Calcitonin Gene-related Peptide Research Articles

Alleviation of migraine related pain and anxiety by inhibiting calcium-stimulating AC1-dependent CGRP in the insula of adult rats.

Recent animal and clinical findings consistently highlight the critical role of calcitonin gene-related peptide (CGRP) in chronic migraine (CM) and related emotional responses. CGRP antibodies and receptor antagonists have been approved for CM treatment. However, the underlying CGRP-related signaling pathways in the pain-related cortex remain poorly understood. The SD rats were used to establish the CM model by dural infusions of inflammatory soup. Periorbital mechanical thresholds were assessed using von-Frey filaments, and anxiety-like behaviors were observed via open field and elevated plus maze tests. Expression of c-Fos, CGRP and NMDA GluN2B receptors was detected using immunofluorescence and western blotting analyses. The excitatory synaptic transmission was detected by whole-cell patch-clamp recording. A human-used adenylate cyclase 1 (AC1) inhibitor, hNB001, was applied via insula stereotaxic and intraperitoneal injections in CM rats. The insular cortex (IC) was activated in the migraine model rats. Glutamate-mediated excitatory transmission and NMDA GluN2B receptors in the IC were potentiated. CGRP levels in the IC significantly increased during nociceptive and anxiety-like activities. Locally applied hNB001 in the IC or intraperitoneally alleviated periorbital mechanical thresholds and anxiety behaviors in migraine rats. Furthermore, CGRP expression in the IC decreased after the hNB001 application. Our study indicated that AC1-dependent IC plasticity contributes to migraine and AC1 may be a promising target for treating migraine in the future.

MicroRNA profiling in women with migraine: effects of CGRP-targeting treatment

BackgroundMigraine lacks biomarkers that can trace the biological pathways of the disease and predict the effectiveness of treatments. Monoclonal antibodies targeting calcitonin gene-related peptide pathway – including erenumab – offer the opportunity of investigating potential migraine biomarkers due to their specific mechanism of action in preventing both episodic (EM) and chronic (CM) migraine. Our study aims at evaluating the expression levels of circulating microRNAs (miRNAs) according to migraine type, before and after treatment with erenumab and based on treatment response, in order to identify miRNAs with potential role as epigenetic biomarkers.MethodsThe study included women aged 25–50 years with EM or CM treated with erenumab according to clinical indications. MiRNAs expression levels were assessed before (baseline) and after a 16-week treatment with erenumab, 140 mg every four weeks (post-treatment). An extensive miRNAs profiling was performed by qRT-PCR in small, pooled groups of ≤ 8 women each, classified according to migraine frequency (EM and CM) and the degree of response to erenumab. The expression levels of selected miRNAs were also validated using single miRNA assays in each woman with EM and CM.ResultsDuring the study, 36 women with migraine (19 with EM and 17 with CM) out of 40 who were initially screened, performed the assessment of miRNA expression at baseline and post-treatment, Erenumab treatment significantly improved migraine burden in both EM and CM. MiRNA profiling revealed differential expression levels of a wide set of miRNAs (hsa-let-7d-3p, hsa-miR-106b-3p, hsa-miR-122-5p, hsa-miR-143-3p, hsa-miR-144-3p, hsa-miR-16-5p, hsa-miR-181a-5p, hsa-miR-221-3p, hsa-miR-25-3p, hsa-miR-29b-2-5p, hsa-miR-326, miR-363-3p, hsa-miR-424-5p, hsa-miR-485-3p, hsa-miR-532-5p, hsa-miR-543, hsa-miR-629-5p, hsa-miR-660-5p, hsa-miR-92a-3p) depending on treatment response. Among them, single miRNA assays confirmed the progressive decrease of hsa-miR-143-3p expression levels in relation to increasing response to erenumab in women with EM (7 with low, 6 with medium, and 6 with high response; p = 0.02). Additionally, single assays showed higher hsa-miR-34a-5p and hsa-miR-382-5p expression levels at baseline in women with CM compared with those with EM (p = 0.0002 and p = 0.0007, respectively), as well as their expression level decrease in women with CM from baseline to follow-up (p = 0.04 and p = 0.02, respectively).ConclusionsOur study suggests that targeting the CGRP pathway in migraine changes the expression levels of certain miRNAs. These miRNA levels are linked to the levels of response to CGRP receptor blockage. Future research challenges include assigning specific functions to the modulated miRNAs to unravel pathways modulated by the disease and the treatment.Trial registrationThe study was registered in clinicaltrials.gov with code NCT04659226 and in the Novartis database with code CAMG334AIT05T.

