Antithrombin attenuates vascular leakage via inhibiting neutrophil activation in acute lung injury.

OBJECTIVE: To test the hypothesis that restoration of antithrombin plasma concentrations attenuates vascular leakage by inhibiting neutrophil activation through syndecan-4 receptor inhibition in an established ovine model of acute lung injury. DESIGN: Randomized controlled laboratory experiment. SETTING: University animal research facility. SUBJECTS: Eighteen chronically instrumented sheep. INTERVENTIONS: Following combined burn and smoke inhalation injury (40% of total body surface area, third-degree flame burn; 4 × 12 breaths of cold cotton smoke), chronically instrumented sheep were randomly assigned to receive an IV infusion of 6 IU/kg/hr recombinant human antithrombin III or normal saline (n = 6 each) during the 48-hour study period. In addition, six sham animals (not injured, continuous infusion of vehicle) were used to obtain reference values for histological and immunohistochemical analyses. MEASUREMENTS AND MAIN RESULTS: Compared to control animals, recombinant human antithrombin III reduced the number of neutrophils per hour in the pulmonary lymph (p < 0.01 at 24 and 48 hr), alveolar neutrophil infiltration (p = 0.04), and pulmonary myeloperoxidase activity (p = 0.026). Flow cytometric analysis revealed a significant reduction of syndecan-4-positive neutrophils (p = 0.002 vs control at 24 hr). Treatment with recombinant human antithrombin III resulted in a reduction of pulmonary nitrosative stress (p = 0.002), airway obstruction (bronchi: p = 0.001, bronchioli: p = 0.013), parenchymal edema (p = 0.044), and lung bloodless wet-to-dry-weight ratio (p = 0.015). Clinically, recombinant human antithrombin III attenuated the increased pulmonary transvascular fluid flux (12-48 hr: p ≤ 0.001 vs control each) and the deteriorated pulmonary gas exchange (12-48 hr: p < 0.05 vs control each) without increasing the risk of bleeding. CONCLUSIONS: The present study provides evidence for the interaction between antithrombin and neutrophils in vivo, its pathophysiological role in vascular leakage, and the therapeutic potential of recombinant human antithrombin III in a large animal model of acute lung injury.

Antithrombin attenuates myocardial dysfunction and reverses systemic fluid accumulation following burn and smoke inhalation injury: a randomized, controlled, experimental study.

INTRODUCTION: We hypothesized that maintaining physiological plasma levels of antithrombin attenuates myocardial dysfunction and inflammation as well as vascular leakage associated with burn and smoke inhalation injury. Therefore, the present prospective, randomized experiment was conducted using an established ovine model. METHODS: Following 40% of total body surface area, third degree flame burn and 4 × 12 breaths of cold cotton smoke, chronically instrumented sheep were randomly assigned to receive an intravenous infusion of 6 IU/kg/h recombinant human antithrombin (rhAT) or normal saline (control group; n = 6 each). In addition, six sheep were designated as sham animals (not injured, continuous infusion of vehicle). During the 48 h study period the animals were awake, mechanically ventilated and fluid resuscitated according to standard formulas. RESULTS: Compared to the sham group, myocardial contractility was severely impaired in control animals, as suggested by lower stroke volume and left ventricular stroke work indexes. As a compensatory mechanism, heart rate increased, thereby increasing myocardial oxygen consumption. In parallel, myocardial inflammation was induced via nitric oxide production, neutrophil accumulation (myeloperoxidase activity) and activation of the p38-mitogen-activated protein kinase pathway resulting in cytokine release (tumor necrosis factor-alpha, interleukin-6) in control vs. sham animals. rhAT-treatment significantly attenuated these inflammatory changes leading to a myocardial contractility and myocardial oxygen consumption comparable to sham animals. In control animals, systemic fluid accumulation progressively increased over time resulting in a cumulative positive fluid balance of about 4,000 ml at the end of the study period. Contrarily, in rhAT-treated animals there was only an initial fluid accumulation until 24 h that was reversed back to the level of sham animals during the second day. CONCLUSIONS: Based on these findings, the supplementation of rhAT may represent a valuable therapeutic approach for cardiovascular dysfunction and inflammation after burn and smoke inhalation injury.

