Pulmonary oxidant stress in murine sepsis is due to inflammatory cell nitric oxide

OBJECTIVE: Pulmonary oxidant stress is an important pathophysiologic feature of acute lung injury. It is unclear whether nitric oxide contributes to this oxidant stress. Thus, we examined the role of inducible nitric oxide synthase (iNOS) in pulmonary oxidant stress in murine sepsis and the differential contribution of different cellular sources of iNOS. DESIGN: Randomized, controlled animal study. SETTING: Research laboratory of an academic institution. SUBJECTS: Male iNOS+/+, iNOS-/- C57Bl/6 mice, and bone-marrow transplanted iNOS chimeric mice: +to- (wild-type iNOS+/+ donor bone-marrow transplanted into iNOS-/- recipient mice) and the reciprocal -to+ chimeras. INTERVENTIONS: Animals were randomized to sepsis (n = 264), induced by cecal ligation and perforation, vs. naive groups (n = 138). MEASUREMENTS AND MAIN RESULTS: In septic iNOS-/- vs. wild-type iNOS+/+ mice, sepsis-induced pulmonary oxidant stress (33 +/- 11 [mean +/- sem] vs. 365 +/- 48 pg 8-isoprostane/mg protein, p < .01) and nitrosative stress (0.0 +/- 0.0 vs. 0.9 +/- 0.4 micromol 3-nitrotyrosine/mmol para-tyrosine, p < .05) were abolished, despite similar septic increases in pulmonary myeloperoxidase activity in both (86 +/- 20 vs. 83 +/- 12 mU/mg protein, p = .78). In +to- iNOS chimeric mice (iNOS localized only to donor bone-marrow-derived inflammatory cells), cecal ligation and perforation resulted in significant pulmonary oxidant stress (368 +/- 81 pg 8-isoprostane/mg protein) and nitrosative stress (0.6 +/- 0.2 micromol 3-nitrotyrosine/mmol para-tyrosine), similar in degree to septic wild-type mice. In contrast, pulmonary oxidant and nitrosative stresses were absent in septic -to+ iNOS chimeras (iNOS localized only to recipient parenchymal cells), similar to iNOS-/- mice. CONCLUSIONS: In murine sepsis-induced acute lung injury, pulmonary oxidant stress is completely iNOS dependent and is associated with tyrosine nitration. Moreover, pulmonary oxidant stress and nitrosative stress were uniquely dependent on the presence of iNOS in inflammatory cells (e.g., macrophages and neutrophils), with no apparent contribution of iNOS in pulmonary parenchymal cells. iNOS inhibition targeted specifically to inflammatory cells may be an effective therapeutic approach in sepsis and acute lung injury.     

脓毒症大鼠急性肺损伤肺血管通透性、炎症因子、诱导型一氧化氮合酶的变化及意义

目的:观察脓毒症大鼠急性肺损伤肺血管通透性、炎症因子、诱导型一氧化氮合酶(iNOS)的变化特点及其意义.方法:24只雄性SD大鼠随机分为脓毒症组和对照组,脓毒症组按5 mg/kg体重腹腔注射脂多糖(LPS),对照组给予等量生理盐水,于注射后2h、6h经腹主动脉采血、处死,比较动脉血气分析、ELISA法检测的肺组织iNOS、白介素-6(IL-6)含量、右下肺肺组织病理切片计算的病理学肺损伤积分.结果:与对照组比较,脓毒症组大鼠氧合指数显著下降、肺组织iNOS、IL-6含量、病理学肺损伤积分均显著增加,P＜0.01.结论:脓毒症大鼠肺iNOS的过度表达增加了炎症因子的释放、增加肺血管通透性,加重肺损伤.     

Simvastatin attenuates ventilator-induced lung injury in mice

Introduction

Different isoforms of nitric oxide synthases (NOS) and determinants of oxidative/nitrosative stress play important roles in the pathophysiology of pulmonary dysfunction induced by acute lung injury (ALI) and sepsis. However, the time changes of these pathogenic factors are largely undetermined.

