Effects of hyperglycemia on neuronal network function in an in vitro model of the ischemic penumbra.

Introduction: Hyperglycemia is common in acute ischemic stroke, and associated with unfavorable outcome. However, the optimal glucose level is not known and cellular effects of hyperglycemia under hypoxia are largely unclear. We assessed how the extracellular glucose concentration affects cultured neuronal networks under experimental in vitro conditions, to provide a starting point for assessment of mechanisms at the neuronal network and cellular levels.

Methods: We used in vitro cultured rat neuronal networks on micro-electrode arrays (MEAs) and glass coverslips. Twenty-four hours of controlled hypoxia was induced. We measured neuronal network activity during baseline (normoxia, 6 h), 24 h of hypoxia, and 6 h after reoxygenation, defined as the summed number of action potentials in 1 h bins. Apoptosis was determined intermittently with caspase 3/7 staining and microscopy. We compared groups of networks under glucose concentrations of 5.0 mmol/L, 7.0 mmol/L, 9.0 mmol/L, and 12.0 mmol/L.

Results: Overall, during hypoxia, a gradual decrease in neuronal network activity and increase in apoptosis was found. There were faster decrease in activity (p < 0.01) and more apoptosis after 24 h of hypoxia under glucose levels of 12 mmol/L in a single-well MEA set-up (p < 0.05), and more apoptosis in glass coverslips with glucose levels of 12.0 mmol/L in comparison with 5 mmol/L (p = 0.03). These differences were not observed in multi-well MEAs, in which effects of hypoxia were much smaller than in single-well MEAs.

Conclusion: Hyperglycemia was associated with a more rapid decrease of neuronal network activity during and more apoptosis after 24 h of hypoxia in cultured neuronal networks. Loss of neuronal activity and apoptosis probably play a role in poorer outcomes of stroke patients under hyperglycemia. Our model provides a starting point for further assessment of pathomechanisms.