Exposing the developing lung to hyperoxic gas: effects on lung development and markers of oxidative stress and inflammation later in life

Owing to lung immaturity, infants born preterm are often administered high levels of oxygen (hyperoxia). However, prolonged hyperoxia can cause oxidative stress and inflammation and can alter lung development, which may contribute to later lung disease. Previous experimental studies of neonatal hyperoxia have largely used oxygen concentrations greater than 80%. There is a need to study the long-term effects of milder oxygen concentrations, which reflect current clinical practice. Preterm infants are likely to have an immature antioxidant system at birth, but how this immaturity contributes to hyperoxia-induced lung injury is poorly understood. As exposure to hyperoxia induces oxidative stress, it is possible that dietary antioxidant supplementation can prevent or reduce the degree of injury. The aim of studies reported in Chapter 3 was to compare the short-term and long-term effects of exposing neonatal mice to either mild (40% O₂) or moderate (65% O₂) hyperoxia for seven days after birth. Controls breathed room air (21% O₂). Lungs were collected at either postnatal day seven (P7d) or adulthood (P56d), after seven weeks of breathing 21% O₂. Unlike neonatal exposure to 65% O₂, exposure to 40% O₂ did not alter lung structure at either age. However, exposure to both 40% and 65% O₂ led to increases in markers of oxidative stress and the number of immune cells in the lung that persisted into adulthood. The aim of studies described in Chapter 4 was to determine the effect of neonatal hyperoxia in mice lacking the gene for glutathione peroxidase 1 (Gpx1; an endogenous antioxidant enzyme), thereby mimicking an immature antioxidant system. Gpx1 knockout and wild-type mice were exposed to 21% or 40% O₂ for seven days after birth. In the absence of Gpx1 expression, neonatal hyperoxia did not increase oxidative stress or alter lung structure; however, the proportion of lymphocytes in the adult lung was increased. Potential redundancy was observed within the model, with the relative gene expression of Gpx2, Gpx3, Gpx4 and Catalase increased at P56d in the Gpx1 knockout mice exposed to 40% O₂. The aim of studies reported in Chapter 5 was to determine whether dietary antioxidants (tomato juice) could protect the lung from the effects of neonatal exposure to 65% O2. Tomato juice supplementation increased lung antioxidant capacity and reduced markers of oxidative stress and inflammation at P7d, but did not prevent decreased alveolarisation following exposure to 65% O₂. At P56d, the supplementation ameliorated the hyperoxia-induced increase in bronchiolar smooth muscle, but did not alter the increase in immune cells in the lung lumen. Conclusions: This thesis shows for the first time that a mild level of neonatal hyperoxia (40% O₂) can cause persistent increases in oxidative stress and immune cell numbers in the lung, in the absence of structural alterations; thus, mild hyperoxia, thought to be clinically benign, can adversely affect the lung in such a way as to increase the risk of later lung disease. The absence of Gpx1 gene expression does not exacerbate hyperoxia-induced alterations in lung structure or increases in oxidative stress, but does alter immune cells in the lung. Finally, dietary antioxidant supplementation may be beneficial in preventing some of the short-term and long-term alterations in the lung induced by neonatal hyperoxia

Exposing the developing lung to hyperoxic gas: effects on lung development and markers of oxidative stress and inflammation later in life

Owing to lung immaturity, infants born preterm are often administered high levels of oxygen (hyperoxia). However, prolonged hyperoxia can cause oxidative stress and inflammation and can alter lung development, which may contribute to later lung disease. Previous experimental studies of neonatal hyperoxia have largely used oxygen concentrations greater than 80%. There is a need to study the long-term effects of milder oxygen concentrations, which reflect current clinical practice. Preterm infants are likely to have an immature antioxidant system at birth, but how this immaturity contributes to hyperoxia-induced lung injury is poorly understood. As exposure to hyperoxia induces oxidative stress, it is possible that dietary antioxidant supplementation can prevent or reduce the degree of injury. The aim of studies reported in Chapter 3 was to compare the short-term and long-term effects of exposing neonatal mice to either mild (40% O₂) or moderate (65% O₂) hyperoxia for seven days after birth. Controls breathed room air (21% O₂). Lungs were collected at either postnatal day seven (P7d) or adulthood (P56d), after seven weeks of breathing 21% O₂. Unlike neonatal exposure to 65% O₂, exposure to 40% O₂ did not alter lung structure at either age. However, exposure to both 40% and 65% O₂ led to increases in markers of oxidative stress and the number of immune cells in the lung that persisted into adulthood. The aim of studies described in Chapter 4 was to determine the effect of neonatal hyperoxia in mice lacking the gene for glutathione peroxidase 1 (Gpx1; an endogenous antioxidant enzyme), thereby mimicking an immature antioxidant system. Gpx1 knockout and wild-type mice were exposed to 21% or 40% O₂ for seven days after birth. In the absence of Gpx1 expression, neonatal hyperoxia did not increase oxidative stress or alter lung structure; however, the proportion of lymphocytes in the adult lung was increased. Potential redundancy was observed within the model, with the relative gene expression of Gpx2, Gpx3, Gpx4 and Catalase increased at P56d in the Gpx1 knockout mice exposed to 40% O₂. The aim of studies reported in Chapter 5 was to determine whether dietary antioxidants (tomato juice) could protect the lung from the effects of neonatal exposure to 65% O2. Tomato juice supplementation increased lung antioxidant capacity and reduced markers of oxidative stress and inflammation at P7d, but did not prevent decreased alveolarisation following exposure to 65% O₂. At P56d, the supplementation ameliorated the hyperoxia-induced increase in bronchiolar smooth muscle, but did not alter the increase in immune cells in the lung lumen. Conclusions: This thesis shows for the first time that a mild level of neonatal hyperoxia (40% O₂) can cause persistent increases in oxidative stress and immune cell numbers in the lung, in the absence of structural alterations; thus, mild hyperoxia, thought to be clinically benign, can adversely affect the lung in such a way as to increase the risk of later lung disease. The absence of Gpx1 gene expression does not exacerbate hyperoxia-induced alterations in lung structure or increases in oxidative stress, but does alter immune cells in the lung. Finally, dietary antioxidant supplementation may be beneficial in preventing some of the short-term and long-term alterations in the lung induced by neonatal hyperoxia

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

AJP-L00270-2023R1 Supplementary Data

Supplementary Data and Figures</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

Lung morphometry at P7d.

<p>Lung morphometry at P7d.</p

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

Lung morphometry at P56d.

<p>Lung morphometry at P56d.</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