Environmental enrichment alleviates hyperalgesia by modulating central sensitization in a nitroglycerin-induced chronic migraine model of mice.

BACKGROUND: Chronic migraine (CM) is a debilitating neurofunctional disorder primarily affecting females, characterized by central sensitization. Central sensitization refers to the enhanced response to sensory stimulation, which involves changes in neuronal excitability, synaptic plasticity, and neurotransmitter release. Environmental enrichment (EE) can increase the movement, exploration, socialization and other behaviors of mice. EE has shown promising effects in various neurological disorders, but its impact on CM and the underlying mechanism remains poorly understood. Therefore, the purpose of this study was to determine whether EE has the potential to serve as a cost-effective intervention strategy for CM. METHODS: A mouse CM model was successfully established by repeated administration of nitroglycerin (NTG). We selected adult female mice around 8 weeks old, exposed them to EE for 2 months, and then induced the CM model. Nociceptive threshold tests were measured using Von Frey filaments and a hot plate. The expression of c-Fos, calcitonin gene-related peptide (CGRP) and inflammatory response were measured using WB and immunofluorescence to evaluate central sensitization. RNA sequencing was used to find differentially expressed genes and signaling pathways. Finally, the expression of the target differential gene was investigated. RESULTS: Repeated administration of NTG can induce hyperalgesia in female mice and increase the expression of c-Fos and CGRP in the trigeminal nucleus caudalis (TNC). Early exposure of mice to EE reduced NTG-induced hyperalgesia in CM mice. WB and immunofluorescence revealed that EE inhibited the overexpression of c-Fos and CGRP in the TNC of CM mice and alleviated the inflammatory response of microglia activation. RNA sequencing analysis identified that several central sensitization-related signaling pathways were altered by EE. VGluT1, a key gene involved in behavior, internal stimulus response, and ion channel activity, was found to be downregulated in mice exposed to EE. CONCLUSION: EE can significantly ameliorate hyperalgesia in the NTG-induced CM model. The mechanisms may be to modulate central sensitization by reducing the expression of CGRP, attenuating the inflammatory response, and downregulating the expression of VGluT1, etc., suggesting that EE can serve as an effective preventive strategy for CM.

Calcitonin gene­related peptide alleviates hyperoxia­induced human alveolar cell injury via the CGRPR/TRPV1/Ca2+ axis.

Although exogenous calcitonin gene­related peptide (CGRP) protects against hyperoxia­induced lung injury (HILI), the underlying mechanisms remain unclear. The present study attempted to elucidate the molecular mechanism by which CGRP protects against hyperoxia­induced alveolar cell injury. Human alveolar A549 cells were treated with 95% hyperoxia to establish a hyperoxic cell injury model. ELISA was performed to detect the CGRP secretion. Immunofluorescence, quantitative (q)PCR, and western blotting were used to detect the expression and localization of CGRP receptor (CGRPR) and transient receptor potential vanilloid 1 (TRPV1). Cell counting kit­8 and flow cytometry were used to examine the proliferation and apoptosis of treated cells. Digital calcium imaging and patch clamp were used to analyze the changes in intracellular Ca2+ signaling and membrane currents induced by CGRP in A549 cells. The mRNA and protein expression levels of Cyclin D1, proliferating cell nuclear antigen (PCNA), Bcl­2 and Bax were detected by qPCR and western blotting. The expression levels of CGRPR and TRPV1 in A549 cells were significantly downregulated by hyperoxic treatment, but there was no significant difference in CGRP release between cells cultured under normal air and hyperoxic conditions. CGRP promoted cell proliferation and inhibited apoptosis in hyperoxia, but selective inhibitors of CGRPR and TRPV1 channels could effectively attenuate these effects; TRPV1 knockdown also attenuated this effect. CGRP induced Ca2+ entry via the TRPV1 channels and enhanced the membrane non­selective currents through TRPV1 channels. The CGRP­induced increase in intracellular Ca2+ was reduced by inhibiting the phospholipase C (PLC)/protein kinase C (PKC) pathway. Moreover, PLC and PKC inhibitors attenuated the effects of CGRP in promoting cell proliferation and inhibiting apoptosis. In conclusion, exogenous CGRP acted by inversely regulating the function of TRPV1 channels in alveolar cells. Importantly, CGRP protected alveolar cells from hyperoxia­induced injury via the CGRPR/TRPV1/Ca2+ axis, which may be a potential target for the prevention and treatment of the HILI.

