Female-selective mechanisms promoting migraine.

Sexual dimorphism has been revealed for many neurological disorders including chronic pain. Prelicinal studies and post-mortem analyses from male and female human donors reveal sexual dimorphism of nociceptors at transcript, protein and functional levels suggesting different mechanisms that may promote pain in men and women. Migraine is a common female-prevalent neurological disorder that is characterized by painful and debilitating headache. Prolactin is a neurohormone that circulates at higher levels in females and that has been implicated clinically in migraine. Prolactin sensitizes sensory neurons from female mice, non-human primates and humans revealing a female-selective pain mechanism that is conserved evolutionarily and likely translationally relevant. Prolactin produces female-selective migraine-like pain behaviors in rodents and enhances the release of calcitonin gene-related peptide (CGRP), a neurotransmitter that is causal in promoting migraine in many patients. CGRP, like prolactin, produces female-selective migraine-like pain behaviors. Consistent with these observations, publicly available clinical data indicate that small molecule CGRP-receptor antagonists are preferentially effective in treatment of acute migraine therapy in women. Collectively, these observations support the conclusion of qualitative sex differences promoting migraine pain providing the opportunity to tailor therapies based on patient sex for improved outcomes. Additionally, patient sex should be considered in design of clinical trials for migraine as well as for pain and reassessment of past trials may be warranted.

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.

Sex differences in descending control of nociception (DCN) responses after chronic orofacial pain induction in rats and the contribution of kappa opioid receptors.

Descending control of nociception (DCN), a measure of efficiency of descending pain inhibition, can be assessed in animals by the combined application of test and conditioning noxious stimuli. Evidence from pre-clinical and clinical studies indicates that this mechanism of pain control may differ between sexes and might be impaired in many chronic pain states. However, little is known about sex differences in DCN efficiency in models of acute and chronic orofacial pain. Herein, we first evaluated DCN responses in male and female rats by the applying formalin into the upper lip or capsaicin into the forepaw as the conditioning stimulus, followed by mechanical stimulation (Randall-Selitto) of the hind paw as the test stimulus. The same protocol (i.e., capsaicin in the forepaw followed by mechanical stimulation of the hind paw) was evaluated in male and female rats on day 3 after intraoral incision and on day 15 and 30 after chronic constriction injury of the infraorbital nerve (CCI-ION). Additionally, we assessed the effect of the kappa opioid receptor (KOR) antagonist Norbinaltorphimine (nor-BNI) on DCN responses of female nerve-injured rats. This study shows that naïve female rats exhibit less efficient DCN compared to males. Postoperative pain did not alter DCN responses in female and male rats, but CCI-ION induced loss of DCN responses in females but not in males. Systemic pretreatment with nor-BNI prevented the loss of DCN induced by CCI-ION in female rats. The results reveal sex differences in DCN responses and female-specific impairment of DCN following chronic orofacial pain. Moreover, the findings suggest that, at least for females, blocking KOR could be a promising therapeutic approach to prevent maladaptive changes in chronic orofacial pain.

Identification of brain areas in mice with peak neural activity across the acute and persistent phases of post-traumatic headache.

