Intranasal administration of Lactobacillus johnsonii attenuates hyperoxia-induced lung injury by modulating gut microbiota in neonatal mice.

BACKGROUND: Supplemental oxygen impairs lung development in newborn infants with respiratory distress. Lactobacillus johnsonii supplementation attenuates respiratory viral infection in mice and exhibits anti-inflammatory effects. This study investigated the protective effects of intranasal administration of L. johnsonii on lung development in hyperoxia-exposed neonatal mice. METHODS: Neonatal C57BL/6N mice were reared in either room air (RA) or hyperoxia condition (85% O2). From postnatal days 0 to 6, they were administered intranasal 10 µL L. johnsonii at a dose of 1 × 105 colony-forming units. Control mice received an equal volume of normal saline (NS). We evaluated the following four study groups: RA + NS, RA + probiotic, O2 + NS, and O2 + probiotic. On postnatal day 7, lung and intestinal microbiota were sampled from the left lung and lower gastrointestinal tract, respectively. The right lung of each mouse was harvested for Western blot, cytokine, and histology analyses. RESULTS: The O2 + NS group exhibited significantly lower body weight and vascular density and significantly higher mean linear intercept (MLI) and lung cytokine levels compared with the RA + NS and RA + probiotic groups. At the genus level of the gut microbiota, the O2 + NS group exhibited significantly higher Staphylococcus and Enterobacter abundance and significantly lower Lactobacillus abundance compared with the RA + NS and RA + probiotic groups. Intranasal L. johnsonii treatment increased the vascular density, decreased the MLI and cytokine levels, and restored the gut microbiota in hyperoxia-exposed neonatal mice. CONCLUSIONS: Intranasal administration of L. johnsonii protects against hyperoxia-induced lung injury and modulates the gut microbiota.

Uteroplacental insufficiency decreases leptin expression and impairs lung development in growth-restricted newborn rats.

BACKGROUND: The study aimed to analyze the effect of uteroplacental insufficiency (UPI) on leptin expression and lung development of intrauterine growth restriction (IUGR) rats. METHODS: On day 17 of pregnancy, time-dated Sprague-Dawley rats were randomly divided into either an IUGR group or a control group. Uteroplacental insufficiency surgery (IUGR) and sham surgery (control) were conducted. Offspring rats were spontaneously delivered on day 22 of pregnancy. On postnatal days 0 and 7, rats' pups were selected at random from the control and IUGR groups. Blood was withdrawn from the heart to determine leptin levels. The right lung was obtained for leptin and leptin receptor levels, immunohistochemistry, proliferating cell nuclear antigen (PCNA), western blot, and metabolomic analyses. RESULTS: UPI-induced IUGR decreased leptin expression and impaired lung development, causing decreased surface area and volume in offspring. This results in lower body weight, decreased serum leptin levels, lung leptin and leptin receptor levels, alveolar space, PCNA, and increased alveolar wall volume fraction in IUGR offspring rats. The IUGR group found significant relationships between serum leptin, radial alveolar count, von Willebrand Factor, and metabolites. CONCLUSION: Leptin may contribute to UPI-induced lung development during the postnatal period, suggesting supplementation as a potential treatment. IMPACT: The neonatal rats with intrauterine growth restriction (IUGR) caused by uteroplacental insufficiency (UPI) showed decreased leptin expression and impaired lung development. UPI-induced IUGR significantly decreased surface area and volume in lung offspring. This is a novel study that investigates leptin expression and lung development in neonatal rats with IUGR caused by UPI. If our findings translate to IUGR infants, leptin may contribute to UPI-induced lung development during the postnatal period, suggesting supplementation as a potential treatment.

Molecular Mechanisms of Hyperoxia-Induced Neonatal Intestinal Injury.

