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.

Cardiorespiratory failure induced by inhalation of aerosolized fentanyl in anesthetized rats.

Intravenous rapid injection of fentanyl causes respiratory depression (severe apneas), leading to sudden death, which constitutes the deadliest drug reaction among overdoses of synthetic opioids. Here we asked whether acute inhalation of overdose fentanyl would also result in similar respiratory failure and death. The anesthetized and spontaneously breathing rats with tracheal cannulation were exposed to aerosolized fentanyl at 100 mg/m3 (FNTH) or 30 mg/m3 (FNTL) for 10 min. Minute ventilation (VE), electromyography (EMG) of the internal and external intercostal muscles and thyroarytenoid muscles (EMGII, EMGEI, and EMGTA), heart rate and arterial blood pressure were recorded. During the exposure, FNTH and FNTL immediately triggered bradypnea (40 % reduction, p < 0.05) with TE prolonged and then gradually decreased VE by 40 % (P < 0.05) after a brief VE recovery. The initial TE prolongation (apneas) were characterized by the cessation of EMGEI activity with enhanced tonic discharges of EMGTA and EMGII. After termination of the exposure, the cardiorespiratory responses to FNTL returned to the baseline values 30 min later, while those to FNTH were greatly exacerbated (P < 0.05), leading to ventilatory and cardiac arrest occurred 16.4 ± 4.7 min and 19.3 ± 4.5 min respectively after the onset of FNTH. The ventilatory arrest was featured by cessation of both EMGEI and EMGII and augmentation of tonic EMGTA. Our results suggest that acute exposure to an overdose of fentanyl aerosol leads to death through initially inducing a brief central and upper airway obstructive apnea as well as chest wall rigidity followed by gradual severe hypoventilation, bradycardia and hypotension, and eventual cardiorespiratory arrest in anesthetized rats.

Highly Efficient Production of Cellulosic Ethanol from Poplar Using an Optimal C6/C5 Co-Fermentation Strain of Saccharomyces cerevisiae.

Cellulosic ethanol is the key technology to alleviate the pressure of energy supply and climate change. However, the ethanol production process, which is close to industrial production and has a high saccharification rate and ethanol yield, still needs to be developed. This study demonstrates the effective conversion of poplar wood waste into fuel-grade ethanol. By employing a two-step pretreatment using sodium chlorite (SC)-dilute sulfuric acid (DSA), the raw material achieved a sugar conversion rate exceeding 85% of the theoretical value. Under optimized conditions, brewing yeast co-utilizing C6/C5 enabled a yield of 35 g/L ethanol from 10% solid loading delignified poplar hydrolysate. We increased the solid loading to enhance the final ethanol concentration and optimized both the hydrolysis and fermentation stages. With 20% solid loading delignified poplar hydrolysate, the final ethanol concentration reached 60 g/L, a 71.4% increase from the 10% solid loading. Our work incorporates the pretreatment, enzymatic hydrolysis, and fermentation stages to establish a simple, crude poplar waste fuel ethanol process, expanding the range of feedstocks for second-generation fuel ethanol production.

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.

Peripheral neuroplasticity of respiratory chemoreflexes, induced by prenatal nicotinic exposure: Implication for SIDS.

Sudden Infant Death Syndrome (SIDS) occurs during sleep in seemingly healthy infants. Maternal cigarette smoking and hypoxemia during sleep are assumed to be the major causal factors. Depressed hypoxic ventilatory response (dHVR) is observed in infants with high risk of SIDS, and apneas (lethal ventilatory arrest) appear during the fatal episode of SIDS. Disturbance of the respiratory center has been proposed to be involved, but the pathogenesis of SIDS is still not fully understood. Peripherally, the carotid body is critical to generate HVR, and bronchopulmonary and superior laryngeal C-fibers (PCFs and SLCFs) are important for triggering central apneas; however, their roles in the pathogenesis of SIDS have not been explored until recently. There are three lines of recently accumulated evidence to show the disorders of peripheral sensory afferent-mediated respiratory chemoreflexes in rat pups with prenatal nicotinic exposure (a SIDS model) in which acute severe hypoxia leads to dHVR followed by lethal apneas. (1) The carotid body-mediated HVR is suppressed with a reduction of the number and sensitivity of glomus cells. (2) PCF-mediated apneic response is largely prolonged via increased PCF density, pulmonary IL-1ß and serotonin (5-hydroxytryptamine, 5-HT) release, along with the enhanced expression of TRPV1, NK1R, IL1RI and 5-HT3R in pulmonary C-neurons to strengthen these neural responses to capsaicin, a selective stimulant to C-fibers. (3) SLCF-mediated apnea and capsaicin-induced currents in superior laryngeal C-neurons are augmented by upregulation of TRPV1 expression in these neurons. These results, along with hypoxic sensitization/stimulation of PCFs, gain insight into the mechanisms of prenatal nicotinic exposure-induced peripheral neuroplasticity responsible for dHVR and long-lasting apnea during hypoxia in rat pups. Therefore, in addition to the disturbance in the respiratory center, the disorders of peripheral sensory afferent-mediated chemoreflexes may also be involved in respiratory failure and death denoted in SIDS victims.

