Elucidating the Ability of CGRP to Modulate Microvascular Events in Mouse Skin.

Oedema formation and polymorphonuclear leukocyte (neutrophil) accumulation are involved in both acute and chronic inflammation. Calcitonin gene-related peptide (CGRP) is a sensory neuropeptide that is released from stimulated sensory nerves. CGRP is a potent vasodilator neuropeptide, especially when administered to the cutaneous microvasculature, with a long duration of action. Here, we have investigated the ability of vasodilator amounts of CGRP to modulate oedema formation and neutrophil accumulation induced in the cutaneous microvasculature of the mouse. To learn more about the mechanism of action of endogenous CGRP, we have investigated the response to the inflammatory stimulants tumour necrosis factor alpha (TNFα) and carrageenan in three different murine models: a model where sensory nerves were depleted by resiniferatoxin (RTX); a pharmacological method to investigate the effect of a selective CGRP receptor antagonist; and a genetic approach using wildtype (WT) and αCGRP knockout (KO) mice. Our results show that exogenous CGRP potentiates oedema formation induced by substance P (SP) and TNFα. This is further supported by our findings from sensory nerve-depleted mice (in the absence of all neuropeptides), which indicated that sensory nerves are involved in mediating the oedema formation and neutrophil accumulation induced by TNFα, and also carrageenan in cutaneous microvasculature. Furthermore, endogenous CGRP was shown to contribute to this inflammatory response as carrageenan-induced oedema formation is attenuated in WT mice treated with the CGRP receptor antagonist, and in αCGRPKO mice. It is therefore concluded that CGRP can contribute to inflammation by promoting oedema formation in skin, but this response is dependent on the pro-inflammatory stimulus and circumstance.

(-)-Englerin-A Has Analgesic and Anti-Inflammatory Effects Independent of TRPC4 and 5.

Recently, we found that the deletion of TRPC5 leads to increased inflammation and pain-related behaviour in two animal models of arthritis. (-)-Englerin A (EA), an extract from the East African plant Phyllanthus engleri has been identified as a TRPC4/5 agonist. Here, we studied whether or not EA has any anti-inflammatory and analgesic properties via TRPC4/5 in the carrageenan model of inflammation. We found that EA treatment in CD1 mice inhibited thermal hyperalgesia and mechanical allodynia in a dose-dependent manner. Furthermore, EA significantly reduced the volume of carrageenan-induced paw oedema and the mass of the treated paws. Additionally, in dorsal root ganglion (DRG) neurons cultured from WT 129S1/SvIm mice, EA induced a dose-dependent cobalt uptake that was surprisingly preserved in cultured DRG neurons from 129S1/SvIm TRPC5 KO mice. Likewise, EA-induced anti-inflammatory and analgesic effects were preserved in the carrageenan model in animals lacking TRPC5 expression or in mice treated with TRPC4/5 antagonist ML204.This study demonstrates that while EA activates a sub-population of DRG neurons, it induces a novel TRPC4/5-independent analgesic and anti-inflammatory effect in vivo. Future studies are needed to elucidate the molecular and cellular mechanisms underlying EA's anti-inflammatory and analgesic effects.

Sensory nerves mediate spontaneous behaviors in addition to inflammation in a murine model of psoriasis.