Oxygen therapy is important for newborns. However, hyperoxia can cause intestinal inflammation and injury. Hyperoxia-induced oxidative stress is mediated by multiple molecular factors and leads to intestinal damage. Histological changes include ileal mucosal thickness, intestinal barrier damage, and fewer Paneth cells, goblet cells, and villi, effects which decrease the protection from pathogens and increase the risk of necrotizing enterocolitis (NEC). It also causes vascular changes with microbiota influence. Hyperoxia-induced intestinal injuries are influenced by several molecular factors, including excessive nitric oxide, the nuclear factor-κB (NF-κB) pathway, reactive oxygen species, toll-like receptor-4, CXC motif ligand-1, and interleukin-6. Nuclear factor erythroid 2-related factor 2 (Nrf2) pathways and some antioxidant cytokines or molecules including interleukin-17D, n-acetylcysteine, arginyl-glutamine, deoxyribonucleic acid, cathelicidin, and health microbiota play a role in preventing cell apoptosis and tissue inflammation from oxidative stress. NF-κB and Nrf2 pathways are essential to maintain the balance of oxidative stress and antioxidants and prevent cell apoptosis and tissue inflammation. Intestinal inflammation can lead to intestinal damage and death of the intestinal tissue, such as in NEC. This review focuses on histologic changes and molecular pathways of hyperoxia-induced intestinal injuries to establish a framework for potential interventions.

Tumoricidal Activity of Simvastatin in Synergy with RhoA Inactivation in Antimigration of Clear Cell Renal Cell Carcinoma Cells.

Among kidney cancers, clear cell renal cell carcinoma (ccRCC) has the highest incidence rate in adults. The survival rate of patients diagnosed as having metastatic ccRCC drastically declines even with intensive treatment. We examined the efficacy of simvastatin, a lipid-lowering drug with reduced mevalonate synthesis, in ccRCC treatment. Simvastatin was found to reduce cell viability and increase autophagy induction and apoptosis. In addition, it reduced cell metastasis and lipid accumulation, the target proteins of which can be reversed through mevalonate supplementation. Moreover, simvastatin suppressed cholesterol synthesis and protein prenylation that is essential for RhoA activation. Simvastatin might also reduce cancer metastasis by suppressing the RhoA pathway. A gene set enrichment analysis (GSEA) of the human ccRCC GSE53757 data set revealed that the RhoA and lipogenesis pathways are activated. In simvastatin-treated ccRCC cells, although RhoA was upregulated, it was mainly restrained in the cytosolic fraction and concomitantly reduced Rho-associated protein kinase activity. RhoA upregulation might be a negative feedback effect owing to the loss of RhoA activity caused by simvastatin, which can be restored by mevalonate. RhoA inactivation by simvastatin was correlated with decreased cell metastasis in the transwell assay, which was mimicked in dominantly negative RhoA-overexpressing cells. Thus, owing to the increased RhoA activation and cell metastasis in the human ccRCC dataset analysis, simvastatin-mediated Rho inactivation might serve as a therapeutic target for ccRCC patients. Altogether, simvastatin suppressed the cell viability and metastasis of ccRCC cells; thus, it is a potentially effective ccRCC adjunct therapy after clinical validation for ccRCC treatment.

Inhibition of FABP4 attenuates hyperoxia-induced lung injury and fibrosis via inhibiting TGF-ß signaling in neonatal rats.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease characterized by interrupted alveologenesis and alveolar simplification caused by oxygen therapy in premature infants. Metabolic dysfunction is involved in the pathogenesis of BPD. Fatty acid-binding protein 4 (FABP4) is significantly increased in specific lung tissues in patients with BPD. Therefore, we investigated whether BMS309403, an FABP4 inhibitor that can mitigate tissue fibrosis, can regulate pulmonary fibrotic processes in newborn rats exposed to hyperoxia. Newborn rat pups were exposed to room air (RA; 21% O2 ) or 85% O2 from 5 to 14 days of age and were then allowed to recover in RA until 29 days of age. They received intraperitoneal injection with placebo (phosphate-buffered saline [PBS]) or BMS 309403 (0.5 mg or 1.0 mg kg-1 d-1 ) every other day from 4 to 14 days of age then were divided into O2 plus PBS or low dose or high dose and RA plus PBS or low dose or high dose groups. We assessed lung histology and evaluated lung collagen I, FABP4 as well as TGF-ß1 expression at 14 and 29 days of age. In the hyperoxia injury-recovery model, prophylactic BMS309403 treatment reduced mean linear intercept values and FABP4 expression (p < 0.001). Prophylactic BMS309403 treatment mitigated pulmonary fibrosis and TGF-ß1 expression immediately after hyperoxia exposure (p < 0.05). The attenuation of hyperoxia-induced alveolar developmental impairment and pulmonary fibrosis by FABP4 inhibition indicated that such inhibition has potential clinical and therapeutic applications.

Immunization with anti-Tn immunogen in maternal rats protects against hyperoxia-induced kidney injury in newborn offspring.

