Human mesenchymal stem cells reduce the severity of acute lung injury in a sheep model of bacterial pneumonia.

BACKGROUND: Human bone marrow-derived mesenchymal stem (stromal) cells (hMSCs) improve survival in mouse models of acute respiratory distress syndrome (ARDS) and reduce pulmonary oedema in a perfused human lung preparation injured with Escherichia coli bacteria. We hypothesised that clinical grade hMSCs would reduce the severity of acute lung injury (ALI) and would be safe in a sheep model of ARDS. METHODS: Adult sheep (30-40 kg) were surgically prepared. After 5 days of recovery, ALI was induced with cotton smoke insufflation, followed by instillation of live Pseudomonas aeruginosa (2.5×10(11) CFU) into both lungs under isoflurane anaesthesia. Following the injury, sheep were ventilated, resuscitated with lactated Ringer's solution and studied for 24 h. The sheep were randomly allocated to receive one of the following treatments intravenously over 1 h in one of the following groups: (1) control, PlasmaLyte A, n=8; (2) lower dose hMSCs, 5×10(6) hMSCs/kg, n=7; and (3) higher-dose hMSCs, 10×10(6) hMSCs/kg, n=4. RESULTS: By 24 h, the PaO2/FiO2 ratio was significantly improved in both hMSC treatment groups compared with the control group (control group: PaO2/FiO2 of 97±15 mm Hg; lower dose: 288±55 mm Hg (p=0.003); higher dose: 327±2 mm Hg (p=0.003)). The median lung water content was lower in the higher-dose hMSC-treated group compared with the control group (higher dose: 5.0 g wet/g dry [IQR 4.9-5.8] vs control: 6.7 g wet/g dry [IQR 6.4-7.5] (p=0.01)). The hMSCs had no adverse effects. CONCLUSIONS: Human MSCs were well tolerated and improved oxygenation and decreased pulmonary oedema in a sheep model of severe ARDS. TRAIL REGISTRATION NUMBER: NCT01775774 for Phase 1. NCT02097641 for Phase 2.

The selective vasopressin type 1a receptor agonist selepressin (FE 202158) blocks vascular leak in ovine severe sepsis*.

OBJECTIVE: To determine if the selective vasopressin type 1a receptor agonist selepressin (FE 202158) is as effective as the mixed vasopressin type 1a receptor/vasopressin V2 receptor agonist vasopressor hormone arginine vasopressin when used as a titrated first-line vasopressor therapy in an ovine model of Pseudomonas aeruginosa pneumonia-induced severe sepsis. DESIGN: Prospective, randomized, controlled laboratory experiment. SETTING: University animal research facility. SUBJECTS: Forty-five chronically instrumented sheep. INTERVENTIONS: Sheep were anesthetized, insufflated with cooled cotton smoke via tracheostomy, and P. aeruginosa were instilled into their airways. They were then placed on assisted ventilation, awakened, and resuscitated with lactated Ringer's solution titrated to maintain hematocrit ± 3% from baseline levels. If, despite fluid management, mean arterial pressure fell by more than 10 mm Hg from baseline level, an additional continuous IV infusion of arginine vasopressin or selepressin was titrated to raise and maintain mean arterial pressure within no less than 10 mm Hg from baseline level. Effects of combination treatment of selepressin with the selective vasopressin V2 receptor agonist desmopressin were similarly investigated. MEASUREMENTS AND MAIN RESULTS: In septic sheep, MAP fell by ~30 mm Hg, systemic vascular resistance index decreased by ~50%, and ~7 L of fluid were retained over 24 hours; this fluid accumulation was partially reduced by arginine vasopressin and almost completely blocked by selepressin; and combined infusion of selepressin and desmopressin increased fluid accumulation to levels similar to arginine vasopressin treatment. CONCLUSIONS: Resuscitation with the selective vasopressin type 1a receptor agonist selepressin blocked vascular leak more effectively than the mixed vasopressin type 1a receptor/vasopressin V2 receptor agonist arginine vasopressin because of its lack of agonist activity at the vasopressin V2 receptor.

No correlation between initial arterial carboxyhemoglobin level and degree of lung injury following ovine burn and smoke inhalation.

