Increase In Breathing Frequency Research Articles

Glutathione ethyl ester reverses the deleterious effects of fentanyl on ventilation and arterial blood-gas chemistry while prolonging fentanyl-induced analgesia

There is an urgent need to develop novel compounds that prevent the deleterious effects of opioids such as fentanyl on minute ventilation while, if possible, preserving the analgesic actions of the opioids. We report that L-glutathione ethyl ester (GSHee) may be such a novel compound. In this study, we measured tail flick latency (TFL), arterial blood gas (ABG) chemistry, Alveolar-arterial gradient, and ventilatory parameters by whole body plethysmography to determine the responses elicited by bolus injections of fentanyl (75 μg/kg, IV) in male adult Sprague–Dawley rats that had received a bolus injection of GSHee (100 μmol/kg, IV) 15 min previously. GSHee given alone had minimal effects on TFL, ABG chemistry and A-a gradient whereas it elicited changes in some ventilatory parameters such as an increase in breathing frequency. In vehicle-treated rats, fentanyl elicited (1) an increase in TFL, (2) decreases in pH, pO2 and sO2 and increases in pCO2 (all indicative of ventilatory depression), (3) an increase in Alveolar-arterial gradient (indicative of a mismatch in ventilation-perfusion in the lungs), and (4) changes in ventilatory parameters such as a reduction in tidal volume, that were indicative of pronounced ventilatory depression. In GSHee-pretreated rats, fentanyl elicited a more prolonged analgesia, relatively minor changes in ABG chemistry and Alveolar-arterial gradient, and a substantially milder depression of ventilation. GSHee may represent an effective member of a novel class of thiolester drugs that are able to prevent the ventilatory depressant effects elicited by powerful opioids such as fentanyl and their deleterious effects on gas-exchange in the lungs without compromising opioid analgesia.

The physiological effect of early pregnancy on a woman's response to a submaximal cardiopulmonary exercise test.

Given all its systemic adaptive requirements, pregnancy shares several features with physical exercise. In this pilot study, we aimed to assess the physiological response to submaximal cardiopulmonary exercise testing (CPET) in early pregnancy. In 20 healthy, pregnant women (<13 weeks gestation) and 20 healthy, non‐pregnant women, we performed a CPET with stationary cycling during a RAMP protocol until 70% of the estimated maximum heart rate (HR) of each participant. Hemodynamic and respiratory parameters were non‐invasively monitored by impedance cardiography (PhysioFlow®) and a breath‐by‐breath analyzer (OxyconTM). To compare both groups, we used linear regression analysis, adjusted for age. We observed a similar response of stroke volume, cardiac output (CO) and HR to stationary cycling in pregnant and non‐pregnant women, but a slightly lower 1‐min recovery rate of CO (−3.9 [−5.5;‐2.3] vs. −6.6 [−8.2;‐5.1] L min−1 min−1; p = .058) and HR (−38 [−47; −28] vs. −53 [−62; −44] bpm/min; p = .065) in pregnant women. We also observed a larger increase in ventilation before the ventilatory threshold (+6.2 [5.4; 7.0] vs. +3.2 [2.4; 3.9] L min−1 min−1; p < .001), lower PETCO2 values at the ventilatory threshold (33 [31; 34] vs. 36 [34; 38] mmHg; p = .042) and a larger increase of breathing frequency after the ventilatory threshold (+4.6 [2.8; 6.4] vs. +0.6 [−1.1; 2.3] breaths min−1 min−1; p = .015) in pregnant women. In conclusion, we observed a slower hemodynamic recovery and an increased ventilatory response to exercise in early pregnancy.

Differential Contribution of the Retrotrapezoid Nucleus and C1 Neurons to Active Expiration and Arousal in Rats.

Collectively, the retrotrapezoid nucleus (RTN) and adjacent C1 neurons regulate breathing, circulation and the state of vigilance, but previous methods to manipulate the activity of these neurons have been insufficiently selective to parse out their relative roles. We hypothesize that RTN and C1 neurons regulate distinct aspects of breathing (e.g., frequency, amplitude, active expiration, sighing) and differ in their ability to produce arousal from sleep. Here we use optogenetics and a combination of viral vectors in adult male and female Th-Cre rats to transduce selectively RTN (Phox2b+/Nmb+) or C1 neurons (Phox2b+/Th+) with Channelrhodopsin-2. RTN photostimulation modestly increased the probability of arousal. RTN stimulation robustly increased breathing frequency and amplitude; it also triggered strong active expiration but not sighs. Consistent with these responses, RTN innervates the entire pontomedullary respiratory network, including expiratory premotor neurons in the caudal ventral respiratory group, but RTN has very limited projections to brainstem regions that regulate arousal (locus ceruleus, CGRP+ parabrachial neurons). C1 neuron stimulation produced robust arousals and similar increases in breathing frequency and amplitude compared with RTN stimulation, but sighs were elicited and active expiration was absent. Unlike RTN, C1 neurons innervate the locus ceruleus, CGRP+ processes within the parabrachial complex, and lack projections to caudal ventral respiratory group. In sum, stimulating C1 or RTN activates breathing robustly, but only RTN neuron stimulation produces active expiration, consistent with their role as central respiratory chemoreceptors. Conversely, C1 stimulation strongly stimulates ascending arousal systems and sighs, consistent with their postulated role in acute stress responses.SIGNIFICANCE STATEMENT The C1 neurons and the retrotrapezoid nucleus (RTN) reside in the rostral ventrolateral medulla. Both regulate breathing and the cardiovascular system but in ways that are unclear because of technical limitations (anesthesia, nonselective neuronal actuators). Using optogenetics in unanesthetized rats, we found that selective stimulation of either RTN or C1 neurons activates breathing. However, only RTN triggers active expiration, presumably because RTN, unlike C1, has direct excitatory projections to abdominal premotor neurons. The arousal potential of the C1 neurons is far greater than that of the RTN, however, consistent with C1's projections to brainstem wake-promoting structures. In short, C1 neurons orchestrate cardiorespiratory and arousal responses to somatic stresses, whereas RTN selectively controls lung ventilation and arterial Pco2 stability.

