MAGNESIUM SULPHATE EXHIBITS NEUROPROTECTIVE ACTIVITY IN NEONATAL HYPOXIC BRAIN INJURY BY IMPROVING THE SURVIVAL AND DIFFERENTIATION OF PRE-MYELINATING OLIGODENDROCYTES <EM>IN VITRO</EM>

Hypoxic injury to the preterm neonatal brain results in death of vulnerable pre-myelinating oligodendrocytes and arrest of oligodendroglial maturation resulting in myelination deficits. Even though antenatal administration of magnesium sulphate has demonstrated neuroprotective effects in preterm neonates at risk of hypoxic brain injury, its mechanism of action is not fully understood. In this study, we evaluated the effect of magnesium sulphate treatment on pre-myelinating oligodendrocyte survival and differentiation in an in vitro model of hypoxic brain injury. We used a pre-myelinating oligodendrocyte cell line (Mo3.13) which can be differentiated into mature oligodendrocytes over 7 days by treatment with 100 nM PMA. To model hypoxic injury, Mo3.13 cells were exposed to 0.2% oxygen for 48 hours, with and without magnesium sulphate supplementation (1 mM, 2.5 mM and 5 mM). It was observed that magnesium sulphate reduced apoptosis of premyelinating oligodendrocytes on hypoxia exposure as measured by Annexin V-FITC/PI staining. Magnesium sulphate treatment decreased expression of pro-apoptotic protein Bax and increased expression of anti-apoptotic protein Bcl-2 demonstrated by Western blotting. These effects were correlated with changes in mitochondrial membrane polarisation (as measured by the JC1 assay) and intracellular calcium (measured by the Fluo-3 AM staining on flow cytometry). Magnesium sulphate treatment was also able to improve oligodendrocyte differentiation after hypoxic injury as seen by increased myelin basic protein (MBP) expression on flow cytometry. Our findings elucidate a novel mechanism by which antenatal magnesium sulphate treatment affords neuroprotection and improves myelination in hypoxic brain injury.