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.

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.

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.

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.

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.

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.

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.

A novel bronchial artery catheterization technique with preserved blood flow in an ovine model.

The authors devised a novel bronchial artery catheterization technique to deliver agents directly into bronchial circulation with preserved blood flow in an awake ovine model. A polyurethane catheter was inserted into bronchial artery via an incision into the 4th intercostal space. Regional blood flow of the airway was measured by fluorescent microspheres before cannulation, after cannulation, and 7 days after the operative procedure. The blood flows were also measured in a sham group (no cannulation, no ligation, n = 6), cannulation group (bronchial artery cannulation, n = 5), and ligation group (bronchial artery ligation, n = 5) at baseline and 6 hours after burn and smoke inhalation injury. The regional blood flows decreased slightly after cannulation in proximal bronchi, but recovered after 7 days. The regional blood flow increased 10-fold after inhalation injury in bronchi of the sham group. Bronchial artery ligation significantly attenuated the increase of blood flow. However, cannulation preserved regional blood flow and did not prevent the blood flow increases after burn and smoke inhalation injury, thus constituting a novel bronchial artery catheterization technique.

Recombinant human activated protein C attenuates cardiovascular and microcirculatory dysfunction in acute lung injury and septic shock.

INTRODUCTION: This prospective, randomized, controlled, experimental animal study looks at the effects of recombinant human activated protein C (rhAPC) on global hemodynamics and microcirculation in ovine acute lung injury (ALI) and septic shock, resulting from smoke inhalation injury. METHODS: Twenty-one sheep (37 ± 2 kg) were operatively prepared for chronic study and randomly allocated to either the sham, control, or rhAPC group (n = 7 each). The control and rhAPC groups were subjected to insufflation of four sets of 12 breaths of cotton smoke followed by instillation of live Pseudomonas aeruginosa into both lung lobes, according to an established protocol. Healthy sham animals were not subjected to the injury and received only four sets of 12 breaths of room air and instillation of the vehicle (normal saline). rhAPC (24 µg/kg/hour) was intravenously administered from 1 hour post injury until the end of the 24-hour experiment. Regional microvascular blood flow was analyzed using colored microspheres. All sheep were mechanically ventilated with 100% oxygen, and fluid resuscitated with lactated Ringer's solution to maintain hematocrit at baseline levels. RESULTS: The rhAPC-associated reduction in heart malondialdehyde (MDA) and heart 3-nitrotyrosine (a reliable indicator of tissue injury) levels occurred parallel to a significant increase in mean arterial pressure and to a significant reduction in heart rate and cardiac output compared with untreated controls that showed a typical hypotensive, hyperdynamic response to the injury (P < 0.05). In addition, rhAPC significantly attenuated the changes in microvascular blood flow to the trachea, kidney, and spleen compared with untreated controls (P < 0.05 each). Blood flow to the ileum and pancreas, however, remained similar between groups. The cerebral blood flow as measured in cerebral cortex, cerebellum, thalamus, pons, and hypothalamus, was significantly increased in untreated controls, due to a loss of cerebral autoregulation in septic shock. rhAPC stabilized cerebral blood flow at baseline levels, as in the sham group. CONCLUSIONS: We conclude that rhAPC stabilized cardiovascular functions and attenuated the changes in visceral and cerebral microcirculation in sheep suffering from ALI and septic shock by reduction of cardiac MDA and 3-nitrotyrosine.

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.

Inhibition of neuronal nitric oxide synthase in ovine model of acute lung injury.

