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The main role of research in medicine is to provide relevant knowledge which, after successful translation to clinical practice, improves the quality of healthcare. The sex bias which is still present in the majority of research disciplines prefers male subjects despite legislation changes in the US grant agencies and European research programme Horizon 2020. Male subjects (cells, animals) still dominate in preclinical research and it has detrimental consequences for women’s health and the quality of science. Opposite bias exists for data obtained mainly in animal models utilizing female subjects (e.g. research in multiple sclerosis, osteoporosis) with skewed outcomes for men affected by these diseases. Either way, scientists are producing results which compromise half of the population. Assumptions that females as cohorts are more variable and another assumption that the oestrous cycle should be tracked in case the females are enrolled in preclinical studies were proven wrong. Variability of male versus female cohorts are comparable and do not only stem from hormonal levels. The widespread prevalence of sex differences in human diseases ultimately requires detailed experiments performed on both sexes, unless the studies are specifically addressing reproduction or sex-related behaviors.

Cough in chronic respiratory diseases is a common symptom associated with significant comorbidities including visceral pain. Available antitussive therapy still has limited efficacy. Recent advances in the understanding of voltage-gated sodium channels (Na

An excessive, irritable, productive or non-productive coughing associated with airway inflammation belongs to pathological cough. Increased activation of airway vagal nociceptors in pathological conditions results from dysregulation of the neural pathway that controls cough. A variety of mediators associated with airway inflammation overstimulate these vagal airway fibers including C-fibers leading to hypersensitivity and hyperreactivity. Because current antitussives have limited efficacy and unwanted side effects there is a continual demand for the development of a novel more effective antitussives for a new efficacious and safe cough treatment. Therefore, inhibiting the activity of these vagal C-fibers represents a rational approach to the development of effective antitussive drugs. This may be achieved by blocking inflammatory mediator receptors or by blocking the generator potential associated with the specific ion channels. Because voltage-gated sodium channels (NaVs) are absolutely required for action potentials initiation and conduction irrespective of the stimulus, NaVs become a promising neural target. There is evidence that NaV1.7, 1.8 and 1.9 subtypes are predominantly expressed in airway cough-triggering nerves. The advantage of blocking these NaVs is suppressing C-fiber irrespective to stimuli, but the disadvantage is that by suppressing the nerves is may also block beneficial sensations and neuronal reflex behavior. The concept is that new antitussive drugs would have the benefit of targeting peripheral airway nociceptors without inhibiting the protective cough reflex.

Cough is a vital airway reflex that keeps the respiratory tract wisely protected. It is also a sign of many diseases of the respiratory system and it may become a disease in its own right. Even though the efficacy of antitussive compounds is extensively studied in animal models with promising results, the treatment of pathological cough in humans is insufficient at the moment. The limited translational potential of animal models used to study cough causes, mechanisms and possible therapeutic targets stems from multiple sources. First of all, cough induced in the laboratory by mechanical or chemical stimuli is far from natural cough present in human disease. The main objective of this review is to provide a comprehensive summary of animal models currently used in cough research and to address their advantages and disadvantages. We also want to encourage cough researchers to call for precision is research by addressing the sex bias which has existed in basic cough research for decades and discuss the role of specific pathogen-free (SPF) animals.

Chronic cough is a significant clinical problem in many patients. Current cough suppressant therapies are largely ineffective and have many dangerous adverse effects. Therefore, the identification of novel therapeutic targets and strategies for chronic cough treatment may lead to development of novel effective antitussive therapies with fewer adverse effects. The experimental research in the area of airway sensory nerves suggests that there are two main vagal afferent nerve subtypes that can directly activate cough – extrapulmonary airway C-fibres and Aδ-fibres (described as cough receptors) innervating the trachea. There are different receptors on the vagal nerve terminals that can trigger coughing, such as TRP channels and P2X2/3 receptors. However, in many patients with chronic respiratory diseases multiple activation of these receptors could be involved and it is also difficult to target these receptors. For that reason, a strategy that would inhibit cough-triggering nerve afferents regardless of activated receptors would be of great benefit. In recent years huge progress in understanding of voltage-gated sodium channels (NaVs) leads to a hypothesis that selective targeting of NaVs in airways may represent an effective treatment of pathological cough. The NaVs (NaV1.1 – NaV1.9) are essential for initiation and conduction of action potentials in these nerve fibres. Effective blocking of NaVs will prevent communication between airways and central nervous system and that would inhibit provoked cough irrespective to stimuli. This review provides an overview of airway afferent nerve subtypes that have been described in respiratory tract of human and in animal models. Moreover, the review highlights the current knowledge about cough, the sensory nerves involved in cough, and the voltage-gated sodium channels as a novel neural target in regulation of cough.

