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Comparative Study
. 2016 Aug 1;311(2):L481-93.
doi: 10.1152/ajplung.00047.2016. Epub 2016 Jun 24.

Sex-specific differences in neonatal hyperoxic lung injury

Krithika Lingappan  1 , Weiwu Jiang  2 , Lihua Wang  2 , Bhagavatula Moorthy  2
Affiliations
Comparative Study

Sex-specific differences in neonatal hyperoxic lung injury

Krithika Lingappan et al. Am J Physiol Lung Cell Mol Physiol. .

Abstract

Male sex is considered an independent predictor for the development of bronchopulmonary dysplasia (BPD) after adjusting for other confounders. BPD is characterized by an arrest in lung development with marked impairment of alveolar septation and vascular development. The reasons underlying sexually dimorphic outcomes in premature neonates are not known. In this investigation, we tested the hypothesis that male neonatal mice will be more susceptible to hyperoxic lung injury and will display larger arrest in lung alveolarization. Neonatal male and female mice (C57BL/6) were exposed to hyperoxia [95% FiO2, postnatal day (PND) 1-5] and euthanized on PND 7 and 21. Extent of alveolarization, pulmonary vascularization, inflammation, and modulation of the NF-κB pathway were determined and compared with room air controls. Macrophage and neutrophil infiltration was significantly increased in hyperoxia-exposed animals but was increased to a larger extent in males compared with females. Lung morphometry showed a higher mean linear intercept (MLI) and a lower radial alveolar count (RAC) and therefore greater arrest in lung development in male mice. This was accompanied by a significant decrease in the expression of markers of angiogenesis (PECAM1 and VEGFR2) in males after hyperoxia exposure compared with females. Interestingly, female mice showed increased activation of the NF-κB pathway in the lungs compared with males VSports手机版. These results support the hypothesis that sex plays a crucial role in hyperoxia-mediated lung injury in this model. Elucidation of the sex-specific molecular mechanisms may aid in the development of novel individualized therapies to prevent/treat BPD. .

Keywords: bronchopulmonary dysplasia; gender; hyperoxia; lung development; sex. V体育安卓版.

