Superior Effects of Nebulized Epinephrine to Nebulized Albuterol and Phenylephrine in Burn and Smoke Inhalation-Induced Acute Lung Injury.

The severity of burn and smoke inhalation-induced acute lung injury (BSI-ALI) is associated with alveolar and interstitial edema, bronchospasm, and airway mucosal hyperemia. Previously, we have reported beneficial effects of epinephrine nebulization on BSI-ALI. However, the underlying mechanisms of salutary effects of nebulized epinephrine remain unclear. The present study compared the effects of epinephrine, phenylephrine, and albuterol on a model of BSI-ALI. We tested the hypothesis that both α1- and ß2-agonist effects are required for ameliorating more efficiently the BSI-ALI. Forty percent of total body surface area, 3rd-degree cutaneous burn, and 48-breaths of cotton smoke inhalation were induced to 46 female Merino sheep. Postinjury, sheep were mechanically ventilated and cardiopulmonary hemodynamics were monitored for 48 h. Sheep were allocated into groups: control, n = 17; epinephrine, n = 11; phenylephrine, n = 6; and albuterol, n = 12. The drug nebulization began 1 h postinjury and was repeated every 4 h thereafter. In the results, epinephrine group significantly improved oxygenation compared to other groups, and significantly reduced pulmonary vascular permeability index, lung wet-to-dry weight ratio, and lung tissue growth factor-ß1 level compared with albuterol and control groups. Epinephrine and phenylephrine groups significantly reduced trachea wet-to-dry weight ratio and lung vascular endothelial growth factor-A level compared with control group. Histopathologically, epinephrine group significantly reduced lung severity scores and preserved vascular endothelial-cadherin level in pulmonary arteries. In conclusion, the results of our studies suggest that nebulized epinephrine more effectively ameliorated the severity of BSI-ALI than albuterol or phenylephrine, possibly by its combined α1- and ß2-agonist properties.

Club Cell Protein, CC10, Attenuates Acute Respiratory Distress Syndrome Induced by Smoke Inhalation.

OBJECTIVES: To evaluate the dose effects of Recombinant human Club cell 10-kDa protein (rhCC10) on lung function in a well-characterized ovine model of acute respiratory distress syndrome (ARDS) induced by smoke inhalation injury (SII); specifically, the potential of rhCC10 protein to control the inflammatory response and protect pulmonary tissue and function following SII. DESIGN: Randomized, controlled, prospective, and large animal translational studies. SETTING: University large animal intensive care unit. SUBJECTS: Thirty-six adult female sheep were surgically prepared and allocated into five groups (Sham (no SII), n = 6; 1 mg/kg/d CC10, n = 8; 3 mg/kg/d CC10, n = 7; 10 mg/kg/d CC10, n = 8; Control SII, n = 7). INTERVENTIONS: All groups except the sham group were subjected to SII with cooled cotton smoke. Then, the animals were placed on a ventilator, treated with 1, 3, and 10 mg/kg/d of intravenous rhCC10 or vehicle, divided evenly into two administrations per day every 12 h, fluid resuscitated, and monitored for 48 h in a conscious state. MEASUREMENTS AND MAIN RESULTS: The group treated with 10 mg/kg/d rhCC10 attenuated changes in the following variables: PaO2/FiO2 ratio, oxygenation index, and peak inspiratory pressure; neutrophil content in the airway and myeloperoxidase levels; obstruction of the large and small airways; systemic leakage of fluid and proteins, and pulmonary edema. CONCLUSIONS: In this study, high-dose rhCC10 significantly attenuated ARDS progression and lung dysfunction and significantly reduced systemic extravasation of fluid and proteins, normalizing fluid balance. Based on these results, rhCC10 may be considered a novel therapeutic option for the treatment of SII-induced ARDS.

Surgical anatomy of the ovine sural nerve for facial nerve regeneration and reconstruction research.

