Tracheal Instillation Research Articles

Correction: Effet of Combined Nitrogen Dioxide and Carbon Nanoparticle Exposure on Lung Function During Ovalbumin Sensitization in Brown Norway Rat.

[This corrects the article DOI: 10.1371/journal.pone.0045687.].

Cardiovascular Effects of Pulmonary Exposure to Titanium Dioxide Nanoparticles in ApoE Knockout Mice

Existing studies on the inhalation toxicology of titanium dioxide (TiO2) have focused on possible carcinogenic capacity; however researches on the cardiovascular effect are limited, particularly in terms of susceptible animal models. The present study examined the inhalation toxicology of nano-TiO2 in ApoE knockout mice (ApoE-/- mice), an atherosclerosis susceptible animal model. The nano-TiO2 particles used were anatase type and the diameter ranged from 5 to 10 nm. ApoE-/- mice were randomly divided into five groups (high dose group, median dose group, low dose group, PBS vehicle control group and the nontreatment control group), each of which were given tracheal instillation of nano-TiO2 at the dose of 100 microg, 50 microg and 10 microg and PBS solution per week respectively, totally for six weeks, while the nontreatment control group received no tracheal instillation. We measured various indicators of inflammation, endothelial dysfunction and lipid metabolism in serum, and determined plaque formation on the aorta. After six weeks of treatment, there was significant difference between the high dose group and PBS control group in terms of C reactive protein (CRP), nitric oxide (NO), endothelial nitric oxide synthases (eNOS), total cholesterol (TC) and high density lipoprotein cholesterol (HDL-C) in serum. The results also showed ratio of plaque area to luminal area and the ratio of the lipid-rich core area to plaque area in the median and high nano-TiO2 dose group significantly increased respectively in HE stained cross-sections. Our study showed that tracheal instillation of nano-TiO2 particles induced considerable systemic inflammation, endothelial dysfunction and lipid metabolism dysfunction, contributing to the progression of atherosclerosis.

EtOH consumption contributes to a ‘pro‐injury’ environment in the alcoholic lung.

Chronic alcohol consumption increases ICU morbidity and mortality due to acute respiratory distress syndrome (ARDS). One reason is that alcohol regulates alveolar epithelial sodium channels (ENaC) via oxidant injury. We used mice to mimic acute and chronic alcohol consumption and tested whether ethanol (EtOH) would affect lung fluid clearance by altering lung ENaC activity. Chronic ethanol animals were tracheally instilled and imaged in vivo to observe lung fluid clearance. Lung fluid clearance showed a latent increase in clearance rates in mice receiving EtOH. Ethanol mice given an acute tracheal instillation of LPS and imaged for four hours showed lung fluid clearance early compared to control mice. But, ethanol mice with overnight exposure to LPS, showed reduced alveolar fluid clearance like the alveolar flooding seen in ARDS. Together, these data suggest that chronic EtOH consumption leads to altered regulation of ENaC, contributing to a ‘pro‐injury’ environment in the alcoholic lung. Funding support: R00HL09226 awarded to MNH; Children's Center for Development in Lung Biology Grant awarded to MNH.

Surfactant Administration via Thin Catheter During Spontaneous Breathing: Randomized Controlled Trial

The primary aim of this randomized study was to describe the feasibility of early administration of surfactant via a thin catheter during spontaneous breathing (Take Care) and compare early mechanical ventilation (MV) requirement with the InSurE (Intubate, Surfactant, Extubate) procedure. Preterm infants, who were <32 weeks and stabilized with nasal continuous positive airway pressure (nCPAP) in the delivery room, were randomized to receive early surfactant treatment either by the Take Care or InSurE technique. Tracheal instillation of 100 mg/kg poractant α via 5-F catheter during spontaneous breathing under nCPAP was performed in the intervention group. In the InSurE procedure, infants were intubated, received positive pressure ventilation for 30 seconds after surfactant instillation, and placed on nCPAP immediately. One hundred infants in each group were analyzed. The MV requirement in the first 72 hours of life was significantly lower in the Take Care group when compared with the InSurE group (30% vs 45%, P = .02, odds ratio -0.52, 95% confidence interval -0.94 to -0.29). Mean duration of both nCPAP and MV were significantly shorter in the Take Care group (P values .006 and .002, respectively). Bronchopulmonary dysplasia rate was significantly lower among the infants treated with the Take Care technique (relative risk -0.27, 95% confidence interval -0.1 to -0.72) The Take Care technique is feasible for the treatment of respiratory distress syndrome in infants with very low birth weight. It significantly reduces both the need and duration of MV, and thus the bronchopulmonary dysplasia rate in preterm infants.

