• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of ajplungPublished ArticleArchivesSubscriptionsSubmissionsContact UsAJP - Lung Cellular and Molecular PhysiologyAmerican Physiological Society
Am J Physiol Lung Cell Mol Physiol. Oct 2009; 297(4): L641–L649.
PMCID: PMC2770788

Neonatal oxygen adversely affects lung function in adult mice without altering surfactant composition or activity

Abstract

Despite its potentially adverse effects on lung development and function, supplemental oxygen is often used to treat premature infants in respiratory distress. To understand how neonatal hyperoxia can permanently disrupt lung development, we previously reported increased lung compliance, greater alveolar simplification, and disrupted epithelial development in adult mice exposed to 100% inspired oxygen fraction between postnatal days 1 and 4. Here, we investigate whether oxygen-induced changes in lung function are attributable to defects in surfactant composition and activity, structural changes in alveolar development, or both. Newborn mice were exposed to room air or 40%, 60%, 80%, or 100% oxygen between postnatal days 1 and 4 and allowed to recover in room air until 8 wk of age. Lung compliance and alveolar size increased, and airway resistance, airway elastance, tissue elastance, and tissue damping decreased, in mice exposed to 60–80% oxygen; changes were even greater in mice exposed to 100% oxygen. These alterations in lung function were not associated with changes in total protein content or surfactant phospholipid composition in bronchoalveolar lavage. Moreover, surface activity and total and hydrophobic protein content were unchanged in large surfactant aggregates centrifuged from bronchoalveolar lavage compared with control. Instead, the number of type II cells progressively declined in 60–100% oxygen, whereas levels of T1α, a protein expressed by type I cells, were comparably increased in mice exposed to 40–100% oxygen. Thickened bundles of elastin fibers were also detected in alveolar walls of mice exposed to ≥60% oxygen. These findings support the hypothesis that changes in lung development, rather than surfactant activity, are the primary causes of oxygen-altered lung function in children who were exposed to oxygen as neonates. Furthermore, the disruptive effects of oxygen on epithelial development and lung mechanics are not equivalently dose dependent.

Keywords: bronchopulmonary dysplasia, epithelium, hyperoxia, type II cells

bronchopulmonary dysplasia (BPD) is a chronic lung disease often seen in premature infants with very low birth weight (21). At autopsy, lungs of infants who die from BPD are less vascularized, with fewer and larger alveoli (7). Although the pathophysiology of BPD is complex and related in part to gestational age, neonatal hyperoxia is recognized as an important contributing factor to this disease in many infants (see Refs. 3, 12, 17, 37 for review). Premature infants with BPD have low plasma levels of glutathione (59), and hyperoxia in the context of an immature antioxidant defense increases the potential for oxidative stress injury. The use of exogenous surfactant, antenatal steroids, and milder ventilation strategies has markedly increased survival and other improved outcomes for premature infants over the past two decades. However, many patients continue to show decreased lung capacity, even as young adolescents (19, 20, 55). Moreover, these children are often rehospitalized following respiratory infection and are at increased risk for asthma, infection, and other respiratory ailments (60, 67). They also are at higher risk for cerebral palsy, visual and hearing problems, and lower IQ, which are potentially caused by oxidant injury to developing neuronal cells.

A variety of changes at the level of the alveolar epithelium and capillary endothelium have been reported after hyperoxic exposure in animal models. For example, exposure of newborn mice to ≥85% oxygen for 10–14 days disrupts epithelial and endothelial cell proliferation, promotes inflammation, and leads to alveolar dysplasia, fibrosis, and pathological signs of BPD (10, 66). Similarly, premature baboons exposed to hyperoxia exhibit disrupted vascular development, with fewer and larger alveoli that eventually are lined with hyperplastic type II epithelial cells (39, 40). VEGF levels also decline during hyperoxia (33, 41). Since pharmacological inhibition of VEGF disrupts alveolar development (35, 47, 71) and recombinant VEGF partially protects the developing rat lung against hyperoxia (34, 62), the oxygen-dependent loss of VEGF likely contributes to the disrupted endothelial and epithelial development in BPD (1).

Studies in several species of newborn animals exposed to ≥65% oxygen and allowed to recover in room air reveal long-term changes in airway responsiveness and increased lung volumes (13, 16, 46, 58, 70). We previously showed that adult mice exposed as neonates to 100% oxygen for 4 days exhibit increased alveolar size in association with a depletion of type II epithelial cells and an increase of type I cells (50, 70). In contrast, significant changes in endothelium-specific genes were not observed in these mice, implying that vascular defects may be affected only after longer periods of hyperoxia. Since alveolar development occurs over the first 2 wk of life in mice, this short-term (4 day) neonatal oxygen exposure model encompasses the initial phases of alveolar development as relevant for premature infants receiving supplemental oxygen in the immediate postnatal period. The present study extends our prior work in this model (50, 70) to investigate the level of neonatal oxygen exposure sufficient to permanently disrupt lung development and function in adult mice. In addition, experiments directly address whether surfactant-related abnormalities, epithelial cell alterations, or both are present and contribute to deficits in pulmonary function/development in adult mice given neonatal oxygen.

MATERIALS AND METHODS

Exposure of mice to oxygen.

Mice were housed in sterile microisolator cages in a specified pathogen-free environment according to a protocol approved by the University Committee on Animal Resources at the University of Rochester. Newborn C57BL/6J mice from several litters were mixed on the morning of birth and randomly separated into groups. Each group was then exposed to room air (21% oxygen) or 40%, 60%, 80%, or 100% oxygen until postnatal day 4, when the oxygen-exposed pups were returned to room air (70). Specific concentrations of oxygen were achieved with 100% oxygen mixed with medical-grade compressed air as needed, and oxygen levels were directly monitored with an oxygen sensor (model TED-60, Teledyne Analytical Instruments, City of Industry, CA). The mixed gas stream was humidified to 40–70% by passage through deionized water-jacketed Nafion membrane tubing (PermPure) and delivered thorough a 0.22-μm filter before passage into a sealed Lexan polycarbonate chamber [~32 × 14 × 24 inches (30 liters)]. The flow rate was set at 6 l/min, resulting in a complete exchange of gas every 5 min, or 12 changes per hour. Dams were cycled between litters exposed to room air and hyperoxia every 24 h to protect them from acute oxygen toxicity and to ensure that the nutrition provided to the pups exposed to hyperoxia was similar to that provided to the pups exposed to room air.