BACKGROUND: Post-traumatic headache is very common after a mild traumatic brain injury. Post-traumatic headache may persist for months to years after an injury in a substantial proportion of people. The pathophysiology underlying post-traumatic headache remains unknown but is likely distinct from other headache disorders. Identification of brain areas activated in acute and persistent phases of post-traumatic headache can provide insights into the underlying circuits mediating headache pain. We used an animal model of mild traumatic brain injury-induced post-traumatic headache and c-fos immunohistochemistry to identify brain regions with peak activity levels across the acute and persistent phases of post-traumatic headache. METHODS: Male and female C57BL/6 J mice were briefly anesthetized and subjected to a sham procedure or a weight drop closed-head mild traumatic brain injury . Cutaneous allodynia was assessed in the periorbital and hindpaw regions using von Frey filaments. Immunohistochemical c-fos based neural activity mapping was then performed on sections from whole brain across the development of post-traumatic headache (i.e. peak of the acute phase at 2 days post- mild traumatic brain injury), start of the persistent phase (i.e. >14 days post-mild traumatic brain injury) or after provocation with stress (bright light). Brain areas with consistent and peak levels of c-fos expression across mild traumatic brain injury induced post-traumatic headache were identified and included for further analysis. RESULTS: Following mild traumatic brain injury, periorbital and hindpaw allodynia was observed in both male and female mice. This allodynia was transient and subsided within the first 14 days post-mild traumatic brain injury and is representative of acute post-traumatic headache. After this acute post-traumatic headache phase, exposure of mild traumatic brain injury mice to a bright light stress reinstated periorbital and hindpaw allodynia for several hours - indicative of the development of persistent post-traumatic headache. Acute post-traumatic headache was coincident with an increase in neuronal c-fos labeling in the spinal nucleus of the trigeminal caudalis, primary somatosensory cortex, and the nucleus accumbens. Neuronal activation returned to baseline levels by the persistent post-traumatic headache phase in the spinal nucleus of the trigeminal caudalis and primary somatosensory cortex but remained elevated in the nucleus accumbens. In the persistent post-traumatic headache phase, coincident with allodynia observed following bright light stress, we observed bright light stress-induced c-fos neural activation in the spinal nucleus of the trigeminal caudalis, primary somatosensory cortex, and nucleus accumbens. CONCLUSION: Examination of mild traumatic brain injury-induced changes in peak c-fos expression revealed brain regions with significantly increased neural activity across the acute and persistent phases of post-traumatic headache. Our findings suggest mild traumatic brain injury-induced post-traumatic headache produces neural activation along pain relevant pathways at time-points matching post-traumatic headache-like pain behaviors. These observations suggest that the spinal nucleus of the trigeminal caudalis, primary somatosensory cortex, and nucleus accumbens may contribute to both the induction and maintenance of post-traumatic headache.

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

ABSTRACT: 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.

Exploring the neurobiology of the premonitory phase of migraine preclinically - a role for hypothalamic kappa opioid receptors?

BACKGROUND: The migraine premonitory phase is characterized in part by increased thirst, urination and yawning. Imaging studies show that the hypothalamus is activated in the premonitory phase. Stress is a well know migraine initiation factor which was demonstrated to engage dynorphin/kappa opioid receptors (KOR) signaling in several brain regions, including the hypothalamus. This study proposes the exploration of the possible link between hypothalamic KOR and migraine premonitory symptoms in rodent models. METHODS: Rats were treated systemically with the KOR agonist U-69,593 followed by yawning and urination monitoring. Apomorphine, a dopamine D1/2 agonist, was used as a positive control for yawning behaviors. Urination and water consumption following systemic administration of U-69,593 was also assessed. To examine if KOR activation specifically in the hypothalamus can promote premonitory symptoms, AAV8-hSyn-DIO-hM4Di (Gi-DREADD)-mCherry viral vector was microinjected into the right arcuate nucleus (ARC) of female and male KORCRE or KORWT mice. Four weeks after the injection, clozapine N-oxide (CNO) was administered systemically followed by the assessment of urination, water consumption and tactile sensory response. RESULTS: Systemic administration of U-69,593 increased urination but did not produce yawning in rats. Systemic KOR agonist also increased urination in mice as well as water consumption. Cell specific Gi-DREADD activation (i.e., inhibition through Gi-coupled signaling) of KORCRE neurons in the ARC also increased water consumption and the total volume of urine in mice but did not affect tactile sensory responses. CONCLUSION: Our studies in rodents identified the KOR in a hypothalamic region as a mechanism that promotes behaviors consistent with clinically-observed premonitory symptoms of migraine, including increased thirst and urination but not yawning. Importantly, these behaviors occurred in the absence of pain responses, consistent with the emergence of the premonitory phase before the headache phase. Early intervention for preventive treatment even before the headache phase may be achievable by targeting the hypothalamic KOR.

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.

