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.

Omega-7 oil increases telomerase activity and accelerates healing of grafted burn and donor site wounds.

This study investigated the efficacy of Omega-7 isolated from the sea buckthorn oil (Polyvit Co., Ltd, Gangar Holding, Ulaanbaatar, Mongolia) in ovine burn wound healing models. In vitro, proliferation (colony-forming rate) and migration (scratch) assays using cultured primary ovine keratinocytes were performed with or without 0.025% and 0.08% Omega-7, respectively. The colony-forming rate of keratinocytes in the Omega-7 group at 72 and 96 h were significantly higher than in the control (P < 0.05). The percentage of closure in scratch assay in the Omega-7 group was significantly higher than in the control at 17 h (P < 0.05). In vivo, efficacy of 4% Omega-7 isolated from buckthorn oil was assessed at 7 and 14 days in grafted ovine burn and donor site wounds. Telomerase activity, keratinocyte growth factor, and wound nitrotyrosine levels were measured at day 14. Grafted sites: Un-epithelialized raw surface area was significantly lower and blood flow was significantly higher in the Omega-7-treated sites than in control sites at 7 and 14 days (P < 0.05). Telomerase activity and levels of keratinocyte growth factors were significantly higher in the Omega-7-treated sites after 14 days compared to those of control (P < 0.05). The wound 3-nitrotyrosine levels were significantly reduced by Omega-7. Donor sites: the complete epithelialization time was significantly shorter and blood flow at day 7 was significantly higher in the Omega-7-treated sites compared to control sites (P < 0.05). In summary, topical application of Omega-7 accelerates healing of both grafted burn and donor site wounds. Omega-7 should be considered as a cost-efficient and effective supplement therapy for burn wound healing.

Prognostic Value of Electrocardiographic QRS Diminution in Patients Hospitalized With COVID-19 or Influenza.

During the clinical care of hospitalized patients with COVID-19, diminished QRS amplitude on the surface electrocardiogram (ECG) was observed to precede clinical decompensation, culminating in death. This prompted investigation into the prognostic utility and specificity of low QRS complex amplitude (LoQRS) in COVID-19. We retrospectively analyzed consecutive adults admitted to a telemetry service with SARS-CoV-2 (n = 140) or influenza (n = 281) infection with a final disposition-death or discharge. LoQRS was defined as a composite of QRS amplitude <5 mm or <10 mm in the limb or precordial leads, respectively, or a ≥50% decrease in QRS amplitude on follow-up ECG during hospitalization. LoQRS was more prevalent in patients with COVID-19 than influenza (24.3% vs 11.7%, p = 0.001), and in patients who died than survived with either COVID-19 (48.1% vs 10.2%, p <0.001) or influenza (38.9% vs 9.9%, p <0.001). LoQRS was independently associated with mortality in patients with COVID-19 when adjusted for baseline clinical variables (odds ratio [OR] 11.5, 95% confidence interval [CI] 3.9 to 33.8, p <0.001), presenting and peak troponin, D-dimer, C-reactive protein, albumin, intubation, and vasopressor requirement (OR 13.8, 95% CI 1.3 to 145.5, p = 0.029). The median time to death in COVID-19 from the first ECG with LoQRS was 52 hours (interquartile range 18 to 130). Dynamic QRS amplitude diminution is a strong independent predictor of death over not only the course of COVID-19 infection, but also influenza infection. In conclusion, this finding may serve as a pragmatic prognostication tool reflecting evolving clinical changes during hospitalization, over a potentially actionable time interval for clinical reassessment.

Muscarinic receptor antagonist therapy improves acute pulmonary dysfunction after smoke inhalation injury in sheep.