Subarachnoid Hemorrhage Depletes Calcitonin Gene-Related Peptide Levels of Trigeminal Neurons in Rat Dura Mater.

Subarachnoid hemorrhage (SAH) remains a major cause of cerebrovascular morbidity, eliciting severe headaches and vasospasms that have been shown to inversely correlate with vasodilator calcitonin gene-related peptide (CGRP) levels. Although dura mater trigeminal afferents are an important source of intracranial CGRP, little is known about the effects of SAH on these neurons in preclinical models. The present study evaluated changes in CGRP levels and expression in trigeminal primary afferents innervating the dura mater 72 h after experimentally induced SAH in adult rats. SAH, eliciting marked damage revealed by neurological examination, significantly reduced the density of CGRP-immunoreactive nerve fibers both in the dura mater and the trigeminal caudal nucleus in the medulla but did not affect the total dural nerve fiber density. SAH attenuated ex vivo dural CGRP release by ~40% and in the trigeminal ganglion, reduced both CGRP mRNA levels and the number of highly CGRP-immunoreactive cell bodies. In summary, we provide novel complementary evidence that SAH negatively affects the integrity of the CGRP-expressing rat trigeminal neurons. Reduced CGRP levels suggest likely impaired meningeal neurovascular functions contributing to SAH complications. Further studies are to be performed to reveal the importance of impaired CGRP synthesis and its consequences in central sensory processing.

Expression of Calcitonin Gene-Related Peptide and Calcitonin Receptor-like Receptor in Colorectal Adenocarcinoma.

Colorectal cancer is one of the most widespread types of cancer that still causes many deaths worldwide. The development of new diagnostic and prognostic markers, as well as new therapeutic methods, is necessary. The calcitonin gene-related peptide (CGRP) neuropeptide alongside its receptor calcitonin receptor-like receptor (CRLR) could represent future biomarkers and a potential therapeutic target. Increased levels of CGRP have been demonstrated in thyroid, prostate, lung, and breast cancers and may also have a role in colorectal cancer. At the tumor level, it acts through different mechanisms, such as the angiogenesis, migration, and proliferation of tumor cells. The aim of this study was to measure the level of CGRP in colorectal cancer patients' serum by enzyme-linked immunosorbent assay (ELISA) and determine the level of CGRP and CRLR at the tumor level after histopathological (HP) and immunohistochemical (IHC) analysis, and then to correlate them with the TNM stage and with different tumoral characteristics. A total of 54 patients with newly diagnosed colorectal adenocarcinoma were evaluated. We showed that serum levels of CGRP, as well as CGRP and CRLR tumor level expression, correlate with the TNM stage, with local tumor extension, the presence of lymph node metastasis, and distant metastasis, and also with the tumor differentiation degree. CGRP is present in colorectal cancer from the incipient TNM stage, with levels increasing with the stage, and can be used as a diagnostic and prognostic marker and may also represent a potentially new therapeutic target.

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.

Unveiling the therapeutic potential of Dl-3-n-butylphthalide in NTG-induced migraine mouse: activating the Nrf2 pathway to alleviate oxidative stress and neuroinflammation.