Psoriasis is characterized by keratinocyte hyperproliferation, erythema, as well as a form of pruritus, involving cutaneous discomfort. There is evidence from both clinical and murine models of psoriasis that chemical or surgical depletion of small-diameter sensory nerves/nociceptors benefits the condition, but the mechanisms are unclear. Hence, we aimed to understand the involvement of sensory nerve mediators with a murine model of psoriasis and associated spontaneous behaviors, indicative of cutaneous discomfort. We have established an Aldara model of psoriasis in mice and chemically depleted the small-diameter nociceptors in a selective manner. The spontaneous behaviors, in addition to the erythema and skin pathology, were markedly improved. Attenuated inflammation was associated with reduced dermal macrophage influx and production of reactive oxygen/nitrogen species (peroxynitrite and protein nitrosylation). Subsequently, this directly influenced observed behavioral responses. However, the blockade of common sensory neurogenic mechanisms for transient receptor potential (TRP)V1, TRPA1, and neuropeptides (substance P and calcitonin gene-related peptide) using genetic and pharmacological approaches inhibited the behaviors but not the inflammation. Thus, a critical role of the established sensory TRP-neuropeptide pathway in influencing cutaneous discomfort is revealed, indicating the therapeutic potential of agents that block that pathway. The ongoing inflammation is mediated by a distinct sensory pathway involving macrophage activation.-Kodji, X., Arkless, K. L., Kee, Z., Cleary, S. J., Aubdool, A. A., Evans, E., Caton, P., Pitchford, S. C., Brain, S. D. Sensory nerves mediate spontaneous behaviors in addition to inflammation in a murine model of psoriasis.

CGRP Discovery and Timeline.

Calcitonin gene-related peptide (CGRP) was discovered over about 35 years ago through molecular biological techniques. Its activity as a vasodilator and the proposal that it was involved in pain processing were then soon established. Today, we are in the interesting situation of having the approval for the clinical use of antagonists and antibodies that have proved to block CGRP activities and benefit migraine. Despite all, there is still much to learn concerning the relevance of the vasodilator and other activities as well as further potential applications of CGRP agonists and blockers in disease. This review aims to discuss the history and present knowledge and to act as an introductory chapter in this volume.

A Novel α-Calcitonin Gene-Related Peptide Analogue Protects Against End-Organ Damage in Experimental Hypertension, Cardiac Hypertrophy, and Heart Failure.

BACKGROUND: Research into the therapeutic potential of α-calcitonin gene-related peptide (α-CGRP) has been limited because of its peptide nature and short half-life. Here, we evaluate whether a novel potent and long-lasting (t½ ≥7 hours) acylated α-CGRP analogue (αAnalogue) could alleviate and reverse cardiovascular disease in 2 distinct murine models of hypertension and heart failure in vivo. METHODS: The ability of the αAnalogue to act selectively via the CGRP pathway was shown in skin by using a CGRP receptor antagonist. The effect of the αAnalogue on angiotensin II-induced hypertension was investigated over 14 days. Blood pressure was measured by radiotelemetry. The ability of the αAnalogue to modulate heart failure was studied in an abdominal aortic constriction model of murine cardiac hypertrophy and heart failure over 5 weeks. Extensive ex vivo analysis was performed via RNA analysis, Western blot, and histology. RESULTS: The angiotensin II-induced hypertension was attenuated by cotreatment with the αAnalogue (50 nmol·kg-1·d-1, SC, at a dose selected for lack of long-term hypotensive effects at baseline). The αAnalogue protected against vascular, renal, and cardiac dysfunction, characterized by reduced hypertrophy and biomarkers of fibrosis, remodeling, inflammation, and oxidative stress. In a separate study, the αAnalogue reversed angiotensin II-induced hypertension and associated vascular and cardiac damage. The αAnalogue was effective over 5 weeks in a murine model of cardiac hypertrophy and heart failure. It preserved heart function, assessed by echocardiography, while protecting against adverse cardiac remodeling and apoptosis. Moreover, treatment with the αAnalogue was well tolerated with neither signs of desensitization nor behavioral changes. CONCLUSIONS: These findings, in 2 distinct models, provide the first evidence for the therapeutic potential of a stabilized αAnalogue, by mediating (1) antihypertensive effects, (2) attenuating cardiac remodeling, and (3) increasing angiogenesis and cell survival to protect against and limit damage associated with the progression of cardiovascular diseases. This indicates the therapeutic potential of the CGRP pathway and the possibility that this injectable CGRP analogue may be effective in cardiac disease.

Transient receptor potential canonical 5 (TRPC5) protects against pain and vascular inflammation in arthritis and joint inflammation.

