Biomarkers of lung alveolarization and microvascular maturation in response to intermittent hypoxia and/or early antioxidant/fish oil supplementation in neonatal rats

Abstract

Objective: Extremely preterm infants experience frequent intermittent hypoxia (IH) episodes during oxygen therapy which causes significant damage to the lungs and curtails important signaling pathways that regulate normal lung alveolarization and microvascular maturation. We tested the hypothesis that early supplementation with fish oil and/or antioxidants in rats exposed to neonatal IH improves expression of lung biomarkers of alveolarization and microvascular maturation, and reduces IH-induced lung injury.

Study design/methods: From birth (P0) to P14, rat pups were exposed to room air (RA) or neonatal IH during which they received daily oral supplementation with either: (1) olive oil (OO) (control); (2) Coenzyme Q10 (CoQ10) in OO; (3) fish oil; (4) glutathione nanoparticles (nGSH); or (5) fish oil +CoQ10. At P14 pups were placed in RA until P21 with no further treatment. RA controls were similarly treated. Lung growth and alveolarization, histopathology, apoptosis, oxidative stress and biomarkers of alveolarization and microvascular maturation were determined.

Results: Neonatal IH was associated with reduced lung weights and severe histopathological outcomes. These effects were curtailed with fish oil and nGSH. nGSH was also protective against apoptosis, while CoQ10 prevented IH-induced ROS production. Of all treatments, nGSH and CoQ10 + fish oil-induced vascular endothelial growth factor₁₆₅ and CD31 (Platelet endothelial cell adhesion molecule-1), which are associated with angiogenesis. CoQ10 + fish oil improved alveolarization in RA and IH despite evidence of hemorrhage.

Conclusions: The benefits of nGSH and CoQ10 + fish oil suggest an antioxidant effect which may be required to curtail IH-induced lung injury. Further clinical assessment of the effectiveness of nGSH is warranted.

Keywords: alveolarization; glutathione nanoparticles; neonatal intermittent hypoxia; tissue inhibitors of metalloproteinases; vascular endothelial growth factor.

Figure 1.

Representative H&E stained images of lungs from 21-day old rat exposed to room air (RA, top panels), or intermittent hypoxia (IH, lower panels). Images are 20X magnification and the scale bar is 50 μM. Arrows show mild and moderate hemorrhage in the IH groups.

Figure 2.

Effects of antioxidants and/or fish oil supplementation during neonatal intermittent hypoxia (IH) on malondialdehyde (MDA) levels in the lung homogenates (panel A), and representative quantitative analysis of lung apoptosis (Panel B). Corresponding images are presented in Supplementary Figure S2. Data are mean±SEM (n=6 samples/group, Panel A; n=12 measurements/group, Panel B).

Figure 3.

Effects of antioxidants and/or fish oil supplementation during neonatal intermittent hypoxia (IH) on lung MMP-2 (panel A) and MMP-9 (panel B) in the lung homogenates. Data are mean±SEM (n=6 samples/group).

Figure 4.

Effects of antioxidants and/or fish oil supplementation during neonatal intermittent hypoxia (IH) on lung VEGF₁₆₅ and sVEGFR-1 levels in the lung homogenates. Data are mean±SEM (n=6 samples/group).

Figure 5.

Representative IHC stains of CD31 in the lung sections from 21-day old rat exposed to room air (RA, top panels), or intermittent hypoxia (IH, lower panels). Images are 20X magnification and the scale bar is 50 μM.