Abstract 316
Neuroprotective Strategies for Hypoxic-Ischemic Encephalopathy Platform, Sunday, 5/2
Post-insult magnesium administration appears effective against both focal and global hypoxic/ischemic brain injury in neonatal and adult animal models (Marret, S. et. al. (1995) Developmental Medicine & Child Neurology 37, 473-84), and the use of magnesium as a tocolytic has been proposed as potentially neuroprotective in neonate. However, the precise mechanism of magnesium's "protective effect" is not easily explained. Embryonic day 6 chick telencephalon are harvested, cleaned of meninges, and mechanically dissociated; cells are plated on polylysine-coated plastic dishes in the presence of DMEM containing 10% FCS and antibiotics. [Mg++] in the media is 0.8 mM. After five days in culture, an additional 2.5 mM [Mg++] is added to treated cells. Cultures are analyzed after 24 hours of treatment for overall cell viability using the MTT assay, apoptosis using both TUNEL staining and a nucleosomal fragmentation ELISA, and cell proliferation after BrdU incorporation using anti-BrdU ELISA and immunocytochemistry assays. Increased extracellular [Mg++] increases overall cell viability by 51% (p=.013). This is secondary, at least in part, to a 25% increase in the rate of cellular proliferation in the presence of increased extracellular [Mg++], as measured by BrdU incorporation (p=.018). There are no significant change in apoptosis in these cultures. Cells pulse-labeled with BrdU are maintained in culture for three weeks. Many BrdU-positive cells differentiated into GFAP-positive astrocytes, suggesting that the cells proliferating during the initial 24 hour period of our assay are glial precursors. The mechanism of this effect appears at least in part to be secondary to activation of P13 Kinase, as the increases in cell viability and proliferation are blocked by wortmannin, a P13 kinase inhibitor which induces apoptosis in these cultures. In addition, increased extracellular [Mg++] augments wortmannin-induced apoptosis by 24% (p=.03), suggesting that the effect of increased extracellular [Mg++] makes the cells more dependent on growth factors, like IGF-1, that work via P13 kinase. Increased extracellular [Mg++] also activates Akt, an anti-apoptotic kinase activated downstream of P13 kinase. It is not clear if increased extracellular [Mg++] activates P13 Kinase directly, or by augmenting the effect of growth factors on their receptors. This is the first time increased extracellular [Mg++] has been shown to increase cell proliferation in neural cells in culture, and the first time it has been suggested that increased extracellular [Mg++] may alter the regulation of P13 Kinase. These results suggest that increased extracellular [Mg++] could have a dramatic impact in neural development, especially during the period of glial precurser proliferation.
