We studied the following end points: histology, serum cytokine levels, hydroxyproline content, tryptase and proteinase-activated receptor-2 (PAR-2) protein level in lung homogenates, and tryptase and PAR-2 immunohistochemical localization in the lungs
We studied the following end points: histology, serum cytokine levels, hydroxyproline content, tryptase and proteinase-activated receptor-2 (PAR-2) protein level in lung homogenates, and tryptase and PAR-2 immunohistochemical localization in the lungs. Results All septic animals developed acute lung injury. controls ( 0.0001). However, protective ventilation attenuated sepsis-induced lung injury and decreased lung tryptase and PAR-2 protein levels. Immunohistochemical staining confirmed the presence of tryptase and PAR-2 in the lungs. Conclusions Mechanical ventilation modified tryptase and PAR-2 in injured lungs. Increased levels of these proteins were associated with development of sepsis and ventilator-induced pulmonary fibrosis early in the course of sepsis-induced lung injury. Introduction Acute lung injury during the acute respiratory distress syndrome (ARDS) complicates a variety of clinical conditions and is associated with significant morbidity and mortality [1]. Sepsis, the most common cause of ARDS, promotes or interferes with mechanisms involved in tissue repair [2]. Mechanical ventilation (MV) is an essential life support for sepsis/ARDS patients and manipulation of the ventilator strategy is the only confirmed treatment for improving survival [3]. However, MV can cause or aggravate lung injury, an entity referred to as ventilator-induced lung injury (VILI) [4]. Experimental and clinical studies [3-6] have provided 2-Naphthol insights into the physiology of VILI, demonstrating that some patterns of MV result in pulmonary and systemic changes that mimic ARDS and sepsis [2-4]. Many ARDS patients survive the underlying disease but die with pulmonary fibrosis [7,8]. Recent reports suggest that sepsis may trigger the development of persistent fibrosis [9] and that VILI may be a major contributor to lung fibrosis [10,11]. The role of MV as an inciting factor for lung fibrosis is usually poorly comprehended. Pulmonary fibrosis appears to be an important determinant of mortality, regardless of the cause of ARDS [11,12]. Areas of fibrosis are adjacent to inflammation in the early exudative phase of ARDS [13-15]. A feature of lungs in patients with fibrotic lung disease Mouse monoclonal to p53 is the increased number of mast cells [16] and it has been suggested that mast cells may support the continuation of the fibroproliferative process in patients with ARDS [17] by release of mediators. The most abundant product of mast cells is usually tryptase, a serine protease with pleiotropic biological activities [18]. Tryptases consist of -tryptase and -tryptase [19]. -tryptase is the main isoenzyme expressed in human lung. Tryptase upregulates the expression of cytokines [19] and vascular endothelial growth factor (VEGF) [20]. We have previously reported that MV modulates the innate immune response by interfering with Toll-like receptors [6] and increases VEGF by activating the Wnt/-catenin signaling pathways [10]. Tryptase is usually a potent stimulant of the synthesis of type I collagen by fibroblasts [21]. The mechanism by which tryptase exerts its effects is by activating 2-Naphthol a member of the protease-activated receptor (PAR) family, PAR-2 [22]. Currently, no published reports have examined the changes of tryptase and PAR-2 in the context of septic ARDS and VILI. We tested the hypothesis that tryptase content is modulated by MV and could contribute to the early development of pulmonary fibrosis in an experimental, 2-Naphthol clinically relevant animal model of sepsis-induced acute lung injury. Material and methods This protocol was approved by the Animal Care Committee at the Hospital Universitario Dr. Negrn (CEEBA-003/10), in accordance with the European Commission Directive 2010/63/EU for animal experimentation. This study followed the ARRIVE guidelines for reporting preclinical animal research [23]. Animal preparation and experimental protocol A total of 40 male Sprague-Dawley rats (300 to 350 g) were included. Animals were anesthetized with an intraperitoneal injection of ketamine hydrochloride (80 mg/kg) and xylazine (8 mg/kg) [24]. Animals were initially randomized to two groups: non-septic controls (n = 6) and septic (n = 34). Sepsis was induced by cecal ligation and perforation (CLP). CLP is considered the gold standard model for experimental sepsis [25]. A detailed description of this model is provided elsewhere [26]. All septic animals