Neurokinin 1 and 2 receptors are involved in PGE2- and citric acid-induced cough and ventilatory responses.

Exposure to aerosolized citric acid (CA, 150 mM) and prostaglandin E2 (PGE2, 0.43 mM) for 10 min in guinea pigs reportedly produces the distinct cough patterns (Type I vs. II) and ventilatory responses (long-lasting hyperventilation vs. brief tachypnea) even though triggering the same cough numbers. Type I and II coughs are primarily mediated by activation of TRPV1 and EP3 receptors (a PGE2 receptor) of vagal C-fibers respectively. Substance P (SP) and neurokinin A (NKA) released by vagal pulmonary sensory fibers peripherally are capable of affecting CA-induced cough and ventilation via preferentially activating neurokinin 1 and 2 receptors (NK1R and NK2R) respectively. This study aimed to define the impacts of CA- and PGE2-exposure on pulmonary SP and NKA levels and the roles of NK1R and NK2R in modulating CA- and PGE2-evoked cough and ventilatory responses. In unanesthetized guinea pigs, we determined: (1) pulmonary SP and NKA contents induced by the CA- or PGE2-exposure; (2) effects of CP-99994 and SR-48968 (a NK1R and a NK2R antagonist respectively) given by intraperitoneal injection (IP) or aerosol inhalation (IH) on the CA- and PGE2-evoked cough and ventilatory responses; and (3) immunocytochemical expressions of NK1R/NK2R in vagal C-neurons labeled by TRPV1 or EP3 receptors. We found that CA- and PGE2-exposure evoked Type I and II cough respectively associated with different degrees of increases in pulmonary SP and NKA. Applications of CP-99994 and SR-48968 via IP and IH efficiently suppressed the cough responses to CA with less impact on the cough response to PGE2. These antagonists inhibited or blocked the ventilatory response to CA and caused hypoventilation in response to PGE2. Moreover, NK1R and NK2R were always co-expressed in vagal C-neurons labeled by TRPV1 or EP3 receptors. These results suggest that SP and NKA endogenously released by CA- and PGE2-exposure play important roles in generating the cough and ventilatory responses to CA and PGE2, at least in part, via activation of NK1R and NK2R expressed in vagal C-neurons (pulmonary C-neurons).

Systemic 8-OH-DPAT challenge causes hyperventilation largely via activating pre-botzinger complex 5-HT1A receptors.

Systemic 8-OH-DPAT (a 5-HT1A receptor agonist) challenge evokes hyperventilation independent of peripheral 5-HT1A receptors. Though the pre-Botzinger Complex (PBC) is critical in generating respiratory rhythm and activation of local 5-HT1A receptors induces tachypnea via disinhibition of local GABAA neurons, its role in the respiratory response to systemic 8-OH-DPAT challenge is still unclear. In anesthetized rats, 8-OH-DPAT (100 µg/kg, iv) was injected twice to confirm the reproducibility of the evoked responses. The same challenges were performed after bilateral microinjections of (S)-WAY-100135 (a 5-HT1A receptor antagonist) or gabazine (a GABAA receptor antagonist) into the PBC. Our results showed that: 1) 8-OH-DPAT caused reproducible hyperventilation associated with hypotension and bradycardia; 2) microinjections of (S)-WAY-100135 into the PBC attenuated the hyperventilation by ˜60 % without effect on the evoked hypotension and bradycardia; and 3) the same hyperventilatory attenuation was also observed after microinjections of gabazine into the PBC. Our data suggest that PBC 5-HT1A receptors play a key role in the respiratory response to systemic 8-OH-DPAT challenge likely via disinhibiting local GABAergic neurons.