Ethanol Alters Alveolar Fluid Balance via Nadph Oxidase (NOX) Signaling to Epithelial Sodium Channels (ENaC) in the Lung

Chronic alcohol consumption is associated with increased incidence of ICU-related morbidity and mortality, primarily from acute respiratory distress syndrome (ARDS). However, the mechanisms involved are unknown. One explanation is that alcohol regulates epithelial sodium channels (ENaC) via oxidant signaling to promote a pro- injury environment. We used small rodent models to mimic acute and chronic alcohol consumption and tested the hypothesis that ethanol (EtOH) would affect lung fluid clearance by up-regulating ENaC activity in the lung. Fluorescence labeling of rat lung slices and in vivo mouse lung revealed an increase in ROS production in response to acute EtOH exposure. Using western blots and fluorescein-5-maleimide labeling, we conclude that EtOH exposure modifies cysteines of α-ENaC while data from single channel patch clamp analysis confirm that 0.16% EtOH increased ENaC activity in rat alveolar cells. In vivo lung fluid clearance demonstrated a latent increase in fluid clearance in mice receiving EtOH diet. Ethanol mice given a tracheal instillation of LPS demonstrated early lung fluid clearance compared to caloric control mice and C57Bl/6 mice. Standard biochemical techniques reveal that chronic EtOH consumption resulted in greater protein expression of the catalytic gp91phox subunit and the obligate Rac1 protein. Collectively these data suggest that chronic EtOH consumption may lead to altered regulation of ENaC, contributing to a ‘pro-injury’ environment in the alcohol lung.

Neonatal Lung Disease and Respiratory Failure

Neonatal Lung Disease and Respiratory Failure

3,5,4′-tri-O-acetylresveratrol Ameliorates Seawater Exposure-Induced Lung Injury by Upregulating Connexin 43 Expression in Lung

The aim of the present study was to examine the effects of 3,5,4′-tri-O-acetylresveratrol on connexin 43 (Cx43) in acute lung injury (ALI) in rats induced by tracheal instillation of artificial seawater. Different doses (50, 150, and 450 mg/kg) of 3,5,4′-tri-O-acetylresveratrol were administered orally for 7 days before modeling. Four hours after seawater inhalation, histological changes, contents of TNF-α, IL-1β and IL-10, and the expression of Cx43 in lungs were detected. Besides, the gap junction communication in A549 cells and human umbilical vein endothelial cells (HUVECs) challenged by seawater was also evaluated. Histological changes, increased contents of inflammatory factors, upregulation in gene level, and deregulation in protein level of Cx43 in lungs stimulated by seawater were observed. On the other hand, pretreatment with 3,5,4′-tri-O-acetylresveratrol significantly inhibited infiltration of inflammation, development of pulmonary edema, and contents of inflammatory mediators in lungs. Above all, 3,5,4′-tri-O-acetylresveratrol upregulated the expression of Cx43 in both gene and protein levels, and its intermediate metabolite, resveratrol, also enhanced the gap junction communication in the two cell lines. The results of the present study suggested that administration of 3,5,4′-tri-O-acetylresveratrol may be beneficial for treatment of inflammatorycellsin lung.

Topical application of phosphatidyl-inositol-3,5-bisphosphate for acute lung injury in neonatal swine.

