Figure 6.

Reactivation of Wnt-β-catenin signaling reverses the effects of anoxia in cortical RGC cultures. (A) Immunolabeling of β-catenin expression in the CP and VZ of the human cerebral cortex at 22 gw. Abbreviations: CP, cortical plate; LV, lateral ventricle; VZ, ventricular zone. Scale bar: 20 μm. (B) qPCR analysis shows changes in WNT7A mRNA levels in cortical RGCs exposed to distinct hypoxic stimuli versus control conditions (atmospheric, atm O₂), represented by a dashed line. (C) The decrease in AXIN2 and LEF-1 mRNA levels in anoxic versus control RGC cultures reflects a reduction in Wnt-β-catenin activation in anoxic RGCs. (D, E) Western blot demonstrates the decreased β-catenin and LEF-1 protein levels in anoxic RGC cultures. Significant differences between hypoxic and control conditions (*P < 0.05; **P < 0.01) and between 1% and 3% O₂ conditions (^##P < 0.01) are indicated. Error bars show the SEM. (F) Timeline of the experimental procedure. (G) Quantification of proliferating Ki67⁺ cells in control and anoxic cultures in the presence or absence of a Wnt-β-catenin agonist (CHIR999021) or antagonist (XAV939). (H) The percentage of βIII-tubulin⁺ neurons from all cells in cortical RGC cultures exposed to anoxia and the Wnt agonist/antagonist. The number of neurons recovered in cultures treated with the Wnt-β-catenin agonist. (I, J) Western blot of the neuronal marker DCX confirms the βIII-tubulin immunolabeling results. Actin served as a loading control. *P < 0.05, **P < 0.01, show significant differences compared with the non-treated-control condition (DMSO; atm); ^#P < 0.05, ^##P < 0.01 show differences between the respectives treated-anoxic (CHIR/CHIR+XAV; <1%) and treated-control conditions (CHIR/CHIR+XAV; atm). Paired t-test was used to compare control versus anoxic conditions and multiple experimental conditions were compared using ANOVA. A minimum of three different cases were used per condition. Error bars indicate the SEM.