Midazolam-ketamine dual therapy stops cholinergic status epilepticus and reduces Morris water maze deficits

Abstract

Objective: Pharmacoresistance remains an unsolved therapeutic challenge in status epilepticus (SE) and in cholinergic SE induced by nerve agent intoxication. SE triggers a rapid internalization of synaptic γ-aminobutyric acid A (GABAA ) receptors and externalization of N-methyl-d-aspartate (NMDA) receptors that may explain the loss of potency of standard antiepileptic drugs (AEDs). We hypothesized that a drug combination aimed at correcting the consequences of receptor trafficking would reduce SE severity and its long-term consequences.

Methods: A severe model of SE was induced in adult Sprague-Dawley rats with a high dose of lithium and pilocarpine. The GABAA receptor agonist midazolam, the NMDA receptor antagonist ketamine, and/or the AED valproate were injected 40 min after SE onset in combination or as monotherapy. Measures of SE severity were the primary outcome. Secondary outcomes were acute neuronal injury, spontaneous recurrent seizures (SRS), and Morris water maze (MWM) deficits.

Results: Midazolam-ketamine dual therapy was more efficient than double-dose midazolam or ketamine monotherapy or than valproate-midazolam or valproate-ketamine dual therapy in reducing several parameters of SE severity, suggesting a synergistic mechanism. In addition, midazolam-ketamine dual therapy reduced SE-induced acute neuronal injury, epileptogenesis, and MWM deficits.

Significance: This study showed that a treatment aimed at correcting maladaptive GABAA receptor and NMDA receptor trafficking can stop SE and reduce its long-term consequences. Early midazolam-ketamine dual therapy may be superior to monotherapy in the treatment of benzodiazepine-refractory SE.

Keywords: Cholinergic seizures; Epileptogenesis; Hippocampal sclerosis; Neuronal injury; Refractory status epilepticus; Spatial memory.

Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Figures

Figure 1. Midazolam-ketamine dual therapy is more efficient than double-dose midazolam or ketamine in reducing SE severity

A) Experimental flow: A severe form of status epilepticus (SE) was induced by administration of a high dose of lithium + scopolamine methyl bromide (scop. m. b), followed by an injection of a high dose of pilocarpine. Drug(s) or vehicle, and scopolamine (scop.) were injected 40 minutes after seizure onset. In acute studies, animals were implanted 1 week prior SE induction and sacrificed 48 h after SE onset to assess neuronal injury with fluoro-jade B staining. In long-term studies, animals were implanted 4 weeks after SE and monitored for the detection of spontaneous seizures during 2 weeks. They were then studied in the Morris Water Maze and subjected to a battery of behavioral tests. B) The left panels show the compressed EEG from SE control, midazolam, ketamine or midazolam-ketamine animals up to 75 min following treatment. The right panels show the magnified 6-sec EEG traces prior to SE or following SE (marked by vertical lines a-c). Vertical bar = 0.5 mV; horizontal bar = 1 sec. C) This graph shows the ratio of EEG power integral over the first hour to initial EEG power at baseline, before pilocarpine injection. The midazolam-ketamine group (n=9), which lowered the EEG power below pre-pilocarpine baseline, is significantly different from SE control (n=10, **** p

Figure 2. Reduction of neuronal injury in animals treated with midazolam-ketamine dual therapy or double-dose midazolam or ketamine

(A-L) These confocal images show fluoro-jade B staining in CA1 area (A-D), CA3 (E-H) and the hilus (I-L) 48 h following SE in SE control (A, E and I), midazolam (B, F and J), ketamine (C, G and K) and midazolam-ketamine (D, H and L) groups. Bars = 100 microns. (M-O) These graph shows the injury score (median values) in in CA1 (M) and CA3 (N), and the number of fluoro-jade B- positive cells counted by an unbiased stereological method in the hilus (O). Midazolam-ketamine dual therapy reduced neuronal injury compared to SE control in CA1. * p

Figure 3. Midazolam-ketamine dual therapy prevents epileptogenesis

(A) The upper panel shows the compressed 80-sec EEG of a SRS. The lower panel shows the magnified 8-sec EEG traces marked by horizontal lines (a-f) (vertical bar = 0.5 mV; horizontal bar = 20 sec in upper panel and 2 sec in lower panel). (B) Graph showing the number of SRS per week. Dual therapy midazolam-ketamine was more effective than double-dose ketamine in reducing the number of SRS. * p

Figure 4. Midazolam-ketamine dual therapy reduces behavioral deficits

(A-B) Performance in the Morris water maze. Graph A shows the latency to reach the hidden platform (y-axis) on each testing day (x-axis). Data are presented as mean ± SEM. * p<0.05 vs Mz4.5+Ket45; ** p < 0.01 vs Mz4.5+Ket45 and **** p<0.0001 vs Mz4.5+Ket45 by 2-way ANOVA. Graph B shows the latency during the retention test. ** p<0.01 vs valproate 270 mg/kg (by Kruskal-Wallis followed by Dunn's test). (C) Performance in the battery of behavioral tests. Data are presented as median values. ## p<0.01 or #### p<0.0001 vs Mz4.5+Ket45 (Kruskal-Wallis followed by Dunn's test).