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

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

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