Figure 1.
A, Neurons cocultured with astrocytes die in a delayed manner after transient hypoxia: schematic diagram of experimental protocol. Neurons (black cells) are plated directly on the monolayer of astrocytes (yellow stars) and allowed to mature for 9 d before being subjected to hypoxia. After hypoxia (12 h, 0.5% oxygen), the cultures were placed in normoxia, and cell death was quantified by counting PI (+) neurons (red fluorescence). B, C, Superimposed phase contrast/fluorescent images (B) demonstrate that most neurons are viable immediately after hypoxia (scale bars, 20 μm), yet profound neuronal death is appreciated 24 h after the exposure to hypoxia (C). Hypoxia-induced HIF-1α protein in cocultures by Western blot (D, F; arrow, hypoxia-induced HIF-1α; arrowhead, nonspecific band; Reperfus., reperfusion). D, HIF-1α protein is increased immediately after hypoxia but returns to baseline after 6 h of reperfusion (normoxia). HN33 (a transformed neuronal cell line) cell lysates (+) or (−) cobalt served as controls. E–G, The addition of tamoxifen (100 nm) to astrocyte cultures derived from HIF-1α f+/f+::ESRCre mice markedly decreases HIF-1α transcript (E) and protein (F) and inhibits hypoxia-induced expression of HKII (G). Three separate replicates (3 samples/replicate) of exon 2 transcript abundance are shown in E (ANOVA; ***p < 0.001; #p < 0.01). In this and subsequent figures: A, astrocyte; N, neuron as cell type for EsrCre expression; error bars are SD, and flat bars above bar graphs indicate no significant difference.