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Unlabelled: Pulmonary abnormalities occur in 30-80% of fatalities after burn. The objective of our study is to investigate lung pathology in autopsy tissues of pediatric burn patients., Methods: Three scientists with pathology training in pediatric burn care reviewed masked autopsy slides of burned children who died after admission to a burn center from 2002 to 2012 (n=43). Autopsy lung tissue was assigned scores for histologic abnormalities in 9 categories, including alveolar and interstitial fibrosis, hyaline membranes, and type II epithelial cell proliferation. Scores were then tested for correlation with age, TBSA burn, number of days between burn and death, time between burn and admission, and the presence of inhalation injury using analyses with linear models., Results: Type II epithelial cell proliferation was significantly more common in cases with a longer time between burn and admission (p<0.02). Interstitial fibrosis was significantly more severe in cases with longer survival after burn (p<0.01). The scores for protein were significantly higher in cases with longer survival after burn (p<0.03). Enlarged air spaces were significantly more prominent in cases with longer survival after burn (p<0.01), and in cases with the presence of inhalation injury (p<0.01)., Conclusions: Histological findings associated with diffuse alveolar damage (DAD), which is the pathological correlate of the acute respiratory distress syndrome (ARDS), were seen in approximately 42% of autopsies studied. Protein-rich alveolar edema, which is the abnormality that leads to ARDS, may occur from multiple causes, including inhalation injury., (Copyright © 2014 Elsevier Ltd and ISBI. All rights reserved.)

Bronchial circulation plays a critical role in the pathophysiology of burn and smoke inhalation-induced acute lung injury. A 10-fold increase in bronchial blood flow is associated with excessive production of nitric oxide (NO) following smoke inhalation and cutaneous burn. Because an increased release of neuropeptides from the airway has been implicated in smoke inhalation injury, we hypothesized that direct delivery into the bronchial artery of low-dose 7-nitroindazole (7-NI), a specific neuronal NO synthase inhibitor, would attenuate smoke/burn-induced acute lung injury. Eighteen adult female sheep were instrumented for chronic hemodynamic monitoring 5 to 7 days before the injury. The bronchial artery was cannulated via intercostal thoracotomy, while blood flow was preserved. Acute lung injury was induced by 40% total body surface area third-degree cutaneous burn and smoke inhalation (48 breaths of cotton smoke, <40°C) under deep anesthesia. Following injury, animals (35.4 ± 1.1 kg) were divided into three groups: (a) 7-NI group: 1 h after injury, 7-NI (0.01 mg · kg · h, 2 mL · h) was continuously infused into the bronchial artery, n = 6; (b) control group: 1 h after injury, same amount of saline was injected into the bronchial artery, n = 6; (c) sham group: no injury, no treatment, same operation and anesthesia, n = 6. After injury, all animals were ventilated and fluid resuscitated according to an established protocol. The experiment was conducted for 24 h. Injury induced severe pulmonary dysfunction, which was associated with increases in lung edema formation, airway obstruction, malondialdehyde, and nitrate/nitrite. 7-Nitroindazole injection into the bronchial artery reduced the degree of lung edema formation and improved pulmonary gas exchange. The increase in malondialdehyde and nitrate/nitrite in lung tissue was attenuated by treatment. Our data strongly suggest that local airway production of NO contributes to pulmonary dysfunction following smoke inhalation and burn injury. Most mechanisms that drive this pathophysiology reside in the airway.

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.

Systemic inflammatory response syndrome is associated with excessive production of nitric oxide (NO·) and superoxide (O2), forming peroxynitrite, which in turn, acts as a terminal mediator of cellular injury by producing cell necrosis and apoptosis. We examined the effect of the peroxynitrite decomposition catalyst, WW-85, in a sheep model of acute lung injury and septic shock. Eighteen sheep were operatively prepared and randomly allocated to the sham, control, or WW-85 group (n = 6 each). After a tracheotomy, acute lung injury was produced in the control and WW-85 groups by insufflation of four sets of 12 breaths of cotton smoke. Then, a 30-mL suspension of live Pseudomonas aeruginosa bacteria (containing 2 - 5 × 10¹¹ colony-forming units) was instilled into the lungs according to an established protocol. The sham group received only the vehicle (30 mL saline). The sheep were studied in awake state for 24 h and ventilated with 100% oxygen. WW-85 was administered 1 h after injury as bolus infusion (0.1 mg/kg), followed by a continuous infusion of 0.02 mg·kg⁻¹·h⁻¹ until the end of the 24-h experimental period. Compared with injured but untreated controls, WW-85-treated animals had significantly improved gas exchange, reductions in airway obstruction, shunt formation, lung myeloperoxidase concentrations, lung malondialdehyde concentrations, lung 3-nitrotyrosine concentrations, and plasma nitrate-to-nitrite levels. Animals treated with WW-85 exhibited less microvascular leakage and improvements in pulmonary function. These results provide evidence that blockade of the nitric oxide-peroxynitrite pathway improves disturbances from septic shock, as demonstrated in a clinically relevant ovine experimental model.

