Impact of Dietary Tomato Juice on Changes in Pulmonary Oxidative Stress, Inflammation and Structure Induced by Neonatal Hyperoxia in Mice (Mus musculus)

Many preterm infants require hyperoxic gas for survival, although it can contribute to lung injury. Experimentally, neonatal hyperoxia leads to persistent alterations in lung structure and increases leukocytes in bronchoalveolar lavage fluid (BALF). These effects of hyperoxia on the lungs are considered to be caused, at least in part, by increased oxidative stress. Our objective was to determine if dietary supplementation with a known source of antioxidants (tomato juice, TJ) could protect the developing lung from injury caused by breathing hyperoxic gas. Neonatal mice (C57BL6/J) breathed either 65% O2 (hyperoxia) or room air from birth until postnatal day 7 (P7d); some underwent necropsy at P7d and others were raised in room air until adulthood (P56d). In subsets of both groups, drinking water was replaced with TJ (diluted 50:50 in water) from late gestation to necropsy. At P7d and P56d, we analyzed total antioxidant capacity (TAC), markers of oxidative stress (nitrotyrosine and heme oxygenase-1 expression), inflammation (interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) expression), collagen (COL) and smooth muscle in the lungs; we also assessed lung structure. We quantified macrophages in lung tissue (at P7d) and leukocytes in BALF (at P56d). At P7d, TJ increased pulmonary TAC and COL1α1 expression and attenuated the hyperoxia-induced increase in nitrotyrosine and macrophage influx; however, changes in lung structure were not affected. At P56d, TJ increased TAC, decreased oxidative stress and reversed the hyperoxia-induced increase in bronchiolar smooth muscle. Additionally, TJ alone decreased IL-1β expression, but following hyperoxia TJ increased TNF-α expression and did not alter the hyperoxia-induced increase in leukocyte number. We conclude that TJ supplementation during and after neonatal exposure to hyperoxia protects the lung from some but not all aspects of hyperoxia-induced injury, but may also have adverse side-effects. The effects of TJ are likely due to elevation of circulating antioxidant concentrations

Altered lung function at mid-adulthood in mice following neonatal exposure to hyperoxia

Infants born very preterm are usually exposed to high oxygen concentrations but this may impair lung function in survivors in later life. However, the precise changes involved are poorly understood. We determined how neonatal hyperoxia alters lung function at mid-adulthood in mice. Neonatal C57BL/6J mice inhaled 65% oxygen (HE group) from birth for 7 days. They then breathed room air until 11 months of age (P11mo); these mice experienced growth restriction. Controls breathed only room air. To exclude the effects of growth restriction, a group of dams was rotated between hyperoxia and normoxia during the exposure period (HE+DR group). Lung function was measured at P11mo. HE mice had increased inspiratory capacity, work of breathing and tissue damping. HE+DR mice had further increases in inspiratory capacity and work of breathing, and reduced FEV₁₀₀/FVC. Total lung capacity was increased in HE+DR males. HE males had elevated responses to methacholine. Neonatal hyperoxia alters lung function at mid-adulthood, especially in males

Bronchiolar remodeling in adult mice following neonatal exposure to hyperoxia: relation to growth

Preterm infants who receive supplemental oxygen for prolonged periods are at increased risk of impaired lung function later in life. This suggests that neonatal hyperoxia induces persistent changes in small conducting airways (bronchioles). Although the effects of neonatal hyperoxia on alveolarization are well documented, little is known about its effects on developing bronchioles. We hypothesized that neonatal hyperoxia would remodel the bronchiolar walls, contributing to altered lung function in adulthood. We studied three groups of mice (C57BL/6J) to postnatal day 56 (P56; adulthood) when they either underwent lung function testing or necropsy for histological analysis of the bronchiolar wall. One group inhaled 65% O₂ from birth until P7, after which they breathed room air; this group experienced growth restriction (HE+GR group). We also used a group in which hyperoxia-induced GR was prevented by dam rotation (HE group). A control group inhaled room air from birth. At P56, the bronchiolar epithelium of HE mice contained fewer Clara cells and more ciliated cells, and the bronchiolar wall contained ~25% less collagen than controls; in HE+GR mice the bronchiolar walls had ~13% more collagen than controls. Male HE and HE+GR mice had significantly thicker bronchiolar epithelium than control males and altered lung function (HE males: greater dynamic compliance; HE+GR males: lower dynamic compliance). We conclude that neonatal hyperoxia remodels the bronchiolar wall and, in adult males, affects lung function, but effects are altered by concomitant growth restriction. Our findings may partly explain the reports of poor lung function in ex-preterm children and adults

Collagen expression in the lung at P7d and P56d.

<p>The mRNA expression of <i>COL1α1</i> in lung tissue at P7d was significantly greater in the TJ groups compared to the non-TJ groups (<b>A</b>). The area of collagen in the outer bronchiolar wall at P56d was significantly greater in the TJ groups compared to the Air group (<b>B</b>). Data points represent values from individual animals and values with different letters are significantly different from each other (p<0.05). Representative images of lung sections stained for collagen (black staining) are representative of the bronchioles analyzed at P56d (<b>C</b>, Air; <b>D</b>, Hyp; <b>E</b>, Air+TJ; <b>F</b>, Hyp+TJ). Scale bar = 10μm.</p

Oxidative stress markers at P7d.

<p>The relative gene expression of <i>heme oxygenase-1</i> (<i>HO-1</i>) in lung tissue was significantly greater in the hyperoxia groups than in the normoxia groups (<b>A</b>). The relative gene expression of <i>HO-1</i> was significantly lower in the Hyp+TJ group compared to the Hyp group (<b>A</b>). The area of nitrotyrosine staining in the lung parenchyma was significantly greater in the Hyp group than in other treatment groups (<b>B</b>). Data points represent values from individual animals. Values with different letters are significantly different from each other (p<0.05). Immunohistochemical images (<b>C</b>-<b>F</b>) of lung sections stained for nitrotyrosine (brown staining) are representative of lung parenchyma analyzed at P7d (<b>C</b>, Air; <b>D</b>, Hyp; <b>E</b>, Air+TJ; <b>F</b>, Hyp+TJ). Scale bar = 50μm.</p

Oxidative stress markers at P56d.

<p>The relative gene expression of <i>heme oxygenase-1</i> (<i>HO-1</i>) was not significantly different between groups (<b>A</b>). The area of nitrotyrosine staining in the lung parenchyma was significantly greater in non-TJ groups than in TJ groups (<b>B</b>). Data points represent values from individual animals. Values with different letters are significantly different from each other (p<0.05). Immunohistochemical images (<b>C</b>-<b>F</b>) of lung sections stained for nitrotyrosine (brown staining) are representative of lung parenchyma analyzed at P56d (<b>C</b>, Air; <b>D</b>, Hyp; <b>E</b>, Air+TJ; <b>F</b>, Hyp+TJ). Scale bar = 50μm.</p

Macrophages in lung tissue at P7d.

<p>The proportion of macrophages (galectin-3 positive cells) in the lung parenchyma was significantly greater in the Hyp group than in the Air group; the proportion of macrophages was not significantly different between the two TJ groups (<b>A</b>). Data points represent values from individual animals. Values with different letters are significantly different from each other (p<0.05). Immunofluorescent images (<b>B</b>-<b>E</b>) of lung sections stained for galectin-3 (red staining) are representative of lung parenchyma analyzed at P7d (<b>B</b>, Air; <b>C</b>, Hyp; <b>D</b>, Air+TJ; <b>E</b>, Hyp+TJ). Scale bar = 30μm.</p