Apneic Response Research Articles

Role of endogenous nerve growth factor in laryngeal airway hyperreactivity and laryngeal inflammation induced by intermittent hypoxia in rats.

Obstructive sleep apnea, characterized by airway exposure to intermittent hypoxia (IH), is associated with laryngeal airway hyperreactivity (LAH) and laryngeal inflammation. The sensitization of capsaicin-sensitive superior laryngeal afferents (CSSLAs) by inflammatory mediators has been implicated in the pathogenesis of LAH. Nerve growth factor (NGF) is an inflammatory mediator that acts on tropomyosin receptor kinase A (TrkA) and the p75 neurotrophin receptor (p75NTR) to induce lower airway hyperresponsiveness. In this study, we investigated the role of NGF in the development of LAH and laryngeal inflammation induced by IH in anesthetized rats. Compared with rats subjected to room air exposure for 14 days, rats with 14-day IH exposure exhibited augmented reflex apneic responses to the laryngeal provocation of three different chemical stimulants of CSSLAs, resulting in LAH. The apneic responses to laryngeal stimulants were abolished by either perineural capsaicin treatment (a procedure that selectively blocks the conduction of CSSLAs) or denervation of the superior laryngeal nerves, suggesting that the reflex was mediated through CSSLAs. The IH-induced LAH was significantly attenuated by daily treatment with anti-NGF antibody, but was unaffected by daily treatment with immunoglobulin G. IH exposure also induced laryngeal inflammation as evidenced by increases in laryngeal levels of NGF, lipid peroxidation, tumor necrosis factor-α, interleukin-1β, TrkA, and p75NTR. Similarly, IH-induced laryngeal inflammation was significantly reduced by daily treatment with anti-NGF antibody. We concluded that NGF contributes to the development of LAH and laryngeal inflammation induced by IH in rats. The LAH may result from the sensitizing effect of NGF on CSSLAs.

Intravenous Bolus Injection of Fentanyl Triggers an Immediate Central and Upper Airway Obstructive Apnea via Activating Vagal Sensory Afferents.

Intravenous bolus (IVb) injection of fentanyl induces an immediate apnea, but the characteristics of the apnea and relevant mechanism remain unclear. Here, we tested whether IVb injection of fentanyl induced an immediate central and upper airway obstructive apnea associated with chest wall rigidity via activating vagal C-fibers (VCFs) and vagal afferent opioid receptors (ORs). Cardiorespiratory and electromyography of external and internal intercostal, thyroarytenoid and superior pharyngeal constrictor muscles (EMGEI, EMGII, EMGTA and EMGSPC) responses to IVb injection of fentanyl were recorded in anesthetized and spontaneously breathing rats with or without bilateral peri-vagal capsaicin treatment or intra-vagal microinjection of naloxone. Immunohistochemical approach was employed to define the presence of opioid mu-receptor (MOR) expression in vagal C-neurons and a patch clamp technique utilized to determine the evoked current responses of vagal C-neurons to fentanyl in vitro. Fentanyl induced an immediate apnea and subsequent respiratory depression. The apnea was characterized by cessation of EMGEI activity and augmentation of tonic discharges of EMGII, EMGTA, and EMGSPC, i.e., central expiratory apnea, laryngeal closure and pharyngeal constriction/collapse accompanied with chest wall rigidity. The apneic response was abolished by blockade of VCF signal conduction and largely attenuated by antagonism of vagal afferent ORs. The latter significantly alleviated the initial (within 5 min post injection), but not the later, respiratory depression. Vagal C-neurons expressed MORs and were activated by fentanyl. We conclude that IVb injection of fentanyl causes a VCF- and vagal afferent OR-mediated immediate central apnea, upper airway obstruction and chest wall rigidity.

Stimulatory effect of methylglyoxal on capsaicin-sensitive lung vagal afferents in rats: role of TRPA1.

Methylglyoxal (MG), a reactive metabolic byproduct of glycolysis, is a causative of painful diabetic neuropathy. Patients with diabetes are associated with more frequent severe asthma exacerbation. Stimulation of capsaicin-sensitive lung vagal (CSLV) afferents may contribute to the pathogenesis of hyperreactive airway diseases such as asthma. However, the possibility of the stimulatory effect of MG on CSLV afferents and the underlying mechanisms remain unknown. Our results showed that intravenous injection of MG (25 mg/kg, MG25) in anesthetized, spontaneously breathing rats elicited pulmonary chemoreflexes characterized by apnea, bradycardia, and hypotension. The MG-induced apneic response was reproducible and dose dependent. MG25 no longer evoked these reflex responses after perineural capsaicin treatment of both cervical vagi to block C-fibers' conduction, suggesting that the reflexes were mediated through the stimulation of CSLV afferents. Pretreatment with HC030031 [an antagonist of transient receptor potential ankyrin subtype 1 protein (TRPA1)] or AP18 (another TRPA1 antagonist), but not their vehicle, markedly attenuated the apneic response induced by MG25. Consistently, electrophysiological results showed that pretreatment with HC030031 largely attenuated the intense discharge in CSLV afferents induced by injection of MG25 in open-chest and artificially ventilated rats. In isolated CSLV neurons, the perfusion of MG evoked an abrupt and pronounced increase in calcium transients in a concentration-dependent manner. This stimulatory effect on CSLV neurons was also abolished by HC030031 treatment but not by its vehicle. In conclusion, these results suggest that MG exerts a stimulatory effect on CSLV afferents, inducing pulmonary chemoreflexes, and such stimulation is mediated through the TRPA1 activation.NEW & NOTEWORTHY Methylglyoxal (MG) is implicated in the development of painful diabetic neuropathy. A retrospective cohort study revealed an increased incidence of asthma exacerbations in patients with diabetes. This study demonstrated that elevated circulating MG levels stimulate capsaicin-sensitive lung vagal afferents via activation of TRPA1, which in turn triggers respiratory reflexes. These findings provide new information for understanding the pathogenic mechanism of diabetes-associated hyperreactive airway diseases and potential therapy.

