Secondary Crest Formation Research Articles

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.

Short-term perinatal oxygen exposure may impair lung development in adult mice

BackgroundHyperoxia at resuscitation increases oxidative stress, and even brief exposure to high oxygen concentrations during stabilization may trigger organ injury with adverse long-term outcomes in premature infants. We studied the long-term effects of short-term perinatal oxygen exposure on cell cycle gene expression and lung growth in adult mice.MethodsWe randomized mice litters at birth to 21, 40, or 100%O2 for 30 min and recovered in room air for 4 or 12 weeks. Cell cycle gene expression, protein analysis, and lung morphometry were assessed at 4 and 12 weeks.ResultsThe principal component analysis demonstrated a high degree of correlation for cell cycle gene expression among the three oxygen groups. Lung elastin was significantly lower in the 100%O2 groups at 4 weeks. On lung morphometry, radial alveolar count, alveolar number, and septal count were similar. However, the mean linear intercept (MLI) and septal length significantly correlated among the oxygen groups. The MLI was markedly higher in the 100%O2 groups at 4 and 12 weeks of age, and the septal length was significantly lower in the 100%O2 groups at 12 weeks.ConclusionShort-term exposure to high oxygen concentrations lead to subtle changes in lung development that may affect alveolarization. The changes are related explicitly to secondary crest formation that may result in alteration in lung elastin. Resuscitation with high oxygen concentrations may have a significant impact on lung development and long-term outcomes such as BPD in premature infants.

Bacillus Calmette–Guerín vaccination: a novel therapeutic approach to preventing hyperoxic lung injury

Objective: A growing body of evidence suggests that vaccinations play a role in the normal maturation of the immune system and in both the development and balance of immune regulatory pathways that can impact health later in life. This study aimed to evaluate the effects of Bacillus Calmette–Guerín (BCG) vaccine on the hyperoxia-induced neonatal rat lung injury.Methods: Four groups were defined as hyperoxia-exposed BCG-vaccinated, hyperoxia-exposed placebo, room air-exposed control and room air-exposed BCG-vaccinated group. The validity of the hyperoxia-induced lung injury model used in this study was confirmed by histological and immunohistochemical test. Gene expression related with cytokine and growth factor was evaluated by real-time reverse transcription polymerase chain reaction.Result: The mean alveolar surface area and quantification of secondary crest formation in the oxygen-exposed placebo group was significantly lower than that of the oxygen-exposed BCG-vaccinated group. Compared to the oxygen-exposed placebo group, the oxygen-exposed BCG-vaccinated group showed a significantly decreased alveolar septal fibrosis and smooth muscle actin expression. The expression of genes VEGF, FGF-BP1, IL-13, and NFκB1 (p50) in the lungs of the hyperoxia-exposed BCG-vaccinated group was significantly higher than that of the hyperoxia-exposed placebo group.Conclusion: Results suggest that BCG vaccination can protect against neonatal hyperoxic lung injury. These benefits may be interpreted to coincide with its immunomodulatory effects on pro-inflammatory and anti-inflammatory cytokine balance and expression of growth factors.

Chronic lung injury in the neonatal rat: Up-regulation of TGFβ1 and nitration of IGF-R1 by peroxynitrite as likely contributors to impaired alveologenesis

Postnatal alveolarization is regulated by a number of growth factors, including insulin-like growth factor-I (IGF-I) acting through the insulin-like growth factor receptor-1 (IGF-R1). Exposure of the neonatal rat lung to 60% O2 for 14 days results in impairments of lung cell proliferation, secondary crest formation, and alveologenesis. This lung injury is mediated by peroxynitrite and is prevented by treatment with a peroxynitrite decomposition catalyst. We hypothesized that one of the mechanisms by which peroxynitrite induces lung injury in 60% O2 is through nitration and inactivation of critical growth factors or their receptors. Increased nitration of both IGF-I and IGF-R1 was evident in 60% O2-exposed lungs, which was reversible by concurrent treatment with a peroxynitrite decomposition catalyst. Increased nitration of the IGF-R1 was associated with its reduced activation, as assessed by IGF-R1 phosphotyrosine content. IGF-I displacement binding plots were conducted in vitro using rat fetal lung distal epithelial cells which respond to IGF-I by an increase in DNA synthesis. When IGF-I was nitrated to a degree similar to that observed in vivo there was minimal, if any, effect on IGF-I displacement binding. In contrast, nitrating cell IGF-R1 to a similar degree to that observed in vivo completely prevented specific binding of IGF-I to the IGF-R1, and attenuated an IGF-I-mediated increase in DNA synthesis. Additionally, we hypothesized that peroxynitrite also impairs alveologenesis by being an upstream regulator of the growth inhibitor, TGFβ1. That 60% O2-induced impairment of alveologenesis was mediated in part by TGFβ1 was confirmed by demonstrating an improvement in secondary crest formation when 60% O2-exposed pups received concurrent treatment with the TGFß1 activin receptor-like kinase, SB 431542. That the increased TGFβ1 content in lungs of pups exposed to 60% O2 was regulated by peroxynitrite was confirmed by its attenuation by concurrent treatment with a peroxynitrite decomposition catalyst. We conclude that peroxynitrite contributes to the impaired alveologenesis observed following the exposure of neonatal rats to 60% O2 both by preventing binding of IGF-I to the IGF-R1, secondary to nitration of the IGF-R1, and by causing an up-regulation of the growth inhibitor, TGFβ1.

