Nociceptors are functionally male or female: from mouse to monkey to man.

The prevalence of many pain conditions often differs between sexes. In addition to such quantitative distinctions, sexual dimorphism may also be qualitative reflecting differences in mechanisms that promote pain in men and women. A major factor that influences the likelihood of pain perception is the threshold for activation of nociceptors. Peripheral nociceptor sensitization has been demonstrated to be clinically relevant in many pain conditions. Whether peripheral nociceptor sensitization can occur in a sexually dimorphic fashion, however, has not been extensively studied. To address this fundamental knowledge gap, we used patch clamp electrophysiology to evaluate the excitability of dorsal root ganglion neurones from male or female rodents, non-human primates, and humans following exposure to putative sensitizing agents. Previous studies from our laboratory, and others, have shown that prolactin promotes female-selective pain responses in rodents. Consistent with these observations, dorsal root ganglion neurones from female, but not male, mice were selectively sensitized by exposure to prolactin. The sensitizing action of prolactin was also confirmed in dorsal root ganglion neurones from a female macaque monkey. Critically, neurones recovered from female, but not male, human donors were also selectively sensitized by prolactin. In the course of studies of sleep and pain, we unexpectedly observed that an orexin antagonist could normalize pain responses in male animals. We found that orexin B produced sensitization of male, but not female, mouse, macaque, and human dorsal root ganglion neurones. Consistent with functional responses, increased prolactin receptor and orexin receptor 2 expression was observed in female and male mouse dorsal root ganglia, respectively. Immunohistochemical interrogation of cultured human sensory neurones and whole dorsal root ganglia also suggested increased prolactin receptor expression in females and orexin receptor 2 expression in males. These data reveal a functional double dissociation of nociceptor sensitization by sex, which is conserved across species and is likely directly relevant to human pain conditions. To our knowledge, this is the first demonstration of functional sexual dimorphism in human sensory neurones. Patient sex is currently not a common consideration for the choice of pain therapy. Precision medicine, based on patient sex could improve therapeutic outcomes by selectively targeting mechanisms promoting pain in women or men. Additional implications of these findings are that the design of clinical trials for pain therapies should consider the proportions of male or female patients enrolled. Lastly, re-examination of selected past failed clinical trials with subgroup analysis by sex may be warranted.

Opposing Effects on Descending Control of Nociception by µ and κ Opioid Receptors in the Anterior Cingulate Cortex.

BACKGROUND: The efficiency of descending pain modulation, commonly assessed with the conditioned pain modulation procedure, is diminished in patients with chronic pain. The authors hypothesized that the efficiency of pain modulation is controlled by cortical opioid circuits. METHODS: This study evaluated the effects of µ opioid receptor activation in the anterior cingulate cortex on descending control of nociception, a preclinical correlate of conditioned pain modulation, in male Sprague-Dawley rats with spinal nerve ligation-induced chronic pain or in sham-operated controls. Additionally, the study explored the consequences of respective activation or inhibition of κ opioid receptor in the anterior cingulate cortex of naive rats or animals with neuropathic pain. Descending control of nociception was measured as the hind paw withdrawal response to noxious pressure (test stimulus) in the absence or presence of capsaicin injection in the forepaw (conditioning stimulus). RESULTS: Descending control of nociception was diminished in the ipsilateral, but not contralateral, hind paw of rats with spinal nerve ligation. Bilateral administration of morphine in the anterior cingulate cortex had no effect in shams but restored diminished descending control of nociception without altering hypersensitivity in rats with neuropathic pain. Bilateral anterior cingulate cortex microinjection of κ opioid receptor antagonists, including nor-binaltorphimine and navacaprant, also re-established descending control of nociception in rats with neuropathic pain without altering hypersensitivity and with no effect in shams. Conversely, bilateral injection of a κ opioid receptor agonist, U69,593, in the anterior cingulate cortex of naive rats inhibited descending control of nociception without altering withdrawal thresholds. CONCLUSIONS: Anterior cingulate cortex κ opioid receptor activation therefore diminishes descending control of nociception both in naive animals and as an adaptive response to chronic pain, likely by enhancing net descending facilitation. Descending control of nociception can be restored by activation of µ opioid receptors in the anterior cingulate cortex, but also by κ opioid receptor antagonists, providing a nonaddictive alternative to opioid analgesics. Navacaprant is now in advanced clinical trials.