Lung mechanics.

At 8 wk of age, five mice were anesthetized with pentobarbital sodium (40 ml/kg). The trachea was exposed and connected to a computer-controlled small animal mechanical ventilator (flexiVent, SCIREQ, Montreal, PQ, Canada) as previously described (48). Mice were injected with pancuronium (1 mg/kg ip) to paralyze the diaphragm and then ventilated with 8 ml/kg at a rate of 150 breaths/min with positive end-expiratory pressure of 2 cmH2O. Estimated tissue damping and tissue elastance were obtained from the flexiVent by fitting a model to each impedance spectrum (29, 51). After assessment of lung mechanics, lungs were examined for histology and protein expression.

Lung histology and morphometry.

The left lobe was tied off, and the right lobes were inflation fixed through the trachea for 10 min with 10% neutral-buffered formalin at 16 cmH2O pressure. The trachea was tied off, the lungs were removed, and the right lobes were further fixed overnight at 4°C. The fixed lobe was dehydrated in graded alcohol and embedded in paraffin. Sections (5 μm) were prepared and stained with hematoxylin-eosin or Hart's elastin stain (65). Fifteen randomly chosen areas were photographed with a ×10 objective of a microscope (Eclipse 80i, Nikon Instruments, Melville, NY). Mean linear intercepts and mean chord lengths of each image were measured using NIS-Elements AR software (Nikon Instruments). Large airways and vessels identified by the software were excluded from analysis.

Immunohistochemistry.

Paraffin-embedded sections were rehydrated and incubated with primary antibodies against rabbit pro-surfactant protein C (pro-SP-C, 1:100 dilution; Santa Cruz Biotechnology) overnight at 4°C. Immune complexes were captured with fluorescently labeled secondary antibodies before sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Stained sections were visualized with a fluorescence microscope (model E800, Nikon Instruments), and images were captured with a digital camera (SPOT-RT, Diagnostic Instruments, Sterling Heights, MI). Five random images at ×20 magnification were captured from four separate mice, and the number of pro-SP-C-positive cells, normalized to the number of DAPI-positive nuclei per field, was quantified. Images were discarded if they contained >50% airway.

Western blot analysis.

The left lobe was homogenized with a Polytron in ice-cold lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 2 mM EDTA, 25 mM β-glycerophosphate, 25 mM NaF, 17.4 μg/ml PMSF, 2% Triton X-100, 3.0% Igepal CA-630, 9.5 μg/ml aprotonin, 10 μg/ml Igepal, 10 μg/ml pepstatin A, and 10 μg/ml leupeptin. The lysates were incubated on ice for 20 min and then centrifuged at 14,000 rpm for 10 min. Total protein was measured in supernatants using the BCA protein assay reagent kit (Pierce, Rockford, IL). Equivalent amounts of protein were resolved on Tris·HCl SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membrane. After the membranes were blocked in 5% nonfat dry milk, they were incubated overnight with goat anti-platelet endothelial cell adhesion molecule (PECAM, 1:200 dilution; Santa Cruz Biotechnology), hamster anti-T1α (1:2,000 dilution; Iowa Hybridoma Bank), rabbit anti-pro-SP-C (1:500 dilution; Millipore), rabbit anti-Clara cell secretory protein (CCSP, 1:10,000 dilution; kindly provided by Dr. Barry Stripp, Duke University), or anti-β-actin (1:5,000 dilution; Sigma, St. Louis, MO) antibody and then with the appropriate horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). Immune complexes were detected with enhanced chemiluminescence (ECL kit, GE Lifesciences, Piscataway, NJ) and visualized on a Fluorchem gel documentation system (Alpha Innotech, San Leandro, CA) or by exposure to blue-sensitive film (Laboratory Products Sales, Rochester, NY). Band intensities were quantified using Image J software.

Lung surfactant composition and aggregate content.

Bronchoalveolar lavage (BAL) was collected from additional mice by instillation of 1 ml of ice-cold saline 10 times into the lung via the trachea. Recovered BAL fluid was immediately centrifuged at 150 g for 10 min to remove cells, and samples from 10 mice from each concentration of oxygen were pooled for analyses of composition and surface activity. Total phospholipid in cell-free BAL fluid was determined by the assay of Ames (2), and phospholipid classes were defined by thin-layer chromatography with a solvent system of chloroform-methanol-2-propanol-triethylamine-water (30:9:25:25:7, by volume) (63). The total protein content of whole cell-free BAL was measured by the Lowry assay (9) modified by the addition of 15% SDS to allow accurate quantitation in the presence of lipid. Large surfactant aggregates were obtained by centrifugation of cell-free BAL fluid at 12,500 g for 30 min and assessed for composition and surface activity. The content of large aggregates as a percentage of total BAL phospholipid was defined by phosphate assay (2), and large-aggregate protein content (total protein and hydrophobic protein following extraction by chloroform-methanol) was measured by the SDS-modified Lowry assay (9).

Surfactant activity studies.

The surface activity of resuspended large aggregates was assessed during cycling at a physiological rate of 20 cycles/min at 37 ± 0.5°C on a pulsating bubble surfactometer (General Transco, Largo, FL; formerly Electronetics, Buffalo, NY) (22). Briefly, an air bubble, communicating with ambient air, was formed in a 40-μl sample of the dispersed surfactant held in a sample chamber. The bubble was pulsated between maximum and minimum radii of 0.55 and 0.4 mm, and surface tension at minimum bubble radius (minimum surface tension, 50% area compression) was calculated as a function of time of pulsation from the measured pressure drop across the bubble interface with use of the Laplace equation for a spherical interface (22, 24). Surfactant samples were examined at a uniform phospholipid concentration of 1 mg/ml in 150 mM NaCl + 2 mM CaCl2 (pH 7.0).

