Augmented Breaths Research Articles

Augmented Biphasic Breathing Using Sniff and an Oral Positive Expiratory Pressure Device (Sniff-PEP) in Fontan Patients.

The use of an oral positive expiratory pressure device (oPEP) with sniff breathing (Sniff-PEP) mimics biphasic ventilation. Biphasic ventilation increases pulmonary blood flow and cardiac output in Fontan patients. The aim of this study was to assess the effect of Sniff-PEP on Fontan flow velocities. A single-center, pilot, prospective study was carried out in 15 subjects with Fontan circulation enrolled to use the oPEP device for 1month. Subjects were instructed on Sniff-PEP and to use the device for 10-15min 3-4 times a day. Measurements of flow velocity and cardiac output were measured via echocardiogram and quality of life assessments were performed at baseline and 4-6weeks later. The mean age at enrollment was 19.9 ± 8.7years (age range of 10-37years). 7 patients (47%) had dominant left ventricle and 8 (53%) had an open fenestration. There was a statistically significant increase in flow velocities in the hepatic vein from 27.5 ± 7.6 to 35.1 ± 11.3cm/s (p = 0.003), left pulmonary artery from 51.6 ± 16.6 to 57.6 ± 21.1cm/s (p = 0.01), and right pulmonary artery from 43.1 ± 14.2 to 45.8 ± 17.2cm/s (p = 0.04). With chronic use, the mean fenestration gradient slightly decreased from 4.5 ± 1.6 to 4.1 ± 1.9mmHg but the difference was not statistically significant (p = 0.14). oPEP device therapy increased flow velocity in several areas in the Fontan circulation with acute use. Further studies are needed to assess the effects long term.Clinical Trial Registration: URL: https://clinicaltrials.gov . Unique identifier: NCT03251742.

Stabilising function of the human diaphragm in response to involuntary augmented breaths induced with or without lower-limb movements.

What is the central question of this study? Is the stabilising function of the diaphragm altered differentially in response to involuntary augmented breathsinduced with or without lower-limb movements? What is the main finding and its importance? At equivalent levels of ventilation, the diaphragm generated higher passive pressure but moved significantly less during incremental cycle ergometry compared with progressive hypercapnia. Diaphragm excursion velocity and power output did not differ between the two tasks. These findings imply that the power output of the diaphragm during stabilising tasks involving the lower limbs may be preserved via coordinated changes in contractile shortening. Activity of key respiratory muscles, such as the diaphragm, must balance the demands of ventilation with the maintenance of stable posture. Our aim was to test whether the stabilising function of the diaphragm would be altered differentially in response to involuntary augmented breaths induced with or without lower-limb movements. Ten healthy volunteers (age 21 (2) years; mean (SD)) performed progressive CO2 -rebreathe (5% CO2 , 95% O2 ) followed 20min later by incremental cycle exercise (15-30W/min), both in a semi-recumbent position. Ventilatory indices, intrathoracic pressures and ultrasonographic measures of diaphragm shortening were assessed before, during and after each task. From rest to iso-time, inspiratory tidal volume and minute ventilation increased two- to threefold. At equivalent levels of tidal volume and minute ventilation, mean inspiratory transdiaphragmatic pressure ( ) was consistently higher during exercise compared with CO2 -rebreathe due to larger increases in gastric pressure and the passive component of (i.e., mechanical output due to static contractions), and yet diaphragm excursion was consistently lower. This lower excursion during exercise was accompanied by a reduction in excursion time with no difference in the active component of . Consequently, the rates of increase in excursion velocity (excursion/time) and power output (active × velocity) did not differ between the two tasks. In conclusion, the power output of the human diaphragm during dynamic lower-limb exercise appears to be preserved via coordinated changes in contractile shortening. The findings may have significance in settings where the ventilatory and stabilising functions of the diaphragm must be balanced (e.g., rehabilitation).

Augmented Core Breathing Pelvic Floor Muscle Training for Patients with Genitourinary Syndrome in Different Phases of Menopause

Pelvic floor muscle has a role in core breathing for twenty-four hours and offers an influence to modulate the lumbar spine and pelvic floor. Several studies reported on pelvic floor muscle strength. Women with genitourinary syndrome do not seek care for their condition and are dismayed to speak with the health care provider about the condition. In the present work, we evaluated the efficiency of augmented core breathing in the genitourinary syndrome of menopause for pelvic floor muscle strength. We tested the role of augmented core breathing efficacy using pelvic floor muscle training in patients with the genitourinary syndrome. Our data revealed significant beneficial effects of core breathing with pelvic floor muscle training and Kegel’s exercise in patients with genitourinary syndrome in different phase of menopause. KEY WORDS: genitourinary syndrome; menopause; pelvic exerciser; pelvic floor muscle training; Utian quality of life scale.

Unilateral vagotomy alters astrocyte and microglial morphology in the nucleus tractus solitarii of the rat.

