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Nan Fang Yi Ke Da Xue Xue Bao = Journal... Jun 2022To investigate the roles of angiotensin-converting enzyme 2 (ACE2) in ozone-induced pulmonary inflammation and airway remodeling in mice.
OBJECTIVE
To investigate the roles of angiotensin-converting enzyme 2 (ACE2) in ozone-induced pulmonary inflammation and airway remodeling in mice.
METHODS
Sixteen wild-type (WT) C57BL/6J mice and 16 ACE2 knock-out (KO) mice were exposed to either filtered air or ozone (0.8 ppm) for 3 h per day for 5 consecutive days. Masson's staining and HE staining were used to observe lung pathologies. Bronchoalveolar lavage fluid (BALF) was collected and the total cell count was determined. The total proteins and cytokines in BALF were determined by BCA and ELISA method. The transcription levels of airway remodeling-related indicators in the lung tissues were detected using real-time quantitative PCR. The airway resistance of the mice was measured using a small animal ventilator with methacholine stimulation.
RESULTS
Following ozoneexposure ACE2 KO mice had significantly higher lung pathological scores than WT mice ( < 0.05). Masson staining results showed that compared with ozone-exposed WT mice, ozone-exposed ACE2 KO mice presented with significantly larger area of collagen deposition in the bronchi [(19.62±3.16)% (6.49±1.34)%, < 0.05] and alveoli [(21.63±3.78)% (4.44±0.99)%, < 0.05]. The total cell count and total protein contents in the BALF were both higher in ozone-exposed ACE2 KO mice than in WT mice, but these differences were not statistically significant ( > 0.05). The concentrations of IL-6, IL-1β, TNF-, CXCL1/KC and MCP-1 in the BALF were all higher in ozone-exposed ACE2 KO mice than in ozone-exposed WT mice, but only the difference in IL-1β was statistically significant ( < 0.05). The transcription levels of MMP-9, MMP-13, TIMP 4, COL1A1, and TGF-β in the lung tissues were all significantly higher in ozone-exposed ACE2 KO mice ( < 0.01). No significant difference was found in airway resistance between ozone-exposed ACE KO mice and WT mice after challenge with 0, 10, 25, or 100 mg/mL of methacholine.
CONCLUSION
ACE2 participates in ozone-induced lung inflammation and airway remodeling in mice.
Topics: Airway Remodeling; Angiotensin-Converting Enzyme 2; Animals; Methacholine Chloride; Mice; Mice, Inbred C57BL; Mice, Knockout; Ozone; Pneumonia
PubMed: 35790436
DOI: 10.12122/j.issn.1673-4254.2022.06.09 -
American Journal of Respiratory Cell... Oct 2022Asthma is a common respiratory disease characterized, in part, by excessive airway smooth muscle (ASM) contraction (airway hyperresponsiveness). Various GABAR...
Asthma is a common respiratory disease characterized, in part, by excessive airway smooth muscle (ASM) contraction (airway hyperresponsiveness). Various GABAR (γ-aminobutyric acid type A receptor) activators, including benzodiazepines, relax ASM. The GABAR is a ligand-operated Cl channel best known for its role in inhibitory neurotransmission in the central nervous system. Although ASM cells express GABARs, affording a seemingly logical site of action, the mechanism(s) by which GABAR ligands relax ASM remains unclear. PI320, a novel imidazobenzodiazepine designed for tissue selectivity, is a promising asthma drug candidate. Here, we show that PI320 alleviates methacholine (MCh)-induced bronchoconstriction and relaxes peripheral airways preconstricted with MCh using the forced oscillation technique and precision-cut lung slice experiments, respectively. Surprisingly, the peripheral airway relaxation demonstrated in precision-cut lung slices does not appear to be GABAR-dependent, as it is not inhibited by the GABAR antagonist picrotoxin or the benzodiazepine antagonist flumazenil. Furthermore, we demonstrate here that PI320 inhibits MCh-induced airway constriction in the absence of external Ca, suggesting that PI320-mediated relaxation is not mediated by inhibition of Ca influx in ASM. However, PI320 does inhibit MCh-induced intracellular Ca oscillations in peripheral ASM, a key mediator of contraction that is dependent on sarcoplasmic reticulum Ca mobilization. Furthermore, PI320 inhibits peripheral airway constriction induced by experimentally increasing the intracellular concentration of inositol triphosphate (IP). These novel data suggest that PI320 relaxes murine peripheral airways by inhibiting intracellular Ca mobilization in ASM, likely by inhibiting Ca release through IPRs (IP receptors).