Effectiveness and tolerability of eptinezumab in treating patients with migraine resistant to conventional preventive medications and CGRP (receptor) antibodies: a multicentre retrospective real-world analysis from Germany.

Eptinezumab is a monoclonal antibody that targets calcitonin gene-related peptide (CGRP mAb) and is used for migraine prophylaxis. Efficacy data are mainly from clinical trials, real-world data are hardly available yet. Reimbursement policy in Germany leads to eptinezumab mainly being used in patients having failed pre-treatment with other CGRP mAb. To date, it is unclear whether eptinezumab is efficacious and well tolerated in this population and how the treatment response differs from patients who are naive to CGRP mAbs. We analysed clinical routine data of 79 patients (episodic migraine (EM): n = 19; chronic migraine (CM): n = 60) from four different centres in Germany. All patients were treated with eptinezumab (100mg). Differences in monthly headache (MHD), migraine (MMD) and acute medication days (AMD) after three months were analysed. The correlation of response with the number of CGRP mAb failures was evaluated. Significance level has been corrected (alpha = 0.017). After three months MHD, MMD and AMD were significantly reduced. In EM, the median reduction for MHD was 4.0 days (IQR: -6.5 to -1.0; p = 0.001), for MMD 3.0 days (IQR: -5.5 to -1.5; p < 0.001) and for AMD 2.0 days (IQR: -5.0 to -0.5; p = 0.006). In CM, median reduction of MHD was 4 days (IQR: -8.0 to 0.0; p < 0.001), 3.0 days (IQR: -6.0 to-1.0; p < 0.001) for MMD and 1.0 day (IQR: -5.0 to 0.0; p < 0.001) for AMD. All patients were resistant to conventional preventive therapies and most to CGRP mAbs. Fourteen patients had never received a CGRP mAb and 65 patients had received at least one mAb without sufficient effectiveness and/or intolerability (one: n = 20, two: n = 28, three: n = 17). There was a significant association between the number of prior therapies and the 30% MHD responder rate (none: 78.6%, one: 45.0%, two: 32.1%, three: 23.5%, p = 0.010). Regarding tolerability, 10.4% (8/77) reported mild side effects. The effectiveness of eptinezumab is significantly reduced in patients who have not previously responded to other CGRP mAbs. However, limitations such as the retrospective nature of the analysis, the small sample size and the short treatment period with only the lower dose of eptinezumab must be considered when interpreting the results.

The nHA/PDA/CMCS 3D printed scaffold loaded with SDF-1α/CGRP promoting the regeneration of inactivated bone

The repair of bone tumors is an urgent problem to be solved in clinical treatment. In this study, nano-hydroxyapatite (nHA), polydopamine (PDA) and carboxymethyl chitosan (CMCS) composite scaffolds loaded with stromal cell-derived factor-1α (SDF-1α) and calcitonin gene-related peptide (CGRP) were constructed by 3D printing. It was found that the chemotaxis of nHA/PDA/CMCS scaffold loaded with SDF-1α/CGRP factors to rat periosteal-derived stem cells (rPDSCs) was fivefold higher than that without factors loading, and the constructed scaffold had good biocompatibility and promoted the reactivation of bone inactivated by microwave thermal ablation.

Meningeal lymphatic CGRP signaling governs pain via cerebrospinal fluid efflux and neuroinflammation in migraine models.

Recently developed antimigraine therapeutics targeting calcitonin gene-related peptide (CGRP) signaling are effective, though their sites of activity remain elusive. Notably, the lymphatic vasculature is responsive to CGRP signaling, but whether meningeal lymphatic vessels (MLVs) contribute to migraine pathophysiology is unknown. Mice with lymphatic vasculature deficient in the CGRP receptor (CalcrliLEC mice) treated with nitroglycerin-mediated (NTG-mediated) chronic migraine exhibit reduced pain and light avoidance compared with NTG-treated littermate controls. Gene expression profiles of lymphatic endothelial cells (LECs) isolated from the meninges of Rpl22HA/+;Lyve1Cre RiboTag mice treated with NTG revealed increased MLV-immune interactions compared with cells from untreated mice. Interestingly, the relative abundance of mucosal vascular addressin cell adhesion molecule 1-interacting (MAdCAM1-interacting) CD4+ T cells was increased in the deep cervical lymph nodes of NTG-treated control mice but not in NTG-treated CalcrliLEC mice. Treatment of cultured hLECs with CGRP peptide in vitro induced vascular endothelial-cadherin (VE-cadherin) rearrangement and reduced functional permeability. Likewise, intra cisterna magna injection of CGRP caused rearrangement of VE-cadherin, decreased MLV uptake of cerebrospinal fluid (CSF), and impaired CSF drainage in control mice but not in CalcrliLEC mice. Collectively, these findings reveal a previously unrecognized role for lymphatics in chronic migraine, whereby CGRP signaling primes MLV-immune interactions and reduces CSF efflux.

