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Characterization of anoxia-induced neuronal death in hippocampal neurons

University of British Columbia

Cerebral ischemia occurs when the blood supply to the brain, in whole or part, is reduced, resulting in a decrease in the supply of oxygen and glucose to the brain. This type of insult typically results in metabolic dysfunction and widespread neuronal death in affected areas of the brain. A number of in vitro models have been previously described which utilize variations in oxygen and glucose supply, or metabolic inhibition, to simulate ischemia or anoxia. In the present study, a new model of anoxia-induced neuronal death has been developed and characterized. In this model, postnatal hippocampal neuronal cultures are exposed to 5 minutes of anoxia produced by the addition of 2 mM sodium dithionite, an oxygen scavenger. The viability of neuronal cultures was determined using trypan blue exclusion in order to assess the potential of various maneuvers to provide neuroprotection. Using this model, we found that 5 min of anoxia induced by dithionite produces severe neurotoxicity in postnatal hippocampal neurons. This neuronal death is produced by the activation of NMDA and AMPA receptors by glutamate, and can be attenuated by the presence of the non-competitive NMDA receptor antagonist MK-801 during anoxia, or the competitive NMDA and AMPA receptor antagonists D-APV and CNQX, respectively, when present postinsult. We observed two phases of neuronal death: 1) an acute phase which occurs within 4h and morphologically resembles necrosis, and 2) a delayed phase occurring over a period of 24h which is unmasked by perfusing the cultures post-anoxia for 5 minutes in normoxic buffer. Substitution of external Na ⁺ and Cl ⁻ with impermeant ions can mitigate a portion of acute neuronal death, however removal of external Ca²⁺ increases neuronal susceptibility to anoxia. Free radicalmediated damage does not appear to play a role in the acute phase of cell death. Induction of neuronal death by this method is rapid, reliable and consistently produces at least 60% neuronal death by 4 hours post-insult. This model has the advantages of short insult duration, rapid development of neuronal death, and the capacity to produce a delayed neuronal death which may occur via an alternate pathway.

Thesis/Dissertation

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2009-08-05

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http://hdl.handle.net/2429/11703

Neuroscience

University of British Columbia

UBCV

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