Figure 1. OGD induces delayed neuronal death of mature hippocampal neurons in culture.

(A) OGD causes hippocampal neuronal cell death, as determined by analysis of nuclear morphology. After incubation under OGD conditions for 1 h (n = 3), 1 h30 (n = 12) and 2 h (n = 10), cells were returned to the 5% CO₂ incubator for 24 h. Cell viability was then assessed by analysis of the nuclear morphology. Pyknotic nuclei (arrows) were counted as dead cells. The results were expressed as the percentage of dead cells relatively to the total cell number. The right panel depicts nuclear morphology of neurons subjected to control and 2 h OGD. (B) Time-course of OGD-induced neuronal death, as determined by LDH release. Cells were subjected to OGD for 2 h and the LDH release was assessed 0 h (n = 6), 4 h (n = 5), 7 h (n = 3), 14 h (n = 6), 18 h (n = 6) and 24 h (n = 13) after the stimulus. (C–D) OGD induces cleavage of spectrin and the formation of spectrin breakdown products (SBDPs). SBDPs protein levels were analyzed by Western blot 7 h (C) and 24 h (D) after 2 h of OGD. The calpain inhibitor MDL 28170 (50 µM) was added 30 min prior to stimulation and kept during the stimulation and post-stimulation period (24 h). Actin was used as loading control. Bars represent the mean ± SEM of five (C) or ten (D) independent experiments performed in distinct preparations. *p<0.05, ***p<0.001, as determined by the Student's t-test (C) and One-way ANOVA followed by Dunn's Multiple Comparison Test (D; *p<0.05 for the OGD condition compared to control, ##p<0.01 for the OGD+MDL28170 condition compared to OGD).