OBJECTIVE: Acute respiratory distress syndrome/acute lung injury is a serious complication of burn patients with concomitant smoke inhalation injury. Nitric oxide has been shown to play a major role in pulmonary dysfunction from thermal damage. In this study, we have tested the hypothesis that inhibition of neuronal nitric oxide synthase could ameliorate the severity of acute lung injury using our well-established ovine model of cutaneous burn and smoke inhalation. DESIGN: Prospective, randomized, controlled, experimental animals study. SETTING: Investigational intensive care unit at university hospital. SUBJECTS: Adult female sheep. INTERVENTIONS: Female sheep (n = 16) were surgically prepared for the study. Seven days after surgery, all sheep were randomly allocated into three study groups: sham (noninjured, nontreated, n = 6); control (injured, treated with saline, n = 6); and neuronal nitric oxide synthase (injured, treated with specific neuronal nitric oxide synthase inhibitor, ZK 234238 (n = 4). Control and neuronal nitric oxide synthase groups were given a cutaneous burn (40% of total body surface, third degree) and insufflated with cotton smoke (48 breaths, <40 degrees C) under halothane anesthesia. Animals in sham group received fake injury also under halothane anesthesia. After injury or fake injury procedure, all sheep were placed on ventilators and resuscitated with lactated Ringer's solution. Neuronal nitric oxide synthase group was administered with continuous infusion of ZK 234238 started 1 hr postinjury with a dose of 100 microg/kg/hr. Sham and control groups received same amount of saline. MEASUREMENTS AND MAIN RESULTS: Cardiopulmonary hemodynamics monitored during the 24-hr experimental time period was stable in the sham group. Control sheep developed multiple signs of acute lung injury. This pathophysiology included decreased pulmonary gas exchange and lung compliance, increased pulmonary edema, and inflammatory indices, such as interleukin-8. Treatment of injured sheep with neuronal nitric oxide synthase inhibitor attenuated all the observed pulmonary pathophysiology. CONCLUSIONS: The results provide definitive evidence that inhibition of neuronal nitric oxide synthase-derived excessive nitric oxide may be a novel and beneficial treatment strategy for pulmonary pathology in burn victims with smoke inhalation injury.

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.

Unilateral developments of osteoarthritis and Charcot's joint in a patient with neurofibromatosis.

BACKGROUND: Precise mechanism of developing neuropathic arthropathy known as Charcot's joint is not fully understood. CASE REPORT: A 55-year-old Japanese woman with neurofibromatosis-1 complained of right gonalgia in December 2001. Physical examination revealed a huge tumor in the right lower leg without signs of inflammation. Laboratory findings were unremarkable. Radiographic examination disclosed the presence of osteoarthropathy in the right knee joint. In contrast, radiologic findings of the right foot and ankle were compatible with neuropathic arthropathy. Further investigations could not reveal abnormal findings in the nervous system. To improve patient's quality of life, partial resection of the tumor was performed. The resected tissues were compatible with neurofibromatosis without malignant transformation. The patient newly noticed pains in the right ankle and tarsal joints one year after the operation. Restricted mobility and insufficient blood supply in the right knee arising from the huge tumor might accelerate development of osteoarthropathy through malnutrition of the chondrocytes. Because the patient did not experience the arthralgia before the operation, the tumor might damage the peripheral nerves unabling to receive afferent signals from such joints resulting in neuropathic arthropathy. CONCLUSIONS: The damaged peripheral nerves might be contributory to developing or accelerating neuropathic arthropathy.

Lung-protective effects of the metalloporphyrinic peroxynitrite decomposition catalyst WW-85 in interleukin-2 induced toxicity.

Recombinant interleukin-2 (IL-2) therapy for malignancy is associated with a pulmonary vascular leakage syndrome (VLS) similar to that seen in sepsis. We investigated the possibility that the IL-2-induced VLS may be associated with the release of peroxynitrite (ONOO(-)), and used a model of IL-2-induced VLS in sheep to test the effects of the ONOO(-) decomposition catalyst WW-85. Eighteen sheep were chronically instrumented and randomly divided into three groups (n=6 per group): sham: lactated Ringer's solution, control: IL-2, and treatment: IL-2 and WW-85. Treatment with WW-85 significantly improved lung transvascular fluid flux, decreased lipid peroxidation, limited iNOS as well as PAR intensity, prevented tachycardia, and attenuated the increase in core body temperature resulting from IL-2 treatment. These findings suggest that ONOO(-) plays a pivotal role in the pathology of IL-2-induced pulmonary VLS, and that WW-85 may become a useful treatment option.

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.