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Inspiratory resistive breathing (IRB) is characterized by large negative intrathoracic pressures and was shown to induce pulmonary inflammation in previously healthy rats. Matrix metalloproteinases (MMP)-9 and -12 are induced by inflammation and mechanical stress in the lung. We hypothesized that IRB induces MMP-9 and -12 in the lung. Anesthetized, tracheostomized rats breathed spontaneously through a two-way valve, connected to an inspiratory resistance, with the tidal inspiratory tracheal pressure set at 50% of the maximum. Quietly breathing animals served as controls. After 3 and 6 h of IRB, respiratory mechanics were measured, bronchoalveolar lavage (BAL) was performed, lung injury score was estimated, and lung MMP-9 was estimated by zymography and ELISA. MMP-9 and MMP-12 immunohistochemistry was performed. Isolated normal alveolar macrophages were incubated with BAL from rats that underwent IRB. After 18 h, MMP-9 and -12 levels were measured in supernatants, and immunocytochemistry was performed. Macrophages were treated with IL-1β, IL-6, or TNF-α, and MMP-9 in supernatants was measured. After 6 h of IRB, leukocytes in BAL increased, and IL-1β and IL-6 levels were elevated. Elasticity and injury score were increased after 3 and 6 h of IRB. Lung MMP-9 levels increased after 6 h of IRB. MMP-9 and MMP-12 were detected in alveolar macrophages and epithelial (bronchial/alveolar) cells after 3 and 6 h of IRB. MMP-9 and MMP-12 were found in supernatants after treatment with 6 h of IRB BAL. Cytosolic immunostaining was detected after treatment with 3 and 6 h of IRB BAL. All cytokines induced MMP-9 in culture supernatants. In conclusion, IRB induces MMP-9 and -12 in the lung of previously healthy rats.

Inspiratory resistive breathing (IRB) induces cytokine expression in the diaphragm. The mechanism of this cytokine induction remains elusive. The roles of MAPKs and NF-κB and the impact of oxidative stress in IRB-induced cytokine upregulation in the diaphragm were studied. Wistar rats were subjected to IRB (50% of maximal inspiratory pressure) via a two-way nonrebreathing valve for 1, 3, or 6 h. Additional groups of rats subjected to IRB for 6 h were randomly assigned to receive either solvent or N-acetyl-cysteine (NAC) or inhibitors of NF-κB (BAY-11–7082), ERK1/2 (PD98059), and P38 MAPK (SB203580) to study the effect of oxidative stress, NF-κB, and MAPKs in IRB-induced cytokine upregulation in the diaphragm. Quietly breathing animals served as controls. IRB upregulated cytokine (IL-6, TNF-α, IL-10, IL-2, IL-1β) protein levels in the diaphragm and resulted in increased activation of MAPKs (P38, ERK1/2) and NF-κB. Inhibition of NF-κB and ERK1/2 blunted the upregulation of all cytokines except that of IL-6, which was further increased. P38 inhibition attenuated all cytokine (including IL-6) upregulation. Both P38 and ERK1/2 inhibition decreased NF-κB/p65 subunit phosphorylation. NAC pretreatment blunted IRB-induced cytokine upregulation in the diaphragm and resulted in decreased ERK1/2, P38, and NF-κB/p65 phosphorylation. In conclusion, IRB-induced cytokine upregulation in the diaphragm is under the regulatory control of MAPKs and NF-κB. IL-6 is regulated differently from all other cytokines through a P38-dependent and NF-κB independent pathway. Oxidative stress is a stimulus for IRB-induced cytokine upregulation in the diaphragm.

Resistive breathing is associated with large negative intrathoracic pressures. Increased mechanical stress induces high-permeability pulmonary edema and lung inflammation.To determine the effects of resistive breathing on the healthy lung.Anesthetized rats breathed through a two-way nonrebreathing valve. The inspiratory line was connected to a resistance setting peak inspiratory tracheal pressure at 50% of maximum (inspiratory resistive breathing), while 100% oxygen was supplied to prevent hypoxemia. Quietly breathing animals (100% oxygen) served as controls. Lung injury was evaluated after 3 and 6 hours of resistive breathing.After both 3 and 6 hours of resistive breathing, lung permeability was increased, as assessed by (99m)Tc-diethylenetriaminepentaacetic acid scintigraphy and Evans blue dye extravasation. Tissue elasticity, measured on the basis of static pressure-volume curves and by the low-frequency forced oscillation technique, was also increased. After both 3 and 6 hours of resistive breathing, gravimetric measurements revealed the presence of pulmonary edema and analysis of bronchoalveolar lavage showed increased total protein content, whereas the total cell count was elevated only after 6 hours of resistive breathing. Cytokine levels were assessed in bronchoalveolar lavage fluid and lung tissue by ELISA and were increased after 6 hours compared with controls. Western blot analysis showed early activation of Src kinase via phosphorylation (at 30 min), and Erk1/2 and IκBα (nuclear factor-κB inhibitor) were phosphorylated at 3 and 6 hours. Pathology revealed the presence of lung injury after resistive breathing.Resistive breathing induces acute lung injury and inflammation.