Effects of the Dual FAAH/MAGL Inhibitor AKU-005 on Trigeminal Hyperalgesia in Male Rats.

The inhibition of endocannabinoid hydrolysis by enzymatic inhibitors may interfere with mechanisms underlying migraine-related pain. The dual FAAH/MAGL inhibitor AKU-005 shows potent inhibitory activity in vitro. Here, we assessed the effect of AKU-005 in a migraine animal model based on nitroglycerin (NTG) administration. Male rats were treated with AKU-005 (0.5 mg/kg, i.p.) or vehicle 3 h after receiving NTG (10 mg/kg, i.p.) or NTG vehicle. One hour later, rats were subjected to the open field test followed by the orofacial formalin test. At the end of the test, we collected serum samples for assessing calcitonin gene-related peptide (CGRP) levels as well as meninges, trigeminal ganglia, and brain areas to assess mRNA levels of CGRP and pro-inflammatory cytokines, and endocannabinoid and related lipid levels. AKU-005 reduced NTG-induced hyperalgesia during the orofacial formalin test but did not influence NTG-induced changes in the open field test. It significantly reduced serum levels of CGRP, CGRP, and pro-inflammatory cytokine mRNA levels in the meninges, trigeminal ganglia, and central areas. Surprisingly, AKU-005 caused no change in endocannabinoids and related lipids in the regions evaluated. The present findings suggest that AKU-005 may have anti-migraine effects by reducing CGRP synthesis and release and the associated inflammatory events. This effect, however, does not seem mediated via an interference with the endocannabinoid pathway.

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

BackgroundChronic 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.MethodsA 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.ResultsRepeated 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.ConclusionEE 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.

New perspectives on migraine treatment: a review of the mechanisms and effects of complementary and alternative therapies.

Migraine is a prevalent and disabling neurovascular disorder, with women being more susceptible, characterized by unilateral throbbing headache, often accompanied by nausea and vomiting, and often associated with various comorbidities such as brain and cardiovascular diseases, which can have a serious impact on quality of life. Although nonsteroidal anti-inflammatory drugs (NSAIDs) are the main first-line medications for the treatment of pain, long-term use often leads to side effects and drug addiction, which emphasizes the need to investigate alternative pain management strategies with fewer adverse effects. Complementary and alternative medicine is a viable pain intervention often used in conjunction with traditional medications, including acupuncture, herbs, moxibustion, transcutaneous electrical stimulation, bio-supplements, and acupressure, which offer non-pharmacological alternatives that are now viable pain management options. This review focuses on the mechanistic doctrine of migraine generation and the role and potential mechanisms of Complementary and Alternative Therapies (CAT) in the treatment of migraine, summarizes the research evidences for CAT as an adjunct or alternative to conventional therapies for migraine, and focuses on the potential of novel migraine therapies (calcitonin gene-related peptide (CGRP) antagonists and pituitary adenylyl cyclase-activating peptide (PACAP) antagonists) with the aim of evaluating CAT therapies as adjunctive or alternative therapies to conventional migraine treatment, thereby providing a broader perspective on migraine management and the design of treatment programs for more effective pain management.

Naringenin relieves paclitaxel-induced pain by suppressing calcitonin gene-related peptide signalling and enhances the anti-tumour action of paclitaxel.

