Article Figures & Data
Figures
- 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.
- Figure 2.
Loss of HIF-1α function in astrocytes and neurons has opposite effects on hypoxia-induced neuronal death. A, A schematic diagram illustrating that neurons derived from HIF-1αf+/f+::ESRCre embryos were plated directly onto a monolayer of astrocytes, which were derived from C57BL/6J mice. Using this approach, tamoxifen induced loss of HIF-1 function selectively in neurons in cocultures (indicated by blue cells). B, As can be appreciated in the phase contrast images, hypoxia (12 h, 0.5% O2) induced death in the vast majority of neurons. Hypoxia for shorter time periods demonstrated that loss of HIF-1 in neurons enhances neuronal susceptibility to hypoxia-induced neuronal death (D; ***p < 0.001, ANOVA). Scale bars, 20 μm. C, A schematic diagram illustrating that tamoxifen induces loss of HIF-1 function selectively in astrocytes (red cells) in cocultures in which astrocytes are derived from HIF-1αf+/f+::EsrCre mice. Loss of astrocyte HIF-1α markedly protected the overlying neurons from hypoxia-induced cell death (B, E) (***p < 0.001, ANOVA; NS, nonsignificant difference). These experiments with selective loss of HIF-1 in neurons or astrocytes are representative examples of the replicates.
- Figure 3.
A, Astrocytic HIF-1α induces neuronal death by both iNOS-dependent and -independent mechanisms. iNOS was greatly induced by hypoxia in astrocytes, which was essentially eliminated with loss of HIF-1 function (ANOVA; ***p < 0.001; #p < 0.05). B, AG, a selective iNOS inhibitor, protects neurons from hypoxia-induced cell death (***p < 0.001; ANOVA) in cocultures in a dose-dependent manner with a similar efficacy as l-NAME, a nonselective NOS inhibitor. Greater neuronal protection is provided by 50–100 μm AG and l-NAME compared with 25 μm AG (###p < 0.001; ANOVA). Compared across multiple independent experiments, loss of HIF-1 in astrocytes is more protective compared with treatment with l-NAME (D) (p < 0.01; comparing y-intercepts following linear regression analysis). To control for a variable amount of hypoxia-induced neuronal death between experiments, the percentages of live neurons in the test groups (red diamonds, HIF-1 loss; blue box, l-NAME) were plotted as a function of neuronal death observed in hypoxia controls (black triangles). C, Experimental methods for media transfer experiments. After hypoxia and reperfusion for 6 h, media from cocultures, which in some cases lacked astrocyte HIF-1 function (red stars/astrocytes), was placed onto normoxia wild-type cocultures, and neuronal death was measured (C). E, Modest (∼20%) cell death was induced by media that was obtained from hypoxia/reperfusion cultures, which was eliminated by loss of HIF-1 function in astrocytes (***p < 0.001; ANOVA).
- Figure 4.
Loss of HIF-1α function in both astrocytes and neurons reveals an adaptive role for HIF-1α function in neurons during hypoxic stress. A, A schematic diagram illustrating that neurons derived from HIF-1αf+/f+::ESRCre embryos were plated directly onto a monolayer of astrocytes, which were also derived from HIF-1αf+/f+::ESRCre mice. Using this approach, tamoxifen induced loss of HIF-1 function in both neurons (blue) and astrocytes (red). B, A schematic diagram of the approach used to induce selective loss of HIF-1 function in astrocytes. C, In agreement with the data in Figure 2, loss of astrocyte HIF-1α markedly protected the overlying neurons from hypoxia-induced cell death (***p < 0.001; ANOVA). Although loss of HIF-1 in both neurons and astrocytes (red and blue striped) also attenuated neuronal death, neuronal survival was significantly less than the loss of HIF-1α in astrocytes alone (red; C) (**p < 0.01; ANOVA). The experiment with loss of HIF-1α in both cell types is a representative example of three independent separate replicates.
Additional Files
Supplemental Data
Files in this Data Supplement:
- supplemental material - Supplemental Legend
- supplemental material - Supplemental Table 1
- supplemental material - Supplemental Table 2
- supplemental material - Supplemental Figure 1
- supplemental material - Supplemental Figure 2
- supplemental material - Supplemental Figure 3