OBJECTIVES: Inhalation injury contributes to the morbidity and mortality of burn victims. In humans and in an ovine model of combined smoke inhalation and burn injury, bronchospasm and acute airway obstruction contribute to progressive pulmonary insufficiency. This study tests the hypothesis that muscarinic receptor antagonist therapy with tiotropium bromide, an M1 and M3 muscarinic receptor antagonist, will decrease the airway constrictive response and acute bronchial obstruction to improve pulmonary function compared to injured animals without treatment. DESIGN: Randomized, prospective study involving 32 sheep. SETTING: Large-animal intensive care research laboratory. INTERVENTIONS: The study consisted of six groups: a sham group (n=4, instrumented noninjured), a control group (n=6, injured and not treated), and tiotropium bromide-treated groups, including both preinjury and postinjury nebulization protocols. Treatments for these groups included nebulization with 36 µg of tiotropium bromide 1 hr before injury (n=6) and postinjury nebulization protocols of 18 µg (n=6), 36 µg (n=6), and 72 µg (n=4) administered 1 hr after injury. All treated groups received an additional 14.4 µg every 4 hrs for the 24-hr study period. MAIN RESULTS: Pretreatment with tiotropium bromide significantly attenuated the increases in ventilatory pressures, pulmonary dysfunction, and upper airway obstruction that occur after combined smoke inhalation and burn injury. Postinjury treatments with tiotropium bromide were as effective as pretreatment in preventing pulmonary insufficiency, although a trend toward decreased obstruction was present only in all post-treatment conditions. There was no improvement noted in pulmonary function in animals that received a higher dose of tiotropium bromide. CONCLUSIONS: This study describes a contribution of acetylcholine to the airway constrictive and lumenal obstructive response after inhalation injury and identifies low-dose nebulization of tiotropium bromide as a potentially efficacious therapy for burn patients with severe inhalation injury.

Acute secretory cell toxicity and epithelial exfoliation after smoke inhalation injury in sheep: an electron and light microscopic study.

Smoke inhalation injury promotes exfoliation of the upper airway columnar epithelium. Tracheal tissues from sheep 30 min after smoke exposure show intact epithelial areas, areas of epithelial disruption with loss of columnar cells and areas denuded of columnar cells. In intact areas detaching ciliated cells can be seen raised above the apical surface. This study aims to assess cell-specific toxicity by examining intact epithelium after inhalation injury. The junctional adhesion integrity between columnar and basal cells and the type of cells initially being displaced were also studied using light (LM) and transmission electron microscopy (TEM). TEM assessment of intact areas of sheep tracheal tissue (n=3) 30 min after exposure showed secretory cell toxicity including extrusion of cytoplasmic contents. In cells with severe secretory cell cytoplasmic disruption, loss of desmosomal junctions between the secretory and adjacent ciliated cells was evident. The number of desmosomes visible between columnar cells and basal cells was reduced (2.8 ± 1.8) in smoke-exposed animals compared to those in uninjured animals (5.0 ± 2.7), p=0.008. Serial sections of intact regions found 52 cells being displaced from the epithelium. All detaching cells were identified as ciliated cells. These studies show that the acute effects of inhalation injury include selective secretory cell toxicity which is associated with loss of junctional adhesion mechanisms and displacement of ciliated cells. Improved understanding of acute hypersecretory responses and epithelial integrity after exposure to toxic agents may improve understanding of epithelial fragility in airway disease.

Substance P antagonist CP-96345 blocks lung vascular leakage and inflammation more effectively than its stereoisomer CP-96344 in a mouse model of smoke inhalation and burn injury.

The recently developed murine model of smoke inhalation and burn (SB) injury was used to study the effect of the substance-P antagonist CP96345. C57BL/6 mice were pre-treated with an i.v. dose of a specific NK-1 receptor antagonist, CP9635, or its inactive enantiomer, CP96344, (10 mg/Kg) 1 h prior to SB injury per protocol (n = 5). Mice were anesthetized and exposed to cooled cotton smoke, 2X 30 s, followed by a 40% total body surface area flame burn per protocol. At 48 h after SB injury Evans Blue (EB) dye and myeloperoxidase (MPO) were measured in lung after vascular perfusion. Lungs were also analyzed for hemoglobin (Hb) and wet/dry weight ratio. In the current study, CP96345 pre-treatment caused a significant decrease in wet/dry weight ratio (23%, p = 0.048), EB (31%, p = 0.047), Hb (46%, p = 0.002), and MPO (54%, p = 0.037) levels following SB injury compared to animals with SB injury alone. CP-96344 pre-treatment caused an insignificant decrease in wet/dry weight ratio (14%, p = 0.18), EB (16%, p = 0.134), Hb (9%, p = 0.39), and an insignificant increase in MPO (4%, p = 0.79) as compared to mice that received SB injury alone. As expected, levels of EB, Hb, MPO, and wet/dry weight ratios were all significantly (p < 0.05) increased 48 h following SB injury alone compared to respective sham animals. In conclusion, the current study indicates that pre-treatment with a specific NK-1R antagonist CP-96345 attenuates the lung injury and inflammation induced by SB injury in mice.