Selective V(1a) agonism attenuates vascular dysfunction and fluid accumulation in ovine severe sepsis.

Vasopressin analogs are used as a supplement to norepinephrine in septic shock. The isolated effects of vasopressin agonists on sepsis-induced vascular dysfunction, however, remain controversial. Because V(2)-receptor stimulation induces vasodilation and procoagulant effects, a higher V(1a)- versus V(2)-receptor selectivity might be advantageous. We therefore hypothesized that a sole, titrated infusion of the selective V(1a)-agonist Phe(2)-Orn(8)-Vasotocin (POV) is more effective than the mixed V(1a)-/V(2)-agonist AVP for the treatment of vascular and cardiopulmonary dysfunction in methicillin resistant staphylococcus aureus pneumonia-induced, ovine sepsis. After the onset of hemodynamic instability, awake, chronically instrumented, mechanically ventilated, and fluid resuscitated sheep were randomly assigned to receive continuous infusions of either POV, AVP, or saline solution (control; each n = 6). AVP and POV were titrated to maintain mean arterial pressure above baseline - 10 mmHg. When compared with that of control animals, AVP and POV reduced neutrophil migration (myeloperoxidase activity, alveolar neutrophils) and plasma levels of nitric oxide, resulting in higher mean arterial pressures and a reduced vascular leakage (net fluid balance, chest and abdominal fluid, pulmonary bloodless wet-to-dry-weight ratio, alveolar and septal edema). Notably, POV stabilized hemodynamics at lower doses than AVP. In addition, POV, but not AVP, reduced myocardial and pulmonary tissue concentrations of 3-nitrotyrosine, VEGF, and angiopoietin-2, thereby leading to an abolishment of cumulative fluid accumulation (POV, 9 ± 15 ml/kg vs. AVP, 110 ± 13 ml/kg vs. control, 213 ± 16 ml/kg; P < 0.001 each) and an attenuated cardiopulmonary dysfunction (left ventricular stroke work index, PaO(2)-to-FiO(2) ratio) versus control animals. Highly selective V(1a)-agonism appears to be superior to unselective vasopressin analogs for the treatment of sepsis-induced vascular dysfunction.

Predictive role of arterial carboxyhemoglobin concentrations in ovine burn and smoke inhalation-induced lung injury.

Inhalation injury frequently occurs in burn patients and contributes to the morbidity and mortality of these injuries. Arterial carboxyhemoglobin has been proposed as an indicator of the severity of inhalation injury; however, the interrelation between arterial carboxyhemoglobin and histological alterations has not yet been investigated. Chronically instrumented sheep were subjected to a third degree burn of 40% of the total body surface area and inhalation of 48 breaths of cotton smoke. Carboxyhemoglobin was measured immediately after injury and correlated to clinical parameters of pulmonary function as well as histopathology scores from lung tissue harvested 24 hours after the injury. The injury was associated with a significant decline in pulmonary oxygenation and increases in pulmonary shunting, lung lymph flow, wet/dry weight ratio, congestion score, edema score, inflammation score, and airway obstruction scores. Carboxyhemoglobin was negatively correlated to pulmonary oxygenation and positively correlated to pulmonary shunting, lung lymph flow, and lung wet/dry weight ratio. No significant correlations could be detected between carboxyhemoglobin and histopathology scores and airway obstruction scores. Arterial carboxyhemoglobin in sheep with combined burn and inhalation injury are correlated with the degree of pulmonary failure and edema formation, but not with certain histological alterations including airway obstruction scores.

Molecular biological effects of selective neuronal nitric oxide synthase inhibition in ovine lung injury.

