Protective effect of chorioamnionitis on the development of bronchopulmonary dysplasia triggered by postnatal systemic inflammation in neonatal rats

Abstract

Background: Prenatal or postnatal systemic inflammation can contribute to the development of bronchopulmonary dysplasia (BPD). We investigated whether prenatal intra-amniotic (i.a.) inflammation or early postnatal systemic inflammation can induce BPD in a rat model.

Methods: One microgram of lipopolysaccharide (LPS) or vehicle was injected into the amniotic sacs 2 d before delivery (E20). After birth, 0.25 mg/kg of LPS or vehicle was injected into the peritoneum of pups on postnatal day (P)1, P3, and P5. On P7 and P14, peripheral blood (PB), bronchoalveolar lavage fluid (BALF), and lung tissue were obtained and analyzed.

Results: Postnatal i.p. injections of LPS significantly increased neutrophil counts in PB and BALF on P7 and P14. Similarly, proinflammatory cytokine and angiogenic factor transcript levels were increased in the lung by i.p. LPS on P7. Alveolar and pulmonary vascular development was markedly disrupted by i.p. LPS on P14. However, pretreatment with i.a. LPS significantly negated the detrimental effects of postnatal i.p. LPS on PB and BALF neutrophil counts and on lung proinflammatory cytokine expression and histopathological changes.

Conclusion: Exposure to early postnatal systemic LPS induces BPD, an arrest in alveolarization, in neonatal rats. Preceding exposure to i.a. LPS protects the lungs against BPD triggered by postnatal systemic inflammation.

Figures

Figure 1

Schematic outline of the experimental protocol. Intra-amniotic LPS (1.0 μg/sac) or vehicle was administered on E20. Postnatal i.p. LPS (0.25 mg/kg/d) or vehicle was administered on P1, P3, and P5. BALF and PB collection and lung harvesting were performed on P7 and P14. BALF, bronchoalveolar lavage fluid; IA, intra-amniotic; IP, intraperitoneal; PB, peripheral blood; V, vehicle.

Figure 2

Survival rates in the experimental groups. Fetal death mostly occurred in the intra-amniotic (i.a.) LPS-treated groups. After birth, most of the deaths occurred in the i.p. LPS-treated groups. Nearly half of the rats in the i.p. LPS-treated groups died during the first several days after birth. In all groups, no further mortality occurred beyond P7. Gray solid line, V+V; gray dashed line, LPS+V; black dashed line, V+LPS; black solid line, LPS+LPS. Group abbreviation: V+V, i.a. vehicle followed by i.p. vehicle-treated group; LPS+V, i.a. LPS followed by i.p. vehicle-treated group; V+LPS, i.a. vehicle followed by i.p. LPS-treated group; LPS+LPS, i.a. LPS followed by i.p. LPS-treated group. The arrowhead indicates i.a. LPS (1.0 μg/sac), and the arrows indicate postnatal i.p. LPS (0.25 mg/kg/d) administrations.

Figure 3

Bronchoalveolar lavage fluid and peripheral blood white blood cell and neutrophil counts. (a, b, d, e) Gray bars denote P7 and black bars P14. (a) The total BALF WBC counts were higher in the i.p. LPS-treated groups than in the i.p. vehicle-treated groups on P7 and P14. (b) The V+LPS group had higher BALF neutrophil counts than in other groups on P7 and in the V+V group on P14. (c) Representative light microscopic images of the cytospin BALF samples on P7. A marked increase in the number of neutrophils in the BALF cytospin sample from the V+LPS group is observed compared with the other groups. The arrows indicate neutrophils. Diff-Quik (Sysmex, Kobe, Japan) stained. Original magnification ×400. The bar indicates 50 μm. (d, e) The total PB WBC and neutrophil counts were highest in the V+LPS group on P7. On P14, the total WBC and neutrophil counts in PB were reduced compared with P7, and the V+V group had the highest number of total WBC and neutrophil counts in PB. Group abbreviation: V+V, i.a. vehicle followed by i.p. vehicle-treated group; LPS+V, i.a. LPS followed by i.p. vehicle-treated group; V+LPS, i.a. vehicle followed by i.p. LPS-treated group; LPS+LPS, i.a. LPS followed by i.p. LPS-treated group. The data are the mean ± SEM (N = 5–6 in each group). *P < 0.05 vs. V+V, ** P < 0.05 vs. LPS+V, †P < 0.05 vs. LPS+LPS, ‡P < 0.05 vs. V+LPS. BALF, bronchoalveolar lavage fluid; PB, peripheral blood; V, vehicle; WBC, white blood cell.

