Calcitonin Gene-related Peptide Production Research Articles

Glycerol Trinitrate Acts Downstream of Calcitonin Gene-Related Peptide in Trigeminal Nociception-Evidence from Rodent Experiments with Anti-CGRP Antibody Fremanezumab.

Calcitonin gene-related peptide (CGRP) and nitric oxide (NO) have been recognized as important mediators in migraine but their mechanisms of action and interaction have not been fully elucidated. Monoclonal anti-CGRP antibodies like fremanezumab are successful preventives of frequent migraine and can be used to study CGRP actions in preclinical experiments. Fremanezumab (30 mg/kg) or an isotype control monoclonal antibody was subcutaneously injected to Wistar rats of both sexes. One to several days later, glyceroltrinitrate (GTN, 5 mg/kg) mimicking nitric oxide (NO) was intraperitoneally injected, either once or for three consecutive days. The trigeminal ganglia were removed to determine the concentration of CGRP using an enzyme-linked immunosorbent assay (ELISA). In one series of experiments, the animals were trained to reach an attractive sugar solution, the access to which could be limited by mechanical or thermal barriers. Using a semi-automated registration system, the frequency of approaches to the source, the residence time at the source, and the consumed solution were registered. The results were compared with previous data of rats not treated with GTN. The CGRP concentration in the trigeminal ganglia was generally higher in male rats and tended to be increased in animals treated once with GTN, whereas the CGRP concentration decreased after repetitive GTN treatment. No significant difference in CGRP concentration was observed between animals having received fremanezumab or the control antibody. Animals treated with GTN generally spent less time at the source and consumed less sugar solution. Without barriers, there was no significant difference between animals having received fremanezumab or the control antibody. Under mechanical barrier conditions, all behavioral parameters tended to be reduced but animals that had received fremanezumab tended to be more active, partly compensating for the depressive effect of GTN. In conclusion, GTN treatment seems to increase the production of CGRP in the trigeminal ganglion independently of the antibodies applied, but repetitive GTN administration may deplete CGRP stores. GTN treatment generally tends to suppress the animals' activity and increase facial sensitivity, which is partly compensated by fremanezumab through reduced CGRP signaling. If CGRP and NO signaling share the same pathway in sensitizing trigeminal afferents, GTN and NO may act downstream of CGRP to increase facial sensitivity.

Mediation of the JNC/ILC2 pathway in DBP-exacerbated allergic asthma: A molecular toxicological study on neuroimmune positive feedback mechanism

BackgroundDibutyl phthalate (DBP), a commonly used plasticizer, has been found to be strongly linked to a consistently high prevalence of allergic diseases, particularly allergic asthma. Previous animal experiments have demonstrated that exposure to DBP can worsen asthma by triggering the production of calcitonin gene-related peptide (CGRP), a neuropeptide in the lung tissue. However, the precise neuroimmune mechanism and pathophysiology of DBP-exacerbated allergic asthma with the assistance of CGRP remain unclear. ObjectiveThe present study was to investigate the potential pathophysiological mechanism in DBP-exacerbated asthma from the perspective of neural-immune interactions. Methods and resultsC57BL/6 mice were orally exposed to different concentrations (0.4, 4, 40 mg/kg) of DBP for 28 days. They were then sensitized with OVA and nebulized with OVA for 7 consecutive excitations. To investigate whether DBP exacerbates allergic asthma in OVA induced mice, we analyzed airway hyperresponsiveness and lung histopathology. To investigate the activation of JNC and TRPV1 neurons and the release of CGRP by JNC cells, we measured the levels of TRPV1 channels, calcium inward flow, and downstream neuropeptide CGRP. Results showed that TRPV1 expression, inward calcium flux, and CGRP levels were significantly elevated in the lung tissues of the 40DBP + OVA group, suggesting the release of CGRP by JNC cells. To counteract the detrimental effects of DBP mediated by CGRP, we employed olcegepant (also known as BIBN-4096), a CGRP receptor specific antagonist. Results revealed that 40DBP + OVA + olcegepant led to notable decreases in TRPV1, calcium inward flow, and CGRP expression in lung tissues compare with 40DBP + OVA, further supporting the efficacy of olcegepant. Additionally, we also conducted ILC2 flow sorting and observed that neuropeptide CGRP-activated ILC2 cells have a crucial role as key effector cells in DBP-induced neuroimmune positive feedback regulation. Finally, we examined the protein expression of CGRP, GATA3 and P-GATA3, and found that significant upregulations of CGRP and P-GATA3 in the 40DBP + OVA group, suggest that GATA3 acted as a key regulator of CGRP-activated ILC2. ConclusionThe aforementioned studies indicate that exposure to DBP can exacerbate allergic asthma, leading to airway inflammation. This exacerbation occurs through the activation of TRPV1 in JNC, resulting in the release of CGRP. The excessive release of CGRP further promotes the release of Th2 cytokines by inducing the activation of ILC2 through GATA phosphorylation. Consequently, this process contributes to the development of airway inflammation and allergic asthma. The increased production of Th2 cytokines also triggers the production of IgE, which interacts with FcεRI on JNC neurons, thereby mediating neuro-immune positive feedback regulation.

