Neonatal Rat Lung Research Articles

6813 Des-Ciclesonide: A Novel Selective Glucocorticoid Receptor Modulator With Therapeutic Potential for Bronchopulmonary Dysplasia, a Chronic Lung Disease of Prematurity

Abstract Disclosure: N. El Khoury: None. J. Jaumotte: None. C. Min: None. C. Madigan: None. J. Wang: None. U. Chandran: None. V. Sampath: None. T.J. Cole: None. P. Monaghan-Nichols: None. R. Houtman: None. K.W. Nettles: None. D.B. DeFranco: None. Infants born before 30 weeks gestation are at risk of developing bronchopulmonary dysplasia (BPD), a prevalent chronic lung disease of prematurity. Glucocorticoids (GCs) such as dexamethasone (DEX) have been a mainstay of BPD prevention as they reduce lung inflammation and injury. However, their use is encumbered with several adverse effects including hyperglycemia, impaired somatic and brain growth and cerebral palsy. These major concerns have resulted in the AAP recommending caution in GC use in preterm infants.Ciclesonide (CIC), an inhaled GC prodrug approved for the treatment of asthma and allergic rhinitis in children, is converted to the potent glucocorticoid receptor (GR) agonist des-ciclesonide (DES) by enzymes enriched in the airways. We previously reported that s.c. delivery of CIC in neonatal rats activated GR transcriptional responses in lung but did not trigger the adverse effects on somatic growth, brain weight or cerebral white matter loss caused by DEX leading us to propose CIC as a potential new pharmacotherapy for BPD. To determine whether limited systemic metabolism of CIC in neonatal rats was solely responsible for its enhanced safety profile, we assessed various outcomes in neonatal rats followed s.c. delivery of its active metabolite DES. Consistent with previous studies, five daily s.c. injections of DEX led to a reduction in body and brain weight as well as reduced serum levels of insulin-like growth factor-1 and chronic hyperglycemia. Surprisingly, analogous s.c. injection of neonatal rats with DES, which has a higher binding affinity for GR than DEX, did not trigger any of these adverse effects. However, DES is as effective as DEX in reducing the expression of various proinflammatory cytokine mRNAs in neonatal rat lung induced by an 11-day systemic treatment with bleomycin. Bulk RNAseq analyses revealed transcriptional responses of neonatal lung and liver to DES, although less robust than that observed to DEX (i.e., 25% and 13% in lung and liver, respectively). Nonetheless, a NAPing assay for coregulator peptide binding showed increased binding of all the DES responsive peptide interactions relative to DEX and cortisol. All-atom molecular dynamics simulations revealed site-specific H-bonding changes within the GR ligand binding domain (LBD) caused by the bulky C16/C17substitution within DES, which was translated into broader changes in LBD structure, specifically revealed as differences in RMSF in helix 7/8 and the surfaces interacting with the NCOA2 peptide. Furthermore, network pathway analysis illustrated that DES mobility at helix 7/8 uniquely generated a meaningful allosteric signaling pathway between Q642 and NCOA2 peptide. In conclusion, the pharmacokinetic properties of the GR prodrug CIC coupled with the tissue-selective pharmacodynamic properties of its active metabolite DES make this FDA approved drug a potential new therapeutic agent for BPD. Presentation: 6/3/2024

Long noncoding RNA signatures in intrauterine infection/inflammation-induced lung injury: an integrative bioinformatics study