Statistical analyses.

Values means ± SD. Group means were compared by ANOVA, and individual conditions were compared post hoc using Fisher's procedure with Statview statistical software (Abacus Concepts, Piscataway, NJ). P < 0.05 was considered significant.

RESULTS

Neonatal oxygen alters lung development and function in adult mice.

Newborn mice were exposed to room air (21% oxygen) or 40%, 60%, 80%, or 100% oxygen between postnatal days 1 and 4 and returned to room air until 8 wk of age, when changes in lung structure were studied. Alveolar simplification was clearly evident in adult mice exposed to 60% and 80% oxygen and was demonstrably greater in mice exposed to 100% oxygen (Fig. 1, A–E). Alveolar size was quantified by measurement of mean linear intercept and mean chord length of individual alveoli. Alveolar size increased by 15% in adult mice exposed to 60% or 80% oxygen and by 35% in mice exposed to 100% oxygen (Fig. 1F; P < 0.03–0.008). Consistent with these histological findings, lung compliance increased in mice exposed to 60% and 80% oxygen as neonates, with further increases in mice exposed to 100% oxygen (Fig. 2A). In contrast to alveolar size and compliance, neonatal oxygen exposure reduced tissue damping, tissue elastance, airway elastance, and airway resistance, but the effects were not equivalently dose dependent (Fig. 2, B–E). For example, tissue damping, a measure of energy dissipated into lung tissues, decreased in mice exposed to 60% oxygen and was not significantly suppressed further with 80% or 100% oxygen. On the other hand, tissue and airway elastance, a measure of energy conservation, was not significantly altered at 60% oxygen but was significantly reduced at 80% and 100% oxygen. Airway resistance was statistically unchanged in mice exposed to 60% or 80% oxygen but was significantly reduced in mice exposed to 100% oxygen.

Fig. 1.
Neonatal oxygen promotes alveolar simplification in adult mice. A–E: representative images of hematoxylin-eosin sections of lungs obtained from adult mice exposed to 21% oxygen (room air) or 40%, 60%, 80%, or 100% oxygen between postnatal days ...
Fig. 2.
Neonatal oxygen disrupts normal pulmonary mechanics. A–E: lung compliance, tissue damping, tissue elastance, airway elastance, and airway resistance in adult mice exposed to 21% oxygen (room air) or 40%, 60%, 80%, or 100% oxygen [fraction of inspired ...

Neonatal oxygen disrupts epithelial development.

Our prior work showed fewer alveolar type II epithelial cells and more type I epithelial cells, as defined by reduced expression of pro-SP-C (type II cell marker) and increased expression of T1α protein (type I cell marker) (70), in adult mice exposed to 100% oxygen as neonates. To determine the amount of oxygen needed to alter alveolar epithelial cell development, the expression of cell-specific markers was investigated by Western blotting of lung homogenates prepared from adult mice exposed as neonates to different levels of hyperoxia. Oxygen exposure suppressed expression of the 15-kDa pro-SP-C precursor, with significant changes observed at ≥60% oxygen (Fig. 3, A and B). Quantitation of band intensities revealed that mean levels of pro-SP-C were reduced to the greatest extent in mice exposed to 100% oxygen, although differences were not statistically significant compared with 60% or 80% oxygen (Fig. 3B). A similar pattern of reduction was seen with the expression of CCSP, a protein expressed by airway Clara cells (Fig. 3, A and D). Exposure to ≥40% oxygen increased the expression of T1α in all groups compared with controls (Fig. 3, A and C), whereas expression of PECAM (or CD31) was not significantly altered by oxygen (Fig. 3, A and E).

Fig. 3.
Neonatal oxygen alters expression of epithelial cell-specific markers in adult mice. A: immunoblots of lungs of adult mice exposed to 21% oxygen (room air) or 40%, 60%, 80%, or 100% oxygen and treated with antibodies against pro-surfactant protein C (pro-SP-C), ...

Immunohistochemistry was used to further assess changes in the number of type II cells associated with reduced levels of pro-SP-C in hyperoxia. Pro-SP-C-positive cells were readily observed in adult mice exposed to room air at birth and were depleted in mice exposed to supplemental oxygen (Fig. 4, A–E). The numbers of pro-SP-C-positive cells were quantified in tissue sections and normalized to the number of DAPI-positive nuclei in the image. The proportion of type II cells in adult mice exposed to room air at birth was 22.1 ± 0.3% (Fig. 4F). This percentage progressively declined in an oxygen dose-dependent manner, reaching a nadir of 10.2 ± 0.1% in mice exposed to 100% oxygen as neonates (P < 0.0001). Although the total number of DAPI-positive cells counted in mice exposed to room air (261 ± 22) and 40% (240 ± 41.4) and 60% (249 ± 56.6) oxygen was not different (P = 0.3), it significantly declined in mice exposed to 80% (220 ± 46.5) and 100% (214.9 ± 70.6) oxygen (P < 0.03). However, the 15–20% reduction in total DAPI-positive alveolar cells in mice exposed to 80% and 100% oxygen did not account for the proportionally greater loss of type II cells in the lungs of these animals (Fig. 4F).

Fig. 4.
Neonatal hyperoxia suppresses the number of type II epithelial cells in adult lungs. A–E: sections of lungs obtained from adult mice exposed to 21% oxygen (room air) or 40%, 60%, 80%, or 100% oxygen were stained with antibodies against pro-SP-C ...

Neonatal oxygen does not affect surfactant composition or biophysical activity.

