Spontaneous recurrent seizures in an intra-amygdala kainate microinjection model of temporal lobe epilepsy are differentially sensitive to antiseizure drugs

Abstract

The discovery and development of novel antiseizure drugs (ASDs) that are effective in controlling pharmacoresistant spontaneous recurrent seizures (SRSs) continues to represent a significant unmet clinical need. The Epilepsy Therapy Screening Program (ETSP) has undertaken efforts to address this need by adopting animal models that represent the salient features of human pharmacoresistant epilepsy and employing these models for preclinical testing of investigational ASDs. One such model that has garnered increased interest in recent years is the mouse variant of the Intra-Amygdala Kainate (IAK) microinjection model of mesial temporal lobe epilepsy (MTLE). In establishing a version of this model, several methodological variables were evaluated for their effect(s) on pertinent quantitative endpoints. Although administration of a benzodiazepine 40 min after kainate (KA) induced status epilepticus (SE) is commonly used to improve survival, data presented here demonstrates similar outcomes (mortality, hippocampal damage, latency periods, and 90-day SRS natural history) between mice given midazolam and those that were not. Using a version of this model that did not interrupt SE with a benzodiazepine, a 90-day natural history study was performed and survival, latency periods, SRS frequencies and durations, and SRS clustering data were quantified. Finally, an important step towards model adoption is to assess the sensitivities or resistances of SRSs to a panel of approved and clinically used ASDs. Accordingly, the following ASDs were evaluated for their effects on SRSs in these mice: phenytoin (20 mg/kg, b.i.d.), carbamazepine (30 mg/kg, t.i.d.), valproate (240 mg/kg, t.i.d.), diazepam (4 mg/kg, b.i.d.), and phenobarbital (25 and 50 mg/kg, b.i.d.). Valproate, diazepam, and phenobarbital significantly attenuated SRS frequency relative to vehicle controls at doses devoid of observable adverse behavioral effects. Only diazepam significantly increased seizure freedom. Neither phenytoin nor carbamazepine significantly altered SRS frequency or freedom under these experimental conditions. These data demonstrate that SRSs in this IAK model of MTLE are pharmacoresistant to two representative sodium channel-inhibiting ASDs (phenytoin and carbamazepine) and partially sensitive to GABA receptor modulating ASDs (diazepam and phenobarbital) or a mixed-mechanism ASD (valproate). Accordingly, this model is being incorporated into the NINDS-funded ETSP testing platform for treatment resistant epilepsy.

Keywords: Antiepileptic drugs; Clusters; Latent period; Pharmacoresistance; Seizures.

Fig. 1.

Microinjection of kainic acid (KA) into the mouse amygdala induces status epilepticus (SE) and the subsequent development of spontaneous recurrent seizures (SRS). A) Schematic showing coordinates for injection cannula and CA1 electrode implantation sites. Anatomic reference images courtesy of the Allen Mouse Brain Atlas (Lein et al., 2007). B) Gamma-band power resulting from microinjection of KA for mice given midazolam (N = 9, Blue) or saline (N = 9, Red) 40 min after detection of their first seizure. All mice EEG recordings were aligned to time = 0 when their first seizures were detected and time = 40 min when either 8 mg/kg midazolam or saline was administered. Data are represented by the mean (solid lines) and SEM (shaded area) for the Log10 of gamma frequency power (db). One-minute-long representative EEG traces for a saline-treated (left) and midazolam-treated (right) IAK mouse are shown below. Relative to time zero, traces were captured at 30 min (before saline or midazolam treatment), 2 h (approximate time of largest midazolam effect), and 4 h (after recovery from midazolam). Note the attenuation (at 2 h) and recovery (at 4 h) of large amplitude EEG spikes in the midazolam group. Scale bar represents 2 mV and 10 s. C) Representative EEG seizure recorded from an IAK mouse with an ipsilateral CA1 hippocampal depth electrode during a Racine stage 4–5 behavioral seizure. Scale bar represents 2 mV and 10 s. Time points (seconds) above the trace correspond to the expanded traces below (total duration = 2 s; bottom scale bar represents 2 mV and 0.5 s). Similar to EEG waveforms documented in other seizure models (e.g. see (Williams et al., 2009)), seizure initiation begins with a large EEG spike (4 s), followed by progressive high amplitude high frequency EEG spikes (12, 20, and 30 s), large amplitude waves containing multiple EEG spikes (40 s), and finally post-ictal depression (50 s).