Neuronal TRPV1-CGRP axis regulates bone defect repair through Hippo signaling pathway

Transient receptor potential vanilloid type 1 (TRPV1) is highly expressed on sensory neurons where it serves as a polymodal receptor for detecting physical and chemical stimuli. However, the role of TRPV1 in bone metabolism remains largely unclear. This study aimed to investigate the underlying mechanism of neuronal TRPV1 in regulating bone defect repair. In vivo experiment verified that TRPV1 activation could trigger dorsal root ganglion (DRG) producing the neuropeptide calcitonin gene-related peptide (CGRP) in mice. The accelerated bone healing of femoral defect in this process was observed compared to the control group (p < 0.05). Conversely, Trpv1 knockdown led to reduced CGRP expression in DRG and nerves innervating femur bone tissue, following impaired bone formation and osteogenic capability in the defect region (p < 0.05), which could be rescued by local CGRP treatment. In vitro, results revealed that TRPV1 function in DRG neurons contributed essentially to the regulation of osteoblast physiology through affecting the production and secretion of CGRP. The capsaicin-activated neuronal TRPV1-CGRP axis could enhance the proliferation, migration and differentiation of osteoblasts (p < 0.05). Furthermore, we found that the promoting role of neuronal TRPV1 in osteogenesis was associated with Hippo signaling pathway, reflected by the phosphorylation protein level of large tumor suppressor 1 (LATS1), MOB kinase activator 1 (MOB1) and Yes-associated protein (YAP), as well as the subcellular location of YAP. Our study clarified the effects and intrinsic mechanisms of neuronal TRPV1 on bone defect repair, which might offer us a therapeutic implication for bone disorders.

Peripheral CCL2-CCR2 signalling contributes to chronic headache-related sensitization.

Migraine, especially chronic migraine, is highly debilitating and still lacks effective treatment. The persistent headache arises from activation and sensitization of primary afferent neurons in the trigeminovascular pathway, but the underlying mechanisms remain incompletely understood. Animal studies indicate that signalling through chemokine C-C motif ligand 2 (CCL2) and C-C motif chemokine receptor 2 (CCR2) mediates the development of chronic pain after tissue or nerve injury. Some migraine patients had elevated CCL2 levels in CSF or cranial periosteum. However, whether the CCL2-CCR2 signalling pathway contributes to chronic migraine is not clear. Here, we modelled chronic headache with repeated administration of nitroglycerin (NTG, a reliable migraine trigger in migraineurs) and found that both Ccl2 and Ccr2 mRNA were upregulated in dura and trigeminal ganglion (TG) tissues that are implicated in migraine pathophysiology. In Ccl2 and Ccr2 global knockout mice, repeated NTG administration did not evoke acute or persistent facial skin hypersensitivity as in wild-type mice. Intraperitoneal injection of CCL2 neutralizing antibodies inhibited chronic headache-related behaviours induced by repeated NTG administration and repetitive restraint stress, suggesting that the peripheral CCL2-CCR2 signalling mediates headache chronification. We found that CCL2 was mainly expressed in TG neurons and cells associated with dura blood vessels, whereas CCR2 was expressed in subsets of macrophages and T cells in TG and dura but not in TG neurons under both control and disease states. Deletion of Ccr2 gene in primary afferent neurons did not alter NTG-induced sensitization, but eliminating CCR2 expression in either T cells or myeloid cells abolished NTG-induced behaviours, indicating that both CCL2-CCR2 signalling in T cells and macrophages are required to establish chronic headache-related sensitization. At cellular level, repeated NTG administration increased the number of TG neurons that responded to calcitonin-gene-related peptide (CGRP) and pituitary adenylate cyclase activating polypeptide (PACAP) as well as the production of CGRP in wild-type but not Ccr2 global knockout mice. Lastly, co-administration of CCL2 and CGRP neutralizing antibodies was more effective in reversing NTG-induced behaviours than individual antibodies. Taken together, these results suggest that migraine triggers activate CCL2-CCR2 signalling in macrophages and T cells. This consequently enhances both CGRP and PACAP signalling in TG neurons, ultimately leading to persistent neuronal sensitization underlying chronic headache. Our work not only identifies the peripheral CCL2 and CCR2 as potential targets for chronic migraine therapy, but also provides proof-of-concept that inhibition of both peripheral CGRP and CCL2-CCR2 signalling is more effective than targeting either pathway alone.

