Periorbital Allodynia Research Articles

Distinct expression profile reveals glia involvement in the trigeminal system attributing to post-traumatic headache

BackgroundPost-traumatic headache (PTH) is a common comorbid symptom affecting at least one-third of patients with mild traumatic brain injury (mTBI). While neuroinflammation is known to contribute to the development of PTH, the cellular mechanisms in the trigeminal system crucial for understanding the pathogenesis of PTH remain unclear.MethodsA non-invasive repetitive mTBI (4 times with a 24-h interval) was induced in male mice and effect of mTBI was tested on either bregma or pre-bregma position on the head. Periorbital allodynia and spontaneous pain behavior were assessed using von Frey test and grimace score, respectively. Quantitative PCR was used to assess extent of mTBI pathology. RNA sequencing was performed to obtain transcriptomic profile of the trigeminal ganglion (TG), trigeminal nucleus caudalis (Sp5C) and periaqueductal gray (PAG) at 7 days post-TBI. Subsequently, quantitative PCR, in situ hybridization and immunohistochemistry were used to examine mRNA and protein expression of glia specific markers and pain associated molecules.ResultsThe repetitive impacts at the bregma, but not pre-bregma site led to periorbital hypersensitivity, which was correlated with enhanced inflammatory gene expression in multiple brain regions. RNA sequencing revealed mTBI induced distinct transcriptomic profiles in the peripheral TG and central Sp5C and PAG. Using gene set enrichment analysis, positive enrichment of non-neuronal cells in the TG and neuroinflammation in the Sp5C were identified to be essential in the pathogenesis of PTH. In situ assays also revealed that gliosis of satellite glial cells in the TG and astrocytes in the Sp5C were prominent days after injury. Furthermore, immunohistochemical study revealed a close interaction between activated microglia and reactive astrocytes correlating with increased calretinin interneurons in the Sp5C.ConclusionsTranscriptomics analysis indicated that non-neuronal cells in peripheral TG and successive in situ assays revealed that glia in the central Sp5C are crucial in modulating headache-like symptoms. Thus, selective targeting of glia cells can be a therapeutic strategy for PTH attributed to repetitive mTBI.

Formoterol dynamically alters endocannabinoid tone in the periaqueductal gray inducing headache

BackgroundHeadache is a pain disorder present in populations world-wide with a higher incidence in females. Specifically, the incidences of medication overuse headache (MOH) have increased worldwide. Comorbidities of MOH include photosensitivity, anxiety, “brain fog”, and decreased physical activity. The FDA-approved long-lasting selective β2-adrenergic receptor agonist, formoterol, is currently approved for use in severe asthma and chronic obstructive pulmonary disease. Recently, interest in repurposing formoterol for use in other disorders including Alzheimer’s disease, and neuropathic pain after spinal cord injury and traumatic brain injury has gained traction. Thus, revisiting known side-effects of formoterol, like headache and anxiety, could inform treatment paradigms. The endocannabinoid (eCB) system is implicated in the etiology of preclinical headache, with observed decreases in the circulating levels of endogenous cannabinoids, referred to as Clinical Endocannabinoid Deficiency. As cross-talk between the eCB system and adrenergic receptors has been reported, this study investigated the role of the eCB system and ability of formoterol to induce headache-like periorbital allodynic behavior.MethodsFemale 8-week-old C57Bl/6J mice were treated daily with formoterol (0.3 mg/kg, i.p.) for up to 42-days, during which they were assessed for periorbital allodynia, open field/novel object recognition, and photosensitivity. At the end of the study, the periaqueductal grey (PAG), a brain region known to contribute to both headache induction and maintenance, was collected and subjected to LC-MS to quantify endocannabinoid levels.ResultsMice exhibited periorbital allodynia at nearly all time points tested and photosensitivity from 28-days onward. Levels of endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), along with cannabinoid receptor 1 (CB1R) expression were altered by both age and upon treatment with formoterol. Administration of FAAH/MAGL inhibitors, to target the eCB system, and a non-selective cannabinoid receptor agonist, WIN 55,212 reversed the formoterol-induced periorbital allodynia.ConclusionsThese results suggest that formoterol is dysregulates eCB tone to drive headache-like periorbital allodynic behaviors. These results could help inform preventative treatment options for individuals receiving formoterol, as well as provide information on the interaction between the eCB and adrenergic system.Graphical