BACKGROUND: Neonatal hyperoxia increases oxidative stress and adversely disturbs glomerular and tubular maturity. Maternal Tn immunization induces anti-Tn antibody titer and attenuates hyperoxia-induced lung injury in neonatal rats. METHODS: We intraperitoneally immunized female Sprague-Dawley rats (6 weeks old) with Tn immunogen (50 µg/dose) or carrier protein five times at biweekly intervals on 8, 6, 4, 2, and 0 weeks before the delivery day. The pups were reared for 2 weeks in either room air (RA) or in 85% oxygen-enriched atmosphere (O2), thus generating four study groups, namely carrier protein + RA, Tn vaccine + RA, carrier protein + O2, and Tn vaccine + O2. On postnatal day 14, the kidneys were harvested for the oxidative stress marker 8-hydroxy-2'-deoxyguanosine (8-OHdG), nuclear factor-κB (NF-κB), and collagen expression and histological analyses. RESULTS: Hyperoxia reduced body weight, induced tubular and glomerular injuries, and increased 8-OHdG and NF-κB expression and collagen deposition in the kidneys. By contrast, maternal Tn immunization reduced kidney injury and collagen deposition in neonatal rats. Furthermore, kidney injury attenuation was accompanied by a reduction in 8-OHdG and NF-κB expression. CONCLUSION: Maternal Tn immunization protects against hyperoxia-induced kidney injury in neonatal rats by attenuating oxidative stress and NF-κB activity. IMPACT: Hyperoxia increased nuclear factor-κB (NF-κB) activity and collagen deposition in neonatal rat kidney. Maternal Tn immunization reduced kidney injury as well as collagen deposition in neonatal rats. Maternal Tn immunization reduced kidney injury and was associated with a reduction in 8-hydroxy-2'-deoxyguanosine and NF-κB activity. Tn vaccine can be a promising treatment modality against hyperoxia-induced kidney injury in neonates.

Maternal antibiotic exposure disrupts microbiota and exacerbates hyperoxia-induced lung injury in neonatal mice.

BACKGROUND: Perinatal antibiotic treatment alters intestinal microbiota and augments hyperoxia-induced lung injury in mice offspring. The effect of maternal antibiotic treatment (MAT) during pregnancy on the lung microbiota and its relationship with lung injury remains unknown. METHODS: We fed timed-pregnant C57BL/6N mice sterile drinking water containing antibiotics from gestational day 15 to delivery. Neonatal mice were reared in either room air (RA) or hyperoxia (85% O2) from postnatal days 1 to 7. Four study groups were obtained: control + RA, control + O2, MAT + RA, and MAT + O2. On postnatal day 7, lung and intestinal microbiota were sampled from the left lung and lower gastrointestinal tract. The right lung was harvested for histology and cytokine analysis. RESULTS: MAT during pregnancy significantly reduced the total number of commensal bacteria in the intestine and birth body weight of newborn mice compared with control newborn mice. Neonatal hyperoxia exposure impaired alveolarization and angiogenesis, which was exacerbated by MAT. Neonatal hyperoxia altered the composition and diversity of intestinal and lung microbiota and MAT further exacerbated neonatal hyperoxia-induced intestinal and lung dysbiosis. CONCLUSIONS: MAT during pregnancy exacerbates hyperoxia-induced lung injury probably through the modulation of intestinal and lung microbiota in neonatal mice. IMPACT: MAT during pregnancy reduced the total number of commensal bacteria in the intestine. Neonatal hyperoxia altered the composition and diversity of intestinal and lung microbiota. MAT exacerbated neonatal hyperoxia-induced intestinal and lung dysbiosis. Neonatal hyperoxia exposure impaired alveolarization and angiogenesis, which was exacerbated by MAT. Avoiding and carefully using antibiotics during pregnancy is a potential therapeutic target for preventing lung injury in hyperoxia-exposed infants.

Prolonged exposure to traffic-related particulate matter and gaseous pollutants implicate distinct molecular mechanisms of lung injury in rats.