Chemotherapy-induced peripheral neuropathy (CIPN) commonly causes neuropathic pain, but its pathogenesis remains unclear, and effective therapies are lacking. Naringenin, a natural dihydroflavonoid compound, has anti-inflammatory, anti-nociceptive and anti-tumour activities. However, the effects of naringenin on chemotherapy-induced pain and chemotherapy effectiveness remain unexplored. Female and male mouse models of chemotherapy-induced pain were established using paclitaxel. Effects of naringenin were assessed on pain induced by paclitaxel or calcitonin gene-related peptide (CGRP) and on CGRP expression in dorsal root ganglia (DRG) and spinal cord tissue. Additionally, we examined peripheral macrophage infiltration, glial activation, c-fos expression, DRG neuron excitability, microglial M1/M2 polarization, and phosphorylation of spinal NF-κB. Furthermore, we investigated the synergic effect and related mechanisms of naringenin and paclitaxel on cell survival of cancer cells in vitro. Systemic administration of naringenin attenuated paclitaxel-induced pain in both sexes. Naringenin reduced paclitaxel-enhanced CGRP expression in DRGs and the spinal cord, and alleviated CGRP-induced pain in naïve mice of both sexes. Naringenin mitigated macrophage infiltration and reversed paclitaxel-elevated c-fos expression and DRG neuron excitability. Naringenin decreased spinal glial activation and NF-κB phosphorylation in both sexes but influenced microglial M1/M2 polarization only in females. Co-administration of naringenin with paclitaxel enhanced paclitaxel's anti-tumour effect, impeded by an apoptosis inhibitor. Naringenin's anti-nociceptive mechanism involves CGRP signalling and neuroimmunoregulation. Furthermore, naringenin facilitates paclitaxel's anti-tumour action, possibly involving apoptosis. This study demonstrates naringenin's potential as a supplementary treatment in cancer therapy by mitigating side effects and potentiating efficacy of chemotherapy.

Prospective evaluation of aura during anti-calcitonin gene-related peptide monoclonal antibody therapy after 52 weeks of treatment

Background: Clinical studies have shown the efficacy and safety of monoclonal antibodies (mAbs) against calcitonin gene-related peptide (anti- CGRP) in migraine patients with and without aura. Early evidence from post hoc and small subgroup analyses suggests that anti-CGRP mAbs reduce the frequency and intensity of aura. Herein, we prospectively assessed the changes in aura after 12 months of anti-CGRP mAb treatment and performed a literature review.Methods: All outpatients treated with anti-CGRP mAbs for one year in two tertiary Headache Centers and who experienced ≥1 episode of aura/month were enrolled. The study reports data from one month before (baseline) and the last three months (months 10, 11, 12) of treatment.Results: Data from 13 patients with a diagnosis of migraine with and without aura were collected. The mean duration from aura onset was 17.8±7.9 years. At baseline nine patients (69.2%) reported visual aura, and four (30.8%) visual and sensory aura. Mean duration of aura episodes was of 34.2±15.7 minutes. At baseline, the mean number of monthly migraine days (MMDs) was 22.3±7.5, and the mean number of MMDs preceded by aura was 9.15±9.0. At month 12 of treatment, there was a significant reduction of MMDs (6.2±9.0, p=0.002) and MMDs with aura (2.6±2.7, p=0.015). Three patients reported episodes of aura without subsequent headache, a phenomenon that was absent prior to treatment. We identified 14 studies that reported changes in aura during anti-CGRP mAbs treatment.Conclusions: This prospective study shows that anti-CGRP mAbs reduce the number of migraine attacks with aura consistently with the reduction of MMDs. Randomized studies with anti-CGRP mAbs specifically assessing migraine aura are required.

The impact of the migraine treatment onabotulinumtoxinA on inflammatory and pain responses: Insights from an animal model.

