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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.)

To investigate the efficacy of sea buckthorn (SBT) seed oil - a rich source of substances known to have anti-atherogenic and cardioprotective activity, and to promote skin and mucosa epithelization - on burn wound healing, five adult sheep were subjected to 3rd degree flame burns. Two burn sites were made on the dorsum of the sheep and the eschar was excised down to the fascia. Split-thickness skin grafts were harvested, meshed, and fitted to the wounds. The autograft was placed on the fascia and SBT seed oil was topically applied to one recipient and one donor site, respectively, with the remaining sites treated with vehicle. The wound blood flow (LASER Doppler), and epithelization (ultrasound) were determined at 6, 14, and 21 days after injury. 14 days after grafting, the percentage of epithelization in the treated sites was greater (95 ± 2.2% vs. 83 ± 2.9%, p<0.05) than in the untreated sites. Complete epithelization time was shorter in both treated recipient and donor sites (14.20 ± 0.48 vs. 19.60 ± 0.40 days, p<0.05 and 13.40 ± 1.02 vs. 19.60 ± 0.50 days, p<0.05, respectively) than in the untreated sites, confirmed by ultrasound. In conclusion, SBT seed oil has significant wound healing activity in full-thickness burns and split-thickness harvested wounds., (Copyright © 2013 Elsevier Ltd and ISBI. All rights reserved.)

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

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.

More than 20,000 burn injury victims suffer from smoke inhalation injury in the United States annually. In an ovine model of acute lung injury, γ-tocopherol had a beneficial effect when nebulized into the airway. We hypothesize that γ-tocopherol scavenges reactive oxygen species (ROS) and reactive nitrogen species resulting from burn and smoke inhalation injury and that these ROS/reactive nitrogen species activate the arginase pathway, leading to increased collagen deposition and decreased pulmonary function. To test this hypothesis, ewes were operatively prepared for chronic study, then they were randomly divided into groups (n = 8): uninjured, injured, or injured with nebulization (γ-tocopherol [950 mg/g] and α-tocopherol [40 mg/g] from hours 3 to 48 after the injury). The injury, under deep anesthesia, consisted of a 20% total body surface burn and 36 breaths of cotton smoke; all animals were killed after 3 weeks. Treatment increased lung γ-tocopherol at 3 weeks after γ-tocopherol nebulization compared with injured sheep (1.75 ± 0.62 nmol/g vs. 0.45 ± 0.06, P < 0.05). The expression of dimethylarginine dimethylaminohydrolase-2, which degrades asymmetrical dimethylarginine, a nitric oxide synthase inhibitor, significantly increases with γ-tocopherol treatment compared with injured sheep (P < 0.05). Arginase activity (0.15 ± 0.02 μM urea/μg protein vs. 0.24 ± 0.009, P < 0.05), ornithine aminotransferase (11,720 ± 888 vs. 13,170 ± 1,775), and collagen deposition (0.62 ± 0.12 μM hydroxyproline/μg protein vs. 1.02 ± 0.13, P < 0.05) significantly decrease with γ-tocopherol compared with injured animals without γ-tocopherol. The decreases in arginase and collagen with γ-tocopherol are associated with significantly increased diffusion capacity (P < 0.05) and decreased lung wet-to-dry ratio (P < 0.05). Smoke-induced chronic pulmonary dysfunction is mediated through the ROS/asymmetrical dimethylarginine/arginase pathway, and ROS scavengers such as γ-tocopherol may be a potential therapeutic management of burn patients with inhalation injury.

Poly(ADP-ribose) polymerase (PARP) is well known to be an enzyme that repairs damaged DNA and also induces cell death when overactivated. It has been reported that PARP plays a significant role in burn and smoke inhalation injury, and the pathophysiology is thought to be localized in the airway during early stages of activation. Therefore, we hypothesized that local inhibition of PARP in the airway by direct delivery of low dose PJ-34 [poly(ADP-ribose) polymerase inhibitor] into the bronchial artery would attenuate burn and smoke-induced acute lung injury. The bronchial artery in sheep was cannulated in preparation for surgery. After a 5-7 day recovery period, sheep were administered a burn and inhalation injury. Adult female sheep (n=19) were divided into four groups following the injury: (1) PJ-34 group A: 1h post-injury, PJ-34 (0.003mg/kg/h, 2mL/h) was continuously injected into the bronchial artery, n=5; (2) PJ-34 group B: 1h post-injury, PJ-34 (0.03mg/kg/h, 2mL/h) was continuously injected into bronchial artery, n=4; (3) CONTROL GROUP: 1h post-injury, an equivalent amount of saline was injected into the bronchial artery, n=5; (4) Sham group: no injury, no treatment, same operation and anesthesia, n=5. After injury, all animals were placed on a ventilator and fluid resuscitated equally. Pulmonary function as evaluated by measurement of blood gas analysis, pulmonary mechanics, and pulmonary transvascular fluid flux was severely deteriorated in the control group. However, the above changes were markedly attenuated by PJ-34 infusion into the bronchial artery (P/F ratio at 24h: PJ-34 group A 398±40*, PJ-34 group B 438±41*†‡, Control 365±58*, Sham 547±47; * vs. sham [p<0.05], † vs. control [p<0.05], ‡ vs. PJ-34 group A [p<0.05]). Our data strongly suggest that local airway production of poly(ADP-ribose) polymerase contributes to pulmonary dysfunction following smoke inhalation and burn., (Copyright © 2012 Elsevier Ltd and ISBI. All rights reserved.)

Unlabelled: Smoke inhalation injury frequently increases the risk of pneumonia and mortality in burn patients. The pathophysiology of acute lung injury secondary to burn and smoke inhalation is well studied, but long-term pulmonary function, especially the process of lung tissue healing following burn and smoke inhalation, has not been fully investigated. By contrast, early burn excision has become the standard of care in the management of major burn injury. While many clinical studies and small-animal experiments support the concept of early burn wound excision, and show improved survival and infectious outcomes, we have developed a new chronic ovine model of burn and smoke inhalation injury with early excision and skin grafting that can be used to investigate lung pathophysiology over a period of 3 weeks., Materials and Methods: Eighteen female sheep were surgically prepared for this study under isoflurane anesthesia. The animals were divided into three groups: an Early Excision group (20% TBSA, third-degree cutaneous burn and 36 breaths of cotton smoke followed by early excision and skin autografting at 24h after injury, n=6), a Control group (20% TBSA, third-degree cutaneous burn and 36 breaths of cotton smoke without early excision, n=6) and a Sham group (no injury, no early excision, n=6). After induced injury, all sheep were placed on a ventilator and fluid-resuscitated with Lactated Ringers solution (4 mL/% TBS/kg). At 24h post-injury, early excision was carried out to fascia, and skin grafting with meshed autografts (20/1000 in., 1:4 ratio) was performed under isoflurane anesthesia. At 48 h post-injury, weaning from ventilator was begun if PaO(2)/FiO(2) was above 250 and sheep were monitored for 3 weeks., Results: At 96 h post-injury, all animals were weaned from ventilator. There are no significant differences in PaO(2)/FiO(2) between Early Excision and Control groups at any points. All animals were survived for 3 weeks without infectious complication in Early Excision and Sham groups, whereas two out of six animals in the Control group had abscess in lung. The percentage of the wound healed surviving area (mean ± SD) was 74.7 ± 7.8% on 17 days post-surgery in the Early Excision group. Lung wet-to-dry weight ratio (mean ± SD) was significantly increased in the Early Excision group vs. Sham group (p<0.05). The calculated net fluid balance significantly increased in the early excision compared to those seen in the Sham and Control groups. Plasma protein, oncotic pressure, hematocrit of % baseline, hemoglobin of % baseline, white blood cell and neutrophil were significantly decreased in the Early Excision group vs. Control group., Conclusions: The early excision model closely resembles practice in a clinical setting and allows long-term observations of pulmonary function following burn and smoke inhalation injury. Further studies are warranted to assess lung tissue scarring and measuring collagen deposition, lung compliance and diffusion capacity., (Published by Elsevier Ltd.)