The nucleus tractus solitarii (nTS) is the initial site of integration of sensory information from the cardiorespiratory system and contributes to reflex responses to hypoxia. Afferent fibers of the bilateral vagus nerves carry input from the heart, lungs, and other organs to the nTS where it is processed and modulated. Vagal afferents and nTS neurons are integrally associated with astrocytes and microglia that contribute to neuronal activity and influence cardiorespiratory control. We hypothesized that vagotomy would alter glial morphology and cardiorespiratory responses to hypoxia. Unilateral vagotomy (or sham surgery) was performed in rats. Prior to and seven days after surgery, baseline and hypoxic cardiorespiratory responses were monitored in conscious and anesthetized animals. The brainstem was sectioned and caudal, mid-area postrema (mid-AP), and rostral sections of the nTS were prepared for immunohistochemistry. Vagotomy increased immunoreactivity (-IR) of astrocytic glial fibrillary acidic protein (GFAP), specifically at mid-AP in the nTS. Similar results were found in the dorsal motor nucleus of the vagus (DMX). Vagotomy did not alter nTS astrocyte number, yet increased astrocyte branching and altered morphology. In addition, vagotomy both increased nTS microglia number and produced morphologic changes indicative of activation. Cardiorespiratory baseline parameters and hypoxic responses remained largely unchanged, but vagotomized animals displayed fewer augmented breaths (sighs) in response to hypoxia. Altogether, vagotomy alters nTS glial morphology, indicative of functional changes in astrocytes and microglia that may affect cardiorespiratory function in health and disease.

Astrocytes and Microglia in the Brainstem Nucleus Tractus Solitarii React to Unilateral Vagotomy

The nucleus tractus solitarii (nTS) of the brainstem plays an important role in cardiorespiratory homeostasis, including the reflex response to hypoxia. The nTS receives, integrates, and modulates sensory afferent input from the viscera. Alterations in afferent input and nTS function occur in many cardiorespiratory conditions, including obstructive sleep apnea, hypertension, and heart failure. The vagus nerves provide sensory input to the nTS. Vagal afferents release glutamate to activate nTS neurons as well as astrocytes, modifying nTS activity and cardiorespiratory output. However, the specific role of nTS astrocytes and other glia in cardiorespiratory modulation is unknown. We hypothesized that decreased afferent input (vagotomy) modifies astrocyte morphology and is associated with impaired cardiorespiratory responses to hypoxia. Male Sprague-Dawley rats (5-7 weeks old) underwent either right cervical vagotomy caudal to the nodose ganglion (n=9), sham surgery (n=8), or no surgery/control (n=8). Prior to surgery and one week post-surgery, cardiorespiratory parameters (respiration, heart rate, blood pressure, oxygen saturation) were recorded in conscious and anesthetized animals under baseline and hypoxic (8-12% O2) conditions. Rats were then euthanized and coronal brainstem sections from three areas of the cardiorespiratory nTS were examined using immunohistochemistry. Astrocytes were identified with glial fibrillary acidic protein (GFAP) and S100 calcium binding protein B (S100B), microglia with ionized calcium-binding protein 1 (IBA-1), and glutamatergic synapses with vesicular glutamate transporter 2 (VGLUT2). Immunoreactivity (-IR) was compared among experimental groups and between the manipulated and intact sides. At the level of the area postrema (mid-AP), vagotomy increased GFAP-IR compared to other groups, and on the manipulated compared to the intact side. Similar changes were seen in the dorsal motor nucleus of the vagus (DMX), where vagal efferent cell bodies are located. Astrocyte numbers were not altered by vagotomy, but Sholl analysis revealed that vagotomy increased astrocyte branching and altered branch patterns. Vagotomy also increased both IBA-1-IR and microglial numbers in the nTS (and DMX). Microglia in vagotomized animals exhibited decreased cell area and increased density, indicative of activation (analyzed with FracLac). VGLUT2-IR particle counts were not different among groups or between sides, suggesting a lack of glutamate synapse pruning resulting from vagotomy. In general, cardiorespiratory parameters under baseline and hypoxic conditions were not different among groups. However, vagotomy decreased the number of augmented breaths (sighs) during hypoxia. These data demonstrate that decreased afferent input via vagotomy produces both astrocyte reactivity (increased GFAP and altered branching) and microglial activation (increased numbers and amoeboid morphology). This likely indicates altered glial activity and contribution to nTS function and cardiorespiratory reflex responses. Further studies are needed to determine the specific contribution of glial changes in the brainstem to homeostasis and cardiorespiratory disease.