Hypoxemic respiratory failure of the neonatal organism involves increased acid sphingomyelinase (aSMase) activity and production of ceramide, a second messenger of a pro-inflammatory pathway that promotes increased vascular permeability, surfactant alterations and alveolar epithelial apoptosis. We comparatively assessed the benefits of topical aSMase inhibition by either imipramine (Imi) or phosphatidylinositol-3,5-bisphosphate (PIP2) when administered into the airways together with surfactant (S) for fortification. In this translational study, a triple-hit acute lung injury model was used that entails repeated airway lavage, injurious ventilation and tracheal lipopolysaccharide instillation in newborn piglets subject to mechanical ventilation for 72 hrs. After randomization, we administered an air bolus (control), S, S+Imi, or S+PIP2. Only in the latter two groups we observed significantly improved oxygenation and ventilation, dynamic compliance and pulmonary oedema. S+Imi caused systemic aSMase suppression and ceramide reduction, whereas the S+PIP2 effect remained compartmentalized in the airways because of the molecule's bulky structure. The surfactant surface tensions improved by S+Imi and S+PIP2 interventions, but only to a minor extent by S alone. S+PIP2 inhibited the migration of monocyte-derived macrophages and granulocytes into airways by the reduction of CD14/CD18 expression on cell membranes and the expression of epidermal growth factors (amphiregulin and TGF-β1) and interleukin-6 as pro-fibrotic factors. Finally we observed reduced alveolar epithelial apoptosis, which was most apparent in S+PIP2 lungs. Exogenous surfactant “fortified” by PIP2, a naturally occurring surfactant component, improves lung function by topical suppression of aSMase, providing a potential treatment concept for neonates with hypoxemic respiratory failure.

Surfactant deactivation in a pediatric model induces hypovolemia and fluid shift to the extravascular lung compartment

Surfactant deficiency is the pivotal abnormality in Neonatal and Acute Respiratory Distress Syndrome. Surfactant deactivation can produce hypoxemia, loss of lung compliance, and pulmonary edema, but its circulatory consequences are less understood. To describe the sequential hemodynamic changes and pulmonary edema formation after surfactant deactivation in piglets. Surfactant deactivation was induced by tracheal instillation of polysorbate 20 in 15 anesthetized and mechanically ventilated Large White piglets. The hemodynamic consequences of surfactant deactivation were assessed at 30, 120, and 240min by transpulmonary thermodilution and traditional methods. Surfactant deactivation caused hypoxemia, reduced lung compliance, and progressively increased lung water content (P<0.01). Early hypovolemia was observed, with reductions of the global end-diastolic volume and stroke volume (P<0.05). Reduced cardiac output was observed at the end of the study (P<0.05). Standard monitoring was unable to detect these early preload alterations. Surprisingly, the bronchoalveolar protein content was greatly increased at the end of the study compared with baseline levels (P<0.01). This finding was inconsistent with the notion that the pulmonary edema induced by surfactant deactivation was exclusively caused by high surface tension. Hypovolemia develops early after surfactant deactivation, in part due to the resulting fluid shift from the intravascular compartment to the lungs.

Effet of Combined Nitrogen Dioxide and Carbon Nanoparticle Exposure on Lung Function During Ovalbumin Sensitization in Brown Norway Rat

The interaction of particulate and gaseous pollutants in their effects on the severity of allergic inflammation and airway responsiveness are not well understood. We assessed the effect of exposure to NO2 in the presence or absence of repetitive treatment with carbon nanoparticle (CNP) during allergen sensitization and challenges in Borwn-Norway (BN) rat, in order to assess their interactions on lung function and airway responses (AR) to allergen and methacholine (MCH), end-expiratory lung volume (EELV), bronchoalveolar lavage fluid (BALF) cellular content, serum and BALF cytokine levels and histological changes. Animals were divided into the following groups (n = 6): Control; CNP (Degussa-FW2): 13 nm, 0.5 mg/kg instilled intratracheally ×3 at 7-day intervals; OVA: ovalbumin-sensitised; OVA+CNP: both sensitized and exposed to CNP. Rats were divided into equal groups exposed either to air or to NO2, 10 ppm, 6 h/d, 5d/wk for 4 weeks. Exposure to NO2, significantly enhanced lung inflammation and airway reactivity, with a significantly larger effect in animals sensitized to allergen, which was related to a higher expression of TH1 and TH2-type cytokines. Conversely, exposure to NO2 in animals undergoing repeated tracheal instillation of CNP alone, increased BALF neutrophilia and enhanced the expression of TH1 cytokines: TNF-α and IFN-γ, but did not show an additive effect on airway reactivity in comparison to NO2 alone. The exposure to NO2 combined with CNP treatment and allergen sensitization however, unexpectedly resulted in a significant decrease in both airway reactivity to allergen and to methacholine, and a reduction in TH2-type cytokines compared to allergen sensitization alone. EELV was significantly reduced with sensitization, CNP treatment or both. These data suggest an immunomodulatory effect of repeated tracheal instillation of CNP on the proinflammatory effects of NO2 exposure in sensitized BN rat. Furthermore, our findings suggest that NO2, CNP and OVA sensitization may significantly slow overall lung growth in parenchymally mature animals.

