Unique behavioral characteristics and microRNA signatures in a drug resistant epilepsy model

Abstract

Background: Pharmacoresistance is a major issue in the treatment of epilepsy. However, the mechanism underlying pharmacoresistance to antiepileptic drugs (AEDs) is still unclear, and few animal models have been established for studying drug resistant epilepsy (DRE). In our study, spontaneous recurrent seizures (SRSs) were investigated by video-EEG monitoring during the entire procedure.

Methods/principal findings: In the mouse pilocarpine-induced epilepsy model, we administered levetiracetam (LEV) and valproate (VPA) in sequence. AED-responsive and AED-resistant mice were naturally selected after 7-day treatment of LEV and VPA. Behavioral tests (open field, object exploration, elevated plus maze, and light-dark transition test) and a microRNA microarray test were performed. Among the 37 epileptic mice with SRS, 23 showed significantly fewer SRSs during administration of LEV (n = 16, LEV sensitive (LS) group) or VPA (n = 7, LEV resistant/VPA sensitive (LRVS) group), while 7 epileptic mice did not show any amelioration with either of the AEDs (n = 7, multidrug resistant (MDR) group). On the behavioral assessment, MDR mice displayed distinctive behaviors in the object exploration and elevated plus maze tests, which were not observed in the LS group. Expression of miRNA was altered in LS and MDR groups, and we identified 4 miRNAs (miR-206, miR-374, miR-468, and miR-142-5p), which were differently modulated in the MDR group versus both control and LS groups.

Conclusion: This is the first study to identify a pharmacoresistant subgroup, resistant to 2 AEDs, in the pilocarpine-induced epilepsy model. We hypothesize that modulation of the identified miRNAs may play a key role in developing pharmacoresistance and behavioral alterations in the MDR group.

Figure 1. The schematic representation of the study protocol and a representative electroencephalogram data.

(A) Sixty days after the pilocarpine induction of status epilepticus (SE), continuous video EEG monitoring was started. After 7 days of baseline recording, levetiracetam (LEV) was administered intraperitoneally (15 mg/kg/day) for 7 days. After a washout period of 7 days, valproate (VPA) was given intraperitoneally (30 mg/kg/day) for 7 days. Behavioral tests were performed following a 7-day washout period starting after the last administration of an AED. After all behavioral tests were completed, mice were sacrificed, and a microRNA microarray was performed. Control group is not illustrated because it was only exposed to mehylscopolamine and did not received pilocarpine. (B) Representative electroencephalogram (EEG) traces of SRS. Abbreviations: AED: antiepileptic drug, d: days, SRS: spontaneous recurrent seizure, LS: levetiracetam sensitive, LRVS: levetiracetam resistant/valproate sensitive, MDR: multidrug resistant.

Figure 2. Behavior of mice in each group in the open field and object exploration test.

Data are shown as mean ± SEM. (A–B) Open field test (control, n = 6; LS group, n = 11; MDR group, n = 7). (A) illustrates the total distance that the mice moved during the 10 min of the open field test. (B) illustrates the time that mice spent in the aversive center of the open field during 5 min. (C–D) Object exploration test (control, n = 6; LS group, n = 8; MDR group, n = 5). (C) illustrates the total distance that the mice moved during 5 min after one object was placed in the center of the box. (D) illustrates the number of entries into the center during 5 min after one object was placed in the center of the box. (*) represents a significant individual difference when compared to the normal group (p<0.05, Mann-Whitney U test).

Figure 3. Behavior of mice in each group in the elevated plus maze test.

Data are shown as mean ± SEM for the elevated plus maze test (control, n = 10; LS group, n = 12; MDR group, n = 6). (A) illustrates the time that mice spent in the aversive open arms of the maze during 5 min of the test. (B) illustrates the time that mice spent in the closed arms of the maze during the test. (C) illustrates the number of crossings observed during the test. (D) illustrates the time that mice spent in the center of the maze during the test. (*) represents a significant individual difference when compared to the normal group (p<0.05, Mann-Whitney U test). (†) represents a significant individual difference when compared to the LS group (p<0.05, Mann-Whitney U test).

Figure 4. Behavior of mice in each group in the light-dark transition test.

Data are shown as mean ± SEM for the light-dark transition test (control, n = 7; LS group, n = 11; MDR group, n = 7). (A) illustrates the time that mice spent in the light compartment during 5 min of the test. (B) illustrates the time that mice spent in the dark compartment during the test. (C) illustrates the time spent prior to the first entry to the light compartment. (D) illustrates the number of crossings between the light and dark compartments during the test. (*) represents a significant individual difference when compared to the normal group (p<0.05, Mann-Whitney U test).

Figure 5. Venn diagram describing miRNA expression patterns in each group.

The Venn diagram represents the number of differently expressed miRNAs observed in the comparisons among the three groups (control, LS, and MDR groups). The tables give the specific miRNAs that showed significantly different expression levels among groups. (>>) represents a significant intergroup difference of more than twofold changes and a P-value <0.05. (>) represents non-significant intergroup differences in miRNA expression levels.