OBJECTIVE: Transient receptor potential canonical 5 (TRPC5) is functionally expressed on a range of cells including fibroblast-like synoviocytes, which play an important role in arthritis. A role for TRPC5 in inflammation has not been previously shown in vivo. We investigated the contribution of TRPC5 in arthritis. METHODS: Male wild-type and TRPC5 knockout (KO) mice were used in a complete Freund's adjuvant (CFA)-induced unilateral arthritis model, assessed over 14 days. Arthritis was determined by measurement of knee joint diameter, hindlimb weightbearing asymmetry and pain behaviour. Separate studies involved chronic pharmacological antagonism of TRPC5 channels. Synovium from human postmortem control and inflammatory arthritis samples were investigated for TRPC5 gene expression. RESULTS: At baseline, no differences were observed. CFA-induced arthritis resulted in increased synovitis in TRPC5 KO mice assessed by histology. Additionally, TRPC5 KO mice demonstrated reduced ispilateral weightbearing and nociceptive thresholds (thermal and mechanical) following CFA-induced arthritis. This was associated with increased mRNA expression of inflammatory mediators in the ipsilateral synovium and increased concentration of cytokines in synovial lavage fluid. Chronic treatment with ML204, a TRPC5 antagonist, augmented weightbearing asymmetry, secondary hyperalgesia and cytokine concentrations in the synovial lavage fluid. Synovia from human inflammatory arthritis demonstrated a reduction in TRPC5 mRNA expression. CONCLUSIONS: Genetic deletion or pharmacological blockade of TRPC5 results in an enhancement in joint inflammation and hyperalgesia. Our results suggest that activation of TRPC5 may be associated with an endogenous anti-inflammatory/analgesic pathway in inflammatory joint conditions.

The sympathetic nervous system is controlled by transient receptor potential vanilloid 1 in the regulation of body temperature.

Transient receptor potential vanilloid 1 (TRPV1) is involved in sensory nerve nociceptive signaling. Recently, it has been discovered that TRPV1 receptors also regulate basal body temperature in multiple species from mice to humans. In the present study, we investigated whether TRPV1 modulates basal sympathetic nervous system (SNS) activity. C57BL6/J wild-type (WT) mice and TRPV1 knockout (KO) mice were implanted with radiotelemetry probes for measurement of core body temperature. AMG9810 (50 mg/kg) or vehicle (2% DMSO/5% Tween 80/10 ml/kg saline) was injected intraperitoneally. Adrenoceptor antagonists or vehicle (5 ml/kg saline) was injected subcutaneously. In WT mice, the TRPV1 antagonist, AMG9810, caused significant hyperthermia, associated with increased noradrenaline concentrations in brown adipose tissue. The hyperthermia was significantly attenuated by the ß-adrenoceptor antagonist propranolol, the mixed α-/ß-adrenoceptor antagonist labetalol, and the α1-adrenoceptor antagonist prazosin. TRPV1 KO mice have a normal basal body temperature, indicative of developmental compensation. d-Amphetamine (potent sympathomimetic) caused hyperthermia in WT mice, which was reduced in TRPV1 KO mice, suggesting a decreased sympathetic drive in KOs. This study provides new evidence that TRPV1 controls thermoregulation upstream of the SNS, providing a potential therapeutic target for sympathetic hyperactivity thermoregulatory disorders.

Superoxide generation and leukocyte accumulation: key elements in the mediation of leukotriene B4-induced itch by transient receptor potential ankyrin 1 and transient receptor potential vanilloid 1.

