Increased Panx1 channel activity in EAE.

<p>(<b>A</b>) Bar histograms of the mean ± s.e.m. values of the relative intensity of YoPro uptake (test/control) recorded from Panx1 wild type (WT) and Panx1 knockout (KO) spinal cord slices of control and EAE mice. **P<0.005, ***P<0.001 (ANOVA followed by Newman-Keuls multiple comparison test). Nine Panx1 WT non-EAE, 9 Panx1 WT EAE, 8 Panx1 KO non-EAE, and 13 Panx1 KO EAE mice were used, with a minimum of 167 slices examined per group. Samples from EAE mice were then separated into mildly affected (flaccid tail, hind-limb weakness, clinical score range 0–3) and severely affected (hind-limb paralysis with possible trunk and forelimb involvement, clinical score range 4–7). (<b>B</b>) Bar histograms of the mean ± s.e.m. values of the relative intensity of propidium iodide (P.I.) uptake (test/control) recorded from Panx1 wild type (WT) and Panx1 knockout (KO) spinal cord slices of control and EAE mice. ***P<0.001, (ANOVA followed by Newman-Keuls post hoc comparison). Seven Panx1 WT non-EAE, 5 Panx1 WT EAE, 5 Panx1 KO non-EAE, and 7 Panx1 KO EAE mice were used, with a minimum of 71 slices per group examined. (<b>C</b>) Bar histograms of the mean ± s.e.m. values of ATP (pmol/mg protein) recorded from ACSF bathing Panx1 wild-type (WT) and Panx1 knockout (KO) spinal cord slices of control and EAE mice. In parentheses are the numbers of animals used. (<b>D</b>) Bar histograms of the mean ± s.e.m. values of Panx1 transcript (normalized to ribosomal 18S) measured from RNA samples extracted from cerebellum of naïve and EAE Panx1 WT mice. Samples are from 3 naïve and 3 EAE mice. ***P<0.001 and *P<0.05 (ANOVA followed by Newman-Keuls multiple comparison test); P value in part <b>D</b> was obtained using unpaired t-test analysis.</p

Deficient IL-1β release from activated Panx1 KO splenic macrophages.

<p>Bar histograms showing the mean ± s.e.m. values of interleukin-1β (IL-1β) measured from media bathing Panx1 wild type (WT) and Panx1 knockout (KO) macrophage cultures that were untreated, treated with lipopolysaccharide (LPS) and treated with LPS and stimulated with 5 mM ATP. Samples were obtained from 9 mice pooled into three groups and ELISA run in triplicates. ***P<0.001 (ANOVA followed by Newman-Keuls multiple comparison test).</p

Blockade of Panx1 channels ameliorates signs of EAE.

<p>Time course of the mean ± s.e.m. values of neurological scores recorded from rats (<b>A, B</b>) and mice (<b>C, D</b>) with EAE. (<b>A</b>) Daily intraperitoneal injections of 5 mg/kg mefloquine (MFQ) but not 1 mg/kg MFQ improved EAE outcome in rats. (<b>B–D</b>) Daily injections of MFQ (5 mg/kg) administered to (<b>B</b>) rats and (<b>C</b>) mice at 7 days post immunization (dpi) till, respectively 14 and 20 dpi, and to (<b>D</b>) mice starting at 14 dpi till 38 dpi is shown to ameliorate the EAE symptoms. P values were calculated from all data points using Mann-Whitney test. (<b>E</b>) Bar histograms showing the mean ± s.e.m. values of conduction latency in the corticospinal pathway of mice with EAE untreated (black bar) and treated (gray bar) with MFQ. In parentheses are the numbers of animals used. P values were obtained from unpaired t-test.</p

Delayed onset of EAE in Panx1 KO mice.

