Mice Treatment

Third Military Medical University, an experimental animal center, provided 8–10weeks (18±2g) C57BL/6 male mice. They were kept at 25°C, with 45% humidity, and exposed to a 12-h light −12-h dark cycle. The hypoxia group was exposed to an altitude of 6,000m for 7days. The normoxic control group was kept at the normal atmosphere of about 300m for 7days. All procedures followed the standards of experimental animal ethics.

After the hypoxia treatment, a small amount of chloral hydrate (10%, w/v) was used to inject the mice to narcosis, which were then perfused with 400mL of normal saline (0.9%, v/v) intracardially; the hippocampus was harvested for western blot and quantitative real-time PCR (qRT-PCR). This was followed by perfusing with 400mL of normal saline (0.9%, v/v) and 350mL of paraformaldehyde (4%, v/v). Finally, we decapitated the mice and fixed their brains in paraformaldehyde (4%, v/v) overnight. The brains were dewatered using 30% sucrose in the same solution; sections of brain serial coronal (30μm) were collected with a cryostat (CM1900, Leica Microsystems, Germany) at −20°C and used further for immunofluorescence (IF).