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. Author manuscript; available in PMC: 2022 Oct 1.
Published in final edited form as: Pain. 2021 Oct 1;162(10):2499–2511. doi: 10.1097/j.pain.0000000000002353

Protein kinase C δ as a neuronal mechanism for headache in a chronic intermittent nitroglycerin model of migraine in mice

Ying He 1, Zuoxiao Shi 1, Yavnika Kashyap 1, Robert O Messing 2, Zaijie Jim Wang 1,3,4
PMCID: PMC8448952  NIHMSID: NIHMS1709405  PMID: 34108435

Introduction

As the third most prevalent disease in the world, migraine is a primary neurological disorder characterized by recurrent episodes of intense and debilitating headaches. Despite its high prevalence affecting over 1 billion people worldwide suffer from migraine at all ages, with predominance in females [33], the pathophysiology underlying migraine is not completely understood. Clinically, migraine is manifested as severe throbbing pain or an intense pulsing sensation, usually on one side of the head. It's often accompanied by nausea, vomiting, and sensitivity to light, sound or smell [11]. Before or during the headache attack, migraine aura may occur as transient focal neurological deficits with visual, sensory, language/speech, or motor abnormalities. On the other hand, the most common types of migraine headaches are those without aura, previously known as common migraines. A typical migraine attack can last from 4 to 72 hours, and pain can be so severe that it can significantly interfere with patients’ daily activities. While there is no cure for migraine, patients are treated with the aim to alleviate or prevent the symptoms. Current pharmacological treatments for acute migraine attacks include ergot alkaloids (e.g., dihydroergotamine mesylate, ergotamine), triptans (e.g., sumatriptan, naratriptan), acetaminophen, nonsteroidal anti-inflammatory drugs (NSAID) and opioids [29]. In addition, tricyclic antidepressants, β–blockers and anticonvulsants have been applied as preventive strategies for chronic migraine [4]. Existing medications, however, cannot fully control migraine and may even generate medication-overuse headaches [9].

A reliable preclinical model of migraine is imperative for mechanistic studies of migraine, as well as translational research of anti-migraine drug discovery. Intravenous infusion of nitroglycerin (NTG) is a commonly used to produce migraine attacks similar to spontaneous migraine in humans [28]. By mobilizing intracellular nitric oxide (NO), NTG triggers headache in healthy subjects and migraine without aura in 80% of migraineurs that are indistinguishable from spontaneous attacks [35]. There has been considerable effort to model NTG-induced migraine headache in mice. A transient thermal hyperalgesia and mechanical allodynia were observed at the plantar surface of the mouse hindpaw 30 - 60 min after an acute intraperitoneal injection of NTG, which was responsive to the acute migraine drug sumatriptan [2]. In addition, NTG can be administered repeated to simulate chronic state of migraine. Topiramate, a migraine preventative drug, was reported to attenuate hindpaw mechanical hypersensitivity in mice treated with repeated intermittent NTG [30]. These studies, however, have focused on evoked pain behaviors at hindpaws to indicate cephalic pain. However, the most prevalent spontaneous headache was not directly characterized. Furthermore, few studies have studied the affective migraine pain that is most relevant for human patients [8].

In the present study, we determined affective pain component in a mouse model of chronic NTG-induced migraine, using the conditioned place preference (CPP) paradigm that we have established for studying other chronic pain conditions in mice [14; 15; 17]. Here we proposed to validate the method for studying affective pain behavior and its chronicity in migraine. Moreover, we investigated the role of protein kinase C (PKC) isoforms in the pathophysiology of migraine.

2. Materials and methods

2.1. Materials

Nitroglycerin (NTG, NDC 0517-4810-25) was purchased from American Regent (Shirely, NY). α-CGRP (8-37) (mouse, rat) was purchased from Bachem (Torrance, CA). Topiramate was purchased from Sigma-Aldrich (San Louis, MO). Sumatriptan (Injection USP) was purchased from AuroMedics (East Windsor, NJ). Myristoylated peptide inhibitors of PKC isoforms [PKCδ: δV1-1 (SFNSYELGSL) and PKCε: εV1-2 (EAVSLKPT)] were synthesized according to previously published sequences [5] and verified by mass spectrometry by the Protein Research Laboratory, University of Illinois at Chicago [15].

2.2. Animals

Adult female mice were used in the study. C57BL/6 mice (25–30 g) were purchased from Charles River Laboratories. Breeders of transgenic PKCδ mice and PKCε mice were provided by Dr. Robert O. Messing, University of Texas at Austin. After arriving at our laboratory, PKCδ mice and PKCε mice were backcrossed with C57BL/6 mice for 10 generations. Heterozygous breeding was used to generate female homozygous PKCδ-null mice, PKCε-null mice, and the corresponding littermate wild-type (WT) female control mice for the study. Unless otherwise stated, female PKCδ mice and PKCε mice ages ranging from 8 to 16 weeks old were used. Mice were maintained on a 14/10 h light/dark cycle (5:00 am on/7:00 pm off) with food and water provided ad libitum before experimental procedures. All animal experiments were performed during the light cycle. Mice were randomly divided into experimental groups according to a computer-generated randomization list. All procedures were performed in accordance with the International Association for the Study of Pain and the National Institutes of Health's Guide for the Care and Use of Laboratory Animals after approval by the University of Illinois Institutional Animal Care and Use Committee. For all behavioral and biochemical tests, the experimenters were blinded to the genotype and treatment information.

