Protease activated receptor 2 (PAR2) activation causes migraine-like pain behaviors in mice

Abstract

Background

Pain is the most debilitating symptom of migraine. The cause of migraine pain likely requires activation of meningeal nociceptors. Mast cell degranulation, with subsequent meningeal nociceptor activation, has been implicated in migraine pathophysiology. Degranulating mast cells release serine proteases that can cleave and activate protease activated receptors. The purpose of these studies was to investigate whether protease activated receptor 2 is a potential generator of nociceptive input from the meninges by using selective pharmacological agents and knockout mice.

Methods

Ratiometric Ca⁺⁺ imaging was performed on primary trigeminal and dural cell cultures after application of 2at-LIGRL-NH₂, a specific protease activated receptor 2 agonist. Cutaneous hypersensitivity and facial grimace was measured in wild-type and protease activated receptor 2^−/− mice after dural application of 2at-LIGRL-NH₂ or compound 48-80, a mast cell degranulator. Behavioral experiments were also conducted in mice after dural application of 2at-LIGRL-NH₂ (2AT) in the presence of either C391, a selective protease activated receptor 2 antagonist, or sumatriptan.

Results

2at-LIGRL-NH₂ evoked Ca²⁺ signaling in mouse trigeminal neurons, dural fibroblasts and in meningeal afferents. Dural application of 2at-LIGRL-NH₂ or 48-80 caused dose-dependent grimace behavior and mechanical allodynia that were attenuated by either local or systemic application of C391 as well as in protease activated receptor 2^−/− mice. Nociceptive behavior after dural injection of 2at-LIGRL-NH₂ was also attenuated by sumatriptan.

Conclusions

Functional protease activated receptor 2 receptors are expressed on both dural afferents and fibroblasts and activation of dural protease activated receptor 2 produces migraine-like behavioral responses. Protease activated receptor 2 may link resident immune cells to meningeal nociceptor activation, driving migraine-like pain and implicating protease activated receptor 2 as a therapeutic target for migraine in humans.

Introduction

Migraine is a common headache disorder that affects millions of people worldwide (1). The etiology of migraine disorders is not well understood. The few treatments available have poor efficacy with undesired side effects (2). While the etiology of migraines is not well understood, there is strong evidence linking afferents from the trigeminal nerve that innervate the dura mater to the generation of pain and driving a key feature of the sensory component of a migraine attack (3–5). How these dural afferents are stimulated during a migraine attack is not known, but discoveries along these lines could lead to new therapeutics. The dura contains mast cells, which are immune cells that can release pro-inflammatory factors when stimulated and have been implicated in migraine etiology (6–9). One class of proteins released during mast cell degranulation is serine proteases, such as trypsin, tryptase, and elastase. These proteases can activate protease activated receptors (PARs), such as PAR2.

PAR2 is a G-protein coupled receptor (GPCR) from the PAR [1–4] family of receptors. PAR2 is activated when its extracellular N-terminus is cleaved at specific sites containing the amino acid serine, and the exposed tethered peptide then acts as a ligand for the receptor. Due to the permanent nature of the protease cleavage of the receptor, PARs are typically recycled into the cell via endocytosis and replaced with a functional receptor (10–11). PAR2 has previously been implicated in a variety of other pain disorders, including visceral pain disorders, irritable bowel syndrome, Crohn’s disease, and bone cancer pain (12–14). PAR2 has previously been investigated for playing a role in facial and migraine pain (15–16); however, these studies used a peptide agonist of PAR2, SLIGRL-NH₂, which is also an agonist of mas-related G protein coupled receptors (Mrgprs) (17). Because Mrgprs are sensory neuron specific GPCRs that can stimulate pain and/or itch behaviors, this clouds the interpretation of these previous studies. We have developed highly selective PAR2 agonists and antagonists that are devoid of activity at Mrgprs or other receptors (18–19).

The goal of the experiments described here was to definitively determine if PAR2 is linked to migraine-like pain in a mouse model. We find that functional PAR2 is expressed by cells in the meninges and in neurons that innervate the meninges. PAR2 activation and mast cell degranulation in the meninges causes migraine-like pain behaviors that are completely absent in PAR2^−/− mice and attenuated by a PAR2 antagonist. We conclude that PAR2 is linked to migraine-like pain and is a potential therapeutic target for disrupting communication between resident meningeal immune cells and nociceptors that innervate this tissue.