Continuous opioid administration in adult rats causes differential tolerance to develop in measures of respiratory, behavioral, and analgesic effects

Opioid drugs cause potent suppression of augmented breaths (ABs). Since many physiological effects of opioids demonstrate tolerance, in this study we sought to determine the temporal pattern and degree of any such tolerance in the suppression of ABs during 6 days of chronic opioid administration. Adult male rats (average 439.8 ± 19 g) received a continuous infusion of either saline (SAL, 0.01 ml/hr, n=5) or methadone (MET, 6 mg/kg/day, n=5) over 6 days via subcutaneously implanted osmotic pumps (Alzet, model 2ML1). Another control group received the cannabinoid receptor agonist WIN 55,212‐2 (WIN, 40 mg/kg/day, n=5), as a means of examining the potential confounding effect of altered behavioral activity on variables of interest. Animals were monitored before surgical implantation (D0), and again on days 2, 4 and 6 post‐implant (D2, D4, D6, respectively). Respiratory variables were measured using whole‐body plethysmography, pain tolerance assessed via tail flick latency (TF), and rearing behavior recorded as an index of behavioral alteration. All outcome variables of interest were analyzed using a mixed model ANOVA and appropriate post‐hoc comparisons, with monitoring day being the within‐animal factor and experimental group being the between‐animal factor considered. We found no differences in baseline measurements between experimental groups. During treatment, MET animals experienced a powerful suppression of ABs (D0 = 6.2 ± 0.383 ABs/15 min, vs. D2 = 1.4 ± 0.447, p<0.001), with tolerance developing across D4 and D6 (3.0 ± 0.523, p<0.001 and 5.0 ± 0.469, p=0.083 vs D0, respectively). No suppression of ABs was observed in either SAL or WIN animals across D0‐D6. Rearing behavior was suppressed in MET (P<0.001 for D2‐D6 vs. D0) and WIN animals across D2‐D6 (P<0.001 for D2‐D6 vs. D0), but not in SAL. However, MET animals demonstrated tolerance in rearing behavior over the 6 days of treatment, increasing back towards baseline on D6 vs. D2, P<0.001). In contrast, WIN animal rearing behavior continued to decrease across D2‐D6 vs baseline. TF latency was prolonged in MET (D0 = 2.812± 0.246 s vs 11.520± 0.389 s, 10.796± 0.152 s, & 10.739± 0.281 s on D2, D4, D6 respectively, all p<0.001), and no significant tolerance was observed across 6 days of treatment in MET animals. TF latency was prolonged in WIN only on D2 vs D0 (6.162± 0.389 s vs 3.826± 0.246, P= 0.002) to a lesser degree than in the MET animals, with no effect observed in SAL animals (p<0.001). We conclude that continuous opioid administration causes profound suppression of ABs with significant tolerance developing towards 6 days of treatment. This tolerance has a temporal pattern dissociated from behavioral effects, and markedly dissimilar to that of the analgesic effect. WIN does not cause any significant alteration of AB production despite a progressively profound suppression of behavioral activity. We propose that the mechanisms of suppression of ABs during opioid treatment follows an opioid‐receptor‐mediated pathway that is distinct from those mediating either analgesic or behavioral effects. It is therefore promising that targeted adjunct therapies to prevent the suppression of ABs during opioid treatment may be developed, that spare therapeutically desirable analgesic effects.

Differential activation of the human costal and crural diaphragm during voluntary and involuntary breaths.

The diaphragm is the primary muscle that generates the negative intrathoracic pressure to drive inspiratory airflow. The diaphragm consists of two parts, the costal and crural portions, with different roles during inspiration in animals, particularly when the stimulus to breathe is increased. In this study, the neural drive to the costal and crural portions of the diaphragm was assessed in nine healthy participants [8 male, aged 32 ± 13 yr (mean ± SD)]. Inspiratory electromyographic activity (EMG) was recorded from the costal diaphragm by using an intramuscular electrode and from the crural diaphragm with a multipair gastroesophageal catheter. Participants performed voluntary augmented breaths at 120%, 140%, and 160% of their tidal volume and also underwent progressive hypercapnia to induce involuntary breathing. Irrespective of the task, the increase in crural activity (normalized to quiet breathing) was only ~60% of the increase in costal activity (slope: 0.56 ± 0.30, P < 0.001). The onset and peak timing of EMG activity was similar for the costal and crural diaphragm during quiet breathing. Thus, when stimulated by either a voluntary or involuntary drive to breathe above tidal volume, the neural drive to the diaphragm was greater to the costal than to the crural portion.NEW & NOTEWORTHY Simultaneous electromyographic recordings from the human costal and crural diaphragm during voluntary augmented breathing and involuntary rebreathing show that the increase in inspiratory crural diaphragm activity was ~60% of the increase in costal diaphragm activity. However costal to crural diaphragm activation did not differ between the two tasks. The dissociation in the amplitude of activation of the costal and crural diaphragm becomes apparent only as the drive to breathe increases above tidal breathing.

