High-dose acetylsalicylic acid is superior to low-dose as well as to clopidogrel in preventing lipopolysaccharide-induced lung injury in mice

BACKGROUND: Use of aspirin (acetylsalicylic acid [ASA]) was found to improve outcome in animal models of acute lung injury (ALI) or its more severe form, acute respiratory distress syndrome. In patients with acute respiratory distress syndrome, data indicating a protective effect of ASA are less convincing. We hypothesize that ASA in a high dose is superior to low-dose ASA in preventing lung injury. Also, the effect on lung injury of inhibiting platelet activation by clopidogrel was investigated. METHODS: Acute lung injury was induced by intranasal instillation of 10 μg lipopolysaccharide (LPS). Before LPS, BALB/c mice were pretreated with either high dose of ASA (100 μg/g intraperitoneally, low-dose ASA (12.5 μg/g i.p), clopidogrel (50 μg/g i.p), or clopidogrel in combination with low dose of ASA. Controls received vehicle or LPS without intervention. Five hours after LPS, bronchoalveolar lavage fluid (BALF) and plasma were obtained. MEASUREMENTS AND MAIN RESULTS: All treatment regimens reduced neutrophil influx in the BALF compared with LPS controls (high-dose ASA 75% ± 2% [mean ± SD], low-dose ASA 86% ± 3%, clopidogrel 82% ± 1%, and low-dose ASA-clopidogrel 82% ± 3% vs. LPS control 88% ± 2%; P ≤ 0.05). High-dose ASA reduced BALF levels of protein compared with LPS controls (median [interquartile range], 0.2 [15] vs. 75 [20] pg/mL; P < 0.01), to a greater extent than after low-dose ASA (48 [32] pg/mL), clopidogrel (37 [23] pg/mL), or low-dose ASA-clopidogrel (57 [8] pg/mL). CONCLUSIONS: High-dose ASA is superior to low-dose ASA, clopidogrel, and to a combination of clopidogrel and low-dose ASA in attenuating LPS-induced lung injury in mice, suggesting high-dose ASA to be the antiplatelet therapy of choice in further research on preventing ALI

TLR2 deficiency aggravates lung injury caused by mechanical ventilation

Innate immunity pathways are found to play an important role in ventilator-induced lung injury. We analyzed pulmonary expression of Toll-like receptor 2 (TLR2) in humans and mice and determined the role of TLR2 in the pathogenesis of ventilator-induced lung injury in mice. Toll-like receptor 2 gene expression was analyzed in human bronchoalveolar lavage fluid (BALF) cells and murine lung tissue after 5 h of ventilation. In addition, wild-type (WT) and TLR2 knockout (KO) mice were ventilated with either lower tidal volumes (VT) of 7 mL/kg with positive end-expiratory pressure (PEEP) or higher VT of 15 mL/kg without PEEP for 5 h. Spontaneously breathing mice served as controls. Total protein and immunoglobulin M levels in BALF, neutrophil influx into the alveolar compartment, and interleukin 6 (IL-6), IL-1β, and keratinocyte-derived chemokine concentrations in lung tissue homogenates were measured. We observed enhanced TLR2 gene expression in BALF cells of ventilated patients and in lung tissue of ventilated mice. In WT mice, ventilation with higher VT without PEEP resulted in lung injury and inflammation with higher immunoglobulin M levels, neutrophil influx, and levels of inflammatory mediators compared with controls. In TLR2 KO mice, neutrophil influx and IL-6, IL-1β, and keratinocyte-derived chemokine were enhanced by this ventilation strategy. Ventilation with lower VT with PEEP only increased neutrophil influx and was similar in WT and TLR2 KO mice. In summary, injurious ventilation enhances TLR2 expression in lungs. Toll-like receptor 2 deficiency does not protect lungs from ventilator-induced lung injury. In contrast, ventilation with higher VT without PEEP aggravates inflammation in TLR2 KO mic

The Extent of Ventilator-Induced Lung Injury in Mice Partly Depends on Duration of Mechanical Ventilation