BACKGROUND: Migraine stands as a prevalent primary headache disorder, with prior research highlighting the significant involvement of oxidative stress and inflammatory pathways in its pathogenesis and chronicity. Existing evidence indicates the capacity of Dl-3-n-butylphthalide (NBP) to mitigate oxidative stress and inflammation, thereby conferring neuroprotective benefits in many central nervous system diseases. However, the specific therapeutic implications of NBP in the context of migraine remain to be elucidated. METHODS: We established a C57BL/6 mouse model of chronic migraine (CM) using recurrent intraperitoneal injections of nitroglycerin (NTG, 10 mg/kg), and prophylactic treatment was simulated by administering NBP (30 mg/kg, 60 mg/kg, 120 mg/kg) by gavage prior to each NTG injection. Mechanical threshold was assessed using von Frey fibers, and photophobia and anxious behaviours were assessed using a light/dark box and elevated plus maze. Expression of c-Fos, calcitonin gene-related peptide (CGRP), Nucleus factor erythroid 2-related factor 2 (Nrf2) and related pathway proteins in the spinal trigeminal nucleus caudalis (SP5C) were detected by Western blotting (WB) or immunofluorescence (IF). The expression of IL-1ß, IL-6, TNF-α, Superoxide dismutase (SOD) and malondialdehyde (MDA) in SP5C and CGRP in plasma were detected by ELISA. A reactive oxygen species (ROS) probe was used to detect the expression of ROS in the SP5C. RESULTS: At the end of the modelling period, chronic migraine mice showed significantly reduced mechanical nociceptive thresholds, as well as photophobic and anxious behaviours. Pretreatment with NBP attenuated nociceptive sensitization, photophobia, and anxiety in the model mice, reduced expression levels of c-Fos and CGRP in the SP5C and activated Nrf2 and its downstream proteins HO-1 and NQO-1. By measuring the associated cytokines, we also found that NBP reduced levels of oxidative stress and inflammation. Most importantly, the therapeutic effect of NBP was significantly reduced after the administration of ML385 to inhibit Nrf2. CONCLUSIONS: Our data suggest that NBP may alleviate migraine by activating the Nrf2 pathway to reduce oxidative stress and inflammation in migraine mouse models, confirming that it may be a potential drug for the treatment of migraine.

Insulin eye drops improve corneal wound healing in STZ-induced diabetic mice by regulating corneal inflammation and neuropeptide release.

INTRODUCTION: In recent years, insulin eye drops have attracted increasing attention from researchers and ophthalmologists. The aim of this study was to investigate the efficacy and possible mechanism of action of insulin eye drops in diabetic mice with corneal wounds. METHODS: A type 1 diabetes model was induced, and a corneal epithelial injury model of 2.5 mm was established. We used corneal fluorescein staining, hematoxylin-eosin (H-E) staining and the Cochet-Bonnet esthesiometer to examine the process of wound healing. Subsequently, the expression levels of Ki-67, IL-1ß, ß3-tubulin and neuropeptides, including substance P (SP) and calcitonin gene-related peptide (CGRP), were examined at 72 h after corneal injury. RESULTS: Fluorescein staining demonstrated an acceleration of the recovery of corneal epithelial injury in diabetic mice compared with the saline treatment, which was further evidenced by the overexpression of Ki-67. Moreover, 72 h of insulin application attenuated the expression of inflammatory cytokines and neutrophil infiltration. Remarkably, the results demonstrated that topical insulin treatment enhanced the density of corneal epithelial nerves, as well as neuropeptide SP and CGRP release, in the healing cornea via immunofluorescence staining. CONCLUSIONS: Our results indicated that insulin eye drops may accelerate corneal wound healing and decrease inflammatory responses in diabetic mice by promoting nerve regeneration and increasing levels of neuropeptides SP and CGRP.

Synovial fibroblast gene expression is associated with sensory nerve growth and pain in rheumatoid arthritis.

It has been presumed that rheumatoid arthritis (RA) joint pain is related to inflammation in the synovium; however, recent studies reveal that pain scores in patients do not correlate with synovial inflammation. We developed a machine-learning approach (graph-based gene expression module identification or GbGMI) to identify an 815-gene expression module associated with pain in synovial biopsy samples from patients with established RA who had limited synovial inflammation at arthroplasty. We then validated this finding in an independent cohort of synovial biopsy samples from patients who had early untreated RA with little inflammation. Single-cell RNA sequencing analyses indicated that most of these 815 genes were most robustly expressed by lining layer synovial fibroblasts. Receptor-ligand interaction analysis predicted cross-talk between human lining layer fibroblasts and human dorsal root ganglion neurons expressing calcitonin gene-related peptide (CGRP+). Both RA synovial fibroblast culture supernatant and netrin-4, which is abundantly expressed by lining fibroblasts and was within the GbGMI-identified pain-associated gene module, increased the branching of pain-sensitive murine CGRP+ dorsal root ganglion neurons in vitro. Imaging of solvent-cleared synovial tissue with little inflammation from humans with RA revealed CGRP+ pain-sensing neurons encasing blood vessels growing into synovial hypertrophic papilla. Together, these findings support a model whereby synovial lining fibroblasts express genes associated with pain that enhance the growth of pain-sensing neurons into regions of synovial hypertrophy in RA.