Fire victims often suffer from burn injury and concomitant inhalation trauma, the latter significantly contributing to the morbidity and mortality in these patients. Measurement of blood carboxyhemoglobin levels has been proposed as a diagnostic marker to verify and, perhaps, quantify the degree of lung injury following inhalation trauma. However, this correlation has not yet been sufficiently validated. A total of 77 chronically instrumented sheep received sham injury, smoke inhalation injury, or combined burn and inhalation trauma following an established protocol. Arterial carboxyhemoglobin concentrations were determined directly after injury and correlated to several clinical and histopathological determinants of lung injury that were detected 48 hours post-injury. The injury induced severe impairment of pulmonary gas exchange and increases in transvascular fluid flux, lung water content, and airway obstruction scores. No significant correlations were detected between initial carboxyhemoglobin levels and all measured clinical and histopathological determinants of lung injury. In conclusion, the amount of arterial carboxyhemoglobin concentration cannot predict the degree of lung injury at 48 hours after ovine burn and smoke inhalation trauma.

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.

Time course of early histopathological lung changes in an ovine model of acute lung injury and pulmonary infection.

Large animal models are valuable tools in biological and medical lung research. Despite the existence of established large animal models, the scientific progress requires more detailed description and expansion of established methods. Previously, we established an ovine model of acute lung injury and subsequent bacterial instillation into the lungs. The current study was designed to assess the time course of early lung histopathological alterations in a large animal model. Injury was induced by smoke inhalation and instillation of live Pseudomonas aeruginosa into the lungs. After 4, 8, 12, 18, and 24 hours, respectively, lung tissue was harvested and histopathological changes were evaluated (n = 4 each). Additional four sheep received no injury and only lung tissue was taken. In injured animals, bronchial obstruction score increased over time and was significantly elevated from 12 to 24 hours (P < .05 versus no injury). Inflammation score was significantly increased at 12 and 18 hours (P < .05 versus no injury). Hemorrhage score was increased at 8 and 12 hours (P < .05 versus no injury). Alveolar edema score was significantly higher in injured sheep at 8, 18, and 24 hours (P < .05 each versus no injury). In conclusion, bronchial obstruction and alveolar edema scores significantly increased over time and reached a plateau, while both inflammation and hemorrhage scores were transiently increased peaking around the 12-hour time point. This information improves the understanding of lung histopathological alterations following acute lung injury and pulmonary infection and may help optimizing the timing of study interventions and evaluation time points in future experiments with this model.

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.

Unlike arginine vasopressin, the selective V1a receptor agonist FE 202158 does not cause procoagulant effects by releasing von Willebrand factor.

OBJECTIVE: To compare the effects on von Willebrand factor release of the mixed vasopressin type 1a and type 2 receptor agonist arginine vasopressin and the selective vasopressin type 1a receptor agonist FE 202158, [Phe2,Ile3,Hgn4,Orn(iPr)8]vasopressin, at doses required for the treatment of septic shock. DESIGN: Prospective, randomized, controlled laboratory experiment. SETTING: University animal research facility. SUBJECTS: Twenty-four chronically instrumented sheep. INTERVENTIONS: After a 5-day recovery from instrumentation, sheep were randomly assigned to receive a single intravenous bolus of the selective vasopressin type 2 receptor agonist desmopressin (1 nmol·kg(-1)) or continuous intravenous infusions of arginine vasopressin (3 pmol·kg(-1)·min(-1)), the selective vasopressin type 1a receptor agonist FE 202158 (10 pmol·kg(-1)·min(-1)), or vehicle (0.9% NaCl) (n = 6 each). MEASUREMENTS AND MAIN RESULTS: The von Willebrand factor antigen activity relative to hemoglobin concentration (vWF:Ag/Hb ratio) was measured at different time points during the 120-min study period. Maximal vWF:Ag/Hb ratio expressed as percentage of baseline level was significantly increased compared to vehicle-infused animals (3 ± 2%) in the desmopressin (40 ± 6%, p < .001) and arginine vasopressin groups (25 ± 4%, p < .001). The ratio for the FE 202158 group was not statistically different from the sham group (9 ± 2%, p = .208). Notably, maximal vWF:Ag/Hb ratio was lower in the FE 202158 than the arginine vasopressin group (p < .005). CONCLUSIONS: Unlike the mixed vasopressin type 1a receptor/vasopressin type 2 receptor agonist arginine vasopressin, the selective vasopressin type 1a receptor agonist FE 202158 does not release von Willebrand factor. Because von Willebrand factor is involved in coagulatory and inflammatory pathways during septic shock, future studies should clarify the role of the vasopressin type 2 receptor-mediated von Willebrand factor increase by arginine vasopressin and the potential benefit of selective vasopressin type 1a receptor-agonists like FE 202158.

