Milena V. de Oliveira

Bone marrow-derived mononuclear cells vs. mesenchymal stromal cells in experimental allergic asthma.

We compared the effects of bone marrow-derived mononuclear cells (BMMCs) and mesenchymal stromal cells (MSCs) on airway inflammation and remodeling and lung mechanics in experimental allergic asthma. C57BL/6 mice were sensitized and challenged with ovalbumin (OVA group). A control group received saline using the same protocol. Twenty-four hours after the last challenge, groups were further randomized into subgroups to receive saline, BMMCs (2×10(6)) or MSCs (1×10(5)) intratracheally. BMMC and MSC administration decreased cell infiltration, bronchoconstriction index, alveolar collapse, collagen fiber content in the alveolar septa, and interleukin (IL)-4, IL-13, transforming growth factor (TGF)-β and vascular endothelial growth factor (VEGF) levels compared to OVA-SAL. Lung function, alveolar collapse, collagen fiber deposition in alveolar septa, and levels of TGF-β and VEGF improved more after BMMC than MSC therapy. In conclusion, intratracheal BMMC and MSC administration effectively modulated inflammation and fibrogenesis in an experimental model of asthma, but BMMCs was associated with greater benefit in terms of reducing levels of fibrogenesis-related growth factors.

Effects of bone marrow-derived mononuclear cells from healthy or acute respiratory distress syndrome donors on recipient lung-injured mice.

Objective:The advantage of using autologous bone marrow–derived mononuclear cells to treat acute respiratory distress syndrome patients is to prevent immunological rejection. However, bone marrow–derived mononuclear cells may be altered by different acute respiratory distress syndrome etiologies, resulting in questionable efficacy and thus limited clinical application. We aimed to investigate the effects of bone marrow–derived mononuclear cells obtained from healthy and acute respiratory distress syndrome donors on pulmonary and extrapulmonary acute respiratory distress syndrome. Design:Prospective, randomized, controlled experimental study. Setting:University research laboratory. Subjects:Two hundred and twenty-five C57BL/6 mice. Interventions:Acute respiratory distress syndrome was induced by Escherichia coli lipopolysaccharide intratracheally (ARDSp) or intraperitoneally (ARDSexp). Control mice (Healthy) received saline solution intratracheally (Cp) or intraperitoneally (Cexp). After 24 hours, whole bone marrow cells were analyzed in vitro: 1) colony-forming unit–fibroblasts and 2) hematopoietic stem cells, neutrophils, T helper lymphocytes, B lymphocytes, and nonhematopoietic precursors. After cell characterization, all groups received saline or bone marrow–derived mononuclear cells (2 × 106), obtained from Cp, Cexp, ARDSp, and ARDSexp donor mice, IV, on day 1. Measurements and Main Results:On day 1, in ARDSp, different patterns of colony formation were found, with nonstromal cells (mainly neutrophils) predominating over fibroblastoid colonies. In ARDSexp, irregular colony-forming unit–fibroblasts morphology with dispersed proliferating colonies and a greater number of hematopoietic stem cells were observed. In ARDSp, colony-forming unit–fibroblasts count was higher but not measurable in ARDSexp. In ARDSp, monocytes and T lymphocytes were increased and hematopoietic precursor cells reduced, with no significant changes in ARDSexp. On day 7, bone marrow–derived mononuclear cells improved survival and attenuated changes in lung mechanics, alveolar collapse, inflammation, pulmonary fibrosis, and apoptosis in the lung and distal organs, regardless of donor type. Conclusions:Bone marrow–derived mononuclear cells from ARDSp and ARDSexp donors showed different characteristics but were as effective as cells obtained from healthy donors in reducing inflammation and remodeling, suggesting the utility of autologous transplant of bone marrow–derived mononuclear cells in the clinical setting.

Therapeutic effects of LASSBio-596 in an elastase-induced mouse model of emphysema.

Emphysema is an intractable pulmonary disease characterized by an inflammatory process of the airways and lung parenchyma and ongoing remodeling process in an attempt to restore lung structure. There is no effective drug therapy that regenerates lung tissue or prevents the progression of emphysema; current treatment is aimed at symptomatic relief. We hypothesized that LASSBio-596, a molecule with potent anti-inflammatory and immunomodulatory effects, might reduce pulmonary inflammation and remodeling and thus improve lung function in experimental emphysema. Emphysema was induced in BALB/c mice by intratracheal administration of porcine pancreatic elastase (0.1 IU) once weekly during 4 weeks. A control group received saline using the same protocol. After the last instillation of saline or elastase, dimethyl sulfoxide, or LASSBio-596 were administered intraperitoneally, once daily for 8 days. After 24 h, in elastase-induced emphysema animals, LASSBio-596 yielded: (1) decreased mean linear intercept, hyperinflation and collagen fiber content, (2) increased elastic fiber content, (3) reduced number of M1 macrophages, (4) decreased tumor necrosis factor-α, interleukin-1β, interleukin-6, and transforming growth factor-β protein levels in lung tissue, and increased vascular endothelial growth factor. These changes resulted in increased static lung elastance. In conclusion, LASSBio-596 therapy reduced lung inflammation, airspace enlargement, and small airway wall remodeling, thus improving lung function, in this animal model of elastase-induced emphysema.