Methods

Twenty-four chronically instrumented sheep were subjected to inhalation of 48 breaths of cotton smoke and instillation of live Pseudomonas aeruginosa into both lungs and were euthanized at 4, 8, 12, 18, and 24 hours post-injury. Additional sheep received sham injury and were euthanized after 24 hrs (control). All animals were mechanically ventilated and fluid resuscitated. Lung tissue was obtained at the respective time points for the measurement of neuronal, endothelial, and inducible NOS (nNOS, eNOS, iNOS) mRNA and their protein expression, calcium-dependent and -independent NOS activity, 3-nitrotyrosine (3-NT), and poly(ADP-ribose) (PAR) protein expression.

Results

The injury induced severe pulmonary dysfunction as indicated by a progressive decline in oxygenation index and concomitant increase in pulmonary shunt fraction. These changes were associated with an early and transient increase in eNOS and an early and profound increase in iNOS expression, while expression of nNOS remained unchanged. Both 3-NT, a marker of protein nitration, and PAR, an indicator of DNA damage, increased early but only transiently.

Conclusions

Identification of the time course of the described pathogenetic factors provides important additional information on the pulmonary response to ALI and sepsis in the ovine model. This information may be crucial for future studies, especially when considering the timing of novel treatment strategies including selective inhibition of NOS isoforms, modulation of peroxynitrite, and PARP.     

Inhaled activated protein C: a new therapy for the prevention of ventilator-induced lung injury?

Systemic administration of activated protein C (APC) has been shown to reduce pulmonary inflammation in preclinical models of acute lung injury. However, there is only limited information concerning the effects of inhaled APC in modulating the severity of pulmonary inflammation. In a study reported in this issue of Critical Care, Maniatis and colleagues show that pretreatment of mice with inhaled APC is protective against ventilator-induced lung injury. While the mechanisms responsible for this effect require additional elucidation, inhaled APC appears to be a potentially useful intervention in diminishing the severity of ventilator-induced lung injury and other forms of acute lung injury.     

Toll-like receptor 2 participates in the response to lung injury in a murine model of pulmonary contusion

Blunt chest trauma resulting in pulmonary contusion with an accompanying acute inflammatory response is a common but poorly understood injury. We report that Toll-like receptor (TLR) 2 participates in the inflammatory response to lung injury. To show this, we use a model of pulmonary contusion in the mouse that is similar to that observed clinically in humans based on histologic, morphologic, and biochemical criteria of acute lung injury. The inflammatory response to pulmonary contusion in our mouse model is characterized by pulmonary edema, neutrophil transepithelial migration, and increased expression of the innate immunity proinflammatory cytokines IL 1beta and IL 6, the adhesion intracellular adhesion molecule 1, and chemokine (CXC motif) ligand 1. Compared with wild-type animals, contused Tlr2(-/-) mice have significantly reduced pulmonary edema and neutrophilia. These findings are associated with decreased levels of circulating chemokine (CXC motif) ligand 1. In contrast, systemic IL 6 levels remain elevated in the TLR2-deficient phenotype. These results show that TLR2 has a primary role in the neutrophil response to acute lung injury. We suggest that an unidentified noninfectious ligand generated by pulmonary contusion acts via TLR2 to generate inflammatory responses.     

The inhibition of inducible nitric oxide synthase in ovine sepsis model

Excessive NO has been shown to play a major role in the pathogenesis of multiple organ dysfunctions in septic condition. Burn injury, especially if it is associated with smoke inhalation, is often complicated by subsequent development of pneumonia or sepsis that determine the outcome. In the present study, we developed an ovine sepsis model, created by exposing sheep to smoke inhalation followed by instillation of bacteria into the airway, that closely mimics human sepsis and pneumonia. We hypothesized that the inhibition of iNOS-derived excessive NO might be beneficial in treating the cardiopulmonary derangement in this model. Female sheep (n = 18) were surgically prepared for the study and given a tracheostomy. This was followed by insufflation of 48 breaths of cotton smoke (< 40 degrees C) into the airway of each animal and subsequent instillation of live Pseudomonas aeruginosa (5 x 10(11) colony forming units) into each sheep's lung. All sheep were mechanically ventilated using 100% O2. Continuous infusion of BBS-2 (100 microg/kg/h), an iNOS inhibitor, was started 1 h after insult. The administration of BBS-2 improved pulmonary gas exchange (PaO2/FiO2 and pulmonary shunt fraction) and partially reduced airway obstruction and an increase in ventilatory pressures. The lung water content was not affected by iNOS inhibition. The hypotension seen in nontreated animals was not ameliorated either. The increase in plasma concentration of nitrate and nitrite was inhibited by BBS-2. The results of present study show that iNOS may be partially involved in the pathogenesis of acute lung injury induced by smoke inhalation followed by bacterial instillation in the airway.     