Ventilatory Response at Rest and During Maximal Exercise Testing in Patients with Severe Obesity Before and After Sleeve Gastrectomy

IntroductionSleeve gastrectomy (SG) has become a widespread treatment option in patients affected by severe obesity. However, studies investigating the impact of the subsequent weight loss on the ventilatory response at rest and during physical exercise are lacking.MethodsThis is an observational study on 46 patients with severe obesity (76% females), comparing parameters of ventilatory function 1 month before and 6 months after SG. Patients were first evaluated by resting spirometry and subsequently with an incremental, maximal cardiopulmonary exercise test (CPET) on treadmill.ResultsThe important weight loss of 26.35 ± 6.17% of body weight (BMI from 43.59 ± 5.30 to 32.27 ± 4.84 kg/m2) after SG was associated with a significant improvement in lung volumes and flows during forced expiration at rest, while resting ventilation and tidal volume were reduced (all p ≤ 0.001). CPET revealed decreased ventilation during incremental exercise (p < 0.001), with a less shallow ventilatory pattern shown by a lower increase of breathing frequency (∆BFrest to ATp = 0.028) and a larger response of tidal volume (∆TVAT to Peakp < 0.001). Furthermore, a concomitant improvement of the calculated dead space ventilation, VE/VCO2 slope and peripheral oxygen saturation was shown (all p ≤ 0.002). Additionally, the increased breathing reserve at peak exercise was associated with a lower absolute oxygen consumption but improved exercise capacity and tolerance (all p < 0.001).ConclusionThe weight loss induced by SG led to less burdensome restrictive limitations of the respiratory system and to a reduction of ventilation at rest and during exercise, possibly explained by an increased ventilatory efficiency and a decrease in oxygen demands.

Characterizing the Action of Arginine Vasopressin at Hypoglossal Motoneurons In Neonatal Mice

Vasopressin, traditionally considered a hormone, also acts as an excitatory neurotransmitter. The effects of vasopressin have been characterized in brainstem motoneurons, including hypoglossal motoneurons (XII MNs). The XII motor nucleus receives inspiratory rhythm from the preBötzinger Complex where it is relayed to the muscles of the tongue via the XII nerve. With some variation across postnatal maturation, the vasopressin V1a receptor gene is expressed from birth to adulthood in the XII motor nucleus, and is enriched in the XII nucleus in adults compared to other cranial motoneuron populations. Previous research demonstrated that vasopressin, acting via V1a receptors on preBötzinger Complex, can stimulate breathing in adult rats by increasing breathing frequency as well as diaphragm and genioglossus (primary tongue protruder) muscle activity. Whether vasopressin can also act directly at XII MNs to potentiate inspiratory bursting has not been well characterized. Thus, the aim of this research was to characterize vasopressin effects on XII MN inspiratory bursting behavior. We generated rhythmic, transverse medullary slices (600μM) from CD1 mice of either sex (postnatal days 0–5), and recorded inspiratory activity from XII nerve rootlets using a suction electrode. We then bath applied arginine vasopressin (0.01μM and 0.1μM, AVP, V1a receptor agonist), or locally injected AVP (0.1μM and 1.0μM). In doing so, we were able to assess the effects of arginine vasopressin on XII inspiratory burst amplitude. Bath applied AVP increased inspiratory burst amplitude by 43.4%±12.5% (n=9) when applied at 0.01μM, and 56.4%±18.1% at 0.1μM. When locally applied (n=7), AVP at 0.1μM increased inspiratory burst amplitude 38.8%±6.2%, and AVP at 1.0μM caused a 26.5%±6.3% increase in inspiratory burst amplitude with a notable increase in tonic activity. In a separate set of experiments (n=8) to test whether AVP acts via V1a receptors, local AVP increased inspiratory burst amplitude by 40.2%±9.3%. Local pre‐application of the V1a receptor antagonist ((d(CH2)51Tyr(Me)2Arg8) Vasopressin, 500nM) for 60 s attenuated the response to AVP (6.7%±14.7% decrease in inspiratory burst amplitude), but the AVP response was recovered after 15 minutes with an increase in XII inspiratory burst amplitude of 22.3%±4.1%. Overall, our results support that AVP can increase XII inspiratory burst amplitude via V1a receptor activation. Further research will need to determine whether AVP plays a role in maintaining airway patency in vivo.Support or Funding InformationKenneth A. Suarez Research Fellowship, Midwestern University

Differential Lung Responses to Early Life Ozone Exposure in Male and Female Rat Offspring