2.3. NTG-induced migraine and drug administration

Mice received NTG (10 mg/kg, i.p.) every 2 days for total 4 injections (days 0, 2, 4, and 6). Control mice received equal volume and number of vehicle injections. Topiramate or α-CGRP (8-37) was administered systemically (i.v.) during the single trial conditioning.

2.4. Assessment of spontaneous ongoing pain

The conditioned place preference (CPP) method was employed to unmask the ongoing spontaneous pain as we have previously described [14] with some modifications. On Day −1, mice were placed into the middle chamber and allowed to freely explore the environment with access to all chambers for 30 min. Meanwhile, a pre-conditioning bias test was performed by recording mice movement and duration spent in each chamber in the first 15 min. Animals spending more than 80% or less than 20% of the total time in an end-chamber had preexisting chamber preference and were eliminated (~ 10% of total animals) from the experiment. On the conditioning day, mice first received saline (i.v.) was paired with a randomly chosen end chamber in the morning. In the afternoon, 4 h after the saline injection, mice were treated with either α-CGRP (8-37) (0.8 mg/kg, i.v.) or topiramate (60 mg/kg, i.v.) and paired with the other end chamber. During the conditioning, mice were allowed to stay only in the paired chamber without access to other compartments for 30 min immediately following vehicle or drug injection. In order to identify PKC mechanism, additional groups of mice were paired with a randomly chosen end chamber for 30 min immediately after saline (i.t.) in the morning, and 4 h later, with the opposite end chamber for 30 min after the treatment with the PKCδ inhibitor or the PKCε inhibitor (3 nmol/5 μL saline, i.t.) in the afternoon. The doses and administration schedule of these inhibitors were based on previous studies [15; 16]. On the testing day, 20 h after the afternoon pairing, mice were placed in the middle chamber of the CPP apparatus with all doors open so animals can have free access to all chambers. Movement and duration of each mouse spent in each chamber were recorded for 15 min for the analysis of chamber preference. Difference scores were calculated as (test time-preconditioning time) spent in the drug chamber.

2.5. Assessment of mechanical sensitivity

Mice were placed in individual Plexiglas containers with wire mesh platform. Mechanical sensitivity was assessed using von Frey filaments (Stoelting, IL), by pressing upward to the midplantar surface of the left hindpaw for 5 s or until a withdrawal response occurred. Using the “up-down” algorithm, 50% probability of paw withdrawal threshold was determined [14; 35].

2.6. Immunohistochemistry

After euthanization and perfusion, bilateral trigeminal ganglia (TG) were dissected by cutting the connective tissue which anchors the TG to the skull. The tissues were fixed, sectioned (10 μm), permeabilized, and incubated with a primary antibody for PKCδ or PKCε (1:500, Santa Cruz Biotechnology), followed by another incubation with Alexa 488-labeled secondary anti-rabbit IgG antibodies (1:500, Invitrogen) as described previously [15]. Images were captured by confocal microscopy (Zeiss LSM 700). The fluorescent density ratio (membrane vs. cytosol) was calculated from the intensity profile across each cell (representative cells and analyses were shown and indicated by dash line) [17].

2.7. Statistical Analysis

All data are presented as Mean ± SEM. To analyze the CPP data, two-way ANOVA (pairing vs. treatment) was applied followed by Bonferroni post hoc test. Difference scores were analyzed using paired t test by computing the differences between test time and preconditioning time for each mouse. Statistical significance was established at the 95% confidence limit.

3. Results

3.1. Presence of ongoing spontaneous pain in mice treated with NTG

Non-evoked spontaneous pain is a primary complaint in migraine patients, manifested as spontaneous migraine attacks. Although NTG has been used to study migraine pain, most studies have determined paw allodynia as a readout for pain using von Frey probing on hindpaws [2; 30]. An operant behavior test for determining spontaneous ongoing migraine pain is still lacking for the NTG model. In this study, we first aimed to validate a behavioral assessment to capture the affective pain component in migraine and detect non-evoked ongoing pain by negative reinforcement, since pain relief-induced conditioned place preference (CPP) is expected to suppress an aversive state to reveal the presence of ongoing pain in mice [14].