Hyperoxia-induced lung injury is well known in animal and human studies. The respiratory epithelium including sensory nerve endings is a major target for oxidative injury that manifested in lung function changes including cough. On the basis of available information we supposed that hyperoxia alone or in combination with primary lung tissue injury should have a damaging effect on lungs, including the airway nerve endings with the changes in the sensitivity of the central and peripheral neuronal pathways regulating cough. We have previously demonstrated that long-term exposure to 100% oxygen inhibits the cough reflex in cat. This review article summarizes the effect of hyperoxia on the cough reflex in guinea pig model using different concentrations of oxygen and different time of exposure. We also present information on the potential role of antioxidants in reversal of the detrimental effects of hyperoxia on coughing and additional analysis of experiments from previously published studies were obtained and analysed for the cough reflex sensitivity.

Esophageal sensory and motor nerves contribute to the symptoms of eosinophilic esophagitis, however, the mechanisms of this neural dysfunction are essentially unknown. We addressed the hypothesis that eosinophilic inflammation in the esophagus alters production of the key regulators of neural function neurotrophins and neurotrophic factors. We developed and optimized the model of allergic eosinophilic inflammation of esophageal mucosa induced by localized administration of allergen ovalbumin into the esophagus in ovalbumin-sensitized guinea pigs. We evaluated changes in expression of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF) and artemin in esophageal mucosa by quantitative RT-PCR. We found that the administration of ovalbumin into the esophageal wall of sensitized animals induced a massive eosinophilic infiltration restricted to esophageal mucosa (3 ± 1 vs. 97 ± 23 eosinophils per high power filed). This inflammatory response altered the expression profile of selected neurotrophic factors. The BDNF mRNA was increased (to 200%), artemin mRNA was decreased (to 50%) while NGF and GDNF was not changed. We conclude that a strong eosinophilic inflammation can be induced in guinea pigs and alters the expression of neurotrophins and neurotrophic factors. Our findings will aid mechanistic studies of neural dysfunction in eosinophilic esophagitis.

Pathological cough is an unwanted and debilitating symptom of a range of chronic respiratory diseases. Currently used antitussive therapies are ineffective and act largely through the central nervous system, having a number of dangerous adverse effects. There is an urgent need for new, better peripherally acting antitussive drugs with minimal adverse effects. Significant progress has recently been achieved in the understanding of voltage-gated sodium channels (NaVs), which points to the biological plausibility that blocking specific NaV sybtypes (NaV1.1 - NaV1.9) in the airway sensory nerves may lead to the inhibition of pathological coughing. In this study we investigated the effect of lidocaine, a non-selective NaVs blocker, on citric acid- and capsaicin-induced cough in the awake naïve guinea pig experimental model. We found that pre-inhalation followed by continuous inhalation of nebulized lidocaine (10 mM) during citric acid (0.8 M) cough challenge was effective in suppressing the cough response (number of coughs, median [IQR]) - (5 [3.8-6.3] pre-lidocaine vs. 1 [0.3-3.8] post-lidocaine; p = 0.002, n = 11). Likewise, lidocaine during capsaicin (50 μM) cough challenge reduced the number of coughs (6 [4.1-7.9] pre-lidocaine vs. 1 [0.5-1.5] post-lidocaine; p = 0.0005, n = 12). We conclude that nebulized lidocaine effectively blocks chemically induced cough. These findings lend support to the notion that NaVs may become a novel neural target in antitussive treatment.

Pathological cough is an unwanted and debilitating symptom of a range of chronic respiratory diseases. Currently used antitussive therapies are ineffective and act largely through the central nervous system, having a number of dangerous adverse effects. There is an urgent need for new, better peripherally acting antitussive drugs with minimal adverse effects. Significant progress has recently been achieved in the understanding of voltage-gated sodium channels (NaVs), which points to the biological plausibility that blocking specific NaV sybtypes (NaV1.1 – NaV1.9) in the airway sensory nerves may lead to the inhibition of pathological coughing. In this study we investigated the effect of lidocaine, a non–selective NaVs blocker, on citric acid– and capsaicin–induced cough in the awake naive guinea pig experimental model. We found that pre–inhalation followed by continuous inhalation of nebulized lidocaine (10 mM) during citric acid (0.8 M) cough challenge was effective in suppressing the cough response (number of coughs, median [IQR]) – (5 [3.8–6.3] pre–lidocaine vs. 1 [0.3–3.8] post–lidocaine; p = 0.002, n = 11). Likewise, lidocaine during capsaicin (50 μM) cough challenge reduced the number of coughs (6 [4.1–7.9] pre–lidocaine vs. 1 [0.5–1.5] post–lidocaine; p = 0.0005, n = 12). We conclude that nebulized lidocaine effectively blocks chemically induced cough. These findings lend support to the notion that NaVs may become a novel neural target in antitussive treatment.