Materials and methods

Animals

Male, 20–30 gram mice were used in this study, ICR (Taconic, Envigo), C57BL/6J (Jackson Laboratories), and PAR2^−/− on a C57Bl/6J background (Jackson Laboratories). The animals were housed in a climate-controlled room with a 12-hour light/dark cycle, and given food and water ad libitum. Animals were separated into experimental groups using a block randomization protocol with block size dependent on the number of experimental groups; for example, four blocks for experiments with vehicle and three doses of 2AT. A statistical power analysis was performed for sample size estimation using GPower 3.1 for a repeated measures group comparison. The effect size of this study was considered f = 0.275 based on data from previous similar studies. With an alpha of 0.05 and power at 0.80, the projected sample size needed with this effect size was approximately eight per group. All experiments and procedures were performed in accordance with the guidelines recommended by the National Institute of Health, the International Association for the study of Pain, the National Centre for the Replacement, Refinement, and Reduction of Animals in Research ARRIVE guidelines, and were approved by the Institutional Animal Care and Use Committee at University of Texas at Dallas.

Supra-dural injections

While under brief 4% isoflurane anesthesia, 2AT, C391, and 48/80 were injected onto the dura using a procedure first developed by Burgos-Vega et al. (20). Hair was removed near the injection site. Mouse dural injectors were created by modifying a commercially-available cannula (Invivo1, part #C313I/SPC, Internal Cannula, Standard, 28 gauge, I.D. 0.18 mm, O.D. 0.35 mm). The projection depth of the internal cannula was determined by adjusting the location of the outer plastic pedestal manually down the length of the injector in order to shorten the projection. A digital caliper was used to measure the injector projection length (0.5 mm to 0.65 mm), which is adjusted based on the weight of the mice. The projection length allows soft tissue to be displaced bypassing the unfused suture plates, while limiting the length to maintain dural integrity. Modified injectors were attached to a 10 µl glass syringe cemented needle (Hamilton Company, 700 series) via Tygon tubing (Cole-Palmer, Item # EW-96460-16). Supradural injections are given ∼9.0 mm posterior to the bregma on the junction of the sagittal and lambdoid sutures. Using the injector, the cranial bone sutures at bregma and lambda are identified via topographical features of the cranial plates. The junction of the sagittal and lambdoidal sutures is located by inserting the injector through the skin and gently probing the lambdoid suture line where it intersects the sagittal suture line using the tip of injector and verified by re- positioning the injector along the skull. The injector is gently placed between the junction of the sagittal and lambdoidal sutures using light downward force to allow for injection. Then 5 µl of solution is injected onto the dura. The animals were removed from the anesthesia and allowed to recover before being returned to their home cages.

Chemicals

2AT and C391 were made as previously described (21). Compound 48/80 was purchased from Sigma-Aldrich. Sumatriptan was purchased from Tocris. Wheat germ agglutinin conjugated to Alexa-Fluor 555 was purchased from Life Technologies and dissolved in synthetic interstitial fluid (SIF) (NaCl 107.8 mM, KCl 3.5 mM, CaCl₂.2H₂O 1.53 mM, MgSO₄.7H₂O 0.7 mM, NaH₂PO₄.2H₂O 1.67 mM, NaHCO₃ 26.2 mM, C₆H₁₁NaO₇ 9.65 mM, Sucrose 7.6 mM, Glucose 5.55 mM, pH 7.4, 310 mOsm). Vehicle controls were SIF with 1% DMSO for supra-dural injections or saline (0.9%) for intravenous injections.

Behavioral assays

Male and female adult ICR mice were habituated to tapered single poly-coated paper cylindrical facial testing chambers (Choice 4 oz. paper cups; 6.5 cm top diameter, 4.5 cm bottom diameter, 72.5 cm length) while contained in clear acrylic compartments (5 cm length × 7.6 cm width × 23 cm height) for two hours each day for three days, prior to baseline. Calibrated von Frey filaments were applied to the midline of the forehead, at the level of the eyes, and a response was indicated by a sharp withdrawal of the head. For hindpaw withdrawal threshold testing, mice were acclimated to suspended Plexiglas chambers (9 × 5 × 5 cm high) with a wire mesh bottom (1 cm²). Withdrawal thresholds to probing the face and hind-paws were determined at 1, 3, 5, 24, and 48 hours after administration. In experiments where both cranial and paw withdrawal thresholds were measured, mice were tested for hind paw mechanical sensitivity then immediately transferred to the enclosures to have facial mechanical sensitivity measured. Paw withdrawal (PW) thresholds were determined by applying von Frey filaments to the plantar aspect of the hind-paws, and a response was indicated by a withdrawal of the paw. The withdrawal thresholds were determined by the Dixon up-down method (22) by blinded observers. Maximum filament strengths were 1 and 2 g for the face and hind-paws, respectively.

