Hyperoxia-induced Lung Damage Research Articles

Transplantation of alveolar macrophages improves the efficacy of endothelial progenitor cell therapy in mouse model of bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a severe complication of preterm births, which develops due to exposure to supplemental oxygen and mechanical ventilation. Published studies demonstrated that the number of endothelial progenitor cells (EPC) is decreased in mouse and human BPD lungs and that adoptive transfer of EPC is an effective approach in reversing the hyperoxia-induced lung damage in mouse model of BPD. Recent advancements in macrophage biology identified the specific subtypes of circulating and resident macrophages mediating the developmental and regenerative functions in the lungs. Several studies reported the successful application of macrophage therapy in accelerating the regenerative capacity of damaged tissues and enhancing the therapeutic efficacy of other transplantable progenitor cells. In the present study, we explored the efficacy of combined cell therapy with EPC and resident alveolar macrophages (rAM) in hyperoxia-induced BPD mouse model. rAM and EPC were purified from neonatal mouse lungs and were used for adoptive transfer to the recipient neonatal mice exposed to hyperoxia. Adoptive transfer of rAM alone did not result in engraftment of donor rAM into the lung tissue but increased the mRNA level and protein concentration of proangiogenic CXCL12 chemokine in recipient mouse lungs. Depletion of rAM by chlodronate-liposomes decreased the retention of donor EPC after their transplantation into hyperoxia-injured lungs. Adoptive transfer of rAM in combination with EPC enhanced the therapeutic efficacy of EPC as evidenced by increased retention of EPC, increased capillary density, improved arterial oxygenation, and alveolarization in hyperoxia-injured lungs. Dual therapy with EPC and rAM has promise in human BPD.NEW & NOTEWORTHY Recent studies demonstrated that transplantation of lung-resident endothelial progenitor cells (EPC) is an effective therapy in mouse model of bronchopulmonary dysplasia (BPD). However, key factors regulating the efficacy of EPC are unknown. Herein, we demonstrate that transplantation of tissue-resident alveolar macrophages (rAM) increases CXCL12 expression in neonatal mouse lungs. rAM are required for retention of donor EPC in hyperoxia-injured lungs. Co-transplantation of rAM and EPC improves the efficacy of EPC therapy in mouse BPD model.

Effect of human epididymis protein 4 on hyperoxia-induced bronchial dysplasia in newborn rats

Objective The study aimed to elucidate the role and the underlying mechanism of human epididymis protein 4 (HE4) in the pathogenesis of hyperoxia-induced bronchial dysplasia in newborn rats. Methods Forty neonatal Sprague–Dawley (SD) rats were separated into two groups: a normal control group (20.8% oxygen concentration) and a hyperoxia-induced group (85% oxygen concentration). Three time intervals of 24 h, 3 days and 7 days were chosen for each group. Haematoxylin–eosin staining was used to identify the pathological alterations in the lung tissue of the SD rats. Enzyme-linked immunosorbent assay was used to evaluate plasma protein levels. Real-time reverse transcription polymerase chain reaction was used to determine messenger RNA (mRNA) expression. Results In newborn SD rats, hyperoxia intervention within 7 days may result in acute lung damage. In the plasma and tissue of newborn SD rats, hyperoxia induction may raise levels of HE4, matrix metalloproteinases (MMP) 9 and tissue inhibitors of metalloproteinases (TIMP) 1. We discovered that the HE4 protein activates the phosphorylation of extracellular regulated protein kinases (ERK) and p65, activates the downstream MMP9 signalling pathway, inhibits MMP9 mRNA expression, inhibits protein activity, reduces type I collagen degradation, increases collagen secretion and promotes matrix remodelling and fibrosis in neonatal rat primary alveolar type II epithelial cells by overexpressing and silencing the HE4 gene. Conclusion Through the ERK, MMP9 and TIMP1 signalling pathways, HE4 mediates the pathophysiological process of hyperoxia-induced lung damage in rats. Lung damage and lung basal remodelling are mediated by HE4 overexpression.