Trimethoxyflavanone relieves Paclitaxel-induced neuropathic pain via inhibiting expression and activation of P2X7 and production of CGRP in mice

Paclitaxel (PTX) is an anticancer drug used to treat solid tumors, but one of its common adverse effects is chemotherapy-induced peripheral neuropathy (CIPN). Currently, there is limited understanding of neuropathic pain associated with CIPN and effective treatment strategies are inadequate. Previous studies report the analgesic actions of Naringenin, a dihydroflavonoid compound, in pain. Here we observed that the anti-nociceptive action of a Naringenin derivative, Trimethoxyflavanone (Y3), was superior to Naringenin in PTX-induced pain (PIP). An intrathecal injection of Y3 (1 μg) reversed the mechanical and thermal thresholds of PIP and suppressed the PTX-induced hyper-excitability of dorsal root ganglion (DRG) neurons. PTX enhanced the expression of ionotropic purinergic receptor P2X7 (P2X7) in satellite glial cells (SGCs) and neurons in DRGs. The molecular docking simulation predicts possible interactions between Y3 and P2X7. Y3 reduced the PTX-enhanced P2X7 expression in DRGs. Electrophysiological recordings revealed that Y3 directly inhibited P2X7-mediated currents in DRG neurons of PTX-treated mice, suggesting that Y3 suppressed both expression and function of P2X7 in DRGs post-PTX administration. Y3 also reduced the production of calcitonin gene-related peptide (CGRP) in DRGs and at the spinal dorsal horn. Additionally, Y3 suppressed the PTX-enhanced infiltration of Iba1-positive macrophage-like cells in DRGs and overactivation of spinal astrocytes and microglia. Therefore, our results indicate that Y3 attenuates PIP via inhibiting P2X7 function, CGRP production, DRG neuron sensitization, and abnormal spinal glial activation. Our study implies that Y3 could be a promising drug candidate against CIPN-associated pain and neurotoxicity.

Gut microbiota modulates visceral sensitivity through calcitonin gene-related peptide (CGRP) production

ABSTRACT Abdominal pain is common in patients with gastrointestinal disorders, but its pathophysiology is unclear, in part due to poor understanding of basic mechanisms underlying visceral sensitivity. Accumulating evidence suggests that gut microbiota is an important determinant of visceral sensitivity. Clinical and basic research studies also show that sex plays a role in pain perception, although the precise pathways are not elucidated. We investigated pain responses in germ-free and conventionally raised mice of both sexes, and assessed visceral sensitivity to colorectal distension, neuronal excitability of dorsal root ganglia (DRG) neurons and the production of substance P and calcitonin gene-related peptide (CGRP) in response to capsaicin or a mixture of G-protein coupled receptor (GPCR) agonists. Germ-free mice displayed greater in vivo responses to colonic distention than conventional mice, with no differences between males and females. Pretreatment with intracolonic capsaicin or GPCR agonists increased responses in conventional, but not in germ-free mice. In DRG neurons, gut microbiota and sex had no effect on neuronal activation by capsaicin or GPCR agonists. While stimulated production of substance P by DRG neurons was similar in germ-free and conventional mice, with no additional effect of sex, the CGRP production was higher in germ-free mice, mainly in females. Absence of gut microbiota increases visceral sensitivity to colorectal distention in both male and female mice. This is, at least in part, due to increased production of CGRP by DRG neurons, which is mainly evident in female mice. However, central mechanisms are also likely involved in this process.