OBJECTIVE: 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. BACKGROUND: 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. METHODS: 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. RESULTS: 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. INTERPRETATION: 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.

CGRP monoclonal antibody prevents the loss of diffuse noxious inhibitory controls (DNIC) in a mouse model of post-traumatic headache.

AIM: Determine the role of calcitonin-gene related peptide in promoting post-traumatic headache and dysregulation of central pain modulation induced by mild traumatic brain injury in mice. METHODS: Mild traumatic brain injury was induced in lightly anesthetized male C57BL/6J mice by a weight drop onto a closed and unfixed skull, which allowed free head rotation after the impact. We first determined possible alterations in the diffuse noxious inhibitory controls, a measure of net descending pain inhibition called conditioned pain modulation in humans at day 2 following mild traumatic brain injury. Diffuse noxious inhibitory control was assessed as the latency to a thermally induced tail-flick that served as the test stimulus in the presence of right forepaw capsaicin injection that provided the conditioning stimulus. Post-traumatic headache-like behaviors were assessed by the development of cutaneous allodynia in the periorbital and hindpaw regions after mild traumatic brain injury. We then determined if intraperitoneal fremanezumab, an anti-calcitonin-gene related peptide monoclonal antibody or vehicle administered 2 h after sham or mild traumatic brain injury induction could alter cutaneous allodynia or diffuse noxious inhibitory control responses on day 2 post mild traumatic brain injury. RESULTS: In naïve and sham mice, capsaicin injection into the forepaw elevated the latency to tail-flick, reflecting the antinociceptive diffuse noxious inhibitory control response. Periorbital and hindpaw cutaneous allodynia, as well as a loss of diffuse noxious inhibitory control, was observed in mice 2 days after mild traumatic brain injury. Systemic treatment with fremanezumab blocked mild traumatic brain injury-induced cutaneous allodynia and prevented the loss of diffuse noxious inhibitory controls in mice subjected to a mild traumatic brain injury. INTERPRETATION: Sequestration of calcitonin-gene related peptide in the initial stages following mild traumatic brain injury blocked the acute allodynia that may reflect mild traumatic brain injury-related post-traumatic headache and, additionally, prevented the loss of net descending inhibition within central pain modulation pathways. As loss of conditioned pain modulation has been linked to multiple persistent pain conditions, dysregulation of descending modulatory pathways may contribute to the persistence of post-traumatic headache. Additionally, evaluation of the conditioned pain modulation/diffuse noxious inhibitory controls response may serve as a biomarker of vulnerability for chronic/persistent pain. These findings suggest that early anti-calcitonin-gene related peptide intervention has the potential to be effective both for the treatment of mild traumatic brain injury-induced post-traumatic headache, as well as inhibiting mechanisms that may promote post-traumatic headache persistence.

Preclinical Assessment of the Analgesic Pharmacology of NKTR-181 in Rodents.

OBJECTIVE: Pharmacological evaluation of the mu-opioid receptor (MOR) agonist properties of NKTR-181 in rodent models. METHODS: Graded noxious stimulus intensities were used in rats to establish the antinociceptive potency and efficacy of NKTR-181 relative to morphine, fentanyl, and oxycodone. Characteristics of MOR agonist actions, as measured by antinociceptive tolerance and cross-tolerance, as well as opioid-induced hyperalgesia (OIH) and naloxone-precipitated withdrawal in NKTR-181- and morphine-dependent in mice, were compared. RESULTS: NKTR-181 showed dose- and time-related antinociception with similar maximal effects to morphine in the rat and mouse hot-water tail-flick test. No sex or species differences were observed in NKTR-181 or morphine antinociception. Rats treated with NKTR-181 and morphine exhibited decreases in both potency and maximal efficacy as nociceptive stimulus intensity was increased from a water temperature of 50 °C to 54 °C. Evaluation of antinociception at a high stimulus intensity revealed that oxycodone and fentanyl exhibited greater efficacy than either NKTR-181 or morphine. The relative potency difference between NKTR-181 and morphine across all tail-flick studies was determined to be 7.6-fold (90% confidence interval, 2.6, 21.5). The peak antinociceptive effect of NKTR-181 was delayed compared to that of the other opioids and cumulative drug effects were not observed. Repeated treatment with escalating, approximately equi-analgesic doses of NKTR-181 or morphine, produced antinociceptive tolerance and cross-tolerance. Under these pharmacological conditions, OIH and naloxone-precipitated physical dependence were similar for NKTR-181 and morphine. CONCLUSIONS: NKTR-181 had a slower onset, but similar efficacy, to morphine in the models studied supporting reduced abuse potential while maintaining analgesic effect in comparison with current opioids.