BackgroundIntrauterine infection/inflammation can result in fetal and neonatal lung injury. However, the biological mechanisms of intrauterine infection/inflammation on fetal and neonatal lung injury and development are poorly known. To date, there are no reliable biomarkers for improving intrauterine infection/inflammation-induced lung injury.MethodsAn animal model of intrauterine infection/inflammation-induced lung injury was established with pregnant Sprague–Dawley rats inoculated with Escherichia coli suspension. The intrauterine inflammatory status was assessed through the histological examination of the placenta and uterus. A serial of histological examinations of the fetal and neonatal rats lung tissues were performed. The fetal and neonatal rat lung tissues were harvested for next generation sequencing at embryonic day 17 and postnatal day 3, respectively. Differentially expressed mRNAs and lncRNAs were identified by conducting high-throughput sequencing technique. The target genes of identified differentially expressed lncRNAs were analyzed. Homology analyses for important differentially expressed lncRNAs were performed.ResultsThe histopathological results showed inflammatory infiltration, impaired alveolar vesicular structure, less alveolar numbers, and thickened alveolar septa in fetal and neonatal rat lung tissues. Transmission electron micrographs revealed inflammatory cellular swelling associated with diffuse alveolar damage and less surfactant-storing lamellar bodies in alveolar epithelial type II cells. As compared with the control group, there were 432 differentially expressed lncRNAs at embryonic day 17 and 125 differentially expressed lncRNAs at postnatal day 3 in the intrauterine infection group. The distribution, expression level, and function of these lncRNAs were shown in the rat genome. LncRNA TCONS_00009865, lncRNA TCONS_00030049, lncRNA TCONS_00081686, lncRNA TCONS_00091647, lncRNA TCONS_00175309, lncRNA TCONS_00255085, lncRNA TCONS_00277162, and lncRNA TCONS_00157962 may play an important role in intrauterine infection/inflammation-induced lung injury. Fifty homologous sequences in Homo sapiens were also identified.ConclusionsThis study provides genome-wide identification of novel lncRNAs which may serve as potential diagnostic biomarkers and therapeutic targets for intrauterine infection/inflammation-induced lung injury.

Combination of pioglitazone, a PPARγ agonist, and synthetic surfactant B-YL prevents hyperoxia-induced lung injury in adult mice lung explants

IntroductionHyperoxia-induced lung injury is characterized by acute alveolar injury, disrupted epithelial-mesenchymal signaling, oxidative stress, and surfactant dysfunction, yet currently, there is no effective treatment. Although a combination of aerosolized pioglitazone (PGZ) and a synthetic lung surfactant (B-YL peptide, a surfactant protein B mimic) prevents hyperoxia-induced neonatal rat lung injury, whether it is also effective in preventing hyperoxia-induced adult lung injury is unknown. MethodUsing adult mice lung explants, we characterize the effects of 24 and 72-h (h) exposure to hyperoxia on 1) perturbations in Wingless/Int (Wnt) and Transforming Growth Factor (TGF)-β signaling pathways, which are critical mediators of lung injury, 2) aberrations of lung homeostasis and injury repair pathways, and 3) whether these hyperoxia-induced aberrations can be blocked by concomitant treatment with PGZ and B-YL combination. ResultsOur study reveals that hyperoxia exposure to adult mouse lung explants causes activation of Wnt (upregulation of key Wnt signaling intermediates β-catenin and LEF-1) and TGF-β (upregulation of key TGF-β signaling intermediates TGF-β type I receptor (ALK5) and SMAD 3) signaling pathways accompanied by an upregulation of myogenic proteins (calponin and fibronectin) and inflammatory cytokines (IL-6, IL-1β, and TNFα), and alterations in key endothelial (VEGF-A and its receptor FLT-1, and PECAM-1) markers. All of these changes were largely mitigated by the PGZ + B-YL combination. ConclusionThe effectiveness of the PGZ + B-YL combination in blocking hyperoxia-induced adult mice lung injury ex-vivo is promising to be an effective therapeutic approach for adult lung injury in vivo.

Inhibition of miR-21 improves pulmonary vascular responses in bronchopulmonary dysplasia by targeting the DDAH1/ADMA/NO pathway.

miR-21 has been confirmed to be overexpressed in neonatal rat lungs with hyperoxia-mediated bronchopulmonary dysplasia (BPD). The specific function of miR-21 in BPD is still unclear. We established the hyperoxia-induced BPD rat model in vivo and the hyperoxia-induced pulmonary microvascular endothelial cells (PMVECs) model in vitro. Transwell assay was utilized to detect the migratory capability of PMVECs. Tube formation assay was utilized to measure angiogenesis ability. ELISA was utilized to test nitric oxide (NO) production and the intracellular and extracellular Asymmetric Dimethylarginine (ADMA) concentration. Furthermore, the interaction between miR-21 and dimethylarginine dimethylaminohydrolase 1 (DDAH1) was evaluated using luciferase reporter assay. We found that miR-21 expression in PMVECs was increased by hyperoxia stimulation. Inhibition of miR-21 improved the migratory and angiogenic activities of PMVECs and overexpression of miR-21 exerted the opposite effects. Furthermore, knockdown of miR-21 increased NO production and decreased intracellular and extracellular ADMA concentration in hyperoxia-treated PMVECs. Next we proved that miR-21 could bind to DDAH1 and negatively regulate its expression. Rescues assays showed that DDAH1 knockdown reversed the effects of miR-21 depletion on hyperoxia-mediated PMVEC functions, NO production, and ADMA concentration. Importantly, miR-21 downregulation restored alveolarization and vascular density in BPD rats. This study demonstrates that inhibition of miR-21 improves pulmonary vascular responses in BPD by targeting the DDAH1/ADMA/NO pathway.