<p>(<b>A - right</b>) Time course of clinical signs recorded from Panx1 wild type (WT) and Panx1 knockout (KO) mice immunized for MOG. (<b>A-left</b>) The graph depicts the initial acute phase of the disease (blue rectangle in <b>A</b>) showing that up to day 12 post-immunization, a significant difference in clinical scores was detected between the two genotypes. Symbols represent mean ± s.e.m. Twenty two Panx1 WT and 22 Panx1 KO female mice were immunized. Thirteen days post-immunization (dpi), 5 animals of each genotype were used for histopathology; 6 Panx1 WT and 2 Panx1 KO died or were euthanized due to the severity of EAE symptoms. *P<0.01, unpaired t-test. (<b>B</b>) Hematoxylin & eosin stained sections of spinal cord from Panx1 WT and Panx1 KO mice obtained at the acute phase of EAE (12–13 dpi). Ventral funiculus is outlined in white box and inflammatory lesions are outlined in cyan. Bar histograms on the right represent the mean ± s.e.m values of the percent lesion area in each genotype. Note that Panx1 KO EAE spinal cords exhibit significantly less infiltrating cells than spinal cords from WT mice. Five Panx1 WT and 5 Panx1 KO mice were used for histology; three spinal cord sections from sacral to thoracic areas were analyzed from each mouse. (<b>C</b>) Hematoxylin & eosin stained sections of spinal cord from Panx1 WT and Panx1 KO mice obtained at the chronic phase of EAE (35 dpi). Bar histograms on the right represent the mean ± s.e.m values of the percent lesion area in each genotype. At this stage of disease, Panx1 WT and Panx1 KO spinal cords exhibit similar extent of lesion areas. Five Panx1 WT and 5 Panx1 KO mice were used for histology. Three sections from sacral to thoracic spinal cords were analyzed from each mouse. Quantification of lesions is presented as percent area of ventral funiculus white matter occupied by inflammatory cells. P values were obtained using unpaired t-test.</p

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

<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

Early phagocytic impairment is related to reduced expression of phagocytosis receptors and reduced motility.

<p>(<b>A</b>) Experimental design and expression of phagocytosis and purinergic receptors by RTqPCR in FACS-sorted microglia from control and KA mice at 1 dpi (<i>n</i> = 3 from 8 pooled hippocampi). HPRT was used as a reference gene. (<b>B</b>) Experimental design and representative projections of 2-photon microscopy images of microglia at t0 (cyan) and 15 min later (magenta) from the DG of controls and KA-treated mice (1 dpi). (<b>C</b>) Motility of microglial processes by 2-photon microscopy in acute slices from CX3CR1^GFP/+ mice after in vivo injection of KA (1 dpi; <i>n</i> = 4–5 cells from 3–4 mice per group). (<b>D</b>) Retraction and protraction of microglial processes by 2-photon microscopy in acute slices from CX3CR1^GFP/+ mice after in vivo injection of KA (1 dpi). (<b>E</b>) Experimental design and representative projections of 2-photon images of microglia at t0 (cyan) and 13.5 min (magenta) in the cortex of controls and KA-treated mice (1 dpi). (<b>F</b>) Motility of microglial processes by 2-photon microscopy in the living cortex of CX3CR1^GFP/+ mice after the injection of KA (1 dpi; <i>n</i> = 6 cells from 3 mice per group). (<b>G</b>) Retraction and protraction of microglial processes by 2-photon microscopy in the living cortex of CX3CR1^GFP/+ mice after the injection of KA. Bars represent mean ± SEM. * indicates <i>p</i> < 0.05, ** indicates <i>p</i> < 0.01, and *** indicates <i>p</i> < 0.001 by Student´s <i>t</i> test (A, C, D). Scale bars = 20 μm (B), 50 mm (E). z = 50 μm (A), 40 μm (B). 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

Microglial phagocytosis is impaired early (1 dpi) due to MTLE seizures in vivo.