Apneic response to fentanyl in adult rats: Role of laryngeal afferents.

Intravenous (systemic) bolus injection of fentanyl (FNT) reportedly induces an immediate vagal-mediated apnea; however, the precise origin of vagal afferents responsible for this apnea remains unknown. We tested whether intralaryngeal (local) application of FNT would also trigger an apnea and whether the apneic response to both local and systemic administration of FNT was laryngeal afferent-mediated. Cardiorespiratory responses to FNT were recorded in anesthetized male adult rats with and without bilateral sectioning of the superior laryngeal nerve (SLNx) or peri-SLN capsaicin treatment (SLNcap) to block local C-fiber signal conduction. Opioid mu-receptor (MOR)-immunoreactivity was detected in laryngeal C- and myelinated neurons. We found that local and systemic administration of FNT elicited an immediate apnea. SLNx, rather than SLNcap, abolished the apneic response to local FNT application though MORs were abundantly expressed in both laryngeal C- and myelinated neurons. Importantly, SLNx failed to affect the apneic response to systemic FNT administration. These results lead to the conclusion that laryngeal afferents' MORs are responsible for the apneic response to local, but not systemic, administration of FNT.

Chronic Vagotomy Alters Glutamate Transmission in the Nucleus Tractus Solitarii (nTS)

The nTS is the first site of integration and processing of sensory afferent information and is one of the crucial brainstem nuclei involved in cardiorespiratory reflexes. This nucleus receives afferent information from many visceral structures in the thorax and abdomen via the peripheral branches of the vagus nerve. Vagal afferents release glutamate (Glu) which initiates signaling processes that leads to chemo- and baroreflex-mediated changes in sympathetic and phrenic nerve activity. Previous studies by us have shown that decreasing afferent input via unilateral vagotomy augmented nTS astrocyte complexity. Astrocytes contribute to regulation of Glu concentration by removing Glu from the synaptic cleft via excitatory amino acid transporters (EAATs). We hypothesized that vagotomy attenuates Glu signaling in the nTS due to increased Glu uptake by greater EAAT expression or function. Surgical transection or sham surgery of the right cervical vagus nerve caudal to the nodose ganglion was performed in male Sprague-Dawley rats. One week after surgery, mean arterial pressure (MAP), heart rate (HR), and splanchnic sympathetic and phrenic nerve activity (SSNA and PhrNA) were recorded in anesthetized and ventilated rats. Intact vagus nerves and the right phrenic nerve were acutely transected. Nanoinjections of Glu (30 nl, 1-10 mM) into the nTS decreased MAP, HR, SSNA and PhNA in both sham and vagotomy groups. Vagotomy blunted the HR, SSNA and PhrNA responses, indicating that Glu signaling was attenuated. To determine if increased EAAT function contributed to blunted Glu responses, the EAAT blocker TFB-TBOA (10nM, 60nl) was nanoinjected into the nTS. EAAT blockade mimicked the effect of Glu, producing similar depressor, bradycardic, and apneic responses in both groups. TBOA also enhanced the cardiorespiratory responses to Glu injection in both groups. EAAT blockade did not restore Glu responses in the vagotomy group compared to sham. Taken together, these data suggest that increased EAAT function did not account for the attenuated Glu signaling in the nTS. Further studies are needed to uncover the mechanisms by which vagotomy affects Glu signaling in the nTS. NIH R01 HL098602 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The superior laryngeal nerve is involved in a brief apneic response to intralaryngeal, but not in the lethal ventilatory arrest to intravenous, administration of fentanyl (FNT) in rats