Figure 4

Alveolar development and morphometric data. (a) Representative light microscopic images of the rat lungs on P7 and P14. Apparently thinned septal walls were observed in the V+LPS group compared with other groups on P7, and markedly large and simple airspaces were observed in the V+LPS group compared with other groups on P14. Hematoxylin and eosin stained; original magnification ×100. Bars, 200 μm. (b–d) Gray bars denote P7 and black bars P14. (b) The mean cord length (Lm), indicating the average alveolar size, was greater in the i.p. LPS-treated groups than in the i.p. vehicle-treated groups on P7. The Lm was decreased on P14 as the lungs developed in all groups, but V+LPS group had the highest Lm among the groups. (c) The alveolar surface area (SA) was smaller in the V+LPS group than in the i.p. vehicle-treated groups on P7. The SA increased on P14 as the lungs developed in all groups, but the i.p. LPS-treated groups had a smaller SA than the i.p. vehicle-treated groups. (d) The alveolar wall thickness (WT) was thinner in the i.p. LPS-treated groups than in the V+V group on P7. The WT was thinned on P14 as the lungs developed in all groups except the V+LPS group, and the V+LPS group had the thickest WT on P14. Group abbreviation: V+V, i.a. vehicle followed by i.p. vehicle-treated group; LPS+V, i.a. LPS followed by i.p. vehicle-treated group; V+LPS, i.a. vehicle followed by i.p. LPS-treated group; LPS+LPS, i.a. LPS followed by i.p. LPS-treated group. The data are the mean ± SEM (N = 6–7 in each group). *P <0.05 vs. V+V, **P < 0.05 vs. LPS+V, †P < 0.05 vs. LPS+LPS. V, vehicle.

Figure 5

Pulmonary vascular development. (a) Representative light microscopic images of von Willebrand factor (vWF) immunohistochemistry of rat lung sections on P7 and P14. Apparently fewer blood vessels and decreased vWF staining were observed in the V+LPS group compared with the other groups on P7 and P14. The arrows indicate vessels. vWF staining was visualized with a DAB reaction (brown color). Light hematoxylin and eosin staining was used as a counterstain. Original magnification, ×400. Bars = 50 μm. (b, c) Gray bars denote P7 and black bars P14. (b) The number of blood vessels per HPF was significantly lower in the i.p. LPS-treated groups than in the i.p. vehicle-treated groups on P7 and P14. On P14, the V+LPS group had a significantly lower number of vessels per HPF than the LPS+LPS group. (c) The vascular density, which was expressed as the ratio of the vWF-positive area to the total area of the lung parenchyma, was significantly lower in the i.p. LPS-treated groups than in the i.p. vehicle-treated groups on P7 and P14. The V+LPS group had a significantly lower vascular density than the LPS+LPS group on P7 and P14. Group abbreviation: V+V, i.a. vehicle followed by i.p. vehicle-treated group; LPS+V, i.a. LPS followed by i.p. vehicle-treated group; V+LPS, i.a. vehicle followed by i.p. LPS-treated group; LPS+LPS, i.a. LPS followed by i.p. LPS-treated group. The data are the mean ± SEM (N = 6–7 in each group). *P < 0.05 vs. V+V, **P < 0.05 vs. LPS+V; †P < 0.05 vs. LPS+LPS. HPF, high-powered field; V, vehicle. DAB, diaminobenzidine.

Figure 6

Inflammatory cytokine and angiogenic factor mRNA expression in lung tissue. The transcript levels of proinflammatory cytokines (TNF-α, IL-1β, IL-6, IL-12A) and angiogenic factors (VEGF, HIF-1α) on (a) P7 (b) and P14. The transcript levels of TNF-α, IL-1β, IL-6, IL-12A, VEGF, and HIF-1α in the lung tissue were significantly higher in the V+LPS group than in the V+V group on P7. The transcript levels of TNF-α, IL-12A, VEGF remained significantly higher in the V+LPS group than in the V+V group on P14. The mRNA expression of each group is presented as the relative value to that of the V+V group. Black bars, V+V; dark gray bars, LPS+V; gray bars, V+LPS; light gray bars, LPS+LPS. Group abbreviation: V+V, i.a. vehicle followed by i.p. vehicle-treated group; LPS+V, i.a. LPS followed by i.p. vehicle-treated group; V+LPS, i.a. vehicle followed by i.p. LPS-treated group; LPS+LPS, i.a. LPS followed by i.p. LPS-treated group. The data are the mean ± SEM (N = 6–7 in each group). *P < 0.05 vs. V+V. HIF, hypoxia-inducible factor.