<p>(<b>A</b>) Hippocampal electroencephalographic recordings of mice injected in the ipsilateral side (I) with KA (50 nL, 20 mM) during status epilepticus (0 dpi) and during a spontaneous seizure occurring in the chronic phase of MTLE (49 dpi). The contralateral hippocampus (C) is shown for comparison purposes. (<b>B</b>) Representative confocal z-stacks of saline and KA (1 dpi) hippocampi labeled with DAPI (nuclear morphology, white), activated caspase 3 (act-casp3⁺, red, for apoptotic cells), and fms-EGFP (cyan, microglia). (<b>C</b>) Number of apoptotic cells (pyknotic/karyorrhectic and act-casp3⁺) in the septal DG (<i>n</i> = 3−9 per time point and treatment). The volume of the septal DG is shown in <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s016" target="_blank">S3B Fig</a></b>. (<b>D</b>) Representative confocal image of a nonphagocytosed apoptotic (pyknotic and act-casp3⁺, arrowhead) cell in the SGZ (orthogonal projection, left; and 3-D-rendered image, right). M, microglial cell body. (<b>E</b>) Representative 3-D-rendered confocal z-stack of apoptotic (pyknotic and act-casp3⁺) cells, phagocytosed (arrow) or not (arrowheads) in the septal DG of mice treated with KA at 1 dpi. M, microglial cell body. (<b>F</b>) Representative 3-D-rendered confocal z-stack of an apoptotic (pyknotic), nonphagocytosed cells (arrowhead) in the DG of mice treated with KA at 1 dpi. The arrow points to a semiengulfed apoptotic cell. M, microglial cell body. (<b>G</b>) Ph index in the septal DG (in % of apoptotic cells) after KA. Phagocytosis by astrocytes and neuroblasts is shown in <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s016" target="_blank">S3C and S3E Fig</a></b>. (<b>H</b>) Weighted Ph capacity of DG microglia (in ppu). (<b>I</b>) Histogram showing the Ph capacity distribution of microglia (in % of cells) in the DG. (<b>J</b>) Total number of microglial cells (fms-EGFP⁺) in the septal DG. Microglial density is shown in <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s016" target="_blank">S3A Fig</a></b>. (<b>K</b>) Ph/A coupling (in fold change) in the septal DG. (<b>L</b>) Histogram showing the distribution of the distance (in μm) of apoptotic cells (in %) to microglial processes. The average distance of apoptotic cells to microglia is shown in <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s016" target="_blank">S3F Fig</a></b>. Bars represent mean ± SEM except in L, where they indicate the sum of cells in each distance slot. * indicates <i>p</i> < 0.05, ** indicates <i>p</i> < 0.01, and *** indicates <i>p</i> < 0.001 by Holm-Sidak posthoc test after two-way ANOVA (H–K) or one-way ANOVA (C, G, where a significant interaction time x treatment was found) were significant at <i>p</i> < 0.05. Scale bars = 50 μm (B), 10 μm (D–F). z = 25 μm (B), 13.9 μm (D), 14.1 μm (E), 8.4 μm (F). 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

ATP impairs microglial phagocytosis in vivo.

<p>(<b>A</b>) Representative confocal z-stacks of saline, 100 mM ATP and 100 mM ATPγS (2 hpi) DG labeled with DAPI (nuclear morphology, white), activated caspase 3 (act-casp3⁺, red, for apoptotic cells), and fms-EGFP (cyan, microglia). Arrow points to a phagocytosed apoptotic cell, whereas arrowheads point to nonphagocytosed apoptotic cells. Activated-caspase 3 puncta within microglia are labeled with a round-ended arrow. (<b>B, H</b>) Experimental designs (<b>B</b>, 100 mM of ATP and ATPγS, 2 h; <b>H</b>, 10 and 100 mM ATP, 4 h; <i>n</i> = 3–4 per group) and number of apoptotic (pyknotic/karyorrhectic and act-casp3⁺) in the septal DG (<i>n</i> = 3–4 per group). No changes in the volume of the DG were found in either experiment (<b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s024" target="_blank">S11C Fig</a></b>). (<b>C, I</b>) Ph index in the septal DG (in % of apoptotic cells). (<b>D, J</b>) Weighted Ph capacity of hippocampal microglia (in ppu). (<b>E, K</b>) Histogram showing the Ph capacity distribution of microglia (in % of cells) in the septal DG. (<b>F, L</b>) Total number of microglial cells (fms-EGFP⁺) in the septal DG. (<b>G, M</b>) Ph/A coupling (in fold change) in the septal DG. Bars represent mean ± SEM, * indicates <i>p</i> < 0.05, ** indicates <i>p</i> < 0.01, and *** indicates <i>p</i> < 0.001 by Holm-Sidak posthoc test after one-way ANOVA were significant at <i>p</i> < 0.05. Scale bars = 50 μm, z = 11.9 μm (control, ATP), 9.8 μm (ATPγs). Inserts are single plane images of the corresponding confocal z-stacks. 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

Microglial phagocytosis impairment is unrelated to monocytes.