辛伐他汀纳米粒通过调节诱导型一氧化氮合酶/内皮型一氧化氮合酶系统对小鼠脓毒症相关急性肺损伤的影响

目的探讨辛伐他汀纳米粒对脓毒症相关急性肺损伤小鼠肺组织中诱导型一氧化氮合酶（iNOS）/内皮型一氧化氮合酶（eNOS）平衡的调节以及对脓毒症小鼠预后的影响。 方法将90只C57 / BL6小鼠分为假手术组、脓毒症组、灌胃组、静脉制剂组及纳米粒组,每组各18只。采用盲肠结扎穿孔术（CLP）建立脓毒症小鼠模型;灌胃组小鼠通过灌胃针,给予辛伐他汀口服制剂灌胃治疗后进行CLP术;静脉制剂组及纳米粒制剂组小鼠CLP术后,立即分别通过尾静脉注射预配置好的辛伐他汀静脉制剂和辛伐他汀纳米粒制剂。其中每组12只小鼠用于7 d生存评估,另外6只用于24 h时间点标本采集。每24小时观察小鼠的生存情况,然后记算各组小鼠每日生存情况。采用苏木素-伊红（HE）染色观察5组小鼠病理变化并计算肺损伤病理评分,免疫组织化学法检测5组小鼠肺组织iNOS、eNOS表达水平。 结果Kaplan-Meier生存曲线结果显示,5组小鼠7 d生存情况比较,差异有统计学意义（χ² = 3.780,P < 0.001）。进一步两两比较发现,脓毒症组及灌胃组小鼠的7 d生存情况均较假手术组明显下降（P均< 0.001）,而纳米粒组小鼠的7 d生存情况显著优于脓毒症组（P = 0.001）。HE染色结果显示,假手术组小鼠肺组织未见明显病理征象;脓毒症组小鼠肺组织弥漫性中性粒细胞浸润、肺泡腔变小、肺泡间中隔增厚、肺间质弥漫性水肿、细胞排列紊乱、部分肺组织完整性遭破坏;灌胃组小鼠病理所示与脓毒症组相似;静脉制剂组及纳米粒组小鼠中性粒细胞渗出均较脓毒症组损伤减少、肺泡完整性较好、损伤程度较轻。5组小鼠肺损伤病理评分、iNOS及eNOS表达水平比较,差异均有统计学意义（F = 889.200、9.633、6.918,P均< 0.05）。进一步两两比较发现,脓毒症组小鼠的肺损伤病理评分、iNOS及eNOS表达水平与假手术组比较,差异均有统计学意义（P均< 0.05）;静脉制剂组及纳米粒组小鼠病理评分、iNOS及eNOS表达水平与脓毒症组比较,差异均有统计学意义（P均< 0.05）,且纳米粒组小鼠的肺损伤病理评分较静脉制剂组显著降低（P < 0.05）。 结论不同的辛伐他汀制剂具有不同的效应,其中纳米粒制剂对于脓毒症相关的肺损伤最具保护价值,建立eNOS与iNOS之间的平衡,可以成为具有保护效应的重要处理位点。     

Inhaled activated protein C protects mice from ventilator-induced lung injury

Introduction  

Activated Protein C (APC), an endogenous anticoagulant, improves tissue microperfusion and endothelial cell survival in systemic inflammatory states such as sepsis, but intravenous administration may cause severe bleeding. We have thus addressed the role of APC delivered locally by inhalation in preventing acute lung injury from alveolar overdistention and the subsequent ventilator-induced lung injury (VILI). We also assessed the effects of APC on the activation status of Extracellular- Regulated Kinase 1/2 (ERK) pathway, which has been shown to be involved in regulating pulmonary responses to mechanical stretch.     