The underlying mechanisms of itch are poorly understood. We have investigated a model involving the chemoattractant leukotriene B4 (LTB4) that is up-regulated in common skin diseases. Intradermal injection of LTB4 (0.1 nmol/site) into female CD1 mice induced significant scratching movements (used as an itch index) compared with vehicle-injected (0.1% bovine serum albumin-saline) mice. Intraperitoneal transient receptor potential (TRP) channel antagonist treatment significantly inhibited itch as follows: TRP vanilloid 1 (TRPV1) antagonist SB366791 (0.5 mg/kg, by 97%) and the TRP ankyrin 1 (TRPA1) antagonists TCS 5861528 (10 mg/kg; 82%) and HC-030031 (100 mg/kg; 76%). Leukotriene B4 receptor 2 antagonism by LY255283 (5 mg/kg i.p.; 62%) reduced itch. Neither TRPV1-knockout (TRPV1-KO) nor TRPA1-knockout (TRPA1-KO mice exhibited LTB4-induced itch compared with their wild-type counterparts. The reactive oxygen species scavengers N-acetylcysteine (NAC; 204 mg/kg i.p.; 86%) or superoxide dismutase (SOD; 10 mg/kg i.p.; 83%) also inhibited itch. LTB4-induced superoxide release was attenuated by TCS 5861528 (56%) and HC-030031 (66%), NAC (58%), SOD (50%), and LY255283 (59%) but not by the leukotriene B4 receptor 1 antagonist U-75302 (9 nmol/site) or SB366791. Itch, superoxide, and myeloperoxidase generation were inhibited by the leukocyte migration inhibitor fucoidan (10 mg/kg i.v.) by 80, 61, and 34%, respectively. Myeloperoxidase activity was also reduced by SB366791 (35%) and SOD (28%). TRPV1-KO mice showed impaired myeloperoxidase release, whereas TRPA1-KO mice exhibited diminished production of superoxide. This result provides novel evidence that TRPA1 and TRPV1 contribute to itch via distinct mechanisms.

TRPV1 deletion enhances local inflammation and accelerates the onset of systemic inflammatory response syndrome.

The transient receptor potential vanilloid 1 (TRPV1) is primarily localized to sensory nerve fibers and is associated with the stimulation of pain and inflammation. TRPV1 knockout (TRPV1KO) mice show enhanced LPS-induced sepsis compared with wild type (WT). This implies that TRPV1 may have a key modulatory role in increasing the beneficial and reducing the harmful components in sepsis. We investigated immune and inflammatory mechanisms in a cecal ligation and puncture (CLP) model of sepsis over 24 h. CLP TRPV1KO mice exhibited significant hypothermia, hypotension, and organ dysfunction compared with CLP WT mice. Analysis of the inflammatory responses at the site of initial infection (peritoneal cavity) revealed that CLP TRPV1KO mice exhibited: 1) decreased mononuclear cell integrity associated with apoptosis, 2) decreased macrophage tachykinin NK(1)-dependent phagocytosis, 3) substantially decreased levels of nitrite (indicative of NO) and reactive oxygen species, 4) increased cytokine levels, and 5) decreased bacteria clearance when compared with CLP WT mice. Therefore, TRPV1 deletion is associated with impaired macrophage-associated defense mechanisms. Thus, TRPV1 acts to protect against the damaging impact of sepsis and may influence the transition from local to a systemic inflammatory state.

Early postnatal, but not late, exposure to chemical ambient pollutant 1,2-naphthoquinone increases susceptibility to pulmonary allergic inflammation at adulthood.

High diesel exhaust particle levels are associated with increased health effects; however, knowledge on the impact of its chemical contaminant 1,2-naphthoquinone (1,2-NQ) is limited. We investigated whether postnatal and adult exposures to 1,2-NQ influence allergic reaction and the roles of innate and adaptive immunity. Male neonate (6 days) and adult (56 days) C57Bl/6 mice were exposed to 1,2-NQ (100 nM; 15 min) for 3 days, and on day 59, they were sensitized and later challenged with ovalbumin (OVA). Airway hyper-responsiveness (AHR) and production of cytokines, immunoglobulin E (IgE) and leukotriene B4 (LTB4) were measured in the airways. Postnatal exposure to 1,2-NQ activated dendritic cells in splenocytes by increasing expressing cell surface molecules (e.g., CD11c). Co-exposure to OVA effectively polarized T helper (Th) type 2 (Th2) by secreting Th2-mediated cytokines. Re-stimulation with unspecific stimuli (PMA and ionomycin) generated a mixed Th1 (CD4(+)/IFN-γ(+)) and Th17 (CD4(+)/IL-17(+)) phenotype in comparison with the vehicle-matched group. Postnatal exposure to 1,2-NQ did not induce eosinophilia in the airways at adulthood, although it evoked neutrophilia and exacerbated OVA-induced eosinophilia, Th2 cytokines, IgE and LTB4 production without affecting AHR and mast cell degranulation. At adulthood, 1,2-NQ exposure evoked neutrophilia and increased Th1/Th2 cytokine levels, but failed to affect OVA-induced eosinophilia. In conclusion, postnatal exposure to 1,2-NQ increases the susceptibility to antigen-induced asthma. The mechanism appears to be dependent on increased expression of co-stimulatory molecules, which leads to cell presentation amplification, Th2 polarization and enhanced LTB4, humoral response and Th1/Th2 cytokines. These findings may be useful for future investigations on treatments focused on pulmonary illnesses observed in children living in heavy polluted areas.