The protocol originally developed by Mogil and colleagues for testing facial grimacing in mice was utilized for this study (23). Mice were placed individually on a tabletop in cubicles (9 × 5 × 5 cm high) with two walls of transparent acrylic glass and two side walls of removable stainless steel. Two high-resolution (1920 × 1080) digital video cameras (High-definition Handycam Camcorder, model HDR-CX100, Sony, San Jose, CA) were placed immediately outside both acrylic glass walls to maximize the opportunity for clear head shots. The animals were then recorded for 20 minutes and the photographs that included views of the mouse face were extracted from each recording and scored by blinded scorers. The scorers assessed facial expressions such as orbital tightening (closing or narrowing of the eyelid and orbital area), ear position (outward or backwards rotation of the ears), nose bulging (bulging of the bridge of the nose and vertical wrinkles on the side of the nose), cheek bulging (bulging of the cheeks), and whisker change (whiskers are pulled forward or back against the cheek); scoring either a 0, 1, or 2 depending on the magnitude of the expression (Supplementary Figure 1). The scores were averaged at each time-point by group.

Cell culture

Mice were anesthetized with isoflurane and sacrificed by decapitation and the TG or dura mater were removed and prepared for culture as previously described (24). For cultures with back-labelled TG neurons, TGs were taken 7 days following supra-dural application of the retrograde tracer, wheat germ agglutinin conjugated Alexa-Fluor 555 nm. After removal, tissue was placed in ice-cold Hanks balanced-salt solution (divalent free). Trigeminal tissue was dissociated enzymatically with collagenase A (1 mg/ml, 25 min, Roche, Indianapolis, IN) and collagenase D (1 mg/ml, Roche, Indianapolis, IN) with papain (30 units/ml) for 20 min at 37℃. Dural tissue was dissociated enzymatically with collagenase A (1 mg/ml, 30 min, Roche, Indianapolis, IN) and collagenase D (1 mg/ml, Roche, Indianapolis, IN) with papain (30 units/ml) for 25 min at 37℃. The tissues were then triturated through fire-polished Pasteur pipettes, and trigeminal cells were plated on poly-D-lysine (Becton Dickinson) and dural cells plated on untreated plates. After several hours at room temperature to allow adhesion, cells were placed in a room-temperature, humidified chamber in Liebovitz L-15 medium supplemented with 10% FBS, 10 mM glucose, 10 mM HEPES and 50 U/ml penicillin/streptomycin. Trigeminal cells were tested within 24 hours post plating and dural cell cultures were tested 2–4 days post plating.

Ca²⁺ imaging

Ca²⁺ was measured using digital imaging microscopy. Twenty four hours after plating, TG or meningeal cultures were washed with HBSS and loaded for 45 min in 5 μM Fura2-AM in HBSS. Fura2 fluorescence was observed on an Olympus IX70 microscope (Waltham, MA, USA) with a 40× oil immersion objective after alternating excitation between 340 and 380 nm by a 75 W Xenon lamp linked to a Delta Ram V illuminator (PTI, London, Ontario, Canada) and a gel optic line. Images were captured with a high-speed camera using Olympus software. Back-labeled TG neurons were detected by excitation at 560 nm prior to imaging. Ca²⁺ signaling response for each individual cell in the field of view was calculated from captured images by the ratio of 340 nm/380 nm. A cell was considered to respond to a stimulus when there was a 10% increase in the 340 nm/380 nm ratio. A minimum of one ratio per 2 seconds was calculated for all experiments.

Statistical analysis

All data are presented as means ± SEM unless otherwise noted. Statistical evaluation was performed using repeated measures two-way analysis of variance (ANOVA) followed by post-hoc Newman-Keuls test with an a priori significance level of α < 0.05. Statistical analysis was done using Graph Pad Prism V7 for PC or Apple.

