Identification of cough-variant asthma phenotypes based on clinical and pathophysiologic data.

BACKGROUND: Cough-variant asthma (CVA) may respond differently to antiasthmatic treatment. There are limited data on the heterogeneity of CVA. OBJECTIVE: We aimed to classify patients with CVA using cluster analysis based on clinicophysiologic parameters and to unveil the underlying molecular pathways of these phenotypes with transcriptomic data of sputum cells. METHODS: We applied k-mean clustering to 342 newly physician-diagnosed patients with CVA from a prospective multicenter observational cohort using 10 prespecified baseline clinical and pathophysiologic variables. The clusters were compared according to clinical features, treatment response, and sputum transcriptomic data. RESULTS: Three stable CVA clusters were identified. Cluster 1 (n = 176) was characterized by female predominance, late onset, normal lung function, and a low proportion of complete resolution of cough (60.8%) after antiasthmatic treatment. Patients in cluster 2 (n = 105) presented with young, nocturnal cough, atopy, high type 2 inflammation, and a high proportion of complete resolution of cough (73.3%) with a highly upregulated coexpression gene network that related to type 2 immunity. Patients in cluster 3 (n = 61) had high body mass index, long disease duration, family history of asthma, low lung function, and low proportion of complete resolution of cough (54.1%). TH17 immunity and type 2 immunity coexpression gene networks were both upregulated in clusters 1 and 3. CONCLUSION: Three clusters of CVA were identified with different clinical, pathophysiologic, and transcriptomic features and responses to antiasthmatics treatment, which may improve our understanding of pathogenesis and help clinicians develop individualized cough treatment in asthma.

Enhanced Diaphragm Muscle Function upon Satellite Cell Transplantation in Dystrophic Mice.

The diaphragm muscle is essential for breathing, and its dysfunctions can be fatal. Many disorders affect the diaphragm, including muscular dystrophies. Despite the clinical relevance of targeting the diaphragm, there have been few studies evaluating diaphragm function following a given experimental treatment, with most of these involving anti-inflammatory drugs or gene therapy. Cell-based therapeutic approaches have shown success promoting muscle regeneration in several mouse models of muscular dystrophy, but these have focused mainly on limb muscles. Here we show that transplantation of as few as 5000 satellite cells directly into the diaphragm results in consistent and robust myofiber engraftment in dystrophin- and fukutin-related protein-mutant dystrophic mice. Transplanted cells also seed the stem cell reservoir, as shown by the presence of donor-derived satellite cells. Force measurements showed enhanced diaphragm strength in engrafted muscles. These findings demonstrate the feasibility of cell transplantation to target the diseased diaphragm and improve its contractility.

Novel regenerative drug, SPG302 promotes functional recovery of diaphragm muscle activity after cervical spinal cord injury.

Spinal cord hemisection at C2 (C2 SH), sparing the dorsal column is widely used to investigate the effects of reduced phrenic motor neuron (PhMN) activation on diaphragm muscle (DIAm) function, with reduced DIAm activity on the injured side during eupnoea. Following C2 SH, recovery of DIAm EMG activity may occur spontaneously over subsequent days/weeks. Various strategies have been effective at improving the incidence and magnitude of DIAm recovery during eupnoea, but little is known about the effects of C2 SH on transdiaphragmatic pressure (Pdi ) during other ventilatory and non-ventilatory behaviours. We employ SPG302, a novel type of pegylated benzothiazole derivative, to assess whether enhancing synaptogenesis (i.e., enhancing spared local connections) will improve the incidence and the magnitude of recovery of DIAm EMG activity and Pdi function 14 days post-C2 SH. In anaesthetised Sprague-Dawley rats, DIAm EMG and Pdi were assessed during eupnoea, hypoxia/hypercapnia and airway occlusion prior to surgery (C2 SH or sham), immediately post-surgery and at 14 days post-surgery. In C2 SH rats, 14 days of DMSO (vehicle) or SPG302 treatments (i.p. injection) occurred. At the terminal experiment, maximum Pdi was evoked by bilateral phrenic nerve stimulation. We show that significant EMG and Pdi deficits are apparent in C2 SH compared with sham rats immediately after surgery. In C2 SH rats treated with SPG302, recovery of eupneic, hypoxia/hypercapnia and occlusion DIAm EMG was enhanced compared with vehicle rats after 14 days. Treatment with SPG302 also ameliorated Pdi deficits following C2 SH. In summary, SPG302 is an exciting new therapy to explore for use in spinal cord injuries. KEY POINTS: Despite advances in our understanding of the effects of cervical hemisection (C2 SH) on diaphragm muscle (DIAm) EMG activity, very little is understood about the impact of C2 SH on the gamut of ventilatory and non-ventilatory transdiaphragmatic pressures (Pdi ). Recovery of DIAm activity following C2 SH is improved using a variety of approaches, but very few pharmaceuticals have been shown to be effective. One way of improving DIAm recovery is to enhance the amount of latent local spared connections onto phrenic motor neurons. A novel pegylated benzothiazole derivative enhances synaptogenesis in a variety of neurodegenerative conditions. Here, using a novel therapeutic SPG302, we show that 14 days of treatment with SPG302 ameliorated DIAm EMG and Pdi deficits compared with vehicle controls. Our results show that SPG302 is a compound with very promising potential for use in improving functional outcomes post-spinal cord injury.