TRPV1 antagonism by capsazepine modulates innate immune response in mice infected with Plasmodium berghei ANKA.

Thousands of people suffer from severe malaria every year. The innate immune response plays a determinant role in host's defence to malaria. Transient receptor potential vanilloid 1 (TRPV1) modulates macrophage-mediated responses in sepsis, but its role in other pathogenic diseases has never been addressed. We investigated the effects of capsazepine, a TRPV1 antagonist, in malaria. C57BL/6 mice received 10(5) red blood cells infected with Plasmodium berghei ANKA intraperitoneally. Noninfected mice were used as controls. Capsazepine or vehicle was given intraperitoneally for 6 days. Mice were culled on day 7 after infection and blood and spleen cell phenotype and activation were evaluated. Capsazepine decreased circulating but not spleen F4/80(+)Ly6G(+) cell numbers as well as activation of both F4/80(+)and F4/80(+)Ly6G(+) cells in infected animals. In addition, capsazepine increased circulating but not spleen GR1(+) and natural killer (NK) population, without interfering with natural killer T (NKT) cell numbers and blood NK and NKT activation. However, capsazepine diminished CD69 expression in spleen NKT but not NK cells. Infection increased lipid peroxidation and the release of TNFα and IFNγ, although capsazepine-treated group exhibited lower levels of lipid peroxidation and TNFα. Capsazepine treatment did not affect parasitaemia. Overall, TRPV1 antagonism modulates the innate immune response to malaria.

Evidence that inositol 1,4,5-trisphosphate 3-kinase and inositol 1,3,4,5-tetrakisphosphate are negative regulators of platelet function.

Background: Inositol 1,3,4,5-tetrakisphosphate (IP4) is formed from inositol 1,4,5-trisphosphate (IP3) by IP3 3-kinase (ITPK) in most cells. Its function is unknown but has been suggested to be involved in Ca2+ entry, IP3 regulation, and phosphoinositide 3-kinase antagonism. Objectives: To better elucidate a function for IP4, we tested a specific inhibitor of ITPK (GNF362) on platelets, the effects of IP4 directly in permeabilized platelets and its effect on phosphatidylinositol 3,4,5-trisphosphate (PIP3) binding to pleckstrin-homology (PH) domain-containing proteins in platelets. Methods: Human platelets were utilized in whole blood for thrombus formation, in platelet-rich plasma and washed suspensions for aggregation, and for Ca2+ studies, or resuspended in high K+ and low Na+ buffers for permeabilization experiments. Phosphorylation of AKT-Ser473 and Rap1-GTP formation were measured by Western blotting and PIP3 binding using PIP3 beads. Results: GNF362-enhanced platelet aggregation stimulated by low concentrations of ADP, collagen, thrombin, U46619, and thrombus formation in collagen-coated capillaries. GNF362 induced a transient elevation of Ca2+ concentration, elevated basal levels of IP3, and enhanced the peak height of Ca2+ elevated by agonists. In permeabilized platelets, IP4 inhibited GTPγS induced formation of AKT-Ser473 phosphorylation and platelet aggregation. IP4 reduced GTPγS-stimulated Rap1-GTP levels and potently reduced extraction of RASA3 and BTK by PIP3 beads. Conclusion: ITPK and IP4 are negative regulators of platelet function. IP4 regulation of PH domain-containing proteins may represent a pathway by which platelet activation may be controlled during thrombosis.