A novel, injury-free rodent model of vulnerability for assessment of acute and preventive therapies reveals temporal contributions of CGRP-receptor activation in migraine-like pain.

AIM: Development and characterization of a novel injury-free preclinical model of migraine-like pain allowing mechanistic assessment of both acute and preventive treatments. METHODS: A "two-hit" hyperalgesic priming strategy was used to induce vulnerability to a normally subthreshold challenge with umbellulone, a transient receptor potential ankyrin 1 (TRPA1) activator, in uninjured female and male C57BL/6 mice. Priming (i.e. the first hit) was induced by three consecutive daily episodes of restraint stress; repeated umbellulone was also evaluated for potential priming effects. Sixteen days after the first restraint stress, mice received inhalational umbellulone (i.e. the second hit) to elicit migraine-like pain. Medications currently used for acute or preventive migraine therapy including propranolol (a beta blocker) and sumatriptan (5HT1B/D agonist), as well as olcegepant, an experimental calcitonin gene related peptide (CGRP) receptor antagonist and nor-Binaltorphimine (nor-BNI), an experimental long-acting kappa opioid receptor (KOR) antagonist, were investigated for their efficacy to block priming and prevent or reverse umbellulone-induced allodynia in primed animals. To assess migraine-like pain, cutaneous allodynia was determined by responses to periorbital or hindpaw probing with von Frey filaments. RESULTS: Repeated restraint stress, but not umbellulone exposure, produced transient cutaneous allodynia that resolved within 16 d. Restraint stress produced long-lasting priming that persisted beyond 16 d, as demonstrated by reinstatement of cutaneous allodynia following inhalational umbellulone challenge. Pretreatment with propranolol or nor-BNI prior to restraint stress prevented both transient cutaneous allodynia and priming, demonstrated by a lack of umbellulone-induced cutaneous allodynia. Following establishment of restraint stress priming, olcegepant, but not propranolol or nor-BNI, prevented umbellulone-induced cutaneous allodynia. When administered 1 h after umbellulone, sumatriptan, but not olcegepant, reversed umbellulone-induced cutaneous allodynia in restraint stress-primed rats. CONCLUSION: We have developed a novel injury-free model with translational relevance that can be used to study mechanisms relevant to migraine-like pain and to evaluate novel acute or preventive treatments. Restraint stress priming induced a state of vulnerability to a subthreshold stimulus that has been referred to as "latent sensitization". The development of latent sensitization could be prevented by blockade of stress pathways with propranolol or with a kappa opioid receptor antagonist. Following establishment of latent sensitization, subthreshold stimulation with umbellulone reinstated cutaneous allodynia, likely from activation of meningeal TRPA1-expressing nociceptors. Accordingly, in restraint stress-primed animals, sumatriptan reversed umbellulone-induced cutaneous allodynia, supporting peripheral sites of action, while propranolol and nor-BNI were not effective. Surprisingly, olcegepant was effective in mice with latent sensitization when given prior to, but not after, umbellulone challenge, suggesting time-dependent contributions of calcitonin gene-related peptide receptor signaling in promoting migraine-like pain in this model. Activation of the calcitonin gene-related peptide receptor participates in initiating, but has a more limited role in maintaining, pain responses, supporting the efficacy of small molecule calcitonin gene-related peptide antagonists as preventive medications. Additionally, the effectiveness of sumatriptan in reversal of established pain thus suggests modulation of additional, non-calcitonin gene-related peptide receptor-mediated nociceptive mechanisms. Kappa opioid receptor antagonists may represent a novel preventive therapy for stress-related migraine.

