Protective Effects of Hydrogen-Rich Saline on Rats with Smoke Inhalation Injury.

OBJECTIVE: To explore the protective effects of hydrogen-rich saline on rats with smoke inhalation injury. METHODS: 36 healthy male Sprague-Dawley rats were randomly divided into 3 groups (n = 12 per group): sham group (S), inhalation injury plus normal saline treatment group (I+NS), and inhalation injury plus hydrogen-rich saline treatment group (I+HS). 30 min after injury, normal saline and hydrogen-rich saline were injected intraperitoneally (5 mL/kg) in I+NS group and I+HS group, respectively. All rats were euthanized and blood and organ specimens were collected for determination 24 h after inhalation injury. RESULTS: Tumor necrosis factor-alpha (TNF-α) levels, malondialdehyde (MDA) concentrations, nuclear factor kappa B (NF-κB) p65 expression, and apoptosis index (AI) in I+HS group were significantly decreased (P < 0.05), while superoxide dismutase (SOD) activities were increased compared with those in I+NS group; and a marked improvement in alveolar structure was also found after hydrogen-rich saline treatment. CONCLUSIONS: Hydrogen-rich saline treatment exerts protective effects in acute lung injury induced by inhalation injury, at least in part through the activation of anti-inflammatory and antioxidant pathways and inhibition of apoptosis.

Inflammatory role of AMP-activated protein kinase signaling in an experimental model of toxic smoke inhalation injury.

OBJECTIVE: The molecular mechanisms underlying lung inflammation in toxic smoke inhalation injury are unknown. We investigated the signaling pathway responsible for the induction of interleukin 8 by wood smoke extract in lung epithelial cells and lung inflammation induced by wood smoke exposure in mice. DESIGN: A randomized, controlled study. SETTING: A research laboratory. INTERVENTIONS AND MAIN RESULTS: Exposure of primary human bronchial epithelial cells to wood smoke extract sequentially activated NADPH oxidase and increased intracellular reactive oxygen species level; activated AMP-activated protein kinase, extracellular signal-regulated kinase and Jun N-terminal kinase (two mitogen-activated protein kinases), and nuclear factor-κB and signal transducer and activator of transcription protein 3 (two transcription factors); and induced interleukin-8. Inhibition of NADPH oxidase activation with apocynin or siRNA targeting p47(phox ) (a subunit of NADPH oxidase) attenuated the increased intracellular reactive oxygen species level, AMP-activated protein kinase activation, and interleukin-8 induction. Removal of intracellular reactive oxygen species by N-acetyl-cysteine reduced the activation of AMP-activated protein kinase, extracellular signal-regulated kinase and Jun N-terminal kinase, and interleukin-8 induction. Prevention of AMP-activated protein kinase activation by Compound C or AMP-activated protein kinase siRNA lessened the activation of Jun N-terminal kinase, extracellular signal-regulated kinase, nuclear factor-κB, signal transducer and activator of transcription protein 3 and interleukin-8 induction. Inhibition of Jun N-terminal kinase and extracellular signal-regulated kinase activation by inhibitors reduced the activation of nuclear factor-κB and signal transducer and activator of transcription protein 3 and interleukin-8 induction. Abrogation of nuclear factor-κB and signal transducer and activator of transcription protein 3 activation by inhibitors attenuated the interleukin-8 induction. Additionally, acute exposure of mice to wood smoke promoted AMP-activated protein kinase phosphorylation and expression of macrophage inflammatory protein 2 (an interleukin-8 homolog) in lung epithelial cells and lungs and lung inflammation, all of which were reduced by Compound C treatment. CONCLUSIONS: Interleukin-8 induction by wood smoke extract in lung epithelial cells is mediated by novel NADPH oxidase-dependent, reactive oxygen species-sensitive AMP-activated protein kinase signaling with Jun N-terminal kinase and extracellular signal-regulated kinase as the downstream kinases and nuclear factor-κB and signal transducer and activator of transcription protein 3 as the downstream transcription factors. This AMP-activated protein kinase signaling is likely important for inducing lung inflammation with toxic smoke exposure in mice.

Circulating proteasomes after burn injury.

