PACAP activates MRGPRX2 on meningeal mast cells to drive migraine-like pain.

Migraine ranks among the most prevalent disorders worldwide, leading to disability and decreased quality of life in patients. Recently, neurogenic inflammation has been recognized as a potential underlying pathology contributing to the migraine pain pathway. Mast cells reside in the meninges and have been implicated in contributing to the pathophysiology of migraine. Here we report for the first time that the mouse Mas-Related G-protein-coupled Receptor B2 (MrgprB2), is expressed on meningeal connective tissue mast cells and contributes to Pituitary Adenylate Cyclase Activating Peptide (PACAP)-induced migraine-like pain behavior. We also found that PACAP was able to dose-dependently lead to enzyme release from human mast cells via activation of MRGPRX2; the human homolog of MrgprB2. Using a transgenic MRGPRX2 mouse, we observed significant increases in PACAP-induced migraine-like pain behavior in MRGPRX2+ mice vs mice lacking the receptor. These results reveal both MrgprB2 and MRGPRX2 as important contributors to neuropeptide-induced migraine pain.

Roles of mast cells and their interactions with the trigeminal nerve in migraine headache.

Migraine pain is characterized by an intense, throbbing pain in the head area and possesses complex pathological and physiological origins. Among the various factors believed to contribute to migraine are mast cells (MCs), resident tissue immune cells that are closely associated with pain afferents in the meninges. In this review, we aim to examine and discuss recent findings on the individual roles of MCs and the trigeminal nerve in migraine, as well as the various connections between their mechanisms with an emphasis on the contributions those relationships make to migraine. This is seen through MC release of histamine, among other compounds, and trigeminal nerve release of calcitonin-gene-related-peptide (CGRP) and pituitary adenylate cyclase activating peptide-38 (PACAP-38), which are peptides that are thought to contribute to migraine. Secondly, we illustrate the bi-directional relationship of neurogenic inflammation as well as highlight the role of MCs and their effect on the trigeminal nerve in migraine mechanisms. Lastly, we discuss potential new targets for clinical interventions of MC- and trigeminal nerve-mediated migraine, and present future perspectives of mechanistic and translational research.

Peripheral mechanisms of chronic pain.

Acutely, pain serves to protect us from potentially harmful stimuli, however damage to the somatosensory system can cause maladaptive changes in neurons leading to chronic pain. Although acute pain is fairly well controlled, chronic pain remains difficult to treat. Chronic pain is primarily a neuropathic condition, but studies examining the mechanisms underlying chronic pain are now looking beyond afferent nerve lesions and exploring new receptor targets, immune cells, and the role of the autonomic nervous system in contributing chronic pain conditions. The studies outlined in this review reveal how chronic pain is not only confined to alterations in the nervous system and presents findings on new treatment targets and for this debilitating disease.

Identification of the NRF2 transcriptional network as a therapeutic target for trigeminal neuropathic pain.

Trigeminal neuralgia, historically dubbed the "suicide disease," is an exceedingly painful neurologic condition characterized by sudden episodes of intense facial pain. Unfortunately, the only U.S. Food and Drug Administration (FDA)-approved medication for trigeminal neuralgia carries substantial side effects, with many patients requiring surgery. Here, we identify the NRF2 transcriptional network as a potential therapeutic target. We report that cerebrospinal fluid from patients with trigeminal neuralgia accumulates reactive oxygen species, several of which directly activate the pain-transducing channel TRPA1. Similar to our patient cohort, a mouse model of trigeminal neuropathic pain also exhibits notable oxidative stress. We discover that stimulating the NRF2 antioxidant transcriptional network is as analgesic as inhibiting TRPA1, in part by reversing the underlying oxidative stress. Using a transcriptome-guided drug discovery strategy, we identify two NRF2 network modulators as potential treatments. One of these candidates, exemestane, is already FDA-approved and may thus be a promising alternative treatment for trigeminal neuropathic pain.

The role of Mrgprs in pain.

Owing to their functional diversity, the Mas-related G-protein-coupled receptor (Mrgpr) family has a role in both itch and pain modulation. While primarily linked to pruritis, Mrgprs were originally characterized in small-diameter nociceptive neurons of dorsal root ganglia (DRG) and trigeminal ganglia. This review will focus on the role Mrgpr's have in pain physiology, discussing recent discoveries as well as how Mrgpr's may provide a new target for the treatment of pathological pain.

A Pungent and Painful Toxin.

In this issue of Cell, King et al. (2019) have discovered a cell penetrating peptide isolated from the venom of the Australian Black Rock scorpion that activates the TRPA1 receptor in a unique way to induce pain. Their findings offer new insights into how animals evolved venoms to target specific ion channel functions.

