Unrestrained Whole-body Plethysmography Research Articles

Adapting Real-Time Lung Function Measurements for SARS-CoV-2 Infection Studies in Syrian Hamsters.

Pulmonary function examinations are critical to assess respiratory disease severity in patients. In preclinical rodent models of viral respiratory infections, however, disease is frequently evaluated based on virological, pathological and/or surrogate clinical parameters, which are not directly associated with lung function. To bridge the gap between preclinical and clinical readouts, we aimed to apply unrestrained whole-body plethysmography (WBP) measurements in a SARS-CoV-2 Syrian hamster challenge model. While WBP measurements are frequently used for preclinical research in mice and rats, results from studies in hamsters are still limited. During unrestrained WBP measurements, we obtained highly variable breathing frequency values outside of the normal physiological range for hamsters. Importantly, we observed that animal movements were recorded as breaths during WBP measurements. By limiting animal movement through either mechanical or chemical restraint, we improved the reliability of the lung function readout and obtained breathing frequencies that correlated with clinical signs when comparing two different variants of SARS-CoV-2 post-inoculation. Simultaneously, however, new sources of experimental variation were introduced by the method of restraint, which demands further optimalization of WBP measurements in Syrian hamsters. We concluded that WBP measurements are a valuable refinement either in combination with video recordings or if average values of measurements lasting several hours are analyzed.

Examining the relationship between Locus coeruleus (LC) activity and breathing in response to chemical exposure

Objective: The control of breathing must continuously adapt in response to homeostatic changes. This is particularly important in the context of chemical exposure to substances such as opioids and anesthetic gases known to suppress the drive for breathing. While these agents suppress breathing through different modalities, how they affect neuromodulatory networks capable of influencing breathing remains unknown. This ongoing study aims to understand how exposure to the ultrapotent synthetic opioid (UPSO), fentanyl, and the anesthetic gas, isoflurane, affect the relationship between LC activity and breathing. Hypothesis: We hypothesize that fentanyl and isoflurane have divergent effects on the relationship between LC activity and breathing. Methods: Measurements were made using unrestrained whole-body plethysmography and fiber photometry in NET-cre positive mice expressing GCaMP8s while breathing room air (FiO2=21%) before and following the administration of either fentanyl (0.7 mg/kg i.p.) or exposure to isoflurane (1.5% inhaled). Post-hoc analyses were conducted using MATLAB. Results: Our analyses showed that awake mice (N=8) exhibited two types of LC activity. Type 1 LC activity coincided with high frequency respiratory transients (HFRTs); whereas Type 2 activity did not appear to coincide with changes in breathing. Both fentanyl (N=5) and isoflurane (N=3) suppressed breathing with a greater degree occurring with the UPSO. Furthermore, the frequency of LC activity increased during opioid-induced respiratory depression (OIRD) where both types of LC activity were evident. Isoflurane anesthetized mice showed decreased Type 1 LC activity and a reduction in the duration of Type 2 activity while breathing either room air or hypoxia (FiO2=10%). Conclusion: While isoflurane suppressed Type 1 LC activity in room air and hypoxia, fentanyl which caused a larger suppression in breathing, preserved Type 1 LC activity. These differential effects suggest that the contribution of LC activity to breathing fundamentally changes when exposed to either anesthesia or fentanyl, which may be relevant to understanding the physiological consequences of exposure to either agent. This work was supported by NIH: R01HL163965, R01DA057767. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Fourteen-day E-Cigarette Exposure Disrupts Ventilation Patterns and Serum IL-1B Levels in Adolescent Rats