Time course of nitric oxide synthases,nitrosative stress,and poly(ADP ribosylation) in an ovine sepsis model

Introduction

Different isoforms of nitric oxide synthases (NOS) and determinants of oxidative/nitrosative stress play important roles in the pathophysiology of pulmonary dysfunction induced by acute lung injury (ALI) and sepsis. However, the time changes of these pathogenic factors are largely undetermined.

Methods

Twenty-four chronically instrumented sheep were subjected to inhalation of 48 breaths of cotton smoke and instillation of live Pseudomonas aeruginosa into both lungs and were euthanized at 4, 8, 12, 18, and 24 hours post-injury. Additional sheep received sham injury and were euthanized after 24 hrs (control). All animals were mechanically ventilated and fluid resuscitated. Lung tissue was obtained at the respective time points for the measurement of neuronal, endothelial, and inducible NOS (nNOS, eNOS, iNOS) mRNA and their protein expression, calcium-dependent and -independent NOS activity, 3-nitrotyrosine (3-NT), and poly(ADP-ribose) (PAR) protein expression.

Results

The injury induced severe pulmonary dysfunction as indicated by a progressive decline in oxygenation index and concomitant increase in pulmonary shunt fraction. These changes were associated with an early and transient increase in eNOS and an early and profound increase in iNOS expression, while expression of nNOS remained unchanged. Both 3-NT, a marker of protein nitration, and PAR, an indicator of DNA damage, increased early but only transiently.

Conclusions

Identification of the time course of the described pathogenetic factors provides important additional information on the pulmonary response to ALI and sepsis in the ovine model. This information may be crucial for future studies, especially when considering the timing of novel treatment strategies including selective inhibition of NOS isoforms, modulation of peroxynitrite, and PARP.     

Contractions to histamine in pulmonary and mesenteric arteries from endotoxemic rabbits: modulation by vascular expressions of inducible nitric-oxide synthase and histamine H1-receptors

The inducible isoform of nitric-oxide synthase (iNOS) is highly expressed after induction of endotoxemia and contributes to vascular hypocontractility in endotoxemia. Circulating levels of histamine are elevated in animal models of sepsis and in patients with septic shock. This study assessed whether the vascular effects of histamine play a significant role in the pathophysiology of endotoxemic shock despite the hyporesponsiveness to vasoconstrictors associated with iNOS up-regulation. Rabbits were rendered endotoxemic by lipopolysaccharide (LPS; 100 microg/kg, i.v.). In mesenteric arteries taken from animals at 6 h of LPS administration, the contractile response to histamine was significantly impaired but histamine-evoked contractions in pulmonary arteries were unchanged. Western blot revealed a drastic increase in iNOS expression in mesenteric vessels after LPS, but endotoxin-induced iNOS increase was not so marked in pulmonary vessels. On the other hand, expression of endothelial nitric-oxide synthase was suppressed under LPS challenge in both types of vessels. In the presence of NG-nitro-l-arginine or (S)-ethylisothiourea used for iNOS inhibition, histamine-evoked contractions of endotoxemic pulmonary and mesenteric vessels were significantly enhanced. This was possibly associated with a dramatic increase in H1-receptor expression at the gene and protein levels, as determined by Northern blot and immunoblot analyses. Furthermore, we found that LPS-induced endotoxemia caused prominent increases in production of histamine through induction of histidine decarboxylase in tissues, including blood vessels. From these results, we propose that histamine may contribute to the development of endotoxin-induced pulmonary hypertension.     