Female C57BL/6 mice were subjected to four intermittent treatments with NTG (10 mg/kg, i.p., every 48 h) to develop migraine like behaviors. To study spontaneous migraine pain, we determined α-CGRP (8-37)-induced CPP in NTG-treated animals. Systemic administration of α-CGRP (8-37), a clinically efficacious CGRP receptor antagonist, is effective in attenuating migraine headache [12; 26; 31]. NTG-treated and vehicle-treated mice were subjected to the conditioning of α-CGRP (8-37) (0.8 mg/kg, i.v.) (Fig. 1A). When tested 24 h later, NTG-treated mice spent significantly more time in the chamber that was paired with α-CGRP (8-37) (433 ± 47 s) than that in the saline-paired chamber (309 ± 16 s; P < 0.05, Fig. 1B). On the other hand, vehicle-treated control mice did not show preference in α-CGRP (8-37)-paired chamber (370 ± 27 s) or saline chamber (398 ± 24 s; P > 0.05, Fig. 1B). Moreover, there was no pre-conditioning bias in these experimental mice (P > 0.05, Fig. 1B). These data demonstrated a strong chamber preference induced by α-CGRP (8-37) (i.e. conditioned place preference) in the mice that were treated with NTG. We further analyzed the time of each mouse spent in α-CGRP (8-37)-paired chamber before and after the conditioning. The significant difference score was observed in NTG treated mice (P < 0.001), but not in vehicle treated control mice, confirming that α-CGRP (8-37) elicited CPP in these mice (Fig. 1C), confirming that migraine was accompanied by non-evoked spontaneous pain in chronic NTG-treated mice. These findings suggested the presence of ongoing pain in chronic-NTG model of migraine in mice, which is consistent with the clinical observation that NTG is a known trigger of migraine like headaches and accompanying symptoms in humans [35]. As reported previously, these mice also exhibited significantly decreased threshold to von Frey filament probing (0.03 ± 0.01 g vs. 1.33 ± 0.15 g, P < 0.001, Fig. 1D) on day 8, suggesting the persistence of evoked mechanical allodynia in chronic NTG-treated mice.

Figure 1.

Figure 1.

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Nitroglycerine (NTG) induced migraine-like pain behaviors in female C57Bl/6 mice. (A) Procedure for evaluating the conditioned place preference (CPP) using a single-trial conditioning on the day of last NTG treatment. (B) The peptide antagonist of calcitonin gene related peptide (CGRP), α-CGRP (8-37) (0.8 mg/kg, i.v.) effectively elicited pain relief-induced CPP and blocked ongoing pain in NTG-treated mice. (C) Difference score analysis (test time-preconditioning time spent in the drug paired chamber) confirmed α-CGRP (8-37)-induced CPP in NTG-treated mice. (D) After four intermittent treatments with NTG, mice displayed significantly reduced paw withdrawal threshold to von Frey filaments probing when compared with the vehicle-treated control mice. * P < 0.05, *** P < 0.001, n = 6 for each group.

3.2. No suppression of ongoing spontaneous migraine pain by systemic topiramate

Topiramate is known to be effective in preventing, but not treating, migraine headaches [32]. We further employed topiramate as a negative control to validate the CPP testing paradigm. As topiramate is futile in stopping already-occurred migraine headaches, we hypothesized that topiramate would not produce CPP. In the same chronic intermittent NTG model of migraine, mice received topiramate (60 mg/kg, i.v.) in the single trial conditioning to assess CPP (Fig. 2A). In contrast to α-CGRP (8-37), topiramate failed to generate chamber preference in NTG-treated mice, as mice spent similar amount of time in saline- (388 ± 24 s) and topiramate-paired (332 ± 18 s) chambers (P > 0.05, Fig. 2B). Meanwhile, vehicle-treated control mice showed comparable chamber staying pattern (373 ± 16s in saline-paired chamber vs. 322 ± 17 s in topiramate-paired chamber, P > 0.05). Analysis of difference scores did not reveal any topiramate chamber preference in either NTG- or vehicle-treated mice (Fig. 2C). Therefore, systemic topiramate did not block ongoing spontaneous pain in mice with migraine, as expected for its clinical efficacy in preventing, not treating, migraine headache once it occurs. These data further validate the specificity of the CPP model in studying affective pain in NTG-induced chronic headache.

Figure 2.

Figure 2.

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NTG-induced ongoing pain was not attenuated by systemic administration of topiramate. (A) Protocol for evaluation of CPP using topiramate as the pairing agent. (B) Topiramate (60 mg/kg, i.v.) failed to generate CPP in NTG-treated mice. (C) Difference score analysis confirmed that topiramate did not induce CPP or suppress ongoing pain in NTG-treated mice. n = 6 for each group.