Migraine, a prevalent and debilitating disease, involves complex pathophysiology possibly including inflammation and heightened pain sensitivity. The current study utilized the complete Freund's adjuvant (CFA) model of inflammation, with onabotulinumtoxinA (BoNT/A) as a treatment of interest due to its use in clinical migraine management. Using an animal model, the study sought to investigate the role of BoNT/A in modulating CFA-induced inflammation, alterations in pain sensitivity, and the regulation of calcitonin gene-related peptide (CGRP) release. Further, we aimed to assess the changes in SNAP-25 through western blot analysis to gain insights into the mechanistic action of BoNT/A. BoNT/A or control was administered subcutaneously at the periorbital region of rats 3 days before the induction of inflammation using CFA. Periorbital mechanical sensitivity was assessed post-inflammation, and alterations in CGRP release were evaluated. Changes in SNAP-25 levels were determined using western blot analysis. Upon CFA-induced inflammation, there was a marked increase in periorbital mechanical sensitivity, with the inflammation side showing increased sensitivity compared to other periorbital areas. BoNT/A did decrease the withdrawal thresholds in the electronic von Frey test. Despite not being able to observe differences in pain thresholds or CGRP release, BoNT/A reduced baseline release under CFA inflamed conditions. Analysis of SNAP-25 levels in the trigeminal ganglion revealed both intact and cleaved forms that were notably elevated in BoNT/A-treated animals. These findings, derived from western blot analysis, suggest an effect on neurotransmitter release. Our investigation highlights the role of BoNT/A in reducing baseline CGRP in the context of inflammation and its involvement in SNAP-25 cleavage. In contrast, BoNT/A did not appear to alter facial pain sensitivity induced by inflammation, suggesting that mechanisms other than baseline CGRP could be implicated in the elevated thresholds in the CFA model.

Treatment resistance in inclusion body myositis: the role of mast cells

Inclusion body myositis is the commonest acquired myopathy in those over 50 years of age. Although it is classified as an idiopathic inflammatory myopathy and the most frequent finding on muscle biopsy in inclusion body myositis is an endomysial inflammatory infiltrate, it is clinically distinct from other myositis, including a lack of response to immunosuppressive medication. Neurogenic changes are commonly reported in inclusion body myositis and inflammatory changes are observed in muscle following neurogenic injury. The objective of our study was to explore whether neurogenic inflammation plays a role in the pathogenesis of inclusion body myositis, possibly explaining its resistance to immunosuppression. The number of mast cells and presence of neuropeptides, substance P and calcitonin gene-related peptide, were assessed in 48 cases of inclusion body myositis, 11 cases of steroid responsive myositis, two cases of focal myositis associated with neurogenic injury, and ten normal controls. The number of mast cells in inclusion body myositis focal and myositis associated to neurogenic injury were significantly greater than that observed in steroid responsive myositis. Our findings suggest that neurogenic inflammation mediated through mast cells may play a role in the pathogenesis of inclusion body myositis, and focal myositis associated to neurogenic injury, and thus, explain in some part its lack of response to immunosuppressive treatments.

Pharmacokinetics and Safety of Atogepant Co-administered with Quinidine Gluconate in Healthy Participants: A Phase 1, Open-Label, Drug-Drug Interaction Study.

Atogepant, an oral calcitonin gene-related peptide receptor antagonist, is approved for the preventive treatment of migraine. Atogepant is a substrate of P-glycoprotein (P-gp), breast cancer resistance protein, organic anion transporting polypeptide transporters, and cytochrome P450 (CYP)3A4 and 2D6. Quinidine is a strong P-gp and CYP2D6 inhibitor. A phase 1 open-label study evaluated the effect of P-gp and CYP2D6 inhibition by quinidine on the pharmacokinetics of atogepant, and the safety and tolerability of atogepant and quinidine gluconate (QG) when co-administered and when given alone in 33 healthy adults. There was no significant change in the atogepant maximum plasma concentration with QG co-administration. The overall systemic exposure, the area under the plasma concentration-time curve (from time 0 to time t or to infinity), of atogepant increased by 25% when co-administered with QG. However, such an increase was not considered clinically relevant. Atogepant did not alter the mean plasma concentration of quinidine at steady state. The incidence of treatment-emergent adverse events (TEAEs) was highest when QG was administered alone (42.4%), which was primarily due to QT prolongation. Most TEAEs reported were mild in severity and resolved within 1-2 days. Co-administration of atogepant with QG did not result in any unexpected tolerability findings in this phase 1 study in healthy participants. The increase in atogepant exposure during QG co-administration could be due to inhibition of CYP2D6 (a minor contributor to atogepant clearance) as well as inhibition of P-gp.

Calcitonin gene‑related peptide alleviates hyperoxia‑induced human alveolar cell injury via the CGRPR/TRPV1/Ca2+ axis.

Although exogenous calcitonin gene‑related peptide (CGRP) protects against hyperoxia‑induced lung injury (HILI), the underlying mechanisms remain unclear. The present study attempted to elucidate the molecular mechanism by which CGRP protects against hyperoxia‑induced 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 kit‑8 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), Bcl‑2 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 non‑selective currents through TRPV1 channels. The CGRP‑induced 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 hyperoxia‑induced injury via the CGRPR/TRPV1/Ca2+ axis, which may be a potential target for the prevention and treatment of the HILI.