Respire: Virtual Reality Art with Musical Agent Guided by Respiratory Interaction

Respire is an immersive art piece that brings together three components: an immersive virtual reality (VR) environment, embodied interaction (via a breathing sensor) and a musical agent system to generate unique experiences of augmented breathing. The breathing sensor controls the user’s vertical elevation of the point of view under and over the virtual ocean. The frequency and patterns of breathing data guide the arousal of the musical agent, and the waviness of a virtual ocean in the environment. Respire proposes an intimate exploration of breathing through an intelligent mapping of breathing data to the parameters of visual and sonic environments.

Stimulatory Effect of 5-Hydroxytryptamine (5-HT) on Rat Capsaicin-Sensitive Lung Vagal Sensory Neurons via Activation of 5-HT3 Receptors.

5-hydroxytryptamine (5-HT) is an inflammatory mediator known to be released in lung. Capsaicin-sensitive lung vagal (CSLV) afferents function as a primary sensor for detecting chemical stimuli and produce consequent reflexes during lung inflammation. To characterize the effect of 5-HT on CSLV afferents, responses of cardiorespiratory reflexes and single-unit C-fiber afferents to right-atrial injections of 5-HT were investigated in anesthetized Sprague-Dawley rats. Bolus injection of 5-HT (8 μg/kg) caused an immediate augmented breath and apnea, accompanied by hypotension and bradycardia. These initial responses were then followed by a brief pressor response and a more sustained depressor response. After a perineural treatment of both cervical vagi with capsaicin to block the conduction of C fibers, 5-HT still triggered the augmented breath, but no longer evoked the apnea, bradycardia and hypotension, indicating an involvement of C-fiber activation. The remaining augmented breath induced by 5-HT after perineural capsaicin treatment was totally eliminated by vagotomy. To further study the effect of 5-HT on CSLV afferents, activities arising from these afferents were determined using the single-fiber recording technique. Right-atrial injection of 5-HT evoked an intense discharge in CSLV afferents in a dose-dependent manner. The highest dose of 5-HT (16 μg/kg) activated 79% (19/24) of CSLV afferents which were also sensitive to capsaicin (0.8 μg/kg). The pretreatment of tropisetron, a selective antagonist of the 5-HT3 receptor, completely blocked CSLV-afferents stimulation induced by 5-HT but did not affect that by capsaicin. Furthermore, a similar afferent response of CSLV afferents was mimicked by phenylbiguanide, a selective agonist of the 5-HT3 receptor. In isolated rat lung vagal C neurons, 5-HT induced intense calcium transients in a dose-dependent manner. The highest concentration (3 μM) of 5-HT activated 67% (18/27) of the CSLV neurons. The 5-HT-induced response was totally abolished by pretreatment of tropisetron. In conclusion, 5-HT exerts an intense stimulatory effect on lung C-fiber terminals mediated through an activation of the 5-HT3 receptor, which may contribute to the airway hypersensitivity under lung inflammation.

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.

Impact of Dietary Folate on Respiratory Recovery after Cervical SCI

While much is known regarding folate's role in neural development, little is known concerning its effects in the adult central nervous system (CNS), particularly following injury or trauma. There is some evidence that folate enhances spinal sensory axon regrowth into a peripheral nerve graft after dorsal column injury. Further, 6 weeks after thoracic spinal contusion, folate supplementation elicits small but significant improvements in locomotor function in rats. Although surveys of individuals with chronic spinal cord injury (SCI) report reduced dietary folate intake, the impact folate intake on functional recovery after SCI is not known. Since respiratory dysfunction is the leading cause of death after cervical SCI, we investigated the effects of folate supplementation and deprivation on respiratory motor function after spinal hemisection at C2 (C2Hx). We hypothesized that folate supplementation would improve, while folate deprivation would hinder breathing recovery after C2Hx. In adult male Sprague Dawley rats with C2Hx, we compared the effects of a: 1) normal diet (4mg/kg folate in chow), 2) normal diet with supplemental folate injections (80ug/kg/day), and 3) folate deprivation (0 mg/kg folate in chow). Animals received their respective diets beginning 1 month prior to injury, and continued through the study duration. Intraperitoneal folate injections in the supplemented group were initiated 3 days prior to C2Hx and continued daily for 2 weeks post‐injury. Bilateral diaphragm electromyography (EMG) recordings were made in urethane anesthetized spontaneously breathing rats at 2 and 8 weeks post‐injury during normoxia (arterial PO2 ~90mmHg, PCO2 ~45mmHg), maximal chemoreceptor stimulation (10.5% O2/7% CO2), spontaneous augmented breaths (sighs), and sustained airway occlusion (40 sec). We did not see any consistent folate effects on ipsilateral or contralateral diaphragm EMG function at either 2 or 8 weeks post injury (P&gt;0.05 for all). Sprouting of serotonergic projections near retrogradely labeled phrenic motoneurons were assessed via immunofluorescence, and quantification is in progress. Although we found no evidence that folate supplementation or deprivation impacts diaphragm function following C2Hx, this does not discount folate's potential role in mediating spinal respiratory motor plasticity or functional recovery in other models of CNS injury. For example, C2Hx severs all ipsilateral axons, with a &gt;1mm gap. Since axonal growth and synapse formation is an innately slow process, the duration of this study may not have been sufficient to observe functional recovery mediated via axonal regrowth. Further studies are needed to rule out any role for folate in functional recovery following cervical SCI.Support or Funding InformationMcKnight Brain Institute, NIH SPARC OT 2OD023854, K12 HD055929, NIH T32 HD043730 and HL69064This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Post-sigh sleep apneas in mice: Systematic review and data-driven definition.

