Oral antioxidant edaravone protects against cognitive deficits induced by chronic hypobaric hypoxia at high altitudes

Abstract

Chronic hypobaric hypoxia at high altitudes can impair cognitive functions, especially causing deficits in learning and memory, which require therapeutic intervention. Here, we showed that mice subjected to hypobaric hypoxia (simulating an altitude of 5000 m) for one month experienced significant cognitive impairment, accompanied by increased biomarker levels of oxidative stress in the brain and blood. Oral administration of a novel formulation of edaravone, a free radical scavenger approved for the treatment of ischaemic stroke and amyotrophic lateral sclerosis, significantly alleviated oxidative stress and cognitive impairments caused by chronic hypobaric hypoxia. Furthermore, oral edaravone treatment also mitigated neuroinflammation and restored hippocampal neural stem cell exhaustion. Additionally, periostin (Postn) is vital in the cognitive deficits caused by chronic hypobaric hypoxia and may be a molecular target of edaravone. In conclusion, our results suggest that oxidative stress plays a crucial role in the cognitive deficits caused by chronic hypobaric hypoxia and that oral edaravone is a potential medicine for protecting against cognitive deficits caused by chronic hypobaric hypoxia in high-altitude areas.

Fig. 1. Edaravone alleviated locomotor and cognitive impairments caused by chronic hypobaric hypoxia.

A Study design: Eight- to nine-week-old C57BL/6 male mice were randomly allocated to the following groups: plain+vehicle, HA+vehicle, HA+treatment and HA+prevention. All mice underwent a battery of behavioural tests from Days 36–44 and were sacrificed on Day 45. B Changes in body weight from Day 0 to Day 35 in the plain+vehicle, HA+vehicle, HA+treatment and HA+prevention groups. C, D Representative trajectory diagrams (C) and total distance travelled (D) of mice in the open field test. E, F Representative heatmaps (E) and discrimination ratios (F) of mice in the plain+vehicle, HA+vehicle, HA+treatment and HA+prevention groups in the novel object recognition test. “N” and “F” represent “novel object” and “familiar object”, respectively. G–I Diagram of the Barnes maze (G) and latency to enter the target hole (H) and errors made (I) during the learning acquisition period. Data are presented as mean ± SEM. Statistical comparisons were performed by two-way (B, H, I) and one-way (D, F) ANOVA with Tukey’s post hoc test. N = 9/group. *P < 0.05, **P < 0.01, ***P < 0.001. HA, high altitude; NORT, novel object recognition test; OFT, open field test.

Fig. 2. Edaravone abrogated alterations in the plasma protein expression profile caused by hypobaric hypoxia.

A Hierarchical clustering heatmap of differentially expressed proteins in plasma between the plain+vehicle and HA+vehicle mice. B, C Volcano plot (B) and significantly enriched biological processes (C) of differentially expressed proteins between the HA+vehicle and plain+vehicle groups. D, E Volcano plot (D) and significantly enriched biological processes (E) of differentially expressed proteins between the HA+treatment and HA+vehicle groups. HA, high altitude. N = 3/group.

Fig. 3. Edaravone reduced oxidative stress and inflammation in the brain caused by chronic hypobaric hypoxia.

A–D Levels of MDA (A), SOD (B), GSH/GSSG ratio (C), and GSH-Px (D) in the brain tissues of plain+vehicle, HA+vehicle, HA+treatment and HA+prevention mice. Data are presented as mean ± SEM. N = 8/group. E Quantitative analysis of Iba1⁺ immunofluorescence staining within the dentate gyrus of plain+vehicle, HA+vehicle, HA+treatment and HA+prevention mice. N = 9/group. F–M Representative images of Iba1⁺ cells in the dentate gyrus region. Scale bar = 100 µm. Data are presented as mean ± SEM. Statistical comparisons were performed by one-way ANOVA with Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. GSH/GSSG, reduced glutathione/oxidized glutathione disulfide; GSH-Px, glutathione peroxidase; HA, high altitude; Iba1, ionized calcium-binding adapter molecule-1; MDA, malondialdehyde; SOD, superoxide dismutase.

Fig. 4. Edaravone restored hippocampal neural stem cell exhaustion caused by chronic hypobaric hypoxia.

A–H Representative images of Sox2⁺/GFAP⁺ colabelled radial glia-like neural progenitor cells in the dentate gyrus region. The arrows indicate Sox2⁺/GFAP⁺ colabelled cells. Scale bar = 100 µm. Q, R Quantitative analysis of Sox2⁺/GFAP⁺ (Q) and Sox2⁺ (R) immunofluorescence staining within the dentate gyrus of plain+vehicle, HA+vehicle, HA+treatment and HA+prevention mice. I–P Representative images of DCX⁺ cells in the dentate gyrus region. Scale bar = 100 µm. S Quantitative analysis of DCX⁺ immunofluorescence staining within the dentate gyrus of plain+vehicle, HA+vehicle, HA+treatment and HA+prevention mice. Data are presented as mean ± SEM. Statistical comparisons were performed by one-way ANOVA with Tukey’s post hoc test. N = 9/group. *P < 0.05, **P < 0.01, ***P < 0.001. DCX, doublecortin; GFAP, glial fibrillary acidic protein; HA, high altitude; Sox2, sex determining region Y-Box 2.

Fig. 5. Edaravone abrogated alterations in brain gene expression induced by hypobaric hypoxia.

A Hierarchical clustering heatmap of differentially expressed genes in the hippocampus of plain+vehicle, HA+vehicle, HA+treatment and HA+prevention mice. B Venn diagram of differentially expressed genes regulated by chronic hypobaric hypoxia and edaravone treatment and prevention. C–E Significantly enriched biological processes of the differentially expressed genes between the plain+vehicle and HA+vehicle groups (C), between the HA+treatment and HA+vehicle groups (D), and between the HA+prevention and HA+vehicle groups (E) analysed by Gene Ontology analysis. N = 3/group. HA, high altitude.

Fig. 6. Postn was detrimental for mice in chronic hypobaric hypoxia, and edaravone can regulate its expression.

A Venn diagram of the 16 differentially expressed molecules altered in both the hippocampus and plasma regulated by chronic hypobaric hypoxia. N = 3/group. B Hierarchical clustering heatmap of the 16 differentially expressed genes in the hippocampus among plain+vehicle, HA+vehicle, HA+treatment and HA+prevention groups. N = 3/group. C–K Representative western blots (C) and quantitative analysis (D–K) for the protein expressions of LOXL1, Postn, ISG15, Sod3, Prdx4, ALDH1a1, Prdx6, and GSTM1 in plain+vehicle, HA+vehicle, HA+treatment and HA+prevention mice. Data are presented as mean ± SEM. Statistical comparisons were performed by one-way ANOVA with Turkey post hoc test. N = 8/group. *P < 0.05, **P < 0.01, ***P < 0.001. ALDH1a1, aldehyde dehydrogenase 1a1; GSTM1, glutathione S-transferase M1; HA, high altitude; ISG15, interferon stimulated gene 15; LOXL1, lysyl oxidase like; Postn, periostin; Prdx, peroxiredoxin; Sod3, superoxide dismutase-3.