Neuronal nitric oxide synthase is critically involved in the pathogenesis of acute lung injury resulting from combined burn and smoke inhalation injury. We hypothesized that 7-nitroindazole, a selective neuronal nitric oxide synthase inhibitor, blocks central molecular mechanisms involved in the pathophysiology of this double-hit insult. Twenty-five adult ewes were surgically prepared and randomly allocated to 1) an uninjured, untreated sham group (n = 7), 2) an injured control group with no treatment (n = 7), 3) an injury group treated with 7-nitroindazole from 1-h postinjury to the remainder of the 24-h study period (n = 7), or 4) a sham-operated group subjected only to 7-nitroindazole to judge the effects in health. The combination injury was associated with twofold increased activity of neuronal nitric oxide synthase and oxidative/nitrosative stress, as indicated by significant increases in plasma nitrate/nitrite concentrations, 3-nitrotyrosine (an indicator of peroxynitrite formation), and malondialdehyde lung tissue content. The presence of systemic inflammation was evidenced by twofold, sixfold, and threefold increases in poly(ADP-ribose) polymerase, IL-8, and myeloperoxidase lung tissue concentrations, respectively (each P < 0.05 vs. sham). These molecular changes were linked to tissue damage, airway obstruction, and pulmonary shunting with deteriorated gas exchange. 7-Nitroindazole blocked, or at least attenuated, all these pathological changes. Our findings suggest 1) that nitric oxide formation derived from increased neuronal nitric oxide synthase activity represents a pivotal reactive agent in the patho-physiology of combined burn and smoke inhalation injury and 2) that selective neuronal nitric oxide synthase inhibition represents a goal-directed approach to attenuate the degree of injury.

A murine model of sepsis following smoke inhalation injury.

Acute lung injury (ALI) by smoke inhalation with subsequent pneumonia and sepsis represents a major cause of morbidity and mortality in burn patients. The aim of the present study was to develop a murine model of ALI and sepsis to enhance the knowledge of mechanistic aspects and pathophysiological changes in patients with these injuries. In deeply anesthetized female C57BL/6 mice, injury was induced by four sets of cotton smoke using an inhalation chamber. Afterward, live Pseudomonas aeruginosa (3.2x10(7) colony-forming units) was administered intranasally. The indicated dose of bacteria was determined based on the results of a dose-response study (n=47). The following study groups were monitored for survival over 96h: (1) sham injury group, (2) only smoke inhalation group, (3) only bacteria group, and (4) smoke inhalation plus bacteria group. Each group included 10 mice. The survival rates were 100%, 90%, 30%, and 10%, respectively. The double hit injury was associated with excessive releases of pro-inflammatory cytokines in the plasma, and enhanced neutrophil accumulation, increased lipid peroxidation, and excessive formation of reactive nitrogen species in the lung. In mice receiving only smoke inhalation injury, no systemic cytokine release and increased lung tissue lipid peroxidation were observed. However, smoke alone significantly increased neutrophil accumulation and formation of reactive nitrogen species in lung tissue. In conclusion, bacterial pneumonia is predominantly responsible for mortality and morbidity in this novel murine model of smoke inhalation and pulmonary sepsis. Reactive oxygen and nitrogen species mediate the severity of lung injury.

Role of different nitric oxide synthase isoforms in a murine model of acute lung injury and sepsis.

Excessive production of nitric oxide (NO) by NO synthase (NOS) with subsequent formation of peroxynitrite and poly(adenosine diphosphate ribose) is critically implemented in the pathophysiology of acute lung injury and sepsis. To elucidate the roles of different isoforms of NOS, we tested the effects of non-selective NOS inhibition and neuronal NOS (nNOS)- and inducible NOS (iNOS)-gene deficiency on the pulmonary oxidative and nitrosative stress reaction in a murine sepsis model. The injury was induced by four sets of cotton smoke using an inhalation chamber and subsequent intranasal administration of live Pseudomonas aeruginosa (3.2x10(7) colony-forming units). In wild type mice, the injury was associated with excessive releases of pro-inflammatory cytokines in the plasma, enhanced neutrophil accumulation, increased lipid peroxidation, and excessive formation of reactive nitrogen species and vascular endothelial growth factor in the lung. Both nNOS- and iNOS-gene deficiency led to significantly reduced oxidative and nitrosative stress markers in the lung, but failed to significantly improve survival. Treatment with a non-selective NOS inhibitor failed to reduce the oxidative and nitrosative stress reaction to the same extent and even tended to increase mortality. In conclusion, the current study demonstrates that both nNOS and iNOS are partially responsible for the pulmonary oxidative and nitrosative stress reaction in this model. Future studies should investigate the effects of specific pharmacological inhibition of nNOS and iNOS at different time points during the disease process.