Activated protein C suppresses adrenomedullin and ameliorates lipopolysaccharide-induced hypotension

Activated protein C (APC) is an important modulator of vascular function that has antithrombotic and anti-inflammatory properties. Studies in humans have shown modulation of endotoxin-induced hypotension by recombinant human APC, drotrecogin alfa (activated), however, the mechanism for this effect is unclear. We have found that APC suppresses the induction of the potent vasoactive peptide adrenomedullin (ADM) and could downregulate lipopolysaccharide (LPS)-induced ADM messenger RNA (mRNA) and nitrite levels in cell culture. This effect was dependent on signaling through protease-activated receptor 1. Addition of 1400W, an irreversible inducible nitric oxide synthase (iNOS) inhibitor, inhibited LPS-induced ADM mRNA, suggesting that ADM induction is NO mediated. Furthermore, in a rat model of endotoxemia, APC (100 microg/kg, i.v.) prevented LPS (10 mg/kg, i.v.)-induced hypotension, and suppressed ADM mRNA and protein expression. APC also inhibited iNOS mRNA and protein levels along with reduction in NO by-products (NOx). We also observed a significant reduction in iNOS-positive leukocytes adhering to vascular endothelium after APC treatment. Moreover, we found that APC inhibited the expression of interferon-gamma (IFN-gamma), a potent activator of iNOS. In a human study of LPS-induced hypotension, APC reduced the upregulation of plasma ADM levels, coincident with protection against the hypotensive response. Overall, we demonstrate that APC blocks the induction of ADM, likely mediated by IFN-gamma and iNOS, and suggests a mechanism that may account for ameliorating LPS-induced hypotension. Furthermore, our data provide a new understanding for the role of APC in modulating vascular response to insult.     

Tryptase is involved in the development of early ventilator-induced pulmonary fibrosis in sepsis-induced lung injury

IntroductionMost patients with sepsis and acute lung injury require mechanical ventilation to improve oxygenation and facilitate organ repair. Mast cells are important in response to infection and resolution of tissue injury. Since tryptase secreted from mast cells has been associated with tissue fibrosis, we hypothesized that tryptase would be involved in the early development of ventilator-induced pulmonary fibrosis in a clinically relevant model of sepsis-induced lung injury.MethodsProspective, randomized, controlled animal study using Sprague-Dawley rats. Sepsis was induced by cecal ligation and perforation. Animals were randomized to spontaneous breathing or two ventilatory strategies for 4 h: protective ventilation with tidal volume (VT) = 6 ml/kg plus 10 cmH₂O positive end-expiratory pressure (PEEP) or injurious ventilation with VT = 20 ml/kg plus 2 cmH₂O PEEP. Healthy, non-ventilated animals served as non-septic controls. 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.ResultsAll septic animals developed acute lung injury. Animals ventilated with high VT had a significant increase of pulmonary fibrosis, hydroxyproline content, tryptase and PAR-2 protein levels compared to septic controls (P <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.ConclusionsMechanical 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.     

NO和iNOS在内毒素诱导的大鼠急性肺损伤的作用及大黄对其影响

目的 主要探讨一氧化氮（ＮＯ）和诱导型一氧化氮合酶（ｉＮＯＳ）在内毒素（ＬＰＳ）诱导的大鼠急性肺损伤（ＡＬＩ）的作用机制及大黄对其影响。方法 在雄性Ｗｉｓｔａｒ大鼠利用舌下静脉注射ＬＰＳ复制ＡＬＩ动物模型,动物分为４组：ＬＰＳ组,对照组,大黄治疗组,地塞米松组。观察大体标本,组织病理以及生物学标志：肺湿／干重比,肺泡灌洗液中性粒细胞比,蛋白含量,肺血管通透性和肺泡通透性指数。同时测定血浆ＮＯ和肺组     

Toll-like receptor 4-dependent responses to lung injury in a murine model of pulmonary contusion