We hypothesize that the nebulization of γ-tocopherol (g-T) in the airway of our ovine model of acute respiratory distress syndrome will effectively improve pulmonary function following burn and smoke inhalation after 96 h. Adult ewes (n = 14) were subjected to 40% total body surface area burn and were insufflated with 48 breaths of cotton smoke under deep anesthesia, in a double-blind comparative study. A customized aerosolization device continuously delivered g-T in ethanol with each breath from 3 to 48 h after the injury (g-T group, n = 6), whereas the control group (n = 5) was nebulized with only ethanol. Animals were weaned from the ventilator when possible. All animals were killed after 96 h, with the exception of one untreated animal that was killed after 64 h. Lung g-T concentration significantly increased after g-T nebulization compared with the control group (38.5 ± 16.8 vs. 0.39 ± 0.46 nmol/g, P < 0.01). The PaO(2)/FIO(2) ratio was significantly higher after treatment with g-T compared with the control group (310 ± 152 vs. 150 ± 27.0, P < 0.05). The following clinical parameters were improved with g-T treatment: pulmonary shunt fraction, peak and pause pressures, lung bloodless wet-to-dry weight ratios (2.9 ± 0.87 vs. 4.6 ± 1.4, P < 0.05), and bronchiolar obstruction (2.0% ± 1.1% vs. 4.6% ± 1.7%, P < 0.05). Nebulization of g-T, carried by ethanol, improved pulmonary oxygenation and markedly reduced the time necessary for assisted ventilation in burn- and smoke-injured sheep. Delivery of g-T into the lungs may be a safe, novel, and efficient approach for management of acute lung injury patients who have sustained oxidative damage to the airway.

Introduction: Autograft take and rapid wound closure is essential for the survival of severely burned patients. Loss of skin grafts typically occurs during the first few days after coverage, mainly due to shear forces and inadequate contact with the wound bed. Slow-clotting fibrin sealant, applied with a spray-on device, has been shown to improve healing of skin grafts in large wounds. However, its use in burn wounds has not been studied so far., Study Aim: To evaluate the effectiveness of sprayed fibrin sealant in excised and grafted full-thickness burns., Material and Methods: Ten female Yorkshire pigs (30-45 kg) received a full-thickness contact burn of approximately 15% total body surface area. The burns were excised to the level of the muscular fascia after 24 h and covered with meshed skin autograft (mesh ratio 1:3). Wounds were randomized to either fibrin sealant (n=20) or standard skin staples (n=16) for graft fixation. Fibrin sealant was used as a slow-clotting spray (4 IU thrombin/ml). Outcome measurements included clinical scoring at days 2, 5, 9 and 14 postoperatively, planimetric analysis of wound closure, and histological examination of epidermal and dermal thickness 14 days after autografting., Results: In the fibrin sealant group, graft adherence scores were significantly increased (p<0.02) and graft dislocation scores significantly decreased (p<0.01) at days 2 and 5 postoperatively, when compared to controls. Planimetric analysis of remaining open mesh interstices showed acceleration of wound closure in the fibrin sealant group but did not reach statistical significance (day 14 p=0.04 at significance level p<0.025). Wound contraction, occurrence of hematoma, and dermal as well as epidermal thickness were not different between the groups at 14 days postoperatively., Conclusion: The results indicate that the use of slow-clotting fibrin sealant spray for autograft fixation is advantageous over skin staples. Easy handling and reduced graft dislocation at early time points are key qualities of this method., (Copyright © 2011. Published by Elsevier Ltd.)

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.

We investigated the effect of the angiotensin-converting enzyme (ACE) inhibitor captopril in a clinically relevant ovine model of smoke and burn injury, with special reference to oxidative stress and activation of poly(ADP-ribose) polymerase, in the lung and in circulating leukocytes. Female, adult sheep (28-40 kg) were divided into three groups. After tracheostomy and under deep anesthesia, both vehicle-control-treated (n = 5) and captopril-treated (20 mg/kg per day, i.v., starting 0.5 h before the injury) (n = 5) groups were subjected to 2 × 20%, third-degree burn injury and were insufflated with 48 breaths of cotton smoke. A sham group not receiving burn/smoke was also studied (n = 5). Animals were mechanically ventilated and fluid resuscitated for 24 h in the awake state. Burn and smoke injury resulted in an upregulation of ACE in the lung, evidenced by immunohistochemical determination and Western blotting. Burn and smoke injury resulted in pulmonary dysfunction, as well as systemic hemodynamic alterations. Captopril treatment of burn and smoke animals improved PaO2/FiO2 ratio and pulmonary shunt fraction and reduced the degree of lung edema. There was a marked increase in PAR levels in circulating leukocytes after burn/smoke injury, which was significantly decreased by captopril. The pulmonary level of ACE and the elevated pulmonary levels of transforming growth factor β in response to burn and smoke injury were significantly decreased by captopril treatment. Our results suggest that the ACE inhibitor captopril exerts beneficial effects on the pulmonary function in burn/smoke injury. The effects of the ACE inhibitor may be related to the prevention of reactive oxygen species-induced poly(ADP-ribose)polymerase overactivation. Angiotensin-converting enzyme inhibition may also exert additional beneficial effects by inhibiting the expression of the profibrotic mediator transforming growth factor β.