Results

Selective PAR2 activation stimulates Ca²⁺ signaling in TG neurons that innervate the meninges

We first sought to assess whether the specific PAR2 agonist, 2AT, activates Ca²⁺ signaling in TG neurons. TG cell cultures were perfused for 30 seconds with 2AT (1 µM) followed 5 min later by ATP (100 µM) and then, 5 min later, KCl (50 µM) with Ca²⁺ signaling measured for 180 seconds after each perfusion. Positive responses were characterized by a minimum of a 10% increase in the 340 nm/380 nm ratio and only cells that responded to 50 mM KCl were classified as neurons. We first examined TG neurons that innervate the meninges using a retrograde tracer that was injected prior to the removal of TG neurons from mice (Figure 1(a)) and recorded the 340 nm/380 nm ratio from these cultured neurons (Figure 1(b)). We then looked at the entire TG population of neurons and found that 18.1% (10 out of 55 neurons) of all TG neurons responded to 2AT and 38.2% (21 out of 55 neurons) responded to ATP (Figure 1(c)). The magnitude of response to 2AT and ATP was similar in TG neurons (Figure 1(d)). These neurons were perfused with 2AT, ATP and KCl solutions. Of the back-labeled neurons, 43.6% (8 out of 18 neurons) responded to 2AT and 65.3% (12 out of 18 neurons) responded to ATP (Figure 1(e)), indicating that a large population of meningeal-projecting neurons likely express PAR2. Dural afferents responding to 2AT showed about a 50% increase in the 340/380 ratio, suggesting a vigorous cellular signaling response upon PAR2 activation in these cells (Figure 1(f)).

Figure 1.

2AT application to cultured trigeminal neurons results in increased intracellular calcium. (a) Representative image showing a trigeminal cell culture stained with the calcium indicator dye, Fura-2AM and the retrograde tracer, WGA-conjugated AlexaFlur 555. The top image shows cells fluorescing at 380 nm and the bottom image shows the same cells fluorescing at 555 nm. The cells stained with the WGA-conjugated AlexaFlur 555 are trigeminal neurons with afferents that innervated the dura. (b) Graph showing the traces from two neurons outlined (red and yellow) in Figure 1(a). (c) Graph showing the percent of primary trigeminal neurons that respond to 2AT (1 µM), ATP (100 µM), and KCl (50 mM). (d) Graph showing the average difference in ratio from trigeminal neurons in response to 2AT (1 µM), ATP (100 µM), and KCl (50 mM). (e) Graph showing the percent of backlabelled neurons that enervate the dura that respond to 2AT (1 µM), ATP (100 µM), and KCl (50 mM). (d) Graph showing the average difference in ratio from backlabelled neurons in response to 2AT (1 µM), ATP (100 µM), and KCl (50 mM).

2AT also activates Ca²⁺ signaling in non-neuronal cells from TG and dura

In the course of performing experiments to look at neuronal responses, we found non-neuronal cells that responded to 2AT. To examine this in more detail, we analyzed responses in cells that did not respond to perfusion with 50 mM KCl. Of these cells in TG cultures, 30.1% (43 of 142 cells) of cells responded to 1 µM 2AT, while 43.6% (63 of 142 cells) of cells responded to ATP (Figure 2(a)). The magnitude of Ca²⁺ response in these cells was less than that seen in back labelled dural afferents (Figure 2(b)). In TG cultures, all of these cells had a fibroblast-like morphology. To examine whether similar PAR2 expressing cells are found in the dura, we cultured dural fibroblasts and perfused them with 2AT (1 µM) and ATP (100 µM). In all, 22.9% (35 of 153 cells) of dural fibroblasts responded to 2AT, while 75.4% (116 of 153 cells) of cells responded to ATP (Figure 2(c)). The magnitude of the Ca2+ response in these cells was similar to the cells with fibroblast-like morphology tested from TG cultures (Figure 2(d)).

Figure 2.