Characterization and preclinical evaluation of a protease activated receptor 2 (PAR2) monoclonal antibody as a preventive therapy for migraine.

AIM: Migraine pain is thought to result from activation of meningeal nociceptors that might involve dural mast cell degranulation and release of proteases and pronociceptive mediators. Tryptase, the most abundant dural mast cell protease, has been demonstrated to stimulate dural mast cells, as well as trigeminal nociceptors by activating the protease activated receptor 2. Mast cell or neuronal protease activated receptors 2 may therefore represent a novel target for migraine treatment. In this study, we characterized and evaluated a novel protease activated receptor 2 monoclonal antibody as a preventive anti-migraine pain therapy in preclinical models. METHODS: Flow cytometry, immunocytochemistry, calcium imaging, Homogeneous Time Resolved Technology (HTRF) epitope competition assay and serum pharmacokinetic (PK) assay in rats were performed to confirm the activity, specificity and in vivo stability of PAR650097, a novel anti- protease activated receptor 2 monoclonal antibody. In vivo assessment was performed in female C57BL/6J mice by evaluation of PAR650097 in preventing cutaneous allodynia elicited by (a) supradural injection of the protease activated receptor 2 agonist, Ser-Leu-Ile-Gly-Arg-Leu-amide trifluoroacetate (SLIGRL), or calcitonin gene-related (CGRP) peptide, and (b) induction of latent sensitization by priming with three daily episodes of restraint stress followed by challenge with a subthreshold inhalational exposure to umbellulone (UMB), a transient receptor potential ankyrin 1 (TRPA1) agonist. PAR650097 was administered as a pretreatment prior to the first restraint stress, umbellulone exposure, SLIGRL or calcitonin gene-related peptide injection. Additionally, fremanezumab, a calcitonin gene-related peptide antibody was administered as pre-treatment prior to supradural administration of calcitonin gene-related peptide or SLIGRL. RESULTS: In vitro, PAR650097 demonstrated rapid interaction with protease activated receptor 2, enabling it to fully inhibit protease-induced protease activated receptor 2 activation, in human and mouse cells, with high potency. Furthermore, PAR650097 was highly selective for protease activated receptor 2, demonstrating no affinity for protease activated receptor 1 protein and no functional effect on the activation of cellular protease activated receptor 1 with thrombin. In addition, PAR650097 had an acceptable PK profile, compatible with testing the effects of selective protease activated receptor 2 inhibition in vivo. In vivo, PAR650097 blocked cutaneous allodynia induced by either supradural SLIGRL or calcitonin gene-related peptide. Fremanezumab abolished cutaneous allodynia induced by supradural CGRP, and partially attenuated cutaneous allodynia induced by SLIGRL. Administration of PAR650097, before the first restraint stress episode, did not prevent the acute stress-induced cutaneous allodynia or restraint stress priming revealed by cutaneous allodynia induced by inhalational umbellulone. In contrast, PAR650097 prevented expression of cutaneous allodynia when given before the umbellulone challenge in restraint stress-primed animals. CONCLUSION: PAR650097 specifically inhibits endogenously expressed protease activated receptor 2 in human and mouse cells with high potency. This antibody has an acceptable PK profile in rodents and effectively blocked SLIGR-induced cutaneous allodynia. PAR650097 additionally prevented cutaneous allodynia induced by supradural calcitonin gene-related peptide, indicating that the protease activated receptor 2 receptor is a downstream consequence of calcitonin gene-related peptide actions. Fremanezumab effectively blocked calcitonin gene-related peptide-induced cutaneous allodynia and only partially reduced cutaneous allodynia induced by a protease activated receptor 2 activator, suggesting both calcitonin gene-related peptide-dependent and -independent mechanisms in promoting migraine pain. While PAR650097 did not prevent stress-induced cutaneous allodynia or priming, it effectively prevented cutaneous allodynia induced by a TRPA1 agonist in animals with latent sensitization. Activation of protease activated receptor 2, therefore, contributes to both calcitonin gene-related peptide-dependent and -independent mechanisms in promoting migraine-like pain. Therapeutic targeting of protease activated receptor 2 receptors may represent an anti-migraine pain strategy with a potentially broad efficacy profile.

