Kopruszinski, Caroline

AbstractMigraine 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.

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.

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.

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.

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 (5HT1 B/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.

Female selective nociceptor sensitization and opioid-induced hyperalgesia result from an imbalance of prolactin receptor isoforms.

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.

Background Stress is the most commonly reported migraine trigger. Dynorphin, an endogenous opioid peptide acting preferentially at kappa opioid receptors (KORs), is a key mediator of stress responses. The aim of this study was to use an injury-free rat model of functional cephalic pain with features of migraine and medication overuse headache (MOH) to test the possible preventive benefit of KOR blockade on stress-induced cephalic pain. Methods Following sumatriptan priming to model MOH, rats were hyper-responsive to environmental stress, demonstrating delayed cephalic and extracephalic allodynia and increased levels of CGRP in the jugular blood, consistent with commonly observed clinical outcomes during migraine. Nor-binaltorphimine (nor-BNI), a long-acting KOR antagonist or CYM51317, a novel short-acting KOR antagonist, were given systemically either during sumatriptan priming or immediately before environmental stress challenge. The effects of KOR blockade in the amygdala on stress-induced allodynia was determined by administration of nor-BNI into the right or left central nucleus of the amygdala (CeA). Results KOR blockade prevented both stress-induced allodynia and increased plasma CGRP. Stress increased dynorphin content and phosphorylated KOR in both the left and right CeA in sumatriptan-primed rats. However, KOR blockade only in the right CeA prevented stress-induced cephalic allodynia as well as extracephalic allodynia, measured in either the right or left hindpaws. U69,593, a KOR agonist, given into the right, but not the left, CeA, produced allodynia selectively in sumatriptan-primed rats. Both stress and U69,593-induced allodynia were prevented by right CeA U0126, a mitogen-activated protein kinase inhibitor, presumably acting downstream of KOR. Conclusions Our data reveal a novel lateralized KOR circuit that mediated stress-induced cutaneous allodynia and increased plasma CGRP in an injury-free model of functional cephalic pain with features of migraine and medication overuse headache. Selective, small molecule, orally available, and reversible KOR antagonists are currently in development and may represent a novel class of preventive therapeutics for migraine.

Objective The objective of this study was the determination of the role of calcitonin gene-related peptide (CGRP) in the induction of medication overuse headache (MOH)-related migraine in an injury-free preclinical model. Methods Rats were primed by a 7-day period of exposure to acute migraine therapies including sumatriptan and morphine. After an additional 14-day drug-free period, rats were exposed to putative migraine triggers including bright light stress (BLS) or nitric oxide (NO) donor in the presence or absence of TEV48125, a fully humanized CGRP antibody. Cutaneous allodynia (CA) was used as an outcome measure and CGRP blood and cerebrospinal fluid (CSF) levels were measured. Results BLS and NO donor challenge evoked delayed, long-lasting CA selectively in rats that were previously treated with sumatriptan or morphine. BLS produced a significant increase in CGRP in the plasma, but not CSF, in animals that were previously exposed to sumatriptan compared to saline controls. TEV48125 did not modify baseline tactile thresholds or produce behavioral side effects, but significantly inhibited both BLS- and NO donor-induced CA in animals that were previously primed with sumatriptan or morphine; an isotype control protein that does not bind CGRP had no effect. Interpretation These data suggest that acute migraine medications may promote MOH in susceptible individuals through CGRP-dependent mechanisms and that anti-CGRP antibodies may be a useful clinical strategy for the treatment of MOH.