Rapid intravenous (IV) injection of FNT induces a sudden ventilatory arrest within a few minutes or even seconds, which is the major cause of overdose FNT‐induced death. Our previous studies have shown that right atrial or IV bolus injection of FNT in anesthetized rats produced a vagal‐mediated apnea or a lethal ventilatory arrest in a dose‐dependent manner (Zhuang et al, AJP 2012 and EB Meeting 2022). The superior laryngeal nerve (SLN) containing the laryngeal sensory afferents also travels in the vagus nerve and is capable of triggering the apneic response associated with hypertension and bradycardia upon activation. Here, we asked whether local administration of FNT into the larynx affected cardiorespiratory activities via acting on the SLN and to what extent the sudden ventilatory arrest induced by IV injection of FNT depended on the SLN integrity. FNT at 15 µg was either sprayed (5‐10 µl) into the larynx or bolus IV‐injected (50 µl) in the anesthetized rats with and without bilateral SLN transection. We found that FNT sprayed into the larynx induced an immediate brief apnea (~3.5‐fold of baseline TE) and laryngeal constriction (but not closure) associated with hypertension and bradycardia. The cardiorespiratory response disappeared after transection of SLN. Interestingly, the same dose of FNT given by IV injection induced a ventilatory arrest (apneic duration >8‐fold longer than that produced by the local administration) associated with laryngeal closure, hypotension and bradycardia. These responses to IV injection of FNT were not significantly affected by SLN transection. Our data suggest that the SLN is responsible for the brief apneic response to laryngeal application of FNT, but not for the lethal ventilatory arrest induced by rapid IV injection of FNT.

Intralaryngeal application of ATP evokes apneic response mainly via acting on P2X3 (P2X2/3) receptors of the superior laryngeal nerve in postnatal rats.

Aerosolized adenosine 5'-triphosphate (ATP) induces cough and bronchoconstriction by activating vagal sensory fibers' P2X3 and P2X2/3 receptors (P2X3R and P2X2/3R). The goal of this study is to determine the effect of these receptors on the superior laryngeal nerve (SLN)-mediated cardiorespiratory responses to ATP challenge. We compared the cardiorespiratory responses to intralaryngeal perfusion of either ATP or α,β-methylene ATP in rat pups before and after 1) intralaryngeal perfusion of A-317491 (a P2X3R and P2X2/3R antagonist); 2) bilateral section of the SLN; and 3) peri-SLN treatment with capsaicin (to block conduction in superior laryngeal C-fibers, SLCFs) or A-317491. The immunoreactivity (IR) of P2X3R and P2X2R was determined in laryngeal sensory neurons of the nodose/jugular ganglia. Lastly, a whole cell patch clamp recording was used to determine ATP- or α,β-methylene ATP (α,β-mATP)-induced currents without and with A-317491 treatment. It was found that intralaryngeal perfusion of both ATP and α,β-mATP induced immediate apnea, hypertension, and bradycardia. The apnea was eliminated and the hypertension and bradycardia were blunted by intralaryngeal perfusion of A-317491 and peri-SLN treatment with either A-317491 or capsaicin, although all of the cardiorespiratory responses were abolished by bilateral section of the SLN. P2X3R- and P2X2R-IR were observed in nodose and jugular ganglionic neurons labeled by fluoro-gold (FG). ATP- and α,β-mATP-induced currents recorded in laryngeal C-neurons were reduced by 75% and 95%, respectively, by the application of A-317491. It is concluded that in anesthetized rat pups, the cardiorespiratory responses to intralaryngeal perfusion of either ATP or α,β-mATP are largely mediated by the activation of SLCFs' P2X3R-P2X2/3R.NEW & NOTEWORTHY Aerosolized ATP induces cough and bronchoconstriction via activating P2X3 and P2X2/3 receptors (P2X3R and P2X2/3R) localized on vagal pulmonary sensory fibers. The superior laryngeal nerve (SLN), particularly SLN C-fibers (SLCFs), is involved in generating apnea, hypertension, and bradycardia. This study demonstrates for the first time that either ATP or α,β-mATP applied onto the laryngeal mucosa elicit these cardiorespiratory responses predominately through the activation of P2X3R-P2X2/3R localized on SLCFs.

HIF-1α-Mediated, NADPH Oxidase-Derived ROS Contributes to Laryngeal Airway Hyperreactivity Induced by Intermittent Hypoxia in Rats.

Obstructive sleep apnea, similar to intermittent hypoxia (IH) during sleep, is associated with laryngeal airway hyperreactivity (LAH). IH-induced laryngeal oxidative stress may contribute to LAH, but the underlying mechanism remains unknown. Conscious rats were subjected to repetitive 75 s cycles of IH for 7 or 14 consecutive days. Reflex apneic responses to laryngeal provocations with chemical stimulants were measured to reflect laryngeal reflex reactivity. Compared with control rats, rats exposed to IH for 14 days, but not for 7 days, displayed enhanced apneic response to laryngeal chemical stimulants. The apneic response to chemical stimulants, but not to mechanical stimulation, was totally abolished by perineural capsaicin treatment of superior laryngeal nerves (SLNs) or by the sectioning of the SLNs, suggesting that the reflex was mediated through capsaicin-sensitive SLNs. Daily intraperitoneal administration of N-acetyl-L-cysteine [NAC, a reactive oxygen species (ROS) scavenger], apocynin (an inhibitor of NADPH oxidase) or YC-1 (an inhibitor of HIF-1α), but not their vehicles, largely attenuated this augmented apneic response in 14 days IH rats. Laryngeal lipid peroxidation (an index of oxidative stress) was elevated in 7 days IH rats and 14 days IH rats, and was abolished by any of these three pharmacologic interventions. The protein expression of HIF-1α (an index of HIF-1 activation) and p47phox subunit in the membrane fraction (an index of NADPH oxidase activation) in the laryngeal tissues increased in 14 days IH rats; the former was reduced by NAC, whereas the latter was inhibited by YC-1. These results suggest that 14 days of IH exposure may sensitize capsaicin-sensitive SLNs and result in exaggerated apneic reflex response to laryngeal chemical stimulants. This phenomenon depends on the action of HIF-1α-mediated, NADPH oxidase-derived ROS.