Muscarinic receptor antagonist therapy improves acute pulmonary dysfunction after smoke inhalation injury in sheep.

OBJECTIVES: Inhalation injury contributes to the morbidity and mortality of burn victims. In humans and in an ovine model of combined smoke inhalation and burn injury, bronchospasm and acute airway obstruction contribute to progressive pulmonary insufficiency. This study tests the hypothesis that muscarinic receptor antagonist therapy with tiotropium bromide, an M1 and M3 muscarinic receptor antagonist, will decrease the airway constrictive response and acute bronchial obstruction to improve pulmonary function compared to injured animals without treatment. DESIGN: Randomized, prospective study involving 32 sheep. SETTING: Large-animal intensive care research laboratory. INTERVENTIONS: The study consisted of six groups: a sham group (n=4, instrumented noninjured), a control group (n=6, injured and not treated), and tiotropium bromide-treated groups, including both preinjury and postinjury nebulization protocols. Treatments for these groups included nebulization with 36 µg of tiotropium bromide 1 hr before injury (n=6) and postinjury nebulization protocols of 18 µg (n=6), 36 µg (n=6), and 72 µg (n=4) administered 1 hr after injury. All treated groups received an additional 14.4 µg every 4 hrs for the 24-hr study period. MAIN RESULTS: Pretreatment with tiotropium bromide significantly attenuated the increases in ventilatory pressures, pulmonary dysfunction, and upper airway obstruction that occur after combined smoke inhalation and burn injury. Postinjury treatments with tiotropium bromide were as effective as pretreatment in preventing pulmonary insufficiency, although a trend toward decreased obstruction was present only in all post-treatment conditions. There was no improvement noted in pulmonary function in animals that received a higher dose of tiotropium bromide. CONCLUSIONS: This study describes a contribution of acetylcholine to the airway constrictive and lumenal obstructive response after inhalation injury and identifies low-dose nebulization of tiotropium bromide as a potentially efficacious therapy for burn patients with severe inhalation injury.

Effects of early neuronal and delayed inducible nitric oxide synthase blockade on cardiovascular, renal, and hepatic function in ovine sepsis.

BACKGROUND: Recent evidence suggests that nitric oxide produced via the neuronal nitric oxide synthase is involved mainly in the early response to sepsis, whereas nitric oxide derived from the inducible nitric oxide synthase is responsible during the later phase. We hypothesized that early neuronal and delayed inducible nitric oxide synthase blockade attenuates multiple organ dysfunctions during sepsis. METHODS: Sheep were randomly allocated to sham-injured, nontreated animals (n = 6); injured (48 breaths of cotton smoke and instillation of Pseudomonas aeruginosa into the lungs), nontreated animals (n = 7); and injured animals treated with a neuronal nitric oxide synthase inhibitor from 1 to 12 h and an inducible nitric oxide synthase inhibitor from 12 to 24 h postinjury (n = 6). RESULTS: The injury induced arterial hypotension, vascular leakage, myocardial depression, and signs of renal and hepatic dysfunctions. The treatment significantly attenuated, but did not fully prevent, the decreases in mean arterial pressure and left ventricular stroke work index. Although the elevation of creatinine levels was partially prevented, the decreases in urine output and creatinine clearance were not affected. The injury-related increases in bilirubin levels, international normalized ratio, and lipid peroxidation in liver tissue were significantly attenuated. Although plasma nitrite/nitrate levels were significantly increased versus baseline from 12-24 h in controls, plasma nitrite/nitrate levels were not increased in treated animals. CONCLUSIONS: The combination treatment shows potential benefit on sepsis-related arterial hypotension and surrogate parameters of organ dysfunctions in sheep. It may be crucial to identify the time course of expression and activation of different nitric oxide synthase isoforms in future investigations.

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.

Combined neuronal and inducible nitric oxide synthase inhibition in ovine acute lung injury.