Protein Kinase G-I Deficiency Induces Pulmonary Hypertension through Rho A/Rho Kinase Activation

Protein kinase G (PKG) plays an important role in the regulation of vascular smooth cell contractility and is a critical mediator of nitric oxide signaling, which regulates cardiovascular homeostasis. PKG-I-knockout (Prkg1(-/-)) mice exhibit impaired nitric oxide/cGMP-dependent vasorelaxation and systemic hypertension. However, it remains unknown whether PKG-I deficiency induces pulmonary hypertension. In this study, we characterized the hypertensive pulmonary phenotypes in Prkg1(-/-) mice and delineated the underlying molecular basis. We observed a significant increase in right ventricular systolic pressure in Prkg1(-/-) mice in the absence of systemic hypertension and left-sided heart dysfunction. In addition, we observed marked muscularization of distal pulmonary vessels in Prkg1(-/-) mice. Microangiography revealed impaired integrity of the pulmonary vasculature in Prkg1(-/-) mice. Mechanistically, PKG-I-mediated phosphorylation of Rho A Ser188 was markedly decreased, and the resultant Rho A activation was significantly increased in Prkg1(-/-) lung tissues, which resulted in Rho kinase activation. The i.t. administration of fasudil, a Rho kinase inhibitor, reversed the hypertensive pulmonary phenotype in Prkg1(-/-) mice. Taken together, these data show that PKG-I deficiency induces pulmonary hypertension through Rho A/Rho kinase activation-mediated vasoconstriction and pulmonary vascular remodeling.

Animal Model Methodology: Immunocompetent or Leucopenic Rats, Which Is the Best? Results from a Model of Experimental Pneumonia due to Derepressed Cephalosporinase-Producing Enterobacter cloacae

Background: The aim of this study was to compare the bactericidal activity of cefepime plus amikacin against experimental pneumonia induced by a stably derepressed cephalosporinase-producing Enterobacter cloacae strain in immunocompetent and leucopenic rats. Methods: Sixty Wistar rats were used. Leucopenia was induced in half of them by a single intravenous administration of 30 mg/kg cyclophosphamide, while the remaining rats received the same volume of saline. All rats were infected 96 h later by tracheal instillation of 8 log₁₀ colony-forming units of E. cloacae. Twelve rats (6 immunocompetent and 6 leucopenic) were sacrificed 6 h later to assess the initial bacterial burden to the lungs. Then, the remaining 48 rats received a combination of 60 mg/kg cefepime twice a day and 25 mg/kg amikacin once a day given intraperitoneally or the same volume of saline. Six rats per group (leucopenic or not, treated or not) were sacrificed 12 and 30 h after therapy started. Results: Spontaneous bacterial clearance with time was observed only in immunocompetent rats. Compared to untreated animals, antibiotic administration induced a decrease in lung bacterial titres in immunocompetent and leucopenic rats. The difference was statistically significant only in leucopenic rats. Conclusions: The use of leucopenic rats reduced spontaneous bacterial clearance in the lungs and increased the bactericidal effect of the antibiotic combination and ultimately the confidence in the reliability of the results.

