Temporal Changes of Nitric Oxide Synthase Activity during Epileptogenesis in the Low Magnesium [italic]In Vitro[/italic] Seizure Model
Abstract number :
3.033
Submission category :
Year :
2001
Submission ID :
400
Source :
www.aesnet.org
Presentation date :
12/1/2001 12:00:00 AM
Published date :
Dec 1, 2001, 06:00 AM
Authors :
E.E. Elmenshawy, M.D., Neurology, U. N. C.; El Mansoura U. Faculty of Medicine, Chapel Hill, NC; R. Meeker, Ph.D., Neurology, University of North Carolina School of Medicine, Chapel Hill, NC; R.S. Greenwood, M.D., Neurology, University of North Carolina,
RATIONALE: Reactive oxygen species (ROS) contribute to cell injury in multiple neurodegenerative disorders, CNS trauma and stroke. In epilepsy, the relationship between seizure and cell death is not yet clear but the generation of nitric oxide (NO) and ROS might facilitate cell death. Using the low magnesium (Mg++) seizure model established by Sombati and Delorenzo (1995), we examined nitric oxide synthase activity (NOS) in cultured neurons during and after exposure to Mg++-free medium.
METHODS: Fetal cortical-hippocampal neurons were grown in culture for 10 - 16 days. These cultures were exposed to Mg++-free medium and cell death was measured at 0, 0.5, 1, 3 and 6 hours using ethidium staining of the nucleus. The cell death was then correlated with changes in intracellular calcium (Ca++) using the Ca++indication dye, Fluo-3, and the number of neurons expressing NADPH diaphorase activity, a marker for NOS.
RESULTS: A significant increase in cell death was noticed as early as half an hour after Mg++- free medium exposure. A small immediate increase in intracellular Ca++ was noticed followed by a slow increase beginning at 1.2 minutes and a delayed Ca++ deregulation (DCD) after 17.8 minutes of low Mg++ exposure. In addition, 10% of the imaged neurons in this condition died. Neuronal cultures pretreated with Mg++-free medium for 3 hours and then returned to normal medium failed to show DCD. NOS activity was found to increase as early as one minute after removal of Mg++ and peaked at 10 minutes followed by a decline. Some of the cells expressing high NOS activity showed swelling and beading of dendrites. The addition of a broad-spectrum NOS inhibitor, nitro-L-arginine methyl ester (L-NAME) to 0 Mg++ treated neuronal culture decreased cell death to the control level.
CONCLUSIONS: Our results suggest that high NOS expressing cells are more susceptible to cell death during epileptogenesis. However, after the development of seizure activity, NOS activity is low and neurons are resistant to DCD.
Support: Egyptian Ministry of Higher Education