Characterization of a Mouse Model of Emphysema Induced by Multiple Instillations of Low-Dose Elastase

Many experimental models have been proposed to study the pathophysiological features of emphysema, as well as to search for new therapeutic approaches for acute or chronically injured lung parenchyma. We aimed to characterize an emphysema model induced by multiple instillations of elastase by tracking changes in inflammation, remodeling, and cardiac function after each instillation. Forty-eight C57BL/6 mice were randomly assigned across two groups. Emphysema (ELA) animals received 1, 2, 3, or 4 intratracheal instillations of pancreatic porcine elastase (PPE, 0.2 IU) with a 1-week interval between them. Controls (C) received saline following the same protocol. Before and after implementation of the protocol, animals underwent echocardiographic analysis. After the first instillation of PPE, the percentage of mononuclear cells in the lung parenchyma increased compared to C (p = 0.0001). The second instillation resulted in hyperinflated alveoli, increased mean linear intercept, and reduced elastic fiber content in lung parenchyma compared to C (p = 0.0197). Following the third instillation, neutrophils and collagen fiber content in alveolar septa and airways increased, whereas static lung elastance was reduced compared to C (p = 0.0094). After the fourth instillation, the percentage of M1 macrophages in lungs; levels of interleukin-1β (IL-1β), keratinocyte-derived chemokine, hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF); and collagen fiber content in the pulmonary vessel wall were increased compared to C (p = 0.0096). At this time point, pulmonary arterial hypertension was apparent, with increased diastolic right ventricular wall thickness. In conclusion, the initial phase of emphysema was characterized by lung inflammation with predominance of mononuclear cells, whereas at the late stage, impairment of pulmonary and cardiovascular functions was observed. This model enables analysis of therapies at different time points during controlled progression of emphysema. Accordingly, early interventions could focus on the inflammatory process, while late interventions should focus on restoring cardiorespiratory function.

Comparison between Variable and Conventional Volume-Controlled Ventilation on Cardiorespiratory Parameters in Experimental Emphysema.

Emphysema is characterized by loss of lung tissue elasticity and destruction of structures supporting alveoli and capillaries. The impact of mechanical ventilation strategies on ventilator-induced lung injury (VILI) in emphysema is poorly defined. New ventilator strategies should be developed to minimize VILI in emphysema. The present study was divided into two protocols: (1) characterization of an elastase-induced emphysema model in rats and identification of the time point of greatest cardiorespiratory impairment, defined as a high specific lung elastance associated with large right ventricular end-diastolic area; and (2) comparison between variable (VV) and conventional volume-controlled ventilation (VCV) on lung mechanics and morphometry, biological markers, and cardiac function at that time point. In the first protocol, Wistar rats (n = 62) received saline (SAL) or porcine pancreatic elastase (ELA) intratracheally once weekly for 4 weeks, respectively. Evaluations were performed 1, 3, 5, or 8 weeks after the last intratracheal instillation of saline or elastase. After identifying the time point of greatest cardiorespiratory impairment, an additional 32 Wistar rats were randomized into the SAL and ELA groups and then ventilated with VV or VCV (n = 8/group) [tidal volume (VT) = 6 mL/kg, positive end-expiratory pressure (PEEP) = 3 cmH2O, fraction of inspired oxygen (FiO2) = 0.4] for 2 h. VV was applied on a breath-to-breath basis as a sequence of randomly generated VT values (mean VT = 6 mL/kg), with a 30% coefficient of variation. Non-ventilated (NV) SAL and ELA animals were used for molecular biology analysis. The time point of greatest cardiorespiratory impairment, was observed 5 weeks after the last elastase instillation. At this time point, interleukin (IL)-6, cytokine-induced neutrophil chemoattractant (CINC)-1, amphiregulin, angiopoietin (Ang)-2, and vascular endothelial growth factor (VEGF) mRNA levels were higher in ELA compared to SAL. In ELA animals, VV reduced respiratory system elastance, alveolar collapse, and hyperinflation compared to VCV, without significant differences in gas exchange, but increased right ventricular diastolic area. Interleukin-6 mRNA expression was higher in VCV and VV than NV, while surfactant protein-D was increased in VV compared to NV. In conclusion, VV improved lung function and morphology and reduced VILI, but impaired right cardiac function in this model of elastase induced-emphysema.

