Time course of the distribution of AO-stained apoptotic cells in the irradiated brains of wt and p53^–/–embryos.

<p>Wt (A, B, C, D) and p53^{<b>–/–</b>}embryos (E, F, G, H) were irradiated with 10 Gy of gamma rays and stained with AO at 3 h (A, E), 12 h (B, F), 24 h (C, G), and 42 h (D, H) after irradiation. Images of clusters of AO-positive spots at higher magnifications (white arrows in squares in B and F) are shown in boxes (I and J) for detailed views of the rosette-shaped clusters of apoptotic neurons, which were fewer and smaller in the OT of p53^{<b>–/–</b>}embryos. Also shown are a bright-field counterpart image for AO-stained fluorescence images (K) and a schematic diagram illustrating the structure of the embryonic medaka brain at stage 30 (L). CE, cerebellum; OT, optic tectum; TE, telencephalon. Scale bars = 50 μm.</p

Time course of the numbers of AO-positive rosette-shaped clusters in the OT of irradiated wt and p53^–/–embryos.

<p>The numbers of AO-positive rosette-shaped clusters in the OT were counted at various times after gamma-ray irradiation (10 Gy). Error bars show the SEM (<i>n</i> = 3). Statistical differences between the means for wt (solid line) and p53^{<b>–/–</b>}embryos (broken line) were evaluated using Student’s unpaired <i>t</i> tests after <i>F</i> tests. *<i>p</i> < 0.05; **<i>p</i> < 0.01.</p

Distribution of ApoE-expressing microglia during phagocytosis shown by WISH.

<p>Activated microglia were identified as ApoE-expressing cells by WISH in nonirradiated control embryos (A), irradiated embryos at 24 h after irradiation (E), and at 42 h after irradiation (J). Frontal plastic sections including the eyes and the OT at the ‘a’ and ‘b’ levels of the brain in A, E, and J are shown in B, F, and K, and C, G, and L, respectively. A few ApoE-expressing cells were present in the retina of wt nonirradiated embryos (arrowhead in C), in the TE (arrowheads in B), and the OT (arrowhead in C). At 24 h after irradiation, hypertrophic and rounded ApoE-expressing microglia (H) appeared in the TE (arrowhead in F), retina (arrowhead in F, G), and OT (arrowhead in G). Unstained round areas were present in the TE, retina, and marginal regions of the OT (open arrowheads in F and G). At 42 h after irradiation, the number of ApoE-expressing microglia had increased markedly (arrowheads in K and L) and they showed a branched morphology (M). The numbers of unstained areas in the TE and OT (open arrowheads in K and L) decreased and they were small, not hypertrophied. Three-dimensional images were constructed from serial sections of WISH-stained nonirradiated (D), embryos at 24 h (I) and at 42 h after irradiation (N). ApoE-expressing microglia are in red and the unstained round areas appear white. MES, mesencephalon; OT, optic tectum; TE, telencephalon. Scale bars = 50 μm.</p

Increased numbers of ApoE-expressing cells were present in p53^-/- embryos at the late phase of phagocytosis.

<p>The p53^{<b>–/–</b>}embryos were irradiated with 10 Gy of gamma rays and frontal sections of heads including the eyes embedded in plastic resin were cut at the level of the solid line in A and subjected to Nissl staining with cresyl violet. Clusters of apoptotic nuclei were present at 24 h after irradiation (arrowhead in B) and images at higher magnification of the squares with dotted outlines in B are shown in C with an arrowhead. An EM image of phagocytosed apoptotic nuclei in a microglial phagosome (D) showed complete digestion of apoptotic nuclei in microglial phagosome as in wt embryos at 42 h after irradiation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127325#pone.0127325.g003" target="_blank">Fig 3I</a>). Activated microglia were identified as ApoE-expressing cells by WISH in irradiated p53^{<b>–/—</b>}embryos 24 h after irradiation (E). Frontal plastic sections including the eyes and the OT of WISH-stained p53^{<b>–/–</b>}embryos (stage 30) at the ‘a’ and ‘b’ levels of the brain in E are shown in F and G, respectively. The numbers of ApoE-expressing microglia increased (arrowheads in F and G) and they showed a branched morphology, not hypertrophy (a higher magnification is shown in H). The 3D reconstructed images showed dramatically increased numbers of ApoE-expressing cells in the irradiated p53^{<b>–/–</b>}embryos (I), as in wt embryos 42 h after irradiation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127325#pone.0127325.g004" target="_blank">Fig 4N</a>). MES, mesencephalon; OT, optic tectum; TE, telencephalon. Scale bar in (D) = 2 μm; Scale bars in (B–G) = 50 μm.</p

Histological analyses of the time course of neuronal damage in irradiated wt brains.