Topics: Animals; Asthma; Calcium; Calcium Signaling; Flumazenil; Inositol; Ligands; Lung; Methacholine Chloride; Mice; Muscle Contraction; Muscle, Smooth; Picrotoxin; gamma-Aminobutyric Acid
PubMed: 35776523
DOI: 10.1165/rcmb.2022-0084OC -
Methods in Molecular Biology (Clifton,... 2022Asthma has been the most prevalent chronic respiratory disease (Mensah et al. J Allergy Clin Immunol 142:744-748, 2018). To explore pathogenic mechanism or new...
Asthma has been the most prevalent chronic respiratory disease (Mensah et al. J Allergy Clin Immunol 142:744-748, 2018). To explore pathogenic mechanism or new treatments of asthma, mice have been utilized to model the disease. Eosinophilic airway inflammation, allergen specific-IgE, and airway hyperresponsiveness have been characteristic features of allergic asthma (Drake et al. Pulm Ther 5:103-115, 2019). In mouse models, airway hyperresponsiveness to inhaled broncho-constrictor agents such as methacholine chloride (MCh) has been a key disease marker (Alessandrini et al. Front Immunol 11:575936, 2020). A variety of systems to assess airway reactivity in mice are currently available. Here, three distinct systems are described as these have been used in many publications. In the first system, an invasive system in which mice are anesthetized and intubated followed by mechanical ventilation, lung resistance (R), dynamic compliance (C), and other respiratory parameters with MCh challenge are measured. In the second system, a noninvasive system equipped with a chamber in which mice can move freely and spontaneously breathe, changes in airways with MCh challenge are measured as enhanced pause (Penh) values. In the third system, in vitro airway smooth muscle (ASM) reactivity is monitored in an extracted mouse tracheal duct with a cholinergic agonist challenge or electrical stimulation. Each of these systems has unique features, benefits, or disadvantages.
Topics: Animals; Asthma; Bronchial Hyperreactivity; Disease Models, Animal; Eosinophilia; Immunoglobulin E; Methacholine Chloride; Mice; Mice, Inbred BALB C; Ovalbumin; Respiration Disorders; Respiratory Hypersensitivity
PubMed: 35771466
DOI: 10.1007/978-1-0716-2364-0_7 -
Respiratory Physiology & Neurobiology Oct 2022BALB/c mice from both sexes underwent one of two nebulized methacholine challenges that were preceded by a period of 20 min either with or without tone induced by...
BALB/c mice from both sexes underwent one of two nebulized methacholine challenges that were preceded by a period of 20 min either with or without tone induced by repeated contractions of the airway smooth muscle. Impedance was monitored throughout and the constant phase model was used to dissociate the impact of tone on conducting airways (R - Newtonian resistance) versus the lung periphery (G and H - tissue resistance and elastance). The effect of tone on smooth muscle contractility was also tested on excised tracheas. While tone markedly potentiated the methacholine-induced gains in H and G in both sexes, the gain in R was only potentiated in males. The contractility of female and male tracheas was also potentiated by tone. Inversely, the methacholine-induced gain in hysteresivity (G/H) was mitigated by tone in both sexes. Therefore, the tone-induced muscle hypercontractility impacts predominantly the lung periphery in vivo, but also promotes further airway narrowing in males while protecting against narrowing heterogeneity in both sexes.
Topics: Animals; Female; Lung; Male; Methacholine Chloride; Mice; Mice, Inbred BALB C; Muscle Contraction; Muscle, Smooth; Trachea
PubMed: 35716869
DOI: 10.1016/j.resp.2022.103938 -
Biomedicine & Pharmacotherapy =... Aug 2022Veronica persica is a flowering plant belonging to the family Scrophulariaceae. Here, we aimed to evaluate the pharmacological activity of the ethanol extract of...