The structure of primary afferent nerve terminals profoundly influences their function. While the complex vagal airway nerve terminals (stretch receptors, cough receptors and neuroepithelial bodies) were thoroughly characterized, much less is known about the structure of airway nerves that do not form distinct complex terminals (often termed free nerve fibers). We selectively induced expression of GFP in vagal afferent nerves in the mouse by transfection with AAV-GFP virus vector and visualized nerve terminals in the trachea by whole organ confocal imaging. Based on structural characteristics we identified four types of vagal afferent nerve fiber terminals in the trachea. Importantly, we found that distinct compartments of tracheal tissue are innervated by distinct nerve fiber terminal types in a non-overlapping manner. Thus, separate terminal types innervate tracheal epithelium vs. anterolateral tracheal wall containing cartilaginous rings and ligaments vs. dorsal wall containing smooth muscle. Our results will aid the study of structure-function relationships in vagal airway afferent nerves and regulation of respiratory reflexes.

The diseases of the esophagus and nose are among the major factors contributing to chronic cough although their role in different patient populations is debated. Studies in animal models and in humans show that afferent C-fiber activators applied on esophageal or nasal mucosa do not initiate cough, but enhance cough induced by inhaled irritants. These results are consistent with the hypothesis that activation of esophageal and nasal C-fibers contribute to cough reflex hypersensitivity observed in chronic cough patients with gastroesophageal reflux disease (GERD) and chronic rhinitis, respectively. The afferent nerves mediating cough sensitization from the esophagus are probably the neural crest-derived vagal jugular C-fibers. In addition to their responsiveness to high concentration of acid typical for gastroesophageal reflux (pH < 5), esophageal C-fibers also express receptors for activation by weakly acidic reflux such as receptors highly sensitive to acid and receptors for bile acids. The nature of sensory pathways from the nose and their activators relevant for cough sensitization are less understood. Increased cough reflex sensitivity was also reported in many patients with GERD or rhinitis who do not complain of cough indicating that additional endogenous or exogenous factors may be required to develop chronic coughing in these diseases.

Sphingosine-1-phosphate (S1P) is an inflammatory mediator increased in the tissue in the number of inflammatory conditions. Preliminary data indicate that the vagal afferent neurons express several S1P receptors including S1PR2-3. We therefore evaluated the hypothesis that S1P induces cough and/or enhances the cough evoked by other tussive stimuli (TRPA1 and TRPV1 activators) in naive guinea pigs. Inhalation of S1P in the concentrations of 0.1 mM and 1 mM did not evoke cough. Preinhalation and continuing inhalation of S1P (1 mM) during citric acid (0.2 M) challenge did not enhance citric acid-induced cough. Preinhalation of S1P and continuing inhalation during AITC (3 mM) challenge also did not enhance AITC-induced cough. We conclude that S1P was not efficient to evoke cough in awake naive guinea pigs. S1P was also not effective to sensitize the cough evoked by stimulation of TRPV1 and TRPA1 receptors. Nonetheless it cannot be excluded that S1P influences cough in the context of inflamed airways when the cough-mediating nerves undergo sensory neuroplasticity.