Hypersensitivity of Airway Reflexes Induced by Hydrogen Sulfide: Role of TRPA1 Receptors.

The activation of capsaicin-sensitive lung vagal (CSLV) afferents can elicit airway reflexes. Hypersensitivity of these afferents is known to contribute to the airway hypersensitivity during airway inflammation. Hydrogen sulfide (H2S) has been suggested as a potential therapeutic agent for airway hypersensitivity diseases, such as asthma, because of its relaxing effect on airway smooth muscle and anti-inflammatory effect. However, it is still unknown whether H2S affects airway reflexes. Our previous study demonstrated that exogenous application of H2S sensitized CSLV afferents and enhanced Ca2+ transients in CSLV neurons. The present study aimed to determine whether the H2S-induced sensitization leads to functional changes in airway reflexes and elevates the electrical excitability of the CSLV neurons. Our results showed that, first and foremost, in anesthetized, spontaneously breathing rats, the inhalation of aerosolized sodium hydrosulfide (NaHS, a donor of H2S; 5 mg/mL, 3 min) caused an enhancement in apneic response evoked by several stimulants of the CSLV afferents. This enhancement effect was found 5 min after NaHS inhalation and returned to control 30 min later. However, NaHS no longer enhanced the apneic response after perineural capsaicin treatment on both cervical vagi that blocked the conduction of CSLV fibers. Furthermore, the enhancing effect of NaHS on apneic response was totally abolished by pretreatment with intravenous HC-030031 (a TRPA1 antagonist; 8 mg/kg), whereas the potentiating effect was not affected by the pretreatment with the vehicle of HC-030031. We also found that intracerebroventricular infusion pretreated with HC-030031 failed to alter the potentiating effect of NaHS on the apneic response. Besides, the cough reflex elicited by capsaicin aerosol was enhanced by inhalation of NaHS in conscious guinea pigs. Nevertheless, this effect was entirely eliminated by pretreatment with HC-030031, not by its vehicle. Last but not least, voltage-clamp electrophysiological analysis of isolated rat CSLV neurons showed a similar pattern of potentiating effects of NaHS on capsaicin-induced inward current, and the involvement of TRPA1 receptors was also distinctly shown. In conclusion, these results suggest that H2S non-specifically enhances the airway reflex responses, at least in part, through action on the TRPA1 receptors expressed on the CSLV afferents. Therefore, H2S should be used with caution when applying for therapeutic purposes in airway hypersensitivity diseases.

Intralaryngeal application of ATP evokes apneic response mainly via acting on P2X3/P2X2/3 receptors of the superior laryngeal nerve (SLN) in postnatal rats

ATP is reportedly capable of stimulating bronchopulmonary C‐ and/or Aδ‐fibers to evoke apnea, bronchoconstriction, and bradycardia via acting on P2X (P2X2/3) receptors. Stimulation of the SLN evokes apnea, bradycardia, and hypertension, especially in early life. Although P2X3/P2X2/3 receptors are present in the nodose and jugular (N/J) ganglion neurons, it is undetermined whether ATP applied onto the larynx mucosa can elicit the cardiorespiratory responses via activation of P2X3/P2X2/3 receptors of the SLN. To address this issue, the cardiorespiratory responses to intralaryngeal perfusion of ATP (a non‐selective P2‐receptor agonist, 5–10 mg/ml) or α,β‐methylene ATP (a selective P2X‐receptor agonist, 2.5–5 mg/ml) were recorded in anesthetized rat pups (P14‐P18) under the following conditions: 1) before and after bilateral SLN section and 2) immediately after 5 min intralaryngeal pretreatment with saline (Ctrl) or saline containing A‐317491 (a P2X3/P2X2/3 receptor antagonist, 2.5–5 mg/ml). The immunoreactivity (IR) of P2X3 receptor was then identified in N/J ganglion neurons retrogradely traced by DiI that was previously injected into the SLN 10 days ago. Whole‐cell patch clamp recording was used to determine the ATP‐ or α,β‐methylene ATP‐induced currents of N/J neurons in primary culture, particularly those labeled by DiI. We found that intralaryngeal perfusion of ATP and α,β‐methylene ATP induced similar apnea (latency < 1.0 s), hypertension, and bradycardia, which were eliminated after bilateral section of the SLN. A‐317491 but not saline pretreatment attenuated the apneic response to ATP and α,β‐methylene ATP by 63% and 76% respectively and failed to affect the evoked hypertension and bradycardia. P2X3 receptor‐IR was identified in ~35% of both N/J ganglion neurons. 12% of these neurons were labeled by DiI, ~60% of which expressed P2X3 receptor‐IR. Both ATP‐ and α,β‐methylene ATP‐induced currents were recorded in DiI‐labeled neurons. The former was reduced by 75% and the latter abolished by A‐317491. We conclude that ATP and α,β‐methylene ATP applied onto the laryngeal mucosa elicit an apnea predominately through activation of P2X3/P2X2/3 receptors of the SLN.

