Despite accounting for 6 million deaths every year worldwide[1], ischaemic stroke treatment remains inadequate. Prior exposure to a sub-lethal ischaemic event (ischaemic preconditioning, IPC) reduces the damage caused by a subsequent ischaemic insult[2]. This protection involves hypoxia-inducible factor-1α (HIF1α), but the precise molecular mechanisms are unclear[3]. We are developing in-vitro models to elucidate the mechanisms of IPC-induced tolerance to ischaemic stroke. Model1: Primary cultures (embryonic day 17-18 rat cortices; serum-free media) predominantly containing neurons (~75% TuJ1+) were subject to IPC [2 or 4 hour oxygen-glucose deprivation (OGD); 0.3% O2] then ‘reperfusion’ (restored glucose and oxygen, 21% O2; 24 h), followed by 6 hour OGD (0.3% O2). There was no cytotoxicity of 2 h and 4 h OGD, with or without reperfusion, validating these as sub-lethal IPC protocols. In contrast, 6 h OGD showed elevated LDH (27±5% v 5±2% control), reduced MTT (57±4% of control), and fewer Tuj1+ cells (~50% of control). These effects were exacerbated with 24 hour reperfusion (LDH: 43±5% v 5%±1%; MTT: 27±4%; ~20% Tuj1+), simulating ischaemic injury. HIF1α was stabilised and a group of hypoxia genes (Glut1, Vegf, Bnip3, and Phd2) were upregulated by OGD for either 2, 4 or 6 hours. Preconditioning the neurons with 2 or 4 hours OGD followed by reperfusion, significantly improved neuron’s viability after 6 hours OGD (2 h – LDH[SJ1] : 20±2%; [SJ2] MTT: 73±2% of control; 4 h – LDH: 20±2%; MTT: 81±4%), compared to non-PC controls (6 h OGD insult – LDH: 35±2%; MTT: 52±3%). Model2: High purity primary astrocyte cultures (postnatal day 1-3 rat cortices; ~98% GFAP+) showed no cytotoxicity for up to 6 h in OGD (0.3% O2). Prolonged OGD produced elevated LDH (24 h: 36±2%, 48 h: 66±3% v 7±3% control), morphological changes (i.e. dystrophic astrocytes with extended processes), increased intensity of GFAP immunostaining per cell, and reduced cell numbers. In conclusion, our data demonstrate that astrocytes resist OGD-induced degeneration for a longer period than neurons. This OGD-induced neuronal damage was reduced by IPC, and deducing the mechanisms underpinning this may offer therapeutic insights for treating ischaemic stroke.