BACKGROUND: Exposure to air pollution exerts direct effects on respiratory organs; however, molecular alterations underlying air pollution-induced pulmonary injury remain unclear. In this study, we investigated the effect of air pollution on the lung tissues of Sprague-Dawley rats with whole-body exposure to traffic-related PM1 (particulate matter < 1 µm in aerodynamic diameter) pollutants and compared it with that in rats exposed to high-efficiency particulate air-filtered gaseous pollutants and clean air controls for 3 and 6 months. Lung function and histological examinations were performed along with quantitative proteomics analysis and functional validation. RESULTS: Rats in the 6-month PM1-exposed group exhibited a significant decline in lung function, as determined by decreased FEF25-75% and FEV20/FVC; however, histological analysis revealed earlier lung damage, as evidenced by increased congestion and macrophage infiltration in 3-month PM1-exposed rat lungs. The lung tissue proteomics analysis identified 2673 proteins that highlighted the differential dysregulation of proteins involved in oxidative stress, cellular metabolism, calcium signalling, inflammatory responses, and actin dynamics under exposures to PM1 and gaseous pollutants. The presence of PM1 specifically enhanced oxidative stress and inflammatory reactions under subchronic exposure to traffic-related PM1 and suppressed glucose metabolism and actin cytoskeleton signalling. These factors might lead to repair failure and thus to lung function decline after chronic exposure to traffic-related PM1. A detailed pathogenic mechanism was proposed to depict temporal and dynamic molecular regulations associated with PM1- and gaseous pollutants-induced lung injury. CONCLUSION: This study explored several potential molecular features associated with early lung damage in response to traffic-related air pollution, which might be used to screen individuals more susceptible to air pollution.

Anti-Tn Monoclonal Antibody Ameliorates Hyperoxia-Induced Kidney Injury by Suppressing Oxidative Stress and Inflammation in Neonatal Mice.

The Tn antigen, an N-acetylgalactosamine structure linked to serine or threonine, has been shown to induce high-specificity, high-affinity anti-Tn antibodies in mice. Maternal immunization with the Tn vaccine increases serum anti-Tn antibody titers and attenuates hyperoxia-induced kidney injury in neonatal rats. However, immunizing mothers to treat neonatal kidney disease is clinically impractical. This study is aimed at determining whether anti-Tn monoclonal antibody treatment ameliorates hyperoxia-induced kidney injury in neonatal mice. Newborn BALB/c mice were exposed to room air (RA) or normobaric hyperoxia (85% O2) for 1 week. On postnatal days 2, 4, and 6, the mice were injected intraperitoneally with PBS alone or with anti-Tn monoclonal antibodies at 25 µg/g body weight in 50 µL phosphate-buffered saline (PBS). The mice were divided into four study groups: RA + PBS, RA + anti-Tn monoclonal antibody, O2 + PBS, and O2 + anti-Tn monoclonal antibody. The kidneys were excised for histology, oxidative stress, cytokine, and Western blot analyses on postnatal day 7. The O2 + PBS mice exhibited significantly higher kidney injury scores, 8-hydroxy-2'-deoxyguanosine (8-OHdG) and nuclear factor-κB (NF-κB) expression, and cytokine levels than did the RA + PBS mice or RA + anti-Tn mice. Anti-Tn monoclonal antibody treatment reduced kidney injury and cytokine levels to normoxic levels. The attenuation of kidney injury was accompanied by a reduction of oxidative stress and NF-κB expression. Therefore, we propose that anti-Tn monoclonal antibody treatment ameliorates hyperoxia-induced kidney injury by suppressing oxidative stress and inflammation in neonatal mice.

Cathelicidin attenuates hyperoxia-induced intestinal injury through inhibition of NF-κB activity in newborn rats.

Supplemental oxygen is often used to treat neonates with respiratory disorders. Preclinical studies have demonstrated that neonatal hyperoxia injures the distal small intestine and activates nuclear factor-κB (NF-κB). Cathelicidin inhibits NF-κB activity and ameliorates lipopolysaccharide-induced intestinal barrier disruption in rats. Sprague-Dawley rat pups were reared in either room air (RA) or hyperoxia (85% O2) and were randomly treated with low-dose cathelicidin (4 mg/kg, LDC) and high-dose cathelicidin (HDC, 8 mg/kg) in 0.05 mL of normal saline (NS) administered intraperitoneally on postnatal days 1-6. The following six groups were obtained: RA + NS, RA + LDC, RA + HDC, O2 + NS, O2 + LDC, and O2 + HDC. The animals were sacrificed and the terminal ileum was removed for Western blot and histological analyses on postnatal day 7. The hyperoxia-reared rats exhibited significantly lower body weights, higher intestinal injury scores, lower occludin and ZO-1 expression, higher intestinal permeability and inducible IκB kinase inhibitor (IKKi) and NF-κB expression than the RA-reared rats. Cathelicidin treatment attenuated intestinal injury as evidenced by lower intestinal injury scores and intestinal permeability and higher intestinal barrier protein expression. The decrease in intestinal injury was accompanied by a decrease in IKKi and NF-κB. Cathelicidin attenuated hyperoxia-induced intestinal injury in the newborn rats, likely through NF-κB activity inhibition.