Visceral pain is initiated by activation of primary afferent neurons among which the capsaicin-sensitive (TRPV1-positive) neurons play an important role. The stomach is a common source of visceral pain. Similar to other organs, the stomach receives dual spinal and vagal afferent innervation. Developmentally, spinal dorsal root ganglia (DRG) and vagal jugular neurons originate from embryonic neural crest and vagal nodose neurons originate from placodes. In thoracic organs the neural crest- and placodes-derived TRPV1-positive neurons have distinct phenotypes differing in activation profile, neurotrophic regulation and reflex responses. It is unknown to whether such distinction exists in the stomach. We hypothesized that gastric neural crest- and placodes-derived TRPV1-positive neurons express phenotypic markers indicative of placodes and neural crest phenotypes. Gastric DRG and vagal neurons were retrogradely traced by DiI injected into the rat stomach wall. Single-cell RT-PCR was performed on traced gastric neurons. Retrograde tracing demonstrated that vagal gastric neurons locate exclusively into the nodose portion of the rat jugular/petrosal/nodose complex. Gastric DRG TRPV1-positive neurons preferentially expressed markers PPT-A, TrkA and GFRα3 typical for neural crest-derived TRPV1-positive visceral neurons. In contrast, gastric nodose TRPV1-positive neurons preferentially expressed markers P2X2 and TrkB typical for placodes-derived TRPV1-positive visceral neurons. Differential expression of neural crest and placodes markers was less pronounced in TRPV1-negative DRG and nodose populations. There are phenotypic distinctions between the neural crest-derived DRG and placodes-derived vagal nodose TRPV1-positive neurons innervating the rat stomach that are similar to those described in thoracic organs.

Cough in respiratory diseases is attributed to the activation of airway C-fibers by inflammation. Inflammatory mediators can act on multiple receptors expressed in airway C-fibers, nonetheless, the action potential initiation in C-fibers depends on a limited number of voltage-gated sodium channel (NaV1) subtypes. We have recently demonstrated that NaV1.8 substantially contributes to the action potential initiation in the airway C-fiber subtype implicated in cough. We therefore hypothesized that the NaV1.8 blocker A-803467 inhibits cough. We evaluated the cough evoked by the inhalation of C-fiber activator capsaicin in awake guinea pigs. Compared to vehicle, intraperitoneal or inhaled A-803467 caused 30-50% inhibition of cough at the doses that did not alter respiratory rate. We conclude that the NaV1.8 blocker A-803467 inhibits cough in a manner consistent with its action on the C-fiber nerve terminals in the airways. Targeting voltage-gated sodium channels mediating action potential initiation in airway C-fibers may offer a means of cough inhibition that is independent of the stimulus.

Eighteen healthy volunteers with normal lung function were tested for cough. Before and after nasal administration of thymol (0.025 ml, 10(-3) M) into both nostrils, urge-to-cough, cough threshold, cumulative and total count of coughs per provocation were estimated during standardized and validated capsaicin cough challenge. Nasal thymol challenges induced pleasant olfactory sensation and in 6 out of the 18 subjects also mild cooling sensation. Cough threshold was not influenced when compared with intranasal saline and vehicle challenges (12.5 vs. 13.2 vs. 10.2 μM of capsaicin to induce two or more coughs (C2), respectively), but the total count of coughs after nasal thymol challenge was significantly lower than that obtained after saline or vehicle (19 vs. 20 vs. 14 coughs/provocation, respectively; p

TRPA1 receptor is activated by endogenous inflammatory mediators and exogenous pollutant molecules relevant to respiratory diseases. Previous studies have implicated TRPA1 as a drug target for antitussive therapy. Here we evaluated the relative efficacy of TRPA1 activation to evoke cough. In conscious guinea pigs the TRPA1 agonist allyl-isothiocyanate (AITC) evoked cough with a maximally effective concentration of 10mM that was abolished by the selective TRPA1 antagonist AP-18. AITC (10mM) was approximately 3-times less effective in inducing cough than capsaicin (50 μM). Ex vivo single fiber extracellular recordings revealed that, similarly to capsaicin, AITC evoked activation in airway jugular C-fibers, but not in airway nodose Aδ-fibers. Consistent with the cough studies, AITC was approximately 3-times less effective than capsaicin in evoking sustained activation of the jugular C-fibers. Another TRPA1 agonist, cinnamaldehyde, was approximately twofold more effective than AITC in inducing cough. However, the cinnamaldehyde (10mM)-induced cough was only partially inhibited by the TRPA1 antagonist AP-18, and was abolished by combination of AP-18 and the TRPV1 antagonist I-RTX. We conclude that in naïve guinea pigs, TRPA1 activation initiates cough that is relatively modest compared to the cough initiated by TRPV1, likely due to lower efficacy of TRPA1 stimulation to induce sustained activation of airway C-fibers.