The objective of the study is to test whether circulating proteasomes are increased in burn patients and to assess whether possible alterations are associated with severity of injury, organ failure, and/or clinically relevant outcomes. In this study, plasma was obtained from burn patients on days 0 (admission, n = 50), 1 (n = 36), 3 (n = 35), 5 (n = 28), 7 (n=34), and 30 (n = 10) (controls: 40 volunteers). The 20S/26S proteasome levels were measured by enzyme-linked immunosorbent assay. Proteasome peptidase activity was assessed using a chymotryptic-like peptide substrate in combination with epoxomicin (specific proteasome inhibitor). Percentage of TBSA burned, presence of inhalation injury, development of sepsis/multiple organ failure, and sequential organ failure assessment scores were documented. On admission, plasma proteasome activity was higher in patients than in controls (P = .011). 26S proteasomes were not detectable. The 20S proteasome concentrations (median [25th/75th percentile]) peaked on day 0 (673 [399/1566] ng/mL; control: 195 [149/249] ng/mL, P < .001), gradually declined within 7 days, and fully returned to baseline at day 30 (116.5 [78/196] ng/mL). Elevated 20S proteasomes were associated with the presence of inhalation injury and correlated linearly with %TBSA in patients without inhalation injury. Initial 20S proteasome concentrations discriminated the presence of inhalation injury in patients with (sensitivity 0.88 and specificity 0.71) and without (sensitivity 0.83 and specificity 0.97) cutaneous burns but did not discriminate sepsis/multiple organ failure development or survival. Circulating 20S proteasome is a biomarker of tissue damage. The 20S proteasome plasma concentrations in patients with burns and/or inhalation injury are unlikely to predict outcomes but may be useful for the diagnosis of inhalation injury.

Proteasomes in human bronchoalveolar lavage fluid after burn and inhalation injury.

The purpose of this study was to determine whether 26S proteasome is detectable in human bronchoalveolar lavage fluid (BALF) and whether burn and inhalation injury is accompanied by changes in BALF proteasome content or activity. BALF was obtained on hospital admission from 28 patients with burn and inhalation injury (controls: 10 healthy volunteers). Proteasome concentrations were quantified by enzyme-linked immunosorbent assay, and their native molecular mass was assessed by gel filtration. Proteasome peptidase activity was measured using a chymotryptic-like peptide substrate in combination with epoxomicin (specific proteasome inhibitor). BALF protein was increased in patients (P<.001) and correlated positively with the degree of inhalation injury. The 20S/26S proteasomes were detectable in all BALF by enzyme-linked immunosorbent assay. Gel filtration confirmed the presence of intact 20S and 26S proteasome that was stable without soluble ATP/Mg. In all BALF chymotryptic-like activity was detectable and could be inhibited with epoxomicin by 60 to 70% (P<.01). Absolute amounts of 20S/26S proteasomes and proteasome activity were increased in patients (P<.001 for all). The relative BALF composition after injury was characterized by increased concentrations of 20S proteasome/mg protein (P=.0034 vs volunteers), decreased concentrations of 26S proteasome/mg protein (P=.041 vs volunteers), and reduced specific proteasome activity (P=.044 vs volunteers). The 26S proteasome per milligram and specific proteasome activity were even further reduced in patients who developed ventilator-associated pneumonia (P=.045 and P=.03 vs patients without ventilator-associated pneumonia). This study supports the novel concept that extracellular proteasomes could play a pathophysiological role in the injured lung and suggests that insufficient proteasome function may increase susceptibility for pulmonary complications.

Mechanisms of toxic smoke inhalation and burn injury: role of neutral endopeptidase and vascular leakage in mice.

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.

Pulmonary expression of nitric oxide synthase isoforms in sheep with smoke inhalation and burn 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.

gamma-Tocopherol nebulization by a lipid aerosolization device improves pulmonary function in sheep with burn and smoke inhalation injury.

Fire accident victims who sustain both thermal injury to skin and smoke inhalation have gross evidence of systemic and pulmonary oxidant damage and acute lung injury. We hypothesized that gamma-tocopherol (gT), a reactive O(2) and N(2) scavenger, when delivered into the airway, would attenuate lung injury induced by burn and smoke inhalation. Acute lung injury was induced in chronically prepared, anesthetized sheep by 40% total burn surface area, third-degree skin burn and smoke insufflation (48 breaths of cotton smoke, <40 degrees C). The study groups were: (1) Sham (not injured, flaxseed oil (FO)-nebulized, n=6); (2) SA-neb (injured, saline-nebulized, n=6); (3) FO-neb (injured, FO-nebulized, n=6); and (4) gT+FO-neb (injured, gT and FO-nebulized, n=6). Nebulization was started 1 h postinjury, and 24 ml of FO with or without gT (51 mg/ml) was delivered into airways over 47 h using our newly developed lipid aerosolization device (droplet size: 2.5-5 microm). The burn- and smoke inhalation-induced pathological changes seen in the saline group were attenuated by FO nebulization; gT addition further improved pulmonary function. Pulmonary gT delivery along with a FO source may be a novel effective treatment strategy in management of patients with acute lung injury.