Protective Effects of Resveratrol Against Airway Hyperreactivity, Oxidative Stress, and Lung Inflammation in a Rat Pup Model of Bronchopulmonary Dysplasia

Oxygen therapy provides an important treatment for preterm and low-birth-weight neonates, however, it has been shown that prolonged exposure to high levels of oxygen (hyperoxia) is one of the factors contributing to the development of bronchopulmonary dysplasia (BPD) by inducing lung injury and airway hyperreactivity. There is no effective therapy against the adverse effects of hyperoxia. Therefore, this study was undertaken to test the hypothesis that natural phytoalexin resveratrol will overcome hyperoxia-induced airway hyperreactivity, oxidative stress, and lung inflammation. Newborn rats were exposed to hyperoxia (fraction of inspired oxygen - FiO2>95 % O2) or ambient air (AA) for seven days. Resveratrol was supplemented either in vivo (30 mg·kg-1·day-1) by intraperitoneal administration or in vitro to the tracheal preparations in an organ bath (100 μM). Contractile and relaxant responses were studied in tracheal smooth muscle (TSM) using the in vitro organ bath system. To explain the involvement of nitric oxide in the mechanisms of the protective effect of resveratrol against hyperoxia, a nitric oxide synthase inhibitor – Nω-nitro-L-arginine methyl ester (L-NAME), was administered in some sets of experiments. The superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and the tumor necrosis factor-alpha (TNF-α) and interleukin-1β (IL-1β) levels in the lungs were determined. Resveratrol significantly reduced contraction and restored the impaired relaxation of hyperoxia-exposed TSM (p<0.001). L-NAME reduced the inhibitory effect of resveratrol on TSM contractility, as well as its promotion relaxant effect (p<0.01). Resveratrol preserved the SOD and GPx activities and decreased the expression of TNF-α and IL-1β in hyperoxic animals. The findings of this study demonstrate the protective effect of resveratrol against hyperoxia-induced airway hyperreactivity and lung damage and suggest that resveratrol might serve as a therapy to prevent the adverse effects of neonatal hyperoxia. Keywords: Bronchopulmonary dysplasia • Hyperoxia • Airway hyperreactivity • Resveratrol • Pro-inflammatory cytokines

Hyperoxia induces alveolar epithelial cell apoptosis by regulating mitochondrial function through small mothers against decapentaplegic 3 (SMAD3) and extracellular signal-regulated kinase 1/2 (ERK1/2)

ABSTRACT Oxygen therapy and mechanical ventilation are widely used to treat and manage neonatal emergencies in critically ill newborns. However, they are often associated with adverse effects and result in conditions such as chronic lung disease and bronchopulmonary dysplasia. Hence, aclear understanding of the mechanisms underlying hyperoxia-induced lung damage is crucial in order to mitigate the side effects of oxygen-based therapy. Here, we have established an in vitro model of hyperoxia-induced lung damage in type II alveolar epithelial cells (AECIIs) and delineated the molecular basis of oxygen therapy-induced impaired alveolar development. Thus, AECIIs were exposed to a hyperoxic environment and their cell viability, cell cycle progression, apoptosis, mitochondrial integrity and dynamics, and energy metabolism were assessed. The results showed that hyperoxia has no significant effect as an inhibitor of SMAD3 and ERK1/2 in AECIIs, but leads to significant inhibition of cell viability. Further, hyperoxia was found to promote AECII apoptosis and mitochondrial, whereas chemical inhibition of SMAD3 or ERK1/2 further exacerbated the detrimental effects of hyperoxia in AECIIs. Overall, these findings presented herein demonstrate the critical role of SMAD/ERK signaling in the regulation of AECII behavior in varying oxygen environments. Thus, this study offers novel insights for the prevention of neonatal lung dysfunction in premature infants.

Constitutive transgenic α-Klotho overexpression enhances resilience to and recovery from murine acute lung injury.