Electrophysiological effects of BDNF and TrkB signaling at type-identified diaphragm neuromuscular junctions.

Previous studies show that synaptic quantal release decreases during repetitive stimulation, i.e., synaptic depression. Neurotrophin brain-derived neurotrophic factor (BDNF) enhances neuromuscular transmission via activation of tropomyosin-related kinase receptor B (TrkB). We hypothesized that BDNF mitigates synaptic depression at the neuromuscular junction and that the effect is more pronounced at type IIx and/or IIb fibers compared to type I or IIa fibers given the more rapid reduction in docked synaptic vesicles with repetitive stimulation. Rat phrenic nerve-diaphragm muscle preparations were used to determine the effect of BDNF on synaptic quantal release during repetitive stimulation at 50 Hz. An ∼40% decline in quantal release was observed during each 330-ms duration train of nerve stimulation (intratrain synaptic depression), and this intratrain decline was observed across repetitive trains (20 trains at 1/s repeated every 5 min for 30 min for 6 sets). BDNF treatment significantly enhanced quantal release at all fiber types (P < 0.001). BDNF treatment did not change release probability within a stimulation set but enhanced synaptic vesicle replenishment between sets. In agreement, synaptic vesicle cycling (measured using FM4-64 fluorescence uptake) was increased following BDNF [or neurotrophin-4 (NT-4)] treatment (∼40%; P < 0.05). Conversely, inhibiting BDNF/TrkB signaling with the tyrosine kinase inhibitor K252a and TrkB-IgG (which quenches endogenous BDNF or NT-4) decreased FM4-64 uptake (∼34% across fiber types; P < 0.05). The effects of BDNF were generally similar across all fiber types. We conclude that BDNF/TrkB signaling acutely enhances presynaptic quantal release and thereby may serve to mitigate synaptic depression and maintain neuromuscular transmission during repetitive activation.NEW & NOTEWORTHY Neurotrophin brain-derived neurotrophic factor (BDNF) enhances neuromuscular transmission via activation of tropomyosin-related kinase receptor B (TrkB). Rat phrenic nerve-diaphragm muscle preparations were used to determine the rapid effect of BDNF on synaptic quantal release during repetitive stimulation. BDNF treatment significantly enhanced quantal release at all fiber types. BDNF increased synaptic vesicle cycling (measured using FM4-64 fluorescence uptake); conversely, inhibiting BDNF/TrkB signaling decreased FM4-64 uptake.

Acute intrathecal BDNF enhances functional recovery after cervical spinal cord injury in rats.