OBJECTIVE: Acute lung injury with subsequent pneumonia and sepsis represents a major cause of morbidity and mortality in thermally injured patients. Production of nitric oxide by the neuronal and inducible nitric oxide synthase may be critically involved in the pathophysiology of the disease process at different time points, and thus specific inhibition at different times may represent an effective treatment regimen. DESIGN: Prospective, controlled, randomized trial. SETTING: University research laboratory. SUBJECTS: Eighteen chronically instrumented, adult, female sheep. INTERVENTIONS: Following baseline measurements, the animals were allocated to either sham-injured, nontreated controls (sham), injured, nontreated controls (control), or injured animals treated with continuous infusion of 7-nitroindazole, a specific neuronal nitric oxide synthase inhibitor, during the first 12 hrs postinjury and infusion of BBS-2, a specific inducible nitric oxide synthase inhibitor, during the next 12 hrs. Injury was induced by 48 breaths of cotton smoke and subsequent instillation of Pseudomonas aeruginosa into the lungs. All sheep were mechanically ventilated and fluid resuscitated for the entire duration of the 24-hr experiment. MEASUREMENTS AND MAIN RESULTS: The injury induced severe pulmonary dysfunction, which was associated with increases in lung edema formation, airway obstruction, and vascular endothelial growth factor, 3-nitrotyrosine, and poly(adenosine diphosphate ribose) expression in lung tissue. The treatment reduced the degree of airway obstruction and improved pulmonary gas exchange, whereas the development of lung edema was not affected. The increases in lung tissue vascular endothelial growth factor, 3-nitrotyrosine, and poly(ribose) expression were attenuated by the treatment. CONCLUSIONS: The combination of early neuronal nitric oxide synthase and delayed inducible nitric oxide synthase inhibition shows potential benefit in ovine acute lung injury by reducing nitrosative stress in the lung and limiting the degree of airway obstruction.

Pulmonary vascular permeability changes in an ovine model of methicillin-resistant Staphylococcus aureus sepsis.

INTRODUCTION: Endothelial dysfunction is a hallmark of sepsis, associated with lung transvascular fluid flux and pulmonary dysfunction in septic patients. We tested the hypothesis that methicillin-resistant Staphylococcus aureus (MRSA) sepsis following smoke inhalation increases pulmonary transvascular fluid flux via excessive nitric oxide (NO) production. METHODS: Ewes were chronically instrumented, and randomised into either a control or MRSA sepsis (MRSA and smoke inhalation) group. RESULTS: Pulmonary function remained stable in the control group, whereas the MRSA sepsis group developed impaired gas exchange and significantly increased lung lymph flow, permeability index and bloodless wet-to-dry weight-ratio (W/D ratio). The plasma nitrate/nitrite (NOx) levels, lung inducible nitric oxide synthases (iNOS) and endothelial nitric oxide synthases (eNOS), vascular endothelial growth factor (VEGF) protein expressions and poly-(ADP)-ribose (PAR) were significantly increased by MRSA challenge. CONCLUSIONS: These results provide evidence that excessive NO production may mediate pulmonary vascular hyperpermeability in MRSA sepsis via up regulation of reactive radicals and VEGF.

Assessment of vascular permeability in an ovine model of acute lung injury and pneumonia-induced Pseudomonas aeruginosa sepsis.

OBJECTIVE: To assess the time changes and mechanism of pulmonary and peripheral vascular permeability in sheep with acute lung injury and sepsis. DESIGN: Prospective, controlled, randomized trial. SETTING: University research laboratory. SUBJECTS: A total of 21 chronically instrumented, adult female sheep. INTERVENTIONS: Sheep were instrumented with lung and prefemoral lymph fistulas and allocated to either an uninjured control group (n = 5) or sepsis group (n = 5). The sheep in the sepsis group received cotton smoke inhalation injury followed by instillation of Pseudomonas aeruginosa into the lungs. All sheep were mechanically ventilated and fluid resuscitated for the entire duration of the 24-hr experiment. Additional sheep (n = 11) received injury and were killed at different time points for the measurement of vascular endothelial growth factor in lung tissue. MEASUREMENTS AND MAIN RESULTS: The injury induced a hypotensive-hyperdynamic circulation; increases in pulmonary capillary pressure, net fluid balance, lung and prefemoral lymph flow and protein content, lung water content, abdominal and thoracic fluid and protein content, neutrophil accumulation in the lung, and vascular endothelial growth factor expression in lung tissue; and decreases in PaO2/FiO2 ratio, plasma protein concentration, plasma oncotic pressure, and myocardial contractility. CONCLUSIONS: Lung edema formation in this model was the result of marked increases in both pulmonary microvascular permeability and pressure. Pulmonary vascular hyperpermeability peaked 12 hrs postinjury and was related to vascular endothelial growth factor overexpression. Early myocardial failure was a potential contributor to the constant increase in pulmonary capillary pressure. The sepsis-induced increase in peripheral microvascular permeability was associated with significant accumulation of fluid and protein in the third space.