E-cigarettes are devices used to deliver vaporized liquids often containing nicotine and other chemicals. The purpose of this study was to investigate the effects of 14 days of e-cigarette vapor exposure in adolescent rats on lung function and inflammatory cytokine expression. Seventeen male long-evans rats were assigned to vape or air groups. The animals were exposed to either air or 5% nicotine vapor using a whole-body exposure chamber, once a day for ten minutes for fourteen consecutive days. Ventilation recordings were completed on day 0 (before exposure) and day 15 (after exposure) using unrestrained whole-body plethysmography. Both lung tissue and blood was collected for molecular assays. Baseline and post-exposure ventilation data were compared between air and vape groups across three different parameters: frequency, tidal volume, and minute ventilation. These parameters were compared resulting in three distinct 2x2 (time x treatment) Mixed Model ANOVAs. Between baseline and post-treatment measurements there was a significant decrease in values in minute ventilation (p=0.0312) and tidal volume (p=0.0031). Between treatment groups there was a significant difference in minute ventilation (p=0.0128) and frequency (p=0042). There were also significant alterations to the presence of IL-1B in the serum (p=0.0003), but not lung tissue (p=0.6525). In conclusion, following two weeks of e-cigarette vapor exposure, ventilation patterns were altered in the vapor exposed animals which decreased in tidal volume and minute ventilation suggesting possible impairment of lung function and e-cigarette exposure modified the expression of pro-inflammatory cytokine, IL-1B.

Aminopyridines Restore Ventilation and Reverse Respiratory Acidosis at Late Stages of Botulism in Mice.

Botulinum neurotoxin (BoNT) is a potent protein toxin that causes muscle paralysis and death by asphyxiation. Treatments for symptomatic botulism are intubation and supportive care until respiratory function recovers. Aminopyridines have recently emerged as potential treatments for botulism. The clinically approved drug 3,4-diaminopyridine (3,4-DAP) rapidly reverses toxic signs of botulism and has antidotal effects when continuously administered in rodent models of lethal botulism. Although the therapeutic effects of 3,4-DAP likely result from the reversal of diaphragm paralysis, the corresponding effects on respiratory physiology are not understood. Here, we combined unrestrained whole-body plethysmography (UWBP) with arterial blood gas measurements to study the effects of 3,4-DAP, and other aminopyridines, on ventilation and respiration at terminal stages of botulism in mice. Treatment with clinically relevant doses of 3,4-DAP restored ventilation in a dose-dependent manner, producing significant improvements in ventilatory parameters within 10 minutes. Concomitant with improved ventilation, 3,4-DAP treatment reversed botulism-induced respiratory acidosis, restoring blood levels of CO2, pH, and lactate to normal physiologic levels. Having established that 3,4-DAP-mediated improvements in ventilation were directly correlated with improved respiration, we used UWBP to quantitatively evaluate nine additional aminopyridines in BoNT/A-intoxicated mice. Multiple aminopyridines were identified with comparable or enhanced therapeutic efficacies compared with 3,4-DAP, including aminopyridines that selectively improved tidal volume versus respiratory rate and vice versa. In addition to contributing to a growing body of evidence supporting the use of aminopyridines to treat clinical botulism, these data lay the groundwork for the development of aminopyridine derivatives with improved pharmacological properties. SIGNIFICANCE STATEMENT: There is a critical need for fast-acting treatments to reverse respiratory paralysis in patients with botulism. This study used unrestrained, whole-body plethysmography and arterial blood gas analysis to show that aminopyridines rapidly restore ventilation and respiration and reverse respiratory acidosis when administered to mice at terminal stages of botulism. In addition to supporting the use of aminopyridines as first-line treatments for botulism symptoms, these data are expected to contribute to the development of new aminopyridine derivatives with improved pharmacological properties.

A Single 10-Minute E-cigarette Vapor Exposure Reduces Tidal Volume and Minute Ventilation in Normoxia and Normobaric Hypoxia in Adult Rats.