Oxytocin shortens spreading depolarization-induced periorbital allodynia

BackgroundMigraine is among the most prevalent and burdensome neurological disorders in the United States based on disability-adjusted life years. Cortical spreading depolarization (SD) is the most likely electrophysiological cause of migraine aura and may be linked to trigeminal nociception. We previously demonstrated, using a minimally invasive optogenetic approach of SD induction (opto-SD), that opto-SD triggers acute periorbital mechanical allodynia that is reversed by 5HT1B/1D receptor agonists, supporting SD-induced activation of migraine-relevant trigeminal pain pathways in mice. Recent data highlight hypothalamic neural circuits in migraine, and SD may activate hypothalamic neurons. Furthermore, neuroanatomical, electrophysiological, and behavioral data suggest a homeostatic analgesic function of hypothalamic neuropeptide hormone, oxytocin. We, therefore, examined the role of hypothalamic paraventricular nucleus (PVN) and oxytocinergic (OXT) signaling in opto-SD-induced trigeminal pain behavior.MethodsWe induced a single opto-SD in adult male and female Thy1-ChR2-YFP transgenic mice and quantified fos immunolabeling in the PVN and supraoptic nucleus (SON) compared with sham controls. Oxytocin expression was also measured in fos-positive neurons in the PVN. Periorbital mechanical allodynia was tested after treatment with selective OXT receptor antagonist L-368,899 (5 to 25 mg/kg i.p.) or vehicle at 1, 2, and 4 h after opto-SD or sham stimulation using von Frey monofilaments.ResultsOpto-SD significantly increased the number of fos immunoreactive cells in the PVN and SON as compared to sham stimulation (p < 0.001, p = 0.018, respectively). A subpopulation of fos-positive neurons also stained positive for oxytocin. Opto-SD evoked periorbital mechanical allodynia 1 h after SD (p = 0.001 vs. sham), which recovered quickly within 2 h (p = 0.638). OXT receptor antagonist L-368,899 dose-dependently prolonged SD-induced periorbital allodynia (p < 0.001). L-368,899 did not affect mechanical thresholds in the absence of opto-SD.ConclusionsThese data support an SD-induced activation of PVN neurons and a role for endogenous OXT in alleviating acute SD-induced trigeminal allodynia by shortening its duration.

Inhibition of 2-AG hydrolysis alleviates posttraumatic headache attributed to mild traumatic brain injury

BackgroundPosttraumatic headache (PTH) is a common and debilitating symptom following repetitive mild traumatic brain injury (rmTBI), and it mainly resembles a migraine-like phenotype. While modulation of the endocannabinoid system (ECS) is effective in treating TBI and various types of pain including migraine, the role of augmentation of endocannabinoids in treating PTH has not been investigated.MethodsRepetitive mild TBI was induced in male C57BL/6J mice using the non-invasive close-head impact model of engineered rotational acceleration (CHIMERA). Periorbital allodynia was assessed using von Frey filaments and determined by the “Up-Down” method. Immunofluorescence staining was employed to investigate glial cell activation and calcitonin gene-related peptide (CGRP) expression in the trigeminal ganglion (TG) and trigeminal nucleus caudalis (TNC) of the rmTBI mice. Levels of 2-arachidonoyl glycerol (2-AG), anandamide (AEA), and arachidonic acid (AA) in the TG, medulla (including TNC), and periaqueductal gray (PAG) were measured by mass spectrometry. The therapeutic effect of endocannabinoid modulation on PTH was also assessed.ResultsThe rmTBI mice exhibited significantly increased cephalic pain hypersensitivity compared to the sham controls. MJN110, a potent and selective inhibitor of the 2-AG hydrolytic enzyme monoacylglycerol lipase (MAGL), dose-dependently attenuated periorbital allodynia in the rmTBI animals. Administration of CGRP at 0.01 mg/kg reinstated periorbital allodynia in the rmTBI animals on days 33 and 45 post-injury but had no effect in the sham and MJN110 treatment groups. Activation of glial cells along with increased production of CGRP in the TG and TNC at 7 and 14 days post-rmTBI were attenuated by MJN110 treatment. The anti-inflammatory and anti-nociceptive effects of MJN110 were partially mediated by cannabinoid receptor activation, and the pain-suppressive effect of MJN110 was completely blocked by co-administration of DO34, an inhibitor of 2-AG synthase. The levels of 2-AG in TG, TNC and PAG were decreased in TBI animals, significantly elevated and further reduced by the selective inhibitors of 2-AG hydrolytic and synthetic enzymes, respectively.ConclusionEnhancing endogenous levels of 2-AG appears to be an effective strategy for the treatment of PTH by attenuating pain initiation and transmission in the trigeminal pathway and facilitating descending pain inhibitory modulation.

Effect of peppermint essential oil (Mentha piperita L.) in migraine-like responses in female rats

Migraine is a severe and disabling neurological disorder characterized by headache, photophobia, phonophobia, nausea and vomiting. 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. Pharmacological treatment of migraine has advanced in the past years but is still considered unsatisfactory for a significant number of patients. There is growing evidence that essential oils may provide benefit for migraineurs. Herein it was tested the hypothesis that peppermint essential oil (Mentha piperita L.) could reduce migraine-related responses in rats. The model consisted in the injection of calcitonin-gene-related peptide (CGRP) in the trigeminal ganglion (TG) of female rats to induce the development of immediate periorbital cutaneous allodynia and late photosensitivity (24 h after CGRP). Inhalation of the peppermint essential oil during 15 minutes before CGRP injection in the TG did not reduce periorbital allodynia and photosensitivity of female rats. However, when the exposure occurred after CGRP injection, peppermint essential oil caused a significant reduction in both parameters. Likewise, intranasal application of menthol, a major component of peppermint essential oil caused a significant reduction of periorbital allodynia induced by CGRP. In conclusion, peppermint essential oil and menthol may represent a safe, low cost and noninvasive adjuvant abortive therapy for headache pain in migraine patients. However, further high-quality clinical studies are clearly warranted to determine efficacy, safety and to establish their best treatment regimen.