Sleep apneas can be categorized as post-sigh (prevailing in non-rapid eye movement sleep) or spontaneous (prevailing in rapid eye movement sleep) according to whether or not they are preceded by an augmented breath (sigh). Notably, the occurrence of these apnea subtypes changes differently in hypoxic/hypercapnic environments and in some genetic diseases, highlighting the importance of an objective discrimination. We aim to: (a) systematically review the literature comparing the criteria used in categorizing mouse sleep apneas; and (b) provide data-driven criteria for this categorization, with the final goal of reducing experimental variability in future studies. Twenty-two wild-type mice, instrumented with electroencephalographic/electromyographic electrodes, were placed inside a whole-body plethysmographic chamber to quantify sleep apneas and sighs. Wake-sleep states were scored on 4-s epochs based on electroencephalographic/electromyographic signals. Literature revision showed that highly different criteria were used for post-sigh apnea definition, the intervals for apnea occurrence after sigh ranging from 1 breath up to 20 s. In our data, the apnea occurrence rate during non-rapid eye movement sleep was significantly higher than that calculated before the sigh only in the 1st and 2nd 4-s epochs following a sigh. These data suggest that, in mice, apneas should be categorized as post-sigh only if they start within 8 s from a sigh; the choice of shorter or longer time windows might underestimate or slightly overestimate their occurrence rate, respectively.

Ventilatory constraints influence physiological dead space in heart failure.

What is the central question of this study? The goal of this study was to investigate the effect of alterations in tidal volume and alveolar volume on the elevated physiological dead space and the contribution of ventilatory constraints thereof in heart failure patients during submaximal exercise. What is the main finding and its importance? We found that physiological dead space was elevated in heart failure via reduced tidal volume and alveolar volume. Furthermore, the degree of ventilatory constraints was associated with physiological dead space and alveolar volume. Patients who have heart failure with reduced ejection fraction (HFrEF) exhibit impaired ventilatory efficiency [i.e. greater ventilatory equivalent for carbon dioxide ( ) slope] and elevated physiological dead space (VD /VT ). However, the impact of breathing strategy on VD /VT during submaximal exercise in HFrEF is unclear. The HFrEF (n= 9) and control (CTL, n= 9) participants performed constant-load cycling exercise at similar ventilation ( ). Inspiratory capacity, operating lung volumes and arterial blood gases were measured during submaximal exercise. Arterial blood gases were used to derive VD /VT , alveolar volume, dead space volume, alveolar ventilation and dead space ventilation. During submaximal exercise, HFrEF patients had greater slope and VD /VT than CTL subjects (P= 0.01). At similar , HFrEF patients had smaller tidal volumes and alveolar volumes (HFrEF 1.11± 0.33litres versus CTL 1.66± 0.37 litres; both P≤ 0.01), whereas dead space volume was not different (P= 0.47). The augmented breathing frequency in HFrEF patients resulted in greater dead space ventilation compared with CTL subjects (HFrEF 15± 4lmin-1 versus CTL 10± 5lmin-1 ; P= 0.048). The HFrEF patients exhibited greater increases in expiratory reserve volume and lower inspiratory capacity (as a percentage of predicted) than CTL subjects (both P< 0.05), which were significantly related to VD /VT and alveolar volume in HFrEF patients (all P< 0.03). In HFrEF, the reduced tidal volume and alveolar volume elevate physiological dead space during submaximal exercise, which is worsened in those with the greatest ventilatory constraints. These findings highlight the negative consequences of ventilatory constraints on physiological dead space during submaximal exercise in HFrEF.

Neural mechanisms for sigh generation during prenatal development.

The respiratory network of the preBötzinger complex (preBötC), which controls inspiratory behavior, can in normal conditions simultaneously produce two types of inspiration-related rhythmic activities: the eupneic rhythm composed of monophasic, low-amplitude, and relatively high-frequency bursts, interspersed with sigh rhythmic activity, composed of biphasic, high-amplitude, and lower frequency bursts. By combining electrophysiological recordings from transverse brainstem slices with computational modeling, new advances in the mechanisms underlying sigh production have been obtained during prenatal development. The present review summarizes recent findings that establish when sigh rhythmogenesis starts to be produced during embryonic development as well as the cellular, membrane, and synaptic properties required for its expression. Together, the results demonstrate that although generated by the same network, the eupnea and sigh rhythms have different developmental onset times and rely on distinct network properties. Because sighs (also known as augmented breaths) are important in maintaining lung function (by reopening collapsed alveoli), gaining insight into their underlying neural mechanisms at early developmental stages is likely to help in the treatment of prematurely born babies often suffering from breathing deficiencies.