The role that early life events (intrauterine growth restriction) and environmental exposures (air pollution) play in development of childhood asthma are only partly understood. We previously showed that exposure of Long‐Evans rats to the oxidant air pollutant ozone (O3) on gestational days (GD) 5 and 6; 0.8 ppm x 4h] resulted in growth restriction at GD21. The aim of this study was to determine whether gestational ± repeated peri‐adolescent O3‐exposure of offspring (0.4–0.8 ppm x 4h; once/week at 5, 6, and 7 weeks‐of‐age) would alter lung growth or acute ventilatory response to O3 exposure.Immediately post‐exposure (0h), baseline (BL) spontaneous ventilation [i.e., breathing frequency (f), tidal volume (TV), and PenH, a unitless parameter suggestive of bronchoconstriction] were obtained using whole body plethysmography (WBP) with EMKA iox 2 software (SCIREQ, Montreal, CANADA) followed by a 6‐min 6%‐CO2 challenge to enhance tidal breathing. At 4h, the left lung lobe was fixed (at 20 cm H2O) for volume displacement and histology. Right lobes were lavaged to assessed lung injury and inflammation. Two‐way ANOVAs were used to assess male (M) and female (F) responses separately; O3‐exposed groups were compared to Air‐exposed dams + postnatal (PN)‐Airx3)].By week 7, in males, dam O3‐exposure resulted in augmented body “height” (skull base‐to‐tail head); but not body weight or weight adjusted by height (gm/cm). In females, PN‐O3x3 exposure was associated with reduced weight/height. By the 3rd PN exposure, both M &amp; F O3‐exposed rats continued to exhibit some degree of dyspnea at 0h, with corresponding increases in BL Penh (F: 5.2–5.6 fold, M: 3.9–4.8 fold). Significant reduction in BL TVcorr (corrected for “height”) was apparent only in F O3+O3x3 rats. CO2 challenge increased breathing frequency in all rats, and F rats were more responsive in terms of enhanced TV increases (A+Ax3 and O3+Ax3 offspring increased 56 and 68%, respectively). M A+Ax3 and O3+Ax3‐exposed rats increased 45%. By contrast, O3‐exposed F A+O3x3 and O3+O3x3 rats only had 25 and 40% increases, respectively; while M A+O3x3 and O3+O3x3 rats increased 24%. Based on enhanced TVcorr, values, both M and F O3‐exposed offspring developed a “classic” short‐and‐shallow respiratory pattern acutely following exposure. In F but not M offspring, CO2‐enhanced TV was inversely correlated with PenH. WBP parameters returned to BL within 2–4 minutes of CO2 discontinuation. At necropsy, dam O3‐exposure was associated with lower lung (fixed) volumes in females. For both sexes, lung injury (increased protein leakage) and inflammation (neutrophils and cytokines in lavage fluid) were increased chiefly by PN‐O3 exposure. Protein leakage increased proportionately with PenH. Histopathologic changes are currently being analyzed.In summary, female offspring, especially O3+O3x3 rats appear to have differences in lung function growth and acute O3 responses. The 6‐min 6%‐CO2 protocol was well tolerated and increased TV to approximately 50% of a maximal breath (FEV1). This approach may provide a sensitive, yet non‐invasive method to document differential lung growth trajectories in rodent models of early life stressors. (Abstract does not reflect USEPA policy).Support or Funding InformationEPA funding; A‐E and SHC programs

Progressive cardiorespiratory dysfunction in Kv1.1 knockout mice may provide temporal biomarkers of pending sudden unexpected death in epilepsy (SUDEP): The contribution of orexin.

Immediately preceding sudden unexpected death in epilepsy (SUDEP), patients experienced a final generalized tonic-clonic seizure (GTCS), rapid ventilation, apnea, bradycardia, terminal apnea, and asystole. Whether a progressive pathophysiology develops and increases risk of SUDEP remains unknown. Here, we determined (a) heart rate, respiratory rate, and blood oxygen saturation (SaO2 ) in low-risk and high-risk knockout (KO) mice; and (b) whether blocking receptors for orexin, a cardiorespiratory neuromodulator, influences cardiorespiratory function mice or longevity in high-risk KO mice. Heart rate and SaO2 were determined noninvasively with ECGenie and pulse oximetry. Respiration was determined with noninvasive airway mechanics technology. The role of orexin was determined within subject following acute treatment with a dual orexin receptor antagonist (DORA, 100mg/kg). The number of orexin neurons in the lateral hypothalamus was determined with immunohistochemistry. Intermittent bradycardia was more prevalent in high-risk KO mice, an effect that may be the result of increased parasympathetic drive. High-risk KO mice had more orexin neurons in the lateral hypothalamus. Blocking of orexin receptors differentially influenced heart rate in KO, but not wild-type (WT) mice. When DORA administration increased heart rate, it also decreased heart rate variability, breathing frequency, and/or hypopnea-apnea. Blocking orexin receptors prevented the methacholine (MCh)-induced increase in breathing frequency in KO mice and reduced MCh-induced seizures, via a direct or indirect mechanism. DORA improved oxygen saturation in KO mice with intermittent hypoxia. Daily administration of DORA to high-risk KO mice increased longevity. High-risk KO mice have a unique cardiorespiratory phenotype that is characterized by progressive changes in five interdependent endpoints. Blocking of orexin receptors attenuates some of these endpoints and increases longevity, supporting the notion that windows of opportunity for intervention exist in this preclinical SUDEP model.