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,ß-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,ß-methylene ATP (α,ß-mATP)-induced currents without and with A-317491 treatment. It was found that intralaryngeal perfusion of both ATP and α,ß-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,ß-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,ß-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,ß-mATP applied onto the laryngeal mucosa elicit these cardiorespiratory responses predominately through the activation of P2X3R-P2X2/3R localized on SLCFs.

Cross-effect of TRPV1 and EP3 receptor on coughs and bronchopulmonary C-neural activities.

Prostaglandin E2 (PGE2)-induced coughs in vivo and vagal nerve depolarization in vitro are inhibited by systemic and local administration of prostaglandin EP3 receptor (L-798106) and TRPV1 antagonists (JNJ 17203212). These results indicate a modulating effect of TRPV1 on the EP3 receptor-mediated cough responses to PGE2 likely through the vagal sensory nerve. This study aimed to determine whether 1) inhalation of aerosolized JNJ 17203212 and L-798106 affected cough responses to citric acid (CA, mainly stimulating TRPV1) and PGE2; 2) TRPV1 and EP3 receptor morphologically are co-expressed and electrophysiologically functioned in the individual of vagal pulmonary C-neurons (cell bodies of bronchopulmonary C-fibers in the nodose/jugular ganglia); and 3) there was a cross-effect of TRPV1 and EP3 receptor on these neural excitations. To this end, aerosolized CA or PGE2 was inhaled by unanesthetized guinea pigs pretreated without or with each antagonist given in aerosol form. Immunofluorescence was applied to identify the co-expression of TRPV1 and EP3 receptor in vagal pulmonary C-neurons (retrogradely traced by DiI). Whole-cell voltage patch clamp approach was used to detect capsaicin (CAP)- and PGE2-induced currents in individual vagal pulmonary C-neurons and determine the effects of the TRPV1 and EP3 receptor antagonists on the evoked currents. We found that PGE2-induced cough was attenuated by JNJ 17203212 or L-798106 and CA-evoked cough greatly suppressed only by JNJ 17203212. Approximately 1/4 of vagal pulmonary C-neurons co-expressed EP3 with a cell size < 20 µm. Both CAP- and PGE2-induced currents could be recorded in the individuals of some vagal pulmonary C-neurons. The former was largely inhibited only by JNJ 17203212, while the latter was suppressed by JNJ 17203212 or L-798106. The similarity of the cross-effect of both antagonists on cough and vagal pulmonary C-neural activity suggests that a subgroup of vagal pulmonary C-neurons co-expressing TRPV1 and EP3 receptor is, at least in part, responsible for the cough response to PGE2.

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).

DT-0111: a novel drug-candidate for the treatment of COPD and chronic cough.

BACKGROUND: Extracellular adenosine 5'-triphosphate (ATP) plays important mechanistic roles in pulmonary disorders in general and chronic obstructive pulmonary disease (COPD) and cough in particular. The effects of ATP in the lungs are mediated to a large extent by P2X2/3 receptors (P2X2/3R) localized on vagal sensory nerve terminals (both C and Aδ fibers). The activation of these receptors by ATP triggers a pulmonary-pulmonary central reflex, which results in bronchoconstriction and cough, and is also proinflammatory due to the release of neuropeptides from these nerve terminals via the axon reflex. These actions of ATP in the lungs constitute a strong rationale for the development of a new class of drugs targeting P2X2/3R. DT-0111 is a novel, small, water-soluble molecule that acts as an antagonist at P2X2/3R sites. METHODS: Experiments using receptor-binding functional assays, rat nodose ganglionic cells, perfused innervated guinea pig lung preparation ex vivo, and anesthetized and conscious guinea pigs in vivo were performed. RESULTS: DT-0111 acted as a selective and effective antagonist at P2X2/3R, that is, it did not activate or block P2YR; markedly inhibited the activation by ATP of nodose pulmonary vagal afferents in vitro; and, given as an aerosol, inhibited aerosolized ATP-induced bronchoconstriction and cough in vivo. CONCLUSIONS: These results indicate that DT-0111 is an attractive drug-candidate for the treatment of COPD and chronic cough, both of which still constitute major unmet clinical needs. The reviews of this paper are available via the supplementary material section.

