Brain adaptation to hypoxia and hyperoxia in mice

Abstract

Aims: Hyperoxic breathing might lead to redox imbalance and signaling changes that affect cerebral function. Paradoxically, hypoxic breathing is also believed to cause oxidative stress. Our aim is to dissect the cerebral tissue responses to altered O₂ fractions in breathed air by assessing the redox imbalance and the recruitment of the hypoxia signaling pathways.

Results: Mice were exposed to mild hypoxia (10%O₂), normoxia (21%O₂) or mild hyperoxia (30%O₂) for 28 days, sacrificed and brain tissue excised and analyzed. Although one might expect linear responses to %O₂, only few of the examined variables exhibited this pattern, including neuroprotective phospho- protein kinase B and the erythropoietin receptor. The major reactive oxygen species (ROS) source in brain, NADPH oxidase subunit 4 increased in hypoxia but not in hyperoxia, whereas neither affected nuclear factor (erythroid-derived 2)-like 2, a transcription factor that regulates the expression of antioxidant proteins. As a result of the delicate equilibrium between ROS generation and antioxidant defense, neuron apoptosis and cerebral tissue hydroperoxides increased in both 10%O₂ and 30%O₂, as compared with 21%O₂. Remarkably, the expression level of hypoxia-inducible factor (HIF)-2α (but not HIF-1α) was higher in both 10%O₂ and 30%O₂ with respect to 21%O₂ INNOVATION: Comparing the in vivo effects driven by mild hypoxia with those driven by mild hyperoxia helps addressing whether clinically relevant situations of O₂ excess and scarcity are toxic for the organism.

Conclusion: Prolonged mild hyperoxia leads to persistent cerebral damage, comparable to that inferred by prolonged mild hypoxia. The underlying mechanism appears related to a model whereby the imbalance between ROS generation and anti-ROS defense is similar, but occurs at higher levels in hypoxia than in hyperoxia.

Keywords: Hypoxia-inducible factor; In vivo hyperoxia; In vivo hypoxia; Neurons; Oxidative injury.

Graphical abstract

Fig. 1

Redox imbalance at the end of the exposure to hypoxia (10%O₂), normoxia (21%O₂) and hyperoxia (30%O₂) for 28 days (n=7, 6 and 6, respectively). Panel A. Expression level of NADPH oxidase subunit 4 (NOX4). Panel B. d-ROMs test to estimate the pro-oxidant capacity of plasma samples towards a chromogenic indicator, expressed in U CARR units. Panel C. Lipotiss test to estimate the hydroperoxide level in cerebral tissue. Data are expressed as box plots indicating the 25th percentile, the median and the 75th percentile, with whiskers indicating the max and min values. The inset reports the ANOVA test. *, P<0.05; **, P<0.01; ***, P<0.001 (Tukey multiple comparison post-test).

Fig. 2

Hypoxia signaling. Panel A. Expression level of cytosolic (left) and nuclear (right) hypoxia-inducible factor (HIF)−1α. Panel B. Expression level of cytosolic (left) and nuclear (right) HIF-2α. Data expressed as described in Fig. 1.

Fig. 3

Protective mechanisms. Panel A. Expression level of cytosolic (left) and nuclear (right) nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Panel B. Ratio between the expression levels of total protein kinase B (Akt) and the phospho-Akt-Ser⁴⁷³ (p-Akt). Data expressed as described in Fig. 1. Panel C. Tissue expression level of erythropoietin (EPO, left) and of the EPO receptor (EPO-R, right).

Fig. 4

Markers of vascularization. Panel A. Expression level of tissue vascular endothelial growth factor (VEGF). Panel B. Expression level of tissue VEGF receptor 2. Panel C. Expression level of hematopoietic progenitor cell antigen (CD34). Panel D. Expression level of platelet endothelial cell adhesion molecule (PECAM-1). Data expressed as described in Fig. 1.

Fig. 5

Brain damage. Panel A. Representative immunofluorescence pictures obtained in cerebral tissue samples from hypoxia (10%O₂), normoxia (21%O₂) and hyperoxia (30%O₂) for 28 days. The green (neurons), red (TdT positive) and blue (nuclei) channels are displayed. The white arrows identify spots associated with apoptosis in neurons (green+red+blue), while purple arrows identify spots associated with apoptosis in non-neuronal cells (red+blue). The bar represents 50 µm. Panel B. Percent of neuronal (left) and glial (right) cells that are positive for TdT as counted in 5 sections from each specimen. Data expressed as described in Fig. 1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)