Wearing body armour and backpack loads increase the likelihood of expiratory flow limitation and respiratory muscle fatigue during marching

The effect of load carriage on pulmonary function was investigated during a treadmill march of increasing intensity. 24 male infantry soldiers marched on six occasions wearing either: no load, 15 kg, 30 kg, 40 kg or 50 kg. Each loaded configuration included body armour which was worn as battle-fit or loose-fit (40 kg only). FVC and FEV1 were reduced by 6 to 15% with load. Maximal mouth pressures were reduced post load carriage by up to 11% (inspiratory) and 17% (expiratory). Increased ventilatory demands associated with carrying increased mass were met by increases in breathing frequency (from 3 to 26 breaths·min−1) with minimal changes to tidal volume. 72% of participants experienced expiratory flow limitation whilst wearing the heaviest load. Loosening the armour had minimal effects on pulmonary function. It was concluded that as mass and exercise intensity are increased, the degree of expiratory flow limitation also increases. Practitioner Summary: This study investigated the effect of soldier load carriage on pulmonary function, to inform the trade-off between protection and burden. Load carriage caused an inefficient breathing pattern, respiratory muscle fatigue and expiratory flow limitation during marching. These effects were exacerbated by increases in mass carried and march intensity.

Low activation threshold of juxtapulmonary capillary (J) receptors of the lung

Respiratory reflexes arising from stimulating juxtapulmonary capillary (J) receptors by increasing doses of phenyl diguanide (PDG) were examined in 18 spontaneously breathing cats. In 60% an immediate and four-fold increase in breathing frequency (fR) was produced by doses as small as 5.1 ± μg/kg (range: 3.5–7.5) thus establishing that a significant increase in fR is produced by J receptors by stimulating them with minimal or threshold doses of PDG. In response to similar minimal doses of PDG J receptor afferent activity increased accompanied by acceleration of breathing rate.The response to supra threshold doses was either an apnoea followed by rapid shallow breathing (rsb) or to an apnoea preceded by rsb or only to rsb. Respiratory excursions counted from high-speed run records of intrapleural pressure revealed that the apnoeic response obtained in some cases was a phase of high-frequency breathing and not its suspension.These findings using a chemical stimulus demonstrate that J receptors, with some variability, have a very low threshold for stimulation resulting in notable respiratory acceleration. Thus their afferent output could increase significantly at low intensities of their physiological stimuli such as rise in cardiac output and incipient pulmonary congestion that are generated with mild exercise, to give rise to augmented breathing which is consequently seen.

Respiratory frequency plasticity during development

Respiratory frequency plasticity is a long-lasting increase in breathing frequency due to a perturbation. Mechanisms underlying respiratory frequency are poorly understood, and there is little evidence of frequency plasticity in neonates. This hybrid review/research article discusses available literature regarding frequency plasticity and highlights potential research opportunities. Also, we include data demonstrating a model of frequency plasticity using isolated neonatal rat brainstem-spinal cord preparations. Specifically, substance P (SubP) application induced a long-lasting (>60 min) increase in spontaneous respiratory motor burst frequency, particularly in brainstem-spinal cords with the pons attached; there were no male/female differences. SubP-induced frequency plasticity is dependent on the application pattern, such that intermittent (rather than sustained) SubP applications induce more frequency plasticity. SubP-induced frequency plasticity was blocked by a neurokinin-1 receptor antagonist. Thus, the newborn rat respiratory control system has the capacity to express frequency plasticity. Identifying mechanisms that induce frequency plasticity may lead to novel methods to safely treat breathing disorders in premature and newborn infants.

Muscle Mechanoreflex Control of Ventilation and Inspiratory Drive in Patients with Chronic Obstructive Pulmonary Disease