Time course of the inflammatory and oxidative stress response to pulmonary infection in mice.

The pathophysiological response to pulmonary infection includes a surge of proinflammatory cytokines and excessive production of nitric oxide (NO), but the time changes are not sufficiently defined. The current study was designed to assess the time course of proinflammatory cytokines and NO production in a murine model of pulmonary infection. The injury was induced by intranasal administration of live Pseudomonas aeruginosa (3.2 × 10(7) colony-forming units) in C57BL/6 wild-type mice. The animals were euthanized at 3, 6, 9, 12, and 15 hours postinjury. Additional mice received sham injury (0 hours; control). Lung tissue and plasma samples were harvested at the respective time points. The injury induced an early increase in interleukin (IL)-1 ß protein in lung tissue that persisted during the entire study period with a peak at the 9-hour time point. The increases in TNF-α and IL-6 proteins in lung tissue were less intense, but showed a peak about 9 hours postinjury. The plasma levels of IL-1 ß and tumor necrosis factor (TNF)-α protein were not elevated during the experimental period, but only an increase in plasma levels of IL-6 plasma protein was detected. These findings compensate for the limitations of previous experiments with similar infection models and improve the understanding of pathophysiologic alterations in response to pulmonary infection. In addition, the identification of the time changes of the described pathogenetic factors may enhance the timing of innovate therapeutic approaches in future experiments.

Beneficial pulmonary effects of a metalloporphyrinic peroxynitrite decomposition catalyst in burn and smoke inhalation injury.

During acute lung injury, nitric oxide (NO) exerts cytotoxic effects by reacting with superoxide radicals, yielding the reactive nitrogen species peroxynitrite (ONOO(-)). ONOO(-) exerts cytotoxic effects, among others, by nitrating/nitrosating proteins and lipids, by activating the nuclear repair enzyme poly(ADP-ribose) polymerase and inducing VEGF. Here we tested the effect of the ONOO(-) decomposition catalyst INO-4885 on the development of lung injury in chronically instrumented sheep with combined burn and smoke inhalation injury. The animals were randomized to a sham-injured group (n = 7), an injured control group [48 breaths of cotton smoke, 3rd-degree burn of 40% total body surface area (n = 7)], or an injured group treated with INO-4885 (n = 6). All sheep were mechanically ventilated and fluid-resuscitated according to the Parkland formula. The injury-related increases in the abundance of 3-nitrotyrosine, a marker of protein nitration by ONOO(-), were prevented by INO-4885, providing evidence for the neutralization of ONOO(-) action by the compound. Burn and smoke injury induced a significant drop in arterial Po(2)-to-inspired O(2) fraction ratio and significant increases in pulmonary shunt fraction, lung lymph flow, lung wet-to-dry weight ratio, and ventilatory pressures; all these changes were significantly attenuated by INO-4885 treatment. In addition, the increases in IL-8, VEGF, and poly(ADP-ribose) in lung tissue were significantly attenuated by the ONOO(-) decomposition catalyst. In conclusion, the current study suggests that ONOO(-) plays a crucial role in the pathogenesis of pulmonary microvascular hyperpermeability and pulmonary dysfunction following burn and smoke inhalation injury in sheep. Administration of an ONOO(-) decomposition catalyst may represent a potential treatment option for this injury.

Specific inhibition of nitric oxide synthases at different time points in a murine model of pulmonary sepsis.