Normal lungs do not express α-Klotho (Klotho) protein but derive cytoprotection from circulating soluble Klotho. It is unclear whether chronic supranormal Klotho levels confer additional benefit. To address this, we tested the age-related effects of modest Klotho overexpression on acute lung injury (ALI) and recovery. Transgenic Klotho-overexpressing (Tg-Kl) and wild-type (WT) mice (2 and 6 mo old) were exposed to hyperoxia (95% O2; 72 h; injury; Hx) then returned to normoxia (21% O2; 24 h; recovery; Hx-R). Control mice were kept in normoxia. Renal and serum Klotho, lung histology, and bronchoalveolar lavage fluid oxidative damage markers were assessed. Effects of hyperoxia on Klotho release were tested in human embryonic kidney cells stably expressing Klotho. A549 lung epithelial cells transfected with Klotho cDNA or vector were exposed to cigarette smoke; lactate dehydrogenase and double-strand DNA breaks were measured. Serum Klotho decreased with age. Hyperoxia suppressed renal Klotho at both ages and serum Klotho at 2 mo of age. Tg-Kl mice at both ages and 2-mo-old WT mice survived Hx-R; 6-mo-old Tg-Kl mice showed lower lung damage than age-matched WT mice. Hyperoxia directly inhibited Klotho expression and release in vitro; Klotho transfection attenuated cigarette smoke-induced cytotoxicity and DNA double-strand breaks in lung epithelial cells. Young animals with chronic high baseline Klotho expression were more resistant to ALI. Chronic constitutive Klotho overexpression in older Tg-Kl animals attenuated hyperoxia-induced lung damage and improves survival and short-term recovery despite an acute reduction in serum Klotho during injury. We conclude that chronic enhancement of Klotho expression increases resilience to ALI.

Hyperoxia-induced bronchopulmonary dysplasia: better models for better therapies.

ABSTRACTBronchopulmonary dysplasia (BPD) is a chronic lung disease caused by exposure to high levels of oxygen (hyperoxia) and is the most common complication that affects preterm newborns. At present, there is no cure for BPD. Infants can recover from BPD; however, they will suffer from significant morbidity into adulthood in the form of neurodevelopmental impairment, asthma and emphysematous changes of the lung. The development of hyperoxia-induced lung injury models in small and large animals to test potential treatments for BPD has shown some success, yet a lack of standardization in approaches and methods makes clinical translation difficult. In vitro models have also been developed to investigate the molecular pathways altered during BPD and to address the pitfalls associated with animal models. Preclinical studies have investigated the efficacy of stem cell-based therapies to improve lung morphology after damage. However, variability regarding the type of animal model and duration of hyperoxia to elicit damage exists in the literature. These models should be further developed and standardized, to cover the degree and duration of hyperoxia, type of animal model, and lung injury endpoint, to improve their translational relevance. The purpose of this Review is to highlight concerns associated with current animal models of hyperoxia-induced BPD and to show the potential of in vitro models to complement in vivo studies in the significant improvement to our understanding of BPD pathogenesis and treatment. The status of current stem cell therapies for treatment of BPD is also discussed. We offer suggestions to optimize models and therapeutic modalities for treatment of hyperoxia-induced lung damage in order to advance the standardization of procedures for clinical translation.

Knockdown of miR-203a-3p alleviates the development of bronchopulmonary dysplasia partly via the up-regulation of vascular endothelial growth factor A.

Bronchopulmonary dysplasia (BPD) is characterized by impaired vascular and alveolar development, and the underlying molecular mechanisms have remained elusive. MicroRNAs are important players in various biological functions including the pathogenesis of BPD. The present study aimed to examine the expression of miR-203a-3p in the peripheral blood of BPD patients and elucidate the mechanisms underlying miR-203a-3p-mediated progression of BPD. We examined the expression of miR-203a-3p in the peripheral blood of BPD patients and found that miR-203a-3p was up-regulated in the patients. Additionally, the mRNA expression levels of vascular endothelial growth factor A (VEGFA) and hypoxia-inducible factor-1alpha were down-regulated in the BPD patients. Further in vitro studies showed that miR-203a-3p suppressed the expression of VEGFA in RLE-6TN cells by targeting the VEGFA 3' untranslated region. Overexpression of miR-203a-3p inhibited the viability of RLE-6TN cells and induced cell apoptosis, whereas the knockdown of miR-203a-3p exerted opposite effects. VEGFA treatment significantly attenuated the increase in the RLE-6TN cell apoptotic rates induced by miR-203a-3p overexpression; while VEGFA knockdown significantly increased the cell apoptotic rates of RLE-6TN cells, which was partially reversed by the treatment with miR-203a-3p inhibitor. Furthermore, miR-203a-3p was up-regulated, whereas VEGFA was down-regulated in the lung tissues of BPD rats, and sequestration of the expression of miR-203a-3p prevented hyperoxia-induced lung damage, increased VEGFA mRNA and protein expression levels, and promoted the protein expression of ERK, PI3K, and p38 in the lung tissues of BDP rats. In summary, the findings of our study indicate that miR-203a-3p knockdown alleviates hyperoxia-induced lung tissue damage in the BPD rat model, and its effect may be associated with the up-regulation of VEGF.

