Lung-Specific Extracellular Superoxide Dismutase Improves Cognition of Adult Mice Exposed to Neonatal Hyperoxia

Abstract

Lung and brain development is often altered in infants born preterm and exposed to excess oxygen, and this can lead to impaired lung function and neurocognitive abilities later in life. Oxygen-derived reactive oxygen species and the ensuing inflammatory response are believed to be an underlying cause of disease because over-expression of some anti-oxidant enzymes is protective in animal models. For example, neurodevelopment is preserved in mice that ubiquitously express human extracellular superoxide dismutase (EC-SOD) under control of an actin promoter. Similarly, oxygen-dependent changes in lung development are attenuated in transgenic Sftpc ^EC-SOD mice that over-express EC-SOD in pulmonary alveolar epithelial type II cells. But whether anti-oxidants targeted to the lung provide protection to other organs, such as the brain is not known. Here, we use transgenic Sftpc ^EC-SOD mice to investigate whether lung-specific expression of EC-SOD also preserves neurodevelopment following exposure to neonatal hyperoxia. Wild type and Sftpc ^EC-SOD transgenic mice were exposed to room air or 100% oxygen between postnatal days 0-4. At 8 weeks of age, we investigated neurocognitive function as defined by novel object recognition, pathologic changes in hippocampal neurons, and microglial cell activation. Neonatal hyperoxia impaired novel object recognition memory in adult female but not male mice. Behavioral deficits were associated with microglial activation, CA1 neuron nuclear contraction, and fiber sprouting within the hilus of the dentate gyrus (DG). Over-expression of EC-SOD in the lung preserved novel object recognition and reduced the observed changes in neuronal nuclear size and myelin basic protein fiber density. It had no effect on the extent of microglial activation in the hippocampus. These findings demonstrate pulmonary expression of EC-SOD preserves short-term memory in adult female mice exposed to neonatal hyperoxia, thus suggesting anti-oxidants designed to alleviate oxygen-induced lung disease such as in preterm infants may also be neuroprotective.

Keywords: anti-oxidants; hyperoxia; long-term consequences; mice; neonatal; neurocognitive; pulmonary.

Figure 1

Human EC-SOD protein is detected in the lungs, but not brains, of transgenic Sftpc^EC−SOD mice. Lung and brain tissues were harvested on postnatal day 4 and at 8 weeks of age from wild type (WT) and Sftpc^EC−SOD transgenic (Tg) mice birthed into room air. Lung and brain homogenates were immunoblotted with antibodies to detect human (h) EC-SOD, mouse (m) EC-SOD, and β-actin (loading control). Each lane represents an individual mouse.

Figure 2

Over-expression of EC-SOD in the lung preserves the ability to learn in adult female mice exposed to neonatal hyperoxia. Novel object recognition testing was evaluated separately in female (A,B) and male (C,D) adult wild type (WT) and Sftpc^EC−SOD transgenic (Tg) mice that were exposed to room air (RA) or 100% oxygen (O₂) between postnatal days (PND) 0–4. A recognition index (A,C) based on the proportion of total time spent in contact with the novel object, as well as a time per approach index (B,D) based on the proportion of total time spent approaching the non-novel object were measured to assess learning. Neonatal hyperoxia significantly decreased recognition of the familiar object in female WT mice (A, ^*P < 0.0001) but not in Tg mice (B, P = 0.28). Neonatal hyperoxia significantly increased recognition of the familiar object in female WT (C, ^*P < 0.002) but not in Tg mice (D, P = 0.25). n = 9–10 mice per group. Values from individual mice are represented as circles.

Figure 3

Exposure to neonatal hyperoxia does not affect locomotor activity in adult mice. Locomotor activity testing was evaluated in adult (A) female and (B) male wild type (WT) and Sftpc^EC−SOD transgenic (Tg) mice (8–10 weeks of age) exposed to room air (RA) or 100% oxygen (O₂) during postnatal days (PND) 0–4. Jumps, vertical counts, ambulatory episodes, stereotypic counts, and resting time were quantified in three 60-min sessions occurring once per day on three consecutive days (n = 9–10 mice per group, ^*P < 0.05 when compared to WT mice exposed to room air as neonates).

Figure 4

Over-expression of EC-SOD in the lung does not block hyperoxia-induced microglial priming in CA1 of the adult hippocampus. (A) CA1 field of the hippocampus showing neuronal nuclei, Iba1⁺ microglia, and GFAP⁺ astrocytes. Scale bar = 200 μm. (B) Neonatal hyperoxia stimulates Iba1 expression in both WT and EC-SOD mice; ^*P < 0.05. (C) Sholl analysis heat map of microglial processes illustrating the number of intersections with concentric circles from the soma to most distal branch. (D,E) Sholl analyses demonstrating no differences in microglial arborization in either adult WT or Sftpc^EC−SOD transgenic mice exposed to room air or neonatal hyperoxia. (n = 4–5 mice per group except panel B where n = 3 mice per group). Data from individual mice are represented as circle (room air) or squares (hyperoxia) in (B).

Figure 5

Over-expression of EC-SOD in the lung protects against hyperoxia-induced changes in neuronal morphology in CA1 and DG of the hippocampus. (A) Over-expression of EC-SOD has no effect on CA1 nuclear size in the CA1 field. Distribution and Gaussian curve fit of neuronal nuclear size in the CA1 field performed in room air-treated mice (WT, 72.80 μm² ± 11.04 vs. EC-SOD, 73.83 μm² ± 9.97; p = 0.74 for comparison of Gaussian curves). (B) Over-expression of EC-SOD in the lung blocks hyperoxia-induced nuclear shrinkage in exposed CA1 neurons (WT, 59.38 μm² ± 12.09 vs. EC-SOD, 65.23 μm² ± 14.06; ^****p < 0.0001 for comparison of Gaussian curves). (C) Transgene expression of EC-SOD in the lung inhibits hyperoxia-induced fiber sprouting in the hilus of the dentate gyrus (DG). Skeleton analysis of MBP⁺ neurites in the DG hilus. Scale bar = 400 μm. (D) Histogram and Gaussian curve fit of branch lengths in the DG with no differences in room air-treated mice (WT, 3.23 μm ± 1.48 vs. EC-SOD, 3.36 μm ± 1.74; p = 0.27 for comparison of Gaussian curves). (E) Neonatal hyperoxia induces neurite sprouting in adult WT when compared to adult Sftpc^EC−SOD mice (WT, 3.79 μm ± 1.84 vs. EC-SOD, 3.33 μm ± 1.75; ^**p < 0.01 for comparison of Gaussian curves). (n = 4–5 mice per group).