Background: Aspirin and its major metabolite salicylic acid (SA), confer protection against glutamate neurotoxicity via COX dependent and independent mechanisms. There is also evidence that SA may protect against hypoxia-induced neuronal cell damage which is associated with increased levels of reactive oxygen species (ROS). Here we investigated the effects of chronically increased levels of SA on the ability of neuronal cells to survive hypoxia-induced neurotoxicity in-vitro and evaluated the underlying mechanisms.

Materials and methods: Human neuronal IMR-32 cells were cultured with or without SA (0.1, 1 and 10µM) for a total of 7 months. In-vitro hypoxia was induced for 3 hours using our recently described system. Cell morphology was evaluated by brightfield microscopy, cell damage was measured by lactate dehydrogenase (LDH) assays and cell metabolism was quantified by colorimetric MTS assays. Production of ROS was analyzed using fluorometric assays. Westernblotting for catalase (CAT) and glutathione peroxidase (GPX) was performed

Results: Long term incubation of neuronal IMR-32 cells with clinically relevant concentrations of SA (0.1, 1 and 10µM) did not change morphology or proliferative potential of the cultures. However, neuronal cells that were exposed to chronic SA treatment showed statistically significant protection against hypoxia/reoxygenation injury (P<0.001 vs. hypoxia for all SA concentrations) and 1µM SA also significantly increased metabolic activity of IMR-32 cells under normoxic as well as hypoxic conditions (P<0.01 vs. the respective normoxia/hypoxia control). Biochemical analyses using cell-free systems revealed that the activity CAT which is protecting cells from oxidative stress, is regulated by SA. However, compared to control cells, protein expression of CAT, GPX and hypoxia-induced ROS generation were not altered in IMR-32 cells exposed to SA. Moreover inhibiting CAT activity in culture modified the hypoxia-induced cell damage. 

Conclusion:
Long term treatment with salicylic acid induces neuronal resistance to hypoxia / reoxygenation injury and this effect may depend partially on catalase and ROS.

Long term application of salicylic acid protects neuronal cells from hypoxia/reoxygenation injury in-vitro