The prolactin receptor long isoform regulates nociceptor sensitization and opioid-induced hyperalgesia selectively in females.

Pain is more prevalent in women for reasons that remain unclear. We have identified a mechanism of injury-free nociceptor sensitization and opioid-induced hyperalgesia (OIH) promoted by prolactin (PRL) in females. PRL signals through mutually inhibitory long (PRLR-L) and short (PRLR-S) receptor isoforms, and PRLR-S activation induces neuronal excitability. PRL and PRLR expression were higher in females. CRISPR-mediated editing of PRLR-L promoted nociceptor sensitization and allodynia in naïve, uninjured female mice that depended on circulating PRL. Opioids, but not trauma-induced nerve injury, decreased PRLR-L promoting OIH through activation of PRLR-S in female mice. Deletion of both PRLR-L and PRLR-S (total PRLR) prevented, whereas PRLR-L overexpression rescued established OIH selectively in females. Inhibition of circulating PRL with cabergoline, a dopamine D2 agonist, up-regulated PRLR-L and prevented OIH only in females. The PRLR-L isoform therefore confers protection against PRL-promoted pain in females. Limiting PRL/PRLR-S signaling pharmacologically or with gene therapies targeting the PRLR may be effective for reducing pain in a female-selective manner.

The opposing contribution of neurotrophin-3 and nerve growth factor to orofacial heat hyperalgesia in rats.

It has been proposed that neurotrophin-3 acts in a manner that is opposed to nerve growth factor, especially in the modulation of heat hyperalgesia. Injury to the constriction of the infraorbital nerve (CION) is a well-established model of trigeminal neuropathic pain that leads to robust heat, cold, and mechanical hyperalgesia. Here, we assessed the effect of local neurotrophin-3 treatment on CION-induced hyperalgesia, and we examined some mechanisms related to the effect of neurotrophin-3. Neurotrophin-3 (1 µg/50 µl) injected into the upper lip of CION rats caused a significant and long-lasting reduction of CION-induced heat hyperalgesia, but failed to affect cold and mechanical hyperalgesia. Increased levels of neurotrophin-3 were detected in the injured nerve at the time point that represents the peak of heat hyperalgesia. The anti-hyperalgesic effect of neurotrophin-3 was markedly reduced in the presence of an antagonist of TrkA receptors (K-252a, 1 µg/50 µl). Moreover, association of lower doses of neurotrophin-3 with an antibody anti-nerve growth factor resulted in a synergistic anti-hyperalgesic effect in CION rats. Local injection of nerve growth factor (3 µg/50 µl) or the TRPV1 agonist capsaicin (1 µg/50 µl), but not neurotrophin-3 injection (1 µg/50 µl), resulted in long-lasting facial heat hyperalgesia, which was both significantly reduced by previous neurotrophin-3 local treatment. In conclusion, we suggest that neurotrophin-3 is a potent modulator of facial heat hyperalgesia, which may exert an inhibitory influence on the trkA pathway. Neurotrophin-3 treatment may represent a promising approach, especially in pain conditions associated with increased levels of nerve growth factor.

Cannabinoids induce latent sensitization in a preclinical model of medication overuse headache.

