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

Abstract

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.

Fig 1. Behavioural and video-EEG read-outs after IN KA.

A) Mortality after SE induced by IN KA administration in C57BL6/J and C57BL6/J CX3CR1^+/eGFP mice. B) Distribution of Racine scores in C57BL6/J and C57BL6/J CX3CR1^+/eGFP mice. The majority of surviving mice reached stages 6 (>80% in C57BL6/J and ≈50% in C57BL6/J CX3CR1^+/eGFP). Values indicate the number of mice at each Racine stage. C) Racine mean score over the 2 hours following IN KA. Identical symbols indicate mice assessed on the same day. D) Video-EEG was used to quantify duration of SE (black bars) and spike activity (grey bars) within the 4 hours following IN KA. E) Correlation between the Racine mean score and the time spent in EEG SE. (F-I) Examples of EEG recordings and analysis during baseline activity (F), status epilepticus (G-H) and spike activity (I). Time joint-frequency analysis and single frequency profile are also provided.

Fig 2. Neurodegeneration and neuroinflammatory gene profile after IN KA induced seizures.

A-C) 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. D-F) 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 materials and methods 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.

Fig 3. Time-dependent microgliosis and astrogliosis following IN KA induced seizures.

Analysis performed in mice experiencing stage 5 or higher. A-D) 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 E-F) 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. G-I) 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. J) 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.

Fig 4. Video-EEG analysis during seizure progression.

A) Heat map (blue to red) and raw numbers indicate the sum of durations (in seconds) of spike activity recorded for each animal in the given recording session. B-C) Examples of EEG recordings during the chronic phase (e.g., SE in B and spike activity in C).

Fig 5. Lack of long-term microglia and astrocytes reactivity.

A-C) Representative images of IBA1 immunostaining in CA1 region in control implanted mice, IN KA mice and mouse #C (see below). Scale bar = 50 μm. D-E) 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 (red data in D). F-H) Representative images of GFAP immunostaining in control implanted mice, KA- mice and mouse C. Scale bar = 50 μm. I) 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.