Resveratrol alleviates alveolar epithelial cell injury induced by hyperoxia by reducing apoptosis and mitochondrial dysfunction.

Bronchopulmonary dysplasia is a severe and long-term pulmonary disease in premature infants. Hyperoxia-induced acute lung injury plays a critical role in bronchopulmonary dysplasia. Resveratrol is a polyphenolic phytoalexin and a natural agonist of Sirtuin 1. Many studies have shown that resveratrol has a protective effect on hyperoxia-induced lung damage, but its specific protective mechanism is still not clear. Further exploration of the possible protective mechanism of resveratrol was the main goal of this study. In this study, human alveolar epithelial cells were used to establish a hyperoxia-induced acute lung injury cell model, and resveratrol (Res or R), the Sirtuin 1 activator SRT1720 (S) and the Sirtuin 1 inhibitor EX-527 (E) were administered to alveolar epithelial cells, which were then exposed to hyperoxia to investigate the role of Res in mitochondrial function and apoptosis. We divided human alveolar epithelial cells into the following groups: (1) the control group, (2) hyperoxia group, (3) hyperoxia+Res20 group, (4) hyperoxia+Res20+E5 group, (5) hyperoxia+Res20+E10 group, (6) hyperoxia+S2 group, (7) hyperoxia+S2+E5 group, and (8) hyperoxia+S2+E10 group. Hyperoxia-induced cell apoptosis and mitochondrial dysfunction were alleviated by Res and SRT1720. Res and SRT1720 upregulated Sirtuin 1, PGC-1α, NRF1, and TFAM but decreased the expression of acetyl-p53 in human alveolar epithelial cells that were exposed to hyperoxia. These findings revealed that Res may alleviated hyperoxia-induced mitochondrial dysfunction and apoptosis in alveolar epithelial cells through the SIRT1/PGC-1a signaling pathway. Thus, Sirtuin 1 upregulation plays an important role in lung protection.

Inhaled vitamin A is more effective than intramuscular dosing in mitigating hyperoxia-induced lung injury in a neonatal rat model of bronchopulmonary dysplasia.

Prevention of bronchopulmonary dysplasia (BPD) in premature-birth babies continues to be an unmet medical need. Intramuscular vitamin A is currently employed in preterm neonates to prevent BPD but requires intramuscular injections in fragile neonates. We hypothesized that noninvasive inhaled delivery of vitamin A, targeted to lung, would be a more effective and tolerable strategy. We employed our well-established hyperoxia-injury neonatal rat model, exposing newborn rats to 7 days of constant extreme (95% O2) hyperoxia, comparing vitamin A dosed every 48 h via either aerosol inhalation or intramuscular injection with normoxic untreated healthy animals and vehicle-inhalation hyperoxia groups as positive and negative controls, respectively. Separately, similar vitamin A dosing of normoxia-dwelling animals was performed. Analyses after day 7 included characterization of alveolar histomorphology and protein biomarkers of alveolar maturation [surfactant protein C (SP-C), peroxisome proliferator-activated receptor (PPAR) γ, cholinephosphate cytidylyl transferase, vascular endothelial growth factor and its receptor, FLK-1, and retinoid X receptors (RXR-α, -β, and -γ], apoptosis (Bcl2 and Bax) key injury repair pathway data including protein markers (ALK-5 and β-catenin) and neutrophil infiltration, and serum vitamin A levels. Compared with intramuscular dosing, inhaled vitamin A significantly enhanced biomarkers of alveolar maturation, mitigated hyperoxia-induced lung damage, and enhanced surfactant protein levels, suggesting that it may be more efficacious in preventing BPD in extremely premature infants than the traditionally used IM dosing regimen. We speculate lung-targeted inhaled vitamin A may also be an effective therapy against other lung damaging conditions leading to BPD or, more generally, to acute lung injury.