Hypoxia induced tightly coupled augmented bursts in phrenic and splanchnic sympathetic nerve activity are eliminated by vagotomy and uncoupled by blockade of the paraventricular nucleus of the hypothalamus

Previous experiments in conscious rats verified that hypoxia decreased mean arterial pressure (MAP), increased heart rate (HR) and increased frequency (F) and amplitude (amp) of diaphragm EMG (dia EMG). In addition to increased eupneic breathing, regularly spaced high amp, longer duration bursts in dia EMG were indicative of augmented breaths. In a separate group of conscious rats, hypoxia‐induced increases in integrated splanchnic sympathetic nerve activity (SSNA) included high amp bursts of SSNA at a frequency similar to that of augmented breaths (4 – 6 bursts/min). Current experiments in inactin anesthetized (100 mg/kg) male Sprague Dawley rats evaluated, in the same animal, the relationship between hypoxic responses in phrenic (Ph) nerve activity (PNA) and SSNA and the role of vagal afferent nerves (n = 6) and the hypothalamic paraventricular nucleus (PVN, n = 3) in augmented bursts. Inspired O2 concentration (nose cone) in freely breathing rats was initially 100% (Control, Con). To produce hypoxia, inspired O2 was slowly decreased while measuring arterial O2 saturation (sat; Mouse Ox foot clip) until PNA stabilized with regularly spaced augmented bursts. PNA and SSNA were recorded during 3 states: O2 sat levels of 100 ±2% (Control, con); 46 ± 2 % (Hypoxia, Hx); and 41 ± 2% (hypoxia after vagotomy, Hx‐VX), with recovery periods at 100% inspired O2 in between. During each of the 3 states, MAP was increased (i.v. phenylephrine, 5 μg/kg) equivalently (+48 ± 2; +52 ± 2; +48 ± 8 mmHg, respectively) and arterial baroreflex decreases in HR (−19 ± 3; −10 ±3; −17 ± 7 beats/min) and SSNA (−72 ± 4; −76 ± 10; −86 ± 24 %) were similar among the 3 states. Consistent with efferent and afferent denervation, VX alone increased HR (+ 21 ± 6 beats/min), SSNA (+69 ± 7 %), and PNA (+21 ± 9 %, P = 0.06), and decreased Ph F (−13 ± 4 breaths/min). Before and after VX, Hx decreased MAP (Hx, −47 ±5 mmHg; Hx‐VX, −60 ± 3mmHg ) and increased HR (Hx, +52 ±11; Hx‐VX, +30 ± 8 beats/min ); SSNA (Hx, +31 ± 10; Hx‐VX, +54 ± 11 %); PNA (P = 0.15; HxCon, +34 ± 16; HxVX, +32 ±22%); and Ph‐F (Hx, +17 ± 3; Hx‐VX, +14 ± 4). Augmented bursts were rare in the control state (100% O2). Prior to vagotomy, during Hx high amp bursts in SSNA (148 ± 12% of adjacent) coincided with high amp bursts in PNA (188 ±16% of adjacent) at a frequency of 5 ± 1/min and were eliminated by vagotomy. In 3 additional rats the PVN was first blocked with muscimol (2mM, 60 nl bilaterally) and the protocol repeated. Although high amplitude bursts in PNA were observed during Hx (~ 2/min) and were eliminated by VX, bursts in SSNA were not tightly coupled with bursts in PNA in 2 of the 3 rats with PVN muscimol injections. The tight correlation between high amplitude bursts in PNA and SSNA during hypoxia suggests CNS integration in initiating augmented responses in autonomic and respiratory efferent nerves. Vagal afferents, likely from pulmonary stretch receptors, are essential for initiating augmented discharge of the phrenic nerve during hypoxia and preliminary data suggests that integration within the PVN contributes to coupling of augmented phrenic bursts with sympathetic outflow.Support or Funding InformationNIH R01 HL98602This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Spontaneous respiratory plasticity following unilateral high cervical spinal cord injury in behaving rats

