Modelling hypoxic-ischaemic injury on neuronal cells

Neuroblastoma SH-SY5Y cells were differentiated into neuron-like cells (NLCs) using brain-derived neurotrophic factor (BDNF) and retinoic acid (RA). The presence of functional N-methyl-D-aspartate (NMDA) neurons was assessed by treating the NLCs with graded concentrations of NMDA (31.25 to 500 micromolar) for 3 h. Cell viability (using MTT assay) and cytotoxicity (LDH assay) were measured thereafter. To model hypoxic-ischaemic injury in NLCs, the cells were initially exposed to oxygen and glucose deprivation (OGD). OGD was achieved using glucose-free media containing a hypoxia-mimetic, cobalt chloride, at graded concentrations (0.25 to 2mM) for 3 h, following which the secretion of proinflammatory cytokines and chemokine were measured (TNF-alpha and CCL2) alongside cell death (by measuring the release of LDH). Thereafter, a phase II experiment was conducted where 1mM cobalt chloride was used to induce hypoxia during OGD, followed by reperfusion at different times within 24 hours (1, 3, 6, 12, 18, and 24 h). The secretion of various proteins (TNF-alpha, CCL2, vascular endothelial growth factor VEGF, BDNF, and nerve growth factors NGF) was also measured in addition to assessment of cell death.