3.3. Persistent ongoing spontaneous pain in mice with chronic migraine

To evaluate the chronicity of NTG-induced ongoing pain in the mouse model of migraine, we extended the CPP protocol to go beyond the acute effect of NTG. In the next series of experiments, NTG was given to the mice every other day for 4 treatments (10 mg/kg, i.p., on days 0, 2, 4 and 6; Fig. 3A), and we then waited for 2 more days, before pairing these mice with saline and α-CGRP (8-37). Chronic NTG-pretreated mice displayed a robust preference for α-CGRP (8-37)-paired chamber (531 ± 49 s) over the saline-paired chamber (289 ± 46 s, P < 0.001, Fig. 3B), indicative of α-CGRP (8-37)-induced CPP in NTG-treated mice, 48 h post the last dose of NTG. In contrast, vehicle-pretreated mice spent similar amount of time in saline-paired chamber (420 ± 49 s) and α-CGRP (8-37)-paired chamber (374 ± 26 s) (P > 0.05, Fig. 3B). Supported further by the significant difference score observed in NTG-pretreated mice (P < 0.01, Fig. 3C), these data revealed the presence of ongoing pain in NTG-treated mice, 48 h post the last dose of NTG. Together with the findings from the CPP test followed immediately after the NTG treatment (Fig. 1), it is evident that ongoing spontaneous pain can be reproducibly revealed and appears to be persistent in this mouse model of migraine resembling chronic migraine in humans [6; 25].

Figure 3.

Figure 3.

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Chronic NTG produced long-lasting ongoing pain behaviors in mice. (A) Modified protocol for evaluation of CPP by extending the test up to 72 h post last NTG treatment. (B) The single-trial conditioning of α-CGRP (8-37), when performed two days after the last treatment of NTG, elicited chamber preference in NTG-treated mice, but not vehicle-treated control mice. (C) Difference score analysis (test time-preconditioning time spent in the drug paired chamber) confirmed that NTG-treated mice exhibited CPP to α-CGRP (8-37) 72 h post the last administration of NTG. ** P < 0.01, *** P < 0.001, n = 6 for each group.

3.4. Evaluation of sumatriptan for ongoing spontaneous pain in migraine

The antimigraine drug sumatriptan has been reported to alleviate NTG-induced allodynia in mice [2]. We confirmed the allodynic effect of acute NTG (10 mg/kg, i.p.), which was reversed by sumatriptan (600 μg/mL, i.p.) within 30 min after its administration (Fig. 4A). We further determined the effectiveness of sumatriptan for ongoing spontaneous pain in the mouse model of migraine. Using the CPP paradigm established in Fig. 1A, sumatriptan (600 μg/mL, i.p.) was applied as the pairing agent. No chamber preference was elicited in either NTG- or vehicle-treated mice (P > 0.05, Fig. 4B-C). On day 6, NTG-treated mice exhibited significant mechanical allodynia on hindaw and the 4th NTG dose did not bring the threshold significantly lower, since it was already at the lowest detectable level. Sumatriptan partially reversed the mechanical allodynia in NTG-treated mice (Fig. 4D).

Figure 4.

Figure 4.

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Evaluation of sumatriptan in the mouse model of chronic migraine. (A) Sumatriptan reversed NTG-induced mechanical hypersensitivity 30 min after administration in naïve mice. Mechanical sensitivity was determined before and 3h after NTG administration. After the von Frey test, mice received sumatriptan (600 ug/mL, i.p.) and were re-tested 30 min later.

(B-D) Using the CPP paradigm in Fig. 1A: (B) Sumatriptan (600 ug/mL, i.p.) did not produce CPP in NTG- treated or vehicle-treated mice. NTG-treated mice and vehicle-treated mice showed no chamber preference. (C) Difference scores (test time-preconditioning time spent in the sumatriptan chamber) confirmed the absence of chamber preference. (D) Mechanical sensitivity was determined before and 3h after the NTG treatment on Day 6. Sumatriptan partially reversed NTG-induced mechanical hypersensitivity 30 min after the administration.

(E-G) Using the CPP paradigm in Fig. 3A: (E) NTG-treated mice and vehicle-treated control mice spent similar amount of time in saline- or sumatriptan-paired chambers. (F) Difference scores (test time-preconditioning time spent in the sumatriptan chamber) confirmed the absence of CPP. (G) When tested 48 h after the last dose of NTG, sumatriptan did not affect the mechanical sensitivity 30 min after the administration. * P < 0.05, ** P < 0.01, *** P < 0.001, n = 6 for each group.

After being conditioned with sumatriptan for 30 min on day 8 using the CPP paradigm in Fig. 3A, neither NTG- nor vehicle-pretreated mice generated CPP to sumatriptan (P > 0.05, Fig. 4E-F). In addition, sumatriptan failed to attenuate the long-lasting mechanical hypersensitivity after chronic NTG treatment on day 8 (Fig. 4G). These data are consistent with findings in humans that sumatriptan, a drug for acute migraine, is ineffective in attenuating chronic migraine [34] .