PS-209 Bcg Vaccination Can Prevent Hyperoxic Lung Injury?

<h3>Introduction</h3> The aim of this study effects of BCG vaccine on the histopathologic and gene expression changes seen in hyperoxia induced neonatal rat lung injury. <h3>Method</h3> Twenty-three rat pubs were divided into four groups: air-exposed control group (n = 5), hyperoxia-exposed placebo group (n = 7), hyperoxia-exposed BCG-vaccinated group (n = 7), and air-exposed BCG-vaccinated group (n = 4). neonatal hyperoxic lung injury model was established according to the previous studies. Measurement of alveolar surface area, quantification of secondary crest formation, microvessel count, evaluation of alveolar septal fibrosis, and smooth muscle actin immunostaining were performed to assess hyperoxia-induced changes in lung morphology. The gene expression level was evaluated by RT-PCR. <h3>Results</h3> The alveolar surface areas were significant different between the oxygen exposed placebo group and oxygen exposed BCG vaccinated group (alveolar surface area; 0.29 ± 0.02 µm² and 0.52 ± 0.04 µm² p &lt; 0.05 respectively). Number of crests and microvessel count was found to be significantly increased in the oxygen exposed BCG vaccinated group compared with the animals in the oxygen exposed placebo group (p &lt; 0.05). Exposure to hyperoxia resulted in a significant decrease in mean alveolar surface area and number of crests formed compared with air-exposed animals (p &lt; 0.05). The degree of fibrosis was found to be significantly increased in the oxygen exposed placebo group compared with the animals in the oxygen exposed BCG vaccinated group (degree of fibrosis: 1.88 ± 0.33 and 0.91 ± 0.66 p &lt; 0.05 respectively). Immunostaining for SMA demonstrated hyperoxia-exposed animals with BCG vaccine in a significantly decrese in smooth muscle content compared with hyperoxia-exposed placebo animals (p &lt; 0.05). The expression of VEGF, FGF1, IL13, NFκB1 and TNFα in the lungs of vaccinated animals was significantly higher than that of non-vaccinated animals (p &lt; 0.05). <h3>Conclusion</h3> Our results suggest that BCG vaccination can be a new protective strategy against neonatal hyperoxic lung injury. These benefical effects may be interpreted with its immunomodulatory effects on proinflammatory-antiinflammatory cytocin balance and expression of growth factors.

The IGF-I/IGF-R1 pathway regulates postnatal lung growth and is a nonspecific regulator of alveologenesis in the neonatal rat

IGF-I, IGF-II, and the IGF-I receptor are widely distributed throughout the neonatal rat lung on days 4, 7, 10, and 14 of life, with a similar abundance at each of these time points. Injection of 20 μg/g of a truncated soluble IGF-I receptor on days 2 and 5 of life, to decoy ligand away from the endogenous IGF-I receptor, reduced lung weight and lung-to-body weight ratio, reduced lung tissue fraction, and impaired alveolar formation, as assessed by secondary crest formation and mean linear intercepts on day 7 of life. Lung procollagen I content and elastin fiber density were also reduced. Injection of 100 μg/day of neutralizing anti-IGF-I, to prevent IGF-I from binding to the IGF-I receptor, on days 3, 4, and 5 of life reduced tissue fraction and elastin fiber density and impaired alveolar formation on day 6 of life. Both interventions reduced total lung cell and secondary crest cell DNA synthesis and small vessel counts per unit area, but these effects were lost after normalization to the reduced tissue fraction. These findings are consistent with a role for IGF-I binding to the IGF-I receptor in postnatal lung growth and on alveologenesis through a nonspecific positive effect on DNA synthesis. Injection of 100 μg/day of neutralizing anti-IGF-II, to prevent IGF-II from binding to the IGF-I receptor, on days 3, 4, and 5 of life had no effect on total lung cell DNA synthesis per unit area on day 6 of life, and a role for IGF-II in postnatal alveologenesis was not further pursued.

