Fibrin biomatrix-conjugated platelet-derived growth factor AB accelerates wound healing in severe thermal injury.

Controlled delivery of growth factors from biodegradable biomatrices could accelerate and improve impaired wound healing. The study aim was to determine whether platelet-derived growth factor AB (PDGF.AB) with a transglutaminase (TG) crosslinking substrate site released from a fibrin biomatrix improves wound healing in severe thermal injury. The binding and release kinetics of TG-PDGF.AB were determined in vitro. Third-degree contact burns (dorsum of Yorkshire pigs) underwent epifascial necrosectomy 24 h post-burn. Wound sites were covered with autologous meshed (3:1) split-thickness skin autografts and either secured with staples or attached with sprayed fibrin sealant (FS; n = 8/group). TG-PDGF.AB binds to the fibrin biomatrix using the TG activity of factor XIIIa, and is subsequently released through enzymatic cleavage. Three doses of TG-PDGF.AB in FS (100 ng, 1 µg and 11 µg/ml FS) were tested. TG-PDGF.AB was bound to the fibrin biomatrix as evidenced by western blot analysis and subsequently released by enzymatic cleavage. A significantly accelerated and improved wound healing was achieved using sprayed FS containing TG-PDGF.AB compared to staples alone. Low concentrations (100 ng-1 µg TG-PDGF.AB/ml final FS clot) demonstrated to be sufficient to attain a nearly complete closure of mesh interstices 14 days after grafting. TG-PDGF.AB incorporated in FS via a specific binding technology was shown to be effective in grafted third-degree burn wounds. The adhesive properties of the fibrin matrix in conjunction with the prolonged growth factor stimulus enabled by this binding technology could be favourable in many pathological situations associated with wound-healing disturbances. Copyright © 2013 John Wiley & Sons, Ltd.

Pulmonary histopathologic abnormalities and predictor variables in autopsies of burned pediatric patients.

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.

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

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

Isotopic study of L-Arginine kinetics in the lung during pseudomonas sepsis in an ovine model.

UNLABELLED: The objective of the study is to investigate how L-Arginine pulmonary metabolism is altered in response Pseudomonas aeruginosa (P. aeruginosa) induced septic conditions using an ovine model. METHODS: Seven female sheep were infused with a primed-constant infusion of L-[(15)N2-guanidino, 5, 5, (2)H2] L-Arginine for 28 hs. After the initial 4 hs of the L-Arginine infusion, a continuous infusion of live Pseudomonas aeruginosa bacteria started for 24 hs. A NO synthase (NOS) inhibitor, N(G)-Methyl-L-arginine (L-NMA), infusion was added during the last 4 hs of the bacterial infusion. Blood samples were taken at specific time points for isotopic enrichment during control, septic and NOS blocking phases of the study. RESULTS: We observed that the level of total delivery of L-Arginine to the lungs was significantly decreased in septic phase after 24 hours of pseudomonas infusion. In contrast, the fractional uptake and metabolism of L-Arginine by the lungs was doubled during septic phase relative to the control phase (MARG-basal = 100% vs. MARG-septic = 220 ± 56%, P < 0.05). NO production in the lungs was also significantly increased. Infusion of L-NMA markedly blunted this elevated NO production and attenuated the total arginine metabolized in the septic lungs (MARG-septic = 220 ± 56% vs. MARG-NO blocking = -25 ± 20%; P < 0.05). We demonstrated sepsis induced by P. aeruginosa infusion caused an increase in the fractional uptake and metabolic rate of arginine in the lungs. Furthermore, our data suggests that arginine was mainly consumed via arginine - NO pathway, which might be responsible for this enhanced arginine metabolic activity in the septic lungs.

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

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

Healing efficacy of sea buckthorn (Hippophae rhamnoides L.) seed oil in an ovine burn wound model.

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.

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

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

Isotopic study of L-Arginine kinetics in the lung during pseudomonas sepsis in an ovine model.

UNLABELLED: The objective of the study is to investigate how L-Arginine pulmonary metabolism is altered in response Pseudomonas aeruginosa (P. aeruginosa) induced septic conditions using an ovine model. METHODS: Seven female sheep were infused with a primed-constant infusion of L-[(15)N2-guanidino, 5, 5, (2)H2] L-Arginine for 28 hs. After the initial 4 hs of the L-Arginine infusion, a continuous infusion of live Pseudomonas aeruginosa bacteria started for 24 hs. A NO synthase (NOS) inhibitor, N(G)-Methyl-L-arginine (L-NMA), infusion was added during the last 4 hs of the bacterial infusion. Blood samples were taken at specific time points for isotopic enrichment during control, septic and NOS blocking phases of the study. RESULTS: We observed that the level of total delivery of L-Arginine to the lungs was significantly decreased in septic phase after 24 hours of pseudomonas infusion. In contrast, the fractional uptake and metabolism of L-Arginine by the lungs was doubled during septic phase relative to the control phase (MARG-basal = 100% vs. MARG-septic = 220 ± 56%, P < 0.05). NO production in the lungs was also significantly increased. Infusion of L-NMA markedly blunted this elevated NO production and attenuated the total arginine metabolized in the septic lungs (Mnitrate-septic = 43.6 ± 5.7 vs. Mnitrate-septic + L-NMA = 13.4 ± 5.1 umol/kg/min; p < 0.05). We demonstrated sepsis induced by P. aeruginosa infusion caused an increase in the fractional uptake and metabolic rate of arginine in the lungs. Furthermore, our data suggests that arginine was mainly consumed via arginine - NO pathway, which might be responsible for this enhanced arginine metabolic activity in the septic lungs.

