Deficits in lung alveolarization and function after systemic maternal inflammation and neonatal hyperoxia exposure

Abstract

Systemic maternal inflammation contributes to preterm birth and is associated with development of bronchopulmonary dysplasia (BPD). Infants with BPD exhibit decreased alveolarization, diffuse interstitial fibrosis with thickened alveolar septa, and impaired pulmonary function. We tested the hypothesis that systemic prenatal LPS administration to pregnant mice followed by postnatal hyperoxia exposure is associated with prolonged alterations in pulmonary structure and function after return to room air (RA) that are more severe than hyperoxia exposure alone. Timed-pregnant C3H/HeN mice were dosed with LPS (80 microg/kg) or saline on gestation day 16. Newborn pups were exposed to RA or 85% O2 for 14 days and then to RA for an additional 14 days. Data were collected and analyzed on postnatal days 14 and 28. The combination of prenatal LPS and postnatal hyperoxia exposure generated a phenotype with more inflammation (measured as no. of macrophages per high-power field) than either insult alone at day 28. The combined exposures were associated with a diffuse fibrotic response [measured as hydroxyproline content (microg)] but did not induce a more severe developmental arrest than hyperoxia alone. Pulmonary function tests indicated that hyperoxia, independent of maternal exposure, induced compliance decreases on day 14 that did not persist after RA recovery. Either treatment alone or combined induced an increase in resistance on day 14, but the increase persisted on day 28 only in pups receiving the combined treatment. In conclusion, the combination of systemic maternal inflammation and neonatal hyperoxia induced a prolonged phenotype of arrested alveolarization, diffuse fibrosis, and impaired lung mechanics that mimics human BPD. This new model should be useful in designing studies of specific mechanisms and interventions that could ultimately be utilized to define therapies to prevent BPD in premature infants.

Fig. 1.

Pup right lung weight at day 28. Pups were exposed to prenatal saline or LPS and postnatal room air (open bars) or 85% O₂ for 14 days (solid bars) and then returned to room air for 14 days. Data were analyzed by 2-way ANOVA and modified t-test post hoc (n = 9). An interaction between prenatal LPS and postnatal hyperoxia exposure was observed. Values are means ± SE. Identical symbols above bars indicate statistical significance between groups: *P ≤ 0.05; §,†,‡P ≤ 0.01.

Fig. 2.

Histological lung sections from pups exposed to prenatal saline or LPS and postnatal room air or 85% O₂. Top: histological sections from pups euthanized at day 14 (D14). Bottom: histological sections from pups euthanized at day 28 (14 days in O₂ followed by return to room air for 14 days; D28). Inflation-fixed lung sections were stained with hematoxylin and eosin. Photomicrographs are representative of 6 mice per group. Magnification ×100.

Fig. 3.

Ratios of total perimeter per high-power field (HPF) to septal wall thickness. Top: pups were exposed to prenatal saline or LPS and postnatal room air (open bars) or 85% O₂ (solid bars) for 14 days, and ratio was measured at day 14. Bottom: pups were exposed to prenatal saline or LPS and postnatal room air (open bars) or 85% O₂ (solid bars) for 14 days, followed by 14 days recovery, and ratio was measured at day 28. Data were analyzed by 2-way ANOVA with modified t-test post hoc (n = 6 per group). Effects of prenatal LPS, postnatal hyperoxia exposure, and an interaction between prenatal LPS and postnatal hyperoxia exposure were observed. Values are means ± SE. Identical symbols above bars indicate statistical significance between groups: *P ≤ 0.05; $,§,†,‡P ≤ 0.01.

Fig. 4.

Collagen deposition in lung tissues. A: histological lung sections from pups exposed as described in methods were stained with Picro-Sirius red and assessed at day 28. B and C: positive-stained areas from lungs exposed to prenatal saline or LPS and postnatal room air (open bars) or 85% O₂ were quantified for number and total area. Five fields for each slide were averaged, and data were analyzed by 2-way ANOVA and modified t-test post hoc (n = 6 per group). Effects of prenatal LPS and postnatal hyperoxia exposure were observed in the numbers of Sirius red (SR) spots and total Sirius red area. Values are means ± SE. Identical symbols above bars indicate statistical significance between groups: *,**P ≤ 0.05; $,§,†,‡,††P ≤ 0.01.

Fig. 5.

Hydroxyproline contents in pup right lungs. Pups were exposed as described in methods, and hydroxyproline content was assessed at day 14 (top) and day 28 (bottom). Data were analyzed by 2-way ANOVA and modified t-test post hoc (n = 5–7 mice). An effect of hyperoxia and an interaction between LPS and hyperoxia on hydroxyproline contents were identified. Values are means ± SE. Identical symbols above bars indicate statistical significance between groups: *,**P ≤ 0.05; $,§,†P ≤ 0.01.

Fig. 6.

Macrophage accumulation in lung tissues. Immunohistochemical assessment of macrophage numbers was performed on tissue sections obtained from pups exposed to prenatal saline or LPS and postnatal room air (open bars) or 85% O₂ (solid bars) for 14 days (top) or 28 days (bottom). [See photomicrographs in supplemental data.] Inflation-fixed lung sections were stained with Mac3 antibodies, and number of macrophages was quantified by digital analyses of the sections. Macrophages were counted in 5 fields for each slide and averaged. Data were analyzed by 2-way ANOVA and modified t-test post hoc (n = 4–5 mice per group and time point). On day 14, there was an effect of prenatal LPS, an effect of postnatal hyperoxia, and an interaction between prenatal LPS and postnatal hyperoxia. On day 28, statistical analyses indicated persistent effects of prenatal LPS and postnatal hyperoxia exposure and an interaction between prenatal LPS and postnatal hyperoxia macrophage numbers. Values are means ± SE. Identical symbols above bars indicate statistical significance between groups: *P ≤ 0.05; §,†,‡P ≤ 0.01.

Fig. 7.

Pulmonary function tests. Pulmonary resistance and compliance were measured in pups exposed to prenatal saline or LPS and postnatal room air (open bars) or 85% O₂ (solid bars). Top: measurements on day 14; middle: measurements on day 28 following room air recovery; bottom: central airway resistance (Rn) on day 28. Data were analyzed by 2-way ANOVA and modified t-test post hoc (n = 5–6 mice per treatment group). Effects of prenatal LPS and an interaction between prenatal LPS and postnatal hyperoxia on total airway resistance were observed. Effects of prenatal LPS and postnatal O₂ and an interaction of prenatal LPS and postnatal O₂ on central airway resistance were also observed. Values are means ± SE. Identical symbols above bars indicate statistical significance between groups: *,**,|P ≤ 0.05; §,†,‡P ≤ 0.01.