Maternal inflammation exacerbates neonatal hyperoxia-induced kidney injury in rat offspring.

BACKGROUND: Preclinical studies have demonstrated that maternal inflammation or neonatal hyperoxia adversely affects kidney maturation. This study explored whether prenatal lipopolysaccharide (LPS) exposure can augment neonatal hyperoxia-induced kidney injury. METHODS: Pregnant Sprague-Dawley rats received intraperitoneal injections of LPS (0.5 mg/kg) in normal saline (NS) or NS on 20 and 21 days of gestation. The pups were reared in room air (RA) or 2 weeks of 85% O2, creating the four study groups, NS + RA, NS + O2, LPS + RA, and LPS + O2. Kidneys were taken for oxidase stress and histological analyses. RESULTS: The rats exposed to maternal LPS or neonatal hyperoxia exhibited significantly higher kidney injury score, lower glomerular number, higher toll-like receptor 4 (TLR4), myeloperoxidase (MPO), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) expressions, and higher MPO activity compared with the rats exposed to maternal NS and neonatal RA. The rats exposed to both maternal LPS and neonatal hyperoxia exhibited significantly lower glomerular number, higher kidney injury score, TLR4, MPO, and 8-OHdG expressions compared with the rats exposed to maternal LPS or neonatal hyperoxia. CONCLUSION: Maternal inflammation exacerbates neonatal hyperoxia-induced kidney injury and the underlying mechanism may be related to activation of TLR4 and increased oxidative stress.

Anti-thymic stromal lymphopoietin antibody suppresses airway remodeling in asthma through reduction of MMP and CTGF.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) mediates immune reaction in patients with asthma. Matrix metalloproteinase (MMP), connective tissue growth factor (CTGF), and transforming growth factor-ß (TGF-ß) are inflammatory mediators whose responses to the anti-TSLP antibody are unknown. This study examined the effect of an anti-TSLP antibody on MMP, CTGF, TGF-ß, and airway structural changes in airway remodeling in asthma. METHODS: Mice were randomly divided into phosphate-buffered-saline-challenged (PBS), ovalbumin-challenged (OVA), and ovalbumin-challenged with anti-TSLP antibody (OVA + anti-TSLP) groups. Airway responsiveness and serum ovalbumin-specific immunoglobulin E were measured. Differential cell counts and MMP-2 and MMP-9 were evaluated in bronchoalveolar lavage fluid (BALF). Airway structural changes were quantified using morphometric analysis and presentation by immunohistochemistry staining. Lung CTGF, TGF-ß, and TSLP were analyzed using western blot. RESULTS: Airway responsiveness was significantly lower in OVA + anti-TSLP and PBS groups than in OVA group. Airway structural changes exhibited less smooth muscle thickness in OVA + anti-TSLP and PBS groups than in OVA group. MMP-2 and MMP-9 in BALF and CTGF, TGF-ß, and TSLP in lungs significantly decreased in OVA + anti-TSLP and PBS groups compared with OVA group. CONCLUSION: Anti-TSLP antibody exerts the preventive effect of decreasing airway structural changes through reduction of MMP, TGF-ß, and CTGF in airway remodeling of asthma.

Cathelicidin attenuates hyperoxia-induced kidney injury in newborn rats.

