Establishing a human in vitro model of ischaemia to examine therapeutic strategies of stroke

Introduction: Stroke is the third leading cause of death in major industrialized countries, and it is the main reason for disability. Glutamate excitotoxicity plays a critical role in brain ischemia. Upon ischemia large amounts of glutamate are released acting subsequently on NMDA receptors.

Methods: A human in-vitro model of hypoxia/ischemia originating from embryonic NTera2 teratocarcinoma (NT2) cells has been developed and used to investigate the molecular mechanisms associated with hypoxia/ischemia. Human NT2 cells were differentiated into postmitotic NT2 neurons (NT2-N) by treatment with retinoic acid and mitose inhibitors.

Results: The expression of the NMDA receptor subunits was found dependent on the stage of neuronal differentiation. Inmunocytochemical analysis revealed a high expression of NR2B and NR2C but less of NR1 and NR2A in 11-week-old differentiated NT2-N. NT2-N bear GABAA receptors in addition to NMDA receptors. Receptor function was examined using the patch-clamp technique in the whole-cell mode. NMDA (plus 10 glycine, each) and GABA induced inward currents with an EC50 of approximately 43 and 10µM, respectively. The NMDA responses were antagonized by memantine, an open channel blocker of the NMDA receptor and required the presence of glycine as co-agonist.

To study the effect of hypoxia/ischemia on neuronal survival, NT2 neurons were exposed to reduced oxygen concentration (1%) and/or glucose deprivation for 3h. Ischemia was associated with a significant decrease in mitochondrial membrane potential detected by the fluorescent probe JC-1. The NMDA-induced currents of ischemic cells showed a significantly lower EC50 of approximately 23µM. Necrotic cell death was assessed by using propidium iodide and analysed with flow cytometry. Necrosis was higher upon ischemia than after either aglycemia or hypoxia. Memantine and CGS19755 (a competitive NMDA receptor antagonist) significantly reduced ischemia-induced cell death. In contrast, 5,7-dichlorokynurenic acid (DKCA), an antagonist at the glycineB binding site of the NMDA receptor did not reveal any significant neuroprotective effect.

Conclusions: The present results verify cultured NT2 neurons as human in vitro model for studying the molecular mechanisms associated with hypoxic/ischemic injury. Interestingly, neuroprotection could be achieved with NMDA receptor antagonists but not with an antagonist at the glycineB binding site of the NMDA receptor.