Rationale: Inspiratory Resistive Breathing (IRB) is an experimental model of increased workload for the respiratory muscles. Contraction of peripheral skeletal muscles leads to muscle glycogen depletion and a compensatory increase in glucose transport possibly through a AMPK-dependent pathway that facilitates GLUT4 translocation to plasma membrane. Objectives: To examine the pathways that regulate glucose transport to the diaphragm during IRB. Methods: Anesthetized, tracheostomized, spontaneously breathing rats were subjected to 1, 3 or 6 hours of moderate IRB (50% of the maximum inspiratory pressure) via a two way-non rebreathing valve. Quietly breathing animals served as controls. AMPK, Acetyl-CoA Carboxylase (ACC), CAMKK and STAT3 were evaluated by immunoblotting, GLUT4 expression and localization with immunohistochemistry, glycogen and glucose content spectrophotometrically. Results: IRB for 1h resulted in diaphragmatic glycogen depletion. Diaphragmatic glucose content increased at 6h of IRB, coinciding with an increase in both AMPK and ACC (its downstreat target) phosphorylation and in GLUT4 expression and translocation to the sarcolemma. CAMKK phosphorylation, a possible AMPK upstream regulator, was increased at 3h of IRB preceding AMPK activation. IRB for 6h resulted in glycogen depletion in the liver and increased STAT3 phosphorylation in both liver and diaphragm suggesting an active IL6-sugnalling. IRB-induced diaphragmatic IL6-upregulation was evidenced from the 1st hour of IRB. Conclusions: IRB leads to diaphragmatic and liver glycogen depletion, increased diaphragmatic GLUT4 expression and AMPK activation. Evidence exists for active IL6 signalling in the liver.

Resistive breathing (encountered in chronic obstructive pulmonary disease and asthma) results in cytokine upregulation and decreased nitric oxide (NO) levels in the strenuously contracting diaphragm. NO can regulate gene expression. We hypothesized that endogenously produced NO downregulates cytokine production triggered by strenuous diaphragmatic contraction. Wistar rats treated with vehicle, the nonselective NO synthase inhibitor NG-nitro-l-arginine-methylester (l-NAME), or the NO donor diethylenetriamine-NONOate (DETA) were subjected to inspiratory resistive breathing (IRB; 50% of maximal inspiratory pressure) for 6 h or sham operation. Additional groups of rats were subjected to IRB for 6 h with concurrent administration of l-NAME and inhibitors of NF-κB (BAY-11-7082), ERK1/2 (PD98059), or P38 (SB203580). Inhibition of NO production (with l-NAME) resulted in upregulation of IRB-induced diaphragmatic IL-6, IL-10, IL-2, TNF-α, and IL-1β levels by 50%, 53%, 60%, 47%, and 45%, respectively. In contrast, the NO donor (DETA) attenuated the IRB-induced cytokine upregulation to levels characteristic of quietly breathing animals. l-NAME augmented IRB-induced activation of MAPKs (P38 and ERK1/2) and NF-κB, whereas DETA triggered the opposite effect. NF-κB and ERK1/2 inhibition in l-NAME-treated animals blunted the l-NAME-induced cytokine upregulation except IL-6, whereas P38 inhibition blunted all (including IL-6) cytokine upregulation. NO downregulates IRB-induced cytokine production in the strenuously contracting diaphragm through its action on MAPKs and NF-κB.

Introduction and objectives Inspiratory resistive breathing (IRB), a hallmark of obstructive pulmonary diseases, is characterised by large negative intrathoracic pressures. IRB is shown to induce pulmonary inflammation in previously healthy rats. Src is a multifunctional kinase that is activated by phosphorylation upon mechanical stress and plays a significant role in inflammatory processes. The aim of our study was to investigate the role of Src in IRB-induced pulmonary inflammation. Methods Anaesthetised, tracheostomised rats were breathing spontaneously through a 2-way non rebreathing valve. The inspiratory port was connected to a resistance, setting peak tidal tracheal pressure at 50% of maximum (IRB). Quietly breathing animals served as controls. After 6 h of IRB, the mechanics of the respiratory system were assessed with the forced oscillation technique. Bronchoalveolar lavage (BAL) was performed to measure total and differential cell count and total protein levels. Phosphorylation of Src and ERK was detected in lung tissue samples by Western blot analysis at 30 min, 3 and 6 h of IRB. The Src inhibitor PP2 was administered intraperitoneally (1 mg/kg), 30 min prior to IRB, in a subgroup of animals. Results After 6 h of IRB, increased tissue elasticity was measured, compared to control. Increased BAL cellularity was also found (2-fold increase to control), due to raised numbers of both macrophages and neutrophils. Total protein levels were elevated in BAL fluid. Src activation was detected at 30 min of IRB (3-fold increase to control), while ERK was phosphorylated at 3 and 6 h. Inhibition of Src kinase attenuated the increase in tissue elasticity after 6 h of IRB. Following inhibition of Src kinase, the total cell number after 6 h of IRB was not increased compared to control. Neither macrophage nor neutrophil count was elevated after 6 h of IRB, following Src inhibition. Total protein levels were not altered by Src inhibition. Src inhibition attenuated the activation of ERK only at 3 h of IRB. Conclusion Src kinase activation partly mediates IRB-induced pulmonary inflammation.