The present study investigated the effects of a single 10-minute exposure to e-cigarette vapor on ventilation in adult male Long-Evans rats. Ventilation was recorded using awake, unrestrained whole-body plethysmography. Baseline recordings were taken the day before full-body exposure to either room air (n = 9; air control group) or e-cigarette vapor (n = 9; treatment group). Post-exposure recordings were taken immediately after the 10-minute room air or vapor exposure. As part of the ventilation protocol, in addition to recording the subject's ventilation in room air, the subjects were also exposed to 10% oxygen (balanced with nitrogen) to assess the effects of e-cigarette vapor on an increased drive to breathe. Ventilation data were analyzed using a 2x2x2 mixed-model ANOVA measuring treatment (vape vs. air) x time (baseline vs. post-treatment) x condition (normoxia vs. hypoxia) for breathing frequency, tidal volume, and minute ventilation. Breathing frequency increased in both treatment groups (air and vape) with exposure to normobaric hypoxia (p < 0.001), with no effect of time (baseline vs. post-treatment) for either group. Tidal volume increased in both treatment groups (air and vape) with exposure to normobaric hypoxia (p < 0.001), and an effect of time (baseline vs. post-treatment) was observed (p = 0.010) for the vape group. Minute ventilation increased in both treatment groups (air and vape) with exposure to normobaric hypoxia (p < 0.001), and an effect of time (baseline vs. post-treatment) was observed (p < 0.001) for the vape group. In conclusion, immediately following a single 10-minute e-cigarette vapor exposure, both tidal volume and minute ventilation were reduced during normoxia and normobaric hypoxia, indicating a decrease in ventilation after a single 10-minute e-cigarette vapor exposure. Furthermore, this exposure also blunted the physiological response to acute hypoxia exposure. Subjects in the vape group, while breathing more rapidly as expected, experienced shallower breathing than the air group during hypoxia. The findings in this study confirm that vaping could result in reduced lung function.

Modulation of opioid overdose susceptibility due to context-dependent tolerance

The US is in the midst of an opioid epidemic, which is primarily being driven by repeat opioid use (ROU). The primary cause of opioid overdose is opioid-induced respiratory depression (OIRD). It has been shown that the risk of overdosing, and subsequently death, can change depending on the environment in which the user takes the drug. This phenomenon is called context-dependent tolerance. However, how OIRD changes depending on the location of drug delivery is not known. The objective of this pilot study is to investigate context-dependent respiratory tolerance to OIRD and overdose. We repeatedly paired an injection of fentanyl (0.7 mg/kg/day i.p.) with a distinctive environmental context (fentanyl-paired (FP) environment) and saline with another distinctive context (saline-paired (SP) environment) in adult mice for five days while monitoring breathing via unrestrained whole-body plethysmography. The ROU protocol was followed by two test days where fentanyl was administered in both environments and breathing was measured. We found that minute ventilation was greater during OIRD in the FP environment as compared to the SP environment (n=4). In the second set of mice (n=6?), we tested overdose susceptibility following ROU by administering a high dose of fentanyl (5mg/kg) in both contexts. In contrast to the FP environment where no mice overdosed (n=0/6), n=4/6 mice overdosed in the SP environment. These data suggest a greater susceptibility to OIRD and overdose when in an environment not previously paired with fentanyl administration. This context-dependent effect on OIRD may be a significant psychophysiological factor driving opioid-related deaths in the ongoing opioid epidemic. NIH: R01NS107421; R01HL163965; R01DA057767. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Pinus sylvestris L. and Syzygium aromaticum (L.) Merr. & L. M. Perry Essential Oils Inhibit Endotoxin-Induced Airway Hyperreactivity despite Aggravated Inflammatory Mechanisms in Mice.

