Calcitonin Gene-related Peptide In Trigeminal Ganglion Research Articles

The Anti-Calcitonin Gene-Related Peptide (Anti-CGRP) Antibody Fremanezumab Reduces Trigeminal Neurons Immunoreactive to CGRP and CGRP Receptor Components in Rats.

Treatment with the anti-CGRP antibody fremanezumab is successful in the prevention of chronic and frequent episodic migraine. In preclinical rat experiments, fremanezumab has been shown to reduce calcitonin gene-related peptide (CGRP) release from trigeminal tissues and aversive behaviour to noxious facial stimuli, which are characteristic pathophysiological changes accompanying severe primary headaches. To further decipher the effects of fremanezumab that underlie these antinociceptive effects in rats, immunohistochemistry and ELISA techniques were used to analyse the content and concentration of CGRP in the trigeminal ganglion, as well as the ratio of trigeminal ganglion neurons which are immunoreactive to CGRP and CGRP receptor components, 1-10 days after subcutaneous injection of fremanezumab (30 mg/kg) compared to an isotype control antibody. After fremanezumab treatment, the fraction of trigeminal ganglion neurons which were immunoreactive to CGRP and the CGRP receptor components calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) was significantly lowered compared to the control. The content and concentration of CGRP in trigeminal ganglia were not significantly changed. A long-lasting reduction in CGRP receptors expressed in trigeminal afferents may contribute to the attenuation of CGRP signalling and antinociceptive effects of monoclonal anti-CGRP antibodies in rats.

NGF-Induced Upregulation of CGRP in Orofacial Pain Induced by Tooth Movement Is Dependent on Atp6v0a1 and Vesicle Release.

The nerve growth factor (NGF) and calcitonin gene-related peptide (CGRP) play a crucial role in the regulation of orofacial pain. It has been demonstrated that CGRP increases orofacial pain induced by NGF. V-type proton ATPase subunit an isoform 1 (Atp6v0a1) is involved in the exocytosis pathway, especially in vesicular transport in neurons. The objective was to examine the role of Atp6v0a1 in NGF-induced upregulation of CGRP in orofacial pain induced by experimental tooth movement. Orofacial pain was elicited by ligating closed-coil springs between incisors and molars in Sprague–Dawley rats. Gene and protein expression levels were determined through real-time polymerase chain reaction, immunostaining, and fluorescence in situ hybridization. Lentivirus vectors carrying Atp6v0a1 shRNA were used to knockdown the expression of Atp6v0a1 in TG and SH-SY5Y neurons. The release of vesicles in SH-SY5Y neurons was observed by using fluorescence dye FM1-43, and the release of CGRP was detected by Enzyme-Linked Immunosorbent Assy. Orofacial pain was evaluated through the rat grimace scale. Our results revealed that intraganglionic administration of NGF and Atp6v0a1 shRNA upregulated and downregulated CGRP in trigeminal ganglia (TG) and trigeminal subnucleus caudalis (Vc), respectively, and the orofacial pain was also exacerbated and alleviated, respectively, following administration of NGF and Atp6v0a1 shRNA. Besides, intraganglionic administration of NGF simultaneously caused the downregulation of Atp6v0a1 in TG. Moreover, the release of vesicles and CGRP in SH-SY5Y neurons was interfered by NGF and Atp6v0a1 shRNA. In conclusion, in the orofacial pain induced by experimental tooth movement, NGF induced the upregulation of CGRP in TG and Vc, and this process is dependent on Atp6v0a1 and vesicle release, suggesting that they are involved in the transmission of nociceptive information in orofacial pain.

N/OFQ modulates orofacial pain induced by tooth movement through CGRP-dependent pathways

BackgroundNociceptin/orphanin FQ (N/OFQ) has been revealed to play bidirectional roles in orofacial pain modulation. Calcitonin gene-related peptide (CGRP) is a well-known pro-nociceptive molecule that participates in the modulation of orofacial pain. We aimed to determine the effects of N/OFQ on the modulation of orofacial pain and on the release of CGRP.MethodsOrofacial pain model was established by ligating springs between incisors and molars in rats for the simulation of tooth movement. The expression level of N/OFQ was determined and pain level was scored in response to orofacial pain. Both agonist and antagonist of N/OFQ receptor were administered to examine their effects on pain and the expression of CGRP in trigeminal ganglia (TG). Moreover, gene therapy based on the overexpression of N/OFQ was delivered to validate the modulatory role of N/OFQ on pain and CGRP expression.ResultsTooth movement elicited orofacial pain and an elevation in N/OFQ expression. N/OFQ exacerbated orofacial pain and upregulated CGRP expression in TG, while UFP-101 alleviated pain and downregulated CGRP expression. N/OFQ-based gene therapy was successful in overexpressing N/OFQ in TG, which resulted in pain exacerbation and elevation of CGRP expression in TG.ConclusionsN/OFQ exacerbated orofacial pain possibly through upregulating CGRP.