Intravitreal Bevacizumab Is Associated With Prolonged Ventilatory Support in Preterm Infants With Bronchopulmonary Dysplasia

Intravitreal bevacizumab (IVB), an anti-vascular endothelial growth factor (VEGF) antibody, is a widely adopted treatment for retinopathy of prematurity (ROP). Although animal studies have demonstrated that IVB inhibits alveologenesis in neonatal rat lung, the clinical influence of IVB on respiratory outcomes has not been studied. Does IVB affect the respiratory outcome in preterm infants with bronchopulmonary dysplasia? We retrospectively assessed very low birth weight (VLBW) preterm infants admitted to our neonatal ICU between January 2016 and June 2021. Furthermore, we evaluated the short-term respiratory outcomes after IVB therapy in VLBW preterm infants requiring ventilatory support at 36weeks' postmenstrual age (PMA). One hundred seventy-four VLBW preterm infants with bronchopulmonary dysplasia were recruited. Eighty-eight infants showed ROP onset before being ventilator free, and 78 infants received a diagnosis of the most severe ROP before being ventilator free. Among them, 32 received a diagnosis with type 1 ROP and received IVB treatment. After adjusting for gestational age, birth body weight, and baseline respiratory status, we discovered that IVB is associated significantly with prolonged ventilatory support and a lower likelihood of becoming ventilator free (hazard ratio, 0.53; P= .03). IVB may have a short-term respiratory adverse effect in patients requiring ventilatory support at 36weeks' PMA. Therefore, long-term follow-up for respiratory outcomes may be considered in VLBW infants who receive IVB treatment.

Comparison of coenzyme Q10 or fish oil for prevention of intermittent hypoxia-induced oxidative injury in neonatal rat lungs

BackgroundNeonatal intermittent hypoxia (IH) results in oxidative distress in preterm infants with immature antioxidant systems, contributing to lung injury. Coenzyme Q10 (CoQ10) and fish oil protect against oxidative injury. We tested the hypothesis that CoQ10 is more effective than fish oil for prevention of IH-induced lung injury in neonatal rats.MethodsNewborn rats were exposed to two clinically relevant IH paradigms at birth (P0): (1) 50% O2 with brief hypoxia (12% O2); or (2) room air (RA) with brief hypoxia (12% O2), until P14 during which they were supplemented with daily oral CoQ10, fish oil, or olive oil from P0 to P14. Pups were studied at P14 or placed in RA until P21 with no further treatment. Lungs were assessed for histopathology and morphometry; biomarkers of oxidative stress and lipid peroxidation; and antioxidants.ResultsOf the two neonatal IH paradigms 21%/12% O2 IH resulted in the most severe outcomes, evidenced by histopathology and morphometry. CoQ10 was effective for preserving lung architecture and reduction of IH-induced oxidative stress biomarkers. In contrast, fish oil resulted in significant adverse outcomes including oversimplified alveoli, hemorrhage, reduced secondary crest formation and thickened septae. This was associated with elevated oxidants and antioxidants activities.ConclusionsData suggest that higher FiO2 may be needed between IH episodes to curtail the damaging effects of IH, and to provide the lungs with necessary respite. The negative outcomes with fish oil supplementation suggest oxidative stress-induced lipid peroxidation.

Pulmonary ACE2 expression in neonatal and adult rats.

Children show a distinct presentation of COVID‐19, characterized by a lower incidence and mild phenotype, but the reason for this is still unknown. The angiotensin‐converting enzyme 2 (ACE2) functions as the primary cell entry receptor for Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and is thought to cause distinct clinical features between children and old people. The primary purpose of this study was to determine whether differences exist in the level of expression and distribution of ACE2 between neonatal and adult rat lungs. The lung tissues from rats of various ages were used to investigate the expression patterns of ACE2. Western blot, immunohistochemistry, and immunofluorescence were used to quantify or identify the localization of ACE2 in rat lungs. ACE2 was homogenously expressed in fewer alveolar type II (AT2) cells in the neonatal lung, with no polarization to the alveolar space and additional expression in pulmonary endothelium when compared to adult rat lungs. These findings suggest that the patterns of ACE2 distribution and cellular localization in rat lungs change with age.