Effects of the bradykinin B2 receptor antagonist icatibant on microvascular permeability after thermal injury in sheep.

Peptide kinins are potent vasoactive agents in the microcirculation that might be released after burn injury. The present study was designed to test the hypothesis that Icatibant (JE 049), a potent, selective peptidomimetic bradykinin-B2 receptor antagonist, would reduce the cardiovascular pathology occurring in sheep exposed to 40% total body surface area (TBSA), third-degree burn. Female sheep were surgically prepared for chronic study. After 5 to 7 days' recovery from the operative procedure, they were randomized to five groups: sham (n = 6, noninjured, nontreated), medicated sham (n = 4, noninjured, treated with 20 microg kg h Icatibant), control (n = 7, 40% TBSA third-degree burn, nontreated), Icatibant-4 (n = 6, 40% TBSA third-degree burn, treated with 4 microg kg h Icatibant [low dose]), Icatibant-20 (n = 8, 40% TBSA third-degree burn, treated with 20 microg kg h Icatibant [high dose]). Prefemoral lymph flow (milliliters per hour) remained constant in the sham and medicated sham groups but increased after injury: control (0 h, 3.9 +/- 0.5; 24 h, 28 +/- 4.2; 48 h, 33.0 +/- 8.1). The increased fluid flux was associated with enhanced protein flux. Both low and high doses of Icatibant significantly reduced the microvascular fluid flux: Icatibant-4 (0 h, 5.3 +/- 0.6; 24 h, 17.5 +/- 3.5; 48 h, 20.3 +/- 3.4); Icatibant-20 (0 h, 5.3 +/- 1.1; 24 h, 15.2 +/- 2; 48 h, 17.6 +/- 4.1). Total prefemoral protein leak was reduced in all treatment groups. The low dose of Icatibant significantly reduced prefemoral lymph flow without adversely affecting the hemodynamic changes observed after burn injury in sheep, suggesting that the bradykinin antagonist would reduce edema formation and improve fluid management of thermally injured patients.

Advantages and pitfalls of combining intravenous antithrombin with nebulized heparin and tissue plasminogen activator in acute respiratory distress syndrome.

BACKGROUND: Pulmonary coagulopathy has become an important therapeutic target in adult respiratory distress syndrome (ARDS). We hypothesized that combining intravenous recombinant human antithrombin (rhAT), nebulized heparin, and nebulized tissue plasminogen activator (TPA) more effectively improves pulmonary gas exchange compared with a single rhAT infusion, while maintaining the anti-inflammatory properties of rhAT in ARDS. Therefore, the present prospective, randomized experiment was conducted using an established ovine model. METHODS: Following burn and smoke inhalation injury (40% of total body surface area, third-degree flame burn, and 4 × 12 breaths of cold cotton smoke), 18 chronically instrumented sheep were randomly assigned to receive intravenous saline plus saline nebulization (control), intravenous rhAT (6 IU/kg/h) started 1 hour after injury plus saline nebulization (AT i.v.) or intravenous rhAT combined with nebulized heparin (10,000 IU every 4 hours, started 2 hours after injury), and nebulized TPA (2 mg every 4 hours, started 4 hours after injury) (triple therapy, n = 6 each). All animals were mechanically ventilated and fluid resuscitated according to standard protocols during the 48-hour study period. RESULTS: Both treatment approaches attenuated ARDS compared with control animals. Notably, triple therapy was associated with an improved PaO2/FiO2 ratio (p = 0.007), attenuated pulmonary obstruction (p = 0.02) and shunting (p = 0.025), as well as reduced ventilatory pressures (p < 0.05 each) versus AT i.v. at 48 hours. However, the anti-inflammatory effects of sole AT i.v., namely, the inhibition of neutrophil activation (neutrophil count in the lymph and pulmonary polymorphonuclear cells, p < 0.05 vs. control each), pulmonary transvascular fluid flux (lymph flow, p = 0.004 vs. control), and systemic vascular leakage (cumulative net fluid balance, p < 0.001 vs. control), were abolished in the triple therapy group. CONCLUSION: Combining intravenous rhAT with nebulized heparin and nebulized TPA more effectively restores pulmonary gas exchange, but the anti-inflammatory effects of sole rhAT are abolished with the triple therapy. Interferences between the different anticoagulants may represent a potential explanation for these findings.