Intermittent CPAP limits hyperoxia-induced lung damage in a rabbit model of bronchopulmonary dysplasia.

A significant proportion of preterm infants develop bronchopulmonary dysplasia (BPD) leading to poor lifelong respiratory health. Limited treatment options exist with continuous positive airway pressure (CPAP) ventilation being one of the few associated with diminished BPD. However, little is known about the effect of the distending pressure of CPAP on the developing lung exposed to hyperoxia. We aimed to identify the functional and structural effects of CPAP in a preterm hyperoxia rabbit model of BPD. Premature rabbit pups were randomized to normoxia, hyperoxia (≥95% O2), or hyperoxia plus 4 h daily CPAP [fraction of inspired oxygen (FiO2) 0.95, 5 cmH2O]. On day 7 postdelivery we performed invasive pressure-volume- and forced oscillation-based pulmonary function tests, before lung harvest for histological evaluation. Alveolar and vascular morphology, airway smooth muscle content, respiratory epithelium height, extracellular matrix components, and inflammatory cytokine expression were quantified. Hyperoxia-reared pups had restrictive lungs: alveolar walls were thickened, with the lung parenchymal tissue, collagen content, and airway smooth muscle content increased. In addition, peripheral pulmonary artery wall thickness was increased. CPAP increased alveolar recruitment and limited the structural effect of hyperoxia on the respiratory epithelium and pulmonary arteries. Additionally, CPAP improved lung function, mitigating hyperoxia-associated changes to respiratory system resistance, tissue damping, and tissue elastance. Hyperoxia disrupted functional and structural lung development. Daily intermittent CPAP limited hyperoxia-associated decreased lung function and attenuated structural changes to pulmonary arteries and respiratory epithelium while having no structural alveolar consequences. The mechanism by which CPAP has these beneficial effects needs further investigation.

34 - The role of myeloperoxidase in hyperoxia-induced bronchopulmonary dysplasia

Bronchopulmonary dysplasia (BPD) is the most common chronic pulmonary disease of premature neonates. While ventilation with supplemental O2 is required for development and survival of the premature neonate, it increases the risk of BPD. Both hyperoxia-induced oxidative stress and transient hypoxia resulting from apneic events increase recruitment and activation of myeloid cells to the lungs. Activated myeloid cells release myeloperoxidase (MPO), which oxidizes chloride ions to hypochlorous acid causing oxidative damage and cell death. If myeloid cell recruitment, activation, and MPO release contribute to the pathogenesis of BPD, then inhibiting MPO oxidant production should protect against hyperoxia-induced lung injury. N-acetyl lysyltyrosylcysteine amide (KYC) is a tripeptide inhibitor of MPO that prevents the generation of toxic oxidants by shuttling high energy peroxy radicals in the iron-heme active site into the glutathione pathway. In order to test the hypothesis that inhibiting MPO prevents hyperoxia-induced lung damage, Sprague-Dawley neonatal rat pups were exposed to ten days (P1-P10) of hyperoxia (>90% O2) and injected twice daily with either 5 mg/kg KYC or vehicle control (PBS). KYC treatment reduced myeloid cell counts and MPO and Cl-Tyr levels in the lungs of hyperoxic neonatal pups. Additionally, KYC-treated pups had increased microvascular and alveolar complexity as well as nuclear factor (erythroid-derived 2)-like 2 (Nrf2) levels. These data provide evidence that myeloid cell recruitment and activation and MPO-induced oxidative damage are significant contributors to hyperoxia-induced lung injury in the developing lungs, and that KYC is an effective inhibitor of MPO-dependent oxidative damage to neonatal lungs.

N-methyl-D-aspartate receptor activation mediates lung fibroblast proliferation and differentiation in hyperoxia-induced chronic lung disease in newborn rats.

