Figure 3.

Induction of neurotoxic stress in acute hippocampal slices

(A) Trypan blue assay of cell damage following CoCl₂ or NMDA exposure. Representative bright-field images of trypan blue-stained acute hippocampal slices at baseline, and after 30, 60, and 120 min in control conditions (control T0, T30, T60, T120, respectively), or following 30 min exposure to 500 μM iPUT in combination with 1 mM CoCl₂ (iPUT + CoCl₂ T30), or to 500 μM iPUT in combination with 250 μM NMDA (iPUT + NMDA T30). Scale bars, 500 μm.

(B) Quantification of CA1 neuron injury at baseline (control T0) and after 30 min incubation in control conditions and in the presence of iPUT with CoCl₂ and iPUT with NMDA (control T0, control T30, iPUT + CoCl₂ T30, and iPUT + NMDA T30, respectively) based on CA1 stratum pyramidale trypan blue absorbance measurements (n = 3 animals per each experimental condition).

(C) Measurements of HIF-1α stabilization following 30 min exposure of acute slices to iPUT with CoCl₂. Left: representative western blot analysis of protein content of HIF-1α and β-actin (loading control) in acute hippocampal slices incubated for 30 min in the absence or presence of CoCl₂. Right: densitometric analysis of HIF-1α band normalized to β-actin band (n = 5 for control condition, n = 3 for iPUT + CoCl₂ exposure). Data information: (B and C) data are presented as mean ± SEM. (B) Statistical analysis: one-way ANOVA: no effect of treatment, F(3,8) = 0.117, p = 0.947. (C) ∗p < 0.05, Student’s t test.