Transient Receptor Potential Canonical 5 (TRPC5): Regulation of Heart Rate and Protection against Pathological Cardiac Hypertrophy.

TRPC5 is a non-selective cation channel that is expressed in cardiomyocytes, but there is a lack of knowledge of its (patho)physiological role in vivo. Here, we examine the role of TRPC5 on cardiac function under basal conditions and during cardiac hypertrophy. Cardiovascular parameters were assessed in wild-type (WT) and global TRPC5 knockout (KO) mice. Despite no difference in blood pressure or activity, heart rate was significantly reduced in TRPC5 KO mice. Echocardiography imaging revealed an increase in stroke volume, but cardiac contractility was unaffected. The reduced heart rate persisted in isolated TRPC5 KO hearts, suggesting changes in basal cardiac pacing. Heart rate was further investigated by evaluating the reflex change following drug-induced pressure changes. The reflex bradycardic response following phenylephrine was greater in TRPC5 KO mice but the tachycardic response to SNP was unchanged, indicating an enhancement in the parasympathetic control of the heart rate. Moreover, the reduction in heart rate to carbachol was greater in isolated TRPC5 KO hearts. To evaluate the role of TRPC5 in cardiac pathology, mice were subjected to abdominal aortic banding (AAB). An exaggerated cardiac hypertrophy response to AAB was observed in TRPC5 KO mice, with an increased expression of hypertrophy markers, fibrosis, reactive oxygen species, and angiogenesis. This study provides novel evidence for a direct effect of TRPC5 on cardiac function. We propose that (1) TRPC5 is required for maintaining heart rate by regulating basal cardiac pacing and in response to pressure lowering, and (2) TRPC5 protects against pathological cardiac hypertrophy.

Regulation of myofibroblast differentiation and bleomycin-induced pulmonary fibrosis by adrenomedullin.

Myofibroblast differentiation induced by transforming growth factor-ß (TGF-ß) is characterized by the expression of smooth muscle α-actin (SMA) and extracellular matrix proteins. We and others have previously shown that these changes are regulated by protein kinase A (PKA). Adrenomedullin (ADM) is a vasodilator peptide that activates cAMP/PKA signaling through the calcitonin-receptor-like receptor (CRLR) and receptor-activity-modifying proteins (RAMP). In this study, we found that recombinant ADM had little effect on cAMP/PKA in quiescent human pulmonary fibroblasts, whereas it induced a profound activation of cAMP/PKA signaling in differentiated (by TGF-ß) myofibroblasts. In contrast, the prostacyclin agonist iloprost was equally effective at activating PKA in both quiescent fibroblasts and differentiated myofibroblasts. TGF-ß stimulated a profound expression of CRLR with a time course that mirrored the increased PKA responses to ADM. The TGF-ß receptor kinase inhibitor SB431542 abolished expression of CRLR and attenuated the PKA responses of cells to ADM but not to iloprost. CRLR expression was also dramatically increased in lungs from bleomycin-treated mice. Functionally, ADM did not affect initial differentiation of quiescent fibroblasts in response to TGF-ß but significantly attenuated the expression of SMA, collagen-1, and fibronectin in pre-differentiated myofibroblasts, which was accompanied by decreased contractility of myofibroblasts. Finally, sensitization of ADM signaling by transgenic overexpression of RAMP2 in myofibroblasts resulted in enhanced survival and reduced pulmonary fibrosis in the bleomycin model of the disease. In conclusion, differentiated pulmonary myofibroblasts gain responsiveness to ADM via increased CRLR expression, suggesting the possibility of using ADM for targeting pathological myofibroblasts without affecting normal fibroblasts.

Schwann cell-specific JAM-C-deficient mice reveal novel expression and functions for JAM-C in peripheral nerves.