The lack of a clinically relevant animal models for research in facial nerve reconstruction is challenging. In this study, we investigated the surgical anatomy of the ovine sural nerve as a potential candidate for facial nerve reconstruction, and performed its histological quantitative analysis in comparison to the buccal branch (BB) of the facial nerve using cadaver and anesthetized sheep. The ovine sural nerve descended to the lower leg along the short saphenous vein. The length of the sural nerve was 14.3 ± 0.5 cm. The distance from the posterior edge of the lateral malleolus to the sural nerve was 7.8 ± 1.8 mm. The mean number of myelinated fibers in the sural nerve was significantly lower than that of the BB (2,311 ± 381vs. 5,022 ± 433, respectively. p = 0.003). The number of fascicles in the sural nerve was also significantly lower than in the BB (10.5 ± 1.7 vs. 21.3 ± 2.7, respectively. p = 0.007). The sural nerve was grafted to the BB with end-to-end neurorrhaphy under surgical microscopy in cadaver sheep. The surgical anatomy and the number of fascicles of the ovine sural nerve were similar of those reported in humans. The results suggest that the sural nerve can be successfully used for facial nerve reconstruction research in a clinically relevant ovine model.

Lack of durable protection against cotton smoke-induced acute lung injury in sheep by nebulized single chain urokinase plasminogen activator or tissue plasminogen activator.

BACKGROUND: Airway fibrin casts are clinically important complications of severe inhalational smoke-induced acute lung injury (ISIALI) for which reliable evidence-based therapy is lacking. Nebulized anticoagulants or a tissue plasminogen activator; tPA, has been advocated, but airway bleeding is a known and lethal potential complication. We posited that nebulized delivery of single chain urokinase plasminogen activator, scuPA, is well-tolerated and improves physiologic outcomes in ISIALI. To test this hypothesis, we nebulized scuPA or tPA and delivered these agents every 4 h to sheep with cotton smoke induced ISIALI that were ventilated by either adaptive pressure ventilation/controlled mandatory ventilation (APVcmv; Group 1, n = 14) or synchronized controlled mandatory ventilation (SCMV)/limited suctioning; Group 2, n = 32). Physiologic readouts of acute lung injury included arterial blood gas analyses, PaO2/FiO2 ratios, peak and plateau airway pressures, lung resistance and static lung compliance. Lung injury was further assessed by histologic scoring. Biochemical analyses included determination of antigenic and enzymographic uPA and tPA levels, plasminogen activator and plasminogen activator inhibitor-1 activities and D-dimer in bronchoalveolar lavage (BAL). Plasma levels of uPA, tPA antigens, D-dimers and α-macroglobulin-uPA complex levels were also assessed. RESULTS: In Group 1, tPA at the 2 mg dose was ineffective, but at 4 mg tPA or scuPA, the PaO2/FiO2 ratios, peak/plateau pressures improved during evolving injury (p < 0.01) without significant differences at 48 h. To improve delivery of the interventions, the experiments were repeated in Group 2 with limited suctioning/SCMV, which generally increased PAs in (BAL). In Group 2, tPA was ineffective, but scuPA (4 or 8 mg) improved physiologic outcomes (p < 0.01) and plateau pressures remained lower at 48 h. Airway bleeding occurred at 8 mg tPA. BAL plasminogen activator (PA) levels positively correlated with physiologic outcomes at 48 h. CONCLUSIONS: Physiologic outcomes improved in sheep in which better delivery of the PAs occurred. The benefits of nebulized scuPA were achieved without airway bleeding associated with tPA, but were transient and largely abrogated at 48 h, in part attributable to the progression and severity of ISIALI.

Fms-Like Tyrosine Kinase-3 Ligand Attenuates Local and Systemic Infection in a Model of Post-Burn Pneumonia.