Spinal trigeminal subnucleus pars muralis: An interface for apneic reflex

Noxious stimulation of the nasal cavity elicits protective reflexes for the upper respiratory tract, including sneezing, mucus secretion, apnea and cardiovascular reflexes. Yet, no studies have shown the full circuit responsible for the apneic response upon noxious stimulation of the nasal mucosa. Here, we show that injection of cholera toxin beta subunit into the nasal mucosa leads to terminal labeling in the transition zone between the interpolaris and caudalis subnuclei of the trigeminal sensory complex, i.e. subnucleus muralis (SpVm). We also provide physiological evidence that ammonia vapor delivered into the nasal cavity induces an apneic reaction and activates some SpVm cells. Subnucleus muralis cells also respond to mechanical stimulation of the narial pad and the nasal mucosa. Yet, none of the muralis cells responded to vibrissa deflection. Electrolytic lesion of SpVm or unilateral transection of the anterior ethmoidal nerve prevent apnea induced by ammonia delivery to the nasal epithelium, demonstrating their crucial role in the apneic reflex. We also found direct projections from SpVm to the respiratory centers, particularly the preBötzinger complex (preBötC). Inhibition of inspiratory preBötC cells and increased firing of expiratory preBötC cells are associated with ammonia‐induced apnea.Anatomical and physiological data presented here support the hypothesis that nasal induced apnea may be mediated by direct projections from nasal innervated Subnucleus muralis neurons to the PreBötC complex.

Astrocytic glutamate transporters reduce the neuronal and physiological influence of metabotropic glutamate receptors in nucleus tractus solitarii.

Astrocytic excitatory amino acid transporters (EAATs) are critical to restraining synaptic and neuronal activity in the nucleus tractus solitarii (nTS). Relief of nTS EAAT restraint generates two opposing effects, an increase in neuronal excitability that reduces blood pressure and breathing and an attenuation in afferent [tractus solitarius (TS)]-driven excitatory postsynaptic current (EPSC) amplitude. Although the former is due, in part, to activation of ionotropic glutamate receptors, there remains a substantial contribution from another unidentified glutamate receptor. In addition, the mechanism(s) by which EAAT inhibition reduced TS-EPSC amplitude is unknown. Metabotropic glutamate receptors (mGluRs) differentially modulate nTS excitability. Activation of group I mGluRs on nTS neuron somas leads to depolarization, whereas group II/III mGluRs on sensory afferents decrease TS-EPSC amplitude. Thus we hypothesize that EAATs control postsynaptic excitability and TS-EPSC amplitude via restraint of mGluR activation. To test this hypothesis, we used in vivo recording, brain slice electrophysiology, and imaging of glutamate release and TS-afferent Ca2+. Results show that EAAT blockade in the nTS with (3S)-3-[[3-[[4-(trifluoromethyl)benzoyl]amino]phenyl]methoxy]-l-aspartic acid (TFB-TBOA) induced group I mGluR-mediated depressor, bradycardic, and apneic responses that were accompanied by neuronal depolarization, elevated discharge, and increased spontaneous synaptic activity. Conversely, upon TS stimulation TFB-TBOA elevated extracellular glutamate to decrease presynaptic Ca2+ and TS-EPSC amplitude via activation of group II/III mGluRs. Together, these data suggest an important role of EAATs in restraining mGluR activation and overall cardiorespiratory function.

Liquiritin apioside attenuates laryngeal chemoreflex but not mechanoreflex in rat pups.

