Aim: 

Understanding neuronal response to conditions of impaired energy metabolism (hypoxia/ischaemia) is crucial in developing new neuroprotection models. We have used primary cultures of cerebellar granule neurones to assess the neuronal damage resulting from 3-hr exposure to oxygen-deprivation (OD–5% dissolved oxygen), glucose-deprivation (GD), or both in combination (oxygen-glucose deprivation; OGD).

Methods: 

Neuronal viability and survival was assessed by use of a combination of fluorescent dyes (cytosolic calcein-AM and nuclear dyes Hoechst33342 and propidium iodide (PI)) and ATP measurements (luminescent assay).

Results: 

Significant differences were evident when analysing in detail the dynamic response of the neuronal cultures during this period of metabolic stress. Exposure to OGD induced a degree of cellular death (%PI-positive neurones), larger than that evoked by either OD and GD combined (20.6 ± 0.1% in OGD vs. 9.9 ± 0.1% in GD and 2.9 ± 0.1% in OD). However, morphological inspection of the 3 culture conditions showed that while in OD and GD there were less than 10% and 40% morphologically compromised cells, in OGD these accounted for more than 80%. To investigate this process, we defined, using the cytosolic and nuclear fluorescent dyes (calcein and Hoechst, respectively) a nuclear/ cytosolic ratio (NCR) parameter allowing us to identify and separate a population of neurones metabolically impaired (i.e., swollen, with decreased NCR). We also show that the temporal dynamics of this population in OGD cultures reflected the differential decrease in ATP during this period.

Conclusion: 

Understanding if and how these metabolically impaired cells can be rescued could open new therapeutic avenues.