Effect of MTA under OGD and AMPA excitotoxicity.

<p>Panels a and b: the effect of APV (100 µM) or MTA in OGD conditions in rat mixed cultures of neurons and astrocytes. Cell death was expressed as the LDH activity at the beginning of any treatment: *<i>p</i><0.05 compared to IAA-treated cells; # <i>p</i><0.05 compared to control; ** <i>p</i><0.01 compared to IAA-treated cells; ## <i>p</i><0.01 compared to control. <b>c</b>) primary oligodendrocytes derived from rat optic nerve subjected to AMPA excitotoxicity (10 µM and 100 µM) in which cell death was measured with calcein-AM, as indicated in Methods. *p<0.05 compared with cells treated with AMPA (Unpaired t-test). Values represent the average ± SEM and were obtained from at least three independent experiments performed in duplicates.</p

Effect of MTA and A_2AR antagonists in an acute mouse model of PD.

<p>The effect of MTA (30 mg/kg), MSX-3 (9 mg/kg) or both in combination on the survival of dopaminergic neurons (TH⁺ cells) within the <i>substantia nigra</i> of the brain of mice treated with MPTP. a) Representative images of the neurodegeneration of dopaminergic cells in the different groups. b) Stereological counts of TH⁺ neurons in control and drug-treated animals (n = 6 per group). The data represent the mean±SEM: *<i>p</i><0.05 comparing with the MPTP group; # <i>p</i><0.05 comparing with the control group (One-way ANOVA, LSD test as a post-hoc). Scale bar: 50 µm.</p

MTA effects in chronic pilocarpine-induced status epilepticus (SE).

<p>a–f: Representative images of Neu N immunoreactivity in the hippocampus are shown 3 days after sham treatment (a) or pilocarpine-induced SE (b, c), or 30 days after SE (d–f). MTA (30 mg/kg) was administered pre-SE (d) or post-SE (c,f) induction. Cell loss is already apparent by 3 days (arrows in b) and is marked by 30 days (e) after SE, but it appears attenuated in MTA-treated animals at both timepoints (c,d,f). g–h) Bar plots show quantification of NeuN-positive cells in CA3(g) or CA1(h) 30 days after SE in animals pre-treated with MTA. The data represent the mean ± SEM. *p<0.05; **p<0.001; ANOVA with Tukey HSD post –hoc test.</p

Effective permeability (Pe) values in the PAMPA assay and in the <i>in vitro</i> model of BBB.

<p>*<i>transport confirmed by HPLC-MS.</i></p

Effect of MTA on brain ischemia.

<p>(a) Representative TTC stained sections of vehicle and MTA treated animals (30 mg/kg/twice daily, i.p.) 3 days after the induction of transient focal ischemia. Histogram (right) showing the infarct volume calculated from TTC stained slices in vehicle- and MTA-treated rats (n = 5 in each group). b) Representative microphotographs of Fluoro Jade C staining after rat transient forebrain ischemia (n = 5 in the vehicle group; n = 6 in the MTA-treated group). MTA (30 mg/kg) was administered 30 min after triggering ischemia. Quantification of Fluoro Jade positive cells per mm length of CA1 pyramidal layer (right). The data represents the mean ± SEM: **p<0.01 compared to the vehicle (Student t-test). Scale bar 100 µm.</p

Effect of MTA under NMDA excitotoxicity.

<p>Effect of MTA co-treatment (a) or MTA pre-treatment (b) on NMDA-induced caspase 3 activation in rat pure primary neuronal cultures. Effect of MTA co-treatment (c) or MTA pre-treatment (d) on NMDA-induced caspase 3 activation in rat mixed astrocyte-neuron cultures. Caspase 3 activity (units of fluorescence per milligram of protein per hour) was determined in cells treated with 300 µM NMDA, in the presence or absence of MTA (panels a and b: 100, 250 and 500 µM; panels c and d: 250 µM) or 10 µM MK-801 (MK; NMDA receptor antagonist). The results are expressed as the mean ± SEM of at least four independent experiments performed in triplicate: *p<0.05, **p<0.01***<i>p</i> <0.001 compared with cells treated with NMDA; # <i>p</i><0.05, ## <i>p</i><0.01, ### <i>p</i><0.001 compared with vehicle cells. One-way analysis of variance (ANOVA) and Bonferroni's t-test for multiple comparisons.</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

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