A13 GUT MICROBIOTA MODULATES CGRP PRODUCTION BY DRG NEURONS IN FEMALE MICE

Abstract Background Abdominal pain is a common complaint in patients with chronic gastrointestinal disorders. Accumulating evidence suggests that gut microbiota is an important determinant of gut function, including visceral sensitivity. Germ-free (GF) mice have been shown to display visceral hypersensitivity, which normalizes after colonization. Thus, gut microbiota is involved in the regulation of gut nociception but the underlying mechanisms remain poorly understood. Aims To investigate the role of gut microbiota in abdominal pain. Methods Method: Adult female and male conventionally raised (SPF) or GF mice were used. Their visceral sensitivity was assessed by visceromotor responses to colorectal distension, at baseline (vehicle: Tween 80 10%, Ethanol 10%, saline 80%) and after intracolonic administration of a TRPV1 agonist capsaicin (30 μg), or a mixture of G-protein coupled receptors agonists (GPCR: bradykinin, histamine and serotonin; 30 μg). Neuronal excitability of dorsal root ganglia (DRG) neurons was assessed by calcium imaging using a fluorescent probe Fluo-4 (1 mM) after stimulation with capsaicin (12.5 nM, 125 nM and 1250 nM) or GPCR agonists (0.3 μM, 3 μM and 30 μM). The neuronal production of substance P (SP) and calcitonin gene-related peptide (CGRP) in response to capsaicin (1250 nM) or GPCR agonists (30 μM) was measured by ELISA. Results At baseline, GF male mice exhibited higher responses to colorectal distension compared to SPF males, while SPF and GF females displayed similar visceral sensitivity. In contrast, both intracolonic capsaicin and GPCR agonists increased visceral sensitivity in GF females compared to SPF females, while responses were comparable in male groups. DRG neuronal activation after stimulation with capsaicin or GPCR agonists was similar in SPF and GF mice of both sexes. While stimulated production of SP by DRG neurons was similar in SPF and GF mice, regardless of sex, the production of CGRP in response to GPCR agonists was higher in GF female than SPF female mice. Conclusions Our data suggest that visceral sensitivity in vivo differs according to the gut microbiota status, sex and the activation of TRPV1 and GPCR pathways. At the level of DRG neurons, the absence of gut microbiota does not affect the neuronal activation or production of SP. However, GPCR agonists-stimulated release of CGRP is higher in GF female compared to SPF female mice. All together, our data demonstrate that the gut microbiota modulates visceral sensitivity by regulating the production of CGRP in the sensory neurons, especially in female mice. Further mechanistic studies are needed to investigate the role of gut microbiota in visceral sensitivity. Funding Agencies CIHR

Beyond Thermal Sensation: Roles of Transient Receptor Potential Vanilloid Subfamily Member 1 and Spicy Food in Cardiometabolic Diseases

Beyond Thermal Sensation: Roles of Transient Receptor Potential Vanilloid Subfamily Member 1 and Spicy Food in Cardiometabolic Diseases

Nerve Growth Factor in Breast Cancer Cells Promotes Axonal Growth and Expression of Calcitonin Gene-related Peptide in a Rat Model of Spinal Metastasis.

Bone metastasis commonly causes severe pain. Nerve growth factor (NGF) contributes to pain, and promotes the production of pain-associated neuropeptides, such as calcitonin gene-related peptide (CGRP), from sensory nerve endings. We hypothesized that breast cancer cells have NGF levels that promote axonal growth from dorsal root ganglia (DRGs) neurons, and increase their CGRP production associated with pain from spinal metastases. Expression of NGF by the cultured rat breast adenocarcinoma cell line CRL-1666 was determined using an enzyme-linked immunosorbent assay (ELISA). We constructed a rat model of spinal metastasis by implanting CRL-1666 into L6 vertebrae and determined the change in CGRP expression in DRG neurons innervating vertebrae immunohistochemically. NGF was expressed by CRL-1666. When DRG cells were co-cultured with CRL-1666, there were more CGRP-ir neurons and with a greater average length of axon growth than in cultures without CRL-1666 (p<0.05). In the rat model of metastasis, there were more CGRP-ir DRG neurons innervating vertebra treated with CRL-1666 than in vertebrae from sham surgery control rats (p<0.05). NGF from breast cancer may mediate spinal bone pain from metastasis via axonal growth and up-regulation of pain-associated neuropeptides.