BackgroundPrevious studies have suggested that endogenous glutamate and its N-methyl-D-aspartate receptors (NMDARs) play important roles in hyperoxia-induced acute lung injury in newborn rats. We hypothesized that NMDAR activation also participates in the development of chronic lung injury after withdrawal of hyperoxic conditions.MethodsIn order to rule out the anti-inflammatory effects of NMDAR inhibitor on acute lung injury, the efficacy of MK-801 was evaluated in vivo using newborn Sprague-Dawley rats treated starting 4 days after cessation of hyperoxia exposure (on postnatal day 8). The role of NMDAR activation in hyperoxia-induced lung fibroblast proliferation and differentiation was examined in vitro using primary cells derived from the lungs of 8-day-old Sprague-Dawley rats exposed to hyperoxic conditions.ResultsHyperoxia for 3 days induced acute lung injury in newborn rats. The acute injury almost completely disappeared 4 days after cessation of hyperoxia exposure. However, pulmonary fibrosis, impaired alveolarization, and decreased pulmonary compliance were observed on postnatal days 15 and 22. MK-801 treatment during the recovery period was found to alleviate the chronic damage induced by hyperoxia. Four NMDAR 2 s were found to be upregulated in the lung fibroblasts of newborn rats exposed to hyperoxia. In addition, the proliferation and upregulation of alpha-smooth muscle actin and (pro) collagen I in lung fibroblasts were detected in hyperoxia-exposed rats. MK-801 inhibited these changes.ConclusionsNMDAR activation mediated lung fibroblast proliferation and differentiation and played a role in the development of hyperoxia-induced chronic lung damage in newborn rats.

Evaluation of Etanercept Treatment in Newborn Rat Model with Hyperoxic Lung Injury

ABSTRACTBackground: Many factors contribute to the development of BPD basically by increasing inflammation in preterm lungs. However, premature neonates have insufficient anti-inflammatory capacity. We aimed to evaluate the effect of etanercept, an anti-TNF agent, on BPD development in newborn rat model with hyperoxia-induced lung injury. Methods: Thirty-two newborn rats were divided into 3 groups as control group (Group 1, n = 11), hyperoxia + placebo group (Group 2, n = 10), and hyperoxia + etanercept group (Group 3, n = 11). Histopathological and biochemical analysis were performed in order to assess inflammation and oxidative stress. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activities, and malondialdehyde (MDA) levels were studied, histopathological scoring and radial alveolar count were applied in lung tissue. Lamellar body membrane protein, vascular endothelial growth factor (VEGF), nuclear factor-kappaB (NF-κB) gene expressions were studied in immunohistochemical evaluation of tissue samples. All three groups were compared with each other in terms of all parameters. Results: SOD and GSH-Px activities were significantly higher, whereas MDA levels were lower in group 3, compared to group 2 (p < 0.001). Histopathological scores were lower, lamellar body membrane protein expression and radial alveolar count were higher in group 3 (p < 0.05). NF-κB expression was higher in group 2, but lower in group 3 in comparison with group 1. Expression of VEGF was decreased in group 2 but came close to group 1 with etanercept treatment in group 3. Conclusions: We found etanercept treatment to be protective in newborn rats with hyperoxia-induced lung damage.

Mesenchymal stem cells in combination with erythropoietin repair hyperoxia-induced alveoli dysplasia injury in neonatal mice via inhibition of TGF-β1 signaling.

The aim of the present study is to investigate the protection effects of bone marrow mesenchymal stem cells (MSCs) in combination with EPO against hyperoxia-induced bronchopulmonary dysplasia (BPD) injury in neonatal mice. BPD model was prepared by continuous high oxygen exposure, 1×106 bone marrow MSCs and 5000U/kg recombinant human erythropoietin (EPO) were injected respectively. Results showed that administration of MSCs, EPO especially MSCs+EPO significant attenuated hyperoxia-induced lung damage with a decrease of fibrosis, radical alveolar counts and inhibition of the occurrence of epithelial-mesenchymal transition (EMT). Furthermore, MSCs+EPO co-treatment more significantly suppressed the levels of transforming growth factor-β1(TGF-β1) than MSCs or EPO alone. Collectively, these results suggested that MSCs, EPO in particular MSCs+EPO co-treatment could promote lung repair in hyperoxia-induced alveoli dysplasia injury via inhibition of TGF-β1 signaling pathway to further suppress EMT process and may be a promising therapeutic strategy.

Intranasal administration with recombinant Lactococcus lactis expressing heme oxygenase-1 reduces hyperoxia-induced lung inflammation in rat pups.