Background The TRPA1 receptor is directly activated by a wide range of chemicals including many endogenous molecules relevant for esophageal pathophysiology. We addressed the hypothesis that the TRPA1 agonists differentially activate esophageal nociceptive subtypes depending on their embryological source (neural crest or epibranchial placodes). Methods Single cell RT-PCR and whole cell patch clamp recordings were performed on the vagal neurons retrogradely labeled from the guinea pig esophagus. Extracellular recordings were made in the isolated innervated esophagus preparation ex vivo. Key Results Single cell RT-PCR revealed that the majority of the nodose (placodes-derived) and jugular (neural crest-derived) TRPV1-positive esophageal nociceptors express TRPA1. Single fiber recording showed that the TRPA1 agonists allyl-isothiocyanate (AITC) and cinnamaldehyde were effective in inducing robust action potential discharge in the nerve terminals of nodose nociceptors, but had far less effect in jugular nociceptors (approximately fivefold less). Higher efficacy of the TRPA1 agonists to activate nodose nociceptors was confirmed in the isolated esophagus-labeled vagal neurons in the whole cell patch clamp studies. Similarly to neural crest-derived vagal jugular nociceptors, the spinal DRG nociceptors that are also neural crest-derived were only modestly activated by allyl-isothiocyanate. Conclusions & Inferences We conclude that the TRPA1 agonists are substantially more effective activators of the placodes-derived than the neural crest-derived esophageal nociceptors. Our data predict that in esophageal diseases the presence of endogenous TRPA1 activators will be preferentially signaled by the vagal nodose nociceptors.

The aim of the study was to investigate the effects of nasal histamine on the intensity of cough reflex and the effects of intensified nasal breathing following the nasal histamine challenge on cough sensitivity (CS) in humans. 20 volunteers (mean age 23, nonsmokers, no history of nasal or respiratory system diseases and atopy) were recruited to the study. Baseline CS was determined in all subjects. 2 days later the subjects (n=20) were challenged with nasal histamine (8 mg/ml, 0.1 ml) and the number of coughs was determined after four consecutive single-breath inhalations of capsaicin C2 concentration during the period of the most intensive nasal symptoms (sneezing, itching of the nose). The same capsaicin C2 challenge was performed after nasal saline challenge two days later. One week later CS was determined after nasal histamine challenge with subsequent 10 min of intensified breathing (5–6 sniff-like aspirations to total lung capacity per minute) through the nose and mouth in randomized order in ten volunteers, and with a two-day interval between the challenges. The same manoeuvres after intranasal saline challenge were performed in volunteers of the control group (n=10). The number of coughs after nasal histamine was significantly higher than that after intranasal saline challenge (8(6–10) vs 5(3–7); p=0.038(. CS was not significantly different between the baseline challenge and challenges after nasal histamine followed by the intensified nasal and mouth breathing ((2.21 (1.8–2.62) vs 2.04 (1.57–2.51) vs 2.05 (1.6–2.5) log10 of capsaicin concentration in μmol.l−1; p=0.09). Conclusions: During the period of maximum nasal symptoms after nasal histamine challenge the cough response to inhaled capsaicin was enhanced. Capsaicin cough sensitivity measured after a 10-min. intensified nasal breathing after nasal histamine challenge, compared to a previous measurement of CS, remain unchanged.

Stimulation of afferent nerves in upper airways may contribute to the pathogenesis of chronic cough in chronic disorders of nose and/or sinuses. We tested the hypothesis that stimulation of the nasal afferent nerves enhances experimentally-induced cough. Intranasal administration of capsaicin (50 microM, 25 microl) did not evoke cough in anaesthetized cats, but enhanced cough induced by mechanical stimulation of the tracheobronchial mucosa (number of coughs, median [IQR]) (6.5 [5.5-8.5] versus 10 [7-14]; P = 0.028, n = 13). In contrast, intranasal histamine (16 mM, 25 microl) had no effect. Intranasal capsaicin (50 microM, 15 microl) did not evoke cough, but enhanced cough evoked by mechanical stimulation of the tracheobronchial mucosa (1 [1-3] versus 3 [2-4]; P = 0.0037, n = 15) in anaesthetized guinea pigs and cough induced by inhalation of citric acid (0.3M, 2 min) in awake guinea pigs (3 [2-5] versus 5 [3-7], P ? 0.0026, n = 23). We conclude that stimulation of nasal afferent nerves with capsaicin enhances experimentally-induced cough. Our results suggest that afferent inputs from the nose interact with the cough reflex pathways in a manner that enhances cough.