A9 DORSAL ROOT GANGLIA NEURONAL RESPONSES AND SUBSTANCE P PRODUCTION ARE HIGHER IN MALE MICE

Abstract Background Abdominal pain is a common complaint in patients with chronic gastrointestinal disorders. Accumulating evidence suggests that gut microbiota is an important determinant of gut function, including visceral sensitivity. Germ-free (GF) mice have been shown to display visceral hypersensitivity, which normalizes after colonization. Sex also appears to play a key role in visceral sensitivity, as women report more abdominal pain than men. Thus, both gut bacteria and sex are important in the regulation of gut nociception, but the underlying mechanisms remain poorly understood. Aims To investigate the role of gut microbiota and sex in abdominal pain. Methods We used primary cultures of sensory neurons from dorsal root ganglia (DRG) of female and male conventionally raised (SPF) or germ-free (GF) mice (7–18 weeks old). To study the visceral afferent activity in vitro, calcium mobilization in DRG sensory neurons was measured by inverted fluorescence microscope using a fluorescent calcium probe Fluo-4 (1mM). Two parameters were considered i) the percentage of responding neurons ii) the intensity of the neuronal response. First, DRG sensory neurons were stimulated by a TRPV1 agonist capsaicin (12.5nM, 125nM and 1.25µM) or by a mixture of G-protein coupled receptors agonist (GPCR: bradykinin, histamine and serotonin; 1µM, 10µM and 100µM). We next measured the neuronal production of substance P and calcitonin gene-related peptide (CGRP), two neuropeptides associated with nociception, in response to capsaicin (1.25µM) or GPCR agonists (100µM) by ELISA and EIA, respectively. Results The percentage of neurons responding to capsaicin and GPCR agonists was similar in male and female SPF and GF mice. However, the intensity of the neuronal response was higher in SPF male compared to SPF female in response to capsaicin (125nM: p=0.0336; 1.25µM: p=0.033) but not to GPCR agonists. Neuronal activation was similar in GF and SPF mice of both sexes after administration of capsaicin or GPCR agonists. Furthermore, substance P and CGRP production by sensory neurons induced by capsaicin or GPCR agonists was similar in SPF and GF mice, regardless of sex. However, while the response to capsaicin was similar, the GPCR agonists-induced production of substance P was higher in SPF male mice compared to SPF females (p=0.003). The GPCR agonists-induced production of CGRP was similar in SPF male and female mice. Conclusions Our data suggest that at the level of DRG neurons, the absence of gut microbiota does not predispose to visceral hypersensitivity. The intensity of DRG neuronal responses to capsaicin and the GPCR agonists-induced production of substance P are higher in male compared to female mice, in contrast to previously published studies in various models of acute and chronic pain. Further studies are thus needed to investigate the role of sex in visceral sensitivity. Funding Agencies CIHR

Inhibition of NR2A reduces calcitonin gene-related peptide gene expression induced by cortical spreading depression in rat amygdala

Despite robust evidence on the role of calcitonin gene-related peptide (CGRP) in migraine via both central and peripheral actions, relatively less is known about how CGRP in the limbic system is involved in migraine progression. This study investigated whether CGRP production machinery exists in the two key limbic regions including hippocampus and amygdala using cortical spreading depression (CSD) as a model of migraine and whether such alteration by CSD is sensitive to N-methyl-d-aspartate (NMDA) receptor regulation in rats. A single or repetitive CSD was induced by topical application of KCl and monitored using electrophysiological methods. The NR2A-containing NMDA receptor antagonist, NVP-AAM077, or its vehicle, was perfused into the contralateral cerebroventricular ventricle of rat. Quantitative PCR was used to measure CGRP mRNA levels in the ipsilateral and contralateral hippocampus and amygdala after CSD events and compared to respective sham treatments. The results showed that neither a single CSD nor repetitive CSD affected CGRP mRNA levels in both the contralateral and ipsilateral hippocampus at 24 h post CSD induction. Differently, significant elevation of CGRP gene expression was observed in the ipsilateral amygdala at 24 h post multiple CSD, but not contralateral side, and not post-single CSD. Further results showed that the CSD-induced CGRP gene expression in the amygdala was markedly reduced by NVP-AAM077 and this reduction corresponded to a reduced cortical susceptibility to CSD in rats. We conclude that repetitive CSD events induce CGRP gene expression in amygdala, which is sensitive to NR2A regulation.