Ciclesonide activates glucocorticoid signaling in neonatal rat lung but does not trigger adverse effects in the cortex and cerebellum

Synthetic glucocorticoids (sGCs) such as dexamethasone (DEX), while used to mitigate inflammation and disease progression in premature infants with severe bronchopulmonary dysplasia (BPD), are also associated with significant adverse neurologic effects such as reductions in myelination and abnormalities in neuroanatomical development. Ciclesonide (CIC) is a sGC prodrug approved for asthma treatment that exhibits limited systemic side effects. Carboxylesterases enriched in the lower airways convert CIC to the glucocorticoid receptor (GR) agonist des-CIC. We therefore examined whether CIC would likewise activate GR in neonatal lung but have limited adverse extra-pulmonary effects, particularly in the developing brain. Neonatal rats were administered subcutaneous injections of CIC, DEX or vehicle from postnatal days 1–5 (PND1-PND5). Systemic effects linked to DEX exposure, including reduced body and brain weight, were not observed in CIC treated neonates. Furthermore, CIC did not trigger the long-lasting reduction in myelin basic protein expression in the cerebral cortex nor cerebellar size caused by neonatal DEX exposure. Conversely, DEX and CIC were both effective at inducing the expression of select GR target genes in neonatal lung, including those implicated in lung-protective and anti-inflammatory effects. Thus, CIC is a promising, novel candidate drug to treat or prevent BPD in neonates given its activation of GR in neonatal lung and limited adverse neurodevelopmental effects. Furthermore, since sGCs such as DEX administered to pregnant women in pre-term labor can adversely affect fetal brain development, the neurological-sparing properties of CIC, make it an attractive alternative for DEX to treat pregnant women severely ill with respiratory illness, such as with asthma exacerbations or COVID-19 infections.

Recombinant adiponectin protects the newborn rat lung from lipopolysaccharide-induced inflammatory injury.

Preterm infants are at high risk for developing bronchopulmonary dysplasia and pulmonary hypertension from inflammatory lung injury. In adult models, adiponectin (APN)—an adipocyte‐derived hormone—protects the lung from inflammatory injury and pulmonary vascular remodeling. Cord blood APN levels in premature infants born < 26 weeks gestation are 5% of the level in infants born at term. We previously reported the expression profile of APN and its receptors in neonatal rat lung homogenates during the first 3 weeks of postnatal development. Here, we characterize the expression profile of APN and its receptors in specific lung cells and the effects of exogenous recombinant APN (rAPN) on lipopolysaccharide‐(LPS)‐induced cytokine and chemokine production in total lung homogenates and specific lung cells. In vitro, rAPN added to primary cultures of pulmonary artery smooth muscle cells attenuated the expression of LPS‐induced pro‐inflammatory cytokines while increasing the expression of anti‐inflammatory cytokines. In vivo, intraperitoneal rAPN (2 mg/kg), given 4 hr prior to intrapharyngeal administration of LPS (5 mg/kg) to newborn rats at postnatal day 4, significantly reduced gene and protein expression of the pro‐inflammatory cytokine IL‐1ß and reduced protein expression of the chemokines monocyte chemoattractant protein (MCP‐1) and macrophage inflammatory protein‐1 alpha (MIP‐1α) in the lung. LPS‐induced histopathological changes in the lung were also decreased. Moreover, rAPN given 20 hr after intrapharyngeal LPS had a similar effect on lung inflammation. These findings suggest a role for APN in protecting the lung from inflammation during early stages of lung development.

TRIM72 mediates lung epithelial cell death upon hyperoxia exposure.