Cryo-EM Structure of the Human Amylin 1 Receptor in Complex with CGRP and Gs Protein.

Inhibition of calcitonin gene-related peptide (CGRP) or its cognate CGRP receptor (CGRPR) has arisen as a major breakthrough in the treatment of migraine. However, a second CGRP-responsive receptor exists, the amylin (Amy) 1 receptor (AMY1R), yet its involvement in the pathology of migraine is poorly understood. AMY1R and CGRPR are heterodimers consisting of receptor activity-modifying protein 1 (RAMP1) with the calcitonin receptor (CTR) and the calcitonin receptor-like receptor (CLR), respectively. Here, we present the structure of AMY1R in complex with CGRP and Gs protein and compare it with the reported structures of the AMY1R complex with rat amylin (rAmy) and the CGRPR in complex with CGRP. Despite similar protein backbones observed within the receptors and the N- and C-termini of the two peptides bound to the AMY1R complexes, they have distinct organization in the peptide midregions (the bypass motif) that is correlated with differences in the dynamics of the respective receptor extracellular domains. Moreover, divergent conformations of extracellular loop (ECL) 3, intracellular loop (ICL) 2, and ICL3 within the CTR and CLR protomers are evident when comparing the CGRP bound to the CGRPR and AMY1R, which influences the binding mode of CGRP. However, the conserved interactions made by the C-terminus of CGRP to the CGRPR and AMY1R are likely to account for cross-reactivity of nonpeptide CGRPR antagonists observed at AMY1R, which also extends to other clinically used CGRPR blockers, including antibodies.

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.

Modification of Mesenchymal Stem/Stromal Cell-Derived Small Extracellular Vesicles by Calcitonin Gene Related Peptide (CGRP) Antagonist: Potential Implications for Inflammation and Pain Reversal.

During the progression of knee osteoarthritis (OA), the synovium and infrapatellar fat pad (IFP) can serve as source for Substance P (SP) and calcitonin gene-related peptide (CGRP), two important pain-transmitting, immune, and inflammation modulating neuropeptides. Our previous studies showed that infrapatellar fat pad-derived mesenchymal stem/stromal cells (MSC) acquire a potent immunomodulatory phenotype and actively degrade Substance P via CD10 both in vitro and in vivo. On this basis, our hypothesis is that CD10-bound IFP-MSC sEVs can be engineered to target CGRP while retaining their anti-inflammatory phenotype. Herein, human IFP-MSC cultures were transduced with an adeno-associated virus (AAV) vector carrying a GFP-labelled gene for a CGRP antagonist peptide (aCGRP). The GFP positive aCGRP IFP-MSC were isolated and their sEVs' miRNA and protein cargos were assessed using multiplex methods. Our results showed that purified aCGRP IFP-MSC cultures yielded sEVs with cargo of 147 distinct MSC-related miRNAs. Reactome analysis of miRNAs detected in these sEVs revealed strong involvement in the regulation of target genes involved in pathways that control pain, inflammation and cartilage homeostasis. Protein array of the sEVs cargo demonstrated high presence of key immunomodulatory and reparative proteins. Stimulated macrophages exposed to aCGRP IFP-MSC sEVs demonstrated a switch towards an alternate M2 status. Also, stimulated cortical neurons exposed to aCGRP IFP-MSC sEVs modulate their molecular pain signaling profile. Collectively, our data suggest that yielded sEVs can putatively target CGRP in vivo, while containing potent anti-inflammatory and analgesic cargo, suggesting the promise for novel sEVs-based therapeutic approaches to diseases such as OA.

Calcitonin gene-related peptide-modulated macrophage phenotypic alteration regulates angiogenesis in early bone healing.