Impact of Different Ventilation Strategies on Driving Pressure, Mechanical Power, and Biological Markers During Open Abdominal Surgery in Rats

BACKGROUND: Intraoperative mechanical ventilation may yield lung injury. To date, there is no consensus regarding the best ventilator strategy for abdominal surgery. We aimed to investigate the impact of the mechanical ventilation strategies used in 2 recent trials (Intraoperative Protective Ventilation [IMPROVE] trial and Protective Ventilation using High versus Low PEEP [PROVHILO] trial) on driving pressure (&Dgr;PRS), mechanical power, and lung damage in a model of open abdominal surgery. METHODS: Thirty-five Wistar rats were used, of which 28 were anesthetized, and a laparotomy was performed with standardized bowel manipulation. Postoperatively, animals (n = 7/group) were randomly assigned to 4 hours of ventilation with: (1) tidal volume (VT) = 7 mL/kg and positive end-expiratory pressure (PEEP) = 1 cm H2O without recruitment maneuvers (RMs) (low VT/low PEEP/RM−), mimicking the low-VT/low-PEEP strategy of PROVHILO; (2) VT = 7 mL/kg and PEEP = 3 cm H2O with RMs before laparotomy and hourly thereafter (low VT/moderate PEEP/4 RM+), mimicking the protective ventilation strategy of IMPROVE; (3) VT = 7 mL/kg and PEEP = 6 cm H2O with RMs only before laparotomy (low VT/high PEEP/1 RM+), mimicking the strategy used after intubation and before extubation in PROVHILO; or (4) VT = 14 mL/kg and PEEP = 1 cm H2O without RMs (high VT/low PEEP/RM−), mimicking conventional ventilation used in IMPROVE. Seven rats were not tracheotomized, operated, or mechanically ventilated, and constituted the healthy nonoperated and nonventilated controls. RESULTS: Low VT/moderate PEEP/4 RM+ and low VT/high PEEP/1 RM+, compared to low VT/low PEEP/RM− and high VT/low PEEP/RM−, resulted in lower &Dgr;PRS (7.1 ± 0.8 and 10.2 ± 2.1 cm H2O vs 13.9 ± 0.9 and 16.9 ± 0.8 cm H2O, respectively; P< .001) and less mechanical power (63 ± 7 and 79 ± 20 J/min vs 110 ± 10 and 120 ± 20 J/min, respectively; P = .007). Low VT/high PEEP/1 RM+ was associated with less alveolar collapse than low VT/low PEEP/RM− (P = .03). E-cadherin expression was higher in low VT/moderate PEEP/4 RM+ than in low VT/low PEEP/RM− (P = .013) or high VT/low PEEP/RM− (P = .014). The extent of alveolar collapse, E-cadherin expression, and tumor necrosis factor-alpha correlated with &Dgr;PRS (r = 0.54 [P = .02], r = −0.48 [P = .05], and r = 0.59 [P = .09], respectively) and mechanical power (r = 0.57 [P = .02], r = −0.54 [P = .02], and r = 0.48 [P = .04], respectively). CONCLUSIONS: In this model of open abdominal surgery based on the mechanical ventilation strategies used in IMPROVE and PROVHILO trials, lower mechanical power and its surrogate &Dgr;PRS were associated with reduced lung damage.

Animal Models of Chronic Obstructive Pulmonary Disease Exacerbations: A Review of the Current Status

Chronic obstructive pulmonary disease (COPD), characterized by airflow limitation and manifested as emphysema and chronic airway obstruction, is a major cause of morbidity and mortality worldwide, resulting in an economic and social burden that is both substantial and increasing. The natural history of COPD involves systemic manifestations, such as skeletal muscle wasting and cardiovascular impairment, and frequent exacerbations. The latter are caused by bacterial or viral infections and have major implications for patients and healthcare systems. There are no effective therapies to prevent or reverse these events. Smoking cessation remains the most effective intervention for reducing disease progression. Animal models of COPD and of COPD exacerbations have been developed to advance understanding of the pathogenesis of COPD and the role of infections in its severity. Cigarette smoke exposure, tracheal instillation of elastase, and genetic manipulation are commonly used to reproduce baseline COPD conditions, each with its advantages and disadvantages. Intratracheal instillation of lipopolysaccharide (LPS), bacteria, or viruses are the most common interventions used to exacerbate baseline COPD. This review highlights the three major animal models used for induction of emphysema and its exacerbations. Further exploration of these models should facilitate identification of new therapeutic approaches for COPD.

Biologic Impact of Mechanical Power at High and Low Tidal Volumes in Experimental Mild Acute Respiratory Distress Syndrome.