Unilateral cervical C2 hemisection (C2Hx) is a classic model of spinal cord injury (SCI) for studying respiratory dysfunction and plasticity. However, most previous studies were performed under anesthesia, which significantly alters respiratory network. Therefore, the goal of this work was to assess spontaneous diaphragm recovery post-C2Hx in awake, freely behaving animals. Adult rats were chronically implanted with diaphragm EMG electrodes and recorded during 8 weeks post-C2Hx. Our results reveal that ipsilateral diaphragm activity partially recovers within days post-injury and reaches pre-injury amplitude in a few weeks. However, the full extent of spontaneous ipsilateral recovery is significantly attenuated by anesthesia (ketamine/xylazine, isoflurane, and urethane). This suggests that the observed recovery may be attributed in part to activation of NMDA receptors which are suppressed by anesthesia. Despite spontaneous recovery in awake animals, ipsilateral hemidiaphragm dysfunction still persists: i) Inspiratory bursts during basal (slow) breathing exhibit an altered pattern, ii) the amplitude of sighs – or augmented breaths – is significantly decreased, and iii) the injured hemidiaphragm exhibits spontaneous events of hyperexcitation. The results from this study offer an under-appreciated insight into spontaneous diaphragm activity and recovery following high cervical spinal cord injury in awake animals.

Compensatory plasticity in diaphragm and intercostal muscle utilization in a rat model of ALS

In SOD1G93A transgenic rat model of ALS, breathing capacity is preserved until late in disease progression despite profound respiratory motor neuron (MN) cell death. To explore mechanisms preserving breathing capacity, we assessed inspiratory EMG activity in diaphragm and external intercostal T2 (EIC2) and T5 (EIC5) muscles in anesthetized SOD1G93A rats at disease end-stage (20% decrease in body mass). We hypothesized that despite significant phrenic motor neuron loss and decreased phrenic nerve activity, diaphragm electrical activity and trans-diaphragmatic pressure (Pdi) are maintained to sustain ventilation. We alternatively hypothesized that EIC activity is enhanced, compensating for impaired diaphragm function. Diaphragm, EIC2 and EIC5 muscle EMGs and Pdi were measured in urethane-anesthetized, spontaneously breathing female SOD1G93A rats versus wild-type littermates during normoxia (arterial PO2 ~90mmHg, PCO2 ~45mmHg), maximal chemoreceptor stimulation (MCS: 10.5% O2/7% CO2), spontaneous augmented breaths and sustained tracheal occlusion. Phrenic MNs were counted in C3-5; T2 and T5 ventrolateral MNs were counted. In end-stage SOD1G93A rats, 29% of phrenic MNs survived (vs. wild-type), yet integrated diaphragm EMG amplitude was normal. Nevertheless, maximal Pdi decreased ~30% vs. wild type (p<0.01) and increased esophageal to gastric pressure ratio (p<0.05), consistent with persistent diaphragm weakness. Despite major T2 and T5 MN death, integrated EIC2 (100% greater than wild type) and EIC5 (300%) EMG amplitudes were increased in mutant rats during normoxia (p<0.01), possibly compensating for decreased Pdi. Thus, despite significant phrenic MN loss, diaphragm EMG activity is maintained; in contrast, Pdi was not, suggesting diaphragm dysfunction. Presumably, increased EIC EMG activity compensated for persistent diaphragm weakness. These adjustments contribute to remarkable preservation of breathing ability despite major respiratory motor neuron death and diaphragm dysfunction.

Effects of the hippocampus on the motor expression of augmented breaths.

Augmented breaths, also known as sighs, constitute the normal repertoire of breathing in freely behaving humans and animals. The breaths are believed to be generated by neurones in the preBötzinger complex but under modulatory influence from higher brain centres, particularly in the limbic system due to the strong correlations between the expression of emotional behaviours such as anxiety and the occurrence of augmented breaths. The current study examines the role of the hippocampus in the motor expression of augmented breaths, and also examines the characteristics of eupneic breaths surrounding a sigh before and after stimulating the hippocampus in urethane anaesthetised Sprague-Dawley rats. Neurochemical microstimulation using the excitatory amino acid, D,L-Homocysteic acid, was used to locate areas in the hippocampus with the potential to modulated the motor expression of augmented breaths. The CA1 neurone cluster of the ventral hippocampus was found to completely suppress the expression of augmented breaths without affecting the intrinsic properties of the breaths. A similar neurone cluster, but in the dorsal field of the hippocampus, was also investigated and found to have no effects over the expression of augmented breaths. The data supports the hypothesis that there is a structural or functional relationship between neurones of the ventral hippocampus and brainstem nuclei that control augmented breaths. The implications of these findings in the context of behaviours are discussed but with due consideration of experimental conditions.

The effects of five clinically relevant narcotic drugs on the generation of spontaneous augmented (sigh) breaths.