Scots pine (SO) and clove (CO) essential oils (EOs) are commonly used by inhalation, and their main components are shown to reduce inflammatory mediator production. The aim of our research was to investigate the chemical composition of commercially available SO and CO by gas chromatography–mass spectrometry and study their effects on airway functions and inflammation in an acute pneumonitis mouse model. Inflammation was evoked by intratracheal endotoxin and EOs were inhaled three times during the 24 h experimental period. Respiratory function was analyzed by unrestrained whole-body plethysmography, lung inflammation by semiquantitative histopathological scoring, myeloperoxidase (MPO) activity and cytokine measurements. α-Pinene (39.4%) was the main component in SO, and eugenol (88.6%) in CO. Both SO and CO significantly reduced airway hyperresponsiveness, and prevented peak expiratory flow, tidal volume increases and perivascular edema formation. Meanwhile, inflammatory cell infiltration was not remarkably affected. In contrast, MPO activity and several inflammatory cytokines (IL-1β, KC, MCP-1, MIP-2, TNF-α) were aggravated by both EOs. This is the first evidence that SO and CO inhalation improve airway function, but enhance certain inflammatory parameters. These results suggest that these EOs should be used with caution in cases of inflammation-associated respiratory diseases.

Reduced expression of SOD2 in the Retrotrapezoid nucleus is associated with enhanced ventilatory chemoreception: implications for breathing disorders in heart failure

Enhanced chemoreflex drive and irregular breathing patterns are both hallmarks in heart failure (HF). The Retrotrapezoid nucleus (RTN), a main area for central chemoreception, contribute to maintain both heightened chemoreflex drive and irregular breathing in experimental HF. Recently, we found that high levels of reactive oxygen species (ROS) within the RTN of HF rats were closely linked to the enhanced central chemoreflex and the generation of breathing disorders. Despite this evidence, the effects of ROS on RTN‐mediated chemoreception and the precise mechanism(s) associated with ROS generation in the RTN has not been previously studied. However, pro/anti‐oxidant enzymes gene screening in RTN from HF rats revealed reduce expression of super oxide dismutase 2 (SOD2). Therefore, in the present study we aimed to determine whether reductions in SOD2 expression in healthy subjects resembles i) high RTN ROS levels, ii) heightened chemoreflex drive, and iii) generation of breathings disorders observed in HF animals. Accordingly, breathing function and central chemoreflex drive were assessed in freely moving heterozygous SOD2+/‐ mice by unrestrained whole‐body plethysmography. ROS levels were determined in brainstem sections containing the RTN using dihydroethidium staining. SOD2 protein expression and tissue localization were determined by immunoblot and immunofluorescence, respectively. SOD2+/‐ mice displayed reduced SOD2 protein expression (65.6 ± 3.2 vs. 100.0 ± 5.8 %; SOD2+/‐ vs. WT, respectively) and high ROS levels at the RTN compared to WT littermates (2.50 ± 0.46 vs. 1.00 ± 0.05 a.u.; SOD2+/‐ vs. WT, respectively). In addition, SOD2+/‐mice displayed an exacerbated ventilatory response to hypercapnia compared to WT mice (3.5 ± 0.2 vs. 1.9 ± 0.2 ml/min/10g; SOD2+/‐ vs. WT, respectively). Importantly, the latter was closely linked to RTN ROS production since a positive correlation (p&lt;0.05) between ROS levels and the hypercapnic ventilatory response was found only in SOD2+/‐animals. Furthermore, SOD2+/‐ mice showed overt signs of disordered breathing while resting at eupneic conditions compared to WT mice as evidenced by large increases in ventilatory instability and apneas/hypoapneas incidence (AHI: 7.7 ± 0.9 vs. 3.4 ± 0.6 events/hr; SOD2+/‐ vs. WT, respectively). Lastly, we found that SOD2 expression is preferentially confined to RTN neurons rather than to medullary astrocytes. Our results shows that reductions in the SOD2 gene expression in healthy rodents mimic the respiratory phenotype observed in experimental HF and suggests that reduced expression of SOD2 within the RTN of HF animals may account for the local increases in ROS production, the enhanced central chemoreflex drive and the generation/maintenance of irregular breathing patterns.