AIM: Evaluation of cannabinoid receptor agonists in a preclinical model of medication overuse headache. METHODS: Female Sprague Dawley rats received graded intraperitoneal doses of WIN55,212-2 or Δ-9-tetrahydrocannabinol (Δ-9-THC). Antinociception (tail-flick test), catalepsy and hypomotility (open field test) and impairment of motor function (rotarod test) were assessed to establish effective dosing. Rats were then treated twice daily with equianalgesic doses of WIN55,212-2 or Δ-9-THC, or vehicle, for 7 days and cutaneous tactile sensory thresholds were evaluated during and three weeks following drug discontinuation. Rats then received a one-hour period of bright light stress (BLS) on two consecutive days and tactile sensory thresholds were re-assessed. RESULTS: WIN55,212-2 and Δ-9-THC produced antinociception as well as hypomotility, catalepsy and motor impairment. Repeated administration of WIN55,212-2 and Δ-9-THC induced generalized periorbital and hindpaw allodynia that resolved within 3 weeks after discontinuation of drug. Two episodes of BLS produced delayed and long-lasting periorbital and hindpaw allodynia selectively in rats previously treated with WIN55,212-2, and Δ-9-THC. INTERPRETATION: Cannabinoid receptor agonists including Δ-9-THC produce a state of latent sensitization characterized by increased sensitivity to stress, a presumed migraine trigger. Overuse of cannabinoids including cannabis may increase the risk of medication overuse headache in vulnerable individuals.

Blockade of peripheral endothelin receptors abolishes heat hyperalgesia and spontaneous nociceptive behavior in a rat model of facial cancer.

OBJECTIVE: To improve understanding of the pathophysiology of cancer-induced facial nociception, by evaluating the contribution of peripheral endothelin receptors in tumor-induced facial heat hyperalgesia, increased spontaneous grooming, as well as ongoing nociception in a rat model of facial cancer. DESIGN: The study was conducted using 396 rats. Facial cancer was induced by inoculating a suspension of Walker-256 cells into the rats' right vibrissal pad. Facial heat hyperalgesia and spontaneous grooming were assessed on day 6, while the conditioned place preference (CPP) test was performed on days 3-6 after tumor cells inoculation. Rats received local injections of the non-peptidic dual ETA/ETB endothelin receptors antagonist, bosentan (10 and 30 µg/50 µL), single or combined injections of peptidic ETA and ETB endothelin receptors antagonists (BQ-123 and BQ-788, at 20 ug/50 µL, each), or of lidocaine (1 mg/50 µl) and morphine (30 µg/50 µL). RESULTS: Bosentan, lidocaine and morphine local treatment all attenuated tumor-induced heat hyperalgesia (p < 0.05) and spontaneous facial grooming (p < 0.05). However, BQ-123 and BQ-788 did not modify tumor-induced heat hyperalgesia or the spontaneous facial grooming (p > 0.05). Whether this difference in effectiveness is due to receptor affinity or to pharmacokinetic factors still needs to be explored. Local injection of bosentan, lidocaine or morphine failed to control ongoing nociception, as evidenced by the absence of CPP in tumor-bearing rats (p > 0.05). CONCLUSION: Endothelins, acting through peripheral ETA and ETB receptors, may play a significant role on the development of heat hyperalgesia and increased spontaneous grooming associated to facial cancer in rats.

Blockade of endothelin receptors reduces tumor-induced ongoing pain and evoked hypersensitivity in a rat model of facial carcinoma induced pain.

Pain reported by patients with head and neck cancer is characterized as persistent pain with mechanical allodynia. Pain management is inadequate for many patients, highlighting the need for improved therapies. We examined the hypothesis that the mixed endothelin ETA and ETB receptor antagonist, bosentan, reduces tumor-induced ongoing pain and evoked hypersensitivity in a rat model of facial cancer pain. Facial cancer was induced by inoculating a suspension of Walker-256 cells into the rat's right vibrissal pad. Tumor-bearing rats developed heat and tactile hypersensitivity along with increased spontaneous grooming behavior. Systemic morphine (2.5mg/kg, s.c.) blocked tumor-induced thermal and tactile hypersensitivity, with a lower dose (0.625mg/kg, s.c.) effective only against thermal hypersensitivity. Systemic bosentan blocked tumor-induced thermal hypersensitivity only at a high (300mg/kg, p.o.) dose, but failed to modify tactile hypersensitivity. Co-administration of the low doses of bosentan and morphine resulted in improved reduction of the tumor-induced heat and tactile hypersensitivity compared to either dose alone. Bosentan (100mg/kg, p.o.) reduced spontaneous grooming and induced conditioned place preference (CPP) selectively in tumor-bearing rats, suggesting that bosentan reduces tumor-induced ongoing pain at a lower dose than required to block tumor-induced hypersensitivity. This study provides evidence that endothelins may mediate tumor-induced ongoing pain and thermal hypersensitivity. In addition, bosentan enhanced morphine's effects on blocking tumor-induced heat and tactile hypersensitivity indicating that endothelin antagonists may be beneficial therapeutic targets that can be used to manage cancer-induced facial pain with opioid-sparing effects.