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.

Role of calcitonin gene-related peptide (CGRP) in ovine burn and smoke inhalation injury.

Concomitant smoke inhalation trauma in burn patients is a serious medical problem. Previous investigations in our sheep model revealed that these injuries lead to significant airway hyperemia, enhanced pulmonary fluid extravasation, and severely impaired pulmonary function. However, the pathophysiological mechanisms are still not fully understood. The lung is innervated by sensory nerves containing peptides such as substance P and calcitonin gene-related peptide. Noxious stimuli in the airways can induce a neurogenic inflammatory response, which has previously been implicated in several airway diseases. Calcitonin gene-related peptide is known to be a potent vasodilator. We hypothesized that calcitonin gene-related peptide is also a mediator of the pulmonary reaction to toxic smoke and planned experiments to evaluate its role in this model. We tested the effects of pretreatment with a specific antagonist of the major receptor for calcitonin gene-related peptide (BIBN4096BS; 32 microg/kg, followed by continuous infusion of 6.4 microg.kg(-1).h(-1)) until the animal was killed 48 h after injury in an established ovine model of burn (40% total body surface, third degree) and smoke inhalation (48 breaths, <40 degrees C) injury. In treated animals (n = 7), the injury-related increases in tracheal blood flow and lung lymph flow were significantly attenuated compared with untreated controls (n = 5). Furthermore, the treatment significantly attenuated abnormalities in respiratory gas exchange. The data suggest that calcitonin gene-related peptide contributes to early airway hyperemia, transvascular fluid flux, and respiratory malfunction following ovine burn and smoke inhalation injury. Future studies will be needed to clarify the potential therapeutic benefit for patients with this injury.

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.

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.

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.

Upper airway mucus deposition in lung tissue of burn trauma victims.

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

Dibromoacetonitrile-induced protein oxidation and inhibition of proteasomal activity in rat glioma cells.

Dibromoacetonitrile (DBAN) is a disinfection byproduct of water chlorination. The present study was designed to investigate the potential oxidative protein modifications and alterations in proteasomal activity induced by DBAN in C6 glioma cells (C6 cells). Cells were exposed to 50-400 ppb DBAN for 24 h or 48 h. Cellular viability and lactate dehydrogenase (LDH) leakage were unaffected at 24 h. However, at 48 h after exposure to high concentrations of DBAN, there was a significant decrease in cell viability accompanied by a significant increase in LDH leakage. Exposure to DBAN for 48 h significantly enhanced formation of reactive oxygen species (ROS) in a concentration-related manner. Incubation of C6 cells for 24h or 48 h caused 1.3-2.4-fold increase in levels of lipid peroxidation as indicated by malondialdehyde (MDA)+4-hydroxy-2(e)-nonenal (4-HNE). Further, DBAN induced a concentration and time-dependent increase (1.6-6-folds) in the levels of protein carbonylation. At 48 h, proteasomal activities were found to decrease to 80%, 72%, 46%, and 34% of control with 50 ppb, 100 ppb, 200 ppb, 400 ppb DBAN, respectively. In conclusion, the present study indicates that exposure of C6 cells to DBAN results in generation of ROS, lipid peroxidation, accumulation of oxidized proteins and inhibition of proteasomal activity.

Assessment of lung inflammation in a mouse model of smoke inhalation and burn injury: strain-specific differences.