Effect of bronchodilators on bronchial gland cell proliferation after inhalation and burn injury in sheep.

The objective of this study is to measure the temporal changes in bronchial submucosal gland (SMG) cell proliferation in sheep after smoke inhalation and burn (S+B) injury, and to assess the effect of bronchodilators on the proliferative response. Archived main bronchial airways from sheep after S+B injury were immunostained for Ki67, and the percentage of ciliated duct and SMG cells expressing nuclear localization of Ki67 was determined for uninjured sheep and in sheep 24, 48, 72, and 96 hours after injury. A semiquantitative measure of lining epithelial exfoliation was made for each tissue. Bronchial tissues from sheep at 48 hours after S+B injury that had been nebulized with albuterol or tiotropium bromide (tiotropium) were examined to assess the effect of bronchodilators on the proliferative response. At 48 through 96 hours after injury, both ciliated duct and SMG cell proliferation were significantly increased compared with that of uninjured animals and animals 24 hours after injury, P <.05. A small increase in proliferation was seen in the SMG cells of albuterol-treated sheep compared with nebulized saline controls, P = .048. SMG cells of tiotropium-treated animals showed a significant increase in Ki67 nuclear staining compared with their study controls, P = .001. Extensive injury to the lining epithelium is associated with a proliferative response in both ciliated duct and SMG cells 24 hours after injury. The increase in proliferation in sheep treated with bronchodilators suggests that therapies for inhalation injury modify the glandular proliferative response. Further study to assess the ability of bronchodilators to enhance epithelial repair is warranted.

Assessment of combined muscarinic antagonist and fibrinolytic therapy for inhalation injury.

This study tests the hypothesis that muscarinic receptor antagonist therapy with tiotropium bromide (tiotropium; TIO), alone or in combination with tissue plasminogen activator (TPA), can attenuate pulmonary dysfunction in sheep after smoke inhalation and burn injury. The study consisted of four study groups, sham (uninjured), control (injured and untreated), TIO (injured and treated with nebulized TIO), and TIO + TPA (injured and treated with nebulized TIO and TPA). Cardiopulmonary and ventilatory parameters were monitored for 48 hours. After killing the animal, airway obstruction, submucosal gland neutrophilia, parenchyma histopathology, and lung wet to dry weight ratios were measured. PaO2/FiO2 was significantly improved in the TIO group compared with the control group at 48 hours, 301 ± 149 vs 99 ± 33, respectively, P < .05. At 48 hours, peak airway pressures in the control, TIO, and TIO + TPA groups were 35 ± 6, 24 ± 7, and 26 ± 10, respectively, with the mean of the TIO group being significantly different from that of the control group, P < .05. A trend of decreased airway obstruction was seen in the treated animals compared with controls; however, the differences were not statistically significant. The TIO and TIO + TPA groups exhibited significant decreases in gland neutrophilia compared with the control group, P < .05. No differences in parenchyma histopathology and lung edema between injured control and treated groups were observed. Nebulization of TIO was effective in improving pulmonary performance and reducing bronchial submucosal gland neutrophilia in sheep after smoke inhalation and burn injury. There was no additive benefit to the inclusion of nebulized TPA with TIO.

Pseudomonas aeruginosa is associated with increased lung cytokines and asymmetric dimethylarginine compared with methicillin-resistant Staphylococcus aureus.

