Figure 6. Astroglial Cx43 hemichannel activity induced by Aβ_25-35-treated microglia followed by hypoxia/reoxygenation accelerates neuronal death caused by opening of neuronal Panx1 hemichannels.

(A) Cell death was monitored as percent of neurons positive to F-Jade staining. Neurons alone (N), or co-cultured with astrocytes (N+A) were treated or untreated with CM-Aβ for 24 h and then subjected to 3 h hypoxia in 5 mM glucose followed by several periods of reoxygenation (0, 1, 12 or 24 h). In some experiments 200 μM Gap26 or Cx43^E2 (1:500 dilution) was applied during the reoxygenation period. Also it is shown data from neurons co-cultured with astrocytes from Cx43^−/− mice. (B) LY diffusion (normalized to control) by astrocytes incubated for 1 h with 200 μM Gap26 (white bar) or by astrocytes exposed to CM-Aβ for 24 h and then subjected to 3 h hypoxia in 5 mM glucose (black bars) followed by 1 h reoxygenation without (middle bar) or with 200 μM Gap26 (right bar). (C) Etd uptake in neurons normalized to control conditions (dashed line) or subjected to 3 h hypoxia in 5 mM glucose followed by 1 h reoxygenation in CM-Ast or not. Also it is shown the effect of La³⁺, Gap26, probenecid (Prob) or ¹⁰panx1 (200 μM each) on Etd uptake of control or treated neuronal cultures. Gap26 (*); effect on neuronal Etd uptake of CM-Ast made in presence of Gap26 during reoxygenation. In some experiments, the effect on neuronal Etd uptake of CM-Ast made in the presence of Cx43^E2 (1:500 dilution) during reoxygenation was studied. (D) Cell death in cultures of neurons subjected to 3 h hypoxia in 5 mM glucose followed by 1 h reoxygenation with the same treatments as in C. *** p < 0.001, ** p < 0.005, compared to control; ^### p < 0.001, compared to hypoxia plus Ast-CM effect. Each value corresponds to mean ± S.E. of at least 4 independent experiments.