Aromatase inhibitors evoke periorbital allodynia in mice via calcitonin gene-related peptide and its receptors in Schwann cells

Background: Treatment with the currently recommended aromatase inhibitors (AIs) for adjuvant endocrine treatment of estrogen receptorpositive breast cancer is associated with debilitating musculoskeletal pain symptoms (AIMS) and headache. Recent evidence suggests that the proalgesic channel transient receptor potential ankyrin 1 (TRPA1) is implicated in AIMS. Here, we investigated the cellular and molecular mechanisms, including TRPA1, implicated in periorbital mechanical allodynia (PMA), a surrogate of headache-like pain, evoked by AIs in mice. Methods: C57BL6/J mice were treated with intragastric letrozole (0.05-0.5 mg/kg), exemestane (1-5 mg/kg) or anastrozole (0.02-0.2 mg/kg) and were evaluated by applying von Frey filaments to the periorbital region over the rostral portion of the eye. Some mice were pretreated (subcutaneous in the periorbital area) with receptor, channel, or enzyme inhibitors. PMA was also investigated in mice with selective silencing of Trpa1 and receptor activity modifying protein 1 [Ramp1, the component of calcitonin gene related peptide (CGRP) receptor required for its functioning] in Schwann cells (Plp-Cre+-Trpa1fl/fl and Plp-Cre+-Ramp1fl/fl mice, respectively) or trigeminal neurons (Adv-Cre+- Trpa1fl/fl and Adv- Cre+- Ramp1fl/fl mice, respectively). Results: Letrozole dose-dependently produced PMA that was attenuated by a TRPA1 antagonist (A967079) or a CGRP receptor antagonist (olcegepant), whereas indomethacin was ineffective. Selective silencing of Trpa1 in both Schwann cells and trigeminal neurons reduced letrozole- evoked PMA. Silencing of Ramp1 in Schwann cells, but not in trigeminal neurons, attenuated PMA. Inhibition of the intracellular pathway known to promote PMA by CGRP action in Schwann cells, including adenylyl cyclase (SQ-22536), nitric oxide synthase (L-NG-Nitro arginine methyl ester), and oxidative stress (N-tert-butyl-a-phenylnitrone) inhibitors reduced letrozole-evoked PMA. PMA evoked by exemestane (1, 5, 10 mg/kg i.g.) or anastrozole (0.02, 0.1, 0.2 mg/kg i.g.) Was also markedly reduced in mice with selective silencing of TRPA1 in Schwann cells and nociceptors. Conclusions: Data indicate that letrozole, targeting TRPA1 in peptidergic nerve terminals, releases CGRP that engages its receptor in adjacent Schwann cells to trigger a complex intracellular pathway that results in TRPA1 activation and the ensuing ROS release to sustain PMA. Should these mechanisms be present in patients, their inhibition may ameliorate cephalic mechanical allodynia associated with aromatase inhibitors-induced headaches.

Oxytocin Receptors on Calvarial Periosteal Innervation: Therapeutic Target for Post-Traumatic Headache?

Following a mild traumatic brain injury (mTBI), the most prevalent and profoundly debilitating occurrence is the emergence of an acute and persistent post-traumatic headache (PTH), for which there are presently no approved treatments. A crucial gap in knowledge exists regarding the consequences of an mTBI, which could serve as a foundation for the development of therapeutic approaches. The activation of trigeminal sensory nerve terminals that innervate the calvarial periosteum (CP)-a densely innervated tissue layer covering the calvarial skull-has been implicated in both migraines and PTHs. We have previously shown that trigeminal oxytocin receptors (OTRs) may provide a therapeutic target for PTHs. This study examined the expression of oxytocin receptors on trigeminal nerves innervating the periosteum and whether these receptors might serve as a therapeutic target for PTHs using a direct application of oxytocin to the periosteum in a rodent model of PTH. We used retrograde tracing and immunohistochemistry to determine if trigeminal ganglion (TG) neurons innervating the periosteum expressed OTRs and/or CGRPs. To model the impact of local inflammation that occurs following an mTBI, we applied chemical inflammatory mediators directly to the CP and assessed for changes in immediate-early gene expression as an indication of neuronal activation. We also determined whether mTBI would lead to expression changes to OTR levels. To determine whether these OTRs could be a viable therapeutic target, we assessed the impact of oxytocin injections into the CP in a mouse model of PTH-induced periorbital allodynia. The results of these experiments demonstrate the following: (1) the cell bodies of CP afferents reside in the TG and express both OTRs and CGRPs; (2) inflammatory chemical stimulation of the periosteum leads to rapid activation of TG neurons (phospho-ERK (p-ERK) expression), (3) mTBI-induced inflammation increased OTR expression compared to the sham group; and (4) administration of oxytocin into the periosteum on day 2 and day 40 blocked cutaneous allodynia for up to one hour post-administration for both acute and persistence phases in the PTH model-an effect that was preventable by the administration of an OTR antagonist. Taken together, our observations suggest that periosteal trigeminal afferents contribute to post-TBI craniofacial pain, and that periosteum tissue can be used as a potential local target for therapeutics such as oxytocin.

