Cumulative effects of neonatal hyperoxia on murine alveolar structure and function

Abstract

Background: Bronchopulmonary dysplasia (BPD) results from alveolar simplification and abnormal development of alveolar and capillary structure. Survivors of BPD display persistent deficits in airflow and membrane and vascular components of alveolar gas diffusion. Despite being the defining feature of BPD, various neonatal hyperoxia models of BPD have not routinely assessed pulmonary gas diffusion.

Methods: To simulate the most commonly-utilized neonatal hyperoxia models, we exposed neonatal mice to room air or ≥90% hyperoxia during key stages of distal lung development: through the first 4 (saccular), 7 (early alveolar), or 14 (bulk alveolar) postnatal days, followed by a period of recovery in room air until 8 weeks of age when alveolar septation is essentially complete. We systematically assessed and correlated the effects of neonatal hyperoxia on the degree of alveolar-capillary structural and functional impairment. We hypothesized that the degree of alveolar-capillary simplification would correlate strongly with worsening diffusion impairment.

Results: Neonatal hyperoxia exposure, of any duration, resulted in alveolar simplification and impaired pulmonary gas diffusion. Mean Linear Intercept increased in proportion to the length of hyperoxia exposure while alveolar and total lung volume increased markedly only with prolonged exposure. Surprisingly, despite having a similar effect on alveolar surface area, only prolonged hyperoxia for 14 days resulted in reduced pulmonary microvascular volume. Estimates of alveolar and capillary structure, in general, correlated poorly with assessment of gas diffusion.

Conclusion: Our results help define the physiological and structural consequences of commonly-employed neonatal hyperoxia models of BPD and inform their clinical utility. Pediatr Pulmonol. 2017;52:616-624. © 2016 Wiley Periodicals, Inc.

Keywords: bronchopulmonary dysplasia; hyperoxia; lung function; neonatal; pulmonary diffusion capacity.

Fig. 1

Impact of neonatal hyperoxia on pulmonary diffusion and alveolar volume. (A) Assessment of the diffusion factor for carbon monoxide (DF_CO) at 2 months of age in animals continuously raised in room air (RA), or initially exposed to neonatal hyperoxia (≥90% O₂) for the first 4, 7, or 14 days after birth followed by recovery in RA. (B) Assessment of alveolar volume by neon gas dilution. Values are expressed as means±SEM. ^***P < 0.001 versus RA, ^**P < 0.01 versus RA, ^#P < 0.05 versus O₂ P0-4, ^###P < 0.001 versus O₂ P0-4, ^$$$P < 0.001 versus O₂ P0-7 by one way ANOVA using Tukey’s multiple comparison post-test analysis. Results are representative of three independent experiments. n = 10 (RA), 10 (O₂ P0-P4), 18 (O₂ P0-P7), 9 (O₂ P0-P14).

Fig. 2

Development of Alveolar surface area following neonatal hyperoxia. (A–D) Representative hematoxylin/eosin stained lung sections at 2 months of age in animals continuously raised in (A) RA, or initially exposed to neonatal hyperoxia (≥90% O₂) from (B) P0-4, (C) P0-7, or (D) P0-14, followed by recovery in RA. Assessment of (E) mean linear intercept (MLI), (F) left lung volume by water displacement, and (G) alveolar surface area. Original images obtained with 20× objective. Scale bar = 50μm. Values are expressed as means±SEM. ^***P < 0.001 versus RA, ^###P < 0.001 versus O₂ P0-4, ^$$$P < 0.001 versus O₂ P0-7) by one way ANOVA using Tukey’s multiple comparison post-test analysis. Results are representative of three independent experiments. n = 19 (RA), 10 (O₂ P0-P4), 12 (O₂ P0-P7), 9 (O₂ P0-P14).

Fig. 3

Effect of Neonatal hyperoxia on pulmonary microvascular development. Pulmonary microvascular volume was assessed using stereological analysis on von Willebrand-stained lung sections to identify vessels 20–50μm in diameter. Values are expressed as means±SEM. ^*P < 0.05 versus RA by one way ANOVA using Tukey’s multiple comparison post-test analysis. Results are representative of three independent experiments. n = 11 (RA), 10 (O₂ P0-P4), 12 (O₂ P0-P7), 9 (O₂ P0-P14).

Fig. 4

Correlation between estimates of distal lung alveolar–capillary simplification and functional impairment. Correlation of mean linear intercept (MLI) (A), volume fraction of the alveolar septal wall (B), alveolar surface area (C), pulmonary microvascular volume (D), alveolar surface area per lung volume (E), and pulmonary microvascular volume per lung volume (F) with DF_CO. Analysis performed using simple linear regression.