Aim: Supplemental oxygen is often used to treat neonates with respiratory disorders. Human and animal studies have demonstrated that neonatal hyperoxia increases oxidative stress and induces damage and collagen deposition in kidney during the perinatal period. Cathelicidin LL-37 is one important group of human antimicrobial peptides which exhibits antioxidant activity and its overexpression resists hyperoxia-induced oxidative stress. This study was designed to evaluate the protective effects of cathelicidin in hyperoxia-induced kidney injury in newborn rats. Methods: Sprague-Dawley rat pups were reared in either room air (RA) or hyperoxia (85% O2) and were randomly treated with low-dose (4 mg/kg) and high-dose (8 mg/kg) cathelicidin in normal saline (NS) administered intraperitoneally on postnatal days 1-6. The following six groups were obtained: RA + NS, RA + low-dose cathelicidin, RA + high-dose cathelicidin, O2 + NS, O2 + low-dose cathelicidin, and O2 + high-dose cathelicidin. Kidneys were taken for Western blot and histological analyses on postnatal day 7. Results: The hyperoxia-reared rats exhibited significantly lower body weights and anti-inflammatory M2 macrophages, but the kidney injury scores, oxidative stress marker 8-hydroxy-2'-deoxyguanosine (8-OHdG)-positive cells, pro-inflammatory M1 macrophages, collagen deposition, and NF-κB expression were higher than did the RA-reared rats. Conclusions: Cathelicidin treatment attenuated kidney injury as evidenced by lower kidney injury scores, 8-OHdG-positive cells, collagen deposition, and reversion of hyperoxia-induced M1/M2 macrophage polarization. The role of Cathelicidin in ameliorates kidney injury of the hyperoxia newborn rats was accompanied by decreased NF-κB expression, which probably through the modulating NF-κB activity in the kidney.

3-Methylcholanthrene/Aryl-Hydrocarbon Receptor-Mediated Hypertension Through eNOS Inactivation.

Endothelial nitric oxide synthase (eNOS) modulates vascular blood pressure and is predominantly expressed in endothelial cells and activated through the protein kinase B (Akt/PKB)-dependent pathway. We previously reported that 3-methylcholanthrene (3MC) activates the aryl hydrocarbon receptor (AhR) and reduces PI3K/Akt phosphorylation. This study investigated the mechanism underlying the downregulatory effects of 3-MC on nitric oxide (NO) production occurring through the AhR/RhoA/Akt-mediated mechanism. The mechanism underlying the effects of 3-MC on eNOS activity and blood pressure was examined in vitro and in vivo through genetic and pharmacological approaches. Results indicated that 3-MC modified heat shock protein 90 (HSP90), caveolin-1, dynein, and eNOS mRNA and protein expression through the AhR/RhoA-dependent mechanism in mouse cerebral vascular endothelial cells (MCVECs) and that 3-MC reduced eNOS phosphorylation through the AhR/RhoA-mediated inactivation of Akt1. The upregulation of dynein expression was associated with decreased eNOS dimer formation (eNOS dimer; an activated form of the enzyme). Coimmunoprecipitation assay results indicated that 3-MC significantly reduced the interaction between eNOS and its regulatory proteins, including Akt1 and HSP90, but increased the interaction between eNOS and caveolin-1. Immunofluorescence and Western blot analysis revealed that 3-MC reduced the amount of membrane-bound activated eNOS, and a modified Griess assay revealed that 3-MC concomitantly reduced NO production. However, simvastatin reduced 3-MC-mediated murine hypertension. Our study results indicate that AhR, RhoA, and eNOS have major roles in blood pressure regulation. Statin intervention might provide a potential therapeutic approach for reducing hypertension caused by 3-MC. J. Cell. Physiol. 232: 1020-1029, 2017. © 2016 Wiley Periodicals, Inc.

Neonatal hyperoxia disrupts the intestinal barrier and impairs intestinal function in rats.

Animal studies have demonstrated that neonatal hyperoxia injures the distal small intestine and disrupts the intestinal barrier. This study evaluated the effects of brief hyperoxia exposure on intestinal function in newborn rats. Newborn Sprague-Dawley rat pups were exposed to room air or normobaric hyperoxia (85% O2) for 1week. The rats were euthanized on Postnatal Day 7, and their terminal ilea and sera were collected for histological analyses and intestinal permeability measurements, respectively. Bacterial translocation to the liver and spleen under aerobic and anaerobic conditions was determined. The expression and localization of epithelial injury markers [intestinal fatty acid binding protein (I-FABP)], intestinal barrier proteins [occludin and zonula occludens (ZO)-1], and inflammation biomarkers [Toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB)] were analyzed through immunofluorescence staining. The body weight at birth was comparable between the two groups. On Postnatal Day 7, the rats in the hyperoxic group exhibited significantly lower body weights, higher intestinal injury scores, lower numbers of goblet cells, higher I-FABP expression, lower occludin and ZO-1 expression, higher TLR4 and NF-κB expression, and higher intestinal permeability and bacterial translocation than did those in the room air group. The rats reared in O2-enriched air displayed indistinct tight junction with widening of the paracellular spaces. Hyperoxia exposure injured the distal small intestine, disrupted the intestinal barrier, and impaired intestinal function in newborn rats. Hyperoxia-induced intestinal injury may be attributable to increased activity of the TLR4/NF-κB pathway during the postnatal period.

