Severe but not moderate hyperoxia of newborn mice causes an emphysematous lung phenotype in adulthood without persisting oxidative stress and inflammation

Newborn mice of the C57BL/6 N strain (Charles River, Sulzfeld, Germany) were treated from birth until PND14 with different concentrations of oxygen to reach normoxic (N; 21% O₂), moderate hyperoxic (mH; 50% O₂) and severe hyperoxic (sH; 75% O₂) normobaric air conditions. This study was approved by the local Commission for Animal Protection. Hyperoxic air was created in a polycarbonate chamber equipped with computerized gas flow control units (Aera FC-R7800; Hitachi Metals, Obuke, Japan) and O₂ gas detector (GfG, Dortmund, Germany), which was large enough for three standard cages. Maximum six newborns of each sex per lactating dam were exposed to either N or mH conditions in one experimental cycle or they were exposed to either N or sH conditions in another experimental cycle. Lactating dams were rotated between hyperoxic and normoxic conditions every day to minimize maternal effects. Because of this experimental procedure, our study generated more mice of the N than mH or sH group. Analyses were performed at the PNDs 14, 60 and/or 120. At PND30, 17 female mice each treatment group were individually placed in a standard cage equipped with a running wheel until PND60. Wheel-running activity (PNDs 53–60) was recorded by use of a data acquisition unit connected to the wheel-running unit as described [20]. In addition, we used newborn (PND0) and PND280 mice for the analyses of expression, ROS and lung function. Because of lung structural and functional analysis at different ages, which were not possible by use of the same mouse, the precise number of mice used each group (minimum 12 mice) is given in the legends of tables and figures, respectively.

Pregnant dams were always housed at specific pathogen-free conditions. After giving birth, dames and their newborns were transferred into an experimental room equipped with oxygen chamber and wheel running system, and housed at conventional conditions (21 °C, 45% humidity, 12-h light/dark cycle, ad libitum access to water and food). Newborns were marked by foot tattoos for their identification. General health and body weights of the experimental mice were checked every day till PND15, every 2 days between PNDs 16–30, and every week after PND31. At the PND of analysis, mice were killed by cervical dislocation, and lung and blood were prepared for further analysis.

A bronchoalveolar lavage (BAL) was performed three times with 0.3 ml phosphate-buffered saline and then pooled. After counting the cell number, BAL cells were centrifuged on a slide and stained with the HemaDiff Quick Staining kit (Bioanalytic GmbH, Umkirch, Germany) for cytological investigations. Cell-free BAL fluid was obtained by centrifugation and analyzed for protein with the Pierce™ bicinchoninic acid (BCA) Protein Assay kit (Thermo Fisher Scientific, Rockford, IL), for IgM with the mouse IgM ELISA kit (Bethyl Laboratories, Montgomery, TX) and for soluble receptor for advanced glycation end-products (sRAGE) with the mouse RAGE DuoSet kit (R&D Systems, Minneapolis, MN). Heparinized blood was taken after transection of the vena cava and subjected to automated blood cell count using the scil Vet ABC™ Hematology Analyzer (scil animal care company GmbH, Viernheim, Germany).

Lung histology was assessed by use of the left lung, which was fixed with 4% phosphate-buffered formaldehyde for 15 min at a filling pressure of 20 cmH₂O. After embedding in paraffin, 4-μm-thick sections were cut, dewaxed, rehydrated and incubated in hematoxylin-eosin solutions (Carl Roth, Karlsruhe, Germany) to stain nuclei and tissue proteins. Lung elastic fibers were visualized by staining the sections with Weigert resorcin-fuchsin (0.05% in acid 70% ethanol; Waldeck, Münster, Germany) followed by counterstaining with tartrazine (0.25% in saturated picric acid; Sigma-Aldrich). Images of the stained sections were taken with the Axiovert microscope equipped with Spot Camera (Carl Zeiss, Jena, Germany). Lung histological parameters were analyzed by use of the ImageJ 1.47v software [21]. The mean linear intercept was determined according to the guidelines of Knudsen et al. [22]. The radial alveolar count was determined between most peripheral bronchiole and the nearest pleural surface according to Obayashi et al. [23].