OBJECTIVES: This study aimed to investigate the effect of calcitonin gene-related peptide (CGRP) on the temporal alteration of macrophage phenotypes and macrophage-regulated angiogenesis duringearlybonehealing and preliminarily elucidate the mechanism. METHODS: In vivo, the rat mandibular defect models were established with inferior alveolar nerve transection (IANT) or CGRP receptor antagonist injection. Radiographicandhistologic assessments for osteogenesis, angiogenesis, and macrophage phenotypic alteration within bone defects were performed. In vitro, the effect and mechanism of CGRP on macrophage polarization and phenotypic alteration were analyzed. Then the conditioned medium (CM) from CGRP-treated M1 or M2 macrophages was used to culture human umbilical vein endothelial cells (HUVECs), and the CGRP's effect on macrophage-regulated angiogenesis was detected. RESULTS: Comparable changes following IANT and CGRP blockade within bone defects were observed, including the suppression of early osteogenesis and angiogenesis, the prolonged M1 macrophage infiltration and the prohibited transition toward M2 macrophages around vascular endothelium. In vitro experiments showed that CGRP promoted M2 macrophage polarization while upregulating the expression of interleukin 6 (IL-6), a major cytokine that facilitates the transition from M1 to M2-dominant stage, in M1 macrophages via the activation of Yes-associated protein 1. Moreover, CGRP-treated macrophage-CM showed an anabolic effect on HUVECs angiogenesis compared with macrophage-CM and might prevail over the direct effect of CGRP on HUVECs. CONCLUSIONS: Collectively, our results reveal the effect of CGRP on M1 to M2 macrophage phenotypic alteration possibly via upregulating IL-6 in M1 macrophages, and demonstrate the macrophage-regulated pro-angiogenic potential of CGRP in early bone healing.

Distribution and anti-nociceptive function of endomorphin-1 in the rat cranial sensory ganglia.

Distribution of endomorphin-1 (EM-1) was immunohistochemically investigated in the rat cranial sensory ganglia. Small to medium-sized neurons in the trigeminal (TG), petrosal (PG), and jugular ganglia (JG) expressed EM-1-immunoreactivity. However, EM-1-immunoreactive (-ir) neurons were infrequent in the nodose ganglion. In the brainstem, EM-1-ir varicose fibers were detected in the superficial layer of the medullary dorsal horn and the caudal part of the nucleus tractus solitarius. By trichrome immunofluorescence analysis, approximately 70% of EM-1-ir neurons were also immunoreactive for transient receptor potential vanilloid 1 (TRPV1) in all the examined ganglia. Additionally, 56.8% of EM1-ir TG neurons and approximately 30% of EM-1-ir PG and JG neurons showed calcitonin gene-related peptide (CGRP)-immunoreactivity. By a retrograde tracing method, several TG, PG, and JG neurons innervating the facial and external ear canal skin expressed EM-1-immunoreactivity. However, EM-1-ir neurons innervating the tooth pulp, circumvallate papilla, and pharynx were relatively rare. Thus, EM-1 expression and its coexistence with TRPV1 and CGRP in the cranial sensory neurons may depend on their various peripheral targets. EM1-ir neurons probably project to the superficial layer of the medullary dorsal horn and caudal part of the nucleus tractus solitarius. EM-1 may be involved in nociceptive transmission from the skin.

Presynaptic inhibition of excitatory synaptic transmission from the calcitonin gene-related peptide-containing parabrachial neurons to the central amygdala in mice - unexpected influence of systemic inflammation thereon.