2AT application to cultured trigeminal and dural cells results in increased intracellular calcium. (a) Graph showing the percent of non-neuronal cells from primary trigeminal cell culture that respond to 2AT (1 µM), ATP (100 µM). (b) Graph showing the average difference in ratio from trigeminal cells in response to 2AT (1 µM) and ATP (100 µM). (c) Graph showing the percent of cells from primary dural cell culture that respond to 2AT (1 µM), ATP (100 µM). (d) Graph showing the average difference in ratio from dural cells in response to 2AT (1 µM) and ATP (100 µM). (e) Graph showing the percent response of primary trigeminal cell to 2AT and C391. (f) Graph showing the percent response of primary dural cells to 2AT and C391. Application of C391 with 2AT significantly reduces the calcium signaling of cells in either primary trigeminal or primary dural cell cultures (p < 0.05).

We also assessed whether the PAR2 antagonist C391 inhibited 2AT responses in TG neurons or in dural fibroblasts. Pretreatment of C391 (10 µM) 30 seconds prior to and then with co-perfusion of 2AT (1 µM) was able to significantly attenuate Ca²⁺ signaling responses to both TG neurons (Figure 2(e)) and dural fibroblasts (Figure 2(f)).

Supra-dural application of 2AT produces grimace behaviors and cutaneous mechanical hypersensitivity

Having established that PAR2 activates TG neurons that innervate the dura and resident dural cells, we tested whether PAR2 activation in the dura produces migraine-like behavior in mice. Supra-dural injection of 2AT (10 pmoles) produced facial grimacing (Figure 3(a)) and cranial mechanical hypersensitivity (Figure 3(b)) when compared to vehicle injected animals. A lower dose of 2AT (3 pmoles) did not have an effect. A higher dose of 2AT (30 pmoles) produced transient grimacing (Figure 3(a)) and cranial mechanical hypersensitivity (Figure 3(b)), but the magnitude of this effect was less than that observed with 10 pmoles. This pattern is consistent with the steep concentration response curve that is observed with PAR2 agonists in other systems and suggests rapid desensitization of the receptor response at higher ligand doses, an effect that is dependent on the β-Arrestin pathway engaged by PAR2 signaling (25–26). When the PAR2 antagonist C391 was co-injected supra-durally with 2AT, affective pain behaviors and hindpaw mechanical hypersensitivity were attenuated when compared to animals only given 2AT (Figure 3(c) and 3(d)). C391 was applied to the dura at 10 and 100 pmole concentrations. The 10 pmole concentration of C391 was able to significantly attenuate 2AT-mediated increases in affective pain behaviors and mechanical hypersensitivity with an effect at 1 and 5 hours after injection for mouse grimace scores and with an effect lasting up to 3 hours after injection for mechanical hypersensitivity. The 100 pmole concentration of C391 was able to significantly attenuate 2AT-mediated increases in affective pain behaviors and mechanical hypersensitivity over the entire time course of effect for grimacing and for hindpaw mechanical hypersensitivity.

Figure 3.

Supra-dural application of 2AT causes mechanical and affective pain behaviors. Grimace behaviors and hindpaw allodynia or facial mechanical allodynia were measured up to 48 hours after supra-dural injection. (a) Mouse grimace scores from mice injected with 10 pmols 2AT (n = 8) were significantly increased when compared to vehicle treated mice (n = 8) at 3 and 5 hours after injection (*, p < 0.05). Mice given 30 pmols 2AT (n = 8) also had significantly increased mouse grimaces scores, but only 3 hours after injection (#, p < 0.05) when compared to vehicle treated mice. (b) Cranial withdrawal thresholds from mice injected with 10 pmols 2AT (n = 8) were significantly decreased when compared to vehicle treated mice (n = 8) at 3 and 5 hours after injection (*, p < 0.05). Mice given 30 pmols 2AT (n = 8) also had significantly decreased cranial withdrawal thresholds only 3 hours after injection (#, p < 0.05) when compared to vehicle treated mice. (c) Mice given 100 pmols C391 showed significantly decreased mouse grimace scores up to 5 hours after injection when compared to mice injected with 2AT and vehicle (*, p < 0.05). Mice given 10 pmols C391 showed significantly decreased mouse grimace scores at 1 and 5 hours after injection when compared to mice given 2AT and vehicle (#, p < 0.05). (d) When given C391 (10 or 100 pmols, n = 8) in conjunction with 2AT onto the dura, mice showed significantly reduced hindpaw allodynia when compared to mice given 2AT with vehicle (10 pmols = * and 100 pmols = #, p < 0.05).