3.5. Prominent activation of PKCδ in chronic NTG-treated mice

Since chronic NTG exposure produced robust and persistent ongoing pain in mice as revealed by CPP to α-CGRP (8-37), we employed this CPP paradigm to explore the mechanisms underlying the spontaneous migraine headache. Several isoforms of PKC have been extensively studied for their roles in chronic pain conditions. PKCδ is not one of those isoforms and has only recently been studied in chronic pain. We proposed the role of PKCδ as a cellular mechanism for nociceptor excitability and nociceptive signaling in the development of peripheral neuropathy induced by paclitaxel [15]. Subsequently, we identified a role of PKCδ in promoting neuropathic pain in sickle cell disease [16]. To examine whether PKCδ contributes to migraine pain, we evaluated the activation of PKCδ in the NTG-migraine model. Indicative of its activation, the plasma membrane translocation of PKCδ [15] was determined by immunofluorescent analysis in the trigeminal ganglion (TG) in the mice receiving repeated administration of NTG (10 mg/kg, i.p, every 48 h for 8 days). In vehicle-treated control mice, immunoreactivity (i.r.) of trigeminal ganglionic PKCδ distributed evenly in the cytosol. On the contrary, prominent plasma membrane enrichment of PKCδ was observed in NTG-pretreated mice, mainly in small-diameter neurons (Fig. 5A). Of the cells with diameter less than 30 μm, 73% ± 8% had significant PKCδ translocation (150 cells from 5 slides/mouse, 3 mice/group). As PKCε, another isoform in the novel PKC subfamily, has been extensively studied in chronic pain signaling (e.g., [13; 20]), we also examined the activation of PKCε in the mouse model of migraine. Notably, i.r. of PKCε was not translocated toward the cytoplasmic membrane upon exposure to NTG (Fig. 5B). Furthermore, NTG-induced prevailing membrane translocation of PKCδ was observed in the primary afferent neurons of all the three large divisions of TG, which are the ophthalmic (V1), maxillary (V2) and mandibular (V3) divisions (Fig. 6). We found PKCδ translocation-positive cells in 102/150 (68%), 108/150 (72%), and 121/150 (81%) of small diameter cells (< 30 μm) examined in V1, V2 and V3 regions, respectively (5 slides/mouse, 3 mice/group). These data demonstrated that strong activation of trigeminal PKCδ in the NTG model of migraine in mice, indicating its potential functional involvement in mediating migraine headache.

Figure 5.

Figure 5.

Figure 5.

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Activation of PKCδ in the trigeminal ganglion (TG) neurons in chronic NTG-treated mice. Immunohistochemistry analysis showed plasma membrane translocation of PKCδ (A), but not PKCε (B), on Day 7 in TG of chronic NTG-treated mice. The fluorescent intensity of PKC isoform across representative cells (indicated by dashed arrows) is shown. Green, PKCδ; Cyan, PKCε; Red, NeuN; Blue, DAPI. Scale bars: 50 μm. n = 15 slices from 3 mice.

Figure 6.

Figure 6.

Figure 6.

Figure 6.

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NTG-induced activation of PKCδ in the primary afferent somata in the ophthalmic (V1), maxillary (V2) and mandibular division (V3) of TG. (A) Schematic diagram of a mouse TG. Red circles indicate the regions of three large divisions (V1, V2, and V3) in TG. Blue squares indicate the image taken region. (B) Plasma membrane translocation of PKCδ was observed in TG neurons of V1, V2 and V3 divisions after chronic NTG treatment. The fluorescent intensity of PKCδ across representative cells (indicated by dashed arrows) is shown. Green, PKCδ; Red, NeuN; Blue, DAPI. Scale bars: 50 μm.

3.6. Pharmacological inhibition of PKCδ in mice with chronic migraine

To assess the possible participation of PKCδ in migraine-associated non-evoked ongoing pain, we examined the effect of centrally administered δV1-1, a cell permeable, isoform-selective, peptidergic inhibitor of PKCδ [16]. Indeed, δV1-1 (3.0 nmol) blocked the membrane translocation of TG PKCδ 30 min after the injection (Fig. 7). Of the TG neurons examined (150 cells from 5 slides/mouse, 3 mice/group), the enhanced membrane/cytosolic fluorescent density ratio of PKCδ (4.4 ± 0.4, before the treatment) was suppressed by δV1-1 (2.0 ± 0.6, 30 min after the injection).

Figure 7.

Figure 7.

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Inhibition of chronic NTG-activated PKCδ by δV1-1 in the trigeminal ganglion (TG) neurons. In chronic NTG-treated mice, plasma membrane translocation of TG PKCδ was significantly abolished 30 min after the administration of a selective peptide inhibitor δV1-1 (3 nmol). Green: PKCδ, Red: NeuN, Blue: DAPI. Scale bar: 50 μm. The fluorescent density ratio (membrane vs. cytosol) was calculated from the intensity profile across each cell (representative cells were shown and indicated by dash lines).