A Mast-Cell-Specific Receptor Mediates Neurogenic Inflammation and Pain.

Mast cells can be found in close proximity to peripheral nerve endings where, upon activation, they release a broad range of pro-inflammatory cytokines and chemokines. However, the precise mechanism underlying this so-called neurogenic inflammation and associated pain has remained elusive. Here we report that the mast-cell-specific receptor Mrgprb2 mediates inflammatory mechanical and thermal hyperalgesia and is required for recruitment of innate immune cells at the injury site. We also found that the neuropeptide substance P (SP), an endogenous agonist of Mrgprb2, facilitates immune cells' migration via Mrgprb2. Furthermore, SP activation of the human mast cell led to the release of multiple pro-inflammatory cytokines and chemokines via the human homolog MRGPRX2. Surprisingly, the SP-mediated inflammatory responses were independent of its canonical receptor, neurokinin-1 receptor (NK-1R). These results identify Mrgprb2/X2 as an important neuroimmune modulator and a potential target for treating inflammatory pain.

Identification of a bilirubin receptor that may mediate a component of cholestatic itch.

Various pathologic conditions result in jaundice, a yellowing of the skin due to a buildup of bilirubin. Patients with jaundice commonly report experiencing an intense non-histaminergic itch. Despite this association, the pruritogenic capacity of bilirubin itself has not been described, and no bilirubin receptor has been identified. Here, we demonstrate that pathophysiologic levels of bilirubin excite peripheral itch sensory neurons and elicit pruritus through MRGPRs, a family of G-protein coupled receptors expressed in primary sensory neurons. Bilirubin binds and activates two MRGPRs, mouse MRGPRA1 and human MRGPRX4. In two mouse models of pathologic hyperbilirubinemia, we show that genetic deletion of either Mrgpra1 or Blvra, the gene that encodes the bilirubin-producing enzyme biliverdin reductase, attenuates itch. Similarly, plasma isolated from hyperbilirubinemic patients evoked itch in wild-type animals but not Mrgpra1-/- animals. Removing bilirubin decreased the pruritogenic capacity of patient plasma. Based on these data, targeting MRGPRs is a promising strategy for alleviating jaundice-associated itch.

Central activation of TRPV1 and TRPA1 by novel endogenous agonists contributes to mechanical allodynia and thermal hyperalgesia after burn injury.

The primary complaint of burn victims is an intense, often devastating spontaneous pain, with persistence of mechanical and thermal allodynia. The transient receptor potential channels, TRPV1 and TRPA1, are expressed by a subset of nociceptive sensory neurons and contribute to inflammatory hypersensitivity. Although their function in the periphery is well known, a role for these TRP channels in central pain mechanisms is less well defined. Lipid agonists of TRPV1 are released from peripheral tissues via enzymatic oxidation after burn injury; however, it is not known if burn injury triggers the release of oxidized lipids in the spinal cord. Accordingly, we evaluated whether burn injury evoked the central release of oxidized lipids . Analysis of lipid extracts of spinal cord tissue with HPLC-MS revealed a significant increase in levels of the epoxide and diol metabolites of linoleic acid: 9,10-DiHOME, 12,13-DiHOME, 9(10)-EpOME, and 12(13)-EpOME, that was reduced after intrathecal (i.t.) injection of the oxidative enzyme inhibitor ketoconazole. Moreover, we found that these four lipid metabolites were capable of specifically activating both TRPV1 and TRPA1. Intrathecal injection of specific antagonists to TRPV1 (AMG-517) or TRPA1 (HC-030031) significantly reduced post-burn mechanical and thermal allodynia. Finally, i.t. injection of ketoconazole significantly reversed post-burn mechanical and thermal allodynia. Our data indicate that spinal cord TRPV1 and TRPA1 contributes to pain after burn and identifies a novel class of oxidized lipids elevated in the spinal cord after burn injury. Since the management of burn pain is problematic, these findings point to a novel approach for treating post-burn pain.

The cell biology of acute itch.

Itch, the irritation we feel and the relief that comes from scratching, is an evolutionary warning system and defense against harmful environmental agents. Although once considered a subtype of pain, itch is now recognized as a unique sense, with its own distinct physiology and cell receptors. Here, we discuss recent advances in our understanding of itch and the molecular players that mediate this sensory modality.

Cervical Ectopic Pregnancy Complicated by Escherichia Coli Septicemia: A Case Report.