BACKGROUND: Burn injury induces immunosuppression and promotes infection with opportunistic pathogens. Pneumonia and sepsis are leading causes of post-burn morbidity and mortality. Fms-like tyrosine kinase-3 ligand (Flt3L) improves local and systemic resistance to P aeruginosa-associated burn wound infection. This study evaluates the effects of post-burn prophylactic Flt3L treatment on local and systemic infection and inflammation in a murine model of pneumonia and sepsis. METHODS: Mice received a severe scald burn, were treated with Flt3L or vehicle (CTR) for 5 days, and inoculated trans-nasally with P aeruginosa. Lung, blood, and spleen were harvested at 24 and 48 h postinoculation (p.i.) to assess infection (bacterial burden, bacteremia, distant organ manifestation) and inflammation (interleukin-6 (IL-6) and myeloperoxidase (MPO) levels). Histology correlated infection and inflammation parameters with morphology. Survival at various bacterial concentrations was monitored for 14 days p.i. RESULTS: Bacterial burden was significantly reduced in lung and spleen of Flt3L-treated mice. Flt3L treatment was associated with decreased signs of pulmonary inflammation (reduced wet weight and IL-6 levels), lower incidences of bacteremia and septic distant organ manifestation, and reduced systemic inflammation (IL-6 and MPO). Histologically, reduced alveolar and peribronchiolar neutrophil and lymphocyte infiltration indicated attenuated pulmonary inflammation after Flt3L treatment. Overall survival was comparable between groups for all doses of P aeruginosa, but mortality delayed in the Flt3L-treated group. CONCLUSION: Prophylactic treatment with Flt3L could augment antimicrobial therapy of post-burn pneumonia through improvement of the initial host response to challenge with P aeruginosa, attenuate local, and systemic inflammation as well as septic pathogen dissemination.

Xanthine oxidase contributes to sustained airway epithelial oxidative stress after scald burn.

Respiratory tract infections and pneumonia are major causes of morbidity and mortality in burn victims, however, limited studies have examined the effects of burn injury on airway epithelium. The current study examines the effect of scald burn injury on rat tracheal epithelium at 5 days after injury and tests the hypothesis that treatment with febuxostat (FBX), an inhibitor of xanthine oxidase (XO), can be protective of cell homeostasis. Sprague Dawley rats were randomly divided into uninjured (sham), injured (control) and injured and FBX treated groups, n = 8. Control and FBX treated groups received 60% total body surface area scald burn injury. The FBX group received an i. p. dose (1 mg/kg) at 1 hour after injury and every 24 hours. At 5 days after injury, the animals were sacrificed and tracheal epithelial cell lysates were collected. Malondialdehyde (MDA), ATP, and XO activity were measured. Formation of 8-OHdG in tracheal epithelium was determined using immunohistochemistry (IHC) and immunoreactivity was quantitated. MDA levels were significantly increased in injured control animals (24.8 ± 2.3) compared to sham (7.93 ± 1.2, p = 0.002). FBX treatment attenuated this response (12.6 ± 2.7, p = 0.02). ATP levels were significantly decreased in control (0.7 ± 0.16) compared to sham, (2 ± 0.14, p = 0.01). ATP levels were increased with FBX treatment (1.8 ± 0.1, p = 0.03) compared to controls. There was a significant increase in XO activity in control animals, 1.04 ± 0.06 compared to sham (0.34 ± 0.05, p = 0.03), and this response decreased with FBX treatment 0.46 ± 0.07 (p = 0.04). Immunolabeling of 8-OHdG in control animals was significantly increased (25.1 ± 0.7 compared to the sham group 5.5 ± 1.9 (p = 0.01)), and was decreased with FBX treatment (7.0 ± 2.3 compared to control (p = 0.03)). The current study indicates that lipid peroxidation and ATP depletion persist in tracheal epithelium for 5 days after injury along with increased XO activity and 8-OHdG. These effects were significantly attenuated by FBX treatment, suggesting that reactive oxygen species generated by XO contribute to airway epithelial injury following scald burn.

Nebulized Epinephrine Limits Pulmonary Vascular Hyperpermeability to Water and Protein in Ovine With Burn and Smoke Inhalation Injury.