The loss of type II cells in adult mice exposed to neonatal hyperoxia has the potential to cause surfactant deficiency and, hence, related deficits in lung mechanics or function. However, surfactant dysfunction would not be consistent with the increases in lung compliance observed in mice exposed to 60–100% oxygen as neonates noted earlier (Fig. 2A). To assess oxygen-induced surfactant dysfunction in our model more fully, experiments directly investigated the composition and surface activity of BAL from adult mice exposed to the maximum level of 100% oxygen for 4 days as neonates compared with room air. The amounts of total phospholipid and total protein in cell-free BAL did not differ between adult mice exposed as neonates to 100% oxygen and those exposed to room air (see supplemental Table 1 in the online version of this article). In addition, the phospholipid class composition of whole BAL was not altered by neonatal hyperoxic exposure (see supplemental Table 2). The percent content of centrifuged large surfactant aggregates in cell-free BAL also did not significantly differ between mice exposed as neonates to oxygen and those exposed to room air (see supplemental Table 1), and large aggregates from these animals had equivalent amounts of total protein and hydrophobic protein (see supplemental Table 3). Finally, direct measurements of surface tension lowering for resuspended large aggregates on a pulsating bubble apparatus showed that surfactant biophysical activity was not different between adult mice exposed to neonatal hyperoxia and those exposed to room air (Fig. 5).

Fig. 5.
Neonatal oxygen does not affect surface tension-lowering properties of surfactant obtained from adult lungs. Large aggregates were resuspended in buffered 150 mM NaCl and 2 mM CaCl2 (pH 7.0) and measured for surface activity on a pulsating bubble surfactometer ...

Neonatal oxygen alters elastin deposition.

The observation that neonatal oxygen had not disrupted surfactant biophysical activity suggested that increased lung compliance might be attributed to changes in elastogenic molecules. To test this hypothesis, we stained lungs of adult mice exposed to varying levels of oxygen as neonates with Hart's elastin stain. The lungs of mice exposed to room air or 40% oxygen displayed elastin bundles at tips of secondary crests (Fig. 6, A and B). Dense bundles of elastic fibers lining alveolar walls were evident in mice exposed to 60% and 80% oxygen and were widely observed in mice exposed to 100% oxygen (Fig. 6, C and D). These fibers were not observed in mice exposed to room air or 40% oxygen.

Fig. 6.
Neonatal oxygen alters elastin deposition in adult lungs. A–E: representative images of lungs obtained from adult mice exposed to 21% oxygen (room air) or 40%, 60%, 80%, or 100% oxygen stained with Hart's elastin stain and counterstained with ...

DISCUSSION

It is generally accepted that hyperoxia, ventilation, and immaturity of antioxidant defenses cause oxidative stress, which promotes BPD and disruption of normal lung development (3, 12, 17, 37). The view that oxidative stress detrimentally affects lung development is clinically supported by the observation that premature infants instilled with recombinant Cu,Zn-SOD had improved pulmonary function at 1 yr of corrected age (15). Consistent with this clinical finding, overexpression of extracellular SOD in respiratory epithelial cells of neonatal transgenic mice preserved type II cell proliferation during the first 3 days of exposure to 95% oxygen (5). However, despite its functional importance, little is known about specific levels of oxidative stress that permanently affect normal lung development and function. In the present study, we provide evidence that a 4-day exposure of neonates to >40% oxygen is sufficient to permanently alter alveolar epithelial development and lung function in adult mice. Neonatal exposure to 60% and 80% oxygen generally caused similar changes, whereas exposure to 100% oxygen was significantly more injurious. However, at this maximum exposure level, there were no apparent changes in surfactant phospholipid composition, large surfactant aggregate content in BAL, large surfactant aggregate protein (total or hydrophobic protein), or large surfactant aggregate biophysical activity. Although these studies do not directly address whether surfactant pool sizes were altered, increased lung compliance was associated with the presence of thick bundles of elastin fibers within alveolar walls.

In the present study, 4 days of neonatal exposure to 40% oxygen minimally affected lung structure and function in adult mice, whereas exposure to 60–80% oxygen was modestly disruptive and exposure to 100% oxygen was the most severely disruptive. Some degree of pulmonary oxidative stress will always occur at birth, when the developing lungs are exposed to room air for the first time. However, our results indicate that normal (full-term) mouse lungs have the capacity to detoxify levels of stress produced by exposure to 40% oxygen in terms of the pulmonary variables investigated. When neonatal oxidant stress was increased by exposure to 60–80% oxygen, measurable increases in alveolar size and changes in lung mechanics were observed, as were changes in epithelial cell markers and reductions in the number of type II cells. Because 60% and 80% oxygen caused similar changes in lung structure and function, these levels may perturb lung development via a common pathway. Hypothetically, and as shown in HeLa cells, this range of oxygen tensions is sufficient to inhibit cell proliferation and synthesis of RNA and protein (56). Indeed, hyperoxia suppressed tritiated thymidine or bromodeoxyuridine incorporation in neonatal rodents (49, 66). This transient growth arrest of individual cell types may dysregulate the coordinated proliferation and differentiation of epithelial and mesenchymal cells required for alveolar development. Consistent with this concept, type II cell proliferation was temporally shifted in newborn mice exposed to 100% oxygen for 4 days and returned to room air (70). On the other hand, lung structure and function were most severely affected by 100% oxygen, implying involvement of additional mechanisms such as cell death and inflammation (4). We found that the numbers of alveolar cells were significantly reduced in adult mice exposed to 80% and 100% oxygen. Thus loss of progenitor cells critical for normal development may be occurring in mice exposed to high levels of oxygen.

Our results showing reduced pro-SP-C expression and decreased type II cell numbers in adult mice exposed as neonates to 100% oxygen agree with prior work examining pro-SP-C, pro-SP-B, and enhanced green fluorescent protein controlled by the SP-C promoter in this animal model (70). Since type II cells are progenitor cells for type I cells, short-term neonatal hyperoxia followed by long-term recovery in room air may have stimulated the differentiation of type II cells to type I cells. However, the increased expression of T1α did not inversely correlate across different levels of oxygen with the loss of pro-SP-C. This suggests that the increased expression of T1α may reflect an increase in the number of type I cells as well as an increase in the expression per cell. Despite these changes, surfactant composition and activity were unaltered in adult mice exposed to 100% oxygen as neonates. The various surfactant-related assessments used here have been shown to be able to document surfactant dysfunction in multiple animal models of acute lung injury (14, 25, 27, 44, 45, 54, 57, 69). Acute surfactant dysfunction directly following severe hyperoxic exposure in adult animals is well documented (27, 36, 44, 45), but this was not true for the recovered adult animals here that were exposed to 100% oxygen as neonates. Adult mice with neonatal exposure to 60–100% oxygen had increased, rather than decreased, lung compliance, again not consistent with surfactant dysfunction. Our experiments did not specifically measure the concentrations of specific surfactant apoproteins in BAL or large aggregates or whether surfactant levels were different between the groups of mice. However, the equivalent contents of hydrophobic protein in large aggregates from oxygen-exposed and control mice strongly suggest that SP-B/SP-C was not changed substantially in amount and imply that sufficient amounts of these proteins were produced, despite the loss of type II cells in this model. Similarly, the equivalent total protein contents of large aggregates from oxygen-exposed and control mice are consistent with no substantial change in total apoprotein content, and the identical large-aggregate surface activity further indicates no substantial alteration in the biophysically functional surfactant apoproteins (SP-A, SP-B, and SP-C). Taken together, these findings imply that the altered lung mechanics observed in the adult mice studied here are the result of structural changes associated with impaired development as opposed to surfactant dysfunction.

