Pressure-volume (PV) curves constructed over the entire lung volume range can reliably detect functional changes in mouse models of lung diseases. In the present study, we constructed full-range PV curves in healthy and elastase-treated mice using either a classic manually operated technique or an automated approach using a computer-controlled piston ventilator [flexiVent FX; Scientific Respiratory Equipment (SCIREQ), Montreal, Quebec, Canada]. On the day of the experiment, subjects were anesthetized, tracheotomized, and mechanically ventilated. Following an initial respiratory mechanics scan and degassing of the lungs with 100% O2, full-range PV curves were constructed using either the classic or the automated technique. In control mice, superimposable curves were obtained, and statistical equivalence was attained between the two methodologies. In the elastase-treated ones, where significant changes in respiratory mechanics and lung volumes were expected, very small differences were observed between the two techniques, and the criteria for statistical equivalence were met in two out of four parameters assessed. The automated technique was adapted to rats and used to estimate the functional residual capacity (FRC) by volume subtraction. This novel approach generated FRC estimates consistent with the literature, with added accuracy relative to the existing method in diseased subjects. In conclusion, the automated technique generated full-range PV curves that were equivalent or very close to those obtained with the classic method under physiological or severe pathological conditions. The automation facilitated some technical aspects of the procedure, eased its use across species, and helped derive a more accurate estimate of FRC in preclinical models of respiratory disease.NEW & NOTEWORTHY Partial and full-range pressure-volume (PV) curves are frequently used to characterize lung disease models. Whereas automated techniques exist to construct partial PV curves, a manually operated approach is classically employed to build the full-range ones. In this study, the full-range PV curve technique was automated using a computer-controlled piston ventilator. The automation simplified the technique, facilitated its extension to other species, and inspired a novel way of estimating the functional residual capacity in laboratory rodents.
Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to non-invasively assess some features of the respiratory function in humans as well as in laboratory animals. The present article focuses on the application of the double-chamber plethysmography (DCP) in small animals. It provides background information on the methodology as well as a detailed step-by-step procedure to successfully assess respiratory function in conscious, spontaneously breathing animals in a non-invasive manner. The DCP can be used to monitor the respiratory function of multiple animals in parallel, as well as to identify changes induced by aerosolized substances over a chosen time period and in a repeated manner. Experiments on control and allergic mice are used herein to demonstrate the utility of the technique, explain the associated outcome parameters, as well as to discuss the related advantages and shortcomings. Overall, the DCP provides valid and theoretically sound readouts that can be trusted to evaluate the respiratory function of conscious small animals both at baseline and after challenges with aerosolized substances.
We determined the effect of inhaled corticosteroid, budesonide, on the release of the anti-inflammatory cytokine, interleukin-10 (IL-10), and of pro-inflammatory cytokines, macrophage inflammatory protein-1 α (MIP-1 α ), interferon- γ (IFN- γ ), and granulocyte-macrophage colony-stimulating factor (GM-CSF), from blood monocytes and alveolar macrophages of mild asthmatic subjects in a double-blind, cross-over, placebo-controlled study. Budesonide reduced bronchial hyperresponsiveness and improved baseline FEV1. Alveolar macrophages were obtained by bronchoalveolar lavage performed at the end of each treatment phase. IL-10 from blood monocytes was not altered, but both IL-10 mRNA and protein expression from alveolar macrophages stimulated by lipopolysaccharide and IL-1 β were increased after corticosteroid therapy. By contrast, alveolar macrophages released significantly less MIP-1 α , IFN- γ , and GM-CSF after steroid treatment. In comparison to alveolar macrophages from normal nonasthmatic volunteers, those from asthmatic patients released more MIP-1 α , IFN- γ , and GM-CSF but lower amounts of IL-10 particularly at baseline and after IL-1 β stimulation. The ability of steroids to inhibit pro-inflammatory cytokines but to enhance the anti-inflammatory cytokine such as IL-10 may contribute to their beneficial actions in asthma. Asthma is characterized by alveolar macrophages exhibiting both an enhanced capacity to release pro-inflammatory cytokines and a reduced capacity to produce IL-10.
A combination of pharmacological and genetic approaches was used to determine the role of type 4 cAMP-specific cyclic nucleotide phosphodiesterase 4 (PDE4) in reversing α2-adrenoceptor–mediated anesthesia, a behavioral correlate of emesis in non-vomiting species. Among the family-specific PDE inhibitors, PDE4 inhibitors reduced the duration of xylazine/ketamine–induced anesthesia in mice, with no effect on pentobarbital-induced anesthesia. The rank order of the PDE4 inhibitors tested was 6-(4-pyridylmethyl)-8-(3-nitrophenyl)quinoline (PMNPQ) > (R)-rolipram > (S)-rolipram >> (R)-N-{4-[1-(3-cyclopentyloxy-4-methoxyphenyl)-2-(4-pyridyl)ethyl]phenyl}N′-ethylurea (CT-2450). The specific roles of PDE4B and PDE4D in this model were studied using mice deficient in either subtype. PDE4D-deficient mice, but not PDE4B-deficient mice, had a shorter sleeping time than their wild-type littermates under xylazine/ketamine–induced anesthesia, but not under that induced with pentobarbital. Concomitantly, rolipram-sensitive PDE activity in the brain stem was decreased only in PDE4D-deficient mice compared with their wild-type littermates. While PMNPQ significantly reduced the xylazine/ketamine–induced anesthesia period in wild-type mice and in PDE4B-null mice, it had no effect in PDE4D-deficient mice. These findings strongly support the hypothesis that inhibition of PDE4D is pivotal to the anesthesia-reversing effect of PMNPQ and is likely responsible for emesis induced by PDE4 inhibitors.
Abstract RANTES is a basic 8-kDa polypeptide of the C-C chemokine subfamily with strong chemotactic activity for eosinophils, lymphocytes, and monocytes. We determined the regulation of RANTES production by human airway smooth muscle cells in culture. While TNF-alpha, but not IFN-gamma, increased RANTES mRNA expression and protein release, the combination of TNF-alpha and IFN-gamma caused a greater degree of expression and release in a time- and dose-dependent manner. Sequential treatment of airway smooth muscle cells with TNF-alpha and IFN-gamma showed that IFN-gamma sensitized the cells to the stimulatory effect of TNF-alpha. Using a modified Boyden chamber technique, RANTES separated by reverse-phase liquid chromatography from cell culture supernatants of airway smooth muscle cells stimulated by TNF-alpha and IFN-gamma showed a strong chemoattractant effect on human eosinophils, an effect inhibited by an anti-RANTES Ab. RANTES production induced by TNF-alpha and IFN-gamma was inhibited partly by the Th2-derived cytokines, IL-4, IL-10, and IL-13, as well as by dexamethasone. Our studies indicate that, in addition to contractile responses and mitogenesis, airway smooth muscle cells have synthetic and secretory potential with the release of RANTES. They may participate in chronic airway inflammation by interacting with both Th1- and Th2-derived cytokines to modulate chemoattractant activity for eosinophils, activated T lymphocytes, and monocytes/macrophages.
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