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.

An advanced recording and analysis system for the differentiation of guinea pig cough responses to citric acid and prostaglandin E2 in real time.

Cough number and/or sound have been used to assess cough sensitivity/intensity and to discriminate cough patterns in clinical settings. However, to date, only manual counting of cough number in an offline manner is applied in animal cough studies, which diminishes the efficiency of cough identification and hinders the diagnostic discrimination of cough patterns, especially in animals with pulmonary diseases. This study aims to validate a novel recording/analysis system by which cough numbers are automatically counted and cough patterns are comprehensively differentiated in real time. The experiment was carried out in conscious guinea pigs exposed to aerosolized citric acid (CA, 150 mM) and prostaglandin E2 (PGE2, 0.43 mM). Animal body posture (video), respiratory flow, and cough acoustics (audio) were simultaneously monitored and recorded. Cough number was counted automatically, and cough sound parameters including waveform, duration, power spectral density, spectrogram, and intensity, were analyzed in real time. Our results showed that CA- and PGE2-evoked coughs had the same cough numbers but completely different patterns [individual coughs vs. bout(s) of coughs]. Compared to CA-evoked coughs, PGE2-evoked coughs possess a longer latency, higher cough rate (coughs/min), shorter cough sound duration, lower cough sound intensity, and distinct cough waveforms and spectrograms. A few mucus- and wheeze-like coughs were noted in response to CA but not to PGE2. In conclusion, our recording/analysis system is capable of automatically counting the cough number and successfully differentiating the cough pattern by using valuable cough sound indexes in real time. Our system enhances the objectivity, accuracy, and efficiency of cough identification and count, improves the intensity evaluation, and offers ability for pattern discrimination compared to traditional types of cough identification. Importantly, this approach is beneficial for assessing the efficacy of putative antitussive drugs in animals without or with pulmonary diseases, particularly in cases without significant change in cough number.

Lethal avian influenza A (H5N1) virus replicates in pontomedullary chemosensitive neurons and depresses hypercapnic ventilatory response in mice.

The highly pathogenic H5N1 (HK483) viral infection causes a depressed hypercapnic ventilatory response (dHCVR, 20%↓) at 2 days postinfection (dpi) and death at 7 dpi in mice, but the relevant mechanisms are not fully understood. Glomus cells in the carotid body and catecholaminergic neurons in locus coeruleus (LC), neurokinin 1 receptor (NK1R)-expressing neurons in the retrotrapezoid nucleus (RTN), and serotonergic neurons in the raphe are chemosensitive and responsible for HCVR. We asked whether the dHCVR became worse over the infection period with viral replication in these cells/neurons. Mice intranasally inoculated with saline or the HK483 virus were exposed to hypercapnia for 5 min at 0, 2, 4, or 6 dpi, followed by immunohistochemistry to determine the expression of nucleoprotein of H5N1 influenza A (NP) alone and coupled with 1) tyrosine hydroxylase (TH) in the carotid body and LC, 2) NK1R in the RTN, and 3) tryptophan hydroxylase (TPH) in the raphe. HK483 viral infection blunted HCVR by ∼20, 50, and 65% at 2, 4, and 6 dpi. The NP was observed in the pontomedullary respiratory-related nuclei (but not in the carotid body) at 4 and 6 dpi, especially in 20% of RTN NK1R, 35% of LC TH, and ∼10% raphe TPH neurons. The infection significantly reduced the local NK1R or TPH immunoreactivity and population of neurons expressing NK1R or TPH. We conclude that the HK483 virus infects the pontomedullary respiratory nuclei, particularly chemosensitive neurons in the RTN, LC, and raphe, contributing to the severe depression of HCVR and respiratory failure at 6 dpi. NEW & NOTEWORTHY The H5N1 virus infection is lethal due to respiratory failure, but the relevant mechanisms remain unclear. In this study, we demonstrated a gradual diminution of hypercapnic ventilatory response to a degree, leading to respiratory failure over a 6-day infection. Death was associated with viral replication in the pontomedullary respiratory-related nuclei, especially the central chemosensitive neurons. These results not only provide insight into the mechanisms of the lethality of H5N1 viral infection but also offer clues in the development of corresponding treatments to minimize and prevent respiratory failure.