The prototypical μ‐opioid agonist, morphine, powerfully suppresses spontaneous augmented breaths (ABs). We previously found that this effect occurs at relatively low‐to‐moderate dosages, where traditional parameters of eupneic breathing (VT and f) are not overtly affected. Based upon the heterogeneity of pharmacological properties among commonly prescribed opioid medications, we hypothesized that the suppression of ABs would be significantly different in the presence of opioid drugs other than morphine. To test this, we studied 6 adult male rats (534±47g, range=402–637g) using a randomized cross‐over repeated‐measures design to evaluate the effects of 5 common narcotics on the rate of ABs during spontaneous breathing in unanesthetized and unrestrained adult male rats, monitored using hybrid sealed‐chamber and open‐flow plethysmography. Drugs were administered via s.c. injection at the following doses: Morphine: 0, 1.0, 2.0, 3.0 mg/kg; Fentanyl: 0, 0.02, 0.03, 0.04 mg/kg; Methadone: 0, 1.0, 2.5, 4.0 mg/kg; Buprenorphine: 0, 0.01, 0.03, 0.05 mg/kg; Oxycodone: 0, 0.5, 1.0, 1.5 mg/kg (0 mg/kg = saline sham dose). All animals received all drugs and dosages in randomized order. Doses were chosen to achieve a range up to and including analgesic effectiveness to prolong tail flick latency 2‐to‐3 fold over the sham injection. A tail flick analgesiometer was used to evaluate tail flick latency 15‐minutes post‐injection, followed by respiratory monitoring in ambient room air conditions for 30 minutes. Each animal completed a trial involving one dosage of one drug on a given day, with at least 48 hours between consecutive sessions. Drugs did not depress tidal volume or frequency of eupneic breathing. The baseline number of ABs observed following control injections for each drug condition were not different, ranging from 6.5 ± 1.4 to 7.3 ± 1.6 ABs/15mins, p=0.912. Each drug tested significantly decreased the number of spontaneous ABs in a dose‐dependent manner. The number of ABs observed at the highest drug dosages were 1.5 ± 1.3 (mor), 1.3 ± 1.0 (fen), 0.8 ± 1.2 (met), 0.0 ± 0.0 (bup), and 1.7 ± 1.2 (oxy) ABs/15mins (p&lt;0.001 for all changes vs sham). The relationship between ABs and analgesic effect (i.e. %increase in tail flick latency vs. % of ABs remaining in the breathing rhythm) followed a 2‐parameter single exponential decay model. Significant relationships were identified between analgesic effect and suppression of ABs for all drugs tested (p&lt;0.05) with the exception of oxycodone wherein the relationship narrowly failed to reach statistical significance (p=0.058). The strongest relationship existed within the buprenorphine condition (rs=.764, p&lt;0.001). Buprenorphine also yielded the lowest analgesic effect required to produce a 50% suppression in ABs (observed at 16% prolongation in tail flick latency). Our results demonstrate that the suppression of ABs is a ubiquitous side effect of common narcotic drugs across the low‐moderate analgesic range, and is therefore unlikely to be mitigated by selective avoidance of specific opioids/opiates. Further, the unique potency of buprenorphine in suppressing ABs suggests that complex opioid receptor interactions may be of key mechanistic importance in mediating this effect.Support or Funding InformationCK was supported by a Summer Scholars Research Program Award through the CMU College of Medicine. HB received research support from an Early Career Award through the Office of Research and Sponsored Programs at CMU, and operating funds from the CMU College of Medicine.

Increased ventilatory variability and complexity in patients with hyperventilation disorder.

It has been hypothesized that hyperventilation disorders could be characterized by an abnormal ventilatory control leading to enhanced variability of resting ventilation. The variability of tidal volume (VT) often depicts a nonnormal distribution that can be described by the negative slope characterizing augmented breaths formed by the relationship between the probability density distribution of VT and VT on a log-log scale. The objectives of this study were to describe the variability of resting ventilation [coefficient of variation (CV) of VT and slope], the stability in respiratory control (loop, controller and plant gains characterizing ventilatory-chemoresponsiveness interactions) and the chaotic-like dynamics (embedding dimension, Kappa values characterizing complexity) of resting ventilation in patients with a well-defined dysfunctional breathing pattern characterized by air hunger and constantly decreased PaCO2 during a cardiopulmonary exercise test. Compared with 14 healthy subjects with similar anthropometrics, 23 patients with hyperventilation were characterized by increased variability of resting tidal ventilation (CV of VT median [interquartile]: 26% [19-35] vs. 36% [28-48], P = 0.020; slope: -6.63 [-7.65; -5.36] vs. -3.88 [-5.91; -2.66], P = 0.004) that was not related to increased chemical drive (loop gain: 0.051 [0.039-0.221] vs. 0.044 [0.012-0.087], P = 0.149) but that was related to an increased ventilatory complexity (Kappa values, P < 0.05). Plant gain was decreased in patients and correlated with complexity (with Kappa 5 - degree 5: Rho = -0.48, P = 0.006). In conclusion, well-defined patients suffering from hyperventilation disorder are characterized by increased variability of their resting ventilation due to increased ventilatory complexity with stable ventilatory-chemoresponsiveness interactions.