Supra-dural application of mast cell degranulator 48/80 produces grimace behaviors and cutaneous allodynia

Mast cell degranulation is an endogenous source of serine proteases which activate PAR2. We tested whether dural mast cell degranulation is able to produce affective pain behaviors and mechanical allodynia and if application of C391, the PAR2 antagonist, is able to attenuate pain behaviors resulting from mast cell degranulation. Application of compound 48/80 (6.5 nmole) onto the dura resulted in an increase in mouse grimaces scores and mechanical allodynia for up to 5 hours after injection when compared to vehicle control groups (Figure 4(a) and 4(b)). When C391 (100 pmole) was given in conjuction with 48/80 onto the dura, animals showed attenuated measures of affective and mechanical pain for up to 3 hours when compared to the mice that received only 48/80 (Figure 4(c) and 4(d)). C391 (8 mg/kg) was also administered systemically via intravenous (IV) injection through the tail vein immediately after supra-dural injection of 48/80. The dose was chosen based on previous findings with other PAR2 antagonists given systemically in mouse inflammatory pain models (27). Animals that received IV C391 had significantly reduced measures of affective and mechanical pain behaviors 3 hours after injections when compared to animals given only IV saline after a supra-dural injection of 48/80 (Figure 4(e) and 4(f)). While the effect on mechanical hypersensitivity was transient, IV injection of the PAR2 antagonist shortened the duration of the grimace response.

Figure 4.

Supra-dural application of 48/80 resulted in mechanical allodynia and affective measures of pain. Hindpaw allodynia and grimace behaviors were measured up to 48 hours after supra-dural injection of 48/80 or C391 and intravenous injection of C391. (a) Mouse grimace scores from mice injected with 48/80 (n = 8) were significantly increased when compared to vehicle treated mice (n = 8) up to 5 hours after injection (*, p < 0.05). (b) Paw withdrawal thresholds from mice injected with 48/80 (n = 8) were significantly decreased when compared to vehicle treated mice (n = 8) up to 5 hours after injection (*, p < 0.05). (c) Mice given supra-dural C391 (100 pmols, n = 8) showed significantly decreased mouse grimace scores up to 3 hours after injection when compared to mice injected with 48/80 and vehicle (* = p < 0.05, **** = p < 0.001). (d) When given C391 (100 pmols, n = 8) in conjunction with 48/80 onto the dura, mice showed significantly reduced hindpaw allodynia when compared to mice given 48/80 with vehicle (** = p < 0.05, **** = p < 0.001). (e) Mice given iv C391 showed significantly decreased mouse grimace scores 3 hours after injection when compared to mice injected with 48/80 and vehicle (* = p < 0.05). (f) When given C391 (iv 8 mg/kg, n = 8) immediately after 48/80 was injected onto the dura, mice showed significantly reduced hindpaw allodynia 3 hours after injection when compared to mice given 48/80 with iv vehicle (*, p < 0.05). Note. * are for Figures 4e and 4f specifically.

Supra-dural application of 48/80 to PAR2^−/− mice demonstrates a key role of PAR2 in mast cell-dependent migraine pain behaviors

PAR2^−/− mice were used to determine the extent to which PAR2 signaling is responsible for pain behaviors observed following supra-dural administration of 48/80. Application of 48/80 to the dura of PAR2^−/− mice failed to produce grimace behaviors or changes in mechanical sensitivity, whereas robust effects of 48/80 were observed in wildtype mice (Figure 5(a), 5(b) and 5(c)). To address whether PAR2^−/− mice fail to respond specifically to 48/80 and not to other supra-dural stimuli, we applied IL-6 (0.1 ng) onto the dura. When compared to animals that only received a vehicle injection, PAR2^−/− mice that received IL-6 displayed significantly more pain behaviors (Figure 5(d) and 5(e)). Therefore, PAR2^−/− mice do not respond to dural mast cell degranulation with a migraine-like pain response, although these mice a capable of responding to other types of dural stimuli.

Figure 5.