Evaluation of outcomes of calcitonin gene-related peptide (CGRP)-targeting therapies for acute and preventive migraine treatment based on patient sex.

BACKGROUND: Women show increased prevalence and severity of migraine compared to men. Whether small molecule calcitonin gene-related peptide receptor (CGRP-R) antagonists (i.e., gepants) and monoclonal antibodies targeting either the CGRP-R or the CGRP peptide might show sexually dimorphic outcomes for acute and preventive therapy has not been established. METHODS: We conducted a subpopulation analysis of available published data from FDA reviews to evaluate potential sex differences in the response rates of ubrogepant, rimegepant and zavegepant for acute migraine therapy. Available data from FDA reviews of erenumab, fremanezumab, galcanezumab and eptinezumab, approved CGRP-R and CGRP monoclonal antibodies and of atogepant were examined for prevention outcomes based on patient sex. Preventive outcomes were analyzed separately for patients with episodic migraine and chronic migraine. RESULTS: In women, the three approved gepants produced statistically significant drug effects regardless of dose tested on the FDA mandated co-primary endpoints, the proportion of patients achieving two-hour pain-freedom and the proportion of patients free of their most bothersome symptom at two hours post-dose. In women, the average placebo-subtracted two-hour pain-freedom proportion was 9.5% (CI: 7.4 to 11.6) and the average numbers needed to treat was 11. The free from most bothersome symptom at two hours outcomes were also significant in women. The gepant drugs did not reach statistically significant effects on the two-hour pain-freedom endpoint in the men, with an average drug effect of 2.8% (CI: -2.5 to 8.2) and an average number needed to treat of 36. For freedom from most bothersome symptom at two hours post-dose endpoint, differences were not significant in male patients. The treatment effect in each of the gepant studies was always numerically greater in women than in men. In evaluation of prevention outcomes with the antibodies or atogepant using the change from the specified primary endpoint (e.g., monthly migraine days), the observed treatment effect for episodic migraine patients almost always favored drug over placebo in both women and men. For chronic migraine patients the treatment effects of antibodies were similar in men and women and always favored the drug treated group.Conclusion/Interpretation: Small molecule CGRP-R antagonists are effective in acute migraine therapy in women but available data do not demonstrate effectiveness in men. CGRP-targeting therapies are effective for migraine prevention in both male and female episodic migraine patients but possible sex differences remain uncertain. In male and female chronic migraine patients, CGRP/CGRP-R antibodies were similarly effective. The data highlight possible differential effects of CGRP targeted therapies in different patient populations and the need for increased understanding of CGRP neurobiology in men and women.

Chronic pain recruits hypothalamic dynorphin/kappa opioid receptor signalling to promote wakefulness and vigilance.

Increased vigilance in settings of potential threats or in states of vulnerability related to pain is important for survival. Pain disrupts sleep and conversely, sleep disruption enhances pain, but the underlying mechanisms remain unknown. Chronic pain engages brain stress circuits and increases secretion of dynorphin, an endogenous ligand of the kappa opioid receptor (KOR). We therefore hypothesized that hypothalamic dynorphin/KOR signalling may be a previously unknown mechanism that is recruited in pathological conditions requiring increased vigilance. We investigated the role of KOR in wakefulness, non-rapid eye movement (NREM) sleep and rapid eye movement (REM) sleep in freely moving naïve mice and in mice with neuropathic pain induced by partial sciatic nerve ligation using EEG/EMG recordings. Systemic continuous administration of U69,593, a KOR agonist, over 5 days through an osmotic minipump decreased the amount of NREM and REM sleep and increased sleep fragmentation in naïve mice throughout the light-dark sleep cycle. We used KORcre mice to selectively express a Gi-coupled designer receptor activated by designer drugs (Gi-DREADD) in KORcre neurons of the hypothalamic paraventricular nucleus, a key node of the hypothalamic-pituitary-adrenal stress response. Sustained activation of Gi-DREADD with clozapine-N-oxide delivered in drinking water over 4 days, disrupted sleep in these mice in a similar way as systemic U69,593. Mice with chronic neuropathic pain also showed disrupted NREM and total sleep that was normalized by systemic administration of two structurally different KOR antagonists, norbinaltorphimine and NMRA-140, currently in phase II clinical development, or by CRISPR/Cas9 editing of paraventricular nucleus KOR, consistent with endogenous KOR activation disrupting sleep in chronic pain. Unexpectedly, REM sleep was diminished by either systemic KOR antagonist or by CRISPR/Cas9 editing of paraventricular nucleus KOR in sham-operated mice. Our findings reveal previously unknown physiological and pathophysiological roles of dynorphin/KOR in eliciting arousal. Physiologically, dynorphin/KOR signalling affects transitions between sleep stages that promote REM sleep. Furthermore, while KOR antagonists do not promote somnolence in the absence of pain, they normalized disrupted sleep in chronic pain, revealing a pathophysiological role of KOR signalling that is selectively recruited to promote vigilance, increasing chances of survival. Notably, while this mechanism is likely beneficial in the short-term, disruption of the homeostatic need for sleep over longer periods may become maladaptive resulting in sustained pain chronicity. A novel approach for treatment of chronic pain may thus result from normalization of chronic pain-related sleep disruption by KOR antagonism.