Unilateral C2 hemisection (C2SH) disrupts descending inspiratory-related drive to phrenic motor neurons and thus, silences rhythmic diaphragm muscle (DIAm) activity. There is gradual recovery of rhythmic DIAm EMG activity over time post-C2SH, consistent with neuroplasticity, which is enhanced by chronic (2 wk) intrathecal BDNF treatment. In the present study, we hypothesized that acute (30 min) intrathecal BDNF treatment also enhances recovery of DIAm EMG activity after C2SH. Rats were implanted with bilateral DIAm EMG electrodes to verify the absence of ipsilateral eupneic DIAm EMG activity at the time of C2SH and at 3 days post-C2SH. In those animals displaying no recovery of DIAm EMG activity after 28 days (n = 7), BDNF was administered intrathecally (450 mcg) at C4. DIAm EMG activity was measured continuously both before and for 30 min after BDNF treatment, during eupnea, hypoxia-hypercapnia, and spontaneous sighs. Acute BDNF treatment restored eupneic DIAm EMG activity in all treated animals to an amplitude that was 78% ± 9% of pre-C2SH root mean square (RMS) (P < 0.001). In addition, acute BDNF treatment increased DIAm RMS EMG amplitude during hypoxia-hypercapnia (P = 0.023) but had no effect on RMS EMG amplitude during sighs. These results support an acute modulatory role of BDNF signaling on excitatory synaptic transmission at phrenic motor neurons after cervical spinal cord injury.NEW & NOTEWORTHY Brain-derived neurotrophic factor (BDNF) plays an important role in promoting neuroplasticity following unilateral C2 spinal hemisection (C2SH). BDNF was administered intrathecally in rats displaying lack of ipsilateral inspiratory-related diaphragm (DIAm) EMG activity after C2SH. Acute BDNF treatment (30 min) restored eupneic DIAm EMG activity in all treated animals to 78% ± 9% of pre-C2SH level. In addition, acute BDNF treatment increased DIAm EMG amplitude during hypoxia-hypercapnia but had no effect on EMG amplitude during sighs.

Disproportionate loss of excitatory inputs to smaller phrenic motor neurons following cervical spinal hemisection.

KEY POINTS: Motor units, comprising a motor neuron and the muscle fibre it innervates, are activated in an orderly fashion to provide varying amounts of force. A unilateral C2 spinal hemisection (C2SH) disrupts predominant excitatory input from medulla, causing cessation of inspiratory-related diaphragm muscle activity, whereas higher force, non-ventilatory diaphragm activity persists. In this study, we show a disproportionately larger loss of excitatory glutamatergic innervation to small phrenic motor neurons (PhMNs) following C2SH, as compared with large PhMNs ipsilateral to injury. Our data suggest that there is a dichotomy in the distribution of inspiratory-related descending excitatory glutamatergic input to small vs. large PhMNs that reflects their differential recruitment. ABSTRACT: Excitatory glutamatergic input mediating inspiratory drive to phrenic motor neurons (PhMNs) emanates primarily from the ipsilateral ventrolateral medulla. Unilateral C2 hemisection (C2SH) disrupts this excitatory input, resulting in cessation of inspiratory-related diaphragm muscle (DIAm) activity. In contrast, after C2SH, higher force, non-ventilatory DIAm activity persists. Inspiratory behaviours require recruitment of only smaller PhMNs, whereas with more forceful expulsive/straining behaviours, larger PhMNs are recruited. Accordingly, we hypothesize that C2SH primarily disrupts glutamatergic synaptic inputs to smaller PhMNs, whereas glutamatergic synaptic inputs to larger PhMNs are preserved. We examined changes in glutamatergic presynaptic input onto retrogradely labelled PhMNs using immunohistochemistry for VGLUT1 and VGLUT2. We found that 7 days after C2SH there was an ∼60% reduction in glutamatergic inputs to smaller PhMNs compared with an ∼35% reduction at larger PhMNs. These results are consistent with a more pronounced impact of C2SH on inspiratory behaviours of the DIAm, and the preservation of higher force behaviours after C2SH. These results indicate that the source of glutamatergic synaptic input to PhMNs varies depending on motor neuron size and reflects different functional control - perhaps separate central pattern generator and premotor circuits. For smaller PhMNs, the central pattern generator for inspiration is located in the pre-Bötzinger complex and premotor neurons in the ventrolateral medulla, sending predominantly ipsilateral projections via the dorsolateral funiculus. C2SH disrupts this glutamatergic input. For larger PhMNs, a large proportion of excitatory inputs appear to exist below the C2 level or from contralateral regions of the brainstem and spinal cord.

A new simple score of chronic cough: cough evaluation test.