The nuclear enzyme poly(ADP-ribose)polymerase (PARP) plays a significant role in the pathogenesis of various forms of critical illness. DNA strand breaks induced by oxidative and nitrative stress trigger the activation of PARP, and PARP, in turn, mediates cell death and promotes proinflammatory responses. Until recently, most studies focused on the role of PARP in solid organs such as heart, liver, and kidney. We investigated the effect of burn and smoke inhalation on the levels of poly(ADP-ribosylated) proteins in circulating sheep leukocytes ex vivo. Adult female merino sheep were subjected to burn injury (2× 20% each flank, 3 degrees) and smoke inhalation injury (insufflated with a total of 48 breaths of cotton smoke) under deep anesthesia. Arterial and venous blood was collected at baseline, immediately after the injury and 1 to 24 h after the injury. Leukocytes were isolated with the Histopaque method. The levels of poly(ADP-ribosyl)ated proteins were determined by Western blotting. The amount of reactive oxygen species was quantified by the OxyBlot method. To examine whether PARP activation continues to increase ex vivo in the leukocytes, blood samples were incubated at room temperature or at 37°C for 3 h with or without the PARP inhibitor PJ34. To investigate whether the plasma of burn/smoke animals may trigger PARP activation, burn/smoke plasma was incubated with control leukocytes in vitro. The results show that burn and smoke injury induced a marked PARP activation in circulating leukocytes. The activity was the highest immediately after injury and at 1 h and decreased gradually over time. Incubation of whole blood at 37°C for 3 h significantly increased poly(ADP-ribose) levels, indicative of the presence of an ongoing cell activation process. In conclusion, PARP activity is elevated in leukocytes after burn and smoke inhalation injury, and the response parallels the time course of reactive oxygen species generation in these cells.

Different isoforms of nitric oxide (NO) synthase are critically involved in the development of pulmonary failure secondary to acute lung injury. Here we tested the hypothesis that simultaneous blockade of inducible and neuronal NO synthase effectively prevents the pulmonary lesions in an ovine model of acute respiratory distress syndrome induced by combined burn and smoke inhalation injury. Chronically instrumented sheep were allocated to a sham-injured group (n = 6), an injured and untreated group (n = 6), or an injured group treated with simultaneous infusion of selective inducible and neuronal NO synthase inhibitors (n = 5). The injury was induced by 48 breaths of cotton smoke and a third-degree burn of 40% total body surface area. All sheep were mechanically ventilated and fluid resuscitated. The injury induced severe pulmonary dysfunction as indicated by decreases in PaO2/FiO2 ratio and increases in pulmonary shunt fraction, ventilatory pressures, lung lymph flow, and lung wet/dry weight ratio. The treatment fully prevented the elevations in lymph and plasma nitrate/nitrite levels, pulmonary shunting, ventilatory pressures, lung lymph flow, and wet/dry weight ratio and significantly attenuated the decline in PaO2/FiO2 ratio. In conclusion, simultaneous blockade of inducible and neuronal NO synthase exerts beneficial pulmonary effects in an ovine model of acute respiratory distress syndrome secondary to combined burn and smoke inhalation injury. This novel treatment strategy may represent a useful therapeutic adjunct for patients with these injuries.

Introduction: Although the beneficial effects of inducible nitric oxide synthase (iNOS) inhibition in acute lung injury secondary to cutaneous burn and smoke inhalation were previously demonstrated, the mechanistic aspects are not completely understood. The objective of the present study is to describe the mechanism(s) underlying these favourable effects. We hypothesised that iNOS inhibition prevents formation of excessive reactive nitrogen species and attenuates the activation of poly(ADP) (poly(adenosine diphosphate)) ribose polymerase, thus mitigating the severity of acute lung injury in sheep subjected to combined burn and smoke inhalation., Methods: Adult ewes were chronically instrumented for a 24-h study and allocated to groups: sham: not injured, not treated, n = 6; control: injured, not treated, n = 6; and BBS-2: injured treated with iNOS dimerisation inhibitor BBS-2, n = 6. Control and BBS-2 groups received 40% total body surface area 3rd-degree cutaneous burn and cotton smoke insufflation into the lungs under isoflurane anaesthesia., Results: Treatment with iNOS inhibitor BBS-2 significantly improved pulmonary gas exchange (partial pressure of oxygen in the blood/fraction of inspired oxygen (PaO₂/FiO₂) 409 ± 43 mmHg vs. 233 ± 50 mmHg in controls, p < 0.05) and reduced airway pressures (peak pressure 20 ± 1 cm H₂O vs. 28 ± 2 cm H₂O in controls, p < 0.05) and lung water content (lung wet-to-dry ratio 4.1 ± 0.3 vs. 5.2 ± 0.2 in controls, p < 0.05) 24h after the burn and smoke injury. BBS-2 significantly reduced the increases in lung lymph nitrite/nitrate (10 ± 3 μM vs. 26 ± 6 μM in controls, p < 0.05) and 3-nitrotyrosine (109 ± 11 (densitometry value) vs. 151 ± 18 in controls, p < 0.05). Burn/smoke-induced increases in lung tissue nitrite/nitrate, poly(ADP)ribose polymerase, nuclear factor-κB (NF-κB) activity, myeloperoxidase activity and malondialdehyde formation and interleukin (IL)-8 expression were also attenuated with BBS-2., Conclusions: The results provide strong evidence that BBS-2 ameliorated acute lung injury by inhibiting the inducible nitric oxide synthase/reactive nitrogen species/poly(ADP-ribose) polymerase (iNOS/RNS/PARP) pathway., (Copyright © 2011. Published by Elsevier Ltd.)

Unlabelled: We previously reported bronchial circulation contributes to pulmonary edema and increases shunt fraction following smoke inhalation, and bronchial blood flow significantly increases in inhalation injury. We hypothesized reduction of bronchial blood flow reduces exudation to the airway and ameliorates lung injury from combined burn and smoke insults (B&S injury)., Method: Merino ewes (n=28) randomly divided into three groups: (1) bronchial artery ligated and injured (injury+ligation group); (2) bronchial artery left intact and injured (injury+no ligation group); (3) bronchial artery ligated but not injured (no injury+ligation group) were subjected to a flame burn and inhalation injury under halothane anesthesia. Parameters were analyzed using Scheffe's post hoc test (P<0.05). All Groups were resuscitated with Ringer lactate solution and placed on a ventilator for 48h., Results: Pulmonary gas exchange (PaO(2)/FiO(2)) improved in injury+ligation group. Further, obstruction score, an index of airway cast formation, significantly changed between injury+no ligation group compared to both ligation groups., Conclusion: Bronchial circulation plays a significant role in lung injury after B&S injury, and reduction of bronchial blood flow by bronchial artery ligation reduces bronchial exudates, resulting in improved gas exchange., (Copyright © 2010 Elsevier Ltd and ISBI. All rights reserved.)

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.

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.