ABSTRACT To test concepts developed in our ovine model of acute respiratory distress syndrome, specifically the roles of neuropeptides and other peptide mediators, a recently developed murine model of combined smoke inhalation and burn (SB) injury was extended by applying methods for quantitative assessment of acute inflammation in the lung. Mice received SB injury per protocol, n = 5 to 7 per group. Mice were anesthetized with i.p. ketamine/xylazine, endotracheally intubated, and exposed to cooled cotton smoke (4 x 30 sec for Balb/C, 2 x 30 sec for C57BL/6). After s.c. injection of 1 mL 0.9% saline, each received a 40% total body surface area (TBSA) flame burn. Buprenorphine (0.1 mg/kg) was given i.p. for postoperative analgesia; 0.9% saline was given i.p. at 4 mL/kg per %TBSA burn. Evans Blue dye (EB) was injected i.v. 15 min before sacrifice. Lung wet/dry weight ratio was measured. In other animals, after vascular perfusion with buffered saline, lungs were sampled and analyzed for myeloperoxidase (MPO), using an EIA kit, and for their content of EB dye. There was a significant (p < 0.05) increase in EB dye content, wet/dry weight ratio, and MPO 24 h after injury in Balb/C mice. Similar increases were seen in C57BL/6 mice 48 h after SB injury, but not at 24 h. C57 mice tolerated less smoke inhalation than Balb/C mice, due to postexposure apnea, and required 48 h to show significant increases in these variables. Direct comparison between animals injured by 40% TBSA burn and 2 x 30 sec smoke exposure and sacrificed after 48 h showed significantly greater abnormality in the C57BL/6 mice. The mouse model can be used effectively to assess acute inflammation in the lung.

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.

The water disinfectant byproduct dibromoacetonitrile induces apoptosis in rat intestinal epithelial cells: possible role of redox imbalance.

Chemically induced oxidative stress poses cytotoxic effects on intestinal epithelial cells that may trigger various forms of injuries in intestinal mucosa. Haloacetonitriles, including dibromoacetonitrile (DBAN, a drinking water contaminant and direct acting mutagen and carcinogen), are known to induce GI disorders. Earlier, we showed that dichloroacetonitrile (DBAN analog) alters glutathione status and increases reactive oxygen species in murine macrophage cell line. Therefore, the present study was undertaken to understand the role of redox imbalance and apoptosis in DBAN-induced GI disorders using rat intestinal epithelial (RIE) cells. Cultured confluent monolayers of RIE cells were continuously exposed to DBAN at 50 to 400 ppb (0.6 to 4.4 muM). After 24, 48, and 72 h of the exposure, oxidative stress and apoptosis were determined. At higher exposure regimens (100 to 400 ppb), a concentration- and time-dependent increase in glutathione disulfide levels (1.5 to 4-fold and 1.6 to 5-fold, p < 0.05) was noticed at 48 and 72 h, respectively, as compared to control. Severe depletion of reduced glutathione was also observed at 72 h after DBAN treatment. DBAN-induced oxidative stress was demonstrated at all concentrations by increased malondialdehyde (MDA) levels (1.3 to 3- and 1.8 to 4-fold, p < 0.05) at 48 and 72 h after treatment, respectively. Increase (1.3 to 2-fold, p < 0.05) in 8-hydroxy-2-deoxyguanosine (8OHdG) levels was observed at 48 h after treatment with 100-400 ppb DBAN. At 72 h these levels were 1.7 to 3- fold higher in DBAN-treated RIE cells as compared to control. DBAN-induced apoptosis, evaluated using TUNEL assay and differential staining techniques, indicates an increase in nuclear damage along with various apoptotic features using epifluorescence or light microscopy. The results of the present study suggest that DBAN-induced redox imbalance could lead to apoptosis and overall oxidative stress in RIE cells.

Fetal origin of adverse pregnancy outcome: the water disinfectant by-product chloroacetonitrile induces oxidative stress and apoptosis in mouse fetal brain.