The effects of inhaled nitric oxide on cardiac pathology and energy metabolism in a canine model of smoke inhalation injury.

OBJECTIVE: The effects of inhaled nitric oxide (NO) on cardiac pathology and energy metabolism were studied in a canine model of smoke inhalation injury. MATERIAL AND METHOD: Twenty-one dogs were randomly divided into three groups: four dogs constituted the normal control group (group N), eight dogs subjected to smoke inhalation followed by O(2) inhalation (FiO(2)=0.45) constituted the injury control group (group C), and nine dogs inhaling a mixture of O(2) and 45ppm nitric oxide after smoke exposure served as the treatment group (group T). Myocardial zymograms were continuously measured, and ventricular muscles were examined for histopathology in the end of the experiment. RESULTS: Lactate dehydrogenase (LDH), alpha-hydroxybutyrate dehydrogenase (alpha-HBD), creatine kinase (CK) and glutamic oxalacetic transaminase (GOT) in group T were less than those in group C(P<0.05, or P<0.01). ATP content and energy charge (EC) in group T were higher significantly than those in group C(P<0.01). Light microscopy and electron microscopy indicated that the ventricular pathologic changes in group T were milder than in control group. CONCLUSION: Nitric oxide inhalation relieved myocardial damage and improved energy metabolism.

[The influence of N-acetyl-L-cysteine on pulmonary injury and oxygen stress after smoke inhalation injury].

OBJECTIVE: To investigate the influence of N-acetyl-L-cysteine (NAC) on pulmonary injury and oxygen stress caused by smoke inhalation injury. METHODS: Rats inflicted with smoke inhalation injury were employed as the model. WBC in BALF (bronchoalveolar lavage fluid), MPO (myeloperoxidase) activity, hydrogen peroxide (H(2)O(2)) content, GSH (glutathione) content and total antioxidant (TAO) capacity in pulmonary tissue were determined. RESULTS: Postinjury WBC in BALF, MPO activity in pulmonary tissue and H(2)O(2) content decreased obviously after NAC treatment. But the pulmonary tissue contents of GSH and ATO increased evidently after the treatment with NAC. CONCLUSION: NAC treatment could ameliorate pulmonary oxygen stress after smoke inhalation injury. AS a result, the pulmonary antioxidant capacity was improved.

The effect of inducible nitric oxide synthase (iNOS) inhibition on smoke inhalation injury in sheep.

Recent studies on smoke inhalation injury have been focused on nitric oxide (NO) as an essential factor of progressive lung injury. We studied the effects of inducible nitric oxide synthase (iNOS) inhibition on inhalation injury in sheep. Sheep (n = 14) were prepared surgically for chronic study. After recovery period, the sheep received 48 breaths of cotton smoke. The animals were then randomised into two groups: MEG group [30 mg/kg mercaptoethylguanidine (MEG), selective inhibitor of iNOS and peroxynitrite scavenger, was given 1 h after injury and then 8 h for 41 h, n = 7] and control group (0.9% NaCl, n = 7). All animals were ventilated mechanically, and airway blood flow was measured using colored microspheres. In the control group, following significant increase in airway blood flow, deterioration in the PaO2/FiO2 ratio was observed. Whereas in the MEG group, it was not observed. In addition, the MEG group did not show significant increase in pulmonary vascular resistance and intrapulmonary shunt fraction. Lung wet/dry ratios, a marker of pulmonary edema, were significantly lower in the MEG group. At 48 h after injury, lung tissue-conjugated dienes, an index of lung oxidative tissue injury, were significantly lower in the MEG group than in the control group. Our data suggest that 1) iNOS-NO produced in the airway circulation plays a major role on the significant increase in airway blood flow, which may contribute to the spread of injury from injured airway to the lung parenchyma; 2) iNOS-NO induced in the pulmonary circulation contributes to the loss of hypoxic pulmonary vasoconstriction; and 3) iNOS-NO plays an important role on the lung oxidative tissue injury.