<p>Wt medaka embryos were irradiated with 10 Gy of gamma rays. Frontal sections of plastic-embedded heads were prepared in the plane that included their eyes and subjected to Nissl staining with cresyl violet. Clusters of apoptotic nuclei are shown in the irradiated wt embryos at 12 h (arrowhead in A), 24 h (arrowhead in D), and 42 h after irradiation (arrowhead in G); images at higher magnification in squares with dotted outlines in A, D, and G are shown with arrowheads in B, E, and H, respectively. Scale bars = 50 μm. Electron microscopic images of apoptotic clusters in the dotted-line boxes in B, E, and H are shown in C, F, and I, respectively. Microglia engulfed 10–15 apoptotic neurons into their phagosomes, and the nuclei of the engulfed apoptotic neurons maintained their appearance almost intact to 12 h after irradiation (C). The nuclei of the phagocytosed apoptotic neurons gradually became fragmented during the following 12 h (F). At 42 h after irradiation, degradation of apoptotic nuclei in phagosomes was almost complete (I). Frontal plastic sections (A, B, D, E, G, and H) were prepared at the level of the solid line of the embryonic brain shown in (J). CE, cerebellum; OT, optic tectum; MES, mesencephalon; TE, telencephalon. Scale bars in A, B, D, E, G, and H = 50 μm. Scale bars in C, F, and I = 2 μm.</p

High burn-up operation and MOX burning in LWR; Effects of burn-up and extended cooling period of spent fuel on vitrification and disposal

<p>Looking ahead to final disposal of high-level radioactive waste arising from further utilization of nuclear energy, the effects of high burn-up of light-water reactors (LWR) with UO₂ and MOX fuel and extended cooling period of spent fuel on waste management and disposal were discussed. It was assumed that the waste loading of waste glass is restricted by three factors: heat generation rate, MoO₃ content, and platinum group metal content. As a result of evaluation for effects of extended cooling period, the waste loading of waste glass from both UO₂ and MOX spent fuel could be increased in the current vitrification technology. For the storage of waste glass from MOX spent fuel with higher waste loading, however, those waste glass require long storage period prior to geological disposal because decay heat of ²⁴¹Am contributes significantly. Therefore, the evaluation of effects of Am separation on the storage period was performed. Furthermore, heat transfer calculation was carried out in order to evaluate the temperature of buffer material in a geological repository. The results showed, 70 to 90% of Am separation is sufficiently effective in terms of thermal feasibility of a repository.</p

Effect of edaravone treatment on neuronal degeneration in cerebral cortex after sepsis.

<p>In the vehicle-treated CLP septic group, there was serious neuronal degeneration in cerebral cortex (B) compared with sham control (A). In the edaravone-treated CLP septic group, normal-appearing neuronal findings were obtained in cerebral cortex (C). Tissues were harvested 24 h after surgery. Magnified view is shown in each inset. (D) Estimation of densely-stained cells in cerebral cortex. Counts of densely-stained cells were made in the sections at a final magnification of ×100. Mean of data from eight animals in each group is presented, with SEM by vertical lines. **<i>p</i><0.01 versus control. #<i>p</i><0.05 versus CLP treated with vehicle.</p

Sepsis-induced changes in NADPH oxidase activity, NADPH oxidase components p47<i>^phox</i> and p67<i>^phox</i> expression, and oxidative stress parameter in brain tissues.

<p>(A) Time course of changes in NADPH oxidase activity. Mean of data from six animals in each group is presented, with SEM by vertical lines. *<i>p</i><0.05 versus control. (B) Western immunoblotting showing up-regulation of p47<i>^phox</i> and p67<i>^phox</i> protein expression after CLP. The result is representative of two independent experiments. (C) The mRNA levels for p47<i>^phox</i> and p67<i>^phox</i> were quantified by real-time PCR. The data are expressed as a fold increase above control (sham operation) normalized GAPDH. Mean of data from six animals in each group is presented, with SEM by vertical lines. *<i>p</i><0.05 versus sham-operated control. (D) Levels of 8-OHdG. Mean of data from ten animals in each group is presented, with SEM by vertical lines. *<i>p</i><0.05 versus control.</p

Sepsis-induced increase in iNOS protein expression in brain tissues.

<p>(A) Time course of changes in cerebral expression of iNOS protein after CLP. β-Actin served as a loading control. The result is representative of two independent experiments. (B) Comparison of iNOS protein expression between sham-operated and CLP septic groups at 24 h after surgery. The summarizing data are presented as iNOS/GAPDH expressed relative to the unoperated control. Mean of data from three animals in each group is presented, with SEM by vertical lines. *<i>p</i><0.05.</p

Sepsis-induced increase in extravasation of sodium fluorescein (A) and Evans blue (B) in brains.

<p>Tissues were harvested 24 h after surgery. Mean of data from 4–6 animals in each group is presented, with SEM by vertical lines. *<i>p</i><0.05 versus sham-operated control.</p