Protective effects of early hypoxic post-conditioning in cultured cortical neurons.

Primary Objective: Recent evidence suggests that delayed hypoxic post-conditioning is neuroprotective. The aim of the present study was to test whether early post-conditioning applied immediately after hypoxia could protect cultured neurons from hypoxia/reoxygenation (H/R)-induced injuries.

Methods: Primary cortical neuronal culture depleted of microglia was exposed to H/R. Post-conditioning started immediately after hypoxia and consisted of three cycles of 15-minutes of reoxygenation and 15-minutes of hypoxia. Cell viability assay was performed using Cell Counting Kit-8 (CCK-8). Apoptosis was evaluated by Hoechst 33258 staining, FITC-Annexin V/PI double staining and Western blot assay (testing the cleaved caspase-3 expression). Reactive oxygen species (ROS), intracellular Ca(2+) and mitochondrial membrane potential (MMP) were examined using confocal laser-scanning microscopy.

Main Results: H/R significantly reduced cell viability and increased neuronal apoptosis and necrosis. Furthermore, the expression of cleaved caspase-3, ROS production and intracellular Ca(2+) were increased. MMP was attenuated. Injuries induced by H/R were substantially attenuated by early hypoxic post-conditioning. Changes in cleaved caspase-3 expression, ROS production, intracellular Ca(2+) level and MMP in response to H/R were significantly decreased by the post-conditioning.

Conclusions: The findings demonstrated that early hypoxic post-conditioning could protect neurons against H/R-induced injuries independent of microglial cells, possibly by inhibiting ROS over-production and intracellular Ca(2+) accumulation and maintaining MMP.