Blunt chest trauma resulting in pulmonary contusion with an accompanying acute inflammatory response is a common but poorly understood injury. We previously demonstrated that toll-like receptor 2 (TLR-2) participates in the inflammatory response to lung injury. We hypothesized that the TLR-4, in an MyD88-dependent manner, may also participate in the response to lung injury. To investigate this, we used a model of pulmonary contusion in the mouse that is similar to that observed clinically in humans and evaluated postinjury lung function, pulmonary neutrophil recruitment, and the systemic innate immune response. Comparisons were made between wild-type mice and mice deficient in TLR-4 or MyD88. We found TLR-4-dependent responses to pulmonary contusion that include hypoxemia, edema, and neutrophil infiltration. Increased expression of IL-6 and chemokine (C-X-C motif) ligand 1 in the bronchoalveolar lavage and serum was also dependent on TLR-4 activation. We further demonstrated that these responses to pulmonary contusion were dependent on MyD88, an adapter protein in the signal transduction pathway mediated by TLRs. These results show that TLRs have a primary role in the response to acute lung injury. Lung inflammation and systemic innate immune responses are dependent on TLR activation by pulmonary contusion.     

Regulation of inflammatory responses by activated protein C: the molecular mechanism(s) and therapeutic implications.

Activated protein C (APC), a natural anticoagulant, is formed from protein C by the action of the thrombin-thrombomodulin (TM) complex on the endothelial cell surface. Endothelial protein C receptor augments the activation of protein C by the thrombin/TM system. APC inactivates the activated form of coagulation factors V and VIII in the presence of protein S. Administration of APC reduced the pulmonary vascular injury and hypotension as well as the coagulation abnormalities by inhibiting production of the tumor necrosis factor-alpha (TNF-alpha) in rats given endotoxin (ET). These therapeutic effects of APC could not be attributed to its anticoagulant effects. APC inhibited ET-induced TNF-alpha production in human monocytes by inhibiting activation of nuclear factor K-B and activator protein-1 in vitro. Administration of the human plasma-derived APC ameliorated coagulation abnormalities without any adverse effects in patients with disseminated intravascular coagulation (DIC). Recombinant APC was reported to reduce the mortality of patients with severe sepsis, and the therapeutic effect was more marked in such patients with overt DIC than those without it. These observations strongly suggest that APC plays important roles in the regulation of inflammation as well as coagulation. Both anti-inflammatory and anticoagulant properties of APC might contribute to the therapeutic usefulness in patients with severe sepsis.     

Effects of thermal preconditioning on the ischemia-reperfusion-induced acute lung injury in minipigs

Lung ischemia-reperfusion (I/R) injury plays an important role in many clinical issues. A series of mechanisms after I/R has been uncovered after numerous related studies. Organ preconditioning (PC) is a process whereby a brief antecedent event, such as transient ischemia, oxidative stress, temperature change, or drug administration, bestows on an organ an early or delayed tolerance to further insults by the same or different stressors. In this study, we want to uncover the optimal thermal PC patterns that cause maximal early or delayed protective effect on the subsequent pulmonary I/R with the use of miniature pig model. Twenty-eight 15- to 20-kg weight Lanyu miniature pigs are used and divided into four groups (seven sham operation control [NC], seven PC only [PC], seven I/R [I/R], and seven PC followed by I/R [PC + I/R]). The PC was performed with the animals being anesthetized and, using an alternative hyperthermic (40 degrees C) and normothermic moist air to ventilate their lungs for 15 min, respectively, for 2 cycles, followed by I/R, which consists of 90 min of blocking the perfusion and ventilation of the left lung followed by 240 min of reperfusion. Control animals had a thoracotomy with hilar dissection only. Indicators of lung injury included hemodynamic parameters, blood gas analysis, histopathological (lung pathology, wet/dry weight ratio, myeloperoxidase assay), and molecular biological profiles (interleukin-1beta [IL-1beta], IL-6, tumor necrosis factor-alpha by enzyme-linked immunosorbent assay analysis). Lung tissue heat shock protein 70 (HSP-70) expression was also detected by Western blotting. This model of lung I/R induced significant lung injury with pulmonary hypertension, increased pulmonary vascular resistance, and pulmonary venous hypoxemia at the ischemia side, increased pulmonary tissue injury score and neutrophil infiltration, increased wet/dry ratio, myeloperoxidase assay, tumor necrosis factor-alpha, IL-1beta, and IL-6 assay. This type of thermal PC would not injure the lung parenchyma or tracheal epithelium. Moreover, it could attenuate the I/R-related lung injury, with some of these parameters improved significantly. Increased expression of HSP-70 was also found in the group of PC plus I/R than the I/R only. Less prominent and transient increase in expression of HSP-70 was found in the PC group. We concluded that the intratracheal thermal PC can effectively attenuate I/R-induced lung injury through various mechanisms, including the decrease of various proinflammatory cytokines. The mechanism of its protective effect might be related to the increased expression of HSP-70.     