Previous studies have indicated increased plasma levels of inducible nitric oxide synthase in lung. This study further examines the pulmonary expression of nitric oxide synthase (NOS) isoforms in an ovine model of acute lung injury induced by smoke inhalation and burn injury (S+B injury). Female range bred sheep (4 per group) were sacrificed at 4, 8, 12, 24, and 48 hours after injury and immunohistochemistry was performed in tissues for various NOS isoforms. The study indicates that in uninjured sheep lung, endothelial (eNOS) is constitutively expressed in the endothelial cells associated with the airways and parenchyma, and in macrophages. Similarly, neuronal (nNOS) is constitutively present in the mucous cells of the epithelium and in neurons of airway ganglia. In uninjured lung, inducible (iNOS) was present in bronchial secretory cells and macrophages. In tissue after S+B injury, new expression of iNOS was evident in bronchial ciliated cells, basal cells, and mucus gland cells. In the parenchyma, a slight increase in iNOS immunostaining was seen in type I cells at 12 and 24 hours after injury only. Virtually no change in eNOS or nNOS was seen after injury.

The morbidity and mortality of burn victims increase when burn injury is combined with smoke inhalation. The goal of the present study was to develop a murine model of burn and smoke inhalation injury to more precisely reveal the mechanistic aspects of these pathological changes. The burn injury mouse group received a 40% total body surface area third-degree burn alone, the smoke inhalation injury mouse group received two 30-s exposures of cotton smoke alone, and the combined burn and smoke inhalation injury mouse group received both the burn and the smoke inhalation injury. Animal survival was monitored for 120 h. Survival rates in the burn injury group, the smoke inhalation injury group, and the combined injury group were 70%, 60%, and 30%, respectively. Mice that received combined burn and smoke injury developed greater lung damage as evidenced by histological changes (septal thickening and interstitial edema) and higher lung water content. These mice also displayed more severely impaired pulmonary gas exchange [arterial PO2 (PaO2)/inspired O2 fraction (FiO2)<200]. Lung myeloperoxidase activity was significantly higher in burn and smoke-injured animals compared with the other three experimental groups. Plasma NO2-/NO3-, lung inducible nitric oxide synthase (iNOS) activity, and iNOS mRNA increased with injury; however, the burn and smoke injury group exhibited a higher response. Severity of burn and smoke inhalation injury was associated with more pronounced production of nitric oxide and accumulation of activated leukocytes in lung tissue. The murine model of burn and smoke inhalation injury allows us to better understand pathophysiological mechanisms underlying cardiopulmonary morbidity secondary to burn and smoke inhalation injury.

Previous study in an ovine model of smoke inhalation and burn (S + B) injury has shown distal migration of upper airway mucus. This study examines the localization of an upper airway gland specific mucus, mucin 5B (MUC5B) in lung autopsy tissues of burn-only injury and in victims of S + B injury. We hypothesize that victims with S + B injury would exhibit increased distal migration of MUC5B than that seen in victims of burn-only injury. Autopsy lung tissue from victims of burn injury alone (n = 38) and combined S + B injury (n = 22) were immunostained for MUC5B. No normal lung tissues were included in the study. Semiquantitative analysis of the extent of MUC5B in bronchioles and parenchyma was performed on masked slides. Irrespective of injury conditions, all victims showed MUC5B in bronchioles. Mucin 5B was seen in the parenchyma except in two burn victims. No statistically significant difference was seen in the mean bronchiolar and parenchyma MUC5B scores between S + B and burn-only victims (P > 0.05). No strong statistical correlation of MUC5B scores with days postinjury or to the number of ventilatory days was evident. The percentage of pneumonia, identified histologically, was also similar between study groups. This study did not confirm our results in an ovine model of S + B injury. In contrast, virtually all pediatric burn victims, regardless of concomitant inhalation injury, showed MUC5B in their bronchioles and parenchyma. Increased mucus synthesis and/or impaired mucociliary function may contribute to the pulmonary pathophysiology associated with burn injury.