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.

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.

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.

Chronicling changes in the somatosensory neurons after peripheral nerve injury.

Current drug discovery efforts focus on identifying lead compounds acting on a molecular target associated with an established pathological state. Concerted molecular changes that occur in specific cell types during disease progression have generally not been identified. Here, we used constellation pharmacology to investigate rat dorsal root ganglion neurons using two models of peripheral nerve injury: chronic constriction injury (CCI) and spinal nerve ligation (SNL). In these well-established models of neuropathic pain, we show that the onset of chronic pain is accompanied by a dramatic, previously unreported increase in the number of bradykinin-responsive neurons, with larger increases observed after SNL relative to CCI. To define the neurons with altered expression, we charted the temporal course of molecular changes following 1, 3, 6, and 14 d after SNL injury and demonstrated that specific molecular changes have different time courses during the progression to a pain state. In particular, ATP receptors up-regulated on day 1 postinjury, whereas the increase in bradykinin receptors was gradual after day 3 postinjury. We specifically tracked changes in two subsets of neurons: peptidergic and nonpeptidergic nociceptors. Significant increases occurred in ATP responses in nAChR-expressing isolectin B4+ nonpeptidergic neurons 1 d postinjury, whereas peptidergic neurons did not display any significant change. We propose that remodeling of ion channels and receptors occurs in a concerted and cell-specific manner, resulting in the appearance of bradykinin-responsive neuronal subclasses that are relevant to chronic pain.

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.

Preclinical assessment of onabotulinumtoxinA for the treatment of mild traumatic brain injury-related acute and persistent post-traumatic headache.

OBJECTIVE: Investigation of onabotulinumtoxinA in a murine model of acute and persistent post-traumatic headache. METHODS: Mild traumatic brain injury was induced with a weight drop method. Periorbital and hindpaw cutaneous allodynia were measured for 14 days. Mice were then exposed to bright light stress and allodynia was reassessed. OnabotulinumtoxinA (0.5 U) was injected subcutaneously over the cranial sutures at different post-injury time points. RESULTS: After milt traumatic brain injury, mice exhibited periorbital and hindpaw allodynia that lasted for approximately 14 days. Allodynia could be reinstated on days 14-67 by exposure to stress only in previously injured mice. OnabotulinumtoxinA administration at 2 h after mild traumatic brain injury fully blocked both transient acute and stress-induced allodynia up to day 67. When administered 72 h post-mild traumatic brain injury, onabotulinumtoxinA reversed acute allodynia, but only partially prevented stress-induced allodynia. OnabotulinumtoxinA administration at day 12, when initial allodynia was largely resolved, produced incomplete and transient prevention of stress-induced allodynia. The degree of acute allodynia correlated positively with subsequent stress-induced allodynia. CONCLUSION: Mild traumatic brain injury induced transient headache-like pain followed by long lasting sensitization and persistent vulnerability to a normally innocuous stress stimulus, respectively modeling acute and persistent post-traumatic headache.. Administration of onabotulinumtoxinA following the resolution of acute post-traumatic headache diminished persistent post-traumatic headache but the effects were transient, suggesting that underlying persistent mild traumatic brain injury-induced maladaptations were not reversed. In contrast, early onabotulinumtoxinA administration fully blocked both acute post-traumatic headache as well as the transition to persistent post-traumatic headache suggesting prevention of neural adaptations that promote vulnerability to headache-like pain. Additionally, the degree of acute post-traumatic headache was predictive of risk of persistent post-traumatic headache.