Clinical studies indicate that adenosine contributes to esophageal mechanical hypersensitivity in some patients with pain originating in the esophagus. We have previously reported that the esophageal vagal nodose C fibers express the adenosine A2Areceptor. Here we addressed the hypothesis that stimulation of the adenosine A2Areceptor induces mechanical sensitization of esophageal C fibers by a mechanism involving transient receptor potential A1 (TRPA1). Extracellular single fiber recordings of activity originating in C-fiber terminals were made in the ex vivo vagally innervated guinea pig esophagus. The adenosine A2Areceptor-selective agonist CGS21680 induced robust, reversible sensitization of the response to esophageal distention (10–60 mmHg) in a concentration-dependent fashion (1–100 nM). At the half-maximally effective concentration (EC50: ≈3 nM), CGS21680 induced an approximately twofold increase in the mechanical response without causing an overt activation. This sensitization was abolished by the selective A2Aantagonist SCH58261. The adenylyl cyclase activator forskolin mimicked while the nonselective protein kinase inhibitor H89 inhibited mechanical sensitization by CGS21680. CGS21680 did not enhance the response to the purinergic P2X receptor agonist α,β-methylene-ATP, indicating that CGS21680 does not nonspecifically sensitize to all stimuli. Mechanical sensitization by CGS21680 was abolished by pretreatment with two structurally different TRPA1 antagonists AP18 and HC030031 . Single cell RT-PCR and whole cell patch-clamp studies in isolated esophagus-specific nodose neurons revealed the expression of TRPA1 in A2A-positive C-fiber neurons and demonstrated that CGS21682 potentiated TRPA1 currents evoked by allylisothiocyanate. We conclude that stimulation of the adenosine A2Areceptor induces mechanical sensitization of nodose C fibers by a mechanism sensitive to TRPA1 antagonists indicating the involvement of TRPA1.

The cold-sensitive cation channel TRPM8 is a target for menthol, which is used routinely as a cough suppressant and as an additive to tobacco and food products. Given that cold temperatures and menthol activate neurons through gating of TRPM8, it is unclear how menthol actively suppresses cough. In this study we describe the antitussive effects of (−)-menthol in conscious and anesthetized guinea pigs. In anesthetized guinea pigs, cough evoked by citric acid applied topically to the tracheal mucosa was suppressed by menthol only when it was selectively administered as vapors to the upper airways. Menthol applied topically to the tracheal mucosa prior to and during citric acid application or administered continuously as vapors or as an aerosol to the lower airways was without effect on cough. These actions of upper airway menthol treatment were mimicked by cold air delivered to the upper airways but not by (+)-menthol, the inactive isomer of menthol, or by the TRPM8/TRPA1 agonist icilin administered directly to the trachea. Subsequent molecular analyses confirmed the expression of TRPM8 in a subset of nasal trigeminal afferent neurons that do not coincidently express TRPA1 or TRPV1. We conclude that menthol suppresses cough evoked in the lower airways primarily through a reflex initiated from the nose.

Clinical studies showed that GABA(B) receptor agonists improve symptoms in patients with gastroesophageal reflux disease. One proposed mechanism of this effect is direct inhibition of the gastroesophageal vagal tension mechanosensors by GABA(B) agonists leading to reduction of reflux. In addition to tension mechanosensors, the vagal nodose ganglion supplies the esophagus with nociceptive C-fibers that likely contribute to impairment of esophageal reflex regulation in diseases. We hypothesized that GABA(B) agonists inhibit mechanically-induced activation of vagal esophageal nodose C-fibers in baseline and/or in sensitized state induced by inflammatory mediators. Ex vivo extracellular recordings were made from the esophageal nodose C-fibers in the isolated vagally-innervated guinea pig esophagus. We found that the selective GABA(B) agonist baclofen (100-300 microM) did not inhibit activation of esophageal nodose C-fibers evoked by esophageal distention (10-60 mmHg). The mechanical response of esophageal nodose C-fibers can be sensitized by different pathways including the stimulation of the histamine H(1) receptor and the stimulation the adenosine A(2A) receptor. Baclofen failed to inhibit mechanical sensitization of esophageal nodose C-fibers induced by histamine (100 microM) or the selective adenosine A(2A) receptor agonist CGS21680 (3 nM). Our data suggest that the direct mechanical inhibition of nodose C-fibers in the esophagus is unlikely to contribute to beneficial effects of GABA(B) agonists in patients with esophageal diseases.