Roles of Bronchopulmonary C-fibers in airway Hyperresponsiveness and airway remodeling induced by house dust mite.

BACKGROUND: Asthma is characterized by chronic airway inflammation, airway hyperresponsiveness (AHR), and airway remodeling. While exposure of house dust mites (HDM) is a common cause of asthma, the pathogenesis of the HDM-induced asthma is not fully understood. Bronchopulmonary C-fibers (PCFs) contribute to the neurogenic inflammation, viral infection induced-persistent AHR, and ovalbumin induced collagen deposition largely via releasing neuropeptides, such as substance P (SP). However, PCF roles in the pathogenesis of the HDM-induced asthma remain unexplored. The goal of this study was to determine what role PCFs played in generating these characteristics. METHODS: We compared the following variables among the PCF-intact and -degenerated BALB/c mice with and without chronic HDM exposure (four groups): 1) AHR and pulmonary SP; 2) airway smooth muscle (ASM) mass; 3) pulmonary inflammatory cells; and 4) epithelium thickening and mucus secretion. RESULTS: We found that HDM evoked AHR associated with upregulation of pulmonary SP and inflammation, ASM mass increase, epithelium thickenings, and mucus hypersecretion. PCF degeneration decreased the HDM-induced changes in AHR, pulmonary SP and inflammation, and ASM mass, but failed to significantly affect the epithelium thickening and mucus hypersecretion. CONCLUSION: Our data suggest an involvement of PCFs in the mechanisms by which HDM induces allergic asthma via airway inflammation, AHR, and airway remodeling.

Dietary nutrients associated with preservation of lung function in Hispanic and non-Hispanic white smokers from New Mexico.

BACKGROUND: COPD is the third leading cause of death in the United States. Cigarette smoking accelerates the age-related forced expiratory volume in 1 s (FEV1) decline, an important determinant for the genesis of COPD. Hispanic smokers have lower COPD prevalence and FEV1 decline than non-Hispanic whites (NHWs). PATIENTS AND METHODS: A nutritional epidemiological study was conducted in the Lovelace Smokers cohort (LSC; n=1,829) and the Veterans Smokers cohort (n=508) to identify dietary nutrients (n=139) associated with average FEV1 and its decline and to assess whether nutrient intakes could explain ethnic disparity in FEV1 decline between Hispanics and NHW smokers. RESULTS: Nutrients discovered and replicated to be significantly associated with better average FEV1 included magnesium, folate, niacin, vitamins A and D, eicosenoic fatty acid (20:1n9), eicosapentaenoic acid (20:5n3), docosapentaenoic acid (DPA; 22:5n3), docosahexaenoic acid (22:6n3), and fiber. In addition, greater intakes of eicosenoic fatty acid and DPA were associated with slower FEV1 decline in the LSC. Among omega 3 polyunsaturated fatty acids, DPA is the most potent nutrient associated with better average FEV1 and slower FEV1 decline. Adverse effect of continuous current smoking on FEV1 decline was completely negated in LSC members with high DPA intake (>20 mg/day). Slower FEV1 decline in Hispanics compared to NHWs may be due to the greater protection of eicosenoic fatty acid and DPA for FEV1 decline rather than greater intake of protective nutrients in this ethnic group. CONCLUSION: The protective nutrients for the preservation of FEV1 in ever smokers could lay foundation for designing individualized nutritional intervention targeting "optimal physiological levels" in human to improve lung function in ever smokers. Ethnic disparity in FEV1 decline may be explained by difference in magnitude of protection of dietary intakes of eicosenoic fatty acid and DPA between Hispanics and NHWs.

Preclinical Pharmacology and Pharmacokinetics of Inhaled Hexadecyl-Treprostinil (C16TR), a Pulmonary Vasodilator Prodrug.