Extrasynaptic δGABAA receptors mediate resistance to migraine-like phenotype in rats

BackgroundGABA, a key inhibitory neurotransmitter, has synaptic and extrasynaptic receptors on the postsynaptic neuron. Background GABA, which spills over from the synaptic cleft, acts on extrasynaptic delta subunit containing GABAA receptors. The role of extrasynaptic GABAergic input in migraine is unknown. We investigated the susceptibility to valid migraine-provoking substances with clinically relevant behavioral readouts in Genetic Absence Epilepsy of Rats Strasbourg (GAERS), in which the GABAergic tonus was altered. Subsequently, we screened relevant GABAergic mechanisms in Wistar rats by pharmacological means to identify the mechanisms.MethodsWistar and GAERS rats were administered nitroglycerin (10 mg/kg) or levcromakalim (1 mg/kg). Mechanical allodynia and photophobia were assessed using von Frey monofilaments and a dark-light box. Effects of GAT-1 blocker tiagabine (5 mg/kg), GABAB receptor agonist baclofen (2 mg/kg), synaptic GABAA receptor agonist diazepam (1 mg/kg), extrasynaptic GABAA receptor agonists gaboxadol (4 mg/kg), and muscimol (0.75 mg/kg), T-type calcium channel blocker ethosuximide (100 mg/kg) or synaptic GABAA receptor antagonist flumazenil (15 mg/kg) on levcromakalim-induced migraine phenotype were screened.ResultsUnlike Wistar rats, GAERS exhibited no reduction in mechanical pain thresholds or light aversion following nitroglycerin or levcromakalim injection. Ethosuximide did not reverse the resistant phenotype in GAERS, excluding the role of T-type calcium channel dysfunction in this phenomenon. Tiagabine prevented levcromakalim-induced mechanical allodynia in Wistar rats, suggesting a key role in enhanced GABA spillover. Baclofen did not alleviate mechanical allodynia. Diazepam failed to mitigate levcromakalim-induced migraine phenotype. Additionally, the resistant phenotype in GAERS was not affected by flumazenil. Extrasynaptic GABAA receptor agonists gaboxadol and muscimol inhibited periorbital allodynia in Wistar rats.ConclusionOur study introduced a rat strain resistant to migraine-provoking agents and signified a critical involvement of extrasynaptic δGABAergic receptors. Extrasynaptic δ GABAA receptors, by mediating constant background inhibition on the excitability of neurons, stand as a novel drug target with a therapeutic potential in migraine.Graphical abstract

Unraveling the directional relationship of sleep and migraine-like pain.

Migraine and sleep disorders are common co-morbidities. Patients frequently link their sleep to migraine attacks suggesting a potential causal relationship between these conditions. However, whether migraine pain promotes or disrupts sleep or whether sleep disruption can increase the risk of migraine remains unknown. We assessed the potential impact of periorbital allodynia, a measure consistent with migraine-like pain, from multiple preclinical models on sleep quantity and quality. Additionally, we evaluated the possible consequences of sleep deprivation in promoting susceptibility to migraine-like pain. Following the implantation of electroencephalogram/electromyography electrodes to record sleep, mice were treated with either single or repeated systemic injections of nitroglycerin at the onset of their active phase (i.e. nocturnal awake period). Neither single nor repeated nitroglycerin affected the total sleep time, non-rapid eye movement sleep, rapid eye movement sleep, sleep depth or other measures of sleep architecture. To account for the possible disruptive effects of the surgical implantation of electroencephalogram/electromyography electrodes, we used immobility recordings as a non-invasive method for assessing sleep-wake behaviour. Neither single nor repeated nitroglycerin administration during either the mouse sleep (i.e. daylight) or active (i.e. night) periods influenced immobility-defined sleep time. Administration of an inflammatory mediator mixture onto the dura mater at either sleep or active phases also did not affect immobility-defined sleep time. Additionally, inhalational umbellulone-induced migraine-like pain in restraint-stressed primed mice did not alter immobility-defined sleep time. The possible influence of sleep disruption on susceptibility to migraine-like pain was evaluated by depriving female mice of sleep over 6 h with novel objects, a method that does not increase circulating stress hormones. Migraine-like pain was not observed following acute sleep deprivation. However, in sleep-deprived mice, subthreshold doses of systemic nitroglycerin or dural calcitonin gene-related peptide induced periorbital cutaneous allodynia consistent with migraine-like pain. Our data reveal that while migraine-like pain does not significantly disrupt sleep, sleep disruption increases vulnerability to migraine-like pain suggesting that a therapeutic strategy focused on improving sleep may diminish migraine attacks.

PACAP-38 related modulation of the cranial parasympathetic projection: A novel mechanism and therapeutic target in severe primary headache.

Little is known of how cranial autonomic symptoms (CAS) in cluster headache and migraine may contribute to their severe headache phenotype. This strong association suggests the involvement of the cranial parasympathetic efferent pathway. To investigate its contribution, we studied the role of pituitary adenylate cyclase activating polypeptide-38 (PACAP-38), a potent sensory and parasympathetic neuropeptide, in modulating pre- and post-ganglionic cranial parasympathetic projectionneurons, and their influence on headache-related trigeminal-autonomic responses. Using PACAP-38 and PACAP-38 responsive receptor antagonists, electrophysiological, behavioural and facial neurovascular-blood flow was measured in rats to probe trigeminal- and parasympathetic-neuronal, periorbital thresholds and cranial-autonomic outcomes, as they relate to primary headaches. Sumatriptan attenuated the development of PACAP-38 mediated activation and sensitization of trigeminocervical neurons and related periorbital allodynia. PACAP-38 also caused activation and enhanced responses of dural-responsive pre-ganglionic pontine-superior salivatory parasympathetic neurons. Further, the PACAP-38 responsive receptor antagonists dissected a role of VPAC1 and PAC1 receptors in attenuating cranial-autonomic and trigeminal-neuronal responses to activation of the cranial parasympathetic projection, which requires post-ganglionic parasympathetic neurotransmission. Given the prevailing view that sumatriptan acts to some degree via a peripheral mechanism, our data support that PACAP-38 mediated receptor activation modulates headache-related cranial-autonomic and trigeminovascular responses via peripheral and central components of the cranial parasympathetic projection. This provides a mechanistic rationale for the association of CAS with more severe headache phenotypes in cluster headache and migraine, and supports the cranial parasympathetic projection as a potential novel locus for treatment by selectively targeting PACAP-38 or PACAP-38 responsive VPAC1 /PAC1 receptors.