A Model of Repeated Opioid Use Producing Tolerance to Opioid Induced Respiratory Depression

Repeated opioid users, such as those who suffer from opioid use disorder, are among the most vulnerable to overdose, overdose‐related death, and opioid‑related morbidities in breathing in the ongoing epidemic. Despite the significant progress made in defining the cellular and circuit basis by which opioid induced respiratory depression (OIRD) occurs, very little is known about the impact that repeated opioid use (ROU) has on the control of breathing. This ongoing work aims to define how ROU impacts the OIRD. We developed a mouse model of ROU that involved six consecutive days of fentanyl (0.7mg/kg via i.p. injection) administration. Breathing was measured via unrestrained whole‐body plethysmography prior to and immediately after fentanyl administration on Day 1 and Day 6. Prior to fentanyl administration, tidal volume (TV), respiratory rate (RR) and minute ventilation (Vmin) were similar on Day 1 and Day 6 (n= 7 mice). Peak depression of TV during fentanyl was similar on Day 1 and Day 6 (n=7, P=0.28); whereas, peak depression of RR (Day 1: 145.5±19.66­BPM vs. Day 6: 172.0 ± 18.89,P=0.005) and Vmin (Day 1: 142.0±30.23­microL•min‐1•g‐1 vs. Day 6: 210.1±21.96­microL•min‐1•g‐1, P=0.04) during fentanyl was greater on Day 1 (n= 6 of 7). Moreover, the change in body temperature during OIRD was not different between Day 1 and Day 6 (Day 1: 0.04±0.021oC vs. Day 6: 0.64 ± 0.021oC, P=0.62). Together these data suggest that tolerance to OIRD emerges following six days of ROU and does not involve significant changes in body temperature. The data from this ongoing study provides foundational knowledge to the impact of ROU on breathing and introduces a novel model of ROU that leads to tolerance to OIRD.

Effects of Thymus vulgaris L., Cinnamomum verum J.Presl and Cymbopogon nardus (L.) Rendle Essential Oils in the Endotoxin-induced Acute Airway Inflammation Mouse Model.

Thyme (TO), cinnamon (CO), and Ceylon type lemongrass (LO) essential oils (EOs) are commonly used for inhalation. However, their effects and mechanisms on inflammatory processes are not well-documented, and the number of in vivo data that would be important to determine their potential benefits or risks is low. Therefore, we analyzed the chemical composition and investigated the activity of TO, CO, and LO on airway functions and inflammatory parameters in an acute pneumonitis mouse model. The components of commercially available EOs were measured by gas chromatography–mass spectrometry. Airway inflammation was induced by intratracheal endotoxin administration in mice. EOs were inhaled during the experiments. Airway function and hyperresponsiveness were determined by unrestrained whole-body plethysmography on conscious animals. Myeloperoxidase (MPO) activity was measured by spectrophotometry from lung tissue homogenates, from which semiquantitative histopathological scores were assessed. The main components of TO, CO, and LO were thymol, cinnamaldehyde, and citronellal, respectively. We provide here the first evidence that TO and CO reduce inflammatory airway hyperresponsiveness and certain cellular inflammatory parameters, so they can potentially be considered as adjuvant treatments in respiratory inflammatory conditions. In contrast, Ceylon type LO inhalation might have an irritant effect (e.g., increased airway hyperresponsiveness and MPO activity) on the inflamed airways, and therefore should be avoided.

Complex Regulatory Role of the TRPA1 Receptor in Acute and Chronic Airway Inflammation Mouse Models.

The Transient Receptor Potential Ankyrin 1 (TRPA1) cation channel expressed on capsaicin-sensitive afferents, immune and endothelial cells is activated by inflammatory mediators and exogenous irritants, e.g., endotoxins, nicotine, crotonaldehyde and acrolein. We investigated its involvement in acute and chronic pulmonary inflammation using Trpa1 gene-deleted (Trpa1−/−) mice. Acute pneumonitis was evoked by intranasal Escherichia coli endotoxin (lipopolysaccharide: LPS) administration, chronic bronchitis by daily cigarette smoke exposure (CSE) for 4 months. Frequency, peak inspiratory/expiratory flows, minute ventilation determined by unrestrained whole-body plethysmography were significantly greater, while tidal volume, inspiratory/expiratory/relaxation times were smaller in Trpa1−/− mice. LPS-induced bronchial hyperreactivity, myeloperoxidase activity, frequency-decrease were significantly greater in Trpa1−/− mice. CSE significantly decreased tidal volume, minute ventilation, peak inspiratory/expiratory flows in wildtypes, but not in Trpa1−/− mice. CSE remarkably increased the mean linear intercept (histopathology), as an emphysema indicator after 2 months in wildtypes, but only after 4 months in Trpa1−/− mice. Semiquantitative histopathological scores were not different between strains in either models. TRPA1 has a complex role in basal airway function regulation and inflammatory mechanisms. It protects against LPS-induced acute pneumonitis and hyperresponsiveness, but is required for CSE-evoked emphysema and respiratory deterioration. Further research is needed to determine TRPA1 as a potential pharmacological target in the lung.