Total RNA and protein was isolated from the right lung by use of the TRIzol™ Reagent (Thermo Fisher Scientific). In addition, we prepared tissue lysates in 1% phosphate-buffered sodium dodecyl sulfate solution supplemented with protease inhibitors. Equal protein amounts were separated by gel electrophoresis with subsequent standard immuno-blot procedure using polyclonal rabbit antibodies against SP-B (Santa Cruz, Santa Cruz, CA), SP-C (Santa Cruz), Cu/ZnSOD (Abcam, Cambridge, UK), catalase (Abcam), MnSOD (Rockland, Limerick, PA), PEX14 (Merck-Millipore, Darmstadt, Germany) or GAPDH (Santa Cruz). The expression of the antioxidant enzymes was determined by slot blot procedure using the Minifold slot blot system (GE Healthcare Life Science, Freiburg, Germany) with subsequent antibody detection. The LAS 3000 computer-based imaging system (FUJI Film, Tokyo, Japan) equipped with AIDA 2.0 software (Raytest, Straubenhardt, Germany) was used for signal detection and quantification of the signal intensities. All data were normalized per total protein stain of the blotted proteins, which was performed with 0.5% amido black solution.

Equal amounts of total RNA were reverse transcribed into cDNA with the M-MLV RT Rnase H Minus (Promega, Madison, WI). Then, the cDNA was subjected to quantitative PCR using the iCycler iQ™ Real-Time PCR Detection System (Rio-Rad, Hercules, CA). PCR was performed with mouse-specific primers for SFTPB (sense: 5′-agttctcctgctagacgcac-3′, anti-sense: 5′-ctgttcacacttttgcctgtc-3′), SFTPC (sense: 5′-tcctgatggagagtccaccg-3′, anti-sense: 5′-cagagcccctacaatcaccac-3′) or ELN (sense: 5′-tcctggagccactcttacag-3′, anti-sense: 5′-ctctctctccccaattagcc-3′). Relative mRNA amounts were quantified by use of an external cDNA standard because the normalization per another gene (gapdh, selenbp1, tkt1 were tested as potential house-keeping genes) appeared to be inappropriate.

ROS are formed in mitochondria and to high extent also in other cellular compartments of lung samples [24]. Therefore, total cytoplasmic protein was isolated from lung tissue in ice-cold 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (5 μl∙mg^− 1 wet tissue) containing 1 μg∙ml^− 1 saponine (Sigma, Saint Louis, MO) as mild detergent and 50 μM deferoxamine mesylate (Sigma) as Fe³⁺ chelator. Nuclei were then removed by centrifugation (600 g for 10 min). 10 μl nuclei-free cytoplasmic fraction were mixed after 90 min (Fe³⁺ chelate effect completed) with 90 μl 1 mM 1-hydroxy-3-carboxy-2,2,5,5,-tetramethyl-pyrrolidine (CPH; Noxygen, Elzach, Germany) as a spin trap for O₂^•-. A computer-operated electron paramagnetic resonance spectrophotometer (MiniScope MS100; Magnettech, Berlin, Germany) equipped with MiniScope Control v2.7.3 analysis software (Microtech GmbH, Bad Kreuznach, Germany) measured the time-dependent formation of 3-carboxy-2,2,5,5-tetra-methyl-pyrrolin-1-oxyl (^•CP) at room temperature every 10 min. O₂^•- data were expressed as ^•CP formation rate∙min^− 1.

The buffer-perfused mouse lung system from Hugo Sachs Elektronik-Harvard Apparatus (March-Hugstetten, Germany) analyzed the lung respiratory mechanics. We described this ex vivo method before in detail [25, 26]. In short, each mouse was placed in an artificial thoracic chamber and positive-pressure ventilated (90 breaths∙min^− 1) via tracheal cannulation. Through cannulation of pulmonary artery and left atrium of the heart we ensured a constant perfusion flow of the lung vascular bed (1 ml∙min^− 1 at 37 °C). RPMI 1640 medium (Thermo Fisher Scientific) was used as perfusion buffer and constantly purged with 5% CO₂/95% N₂ to yield pH 7.4 and O₂-reduced conditions. Thereafter, the ventilation was switched to negative pressure ventilation with constant minimal pleural pressure (− 2 cmH₂O) and specific maximal pleural pressures. Since the isolated lung is not surrounded by the pleurae, repeating augmented breaths every 3 min at − 20 cmH₂O maximal pleural pressure stabilize the tidal volume (TV) that would otherwise decline steadily. The PULMODYN® Pulmonary Mechanics Data Acquisition software recorded the lung physiological parameters.