Liquiritin apioside (LA), a main flavonoid component of licorice, reportedly suppresses cough responses to inhalation of aerosolized capsaicin [CAP; a stimulant to transient receptor potential vanilloid 1 (TRPV1)] in conscious guinea pigs via acting on peripheral nerves. However, the evidence of LA having a direct effect on airway sensory fibers is lacking. Considering the important role laryngeal chemoreceptors and mechanoreceptors play in triggering apnea and cough, we studied whether LA suppressed the apneic responses to stimulation of these receptors via directly acting on the superior laryngeal nerve (SLN). Intralaryngeal delivery of chemical [CAP, HCl, and distilled water (DW)] and mechanical [an air-pulse (AP)] stimulations was applied in anesthetized rat pups to evoke the apnea. These stimuli were repeated after intralaryngeal LA treatment or peri-SLN LA treatment to determine the direct effect of LA on the SLN. Our results showed that all stimuli triggered an immediate apnea. Intralaryngeal LA treatment significantly attenuated the apneic response to chemical but not mechanical stimulations. The same attenuation was observed after peri-SLN LA treatment. Owing that TRPV1 receptors of laryngeal C fibers are responsible for the CAP-triggered apneas, the LA impact on the activity of laryngeal C neurons retrogradely traced by DiI was subsequently studied using a patch-clamp approach. LA pretreatment significantly altered the electrophysiological kinetics of CAP-induced currents in laryngeal C neurons by reducing their amplitudes, increasing the rise times, and prolonging the decay times. In conclusion, our results, for the first time, reveal that LA suppresses the laryngeal chemoreceptor-mediated apnea by directly acting on the SLN (TRPV1 receptors of laryngeal C fibers).

Prolongation of bronchopulmonary C-fiber-mediated apnea by prenatal nicotinic exposure in rat pups: role of 5-HT3 receptors.

Prenatal nicotinic exposure (PNE) reportedly sensitizes bronchopulmonary C-fibers (PCFs) and prolongs PCF-mediated apnea in rat pups, contributing to the pathogenesis of sudden infant death syndrome. Serotonin, or 5-hydroxytryptamine (5-HT), induces apnea via acting on 5-HT receptor 3 (5-HT3R) in PCFs, and among the 5-HT3R subunits, 5-HT3B is responsible for shortening the decay time of 5-HT3R-mediated currents. We examined whether PNE would promote pulmonary 5-HT secretion and prolong the apnea mediated by 5-HT3Rs in PCFs via affecting the 5-HT3B subunit. To this end, the following variables were compared between the control and PNE rat pups: 1) the 5-HT content in bronchoalveolar lavage fluid, 2) the apneic response to the right atrial bolus injection of phenylbiguanide (a 5-HT3R agonist) before and after PCF inactivation, 3) 5-HT3R currents and the stimulus threshold of the action currents of vagal pulmonary C-neurons, and 4) the immunoreactivity (IR) and mRNA expression of 5-HT3A and 5-HT3B in these neurons. Our results showed that PNE up-regulated the pulmonary 5-HT concentration and strengthened the PCF 5-HT3R-mediated apnea. PNE significantly facilitated neural excitability by shortening the decay time of 5-HT3R currents, lowering the stimulus threshold, and increasing 5-HT3B IR. In summary, PNE prolongs the apnea mediated by 5-HT3Rs in PCFs, likely by increasing 5-HT3B subunits to enhance the excitability of 5-HT3 channels.-Zhao, L., Gao, X., Zhuang, J., Wallen, M., Leng, S., Xu, F. Prolongation of bronchopulmonary C-fiber-mediated apnea by prenatal nicotinic exposure in rat pups: role of 5-HT3 receptors.

Diving responses elicited by nasopharyngeal irrigation mimic seizure-associated central apneic episodes in a rat model

The spread of epileptic seizure activity to brainstem respiratory and autonomic regions can elicit episodes of obstructive apnea and of central apnea with significant oxygen desaturation and bradycardia. Previously, we argued that central apneic events were not consequences of respiratory or autonomic activity failure, but rather an active brainstem behavior equivalent to the diving response resulting from seizure spread.To test the similarities of spontaneous seizure-associated central apneic episodes to evoked diving responses, we used nasopharyngeal irrigation with either cold water or mist for 10 or 60 s to elicit the diving response in urethane-anesthetized animals with or without kainic acid-induced seizure activity.Diving responses included larger cardiovascular changes during mist stimuli than during water stimuli. Apneic responses lasted longer than 10 s in response to 10 s stimuli or about 40 s in response to 60 s stimuli, and outlasted bradycardia. Repeated 10 s mist applications led to an uncoupling of the apneic episodes (which always occurred) from the bradycardia (which became less pronounced with repetition). These uncoupled events matched the features of observed spontaneous seizure-associated central apneic episodes. The duration of spontaneous central apneic episodes correlated with their frequency, i.e. longer events occurred when there were more events.Based on our ability to replicate the properties of seizure-associated central apneic events with evoked diving responses during seizure activity, we conclude that seizure-associated central apnea and the diving response share a common neural basis and may reflect an attempt by brainstem networks to protect core physiology during seizure activity.

Estrogen Modulates the Sensitivity of Lung Vagal C Fibers in Female Rats Exposed to Intermittent Hypoxia.