Background: We previously showed that thermal injury depletes plasma vitamin E in pediatric burn patients; however, plasma changes may reflect immediate alterations in vitamin E nutriture. Adipose tissue α-tocopherol concentrations are generally accepted to reflect long-term vitamin E status., Objective: To test the hypothesis that thermal injury depletes body stores of vitamin E, α-tocopherol concentrations were measured in adipose tissue samples., Design: Pediatric patients (n = 8) were followed up to 1 y after burn injury. Surgically obtained samples were collected at various intervals and stored at -80°C in a biorepository. α- and γ-Tocopherols, cholesterol, and triglycerides were measured in the same tissue aliquot., Results: During the first week after injury, adipose tissue α-tocopherol concentrations were within the expected normal range of 199 ± 40 nmol/g adipose tissue but were substantially lower at weeks 2 and 3 (133 ± 13 and 109 ± 8 nmol/g adipose tissue, respectively). Individual rates of decrease, estimated by linear regression, showed that adipose tissue α-tocopherol decreased by an average of 6.1 ± 0.6 nmol/g daily. During the first month after injury, adipose tissue triglyceride concentrations also decreased, whereas no changes in cholesterol concentrations occurred., Conclusions: These data emphasize that the burn injury experienced by these pediatric patients altered their metabolism such that vitamin E status diminished during the month after injury. Further studies are needed to evaluate the mechanism and consequences of the observed vitamin E depletion. This trial was registered at clinicaltrials.gov as NCT00675714.

Liposomal gene transfer effectively enhances dermal and epidermal regeneration in burned rodents. To advance this treatment to clinical studies, we investigated the efficacy of liposomal gene transfer in a clinically relevant porcine wound model. Mimicking the clinical scenario, six female Yorkshire pigs (40-50 kg) received up to 12 burns of 50 cm(2) area that were fully excised and covered with skin autograft meshed at 4:1 ratio 24 h post-burn. Animals received control injections (empty liposomes), liposomes (DMRIE-C) containing 1 mg LacZ-cDNA, or liposomes (DMRIE-C) with 1 mg of platelet-derived growth factor (PDGF)-cDNA, or the naked PDGF gene. Serial biopsies were taken from different wound sites at multiple time points up to 12 days post-wounding. Transfection efficacy and transfection rate of LacZ and localization of beta-gal were determined by immunohistochemical and immunofluorescent techniques. RT-PCR and multiplex protein analysis (ELISA) were used to measure levels of growth factor mRNA transcribed and growth factor protein translated. Wound re-epithelialization and graft adhesion was evaluated using planimetric analysis and clinical scores. We found that peak transfection of liposomal beta-galactosidase occurred on day 2, with a fluorescence increase of 154% to baseline (P<0.001). Transfection intensity dropped to 115% above baseline on day 4 (P<0.001) and 109% on day 7. Immunohistochemistry showed a maximum transfection rate of 34% of cells in wound tissue. Gene transfer of liposomal PDGF-cDNA resulted in increased PDGF-mRNA and protein expression on days 2 and 4, and accelerated wound re-epithlialization as well as graft adhesion on day 9 (P<0.05). In this study, we showed that liposomal cDNA gene transfer is possible in a porcine wound model, and by using PDGF-cDNA we further showed that dermal and epidermal regeneration can be improved. These data indicate that liposomal gene transfer can be a new therapeutic approach to improve wound healing in humans.

The bronchial circulation plays a significant role in the pathophysiological changes of burn and smoke-inhalation injury. Bronchial blood flow markedly increases immediately after inhalational injury. This study examines whether the ablation of the bronchial artery attenuates pathophysiological changes and improves survival after burn and smoke-inhalational injury in an ovine model. Acute lung injury was induced by 40% total body surface-area third-degree cutaneous burn and cotton smoke inhalation (48 breaths of cotton smoke, <40 degrees C) under deep anaesthesia. Twelve adult female sheep were divided into two groups: (1) sham (injured, non-ablated bronchial artery, n=6); (2) ablation (injured, ablated bronchial artery, n=6). Ablation of the bronchial artery was performed 72 h before the injury. The experiment was continued for 96 h. Burn and smoke-inhalation injury significantly increased regional blood flow in the bronchi. Ablation of the bronchial artery significantly reduced acute regional blood flow increases in the proximal and distal bronchi. All animals in the ablation group survived to 96 h. Four of these were successfully weaned off the ventilator. Three animals of the sham group met standardised euthanasia criteria at 60 h, while another met the criteria at 78 h. The lung wet-to-dry weight ratio, histology score and myeloperoxidase (MPO) activity were significantly increased by the insult, but ablation of the bronchial artery attenuated these changes. Burn and smoke-inhalation injury induced a significant increase in bronchial blood flow and accelerated airway obstruction, pulmonary vascular changes, pulmonary oedema and pulmonary dysfunction. Ablated bronchial circulation attenuated these pathophysiological changes.

In a recent study, we have shown a rapid inflammatory cell influx across the glandular epithelium and strong proinflammatory cytokine expression at 4 hours after inhalation injury. Studies have demonstrated a significant role of nuclear factor kappa B in proinflammatory cytokine gene transcription. This study examines the acute airway inflammatory response and immunohistochemical detection of p65, a marker of nuclear factor kappa B activation, in sheep after smoke inhalation and burn injury. Pulmonary tissue from uninjured sheep and sheep at 4, 8, 12, 24, and 48 hours after inhalation and burn injury was included in the study. Following immunostaining for p65 and myeloperoxidase, the cell types and the percentage of bronchial submucosal gland cells staining for p65 and the extent of myeloperoxidase stained neutrophils in the bronchial submucosa were determined. Results indicate absence of detection of P65 before 12 hours after injury. At 12 hours after injury, strong perinuclear staining for p65 was evident in bronchial gland epithelial cells, macrophages, and endothelial cells. Bronchial submucosal gland cells showed a significant increase in the percentage of cells stained for p65 compared with uninjured animals and earlier times after injury, P < .05. At 24 and 48 hours after injury, p65 expression was evident in the bronchiolar epithelium, Type II pneumocytes, macrophages, and endothelial cells. Quantitation of the neutrophil influx into the bronchial submucosa showed a significant increase compared with uninjured tissue at 24 and 48 hours after injury, P < .05. In conclusion, immunohistochemical detection of activated p65 preceded the overall inflammatory response measured in the lamina propria. However, detection of p65 did not correlate with a recent study showing rapid emigration of neutrophils at 4 hours postinjury. Together, these results suggest that p65 immunostaining may identify cells that are activated to produce proinflammatory cytokines after injury; however, the immunoexpression may not adequately reflect the temporal activation of gene transcription that may occur with proinflammatory cytokine production with inhalation injury.

