT7 receptor reduces CGRP release in an experimental model for migraine. Headache 2010; 50: 579–587.

37.

Cady

Glenn

Smith

, et al. Calcitonin gene-related peptide promotes cellular changes in trigeminal neurons and glia implicated in peripheral and central sensitization. Mol Pain 2011; 7: 94–94.

38.

Messlinger

Lennerz

Eberhardt

, et al. CGRP and NO in the trigeminal system: Mechanisms and role in headache generation. Headache 2012; 52: 1411–1427.

39.

Araya

Nones

CFM

Ferreira

LEN

, et al. Role of peripheral and central TRPV1 receptors in facial heat hyperalgesia in streptozotocin-induced diabetic rats. Brain Res 2017; 1670: 146–155.

40.

Raghavendra

Tanga

Deleo

. Inhibition of microglial activation attenuates the development but not existing hypersensitivity in a rat model of neuropathy. J Pharmacol Exp Ther 2003; 306: 624–630.

41.

Yong

Wells

Giuliani

, et al. The promise of minocycline in neurology. Lancet Neurol 2004; 3: 744–751.

42.

Padi

SSV

Kulkarni

. Minocycline prevents the development of neuropathic pain, but not acute pain: Possible anti-inflammatory and antioxidant mechanisms. Eur J Pharmacol 2008; 601: 79–87.

43.

Glenn

Sonceau

Wynder

, et al. Histochemical evidence for microglia-like macrophages in the rat trigeminal ganglion. J Anat 1993; 183: 475–481.

44.

Mori

Goshima

Koshizuka

, et al. Iba1-expressing microglia respond to herpes simplex virus infection in the mouse trigeminal ganglion. Mol Brain Res 2003; 120: 52–56.

45.

Lynds

Lyu

, et al. Neuronal plasticity of trigeminal ganglia in mice following nerve injury. J Pain Res 2017; 10: 349–357.

46.

Zhong

Zhou

Ren

, et al. The direction of synaptic plasticity mediated by C-fibers in spinal dorsal horn is decided by Src-family kinases in microglia: The role of tumor necrosis factor-α. Brain Behav Immun 2010; 24: 874–880.

47.

Peng

, et al. Minocycline enhances inhibitory transmission to substantia gelatinosa neurons of the rat spinal dorsal horn. Neuroscience 2016; 319: 183–193.

48.

Itoh

Chiang

, et al. Central sensitization of nociceptive neurons in rat medullary dorsal horn involves purinergic P2X7 receptors. Neuroscience 2011; 192: 721–731.

49.

Rudolphi

Schubert

. Modulation of neuronal and glial cell function by adenosine and neuroprotection in vascular dementia. Behav Brain Res 1997; 83: 123–128.

50.

Nitta

Ogihara

Onishi

, et al. Oral administration of propentofylline, a stimulator of nerve growth factor (NGF) synthesis, recovers cholinergic neuronal dysfunction induced by the infusion of anti-NGF antibody into the rat septum. Behav Brain Res 1997; 83: 201–204.

51.

Bolay

Berman

NEJ

Akcali

. Sex-related differences in animal models of migraine headache. Headache 2011; 51: 891–904.

52.

Bereiter

. Sex differences in brainstem neural activation after injury to the TMJ region. Cells Tissues Organs 2001; 169: 226–237.

53.

Puri

Cui

Liverman

, et al. Ovarian steroids regulate neuropeptides in the trigeminal ganglion. Neuropeptides 2005; 39: 409–417.

54.

Puri

Svojanovsky

, et al. Effects of oestrogen on trigeminal ganglia in culture: Implications for hormonal effects on migraine. Cephalalgia 2006; 26: 33–42.

55.

Kramer

Bellinger

. The effects of cycling levels of 17β-estradiol and progesterone on the magnitude of temporomandibular joint-induced nociception. Endocrinology 2009; 150: 3680–3689.

56.

Stewart

Lipton

Chee

, et al. Menstrual cycle and headache in a population sample of migraineurs. Neurology 2000; 55: 1517–1523.

57.

MacGregor

. Oestrogen and attacks of migraine with and without aura. Lancet Neurol 2004; 3: 354–361.

58.

MacGregor

Frith

Ellis

, et al. Incidence of migraine relative to menstrual cycle phases of rising and falling estrogen. Neurology 2006; 67: 2154–2158.

59.

Slater

Crawford

Kabbouche

, et al. Effects of gender and age on paediatric headache. Cephalalgia 2009; 29: 969–973.

60.

Delaruelle

Ivanova

Khan

, et al. Male and female sex hormones in primary headaches. J Headache Pain 2018; 19: 1–12.