Adapting the scheme proposed in Fig. 3A, CPP test was performed using δV1-1 as the pairing agent. If spontaneous ongoing migraine pain is mediated by PKCδ, it is expected that its inhibition by δV1-1 will suppress ongoing pain and produce CPP in migraine mice similar to the effect of α-CGRP (8-37) as demonstrated above. After pairing with saline or δV1-1 (3.0 nmol) for 30 min, chronic NTG-pretreated mice showed a strong preference for δV1-1-paired chamber (518 ± 61 s) over the saline chamber (326 ± 60 s, P < 0.01, Fig. 8A) when tested on the following day. In contrast, vehicle-pretreated control mice spent similar amount of time in the saline chamber (363 ± 42 s) and the δV1-1 chamber (332 ± 37 s) (P > 0.05). The place preference to the chamber paired with δV1-1 was also illustrated by the significant difference score generated in the NTG-pretreated mice, but not the vehicle-pretreated control mice (P < 0.05, Fig. 8B). Since the PKCδ inhibitor δV1-1 elicited CPP in the mouse model of migraine, these data demonstrated the functional relevance of PKCδ in mediating ongoing pain in migraine.

Figure 8.

Figure 8.

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PKCδ contributes to the maintenance of ongoing spontaneous pain in NTG-treated mice. (A) Inhibition of PKCδ by δV1-1 (3 nmol) induced CPP in chronic NTG-treated mice. (B) Difference score analysis confirmed that NTG-, but not saline-, treated mice showed CPP to PKCδ inhibitor. (C) The PKCε inhibitor, εV1-2 (3 nmol) did not generate CPP in either NTG-treated mice or vehicle-treated control mice. (D) Difference score analysis confirmed the absence of CPP to εV1-2 in chronic NTG-treated mice. * P < 0.05, ** P < 0.01, n = 6.

To rule out the possibility of non-specific targeting, εV1-2, an isoform-selective inhibitor of PKCε, was employed as a pairing agent in the CPP test. Control and NTG-treated mice spent about equal amounts of time in saline- and εV1-2-paired chambers (Fig. 8C, P > 0.05). Analysis of different scores confirmed the absence of chamber preference to PKCε inhibition in chronic NTG-pretreated (Fig. 8D, P > 0.05). These data are consistent with the findings that chronic NTG treatment failed to activate PKCε in TG region (Fig. 5B); Therefore, inhibiting PKCε by εV1-2 did not suppress ongoing pain or generate CPP in chronic NTG-pretreated mice.

3.7. Absence of ongoing spontaneous pain in chronic NTG-treated mice lacking PKCδ

To further investigate the causative role of PKCδ in ongoing spontaneous pain associated with migraine, we generated migraine model in mice lacking PKCδ. Female PKCδ-null (KO) mice as well as wild-type (WT) littermates received repeated administrations of NTG every other day for 4 treatments (10 mg/kg, i.p., on day 0, 2, 4 and 6). PKCδ WT and KO mice showed normal exploratory and locomotor activity after the exposure to NTG. On day 8 (i.e., 48 h after the last dose of NTG), KO and WT mice were subjected to the CCP paradigm to detect spontaneous ongoing pain. In agreement with the finding in C57BL/6 mice (Fig. 3), chronic NTG-treated PKCδ WT mice spent significantly more time in the α-CGRP (8-37)-paired chamber (582 ± 46 s) than in the saline chamber (263 ± 38 s) (P < 0.001, Fig. 9A). After chronic NTG treatment, PKCδ KO mice did not show preference for the saline- (427 ± 91 s) or the αCGRP (8-37)-paired (365 ± 71 s) chamber (P > 0.05). The difference score analysis revealed the existence of spontaneous ongoing pain in WT, but not KO mice of PKCδ after chronic NTG treatment (Fig. 9B).

Figure 9.

Figure 9.

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PKCδ contributes to the generation of ongoing spontaneous pain in NTG-treated mice. (A) In response to repeated administration of NTG, ongoing spontaneous pain was developed in PKCδ wild-type (+/+) mice, but not in mice lacking PKCδ (−/−). (B) Difference score analysis confirmed that wild type, but not PKCδ null mice showed CPP to α-CGRP (8-37) after chronic NTG treatment. (C) Both wild type and PKCε null mice had CPP to α-CGRP (8-37) after chronic NTG treatment. (D) Difference score analysis showed chronic NTG treatment produced ongoing spontaneous pain in both PKCε null and wild type mice. * P < 0.05, *** P < 0.001, n = 6.

