title: Protective effects of early hypoxic post-conditioning in cultured cortical neurons.
abstract: 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.
  
  
  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.
authors.affiliation: Department of Physiology, Pre-Clinic College, Xinjiang Medical University, Urumqi, PR, China.
authors.forename: Qiao-Ling
authors.initials: QL
authors.lastname: Yao
authors.forename: Mei-Fen
authors.initials: MF
authors.lastname: Zhang
authors.forename: Chu-Huai
authors.initials: CH
authors.lastname: Wang
authors.forename: Fen
authors.initials: F
authors.lastname: Hu
authors.forename: Ai-Ping
authors.initials: AP
authors.lastname: Lan
authors.forename: Rui-Xian
authors.initials: RX
authors.lastname: Guo
authors.forename: Pei-Xi
authors.initials: PX
authors.lastname: Chen
authors.forename: Jian-Qiang
authors.initials: JQ
authors.lastname: Feng
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western; D015687:Cell Hypoxia / Q000502:physiology*; D002470:Cell Survival / Q000502:physiology*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000378:metabolism* / Q000503:physiopathology; D009474:Neurons / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D017382:Reactive Oxygen Species