Cutaneous heat and light-induced pain thresholds in post-traumatic headache attributed to mild traumatic brain injury.

OBJECTIVE: The purpose of this study was to characterize cutaneous heat and light-induced pain thresholds in people with post-traumatic headache (PTH) compared with healthy controls (HCs). BACKGROUND: Photophobia and allodynia are common in PTH, and there is emerging evidence to support multimodal sensory dysfunction. METHODS: In this age- and sex-matched cohort study, individuals with PTH (n = 20) and HCs (n = 20), aged 18-65 years, were recruited from an institutional database of research volunteers, from the concussion clinic, and via the use of approved flyers posted on the Mayo Clinic Campus in Scottsdale, Arizona. Participants were assessed using the Allodynia Symptom Checklist (ASC-12), Photosensitivity Assessment Questionnaire (PAQ), State Trait Anxiety Inventory (STAI), and Beck Depression Inventory (BDI). Quantitative sensory testing quantified heat pain thresholds. A light stimulation device quantified light-induced pain thresholds. Subsequently, heat pain thresholds were obtained immediately, 10, and 40 min after a bright light stressor. RESULTS: The mean photophobia symptom severity score, based on the PAQ, was higher in participants with PTH compared with HCs, mean 0.62 (SD = 0.25) versus mean 0.24 (SD = 0.24), p < 0.001. Light-induced pain thresholds were lower in participants with PTH (median = 90.5 lux and quartiles = 17.8 to 378.5) compared with HCs (median = 863.5 lux and quartiles = 519.9 to 4906.5) and were independent from BDI and STAI (p < 0.001). Allodynia scores did not differ between participants with PTH and HCs after adjusting for BDI and STAI scores. Baseline forehead heat pain thresholds were not different, participants with PTH mean 41.9°C (SD = 0.89) versus HCs mean 44.3°C (SD = 0.89), p = 0.061; however, forearm heat pain thresholds were lower in participants with PTH compared with HCs, mean 40.8°C (SD = 0.80) versus mean 44.4°C (SD = 0.80), p = 0.002. The forehead heat pain threshold change from baseline post bright light stressor in participants with PTH versus HCs was different immediately (mean -1.2 (SD = 0.53), p = 0.025), 10 min (mean -1.8 (SD = 0.74), p = 0.015), and 40 min (mean -1.8 (SD = 0.88), p = 0.047). The forearm heat pain threshold change immediately post bright light stressor in participants with PTH versus HCs was different, mean -1.9°C (SD = 0.58), p = 0.001, however, not different at 10 and 40 min. CONCLUSIONS: Photophobia is higher and light-induced pain thresholds are lower in participants with PTH. Exposure to a light stressor reduced heat pain thresholds in participants with PTH immediately post bright light stressor, but not in HCs. This study provides evidence for multimodal sensory dysfunction in people with PTH.

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.

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.

Evaluation of green light exposure on headache frequency and quality of life in migraine patients: A preliminary one-way cross-over clinical trial.