Junctional adhesion molecule-C (JAM-C) is an adhesion molecule expressed at junctions between adjacent endothelial and epithelial cells and implicated in multiple inflammatory and vascular responses. In addition, we recently reported on the expression of JAM-C in Schwann cells (SCs) and its importance for the integrity and function of peripheral nerves. To investigate the role of JAM-C in neuronal functions further, mice with a specific deletion of JAM-C in SCs (JAM-C SC KO) were generated. Compared to wild-type (WT) controls, JAM-C SC KO mice showed electrophysiological defects, muscular weakness, and hypersensitivity to mechanical stimuli. In addressing the underlying cause of these defects, nerves from JAM-C SC KO mice were found to have morphological defects in the paranodal region, exhibiting increased nodal length as compared to WTs. The study also reports on previously undetected expressions of JAM-C, namely on perineural cells, and in line with nociception defects of the JAM-C SC KO animals, on finely myelinated sensory nerve fibers. Collectively, the generation and characterization of JAM-C SC KO mice has provided unequivocal evidence for the involvement of SC JAM-C in the fine organization of peripheral nerves and in modulating multiple neuronal responses.

Enhancing Techniques for Determining Inflammatory Edema Formation and Neutrophil Accumulation in Murine Skin.

Inflammatory edema formation and polymorphonuclear leukocyte (neutrophil) accumulation are common components of cutaneous vascular inflammation, and their assessment is a powerful investigative and drug development tool but typically requires independent cohorts of animals to assess each. We have established the use of a mathematical formula to estimate the ellipsoidal-shaped volume of the edematous wheal or bleb after intradermal injections of substances in mice pretreated intravenously with Evans blue dye (which binds to plasma albumin) to act as an edema marker. Whereas previous extraction of Evans blue dye with formamide is suitable for all strains of mice, we report this quicker and more reliable assessment of edema volume in situ. This therefore allows neutrophil accumulation to be assessed from the same mouse using the myeloperoxidase assay. Importantly, we examined the influence of Evans blue dye on the spectrometry readout at the wavelength at which myeloperoxidase activity is measured. The results indicate that it is feasible to quantify edema formation and neutrophil accumulation in the same mouse skin site. Thus, we show techniques that can assess edema formation and neutrophil accumulation at the same site in the same mouse, allowing paired measurements and reducing the total use of mice by 50%.

Disruption of Atrial Rhythmicity by the Air Pollutant 1,2-Naphthoquinone: Role of Beta-Adrenergic and Sensory Receptors.

The combustion of fossil fuels contributes to air pollution (AP), which was linked to about 8.79 million global deaths in 2018, mainly due to respiratory and cardiovascular-related effects. Among these, particulate air pollution (PM2.5) stands out as a major risk factor for heart health, especially during vulnerable phases. Our prior study showed that premature exposure to 1,2-naphthoquinone (1,2-NQ), a chemical found in diesel exhaust particles (DEP), exacerbated asthma in adulthood. Moreover, increased concentration of 1,2-NQ contributed to airway inflammation triggered by PM2.5, employing neurogenic pathways related to the up-regulation of transient receptor potential vanilloid 1 (TRPV1). However, the potential impact of early-life exposure to 1,2-naphthoquinone (1,2-NQ) on atrial fibrillation (AF) has not yet been investigated. This study aims to investigate how inhaling 1,2-NQ in early life affects the autonomic adrenergic system and the role played by TRPV1 in these heart disturbances. C57Bl/6 neonate male mice were exposed to 1,2-NQ (100 nM) or its vehicle at 6, 8, and 10 days of life. Early exposure to 1,2-NQ impairs adrenergic responses in the right atria without markedly affecting cholinergic responses. ECG analysis revealed altered rhythmicity in young mice, suggesting increased sympathetic nervous system activity. Furthermore, 1,2-NQ affected ß1-adrenergic receptor agonist-mediated positive chronotropism, which was prevented by metoprolol, a ß1 receptor blocker. Capsazepine, a TRPV1 blocker but not a TRPC5 blocker, reversed 1,2-NQ-induced cardiac changes. In conclusion, neonate mice exposure to AP 1,2-NQ results in an elevated risk of developing cardiac adrenergic dysfunction, potentially leading to atrial arrhythmia at a young age.