The objective of the study was to investigate pulmonary responses to Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) using ovine and mice models of sepsis with emphasis on lung cytokine expression, asymmetric dimethylarginine (ADMA) concentration, and the arginase pathway. Sheep were instilled with either MRSA, P. aeruginosa, or saline under deep anesthesia; mechanically ventilated; resuscitated with fluid; and killed after 24 h. Mice were instilled with either MRSA, P. aeruginosa, or saline under deep anesthesia and killed after 8 h. Lungs were assessed for ADMA concentration, arginase activity, oxidative stress, and cytokine expression, and plasma was assessed for nitrate/nitrite concentrations. The severity of lung injury was more pronounced in P. aeruginosa sepsis compared with MRSA. The significant changes in sheep lung function after P. aeruginosa sepsis were associated with significantly increased ADMA concentrations and arginase activity compared with MRSA. However, the plasma concentration of nitrites and nitrates were significantly increased in MRSA sepsis compared with P. aeruginosa sepsis. In the mice model, P. aeruginosa significantly increased lung cytokine expression (IL-1 and IL-13), protein oxidation, and arginase activity compared with MRSA. Our data suggest that the greater expression of cytokines and ADMA concentrations may be responsible for severity of acute lung injury in P. aeruginosa sepsis. The lack of arginase activity may explain the greater nitric oxide production in MRSA sepsis.

Role of neuronal nitric oxide synthase in ovine sepsis model.

Smoke inhalation injury is often complicated with pneumonia, which frequently leads to subsequent development of sepsis. Excessive NO has been shown to mediate many sepsis-related pathological responses. In the present study, we used our well-established ovine smoke inhalation and pneumonia/sepsis model to examine the hypothesis that neuronal NO synthase (NOS) may be primarily responsible for these pathological alterations. We report the beneficial effects of the specific neuronal NOS (nNOS) inhibitor ZK234238. Adult female sheep were surgically prepared for the study. After 5 to 7 days' recovery, sheep were anesthetized and given double injury: 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 via tracheostomy tube. All sheep were mechanically ventilated and fluid resuscitated by lactated Ringer's solution. Sheep were randomly allocated into groups: control (injured not treated, n = 6) and treated (injured, but treated with ZK234238, n = 4). Continuous infusion of ZK234238 (100 microg x kg(-1) x h(-1)) was started 1 h after insult. ZK234238 attenuated the hypotension (at 18 and 24 h) and fall in systemic vascular resistance (at 24 h) seen in control animals. ZK234238 significantly inhibited increased fluid accumulation as well as increased plasma nitrate/nitrite 24 h after injury. Neuronal NOS inhibition significantly reduced lung water content and attenuated inflammatory indices such as lung tissue myeloperoxidase activity, IL-6 mRNA, and reactive nitrogen species. The above results suggest that the nNOS-derived NO may be involved in the pathophysiology of sepsis-related multiorgan dysfunction.

Cardiovascular collapse and vascular permeability changes in an ovine model of methicillin-resistant Staphylococcus aureus sepsis.

Methicillin-resistant Staphylococcus aureus (MRSA) infections with severe outcomes such as sepsis and septic shock are progressively increasing in both the community and in hospital settings. We hypothesized that overexpression of reactive nitrogen and oxygen species and vascular endothelial growth factor (VEGF) play a pivotal role in cardiovascular collapse associated with vascular hyperpermeability in MRSA sepsis. Twelve sheep were surgically prepared and randomized into a control (noninjured; n = 6) and a sepsis (injured; n = 6) group. Animals in the sepsis group were subjected to cotton smoke inhalation and instillation of 2.5 x 10(11) colony-forming units of live MRSA into both lungs. Cardiovascular variables in the control group remained stable, whereas the MRSA sepsis group developed a hypotensive and hyperdynamic circulatory shock state beginning at 6 h associated with significantly increased vascular permeability evidenced by increased prefemoral lymph flow starting at 12 h and permeability index from 12 to 18 h, higher fluid accumulation from 12 to 24 h, and significantly decreased plasma protein concentration and oncotic pressure beginning at 6 h compared with control animals. Myocardial 3-nitrotyrosine (3-NT) protein, poly (adenosine diphosphate-ribose), and VEGF mRNA expressions measured after the 24-h experiment were significantly increased in the injured animals as well. These results evidence that excessive production of reactive radicals and VEGF may play a major role in cardiovascular collapse and vascular hyperpermeability in MRSA sepsis.