Evidence for Status Epilepticus and Pro-Inflammatory Changes after Intranasal Kainic Acid Administration in Mice.

Kainic acid (KA) is routinely used to elicit status epilepticus (SE) and epileptogenesis. Among the available KA administration protocols, intranasal instillation (IN) remains understudied. Dosages of KA were instilled IN in mice. Racine Scale and Video-EEG were used to assess and quantify SE onset. Time spent in SE and spike activity was quantified for each animal and confirmed by power spectrum analysis. Immunohistochemistry and qPCR were performed to define brain inflammation occurring after SE, including activated microglial phenotypes. Long term video-EEG recording was also performed. Titration of IN KA showed that a dose of 30 mg/kg was associated with low mortality while eliciting SE. IN KA provoked at least one behavioral and electrographic SE in the majority of the mice (>90%). Behavioral and EEG SE were accompanied by a rapid and persistent microglial-astrocytic cell activation and hippocampal neurodegeneration. Specifically, microglial modifications involved both pro- (M1) and anti-inflammatory (M2) genes. Our initial long-term video-EEG exploration conducted using a small cohort of mice indicated the appearance of spike activity or SE. Our study demonstrated that induction of SE is attainable using IN KA in mice. Typical pro-inflammatory brain changes were observed in this model after SE, supporting disease pathophysiology. Our results are in favor of the further development of IN KA as a means to study seizure disorders. A possibility for tailoring this model to drug testing or to study mechanisms of disease is offered

MK801-elicited microglial activation is caused by neuronal injury.

<div><p>(<b>A</b>) Comparison of the intrinsic neurotoxicity of GK11 or MK801 on densely seeded and mature cortical cultures. Neuronal cultures were challenged for 48h with increasing concentrations of the NMDAR antagonists. Neuronal suffering was assessed by determining the MAP2-immunopositive surface. Photographs represent typical examples of MAP2-stained neuronal cultures (Scale bar = 20 µm). Bar graph represents quantitative analysis (mean ± SEM from at least three independent experiments). Y axis: % of MAP2-positive surface in neuronal cultures treated with MK801 or GK11 normalized to the sham-treated neuronal cultures. Statistical analyses were performed using two-way ANOVA followed by Fisher’s LSD post-tests. *: p<0.05; **: p<0.01; ***: p<0.001 when compared to controls. $: p<0.05;$ $: p<0.01:$ $$: p<0.001 when compared to GK11 treated cultures. </p> <p>(<b>B</b>) Effects of conditioned media obtained from Sham, MK801 and GK11-treated neuronal cultures on microglial BV-2 cells morphology. Cells treated with non-conditioned medium show a branched morphology with long processes, and only a few amoeboid cells can be seen. Cells treated with conditioned media from control (0.9% NaCl) or 100 µM GK11-treated neuronal cultures displayed similar morphology. In contrast, BV-2 cells treated with conditioned media from 100 µM MK801-treated neuronal culture show only a few short processes, and the majority of the cells had an amoeboid (“activated”) shape (Scale bar = 100 µm). The number of cells with at least one process (see Materials and Methods for further details) was quantified; the results are presented in the bar graph. The results are the mean ± SEM of quantitations performed in 3 experiments. Statistical analyses were performed using one-way ANOVA followed by Fisher’s LSD post-tests. ***:p<0.001 compared to controls; $$ $:p<0.001 compared to GK11-treated cultures.</p></div

Number of mice included in the different analyses (stage≥5 Racine).

<p>Number of mice included in the different analyses (stage≥5 Racine).</p

Time-dependent microgliosis and astrogliosis following IN KA induced seizures.

<p>Analysis performed in mice experiencing stage 5 or higher. <b>A-D)</b> Representative images of IBA1 immunostaining (upper panels) and GFP fluorescence (C57BL6/J CX3CR1^+/eGFP mice; lower panels) in CA1 region in control mice (A), and 24h (B), 72h (C) and 15 days (D) after IN KA. Scale bar = 50 μm. Insets depict enlarged image of individual microglial cells. Scale bar = 10 μm <b>E-F)</b> Quantitative analysis of IBA1 immunostaining in the hippocampus 24h, 72h and 15 days after IN KA-induced SE shows significant microglial activation, including increased cell number and size. <b>G-I)</b> Representative images of GFAP immunostaining in CA1 region in control mice (G), 72h (H) and 15 days (I) after IN KA. Scale bar = 20 μm. <b>J)</b> Quantitative analysis of GFAP immunostaining in the hippocampus shows astrogliosis 72h after IN KA. Results are represented as mean ± SEM (n ≥ 6 per group). Statistical analysis was performed using a non-parametric Kruskal-Wallis one-way analysis of variance followed by Dunn’s post-test. *: p<0.05; **: p<0.01; ***: p<0.001 compared to control condition.</p

Neurodegeneration and neuroinflammatory gene profile after IN KA induced seizures.

<p><b>A-C)</b> Fluoro-Jade C staining is observed 24h after IN KA, diminishing at 72h. Correspondence existed between presence of FJC positive neurons and behavioral score after IN KA (B: Mouse 9, mean-score = 5.8; C: Mouse 28, mean-score = 3.8). Scale bar 50 μm. <b>D-F)</b> qPCR analysis and changes of inflammatory gene levels in mice experiencing stage 5 or higher. Analysis was performed 24h (white bars) and 72h (grey bars) after IN KA. Panels (D) and (E) represent mRNA changes for M1 and M2 microglial markers. Red bars indicate genes that are not detectable under control conditions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150793#sec002" target="_blank">materials and methods</a> section). Results are presented as mean ± SEM (n ≥ 7 / group). Statistical analysis was performed using a non-parametric Mann-Whitney test between control and KA-treated conditions. *: p<0.05; ** p<0.01; ***: p<0.001 compared to controls.</p

Using IPA software and analysis of the literature, the genes deregulated by either MK801 or GK11 treatment were each allocated one main biological function.

