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 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.

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.

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.

MicroCT-based virtual histology evaluation of preclinical medulloblastoma.

PURPOSE: The purpose of this paper is to validate a rapid and cost-effective ex vivo technique, microCT-based virtual histology, as an alternative to MRI imaging for assessing the therapeutic response in genetically engineered mouse models of cancer. PROCEDURES: All animal procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Texas Health Science Center at San Antonio. MRI imaging was performed on 6-week-old, bortezomib-treated genetically engineered Patched1, p53 mice that recapitulate the characteristics of human medulloblastoma. After MRI scans, the same mice were euthanized to collect brain or spine samples for virtual histology staining followed by microCT scanning. RESULTS: Nine-micrometer resolution ex vivo micro X-ray computed tomography (microCT)-based virtual histology images were qualitatively reflective of high-field live animal images obtained with magnetic resonance imaging (MRI) and histopathology. Cerebellar volumes on microCT-based virtual histology correlated closely with MRI cerebellar volumes (R = 0.998). MRI and microCT-based virtual histology both indicated a significant difference between cerebellar volumes of untreated and treated mice (p = 0.02 and p = 0.04, respectively). The ex vivo microCT method also allowed a 7,430-fold improvement in voxel resolution (voxel volume of 729 µm³ for 9-µm isometric resolution microCT vs. 5,416,800 µm³ for 400 × 111 × 122 µm resolution MRI) at a 28% cost savings ($400 vs. $555 per animal). CONCLUSION: The ex vivo, en bloc technique of microCT-based virtual histology matched MRI in reflecting histopathology. MicroCT-based virtual histology proved to be a more cost-effective technique and less labor-intensive. On the other hand, MRI provides ability to perform in vivo imaging, faster scanning and lower radiation dose by sacrificing the spatial resolution. Thus, both in vivo MRI and ex vivo microCT-based virtual histology are effective means of quantitatively evaluating therapeutic response in preclinical models of cerebellar tumors including the childhood cancer, medulloblastoma.