Reduction of Alveolar Type II Cell Volume in Klotho‐deficient Mice With Premature Aging

Klotho mitigates aging in multiple organs. Genetic Klotho deficiency (KL−/−) leads to premature senescence, organ degeneration and death. Age‐related airspace enlargement has been reported in the lungs of KL−/− and haplo‐insufficient (KL+/−) mice (Nature 1997;390:45–51). To examine the relationship between Klotho deficiency and lung structure, we performed morphometry on the right lung of 8–12 wk old KL−/− (n=9) and wild type (KL+/+, n=5) mice. Lungs were fixed by tracheal instillation of 2.5% glutaraldehyde (25cmH2O airway pressure). τhb, harmonic mean thickness of diffusion barrier Mean±SD. Genotype KL+/+ KL−/− Body weight (g) 18.9±3.8 10.1±3.9 * Right lung vol (mL/kg) 25.55±4.21 28.87±8.03 τhb (μm) 0.549±0.028 0.556±0.049 Volume‐to‐lung volume ratio Alveolar ducts 0.045±0.007 0.056±0.020 † Septum 0.113±0.019 0.102±0.004 Capillary blood 0.040±0.007 0.036±0.002 Epithelium–type I 0.018±0.003 0.018±0.001 –type II 0.007±0.001 0.005±0.000 * Interstitium–collagen 0.026±0.004 0.023±0.002 –cell/matrix 0.004±0.001 0.004±0.001 Endothelium 0.018±0.003 0.018±0.001 p≤0.001, p&lt;0.05 vs. WT. KL−/− mice were small and frail. Per kg of body weight, volumes and surface areas of the lung and alveolar components were similar to WT except the volume density of alveolar ducts was 24% higher and that of alveolar type II cells 30% lower. We conclude that Klotho deficiency is associated with diminished reservoir of alveolar progenitor cells, which could contribute to premature aging of the lung.Supported by NIH F32 HL103043, RO1 AG19712

Effects of pulmonary surfactant in treatment of acute lung injury after cardiac surgery in infants

Acute lung injury is a severe complication after cardiac surgery performed with cardiopulmonary bypass in infant patients. Pulmonary surfactant has been successfully used in treating neonatal respiratory distress syndrome for many years. This study focused on exploring the clinical efficacy of exogenous pulmonary surfactant in treating infant patients with acute lung injury after cardiac surgery with the use of extracorporeal circulation. Twenty-three infants with ventricular septal defect (VSD) were enrolled in the study. None of the patients needed oxygen treatment nor ventilation before heart surgery. VSD repair operation was done under cardiopulmonary bypass, and acute lung injury was found postoperatively. Ten infant patients in the treatment group were given pulmonary surfactant by tracheal instillation during mechanical ventilating postoperatively. Thirteen patients in control group were randomly selected in the same period in hospital, and acute lung injury was diagnosed without pulmonary surfactant treatment after cardiopulmonary bypass. Blood samples were obtained from all the patients, and blood gas analysis was performed every 6 hours. Demographics (body weigh, age, gender, VSD diameter) and clinical characteristics (CPB time, oxygenation index, mean airway pressure, pH and PCO2) of all the patients were collected, and statistical analysis was done to compare the data between treatment and control group. In the first 24 hours after heart surgery, compared with the control group, a more increased oxygenation index (from 89.36 ± 12.69 to 285 ± 16.51) was observed in treatment group, and it was from 93.71 ± 11.82 to 133 ± 19.62 in the control group. There was a significant difference in oxygenation index between the two groups (P < 0.05). At the same time, the MAP (from 17.5 ± 3.18 to 10.4 ± 3.37) of the patients in comparison with the control group (from 18.2 ± 2.63 to 13.8 ± 2.55), a more significantly decreased MAP was observed in the treatment group (P < 0.05). There was no significant difference in pH and PCO2 between the two groups. The ventilation time in cardiac care unit was shorter in treatment group (17.43 ± 9.12) h compared with the control group [(30.97 ± 14.85) h, P < 0.05]. ICU stay time of treatment group (3.90 ± 1.34) d was shorter than that of control group [(6.18 ± 1.90) d, P < 0.05].Two infants of the control group died, but none in treatment group died. In this study, a satisfactory curative effect was observed for the treatment of acute lung injury with PS intratracheal instillation after heart surgery under cardiopulmonary bypass in infant patients. It can reduce the duration of mechanical ventilation and cardiac care unit stay and improve prognosis. In addition, this study was a pilot study and the limited sample size was probably the cause of insufficient statistical power. Further study of larger scale is needed.