IntroductionActivation of mechano‐ and metabo‐sensitive skeletal muscles afferents contributes to the regulation of exercise hyperpnea in healthy humans. Patients with chronic obstructive pulmonary disease (COPD) commonly have skeletal muscle dysfunction, which is likely accompanied by alteration in reflex ventilatory responses mediated by activation of skeletal muscle afferents.ObjectiveTo investigate the effect of muscle mechanoreflex activation on ventilation and inspiratory drive in patients with COPD.MethodsThirty four patients with COPD (FEV1 = 48 ± 13% predicted, mean ± SEM) and 20 age‐ and sex‐matched healthy controls participated in the study. Muscle mechanoreceptors were activated by passive knee flexion and extension of the dominant leg. The passive movement was performed at 0.5 Hz for 30 s using an isokinetic dynamometer. Each trial was repeated at least four times. Superficial electrical activity of the vastus lateralis was monitored to confirm the absence of active contractions. Respiratory flow was continuously measured via a differential pressure pneumotachometer. Inspiratory flow (i.e., tidal volume divided by inspiratory time) and superficial electrical activity of the parasternal intercostals multiplied by the respiratory frequency (EMGpara index) provided proxies of the inspiratory drive. All data were interpolated to 1‐s intervals, time‐aligned across repetitions, and ensemble averaged. Baseline data were compared by the Student's t‐test. Responses to passive movement (Δ = passive movement minus baseline) were analyzed via two‐way repeated measures ANOVA, followed by Fisher's post hoc test. Data are reported as mean ± SEM. Statistical significance was set at P &lt; 0.05.ResultsAt rest, patients with COPD presented higher ventilation, inspiratory flow and EMGpara index in comparison with controls. Passive movement increased ventilation, via increase in breathing frequency, as well as increased inspiratory flow and EMGpara index in both groups. However, patients with COPD presented blunted ventilatory (ΔCOPD = 0.527 ± 0.149 vs. Δcontrols = 1.099 ± 0.194 L/min, P = 0.01) and inspiratory flow (ΔCOPD = 0.011 ± 0.007 vs. Δcontrols = 0.036 ± 0.009 L/s, P = 0.03) responses during the first 10 s of the passive movement as compared to controls. The EMGpara index tended to be higher in patients with COPD at this time point (P = 0.06). During the final 10 s of the passive movement, the ventilatory response was similar between groups, whereas inspiratory flow (ΔCOPD = 0.061 ± 0.008 L/s vs. Δcontrols = 0.024 ± 0.011 L/s, P &lt; 0.01) and EMGpara index (ΔCOPD = 54 ± 9 vs. Δcontrols = 24 ± 13 a.u., P = 0.046) responses were greater in patients with COPD as compared to controls.ConclusionsPatients with COPD had a slower ventilatory response and a higher inspiratory drive response to muscle mechanoreflex activation. The results suggest that the muscle mechanoreflex control of ventilation and inspiratory drive is abnormal in COPD which may be involved in the generation of exercise‐related dyspnea.Support or Funding InformationThe study was funded by (FAPESP: 2017/07771‐3). L.C.A. received a scholarship (CAPES).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Microinjection of Gabazine and Kynurenic Acid into the PreBotzinger Complex Alter Cough Phase Durations in the Cat

The preBotzinger complex (preBotC) is important in breathing rhythmogenesis, but its role in cough patterning is unknown. We previously microinjected an excitatory neurotransmitter, 5 mM DL‐Homocysteic acid (DLH), into the preBotC, which altered breathing phase timing, but not cough phase timing. To further understand the role of the preBotC in cough rhythmogenesis, we hypothesized that inhibiting the preBotC would also have no effect on cough phase timing but would inhibit coughing. To test this hypothesis, we bilaterally microinjected 50 mM kynurenic acid (KYN, n=6), a broad spectrum excitatory amino acid blocker, and separately 0.1 mM gabazine (GBZ, n=5), a GABA‐A receptor antagonist, into the preBotC in anesthetized, spontaneously breathing adult cats. We recorded electromyograms (EMG) from parasternal (PS), diaphragm (DIA) and internal oblique (ABD) muscles. Cough was elicited by mechanical stimulation of the tracheobronchial tree. KYN microinjection significantly increased breathing frequency (21±2 to 43±10 breaths/min, p&lt;0.05) by changing both inspiratory (TI) and expiratory (TE) durations (TI 1.0±0.1 to 0.6±0.1, p&lt;0.05; TE 1.8±0.3 to 0.9±0.2 s, p&lt;0.05). DIA EMG magnitude and cough number did not change (4.5±0.6 to 3.9±1.4). However, inspiratory duration decreased (TI 1.1±0.1 to 0.7±0.06 s, p&lt;0.05), and while active expiratory duration (TE1) did not change significantly (TE1 1.0±0.11 to 0.8±0.04 s, n.s.), passive expiratory duration (TE2) was altered but not until 2 hours after injection (TE2 baseline: 0.9±0.2; 20 min after injection: 0.6±0.3 s; 2 hrs after injection: 0.50±0.2 s, p&lt;0.05). Both DIA (55±5 % of baseline) and ABD EMG (56±11 % of baseline) amplitudes decreased during cough. GBZ microinjection had the opposite effect on breathing compared to KYN, where breathing frequency decreased (21.0±4.0 to 12.0±1.0 breaths/minute, p&lt;0.05) by altering both TI and TE (TI 1.1±0.1 to 2.1 ±0.3 s, p&lt;0.05; TE 1.9±0.4 to 2.7±0.4 s, p&lt;0.05). With respect to cough, GBZ decreased cough number (5.1±0.6 to 1.5±0.4, p&lt;0.05) and ABD EMG magnitude (38±4 % of baseline, p&lt;0.05), although PS EMG magnitude did not change significantly (147±34 % of baseline). Microinjection of GBZ increased the duration of all phases of cough (TI 1.1±0.1 to 2.1±0.4 s, p&lt;0.05, TE1 0.9±0.05 to 1.8±0.2 s, p&lt;0.05, TE2 1.1±0.70 to 2.4±0.9 s, p=0.06). These data indicate that the preBotC participates in the regulation of cough phase timing and magnitudes in the anesthetized cat. Disruption of excitatory amino acid neurotransmission with KYN primarily affected inspiratory duration whereas GBZ‐induced blockade of GABA‐A receptor transmission had a generalized effect on cough. These data support an important but more complex role for the preBotC in the regulation of the cough motor pattern than that for breathing.Support or Funding InformationSupported by OT2OD023854‐01, HL131716.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Trpm4 ion channels in pre-Bötzinger complex interneurons are essential for breathing motor pattern but not rhythm.