Role of activated neutrophils in chest trauma-induced septic acute lung injury

More than 50% of severely injured patients have chest trauma. Second insults frequently result in acute lung injury (ALI), with sepsis being the main underlying condition. We aimed to develop a standardized, reproducible, and clinically relevant double-hit mouse model of ALI induced by chest trauma and polymicrobial sepsis and to investigate the pathophysiologic role of activated neutrophils. Lung contusion was applied to C57Bl/6 mice via a focused blast wave. Twenty-four hours later, sepsis was induced by cecal ligation and puncture. For polymorphonuclear leukocyte (PMN) depletion, animals received intravenous injections of PMN-depleting antibody. In response to blunt chest trauma followed by sepsis as well as after sepsis alone, a significant local and systemic inflammatory response with increased cytokine/chemokine levels in lung and plasma was observed. In contrast, lung apoptosis was markedly elevated only after a double hit. Intra-alveolar neutrophils and total bronchoalveolar lavage protein concentrations were markedly increased following isolated chest trauma or the combined insult, but not after sepsis alone. Lung myeloperoxidase activity was enhanced only in response to the double hit accompanied by histological disruption of the alveolar architecture, lung congestion, and marked cellular infiltrates. Neutrophil depletion significantly diminished lung interleukin 1β and interleukin 6 concentrations and reduced the degree of septic ALI. Here we have established a novel and highly reproducible mouse model of chest trauma-induced septic ALI characterizing a clinical relevant double-hit scenario. In particular, the depletion of neutrophils substantially mitigated the extent of lung injury, indicating a pathomechanistic role for neutrophils in chest trauma-induced septic ALI.     

Procalcitonin as a predictive biomarker for total body irradiation-induced bacterial load and lethality in mice

This experimental animal study investigates the effects of combined recombinant human activated protein C (rhAPC) and ceftazidime on cardiopulmonary function in acute lung injury and severe sepsis. Twenty-one sheep (37 ± 2 kg) were operatively prepared and randomly allocated to either the sham, control, or treatment group (n = 7 each). Single treatments of rhAPC or ceftazidime were published previously; therefore, control groups were dispensed in the present study, what may be considered a study limitation. Acute lung injury and sepsis were induced according to an established protocol. The sham group received only the vehicle. The sheep were studied in awake state for 24 h and mechanically ventilated. Recombinant human APC (continuous infusion 24 μg/kg per hour) and ceftazidime (3-g bolus at 1 and 13 h) were intravenously administered. The animals were fluid resuscitated with Ringer's lactate to maintain hematocrit at baseline. Compared with injured controls, the treatment group had a significantly higher PaO?/FIO? ratio, and the onset of acute respiratory distress syndrome was prevented. The increase in pulmonary microvascular shunt fraction and airway obstruction in bronchi and bronchiole, as well as lung 3-nitrotyrosine, lung myeloperoxidase, cardiac 3-nitrotyrosine, and cardiac malondialdehyde levels, was significantly reduced as compared with controls (P < 0.05 each). Treated sheep had significantly improved hemodynamics as reflected by mean arterial pressure, heart rate, cardiac index, and systemic vascular resistance index (P < 0.05 each). In addition, plasma oncotic pressure and urine output were significantly improved (P < 0.05 each). Combined rhAPC and ceftazidime significantly improved cardiopulmonary function, reduced pulmonary and cardiac tissue injury, and prevented the onset of acute respiratory distress syndrome in ovine severe sepsis without obvious adverse effects.