Fig. 2. Cleaved caspase-3 expression in the hippocampus of organotypic hippocampal slice culture derived from C57BL6 neonatal mice pups after anesthetic gas treatment. ( A ) Representative living culture was killed by exposure to glutamate (50 μm), and the image was enhanced with propidium iodide staining, acting as a guide to outline the CA area of the hippocampus in B and D . DG = dentate gyrus. ( B ) Representative photomicrograph of hippocampal culture treated with 0.75% isoflurane (Iso) plus 75% nitrous oxide (N2O) followed by caspase-3 immunostaining. ( C ) High-magnification image derived from the highlighted area of B , indicating that cleaved caspase 3–positive cells are identifiable by black cell body and dendritic staining. ( D ) Representative photomicrograph of hippocampal culture treated with 0.75% Iso plus 60% xenon (Xe) followed by caspase-3 immunostaining. ( E ) High-magnification image derived from the highlighted area of D . ( F ) Data (mean ± SD, n = 15) of cleaved caspase 3–positive cells counted from whole CA area of hippocampus after 0.75% Iso, 75% N2O, 60% Xe, or 0.75% Iso combined with either 75% N2O or 60% Xe. * P < 0.05, ** P < 0.01 versus control. + P < 0.05, ++ P < 0.01 versus Iso plus Xe. Scale bar in A , B , and D = 200 μm; scale bar in C and E = 100 μm.