BACKGROUND: Cervical ectopic pregnancy is an uncommon form of ectopic pregnancy. The spectrum of treatment options includes systemic medical therapy, local injection with methotrexate and/or potassium chloride, surgery, or a combination of these modalities. CASE: A 29-year-old woman, gravida 3, aborta 2, was found to have a cervical ectopic pregnancy on ultrasound at 5+6 weeks' gestation. She presented to the hospital with vaginal bleeding. Treatment was initiated with multidose methotrexate and leucovorin rescue. She subsequently developed Gram-negative septicemia, with blood cultures growing Escherichia coli. This was managed successfully by surgical removal of the ectopic pregnancy and antimicrobial therapy. CONCLUSION: A cervical ectopic pregnancy can be complicated by E. coli septicemia.

Influence of hypophysectomy, ovariectomy and gonadectomy on postoperative hypersensitivity in rats.

Surgical procedures lead to profound and sustained (up to 1-2 weeks) activation of the pituitary gland, resulting in changes in endocrine function. Questions remain on whether activation of the pituitary influences the threshold and development time-course of postoperative pain. To address these questions, we evaluated postoperative hypersensitivity in female and male rats with ablated pituitary and gonadal hormone productions via hypophysectomy, ovariectomy and gonadectomy, respectively. Plantar incision, a model of acute postoperative pain, or sham operation was performed on rat hind paws. Hypophysectomy, ovariectomy and gonadectomy were achieved by surgical disconnection of pituitary, ovaries and testicles, respectively. Postoperative thermal and mechanical hypersensitivity were monitored for 7 days post incision. Hypophysectomy on female and male rats produced statistically similar thermal and mechanical postoperative hypersensitivity thresholds and time-courses as compared to intact estrous female and male rats. Moreover, ovariectomy and gonadectomy did not significantly change postoperative hypersensitivity observed in control female and male animals. Our experiments demonstrate that hypophysectomy, ovariectomy and gonadectomy do not significantly impact postoperative hypersensitivity observed in normal female and male animals. These data suggest that surgery-induced changes in the endocrine system via activation of pituitary and subsequently gonadal tissues have little impact on the threshold and development of postoperative pain in female and male rats.

Persistent Nociception Triggered by Nerve Growth Factor (NGF) Is Mediated by TRPV1 and Oxidative Mechanisms.

Nerve growth factor (NGF) is elevated in certain chronic pain conditions and is a sufficient stimulus to cause lasting pain in humans, but the actual mechanisms underlying the persistent effects of NGF remain incompletely understood. We developed a rat model of NGF-induced persistent thermal hyperalgesia and mechanical allodynia to determine the role of transient receptor potential vanilloid 1 (TRPV1) and oxidative mechanisms in the persistent effects of NGF. Persistent thermal hypersensitivity and mechanical allodynia require de novo protein translation and are mediated by TRPV1 and oxidative mechanisms. By comparing effects after systemic (subcutaneous), spinal (intrathecal) or hindpaw (intraplantar) injections of test compounds, we determined that TRPV1 and oxidation mediate persistent thermal hypersensitivity via peripheral and spinal sites of action and mechanical allodynia via only a spinal site of action. Therefore, NGF-evoked thermal and mechanical allodynia are mediated by spatially distinct mechanisms. NGF treatment evoked sustained increases in peripheral and central TRPV1 activity, as demonstrated by increased capsaicin-evoked nocifensive responses, increased calcitonin gene-related peptide release from hindpaw skin biopsies, and increased capsaicin-evoked inward current and membrane expression of TRPV1 protein in dorsal root ganglia neurons. Finally, we showed that NGF treatment increased concentrations of linoleic and arachidonic-acid-derived oxidized TRPV1 agonists in spinal cord and skin biopsies. Furthermore, increases in oxidized TRPV1-active lipids were reduced by peripheral and spinal injections of compounds that completely blocked persistent nociception. Collectively, these data indicate that NGF evokes a persistent nociceptive state mediated by increased TRPV1 activity and oxidative mechanisms, including increased production of oxidized lipid TRPV1 agonists.

Released lipids regulate transient receptor potential channel (TRP)-dependent oral cancer pain.