Perspective: A Neuro-Hormonal Systems Approach to Understanding the Complexity of Cryptorchidism Susceptibility.

Nonsyndromic cryptorchidism is a common multifactorial, condition with long-term risks of subfertility and testicular cancer. Revealing the causes of cryptorchidism will likely improve prediction and prevention of adverse outcomes. Herein we provide our current perspective of cryptorchidism complexity in a synthesis of cumulative clinical and translational data generated by ourselves and others. From our recent comparison of genome-wide association study (GWAS) data of cryptorchidism with or without testicular germ cell tumor, we identified RBFOX family genes as candidate susceptibility loci. Notably, RBFOX proteins regulate production of calcitonin gene-related peptide (CGRP), a sensory neuropeptide linked to testicular descent in animal models. We also re-analyzed existing fetal testis transcriptome data from a rat model of inherited cryptorchidism (the LE/orl strain) for enrichment of Leydig cell progenitor genes. The majority are coordinately downregulated, consistent with known reduced testicular testosterone levels in the LE/orl fetus, and similarly suppressed in the gubernaculum. Using qRT-PCR, we found dysregulation of dorsal root ganglia (DRG) sensory transcripts ipsilateral to undescended testes. These data suggest that LE/orl cryptorchidism is associated with altered signaling in possibly related cell types in the testis and gubernaculum as well as DRG. Complementary rat and human studies thus lead us to propose a multi-level, integrated neuro-hormonal model of testicular descent. Variants in genes encoding RBFOX family proteins and/or their transcriptional targets combined with environmental exposures may disrupt this complex pathway to enhance cryptorchidism susceptibility. We believe that a systems approach is necessary to provide further insight into the causes and consequences of cryptorchidism.

Amygdalin improves microcirculatory disturbance and attenuates pancreatic fibrosis by regulating the expression of endothelin-1 and calcitonin gene-related peptide in rats

BackgroundThe pathogenesis of chronic pancreatitis (CP) is a complex process of interaction between tissue injury and repair, which involves microcirculatory disturbance. Amygdalin, an effective component extracted from Semen Persicae (a kind of Chinese herbal medicine), can decrease blood viscosity and improve microcirculation. In this study, we investigated the therapeutic effects of amygdalin on pancreatic fibrosis in rats with CP. MethodsThe rat CP model was induced by injecting dibutyltin dichloride (DBTC) into the right caudal vein. Amygdalin was administrated via the penile vein at a dose of 10 mg/(kg d) from the next day, after the induction of CP, once a day for the previous 3 days, and then once every 2 days, until the end of the experiment. Body weight was observed every 7 days. Pancreatic blood flow and histopathological changes were assessed at 28 days. The activation of pancreatic stellate cells (PSCs) was estimated by the expression of α-smooth muscle actin (α-SMA). At the same time, the expression of platelet-derived growth factor-BB (PDGF-BB), transforming growth factor β-1 (TGFβ-1), endothelin-1 (ET-1), and calcitonin gene-related peptide (CGRP) of pancreatic tissues were detected. ResultsTreatment of CP rats with amygdalin improved body weight and pancreatic blood flow, as well as alleviated pancreatic fibrosis and acinar destruction, accompanied by the down-regulation of the expressions of α-SMA, PDGF-BB, TGFβ-1, and ET-1, and the up-regulation of the CGRP's expression. ConclusionAmygdalin could reduce the production of pro-fibrotic cytokines, inhibit the activation of PSCs, and attenuate pancreatic fibrosis in a rat with CP. The mechanism probably includes improving microcirculatory disturbance by regulating the production of ET-1 and CGRP.

Mediating Role of TRPV1 Ion Channels in the Co-exposure to PM2.5 and Formaldehyde of Balb/c Mice Asthma Model

Asthma is a complex pulmonary inflammatory disease that can be promoted by air pollutants such as PM2.5 and formaldehyde (FA). However, existent experimental evidence principally focuses on the negative influence of a single air pollutant, neglecting the possible synergistic effect in biological responses to mixture of these pollutants, a more common situation in our daily life. In this study, allergic Balb/c mice were exposed to a mixture of PM2.5 and FA, and their toxicological effects and mechanisms were explored. It is demonstrated that the combined exposure to PM2.5 and FA can greatly aggravate allergic asthma in mice. When compared with exposure to PM2.5 or FA alone, the co-exposure showed a certain synergistic effect. Increased levels of ROS, inflammatory factors and total serum immunoglobulin E were concomitant with this deterioration. Furthermore, results suggested that co-exposure exacerbated the activation of TRPV1 signal pathways, with an enhancement in substance P and calcitonin gene-related peptide production, which contributed to inflammation in asthma by neurogenic inflammation. The study also proved that capsazepine treatment could reduce the levels of not only pro-inflammatory neuropeptides, but also oxidative stress. It is concluded that co-exposure to PM2.5 and FA exacerbated allergic asthma through oxidative stress and enhanced TRPV1 activation.