Epidemiological studies indicate a relationship between water disinfectant by-products (DBP) and adverse pregnancy outcomes (APO) including neural tube defects. These studies suggest that fetal brain may be vulnerable to DBP during early stages of development. Therefore, we examined several molecular markers commonly known to indicate chemical-induced neurotoxicity during fetal brain development following prenatal exposure to the DBP; chloroacetonitrile (CAN). Pregnant mice, at gestation day 6 (GD6), were treated with a daily oral dose of CAN (25 mg/kg). At GD12, two groups of animals were treated with an i.v. tracer dose of [2-14C]-CAN. These animals were sacrificed at 1 and 24 h after treatment and processed for quantitative in situ micro-whole-body autoradiography. The remaining groups of animals continued to receive CAN. At GD18, control and treated animals were weighed, anesthetized, and fetuses were obtained and their brains were removed for biochemical and immunohistochemical analyses. Whole-body autoradiography studies indicate a significant uptake and retention of [2-14C]-CAN/metabolites (M) in fetal brain (cerebral cortex, hippocampus, cerebellum) at 1 and 24 h. There was a 20% reduction in body weight and a 22% reduction in brain weight of fetuses exposed to CAN compared to controls. A significant increase in oxidative stress markers was observed in various fetal brain regions in animals exposed to CAN compared to controls. This was indicated by a 3- to 4-fold decrease in the ratio of the reduced to oxidized form of glutathione (GSH/GSSG), increased lipid peroxidation (1.3-fold), and increased 8-hydroxy-2-deoxyguanosine levels (1.4-fold). Cupric silver staining indicated a significant increase in the number of degenerating neurons in cortical regions in exposed animals. In animals exposed to CAN there was increase in nuclear DNA fragmentation (TUNEL staining) detected in the cerebral cortex and cerebellum (2-fold increase in apoptotic indices). Caspase-3 activity in cerebral cortex and cerebellum of treated animals were also increased (1.7- and 1.5-fold, respectively). In conclusion, this study indicates that CAN/M crossed the placenta and accumulated in fetal brain tissues where it caused oxidative stress and neuronal apoptosis. These events could predispose the fetus to altered brain development leading to APO as well as behavioral and learning and memory deficits.

Neurological impairment in fetal mouse brain by drinking water disinfectant byproducts.

Developmental exposure to environmental chemicals may have detrimental effects on embryonic brains that could play a major role in the etio-pathology of fetal and adult neurological diseases. The exact mechanism by which prenatal exposures to environmental agents, such as drinking water disinfectant byproducts (DBP), cause neurological impairment in fetus is not known. Our objective is to examine the impact of prenatal exposure to DBP on fetal brain development. Pregnant CD-1 mice, at the sixth day of gestation (GD-6), received a daily (GD-6-GD-18) oral dose of chloroacetonitrile (CAN, 25 ppm), a member of DBP. To assess fetal brain uptake of CAN, several animals were injected with a tracer dose of 2-[(14)C]-CAN (333 microCi/kg, i.v.), at GD-12 and processed for quantitative in situ micro whole-body autoradiography (QIMWBA) at 1 and 24 h after treatment. The remaining animals continued receiving CAN until GD-18 when fetal brains were processed for biochemical determination of oxidative stress (OS) or prepared for histological examinations. The results indicate a rapid placental transfer and fetal brain uptake of 2-[(14)C]-CAN/metabolites in cortical areas and hippocampus. In treated animals 3-fold decrease in glutathione (GSH), 1.3-fold increase in lipid peroxidation and 1.4-fold increase in DNA oxidation were detected as compared to control. DeOlmos cupric silver staining of fetal brains indicated significant increase in cortical neurodegeneration in treated animals. Immunohistochemical labeling (TUNEL) of apoptotic nuclei in the cortices and choroid plexuses were also increased in treated animals as compared to control. In conclusion, CAN crosses the placental and fetal blood-brain barriers and induces OS that triggered apoptotic neurodegenration in fetal brain. Future studies will examine the molecular mechanisms of these events and its impact on neural development of offspring.

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.