Lipid hydroperoxide formation regulates postnatal rat lung cell apoptosis and alveologenesis

An acute increase in oxygen tension after birth imposes an oxidative stress upon the lung. We hypothesized that the resultant increase in reactive oxygen species, specifically lipid hydroperoxides, would trigger postnatal alveologenesis and physiological lung cell apoptosis in the neonatal rat. Neonatal rats were either untreated or treated daily with subcutaneous vehicle or diphenyl phenyl diamine, a scavenger of lipid hydroperoxides and inhibitor of lipid peroxidation, from day 1 to 6 of life. Alveolar formation and physiological lung cell apoptosis were assessed by morphometry, immunohistochemistry, and Western blot analyses on day 7 samples. Substitution experiments were conducted using the prototypic lipid hydroperoxide t-butylhydroperoxide. At a minimum effective dose of 15μg/g body wt, treatment with diphenyl phenyl diamine resulted in a significant increase in tissue fraction and mean linear intercept and significant reductions in small peripheral blood vessels, secondary crest formation, lung and secondary crest cell DNA synthesis, and estimated alveolar number. Decreased numbers of apoptotic type II pneumocytes and mesenchymal cells, and decreased contents of proapoptotic cleaved caspase-3 and -7 and cytoplasmic cytochrome c, and an increase in antiapoptotic Bcl-xL were found in lungs treated with diphenyl phenyl diamine. A prevention of selected changes induced by diphenyl phenyl diamine was observed with concurrent treatment with intraperitoneal t-butylhydroperoxide, at a minimally effective dose of 187μg/g body wt. We conclude that oxidative stress after birth induces lipid hydroperoxide formation, which, in turn, triggers postnatal alveologenesis and physiological lung cell apoptosis in the neonatal rat.

Disrupted postnatal lung development in heme oxygenase-1 deficient mice

BackgroundHeme oxygenase (HO) degrades cellular heme to carbon monoxide, iron and biliverdin. The HO-1 isoform is both inducible and cyto-protective during oxidative stress, inflammation and lung injury. However, little is known about its precise role and function in lung development. We hypothesized that HO-1 is required for mouse postnatal lung alveolar development and that vascular expression of HO-1 is essential and protective during postnatal alveolar development.MethodsNeonatal lung development in wildtype and HO-1 mutant mice was evaluated by histological and molecular methods. Furthermore, these newborn mice were treated with postnatal dexamethasone (Dex) till postnatal 14 days, and evaluated for lung development.ResultsCompared to wildtype littermates, HO-1 mutant mice exhibited disrupted lung alveolar structure including simplification, disorganization and reduced secondary crest formation. These defects in alveolar development were more pronounced when these mice were challenged with Dex treatment. Expression levels of both vascular endothelial and alveolar epithelial markers were also further decreased in HO-1 mutants after Dex treatment.ConclusionsThese experiments demonstrate that HO-1 is required in normal lung development and that HO-1 disruption and dexamethasone exposure are additive in the disruption of postnatal lung growth. We speculate that HO-1 is involved in postnatal lung development through modulation of pulmonary vascular development.