Genome-Wide Analysis of DNA Methylation in Hyperoxia-Exposed Newborn Rat Lung.

PURPOSE: Oxygen therapy is often required to treat newborn infants with respiratory disorders. Prolonged exposure of neonatal rats to hyperoxia reduced alveolar septation, increased terminal air space size, and increased lung fibrosis; these conditions are very similar to those of human bronchopulmonary dysplasia. Epigenetic regulation of gene expression plays a crucial role in bronchopulmonary dysplasia development. METHOD: We reared Sprague-Dawley rat pups in either room air (RA, n = 24) or an atmosphere containing 85% O2 (n = 26) from Postnatal Days 1 to 14. Methylated DNA immunoprecipitation (MeDIP) was used to analyze genome-wide DNA methylation in lung tissues of neonatal rats. Hyperoxia-exposed rats exhibited larger air spaces and thinner septa than RA-exposed rats did on Postnatal Day 14. The rats exposed to hyperoxia exhibited significantly higher mean linear intercepts than did the rats exposed to RA. We applied MeDIP next-generation sequencing for profiling changes in DNA methylation in the rat lungs exposed to hyperoxia and RA. We performed bioinformatics and pathway analyses on the raw sequencing data to identify differentially methylated candidate genes. RESULTS: Our in vivo model revealed that neonatal hyperoxia exposure arrested alveolarization on Postnatal Day 14. We found that the ErbB, actin cytoskeleton, and focal adhesion signaling pathways are epigenetically modulated by exposure to hyperoxia. We demonstrated that hyperoxia exposure contribute in delaying lung development through an epigenetic mechanism by disrupting the expression of genes in lungs that might be involved in alveolarization. CONCLUSIONS: These data indicate that aberrant DNA methylation and deregulation of the actin cytoskeleton and focal adhesion pathways of lung tissues may be involved in the pathophysiology of hyperoxia-induced arrested alveolarization.

Hyperoxia disrupts the intestinal barrier in newborn rats.

Animal studies have demonstrated that neonatal hyperoxia injures the distal small intestine. This study aimed to determine the effects of neonatal hyperoxia exposure on the intestinal morphology and intestinal barrier integrity in newborn rats. Sprague-Dawley rat pups were exposed to either ambient air or hyperoxia. The ambient air and normobaric hyperoxia groups were maintained in room air and 85% O2 for 2weeks, respectively. The rats were euthanized on Postnatal Day 14, and the terminal ileum was collected for histological analyses and oxidative stress measurements. The generation of reactive oxygen species was evaluated by measuring the production of 8-hydroxy-2'-deoxyguanosine (8-OHdG). The expression and localization of epithelial injury markers [intestinal fatty acid binding protein (I-FABP)] and intestinal barrier proteins [occludin and zonula occludens (ZO)-1] were analyzed through immunofluorescence staining and western blotting. The body weight at birth was comparable between the two groups. On Postnatal Day 14, the rats in the hyperoxia group exhibited significantly lower body weight, a higher serum interleukin-6 level, a higher intestinal injury score, higher 8-OHdG and I-FABP expression, and lower occludin and ZO-1 protein expression than did those in the ambient air group. Hyperoxia exposure injured the distal small intestine and disrupted the intestinal barrier in newborn rats. This may be attributable to oxidative stress during the postnatal period.

Microcomputed tomography assessment of lipopolysaccharide-induced acute lung injury in rat.

PURPOSE: High-resolution microcomputed tomography (micro-CT) is an extremely flexible and accurate technique for three-dimensional examination of biological tissues. The aim was to evaluate the feasibility of micro-CT as a noninvasive tool for analyzing the lung structure during lipopolysaccharide (LPS)-induced acute lung injury (ALI) in rats. MATERIALS AND METHODS: ALI was induced in rats by intratracheal instillation of LPS (2 mg/kg) in 0.3 ml saline, and the control treatment consisted of intratracheal instillation of an equal volume of normal saline. Morphological changes were assessed by using micro-CT and a light microscope at 24 and 48 hours after LPS instillation. Total volume is the sum of all pixels marked as the whole lung and total air volume (Air V) is the sum volume of all air in the lung. RESULTS: The saline groups exhibited no major histological abnormalities, whereas the LPS groups exhibited patchy areas of haemorrhage and thickened alveolar walls with inflammatory cell infiltration at 24 and 48 hours. The LPS groups had significantly smaller Air V and percent total air volume (Air V/TV) compared with those of the saline groups at 24 and 48 hours. Air V/TV was strongly negatively correlated with the lung injury score (r = -0.641, P = .004). CONCLUSIONS: Micro-CT is a feasible tool for evaluating the lung structure and lung injury progression during LPS-induced ALI.