PAR2^−/− mice are resistant to the nociceptive effects of supra-dural application of 48/80. Grimace behaviors and paw withdrawal thresholds were measured up to 48 hours after supra-dural injection of 48/80. (a) Mouse grimace scores from PAR2^−/− mice injected with 48/80 (n = 8) were significantly reduced when compared to WT mice (n = 8) for up to 5 hours after injection (*, p < 0.05). (b) Cranial withdrawal thresholds from WT mice injected with 48/80 (n = 8) were significantly different compared to PAR2^−/− mice (n = 8) also injected with 48/80 for up to 24 hours after injection (*, p < 0.05). (c) Paw withdrawal thresholds from WT mice injected with 48/80 (n = 8) were significantly increased when compared to PAR2^−/− mice (n = 8) also injected with 48/80 for up to 24 hours after injection (*, p < 0.05). (d) Mouse grimace scores from PAR2^−/− mice injected with IL-6 (0.1 ng, n = 8) were significantly increased when compared to mice given only vehicle (n = 8) for up to 5 hours after injection (*, p < 0.05). (e) Paw withdrawal thresholds from PAR2^−/− mice injected with IL-6 (n = 8) were significantly decreased when compared to mice given only vehicle (n = 8) for up to 3 hours after injection (*, p < 0.05).

Sumatriptan attenuates 2AT-induced migraine-like pain behaviors

Finally, we assessed whether the behavioral response to 2AT in mice would be attenuated by the standard of care acute migraine therapeutic sumatriptan. Sumatriptan was injected intraperitoneally (IP, 0.6 mg/kg) immediately after supra-dural application of 2AT. When compared to animals given a vehicle injection, animals given sumatriptan displayed significantly less grimacing at 1 and 5 hrs after injection and mechanical hypersensitivity in the hindpaw was attenuated at 1 and 3 hrs after injection (Figure 6(a) and 6(b)).

Figure 6.

Sumatriptan attenuates affective and mechanical pain behaviors after supra-dural application of 2AT. (a) After supra-dural application of 2AT, mouse grimace scores from mice injected with sumatriptan (0.6 mg/kg, n = 8) were significantly reduced when compared to mice given a vehicle injection (n = 8) for up to 5 hours after injection (*, p < 0.05). (b) After supra-dural application of 2AT, paw withdrawal thresholds from mice injected with vehicle (n = 8) were significantly reduced when compared to mice given an injection of sumatriptan (0.6 mg/kg, n = 8) for up to 3 hours after injection (*, p < 0.05).

Discussion

We have shown that a highly specific PAR2 agonist, 2AT, evokes Ca2+ signaling in TG neurons, dural fibroblasts and in back-labeled TG neurons that innervate the cranial meninges, suggesting a rich population of cells in these migraine-relevant tissues are activated downstream of PAR2. Furthermore, when applied to the dura of mice, 2AT or 48/80 caused grimace behaviors and hindpaw allodynia. These nociceptive behaviors were attenuated by the direct or systemic application of C391, a specific PAR2 antagonist, demonstrating that C391 can be effective when administered systemically, which increases its value as a potential therapeutic agent for treating PAR2 mediated migraine pain. When the mast cell degranulator 48/80 was applied to the dura of PAR2^−/− mice, nociceptive behaviors were profoundly reduced compared to wildtype controls. Moreover, sumatriptan, a standard of care migraine medication, was found to attenuate migraine-like pain behaviors produced by PAR2 activation. Collectively, our findings strongly support a role for PAR2 in the generation of migraine-like pain and link this receptor to mast cell degranulation in the context of migraine.

Migraine is a disorder characterized by a large variety of symptoms, the most common being head pain (28). In the current study, we measured spontaneous pain using an affective measure of nociception (mouse grimace) and we also measured cranial or hindpaw allodynia following supra-dural injection of test compounds. These measures of migraine pain are consistent with symptoms reported by human migraine patients, such as cephalic and extracephalic mechanical allodynia, and are blocked by the anti-migraine drug sumatriptan (29). We also measured cranial mechanical hypersensitivity in our early experiments involving 2AT; however, we discontinued using this measurement in preference to the paw mechanical sensitivity in this study due to the strong correlation between the two measures. Importantly, we found that hindpaw measures of mechanical hypersensitivity were consistent with our cranial measurements, with 2AT and hindpaw mechanical hypersensitivity largely paralleled by grimace scale responses to supra-dural injection. We propose that grimace responses are likely the most relevant behavioral measure to assess in this model given that this is a reflection of ongoing pain, which is the primary complaint of migraine patients. Using the grimace scale in conjunction with traditional pain behavioral assays in migraine pain, preclinical studies may enhance translation of preclinical studies to the clinic.