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 effects of neutral endopeptidase (NEP) in acute inflammation in the lung were studied using a newly developed murine model of smoke and burn (SB) injury. C57BL/6 mice were pretreated with an i.v. dose of a specific NEP antagonist CGS-24592 (10 mg/Kg) 1 h prior to SB injury (n = 5-8/group). Mice were anesthetized with i.p. ketamine/xylazine, intubated, and exposed to cooled cotton smoke (2 x 30 s). After s.c. injection of 1 ml 0.9% saline, each received a 40% total body surface area (TBSA) flame burn. Buprenorphene (2 mg/kg) was given i.p. and resuscitated by saline. Evans Blue dye (EB) was injected i.v. 15 min before sacrifice. Lung wet/dry weight ratio was measured. After vascular perfusion, lungs were analyzed for their levels of EB dye and myeloperoxidase (MPO). In mice pretreated with CGS-24592 followed by SB injury the EB levels were significantly higher (61%, p = 0.043) than those with SB injury alone. There was a significant increase (144%, p = 0.035) in EB dye in animals with SB injury alone as compared to shams. In mice pretreated with CGS-24592 prior to SB injury wet/dry weight ratios were significantly (27%, p = 0.042) higher compared to animals with SB injury alone. CGS-24592 pretreatment also caused a significant increase in MPO (29%, p = 0.026) as compared to mice with SB injury alone. In conclusion the current study indicates that specific NEP inhibitor CGS 24592 exacerbates the SB-induced lung injury and inflammation in mice.

Oxidants are involved in the pathogenesis of many disorders caused by burn and smoke inhalation; alpha- and gamma-tocopherols are major tissue antioxidants, and their depletion should reflect oxidant injury. To determine whether plasma and tissue vitamin E levels would thus be depleted in severe burn, prepared sheep were randomly divided into the following groups: non-injured, burn- and smoke-exposed, burned only and smoke-exposed only. All were resuscitated with Ringer's lactate solution, mechanically ventilated and sacrificed at various time intervals. Immediately following injury plasma, lung, trachea, heart and liver tocopherols/lipids were measured and found to be significantly depleted except in the heart. Reduction of tissue gamma-tocopherol appeared earlier than reduction of alpha-tocopherol. Thus animals receiving combined burn and inhalation injury underwent marked oxidative stress, suggesting that vitamin E might be depleted also in humans with burn and smoke inhalation injury, and that appropriate supplementation should be evaluated.

Acute burn wounds often require early excision and adequate coverage to prevent further hypothermia, protein and fluid losses, and the risk of infection. Meshed autologous skin grafts are generally regarded as the standard treatment for extensive full-thickness burns. Graft take and rate of wound healing, however, depend on several endogenous factors. This paper describes a standardized reproducible porcine model of burn and skin grafting which can be used to study the effects of topical treatments on graft take and re-epithelialization. Procedures provide a protocol for successful porcine burn wound experiments with special focus on pre-operative care, anesthesia, burn allocation, excision and grafting, postoperative treatment, dressing application, and specimen collection. Selected outcome measurements include percent area of wound closure by planimetry, wound assessment using a clinical assessment scale, and histological scoring. The use of this standardized model provides burn researchers with a valuable tool for the comparison of different topical drug treatments and dressing materials in a setting that closely mimics clinical reality.

Acute lung injury results in a severe inflammatory response, which leads to priming and activation of leucocytes, release of reactive oxygen and reactive nitrogen species, destruction of pulmonary endothelium, extravasation of protein-rich fluid into the interstitium and formation of oedema. Recently, H2S (hydrogen sulfide) has been shown to decrease the synthesis of pro-inflammatory cytokines, reduce leucocyte adherence to the endothelium and subsequent diapedesis of these cells from the microvasculature in in vivo studies, and to protect cells in culture from oxidative injury. In the present study, we hypothesized that a parenteral formulation of H2S would reduce the lung injury induced by burn and smoke inhalation in a novel murine model. H(2)S post-treatment significantly decreased mortality and increased median survival in mice. H2S also inhibited IL (interleukin)-1beta levels and significantly increased the concentration of the anti-inflammatory cytokine IL-10 in lung tissue. Additionally, H2S administration attenuated protein oxidation following injury and improved the histological condition of the lung. In conclusion, these results suggest that H2S exerts protective effects in acute lung injury, at least in part through the activation of anti-inflammatory and antioxidant pathways.

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.

Due to up-regulation of the parathyroid gland calcium-sensing receptor (CaR), burned children have hypocalcemic hypoparathyroidism, and decreased myocardial contractility. Our aim was to localize the CaR in the heart and measure receptor density changes due to burns. Heart and aorta samples from sheep subjected to 40% burn or sham conditions were probed for CaR via fluorescence microscopy. CaR was localized to endocardial endothelium, myocardial microvasculature, and fibroblasts and vessels of the aortic adventitia. CaR was not found in cardiomyocytes or smooth muscle cells. No differences in density of CaR or beta-adrenergic receptors were noted. No differences in CaR distribution were seen in the myocardium or aorta, in contrast to the parathyroid where burn injury up-regulates CaR. We suggest that CaR has a local, tissue-specific role, and functions in vascular calcium sensing for intravascular calcium deposition or regulation of other calcium channels after trauma or burn.

Objective: We hypothesized that nitric oxide derived from the neuronal nitric oxide synthase (NOS) is responsible for much of the injury resulting from skin burn and smoke inhalation. Therefore, we aimed to examine the effects of selective neuronal NOS inhibition on cardiopulmonary functions and cellular injury in sheep with acute respiratory distress syndrome secondary to combined burn and smoke inhalation injury., Design: Prospective, randomized, controlled laboratory experiment., Setting: Investigational intensive care unit., Subjects: A total of 22 chronically instrumented adult ewes., Interventions: Sheep were randomly assigned to either healthy controls (sham), injured controls (40% third-degree flame burn; 48 breaths of cotton smoke), or an injury group treated with the specific neuronal NOS inhibitor 7-nitroindazole (1 mg x kg(-1) x hr(-1)) from 1 hr postinjury to the end of the 48-hr study period. Hypoxic pulmonary vasoconstriction was assessed as decrease in left pulmonary blood flow in response to single-lung hypoxic challenges (100% nitrogen) at baseline, 24 hrs, and 48 hrs., Measurements and Main Results: The combination injury contributed to a approximately 90% loss of hypoxic pulmonary vasoconstriction and was associated with significant pulmonary shunting and death of one animal. The increase in nitrate/nitrite plasma levels in injured controls (12 hrs: 17 +/- 2 vs. 6 +/- 1 microM in sham animals; p < .001) was linked to increases in inducible NOS messenger RNA and 3-nitrotyrosine formation in lung tissue (48 hrs: 22 +/- 1 vs. 0.8 +/- 0.3 nM in sham animals; p < .001). 7-Nitroindazole treatment prevented the injury-associated changes in inducible NOS messenger RNA, nitrate/nitrite, and 3-nitrotyrosine, thereby attenuating the loss of hypoxic pulmonary vasoconstriction and improving gas exchange. In addition, 7-nitroindazole decreased lung tissue concentrations of hemoxygenase-1 and ameliorated myocardial depression, airway obstruction, pulmonary edema, ventilatory pressures, and histopathologic changes seen in injured controls., Conclusions: The present study provides evidence that neuronal NOS-derived nitric oxide plays a pivotal role in the pathogenesis of acute respiratory distress syndrome resulting from combined 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.