Premature infants often require oxygen (O2) therapy for respiratory distress syndrome; however, excessive use of O2 can cause clinical conditions such as bronchopulmonary dysplasia. Although many treatment methods are currently available, they are not effective in preventing bronchopulmonary dysplasia. Herein, we explored the role of tripartite motif protein 72 (TRIM72), a factor involved in repairing alveolar epithelial wounds, in regulating alveolar cells upon hyperoxia exposure. In this in vivo study, we used Sprague-Dawley rat pups that were reared in room air or 85% O2 for 2 weeks after birth. The lungs were excised for histological analyses, and TRIM72 expression was assessed on postnatal days 7 and 14. For in vitro experiments, RLE-6TN cells (i.e., rat alveolar type II epithelial cells) and A549 cells (i.e., human lung carcinoma epithelial cells) were exposed to 85% O2 for 5 days. The cells were then analyzed for cell viability, and TRIM72 expression was determined. Exposure to hyperoxia reduced body and lung weight, increased mean linear intercept values, and upregulated TRIM72 expression. In vitro study results revealed increased or decreased lung cell viability upon hyperoxia exposure depending on the suppression or overexpression of TRIM72, respectively. Hyperoxia upregulates TRIM72 expression in neonatal rat lung tissue; moreover, it initiates TRIM72-dependent alveolar epithelial cell death, leading to hyperoxia-induced lung injury.

Effects of intravenous and oral di(2-ethylhexyl) phthalate (DEHP) and 20% Intralipid vehicle on neonatal rat testis, lung, liver, and kidney

The highest human exposures to the plasticizer di(2-ethylhexyl) phthalate (DEHP) occur through intravenous (iv) exposure from medical procedures. Rodent toxicity studies, mainly using oral exposures, have identified male reproductive toxicity after developmental exposure to DEHP as the primary concern. Other organs are also affected by DEHP and route may influence the degree of target organ involvement. Cammack et al. (2003) reported a critical study focused on testicular toxicity using oral and iv exposures of neonatal Sprague-Dawley rats to 60, 300, or 600 mg/kg body weight/day DEHP in Intralipid vehicle. The present study followed the same dosing paradigm and included assessment of additional organs to evaluate the potential utility of this design for DEHP alternatives. Reduction of testis weight was observed in all DEHP treatment groups and germ cell and Sertoli cell toxicity was observed at the two highest doses with both routes. Lung granulomas occurred in all iv DEHP groups, possibly related to increased fat particle size in DEHP lipid emulsions. Lung alveolar development was inhibited after both oral and iv high dose DEHP. Toxicity of oral Intralipid vehicle was observed in germ and Sertoli cells. The lack of such effects after iv vehicle exposure suggested that this may be a gut-mediated effect.

Human placenta-derived mesenchymal stem cells attenuate established hyperoxia-induced lung injury in newborn rats

Hyperoxia increases Sonic hedgehog (Shh) expression in neonatal rat lungs. The effect of mesenchymal stem cells (MSCs) on the hedgehog signaling pathway in hyperoxia-induced lung injury is unknown. This study evaluated whether MSCs could inhibit hedgehog signaling and improve established hyperoxia-induced lung injury in newborn rats. Newborn rats were assigned to room air (RA) or hyperoxia (85% O2) groups from postnatal day 4-15, and some received intravenous injection of human MSCs (9×105cells) in 90μL of normal saline (NS) through the tail vein on postnatal day 15. We obtained four study groups as follows: RA+NS, RA+MSCs, O2+NS, and O2+MSCs. Pups from each group were sacrificed on postnatal days 15 and 29, and lungs were removed for histological and Western blot analyses. Neonatal hyperoxia on postnatal days 4-15 reduced the body weight, increased the mean linear intercept, and decreased the vascular density of the rats, and these effects were associated with increased Shh and Smoothened (Smo) expression and decreased Patched expression. By contrast, the MSC-treated hyperoxic pups exhibited improved alveolarization, increased vascularization, and decreased Shh and Smo expression on postnatal day 29. Human MSC treatment reversed established hyperoxia-induced lung injury in newborn rats, probably through suppression of the hedgehog pathway.

Sumoylation of CCAAT-enhancer-binding protein α inhibits lung differentiation in Bronchopulmonary Dysplasia model rats.

