Effect of different levels of intermittent hypoxia on autophagy of hippocampal neurons

Abstract

Purpose: The current study was carried out to assess the effects of different levels of intermittent hypoxia (IH) on autophagy in hippocampal neurons, and explore the extent, frequency and duration of IH for researching on autophagy in hippocampal neurons.

Methods: Hippocampal neurons were exposed to different levels of IH. To analyze the oxygen level of neuronal exposure environment, we detected the oxygen concentration in the chamber by O₂ analyzer, and monitored the oxygen partial pressure (PO₂), carbon dioxide partial pressure (PCO₂), and pH in the culture media by blood gas analyzer. After 4-, 8-, and 12-h IH, the morphology and quantity of neurons, as well as the expression of light chain 3 (LC3)-II positive dots were observed by immunofluorescence. The expression of apoptosis marker protein cleaved caspase-3 and autophagy marker protein LC3 were examined by western blotting.

Results: The oxygen level in the chamber and the neuronal culture media both reached to the values set previously in three models. The level of cleaved caspase-3 and LC3 had no significant changes in IH-1 group. The morphology and quantity had no significant changes, while the levels of cleaved caspase-3 and LC3 were both increased in IH-2 group. The quantity of neurons was reduced significantly, and the chromatin condensed and nuclei fragmented in IH-3 group.

Conclusions: The effects of varying degrees of IH on autophagy in hippocampal neurons are different. The IH model, hypoxia phase (1.5% O₂, 5% CO₂, and balance N₂) for 5 min and reoxygenation phase (21% O₂, 5% CO₂, and balance N₂) for 10 min, may be the best condition for researching on autophagy in hippocampal neurons.

Keywords: Apoptosis; Autophagy; Hippocampal neurons; Intermittent hypoxia.