This article describes the preclinical pharmacology and pharmacokinetics (PK) of hexadecyl-treprostinil (C16TR), a prodrug of treprostinil (TRE), formulated in a lipid nanoparticle (LNP) for inhalation as a pulmonary vasodilator. C16TR showed no activity (>10 µM) in receptor binding and enzyme inhibition assays, including binding to prostaglandin E2 receptor 2, prostaglandin D2 receptor 1, prostaglandin I2 receptor, and prostaglandin E2 receptor 4; TRE potently bound to each of these prostanoid receptors. C16TR had no effect (up to 200 nM) on platelet aggregation induced by ADP in rat blood. In hypoxia-challenged rats, inhaled C16TR-LNP produced dose-dependent (0.06-6 µg/kg), sustained pulmonary vasodilation over 3 hours; inhaled TRE (6 µg/kg) was active at earlier times but lost its effect by 3 hours. Single- and multiple-dose PK studies of inhaled C16TR-LNP in rats showed proportionate dose-dependent increases in TRE Cmax and area under the curve (AUC) for both plasma and lung; similar results were observed for dog plasma levels in single-dose PK studies. In both species, inhaled C16TR-LNP yielded prolonged plasma TRE levels and a lower plasma TRE Cmax compared with inhaled TRE. Inhaled C16TR-LNP was well tolerated in rats and dogs; TRE-related side effects included cough, respiratory tract irritation, and emesis and were seen only after high inhaled doses of C16TR-LNP in dogs. In guinea pigs, inhaled TRE (30 µg/ml) consistently produced cough, but C16TR-LNP (30 µg/ml) elicited no effect. These results demonstrate that C16TR-LNP provides long-acting pulmonary vasodilation, is well tolerated in animal studies, and may necessitate less frequent dosing than inhaled TRE with possibly fewer side effects.

Pulmonary C-fiber degeneration downregulates IFN-γ receptor 1 via IFN-α induction to attenuate RSV-induced airway hyperresponsiveness.

Respiratory syncytial virus (RSV) is a leading cause of respiratory infection in infants. Unfortunately, no effective vaccine or treatment against RSV is currently available. Pulmonary C-fibers (PCFs) are critical for regulating pulmonary inflammation and airway hyperresponsiveness (AHR). We previously reported that IFN-γ partially mediated RSV-induced airway disorders. In this study, we found that PCF degeneration alleviated RSV-induced airway inflammation, especially AHR by downregulating IFN-γ receptor 1 (IFNGR1), but had no effect on IFN-γ induction. In contrast, PCF degeneration actually increased IFN-α/ß levels, as were the levels of STAT1 and phosphorylated STAT1 (pSTAT1). Exogenous IFN-α treatment induced STAT1 activation and downregulated IFNGR1 expression. These results suggest that PCFs affect IFNGR1 expression by inducing IFN-α to regulate IFN-γ-mediated airway inflammation and AHR. Thus, targeting PCFs activation may help control RSV-induced airway disorders, especially AHR, even with the presence of inflammation.

Lethal avian influenza A (H5N1) virus induces ataxic breathing in mice with apoptosis of pre-Botzinger complex neurons expressing neurokinin-1 receptor.

Lethal influenza A (H5N1) induces respiratory failure in humans. Although it also causes death at 7 days postinfection (dpi) in mice, the development of the respiratory failure and the viral impact on pre-Botzinger complex (PBC) neurons expressing neurokinin 1 receptor (NK1R), which is the respiratory rhythm generator, have not been explored. Body temperature, weight, ventilation, and arterial blood pH and gases were measured at 0, 2, 4, and 6 dpi in control, lethal HK483, and nonlethal HK486 viral-infected mice. Immunoreactivities (IR) of PBC NK1R, H5N1 viral nucleoprotein (NP), and active caspase-3 (CASP3; a marker for apoptosis) were detected at 6 dpi. HK483, but not HK486, mice showed the following abnormalities: 1) gradual body weight loss and hypothermia; 2) tachypnea at 2-4 dpi and ataxic breathing with long-lasting apneas and hypercapnic hypoxemia at 6 dpi; and 3) viral replication in PBC NK1R neurons with NK1R-IR reduced by 75% and CASP3-IR colabeled at 6 dpi. Lethal H5N1 viral infection causes tachypnea at the early stage and ataxic breathing and apneas (hypercapnic hypoxemia) leading to death at the late stage. Its replication in the PBC induces apoptosis of local NK1R neurons, contributing to ataxic breathing and respiratory failure.