Excessive production of nitric oxide (NO) by NO synthase (NOS) and a subsequent oxidative stress reaction are thought to be critically involved in the pathophysiology of sepsis. Previous studies suggested that NO production by neuronal NOS (nNOS) and inducible NOS (iNOS) is implemented in the disease process at different time points after the injury. Here we tested the roles of selective pharmacological inhibition of nNOS and iNOS at different time points in a murine model of pulmonary sepsis. The injury was induced by intranasal administration of live Pseudomonas aeruginosa (3.2×10(7) colony-forming units) in C57BL/6 wild-type mice. The animals received no treatment (control) or treatment with a specific nNOS inhibitor (4 or 8h), iNOS inhibitor (4 or 8h), or non-specific NOS inhibitor (4 or 8h). In controls, the injury was associated with excessive releases of pro-inflammatory cytokines in the plasma, enhanced tissue lipid peroxidation, and decreased survival. Non-specific NOS inhibition at either time point did not influence survival and was not further investigated. While nNOS inhibition at 4h was associated with a trend toward improved survival and significantly reduced contents of lung nitrite/nitrate (NO(x)) and liver malondialdehyde, the blockade of nNOS at 8h had no effect on these parameters. In contrast, early iNOS inhibition was associated with a trend toward decreased survival and no effects on lung NO(x) and liver malondialdehyde contents, whereas later iNOS blockade was associated with decreased malondialdehyde content in liver homogenates. In conclusion, pulmonary sepsis in mice may be beneficially influenced by specific pharmacological nNOS inhibition at an earlier time point and iNOS inhibition at a later time points post-injury. Future investigations should identify the time changes of the expression and activation of NOS isoforms.

Role of selective V1a receptor agonism in ovine septic shock.

OBJECTIVE: The vasopressor effect of arginine vasopressin, a mixed V1a/V2 receptor (V1aR/V2R) agonist, is mediated through the V1aR. Because V2R stimulation may aggravate sepsis-induced vasodilation, fluid accumulation, and microvascular thrombosis, a higher V1aR vs. V2R selectivity might be advantageous. The objective of this study was to elucidate the effects of a first-line therapy with the selective V1aR agonist Phe2-Orn8-Vasotocin vs. arginine vasopressin or norepinephrine on cardiopulmonary hemodynamics and organ function in ovine septic shock. DESIGN: Randomized controlled laboratory experiment. SETTING: University animal research facility. SUBJECTS: : Twenty-four chronically instrumented sheep. INTERVENTIONS: After the onset of fecal peritonitis-induced septic shock (mean arterial pressure <60 mm Hg), sheep were randomly assigned to receive first-line treatment with Phe2-Orn8-Vasotocin (0.05 µg·kg·h), arginine vasopressin (0.05 µg·kg·h), or normal saline (each n = 8). In all groups, open-label norepinephrine was additionally titrated up to 1 µg·kg·min to maintain mean arterial pressure at 70 ± 5 mm Hg, if necessary. MEASUREMENTS AND MAIN RESULTS: Compared with single norepinephrine therapy, the selective V1aR agonist Phe2-Orn8-Vasotocin reduced norepinephrine requirements (2-6 hrs: p < .05 each) and fluid accumulation (p = .043). In addition, mean arterial pressure (6-10 hrs: p < .05 each), pulmonary gas exchange (8-10 hrs: p < .05 each), and global oxygen transport (10 hrs: p < .05 each) were improved by Phe2-Orn8-Vasotocin vs. both other groups. Despite similar preload left ventricular stroke work index was higher in Phe2-Orn8-Vasotocin- than in arginine vasopressin-treated animals (10 hrs: p = .02). Metabolic dysfunction (base excess, lactate concentrations) and renal dysfunction (urinary output, creatinine clearance) were attenuated by Phe2-Orn8-Vasotocin infusion when compared with arginine vasopressin and single norepinephrine therapy. Immunohistochemical analyses of lung tissue revealed higher hemeoxygenase-1 and lower 3-nitrotyrosine concentrations in Phe2-Orn8-Vasotocin-treated animals vs. both other groups (p < .05 each). In addition, the selective V1aR agonist Phe2-Orn8-Vasotocin slightly prolonged survival when compared with arginine vasopressin (p = .01) and standard treatment with norepinephrine (p = .003). CONCLUSIONS: Selective V1aR agonism appears to be superior to the V1aR/V2R agonist arginine vasopressin and single norepinephrine infusion for hemodynamic support in septic shock.