Fig. 2.

Midazolam treatment during SE does not significantly affect survival or the development of epilepsy in IAK mice. A) Percentages of those animals developing spontaneous recurrent seizures (SRS), not developing SRS, and died in a cohort of mice that received 8 mg/kg midazolam 40 min after initiation of SE and another cohort of mice that did not receive midazolam. B) Heatmaps depicting the daily number of SRSs experienced by each mouse in both cohorts over a 90-day video-EEG observation period. From top to bottom, mice were rank ordered by the total number of SRSs experienced over 90 days from lowest to highest respectively. Note: video EEG was not recorded for the first 3 days after SE. C) Relative frequency distributions for latent periods and group medians. Open bars represent midazolam treated mice and closed bars represent mice whose SE was uninterrupted. Median latent periods were not significantly different (ns, Mann-Whitney test). D) Weekly averages for SRS daily frequencies over the 90-day natural history observation period. Open bars represent midazolam treated mice and closed bars represent mice whose SE was uninterrupted. There were no significant differences in seizure frequency between these cohorts over the entire period (ns, two-way repeated-measures ANOVA).

Fig. 3.

Midazolam treatment during SE does not significantly affect hippocampal damage 48 h after SE. A) Representative photomicrographs of FluoroJade-B stained neurons in the dorsal hippocampi of midazolam-treated (Left) and non-treated (Right) IAK mice. As depicted in Example 1 of the Midazolam images, the left hemisphere is ipsilateral “I” to the site of KA injection, and the right hemisphere is contralateral “C”; this orientation applies to all images. In both midazolam and no midazolam images, Example 1 represents isolated ipsilateral CA3 damage and Example 2 represents extensive ipsilateral and contralateral damage. B) Quantified percentages of mice with FluoroJade-B positive cells in each subdivision of the ipsilateral and contralateral dorsal hippocampus reveals no significant differences between midazolam-treated and non-treated groups. In both graphs, solid bars represent ipsilateral percentages and open bars represent contralateral percentages.

Fig. 4.

Natural history and characterization of SRSs in this IAK microinjection model of MTLE. A) Percentages of SRS, no SRS, and death for a cohort of 101 IAK mice. B) Survival curve for the 36 mice that died at varying times over the course of the 90-day observation period. Dashed lines highlight 80% survival at 21 days and 65% survival at 90 days. C) Heatmaps depicting the daily number of SRSs experience by each mouse over the 90-day video-EEG observation period. From top to bottom, mice were rank ordered by the total number of SRSs experienced over 90 days from lowest to highest respectively. Note: video EEG was not recorded for the first 3 days after SE. D) Relative frequency distribution of latent periods for all mice in this study. Median latent period was 11 days (Mean latent period was 20 ± 2 days). E) Weekly averages for SRS daily frequencies over the 90-day natural history observation period. Solid bars represent 30 mice whose latent period was < 14 days, and gray bars represent 25 mice whose latent periods were > 14 days. F) Percentages of SRSs that were accompanied by Racine score 4–5 behavioral seizures (93%), score 1–3 behavioral seizures (6%), or without obvious behaviors (1%). G) Weekly averages for SRS durations over the 90-day observation period. Solid bars represent seizure durations for score 4–5 behavioral seizures, and gray bars represent seizure durations for score 1–3 behavioral seizures. For panels F and G, a subset of 1484 SRSs were analyzed from a total of 28 mice.

Fig. 5.

Natural history and characterization of SRS clusters in this IAK microinjection model of MTLE. A) Relative frequency distribution of the number of consecutive days IAK mice experienced SRSs. Median was 3 days. Inset bar graph represents mean (4.3 ± 0.3 days) of this distribution and individual consecutive day events. B) Relative frequency distribution of the number of consecutive days IAK mice did not experienced SRSs. Median was 4 days. Inset bar graph represents mean (6.0 ± 0.4 days) of this distribution and individual consecutive day zero-SRSs periods. C) Relative frequency distribution of the total number of SRSs per seizure cluster. Median was 10 SRSs. Inset bar graph represents mean (24 ± 5 SRSs) of this distribution and individual SRS number per cluster. D) Relative frequency distribution of seizure cluster durations (days). Median was 5 days. Inset bar graph represents mean (6.9 ± 0.7 days) of this distribution and individual seizure cluster durations.