Increased group IV cytosolic phospholipase A2 activity in lungs of sheep after smoke inhalation injury.

Increased phospholipase A2 (PLA2) activity was measured in cytosolic fractions of lungs from sheep exposed to smoke from burning cotton or to synthetic smoke consisting of carbon and acrolein, a cotton smoke toxin. Three peaks of PLA2 activity were identified by heparin-Sepharose chromatography. The heparin-nonbinding PLA2 activity was twofold higher in the extracts from lungs exposed to smoke than in normal lungs. This activity was identified as the group IV 85-kDa cytosolic PLA2 (cPLA2). The activities of the forms of PLA2 that bound to heparin did not change after smoke exposure. Those activities showed a pH optimum of 9.0, required a millimolar Ca2+ concentration for full activity, and were inhibited by 5 mM dithiothreitol. One activity eluted at an NaCl concentration typical for group Ib and V PLA2 and had the expected substrate specificity. The other form of lung PLA2 that bound heparin was a group II PLA2. Lung myeloperoxidase activity increased progressively with increased exposure to smoke. cPLA2 was identified in sheep neutrophils. With 30 breaths of smoke exposure, there was an increase in cPLA2 activity without a difference in immunoreactivity on Western blot, indicating that the increased activity was not due to increased amounts of protein. In conclusion, smoke induces increases in resident lung cell cPLA2 activity that is likely responsible for eicosanoid production, leading to lung inflammation and bronchoconstriction.

Assessment of erythrocyte cholinesterase activity in victims of smoke inhalation.

BACKGROUND: The nature of the toxic gases that cause death from smoke inhalation is incompletely understood, and the mechanisms leading to incapacitation remain to be determined. Thermal degradation products of various compounds, including phosphorous-based fire retardants, are suspected capable of impairing human cholinesterase activity. The aim of this study was to measure the erythrocyte cholinesterase activity in victims of smoke inhalation. METHODS: We prospectively measured the erythrocyte cholinesterase activity in blood samples obtained at the scene of residential fires from 49 fire victims. We compared the results with those in an unmatched group of 45 persons with acute drug poisoning. RESULTS: The median (25th-75th percentiles) erythrocyte cholinesterase activity in the 49 fire victims, 1968 IU/L (1660-2276), was significantly lower than in the 45 control subjects 2460 IU/mL (1968-2890), (p = 0.0004). There was no significant difference of the red blood cell counts or plasma protein levels between the 2 groups, while the hematocrit was significantly greater in the fire victims than in the drug-poisoned patients. There was a significant correlation between blood cyanide and carbon monoxide concentrations in the fire victims (r = 0.494, p = 0.002). There was no correlation between erythrocyte cholinesterase activity and either blood cyanide (r = 0.11, p = 0.44) or blood carbon monoxide concentrations (r = 0.04, p = 0.78). CONCLUSIONS: We found a significantly lower level of erythrocyte cholinesterase activity in victims of residential fires, when compared with a convenience sample of hospitalized poisoned patients. Despite the limitations of the study, investigations of the toxic gases potentially responsible for impairment of cholinesterase activity and the clinical significance of this lower enzymatic activity merit further investigation.

[Determination of placental alkaline phosphatase (PLAP) for detecting the damages of alveolar type I cells caused by smoke inhalation].

The placental alkaline phosphatase (PLAP) is a marker enzyme of alveolar type I cells. To evaluate the damages of alveolar type I cells and its relations with the development of acute lung injury caused by smoke inhalation, the present study was designed to observe the dynamic changes in PLAP contents in plasma and bronchoalveolar lavage fluid (BALF). Following the induction of smoke inhalation injury in rat, the arterial blood gas levels, lung water volume, total protein and albumin contents in BALF, and PLAP contents in plasma and BALF were determined respectively in normal control and injured animals at 2, 6, 12 and 24 h after injury. The pathomorphology of lung tissues was also observed. It was found that after smoke inhalation, animals showed acute respiratory failure and serious pulmonary edema. The total protein and albumin levels in BALF increased markedly. Both PLAP contents in plasma and BALF also increased dramatically, and there was a significant positive correlation between the changes in the PLAP and the total protein contents in BALF. The pathomorphologically serious structural damage of alveolar type I cells were also found. PLAP may be not only a marker of alveolar type I cells injury, but also interrelated with the permeability increase of alveolar-capillary membrane after smoke inhalation.