BACKGROUND: Pharmacological management of migraine can be ineffective for some patients. We previously demonstrated that exposure to green light resulted in antinociception and reversal of thermal and mechanical hypersensitivity in rodent pain models. Given the safety of green light emitting diodes, we evaluated green light as a potential therapy in patients with episodic or chronic migraine. MATERIAL AND METHODS: We recruited (29 total) patients, of whom seven had episodic migraine and 22 had chronic migraine. We used a one-way cross-over design consisting of exposure for 1-2 hours daily to white light emitting diodes for 10 weeks, followed by a 2-week washout period followed by exposure for 1-2 hours daily to green light emitting diodes for 10 weeks. Patients were allowed to continue current therapies and to initiate new treatments as directed by their physicians. Outcomes consisted of patient-reported surveys. The primary outcome measure was the number of headache days per month. Secondary outcome measures included patient-reported changes in the intensity and frequency of the headaches over a two-week period and other quality of life measures including ability to fall and stay asleep, and ability to perform work. Changes in pain medications were obtained to assess potential reduction. RESULTS: When seven episodic migraine and 22 chronic migraine patients were analyzed as separate cohorts, white light emitting diodes produced no significant change in headache days in either episodic migraine or chronic migraine patients. Combining data from the episodic migraine and chronic migraine groups showed that white light emitting diodes produced a small, but statistically significant reduction in headache days from (days ± SEM) 18.2 ± 1.8 to 16.5 ± 2.01 days. Green light emitting diodes resulted in a significant decrease in headache days from 7.9 ± 1.6 to 2.4 ± 1.1 and from 22.3 ± 1.2 to 9.4 ± 1.6 in episodic migraine and chronic migraine patients, respectively. While some improvement in secondary outcomes was observed with white light emitting diodes, more secondary outcomes with significantly greater magnitude including assessments of quality of life, Short-Form McGill Pain Questionnaire, Headache Impact Test-6, and Five-level version of the EuroQol five-dimensional survey without reported side effects were observed with green light emitting diodes. Conclusions regarding pain medications reduction with green light emitting diode exposure were not possible. No side effects of light therapy were reported. None of the patients in the study reported initiation of new therapies. DISCUSSION: Green light emitting diodes significantly reduced the number of headache days in people with episodic migraine or chronic migraine. Additionally, green light emitting diodes significantly improved multiple secondary outcome measures including quality of life and intensity and duration of the headache attacks. As no adverse events were reported, green light emitting diodes may provide a treatment option for those patients who prefer non-pharmacological therapies or may be considered in complementing other treatment strategies. Limitations of this study are the small number of patients evaluated. The positive data obtained support implementation of larger clinical trials to determine possible effects of green light emitting diode therapy.This study is registered with clinicaltrials.gov under NCT03677206.

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.

A new hypothesis linking oxytocin to menstrual migraine.

OBJECTIVE: To highlight the emerging understanding of oxytocin (OT) and oxytocin receptors (OTRs) in modulating menstrual-related migraine (MRM). BACKGROUND: MRM is highly debilitating and less responsive to therapy, and attacks are of longer duration than nonmenstrually related migraine. A clear understanding of the mechanisms underlying MRM is lacking. METHODS: We present a narrative literature review on the developing understanding of the role of OT and the OTR in MRM. Literature on MRM on PubMed/MEDLINE database including clinical trials and basic science publications was reviewed using specific keywords. RESULTS: OT is a cyclically released hypothalamic hormone/neurotransmitter that binds to the OTR resulting in inhibition of trigeminal neuronal excitability that can promote migraine pain including that of MRM. Estrogen regulates OT release as well as expression of the OTR. Coincident with menstruation, levels of both estrogen and OT decrease. Additionally, other serum biochemical factors, including magnesium and cholesterol, which positively modulate the affinity of OT for OTRs, both decrease during menstruation. Thus, during menstruation, multiple menstrually associated factors may lead to decreased circulating OT levels, decreased OT affinity for OTR, and decreased expression of the trigeminal OTR. Consistent with the view of migraine as a threshold disorder, these events may collectively result in decreased inhibition promoting lower thresholds for activation of meningeal trigeminal nociceptors and increasing the likelihood of an MRM attack. CONCLUSION: Trigeminal OTR may thus be a novel target for the development of MRM therapeutics.

C-terminal modified Enkephalin-like tetrapeptides with enhanced affinities at the kappa opioid receptor and monoamine transporters.

A new series of enkephalin-like tetrapeptide analogs modified at the C-terminus by an N-(3,4-dichlorophenyl)-N-(piperidin-4-yl)propionamide (DPP) moiety were designed, synthesized, and tested for their binding affinities at opioid receptors and monoamine transporters to evaluate their potential multifunctional activity for the treatment of chronic pain. Most ligands exhibited high binding affinities in the nanomolar range at the opioid receptors with a slight delta-opioid receptor (DOR) selectivity over mu-opioid receptor (MOR) and kappa-opioid receptor (KOR) and low binding affinities in the micromolar range at the monoamine transporters, SERT and NET. Ligands of which the positions 1 and 4 were substituted by Dmt and Phe(4-X) residues, respectively, showed the excellent binding affinities at three opioid receptors. Among them, Dmt-d-Tic-Gly-Phe(4-F)-DPP was the most promising considering its excellent opioid affinities, particularly unexpected high binding affinity (Ki = 0.13 nM) at the KOR, and moderate interactions with serotonin/norepinephrine reuptake inhibitors (SNRIs). Docking studies revealed that the ligand was a good fit for the KOR binding pocket (binding score = 8,750).