The effect of sex and estrus cycle stage on optogenetic spreading depression induced migraine-like pain phenotypes

BackgroundMigraine is more prevalent in females, raising the possibility that sex and gonadal hormones modulate migraine. We recently demonstrated that minimally invasive optogenetic spreading depolarization (opto-SD) elicits robust periorbital allodynia. The objective of this study was to test the hypothesis that opto-SD induced migraine-like pain behavior is worse in females and varies during the estrus cycle.MethodsSingle or repeated opto-SDs were induced in male and female adult Thy1-ChR2-YFP transgenic mice. Von Frey monofilaments were used to test periorbital mechanical allodynia. Mouse grimace was also examined under increasing light intensity to quantify spontaneous discomfort and light-aversive behavior. Vaginal smears were obtained for estrus cycle staging at the end of behavioral testing.ResultsA multi-variable regression analysis was performed using a male and female cohort to test the effect of independent variables on periorbital allodynia. Opto-SD predicted lower periorbital thresholds as compared with sham stimulation (p < 0.0001). Additionally, female sex predicted lower periorbital thresholds compared with males (p = 0.011). There were significant interactions between opto-SD and time (interaction p = 0.030) as animals tended to recover from opto-SD allodynia over time, and between sex and time (p = 0.020) as females tended to take longer to recover. Proestrus, estrus (PE) and metestrus, diestrus (MD) stages were combined to represent high versus low circulating estradiol relative to progesterone, respectively. Multi-variable regression revealed an effect of estrus cycle (p = 0.015) on periorbital thresholds. In the sham group, PE had lower thresholds than MD. However, there was no interaction between opto-SD and the estrus cycle (p = 0.364). Grimace scores were also examined at incremental light intensities. There was an effect of opto-SD (p < 0.0001), light intensity (p = 0.001) and estrus cycle (p = 0.024) on grimace without interaction among them (three-way ANOVA).ConclusionsFemale sex and estrus stages with high circulating estradiol relative to progesterone lower trigeminal pain thresholds and augment photosensitivity. In females, opto-SD increased pain behavior and photosensitivity irrespective of the estrus stage.Graphical

Preclinical effects of cannabidiol in an experimental model of migraine.

Migraine is a disabling disorder characterized by recurrent headaches, accompanied by abnormal sensory sensitivity and anxiety. Despite extensive historical use of cannabis in headache disorders, there is limited research on the nonpsychoactive cannabidiol (CBD) for migraine and there is no scientific evidence to prove that CBD is an effective treatment. The effects of CBD are examined here using a calcitonin gene-related peptide (CGRP)-induced migraine model that provides measures of cephalic allodynia, spontaneous pain, altered light sensitivity (photophobia), and anxiety-like behavior in C57BL/6J mice. A single administration of CGRP induced facial hypersensitivity in both female and male mice. Repeated CGRP treatment produced progressively decreased levels in basal thresholds of allodynia in females, but not in males. A single CBD administration protected both females and males from periorbital allodynia induced by a single CGRP injection. Repeated CBD administration prevented increased levels of basal allodynia induced by repeated CGRP treatment in female mice and did not lead to responses consistent with migraine headache as occurs with triptans. Cannabidiol, injected after CGRP, reversed CGRP-evoked allodynia. Cannabidiol also reduced spontaneous pain traits induced by CGRP administration in female mice. Finally, CBD blocked CGRP-induced anxiety in male mice, but failed in providing protection from CGRP-induced photophobia in females. These results demonstrate the efficacy of CBD in preventing episodic and chronic migraine-like states with reduced risk of causing medication overuse headache. Cannabidiol also shows potential as an abortive agent for treating migraine attacks and headache-related conditions such as spontaneous pain and anxiety.

Inhibition of persistent sodium current reduces spreading depression-evoked allodynia in a mouse model of migraine with aura.