In addition, we performed the same NTG treatment and CPP testing in PKCε-null mice. When tested 3 days after the final NTG administration, α-CGRP (8-37)-induced place preference was exhibited in both PKCε KO and WT mice (P < 0.05, Fig. 9C). Compared the difference scores in α-CGRP (8-37)-paired chamber, there was no significant difference between the KO and WT mice of PKCε (Fig. 9D). Meanwhile, PKCδ deficiency completely abolished the CPP induced by CGRP inhibition by α-CGRP (8-37) in the mouse model of chronic migraine. Since persistent ongoing spontaneous pain was no longer associated with chronic NTG-treatment in mice lacking PKCδ, these data strongly supported the hypothesis that PKCδ is required for the generation (as in the PKCδ null mice) and maintenance (disrupted by δ1-1) of ongoing migraine pain. The effect appears to be PKC isoform specific, as PKCε inhibition or deletion did not produce the same effect.

Moreover, chronic NTG-induced mechanical allodynia, which was detected in C57BL/6 mice (Fig. 1D and Fig. 4D) and in wild-type mice (Fig. 10A, C), did not develop in mice lacking PKCδ (Fig. 10A) or PKCε (Fig. 10C). On the other hand, evoked mechanical hypersensitivity was detected 3 h after the last dose of NTG on day 6, indicating that NTG was still capable of inducing hindpaw mechanical allodynia, in the PKCδ null mice (Fig. 10B) and the PKCε null mice (Fig. 10D). These observations are in agreement with previous findings that PKCδ and PKCε do not participate in acute pain signaling [17; 36], suggesting that PKCδ and PKCε contributed only to the chronicity of mechanical allodynia induced by NTG.

Figure 10.

Figure 10.

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Participation of PKCδ and PKCε in mediating mechanical allodynia in NTG-treated mice. (A) In response to repeated administration of NTG, PKCδ wild-type (+/+) mice, but not mice lacking PKCδ (−/−) developed mechanical hypersensitivity. (B) Mechanical sensitivity was determined before and 3h after NTG on Day 6 in PKCδ wild-type and null mice. * P < 0.05, *** P < 0.001, n = 6 for PKCδ (+/+) group and n = 4 for PKCδ (−/−) group. Paw withdrawal threshold in PKCδ null mice (−/−) was transiently reduced by NTG. (C) In response to repeated administration of NTG, PKCε wild-type (+/+) mice developed mechanical hypersensitivity. In contrast, mechanical sensitivity in PKCε null mice (−/−) remained unaffected by chronic NTG treatment. (D) Mechanical sensitivity was determined before and 3h after NTG on Day 6 in PKCε wild-type and null mice. Paw withdrawal threshold in PKCε null (−/−) mice was transiently reduced by NTG. * P < 0.05, *** P < 0.001, n = 6 for each group.

4. Discussion

Migraine is viewed as a complex neurological disorder with recurrent disabling headache and a number of other symptoms such as photophobia, phonophobia and osmophobia [22; 23]. Collectively, migraine is believed to be composed of multifaceted function abnormalities in autonomic, affective, cognitive, and sensory neuronal systems [3]. It is generally accepted that cephalic pain in migraine is unpredictable and arises/subsides on its own, underscoring our limited understanding of migraine pathogenesis. In addition, patients tend to experience migraine progression from episodic to chronic state with unremitting pain. As such, deciphering ongoing spontaneous cephalic pain associated with migraine is of vital importance in understanding the pathophysiology of migraine.

In the present study, we tested the hypothesis that affective dimension of migraine headache may be captured by negative reinforcement. To unravel the neurobiological mechanisms underlying migraine headache, there is an urgent need to establish a predictive and reliable animal model. As a donor of NO, NTG has been known as a potent headache inducing agent in humans for more than 100 years [1]. Patients with migraine are hypersensitive to NO. Migraineurs develop significantly stronger headache after NTG infusion than healthy subjects [27]. Furthermore, NTG not only produces an immediate headache during the infusion, but also a delayed headache that is maximal around six to seven hours after the infusion [35]. This delayed headache has the characteristics of typical attacks of migraine without aura (MOA) based on the International Classification of Headache Disorders (ICHD-II) criteria [18]. Therefore, we adapted the use of NTG to trigger migraine attacks in mice.

Distinguished from other studies testing evoked hypersensitivity in hindpaw, we focused on characterizing ongoing pain in this mouse model. Applying the CPP paradigm we previously established and validated in mice [7; 14; 15], we were able to successfully demonstrate the presence of ongoing pain in a mouse model of NTG-induced migraine. We found α-CGRP(8-37) intravenously administered 3-48 h post NTG selectively elicited pain relief and produced CPP (Fig. 1, 3), suggesting the ongoing pain induced by NTG in mice correlated with the onset of MOA in humans. As negative controls, we were able to verify that topiramate, which is known to prevent, but not treat, migraine headaches [32], was ineffective in generate CPP in NTG-treated mice. Similarly, sumatriptan, a drug for acute migraine, is ineffective in attenuating chronic migraine including the affective headache component, as determined by the absence of sumatriptan-induced CPP in chronic NTG-treated mice.