Fig. 6.

SRSs in IAK mice are resistant to phenytoin (40 mg/kg/day) and carbamazepine (90 mg/kg/day). A-B and E-F) Daily average SRS frequencies over 26-day evaluation periods for vehicle (A: saline or E: methylcellulose) and (B) PHT or (F) CBZ treatments. In all panels, open bars represent baseline and washout periods, while gray bars represent vehicle treatment averages (VEH) and solid black bars represent PHT or CBZ treatment averages for both halves of the crossover. C and G) SRS frequency daily averages, and individual mouse data points, for baseline (open bar), vehicle (gray bar), and (C) PHT or (G) CBZ (black bar) treatments. Vehicle and PHT data were not significantly different (ns, Wilcoxon Test). Likewise, Vehicle and CBZ data were not significantly different (ns, Wilcoxon Test). D and H) Comparisons of the percentages of SRS-free mice during vehicle and (D) PHT or (H) CBZ treatments. Solid black bars represent percentages of mice that experienced SRS, and open white bars represent percentages of mice that were SRS-free, during each treatment period. Differences between vehicle and PHT groups were not significant (ns, Fisher’s Exact Test). Likewise, differences between vehicle and CBZ groups were not significant (ns, Fisher’s Exact Test).

Fig. 7.

SRSs in IAK mice are sensitive to valproic acid (VPA) (720 mg/kg/day) and diazepam (DZP) (8 mg/kg/day). A-B and E-F) Daily average SRS frequencies over 26-day evaluation periods for vehicle (A: saline and E: saline) and (B) VPA or (F) DZP treatments. In all panels, open bars represent baseline and washout periods, while gray bars represent vehicle treatment averages (VEH) and solid black bars represent VPA or DZP treatment averages for both halves of the crossover. C and G) SRS frequency daily averages, and individual mouse data points, for baseline (open bar), vehicle (gray bar), and (C) VPA or (G) DZP (black bar) treatments. Vehicle and VPA data were significantly different (**, Wilcoxon Test). Likewise, Vehicle and DZP data were significantly different (**, Wilcoxon Test). D and H) Comparisons of the percentages of SRS-free mice during vehicle and (D) VPA or (H) DZP treatments. Solid black bars represent percentages of mice that experienced SRS, and open white bars represent percentages of mice that were SRS-free, during each treatment period. Differences between vehicle and VPA groups were not significant (ns, Fisher’s Exact Test). However, differences between vehicle and DZP groups were significant (****, Fisher’s Exact Test).

Fig. 8.

SRSs in IAK mice are sensitive to phenobarbital (PB) (50 mg/kg/day and 100 mg/kg/day). A-B and E-F) Daily average SRS frequencies over 26-day evaluation periods for vehicle (A and E: saline) and (B) 25 mg/kg b.i.d. or (F) 50 mg/kg b.i.d. PB treatments. In all panels, open bars represent baseline and washout periods, while gray bars represent vehicle treatment averages (VEH), and solid black bars represent 25 or 50 mg/kg b.i.d. PB treatment averages for both halves of the crossover. C and G) SRS frequency daily averages, and individual mouse data points, for baseline (open bar), vehicle (gray bar), and (C) 25 mg/kg b.i.d. or (G) 50 mg/kg b.i.d. PB (black bar) treatments. For both doses, vehicle and PB data were significantly different (*, Wilcoxon Test). D and H) Comparisons of the percentages of SRS-free mice during vehicle and (D) 25 mg/kg b.i.d. or (H) 50 mg/kg b.i.d. PB treatments. Solid black bars represent percentages of mice that experienced SRS, and open white bars represent percentages of mice that were SRS-free, during each treatment period. Differences between vehicle and PB groups, for either dose, were not significant (ns, Fisher’s Exact Test).