Figure 6

Deletion of bmf confers protection against neuronal injury induced by OGD in cultured primary cortical neurons. Cortical neurons derived from WT and bmf-deficient mice were sham-treated or subjected to either 45 min OGD or a model of excitotoxic NMDA receptor overactivation by exposure to NMDA/glycine (100 μM/10 μM) for 5 min and allowed to recover under normoxic conditions for 24 h. Cell death was assessed 24 h after OGD or NMDA treatment in each model by Hoechst and PI staining. (a and b) Cell death was significantly reduced in bmf-deficient neurons compared with WT, strongly implicating bmf in the apoptotic response to both (a) OGD and (b) excitotoxic NMDA receptor overactivation. Data presented as mean±S.E.M., figures representative of n=3 independent experiments from n=3 independent cultures carried out in triplicate with similar results. *P<0.05 compared with sham-treated control; ^#P<0.05 between treatments (ANOVA, post hoc Tukey’s test). (c) Quantification of the effect of Bmf overexpression (OE) in cortical neurons on cell survival after OGD. Neurons were sham-treated or subjected to 45 min OGD and allowed to recover under normoxic conditions for 24 h. GFP and Hoechst 33358 (1 μg/ml) fluorescence images were acquired to identify transfected cells and quantify nuclear apoptosis. The number of Hoechst-positive cells with condensed nucleus in eGFP-positive transfected neurons was quantified. Data presented as mean±S.E.M. from n=2 independent platings carried out in triplicate and pooled. *P<0.05 compared with sham-treated control; ^#P<0.05 between treatments. (d) Refpresentative images of Bmf and eGFP co-transfected neurons and control eGFP transfected neurons sham-treated or subjected to 45 min OGD and allowed to recover under normoxic conditions for 24 h. Scale bar, 10 μm