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Figures

Fig. 1.
Fig. 1.
PCR analysis of genomic DNA from mouse tails, showing Sry and control (myogenin) bands. M stands for molecular marker. A representative PCR analysis shows expression of Sry, a sex-determining region Y gene, in genomic DNA derived from neonatal male but not in female mice.
Fig. 2.
Fig. 2.
A: body weights [postnatal day (PND) 21] in male and female neonatal mice exposed to hyperoxia (95% FiO2, PND 1–5). In the room air group: n = 12 male, n = 15 female; in the hyperoxia group: n = 7 male, n = 17 female mice. B: lung weight/Body weight ratios (mg/g) in male and female neonatal mice exposed to hyperoxia (95% FiO2, PND 1–5) on PND6 immediately after hyperoxia exposure (n = 4 animals/group). Values are means ± SE. Significant differences room air- and hyperoxia-exposed animals ##P < 0.01.
Fig. 3.
Fig. 3.
Lung architecture (PND 21) in male and female neonatal mice (n = 6/group) exposed to hyperoxia (95% FiO2, PND 1–5). Representative hematoxylin and eosin stained sections form male and female neonatal mice exposed to room air or hyperoxia at 10× (A) and 20× (B) magnification.
Fig. 4.
Fig. 4.
Lung morphometry in male and female neonatal mice (n = 6 mice per group) exposed to hyperoxia (95% FiO2, PND 1–5). A: mean linear intercept (MLI) in male and female neonatal mice exposed to room air or hyperoxia on PND 21. B: radial alveolar count in male and female neonatal mice exposed to room air or hyperoxia on PND 21. Values are means ± SE from 6 individual animals. Significant differences between room air and hyperoxia ###P <0.001. Significant differences between male and female mice: **P < 0.01 and ***P < 0.001.
Fig. 5.
Fig. 5.
Masson's trichrome staining for assessment of lung fibrosis in male and female and neonatal mice (n = 6 mice per group) exposed to hyperoxia (95% FiO2, PND 1–5). Representative sections from male and female neonatal mice exposed to room air or hyperoxia at 10× magnification. Blue staining was seen around airways and blood vessels indicated by arrows.
Fig. 6.
Fig. 6.
Immunohistochemistry and quantitation of pulmonary macrophage recruitment. A: representative immunostained images for lung macrophages. Hyperoxia-induced macrophage recruitment was determined by immunohistochemistry with anti-macrophage antibodies in male and female mice (n = 5 male, room air; n = 6 male, hyperoxia; n = 5 female, room air; n = 10 female, hyperoxia) in room air or hyperoxia (95% FiO2, PND 1–5). Arrows point to brown-staining macrophages. B: quantitative analyses showing number of macrophages per high-power field. Representative quantitative analysis of the hyperoxia effects on macrophage recruitment in lungs of male vs. female mice. Values are means ± SE from 5–10 individual animals. Significant differences between room air and hyperoxia ###P < 0.001. Significant differences between male and female mice: ***P < 0.001.
Fig. 7.
Fig. 7.
Immunohistochemistry and quantitation of pulmonary neutrophil recruitment. A: representative immunostained images for lung neutrophils. Hyperoxia-induced neutrophil recruitment was determined by immunohistochemistry with anti-neutrophil antibodies in male and female mice (n = 5/group) in room air or hyperoxia (95% FiO2, PND 1–5). Arrows point to brown-staining neutrophils. B: quantitative analyses showing number of neutrophils per high-power field. Representative quantitative analysis of the hyperoxia effects on neutrophil recruitment in lungs of male vs. female mice. Values are means ± SE from 5 individual animals. Significant differences between room air and hyperoxia #P < 0.05, ###P < 0.001.
Fig. 8.
Fig. 8.
Real time RT-PCR analysis of mRNA from the lungs on PND7 of male and female mice exposed to room air or hyperoxia. Fold change in mRNA expression over male room air values are shown. Values are means ± SE from 4 individual animals. Significant differences between room air and hyperoxia ###P < 0.001. Significant differences between male and female mice: *P < 0.05, ***P < 0.001.
Fig. 9.
Fig. 9.
Immunohistochemistry and quantitation of pulmonary vessels. A: representative immunostained images for vWF (endothelial-cell specific marker). Effect of hyperoxia on pulmonary vascular development was determined by immunohistochemistry with anti-vWF antibodies in male and female mice (n = 6 mice per group) in room air or hyperoxia (95% FiO2, PND 1–5). Arrows point to brown-staining vessels. B: quantitative analyses showing number of vessels per high-power field in lungs of male vs. female mice. Values are means ± SE from 6 individual animals. Significant differences between room air and hyperoxia ##P < 0.01 and ###P < 0.001. Significant differences between male and female mice: ***P < 0.001.
Fig. 10.
Fig. 10.
Effect of hyperoxia on PECAM1 and Flk-1 protein expression in neonatal mouse lung. Representative Western immunoblots (A) and densitometric analysis of pulmonary PECAM1 (B) and Flk-1 (C) isolated from WT male and female neonatal mice (PND 7) exposed to room air or hyperoxia (95% FiO2, PND 1–5). Under each sample lane is the corresponding beta-actin blot to account for protein loading. Values are means ± SE from 3 individual animals. Significant differences between room air and hyperoxia within each sex: #P < 0.05.
Fig. 11.
Fig. 11.
Sex-specific differences in the effect of hyperoxia on NF-κB pathway in neonatal mouse lung. Representative Western immunoblots and densitometric analysis of pulmonary NF-κB p65 (A), phosphorylated NF-κB [phospho-NF-κB p65 (Ser536)] (B), p-IκB-α (C), and IKK-α/β (D) isolated from WT male and female neonatal mice (PND 7) exposed to room air or hyperoxia (95% FiO2, PND 1–5). Under each sample lane is the corresponding beta-actin blot to account for protein loading. Values are means ± SE from 3 individual animals. Significant differences between room air and hyperoxia within each sex: #P < 0.05 and ###P < 0.001. E: the representative immunostained images for phosphorylated NF-κB (phospho-NF-κB p65) in WT male and female neonatal mice (PND 21, n = 6/group) exposed to room air or hyperoxia (95% FiO2, PND 1–5). Arrows point to brown staining for the protein in bronchial epithelial and alveolar epithelial cells.
Fig. 12.
Fig. 12.
Sex-specific differences in pulmonary CYP1A1 mRNA expression in neonatal mice. Real-time RT-PCR analysis showing expression of pulmonary Cyp1a1 mRNA in male and female neonatal mice (PND7) exposed to room air or hyperoxia (95% FiO2, PND 1–5). Values are means ± SE from 3 individual animals. Significant differences between room air and hyperoxia ###P < 0.001. Significant differences between male and female mice: ***P < 0.001.
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