<p>(<b>A</b>) CD45 staining in saline- and KA-injected mice at 3 dpi. Cell nuclei are shown in white (DAPI), microglia in cyan (fms-EGFP), and CD45 in red. In control mice, the expression of CD45 was dim, showing diffuse cytoplasmic inclusions within microglia. A CD45⁺ cell is shown engulfing an apoptotic cell (arrow, enlarged). In KA mice, CD45 had a higher and more widespread expression in all microglial cells, including a dividing cell (arrowhead, enlarged). A clear distinction between CD45^high and CD45^low cells was not evident. (<b>B</b>) Flow cytometry analysis of the expression of CD45 in fms-EGFP⁺ hippocampal cells from control and KA-treated mice. Gates for CD45^low (cyan) and CD45^high (red) were defined based on the distribution of the fms-EGFP⁺ cells in control (not injected) mice. 3 dpi after the KA injection, more cells were found in the CD45^high gate, although the fms-EGFP⁺ cells were in fact distributed along a continuum of CD45 expression, all of them with higher expression than control mice. At 7 dpi, the expression of CD45 returned to basal levels. The gating strategy is shown in <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s017" target="_blank">S4B Fig</a></b>. (<b>C</b>) Percentage of fms-EGFP⁺ cells that expressed low or high levels of CD45 in control or KA-treated mice determined by flow cytometry (<i>n</i> = 4 per group). (<b>D</b>) Experimental design and representative confocal z-stacks of the hippocampus of CCR2^-/- (CCR2 KO) mice and control WTs (C57BL/6) injected with KA (3 dpi). No obvious differences in the status epilepticus, neuronal damage, microglial morphology, nor in the DG volume (<b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s017" target="_blank">S4C Fig</a></b>), or neutrophil infiltration were found (<b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s017" target="_blank">S4D and S4E Fig</a></b>). (<b>E</b>) Number of apoptotic (pyknotic/karyorrhectic) in the septal DG in WT and CCR2 KO mice 3 dpi after KA (<i>n</i> = 4 per group). (<b>F</b>) Ph index in the septal DG (in % of apoptotic cells) in WT and CCR2^-/- mice 3 dpi after KA. (<b>G</b>) Multinuclearity in WT and CCR2^-/- mice. (<b>H</b>) Size of multinucleated cells in WT and CCR2^-/- mice. (<b>I</b>) Weighted Ph capacity in WT and CCR2^-/- mice. Note that the Ph capacity is higher than in our previous time course (<b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.g004" target="_blank">Fig 4H</a></b>), reflecting an increased number of apoptotic cells in this experiment compared to the previous one, possibly because it was performed in different animal facilities. (<b>J</b>) Weighted PhP (phagoptosis) capacity in the septal DG in WT and CCR2^-/- mice. Data are shown as mean ± SEM. * indicates <i>p</i> < 0.05, ** indicates <i>p</i> < 0.01, and *** indicates <i>p</i> < 0.001 by Holm-Sidak posthoc test, after one-way ANOVA was significant at <i>p</i> < 0.05; only significant interactions are shown. Scale bars = 20 μm (A), 50 μm (D); z = 14.7 μm (A), 12.6 μm (D). 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

Microglial phagocytic response during in vivo acute and chronic inflammatory challenge.

<p>(<b>A</b>) Experimental design and apoptosis in the DG of c57BL/6 fms-EGFP 1-mo mice injected systemically with LPS (1mg/kg; <i>n</i> = 5) or vehicle (saline; <i>n</i> = 4) 8 h prior to sacrifice. Apoptotic cells were identified by pyknosis/karryorhexis. <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.g002" target="_blank">Fig 2A</a></b> was generated from data that was originally published as part of [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.ref009" target="_blank">9</a>]. (<b>B</b>) Weighted Ph capacity of microglia (in parts per unit, ppu) in control and LPS mice. (<b>C</b>) Number of microglial cells in control and LPS mice. (<b>D</b>) Ph/A coupling in the 1-mo mouse hippocampus (in fold change) during acute inflammatory challenge. (<b>E</b>) Experimental design and representative confocal z-stacks of the DG of PND21 Swiss mice fed during gestation and lactation with a diet balanced (Ω3 bal; <i>n</i> = 7) or deficient (Ω3 def; <i>n</i> = 7) in the omega 3 polyunsaturated fatty acid, a diet that induces chronic inflammation in the hippocampus. Microglia were labeled with Iba1 (cyan) and apoptotic nuclei were detected by pyknosis/karyorrhexis (white, DAPI). Arrows point to apoptotic cells engulfed by microglia (M). Scale bars = 50 μm; z = 22.5μm. (<b>F</b>) Number of apoptotic (pyknotic/karyorrhectic) cells in mice fed with Ω3 balanced and deficient diets. (<b>G</b>) Ph index in the PND21 hippocampus (in % of apoptotic cells) in mice fed with Ω3 balanced and deficient diets. (<b>H</b>) Weighted Ph capacity of microglia (in ppu) in PND21 mice. (<b>I</b>) Histogram showing the Ph capacity distribution of microglia (in % of cells) in PND21 mice. (<b>J</b>) Total number of microglial cells (Iba1⁺) in PND21 mice. (<b>K</b>) Ph/A coupling in PND21 mice. Bars represent mean ± SEM. * indicates <i>p</i> < 0.05 and ** indicates <i>p</i> < 0.01 by one-tail Student´s <i>t</i> test. 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