Veronica persica is a flowering plant belonging to the family Scrophulariaceae. Here, we aimed to evaluate the pharmacological activity of the ethanol extract of Veronica persica (EEVP) in an airway inflammation model. We examined airway responsiveness to aerosolized methacholine, serum immunoglobulin (Ig)E levels, and total cell numbers in the lung and bronchoalveolar lavage fluid (BALF). Histological analysis of the lung tissue was performed using hematoxylin-eosin, Masson trichrome, or periodic acid-Schiff staining. Fluorescence-activated cell sorting analysis in the lung and BALF was applied to clarify the changes in immune cell types. Enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction were applied to investigate cytokine levels and gene expression related to airway inflammation. STAT-3/6 phosphorylation was examined in primary bronchial/tracheal epithelial cells using western blot analysis. EEVP significantly suppressed total IgE levels and methacholine-induced increase of Penh value in the HDM-challenged mouse model. EEVP also attenuated the severity of airway remodeling in lung tissues and decreased eosinophil and neutrophil infiltration in the lungs and BALF. EEVP significantly reduced the production of cytokines in BAL and splenocyte culture medium, and the expression of mRNAs related to airway inflammation in the lung tissue. EEVP suppressed IL-4/13-induced STAT-3/6 phosphorylation in the epithelial cells. We showed for the first time that EEVP effectively inhibits eosinophilic airway inflammation by suppressing the expression of inflammatory factors for T cell activation and polarization, and inhibits MCP-1 production of bronchial/tracheal epithelial cells by suppressing STAT-3/6 activation. EEVP may be a potential pharmacological agent to prevent inflammatory airway diseases.
Topics: Animals; Asthma; Cytokines; Ethanol; Immunoglobulin E; Inflammation; Lung; Methacholine Chloride; Mice; Pyroglyphidae; Veronica
PubMed: 35696941
DOI: 10.1016/j.biopha.2022.113264 -
Respiratory Care Aug 2022Methacholine challenge testing (MCT) is a common bronchoprovocation technique used to assess airway hyper-responsiveness. We previously demonstrated that the addition of...
BACKGROUND
Methacholine challenge testing (MCT) is a common bronchoprovocation technique used to assess airway hyper-responsiveness. We previously demonstrated that the addition of a viral filter to the nebulizer exhalation limb substantially reduced expelled particles during MCT. Our aim was to evaluate whether this modification affects the delivered dose of methacholine.
METHODS
A mechanical ventilator was connected to a lung simulator with breathing frequency 15 breaths/min, tidal volume 500 mL, inspiratory-expiratory ratio 1:1, with a sinusoidal waveform. We compared methacholine dose delivery using the Hudson Micro Mist or AeroEclipse II BAN nebulizers powered by either a dry gas source or a compressor system. A filter placed in line between the nebulizer and test lung was weighed before and after 1 min of nebulized methacholine delivery. Mean inhaled mass was measured with and without a viral filter on the exhalation limb. Dose delivery was calculated by multiplying the mean inhaled mass by the respirable fraction (particles < 5 μm) and inhalation time. Unpaired test was used to compare methacholine dose delivery with and without viral filter placement.
RESULTS
The addition of a viral filter did not significantly affect methacholine dose delivery across all devices tested. Using a 50-psi dry gas source, dose delivered with or without a viral filter did not differ with the Hudson (422.3 μg vs 282.0 μg, = .11) or the AeroEclipse nebulizer (563.0 μg vs 657.6 μg, = .59). Using the compressor, dose delivered with and without a viral filter did not differ with the Hudson (974.0 μg vs 868.0 μg, = .03) or the AeroEclipse nebulizer (818.0 μg vs 628.5 μg, = .42).
CONCLUSIONS
The addition of a viral filter to the nebulizer exhalation limb did not affect methacholine dose during bronchoprovocation testing. Routine use of a viral filter should be considered to improve pulmonary function technician safety and infection control measures during the ongoing COVID-19 pandemic.
Topics: Administration, Inhalation; Aerosols; Albuterol; Bronchodilator Agents; COVID-19; Equipment Design; Exhalation; Humans; Methacholine Chloride; Nebulizers and Vaporizers; Pandemics
PubMed: 35610032
DOI: 10.4187/respcare.09703 -
Journal of Applied Physiology... Jul 2022Mice are a valuable model for elegant studies of complex, system-dependent diseases, including pulmonary diseases. Current tools to assess lung function in mice are...