Mitochondrial complex II is essential for hypoxia-induced pulmonary vasoconstriction of intra- but not of pre-acinar arteries

Alveolar hypoxia acutely elicits contraction of pulmonary arteries, leading to a rise in pulmonary arterial pressure (PAP) and shifting blood to better ventilated areas of the lung. The molecular mechanisms underlying this hypoxic pulmonary vasoconstriction (HPV) are still incompletely understood. Here, we investigated the role of succinate dehydrogenase (SDH; synonymous to mitochondrial complex II) in HPV, with particular emphasis on regional differences along the vascular bed and consequences for PAP and perfusion-to-ventilation matching, using mutant mice heterozygous for the SDHD subunit of complex II (SDHD(+/-)). Western blots revealed reduced protein content of complex II subunits SDHA, SDHB, and SDHC in lungs of SDHD(+/-) mice, despite unaffected mRNA content as determined by real-time PCR. Hypoxic pulmonary vasoconstriction of small (20-50 µm) intra-acinar and larger (51-100 µm) pre-acinar arteries was evaluated by videomorphometric analysis of precision-cut lung slices. The hypoxic response was detectable in pre-acinar arteries but absent from intra-acinar arteries of SDHD(+/-) mice. In isolated perfused lungs, basal PAP and its hypoxia-induced increase were indistinguishable between both mouse strains. Arterial oxygenation was measured after provocation of regional ventilatory failure by tracheal fluid instillation in anaesthetized mice, and it declined more in SDHD(+/-) than in wild-type mice. SDHD is required for the formation of a stable mitochondrial complex II and it is selectively important for HPV of intra-acinar vessels. This specialized vascular segment participates in perfusion-to-ventilation matching but does not significantly contribute to the acute hypoxic rise in PAP that results from more proximal vasoconstriction.

Veno-arterial difference of carbon dioxide as a predictor of low cardiac output in an experimental pediatric model

Cardiac output (CO) measurement is not a standard of care for critically ill children, but it can be estimated by indirect methods such as veno-arterial pCO2 difference (ΔVACO2). To determine the correlation between CO and ΔVACO2 and evaluate the usefulness of ΔVACO2 in the diagnosis of low CO in an experimental pediatric model. Thirty piglets weighing 4.8 ± 0.35 kg were anesthetized and monitored with transpulmonary thermodilution. Lung injury was induced with tracheal instillation of Tween 20®. Serial measurements of central venous and arterial blood gases, as well as CO, were obtained at baseline, 1, 2 and 4 h after lung injury induction. Low cardiac output (LCO) was defined as CO lower than 2.5 Llminlm². There was an inverse correlation between CO and ΔVACO2 (r = -0.36, p < 0.01). ΔVACO2 was 14 ± 8 mmHg in LCO state and 8 ± 6 mmHg when this condition was not present (p < 0.01). Area under the receiver operating characteristic (ROC) curves of ΔVACO2 and LCO state was 0.78 (0.68-0.86). The best cut-point was 8.9 mmHg to determine LCO with a sensibility 0.78, specificity 0.7, positive predictive value 0.27 and negative predictive value 0.96. In this model there was an inverse correlation between ΔVACO2 and CO. The best cutoff value to discard LCO was ΔVACO2 of 8.9 mmHg, indicating that under this value the presence of LCO is very unlikely.

Effect of schisandrin B on lung mRNA expression of transforming growth factor-beta1 signal transduction molecule in rat lungs exposed to silica