A distinct role for transient receptor potential ankyrin 1, in addition to transient receptor potential vanilloid 1, in tumor necrosis factor α-induced inflammatory hyperalgesia and Freund's complete adjuvant-induced monarthritis.

OBJECTIVE: To investigate the involvement of transient receptor potential ankyrin 1 (TRPA1) in inflammatory hyperalgesia mediated by tumor necrosis factor α(TNFα) and joint inflammation. METHODS: Mechanical hyperalgesia was assessed in CD1 mice, mice lacking functional TRP vanilloid 1 (TRPV1-/-) or TRPA1 (TRPA1-/-), or respective wildtype (WT) mice. An automated von Frey system was used, following unilateral intraplantar injection of TNFα or intraarticular injection of Freund's complete adjuvant (CFA). Knee swelling and histologic changes were determined in mice treated with intraarticular injections of CFA. RESULTS: TNFα induced cyclooxygenase-independent bilateral mechanical hyperalgesia in CD1 mice. The selective TRPV1 receptor antagonist SB-366791 had no effect on mechanical hyperalgesia when it was coinjected with TNFα, but intrathecally administered SB- 366791 attenuated bilateral hyperalgesia, indicating the central but not peripheral involvement of TRPV1 receptors. A decrease in pain sensitivity was also observed in TRPV1-/- mice. Intraplantar coadministration of the TRPA1 receptor antagonist AP-18 with TNFα inhibited bilateral hyperalgesia. Intrathecal treatment with AP-18 also reduced TNFα-induced hyperalgesia. CFA-induced mechanical hyperalgesia in CD1 mice was attenuated by AP-18 (administered by intraarticular injection 22 hours after the administration of CFA). Furthermore, intraarticular CFA­induced ipsilateral mechanical hyperalgesia was maintained for 3 weeks in TRPA1 WT mice. In contrast, TRPA1-/- mice exhibited mechanical hyperalgesia for only 24 hours after receiving CFA. CONCLUSION: Evidence suggests that endogenous activation of peripheral TRPA1 receptors plays a critical role in the development of TNFα-induced mechanical hyperalgesia and in sustaining the mechanical hyperalgesia observed after intraaarticular injection of CFA. These results suggest that blockade of TRPA1 receptors may be beneficial in reducing the chronic pain associated with arthritis.

The Vascular-Dependent and -Independent Actions of Calcitonin Gene-Related Peptide in Cardiovascular Disease.

The treatment of hypertension and heart failure remains a major challenge to healthcare providers. Despite therapeutic advances, heart failure affects more than 26 million people worldwide and is increasing in prevalence due to an ageing population. Similarly, despite an improvement in blood pressure management, largely due to pharmacological interventions, hypertension remains a silent killer. This is in part due to its ability to contribute to heart failure. Development of novel therapies will likely be at the forefront of future cardiovascular studies to address these unmet needs. Calcitonin gene-related peptide (CGRP) is a 37 amino acid potent vasodilator with positive-ionotropic and -chronotropic effects. It has been reported to have beneficial effects in hypertensive and heart failure patients. Interestingly, changes in plasma CGRP concentration in patients after myocardial infarction, heart failure, and in some forms of hypertension, also support a role for CGRP on hemodynamic functions. Rodent studies have played an important role thus far in delineating mechanisms involved in CGRP-induced cardioprotection. However, due to the short plasma half-life of CGRP, these well documented beneficial effects have often proven to be acute and transient. Recent development of longer lasting CGRP agonists may therefore offer a practical solution to investigating CGRP further in cardiovascular disease in vivo. Furthermore, pre-clinical murine studies have hinted at the prospect of cardioprotective mechanisms of CGRP which is independent of its hypotensive effect. Here, we discuss past and present evidence of vascular-dependent and -independent processes by which CGRP could protect the vasculature and myocardium against cardiovascular dysfunction.