Green Light Exposure Improves Pain and Quality of Life in Fibromyalgia Patients: A Preliminary One-Way Crossover Clinical Trial.

OBJECTIVE: Fibromyalgia is a functional pain disorder in which patients suffer from widespread pain and poor quality of life. Fibromyalgia pain and its impact on quality of life are not effectively managed with current therapeutics. Previously, in a preclinical rat study, we demonstrated that exposure to green light-emitting diodes (GLED) for 8 hours/day for 5 days resulted in antinociception and reversal of thermal and mechanical hypersensitivity associated with models of injury-related pain. Given the safety of GLED and the ease of its use, our objective is to administer GLED as a potential therapy to patients with fibromyalgia. DESIGN: One-way crossover clinical trial. SETTING: United States. METHOD: We enrolled 21 adult patients with fibromyalgia recruited from the University of Arizona chronic pain clinic who were initially exposed to white light-emitting diodes and then were crossed over to GLED for 1 to 2 hours daily for 10 weeks. Data were collected by using paper surveys. RESULTS: When patients were exposed to GLED, but not white light-emitting diodes, they reported a significant reduction in average pain intensity on the 10-point numeric pain scale. Secondary outcomes were assessed by using the EQ-5D-5L survey, Short-Form McGill Pain Questionnaire, and Fibromyalgia Impact Questionnaire and were also significantly improved in patients exposed to GLED. GLED therapy was not associated with any measured side effects in these patients. CONCLUSION: Although the mechanism by which GLED elicits pain reduction is currently being studied, these results supporting its efficacy and safety merit a larger clinical trial.

Supraspinal Opioid Circuits Differentially Modulate Spinal Neuronal Responses in Neuropathic Rats.

BACKGROUND: The anterior cingulate cortex and central nucleus of the amygdala connect widely with brainstem nuclei involved in descending modulation, including the rostral ventromedial medulla. Endogenous opioids in these circuits participate in pain modulation. The hypothesis was that a differential opioidergic role for the brain nuclei listed in regulation of spinal neuronal responses because separable effects on pain behaviors in awake animals were previously observed. METHODS: This study utilized in vivo electrophysiology to determine the effects of morphine microinjection into the anterior cingulate cortex, right or left central nucleus of the amygdala, or the rostral ventromedial medulla on spinal wide dynamic range neuronal responses in isoflurane-anesthetized, male Sprague-Dawley rats. Ongoing activity in the ventrobasal thalamus was also measured. In total, 33 spinal nerve ligated and 26 control age- and weight-matched control rats were used. RESULTS: Brainstem morphine reduced neuronal firing to 60-g von Frey stimulation in control rats (to 65 ± 12% of control response (means ± 95% CI), P < 0.001) with a greater inhibition in neuropathic rats (to 53 ± 17% of control response, P < 0.001). Contrasting anterior cingulate cortex morphine had only marginal modulatory effects on spinal neuronal responses with limited variance in effect between control and neuropathic rats. The inhibitory effects of morphine in the central nucleus of the amygdala were dependent on pain state and laterality; only right-side morphine reduced neuronal firing to 60-g stimulation in neuropathic rats (to 65 ± 14% of control response, P = 0.001). In addition, in neuropathic rats elevated ongoing neuronal activity in the ventral posterolateral thalamus was not inhibited by anterior cingulate cortex morphine, in contrast to evoked responses. CONCLUSIONS: Cumulatively the data support opioid modulation of evoked responses predominately through a lateralized output from the right amygdala, as well as from the brainstem that is enhanced in injured conditions. Minimal modulation of dorsal horn responses was observed after anterior cingulate cortex opioid administration regardless of injury state.