received 10 ml normal saline subcutaneously immediately after CLP for postoperative fluid resuscitation. Eighteen hours after CLP, the peritoneal cavity was reopened in surviving animals and the cecum was excised and removed distal to the ligature. We then washed the peritoneal cavity with 20 ml warm, normal saline, and gently squeezed the abdomen several times. After closing.At the end of the 4-h MV period, peak inspiratory pressure was 28 2 cmH2O in 2-Naphthol the high-VT group and 20 2 cmH2O in the low-VT group ( 0.0001). Histological examination of septic lungs revealed the presence of acute lung injury and pulmonary fibrosis (Figure?1A). and PAR-2 protein levels compared to septic controls ( 0.0001). However, protective ventilation attenuated sepsis-induced lung injury and decreased lung tryptase and PAR-2 protein levels. Immunohistochemical staining confirmed the presence of tryptase and PAR-2 in the lungs. Conclusions Mechanical ventilation modified tryptase and PAR-2 in injured lungs. Increased levels of these proteins were associated with development of sepsis and ventilator-induced pulmonary fibrosis early in the course of sepsis-induced lung injury. Introduction Acute lung injury during the acute respiratory distress syndrome (ARDS) complicates a variety of clinical conditions and is associated with significant morbidity and mortality [1]. Sepsis, the most common cause of ARDS, promotes or interferes with mechanisms involved in tissue repair [2]. Mechanical ventilation (MV) is an essential life support for sepsis/ARDS patients and manipulation of the ventilator strategy is the only proven treatment for improving survival [3]. However, MV can cause or aggravate lung injury, an entity referred to as ventilator-induced lung injury (VILI) [4]. Experimental and clinical studies [3-6] have provided insights into the physiology of VILI, demonstrating that some patterns of MV result in pulmonary and systemic changes that mimic ARDS and sepsis [2-4]. Many ARDS patients survive the underlying disease but die with pulmonary fibrosis [7,8]. Recent reports suggest that sepsis may trigger the development of persistent fibrosis [9] and that VILI may be a major contributor to lung fibrosis [10,11]. The role of MV as an inciting factor for lung fibrosis is poorly understood. Pulmonary fibrosis appears to be an important determinant of mortality, regardless of the cause of ARDS [11,12]. Areas of fibrosis are adjacent to inflammation in the early exudative phase of ARDS [13-15]. A feature of lungs in patients with fibrotic lung disease is the increased number of mast cells [16] and it has been suggested that mast cells may support the continuation of the fibroproliferative process in patients with ARDS [17] by release of mediators. The most abundant product of mast cells is tryptase, a serine protease with pleiotropic biological activities [18]. Tryptases consist of -tryptase and -tryptase [19]. -tryptase is the main isoenzyme expressed in human lung. Tryptase upregulates the expression of cytokines [19] and vascular endothelial growth factor (VEGF) [20]. We have previously reported that MV modulates the innate immune response by interfering with Toll-like receptors [6] and increases VEGF by activating the Wnt/-catenin signaling pathways [10]. Tryptase is a potent stimulant of the synthesis of type I collagen by fibroblasts [21]. The mechanism by which tryptase exerts its effects is by activating a member of the protease-activated receptor (PAR) family, PAR-2 [22]. Currently, no published reports have examined the changes of tryptase and PAR-2 in the context of septic ARDS and VILI. We tested the hypothesis that tryptase content is modulated by MV and could contribute to the early development of pulmonary fibrosis in an experimental, clinically relevant animal model of sepsis-induced acute lung injury. Material and methods This protocol was approved by the Animal Care Committee at the Hospital Universitario Dr. Negrn (CEEBA-003/10), in accordance with the European Commission Directive 2010/63/EU for animal experimentation. This study followed the ARRIVE guidelines for reporting preclinical animal research [23]. Animal preparation and experimental protocol A total of 40 male Sprague-Dawley rats (300 to 350 g) were included. Animals were anesthetized with an intraperitoneal injection of ketamine hydrochloride (80 mg/kg) and xylazine (8 mg/kg) [24]. Animals were initially randomized to two groups: non-septic controls (n = 6) and septic (n = 34). Sepsis was induced by cecal ligation and perforation (CLP). CLP is considered the gold standard model for experimental sepsis [25]. A detailed description of this model is.