We investigated the efficacy of inhibiting persistent Na + currents (I NaP ) in acute rodent models of migraine with aura. Cortical spreading depression (SD) is a slow wave of neuronal and glial depolarization that underlies the migraine aura. Minimally invasive optogenetic SD (opto-SD) causes periorbital mechanical allodynia in mice, suggesting SD activates trigeminal nociceptors. Persistent Na + currents contribute to neuronal intrinsic excitability and have been implicated in peripheral and cortical excitation. We examined a preferential inhibitor of I NaP, GS-458967, on SD-induced periorbital allodynia, SD susceptibility, and formalin-induced peripheral pain. Periorbital mechanical allodynia was tested in male and female Thy1-ChR2-YFP mice after a single opto-SD event using manual von Frey monofilaments. GS-458967 (1 mg/kg, s.c.) or vehicle was dosed immediately after opto-SD induction, and allodynia was tested 1 hour later. The electrical SD threshold and KCl-induced SD frequency were examined in the cortex in male Sprague-Dawley rats after 1 hour pretreatment with GS-458967 (3 mg/kg, s.c.) or vehicle. Effects of GS-458967 (0.5-5 mg/kg, p.o.) on spontaneous formalin hind paw behavior and locomotion were also examined in male CD-1 mice. GS-458967 suppressed opto-SD-induced periorbital allodynia and decreased susceptibility to SD. GS-458967 also diminished early and late phase formalin-induced paw-licking behavior with early phase paw licking responding to lower doses. GS-458967 up to 3 mg/kg had no impact on locomotor activity. These data provide evidence that I NaP inhibition can reduce opto-SD-induced trigeminal pain behavior and support I NaP inhibition as an antinociceptive strategy for both abortive and preventive treatment of migraine.

Effect of Voluntary Exercise on Endogenous Pain Control Systems and Post-traumatic Headache in Mice

Traumatic brain injury (TBI) can cause acute and chronic pain along with motor, cognitive, and emotional problems. Although the mechanisms are poorly understood, previous studies suggest disruptions in endogenous pain modulation may be involved. Voluntary exercise after a TBI has been shown to reduce some consequences of injury including cognitive impairment. We hypothesized, therefore, that voluntary exercise could augment endogenous pain control systems in a rodent model of TBI. For these studies, we used a closed-head impact procedure in male mice modeling mild TBI. We investigated the effect of voluntary exercise on TBI-induced hindpaw nociceptive sensitization, diffuse noxious inhibitory control failure, and periorbital sensitization after bright light stress, a model of post-traumatic headache. Furthermore, we investigated the effects of exercise on memory, circulating markers of brain injury, neuroinflammation, and spinal cord gene expression. We observed that exercise significantly reduced TBI-induced hindpaw allodynia and periorbital allodynia in the first week following TBI. We also showed that exercise improved the deficits associated with diffuse noxious inhibitory control and reduced bright light stress-induced allodynia up to 2 months after TBI. In addition, exercise preserved memory and reduced TBI-induced increases in spinal BDNF, CXCL1, CXCL2, and prodynorphin expression, all genes previously linked to TBI-induced nociceptive sensitization. Taken together, our observations suggest that voluntary exercise may reduce pain after TBI by reducing TBI-induced changes in nociceptive signaling and preserving endogenous pain control systems. PerspectiveThis article evaluates the effects of exercise on pain-related behaviors in a preclinical model of traumatic brain injury (TBI). The findings show that exercise reduces nociceptive sensitization, loss of diffuse noxious inhibitory control, memory deficits, and spinal nociception-related gene expression after TBI. Exercise may reduce or prevent pain after TBI.

ABHD6 and MAGL control 2-AG levels in the PAG and allodynia in a CSD-induced periorbital model of headache.

The high prevalence and severe symptoms of migraines in humans emphasizes the need to identify underlying mechanisms that can be targeted for therapeutic benefit. Clinical Endocannabinoid Deficiency (CED) posits that reduced endocannabinoid tone may contribute to migraine development and other neuropathic pain conditions. While strategies that increase levels of the endocannabinoid n-arachidonoylethanolamide have been tested, few studies have investigated targeting the levels of the more abundant endocannabinoid, 2-arachidonoylgycerol, as an effective migraine intervention. Cortical spreading depression was induced in female Sprague Dawley rats via KCl (potassium chloride) administration, followed by measures of endocannabinoid levels, enzyme activity, and neuroinflammatory markers. Efficacy of inhibiting 2-arachidonoylglycerol hydrolysis to mitigate periorbital allodynia was then tested using reversal and prevention paradigms. We discovered reduced 2-arachidonoylglycerol levels in the periaqueductal grey associated with increased hydrolysis following headache induction. Pharmacological inhibition of the 2-arachidonoylglycerol hydrolyzing enzymes, α/β-hydrolase domain-containing 6 and monoacylglycerol lipase reversed and prevented induced periorbital allodynia in a cannabinoid receptor-dependent manner. Our study unravels a mechanistic link between 2-arachidonoylglycerol hydrolysis activity in the periaqueductal grey in a preclinical, rat model of migraine. Thus, 2-arachidonoylglycerol hydrolysis inhibitors represent a potential new therapeutic avenue for the treatment of headache.

Rodent behavior following a dural inflammation model with anti-CGRP migraine medication treatment.