Neuropeptide FF receptor 2 inhibits capsaicin-induced CGRP Upregulation in mouse trigeminal ganglion

BackgroundStimulation of trigeminovascular pathway is widely used to establish the headache animal model. Headache is a common neurological disorder, in which symptomatic attacks are mediated by calcitonin-gene-related peptide (CGRP). CGRP is synthesized and released from the trigeminal ganglion to transmit pain signals under stimulation. On the other hand, Neuropeptide FF (NPFF) is a candidate transmitter/modulator for migraine, and stimulation of its receptor, NPFFR2, increases the expression and release of CGRP in mice sensory neurons. Here, we investigate the impact of NPFFR2 on trigeminal CGRP level in a capsaicin-induced headache mouse model.MethodsMice were intracisternally injected with capsaicin into the cisterna magna to activate the trigeminovascular pathway and induce headache symptoms. Mice pretreated with Npffr2-shRNA or NPFFR2 knockouts were adopted to test the impact of NPFFR2 on capsaicin-induced CGRP upregulation in trigeminal ganglion. The gene silencing effect of Npffr2-shRNA in trigeminal ganglion was confirmed by real-time PCR. Trigeminal CGRP level was determined by immunofluorescence staining, and the percentage of CGRP-positive cell was calculated after setting the signal intensity threshold by Image J software. Amount of trigeminal CGRP in NPFFR2 overexpressed mice was also measured by CGRP ELISA.FindingsInfusion of capsaicin into the cisterna magna upregulated the CGRP in trigeminal ganglion and induced spontaneous pain behaviors including the reduction of locomotor activity and the increase of freezing behavior. Intracisternal injection of Npffr2-shRNA reduced the mRNA of Npffr2 in trigeminal ganglion. Mice pretreatment with Npffr2-shRNA prevented capsaicin-induced CGRP upregulation in trigeminal ganglion. Similarly, CGRP upregulation was also reduced in NPFFR2 knockout mice. On the contrary, trigeminal CGRP was increased in NPFFR2 overexpressed mice.ConclusionsReducing the level of NPFFR2 leads to the downregulation of capsaicin-induced CGRP in trigeminal ganglion, which would consequently attenuate the activation of trigeminovascular pathway. Thus, NPFFR2 could serve as a potential target for neuromodulation of cephalic pain.

Efficacy of (S)-Lacosamide in preclinical models of cephalic pain.

Migraine is one of the world's most common neurological disorders. Current acute migraine treatments have suboptimal efficacy, and new therapeutic options are needed. Approaches targeting calcitonin gene related peptide (CGRP) signaling are clinically effective, but small molecule antagonists have not been advanced because of toxicity. In this study, we explored the axonal growth/specification collapsin response mediator protein 2 (CRMP2) as a novel “druggable” target for inhibiting CGRP release and for potential relevance for treatment of migraine pain. Collapsin response mediator protein 2 has been demonstrated to regulate N-type voltage-gated Ca2+ channel activity and Ca2+-dependent CGRP release in sensory neurons. The coexpression of CRMP2 with N-type voltage-gated Ca2+ channel and CGRP in trigeminal ganglia (TGs) sensory neurons suggested the possibility of a novel approach to regulate CGRP release in the trigeminal system. Screening protocols surprisingly revealed that (S)-lacosamide ((S)-LCM), an inactive analog of the clinically approved small molecule antiepileptic drug (R)-lacosamide (Vimpat), inhibited CRMP2 phosphorylation by cyclin-dependent kinase 5 in rat TG slices and decreased depolarization-evoked Ca2+ influx in TG cells in culture. (S)-LCM significantly blocked capsaicin-evoked CGRP release from dural nerve terminals in the rat in ex vivo cranial cup preparation. Additionally, cephalic and extracephalic cutaneous allodynia induced in rats by activation of dural nociceptors with a cocktail of inflammatory mediators, was inhibited by oral administration of (S)-LCM. The confirmation of CRMP2 as an upstream mediator of CGRP release, together with the brain penetrance of this molecule suggests (S)-LCM as a potential therapy for acute migraine.

Effects of estrogen on the serotonergic system and calcitonin gene-related peptide in trigeminal ganglia of rats.

The prevalence of migraine is 3-folds higher in females than in males, and it is intricately related to the levels of estrogen. Estrogen may regulate the expression of metabolic enzymes and receptors of serotonin and also calcitonin gene-related peptide (CGRP), which are implicated in migraine pathogenesis. To study the effects of estrogen on the components of serotonin system and CGRP in trigeminal ganglia of ovariectomized (OVX) rats. OVX rats were administered estrogen in silastic tubes and after 48 h, serum estrogen levels were determined. Trigeminal ganglia tissues were used for RT-PCRs of tryptophan hydroxylase (TPH), monoamine oxidase (MAO), serotonin receptors (5-HT1A, 5-HT1B, 5-HT2A), estrogen receptor (ER) and CGRP. Western blots of TPH and MAO were performed. Estradiol administration to OVX rats increased TPH mRNA levels, while decreased MAO mRNA levels in trigeminal ganglia tissue. Western blot data correlate with the gene expression results. The decreased mRNA levels of serotonin receptors following ovariectomy were restored in estrogen-replenished rats. The induced gene expression of ER in OVX rats was restored following estrogen replenishment. Estrogen levels affect the levels of serotonin metabolizing enzymes and its receptors besides CGRP levels. Since TPH and MAO levels regulate circulating and physiologically available serotonin content, the regulation of serotonin metabolizing enzymes suggest a plausible mechanism by which estrogen alleviates migraine in women.