osely monitored with a rectal probe and maintained at 37.0 6 0.5uC with a heating pad during and after surgery until recovery from anesthesia. Transmission Electron Microscopy of Autophagosomes in the Hippocampus after I/R Injury To observe the time course of the I/R-induced formation of autophagosomes and morphologic changes in the organelles by TEM, the rats were transcardially perfused with phosphatebuffered saline followed by PBS containing 4% paraformaldehyde 1, 3, 6, 12 and 24 h after I/R. To study the effects of the propofol and 3-MA by TEM, the rats were sacrificed 12 h after I/R. The brain tissue samples of 1 cubic millimeter that were removed from the ischemic core of the Propofol Prevents Autophagic Cell Death hippocampus were first immersed in 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer, post-fixed in 1% osmium tetroxide in 0.1 mol/L phosphate buffer, dehydrated in graded ethanol series, and flat embedded in Araldite. Ultrathin sections were placed on grids, and double-stained with uranyl acetate and lead citrate. The sections were observed under a Philips CM-120 electron microscope. Histochemical Analyses The rats were deeply anesthetized with pentobarbital and fixed by cardiac perfusion with 4% paraformaldehyde buffered with 0.1 mol/L phosphate buffer containing 4% sucrose for light microscopy. For light microscopy, the brain tissues were quickly removed from the rats and further immersed in the same fixative for 2 hours at 4uC. The samples processed for paraffin embedding were cut into 5-mm thick sections using a semi-motorized rotary microtome and placed on silane-coated glass slides. For routine histological LY-411575 site studies, the paraffin sections were stained with thionine. For the light microscopy observations, semithin sections were cut 1mm thick with an ultramicrotome and stained with thionine. Immunohistochemical Analyses The rats were deeply anesthetized with pentobarbital and then perfused transcardially with 4% paraformaldehyde in 0.1 mol/L PBS 6 or 24 h after I/R. Immunohistochemistry was performed on 18-mm thick cryostat sections. For immunofluorescence labeling, the sections were preincubated for 45 minutes in 15% serum and 0.3% Triton X100 in PBS and then incubated overnight at 4uC with the primary antibody in 1.5% serum and 0.1% Triton in PBS, washed in PBS, and incubated for 2 h in fluorochrome-coupled secondary antibody at room temperature. The sections were then rinsed in PBS and mounted with FluorSave with the nuclear stain 49,69-diamidino-2-phenyl indole dihydrochloride . A LSM 510 Meta confocal microscope was used for the confocal laser microscopy. The confocal images were displayed as individual optical sections. For the double labeling experiments, the immunoreactive signals were sequentially visualized in the same section with two 
distinct filters, with acquisition performed in separated mode. The sections were viewed under high power with a fluorescence microscope with a Nikon digital camera, and the images were visualized in a computer monitor. For the quantification of LC3-II immunostaining, 10 microscopic fields in each section across ischemic hippocampus regions in the ipsilateral hemisphere were analyzed. Three sections were used for each animal. The number of cells with LC3-II immunoreactivity in each field was counted by an examiner who was blind to the experimental conditions. removed the brain tissues from ischemic hippocampus area and the corresponding area of sham-operated rats. We immedia