61.

Greco

Tassorelli

Mangione

, et al. Effect of sex and estrogens on neuronal activation in an animal model of migraine. Headache 2013; 53: 288–296.

62.

Kuzawińska

Lis

Cudna

, et al. Gender differences in the neurochemical response of trigeminal ganglion neurons to peripheral inflammation in mice. Acta Neurobiol Exp (Wars) 2014; 74: 227–232.

63.

Korczeniewska

Husain

Khan

, et al. Differential gene expression in trigeminal ganglia of male and female rats following chronic constriction of the infraorbital nerve. Eur J Pain (United Kingdom) 2018; 22: 875–888.

64.

Dubner

Berta

Qadri

, et al. Microglial signaling in chronic pain with a special focus on caspase 6, p38 MAP kinase, and sex dependence. J Dent Res 2016; 95: 1124–1131.

65.

Taves

Berta

Liu

D-L

, et al. Spinal inhibition of p38 MAP kinase reduces inflammatory and neuropathic pain in male but not female mice: Sex-dependent microglial signaling in the spinal cord. Brain Behav Immun 2016; 55: 70–81.

66.

Noseda

Burstein

. Advances in understanding the mechanisms of migraine-type photophobia. Curr Opin Neurol 2011; 24: 197–202.

67.

Lovati

Mariotti

Giani

, et al. Central sensitization in photophobic and non-photophobic migraineurs: Possible role of retino nuclear way in the central sensitization process. Neurol Sci 2013; 34: 133–135.

68.

Mason

Kaiser

Kuburas

, et al. Induction of migraine-like photophobic behavior in mice by both peripheral and central CGRP mechanisms. J Neurosci 2017; 37: 204–216.

69.

Recober

Kuburas

Zhang

, et al. Role of calcitonin gene-related peptide in light-aversive behavior: Implications for migraine. J Neurosci 2009; 29: 8798–8804.

70.

Kaiser

Kuburas

Recober

, et al. Modulation of CGRP-induced light aversion in wild-type mice by a 5-HT1B/D agonist. J Neurosci 2012; 32: 15439–15449.

71.

Walsh

Cummins

. The open-field test: A critical review. Psychol Bull 1976; 83: 482–504.

72.

Choi

Furay

Evanson

, et al. Bed nucleus of the stria terminalis subregions differentially regulate hypothalamic-pituitary-adrenal axis activity: Implications for the integration of limbic inputs. J Neurosci 2007; 27: 2025–2034.

73.

Gungor

Pare

. CGRP inhibits neurons of the bed nucleus of the stria terminalis: Implications for the regulation of fear and anxiety. J Neurosci 2014; 34: 60–65.

74.

Durham

. Diverse physiological roles of calcitonin gene-related peptide in migraine pathology: Modulation of neuronal-glial-immune cells to promote peripheral and central sensitization. Curr Pain Headache Rep 2016; 20: 48–48.

75.

Summ

Charbit

Andreou

, et al. Modulation of nociceptive transmission with calcitonin gene-related peptide receptor antagonists in the thalamus. Brain 2010; 133: 2540–2548.

76.

Noseda

Kainz

Borsook

, et al. Neurochemical pathways that converge on thalamic trigeminovascular neurons: Potential substrate for modulation of migraine by sleep, food intake, stress and anxiety. PLoS One 2014; 9: e0103929–e0103929.

77.

Edvinsson

Goadsby

. CGRP and its receptors provide new insights into migraine pathophysiology. Nat Rev Neurol 2010; 6: 573–582.

78.

Burstein

Jakubowski

. Neural substrate of depression during migraine. Neurol Sci 2009; 30: 27–31.

79.

Raffaelli

Neeb

Reuter

. Monoclonal antibodies for the prevention of migraine. Expert opinion on Biological Therapy 2019; 19: 1307–1317.

80.

Rosen

Pearlman

Ruff

, et al. 100% response rate to galcanezumab in patients with episodic migraine: A post hoc analysis of the results from Phase 3, randomized, double-blind, placebo-controlled EVOLVE-1 and EVOLVE-2 studies. Headache 2018; 58: 1347–1357.

81.

Ashina

Goadsby

Reuter

, et al. Long-term safety and tolerability of erenumab: Three-plus year results from a five-year open-label extension study in episodic migraine. Cephalalgia 2019; 39: 1455–1464.

82.

Sun

Dodick

Silberstein

, et al. Safety and efficacy of AMG 334 for prevention of episodic migraine: A randomised, double-blind, placebo-controlled, phase 2 trial. Lancet Neurol 2016; 15: 382–390.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.14 MB