To evaluate the effects of a recombinant strain of Lactococcus lactis secreting bioactive heme oxygenase-1 (LL-HO-1) in a model of hyperoxia-induced lung injury in rat pups Intranasal administration with LL-HO-1 significantly reduced hyperoxia-induced lung injury as demonstrated by a decreased wet/dry ratio, myeloperoxidase activity in lung tissue, tumor necrosis factor alpha levels in bronchoalveolar lavage (BAL) fluid, and attenuated lung injury scores. Although expression of HO-1 and NADPH oxidase-1 and production of superoxide in lung tissue were not affected by LL-HO-1 treatment, HO-1 levels in nasal mucosa and interleukin-10 concentrations in BAL fluid significantly increased compared with the hyperoxia control group Intranasal administration of LL-HO-1 protects against hyperoxia-induced lung damage, apparently through attenuation of inflammation.

Cytidine 5′-diphosphocholine ameliorates hyperoxic lung injury in a neonatal rat model

Bronchopulmonary dysplasia (BPD) is an important cause of morbidity. The aim of this study was to evaluate the preventive effect of cytidine 5'-diphosphocholine (CDP-choline) treatment on hyperoxic lung injury in a neonatal rat model. A total of 30 newborn pups were divided into control, hyperoxia, and hyperoxia + CDP-choline groups. After birth, pups in the control group were kept in room air and received saline injections, whereas those in hyperoxia and hyperoxia + CDP-choline groups were exposed to 95% O₂ and received daily injections of saline and CDP-choline throughout postnatal day 10, respectively. Histopathological scoring, radial alveolar count, lamellar body membrane protein expression, fibrosis, proinflammatory cytokine levels, lung tissue and bronchoalveolar lavage (BAL) fluid phospholipid content, and apoptosis were evaluated. Hyperoxia-induced severe lung damage was reduced significantly by CDP-choline treatment. Radial alveolar count and lamellar body membrane protein expression were significantly recovered, and the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling-positive cells, active caspase-3 expression, and tissue proinflammatory cytokine levels were decreased by CDP-choline administration. Lung tissue and BAL phospholipid contents showed significant increases after CDP-choline administration. These data show that CDP-choline ameliorates hyperoxic lung injury in a neonatal rat model. It may therefore be suggested that CDP-choline may be a novel therapeutic option for the prevention of BPD.

Fluoxetine may worsen hyperoxia-induced lung damage in neonatal rats.

Fluoxetine shows controversial lung effects as it prevents pulmonary hypertension in adult rats but exposure during gestation causes pulmonary hypertension in neonatal rats. In the present study, we tested the null hypothesis that the antidepressant drug fluoxetine does not modify the development of bronchopulmonary dysplasia (BPD) in neonatal rats. Experimental categories included I: room air (controls) with daily injection of saline; II: room air with daily injection of 10 mg/kg fluoxetine, i.p., during two weeks; III: 60% oxygen with daily injection of saline; and IV: 60% oxygen with daily injection of 10 mg/kg fluoxetine, i.p., during two weeks. Hyperoxia resulted in significant reduction in alveolar density and an increase in pulmonary endocrine cells, as well as increases in muscle layer areas of bronchi and arteries. Fluoxetine treatment generated a further increase in muscularisation and did not significantly modify the hyperoxia-induced reductions in alveolar density and increases in the endocrine cells. In hyperoxia, Real-Time PCR showed a lower pulmonary expression of vascular endothelial growth factor (VEGF) with no significant changes in the expression of matrix metalloproteinases (MMP) 2 and 12. Fluoxetine did not affect VEGF or MMP-2 expression but it significantly increased MMP-12 mRNA in both normoxic and hyperoxic groups. Zymographic analysis of MMP-2 activity in bronchoalveolar fluid showed a significantly reduced MMP-2 activity in hyperoxia, while fluoxetine treatment restored MMP-2 activity to levels comparable with the normoxic group. In conclusion, our data show that fluoxetine may worsen bronchial and arterial muscularisation during development of BPD and may up-regulate MMP expression or activity.