P2X3 receptor antagonism reduces the occurrence of apnoeas in newborn rats

Hyperreflexia of the peripheral chemoreceptors is a potential contributor of apnoeas of prematurity (AoP). Recently, it was shown that elevated P2X3 receptor expression was associated with elevated carotid body afferent sensitivity. Therefore, we tested whether P2X3 receptor antagonism would reduce AoP known to occur in newborn rats. Unrestrained whole-body plethysmography was used to record breathing and from this the frequency of apnoeas at baseline and following administration of either a P2X3 receptor antagonist - AF-454 (5 mg/kg or 10 mg/kg s.c.) or vehicle was derived. In a separate group, we tested the effects of AF-454 (10 mg/kg) on the hypoxic ventilatory response (10 % FiO2). Ten but not 5 mg/kg AF-454 reduced the frequency of AoP and improved breathing regularity significantly compared to vehicle. Neither AF-454 (both 5 and 10 mg/kg) nor vehicle affected baseline respiration. However, P2X3 receptor antagonism (10 mg/kg) powerfully blunted hypoxic ventilatory response to 10 % FiO2. These data suggest that P2X3 receptors contribute to AoP and the hypoxic ventilatory response in newborn rats but play no role in the drive to breathe at rest.

Glucagon-Like Peptide 1 and Atrial Natriuretic Peptide in a Female Mouse Model of Obstructive Pulmonary Disease.

Glucagon-like peptide-1 (GLP-1) is protective in lung disease models but the underlying mechanisms remain elusive. Because the hormone atrial natriuretic peptide (ANP) also has beneficial effects in lung disease, we hypothesized that GLP-1 effects may be mediated by ANP expression. To study this putative link, we used a mouse model of chronic obstructive pulmonary disease (COPD) and assessed lung function by unrestrained whole-body plethysmography. In 1 study, we investigated the role of endogenous GLP-1 by genetic GLP-1 receptor (GLP-1R) knockout (KO) and pharmaceutical blockade of the GLP-1R with the antagonist exendin-9 to -39 (EX-9). In another study the effects of exogenous GLP-1 were assessed. Lastly, we investigated the bronchodilatory properties of ANP and a GLP-1R agonist on isolated bronchial sections from healthy and COPD mice.Lung function did not differ between mice receiving phosphate-buffered saline (PBS) and EX-9 or between GLP-1R KO mice and their wild-type littermates. The COPD mice receiving GLP-1R agonist improved pulmonary function (P < .01) with less inflammation, but no less emphysema compared to PBS-treated mice. Compared with the PBS-treated mice, treatment with GLP-1 agonist increased ANP (nppa) gene expression by 10-fold (P < .01) and decreased endothelin-1 (P < .01), a peptide associated with bronchoconstriction. ANP had moderate bronchodilatory effects in isolated bronchial sections and GLP-1R agonist also showed bronchodilatory properties but less than ANP. Responses to both peptides were significantly increased in COPD mice (P < .05, P < .01).Taken together, our study suggests a link between GLP-1 and ANP in COPD.