Migraine is a widespread and prevalent disease with a complex pathophysiology, of which neuroinflammation and increased pain sensitivity have been suggested to be involved. Various studies have investigated the presence of different inflammatory markers in migraineurs and investigated the role of inflammation in inflammatory models with complete Freund's adjuvant (CFA) or inflammatory soup added to the dura mater. The aim of the current study was to examine whether application of CFA to the dura mater would cause behavioral alterations that are migraine relevant. In addition, we investigated the potential mitigating effects of fremanezumab, a CGRP (calcitonin gene-related peptide) specific antibody, following CFA application. Male Sprague-Dawley rats were randomly divided into six groups: fresh (n = 7), fresh + carprofen (n = 6), fresh + anti-CGRP (n = 6), sham (n = 7), CFA (n = 16), CFA + anti-CGRP (n = 8). CFA was applied for 15 min on a 3 × 3 mm clearing of the skull exposing the dura mater of male Sprague-Dawley rats. We applied the Light/Dark box and Open Field test, combined with the electronic von Frey test to evaluate outcomes. Finally, we observed CGRP immunoreactivity in the trigeminal ganglion. No differences were observed in the Light/Dark box test. The Open Field test detected behavior differences, notably that sham rats spend less time in the central zone, reared less and groomed more than fresh + carprofen rats. The other groups were not significantly different compared to sham rats, indicating that activation of the TGVS is present in sham surgery and cannot be exacerbated by CFA. However, for the allodynia, we observed specific periorbital sensitization, not observed in the sham animals. This could not be mitigated by fremanezumab, although it clearly reduced the amount of CGRP positive fibers. CFA surgically administered to the dura causes periorbital allodynia and increases CGRP positive fibers in the trigeminal ganglion. Fremanezumab does not reduce periorbital allodynia even though it reduces CGRP positive fibers in the TG. Further work is needed to investigate whether CFA administered to the dura could be used as a non-CGRP inflammatory migraine model.

Kappa opioid receptor agonists produce sexually dimorphic and prolactin-dependent hyperalgesic priming.

Repeated stress produces hyperalgesic priming in preclinical models, but underlying mechanisms remain uncertain. As stress engages kappa opioid receptors (KORs), we hypothesized that repeated administration of KOR agonists might mimic, in part, stress-induced hyperalgesic priming. The potential contribution of circulating prolactin (PRL) and dysregulation of the expression of PRL receptor (PRLR) isoforms in sensory neurons after KOR agonist administration was also investigated. Mice received 3 daily doses of U-69593 or nalfurafine as a "first-hit" stimulus followed by assessment of periorbital tactile allodynia. Sixteen days after the first KOR agonist administration, animals received a subthreshold dose of inhalational umbellulone, a TRPA1 agonist, as the second-hit stimulus and periorbital allodynia was assessed. Cabergoline, a dopamine D2 receptor agonist, was used to inhibit circulating PRL in additional cohorts. Prolactin receptor isoforms were quantified in the V1 region of the trigeminal ganglion after repeated doses of U-69593. In both sexes, KOR agonists increased circulating PRL and produced allodynia that resolved within 14 days. Hyperalgesic priming, revealed by umbellulone-induced allodynia in animals previously treated with the KOR agonists, also occurred in both sexes. However, repeated U-69593 downregulated the PRLR long isoform in trigeminal neurons only in female mice. Umbellulone-induced allodynia was prevented by cabergoline co-treatment during priming with KOR agonists in female, but not male, mice. Hyperalgesic priming therefore occurs in both sexes after either biased or nonbiased KOR agonists. However, a PRL/PRLR-dependence is observed only in female nociceptors possibly contributing to pain in stress-related pain disorders in females.

Dysregulation of serum prolactin links the hypothalamus with female nociceptors to promote migraine.

Migraine headache results from activation of meningeal nociceptors, however, the hypothalamus is activated many hours before the emergence of pain. How hypothalamic neural mechanisms may influence trigeminal nociceptor function remains unknown. Stress is a common migraine trigger that engages hypothalamic dynorphin/kappa opioid receptor (KOR) signalling and increases circulating prolactin. Prolactin acts at both long and short prolactin receptor isoforms that are expressed in trigeminal afferents. Following downregulation of the prolactin receptor long isoform, prolactin signalling at the prolactin receptor short isoform sensitizes nociceptors selectively in females. We hypothesized that stress may activate the kappa opioid receptor on tuberoinfundibular dopaminergic neurons to increase circulating prolactin leading to female-selective sensitization of trigeminal nociceptors through dysregulation of prolactin receptor isoforms. A mouse two-hit hyperalgesic priming model of migraine was used. Repeated restraint stress promoted vulnerability (i.e. first-hit priming) to a subsequent subthreshold (i.e. second-hit) stimulus from inhalational umbellulone, a TRPA1 agonist. Periorbital cutaneous allodynia served as a surrogate of migraine-like pain. Female and male KORCre; R26lsl-Sun1-GFP mice showed a high percentage of KORCre labelled neurons co-localized in tyrosine hydroxylase-positive cells in the hypothalamic arcuate nucleus. Restraint stress increased circulating prolactin to a greater degree in females. Stress-primed, but not control, mice of both sexes developed periorbital allodynia following inhalational umbellulone. Gi-DREADD activation (i.e. inhibition through Gi-coupled signalling) in KORCre neurons in the arcuate nucleus also increased circulating prolactin and repeated chemogenetic manipulation of these neurons primed mice of both sexes to umbellulone. Clustered regularly interspaced short palindromic repeats-Cas9 deletion of the arcuate nucleus KOR prevented restraint stress-induced prolactin release in female mice and priming from repeated stress episodes in both sexes. Inhibition of circulating prolactin occurred with systemic cabergoline, a dopamine D2 receptor agonist, blocked priming selectively in females. Repeated restraint stress downregulated the prolactin receptor long isoform in the trigeminal ganglia of female mice. Deletion of prolactin receptor in trigeminal ganglia by nasal clustered regularly interspaced short palindromic repeats-Cas9 targeting both prolactin receptor isoforms prevented stress-induced priming in female mice. Stress-induced activation of hypothalamic KOR increases circulating prolactin resulting in trigeminal downregulation of prolactin receptor long and pain responses to a normally innocuous TRPA1 stimulus. These are the first data that provide a mechanistic link between stress-induced hypothalamic activation and the trigeminal nociceptor effectors that produce trigeminal sensitization and migraine-like pain. This sexually dimorphic mechanism may help to explain female prevalence of migraine. KOR antagonists, currently in phase II clinical trials, may be useful as migraine preventives in both sexes, while dopamine agonists and prolactin/ prolactin receptor antibodies may improve therapy for migraine, and other stress-related neurological disorders, in females.