Inspiratory breathing movements depend on pre-Bötzinger complex (preBötC) interneurons that express calcium (Ca2+)-activated nonselective cationic current (ICAN) to generate robust neural bursts. Hypothesized to be rhythmogenic, reducing ICAN is predicted to slow down or stop breathing; its contributions to motor pattern would be reflected in the magnitude of movements (output). We tested the role(s) of ICAN using reverse genetic techniques to diminish its putative ion channels Trpm4 or Trpc3 in preBötC neurons in vivo. Adult mice transduced with Trpm4-targeted short hairpin RNA (shRNA) progressively decreased the tidal volume of breaths yet surprisingly increased breathing frequency, often followed by gasping and fatal respiratory failure. Mice transduced with Trpc3-targeted shRNA survived with no changes in breathing. Patch-clamp and field recordings from the preBötC in mouse slices also showed an increase in the frequency and a decrease in the magnitude of preBötC neural bursts in the presence of Trpm4 antagonist 9-phenanthrol, whereas the Trpc3 antagonist pyrazole-3 (pyr-3) showed inconsistent effects on magnitude and no effect on frequency. These data suggest that Trpm4 mediates ICAN, whose influence on frequency contradicts a direct role in rhythm generation. We conclude that Trpm4-mediated ICAN is indispensable for motor output but not the rhythmogenic core mechanism of the breathing central pattern generator.

Reboxetine and hyoscine butylbromide improve upper airway function during nonrapid eye movement and suppress rapid eye movement sleep in healthy individuals.

Recent findings indicate that noradrenergic and antimuscarinic processes are crucial for sleep-related reductions in pharyngeal muscle activity. However, there are few human studies. Accordingly, this study aimed to determine if a combined noradrenergic and antimuscarinic intervention increases pharyngeal dilator muscle activity and improves airway function in sleeping humans. Genioglossus (GG) and tensor palatini electromyography (EMG), pharyngeal pressure, upper airway resistance, and breathing parameters were acquired in 10 healthy adults (5 female) during two overnight sleep studies after 4 mg of reboxetine (REB) plus 20 mg of hyoscine butylbromide (HBB) or placebo using a double-blind, placebo-controlled, randomized, cross-over design. Compared with placebo, peak and tonic GG EMG were lower (Mean ± SD: 83 ± 73 vs. 130 ± 75, p = 0.021 and 102 ± 102 vs. 147 ± 123 % wakefulness, p = 0.021, respectively) but the sleep-related reduction in tensor palatini was less (Median [25th, 75th centiles]: 53[45, 62] vs. 34[28, 38] % wakefulness, p = 0.008) with the drug combination during nonrapid eye movement (non-REM) sleep. These changes were accompanied by improved upper airway function including reduced pharyngeal pressure swings, airway resistance, respiratory load compensation, and increased breathing frequency during N2. REB and HBB significantly reduced rapid eye movement sleep compared with placebo (0.6 ± 1.1 vs. 14.5 ± 6.8 % total sleep time, p < 0.001). Contrary to our hypothesis, GG muscle activity (% wakefulness) during non-REM sleep was lower with REB and HBB. However, sleep-related reductions in tensor palatini activity were less and upper airway function improved. These findings provide mechanistic insight into the role of noradrenergic and antimuscarinic processes on upper airway function in humans and have therapeutic potential for obstructive sleep apnea. Australian New Zealand Clinical Trials Registry, https://www.anzctr.org.au, trial ID: ACTRN12616000469415.

Ventilatory and sensory responses to incremental exercise in adults with a Fontan circulation.

Many adults with single-ventricle congenital heart disease who have undergone a Fontan procedure have abnormal pulmonary function resembling restrictive lung disease. Whether this contributes to ventilatory limitations and increased dyspnea has not been comprehensively studied. We recruited 17 Fontan participants and 17 healthy age- and sex-matched sedentary controls. All participants underwent complete pulmonary function testing followed by a symptom-limited incremental cardiopulmonary cycle exercise test with detailed assessments of dyspnea and operating lung volumes. Fontan participants and controls were well matched for age, sex, body mass index, height, and self-reported physical activity levels (all P > 0.05), although Fontan participants had markedly reduced cardiorespiratory fitness and peak work rates ( P < 0.001). Fontan participants had lower values for most pulmonary function measurements relative to controls with 65% of Fontan participants showing evidence of a restrictive ventilatory defect. Relative to controls, Fontan participants had significantly higher breathing frequency, end-inspiratory lung volume (% total lung capacity), ventilatory inefficiency (high ventilatory equivalent for CO2), and dyspnea intensity ratings at standardized absolute submaximal work rates. There were no between-group differences in qualitative descriptors of dyspnea. The restrictive ventilatory defect in Fontan participants likely contributes to their increased breathing frequency and end-inspiratory lung volume during exercise. This abnormal ventilatory response coupled with greater ventilatory inefficiency may explain the increased dyspnea intensity ratings in those with a Fontan circulation. Interventions that enhance the ventilatory response to exercise in Fontan patients may help optimize exercise rehabilitation interventions, resulting in improved exercise tolerance and exertional symptoms. NEW & NOTEWORTHY This is the first study to comprehensively characterize both ventilatory and sensory responses to exercise in adults that have undergone the Fontan procedure. The majority of Fontan participants had a restrictive ventilatory defect. Compared with well-matched controls, Fontan participants had increased breathing frequency, end-inspiratory lung volume, and ventilatory inefficiency. These abnormal ventilatory responses likely form the mechanistic basis for the increased dyspnea intensity ratings observed in our Fontan participants during exercise.