Sex Differences in Alpha Synuclein Gastrointestinal Aggregation

Parkinson’s Disease (PD) is a neurodegenerative disorder with prodromal symptoms often emerging decades before motor issues, including gastrointestinal (GI) symptoms such as constipation and visceral pain. The pathological hallmark of PD is accumulation of misfolded α-synuclein (aSyn) protein in the brain and periphery, including the GI tract. Men have two times the risk of developing PD compared to women. However, disease progression and mortality are higher in women, who also report worse GI symptoms. Gut calcitonin gene related peptide (CGRP) is involved in modulating intestinal contractions and pain, and CGRP receptor antagonists lead to constipation. We hypothesize that sex differences in GI symptoms of PD are the result of sex specific aSyn aggregation and differential enteric CGRP signaling. Intraperitoneal injections of the pesticide rotenone were given to C57BL/6J mice to facilitate aggregation of aSyn. Immunohistochemistry was performed on colon sections using antibodies to aSyn, protein gene product (PGP) 9.5, and CGRP. Quantities of aSyn were analyzed in myenteric PGP9.5+ neuronal fibers. Vehicle treated females had 48% (mean +/- SEM = 0.66 μm2 +/- 0.04) less immunoreactive (ir) enteric aSyn than males (1.3 μm2 +/- 0.13; [F(1,22) = 4.8, p &lt; 0.05). Rotenone treatment increased aSyn-ir by 56% (1.5 μm2 +/- 0.13) in females and 28% (1.08 μm2 +/- 0.14) in males. When interrogated by region, rotenone treatment increased the average precent area of CGRP-ir in the apical crypt (male 9% +/- 0.6; female 10% +/- 0.6) compared to vehicle (male 7% +/- 0.8; female 7% +/- 0.7). This increase was 14% higher in females compared to males (F(1,12) = 5.6, p &lt; 0.05). In males and females, the average percent area of CGRP-ir in the mid-basal crypt region decreased with rotenone treatment (male 4% +/- 0.4; female 2.6% +/- 0.1) compared to vehicle (male 3%+/-0.2, female 1.7%+/-0.06; [F(1,12) = 19.62, p &lt; 0.001). Microbiota have been shown to modulate visceral pain via the release of CGRP, and PD patients have altered microbiotas. The larger increase in CGRP-ir at the apical region of crypts after rotenone treatment in females may contribute to sensitivity to noxious stimuli from the lumen, including microbiota and their metabolites. In addition, decreased mid-basal CGRP-ir and aSyn increases in females after rotenone treatment provide valuable data towards explaining increased GI symptoms seen in PD females. Females are significantly understudied within the context of PD, however, investigating the underlying pathophysiology of GI symptoms while addressing sex as a variable could provide clues to the sex differences seen in clinical outcomes. Since these symptoms often arise years before motor dysfunction and diagnosis, it is critical to determine sex specific mechanisms of aSyn aggregation and influence on enteric neural involvement in prodromal GI symptoms. SCORE MGH ORWH U54-MH118919. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Pulmonary Fibrosis Augments CGRP-Dependent Hyperpolarization and Vasodilation of Mouse Pulmonary Arteries