A prolactin-dependent sexually dimorphic mechanism of migraine chronification.

Determination of possible sex differences in mechanisms promoting migraine progression and the contribution of prolactin and the prolactin long (PRLR-L) and short (PRLR-S) receptor isoforms. The majority of patients with chronic migraine and medication overuse headache are female. Prolactin is present at higher levels in women and increases migraine. Prolactin signaling at the PRLR-S selectively sensitizes nociceptors in female rodents, while expression of the PRLR-L is protective. Medication overuse headache was modeled by repeated sumatriptan administration in male and female mice. Periorbital and hindpaw cutaneous allodynia served as a surrogate of migraine-like pain. PRLR-L and PRLR-S isoforms were measured in the trigeminal ganglion with western blotting. Possible co-localization of PRLR with serotonin 5HT1B and 5HT1D receptors was determined with RNAscope. Cabergoline, a dopamine receptor agonist that inhibits circulating prolactin, was co-administered with sumatriptan. Nasal administration of CRISPR/Cas9 plasmid was used to edit expression of both PRLR isoforms. PRLR was co-localized with 5HT1B or 5HT1D receptors in the ophthalmic region of female trigeminal ganglion. A single injection of sumatriptan increased serum PRL levels in female mice. Repeated sumatriptan promoted cutaneous allodynia in both sexes but down-regulated trigeminal ganglion PRLR-L, without altering PRLR-S, only in females. Co-administration of sumatriptan with cabergoline prevented allodynia and down-regulation of PRLR-L only in females. CRISPR/Cas9 editing of both PRLR isoforms in the trigeminal ganglion prevented sumatriptan-induced periorbital allodynia in females. We identified a sexually dimorphic mechanism of migraine chronification that involves down-regulation of PRLR-L and increased signaling of circulating prolactin at PRLR-S. These studies reveal a previously unrecognized neuroendocrine mechanism linking the hypothalamus to nociceptor sensitization that increases the risk of migraine pain in females and suggest opportunities for novel sex-specific therapies including gene editing through nasal delivery of CRISPR/Cas9 constructs.

DAGLα Inhibition as a Non-invasive and Translational Model of Episodic Headache.

Recent findings suggested that Clinical Endocannabinoid Deficiency underlies the pathophysiology of pain disorders, including migraine and headache. In models of medication overuse headache induced by sustained administration of sumatriptan or morphine, 2-AG levels were selectively depleted in the periaqueductal gray (PAG) and anandamide (AEA) increased in the cortex suggesting distinct regulation of the endocannabinoid system during headache pain. These results led to the hypothesis that blockade of DAGL, to reduce 2-AG levels would induce headache-like behaviors as a new, translationally relevant model of episodic headache. Our study investigated whether non-selective and selective blockade of DAGL, the main biosynthetic enzyme for 2-AG, induced periorbital and hind-paw allodynia, photophobia, anxiety-like behaviors, responsivity to abortive anti-migraine agents, and 2-AG/AEA levels. Injection of non-selective DAGL (DH376, 10 mg/kg, IP) and selective DAGLα (LEI106, 20 mg/kg, IP) inhibitors, but not DAGLβ agents, induced facial sensitivity in 100% and ∼60% of female and male rats, respectively, without induction of peripheral sensitivity. Notably, male rats showed significantly less sensitivity than female rats after DAGLα inhibition, suggesting sexual dimorphism in this mechanism. Importantly, LEI106 induced periorbital allodynia was attenuated by administration of the clinically available abortive antimigraine agents, sumatriptan and olcegepant. Selective DAGLα inhibition induced significant photophobia as measured by the light-dark box, without anxiety like behaviors or changes in voluntary movement. Analysis of AEA and 2-AG levels at the time of peak pain sensitivity revealed reductions in 2-AG in the visual cortex and periaqueductal gray (PAG), without altering anandamide or significantly increasing diacylglycerol levels. These results provide foundational evidence for DAGL-2AG in the induction of headache-like pain and photophobia without extracephalic allodynia, thus modeling the clinical episodic migraine. Mechanistically, behavioral measures of headache sensitivity after DAGL inhibition suggests that reduced 2-AG signaling in the cortex and PAG, but not the trigeminal nucleus caudalis or trigeminal ganglia, drives headache initiation. Therefore, episodic DAGL inhibition, which reduces the time, cost, and invasiveness of currently accepted models of headache, may fill the need for episodic migraine/headache models mirroring clinical presentation. Moreover, use of this approach may provide an avenue to study the transition from episodic to chronic headache.