The monosynaptic connection from the lateral parabrachial nucleus (LPB) to the central amygdala (CeA) serves as a fundamental pathway for transmitting nociceptive signals to the brain. The LPB receives nociceptive information from the dorsal horn and spinal trigeminal nucleus and sends it to the "nociceptive" CeA, which modulates pain-associated emotions and nociceptive sensitivity. To elucidate the role of densely expressed mu-opioid receptors (MORs) within this pathway, we investigated the effects of exogenously applied opioids on LPB-CeA synaptic transmission, employing optogenetics in mice expressing channelrhodopsin-2 in LPB neurons with calcitonin gene-related peptide (CGRP). A MOR agonist ([D-Ala2,N-Me-Phe4,Glycinol5]-enkephalin, DAMGO) significantly reduced the amplitude of light-evoked excitatory postsynaptic currents (leEPSCs), in a manner negatively correlated with an increase in the paired-pulse ratio. An antagonist of MORs significantly attenuated these effects. Notably, this antagonist significantly increased leEPSC amplitude when applied alone, an effect further amplified in mice subjected to lipopolysaccharide injection 2 h before brain isolation, yet not observed at the 24-h mark. We conclude that opioids could shut off the ascending nociceptive signal at the LPB-CeA synapse through presynaptic mechanisms. Moreover, this gating process might be modulated by endogenous opioids, and the innate immune system influences this modulation.

AMPK activation attenuates central sensitization in a recurrent nitroglycerin-induced chronic migraine mouse model by promoting microglial M2-type polarization.

BACKGROUND: Energy metabolism disorders and neurogenic inflammation play important roles in the central sensitization to chronic migraine (CM). AMP-activated protein kinase (AMPK) is an intracellular energy sensor, and its activation regulates inflammation and reduces neuropathic pain. However, studies on the involvement of AMPK in the regulation of CM are currently lacking. Therefore, this study aimed to explore the mechanism underlying the involvement of AMPK in the central sensitization to CM. METHODS: Mice with recurrent nitroglycerin (NTG)-induced CM were used to detect the expression of AMPK protein in the trigeminal nucleus caudalis (TNC). Following intraperitoneal injection of the AMPK activator 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) and inhibitor compound C, the mechanical pain threshold, activity level, and pain-like behaviors in the mice were measured. The expression of calcitonin gene-related peptide (CGRP) and cytokines, M1/M2 microglia, and NF-κB pathway activation were detected after the intervention. RESULTS: Repeated NTG injections resulted in a gradual decrease in AMPK protein expression, and the negative regulation of AMPK by increased ubiquitin-like plant homeodomain and RING finger domain 1 (UHRF1) expression may counteract AMPK activation by increasing ADP/ATP. AICAR can reduce the hyperalgesia and pain-like behaviors of CM mice, improve the activity of mice, reduce the expression of CGRP, IL-1ß, IL-6, and TNF-α in the TNC region, and increase the expression of IL-4 and IL-10. Moreover, AMPK in TNC was mainly located in microglia. AICAR could reduce the expression of inducible NO synthase (iNOS) in M1 microglia and increase the expression of Arginase 1 (Arg1) in M2 microglia by inhibiting the activation of NF-κB pathway. CONCLUSIONS: AMPK was involved in the central sensitization of CM, and the activation of AMPK reduced neuroinflammation in NTG-induced CM mice. AMPK may provide new insights into interventions for energy metabolism disorders and neurogenic inflammation in migraine.

Activation of CGRP receptor-mediated signaling promotes tendon-bone healing.

Calcitonin gene-related peptide (CGRP), an osteopromotive neurotransmitter with a short half-life, shows increase while calcitonin receptor-like (CALCRL) level is decreased at the early stage in bone fractures. Therefore, the activation of CALCRL-mediated signaling may be more critical to promote the tendon-bone healing. We found CGRP enhanced osteogenic differentiation of BMSCs through PKA/CREB/JUNB pathway, contributing to improved sonic hedgehog (SHH) expression, which was verified at the tendon-bone interface (TBI) in the mice with Calcrl overexpression. The osteoblast-derived SHH and slit guidance ligand 3 were reported to favor nerve regeneration and type H (CD31hiEMCNhi) vessel formation, respectively. Encouragingly, the activation or inactivation of CALCRL-mediated signaling significantly increased or decreased intensity of type H vessel and nerve fiber at the TBI, respectively. Simultaneously, improved gait characteristics and biomechanical performance were observed in the Calcrl overexpression group. Together, the gene therapy targeting CGRP receptor may be a therapeutic strategy in sports medicine.

Is calcitonin gene-related peptide (CGRP) the missing link in food histamine-induced migraine? A review of functional gut-to-trigeminovascular system connections.