Background. Mechanical ventilation (MV) has the potential to initiate ventilator-induced lung injury (VILI). The pathogenesis of VILI has been primarily studied in animal models using more or less injurious ventilator settings. However, we speculate that duration of MV also influences severity and character of VILI. Methods. Sixty-four healthy C57Bl/6 mice were mechanically ventilated for 5 or 12 hours, using lower tidal volumes with positive end-expiratory pressure (PEEP) or higher tidal volumes without PEEP. Fifteen nonventilated mice served as controls. Results. All animals remained hemodynamically stable and survived MV protocols. In both MV groups, PaO2 to FiO2 ratios were lower and alveolar cell counts were higher after 12 hours of MV compared to 5 hours. Alveolar-capillary permeability was increased after 12 hours compared to 5 hours, although differences did not reach statistical significance. Lung levels of inflammatory mediators did not further increase over time. Only in mice ventilated with increased strain, lung compliance declined and wet to dry ratio increased after 12 hours of MV compared to 5 hours. Conclusions. Deleterious effects of MV are partly dependent on its duration. Even lower tidal volumes with PEEP may initiate aspects of VILI after 12 hours of M

The receptor for advanced glycation end products in ventilator-induced lung injury

Mechanical ventilation (MV) can cause ventilator-induced lung injury (VILI). The innate immune response mediates this iatrogenic inflammatory condition. The receptor for advanced glycation end products (RAGE) is a multiligand receptor that can amplify immune and inflammatory responses. We hypothesized that RAGE signaling contributes to the pro-inflammatory state induced by MV. RAGE expression was analyzed in lung brush and lavage cells obtained from ventilated patients and lung tissue of ventilated mice. Healthy wild-type (WT) and RAGE knockout (KO) mice were ventilated with relatively low (approximately 7.5 ml/kg) or high (approximately 15 ml/kg) tidal volume. Positive end-expiratory pressure was set at 2 cm H2O during both MV strategies. Also, WT and RAGE KO mice with lipopolysaccharide (LPS)-induced lung injury were ventilated with the above described ventilation strategies. In separate experiments, the contribution of soluble RAGE, a RAGE isoform that may function as a decoy receptor, in ventilated RAGE KO mice was investigated. Lung wet-to-dry ratio, cell and neutrophil influx, cytokine and chemokine concentrations, total protein levels, soluble RAGE, and high-mobility group box 1 (HMGB1) presence in lung lavage fluid were analyzed. MV was associated with increased RAGE mRNA levels in both human lung brush samples and lung tissue of healthy mice. In healthy high tidal volume-ventilated mice, RAGE deficiency limited inflammatory cell influx. Other VILI parameters were not affected. In our second set of experiments where we compared RAGE KO and WT mice in a 2-hit model, we observed higher pulmonary cytokine and chemokine levels in RAGE KO mice undergoing LPS/high tidal volume MV as compared to WT mice. Third, in WT mice undergoing the LPS/high tidal volume MV, we observed HMGB1 presence in lung lavage fluid. Moreover, MV increased levels of soluble RAGE in lung lavage fluid, with the highest levels found in LPS/high tidal volume-ventilated mice. Administration of soluble RAGE to LPS/high tidal volume-ventilated RAGE KO mice attenuated the production of inflammatory mediators. RAGE was not a crucial contributor to the pro-inflammatory state induced by MV. However, the presence of sRAGE limited the production of pro-inflammatory mediators in our 2-hit model of LPS and high tidal volume M

Pre-Treatment with Allopurinol or Uricase Attenuates Barrier Dysfunction but Not Inflammation during Murine Ventilator-Induced Lung Injury