A Subset of Epithelial Cells with CCSP Promoter Activity Participates in Alveolar Development

Alveolar formation is hallmarked by the transition of distal lung saccules into gas exchange units through the emergence of secondary crests and an exponential increase in surface area. Several cell types are involved in this complex process, including families of epithelial cells that differentiate into alveolar type I and II cells. Subsets of cells expressing Clara cell secretory protein (CCSP) have been identified in both lung and bone marrow compartments, and are described as a progenitor/stem cell pool involved in airway regeneration and alveolar homeostasis. Whether these cells also participate in alveolar formation during postnatal development remains unknown. Based on their regenerative capacity, we asked whether these cells participate in alveogenesis. We used a previously described transgenic mouse model (CCSP-tk) in which Ganciclovir exposure selectively depletes all cells with CCSP promoter activity through intracellular generation of a toxic metabolite of thymidine kinase. Our results showed that Ganciclovir treatment in newborn CCtk mice depleted this cell population in lung airways and bone marrow, and was associated with alveolar hypoplasia and respiratory failure. Hypoplastic lungs had fewer alveolar type I and II cells, with impaired secondary crest formation and decreased vascular endothelial growth factor expression in distal airways. These findings are consistent with a model in which a unique population of cells with CCSP promoter activity that expresses vascular endothelial growth factor participates in alveolar development.

Therapeutic effects of hypercapnia on chronic lung injury and vascular remodeling in neonatal rats

Permissive hypercapnia, achieved using low tidal volume ventilation, has been an effective protective strategy in patients with acute respiratory distress syndrome. To date, no such protective effect has been demonstrated for the chronic neonatal lung injury, bronchopulmonary dysplasia. The objective of our study was to determine whether evolving chronic neonatal lung injury, using a rat model, is resistant to the beneficial effects of hypercapnia or simply requires a less conservative approach to hypercapnia than that applied clinically to date. Neonatal rats inhaled air or 60% O2 for 14 days with or without 5.5% CO2. Lung parenchymal neutrophil and macrophage numbers were significantly increased by hyperoxia alone, which was associated with interstitial thickening and reduced secondary crest formation. The phagocyte influx, interstitial thickening, and impaired alveolar formation were significantly attenuated by concurrent hypercapnia. Hyperoxic pups that received 5.5% CO2 had a significant increase in alveolar number relative to air-exposed pups. Increased tyrosine nitration, a footprint for peroxynitrite-mediated reactions, arteriolar medial wall thickening, and both reduced small peripheral pulmonary vessel number and VEGF and angiopoietin-1 (Ang-1) expression, which were observed with hyperoxia, was attenuated by concurrent hypercapnia. We conclude that evolving chronic neonatal lung injury in a rat model is responsive to the beneficial effects of hypercapnia. Inhaled 5.5% CO2 provided a significant degree of protection against parenchymal and vascular injury in an animal model of chronic neonatal lung injury likely due, at least in part, to its inhibition of a phagocyte influx.

Loss of PECAM-1 Function Impairs Alveolarization

The final stage of lung development in humans and rodents occurs principally after birth and involves the partitioning of the large primary saccules into smaller air spaces by the inward protrusion of septae derived from the walls of the saccules. Several observations in animal models implicate angiogenesis as critical to this process of alveolarization, but all anti-angiogenic treatments examined to date have resulted in endothelial cell (EC) death. We therefore targeted the function of platelet endothelial cell adhesion molecule, (PECAM-1), an EC surface molecule that promotes EC migration and has been implicated in in vivo angiogenesis. Administration of an anti-PECAM-1 antibody that inhibits EC migration, but not proliferation or survival in vitro, disrupted normal alveolar septation in neonatal rat pups without reducing EC content. Three-dimensional reconstruction of lungs showed that pups treated with a blocking PECAM-1 antibody had remodeling of more proximal branches resulting in large tubular airways. Subsequent studies in PECAM-1-null mice confirmed that the absence of PECAM-1 impaired murine alveolarization, without affecting EC content, proliferation, or survival. Further, cell migration was reduced in lung endothelial cells isolated from these mice. These data suggest that the loss of PECAM-1 function compromises postnatal lung development and provide evidence that inhibition of EC function, in contrast to a loss of viable EC, inhibits alveolarization.

