The brain, despite comprising only 2% of body weight, consumes nearly 20% of the body’s oxygen, making it highly susceptible to hypoxic injury. Hypoxia triggers oxidative stress, cell death, and neurodegeneration, central to the pathophysiology of many neurological conditions like ischemic stroke, epilepsy, and Alzheimer’s disease. However, repeated mild hypoxia has been shown to activate endogenous neuroprotective mechanisms, enhancing cellular resilience. Intermittent hypoxic-hyperoxic training (IHHT) is emerging as a promising therapeutic approach to hypoxic injury, yet the mechanisms underlying its neuroprotective effects remain unclear. Proteomic studies aimed to identify these mechanisms are limited. This study focuses on identifying proteins and pathways altered by repeated mild hypoxia in the hippocampus, a brain region essential for memory and learning. Additionally, it aims to reveal sex-specific neuroprotective responses that could guide the development of targeted therapeutic strategies for brain injury. Eight-week-old male and female Sprague-Dawley rats from the ATSU KCOM facility were maintained under controlled conditions (12 hr light/dark cycle, 24°C, 40% humidity, water and food ad libitum). Rats were exposed to either room air (CTL, n = 3/sex) or to five 2-hour episodes of hypoxia (HX, 10% O 2 + 90% N 2 , 24 hours apart, n = 3/sex) using a BioSperix hypoxia chamber. 24 hours after the final exposure, hippocampi were dissected, flash frozen in liquid nitrogen, and stored at -80°C until sent for proteomic analysis (MetwareBio). Samples underwent protein extraction, enzymatic digestion, liquid chromatography, and high-resolution mass spectrometry. Data were analyzed using DIA-NN to identify differentially expressed proteins using a fold change threshold of ≥ 1.5 or ≤ 0.667 and a p-value of ≤ 0.05 (t-test), followed by bioinformatics-based annotation, enrichment, and visualization of affected pathways, protein interactions, subcellular localization, and expression patterns. Proteomics analysis identified a total of 11,333 proteins from 124,965 peptides. Quantitative results showed consistent abundance across biological replicates ensuring high reliability of the differential protein expression data. A total of 91 differentially expressed proteins were found in the male-CTL vs. male-HX group and a total of 52 were found in the female-CTL vs. female-HX group. Key findings include that males upregulate energy metabolism and synaptic plasticity proteins (e.g., PANK1, FXYD6) and females enhance blood-brain barrier integrity and lipid homeostasis proteins (e.g., ENHO, SCP2). Additionally, repeated mild hypoxia reduced Zdhhc8 expression in both male-HX and female-HX groups (compared to their CTLs), reducing neuronal palmitoylation activity and potentially impairing trafficking and signaling pathways. This study successfully used a proteomic approach to investigate the neuroprotective mechanisms activated in the rat hippocampus following repeated mild hypoxia exposure. Males primarily upregulated proteins associated with energy metabolism and synaptic plasticity, suggesting enhanced functional resilience. Females showed an enrichment of proteins related to blood-brain barrier integrity and lipid homeostasis, pointing toward structural and metabolic protection. Our data reveal significant sex-specific differences in the underlying neuroprotective pathways, emphasizing the need for sex-aware therapeutic approaches. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.
Calbaza et al. (Fri,) studied this question.