Ischemia-induced death of astrocytes and neurons in primary culture: pitfalls in quantifying neuronal cell death.

We have demonstrated that both astrocytes and cerebellar granule cell neurons die during an ischemic insult only when there is complete loss of mitochondrial membrane potential. This was determined by comparing the ability of mitochondria to sequester rhodamine 123 with the ability of cells to exclude propidium iodide. We have also demonstrated that in astrocyte cultures cellular LDH loss correlates directly with propidium iodide uptake, i.e. cell death, and inversely with rhodamine 123 uptake. Thus, both LDH loss and rhodamine 123 uptake can be used to quantitatively measure astrocyte cell death in culture; however, this is not the case with neuronal cultures. It was demonstrated that even in highly enriched cerebellar granule cell neuron cultures where astrocytes comprise less than 10% of the total culture protein, approximately 40% of total culture LDH was in the astrocytes. This was also the case with rhodamine 123 sequestration. Caution must therefore be used when using the LDH technique to determine the proportion of neurons that have died in such enriched neuronal cultures.