Consistent with this concept, neonatal oxygen altered the balance of alveolar type I and II cells and the deposition of elastin fibers. Hypothetically, an increase in the number of elongated type I cells at the expense of cuboidal type II cells could allow alveoli to be more expandable and, hence, exhibit increased lung compliance. At the same time, thick bundles of elastin fibers were observed within alveolar walls of mice exposed to ≥60% oxygen. Elastin fibers normally localize to tips of alveolar secondary crests, which are attenuated or lost in rodents exposed to neonatal hyperoxia and are replaced by more simplified alveolar structures lined with thick bundles of elastin fibers during recovery in room air (8, 65). These abnormal elastin bundles may contribute to increased expandability or compliance activity measured on the flexiVent (28, 43, 61). Although there are no animal models to confirm this hypothesis, it is supported by experimental in silico modeling (6). The increased elastin deposition in the lungs following neonatal oxygen exposure shown in the present study provides a possible mechanism for the airway hyperresponsiveness noted in children born prematurely and treated therapeutically with oxygen as neonates, hence laying the groundwork for future studies in this area (32).

Increased compliance was associated with reduced tissue damping, a measure of tissue rigidity, and tissue elastance, a measure of energy conservation. On the other hand, exposure to ≥60% oxygen reduced airway elastance and airway resistance in adult mice (Fig. 2). Hypothetically, the terminal bronchioles have become less rigid and, hence, more likely to collapse upon end expiration as overdistended alveoli collapse. This would lead to gas trapping and wheezing, symptoms often seen in children who were born prematurely (60, 67). Although many models of emphysema demonstrate alveolar simplification leading to loss of alveolar support and resultant decreased airway stenting and increased airway resistance, our data are consistent with other models of emphysema that result in decreased resistance. Tissue inhibitor of metalloproteinase 3-null mice demonstrate alveolar simplification, as well as decreased resistance, compared with wild-type mice (42). Fetal lambs treated with betamethasone and thyroxine demonstrate alveolar simplification and decreased airway resistance (68). These observations may be a result of a decrease in the length of the airway tree or an increase in the airway radius. The alveolar simplification and increased compliance noted in our model also make it relevant to adult pulmonary emphysema (18, 23, 64). Although the present work supports altered alveolarization, rather than surfactant deficiency/dysfunction, as the primary mechanism of abnormal pulmonary mechanics following neonatal oxygen exposure, the loss of alveoli may dominate any effect observed with surfactant function (26, 38).

It is difficult to extrapolate the dose-dependent effects of oxygen in this study to outcomes in children born prematurely, because levels and duration of oxygen supplementation vary widely among premature infants depending on need. Because lung development was moderately and comparably affected by 60–80% oxygen and more severely by 100% oxygen in our study, interventions that target the reduction of oxidant stress in premature infants exposed to >80% oxygen may have greater potential for efficacy. However, there is clearly variability in available animal studies investigating the effects of neonatal hyperoxic exposure. For instance, altered lung compliance and interstitial thickening have been seen in newborn rats exposed to 40% oxygen for 6 days and allowed to recover in room air for 2 wk (11), whereas lung structure and function were minimally affected by 4 days of 40% oxygen in mice in the present study. Although the use of rats vs. mice may influence these differences, they also likely reflect the length of oxygen exposure. This hypothesis is consistent with an observation that 20 days of supplemental oxygen had little effect on forced expired volume in 1 s (FEV1) assessed in 11-yr-old children, but each additional week of exposure was associated with a progressive 3% loss in FEV1 (31). Thus, reducing the length of postnatal hyperoxic exposure for premature infants may be as beneficial as reducing absolute oxygen exposure levels.

Besides hyperoxia, there is growing evidence that pre- and postnatal environmental factors such as tobacco smoke and infection impact the normal structural development and programming of the lung and immune system (30, 52, 53). Reactive oxygen species are likely to play an important role in this process. The present findings using hyperoxia suggest that the developing lung is responsive to three levels of oxidative stress that lead to normal, modest, or severe deficits in lung structure and function. This may explain why children born prematurely or exposed to environmental pollutants display varying degrees of lung deficits. Understanding how different levels of oxidative stress reprogram normal lung development and, hence, immune functions could provide new opportunities for improving human health.

GRANTS

This work was supported in part by March of Dimes Birth Defects Foundation Grant 6-FY08-264, National Heart, Lung, and Blood Institute Grants HL-091968 (M. O'Reilly), HL-81148, HL-59956 (D. Dean), and P50 HL-084945 (S. McGrath-Morrow), and a Flight Attendant Medical Research Institute Young Clinical Scientist Award (S. McGrath-Morrow). The animal inhalation facility was supported by National Institutes of Health Center Grant ES-01247.