Moreover, the current study was able to significantly expand the study window of the NTG migraine model using the CPP testing. Chronic intermittent NTG-trigger migraine maintained long-lasting spontaneous pain as demonstrated by the exhibition of α-CGRP(8-37)-CPP 72 h post the last NTG exposure (Fig. 3). Incorporating two CPP strategies to the NTG model, the present study was able to profile the development of spontaneous ongoing pain. Therefore, chronic NTG treatment triggered persistent spontaneous ongoing pain in mice, strongly indicating it is a good preclinical model to explore the mechanisms of migraine progression from episodic to chronic state.

Furthermore, we identified that PCKδ functioned as a critical mediator in the development and maintenance of persistent spontaneous ongoing pain in migraine. PKCδ belongs to the novel PKC (nPKC) subfamily that requires diacylglycerol but not Ca2+ for activation. This subfamily also includes PKCε that has been extensively studied and found to be essential for a number of chronic pain conditions [21]. In the previous studies, we proposed a role of PKCδ in chronic pain, using experimental models of paclitaxel chemotherapy-induced peripheral neuropathy [15] and sickle cell anemia [16]. While both PKCδ and PKCε participated in mediating NTG-induced mechanical allodynia, we identified a role of PKCδ in promoting ongoing migraine headache. In contrast, PKCε was found not to be involved in the process. These findings were consistent with our previous report for the differential role of PKCδ and PKCε in spontaneous pain vs. evoked hypersensitivity [15; 16]. Along this line, it is reasonable to speculate that PKCδ is an essential player in the neuronal circuit of ongoing migraine headache induced by chronic intermittent NTG treatment, as PKCδ is likely to serve as a point of intracellular signaling convergence for the development of central sensitization. It remains to be investigated whether the PKCδ mechanism exists in other migraine headache conditions. Activation of PKCδ may propagate action potentials, increase the frequency of spontaneous/miniature excitatory postsynaptic currents, deactivate GABAergic inhibitory neurons, and therefore, promotes and potentiates pain signaling [19]. Given the growing understanding of the activation and sensitization of the trigeminovascular pathway in migraine headache, it will be of interest to characterize the function of PKCδ in meningeal nociceptors and central trigeminovascular neurons in future studies.

It has been a matter of debate, if ongoing spontaneous pain in migraine headache can be revealed in animal models. It has been previously demonstrated that systemic α-CGRP(8-37) was able to induced CPP in the rats that have been treated with inflammatory mediator to induce acute migraine attack [8]. We demonstrated here that CPP (i.e. negative reinforcement that is uncovered through the suppression of affective chronic pain) can be induced by non-addictive agents (inhibition of the CGRP receptor by α-CGRP(8-37) or inhibition of PKCδ by δV1-1) in a model of chronic migraine in mice. In addition to identifying PKCδ as a molecular mechanism for migraine pathophysiology, it is also reasonable to propose the translational relevance of the CPP testing paradigm in the chronic NTG-treated mice as a system for preclinical drug discovery research. As we examined here, α-CGRP (8-37), a competitive CGRP receptor antagonist, was effective in alleviating ongoing migraine headache. Humanized monoclonal antibodies targeting CGRP receptor has been approved by FDA as a major groundbreaking advance for the treatment of migraine in adults. On the other hand, topiramate failed to block ongoing cephalic pain in our mouse model of migraine, consistent with the clinical usage of this drug for migraine prevention but not acute attacks. In addition, we found that sumatriptan failed to attenuate ongoing spontaneous pain in migraine (Fig. 4). Sumatriptan and newer triptans, as combination therapy or monotherapy, are the first-line option for the treatment of migraine in patients aged 12 years and older. Sumatriptan needs to be taken timely at the onset of a migraine attack to be effective [10]. While sumatriptan works after acute NTG treatment [24], its responsiveness for chronic migraine, especially for affective pain, is not well characterized. The concept of CPP paradigm for detecting affective pain may facilitate preclinical drug discovery research for new migraine headache therapies.

In summary, we characterized ongoing spontaneous pain in chronic NTG-treated mice that closely mimic migraine in humans. Using the CPP testing paradigm, we uncovered persistent ongoing spontaneous pain that may serve as a model for studying migraine progression from episodic to chronic state. Moreover, we found that PKCδ is a critical cellular mechanism for the generation and maintenance of ongoing migraine pain. Our findings offer new insights into migraine pathophysiology and may ultimately lead to translational research targeting PKCδ or its signaling pathways for preventing and treating migraine headache.

Acknowledgments

Conflict of Interest Statement

The authors have no conflicts of interest to declare. We thank Xiao Guo for assistance during the study. This study was supported in part by a grant from the National Institute of Neurological Disorders and Stroke (NINDS) R01 NS103350-01A1. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NINDS or NIH. The final peer reviewed manuscript is subject to the National Institutes of Health Public Access Policy.

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