OBJECTIVES: To test the hypothesis that nebulized epinephrine ameliorates pulmonary dysfunction by dual action-bronchodilation (ß2-adrenergic receptor agonism) and attenuation of airway hyperemia (α1-adrenergic receptor agonism) with minimal systemic effects. DESIGN: Randomized, controlled, prospective, and large animal translational studies. SETTING: University large animal ICU. SUBJECTS: Twelve chronically instrumented sheep. INTERVENTIONS: The animals were exposed to 40% total body surface area third degree skin flame burn and 48 breaths of cooled cotton smoke inhalation under deep anesthesia and analgesia. The animals were then placed on a mechanical ventilator, fluid resuscitated, and monitored for 48 hours in a conscious state. After the injury, sheep were randomized into two groups: 1) epinephrine, nebulized with 4 mg of epinephrine every 4 hours starting 1 hour post injury, n = 6; or 2) saline, nebulized with saline in the same manner, n = 6. MEASUREMENTS AND MAIN RESULTS: Treatment with epinephrine had a significant reduction of the pulmonary transvascular fluid flux to water (p < 0.001) and protein (p < 0.05) when compared with saline treatment from 12 to 48 hours and 36 to 48 hours, respectively. Treatment with epinephrine also reduced the systemic accumulation of body fluids (p < 0.001) with a mean of 1,410 ± 560 mL at 48 hours compared with 3,284 ± 422 mL of the saline group. Hemoglobin levels were comparable between the groups. Changes in respiratory system dynamic compliance, mean airway pressure, PaO2/FiO2 ratio, and oxygenation index were also attenuated with epinephrine treatment. No considerable systemic effects were observed with epinephrine treatment. CONCLUSIONS: Nebulized epinephrine should be considered for use in future clinical studies of patients with burns and smoke inhalation injury.

Physiologic and molecular changes in the tracheal epithelium of rats following burn injury.

Pneumonia is the leading complication in the critical care of burn victims. Airway epithelial dysfunction compromises host defense against pneumonia. The aim of this study is to test the hypothesis that burn injury alters the physiology of the airway epithelium. A rat model of 60% TBSA third degree scald burn was used. At 24 hours after injury, tracheal epithelial ultrastructure was studied using transmission electron microscopy (TEM) and proliferation was measured by Ki67 immunohistochemistry. Mucociliary clearance (MCC) was measured using fluorescent microspheres. The level of malondialdehyde (MDA), an indicator of lipid peroxidation, was also measured. Changes in epithelial mRNA expression were measured using microarray. Burn injury led to a ten-fold reduction in MCC that was statistically significant (p = 0.007) 24 hours after injury. No significant change was noted in the morphology of tracheal epithelial cells between groups, although a marginal increase in extracellular space was noted in injured animals. Ki67 nuclear expression was significantly reduced (25%, p = 0.008) in injured rats. There was a significant increase in MDA levels in the epithelial lysate of burned animals, p = 0.001. Microarray analysis identified 59 genes with significant differences between sham and injured animals. Burn injury altered multiple important functions in rat tracheal epithelium. The decrease in MCC and cell proliferation may be due to oxidative injury. Mechanistic studies to identify physiological processes associated with changes in airway function may help in designing therapeutic agents to reduce burn-induced airway pathogenesis.

Integrity of airway epithelium in pediatric burn autopsies: Association with age and extent of burn injury.

UNLABELLED: This study examines the structural integrity of the airway epithelium in autopsy tissues from pediatric burn subjects. METHODS: A semi-quantitative score for the degree of airway epithelial integrity was made for seventy- two pediatric burn autopsies. Multivariate ordinal logistic regression was performed to identify relationships between epithelial integrity and conditions related to tissue fixation, time of death after injury, age, total body surface area burn (TBSA), extent of 3rd degree burn, presence of inhalation injury, ventilator days and pneumonia. RESULTS: No significant difference in epithelial integrity scores was identified between burn only cases and those with inhalation injury. Significant correlations with bronchiolar epithelial integrity scores were identified for age, p=0.02, and percent 3rd degree burn, p=0.02. There was no significant relationship between epithelial integrity and time between death and autopsy, p>0.44. CONCLUSIONS: Airway epithelial loss seen in autopsy tissue is not simply an artifact of tissue fixation. The degree of compromise correlates most strongly with age and degree of burn. Further studies are needed to identify physiological or critical care factors following burn injury that contribute to compromise in the structural and functional properties of the airway epithelium.

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.

Predictive Value of IL-8 for Sepsis and Severe Infections After Burn Injury: A Clinical Study.