Mice are a valuable model for elegant studies of complex, system-dependent diseases, including pulmonary diseases. Current tools to assess lung function in mice are either terminal or lack accuracy. We set out to develop a low-cost, accurate, head-out variable-pressure plethysmography system to allow for repeated, nonterminal measurements of lung function in mice. Current head-out plethysmography systems are limited by air leaks that prevent accurate measures of volume and flow. We designed an inflatable cuff that encompasses the mouse's neck preventing air leak. We wrote corresponding software to collect and analyze the data, remove movement artifacts, and automatically calibrate each dataset. This software calculates volume, inspiratory/expiratory time, breaths per minute, mid-expiratory flow, and end-inspiratory pause. To validate the use, we established that our plethysmography system accurately measured tidal breathing, the bronchoconstrictive response to methacholine, sex- and age-associated changes in breathing, and breathing changes associated with house dust mite sensitization. Our estimates of volume, flow, and timing of breaths are in line with published estimates, we observed dose-dependent decreases in volume and flow in response to methacholine ( < 0.05), increased lung volume, and decreased breathing rate with aging ( < 0.05), and that house dust mite sensitization decreased volume and flow ( < 0.05) while exacerbating the methacholine-induced increase in inspiratory time ( < 0.05). We describe an accurate, sensitive, low-cost, head-out plethysmography system that allows for longitudinal studies of pulmonary disease in mice. We describe a low-cost, variable-pressure head-out plethysmography system that can be used to assess lung function in mice. A balloon cuff is inflated around the mouse's neck to prevent air leak, allowing for accurate measurements of lung volume and air flow. Custom software facilitates system calibration, removes movement artifacts, and eases data analysis. The system was validated by measuring tidal breathing, responses to methacholine, and changes associated with house dust mite sensitization, sex, and aging.
Topics: Animals; Bronchoconstriction; Lung; Lung Volume Measurements; Methacholine Chloride; Mice; Plethysmography; Tidal Volume
PubMed: 35608203
DOI: 10.1152/japplphysiol.00835.2021 -
Journal of Applied Physiology... Jun 2022Specific ventilation imaging (SVI) measures the spatial distribution of specific ventilation (SV) in the lung with MRI by using inhaled oxygen as a contrast agent....
Specific ventilation imaging (SVI) measures the spatial distribution of specific ventilation (SV) in the lung with MRI by using inhaled oxygen as a contrast agent. Because of the inherently low signal-to-noise ratio in the technique, multiple switches between inspiring air and O are utilized, and the high spatial resolution SV distribution is determined as an average over the entire imaging period (∼20 min). We hypothesized that a trade-off between spatial and temporal resolution could allow imaging at a higher temporal resolution, at the cost of a coarser, yet acceptable, spatial resolution. The appropriate window length and spatial resolution compromise were determined by generating synthetic data with signal- and contrast-to-noise characteristics reflective of that in previously published experimental data, with a known and unchanging distribution of SV, and showed that acceptable results could be obtained in an imaging period of ∼7 min (80 breaths), with a spatial resolution of ∼1 cm. Previously published data were then reanalyzed. The average heterogeneity of the temporally resolved maps of SV was not different from the previous overall analysis, however, the temporally resolved maps were less effective at detecting the amount of bronchoconstriction resulting from methacholine administration. The results further indicated that the initial response to inhaled methacholine and subsequent inhalation of albuterol were largely complete within ∼22 min and ∼9 min, respectively, although there was a tendency for an ongoing developing effect in both cases. These results suggest that it is feasible to use a shortened SVI protocol, with a modest sacrifice in spatial resolution, to measure temporally dynamic processes. Dynamic imaging providing maps of specific ventilation with a temporal resolution of ∼7 min with a spatial resolution of ∼1 cm using MRI was shown to be practical. The technique provides an ionizing radiation free means of temporally following the spatial pattern of specific ventilation. Reanalysis of previously published data showed that the effects of inhaled methacholine and albuterol were largely complete at ∼22 min and ∼9 min, respectively after administration.
Topics: Albuterol; Bronchoconstriction; Lung; Magnetic Resonance Imaging; Methacholine Chloride; Oxygen
PubMed: 35482322
DOI: 10.1152/japplphysiol.00652.2021 -
Pediatric Allergy and Immunology :... Apr 2022Onset of wheeze is the endpoint often used in the determination of a positive bronchial challenge test (BCT) in young children who cannot perform spirometry. We sought...
