Figure 2

(a) PTZ induced seizure increases the release of cytochrome-c from mitochondria in prenatal rat hippocampal neurons. Western blot analyses (2a) of the cytochrome-c in the primary cultured hippocampal neuronal cells at GD 17.5 from PTZ-induced seizure model during pregnancy. Cells were treated for 20 min with normal media as control (C), media containing 100 mM ethanol (E), media contain 10 mM pentylenetetrazol (PTZ), media contain 1 μM Kainic acid (KA), media contain PTZ and ethanol (E+PTZ), media contain ethanol plus baclofen plus phaclofen (E+B+P), media contain 50 μM baclofen (B), and media contain 100 μM phaclofen (P) respectively. β-actin is taken as loading control in each case. A: Immunoblots of cytochrome-c of hippocampal neuronal cells under different treatment conditions. The immunoblots were labeled with an anti cytochrome-c antibody. B: Density values were expressed as mean ± SEM (n = 4, mean four rat per group) of the corresponding protein of cytochrome-c are presented. The density values on (Y-axis) are expressed as arbitrary units (AU). *P < 0.05 and **P < 0.01 versus control group. (b) Visualization of mitochondrial cytochrome c release and caspase-3 expression in same neuronal cells. In situ analysis of cytochrome-c release and caspase-3 expression was carried out by immunofluorescence technique upon exposure of ethanol and PTZ in hippocampal neuronal cells culture. Primary neuronal cells culture of prenatal rat exposed to PTZ and ethanol for 20 min. Detail procedures are mentioned in materials and methods section. The immunofluorescence of hippocampal cell double stained for cytochrome-c (FITC-labeled cytochrome-c antibody, green) and caspase-3 (TRITC-labeled, red). Yellow color (green + red; merge image) indicates release of cytochrome-c from mitochondria into cytosol and caspase-3 in the same neuronal cells, as detected by confocal microscopy. Arrow indicates the dead cell with release of cytochrome-c and caspse-3 expression. Magnification 40×, Scale bar = 20 μm for A-C.