There is little evidence to support the down-regulation of coughing from the nose. The cough response to citric acid (CA) was studied in anesthetized and conscious guinea pigs after nasal pretreatment with saline, 1% DMSO, allylisothiocyanate (TRPA1 agonist) and allylisothiocyanate +AP-18 (TRPA1 antagonist). Cough was induced by adding citric acid (CA) to the tracheal perfusion in anaesthetized animals, or by inhaling 0.4M CA in conscious animals. The cough response was counted from the dose response curves, airflow traces and cough sound analysis. In conscious animals, nasal allylisothiocyanate induced reproducible, dose dependent nasal symptoms and a significant drop in respiratory rate. Cough induced by CA was suppressed after nasal allylisothiocyanate (p0.05), and this effect was prevented by AP-18 (1mM). In anaesthetized animals, nasal allylisothiocyanate induced a significant drop in respiratory rate. Cough induced subsequently by CA was suppressed when compared to baseline and vehicle responses (p0.05). The reasons for the suppression of CA induced cough by TRPA1 agonist applied to the nose are not clear and remain to be elucidated.

Background Cough, the most important airways defensive mechanism is modulated by many afferent inputs either from respiratory tussigenic areas, but also by afferent drive from other organs. In animal models, modulation of cough by nasal afferent inputs can either facilitate or inhibit the cough response, depending on the type of trigeminal afferents stimulated. Methods In this study we addressed the question of possible bidirectional modulation of cough response in human healthy volunteers by nasal challenges with TRPA1 and TRPM8 agonists respectively. After nasal challenges with isocyanate (AITC), cinnamaldehyde, (−) menthol and (+) menthol (all 10-3 M) nasal symptom score, cough threshold (C2), urge to cough (Cu) and cumulative cough response were measured). Results Nasal challenges with TRPA1 relevant agonists induced considerable nasal symptoms, significantly enhanced urge to cough (p Conclusions In addition to trigeminal afferents expressing TRP channels, olfactory nerve endings, trigemino – olfactoric relationships, the smell perception process and other supramedullar influences should be considered as potential modulators of the cough response in humans.

Clinical studies implicate adenosine acting on esophageal nociceptive pathways in the pathogenesis of noncardiac chest pain originating from the esophagus. However, the effect of adenosine on esophageal afferent nerve subtypes is incompletely understood. We addressed the hypothesis that adenosine selectively activates esophageal nociceptors. Whole cell perforated patch-clamp recordings and single-cell RT-PCR analysis were performed on the primary afferent neurons retrogradely labeled from the esophagus in the guinea pig. Extracellular recordings were made from the isolated innervated esophagus. In patch-clamp studies, adenosine evoked activation (inward current) in a majority of putative nociceptive (capsaicin-sensitive) vagal nodose, vagal jugular, and spinal dorsal root ganglia (DRG) neurons innervating the esophagus. Single-cell RT-PCR analysis indicated that the majority of the putative nociceptive (transient receptor potential V1-positive) neurons innervating the esophagus express the adenosine receptors. The neural crest-derived (spinal DRG and vagal jugular) esophageal nociceptors expressed predominantly the adenosine A1 receptor while the placodes-derived vagal nodose nociceptors expressed the adenosine A1 and/or A2A receptors. Consistent with the studies in the cell bodies, adenosine evoked activation (overt action potential discharge) in esophageal nociceptive nerve terminals. Furthermore, the neural crest-derived jugular nociceptors were activated by the selective A1 receptor agonist CCPA, and the placodes-derived nodose nociceptors were activated by CCPA and/or the selective adenosine A2A receptor CGS-21680. In contrast to esophageal nociceptors, adenosine failed to stimulate the vagal esophageal low-threshold (tension) mechanosensors. We conclude that adenosine selectively activates esophageal nociceptors. Our data indicate that the esophageal neural crest-derived nociceptors can be activated via the adenosine A1 receptor while the placodes-derived esophageal nociceptors can be activated via A1 and/or A2A receptors. Direct activation of esophageal nociceptors via adenosine receptors may contribute to the symptoms in esophageal diseases.

Vagal afferent nerves are essential for optimal neural regulation of visceral organs, but are not often considered important for their defense. However, there are well-defined subsets of vagal afferent nerves that have activation properties indicative of specialization to detect potentially harmful stimuli (nociceptors). This is clearly exemplified by the vagal bronchopulmonary C-fibers that are quiescent in healthy lungs but are readily activated by noxious chemicals and inflammatory molecules. Vagal afferent nerves with similar activation properties have been also identified in the esophagus and probably exist in other visceral tissues. In addition, these putative vagal nociceptors often initiate defensive reflexes, can be sensitized, and have the capacity to induce central sensitization. This set of properties is a characteristic of nociceptors in somatic tissues.