BACKGROUND: Chronic cough has an important impact on physical, social and psychological aspects. A simple and effective method to assess different aspects of chronic cough severity is required. We aimed to develop a simple, self-completed test, Cough Evaluation Test (CET), to evaluate cough severity and its impact on health. METHOD: The items of preliminary CET were generated based on literature review and clinical practice. Items reduction was conducted by modified Delphi method. Patients with chronic cough were recruited to complete CET, Cough Visual Analog Scales (VAS), Mandarin Chinese version of the Leicester Cough Questionnaire (LCQ-MC), and Cough Symptom Score (CSS). Reassessments were performed at 1 week apart before treatment, and after more than 2 weeks treatments. Concurrent validation, internal consistency, repeatability, responsiveness and the minimal important difference (MID) were determined. RESULTS: CET consists of five items with a 5-point Likert scale (1-5 scaling of items, 5-25 score range). The Cronbach's alpha values for CET was 0.80. CET showed a stronger correlation with LCQ-MC (r = - 0.74) compared to that between LCQ-MC with VAS (r = - 0.61). CET also showed a stronger correlation with VAS (r = 0.70) compared to that between VAS with other measures. Intraclass correlation coefficients for CET was 0.84. In patients undergoing treatment, CET scores significantly changed (p < 0.0001). The MID of CET was 2. CONCLUSION: Cough Evaluate Test is a reliable, valid and responsive tool to simply evaluate impact of cough on physical, social and psychological aspects.

Phrenic motor neuron loss in aged rats.

Sarcopenia is the age-related reduction of muscle mass and specific force. In previous studies, we found that sarcopenia of the diaphragm muscle (DIAm) is evident by 24 mo of age in both rats and mice and is associated with selective atrophy of type IIx and IIb muscle fibers and a decrease in maximum specific force. These fiber type-specific effects of sarcopenia resemble those induced by DIAm denervation, leading us to hypothesize that sarcopenia is due to an age-related loss of phrenic motor neurons (PhMNs). To address this hypothesis, we determined the number of PhMNs in young (6 mo old) and old (24 mo old) Fischer 344 rats. Moreover, we determined age-related changes in the size of PhMNs, since larger PhMNs innervate type IIx and IIb DIAm fibers. The PhMN pool was retrogradely labeled and imaged with confocal microscopy to assess the number of PhMNs and the morphometry of PhMN soma and proximal dendrites. In older animals, there were 22% fewer PhMNs, a 19% decrease in somal surface area, and a 21% decrease in dendritic surface area compared with young Fischer 344 rats. The age-associated loss of PhMNs involved predominantly larger PhMNs. These results are consistent with an age-related denervation of larger, more fatigable DIAm motor units, which are required primarily for high-force airway clearance behaviors. NEW & NOTEWORTHY Diaphragm muscle sarcopenia in rodent models is well described in the literature; however, the relationship between sarcopenia and frank phrenic motor neuron (MN) loss is unexplored in these models. We quantify a 22% loss of phrenic MNs in old (24 mo) compared with young (6 mo) Fischer 344 rats. We also report reductions in phrenic MN somal and proximal dendritic morphology that relate to decreased MN heterogeneity in old compared with young Fischer 344 rats.

Impact of glutamatergic and serotonergic neurotransmission on diaphragm muscle activity after cervical spinal hemisection.

Incomplete cervical spinal cord hemisection at C2 (SH) disrupts descending excitatory drive to phrenic motoneurons, paralyzing the ipsilateral diaphragm muscle. Spontaneous recovery over time is associated with increased phrenic motoneuron expression of glutamatergic N-methyl-d-aspartate (NMDA) and serotonergic 5-HT2A receptors. We hypothesized that NMDA and 5-HT2A receptor-mediated neurotransmission play a role in ipsilateral diaphragm muscle activity post-SH. Adult male Sprague-Dawley rats were implanted with bilateral diaphragm EMG electrodes for chronic EMG recordings up to 28 days post-SH (SH 28D). The extent of recovery was calculated by peak root-mean-square (RMS) EMG amplitude. In all animals, absence of ipsilateral activity was verified at 3 days post-SH. Diaphragm EMG activity was also recorded during exposure to hypoxia-hypercapnia (10% O2-5% CO2). In SH animals displaying recovery of ipsilateral diaphragm EMG activity at SH 28D, cervical spinal cord segments containing the phrenic motor nucleus (C3-C5) were surgically exposed and either the NMDA receptor antagonist d-2-amino-5-phosphonovalerate (d-AP5; 100 mM, 30 µl) or 5-HT2A receptor antagonist ketanserin (40 mM, 30 µl) was instilled intrathecally. Following d-AP5, diaphragm EMG amplitude was reduced ipsilaterally, during both eupnea (42% of pre-d-AP5 value; P = 0.007) and hypoxia-hypercapnia (31% of pre-d-AP5 value; P = 0.015), with no effect on contralateral EMG activity or in uninjured controls. Treatment with ketanserin did not change ipsilateral or contralateral RMS EMG amplitude in SH animals displaying recovery at SH 28D. Our results suggest that spinal glutamatergic NMDA receptor-mediated neurotransmission plays an important role in ipsilateral diaphragm muscle activity after cervical spinal cord injury.NEW & NOTEWORTHY Spontaneous recovery following C2 spinal hemisection (SH) is associated with increased phrenic motoneuron expression of glutamatergic and serotonergic receptors. In this study, we show that pharmacological inhibition of glutamatergic N-methyl-d-aspartate (NMDA) receptors blunts ipsilateral diaphragm activity post-SH. In contrast, pharmacological inhibition of serotonergic 5-HT2A receptors does not change diaphragm EMG activity post-SH. Our results suggest that NMDA receptor-mediated glutamatergic neurotransmission plays an important role in enhancing rhythmic respiratory-related diaphragm activity after spinal cord injury.