Bronchopulmonary dysplasia (BPD) is a major cause of mortality and morbidity in premature infants, characterized by alveolar simplification, surfactant deficiency, and respiratory distress. In the present study, we have investigated the functional roles of sumoylated CCAAT/enhancer binding protein alpha (C/EBPα) in the BPD rat model. A significant increase in small ubiquitin‐like modifier 1 (SUMO1) and sumoylated C/EBPα protein levels were observed in BPD rats, and the levels of the sumoylated C/EBPα were associated with the pulmonary surfactant proteins (SPs). In order to confirm the role of sumoylated C/EBPα in BPD rats, SUMO1 was knocked down by lentiviral transfection of neonatal rat lungs with SUMO1‐RNAi‐LV. We found that the expression of C/EBPα and surfactant proteins increased following SUMO1 knockdown. Furthermore, the relatively low decrease in the levels of C/EBPα sumoylation was correlated with reduced glycogen consumption. Besides, co‐immunoprecipitation assays revealed that sumoylation is involved in the regulation of the interaction between C/EBPα and TGFβ2 in the lung. In conclusion, our findings indicate that sumoylation may act as a negative regulator of the C/EBPα‐mediated transactivation in BPD rats.

Terbutaline alleviates the lung injury in the neonatal rats exposed to endotoxin: Potential roles of epithelial sodium channels.

Intrauterine inflammation generates inflammatory mediators that damage the developing bronchoalveolar epithelium, resulting in neonatal lung injury. Lung fluid transport disorders are the main reasons for the development of pulmonary edema, an important pathology of lung injury. Previous studies suggested that epithelial sodium channels (ENaCs) play an important role in lung fluid transport. Here, we investigated whether changes in the expression of ENaCs were observed when neonatal rat lung injury was induced by maternal exposure to endotoxin. We also examined the therapeutic effect of terbutaline nebulizer inhalation on this injury. The results showed that maternal exposure to endotoxin increased the levels of TNF‐α and IL‐1β in bronchoalveolar lavage fluid, suppressed α‐, β‐, γ‐ENaC in the neonatal rat lung, and resulted in the formation of pulmonary edema on postnatal days 1 and 7. Terbutaline up‐regulated the expression of β‐ and γ‐ENaC in the distal lung after 7 days of treatment. The potential signal molecules cAMP, PKA, and CREB expressions were increased after terbutaline treatment. In summary, maternal exposure to endotoxin decreased the expression of ENaCs in neonatal rats which, in turn, may exacerbate pulmonary edema. Inhalation of the β2‐adrenergic receptor agonist terbutaline improved lung liquid clearance. By increasing the expression of sodium ion channels, the effective removal of alveolar fluid provides a new way for the prevention and treatment of neonatal lung injury.

Brain Distribution and Metabolism of Flupirtine, a Nonopioid Analgesic Drug with Antiseizure Effects, in Neonatal Rats.

Flupirtine, a nonopioid analgesic drug, is effective in treating neonatal seizures. However, its brain delivery and pharmacokinetics are unknown in neonatal mammals. The purpose of this study was to determine the pharmacokinetics of flupirtine and the formation of its active metabolite D-13223 in various tissues such as brain in neonate animals. On postnatal day 7, rat pups received 25 mg/kg of flupirtine intraperitoneally. Liver; heart; kidney; lung; spleen; retina; serum; and brain regions hippocampus, cortex, and the remaining brain (devoid of cerebellum) were harvested up to 24-h postdosing. An LC-MS/MS assay was developed to quantify flupirtine and D-13223. Flupirtine was delivered to all tissues assessed, with the highest area under the concentration vs. time curve (AUC0–24h) in liver (488 µg·h/g tissue) and the lowest in spleen (82 µg·h/g tissue). Flupirtine reached the brain, including the hippocampus and cortex, within 1 h of dosing and persisted at 24 h. Flupirtine AUC in various brain regions was approximately 195 µg·h/g tissue. The half-life of flupirtine in various tissues ranged from 3.1 to 5.2 h. D-13223 was formed in vivo and detected in all tissues assessed, with the concentrations being the highest in the liver. Incubation of isolated neonatal rat liver, heart, kidney, lung, spleen, whole eye, serum, or whole brain with flupirtine for 3 h at 37 °C formed D-13223 in all tissues, except serum. D-13223 formation was the highest in isolated liver tissue. Tissue partition coefficients based on isolated tissue uptake correlated well with in vivo tissue:serum drug exposure ratios. Thus, flupirtine reaches the target brain tissues from the systemic route in neonatal rats, and brain tissue forms the active metabolite D-13223.

Impaired Angiogenic Supportive Capacity and Altered Gene Expression Profile of Resident CD146+ Mesenchymal Stromal Cells Isolated from Hyperoxia-Injured Neonatal Rat Lungs.