To investigate the effects of schisandrin B (Sch-B) on expression of transforming growth factor-beta1 (TGF-beta1) and signal transduction molecule mRNA in rat lungs exposed to SiO2, and explore the intervention mechanism of Sch-B on pulmonary fibrosis induced by SiO2. Ninety six Wistar rats were randomly divided into control (normal saline) group, SiO2 group and SiO2 plus Sch-B group. The rats were exposed to SiO2 by direct tracheal instillation to establish the silicotic animal models. SiO2 group and SiO2 plus Sch-B group were treated with 1 ml SiO2 (50 mg/ml) for each rat From the first day after model establishment, SiO2 plus Sch-B group were orally given Sch-B (80 mg/kg) a day, control group and silica group were orally given olive oil. On the 3rd, 7th, 14th and 28th days after treatment, 8 rats in each group were sacrificed and samples were collected. The histo-pathological examination of lung was performed by HE staining. The expression levels of TGF-beta1, TGF-betaR II and Smad4 mRNA in the lung tissues were detected by RT-PCR. The results of histo-pathological examination showed that in SiO2 group, lung tissues were injured obviously; the alveolar inflammation with alveolus interval edema and inflammation cell infiltration appeared on the 3rd and 7th days; the alveolus interval became thicker, became thicker, fibroblast and collagen matrix increased markedly on 14th day; the alveolar structure was damaged, alveolar wall thickened obviously, collagen aggravation and pulmonary fibrosis displayed on 28th day. The alveolar inflammation and pulmonary fibrosis in SiO2 plus Sch-B group were significantly less than those in SiO2 group. The expressions levels of TGF-beta1 TGF-betaR II and Smad4 mRNA (TGF-1beta: 1.03 +/- 0.31, 1.33 +/- 0.39,1.08 +/- 0.26, 0.82 +/- 0.16, TGF-betaR II: 0.65 +/- 0.11, 0.80 +/- 0.16, 0.83 +/- 0.24, 0.62 +/- 0.15, Smad4:0.87 +/- 0.15, 0.68 +/- 0.11, 0.78 +/- 0.19, 0.30 +/- 0.08) in SiO2 group were significantly higher than those in the control group (TGF-beta1:0.59 +/- 0.22, 0.55 +/- 0.25, 0.56 +/- 0.20, 0.55 +/- 0.12, TGR-betaR II :0.28 +/- 0.13, 0.31 +/- 0.15, 0.34 +/- 0.15, 0.27 +/- 0.09, Smad4:0.23 +/- 0.11, 0.40 +/- 0.12, 0.39 +/- 0.12, 0.18 +/- 0.06) (P < 0.01 or P < 0.05), but the expression level of TGF-beta1 mRNA was the highest on the 7th day. The expression levels of TGF-beta1 and Smad4 mRNA (TGF-beta1:0.68 +/- 0.28, 0.88 +/- 0.25, 0.75 +/- 0.11, 0.61 +/- 0.14,Smad4:0.25 +/- 0.12, 0.45 +/- 0.09, 0.44 +/- 0.07, 0.21 +/- 0.04) in SiO2 plus Sch-B group were significantly lower than those in SiO2 group (P < 0.01 or P < 0.05 ), but there were no significant differences of the TGFbetaR II mRNA expression levels between SiO2 group and SiO2 plus Sch-B group. Sch-B can reduce the pulmonary fibrosis induced by SiO2 through inhibition of the mRNA express of TGF-beta1 and Smad4 in the lung tissue, modulating the TGF-beta1/Smad4 signal transduction pathway and inhibiting the target gene activation.

Role of nitric oxide synthases in elastase-induced emphysema

Nitric oxide (NO) in combination with superoxide produces peroxynitrites and induces protein nitration, which participates in a number of chronic degenerative diseases. NO is produced at high levels in the human emphysematous lung, but its role in this disease is unknown. The aim of this study was to determine whether the NO synthases contribute to the development of elastase-induced emphysema in mice. nNOS, iNOS, and eNOS were quantified and immunolocalized in the lung after a tracheal instillation of elastase in mice. To determine whether eNOS or iNOS had a role in the development of emphysema, mice bearing a germline deletion of the eNOS and iNOS genes and mice treated with a pharmacological iNOS inhibitor were exposed to elastase. Protein nitration was determined by immunofluorescence, protein oxidation was determined by ELISA. Inflammation and MMP activity were quantified by cell counts, RT–PCR and zymography in bronchoalveolar lavage fluid. Cell proliferation was determined by Ki67 immunostaining. Emphysema was quantified morphometrically. iNOS and eNOS were diffusely upregulated in the lung of elastase-treated mice and a 12-fold increase in the number of 3-nitrotyrosine-expressing cells was observed. Over 80% of these cells were alveolar type 2 cells. In elastase-instilled mice, iNOS inactivation reduced protein nitration and increased protein oxidation but had no effect on inflammation, MMP activity, cell proliferation or the subsequent development of emphysema. eNOS inactivation had no effect. In conclusion, in the elastase-injured lung, iNOS mediates protein nitration in alveolar type 2 cells and alleviates oxidative injury. Neither eNOS nor iNOS are required for the development of elastase-induced emphysema.