BDNF effects on functional recovery across motor behaviors after cervical spinal cord injury.

Unilateral C2 cervical spinal cord hemisection (SH) disrupts descending excitatory drive to phrenic motor neurons, thereby paralyzing the ipsilateral diaphragm muscle (DIAm) during ventilatory behaviors. Recovery of rhythmic DIAm activity ipsilateral to injury occurs over time, consistent with neuroplasticity and strengthening of spared synaptic inputs to phrenic motor neurons. Localized intrathecal delivery of brain-derived neurotrophic factor (BDNF) to phrenic motor neurons after SH enhances recovery of eupneic DIAm activity. However, the impact of SH and BDNF treatment on the full range of DIAm motor behaviors has not been fully characterized. We hypothesized that all DIAm motor behaviors are affected by SH and that intrathecal BDNF enhances the recovery of both ventilatory and higher force, nonventilatory motor behaviors. An intrathecal catheter was placed in adult, male Sprague-Dawley rats at C4 to chronically infuse artificial cerebrospinal fluid (aCSF) or BDNF. DIAm electromyography (EMG) electrodes were implanted bilaterally to record activity across motor behaviors, i.e., eupnea, hypoxia-hypercapnia (10% O2 and 5% CO2), sighs, airway occlusion, and sneezing. After SH, ipsilateral DIAm EMG activity was evident in only 43% of aCSF-treated rats during eupnea, and activity was restored in all rats after BDNF treatment. The amplitude of DIAm EMG (root mean square, RMS) was reduced following SH during eupnea and hypoxia-hypercapnia in aCSF-treated rats, and BDNF treatment promoted recovery in both conditions. The amplitude of DIAm RMS EMG during sighs, airway occlusion, and sneezing was not affected by SH or BDNF treatment. We conclude that the effects of SH and BDNF treatment on DIAm activity depend on motor behavior. NEW & NOTEWORTHY: This study demonstrates that after unilateral C2 spinal cord hemisection (SH), there are differences in the spontaneous recovery of diaphragm (DIAm) electromyographic activity during ventilatory compared with more forceful, nonventilatory motor behaviors. Furthermore, we show that intrathecal delivery of brain-derived neurotrophic factor (BDNF) at the level of the phrenic motor neuron pool enhances recovery of ipsilateral DIAm activity following SH, exerting main effects on recovery of ventilatory but not higher force, nonventilatory behaviors.

Non-asthmatic eosinophilic bronchitis and its relationship with asthma.

Non-asthmatic eosinophilic bronchitis (NAEB) is an important cause of chronic cough. NAEB shares similar eosinophilic inflammation of airway and response to corticosteroids with asthma. However, in contrast to asthma, NAEB subjects have no airflow obstruction and airway hyperresponsiveness. The pathogenesis of NAEB are involved with many inflammatory cells such as eosinophils, lymphocytes, mast cells, cytokines and inflammatory mediators, includingTh2 cytokines, histamine, leukotrines, prograndins and oxidative stress. The different locations of inflammatory cells and the inflammatory mediators level may explain this difference between EB and asthma. There is no study on dose and duration of treatment. On the prognosis of NAEB, long term follow-up study suggested that NAEB should be a distinct entity rather than an early stage of asthma or COPD. The relapse rate is high after treatment. Assessing sputum eosinophils after treatment is useful to identify those at risk of relapse.

Cervical spinal cord hemisection impacts sigh and the respiratory reset in male rats.