Bronchopulmonary dysplasia (BPD), the most common complication of extreme preterm birth, can be caused by oxygen-related lung injury and is characterized by impaired alveolar and vascular development. Mesenchymal stromal cells (MSCs) have lung protective effects. Conversely, BPD is associated with increased MSCs in tracheal aspirates. We hypothesized that endogenous lung (L-)MSCs are perturbed in a well-established oxygen-induced rat model mimicking BPD features. Rat pups were exposed to 21% or 95% oxygen from birth to postnatal day 10. On day 12, CD146+ L-MSCs were isolated and characterized according to the International Society for Cellular Therapy criteria. Epithelial and vascular repair potential were tested by scratch assay and endothelial network formation, respectively, immune function by mixed lymphocyte reaction assay. Microarray analysis was performed using the Affymetrix GeneChip and gene set enrichment analysis software. CD146+ L-MSCs isolated from rat pups exposed to hyperoxia had decreased CD73 expression and inhibited lung endothelial network formation. CD146+ L-MSCs indiscriminately promoted epithelial wound healing and limited T cell proliferation. Expression of potent antiangiogenic genes of the axonal guidance cue and CDC42 pathways was increased after in vivo hyperoxia, whereas genes of the anti-inflammatory Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and lung/vascular growth-promoting fibroblast growth factor (FGF) pathways were decreased. In conclusion, in vivo hyperoxia exposure alters the proangiogenic effects and FGF expression of L-MSCs. In addition, decreased CD73 and JAK/STAT expression suggests decreased immune function. L-MSC function may be perturbed and contribute to BPD pathogenesis. These findings may lead to improvements in manufacturing exogenous MSCs with superior repair capabilities.

Effects and molecular mechanisms of intrauterine infection/inflammation on lung development

BackgroundIntrauterine infection/inflammation plays an important role in the development of lung injury and bronchopulmonary dysplasia (BPD) in preterm infants, While a multifactorial genesis is likely, mechanisms involved in BPD after intrauterine infection/inflammation are largely unknown. Recent studies have suggested microRNAs (miRNAs) are likely to play a role. Therefore, this study aimed to study the effects and mechanisms of intrauterine infection/inflammation on lung development, and to identify miRNAs related to lung injury and BPD.MethodsAn animal model of intrauterine infection/inflammation was established with pregnant SD rats endocervically inoculated with E.coli. The fetal and neonatal rats were observed at embryonic day (E) 17, 19, 21 and postnatal day (P) 1, 3, 7, 14, respectively. Body weight, lung weight, the expression levels of NLRP3, TNF-α, IL-lβ, IL-6, VEGF, Collagen I, SP-A, SP-B and SP-C in the lung tissues of fetal and neonatal rats were measured. Expression profiles of 1218 kinds of miRNAs in the lungs of neonatal rats were detected by miRNA microarray technique. Target genes of the identified miRNAs were predicted through online software.ResultsIntrauterine infection/inflammation compromised not only weight development but also lung development of the fetal and neonatal rats. The results showed significantly increased expression of NLRP3, TNF-α, IL-1β, IL-6, Collagen I, and significantly decreased expression of VEGF, SP-A, SP-B and SP-C in the fetal and neonatal rat lung tissues in intrauterine infection group compared to the control group at different observation time point (P < 0.05). Forty-three miRNAs with significant differential expression were identified. Possible target genes regulated by the identified miRNAs are very rich.ConclusionsIntrauterine infection/inflammation results in lung histological changes which are very similar to those observed in BPD. Possible mechanisms may include NLRP3 inflammasome activation followed by inflammatory cytokines expression up-regulated, inhibiting the expression of pulmonary surfactant proteins, interfering with lung interstitial development. There are many identified miRNAs which target a wide range of genes and may play an important role in the processes of lung injury and BPD.

The investigation of effect of alpha lipoic acid against damage on neonatal rat lung to maternal tobacco smoke exposure.