Calcitonin gene related peptide (CGRP) hyperpolarizes membrane potential (Vm) of pulmonary arterial smooth muscle cells (SMCs) and endothelial cells (ECs) through PKA-dependent activation of KATP channels. CGRP can diminish the severity of pulmonary fibrosis (PF), however, the effects on vascular signaling were poorly defined. We hypothesized that hyperpolarization to CGRP would be augmented in a mouse model of PF. PF was induced by intratracheal delivery of bleomycin (3 wk), and saline was used as a control (sham). Pulmonary arteries (PAs; 100-150 μm diameter) from male C57BL/6J mice (age, ~5 mo), were cannulated, pressurized to 16 cmH2O, and studied at 37°C. Alternatively, intact endothelial tubes were studied at 32°C following dissociation of SMCs. Vm was recorded continuously using intracellular microelectrodes. At rest, Vm was similar for SMCs (bleomycin = -43±3 mV, sham = -44±3 mV; n = 5/group) and ECs (bleomycin = -45±4 mV, sham = -44±3 mV; n=5/group). Increasing concentrations of CGRP (10−10-10−6 M) evoked hyperpolarization of Vm with greater potency in SMCs (EC50: bleomycin = 2 nM, sham = 15 nM) and ECs (EC50: bleomycin = 3 nM, sham = 8 nM) from PF mice, without changing effcacy (SMCs: bleomycin ΔVm = -24±4 mV, sham ΔVm = -21±3 mV; ECs: bleomycin ΔVm = -26±2 mV, sham ΔVm=23±4 mV). Consistently, vasodilation to CGRP in PAs preconstricted with uridine triphosphate (UTP, 5 μM; EC50) was enhanced in PF (92±6% maximum dilation;) vs. sham (83±4% maximum dilation) mice (n=5/group). Greater vasodilation and hyperpolarization of SMCs to CGRP persisted in endothelium-disrupted PAs and during treatment with L-NAME (10−4 M) indicating that ECs are not required for greater responsiveness to CGRP in SMCs. With no effect on resting Vm, inhibition of KATP channels with 1 μM glibenclamide or PKA with protein kinase inhibitor [PKI-(14-22)-amide, myristoylated; 1 μM] significantly attenuated (p&lt;0.05) hyperpolarization of SMCs and ECs (ΔVm ≤5 mV for all groups). Both glibenclamide and PKI attenuated vasodilation to CGRP in PAs and eliminated differences between groups. In a mouse model of PF, CGRP-dependent hyperpolarization of pulmonary arterial SMCs and ECs is augmented through increased PKA-dependent activation of KATP channels leading to increased vasodilator sensitivity. AHA CDA#931652. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Quantitative Analysis of CGRP-IR Afferent Axons in the Mouse Stomach Using Zeiss Arivis Vision4D for Automated Tracing

Gastric nociceptive afferents detect and respond to noxious stimuli by transmitting signals from the stomach to the brain. Calcitonin gene-related peptide (CGRP) is a marker for nociceptive afferents and is crucial to gastric functions, including gastric acid secretion, gut motility, and inflammation. However, the topographical quantitative analysis of CGRP-immunoreactive (CGRP-IR) axons in the whole stomach is challenging due to the large amount of axon innervation in various targets within the tissue. Manual tracing of axons is time-consuming, labor-intensive, and extremely tedious, while currently existing automatic neuron tracing programs often need help with large file sizes or tracing inaccuracies. In this study, we used Zeiss Arivis Vision4D, a novel and sophisticated software with a user-guided machine learning capability, for automated tracing of CGRP-IR axons in flat-mounts of the whole mouse stomach muscular layers and thick serial sections of the antrum-pylorus region (C57BL/6J, male, 2-3 months old, n=7). We utilized a combination of techniques, including the Zeiss M2 Imager for automated scanning, Zen 3.3 (blue edition) for image processing, and Vision4D for stitching the entire stomach flat-mounts and sections, 3D fiber tracing, and density analysis. Our results demonstrated that: 1) Vision4D detected and traced almost all CGRP-IR axons of different gastric targets (blood vessels, muscles, myenteric neurons) in different regions (fundus, corpus, antrum) of the stomach. 2) The x-y-z tracing allowed the visualization of axons in space, portraying its continuous trajectory across layers in the muscular flat mounts (longitudinal muscle, myenteric plexus, circular muscle) and cross sections (muscles, submucosa, mucosa). 3) Vision4D also enabled quantitative morphological and laminar analysis based on target and layer separations. The axon density heatmap generated from the tracing data showed high CGRP-IR axon innervation in the blood vessels. After the removal of blood vessels, dense innervation was seen in the upper fundus and antrum-pylorus followed by corpus, indicating the abundance of CGRP in these regions. 4) Vision4D is a powerful tool in handling large data sizes (hundreds of gigabytes of optical sections in more than 300 image frames per stomach) and outstanding in its capability to track nerve fibers in a highly accurate and time effcient manner. This has paved the way for data batch processing to be accessible, as well as shown that multi-marker tracing of the same tissue sample is attainable. 5) The tracing data will be integrated into a stomach scaffold for a 3D map of CGRP-IR afferent axons. This study represents, for the first time, the automatically traced CGRP-IR axons using whole stomach preparations and highlights the potential of Vision4D in creating axon tracing models in other organs and species. This study is supported by NIH 1 U01 NS113867-01, NIH R15 1R15HL137143-01A1 and Carl ZEISS Company. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Stressful life events, psychiatric comorbidities and serum neuromediator levels in patients with chronic spontaneous urticaria treated with omalizumab.