Mechanisms involved in facial heat hyperalgesia induced by endothelin-1 in female rats.

OBJECTIVE: Pronociceptive responses to endothelins in the trigeminal system seem to be mediated by ETA and ETB receptors, which have been shown to be expressed in neurons of the trigeminal ganglion of humans and rats. The present study aimed to evaluate the ability of endothelin-1 (ET-1) to induce facial heat hyperalgesia in female rats, the contribution of ETA and ETB receptors to this response, as well as the mechanisms underlying heat hyperalgesia induced by ET-1. DESIGN: ET-1 (100pmol/50µL) was injected into the upper lip and heat hyperalgesia was evaluated for up to 6h. Facial heat hyperalgesia induced by ET-1 was assessed in rats pre-treated locally with BQ-123 or BQ-788 (selective ETA and ETB receptor antagonists, respectively, 30nmol/50µL); BCTC (TRPV1 receptor antagonist; 300µg/50µL); anti-NGF (3µg/50µL); K252a (TrkA inhibitor, 1µg/50µL); or in rats that received intraganglionar resiniferatoxin injection (RTX, 200ng/10µL) to promote C-fibers ablation. RESULTS: ET-1 induced facial heat hyperalgesia that persisted up to 6h and was prevented by BQ-123, BQ-788 or by intraganglionar RTX injection. Likewise, local pre-treatment with BCTC abolished ET-1 induced facial heat hyperalgesia up to 3h. Local pre-treatment with anti-NGF or K252a was effective to prevent ET-1 induced heat hyperalgesia. CONCLUSIONS: In conclusion, ET-1 is able to induce heat hyperagelsia in trigeminal primary afferents of female rats, which is mediated by ETA and ETB receptors. Activation of TRPV1 receptors and NGF-signaling pathways may contribute to heat hyperalgesia induced by ET-1.

Facial pain and anxiety-like behavior are reduced by pregabalin in a model of facial carcinoma in rats.

Pain and anxiety are common symptoms in head and neck cancer patients. The anticonvulsant pregabalin has therapeutic indication for the treatment of pain and anxiety, and may represent a useful drug for both conditions. Thus, the aim of this study was to investigate the relationship between pain and anxiety in rats with facial carcinoma, as the influence of pregabalin treatment in both aspects. Facial carcinoma was induced by subcutaneous inoculation of Walker-256 tumor cells in the vibrissa pad of Wistar rats. On day 6 after inoculation spontaneous facial grooming and conditioned place preference were assessed as non-evoked pain measurements and facial mechanical hyperalgesia were assessed 3 and 6 days after tumor cells inoculation. Moreover, anxiety-like behavior was evaluated on the elevated plus maze and light-dark transition tests at the same time points. The effect of pregabalin treatment (30 mg/kg, p.o.) was evaluated in all tests. Our results demonstrated that pregabalin treatment reduced the spontaneous facial grooming and induced conditioned place preference 6 days post tumor inoculation. Tumor-bearing rats developed mechanical hyperalgesia starting 3 days post tumor induction, which was also significant on day 6, but the anxiety-like behavior was detected only in tumor-bearing rats that developed mechanical hyperalgesia and only six days after tumor cells inoculation. Both, the mechanical hyperalgesia and the anxiety-like behavior related to the tumor were significantly reduced by pregabalin treatment on day 6. Pregabalin treatment resulted in antinociceptive and anxiolytic-like effects on facial tumor-bearing rats and may represent a promising therapeutic option for cancer patients.