The Hypoxic Ventilatory Response is Blocked by AMPK Deletion in Catecholaminergic, but not Adrenergic Cells

During hypoxia, ventilatory adjustments are critical to the maintenance of adequate oxygen (O2) delivery, for example during sleep or ascent to altitude (1). We recently demonstrated that the AMP‐activated protein kinase (AMPK), a ubiquitously expressed metabolic sensor (2), protects against hypoventilation and apnea during hypoxia (3). Here, we further investigated the circuit mechanism by which AMPK deficiency impacts the hypoxic ventilatory response (HVR). Using unrestrained whole‐body plethysmography, we compared the effects on the HVR of mice with dual deletion of the AMPK‐α1/α2 catalytic subunits in all catecholaminergic (TH‐positive) cells with the HVR of mice with AMPK‐α1/α2 deletion targeted to adrenergic cells alone (Phenylethanolamine N‐methyltransferase (PNMT)‐positive). Relative to controls (AMPK‐α1/α2 floxed), mice with TH‐Cre driven AMPK deletion (TH AMPK‐α1/α2 dKO) exhibited hypoventilation, characterized by marked attenuation of minute ventilation (p&lt;0.0001) during each minute of exposures to severe hypoxia (8% O2). By contrast, in mice with PNMT‐Cre driven AMPK deletion (PNMT AMPK‐α1/α2 dKO) the HVR was augmented rather than attenuated, although this only reached significance relative to controls (AMPK‐α1/α2 floxed and PNMT Cre) when taken as averages for the whole 10min period of exposure to severe hypoxia (p&lt;0.001–0.0001). As one might expect given these outcomes, the HVR of PNMT AMPK‐α1/α2 dKO mice was significantly greater than that for TH AMPK‐α1/α2 dKO during each full minute of hypoxia (p&lt;0.0001). Furthermore, the apnea‐duration index (ADI) was significantly augmented in TH AMPK‐α1/α2 dKO mice compared to controls (p&lt;0.01–0.0001). By contrast, PNMT AMPK‐α1/α2 dKO mice exhibited a reduction in the average ADI compared to controls (p&lt;0.05–0.01). This reduction in the ADI for PNMT AMPK‐α1/α2 dKO mice was primarily due to a lower number of apneas during the first 3 to 4 minutes of hypoxic exposure, the ADI being comparable to controls between 4 and 10 min. Accordingly, the ADI of PNMT AMPK‐α1/α2 dKO was attenuated at each time point throughout the 10min exposure to hypoxia when compared to TH AMPK‐α1/α2 dKO mice (p&lt;0.0001). In conclusion, we have uncovered a split in the AMPK‐dependent regulation of the catecholaminergic respiratory network during hypoxia, where the HVR is promoted by AMPK‐dependent modulation of noradrenergic inputs, but opposed by AMPK‐dependent modulation of adrenergic inputs. AMPK may thus protect against hypoventilation and apnea through noradrenergic signaling, and might aid restoration of normal CO2 levels during hypoxic hyperventilation through adrenergic signaling (4).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Prophylactic and therapeutic inhibition of allergic airway inflammation by probiotic Escherichia coli O83

Prophylactic and therapeutic inhibition of allergic airway inflammation by probiotic Escherichia coli O83

Persistent Expression of Serotonin Receptor 5b Alters Breathing Behavior in Male MeCP2 Knockout Mice