The inflammatory response induced by burn injury contributes to increased incidence of infections, sepsis, organ failure, and mortality. Thus, monitoring postburn inflammation is of paramount importance but, so far, there are no reliable biomarkers available to monitor and/or predict infectious complications after burn. As interleukin 8 (IL-8) is a major mediator for inflammatory responses, the aim of our study was to determine whether IL-8 expression can be used to predict postburn sepsis, infections, and mortality. Plasma cytokines, acute-phase proteins, constitutive proteins, and hormones were analyzed during the first 60 days after injury from 468 pediatric burn patients. Demographics and clinical outcome variables (length of stay, infection, sepsis, multiorgan failure [MOF], and mortality) were recorded. A cutoff level for IL-8 was determined using receiver operating characteristic analysis. Statistical significance is set at P < 0.05. Receiver operating characteristic analysis identified a cutoff level of 234 pg/mL for IL-8 for survival. Patients were grouped according to their average IL-8 levels relative to this cutoff and stratified into high (H) (n = 133) and low (L) (n = 335) groups. In the L group, regression analysis revealed a significant predictive value of IL-8 to percent of total body surface area burned and incidence of MOF (P < 0.001). In the H group, IL-8 levels were able to predict sepsis (P < 0.002). In the H group, elevated IL-8 was associated with increased inflammatory and acute-phase responses compared with the L group (P < 0.05). High levels of IL-8 correlated with increased MOF, sepsis, and mortality. These data suggest that serum levels of IL-8 may be a valid biomarker for monitoring sepsis, infections, and mortality in burn patients.

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

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

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

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

Airway obstruction and bacterial invasion in autopsy tissue of pediatric burn victims.

This study measured airway obstruction and bacterial invasion in systematically sampled lung tissue of burn victims at autopsy. Lung tissue from victims of combined smoke inhalation and burn injury (n = 5) and burn injury alone (n = 9) was examined histologically and the degree of bronchial and bronchiolar obstruction was measured. The walls of both bronchi and bronchioles were examined for bacterial invasion. Correlation analysis was performed for the association of airway obstruction with TBSA burn, number of ventilatory days, maximum inspiratory pressure, and days after injury. There was no significant difference in the mean degree of airway obstruction in smoke inhalation and burn victims compared with victims of burn-only injury (P > .05). Increased bronchiolar obstruction scores were detected in victims with pneumonia (55.3 ± 24.2%) compared with victims without pneumonia (9.3 ± 0.2%; P = .03). Bacterial invasion of the bronchial wall was present in one case, and invasion into the walls of bronchioles was seen in five cases. Burned children who died had extensive bronchiolar obstruction whether or not they had smoke inhalation injury. There was bacterial invasion into the airway wall in six of 14 cases (43%). Improved understanding of the mechanisms of airway obstruction is important for improved care of burned children.

Tiotropium bromide suppresses smoke inhalation and burn injury-induced ERK 1/2 and SMAD 2/3 signaling in sheep bronchial submucosal glands.

The effects of tiotropium bromide on ERK 1/2, SMAD 2/3 and NFκB signaling in bronchial submucosal gland (SMG) cells of sheep after smoke inhalation and burn injury (S + B) were studied. We hypothesized that tiotropium would modify intracellular signaling processes within SMG cells after injury. Bronchial tissues were obtained from uninjured (sham, n = 6), S + B injured sheep 48 h after injury (n = 6), and injured sheep nebulized with tiotropium (n = 6). The percentage (mean ± SD) of cells showing nuclear localization of phosphorylated ERK 1/2, pSMAD 2/3, and NFκB (p65) was determined by immunohistochemistry. Nuclear pERK 1/2 staining was increased in injured animals as compared to sham, (66 ± 20 versus 14 ± 9), p = 0.0022, as was nuclear pSMAD, 84 ± 10 versus 20 ± 10, p = 0.0022. There was a significant decrease in pERK 1/2 labeling in the tiotropium group compared to the injured group (31 ± 20 versus 66 ± 20, p = 0.013), and also a decrease in pSMAD labeling, 62 ± 17 versus 84 ± 10, p = 0.04. A significant increase for NFκB (p65) was noted in injured animals as compared to sham (73 ± 16 versus 7 ± 6, p = 0.0022). Tiotropium-treated animals showed decreased p65 labeling as compared to injured (35 ± 17 versus 74 ± 16, p = 0.02). The decrease in nuclear expression of pERK, pSMAD and NFκB molecules in SMG cells with tiotropium treatment is suggestive that their activation after injury is mediated in part through muscarinic receptors.