Burn and smoke inhalation-related multiple organ dysfunction is associated with a severe fall in the plasma concentration of antithrombin. Therefore the aim of the present study was to test the hypothesis that intravenous administration of recombinant human antithrombin in combination with aerosolized heparin will ameliorate acute lung injury in sheep exposed to cutaneous burn and smoke inhalation. Sheep were prepared operatively for study and, 7 days post-surgery, sheep were given a cutaneous burn (40% of total body surface area, third-degree burn) and insufflated with cotton smoke (48 breaths, <40 degrees C) under halothane anaesthesia. After injury, sheep were placed on a ventilator and resuscitated with Ringer's lactate solution. The animals were divided into three groups: sham group (non-injured and non-treated; n=6), saline group (injured and received saline; n=6) and rhAT.iv.+Hep group [injured and treated with rhAT (recombinant human antithrombin) and heparin; n=6]. In the rhAT.iv.+Hep group, rhAT was infused continuously for 48 h starting 1 h post-injury with a dose of 0.34 mg.h(-1).kg(-1) of body weight and heparin (10000 units) was aerosolized every 4 h starting at 1 h post-injury. The experiment lasted 48 h. Haemodynamics were stable in sham group, whereas the saline-treated sheep developed multiple signs of acute lung injury, including decreased pulmonary gas exchange, increased inspiratory pressures, extensive airway obstruction and increased pulmonary oedema. These pathological changes were associated with a severe fall in plasma antithrombin concentration, lung tissue accumulation of leucocytes and excessive production of NO. Treatment of injured sheep with anticoagulants attenuated all of the pulmonary pathophysiology observed. In conclusion, the results provide definitive evidence that anticoagulant therapy may be a novel and effective treatment tool in the management of burn patients with concomitant smoke inhalation injury.

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

Because of their extensive wounds, burn patients are chronically exposed to inflammatory mediators. Thus, burn patients, by definition, already have "systemic inflammatory response syndrome." Current definitions for sepsis and infection have many criteria (fever, tachycardia, tachypnea, leukocytosis) that are routinely found in patients with extensive burns, making these current definitions less applicable to the burn population. Experts in burn care and research, all members of the American Burn Association, were asked to review the literature and prepare a potential definition on one topic related to sepsis or infection in burn patients. On January 20, 2007, the participants met in Tucson, Arizona to develop consensus for these definitions. After review of the definitions, a summary of the proceedings was prepared. The goal of the consensus conference was to develop and publish standardized definitions for sepsis and infection-related diagnoses in the burn population. Standardized definitions will improve the capability of performing more meaningful multicenter trials among burn centers.

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.

To test the hypothesis that burn and smoke injury will deplete tissue alpha-tocopherol and cause its faster plasma disappearance, deuterium-labeled vitamin E was administered to sheep exposed to both surface skin burn and smoke insufflation, which cause injuries similar to those of human victims of fire accidents. Two different protocols were used: (1) deuterated vitamin E was administered orally with food at time 0 (just before injury) or (2) the labeled vitamin E was administered orally with food the day before injury. The animals, which had been operatively prepared seven days before, were anesthetized and then received both 40% body surface area third-degree burn and 48 breaths of cotton smoke or sham injuries. All were resuscitated with Ringer's lactate solution (4 ml/kg/% BSA burn/24 h) and mechanically ventilated. Blood samples were collected at various times after vitamin E dosing. In both studies the depletion of plasma alpha-tocopherol was faster in the injured sheep. The sheep given deuterated vitamin E 24 h before injury had similar maximum alpha-tocopherol concentrations at similar times. The exponential rates of alpha-tocopherol disappearance were 1.5 times greater and half-lives were 12 h shorter (p < 0.05) in the injured sheep. In separate studies, various tissues were obtained from sheep that were sacrificed from 4 to 48 h after injury. The liver alpha-tocopherol concentrations in sheep killed at various times after injury seem to show a linear decrease at a rate of 0.1 nmol alpha-tocopherol/g liver per hour, suggesting that the liver is supplying alpha-tocopherol to maintain the plasma and lung alpha-tocopherol concentrations, but that this injury is so severe the liver is unable to maintain lung alpha-tocopherol concentrations. These findings suggest that alpha-tocopherol should be administered to burn patients to prevent vitamin E depletion and to protect against oxidative stress from burn injury.

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.

Victims of fire accidents who sustain both thermal injury to the skin and smoke inhalation have gross evidence of oxidant injury. Therefore, we hypothesized that delivery of vitamin E, an oxygen superoxide scavenger, directly into the airway would attenuate acute lung injury postburn and smoke inhalation. Sheep (N = 17 female, 35 +/- 5 kg) were divided into 3 groups: (1) injured, then nebulized with vitamin E (B&S, Vitamin E, n = 6); (2) injured, nebulized with saline (B&S, Saline, n = 6); and (3) not injured, not treated (Sham, n = 5). While under deep anesthesia with isoflurane, the sheep were subjected to a flame burn (40% total body surface area, 3rd degree) and inhalation injury (48 breaths of cotton smoke, <40 degrees C). All groups were resuscitated with Ringer lactate solution (4 mL/kg/%burn/24 h) and placed on a ventilator [positive end-expiratory pressure (PEEP) = 5 cm H2O, tidal volume = 15 mL/kg] for 48 h. B&S injury halved the lung alpha-tocopherol concentrations (0.9 +/- 0.1 nmol/g) compared with sham-injured animals (1.5 +/- 0.3), whereas vitamin E treatment elevated the lung alpha-tocopherol concentrations (7.40 +/- 2.61) in the injured animals. B&S injury decreased pulmonary gas exchange (PaO2/FiO2 ratios) from 517 +/- 15 at baseline to 329 +/- 49 at 24 h and to 149 +/- 32 at 48 h compared with sham ratios of 477 +/- 14, 536 +/- 48, and 609 +/- 49, respectively. Vitamin E treatment resulted in a significant improvement of pulmonary gas exchange; ratios were 415 +/- 34 and 283 +/- 42 at 24 and 48 h, respectively. Vitamin E nebulization therapy improved the clinical responses to burn and smoke inhalation-induced acute lung injury.