Aging Effects on Alveolar Sacs Under Mechanical Ventilation.

Alveolar sacs are primarily responsible for gas exchange in the human respiratory system and lose their functionality with aging. Three-dimensional (3D) models of young and old human alveolar sacs were constructed and fluid-solid interaction was employed to investigate the contribution of age-related changes to decline in alveolar sacs function under mechanical ventilation (MV). Simulation results illustrated that compliance and pressure reduced in the alveolar sacs of the elderly adults, and they have to work harder to breathe. Morphological changes were found to be mainly responsible for the decline in alveolar sacs function. Influence of individual differences on the alveolar sacs function was negligible and 95% confidence intervals for compliance and work of breathing (WOB) using measures from different individuals also support this finding. Moreover, higher mortality risk was recorded for elderly adults who undergo MV. Specifically, ventilator devices setting has been identified as a potential parameter for compromising respiratory function in the elderly adults. Volume-controlled ventilation applied less pressure, whereas, pressure-controlled ventilation resulted in higher compliance in the alveolar sacs and decreased WOB. Sensitivity of alveolar sacs to ventilator setting under the volume-controlled mode illustrated that increasing breathing frequency and decreasing the ratio of inhalation to exhalation times and TV caused an increase in alveolar sacs expansion and compliance in older patients. Results from this study can help clinicians to develop individualized and effective ventilator protocols and to improve respiratory function in the elderly adults.

Saline as a vehicle control does not alter ventilation in male CD-1 mice.

Saline (0.9% NaCl) is used in clinical and research settings as a vehicle for intravenous drug administration. While saline is a standard control in mouse studies, there are reports of hyperchloremic metabolic acidosis in high doses. It remains unknown if metabolic acidosis occurs in mice and/or if compensatory increases in breathing frequency and tidal volume accompany saline administration. It was hypothesized that saline administration alters blood pH and the pattern of breathing in conscious CD‐1 male mice exposed to air or hypoxia (10% O2, balanced N2). Unrestrained barometric plethysmography was used to quantify breathing frequency (breaths/min; bpm), tidal volume (VT; mL/breath/10 g body weight (BW)), and minute ventilation (VE; mL/min/10 g BW) in two designs: (1) 11‐week‐old mice with no saline exposure (n = 11) compared to mice with 7 days of 0.9% saline administration (intraperitoneal, i.p.; 10 mL/kg body mass; n = 6). and (2) 17‐week‐old mice tested before (PRE) and after 1 day (POST1, n = 6) or 7 days (POST7, n = 5) of saline (i.p.; 10 mL/kg body mass). There were no differences when comparing frequency, VT, or VE between groups for either design with room air or hypoxia exposures. Hypoxia increased frequency, VT, and VE compared to room air. Moreover, conscious blood sampling showed no differences in pH, pa CO 2, paO2, or HCO3− in mice without or with 7 days of saline. These findings reveal no differences in ventilation following 1 and/or 7 days of saline administration in mice. Therefore, the use of 0.9% saline as a control is supported for studies evaluating the control of breathing in mice.

Hyperoxia‐Induced Bronchopulmonary Dysplasia in Neonatal Rats Acutely and Chronically Alters the Control of Breathing

Infants born prematurely have immature lungs, and the neural mechanisms that control breathing are still developing. Premature infants often require supplemental oxygen therapy (hyperoxia) which acutely improves oxygen saturation but consequently leads to bronchopulmonary dysplasia (BPD; chronic lung disease in infants). Whether hyperoxia‐induced BPD also exacerbates the control of breathing is unknown. Thus, the major objective of this study was to elucidate the short and long‐term effects of hyperoxia‐induced BPD on the control of breathing. Sprague Dawley rat pups were exposed to room air (21% O2/Bal. N2) or hyperoxia (90% O2/Bal. N2) from postnatal day 0–10 (P0–10) and ventilation measured during room air (20 min) and hypoxic (10 min; 12% O2/Bal. N2) conditions at P10, 12, 14, 17, 21, 43, and 60 using plethysmography to test the hypothesis that hyperoxia‐induced BPD would decrease breathing stability and impair development of key brainstem respiratory nuclei. By P20, hyperoxic treated pups had significantly (p&lt;0.05) reduced inspiratory (33%) and expiratory flow rates (24%) compared to control (normoxic‐reared) pups, consistent with BPD and pulmonary histologic data from prior publications. Minute ventilation in hyperoxia‐treated pups was significantly decreased (−20% of control) at P10 but increased thereafter while breathing room air (+50% of control; P12–14), and increased ~25% under hypoxic conditions (P12–14, 60, p&lt;0.05). These changes were initially driven by significant increases in breathing frequency (P12–17; +20–46%) and later by increased tidal volume (P43–60; 50%). Room air and hypoxia breathing frequency variability was significantly lower by 20–35% from P10–21 and elevated by 70% at P60, respectively. Moreover, the cumulative histograms of breathing frequencies binned every 10 breaths/min were significantly different in room air and hypoxia conditions across all ages in hyperoxic pups compared to control pups. Preliminary analyses indicated tidal volume variability across room air and hypoxia is also significantly elevated acutely (P10) by 100% and chronically (P60) by 250% in hyperoxic pups compared to control pups. Additional preliminary data indicate increased expression of an astrocytic marker (GFAP) throughout brainstem respiratory nuclei in P60 rats perinatally treated with hyperoxia, consistent with astrogliosis. These data demonstrate significant and lasting anatomical and physiological changes to the brainstem and the neural control of breathing in a rat model of BPD, suggesting additional potential pathologies in human BPD patients.Support or Funding InformationChildren's Hospital of Wisconsin Research InstituteThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Time course of alterations in chemical control of breathing in conscious spontaneously breathing 6‐OHDA SN‐lesioned Parkinson's Disease rat model