Supplementary Material

[Supplemental Tables]

REFERENCES

1. Abman SH. Bronchopulmonary dysplasia: “a vascular hypothesis.” Am J Respir Crit Care Med 164: 1755–1756, 2001. [PubMed]
2. Ames BN. Assay of inorganic phosphate, total phosphate, and phosphatases. Methods Enzymol 8: 115–118, 1966
3. Askie LM, Henderson-Smart DJ, Irwig L, Simpson JM. Oxygen-saturation targets and outcomes in extremely preterm infants. N Engl J Med 349: 959–967, 2003. [PubMed]
4. Auten RL, Jr, Mason SN, Tanaka DT, Welty-Wolf K, Whorton MH. Anti-neutrophil chemokine preserves alveolar development in hyperoxia-exposed newborn rats. Am J Physiol Lung Cell Mol Physiol 281: L336–L344, 2001. [PubMed]
5. Auten RL, O'Reilly MA, Oury TD, Nozik-Grayck E, Whorton MH. Transgenic extracellular superoxide dismutase protects postnatal alveolar epithelial proliferation and development during hyperoxia. Am J Physiol Lung Cell Mol Physiol 290: L32–L40, 2006. [PMC free article] [PubMed]
6. Bates JH, Davis GS, Majumdar A, Butnor KJ, Suki B. Linking parenchymal disease progression to changes in lung mechanical function by percolation. Am J Respir Crit Care Med 176: 617–623, 2007. [PMC free article] [PubMed]
7. Bhatt AJ, Pryhuber GS, Huyck H, Watkins RH, Metlay LA, Maniscalco WM. Disrupted pulmonary vasculature and decreased vascular endothelial growth factor, Flt-1, and TIE-2 in human infants dying with bronchopulmonary dysplasia. Am J Respir Crit Care Med 164: 1971–1980, 2001. [PubMed]
8. Blanco LN, Frank L. The formation of alveoli in rat lung during the third and fourth postnatal weeks: effect of hyperoxia, dexamethasone, and deferoxamine. Pediatr Res 34: 334–340, 1993. [PubMed]
9. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37: 911–917, 1959. [PubMed]
10. Bonikos DS, Bensch KG, Ludwin SK, Northway WH., Jr Oxygen toxicity in the newborn. The effect of prolonged 100 percent O2 exposure on the lungs of newborn mice. Lab Invest 32: 619–635, 1975. [PubMed]
11. Bucher JR, Roberts RJ. The development of the newborn rat lung in hyperoxia: a dose-response study of lung growth, maturation, and changes in antioxidant enzyme activities. Pediatr Res 15: 999–1008, 1981. [PubMed]
12. Chess PR, D'Angio CT, Pryhuber GS, Maniscalco WM. Pathogenesis of bronchopulmonary dysplasia. Semin Perinatol 30: 171–178, 2006. [PubMed]
13. Dauger S, Ferkdadji L, Saumon G, Vardon G, Peuchmaur M, Gaultier C, Gallego J. Neonatal exposure to 65% oxygen durably impairs lung architecture and breathing pattern in adult mice. Chest 123: 530–538, 2003. [PubMed]
14. Davidson BA, Knight PR, Wang Z, Chess PR, Holm BA, Russo TA, Hutson A, Notter RH. Surfactant alterations in acute inflammatory lung injury from aspiration of acid and gastric particulates. Am J Physiol Lung Cell Mol Physiol 288: L699–L708, 2005. [PubMed]
15. Davis JM, Parad RB, Michele T, Allred E, Price A, Rosenfeld W. Pulmonary outcome at 1 year corrected age in premature infants treated at birth with recombinant human CuZn superoxide dismutase. Pediatrics 111: 469–476, 2003. [PubMed]
16. Denis D, Fayon MJ, Berger P, Molimard M, De Lara MT, Roux E, Marthan R. Prolonged moderate hyperoxia induces hyperresponsiveness and airway inflammation in newborn rats. Pediatr Res 50: 515–519, 2001. [PubMed]
17. Deulofeut R, Golde D, Augusto S. Treatment-by-gender effect when aiming to avoid hyperoxia in preterm infants in the NICU. Acta Paediatr 96: 990–994, 2007. [PubMed]
18. Dillon TJ, Walsh RL, Scicchitano R, Eckert B, Cleary EG, McLennan G. Plasma elastin-derived peptide levels in normal adults, children, and emphysematous subjects. Physiologic and computed tomographic scan correlates. Am Rev Respir Dis 146: 1143–1148, 1992. [PubMed]
19. Doyle LW. Respiratory function at age 8–9 years in extremely low birthweight/very preterm children born in Victoria in 1991–1992. Pediatr Pulmonol 41: 570–576, 2006. [PubMed]
20. Doyle LW, Faber B, Callanan C, Freezer N, Ford GW, Davis NM. Bronchopulmonary dysplasia in very low birth weight subjects and lung function in late adolescence. Pediatrics 118: 108–113, 2006. [PubMed]
21. Eber E, Zach MS. Long term sequelae of bronchopulmonary dysplasia (chronic lung disease of infancy). Thorax 56: 317–323, 2001. [PMC free article] [PubMed]
22. Enhorning G. Pulsating bubble technique for evaluating pulmonary surfactant. J Appl Physiol 43: 198–203, 1977. [PubMed]
23. Guenard H, Diallo MH, Laurent F, Vergeret J. Lung density and lung mass in emphysema. Chest 102: 198–203, 1992. [PubMed]
24. Hall SB, Bermel MS, Ko YT, Palmer HJ, Enhorning G, Notter RH. Approximations in the measurement of surface tension on the oscillating bubble surfactometer. J Appl Physiol 75: 468–477, 1993. [PubMed]
25. Hickman-Davis J, Wang Z, Chess PR, Page GP, Matalon S, Notter RH. Surfactant dysfunction in SP-A-deficient and iNOS-deficient mice with mycoplasma infection. Am J Respir Cell Mol Biol 36: 103–113, 2007. [PMC free article] [PubMed]
26. Higenbottam T. Pulmonary surfactant and chronic lung disease. Eur J Respir Dis Suppl 153: 222–228, 1987. [PubMed]