Effects of Erythropoietin on Hyperoxic Lung Injury in Neonatal Rats

Pulmonary oxygen toxicity is believed to play a prominent role in the lung injury that leads to the development of bronchopulmonary dysplasia (BPD). To determine whether human recombinant erythropoietin (rhEPO) treatment reduces the risk of developing BPD, we investigated the effect of rhEPO treatment on the histopathologic changes seen in hyperoxia-induced lung injury of BPD. Twenty-five rat pups were divided into four groups: air-exposed control group (n = 5), hyperoxia-exposed placebo group (n = 7), hyperoxia-exposed rhEPO-treated group (n = 6), and air-exposed rhEPO-treated group (n = 7). Measurement of alveolar surface area, quantification of secondary crest formation, microvessel count, evaluation of alveolar septal fibrosis, and smooth muscle actin immunostaining were performed to assess hyperoxia-induced changes in lung morphology. Treatment of hyperoxia-exposed animals with rhEPO resulted in a significant increase in the mean alveolar area, number of secondary crests formed, and the microvessel count in comparison with hyperoxia-exposed placebo-treated animals. There was significantly less fibrosis in rhEPO-treated animals. However, treatment of hyperoxia-exposed animals with rhEPO did not result in a significant change in smooth muscle content compared with hyperoxia-exposed placebo treated animals. Our results suggest treatment with rhEPO during hyperoxia exposure is associated with improved alveolar structure, enhanced vascularity, and decreased fibrosis. Therefore, we conclude that treatment of preterm infants with EPO might reduce the risk of developing BPD.

Effect of retinoic acid on oxygen-induced lung injury in the newborn rat

Oxygen-induced lung injury is believed to lead to the development of bronchopulmonary dysplasia (BPD). To determine whether retinoic acid (RA) treatment prevents the development of BPD by minimizing lung injury, we investigated the effect of RA on the histopathologic characteristics of oxygen-induced lung injury in a newborn rat model. Eighteen rat pups were divided into three groups: room air-exposed control group (n=5), oxygen-exposed placebo group (n=7), and RA-treated oxygen-exposed group (n=6). Measurement of alveolar area, quantitation of secondary crest formation, microvessel count, evaluation of alveolar septal fibrosis, and smooth muscle actin (SMA) immunostaining were performed to assess oxygen-induced changes in lung morphology. Treatment of oxygen-exposed animals with RA resulted in a significant increase in mean alveolar area; however, it had no effect on the number of secondary crests and microvessel count. The degree of fibrosis and SMA expression showed a significant decrease in RA-treated animals. We conclude that RA treatment improves alveolar structure and decreases fibrosis in the newborn rat with oxygen-induced lung injury. Extrapolating these findings to humans, we speculate that similar treatment with RA may reduce lung injury in preterm infants at risk for BPD.

Composition of the pulmonary interstitium during normal development of the human fetus.

Normal lung development is dependent on epithelial-mesenchymal interactions. This study was undertaken to examine the structure of the interstitium of the developing human fetal lung, concentrating particularly on the first and second trimesters. Lung tissue was obtained at autopsy from nonmalformed, nonmacerated cases of spontaneous abortion (n = 15), stillbirth (n = 9), and very early neonatal death (n = 5) (range of gestations, 10-42 weeks). Paraffin-embedded tissue sections were examined using immunohistochemical methods to determine expression of collagens I, III, IV, V, and VI; the glycoproteins fibronectin and laminin; and the intermediate filaments vimentin, alpha-smooth muscle actin (alphaSMA), and desmin. Collagens III and VI and cells expressing alphaSMA were present consistently at points of airway branching and secondary crest formation, indicating a role for these components in the initiation and stabilization of airway branches in the developing lung. Desmin expression by stromal cells succeeded alphaSMA temporally, and may represent a marker of terminal smooth muscle differentiation within the airway; it was not detected in the vascular tree. Other components were widely expressed throughout the extracellular matrix, including basement membranes, at all gestations. The spatial and temporal patterns of expression of components of the lung interstitium provide clues to the mechanisms underlying normal human lung development and possible insights into the pathogenesis of fetal and neonatal lung disease.

ORIGIN, EFFECT AND SUPPRESSION OF SECONDARY WAVES

Beach profile formation may be severely affected by secondary waves which, together with the basic wave, always originate from a sinusoidally.moving wave board The ascertainment of this experimental fact is followed by an investigation of the behaviour of the generated waves and their interactions It appears that the many characteristic features, among which the spatial beat phenomenon and the secondary crest formation, are generally in good accordance with the theories of Fontanet [I4j, and Kravtchenko and Santon I 20J It is concluded that an "outer" analysis, eg by plotting the x-tlines of visible peaks, is only of limited use to describe the "inner" character of the complex phenomenon. The subsequent study of the effect of secondary waves on a horizontal sand bed reveals that not only the wave form, but also the sand transport varies spatially, resulting in the formation of bars and troughs This typical behaviour of the onshore-offshore transport is provisionally investigated in a small pulsating water block. Finally, a method is described which suppresses the secondary waves, by using a low rectangular sill on the otherwise horizontal bottom.