While previous work has studied PAR2 signaling in the context of head pain (15–16), these studies did not use selective PAR2 pharmacological agents, transgenic mouse models, or measure animal nociception behaviors consistent with head pain. These issues make confirming the definitive role for PAR2 in head pain difficult. A major goal of our work was to use specific pharmacological and genetic tools to draw a clear link between PAR2 and migraine-like pain behaviors. To this end, we used 2AT, a highly selective and potent PAR2 agonist and C391, a selective PAR2 antagonist, as well as transgenic mice lacking PAR2. The supra-dural injection approach described here, and in our companion paper, opens up the investigation of migraine-like pain in transgenic mouse models. Also, the supra-dural injection approach used here in mice is advantageous for studying the effects of mast cell degranulation in our preclinical model since compound 48/80 is applied in the absence of mast cell degranulation caused by cannula implantation or craniotomies, which are typically used in other preclinical migraine models. We believe that this will be an important step forward for the field as it allows for the harnessing of mouse genetics in behavioral experimental work.

Our work demonstrates a clear link between mast cell degranulation and the generation of migraine-like pain behaviors via activation of PAR2. Mast cell degranulation has been found to be induced by stress, by eating certain foods, by administration of nitric oxide donors, by oxidative stress, and by strong sensory stimuli, all of which are also triggers for migraine attacks (30–34). Mast cell degranulation has also been shown to occur in response to cortical spreading depression, which is suspected to mediate the aura component in certain types of migraine (35). Once mast cells degranulate, they release many pro-nociceptive agents and mast cell degranulation has been associated with many different types of pathological pain (36). In the context of our experiments, it is highly likely that mast cell degranulation releases serine proteases in the dura, because the effect was completely dependent on PAR2 expression. These proteases may be activating PAR2 expressed in dural afferents from trigeminal neurons, as we show that many of these cells express PAR2. Activation of PAR2 in sensory neurons can sensitize the activity of other sensory receptors such as TRPV1, TRPV4, and TRPA1, leading to profound changes in the responsivity of these neurons (37–39). However, besides activating Ca²⁺ signaling in trigeminal neurons, we also observed activation of Ca²⁻ signaling in non-neuronal cells from primary TG and dural cell cultures when stimulated with 2AT. These cells were likely fibroblasts. Previous studies from our lab have shown that dural fibroblasts may play a potential role in headache pathophysiology (40–41). It is possible that dural fibroblasts and dural afferents from trigeminal neurons may act in concert after PAR2 activation to generate migraine pain, with the trigeminal neurons responsible for transmitting pain information, while the dural fibroblasts release pro-nociceptive factors which can sensitize surrounding dural afferents. Future studies using genetic manipulations that allow for cell-type specific ablation of PAR2 expression can be used to elucidate cell types that are required for PAR2-mediated migraine-like pain.

These studies used a novel mouse model of migraine that allows the measurement of pain behaviors in awake and behaving animals to demonstrate a role for PAR2 in migraine-like pain using selective pharmacological tools. We also used PAR2^−/− mice to demonstrate an unequivocal link between this receptor and the generation of migraine-like pain behavior. We conclude that PAR2 is an important contributor to migraine etiology and that further study is warranted to develop this target for migraine therapeutics. Given the association of dural mast cell degranulation with migraine, our results support the hypothesis that PAR2 is a causative factor in the generation of migraine pain, and that this receptor should be considered as a potential therapeutic target of migraine.

Key findings

PAR2 agonist 2AT can activate calcium signaling in cultured primary trigeminal neurons and dural non-neuronal cells.

PAR2 agonist 2AT can elicit migraine-like nociceptive behaviors when applied to the dura of mice.

PAR2 antagonist C391 can reduce migraine-like nociceptive behaviors in mice after dural application of 48/80, a mast cell degranulator.

Supplemental Material

Supplementary Figure -Supplemental material for Protease activated receptor 2 (PAR2) activation causes migraine-like pain behaviors in mice

Supplemental material, Supplementary Figure for Protease activated receptor 2 (PAR2) activation causes migraine-like pain behaviors in mice by Shayne N Hassler, Fatima B Ahmad, Carolina C Burgos-Vega, Scott Boitano, Josef Vagner, Theodore J Price and Gregory Dussor in Cephalalgia