Cervical spinal cord injury impacts ventilatory and non-ventilatory functions of the diaphragm muscle (DIAm) and contributes to clinical morbidity and mortality in the afflicted population. Periodically, integrated brainstem neural circuit activity drives the DIAm to generate a markedly augmented effort or sigh-which plays an important role in preventing atelectasis and thus maintaining lung function. Across species, the general pattern of DIAm efforts during a normal sigh is variable in amplitude and the extent of post-sigh "apnea" (i.e., the post-sigh inter-breath interval). This post-sigh inter-breath interval acts as a respiratory reset, following the interruption of regular respiratory rhythm by sigh. We examined the impact of upper cervical (C2 ) spinal cord hemisection (C2 SH) on the transdiaphragmatic pressure (Pdi ) generated during sighs and the post-sigh respiratory reset in rats. Sighs were identified in Pdi traces by their characteristic biphasic pattern. We found that C2 SH results in a reduction of Pdi during both eupnea and sighs, and a decrease in the immediate post-sigh breath interval. These results are consistent with partial removal of descending excitatory synaptic inputs to phrenic motor neurons that results from C2 SH. Following cervical spinal cord injury, a reduction in the amplitude of Pdi during sighs may compromise the maintenance of normal lung function.

Profile of cough triggers and their relationship with capsaicin cough sensitivity in chronic cough.

BACKGROUND: Cough hypersensitivity is an important part of the neurophysiology of cough, which presents with increased cough response to a lower level of stimuli or triggers. Classification of stimuli might bring about additional insight into the underlying mechanisms and management. OBJECTIVES: This study investigated the profile of cough triggers in chronic cough patients and their relationship with capsaicin cough sensitivity. DESIGN: This was a cross-sectional observational study. METHODS: We enrolled patients with different causes of chronic cough from 2006 to 2021. Cough triggers were defined as cough response to chemical triggers, mechanical triggers, meal triggers, or thermal trigger. Cough sensitivity to capsaicin was evaluated by the capsaicin challenge test, which was expressed as the lowest concentration of capsaicin inducing 5 or more coughing (C5). RESULTS: Among 1211 patients with chronic cough, 1107 (91.4%) patients reported at least one cough trigger. Chemical triggers (66.9%) were the most common cough triggers, followed by thermal exposure (50.6%), mechanical triggers (48.2%), and meal triggers (21.2%). There was no difference in the proportion of chemical triggers among different etiologies. Patients with refractory chronic cough reported the highest prevalence of cough triggers (97.1%). A higher number of meal triggers (34.9%) was associated with gastroesophageal reflux-related cough, and meal triggers and mechanical triggers were more common in refractory chronic cough. Among 254 patients who completed capsaicin challenge test, both the number of total triggers and the number of chemical triggers had a significant but mild correlation with capsaicin cough sensitivity. CONCLUSION: Cough hypersensitivity as reflected by a variety of cough triggers is a common feature in chronic cough patients, but different etiologies present specific profiles of cough triggers, which could not be evaluated comprehensively by capsaicin cough sensitivity.

Postnatal survival of phrenic motor neurons is promoted by BDNF/TrkB.FL signaling.

The number of motor neurons (MNs) declines precipitously during the final trimester before birth. Thereafter, the number of MNs remains relatively stable, with their connections to skeletal muscle dependent on neurotrophins, including brain-derived neurotrophic factor (BDNF) signaling through its high-affinity full-length tropomyosin-related kinase receptor subtype B (TrkB.FL) receptor. As a genetic knockout of BDNF leads to extensive MN loss and postnatal death within 1-2 days after birth, we tested the hypothesis that postnatal inhibition of BDNF/TrkB.FL signaling is important for postnatal phrenic MN (PhMN) survival. In the present study, we used a 1NMPP1-sensitive TrkBF616A mutant mouse to evaluate the effects of inhibition of TrkB kinase activity on phrenic MN (PhMN) numbers and diaphragm muscle (DIAm) fiber cross-sectional area (CSA). Pups were exposed to 1NMPP1 or vehicle (DMSO) from birth to 21 days old (weaning) via the mother's ingestion in the drinking water. Following weaning, the right phrenic nerve was exposed in the neck and the proximal end dipped in a rhodamine solution to retrogradely label PhMNs. After 24 h, the cervical spinal cord and DIAm were excised. Labeled PhMNs were imaged using confocal microscopy, whereas DIAm strips were frozen at ∼1.5× resting length, cryosectioned, and stained with hematoxylin and eosin to assess CSA. We observed an ∼34% reduction in PhMN numbers and increased primary dendrite numbers in 1NMPP1-treated TrkBF616A mice. The distribution of PhMN size (somal surface area) DIAm fiber cross-sectional areas did not differ. We conclude that survival of PhMNs during early postnatal development is sensitive to BDNF/TrkB.FL signaling.NEW & NOTEWORTHY During early postnatal development, BDNF/TrkB signaling promotes PhMN survival. Inhibition of BDNF/TrkB signaling in early postnatal development does not impact PhMN size. Inhibition of BDNF/TrkB signaling in early postnatal development does not impact the number or CSA of DIAm fibers.

