Effective Protection Against Status Epilepticus Caused by Lithium-Pilocarpine: Combination of Midazolam and Lacosamide

Abstract

Aim: Status epilepticus causes the most severe condition related to epilepsy in terms of high mortality rate. Although status epilepticus treatment guidelines specify a treatment process based on three-stage monotherapy, effective control cannot yet be achieved in all cases. In the presented study, with electrophysiological and behavioral tests, it was aimed to investigate the effectiveness of the combination of midazolam (MDZ), one of the most commonly used benzodiazepines in the first-line treatment of status epilepticus, with the second-line antiepileptics levetiracetam (LEV), lacosamide (LCM), valproic acid (VPA), and fosphenytoin (fPHT).

Methods: A status epilepticus model was created with lithium-pilocarpine (5 mEq/kg-320 mg/kg) in adult male Sprague-Dawley rats with implanted electroencephalography (EEG) electrodes. MDZ (9 mg/kg) alone or in dual combinations with antiepileptic drugs (200 mg/kg LEV, 50 mg/kg LCM, 300 mg/kg VPA, 100 mg/kg fPHT) was injected i.p. to the experiment groups with status epilepticus. After video-EEG recordings were taken from the rats during and after status, the effects of drug interactions on cognitive and motor behaviors were examined by applying behavioral tests (open field, Rotarod, radial arm maze, and passive avoidance).

Results: Compared with the untreated status epilepticus group, it was determined that MDZ alone and the combination of four antiepileptic drugs administered with MDZ significantly reduced the mortality rate, spike frequency, and spike amplitude of epileptic seizures and suppressed epileptic seizures at certain levels (p < 0.01). Compared to MDZ monotherapy, it was determined that the mortality rate and spike frequency and amplitude decreased significantly in the MDZ + LCM group (p < 0.01), whereas on the other hand, mortality and spike frequency increased in the MDZ + LEV group (p < 0.01). No negative effects were observed in learning and memory in all treatment groups, but it was determined that the motor functions of the animals treated with MDZ + fPHT were impaired compared to both the control group without any treatment and the MDZ group (p < 0.01).

Conclusion: In the status epilepticus model induced by lithium-pilocarpine, the combination of MDZ + LCM was found to be the most effective polytherapy option in reducing seizures and mortality. Additionally, it was observed that LEV, LCM, and VPA administered together with MDZ did not negatively affect both cognitive and motor functions.

Keywords: fosphenytoin; lithium–pilocarpine; midazolam; status epilepticus; valproic acid.

FIGURE 1

Experimental design. (A) After creating a status epilepticus model in the acute period, MDZ and antiepileptic drug combinations were applied, and video‐EEG recording was performed. (B) Behavioral tests were applied after video‐EEG recordings were made in the chronic period. MDZ: midazolam; seizures began approximately 7–8 min after pilocarpine injection. EEG, electroencephalography.

FIGURE 2

EEG trace examples of midazolam and antiepileptic drug combinations. Baseline recordings show 5 min before induction of status epilepticus with pilocarpine. EEG traces during the status epilepticus period show activity between 10 and 12 min after seizures were initiated by pilocarpine injection. The post‐drug period represents activity between 90 and 92 min after administration of MDZ or its combinations. All groups received pilocarpine. Untreated SE: untreated status epilepticus group (n = 8); MDZ: midazolam group (n = 8); MDZ + LEV: midazolam + levetiracetam group (n = 6); MDZ + LCM: midazolam + lacosamide group (n = 8); MDZ + VPA: midazolam + valproic acid group (n = 6); MDZ + fPHT: midazolam + fosfenitoin group (n = 8). EEG, electroencephalography; fPHT, fosphenytoin; LEV, levetiracetam; LCM, lacosamide; MDZ, midazolam; VPA, valproic acid.

FIGURE 3

Spike frequencies related to status epilepticus during the 120‐min EEG recording period before and after the application of MDZ and antiepileptic drug combinations. Spike frequency values are shown in (A‐E), respectively. Before antiepileptic drug administration (A), at 30th minute (B), 60th minute (C), 90th minute (D) and 120th minute (E). In the experimental groups where status epilepticus was induced with lithium–pilocarpine, 9 mg/kg MDZ alone or in combination with antiepileptic drugs like 200 mg/kg LEV, 50 mg/kg LCM, 300 mg/kg VPA, or 100 mg/kg fPHT were administered intraperitoneally. The graphs show the median (min–max and Q1–Q3) spike frequency values for 1 min of activity, obtained at 30‐min intervals (*p < 0.05 and **p < 0.01 compared to status epilepticus group; #p < 0.05 and ##p < 0.01 compared to MDZ group. Kruskal Wallis followed by Mann Whitney U test). All groups received pilocarpine. Untreated SE: untreated status epilepticus group (n = 8); MDZ: midazolam group (n = 8); MDZ + LEV: midazolam + levetiracetam group (n = 6); MDZ + LCM: midazolam + lacosamide group (n = 8); MDZ + VPA: midazolam + valproic acid group (n = 6); MDZ + fPHT: midazolam + fosfenitoin group (n = 8). EEG, electroencephalography; fPHT, fosphenytoin; LEV, levetiracetam; LCM, lacosamide; MDZ, midazolam; VPA, valproic acid.