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.

Bacterial respiratory tract infections are promoted by systemic hyperglycemia after severe burn injury in pediatric patients.

BACKGROUND: Burns are associated with hyperglycemia leading to increased incidence of infections with pneumonia being one of the most prominent and adverse complications. Recently, various studies in critically ill patients indicated that increased pulmonary glucose levels with airway/blood glucose threshold over 150 mg/dl lead to an overwhelming growth of bacteria in the broncho-pulmonary system, subsequently resulting in an increased risk of pulmonary infections. The aim of the present study was to determine whether a similar cutoff value exists for severely burned pediatric patients. METHODS: One-hundred six severely burned pediatric patients were enrolled in the study. Patients were divided in two groups: high (H) defined as daily average glucose levels >75% of LOS >150 mg/dl), and low (L) with daily average glucose levels >75% of the LOS <150 mg/dl). Incidences of pneumonia, atelectasis, and acute respiratory distress syndrome (ARDS) were assessed. Incidence of infections, sepsis, and respiratory parameters were recorded. Blood was analyzed for glucose and insulin levels. Statistical analysis was performed using Student's t-test and chi-square test. Significance was set at p<0.05. RESULTS: Patient groups were similar in demographics and injury characteristics. Pneumonia in patients on the mechanical ventilation (L: 21%, H: 32%) and off mechanical ventilation (L: 5%, H: 15%), as well as ARDS were significantly higher in the high group (L: 3%, H: 19%), p<0.05, while atelectasis was not different. Patients in the high group required significantly longer ventilation compared to low patients (p<0.05). Furthermore, incidence of infection and sepsis were significantly higher in the high group, p<0.05. CONCLUSION: Our results indicate that systemic glucose levels over 150 mg/dl are associated with a higher incidence of pneumonia confirming the previous studies in critically ill patients.

Advantages and pitfalls of combining intravenous antithrombin with nebulized heparin and tissue plasminogen activator in acute respiratory distress syndrome.

BACKGROUND: Pulmonary coagulopathy has become an important therapeutic target in adult respiratory distress syndrome (ARDS). We hypothesized that combining intravenous recombinant human antithrombin (rhAT), nebulized heparin, and nebulized tissue plasminogen activator (TPA) more effectively improves pulmonary gas exchange compared with a single rhAT infusion, while maintaining the anti-inflammatory properties of rhAT in ARDS. Therefore, the present prospective, randomized experiment was conducted using an established ovine model. METHODS: Following burn and smoke inhalation injury (40% of total body surface area, third-degree flame burn, and 4 × 12 breaths of cold cotton smoke), 18 chronically instrumented sheep were randomly assigned to receive intravenous saline plus saline nebulization (control), intravenous rhAT (6 IU/kg/h) started 1 hour after injury plus saline nebulization (AT i.v.) or intravenous rhAT combined with nebulized heparin (10,000 IU every 4 hours, started 2 hours after injury), and nebulized TPA (2 mg every 4 hours, started 4 hours after injury) (triple therapy, n = 6 each). All animals were mechanically ventilated and fluid resuscitated according to standard protocols during the 48-hour study period. RESULTS: Both treatment approaches attenuated ARDS compared with control animals. Notably, triple therapy was associated with an improved PaO2/FiO2 ratio (p = 0.007), attenuated pulmonary obstruction (p = 0.02) and shunting (p = 0.025), as well as reduced ventilatory pressures (p < 0.05 each) versus AT i.v. at 48 hours. However, the anti-inflammatory effects of sole AT i.v., namely, the inhibition of neutrophil activation (neutrophil count in the lymph and pulmonary polymorphonuclear cells, p < 0.05 vs. control each), pulmonary transvascular fluid flux (lymph flow, p = 0.004 vs. control), and systemic vascular leakage (cumulative net fluid balance, p < 0.001 vs. control), were abolished in the triple therapy group. CONCLUSION: Combining intravenous rhAT with nebulized heparin and nebulized TPA more effectively restores pulmonary gas exchange, but the anti-inflammatory effects of sole rhAT are abolished with the triple therapy. Interferences between the different anticoagulants may represent a potential explanation for these findings.

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

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

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

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