Assessing Functional Recovery of Eupneic Diaphragm Activity Following Unilateral Cervical Spinal Cord Injury in Rats.

Following cSCI, activation of the DIAm can be impacted depending on the extent of the injury. The present manuscript describes a unilateral C2 hemisection (C2SH) model of cSCI that disrupts eupneic ipsilateral diaphragm (iDIAm) electromyographic (EMG) activity during breathing in rats. To evaluate recovery of DIAm motor control, the extent of deficit due to C2SH must first be clearly established. By verifying a complete initial loss of iDIAm EMG during breathing, subsequent recovery can be classified as either absent or present, and the extent of recovery can be estimated using the EMG amplitude. Additionally, by measuring the continued absence of iDIAm EMG activity during breathing after the acute spinal shock period following C2SH, the success of the initial C2SH may be validated. Measuring contralateral diaphragm (cDIAm) EMG activity can provide information about the compensatory effects of C2SH, which also reflects neuroplasticity. Moreover, DIAm EMG recordings from awake animals can provide vital physiological information about the motor control of the DIAm after C2SH. This article describes a method for a rigorous, reproducible, and reliable C2SH model of cSCI in rats, which is an excellent platform for studying respiratory neuroplasticity, compensatory cDIAm activity, and therapeutic strategies and pharmaceuticals.

Sputum Transcriptomics Reveals FCN1+ Macrophage Activation in Mild Eosinophilic Asthma Compared to Non-Asthmatic Eosinophilic Bronchitis.

PURPOSE: Eosinophilic asthma (EA) and non-asthmatic eosinophilic bronchitis (EB) share similar eosinophilic airway inflammation. Unlike EA, EB did not present airway hyperresponsiveness or airflow obstruction. We aimed to compare the mechanism underlying the different manifestations between EA and EB via sputum transcriptomics analysis. METHODS: Induced-sputum cells from newly physician-diagnosed EA, EB patients, and healthy controls (HCs) were collected for RNA sequencing. RESULTS: Bulk RNA sequencing was performed using sputum cells from patients with EA (n = 18), EB (n = 15) and HCs (n = 28). Principal component analysis revealed similar gene expression patterns in EA and EB. The most differentially expressed genes in EB compared with HC were also shared by EA, including IL4, IL5 IL13, CLC, CPA3, and DNASE1L3. However, gene set enrichment analysis showed that the signatures regulating macrophage activation were enriched in EA compared to EB. Sputum cells were profiled using single-cell RNA sequencing. FABP4+ macrophages, SPP1+ macrophages, FCN1+ macrophages, dendritic cells, T cells, B cells, mast cells, and epithelial cells were identified based on gene expression profiling. Analysis of cell-cell communication revealed that interactions between FCN1+ macrophages and other cells were higher in EA than in EB. A wealth of transforming growth factor beta (TGF-ß) and vascular endothelial growth factor (VEGF) interactions between FCN1+ macrophages and other cells have been shown in EA. The gene expression levels of EREG, TGFBI, and VEGFA in FCN1+ macrophages of EA were significantly higher than those of EB. Furthermore, signatures associated with the response to TGF-ß, cellular response to VEGF stimulus and developmental cell growth were enriched in FCN1+ macrophages of EA compared to those of EB. CONCLUSIONS: FCN1+ macrophage activation associated with airway remodeling processes was upregulated in EA compared to that in EB, which may contribute to airway hyperresponsiveness and airflow obstruction.

Profiles of Cough and Associated Risk Factors in Nonhospitalized Individuals With SARS-CoV-2 Omicron Variant Infection: Cross-Sectional Online Survey in China.

