Advancing cough research: Methodological insights into cough challenge in guinea pig models using double chamber vs whole-body plethysmography.

OBJECTIVE: This study compares two methods of citric acid-induced cough in guinea pigs in whole-body plethysmography (WBP) and double chamber plethysmography (DCP) to evaluate their efficacy. METHODS: Sixteen specific pathogen-free (SPF) and sixteen conventionally-bred (CON) animals were exposed to 0.4 M citric acid aerosol. They underwent cough provocation using both DCP and WBP methods. The number of coughs and latency to the first cough were recorded and analysed using statistical methods to determine significant differences between the two techniques. RESULTS: WBP resulted in significantly higher cough counts (WBP vs. DCP: 13±9 vs 2±3 for SPF; 14±8 vs 5±5 for CON; p<0.0001) and shorter latency (WBP vs. DCP: 59±6 s vs 159±14 s for SPF; 77±4 s vs 112±12 s for CON; p<0.0001) compared to DCP in both groups. CONCLUSION: Methodological differences substantially impact cough responses. WBP provides a more reliable and physiologically relevant methodology for cough assessment, suggesting the need for standardized protocols in cough research to enhance translational relevance.

Modelling Duchenne muscular dystrophy in vitro with newly generated, blood cell-derived induced pluripotent stem cell line ORIONi003-A.

Here, we present newly derived in vitro model for modeling Duchenne muscular dystrophy. Our new cell line was derived by reprogramming of peripheral blood mononuclear cells (isolated from blood from pediatric patient) with Sendai virus encoding Yamanaka factors. Derived iPS cells are capable to differentiate in vitro into three germ layers as verified by immunocytochemistry. When differentiated in special medium, our iPSc formed spontaneously beating cardiomyocytes. As cardiomyopathy is the main clinical complication in patients with Duchenne muscular dystrophy, the cell line bearing the dystrophin gene mutation might be of interest to the research community.

Cough response in specific pathogen-free guinea pig animal model.

Laboratory animal science associations recommend the usage of specific pathogen-free (SPF) animals in experiments to achieve appropriate results. However, their non-natural microbiome might represent an obstacle in the successful translation of obtained results into clinical practice, since the microbiome contributes to the regulation of the host's physiology. Inconsistent results, even in repeated experiments in separate animal groups, irrespective of the season or sex led us to assess the cough response of SPF animal variables and compare it to wild-type animals. The dose-response curves in citric acid and capsaicin cough challenge construction shows significantly increased cough threshold in SPF animals compared to wild-type animals of both sexes. The cause of cough response alteration in SPF animals is not presently known and thus their usage in basic cough research remains questionable. The relationship between airway reflexes and the airway microbiome has never been studied hence we propose that it might be microbiome-related.

Influence of combined voltage-gated sodium channel NaV1.7 and NaV1.8 inhibitors on cough in a guinea pig model.

Pathological excessive cough is a serious clinical problem in many patients. It is no doubt that an increased activation and sensitization of airway vagal C-fibres in disease stems from dysregulation of the neural pathways that control cough. Due to the limited efficacy and unwanted side effects of current antitussives, there is a continual demand for the development of a novel more effective antitussive. Since voltage-gated sodium channels (NaVs) are absolutely required for action potentials initiation and conduction irrespective of the stimulus, NaVs became a promising and attractive neural target. Current studies establish that NaV1.7 and NaV1.8 inhibitors have the potential to suppress cough. In this study, we demonstrated that inhaled aerosol of NaV1.7 inhibitor PF-05089771 (10 µM) and NaV1.8 inhibitor A-803467 (1 mM) mixture inhibited the capsaicin-induced cough by ≈ 60 % and citric acid-induced cough by ≈ 65 % at doses that did not modify respiratory rate. Our previous and present studies indicate that NaV1.7 and NaV1.8 may present promising therapeutic targets for antitussive therapy.

Modification of oestrogen signalling pathways influences cough induced by citric acid but not capsaicin in the animal model of both sexes.

To clarify the role of oestrogen signalling and the role of oestrogen receptor alpha (ERα) in the cough pathways we performed a study in which coughing was observed in both sexes animal models after the treatment by selective ERα degrader fulvestrant (ICI 182-780) and inhibitor of oestrogen synthesis danazol. Degradation of ERα with the normal plasma oestrogen levels induced by fulvestrant, significantly augments the cough response of female but not male guinea pigs. These changes were observed in citric acid-induced cough. Female guinea pigs responded with an increased count of cough expulsions per challenge time and we also detected shorter cough latency. The capsaicin-induced cough did not change. A similar response was observed after danazol treatment, which decreased the plasma oestrogen level. Our results indicate that the transient receptor potential vanilloid-1 (TRPV1) channel-mediated cough is resistant to the hypoestrous state, while the citric acid-mediated cough is oestrogen-dependent and hypersensitive during the hypoestrous state.

The effect of the voltage-gated sodium channel NaV1.7 blocker PF-05089771 on cough in the guinea pig.

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 (NaVs) lead to the rational hypothesis that subtype NaV1.7 is involved in initiating cough and thus may present a promising therapeutic target for antitussive therapy. We evaluated the antitussive effect of NaV1.7 blocker PF-05089771 administered systemically and topically in awake guinea pigs using capsaicin cough challenge. Compared to vehicle, peroral or inhaled PF-05089771 administration caused about 50-60 % inhibition of cough at the doses that did not alter respiratory rate. We conclude that the NaV1.7 blocker PF-05089771 inhibits cough in a manner consistent with its electrophysiological effect on airway C-fibre nerve terminals.

Animal models of cough.

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.

Lidocaine, a Non-selective Inhibitor of Voltage-Gated Sodium Channels, Blocks Chemically-Induced Cough in Awake Naïve Guinea Pigs.

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.

The inflammatory molecule sphingosine-1-phosphate is not effective to evoke or sensitize cough in naïve guinea pigs.

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 naïve 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 naïve 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.

Hyperoxia-induced regulation of cough reflex and its effect after antioxidant supplementation.

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.

Structure of vagal afferent nerve terminal fibers in the mouse trachea.

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.

Distinct Expression of Phenotypic Markers in Placodes- and Neural Crest-Derived Afferent Neurons Innervating the Rat Stomach.

BACKGROUND: 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. AIMS: We hypothesized that gastric neural crest- and placodes-derived TRPV1-positive neurons express phenotypic markers indicative of placodes and neural crest phenotypes. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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 reflex sensitization from esophagus and nose.

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.

Modulation of cough response by sensory inputs from the nose - role of trigeminal TRPA1 versus TRPM8 channels.

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<0.05) but no statistically significant modulation of the C2 and cumulative cough response. In contrast, both TRPM8 agonists administered to the nose significantly modulated all parameters including C2 (p<0.05), Cu (p<0.01) and cumulative cough response (p <0.01) documenting strong anti irritating potential of menthol isomers. 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.

Comparison of TRPA1-versus TRPV1-mediated cough in guinea pigs.

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.

Cough and gastroesophageal reflux: insights from animal models.

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.

Mechanisms of the cough associated with rhinosinusitis.

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.