27. Holm BA, Notter RH, Siegle J, Matalon S. Pulmonary physiological and surfactant changes during injury and recovery from hyperoxia. J Appl Physiol 59: 1402–1409, 1985. [PubMed]
28. Ito S, Ingenito EP, Brewer KK, Black LD, Parameswaran H, Lutchen KR, Suki B. Mechanics, nonlinearity, and failure strength of lung tissue in a mouse model of emphysema: possible role of collagen remodeling. J Appl Physiol 98: 503–511, 2005. [PubMed]
29. Ito S, Lutchen KR, Suki B. Effects of heterogeneities on the partitioning of airway and tissue properties in normal mice. J Appl Physiol 102: 859–869, 2007. [PubMed]
30. Kajekar R. Environmental factors and developmental outcomes in the lung. Pharmacol Ther 114: 129–145, 2007. [PubMed]
31. Kennedy JD, Edward LJ, Bates DJ, Martin AJ, Dip SN, Haslam RR, McPhee AJ, Staugas RE, Baghurst P. Effects of birthweight and oxygen supplementation on lung function in late childhood in children of very low birth weight. Pediatr Pulmonol 30: 32–40, 2000. [PubMed]
32. Kim do K, Choi SH, Yu J, Yoo Y, Kim B, Koh YY. Bronchial responsiveness to methacholine and adenosine 5′-monophosphate in preschool children with bronchopulmonary dysplasia. Pediatr Pulmonol 41: 538–543, 2006. [PubMed]
33. Klekamp JG, Jarzecka K, Perkett EA. Exposure to hyperoxia decreases the expression of vascular endothelial growth factor and its receptors in adult rat lungs. Am J Pathol 154: 823–831, 1999. [PMC free article] [PubMed]
34. Kunig AM, Balasubramaniam V, Markham NE, Morgan D, Montgomery G, Grover TR, Abman SH. Recombinant human VEGF treatment enhances alveolarization after hyperoxic lung injury in neonatal rats. Am J Physiol Lung Cell Mol Physiol 289: L529–L535, 2005. [PubMed]
35. Le Cras TD, Markham NE, Tuder RM, Voelkel NF, Abman SH. Treatment of newborn rats with a VEGF receptor inhibitor causes pulmonary hypertension and abnormal lung structure. Am J Physiol Lung Cell Mol Physiol 283: L555–L562, 2002. [PubMed]
36. Loewen GM, Holm BA, Milanowski L, Wild LM, Notter RH, Matalon S. Alveolar hyperoxic injury in rabbits receiving exogenous surfactant. J Appl Physiol 66: 1987–1992, 1989 [PubMed]
37. Madan A, Brozanski BS, Cole CH, Oden NL, Cohen G, Phelps DL. A pulmonary score for assessing the severity of neonatal chronic lung disease. Pediatrics 115: e450–e457, 2005. [PubMed]
38. Mall MA, Harkema JR, Trojanek JB, Treis D, Livraghi A, Schubert S, Zhou Z, Kreda SM, Tilley SL, Hudson EJ, O'Neal WK, Boucher RC. Development of chronic bronchitis and emphysema in β-epithelial Na+ channel-overexpressing mice. Am J Respir Crit Care Med 177: 730–742, 2008. [PMC free article] [PubMed]
39. Maniscalco WM, Watkins RH, O'Reilly MA, Shea CP. Increased epithelial cell proliferation in very premature baboons with chronic lung disease. Am J Physiol Lung Cell Mol Physiol 283: L991–L1001, 2002. [PubMed]
40. Maniscalco WM, Watkins RH, Pryhuber GS, Bhatt A, Shea C, Huyck H. Angiogenic factors and alveolar vasculature: development and alterations by injury in very premature baboons. Am J Physiol Lung Cell Mol Physiol 282: L811–L823, 2002. [PubMed]
41. Maniscalco WM, Watkins RH, Roper JM, Staversky R, O'Reilly MA. Hyperoxic ventilated premature baboons have increased p53, oxidant DNA damage and decreased VEGF expression. Pediatr Res 58: 549–556, 2005. [PubMed]
42. Martin EL, Truscott EA, Bailey TC, Leco KJ, McCaig LA, Lewis JF, Veldhuizen RA. Lung mechanics in the TIMP3 null mouse and its response to mechanical ventilation. Exp Lung Res 33: 99–113, 2007. [PubMed]
43. Massa CB, Allen GB, Bates JH. Modeling the dynamics of recruitment and derecruitment in mice with acute lung injury. J Appl Physiol 105: 1813–1821, 2008. [PMC free article] [PubMed]
44. Matalon S, Holm BA, Loewen GM, Baker RR, Notter RH. Sublethal hyperoxic injury to the alveolar epithelium and the pulmonary surfactant system. Exp Lung Res 14: 1021–1033, 1988. [PubMed]
45. Matalon S, Holm BA, Notter RH. Mitigation of pulmonary hyperoxic injury by administration of exogenous surfactant. J Appl Physiol 62: 756–761, 1987. [PubMed]
46. McGrath-Morrow SA, Cho C, Soutiere S, Mitzner W, Tuder R. The effect of neonatal hyperoxia on the lung of p21Waf1/Cip1/Sdi1-deficient mice. Am J Respir Cell Mol Biol 30: 635–640, 2004. [PubMed]
47. McGrath-Morrow SA, Cho C, Zhen L, Hicklin DJ, Tuder RM. Vascular endothelial growth factor receptor 2 blockade disrupts postnatal lung development. Am J Respir Cell Mol Biol 32: 420–427, 2005. [PubMed]
48. Mutlu GM, Machado-Aranda D, Norton JE, Bellmeyer A, Urich D, Zhou R, Dean DA. Electroporation-mediated gene transfer of the Na+,K+-ATPase rescues endotoxin-induced lung injury. Am J Respir Crit Care Med 176: 582–590, 2007. [PMC free article] [PubMed]
49. Northway WH, Jr, Rezeau L, Petriceks R, Bensch KG. Oxygen toxicity in the newborn lung: reversal of inhibition of DNA synthesis in the mouse. Pediatrics 57: 41–46, 1976. [PubMed]
50. O'Reilly MA, Marr SH, Yee M, McGrath-Morrow SA, Lawrence BP. Neonatal hyperoxia enhances the inflammatory response in adult mice infected with influenza A virus. Am J Respir Crit Care Med 177: 1103–1110, 2008. [PMC free article] [PubMed]