Human mesenchymal stem cells attenuate pulmonary hypertension induced by prenatal lipopolysaccharide treatment in rats.

Intra-amniotic injection of lipopolysaccharide (LPS) induces pulmonary hypertension in newborn rats. This study was designed to test whether human mesenchymal stem cells (MSCs) reduce pulmonary hypertension and alleviate cardiac hypertrophy in prenatal LPS-treated rats. Pregnant Sprague-Dawley rats were injected intraperitoneally with LPS (0.5 mg/kg per day) or untreated on gestational days 20 and 21. Human MSCs (3×10(5) cells and 1×10(6) cells) in 0.03 mL of normal saline (NS) were transplanted intratracheally on postnatal day 5. Four study groups were considered: normal, LPS+NS, LPS+MSCs (3×10(5) cells), and LPS+MSCs (1×10(6) cells). On postnatal day 14, lung and heart tissues were collected for measuring the arterial medial wall thickness (MWT) and ß-myosin heavy chain (ß-MHC) level as markers of pulmonary hypertension and cardiac hypertrophy, respectively. The LPS+NS group exhibited a significantly higher right ventricle (RV)/[left ventricle (LV)+ interventricular septum (IVS)] thickness ratio and MWT, a greater cardiomyocyte width, a greater number of cardiomyocyte nuclei per squared millimeter, and higher ß-MHC expression than those observed in the normal group. Human MSC transplantation (3×10(5) cells and 1×10(6) cells) in LPS-treated rats reduced MWT and the RV/(LV+IVS) thickness ratio to normal levels. This improvement in right ventricular hypertrophy was accompanied by a decrease in toll-like receptor 4 (TLR4), nuclear factor-κB, and tumor necrosis factor-α expression in the heart. Intratracheal human MSCs transplantation can attenuate pulmonary hypertension and right ventricular hypertrophy in prenatal LPS-treated rats; this attenuation may be associated with suppression of TLR4 expression via paracrine pathways.

Bubble CPAP Support after Discontinuation of Mechanical Ventilation Protects Rat Lungs with Ventilator-Induced Lung Injury.

BACKGROUND: Bubble continuous positive airway pressure (BCPAP) has been used in neonates with respiratory distress for decades, but its lung-protective effect and underlying mechanism has not been investigated. OBJECTIVES: To test the hypothesis that BCPAP use after extubation decreases lung injury and that alterations to lung nitric oxide synthase (NOS) 3 expression may be one of the underlying mechanisms. METHODS: We compared gas exchange, lung injury severity, and lung NOS expression among rats with ventilator-induced lung injury (VILI) treated with either BCPAP or spontaneous breathing. After high tidal volume ventilation for 30 min, the rats were randomly divided to three groups: a control group underwent spontaneous breathing (n = 7), and two BCPAP groups were treated with the bubble technique with either a 2.5-mm-diameter (n = 7) or a 5.5-mm-diameter (n = 7) expiratory limb for 2 h. RESULTS: The bubble technique (2.5 and 5.5 mm diameter combined) resulted in a significantly higher PaO2, decreased alveolar protein levels (1.01 vs. 1.43 mg/kg, p < 0.05), a lower lung injury score (3.87 vs. 4.86, p < 0.05), and decreased NOS3 expression (1.99 vs. 3.32, p < 0.05) compared to spontaneous breathing in the control group. BCPAP with a 2.5-mm-diameter and with a 5.5-mm-diameter expiratory limb was not different with regard to gas exchange, alveolar protein levels, and lung injury scores, but there was a trend for lower NOS3 expression in the 5.5-mm group (1.41 vs. 2.56, p = 0.052). CONCLUSIONS: BCPAP decreases lung injury in rats with VILI after stopping mechanical ventilation. Attenuation of lung NOS3 expression may be one of the underlying mechanisms.