BACKGROUND: Cough is a common symptom during and after COVID-19 infection; however, few studies have described the cough profiles of COVID-19. OBJECTIVE: The aim of this study was to investigate the prevalence, severity, and associated risk factors of severe and persistent cough in individuals with COVID-19 during the latest wave of the Omicron variant in China. METHODS: In this nationwide cross-sectional study, we collected information of the characteristics of cough from individuals with infection of the SARS-CoV-2 Omicron variant using an online questionnaire sent between December 31, 2022, and January 11, 2023. RESULTS: There were 11,718 (n=7978, 68.1% female) nonhospitalized responders, with a median age of 37 (IQR 30-47) years who responded at a median of 16 (IQR 12-20) days from infection onset to the time of the survey. Cough was the most common symptom, occurring in 91.7% of participants, followed by fever, fatigue, and nasal congestion (68.8%-87.4%). The median cough visual analog scale (VAS) score was 70 (IQR 50-80) mm. Being female (odds ratio [OR] 1.31, 95% CI 1.20-1.43), having a COVID-19 vaccination history (OR 1.71, 95% CI 1.37-2.12), current smoking (OR 0.48, 95% CI 0.41-0.58), chronic cough (OR 2.04, 95% CI 1.69-2.45), coronary heart disease (OR 1.71, 95% CI 1.17-2.52), asthma (OR 1.22, 95% CI 1.02-1.46), and gastroesophageal reflux disease (GERD) (OR 1.21, 95% CI 1.01-1.45) were independent factors for severe cough (VAS>70, 37.4%). Among all respondents, 35.0% indicated having a productive cough, which was associated with risk factors of being female (OR 1.44, 95% CI 1.31-1.57), having asthma (OR 1.84, 95% CI 1.52-2.22), chronic cough (OR 1.44, 95% CI 1.19-1.74), and GERD (OR 1.22, 95% CI 1.01-1.47). Persistent cough (>3 weeks) occurred in 13.0% of individuals, which was associated with the risk factors of having diabetes (OR 2.24, 95% CI 1.30-3.85), asthma (OR 1.70, 95% CI 1.11-2.62), and chronic cough (OR 1.97, 95% CI 1.32-2.94). CONCLUSIONS: Cough is the most common symptom in nonhospitalized individuals with Omicron SARS-CoV-2 variant infection. Being female, having asthma, chronic cough, GERD, coronary heart disease, diabetes, and a COVID-19 vaccination history emerged as independent factors associated with severe cough, productive cough, and persistent cough.

Predictive value of clinical features for anti-reflux therapy response in suspected gastroesophageal reflux-induced chronic cough.

Background: Not all gastroesophageal reflux-induced cough (GERC) patients respond to anti-reflux treatment. It is not certain whether reflux-related symptoms or other clinical characteristics could indicate a successful response to anti-reflux treatment. In this study, we aimed to investigate the relationship between clinical features and anti-reflux response. Methods: We retrospectively analyzed the clinical characteristics of suspected GERC who had reflux-related symptoms or reflux evidence based on abnormal 24-hour esophageal pH value monitoring, or who had no evidence of other common causes of chronic cough in our chronic cough database with a standard case report form. All patients experienced anti-reflux treatment with proton pump inhibitors (PPIs) plus prokinetic agents for at least 2 weeks and were divided into responders and non-responders based on the treatment response. Results: Among 241 patients with suspected GERC, 146 (60.6%) showed a successful response. There was no significant difference in regard to the proportion of reflux-related symptoms, and results of 24-hour esophageal pH value monitoring between responders and non-responders. Compared with non-responders, responders had higher proportions of nasal itching (21.2% vs. 8.4%; P=0.014), tickle in the throat (51.4% vs. 35.8%; P=0.025) and lower proportion of pharyngeal foreign body sensation (32.9% vs. 54.7%; P=0.001). Multivariate analysis showed that nasal itching [hazard ratio (HR): 1.593, 95% confidence interval (CI): 1.025-2.476, P=0.039], tickle in the throat (HR: 1.605, 95% CI: 1.152-2.238, P=0.005), pharyngeal foreign body sensation (HR: 0.499, 95% CI: 0.346-0.720, P<0.001) and sensitivity to at least one cough trigger (HR: 0.480, 95% CI: 0.237-0.973, P=0.042) were associated with the therapeutic response. Conclusions: Over half of suspected GERC patients benefited from anti-reflux therapy. A few clinical features rather than reflux-related symptoms might indicate a response to anti-reflux treatment. Further study is needed for the predictive value.