Chronic cough in gastroesophageal reflux disease (GERD) has been attributed to irritation of the esophagus and/or upper airways by reflux of gastric content. Animal models have provided insight into both of these putative mechanisms. In patients with chronic cough and GERD, stimuli associated with reflex in the esophagus sensitize the cough reflex. This sensitization can be reproduced in the guinea pig and is most likely mediated by the esophageal afferent nerve fibers carried by the vagus nerves. Studies in animals have identified several subtypes of vagal esophageal C-fibers that may subserve this function. The putative nociceptive vagal C-fibers in the guinea pig esophagus are stimulated by acid and express the TRPV1 and TRPA1 receptors that confer responsiveness to disparate noxious stimuli. Acute and/or chronic irritation of the upper airways by reflux may contribute to cough by stimulation and/or sensitization of the airway afferent nerves. Studies in animals have identified airway nerves that likely initiate cough due to aspirated reflux; have characterized their pharmacology; and have provided insight into changes of their sensitivity. Studies in animal models have also described the neurophysiology of reflexes that protect the airways from reflux. In conclusion, animal models provide mechanistic insight into the modulation of cough from the esophagus and the pharmacology of neural pathways mediating cough in GERD.

The diseases of the nose and paranasal sinuses (rhinosinusitis) often in combination with asthma and gastroesophageal reflux are common causes of chronic cough in patients with normal chest radiograph. The relationships between rhinosinusitis and cough are incompletely understood. We investigated modulation of the cough reflex by the inputs from the nose. We demonstrate that the cough reflex is sensitized by the intranasal administration of sensory nerve activators in animal models and in humans. Cough reflex is also sensitized in the guinea pig model of allergic nasal inflammation and in patients with allergic rhinitis. In patients with allergic rhinitis the cough sensitization is augmented during the allergen season. We conclude that the cough reflex can be sensitized from the nose. Our data indicate that this sensitization is mediated by nasal sensory nerves. We speculate that by inducing the cough reflex sensitization rhinosinusitis contributes to chronic cough. If combined with environmental or endogenous cough triggers, the cough reflex sensitization is predicted to cause excessive coughing. The potential endogenous cough triggers may be associated with rhinosinusitis (postnasal drip, aspiration of nasal secrets) or secondary to a coexistent disease such as asthma or gastroesophageal reflux.

Cough is a phenomenon frequently associated with upper airway diseases and as a reflex is modulated by many afferent inputs either from respiratory tussigenic areas, but also by afferent drive from other organs. Modulation of cough by nasal afferent inputs could either facilitate cough response or inhibit it in animal models, depending on the type of trigeminal afferents which are stimulated. In recent study we focused on afferents expressing TRPA1, TRPM8 & TRPV3 channels -- channels known as relevant for airway irritants (TRPA1), menthol and other cooling substances (TRPM8) and thymol (TRPV3). Particularly menthol and thymol are substances which are frequently used in over-the-counter medication for cough and common cold based on empirical approach. Objective evidence regarding the modulation of cough in humans has never been reported. 60 human healthy volunteers participated in the study, and they have been challenged by intranasal drops containing agonists of selected ion channels: isocyanate (AITC) & cinnamaldehyde for TRPA1, (-) menthol and (+) menthol for TRPM8 and thymol for TRPV3 ion channels in randomized order (all 10-3 M). Nasal symptom score, cough threshold (C2), urge to cough (Cu) and cumulative cough response had been assessed using capsaicin cough challenge tests. Nasal challenges of TRPA1 relevant agonists induced considerable nasal symptoms, significantly enhanced urge to cough (p < 0.05) but modulation of C2 and cumulative cough response did not reach significance level. Both TRPM8 agonists and TRPV3 agonist thymol administered to the nose significantly modulated all parameters including C2 (p

isms of any sensitizing or direct action. Methods: Afferent mechanoand chemosensitivity was assessed in mouse colo-rectum In Vitro. High-threshold nociceptive afferents were characterised by responses to mechanical stimuli (3). Cytokine receptor abundance was assessed using PCR of Dorsal Root Ganglia (DRG) (L6-S1) and specifically in laser-captured colonic DRG neurons. Healthy and PIVH (TNBS 130μg/mL with 28 day recovery) mice were compared (3). Results: In healthy mice TNF-α (100ng/mL) induced potent mechanical hypersensitivity (P