Preconditioning With Intermittent Hypobaric Hypoxia Attenuates Stroke Damage and Modulates Endocytosis in Residual Neurons.

Moderate hypobaric hypoxia induces cerebral ischemic tolerance. We investigated the optimal method for applying hypobaric hypoxia preconditioning at 5,000 m to ischemic brain tissue and combined it with proteomics to determine the mechanisms underlying this effect. Male SD rats were randomly grouped as S (sham, = 20), M (middle cerebral artery occlusion [MCAO], = 28), H2M (intermittent hypobaric hypoxia preconditioned MCAO group, 2 h/day, 10 days, = 20), H6M (intermittent hypobaric hypoxia preconditioned MCAO group, 6 h/day, 10 days, = 28), and HpM (persistent hypobaric hypoxia preconditioned MCAO group, 10 days, = 28). The permanent MCAO model was established based on the Zea Longa method. Infarction was assessed with the modified neurological severity score (mNSS) and 2,3,5-triphenyl tetrazolium chloride staining. The total protein expression of the neuron-specific nuclear protein (NeuN), cysteinyl aspartate specific proteinase 3 (caspase-3), cleaved-caspase-3, and interleukin 6 (IL-6) was determined using western blotting. We assessed the peri-infarct cortex's ultrastructural changes. A label-free proteomic study and western blot verification were performed on the most effective preconditioned group. The H6M group showed a lower infarct volume ( = 0.0005), lower mNSS score ( = 0.0009) than the M group. The H2M showed a lower level of IL-6 ( = 0.0213) than the M group. The caspase-3 level decreased in the H2M ( = 0.0002), H6M ( = 0.0025), and HpM groups ( = 0.0054) compared with that in the M group. Cleaved-caspase-3 expression decreased in the H2M ( = 0.0011), H6M ( < 0.0001), and HpM groups ( < 0.0001) compared with that in the M group. The neurons' ultrastructure and the blood-brain barrier in the peri-infarct tissue improved in the H2M and H6M groups. Immunofluorescence revealed increased NeuN-positive cells in the peri-infarct tissue in the H6M group ( = 0.0003, H6M vs. M). Protein expression of Chmp1a, Arpc5, and Hspa2 factors related to endocytosis were upregulated in the H6M compared with those of the M group ( < 0.05 for all) on western blot verification of label-free proteomics. Intermittent hypobaric hypoxia preconditioning exerts a neuroprotective effect in a rat stroke model. Persistent hypobaric hypoxia stimulation exhibited no significant neuroprotective effect. Intermittent hypoxic preconditioning for 6 h/day for 10 days upregulates key proteins in clathrin-dependent endocytosis of neurons in the cortex.