Cardiopulmonary effects of low-dose arginine vasopressin in ovine acute lung injury.

OBJECTIVE: To elucidate the effects of low-dose arginine vasopressin on cardiopulmonary functions and nitrosative stress using an established model of acute lung injury. DESIGN: Prospective, randomized, controlled laboratory experiment. SETTING: Investigational intensive care unit. SUBJECTS: Eighteen chronically instrumented sheep. INTERVENTIONS: Sheep were randomly assigned to a sham group without injury or treatment, an injury group without treatment (40% total body surface area third-degree burn and 48 breaths of cold cotton smoke), or an injured group treated with arginine vasopressin (0.02 IU·min⁻¹) from 1 hr after injury until the end of the 24-hr study period (each n = 6). All sheep were mechanically ventilated and fluid resuscitated using an established protocol. MEASUREMENTS AND MAIN RESULTS: There were no differences among groups at baseline. The injury was characterized by a severe deterioration of cardiopulmonary function (left ventricular stroke work indexes and Pao2/Fio2 ratio; p < .01 each vs. sham). Compared with controls, arginine vasopressin infusion improved myocardial function, as suggested by higher stroke volume indexes and left ventricular stroke work indexes (18-24 hrs and 6-24 hrs, respectively; p < .05 each). In addition to an improved gas exchange (higher Pao2/Fio2 ratios from 6 to 24 hrs, p < .01 each), pulmonary edema (bloodless wet-to-dry-weight ratio; p = .018), bronchial obstruction (p = .01), and pulmonary shunt fraction (12-24 hrs; p ≤ .001 each) were attenuated in arginine vasopressin-treated animals compared with controls. These changes occurred along with reduced nitrosative stress, as indicated by lower plasma levels of nitrate/nitrite (12-24 hrs, p < .01 each), as well as lower myocardial and pulmonary tissue concentrations of 3-nitrotyrosine (p = .041 and p = .042 vs. controls, respectively). At 24 hrs, pulmonary 3-nitrotyrosine concentrations were negatively correlated with Pao2/Fio2 ratio (r = -.882; p < .001) and myocardial 3-nitrotyrosine content with stroke volume indexes (r = -.701; p = .004). CONCLUSIONS: Low-dose arginine vasopressin reduced nitrosative stress and improved cardiopulmonary functions in sheep with acute lung injury secondary to combined burn and smoke inhalation injury.

Preclinical evaluation of epinephrine nebulization to reduce airway hyperemia and improve oxygenation after smoke inhalation injury.

OBJECTIVE: Acute lung injury secondary to smoke inhalation is a major source of morbidity and mortality in burn patients. We tested the hypothesis that nebulized epinephrine would ameliorate pulmonary dysfunction secondary to acute lung injury by reducing airway hyperemia and edema formation and mediating bronchodilatation in an established, large animal model of inhalation injury. DESIGN: Prospective, controlled, randomized trial. SETTING: University research laboratory. SUBJECTS: Twenty-four chronically instrumented, adult, female sheep. INTERVENTIONS: Following baseline measurements, the animals were allocated to a sham-injured group (n = 5), an injured and saline-treated group (n = 6), or an injured group treated with 4 mg of nebulized epinephrine every 4 hrs (n = 6). Inhalation injury was induced by 48 breaths of cotton smoke. The dose of epinephrine was derived from dose finding experiments (n = 7 sheep). MEASUREMENTS AND MAIN RESULTS: The injury induced significant increases in airway blood flows, bronchial wet/dry weight ratio, airway obstruction scores, ventilatory pressures, and lung malondialdehyde content, and contributed to severe pulmonary dysfunction as evidenced by a significant decline in Pao2/Fio2 ratio and increase in pulmonary shunt fraction. Nebulization of epinephrine significantly reduced tracheal and main bronchial blood flows, ventilatory pressures, and lung malondialdehyde content. The treatment was further associated with significant improvements of Pao2/FIO2 ratio and pulmonary shunting. CONCLUSIONS: Nebulization of epinephrine reduces airway blood flow and attenuates pulmonary dysfunction in sheep subjected to severe smoke inhalation injury. Future studies will have to improve the understanding of the underlying pathomechanisms and identify the optimal dosing for the treatment of patients with this injury.