<div><h3>Introduction</h3><p>Uric acid released from injured tissue is considered a major endogenous danger signal and local instillation of uric acid crystals induces acute lung inflammation via activation of the NLRP3 inflammasome. Ventilator-induced lung injury (VILI) is mediated by the NLRP3 inflammasome and increased uric acid levels in lung lavage fluid are reported. We studied levels in human lung injury and the contribution of uric acid in experimental VILI.</p> <h3>Methods</h3><p>Uric acid levels in lung lavage fluid of patients with acute lung injury (ALI) were determined. In a different cohort of cardiac surgery patients, uric acid levels were correlated with pulmonary leakage index. In a mouse model of VILI the effect of allopurinol (inhibits uric acid synthesis) and uricase (degrades uric acid) pre-treatment on neutrophil influx, up-regulation of adhesion molecules, pulmonary and systemic cytokine levels, lung pathology, and regulation of receptors involved in the recognition of uric acid was studied. In addition, total protein and immunoglobulin M in lung lavage fluid and pulmonary wet/dry ratios were measured as markers of alveolar barrier dysfunction.</p> <h3>Results</h3><p>Uric acid levels increased in ALI patients. In cardiac surgery patients, elevated levels correlated significantly with the pulmonary leakage index. Allopurinol or uricase treatment did not reduce ventilator-induced inflammation, IκB-α degradation, or up-regulation of NLRP3, Toll-like receptor 2, and Toll-like receptor 4 gene expression in mice. Alveolar barrier dysfunction was attenuated which was most pronounced in mice pre-treated with allopurinol: both treatment strategies reduced wet/dry ratio, allopurinol also lowered total protein and immunoglobulin M levels.</p> <h3>Conclusions</h3><p>Local uric acid levels increase in patients with ALI. In mice, allopurinol and uricase attenuate ventilator-induced alveolar barrier dysfunction.</p> </div

S100A8/A9 presence in lung tissue increases in mice with lung injury.

<p>Representative images of immunohistochemical stainings of S100A8 and S100A9 (specific staining in red, background staining in blue) of murine lung sections. Wild-type mice were spontaneously breathing (C), mechanically ventilated for 5 hours with high tidal volume (HV_T MV), received intranasal lipopolysaccharide (LPS; 0.25 mg/kg) followed by spontaneously breathing for 5 hours (LPS), or received intranasal LPS followed by 5 hours of HV_T mechanical ventilation (HV_T MV+LPS). Magnification 10×, detailed view of the HV_T MV+LPS group: magnification 100×.</p

Histopathological changes were reduced in S100A9 knockout mice undergoing 2-hit lung injury.

<p>Representative histological sections of hematoxylin and eosin stained lungs of a wild-type (WT) mouse (A) and a S100A9 knockout (KO) (B) mouse exposed to lipopolysaccharide (LPS) and high tidal volume mechanical ventilation (HV_T MV). Original magnification x20 (in set x40).</p

Histopathology of VILI in mice is affected by allopurinol pre-treatment.

<p>Lung tissue slides of mice pre-treated with vehicle (10% dimethylsulfoxide, dark grey bars), uricase (0.2 mg/kg, light grey, striped bars), or allopurinol (25 mg/kg (white bars) 1 hour before start of mechanical ventilation (VILI). Spontaneously breathing, vehicle pre-treated mice served as controls (C). Lungs were scored for presence of edema, hemorrhage, neutrophil influx, and hyaline membranes. Total scores and edema scores are demonstrated below. Data are presented as mean ± SEM of 4 control mice and n = 9 for the VILI groups. *p<0.05 vs vehicle control H&E staining, magnification ×20.</p

Uric acid levels in murine ventilator-induced lung injury.

<p>Uric acid levels in plasma (<b>A</b>) and bronchoalveolar lavage fluid (BALF) (<b>B</b>) of mice pre-treated with vehicle (10% dimethylsulfoxide, dark grey bars), uricase (0.2 mg/kg, light grey, striped bars), or allopurinol (25 mg/kg (white bars) 1 hour before start of 5 hours of mechanical ventilation (VILI). Spontaneously breathing, vehicle pre-treated mice served as controls (C). Data represent mean ± SEM of 4 control mice and n = 6−9 ventilated mice. **p<0.01 vs. control, #p<0.05 vs. vehicle ventilated.</p

Exogenous S100A8/A9 proteins amplify VILI via Toll-like receptor 4.

<p>Total protein (A), immunoglobulin M (IgM) (B) Neutrophil influx (C), interleukin (IL)-6 (D), keratinocyt-derived chemokine (KC) (E), macrophage inflammatory protein (MIP)-2 (F), IL-1β (G) and tumor necrosis factor (TNF)-α concentrations (H) in C3H-HeN and C3H-HeJ mice exposed to S100A8/A9 (30 µg/mouse) or vehicle intratracheally at start of high tidal volume mechanical ventilation. Mice were ventilated for 5 hours. Data represent mean (SEM) of 7–8 mice per group. *p<0.05, **p<0.01, ***p<0.001 versus vehicle treated mice. ^#p<0.05 S100A8 versus S100A8/A9 treated mice.</p