Nociceptors Boost the Resolution of Fungal Osteoinflammation via the TRP Channel-CGRP-Jdp2 Axis

Candida albicans can enter skeletal tissue through askin wound in an immunocompromised host or by contamination during orthopedic surgery. Such Candida osteomyelitis is accompanied by severe pain and bone destruction. It is established that nociceptor innervation occurs in skin and bone, but the mechanisms of nociceptive modulation in fungal inflammation remain unclear. In this study, we show that C.albicans stimulates Nav1.8-positive nociceptors via the β-glucan receptor Dectin-1 to induce calcitonin gene-related peptide (CGRP). This induction of CGRP is independent of Bcl-10 or Malt-1 butdependent on transient receptor potential cation channel subfamily V member 1 (TRPV1)/transient receptor potential cation channel subfamily A member 1 (TRPA1) ion channels. Hindpaw β-glucan injectionafter Nav1.8-positive nociceptor ablation or inTRPV1/TRPA1 deficiency showed dramatically increased osteoinflammation accompanied by impaired CGRP production. Strikingly, CGRP suppressed β-glucan-induced inflammation and osteoclast multinucleation via direct suppression of nuclear factor-κB (NF-κB) p65 by the transcriptional repressor Jdp2 and inhibition of actin polymerization, respectively. These findings clearly suggest a role for Dectin-1-mediated sensocrine pathways in the resolution of fungal osteoinflammation.

Evaluation of Histological Changes in Back Muscle Injuries in Rats over Time.

Study DesignAnimal model study.PurposeThe purpose of this study was to evaluate the histological variation in the injured muscle and production of calcitonin gene-related peptide in rats over time.Overview of LiteratureVertebral surgery has been reported to cause atrophy of the back muscles, which may result in pain. However, few reports have described the time series histological variation in the injured muscle and changes in the dominant nerve.MethodsWe used 30 male, 8-week-old Sprague-Dawley rats. The right and left sides of the paravertebral muscle were considered as the injured and uninjured sides, respectively. A 115 g weight was dropped from a height of 1 m on the right paravertebral muscle. Hematoxylin and eosin (H&E) staining of the muscle was performed 1–3 weeks after injury for histological evaluation. Fluoro-Gold (FG) was injected into the paravertebral muscle. The L2 dorsal root ganglia on both sides were resected 1, 2, and 3 weeks after injury, and immunohistochemical staining for calcitonin gene-related peptide was performed.ResultsH&E staining of the paravertebral muscle showed infiltration of inflammatory cells and the presence of granulation tissue in the injured part on the ipsilateral side 1 week after injury. Muscle atrophy occurred 3 weeks after injury, but was repaired via spontaneous replacement of muscle cells/fibers. In contrast, compared with the uninjured side, the percentage of cells double-labeled with FG and calcitonin gene-related peptide in FG-positive cells in the dorsal root ganglia of the injured side was significantly increased at each time point throughout the study period.ConclusionsThese results suggest that sensitization of the dominant nerve in the dorsal root ganglia, which may be caused by cicatrix formation, can protract injured muscle pain. This information may be helpful in elucidating the underlying mechanism of persistent pain after back muscle injury.

The influence of migraine and female hormones on capsaicin-induced dermal blood flow.