<p>Each bar represents the number of genes (expressed as the percentage of the total number of deregulated genes after each treatment) assigned to each specific biological function. Although the functions of a significant number of genes affected by the NMDAR antagonists are unknown, the most affected biofunction corresponds to the inflammatory and immune response regulators.</p

RNA Profiling of Mouse Ependymal Cells after Spinal Cord Injury Identifies the Oncostatin Pathway as a Potential Key Regulator of Spinal Cord Stem Cell Fate

Ependymal cells reside in the adult spinal cord and display stem cell properties in vitro. They proliferate after spinal cord injury and produce neurons in lower vertebrates but predominantly astrocytes in mammals. The mechanisms underlying this glial-biased differentiation remain ill-defined. We addressed this issue by generating a molecular resource through RNA profiling of ependymal cells before and after injury. We found that these cells activate STAT3 and ERK/MAPK signaling post injury and downregulate cilia-associated genes and FOXJ1, a central transcription factor in ciliogenesis. Conversely, they upregulate 510 genes, seven of them more than 20-fold, namely Crym, Ecm1, Ifi202b, Nupr1, Rbp1, Thbs2 and Osmr&mdash;the receptor for oncostatin, a microglia-specific cytokine which too is strongly upregulated after injury. We studied the regulation and role of Osmr using neurospheres derived from the adult spinal cord. We found that oncostatin induced strong Osmr and p-STAT3 expression in these cells which is associated with reduction of proliferation and promotion of astrocytic versus oligodendrocytic differentiation. Microglial cells are apposed to ependymal cells in vivo and co-culture experiments showed that these cells upregulate Osmr in neurosphere cultures. Collectively, these results support the notion that microglial cells and Osmr/Oncostatin pathway may regulate the astrocytic fate of ependymal cells in spinal cord injury

Effects of MK801, GK11 and Memantine treatment on glial and microglial activation in the rat cortex.

<div><p>(<b>A</b>) Quantitative analysis of the IBA1-labeled surface in the cingulate cortex 24h after the different drug treatments shows significant microglial activation after high and low doses of MK801 and Memantine treatment, but not after high-dose GK11 administration. The results are presented as the mean ± SEM (n=6 per group). </p> <p>(<b>B</b>) Quantitative analysis of the GFAP-labeled surface (left) and the Optical Density (right) within the labeled surface in the cingulate cortex (96h after the different drug treatments) shows significant astroglial activation after high and low doses of MK801 and Memantine treatment, but only a slight astrogliosis after high-dose GK11 administration. The results are presented as the mean ± SEM (n=3-6 per group). </p> <p>Statistical analyses were performed using one-way ANOVA followed by Fisher’s LSD post-tests. *: p<0.05; **: p<0.01; ***: p<0.001 compared to controls. $: p<0.05;$ $: p<0.1;$ $$: p<0.001 compared to GK11-treated rats; #: p<0.05; ##: p<0.01; ###:p<0.001 compared to MK801-treated rats.</p></div

Lack of long-term microglia and astrocytes reactivity.

<p><b>A-C)</b> Representative images of IBA1 immunostaining in CA1 region in control implanted mice, IN KA mice and mouse #C (see below). Scale bar = 50 μm. <b>D-E)</b> IBA1 analysis shows no significant microglial activation in KA mice compared to control mice. Mouse #C experienced severe SE in and displayed elevated microglia density (<i>red data</i> in D). <b>F-H)</b> Representative images of GFAP immunostaining in control implanted mice, KA- mice and mouse C. Scale bar = 50 μm. <b>I)</b> Quantitative analysis of GFAP immunostaining in the hippocampus shows no difference at 49 days post KA. Results are presented as mean ± SEM. Statistical analyses were performed using a non-parametric Mann-Whitney test.</p

Acute and delayed effects of MK801, GK11 and Memantine treatment at the behavioural and histological levels.

<div><p>(<b>A</b>) Analysis of locomotive behaviour 10 min after the drug administration showed that 5 mg/kg GK11-treated rats exhibited the hyperlocomotive behaviour typically observed shortly after treatment with low dose of a high-affinity NMDAR antagonist. The same tendency was observed after 1 mg/kg MK801treatment. To the contrary, the higher doses of MK801 and Memantine (5 and 50 mg/kg, respectively) induce ataxic effects with reduced maximal speed. (<b>B</b>) Representative traces of individual rats’ routes within the arena during the observation time revealed distinct acute exploratory behaviours after the different treatments. </p> <p>(<b>C</b>, <b>D</b>) 24h after the treatment, GK11-treated rats behaved like control animals, whereas all other treated animals displayed ataxia and reduced exploratory behaviour. </p> <p>Quantitations were performed on 12 rats per group. Statistical analyses were performed using the one-way non-parametric ANOVA statistical test followed by Dunn’s post-tests. *: p<0.05; **: p<0.01; ***:p<0.001 compared to controls. </p> <p>(<b>E</b>) Immunohistological examination of the number of HSP70-positive neurons in the cingulate cortex. Representative images of HSP70 immunostaining: GK11 treatment did not induce any expression of HSP70, whereas a strong signal was observed in pyramidal neurons in MK801- and Memantine-treated rats (scale bar = 250 µm). Inset in MK801 1mg/kg image (scale bar 25 µm). Quantifications were performed in 6 rats per condition. Statistical analyses were performed using the one-way ANOVA statistical test followed by Fisher’s LSD post-tests. *: p<0.05; **: p<0.01; ***:p<0.001 compared to controls. $: p<0.05;$ $: p<0.01;$ $$: p<0.001 compared to GK11-treated rats.</p></div