Introduction: A decrease in alpha-tocopherol (vitamin E) plasma levels in burn patients is typically associated with increased mortality. We hypothesized that vitamin E supplementation (alpha-tocopherol) would attenuate acute lung injury induced by burn and smoke inhalation injury., Materials and Methods: Under deep anesthesia, sheep (33 +/- 5 kg) were subjected to a flame burn (40% total body surface area, third degree) and inhalation injury (48 breaths of cotton smoke, < 40 degrees C). Half of the injured group received alpha-tocopherol (1000 IU vitamin E) orally, 24 h prior to injury. The sham group was neither injured nor given vitamin E. All three groups (n = 5 per group) were resuscitated with Ringer's lactate solution (4 ml/kg/%burn/24 h), and placed on a ventilator (PEEP = 5 cmH2O; tidal volume = 15 ml/kg) for 48 h., Results: Plasma alpha-tocopherol per lipids doubled in the vitamin E treated sheep. Vitamin E treatment prior to injury largely prevented the increase in pulmonary permeability index and moderated the increase in lung lymph flow (52.6 +/- 6.2 ml/min, compared with 27.3 +/- 6.0 ml/min, respectively), increased the PaO2/FiO2 ratio, ameliorated both peak and pause airway pressure increases, and decreased plasma conjugated dienes and nitrotyrosine., Conclusions: Pretreatment with vitamin E ameliorated the acute lung injury caused by burn and smoke inhalation exposure.

Markedly elevated levels of plasma atrial natriuretic peptide (ANP), which exhibit potent diuretic and vasoactive properties, has been well documented in patients with acute lung injury. We examined the physiological effects of additional smoke inhalation on plasma ANP concentrations in an ovine burn model. Seventeen sheep were instrumented to receive fluid and have physiological measurements taken. The burn group (n=8) received 40% body surface area third degree burn and the burn+smoke group (n=9) received the same burn plus 48 breaths of cotton smoke insufflation. The animals were resuscitated according to the Parkland formula with Ringer's lactate in the following 72 h period. Hemodynamic, oxygenation, fluid balance, and plasma ANP levels were serially determined. The effects of smoke inhalation manifested as deteriorated oxygenation, and increased fluid accumulation after a sustained initial shock period of more than 12 h. Plasma ANP levels in the burn+smoke group showed a biphasic elevation, whereas the burn group showed no appreciable changes throughout the whole experimental period. The initial increase in plasma ANP concentrations occurred immediately after injury (from 96+/-10 at baseline to 136+/-17 pg/mL at 3h after injury); thereafter, it decreased towards baseline value, followed by a second increase in the post resuscitation period (183+/-43 pg/mL at 72 h after injury). Decreased urine output and accentuated pulmonary vascular resistance in the combined injury group was observed between the two ANP level peaks, indicating that ANP release modified physiological responses to the burn+smoke injury.

The objective of this study was to determine the relationship between carboxyhemoglobin (COHb) formation, global oxygen transport, and cardiac performance in the acute phase of combined burn and smoke inhalation injury. Following a third degree burn of 20% of the total body surface area, adult sheep were subjected to cotton smoke (4x12 breaths) according to an established protocol. Compared with baseline (BL), the burn injury led to an immediate and sustained COHb-independent depression in myocardial contractility. Despite a progressive increase in COHb formation, up to a maximum of 78+/-3% (P < 0.001 vs BL), smoke inhalation did not further impair these hemodynamic changes. This study demonstrated that in the early stage of combined burn and smoke inhalation injury, the depression in cardiac function is basically triggered by the burn injury, whereas COHb generation secondary to cotton smoke exposure primarily contributes to pulmonary shunting.

Background: Burn and sepsis are associated with hepatic ischemia and reperfusion injury. This study examines the hypothesis that postburn treatment with the vasodilator prostacyclin would be beneficial for hepatic perfusion and oxygenation., Methods: Female pigs (n = 18, 20-25 kg) underwent laparotomy, during which ultrasonic flow probes were placed on the portal vein and the common hepatic artery. Catheters were inserted in the superior mesenteric and left hepatic veins. After 5 days, all animals were anesthetized and 12 of them received 40% total body surface area third-degree burn; 100 microg/kg Escherichia coli lipopolysaccharide (LPS) was intravenously administered 18 hours postburn. Burned animals were randomized to receive a constant infusion of iloprost (20 ng/kg per minute) or an equivalent amount of carrier solution (normal saline). All animals were studied for 42 hours., Results: Burn caused a 2.5-fold increase in hepatic arterial vascular resistance (HAVR) and a 39% decrease in hepatic arterial blood flow (HABF). Postburn administration of iloprost did not improve the hepatic arterial hemodynamics (1.8-fold increase in HAVR and 38% decrease in HABF). Post-LPS, HABF was significantly reduced to 22% of baseline and HAVR was 15-fold increased (P < 0.05 vs. baseline, ANOVA). In contrast, iloprost-treated animals did not show hepatic arterial vasoconstriction, as both HABF and HAVR remained baseline values during the endotoxic phase (P < 0.05 vs. nontreated group, ANOVA). Postburn iloprost treatment yielded a significant improvement in post-LPS portal venous blood flow (PVBF, 79% of baseline vs. 45% of baseline in nontreated animals, P < 0.05, ANOVA). Portal venous pressure showed 16% and 56% increases after burn and endotoxin, respectively. Portal hypertension did not occur in iloprost-treated animals, as portal venous pressure remained within baseline range (P < 0.05 vs. nontreated group, ANOVA). Burn and endotoxemia resulted in a significant decrease of hepatic oxygen delivery (hDO2, 63% and 12% of baseline, respectively) and hepatic oxygen consumption (hVO2, 61% and 21% of baseline, respectively). Only during the postburn endotoxic phase, iloprost improved hDO2 and hVO2 (140% and 79%, respectively; P < 0.05 vs. nontreated group, ANOVA)., Conclusions: Postburn prostacyclin treatment appears to have no beneficial effects on hepatic perfusion early postburn. However, during the late postburn endotoxic phase, prostacyclin seems to significantly improve hepatic total blood flow and oxygenation. In addition, prostacyclin treatment attenuated burn- and endotoxin-induced portal hypertension.

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.