Obstructive sleep apnea is mainly characterized by intermittent hypoxia (IH), which is associated with hyperreactive airway diseases and lung inflammation. Sensitization of lung vagal C fibers (LVCFs) induced by inflammatory mediators may play a central role in the pathogenesis of airway hypersensitivity. In females, estrogen interferes with inflammatory signaling pathways that may modulate airway hyperreactivity. In this study, we investigated the effects of IH on the reflex and afferent responses of LVCFs to chemical stimulants and lung inflammation in adult female rats, as well as the role of estrogen in these responses. Intact and ovariectomized (OVX) female rats were exposed to room air (RA) or IH for 14 consecutive days. On day 15, IH enhanced apneic responses to right atrial injection of chemical stimulants of LVCFs (e.g., capsaicin, phenylbiguanide, and α,β-methylene-ATP) in intact anesthetized females. Rats subjected to OVX prior to IH exposure exhibited an augmented apneic response to the same dose of stimulants compared with rats subjected to other treatments. Apneic responses to the stimulants were completely abrogated by bilateral vagotomy or perivagal capsaicin treatment, which blocked the neural conduction of LVCFs. Electrophysiological experiments revealed that in IH-exposed rats, OVX potentiated the excitability of LVCFs to stimulants. Moreover, LVCF hypersensitivity in rats subjected to OVX prior to IH exposure was accompanied by enhanced lung inflammation, which was reflected by elevated inflammatory cell infiltration in bronchoalveolar lavage fluid, lung lipid peroxidation, and protein expression of inflammatory cytokines. Supplementation with 17β-estradiol (E2) at a low concentration (30 μg/ml) but not at high concentrations (50 and 150 μg/ml) prevented the augmenting effects of OVX on LVCF sensitivity and lung inflammation caused by IH. These results suggest that ovarian hormones prevent the enhancement of LVCF sensitivity and lung inflammation by IH in female rats, which are related to the effect of low-dose estrogen.

Impact of cervical spinal contusion on the upper airway resistance in the rat

Bronchopulmonary C‐fiber induced pulmonary chemoreflex is a critical component of the respiratory defense reflex and characterized by cessation of inspiratory airflow (i.e., apnea) and increase of upper airway resistance (i.e., laryngeal closure). Our previous reports demonstrated that apneic response was attenuated following cervical spinal cord injury; however, it remained unclear whether the upper airway response of pulmonary chemoreflex was also influenced by spinal injury. Accordingly, the present study was designed to investigate the response of laryngeal function to bronchopulmonary C‐fiber activation following unilateral high‐cervical spinal cord contusion in rats. Adult Sprague–Dawley male rats were received laminectomy or unilateral contusion at C2 spinal cord using the MASCIS Impactor (height: 6.25 mm). The respiratory airflow and subglottal pressure in response to intra‐jugular capsaicin (1.5 μg/kg)‐induced pulmonary chemoreflex were measured under spontaneously breathing condition at 3 days, 2 weeks or 6 weeks following spinal surgery. The result demonstrated that high cervical contusion caused a long‐term reduction in the tidal volume without alteration of laryngeal resistance at three time points after injury. However, capsaicin‐induced increase of subglottal pressure was substantially attenuated in contused animals at 3 days post‐injury, and partially recovered at 2 and 6 weeks post‐injury. These results suggested that cervical spinal cord injury not only influence the breathing pattern but also impact the upper airway function. The attenuated laryngeal closure response may unfavorably reduce the ability to prevent inhaling irritants and cause the pulmonary pathological condition following cervical spinal cord injury.Support or Funding InformationMOST 105‐2628‐B‐110‐002‐MY3This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Prenatal Nicotinic Exposure Prolongs the Apneic Response to Activation of Bronchopulmonary C‐fiber 5‐HT3 Receptor with Upregulation of C‐neural 5‐HT3B Expression in Rat Pups

5‐HT3 receptor, an important ionic channel, is heavily expressed in bronchopulmonary C‐fibers (PCFs). Among five 5‐HT3 receptor subunits (5‐HT3A–E), only homomeric 5‐HT3A and heteromeric 5‐HT3AB form functional ion channels. It is reported that the recovery from channel desensitization is much faster in 5‐HT3AB receptor assemblies than that in 5‐HT3A receptor assemblies. Our previous studies have shown that prenatal nicotinic exposure (PNE) significantly prolongs the apneic response to stimulation of PCFs in rat pups. Here, we asked whether PNE would promote 5‐HT secretion in BALF and prolong the 5‐HT3‐mediated apneic response depending on PCFs. We further asked whether this augmented 5‐HT3‐mediated response was associated with alterations of protein and mRNA expressions of 5‐HT3A and/or 5‐HT3B subunits and changes of 5‐HT3 currents in vagal pulmonary C‐neurons. To this end, Ctrl and PNE rat pups were used to detect the differences in: 1) 5‐HT content in BALF; 2) the apneic response to right atrial bolus injection of phenylbiguanide (a selective 5‐HT3 agonist, 10 μg/kg) under PCF intact condition and degenerated condition induced by perivagal capsaicin treatment; 3) 5‐HT3A and 5‐HT3B mRNA (single cell RT‐PCR) and protein expression (immunohistochemistry) in the nodose/jugular ganglionic vagal pulmonary C‐neurons that were retrogradely traced by intratracheal instillation of DiI and marked with TRPV1 expression; and 4) 5‐HT3 currents on these neurons recorded by patch‐clamp in vitro. Our results show that PNE not only upregulates pulmonary 5‐HT concentration but also strengthens the 5‐HT3‐mediated apnea depending on PCF. PNE increases 5‐HT3B but not 5‐HT3A protein expression with little changes in their mRNA level in the vagal pulmonary C‐neurons. Interestingly, PNE significantly accelerates 5‐HT3 channel desensitization and shortens the half‐widths and decay times but has no effect on the rise‐times of these channels. Our results suggest that PNE prolongs PCF 5‐HT3‐mediated apnea likely through altering the formation of 5‐HT subunits (increasing 5‐HT3B) to enhance the sensitivities of 5‐HT3 channels.Support or Funding InformationHL119683 and HL107462This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Prenatal Nicotinic Exposure (PNE) Prolongs Superior Laryngeal C‐Fibers (SLCFs)‐mediated Apnea through Upregulating TRPV1 Receptor