51. Pillow JJ, Korfhagen TR, Ikegami M, Sly PD. Overexpression of TGF-α increases lung tissue hysteresivity in transgenic mice. J Appl Physiol 91: 2730–2734, 2001. [PubMed]
52. Pinkerton KE, Joad JP. The mammalian respiratory system and critical windows of exposure for children's health. Environ Health Perspect 108Suppl 3: 457–462, 2000. [PMC free article] [PubMed]
53. Plopper CG, Smiley-Jewell SM, Miller LA, Fanucchi MV, Evans MJ, Buckpitt AR, Avdalovic M, Gershwin LJ, Joad JP, Kajekar R, Larson S, Pinkerton KE, Van Winkle LS, Schelegle ES, Pieczarka EM, Wu R, Hyde DM. Asthma/allergic airways disease: does postnatal exposure to environmental toxicants promote airway pathobiology? Toxicol Pathol 35: 97–110, 2007. [PubMed]
54. Raghavendran K, Davidson BA, Knight PR, Wang Z, Helinski J, Chess PR, Notter RH. Surfactant dysfunction in lung contusion with and without superimposed gastric aspiration in a rat model. Shock 30: 508–517, 2008. [PMC free article] [PubMed]
55. Robin B, Kim YJ, Huth J, Klocksieben J, Torres M, Tepper RS, Castile RG, Solway J, Hershenson MB, Goldstein-Filbrun A. Pulmonary function in bronchopulmonary dysplasia. Pediatr Pulmonol 37: 236–242, 2004. [PubMed]
56. Rueckert RR, Mueller GC. Effect of oxygen tension on HeLa cell growth. Cancer Res 20: 944–949, 1960. [PubMed]
57. Russo TA, Wang Z, Davidson BA, Genagon SA, Beanan JM, Olsen R, Holm BA, Knight PR, Chess PR, Notter RH. Surfactant dysfunction and lung injury due to the E. coli virulence factor hemolysin in a rat pneumonia model. Am J Physiol Lung Cell Mol Physiol 292: L632–L643, 2007. [PubMed]
58. Schulman SR, Canada AT, Fryer AD, Winsett DW, Costa DL. Airway hyperreactivity produced by short-term exposure to hyperoxia in neonatal guinea pigs. Am J Physiol Lung Cell Mol Physiol 272: L1211–L1216, 1997 [PubMed]
59. Smith CV, Hansen TN, Martin NE, McMicken HW, Elliott SJ. Oxidant stress responses in premature infants during exposure to hyperoxia. Pediatr Res 34: 360–365, 1993. [PubMed]
60. Smith VC, Zupancic JA, McCormick MC, Croen LA, Greene J, Escobar GJ, Richardson DK. Rehospitalization in the first year of life among infants with bronchopulmonary dysplasia. J Pediatr 144: 799–803, 2004. [PubMed]
61. Suki B, Ito S, Stamenovic D, Lutchen KR, Ingenito EP. Biomechanics of the lung parenchyma: critical roles of collagen and mechanical forces. J Appl Physiol 98: 1892–1899, 2005. [PubMed]
62. Thebaud B, Ladha F, Michelakis ED, Sawicka M, Thurston G, Eaton F, Hashimoto K, Harry G, Haromy A, Korbutt G, Archer SL. Vascular endothelial growth factor gene therapy increases survival, promotes lung angiogenesis, and prevents alveolar damage in hyperoxia-induced lung injury: evidence that angiogenesis participates in alveolarization. Circulation 112: 2477–2486, 2005. [PubMed]
63. Touchstone JC, Chen JC, Beaver KM. Improved separation of phospholipids in thin-layer chromatography. Lipids 15: 61–62, 1980
64. Vanoirbeek JA, Rinaldi M, De Vooght V, Haenen S, Bobic S, Gayan-Ramirez G, Hoet PH, Verbeken E, Decramer M, Nemery B, Janssens W. Noninvasive and invasive pulmonary function in mouse models of obstructive and restrictive respiratory diseases. Am J Respir Cell Mol Biol In press [PubMed]
65. Veness-Meehan KA, Pierce RA, Moats-Staats BM, Stiles AD. Retinoic acid attenuates O2-induced inhibition of lung septation. Am J Physiol Lung Cell Mol Physiol 283: L971–L980, 2002. [PubMed]
66. Warner BB, Stuart LA, Papes RA, Wispe JR. Functional and pathological effects of prolonged hyperoxia in neonatal mice. Am J Physiol Lung Cell Mol Physiol 275: L110–L117, 1998 [PubMed]
67. Weisman LE. Populations at risk for developing respiratory syncytial virus and risk factors for respiratory syncytial virus severity: infants with predisposing conditions. Pediatr Infect Dis J 22: S33–S39, 2003. [PubMed]
68. Willet KE, Gurrin L, Burton P, Lanteri CJ, Reese AC, Vij J, Matsumoto I, Jobe AH, Ikegami M, Polk D, Newnham J, Kohan R, Kelly R, Sly PD. Differing patterns of mechanical response to direct fetal hormone treatment. Respir Physiol 103: 271–280, 1996. [PubMed]
69. Wright TW, Notter RH, Wang Z, Harmsen AG, Gigliotti F. Pulmonary inflammation disrupts surfactant function during P. carinii pneumonia. Infect Immun 69: 758–764, 2001 [PMC free article] [PubMed]
70. Yee M, Vitiello PF, Roper JM, Staversky RJ, Wright TW, McGrath-Morrow SA, Maniscalco W, Finkelstein JN, O'Reilly MA. Type II epithelial cells are a critical target for hyperoxia-mediated impairment of postnatal lung development. Am J Physiol Lung Cell Mol Physiol 291: L1101–L1111, 2006. [PubMed]
71. Zhao L, Wang K, Ferrara N, Vu TH. Vascular endothelial growth factor co-ordinates proper development of lung epithelium and vasculature. Mech Dev 122: 877–886, 2005. [PubMed]

Articles from American Journal of Physiology - Lung Cellular and Molecular Physiology are provided here courtesy of American Physiological Society
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Compound
    Compound
    PubChem Compound links
  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...