Background: The authors hypothesized that low tidal volume (VT) would minimize ventilator-induced lung injury regardless of the degree of mechanical power. The authors investigated the impact of power, obtained by different combinations of VT and respiratory rate (RR), on ventilator-induced lung injury in experimental mild acute respiratory distress syndrome (ARDS). Methods: Forty Wistar rats received Escherichia coli lipopolysaccharide intratracheally. After 24 h, 32 rats were randomly assigned to be mechanically ventilated (2 h) with a combination of different VT (6 ml/kg and 11 ml/kg) and RR that resulted in low and high power. Power was calculated as energy (&Dgr;P,L2/E,L) × RR (&Dgr;P,L = transpulmonary driving pressure; E,L = lung elastance), and was threefold higher in high than in low power groups. Eight rats were not mechanically ventilated and used for molecular biology analysis. Results: Diffuse alveolar damage score, which represents the severity of edema, atelectasis, and overdistension, was increased in high VT compared to low VT, in both low (low VT: 11 [9 to 14], high VT: 18 [15 to 20]) and high (low VT: 19 [16 to 25], high VT: 29 [27 to 30]) power groups. At high VT, interleukin-6 and amphiregulin expressions were higher in high-power than in low-power groups. At high power, amphiregulin and club cell protein 16 expressions were higher in high VT than in low VT. Mechanical energy and power correlated well with diffuse alveolar damage score and interleukin-6, amphiregulin, and club cell protein 16 expression. Conclusions: In experimental mild ARDS, even at low VT, high mechanical power promoted ventilator-induced lung injury. To minimize ventilator-induced lung injury, low VT should be combined with low power.

Ghrelin therapy improves lung and cardiovascular function in experimental emphysema

BackgroundEmphysema is a progressive disease characterized by irreversible airspace enlargement followed by a decline in lung function. It also causes extrapulmonary effects, such as loss of body mass and cor pulmonale, which are associated with shorter survival and worse clinical outcomes. Ghrelin, a growth-hormone secretagogue, stimulates muscle anabolism, has anti-inflammatory effects, promotes vasodilation, and improves cardiac performance. Therefore, we hypothesized that ghrelin might reduce lung inflammation and remodelling as well as improve lung mechanics and cardiac function in experimental emphysema.MethodsForty female C57BL/6 mice were randomly assigned into two main groups: control (C) and emphysema (ELA). In the ELA group (n=20), animals received four intratracheal instillations of pancreatic porcine elastase (PPE) at 1-week intervals. C animals (n=20) received saline alone (50 μL) using the same protocol. Two weeks after the last instillation of saline or PPE, C and ELA animals received ghrelin or saline (n=10/group) intraperitoneally (i.p.) daily, during 3 weeks. Dual-energy X-ray absorptiometry (DEXA), echocardiography, lung mechanics, histology, and molecular biology were analysed.ResultsIn elastase-induced emphysema, ghrelin treatment decreased alveolar hyperinflation and mean linear intercept, neutrophil infiltration, and collagen fibre content in the alveolar septa and pulmonary vessel wall; increased elastic fibre content; reduced M1-macrophage populations and increased M2 polarization; decreased levels of keratinocyte-derived chemokine (KC, a mouse analogue of interleukin-8), tumour necrosis factor-α, and transforming growth factor-β, but increased interleukin-10 in lung tissue; augmented static lung elastance; reduced arterial pulmonary hypertension and right ventricular hypertrophy on echocardiography; and increased lean mass.ConclusionIn the elastase-induced emphysema model used herein, ghrelin not only reduced lung damage but also improved cardiac function and increased lean mass. These findings should prompt further studies to evaluate ghrelin as a potential therapy for emphysema.

Distensibility index of the inferior vena cava in experimental acute respiratory distress syndrome.

We determined the accuracy of distensibility index of inferior vena cava (dIVC) for evaluation of fluid responsiveness in rats with acute respiratory distress syndrome (ARDS) and validated this index for use in rat models. In protocol 1, E. coli lipopolysaccharide was administered in Wistar rats (n=7). After 24h, animals were mechanically ventilated, and stroke volume (SV) and dIVC quantified after blood drainage and subsequent volume expansion (albumin 20%). A receiver operating characteristic (ROC) curve was plotted to determine the optimal dIVC cutoff. In protocol 2, rats (n=10) were divided into fluid-responders (SV increase >5%) and nonresponders (SV increase <5%). The dIVC cutoff obtained from protocol 1 was 25%. Fluid responders had a 2.5 relative risk of low dIVC (<25%). The sensitivity, specificity, positive predictive, and negative predictive values for dIVC were 74%, 62%, 59%, and 76%, respectively. In conclusion, a dIVC threshold <25% was associated with positive response after volume expansion and could be used to titrate fluids in endotoxin-induced ARDS.