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Excitotoxic neuronal death mediated by over-activation of glutamate receptors has been implicated in ischemia, hypoglycemia and some neurodegenerative diseases. It involves oxidative stress and is highly facilitated during impairment of energy metabolism. We have shown previously that in vivo systemic glycolysis inhibition with iodoacetate (IOA), exacerbates glutamate excitotoxicity. We have now investigated whether this effect involves oxidative damage to membrane lipids, as evaluated by the presence of thiobarbituric acid-reactive substances. We have also tested whether the ketone body, D-beta-hydroxybutyrate (D-BHB), prevents lipoperoxidation and tissue damage. Results show that glutamate intrastriatal injection in control rats transiently enhances lipoperoxidation, while in IOA-treated animals increased lipoperoxidation is sustained. Treatment with D-BHB significantly reduces striatal lesions and lipoperoxidation. Vitamin E also reduced neuronal damage and lipoperoxidation. Results suggest that glycolysis impairment favors a pro-oxidant condition and situates oxidative damage as an important mediator of in vivo induced excitotoxicity. Results provide evidence for the neuroprotective effect of D-BHB against glutamate toxicity.

The cell line M213-2O CL-4 was derived from cell line M213-2O and further modified to express human glutamate decarboxylase (hGAD-67), the enzyme that synthesizes GABA. Brain transplants of this cell line in animal models of epilepsy have been shown to modulate seizures. However, the mechanisms that underlie such actions are unknown. The purpose of the present study was to characterize this cell line and its responsiveness to several depolarizing conditions, in order to better understand how these cells exert their effects. Intracellular GABA levels were 34-fold higher and GAD activity was 16-fold higher in clone M213-2O CL-4 than in M213-2O. Both cell lines could take up [³H]GABA in vitro, and this uptake was prevented by nipecotic acid. By combining GABA release measurements and calcium imaging in vitro, we found that high extracellular K(+), zero Mg(2+), or glutamate activated M213-2O CL-4 cells and resulted in GABA release. The response to glutamate appeared to be mediated by AMPA/NMDA-like receptors. High KCl-induced GABA release was prevented when a Ca(2+)-free Krebs solution was used, suggesting an exocytotic-like mechanism. These results indicate that the cell line M213-2O CL-4 synthesizes, releases, and takes up GABA in vitro, and can be activated by depolarizing stimuli.

Disorders of the central nervous system (CNS) as a result of trauma or ischemic or neurodegenerative processes still pose a challenge for modern medicine. Due to the complexity of the CNS, and in spite of the advances in the knowledge of its anatomy, pharmacology, and molecular and cellular biology, treatments for these diseases are still limited. The development of cell lines as a source for transplantation into the damaged CNS (cell therapy), and more recently their genetic modification to favor the expression and delivery of molecules with therapeutic potential (ex vivo gene therapy), are some of the techniques used in search of novel restorative strategies. This article reviews the different approaches that have been used and perfected during the last decade to generate cell lines and their use in experimental models of neuronal damage, and evaluates the prospects of applying these methods to treat CNS disorders.

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