Mild-stretch mechanical ventilation upregulates toll-like receptor 2 and sensitizes the lung to bacterial lipopeptide

IntroductionMechanical ventilation (MV) could prime the lung toward an inflammatory response if exposed to another insult such as bacterial invasion. The underlying mechanisms are not so far clear. Toll-like receptors (TLRs) allow the host to recognize selectively bacterial pathogens and in turn to trigger an immune response. We therefore hypothesized that MV modulates TLR2 expression and in turn modifies responsiveness to agonists such as bacterial lipopeptide (BLP).MethodBoth in vitro and in vivo experiments were conducted. First, TLR2 expression and protein were measured in the A549 pulmonary epithelial cell line submitted to 8-hour cyclic stretch (20% elongation; 20/minute rate). After a 24-hour period of cyclic stretch, the inflammatory response of the A549 cells to the synthetic BLP, Pam3CSK4, was tested after 8 hours of exposure. In a second set of experiments, healthy anesthetized and paralyzed rabbits were submitted to 8-hour MV (tidal volume = 12 ml/kg, zero end-expiratory pressure; FIO2 = 50%; respiratory rate = 20/minute) before being sacrificed for TLR2 lung expression assessment. The lung inflammatory response to BLP was then tested in animals submitted to 24-hour MV before being sacrificed 8 hours after the tracheal instillation of Pam3CSK4.ResultsCyclic stretch of human pulmonary epithelial cell lines increased both TLR2 mRNA and protein expression. Cells submitted to cyclic stretch also increased IL-6 and IL-8 secretion in response to Pam3CSK4, a classical TLR2 ligand. A mild-stretch MV protocol induced a 60-fold increase of TLR2 mRNA expression in lung tissue when compared with spontaneously breathing controls. Moreover, the combination of MV and airway exposure to Pam3CSK4 acted synergistically in causing lung inflammation and injury.ConclusionsMild-stretch MV increases lung expression of TLR2 and sensitizes the lung to bacterial TLR2 ligands. This may account for the propensity of mechanically ventilated patients to develop acute lung injury in the context of airway bacterial colonization/infection.

Experimental Validation of Cardiac Index Measurement Using Transpulmonary Thermodilution Technique in Neonatal Total Liquid Ventilation

This study aimed to assess the precision and the interchangeability of cardiac index measurement by transpulmonary thermodilution (TPTD) and pulmonary thermodilution (PTD) devices on a neonatal animal model of acute respiratory distress syndrome under total liquid ventilation (TLV) and conventional mechanical ventilation (CMV). After acute respiratory distress induction by tracheal instillation of hydrochloric acid, transpulmonary (CI(TPTD)) and pulmonary (CI(PTD)) cardiac index were simultaneously measured every 30 minutes for a 240-minute experiment. Reproducibility of both thermodilution techniques was very good to excellent in both groups of ventilation with intrainstrument intraclass correlation coefficient >0.60. Disagreement was found between TPTD and PTD in TLV and CMV. Bland-Altman analysis revealed mean biases of 0.98 L/min/m² (22.8%) with limits of agreement of -1.33 to 3.25 L/min/m² in CMV and 1.28 L/min/m² (17.3%) with limits of agreement of -1.17 to 3.72 L/min/m² in TLV. Bias between TPTD and PTD was not statistically different in TLV than in CMV (p = 0.11). Transpulmonary thermodilution and PTD remained precise but not interchangeable techniques under TLV as well as CMV. Because TLV does not bring additional bias between both thermodilution techniques, we advocate the use of the less-invasive TPTD under TLV as currently recommended in CMV.