BACKGROUND: Pain in the head neck area is an early symptom in oral cancer, supporting the hypothesis that cancer cells control the activities of surrounding nociceptors at the site of the tumor. Several reports implicate TRPV1 and TRPA1 in cancer pain, although there is a large gap in knowledge since the mechanisms for tumor-induced activation of these TRP receptors are unknown. Interestingly, TRP-active lipids such as linoleic acid, arachidonic acid, hydroxyoctadecadienoic acid and hydroxyeicosatetraenoic acid are significantly elevated in the saliva of oral cancer patients compared to normal patients, supporting a possible linkage between these lipids and oral cancer pain. We therefore hypothesize that oral squamous cell carcinomas release certain lipids that activate TRPV1 and/or TRPA1 on sensory neurons, contributing to the development of oral cancer pain. METHODS: Lipid extracts were made from conditioned media of three human oral squamous cell carcinoma (OSCC) cell lines as well as one normal human oral keratinocytes cell line. These were then injected intraplantarly into rat hindpaws to measure spontaneous nocifensive behavior, as well as thermal and mechanical allodynia. For interventional experiments, the animals were pretreated with AMG517 (TRPV1 antagonist) or HC030031 (TRPA1 antagonist) prior to extract injection. RESULTS: These studies demonstrate that lipids released from the three OSCC cell lines, but not the normal cell line, were capable of producing significant spontaneous nocifensive behaviors, as well as thermal and mechanical allodynia. Notably each of the cell lines produced a different magnitude of response for each of three behavioral assays. Importantly, pre-treatment with a TRPVI antagonist blocked lipid-mediated nocifensive and thermal hypersensitivity, but not mechanical hypersensitivity. In addition, pre-treatment with a TRPA1 antagonist only reversed thermal hypersensitivity without affecting lipid-induced nocifensive behavior or mechanical allodynia. CONCLUSIONS: These data reveal a novel mechanism for cancer pain and provide strong direction for future studies evaluating the cellular mechanism regulating the TRP-active lipids by OSCC tumors.

Role of endogenous TRPV1 agonists in a postburn pain model of partial-thickness injury.

Oxidized linoleic acid metabolites (OLAMs) are a class of endogenous transient receptor potential vanilloid 1 (TRPV1) channel agonists released on exposure of tissue to transient noxious temperatures. These lipid compounds also contribute to inflammatory and heat allodynia. Because persistent pain after a burn injury represents a significant clinical challenge for treatment, we developed an in vivo rat model of partial-thickness cutaneous thermal injury and examined whether TRPV1 and specific OLAM metabolites play a role in mediating postburn pain injury. This peripheral model of burn injury had marked thermal allodynia peaking at 24h after thermal injury, with allodynia being maintained for up to 7d. Immunohistochemical characterization of tissue taken from injury sites revealed an increase in leukocyte/macrophage infiltration that was colocalized with TRPV1-positive fibers. Using this peripheral thermal injury model, we found that pharmacological blockade of peripheral TRPV1 receptors reduced thermal allodynia by about 98%. Moreover, there was a significant increase in OLAM levels compared to naive controls in hind paw skin biopsies. Additional studies of the metabolism of [C(14)]-linoleic acid in skin biopsies revealed the role of the cytochrome P450 (CYP) system in mediating the metabolism of linoleic acid after thermal injury. Finally, we demonstrated that direct inhibition of OLAMs using OLAM antibodies and indirect inhibition using the CYP inhibitor ketoconazole significantly reduced postburn thermal allodynia. Collectively, these findings point to a novel role of the OLAMs and CYP-related enzymes in generating postburn allodynia via activation of peripheral TRPV1.

The cytochrome P450 inhibitor, ketoconazole, inhibits oxidized linoleic acid metabolite-mediated peripheral inflammatory pain.

BACKGROUND: Oxidized linoleic acid metabolites (OLAMs) are a class of endogenous agonists to the transient receptor potential V1 (TRPV1) receptor. Although TRPV1 mediates inflammatory heat hyperalgesia, it is not known if the OLAMs contribute to the peripheral activation of this receptor during tissue inflammation. In the present study, we evaluated whether the OLAM system is activated during inflammation and whether cytochrome P450 enzymes mediate OLAM contributions to heat hyperalgesia using the complete Freund's adjuvant (CFA) model of inflammation. RESULTS: Our results demonstrate that the intraplantar (ipl) injection of anti-OLAM antibodies significantly reversed CFA-induced heat hyperalgesia. Moreover, application of lipid extracts from inflamed rat skin to cultured sensory neurons triggered a significant release of iCGRP that is blocked by co-treatment with I-RTX, a TRPV1 antagonist. To determine the role of CYP enzymes in mediating OLAM effects, we used a broad spectrum CYP inhibitor, ketoconazole. Pretreatment with ketoconazole inhibited the release of TRPV1 agonists in lipid extracts from inflamed skin and significantly reversed CFA-induced heat hyperalgesia by a peripheral mechanism of action. Moreover, the ipl injection of linoleic acid to rats 24 hr after CFA evoked spontaneous nocifensive behaviors that were significantly reduced by capsazepine, by knockout of the TRPV1 gene, or by pretreatment with either anti-OLAM antibodies or ketoconazole. CONCLUSIONS: Taken together, our data suggests that OLAMs contribute to inflammatory nociception in the periphery and that cytochrome P450 enzymes play a crucial role in mediating OLAM contributions to inflammatory heat hyperalgesia.