BACKGROUND
Onset of wheeze is the endpoint often used in the determination of a positive bronchial challenge test (BCT) in young children who cannot perform spirometry. We sought to assess several clinical endpoints at the time of a positive BCT in young children with recurrent wheeze compared to findings in school-aged children with asthma.
METHODS
Positive BCT was defined in: (1) preschool children (n = 22) as either persistent cough, wheeze, fall in oxygen saturation (SpO ) of ≥5%, or ≥50% increase in respiratory rate (RR) from baseline; and (2) school-aged children (n = 22) as the concentration of methacholine (MCh) required to elicit a 20% decline in FEV (PC ).
RESULTS
All preschool children (mean age 3.4 years) had a positive BCT (median provocative MCh concentration 1.25 mg/ml [IQR, 0.62, 1.25]). Twenty (91%) school-aged children (mean age 11.3 years) had a positive BCT (median PC 1.25 mg/ml [IQR, 0.55, 2.5]). At the time of the positive BCT, the mean fall in SpO (6.9% vs. 3.8%; p = .001) and the mean % increase in RR (61% vs. 22%; p < .001) were greater among preschool-aged than among school-aged children. A minority of children developed wheeze at time of positive BCT (23% preschool- vs. 15% school-aged children; p = .5).
CONCLUSIONS
The use of wheeze as an endpoint for BCT in preschool children is unreliable, as it rarely occurs. The use of clinical endpoints, such as ≥25% increase in RR or fall in SpO of ≥3%, captured all of our positive BCT in preschool children, while minimizing undue respiratory distress.
Topics: Asthma; Bronchial Provocation Tests; Child; Child, Preschool; Humans; Methacholine Chloride; Respiratory Sounds; Spirometry
PubMed: 35470941
DOI: 10.1111/pai.13767 -
Role of hyperpnea in the relaxant effect of inspired CO on methacholine-induced bronchoconstriction.Journal of Applied Physiology... May 2022Inhaling carbon dioxide (CO) in humans is known to cause inconsistent effects on airway function. These could be due to direct effects of CO on airway smooth muscle or...
Inhaling carbon dioxide (CO) in humans is known to cause inconsistent effects on airway function. These could be due to direct effects of CO on airway smooth muscle or to changes in minute ventilation (V̇e). To address this issue, we examined the responses of the respiratory system to inhaled methacholine in healthy subjects and subjects with mild asthma while breathing air or gas mixtures containing 2% or 4% CO. Respiratory mechanics were measured by a forced oscillation technique at 5 Hz during tidal breathing. At baseline, respiratory resistance (R) was significantly higher in subjects with asthma (2.53 ± 0.38 cmHO·L·s) than healthy subjects (2.11 ± 0.42 cmHO·L·s) ( = 0.008) with room air. Similar values were observed with CO 2% or 4% in the two groups. V̇e, tidal volume (V), and breathing frequency (BF) significantly increased with CO-containing mixtures ( < 0.001) with insignificant differences between groups. After methacholine, the increase in R and the decrease in respiratory reactance (X) were significantly attenuated up to about 50% with CO-containing mixtures instead of room air in both asthmatic ( < 0.001) and controls ( < 0.001). Mediation analysis showed that the attenuation of methacholine-induced changes in respiratory mechanics by CO was due to the increase in V̇e ( = 0.006 for R and = 0.014 for X) independently of the increase in V or BF, rather than a direct effect of CO. These findings suggest that the increased stretching of airway smooth muscle by the CO-induced increase in V̇e is a mechanism through which hypercapnia can attenuate bronchoconstrictor responses in healthy subjects and subjects with mild asthma. The main results of the present study are as follows: ) breathing gas mixtures containing 2% or 4% CO significantly attenuated bronchoconstrictor responses to methacholine, not differently in healthy subjects and subjects with mild asthma, and ) the causal inhibitory effect of CO was significantly mediated via an indirect effect of the increment of V̇e in response to intrapulmonary hypercapnia.
Topics: Airway Resistance; Asthma; Bronchoconstriction; Bronchoconstrictor Agents; Carbon Dioxide; Humans; Hypercapnia; Hyperventilation; Methacholine Chloride
PubMed: 35358399
DOI: 10.1152/japplphysiol.00763.2021