Thermal injury results in reduced plasma levels of arginine (Arg). With reduced Arg availability, NOS produces superoxide instead of NO. We hypothesized that Arg supplementation after burn and smoke inhalation (B + S) injury would attenuate the acute insult to the lungs and, thus, protect pulmonary function. Seventeen Suffolk ewes (n = 17) were randomly divided into three groups: (1) sham injury group (n = 6), (2) B + S injury plus saline treatment (n = 6), and (3) B + S injury plus L-ARG infusion at 57 mg.kg(-1).h(-1) (n = 5). Burn and smoke inhalation injury was induced by standardized procedures, including a 40% area full thickness flame burn combined with 48 breaths of smoke from burning cottons. All animals were immediately resuscitated by Ringer solution and supported by mechanical ventilation for 48 h, during which various variables of pulmonary function were monitored. The results demonstrated that Arg treatment attenuated the decline of plasma Arg concentration after B + S injury. A higher plasma Arg concentration was associated with a less decline in Pao2/Fio2 ratio and a reduced extent of airway obstruction after B + S injury. Histopathological examinations also indicated a remarkably reduced histopathological scores associated with B + S injury. Nitrotyrosine stain in lung tissue was positive after B + S injury, but was significantly reduced in the group with Arg. Therefore, L-Arg supplementation improved gas exchange and pulmonary function in ovine after B + S injury via its, at least in part, effect on reduction of oxidative stress through the peroxynitrite pathway.

Objective: To examine the effects of combined burn and smoke inhalation injury on hypoxic pulmonary vasoconstriction, 3-nitrotyrosine formation, and respiratory function in adult sheep., Design: Prospective, placebo-controlled, randomized, single-blinded trial., Setting: University research laboratory., Subjects: Twelve chronically instrumented ewes., Interventions: Following a baseline measurement, sheep were randomly allocated to either healthy controls (sham) or the injury group, subjected to a 40%, third-degree body surface area burn and 48 breaths of cotton smoke according to an established protocol (n = 6 each). Hypoxic pulmonary vasoconstriction was assessed as changes in pulmonary arterial blood flow (corrected for changes in cardiac index) in response to left lung hypoxic challenges performed at baseline and at 24 and 48 hrs postinjury., Measurements and Main Results: Combined burn and smoke inhalation was associated with increased expression of inducible nitric oxide (NO) synthase, elevated NO2/NO3 (NOx) plasma levels (12 hrs, sham, 6.2 +/- 0.6; injury, 16 +/- 1.6 micromol.L; p < .01) and increased peroxynitrite formation, as indicated by augmented lung tissue 3-nitrotyrosine content (30 +/- 3 vs. 216 +/- 8 nM; p < .001). These biochemical changes occurred in parallel with pulmonary shunting, progressive decreases in Pao2/Fio2 ratio, and a loss of hypoxic pulmonary vasoconstriction (48 hrs, -90.5% vs. baseline; p < .001). Histopathology revealed pulmonary edema and airway obstruction as the morphologic correlates of the deterioration in gas exchange and the increases in airway pressures., Conclusions: This study provides evidence for a severe impairment of hypoxic pulmonary vasoconstriction following combined burn and smoke inhalation injury. In addition to airway obstruction, the loss of hypoxic pulmonary vasoconstriction may help to explain why blood gases are within physiologic ranges for a certain time postinjury and then suddenly deteriorate.

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

We investigated the role of the nuclear enzyme poly (ADP ribose) synthetase (PARS) in the pathogenesis of combined burn and smoke inhalation (burn/smoke) injury in an ovine model. Eighteen sheep were operatively prepared for chronic study. PARS inhibition was achieved by treatment with a novel and selective PARS inhibitor INO-1001. The PARS inhibitor attenuated 1) lung edema formation, 2) deterioration of gas exchange, 3) changes in airway blood flow, 4) changes in airway pressure, 5) lung histological injury, and 6) systemic vascular leakage. Lipid oxidation and plasma nitrite/nitrate (stable breakdown products of nitric oxide) levels were suppressed with the use of INO-1001. We conclude that PARS inhibition attenuates various aspects of the pathophysiological response in a clinically relevant experimental model of burn/smoke inhalation injury.

