Neonatal intermittent hypoxia, fish oil, and/or antioxidant supplementation on gut microbiota in neonatal rats

Abstract

Background: Preterm infants frequently experience intermittent hypoxia (IH) episodes, rendering them susceptible to oxidative stress and gut dysbiosis. We tested the hypothesis that early supplementation with antioxidants and/or fish oil promotes gut biodiversity and mitigates IH-induced gut injury.

Methods: Newborn rats were exposed to neonatal IH from birth (P0) to P14 during which they received daily oral supplementation with: (1) coenzyme Q10 (CoQ10) in olive oil, (2) fish oil, (3) glutathione nanoparticles (nGSH), (4) CoQ10 + fish oil, or (5) olive oil (placebo control). Pups were placed in room air (RA) from P14 to P21 with no further treatment. RA controls were similarly treated. Stool samples were assessed for microbiota and terminal ileum for histopathology and morphometry, total antioxidant capacity, lipid peroxidation, and biomarkers of gut injury.

Results: Neonatal IH induced histopathologic changes consistent with necrotizing enterocolitis, which were associated with increased lipid peroxidation, toll-like receptor, transforming growth factor, and nuclear factor kappa B. Combination of CoQ10 + fish oil and nGSH were most effective for preserving gut integrity, reducing biomarkers of gut injury, and increasing commensal organisms.

Conclusions: Combination of antioxidants and fish oil may confer synergistic benefits to mitigate IH-induced injury in the terminal ileum.

Impact: Antioxidant and fish oil (PUFA) co-treatment was most beneficial for reducing neonatal IH-induced gut injury. The synergistic effects of antioxidant and fish oil is likely due to prevention of IH-induced ROS attack on lipids, thus preserving and augmenting its therapeutic benefits. Combination treatment was also effective for increasing the abundance of the non-pathogenic Firmicutes phylum, which is associated with a healthy gastrointestinal system of the newborn. Extremely low gestational age neonates who are at high risk for frequent, repetitive neonatal IH and oxidative stress-induced diseases may benefit from this combination therapy.

Figure 1.

Representative H&E stained sections from the terminal ileum of 21-day old (P21) neonatal rats exposed to neonatal intermittent hypoxia (IH, upper panel) and room air (RA, lower panel). Arrows indicate location of pathology. OO (olive oil); CoQ10 (coenzyme Q10); fish oil (omega 3 polyunsaturated fatty acids), nGSH (glutathione nanoparticles). Images are 40X magnification, scale bar is 20 μm.

Figure 2.

Morphometric analysis of the terminal ileum H&E stains presented in Figure 1. Data was analyzed using two-way ANOVA. Data are mean±SEM. *p<0.05; **p<0.01 vs Olive Oil RA; §p<0.05; §§p<0.01 vs Olive Oil IH; †p<0.05, ‡p<0.01 vs RA littermates.

Figure 3.

Effect of antioxidants and/or fish oil on lipid peroxidation/malondialdehyde assay (panel A), total antioxidant capacity (panel B), TLR-4 (panel C), and TGFβ1 in the terminal ileum homogenates from neonatal rats exposed to neonatal IH from P0 to P14 and allowed to recover in room air (RA) from P14 to P21. Groups are as described in Figure 1. Data was analyzed using two-way ANOVA. Data are mean±SEM (n=8 samples/group). *p<0.05; **p<0.01 vs Olive Oil RA; §p<0.05; §§p<0.01 vs Olive Oil IH; †p<0.05, ‡p<0.01 vs RA.

Figure 4

Representative immunoreactivity of TLR-4 (red), counterstained with DAPI (blue) in the terminal ileum of P21 neonatal rats exposed to RA (upper panel) and neonatal IH (lower panel). Images are 20X magnification, scale bar is 50 μm.

Figure 5.

Quantitative assessment of pNFkB (panel A), IkB (panel B), TLR-4 (panel C), and TGFβ1 (panel D) immunoreactivity in the terminal ileum from neonatal rats exposed to neonatal IH from P0 to P14 and allowed to recover in (RA) from P14 to P21. Groups are as described in Figure 1. Data are mean±SEM (n=12 measurements/group). *p<0.05; **p<0.01 vs Olive Oil RA; §§p<0.01 vs Olive Oil IH; †p<0.05, ‡p<0.01 vs RA.

Figure 6.

Changes in the relative proportion of the most abundant microorganisms in response to neonatal IH and antioxidant and/or fish oil supplementation. Unspecified organisms and organisms that are <1% of the total, are not shown.