Background Migraine is much more common in females than in males, and occurrence is associated with changes in female sex hormones. Calcitonin gene-related peptide (CGRP) plays a key role in migraine, and variations in female sex hormones may affect CGRP sensitivity and/or production. Objectives Investigate repeatability, gender differences, influence of the menstrual cycle and of migraine on CGRP-dependent changes in dermal blood flow (DBF). Methods CGRP-dependent increases in DBF were assessed using laser Doppler perfusion imaging after topical application of 300 or 1000 µg capsaicin on the forearm of healthy subjects and migraine patients. Results In healthy males, DBF response did not vary over time and was comparable with DBF in male migraineurs. In healthy females, capsaicin-induced DBF responses to both doses of capsaicin were higher during menstruation compared to the late-secretory phase (p < 0.05); this menstrual cycle dependence was absent in female migraine patients. Compared to healthy subjects, female migraineurs displayed a higher DBF response both during menstruation and during the late-secretory phase (p < 0.05). Conclusions An increased capsaicin-induced, CGRP-mediated DBF response was observed during menstruation in healthy women, but in female migraine patients this increased response was not affected by the menstrual cycle.

524 In vitro evaluation of phototoxic properties of systemic antipsoriatics

524 In vitro evaluation of phototoxic properties of systemic antipsoriatics

Calcitonin gene-related peptide in aerobic exercise induces collateral circulation development in rat ischemia myocardium.

Aerobic exercise may offer favorable effects for coronary perfusion in the myocardial ischemia area, although the underlying molecular mechanisms remain unclear. This study was designed to investigate the effect of aerobic exercise on the collateral circulation in the ischemia myocardium and to evaluate calcitonin gene-related peptide (CGRP) changes during this process. Wistar rats were randomly divided into 3 groups of 7 rats each: a sham operated group (Sham), a myocardial ischemia-only group (MI) and a MI plus aerobic exercise group (MI+AE). The rat myocardial ischemia model was established by injecting isoprenaline (2mg/kg, i.p.). The aerobic exercise training consisted of swimming (40min/d, 5d/wk) for 4 weeks. At the end-points, after echocardiographic analysis was performed, blood and ischemia myocardium samples were collected and analyzed by ELISA to quantify the CGRP protein. The dorsal root ganglia were isolated and analyzed by reverse transcription polymerase chain reaction (RT-PCR) to examine the CGRP mRNA expression, and ischemia myocardium capillary density was evaluated by immunohistochemistry. Our data showed that the capillary density in the left ventricle and heart function were significantly decreased with decreased CGRP production in the MI rats, which were reversed by aerobic exercise in the MI+AE rats. These results indicate that aerobic exercise may alleviate myocardial ischemia through collateral circulation development with increased CGRP production. CGRP may play an important role in developing the collateral circulation.

Clinical study of cerebrospinal fluid neuropeptides in patients with primary trigeminal neuralgia

ObjectivesTo investigate the expression levels of calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal peptide (VIP), and β-endorphin in the cerebrospinal fluid (CSF) and peripheral blood of patients with primary trigeminal neuralgia (TN). Patients and methodsWe included 20 patients with primary TN who underwent percutaneous radiofrequency thermocoagulation and collected four types of samples from all of them: sample A: CSF samples; sample B: peripheral blood samples; sample C: peripheral blood samples collected one day before the operation; sample D: peripheral blood samples withdrawn one day after the operation. Another 20 CSF samples of patients with nervous system disease or gynecological disease were collected as a control (sample E). Samples A and B were obtained at the same time. We also evaluated the expression of CGRP, SP, β-endorphin, and VIP by visual analog scale (VAS) scores one day before and one day after the operation. In addition, heart rate (HR) at baseline and at the time of sample collection, mean arterial pressure (MAP), and all side effects of the procedure were recorded. ResultsSignificance were found concerning about CGRP, SP, β-endorphin, and VIP in TN patients and the controls (P<0.001). The expression of CGRP, SP, and VIP in sample A was higher than that in sample E. However, the β-endorphin level in sample A was lower than that in sample E. There was a positive correlation between sample A and B regarding the expression of CGRP, SP, β-endorphin, and VIP (P<0. 01). There was no relationship between the time of disease onset and the expression of CGRP, SP, β-endorphin, and VIP in sample A and sample B (P>0.05). No difference was detected between the neuropeptides levels in samples B and C (P>0.05). Notably, VAS in sample D was significantly lower than that in sample C (P<0.01). Finally, there was no difference between the intraoperative HR and MAP values in the studied samples. ConclusionIn primary TN patients, the blood levels of CGRP, SP, β-endorphin, and VIP were associated with those in CSF samples. There was a significant difference between the levels of the four neuropeptides in CSF and control samples. Our results also indicated that the levels of neuropeptides in blood samples can be tested for those in CSF. The disease onset and duration exerted insignificant effects on the production and release of CGRP, SP, β-endorphin, and VIP.