At PND60, the treatment of newborn mice with neonatal hyperoxia slightly reduced their survival rate which was significant for mice of the mH group and only a trend for those of the sH group (Table 1). This reduction was not associated with differences in physical development (body weight, lung weight) and physical activity in a running wheel (Table 1). However, mice of the mH group showed a slight increase of platelets in the whole blood and number of cells in the BAL fluid (Table 1). Several BAL parameters indicating inflammation and damage of the alveolo-capillary membrane remained unchanged (Table 1). We also did not identify changes of the soluble variant of the cell surface molecule RAGE (sRAGE), which is reduced in BAL samples following alveolar epithelial cell damage and lung emphysema [27].

Higher oxygen supply will increase the mitochondrial formation of O₂^•- in mouse lung tissues [2]. For that reason, we quantified MnSOD and Cu/ZnSOD, which are important intra-cellular O₂^•--scavenging enzymes, as well as catalase, which detoxifies resultant H₂O₂, in lung samples depending on development (age) and treatment. Development-dependent analyses showed increasing protein amounts of all three enzymes with early changes in catalase and late changes in MnSOD and Cu/ZnSOD (Fig. 1a-c). Hyperoxic conditions moderately increased the protein amounts of MnSOD in case of sH conditions (Fig. 1a) and catalase in case of mH conditions (Fig. 1c) when quantified directly after treatment (PND14) but not later. Detailed analysis of the O₂^•- formation did not reveal a correlation of free O₂^•- amount and the amount of selected O₂^•--scavenging enzymes. Lung tissues of PND14 mice commonly formed less free O₂^•-, especially when they were treated with mH from birth (Fig. 1d). However, this effect was abolished later because lung tissues of PND60 mice of the mH treatment group generated more free O₂^•- than those of the N or sH group (Fig. 1d).

Morphometric analyses of lung sections prepared from PND60 mice revealed significant changes in the lung structure of the sH but not mH treatment group. In this regard, we identified a lower alveolar density, thicker alveolar walls and augmented parenchymal airspace sizes with particular augmentation of the large airspaces (Table 2, Additional file 1: Figure S1). As result of these changes, less cell counts per tissue area but not per total area have been counted (Table 2). The amount of elastic fibers remained unchanged (Fig. 2a) but their quality was impaired as indicated by an increase in shortened fibers (Fig. 2b-c).

Functional elastin and pulmonary surfactant significantly contribute to the elasticity of the lung tissue. However, neonatal hyperoxia did also not influence the lung expression of elastin as indicated by unaltered mRNA amounts at PND60 (Fig. 3a). The mRNA and protein amounts of the pro-surfactant proteins (SPs) B and C were also not influenced by neonatal hyperoxia (Fig. 3b-c). In contrast, the lung expressions of elastin and both pro-SPs were significantly influenced by age. In this regard, the mRNA amount of elastin was reduced in fully grown mice at PND120 without influence of neonatal hyperoxia, which was analyzed for sH conditions only (Fig. 3a). The mRNA amounts of the pro-SPs B and C were increased with higher age but also not considerably altered by neonatal hyperoxia (Fig. 3b-c). Comparative analyses of mRNA and protein amount additionally revealed a reverse regulation of mRNA and protein depending on mouse age, in particular for pro-SP-C (Fig. 3b-c).

Lung functional parameters were analyzed ex vivo in mice at PND120. In order to better assess potential lung functional changes caused by neonatal hyperoxia, we performed comparative analyses of untreated PND120 mice and older (PND280) mice in an initial experiment. This comparison revealed an age-related increase of the lung compliance as indicated by changes in the pleural pressure-TV relations (Fig. 4a) accompanied by higher peak airflows for inspiration and expiration (Fig. 4b). Like the untreated PND280 mice, PND120 mice treated with neonatal hyperoxia showed an increase of the lung compliance which was more pronounced in the sH than mH treatment group (Fig. 5a). Unlike the untreated PND280 mice, PND120 mice treated with neonatal hyperoxia did not show higher peak airflows for inspiration and expiration (Fig. 5b). In the case of neonatal sH condition, the peak airflows for inspiration and expiration were significantly reduced (Fig. 5b). Airway resistance and basal pulmonary artery pressure were not influenced by neonatal hyperoxia (Fig. 5d). Only the perfusion flow-dependent pulmonary artery pressure was slightly increased in PND120 mice of the sH group (Fig. 5d). Also, lungs of the sH group were more susceptible to edema formation due to our ex vivo ventilation procedure (Fig. 5e).