Dynamic balance changes between elastase and antiprotease in the early stages after smoke inhalation injury.

Following the use of a rabbit smoke inhalation injury model established in this institute, there were marked reductions in elastase activities in neutrophils and alveolar macrophages; rapid increases in elastase activity in broncho-alveolar lavage fluid (BALF); reductions of serum trypsin inhibitory capacity; a decrease of Pao2 and an increase of PaCO2, and marked increases of lung water volume. Significant correlations were found between the increased extravascular lung water content and the rising elastase activity in BALF. It seems probable that the imbalance between elastase and antiprotease played an important role in the development of acute lung injury after smoke inhalation.

Angiotensin-1-converting enzyme activity as index of pulmonary damage in thermal injury with or without smoke inhalation.

Serum angiotensin-converting enzyme (ACE) activity, plasma renin activity (PRA), and serum aldosterone levels were measured up to four weeks in a population of adults exposed to thermal injury, with or without concomitant exposure to smoke inhalation. In 10 patients, plasma levels of angiotensin-2 and ACE activity in bronchial lavage were also evaluated. Patients with severe burn injury had a significant decline of serum ACE activity while the concentrations of aldosterone and PRA were markedly elevated. Smoke inhalation seemed to counterbalance the decline of serum ACE activity, and, in the last group of patients, ACE concentrations were higher than those recorded in patients suffering only from cutaneous burn. The ACE activity was evidenced in bronchial lavage of patients exposed to smoke inhalation with the highest values present in the first day after the injury. The same patients had also very elevated levels of plasma angiotensin 2. In conclusion, serum ACE activity decreases in burn patients according to the severity of the cutaneous burn; smoke inhalation influences serum levels of the enzyme with concentration values opposite to the low ones present in cutaneous burn. Finally, the enzyme activity has an independent pattern from that of the other components of the renin angiotensin aldosterone system. The evaluation of ACE activity may be a marker of pulmonary damage in smoke inhalation.

Effects of allopurinol on smoke inhalation in the ovine model.

We hypothesized that the pulmonary damage induced by smoke inhalation is the result of ischemic reperfusion injury. We determined the effect of allopurinol (xanthine oxidase inhibitor) on the pulmonary microvascular fluid flux in an ovine model after inhalation of cotton smoke (n = 13) and compared these data with those from untreated similarly smoke-injured (n = 7), as well as sham- (air, n = 9) smoked, animals and sheep given an equivalent dose of CO (n = 7). Smoke injury resulted in an increased lung lymph flow, lymph-to-plasma protein ratio, lung content of polymorphonuclear cells, and extravascular lung water (gravametric), in addition to histological evidence of tissue (pulmonary) edema and destruction. No significant difference was found in these variables between the sheep that were injured with smoke whether or not they were pretreated with allopurinol. The sham-smoked and CO-insufflated animals showed no significant changes in cardiopulmonary function or morphology. We conclude that there are few data to support a role of ischemic reperfusion injury in the pulmonary damage seen after smoke inhalation.

[Experimental study of dynamic changes in elastase-antiprotease in rabbits in the early stage of inhalation injury].

In this experiment, rabbit model with smoke inhalation injury was used. The study was designed to observe the dynamic changes of elastase activities of polymorphonuclear leukocytes (PMN), alveolar macrophages (AM) and bronchoalveolar lavage fluid (BALF); and trypsin inhibitory capacities of serum and BALF (STIC & BTIC). The relationships between these changes and acute lung injury, as well as the concomitant changes of arterial blood gas levels, lung water volume and pathomorphology of trachea and lung tissues were also observed. It was found that after injury the elastase activities of PMN and AM were markedly reduced, and the elastase activity of BALF was rapidly increased. STIC was also reduced. PaO2 progressively dropped and PaCO2 progressively increased. Animals showed respiratory distress. Pathomorphological phagocytes aggregations in lungs, pulmonary edema and pneumorrhagia were found. There were serious destructions of capillary endothelial cells, alveolar epithelial cells, basement membranes and interstitial fibers. The number of elastic fibers of parenchyma decreased. The lung water volume was markedly increased, and there was a significant correlation between the increment of extravascular lung water and the rising of elastase activity of BALF. On the basis of our observation, it is proposed that the imbalance of elastase-antiprotease may play an important role in the development of acute lung injury after smoke inhalation.