Objective: To evaluate the effect of interleukin-1alpha (IL-1alpha) on the mesenteric circulation, intestinal mucosal integrity, and bacterial translocation in a burn/endotoxemia chronic porcine model., Summary Background Data: Major burn and sepsis are associated with a high mortality, ischemia/reperfusion injury to the intestine, and an increased rate of bacterial translocation. Pathologic alterations of IL-1 synthesis, degradation, and binding to receptors have been reported. Manipulation of IL-1-mediated effects might be of therapeutic utility., Methods: Twenty-one female pigs were instrumented with an ultrasonic flow probe on the superior mesenteric artery and a catheter into the superior mesenteric vein. After 5 days, all animals were anesthetized, and 14 received 40% total body surface area third-degree burn. IL-1alpha was administered intravenously at 1,000 ng/kg to seven pigs immediately after burn. Eighteen hours after burn, 100 microg/kg lipopolysaccharide (LPS) was administered intravenously. Systemic and splanchnic hemodynamics were measured and blood samples were drawn for blood gas analysis. Intestinal permeability was assessed every 6 hours by measuring the lactulose/mannitol (L/M) excretion ratio. At the end of the study (42 hours), tissue samples were harvested for bacteriologic cultures., Results: Mesenteric blood flow was significantly decreased after burn and endotoxin. Administration of IL-1alpha significantly improved mesenteric blood flow postburn and post-LPS. Mesenteric oxygen supply and consumption showed a significant reduction after burn. In contrast, animals treated with IL-1alpha showed an increase in postburn mesenteric oxygen supply and consumption. LPS-induced mesenteric hypoxia was also ameliorated by IL-1alpha treatment. Intestinal permeability, as assessed by the L/M ratio, showed a 7- and 10-fold elevation after thermal injury and LPS, respectively. In contrast, IL-1alpha-treated animals showed an increase of only three- and fourfold in the L/M ratio, respectively. Bacterial translocation was significantly increased in the burn/endotoxin group. IL-1alpha significantly reduced the rates of bacterial translocation., Conclusions: IL-1alpha treatment attenuates mesenteric ischemia and reperfusion injury induced by thermal injury and endotoxemia by improving mesenteric blood flow and oxygenation. Subsequently, IL-1alpha reduces intestinal permeability and bacterial translocation after burn and sepsis.

We hypothesized that the antibody neutralization of L-selectin would decrease the pulmonary abnormalities characteristic of burn and smoke inhalation injury. Three groups of sheep (n = 18) were prepared and randomized: the LAM-(1-3) group (n = 6) was injected intravenously with 1 mg/kg of leukocyte adhesion molecule (LAM)-(1-3) (mouse monoclonal antibody against L-selectin) 1 h after the injury, the control group (n = 6) was not injured or treated, and the nontreatment group (n = 6) was injured but not treated. All animals were mechanically ventilated during the 48-h experimental period. The ratio of arterial PO2 to inspired O2 fraction decreased in the LAM-(1-3) and nontreatment groups. Lung lymph flow and pulmonary microvascular permeability were elevated after injury. This elevation was significantly reduced when LAM-(1-3) was administered 1 h after injury. Nitrate/nitrite (NO(x)) amounts in plasma and lung lymph increased significantly after the combined injury. These changes were attenuated by posttreatment with LAM-(1-3). These results suggest that the changes in pulmonary transvascular fluid flux result from injury of lung endothelium by polymorphonuclear leukocytes. In conclusion, posttreatment with the antibody for L-selectin improved lung lymph flow and permeability index. L-selectin appears to be principally involved in the increased pulmonary transvascular fluid flux observed with burn/smoke insult. L-selectin may be a useful target in the treatment of acute lung injury after burn and smoke inhalation.

Advances in local wound management with early excisional therapy have decreased morbidity and mortality of massive third-degree burn patients. Although blood redistribution within burned tissue is of clinical interest, few studies have longitudinally determined the regional blood flow of various layers of the burn wound. We used a conscious ovine model in which animals were subjected to 40% third degree burn. Burned tissue was divided into the four layers (i.e. skin, panniculus carnosus, adipose tissue, and skeletal muscle), and regional blood flow was determined separately, with fluorescent microspheres, while measuring systemic hemodynamics and total burned tissue microvascular fluid flux. The subburn adipose tissue exhibited a remarkable biphasic alteration in regional blood flow, whereas the skin layer showed only decreased blood flow during the whole experimental period. The increase in blood flow to the adipose tissue seems to be related to a sustained fluid filtrate in the postresuscitation period, resulting in edema formation mainly located in the adipose tissue at the endpoint.

This study tested the hypothesis that nitric oxide (NO) synthesized from inducible NO synthase (iNOS) is responsible for the cardiac dysfunction observed after burn and smoke inhalation injury. Twelve sheep received 40% third-degree burn and smoke inhalation under halothane anesthesia. The animals were divided into two groups: a MEG group [iNOS was inhibited with mercaptoethylguanidine (MEG), a selective inhibitor of iNOS, n=6] and a control group (n=6). The control group showed a significant increase in NO(2)(-)/NO(3)(-) (NO(x)) concentration, metabolite of NO, in plasma after 24 h, whereas the MEG group did not. In the control group, cardiac depression was observed immediately after injury associated with hemoconcentration. Cardiac function returned to a normal level within 6 h following injury. In the control group cardiac dysfunction was observed again after 24 h although the hemoconcentration peaked at 24 h after injury and then began to resolve. In the MEG group, cardiac depression and hemoconcentration were not observed. The present data suggest that cardiac depression seen with this combination injury consists of two phases and that the later phase is mediated by iNOS-NO.

Background: The primary goal of this study was to investigate hepatic fatty acid (FA) metabolism after severe thermal injury., Methods: Sixteen pigs were divided into control (n = 8) and burn (n = 8, with 40% full thickness total body surface area burned) groups. Catheters were inserted in the right common carotid artery, portal vein, and hepatic vein for blood sampling. Flow probes were placed around the hepatic artery and portal vein for blood flow measurements. Animals were given pain medication and sedated until the tracer study on day 4 after burn. The pigs were infused for 4 hours with U-13C16-palmitate in order to quantify hepatic FA kinetics and oxidation., Results: Liver triglyceride (TG) content was elevated from 162 +/- 16 (control) to 297 +/- 28 micromol TG/g dry liver wt. (p < .05). Hepatic FA uptake and oxidation were similar between the 2 groups, as were malonyl-coenzyme A (CoA) levels and activities of acetyl-CoA carboxylase and adenosine monophosphate (AMP)-activated protein kinase. In contrast, incorporation of plasma-free fatty acids into hepatic TG was elevated (p < .05) and very low density lipoprotein TG (VLDL-TG) secretion was decreased from 0.17 +/- 0.02 (control) to 0.03 +/- 0.01 micromol/kg per minute in burned pigs (p < .05)., Conclusions: The accumulation of hepatic TG in burned animals is due to inhibition of VLDL-TG secretion and to increased synthesis of hepatic TG. Fatty acids are not channeled to TG because of impaired oxidation.

We investigated the pathophysiological alterations seen with combined burn and smoke inhalation injuries by focusing on pulmonary vascular permeability and cardiopulmonary function compared with those seen with either burn or smoke inhalation injury alone. To estimate the effect of factors other than injury, the experiments were also performed with no injury in the same experimental setting. Lung edema was most severe in the combined injury group. Our study revealed that burn injury does not affect protein leakage from the pulmonary microvasculature, even when burn is associated with smoke inhalation injury. The severity of lung edema seen with the combined injury is mainly due to augmentation of pulmonary microvascular permeability to fluid, not to protein. Cardiac dysfunction after the combined injury consisted of at least two phases. An initial depression was mostly related to hypovolemia due to burn injury. It was improved by a large amount of fluid resuscitation. The later phase, which was indicated to be a myocardial contractile dysfunction independent of the Starling equation, seemed to be correlated with smoke inhalation injury.