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.

Effects of combined arginine vasopressin and levosimendan on organ function in ovine septic shock.

OBJECTIVE: To compare the effects of a first-line therapy of combined arginine vasopressin, levosimendan, and norepinephrine with arginine vasopressin + norepinephrine or norepinephrine alone in ovine septic shock. DESIGN: Prospective, randomized, controlled laboratory experiment. SETTING: University animal research facility. SUBJECTS: Twenty-one chronically instrumented sheep. INTERVENTIONS: After the onset of fecal peritonitis-induced septic shock (mean arterial pressure <60 mm Hg), sheep were randomly assigned to receive first-line treatment with arginine vasopressin (0.5 mU·kg·min), combined arginine vasopressin (0.5 mU·kg·min) and levosimendan (0.2 µg·kg·min), or normal saline (each n = 7) for 24 hrs. In all groups, open-label norepinephrine was additionally titrated to maintain mean arterial pressure at 70 ± 5 mm Hg, if necessary. MEASUREMENTS AND MAIN RESULTS: Arginine vasopressin + levosimendan + norepinephrine improved left ventricular contractility (higher stroke work indices at similar or lower preload) and pulmonary function (Pao2/Fio2 ratio) when compared with the other groups (p < .05 each). Both nonadrenergic treatment strategies reduced open-label norepinephrine doses. However, only arginine vasopressin + levosimendan + norepinephrine limited fluid requirements and positive fluid balance vs. both other groups (p < .05 each). In addition, arginine vasopressin + levosimendan + norepinephrine increased mixed venous oxygen saturation as compared with arginine vasopressin + norepinephrine. Histologic tissue analyses and pulmonary hemeoxygenase-1 activity revealed no differences among groups. Notably, arginine vasopressin + levosimendan + norepinephrine therapy reduced pulmonary 3-nitrotyrosine levels (p = .028 vs. control animals) as well as urinary protein/creatinine ratio (p < .05 each) and slightly prolonged survival when compared with both other groups (4 hrs vs. arginine vasopressin + norepinephrine: p = .013; 7 hrs vs. norepinephrine alone: p = .003). CONCLUSIONS: First-line cardiovascular support with combined arginine vasopressin and levosimendan supplemented with norepinephrine improves myocardial, vascular, pulmonary, and renal function as compared with arginine vasopressin + norepinephrine in septic shock.

Hypoproteinemia does not alter plasma volume expansion in response to a 0.9% saline bolus in awake sheep.

OBJECTIVE: To test the hypothesis that hypoproteinemia reduces plasma volume expansion produced by a bolus of crystalloid solution given to awake sheep. DESIGN: Prospective and observational. SETTING: Laboratory. SUBJECTS: Five female merino sheep (n = 5) weighing 37 ± 3 kg were anesthetized. INTERVENTIONS: Each animal was subjected to a 5-day test period: day 1: 50 mL/min 0.9% saline infusion over 20 mins. Days 2-4: daily plasmapheresis and replacement of the shed plasma with 6 L of 0.9% saline were performed in increments. MEASUREMENTS AND MAIN RESULTS: Fractional plasma volume expansion after rapid infusion of saline on days 1 and 5 was calculated from changes in hemoglobin concentration. There was a significant reduction in total plasma protein concentration after plasmapheresis (p < .05). Colloid osmotic pressures were also significantly lowered (p < .05). A crystalloid infusion of 0.9% saline did not alter any of these values compared with baseline. The hemodynamic measurements did not show significant differences between the experiments. The plasma volume expansion reached approximately 20% at the end of infusion and stayed at 10-15% during the experiments. No difference was found in plasma volume expansion produced by a bolus of 50 mL/min of 0.9% in the hypoproteinemic state when compared with the euproteinemic state (p = .61). No difference in cumulative urinary output was found between the two states. CONCLUSIONS: In contrast to our hypothesis, severe acute hypoproteinemia does not reduce plasma volume expansion in response to 50 mL/min 0.9% saline infusion in nonspleenectomized sheep when compared with the resultant plasma volume expansion after a 50 mL/min of 0.9% infusion in the euproteinemic state.