FIGURE 4

Spike amplitudes related to status epilepticus during the 120‐min EEG recording period before and after the application of MDZ and antiepileptic drug combinations. Spike amplitude values are shown in (A‐E) respectively. Before antiepileptic drug administration (A), at 30th minute (B), 60th minute (C), 90th minute (D) and 120th minute (E). In the experimental groups where status epilepticus was induced with lithium–pilocarpine, 9 mg/kg MDZ alone or in combination with antiepileptic drugs like 200 mg/kg LEV, 50 mg/kg LCM, 300 mg/kg VPA, or 100 mg/kg fPHT were administered intraperitoneally. The graphs show the median (min–max and Q1–Q3) spike amplitude values for 1 min of activity, obtained at 30‐min intervals (*p < 0.05 and **p < 0.01 compared to status epilepticus group; #p < 0.05 and ##p < 0.01 compared to MDZ group. Kruskal Wallis followed by Mann Whitney U test). All groups received pilocarpine. Untreated SE: untreated status epilepticus group (n = 8); MDZ: midazolam group (n = 8); MDZ + LEV: midazolam + levetiracetam group (n = 6); MDZ + LCM: midazolam + lacosamide group (n = 8); MDZ + VPA: midazolam + valproic acid group (n = 6); MDZ + fPHT: midazolam + fosfenitoin group (n = 8). EEG, electroencephalography; fPHT, fosphenytoin; LEV, levetiracetam; LCM, lacosamide; MDZ, midazolam; VPA, valproic acid.

FIGURE 5

Effect of MDZ and antiepileptic drug combinations on seizure parameters. (A) Seizure onset latency shows the median (min–max) values for the time until the onset of seizure activity after 320 mg/kg pilocarpine injection. (B) The initial latency of drug efficacy represents the time until the onset of the first statistically significant decrease in spike frequency, according to the status epilepticus group. (C) Mortality latency shows the median (min–max) values of the animal death time from the physiological saline or drug injections applied at the 30th minute of status epilepticus to the EEG recording on the 14th day. (D) Mortality rate shows the percentage of animal deaths due to status epilepticus in the experimental groups. Because two animals died due to status epilepticus before treatment in the MDZ + LEV and MDZ + VPA groups, the mortality rate in these groups was calculated on n = 6 (*p < 0.05, **p < 0.01, and ***p < 0.001 compared to status epilepticus group; #p < 0.05 and ##p < 0.01 compared to MDZ group. Kruskal Wallis followed by Mann Whitney U test). All groups received pilocarpine. Untreated SE: untreated status epilepticus group (n = 8); MDZ: midazolam group (n = 8); MDZ + LEV: midazolam + levetiracetam group (n = 6); MDZ + LCM: midazolam + lacosamide group (n = 8); MDZ + VPA: midazolam + valproic acid group (n = 6); MDZ + fPHT: midazolam + fosfenitoin group (n = 8). EEG, electroencephalography; fPHT, fosphenytoin; LEV, levetiracetam; LCM, lacosamide; MDZ, midazolam; VPA, valproic acid.

FIGURE 6

Heat map depicting the locomotor behavior of animals in an open field test. A typical representation of the average behavior of each group is shown. The red color highlights areas where more than 5 s were spent. All groups except the control group received pilocarpine. Control: control group (n = 8); MDZ: midazolam group (n = 8); MDZ + LEV: midazolam + levetiracetam group (n = 6); MDZ + LCM: midazolam + lacosamide group (n = 8); MDZ + VPA: midazolam + valproic acid group (n = 6); MDZ + fPHT: midazolam + fosfenitoin group (n = 8). fPHT, fosphenytoin; LEV, levetiracetam; LCM, lacosamide; MDZ, midazolam; VPA, valproic acid.

FIGURE 7

Behavior parameters for open field testing. The increase in the total distance traveled (A) and the number of rearings (B) indicates an increase in locomotor efficiency. An increase in the number of times the central square (C) was entered and the time spent (D) there was evaluated as a measure of the decrease in anxiety‐like behavior (*p < 0.05 and **p < 0.01 compared to the control group; #p < 0.05 compared to the MDZ group. Kruskal Wallis followed by Mann Whitney U test). All groups except the control group received pilocarpine. Control: control group (n = 8); MDZ: midazolam group (n = 8); MDZ + LEV: midazolam + levetiracetam group (n = 6); MDZ + LCM: midazolam + lacosamide group (n = 8); MDZ + VPA: midazolam + valproic acid group (n = 6); MDZ + fPHT: midazolam + fosfenitoin group (n = 8). fPHT, fosphenytoin; LEV, levetiracetam; LCM, lacosamide; MDZ, midazolam; VPA, valproic acid.

FIGURE 8

Motor and cognitive test parameters. Fall latency in the Rotarod test was assessed as an indicator of forced motor activity (A). Avoidance latency in the passive avoidance test reflects a reduction in fear‐based memory (B). In the radial arm maze test, the latency of locating the correct arm (C) was deemed a favorable parameter for spatial memory, whereas the number of incorrect arm entries (D) was seen as a negative parameter (*p < 0.05 and **p < 0.01 compared to control group; #p < 0.05 and ##p < 0.01 compared to MDZ group. Kruskal Wallis followed by Mann Whitney U test). All groups except the control group received pilocarpine. Control: control group (n = 8); MDZ: midazolam group (n = 8); MDZ + LEV: midazolam + levetiracetam group (n = 6); MDZ + LCM: midazolam + lacosamide group (n = 8); MDZ + VPA: midazolam + valproic acid group (n = 6); MDZ + fPHT: midazolam + fosfenitoin group (n = 8). fPHT, fosphenytoin; LEV, levetiracetam; LCM, lacosamide; MDZ, midazolam; VPA, valproic acid.