Respiratory dysfunction in Parkinson's Disease (PD) patients manifests as a variety of altered breathing patterns and chemical control of breathing deficits that are suggested to result from impairment of central respiratory control. Ongoing work in our laboratory has identified time‐dependent alterations in both basal inspiratory motor output and chemical control of breathing in the 6‐hydroxydopamine (6‐OHDA) neurotoxin‐induced unilateral substantia nigra (SN) lesion rat model. With reference to alterations in chemical control of breathing, our studies have been limited to examination of breathing behaviors at specified time points in acute terminal experiments; thus, the time course underlying the progressive nature of the changes observed is not known. Here, we begin to address this issue by examining the hypoxic (HVR; 12% O2, 3 min) and hypercapnic (HCVR; 7% CO2, 5 min) ventilatory responses using whole body plethysmography before and weekly for 4‐weeks after administration of 6‐OHDA into the SN of adult female Sprague‐Dawley rats; vehicle injected rats served as a control. For the HVR, we found that before injection into the SN, minute ventilation (MV) was increased by ~50% increase, which was mediated predominantly by an increase in breathing frequency (fb) but increases in tidal volume (VT) were noted in some rats; similar observations were noted in vehicle injected rats at the time points examined although some variability in magnitude of the responses were noted at various time points (but not in any specific pattern). In contrast, in 6‐OHDA injected rats, a markedly blunted HVR (~0–10% MV increase), in which fb and VT were minimally altered from pre‐hypoxic levels, was noted; this HVR alteration in behavior was stable over the time course examined albeit in a subset of rats, an HVR began to re‐emerge at the 4‐week post‐lesion time point. For the HCVR, we found that before injection into the SN, MV more than doubled due to similar magnitude increases in both fb and VT. In vehicle injected rats, there was a progressive increase in the magnitude of the HCVR ventilatory behaviors (MV, fb, VT) that stabilized over the 3–4 week post injection time point. In contrast in 6‐OHDA injected rats, the peak of the HCVR MV increase was similar at 5‐min of CO2 exposure, but the response during the initial 4‐min of CO2 exposure was blunted, especially at the 2–3 week post‐lesion time point. This deficit appeared to be mediated by attenuated fb and VT responses. These observations suggest that in conscious 6‐OHDA SN‐lesioned rats, chemical control of breathing undergoes progressive alterations albeit ventilatory behaviors associated with the HVR and HCVR appear to be differentially affected. It remains to be determined whether the ventilatory impairments noted in this PD rat model persist beyond the 4‐week time point studied here.Support or Funding InformationNIH NS101737; Thomas Hartman Center for Parkinson's Disease Research at Stony Brook UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neurons in a Subregion of the Medial Parabrachial Nucleus (mPBN) Attenuate the Gain of the Hering‐Breuer (H‐B) Reflex

This study provides new evidence showing that a subregion of the mPBN, which plays a key role in the control of breathing frequency (1), also attenuates the H‐B expiratory facilitatory reflex. Experiments were performed in decerebrate, vagotomized dogs during isocapnic hyperoxia. Phase durations TI and TE were obtained from the phrenic neurogram(PNG). A small concentric bipolar electrode was used to microstimulate the mPBN subregion. Lung inflations and/or weak electrical stimulation of a desheathed cervical vagus nerve were used to activate the slowly adapting pulmonary stretch receptors (PSRs) to produce the H‐B reflex. Pulse‐train PSR stimulation at various frequencies (Fs) were delivered during the E‐phase of test cycles separated by several non‐stimulated control cycles. During every other PSR test cycle, pulse‐train stimulation of the pontine subregion at a predetermined frequency was concurrently delivered to determine its effect on the PSR‐induced increase in TE (e.g., fig A). The effect of the pontine stimulus in the absence of the PSR input was also measured. Plots of TE vs. PSR step Fs have the shape of an increasing exponential function. The effect of the pontine input was a proportional reduction in TE at each of the PSR step Fs, and was shown as an attenuated curve in the TE‐Fs plots (fig B). The relationship between the reduction in the H‐B reflex and the pontine stimulus frequency was linear over the range of 0 to 20 Hz, with a maximum attenuation of the H‐B reflex to ~20% of control (no pontine stimulus) produced by pontine stimulus frequencies in the 20–40 Hz range. These findings suggest that as inputs from the pontine subregion increase breathing frequency, this region concurrently attenuates the H‐B expiratory facilitatory reflex. This mechanism counteracts the PSR‐induced increase in TE, which reduces breathing frequency.Support or Funding InformationSupported by VA grant 2 I01BX000721‐07This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.