This study was carried out to determine the changes in the lungs of the rat pups exposed to tobacco smoke during pregnancy period and to investigate the protective effects of alpha lipoic acid, which is administered during pregnancy, on these changes. Spraque-Dawley female rats were divided into four groups: control, tobacco smoke (TS), tobacco smoke + alpha lipoic acid (TS + ALA) and alpha lipoic acid (ALA). The rats in control group were untreated. Rats were exposed to TS twice a day for one hour starting from eight weeks before mating and during pregnancy. 20 mg / kg of ALA was administered to rats. On 7th and 21st days 7 of the pups from each group were decapitated. Histological, morphometric, biochemical and quantitative real-time RT-PCR analyzes were performed. Histopathological and biochemical changes were observed in TS group. While a significant decrease was observed both in SP-A and VEGF immunoreactivities and mRNA levels, caspase-3 immunoreactivity and TUNEL positive cells were increased in TS group. It is suggested that prenatal TS exposure leads to morphological and histopathological changes on lung development by causing oxidative damage in lungs of neonatal rats and the maternal use of ALA can provide a limited protective effect on the neonatal lung development against this oxidative stress originating from TS. Although pregnant women are increasingly aware on health risks of smoking, environmental tobacco smoke exposure is still a widespread problem. For this reason, it is thought that this damage can be partially reduced by some antioxidant supplements in pregnancy.

The expression of HoxB5 and SPC in neonatal rat lung after exposure to fluoxetine.

ObjectiveApproximately 10% of pregnant women suffer from pregnancy-associated depression. Fluoxetine, as a selective serotonin reuptake inhibitor, is being employed as a therapy for depressive disorders. The present study aimed to determine the effects of fluoxetine on neonatal lung development.MethodsThirty pregnant Wistar rats (weighing 200–250 g) were treated daily with 7 mg/kg fluoxetine from gestation day 0 to gestation day 21, via gavage. The control group received a similar volume of distilled water only. Following delivery, the newborns and their lungs were immediately weighed in both of the groups. The right lung was fixed for histological assessments while the left lung was used for evaluation of the expression of SPC and HoxB5 by the real-time polymerase chain reaction method.ResultsResults have indicated that even though the body weight and the number of neonatal rats in both groups were the same, the lung weight of neonates exposed to fluoxetine was significantly different compared to the control group (P<0.05). Expression of both genes was increased, nonetheless, only elevation of HoxB5 was significant (P<0.05). Histological studies demonstrated that lung tissue in the fluoxetine treatment group morphologically appears to be similar to the pseudoglandular phase, whereas the control group lungs experienced more development.ConclusionAccording to the upregulated expression of HoxB5 concerning histological findings, results of the present study showed that fluoxetine can influence lung growth and may in turn lead to delay in lung development. So establishment of studies to identify the effects of antidepressant drugs during pregnancy is deserved.

Knockdown of placental growth factor (PLGF) mitigates hyperoxia-induced acute lung injury in neonatal rats: Suppressive effects on NFκB signaling pathway

Although supplemental high-level oxygen treatment can promote the survival of premature infants, hyperoxia may adversely induce acute lung injury (ALI) in newborns. Our prior work illustrated that hyperoxic exposure could enhance the release of placental growth factor (PLGF) in the lungs of neonatal rats. We therefore postulated that PLGF contributed to hyperoxic ALI in newborns and evaluated the anti-PLGF treatment mediated by systematic delivery of lentivirus in hyperoxic ALI in this study. Lentivirus particles containing PLGF specific shRNA were injected into neonatal rats prior to hyperoxic exposure (90% oxygen for 72h) to inhibit PLGF expression. Hyperoxia induced oxidative damages in lung tissues as evidenced by the increased malondialdehyde and myeloperoxidase, and the decreased antioxidant superoxide dismutase. Also, hyperoxia caused excessive infiltration of inflammatory cells and overproduction of proinflammatory cytokines (tumor necrosis factor-α, interleukin-1β and interleukin-6) in rat lung tissue. These pathological alterations were partly reversed by PLGF shRNA delivery. The expression levels and activities of metalloproteinase (MMP)-2 and MMP9 were up-regulated in response to hyperoxia, whereas down-regulated when PLGF was inhibited. Moreover, PLGF shRNA inhibited nuclear factor kappa B (NFκB) signaling delivery in hyperoxic rat lungs. Additionally, exogenous PLGF-induced activation of MMPs in rat RLE-6TN alveolar epithelial cells was suppressed by NFκB inhibitor pyrrolidine dithiocarbamate. These results suggest that therapy targeting PLGF may be beneficial for infants with hyperoxic ALI.