Sepsis is a life-threatening event when it occurs in patients suffering from smoke inhalation injury. Pneumonia is one of the most frequent sources of infection in sepsis. Activated leukocytes likely play a role in the pathogenesis of sepsis. Cepharanthin is a biscoclaurine alkaloid that reportedly inhibits the activation of neutrophils. In this study, we investigated the effects of cephranthin on a post-smoke inhalation model of sepsis in sheep. Female sheep (n = 15) were surgically prepared for the study. After 5 days recovery from the operative procedures, tracheostomy was performed in all animals and 48 breaths of cotton smoke (<40 degrees C) were given via a modified bee smoker under halothane anesthesia. After smoke insufflation, Pseudomonas aeruginosa (5 x 109 cfu/kg) was instilled into the airway using a bronchoscope. All of the animals were mechanically ventilated with 100% O(2). Cepharanthin (1.3 mg/kg/h) was infused in five sheep continuously beginning 1 h after the insult and thereafter for the remainder of the 24-h study period. Control animals (n = 6) were treated with 5% dextrose as a vehicle control. Cepharanthin significantly attenuated changes in lung histology as well as in lung wet/dry weight ratio. An in vitro study revealed that cepharanthin inhibited the release of neutrophil elastase from isolated neutrophils stimulated with either formyl-methyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate with an IC(50) of 60 microM. Cepharanthin also inhibited the fMLP-induced increase in intracellular calcium levels of neutrophils. This result indicates cepharanthin inhibits protein kinase C or a more downstream signaling pathway in neutrophil activation. In conclusion, cepharanthin attenuates acute lung injury and septic shock after smoke inhalation in sheep.

Objectives: We investigated the effect of various dietary fats on pulmonary surfactant composition and lung function changes that occur before and after endotoxin infusion in pigs., Methods: Eighteen pigs were assigned to three groups (n = 6 per group) to receive a diet of protein (20% of calories), carbohydrate (20% of calories), and fat (40% of calories). In one group the fat content consisted entirely of palmitic acid. In the second group, fat came from Intralipid, which provided predominantly linoleic acid. The third group was fed fish oil. Pigs were maintained on these diets for 21 d before the experiment. Cardiovascular and pulmonary functions were determined on day 22. Pigs then were infused with endotoxin (80 mg. kg(-1). min(-1)) until the pulmonary arterial pressure reached a pressure similar to that found in trauma victims (45 to 50 mmHg). Cardiovascular and pulmonary function tests were then repeated, the animals killed, and the lungs removed for study., Results: Compliance was reduced in the linoleate and fish-oil groups compared with the palmitate group before and after endotoxin. Compliance changes in pigs fed the linoleate and fish-oil diets were consistent with significant increases in lung wet:dry weight ratios, increased CO(2) retention, histologic evidence of vascular congestion, intra-alveolar edema, and alveolar septa thickening. Changes in surfactant phosphatidylcholine composition between groups were consistent with the notion that increased unsaturated fatty acids could affect surfactant function., Conclusions: We concluded that the common practice of providing calories in the form of polyunsaturated fatty acids to critically ill patients carries the risk of being detrimental to lung function.

BackgroundHuman 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.MethodsAdult 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.ResultsBy 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.ConclusionsHuman MSCs were well tolerated and improved oxygenation and decreased pulmonary oedema in a sheep model of severe ARDS.Trail registration numberNCT01775774 for Phase 1. NCT02097641 for Phase 2.

Abstract: Burn patients often develop respiratory distress and ARDS several days after injury. An ovine model allows experimental study of this problem. In sheep the injury is characterized by intense acute inflammation in the trachea and bronchi from 3 to 48h after injury, with accumulation of neutrophils, fibrin and other plasma proteins, and mucus in airway lumens. We have carried out immunostaining for multiple cytokines in this model, including interleukin-8 (IL-8), Interleukin-1 beta (IL-1β), interleukin-1 alpha (IL-1α), tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor (VEGF). All of these show intense immunostaining in airway mucous glands. IL-1β and VEGF show substantial constitutive staining in the serous cells of mucous glands, while IL-8, IL-1α, and TNF-α show substantially increased expression after injury. This pattern of expression of cytokines in mucous glands, and the apparent release of cytokines into the lumen after injury, are considered potentially highly significant in the progression of injury in this model. In addition, a proinflammatory function of mucous glands might prove to be important in chronic lung diseases such as chronic bronchitis and asthma. [Copyright &y& Elsevier]