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

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

Heterogeneity of the effects of combined nitric oxide synthase inhibition on organ perfusion in ovine sepsis.

INTRODUCTION: Previous studies demonstrated beneficial effects of early neuronal nitric oxide synthase (nNOS) and subsequent inducible NOS (iNOS) inhibition on the development of multiple organ dysfunctions in septic sheep. However, the effects of NOS inhibition on regional blood flow remained undetermined. The current study was conducted to assess the effects of combined NOS inhibition on blood flow to various organs in an ovine sepsis model. METHODS: Eighteen adult, female sheep were randomly allocated to the following groups: (1) sham-injured, non-treated group, (2) injured (smoke inhalation and instillation of Pseudomonas aeruginosa into the lungs), non-treated group (control), and (3) injured, treated group (specific nNOS inhibition from 1 h to 12 h and iNOS inhibition from 12 h to 24 h post-injury). Fluorescent microspheres were injected at baseline and various time points post-injury. At the end of the 24-h experimental period, tissue from various organs was harvested. RESULTS: Blood flow to the ileum was significantly increased in the control group from 12 h to 24 h versus sham (P < 0.05). This increase was attenuated in the treatment group (P < 0.05). In contrast, blood flow to the pancreas was significantly increased in the treatment group after 12 h and 24 h versus both sham and control (P < 0.05). Blood flow to the spleen was significantly lower after 24h in the control group versus sham and treatment (P < 0.05 both). CONCLUSIONS: Combined NOS inhibition significantly influenced the pathologically altered organ perfusion during ovine sepsis. However, this treatment strategy showed heterogeneous effects on organ perfusion, perhaps dependent on the sepsis-related degree of NO production and ensuing changes in regional flow.

Multiple versus single injections of fluorescent microspheres for the determination of regional organ blood flow in septic sheep.

Determination of regional blood flow by the injection of microspheres in sepsis models is crucial for the experimental evaluation of the influence of experimental treatment strategies on organ perfusion. However, multiple injections may critically increase the total quantity of microspheres, thereby restricting regional microcirculation and altering the results of blood flow measurements. This study was designed to compare the results of multiple versus single injections of microspheres in an established ovine sepsis model. Injury was induced by smoke inhalation and instillation of Pseudomonas aeruginosa into the lungs. Twenty sheep were studied for 4, 8, 12, 18, or 24 h, respectively. Microspheres were injected at the end of the study period and the animals were euthanized and organ tissues were harvested. Another four sheep were studied for 24 h and multiple microsphere injections were performed at the above indicated time points in the same animals. Tracheal blood flow significantly increased and blood flow to the pancreas and ileum significantly decreased versus baseline in both groups (P < 0.05 each). Blood flow to the ileum, renal cortex and skin did not significantly change versus baseline in both groups (P > 0.05). Blood flow was higher to the trachea in the multiple injection group at 18 h (P = 0.048) and to the ileum at 12 h (P = 0.049), and lower to the skin at 18 h (P = 0.015). In conclusion, the results indicate that multiple versus single microsphere injections induced no or negligible alterations during ovine sepsis. This finding may help reduce the quantity of animals needed in future experiments.

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

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

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

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

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

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

γ-tocopherol nebulization decreases oxidative stress, arginase activity, and collagen deposition after burn and smoke inhalation in the ovine model.

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.

Administration of a peroxynitrite decomposition catalyst into the bronchial artery attenuates pulmonary dysfunction after smoke inhalation and burn injury in sheep.

Reactive nitrogen species such as peroxynitrite play a significant role in burn and smoke inhalation injury. The bronchial circulation increases more than 10-fold in response to this combination injury. We hypothesized that direct delivery of low-dose WW-85, a peroxynitrite decomposition catalyst, into the bronchial artery would attenuate burn- and smoke inhalation-induced acute lung injury. In adult female sheep (n = 17), the bronchial artery was cannulated in preparation surgery. After a 5- to 7-day recovery period, sheep were subjected to a burn (40% total body surface area, third degree) and inhalation injury (48 breaths of cotton smoke, <40°C). The animals were divided into three groups following the injury: (i) WW-85 group: 1 h after injury, WW-85 (0.002 mg/kg per hour) was continuously infused into the bronchial artery, n = 5; (ii) control group: 1 h after injury, an equivalent amount of saline was injected into the bronchial artery, n = 6; (iii) sham group: no injury, no treatment, same operation and anesthesia, n = 6. All animals were mechanically ventilated and fluid resuscitated equally. In the control group, the injury induced a severe deterioration of pulmonary oxygenation and shunting and an increase in pulmonary microvascular permeability toward sham. The injury was further associated with an increase in reactive nitrogen species in lung tissues of the control group. All these alterations were significantly attenuated in the WW-85 group. We demonstrated that a low dosage of WW-85 directly administered into the bronchial artery attenuated pulmonary dysfunction to the same extent as higher systemically administered doses in previous experiments. Our data strongly suggest that local airway production of peroxynitrite contributes to pulmonary dysfunction following smoke inhalation and burn injury.

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

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

Administration of poly(ADP-ribose) polymerase inhibitor into bronchial artery attenuates pulmonary pathophysiology after smoke inhalation and burn in an ovine model.

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.