Many chronic spontaneous urticaria (CSU) patients have highly stressful life events and exhibit psychiatric comorbidities. Emotional stress can cause or exacerbate urticaria symptoms by causing mast cell degranulation via neuromediators. To investigate the frequency of stressful life events and compare psychiatric comorbidities and serum neuromediator levels in patients with CSU who responded to omalizumab with healthy controls. In this cross-sectional study, we included 42 patients with CSU who received at least 6 months of omalizumab treatment and a control group of 42 healthy controls. Stressful life events were evaluated with the Life Events Checklist for DSM-5 (LEC-5). The Depression Anxiety Stress Scale-42 (DASS-42) was used to evaluate depression, anxiety and stress levels. Serum nerve growth factor (NGF), calcitonin gene-related peptide (CGRP) and substance P (SP) levels were measured using the enzyme-linked immunosorbent assay (ELISA) technique. Twenty-six (62%) patients reported at least one stressful life event a median of 3.5 months before the onset of CSU. There were no significant differences in all three variables in the DASS subscales between the patient and control groups. Serum NGF levels were found to be significantly lower in patients with CSU (p <0.001), whereas CGRP levels were found to be significantly higher (p <0.001). There was no significant difference for SP. The psychological status of patients with CSU who benefited from omalizumab was similar to that of healthy controls. Omalizumab may affect stress-related neuromediator levels.

Use of Calcitonin Gene-Related Peptide Monoclonal Antibodies for the Treatment of Migraines in Individuals With Multiple Sclerosis.

Migraines are a common comorbidity and source of disability in patients with chronic inflammatory diseases like multiple sclerosis (MS). Recently, therapeutic agents for episodic and chronic migraine known as calcitonin gene-related peptide (CGRP) inhibitors have shown to effectively control migraine attacks and improve quality of life in the general population. This study explored the use of these novel agents in individuals with comorbid MS. This was a retrospective, population-based cohort study at the University of South Florida's neurology clinic; it evaluated individuals with both MS and migraine. A total of 27 individuals with MS and chronic or episodic migraine who received treatment with a CGRP monoclonal antibody were identified. Of these, 63% reported a reduction in their migraine frequency of greater than 75%. Concurrent use of a disease-modifying therapy (DMT) for MS occurred in 82% of patients, and in 37% of these, the DMT used was also a monoclonal antibody. Adverse effects from CGRP monoclonal antibodies were mild and occurred in only 11% of patients, and no patient experienced worsening of their MS symptoms during cotreatment over the duration of the study. Our study showed a significant reduction in migraine frequency and a favorable adverse event profile for individuals with comorbid MS who took CGRP monoclonal antibodies and experienced no worsening of MS symptoms. In individuals with MS, CGRP monoclonal antibodies seem to be a safe and effective therapy for episodic or chronic migraine.

Nociceptor sensory neurons promote pneumonic sepsis during carbapenem-resistant Klebsiella pneumoniae lung infection.

Abstract Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a leading cause of Gram-negative pneumonia and pneumonic sepsis for which limited therapeutic options are available. The lungs are densely innervated by nociceptive sensory neurons (nociceptors), which mediate breathing, cough, and bronchoconstriction. However, the role of these neurons in host defense against CRKP-induced pneumonic sepsis is unknown. We found that nociceptors play a host deleterious role to promote pneumonic sepsis. Nociceptor-ablated mice showed an enhanced survival and bacterial clearance after CRKP lung infection. Furthermore, nociceptors ablation resulted in the increased influx of neutrophils and Ly6Chi monocytes and cytokine induction. Depletion of Ly6Chi monocytes, but not of neutrophils, impaired lung bacterial clearance and bacterial dissemination in nociceptor-ablated mice, indicating that Ly6Chi monocytes are a critical cellular population downstream of nociceptors to regulate pneumonic sepsis. Moreover, nociceptive neuropeptide calcitonin gene-related peptide (CGRP) suppressed induction of reactive oxygen species and intracellular bacterial killing. Collectively, our data suggest that the nociceptor neuronal signaling could be a therapeutic target for treating multidrug-resistant CRKP lung infections and pneumonic sepsis. Supported by grants from NIH/NIGMS P20GM113117, American Lung Association, and JCRC Heart Research Award.