Maternal cigarette smoke is a critical factor in the pathogenesis of sudden infant death syndrome (SIDS). Simulation of SLCFs evokes an immediate apnea and cardiovascular instability, which are potentially life‐threatening and implicated in pathogenesis of SIDS. To date, the impact of PNE on SLCFs has not been explored. We tested hypothesis whether PNE could prolong the SLCF‐mediated apnea, and if so, whether this prolongation is associated with an upregulated expression of TRPV1 on nodose/jugular (N/J) ganglionic neurons retrogradely traced from the superior laryngeal nerve (SLN). To this end, we compared following outcomes between Ctrl and PNE rat pups (postnatal days 11–14): 1) the apneic response to intralaryngeal application of capsaicin (10 μg/kg, 50 μl) to stimulate local TRPV1 in anesthetized preparation; 2) immunoreactivity of TRPV1 in laryngeal sensory neurons of N/J ganglia retrogradely traced by DiI; and 3) TRPV1 currents recorded by whole‐cell patch clamp on cultured sensory laryngeal C‐neurons from N/J ganglia retrogradely traced by DiI. Our results showed that PNE: 1) markedly prolonged the SLCF‐mediated apnea; 2) significantly increased the expression TRPV1 receptors in N/J ganglionic C‐neurons; and 3) augmented the amplitudes of TRPV1 currents on these neurons. The results suggest that PNE is able to prolong the SLCF‐mediated apnea likely though upregulating TRPV1 receptor expression, which may contribute to SIDS.Support or Funding InformationSupported by HL‐107462 and HL‐119683

Inflammatory Role of ROS-Sensitive AMP-Activated Protein Kinase in the Hypersensitivity of Lung Vagal C Fibers Induced by Intermittent Hypoxia in Rats.

Obstructive sleep apnea (OSA), manifested by airway exposure to intermittent hypoxia (IH), is associated with excess reactive oxygen species (ROS) production in airways, airway inflammation, and hyperreactive airway diseases. The cause-effect relationship for these events remains unclear. We investigated the inflammatory role of ROS-sensitive AMP-activated protein kinase (AMPK) in IH-induced airway hypersensitivity mediated by lung vagal C fibers (LVCFs) in rats. Conscious rats were exposed to room air (RA) or IH with or without treatment with N-acetyl-L-cysteine (NAC, an antioxidant), Compound C (an AMPK inhibitor), ibuprofen (a cyclooxygenase inhibitor), or their vehicles. Immediately after exposure (24 h), we found that intravenous capsaicin, phenylbiguanide, or α,β-methylene-ATP evoked augmented LVCF-mediated apneic responses and LVCF afferent responses in rats subjected to IH exposure in comparison with those in RA rats. The potentiating effect of IH on LVCF responses decreased at 6 h after and vanished at 12 h after the termination of IH exposure. The potentiating effect of IH on LVCF-mediated apneic and LVCF afferent responses was significantly attenuated by treatment with NAC, compound C, or ibuprofen, but not by their vehicles. Further biochemical analysis revealed that rats exposed to IH displayed increased lung levels of lipid peroxidation (an index of oxidative stress), AMPK phosphorylation (an index of AMPK activation), and prostaglandin E2 (a cyclooxygenase metabolite), compared with those exposed to RA. IH-induced increase in lipid peroxidation was considerably suppressed by treatment with NAC but not by compound C or ibuprofen. IH-induced increase in AMPK phosphorylation was totally abolished by NAC or compound C but not by ibuprofen. IH-induced increase in prostaglandin E2 was considerably prevented by any of these three inhibitor treatments. The vehicles of these inhibitors exerted no significant effect on the three IH-induced responses. These results suggest that 24-h IH exposure sensitizes LVCFs, leading to an exaggerated reflex and afferent responses to chemical stimulants in rats. Moreover, this IH-induced LVCF sensitization is mediated through a cascade of inflammatory responses in the airways involving increases in ROS, AMPK activation, and cyclooxygenase metabolite release.