Microglial phagocytic impairment leads to delayed clearance of apoptotic cells at 1 dpi.

Abstract

<p>(<b>A</b>) Experimental design used to analyze the survival of 3 do cells after the injection of saline (<i>n</i> = 7) or KA (<i>n</i> = 8) in mice. (<b>B</b>) Representative confocal z-stacks of the DG of control and KA-injected mice (1 dpi). The damage induced by KA was evidenced by the presence of cells with abnormal nuclear morphology (DAPI, white), and the altered morphology of microglia (fms-EGFP⁺, cyan). (<b>C</b>) Representative confocal images of 3 do apoptotic (pyknotic, DAPI, white) cells labeled with BrdU (red; arrows) in the SGZ of the hippocampus of saline and KA-injected mice at 1 dpi. In the saline mouse, the BrdU⁺ apoptotic cell, next to a cluster of BrdU⁺ cells, was phagocytosed by a terminal branch of a nearby microglia (fms-EGFP, cyan), whose nucleus was also positive for BrdU. In the KA mouse, the apoptotic BrdU⁺ cell was not phagocytosed by microglia. A nearby apoptotic cell (BrdU^-; arrowhead) was partially engulfed by microglia. (<b>D</b>) Total number of live 3 do BrdU⁺ cells (nonapoptotic) in the septal hippocampus after treatment with KA. The total number of 3 do and 8 do BrdU⁺ cells by a single BrdU injection in saline and KA-injected mice is shown in <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s026" target="_blank">S13A and S13B Fig</a></b>. (<b>E</b>) Total number of apoptotic 3 do BrdU⁺ cells in the septal hippocampus after treatment with KA. (<b>F</b>) Percentage of 3 do BrdU⁺ cells that re-enter cell cycle, assessed by their colabeling with the proliferation marker Ki67 after treatment with KA. Representative confocal z-stacks of BrdU/Ki67 cells are found in <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s026" target="_blank">S13C Fig</a></b>. (<b>G</b>) Percentage of apoptotic BrdU⁺ cells over total apoptotic cells in the septal hippocampus. (<b>H</b>) Estimated clearance of apoptotic cells in the septal hippocampus. The total number of apoptotic BrdU⁺ (from E) present in the tissue was added to the number of estimated apoptotic BrdU⁺ cells that had been cleared. In saline mice, this number was calculated using the clearance time formula shown in Methods with a clearance time of 1.5 h [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.ref009" target="_blank">9</a>]. As the total number of cells should be identical in saline and KA mice, the number of cleared apoptotic cells in KA mice was calculated as the difference between the total (in saline) and the number of present apoptotic cells (in KA). From here, we calculated a new clearance time using the same formula as in saline mice, of 6.3 h. (<b>I</b>) Linear regression analysis of the relationship between apoptosis and phagocytosis (Ph index) in saline and KA-injected mice (6 hpi and 1 dpi). (<b>J</b>) Experimental design used to compare SGZ apoptosis induced by KA at 1 dpi in young (2 mo) and mature (6 mo) mice. (<b>K</b>) Representative epifluorescent tiling image of the hippocampus and surrounding cortex of 2 and 6 mo mice injected with KA at 1 dpi stained with the neuronal activation marker c-fos. The same pattern of expression was found in young and mature mice throughout the DG, CA2, CA1 and the above cortex. (<b>L</b>) Representative confocal z-stacks of the apoptotic (pyknotic, white; act-casp3⁺, red) cells in the SGZ of the hippocampus of 2 mo and 6 mo mice injected with KA (1 dpi). The microglial phagocytosis impairment was similar in the two age groups (<b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s026" target="_blank">S13D Fig</a></b>). (<b>M</b>) Total number of apoptotic cells in the SGZ of 2 and 6 mo mice treated with saline or KA (1 dpi; <i>n</i> = 4–5 per group). Bars show mean ± SEM. * indicates <i>p</i> < 0.05, ** <i>p</i> < 0.01, and *** <i>p</i> < 0.001 by Student´s <i>t</i> test (E, G) or by Holm-Sidak posthoc test after one-way ANOVA (M) was significant at <i>p</i> < 0.05. Scale bars = 50 μm (B), 20 μm (C), 500 μm (K), 25 μm (L). z = 14 μm (B), 12.6 μm (C, sal), 15.4 μm (C, KA), 25 μm (L). Underlying data is shown in <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s001" target="_blank">S1 Data</a></b>.</p