Mutations in the transcription factor methyl-CpG-binding protein 2 (MeCP2) cause the neurodevelopmental disorder Rett syndrome (RTT). Besides many other neurological problems, RTT patients show irregular breathing with recurrent apneas or breath-holdings. MeCP2-deficient mice, which recapitulate this breathing phenotype, show a dysregulated, persistent expression of G-protein-coupled serotonin receptor 5-ht5b (Htr5b) in the brainstem. To investigate whether the persistence of 5-ht5b expression is contributing to the respiratory phenotype, we crossbred MeCP2-deficient mice with 5-ht5b-deficient mice to generate double knockout mice (Mecp2−/y;Htr5b−/−). To compare respiration between wild type (WT), Mecp2−/y and Mecp2−/y;Htr5b−/− mice, we used unrestrained whole-body plethysmography. While the breathing of MeCP2-deficient male mice (Mecp2−/y) at postnatal day 40 is characterized by a slow breathing rate and the occurrence of prolonged respiratory pauses, we found that in MeCP2-deficient mice, which also lacked the 5-ht5b receptor, the breathing rate and the number of pauses were indistinguishable from WT mice. To test for a potential mechanism, we also analyzed if the known coupling of 5-ht5b receptors to Gi proteins is altering second messenger signaling. Tissue cAMP levels in the medulla of Mecp2−/y mice were decreased as compared to WT mice. In contrast, cAMP levels in Mecp2−/y;Htr5b−/− mice were indistinguishable from WT mice. Taken together, our data points towards a role of 5-ht5b receptors within the complex breathing phenotype of MeCP2-deficient mice.

X-Ray based Lung Function measurement-a sensitive technique to quantify lung function in allergic airway inflammation mouse models.

In mice, along with the assessment of eosinophils, lung function measurements, most commonly carried out by plethysmography, are essential to monitor the course of allergic airway inflammation, to examine therapy efficacy and to correlate animal with patient data. To date, plethysmography techniques either use intubation and/or restraining of the mice and are thus invasive, or are limited in their sensitivity. We present a novel unrestrained lung function method based on low-dose planar cinematic x-ray imaging (X-Ray Lung Function, XLF) and demonstrate its performance in monitoring OVA induced experimental allergic airway inflammation in mice and an improved assessment of the efficacy of the common treatment dexamethasone. We further show that XLF is more sensitive than unrestrained whole body plethysmography (UWBP) and that conventional broncho-alveolar lavage and histology provide only limited information of the efficacy of a treatment when compared to XLF. Our results highlight the fact that a multi-parametric imaging approach as delivered by XLF is needed to address the combined cellular, anatomical and functional effects that occur during the course of asthma and in response to therapy.

The effect of prenatal maternal infection on respiratory function in mouse offspring: evidence for enhanced chemosensitivity.

Systemic maternal inflammation is implicated in preterm birth and bronchopulmonary dysplasia (BPD) and may induce morbidities including reduced pulmonary function, sleep-disordered breathing, and cardiovascular disorders. Here we test the hypothesis that antenatal maternal inflammation per se causes altered alveolar development and increased chemoreflex sensitivity that persists beyond infancy. Pregnant C57BL/6 mice were administered lipopolysaccharide (LPS) (150 μg/kg ip) to induce maternal inflammation or saline (SHAM) at embryonic day 16 (randomized). Pups were weighed daily. On days 7, 28, and 60 (D07, D28, and D60), unrestrained wholebody plethysmography quantified ventilation and chemoreflex responses to hypoxia (10%), hypercapnia (7%), and asphyxia (hypoxic hypercapnia). Lungs were harvested to quantify alveolar number, size, and septal thickness. LPS pups had reduced baseline ventilation per unit bodyweight (∼40%, P < 0.001) vs. SHAM. LPS increased ventilatory responses to hypoxia (D07: 66% vs. 28% increase in ventilation; P < 0.001) hypercapnia (170% vs. 88%; P < 0.001), and asphyxia (249% vs. 154%; P < 0.001); hypersensitive hypoxic responsiveness persisted until D60 (P < 0.001). LPS also increased apnea frequency (P < 0.01). LPS caused thicker alveolar septae (D07, P < 0.001), diminished alveolar number (D28, P < 0.001) vs. SHAM, but effects were minimal by D60. Pups delivered from mothers exposed to antenatal inflammation exhibit deficits in lung structure and hypersensitive responses to respiratory stimuli that persist beyond the newborn period. Antenatal inflammation may contribute to impaired gas exchange and unstable breathing in newborn infants and adversely affect long-term health.

Measuring respiratory function in mice using unrestrained whole-body plethysmography.

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.

Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.