Calcitonin Gene-Related Peptide Ameliorates Hyperoxia-Induced Lung Injury in Neonatal Rats

Therapies with prolonged exposure to high-concentration oxygen are common in the treatment of critical pulmonary and cardiac conditions in newborns. However, prolonged exposure to hyperoxia could result in lung damages and developmental disorders manifested as acute lung injury and bronchopulmonary dysplasia, respectively. Calcitonin gene-related peptide (CGRP) has been shown to have a broad regulatory effect on the respiratory system. In this study, we explored the protective effects of CGRP on the hyperoxia-induced lung damage. Newborn Sprague-Dawley rats were randomly divided into three groups: normoxia, hyperoxia, and hyperoxia with CGRP. Hyperoxia groups were exposed to 95% oxygen for 14 days and treated once every other day with saline or CGRP. Hyperoxia exposure reduced the survival rate to 73%, when compared with the 93% survival rate observed in the normoxia group. The survival rate was improved to 84% with CGRP treatment. Treatment with CGRP under hyperoxia significantly alleviated the hyperoxia-induced lung histomorphological changes and the increases in leukocyte counts and total protein levels in bronchoalveolar lavage fluid that reflect the pulmonary microvasular damages. CGRP treatment also restored the decreased activity of superoxide dismutase, while it decreased the increased level of malondialdehyde in the lung tissues. Importantly, CGRP treatment significantly decreased the magnitude of the hyperoxia-mediated increase in the expression levels of tumor necrosis factor-α mRNA and transforming growth factor-β 1 protein. In conclusion, the hyperoxia-induced acute lung injury is associated with both oxidative stress and inflammatory responses, and CGRP may ameliorate the hyperoxia-induced lung injury by down-regulating these processes.

Adiponectin Protects Against Hyperoxic Lung Injury and Vascular Leak

Adiponectin (Ad), an adipokine exclusively secreted by the adipose tissue, has emerged as a paracrine metabolic regulator as well as a protectant against oxidative stress. Pharmacological approaches of protecting against clinical hyperoxic lung injury during oxygen therapy/treatment are limited. We have previously reported that Ad inhibits the NADPH oxidase-catalyzed formation of superoxide from molecular oxygen in human neutrophils. Based on this premise, we conducted studies to determine whether (i) exogenous Ad would protect against the hyperoxia-induced barrier dysfunction in the lung endothelial cells (ECs) in vitro, and (ii) endogenously synthesized Ad would protect against hyperoxic lung injury in wild-type (WT) and Ad-overexpressing transgenic (AdTg) mice in vivo. The results demonstrated that exogenous Ad protected against the hyperoxia-induced oxidative stress, loss of glutathione (GSH), cytoskeletal reorganization, barrier dysfunction, and leak in the lung ECs in vitro. Furthermore, the hyperoxia-induced lung injury, vascular leak, and lipid peroxidation were significantly attenuated in AdTg mice in vivo. Also, AdTg mice exhibited elevated levels of total thiols and GSH in the lungs as compared with WT mice. For the first time, our studies demonstrated that Ad protected against the hyperoxia-induced lung damage apparently through attenuation of oxidative stress and modulation of thiol-redox status.

Glutamate Mediates Hyperoxia-Induced Newborn Rat Lung Injury through N-Methyl-D-Aspartate Receptors

Our laboratory found that the N-methyl-D-aspartate receptor (NMDAR) antagonist, MK-801, was able to decrease hyperoxia-induced lung damage. To further search for direct evidence of glutamate and its NMDARs participating in hyperoxia-induced lung injury, the amount of glutamate in the bronchoalveolar lavage fluid and the expression of NMDAR 2D in lung tissue were tracked in newborn rats that were exposed to 95% oxygen for 1, 3, and 7 days. The protective effect of MK-801 was then observed at different hyperoxia exposure times. As demonstrated by RT-PCR, NMDAR 2D expression was much higher in hyperoxia exposure on the third and the seventh days than in the air control group. The levels of glutamate in the bronchoalveolar lavage fluid on the first and third days of hyperoxia exposure were significantly higher than in the air control group. MK-801 alleviated lung injury and inflammatory reaction induced by 95% O(2) for 3 and 7 days. These results indicate that large amounts of endogenous glutamate from the lungs were released, and its NMDAR were expressed strongly under conditions of high oxygen concentration. We conclude that the endogenous glutamate mediated newborn rat lung damage induced by hyperoxia through NMDARs.