Calcitonin gene-related peptide (CGRP) and histamine plasma concentrations increase during migraine attacks. Both mediators are potent vasodilators, and they have been shown to reciprocally contribute to the release of each other in the trigeminovascular system, possibly driving migraine development. A high-histamine-content diet triggers migraine in patients who have histamine degradation deficiency owing to diaminooxidase (DAO) gene mutations. Therefore, studying functional links between exogenous histamine and CGRP seems promising for the understanding of diet-induced migraine generation. Notably, there is a lack of knowledge about the interplay of the enteric nervous system and the spinal/trigeminal somatosensory system with regard to CGRP and histamine. Based on background evidence, we propose that a functional interconnection between exogenous histamine and CGRP contributes to migraine development.

CGRP sensory neurons promote tissue healing via neutrophils and macrophages.

The immune system has a critical role in orchestrating tissue healing. As a result, regenerative strategies that control immune components have proved effective1,2. This is particularly relevant when immune dysregulation that results from conditions such as diabetes or advanced age impairs tissue healing following injury2,3. Nociceptive sensory neurons have a crucial role as immunoregulators and exert both protective and harmful effects depending on the context4-12. However, how neuro-immune interactions affect tissue repair and regeneration following acute injury is unclear. Here we show that ablation of the NaV1.8 nociceptor impairs skin wound repair and muscle regeneration after acute tissue injury. Nociceptor endings grow into injured skin and muscle tissues and signal to immune cells through the neuropeptide calcitonin gene-related peptide (CGRP) during the healing process. CGRP acts via receptor activity-modifying protein 1 (RAMP1) on neutrophils, monocytes and macrophages to inhibit recruitment, accelerate death, enhance efferocytosis and polarize macrophages towards a pro-repair phenotype. The effects of CGRP on neutrophils and macrophages are mediated via thrombospondin-1 release and its subsequent autocrine and/or paracrine effects. In mice without nociceptors and diabetic mice with peripheral neuropathies, delivery of an engineered version of CGRP accelerated wound healing and promoted muscle regeneration. Harnessing neuro-immune interactions has potential to treat non-healing tissues in which dysregulated neuro-immune interactions impair tissue healing.

Sensory nerve release of CGRP increases tumor growth in HNSCC by suppressing TILs.

BACKGROUND: Perineural invasion (PNI) and nerve density within the tumor microenvironment (TME) have long been associated with worse outcomes in head and neck squamous cell carcinoma (HNSCC). This prompted an investigation into how nerves within the tumor microenvironment affect the adaptive immune system and tumor growth. METHODS: We used RNA sequencing analysis of human tumor tissue from a recent HNSCC clinical trial, proteomics of human nerves from HNSCC patients, and syngeneic orthotopic murine models of HPV-unrelated HNSCC to investigate how sensory nerves modulate the adaptive immune system. FINDINGS: Calcitonin gene-related peptide (CGRP) directly inhibited CD8 T cell activity in vitro, and blocking sensory nerve function surgically, pharmacologically, or genetically increased CD8 and CD4 T cell activity in vivo. CONCLUSIONS: Our data support sensory nerves playing a role in accelerating tumor growth by directly acting on the adaptive immune system to decrease Th1 CD4 T cells and activated CD8 T cells in the TME. These data support further investigation into the role of sensory nerves in the TME of HNSCC and points toward the possible treatment efficacy of blocking sensory nerve function or specifically inhibiting CGRP release or activity within the TME to improve outcomes. FUNDING: 1R01DE028282-01, 1R01DE028529-01, 1P50CA261605-01 (to S.D.K.), 1R01CA284651-01 (to S.D.K.), and F31 DE029997 (to L.B.D.).

Recent advances in the knowledge of the mechanism of reflux hypersensitivity.

Reflux hypersensitivity (RH) is a subtype of gastroesophageal reflux disease. The Rome IV criteria separated RH from the original nonerosive reflux disease subgroup and classified it as a new functional oesophageal disease. Recently, the pathogenesis of RH has become the focus of research. According to the latest research reports, upregulation of acid-sensitive receptors, distribution of calcitonin gene-related peptide-positive nerve fibres, and psychiatric comorbidity have key roles in the pathogenesis of RH. This work reviews the latest findings regarding RH mechanisms.