Acute and Sub-chronic Anticonvulsant Effects of Edaravone on Seizure Induced by Pentylenetetrazole or Electroshock in Mice, Nitric Oxide Involvement

Abstract

Background: Edaravone is an anti-stroke medication that may have nitric oxide (NO) modulating properties. This study evaluated the role of NO in the acute and sub-chronic anticonvulsant effects of edaravone in murine models of seizures induced by intraperitoneal (IP) or intravenous (IV) injections of pentylenetetrazole (PTZ) or electroshock (maximal electroshock seizure [MES]).

Methods: 132 male albino mice were randomly divided into 22 groups (n=6) and given IP injections of vehicle or edaravone either acutely or for eight days (sub-chronically). The seizure was induced by electroshock or PTZ (IP or IV). The following edaravone doses were used: 7.5, 10, 12.5 (acute); 5, 7.5, 10 (sub-chronic) in IP PTZ model; 5, 7.5, 10 in IV PTZ model; and 5, 10 mg/Kg in the MES. To evaluate NO involvement, 216 mice were randomly divided into 36 groups (n=6) and pretreated with vehicle, edaravone, a non-specific nitric oxide synthase (NOS) inhibitor: N(ω)-nitro-L-arginine methyl ester (L-NAME) (5 mg/Kg), a specific nNOS inhibitor: 7-nitroindazole (7-NI) (60 mg/Kg), or a combination of edaravone plus L-NAME or 7-NI, either acutely or for eight days before seizure induction. Doses of edaravone were as follows: in IP PTZ model: 12.5 (acute) and 10 (sub-chronic); in IV PTZ model: 10; and in the MES: 5 mg/Kg. Data were analyzed using the one-way analysis of variance (ANOVA) followed by Tukey's test (SPSS 18). P≤0.05 was considered statistically significant.

Results: In the IP PTZ model, edaravone increased time latencies to seizures (P<0.001), prevented tonic seizures, and death. Edaravone increased the seizure threshold (P<0.001) in the IV PTZ model and shortened the duration of tonic hind-limb extension (THE) in the MES model (P<0.001). In comparison to mice treated with edaravone alone, adding L-NAME or 7-NI reduced seizure time latencies (P<0.001), reduced seizure threshold (P<0.001), and increased THE duration (P<0.001).

Conclusion: Edaravone (acute or sub-chronic) could prevent seizures by modulating NO signaling pathways.

Keywords: Edaravone; Electroshock; Epilepsy; Nitric oxide; Pentylenetetrazole.

Figure 1

(A) Acute pretreatment of mice with edaravone (12.5 mg/Kg, IP) caused significant prolongation of latency times to the onsets of both myoclonic and clonic seizures induced by PTZ (85 mg/Kg, IP) in comparison to the vehicle group (P<0.001). (B) Acute pretreatment of mice with edaravone (12.5 mg/Kg, IP) plus L-NAME (5 mg/Kg, IP) or edaravone (12.5 mg/Kg, IP) plus 7-NI (60 mg/Kg, IP) significantly reduced the latency times to the onsets of both myoclonic and clonic seizures induced by PTZ (85 mg/Kg, IP) in comparison to those for edaravone (12.5 mg/Kg, IP) alone (P<0.001 for both co-administrations of LNAME plus edaravone or 7-NI plus edaravone). (C) Sub-chronic pretreatment of mice with edaravone (5, 7.5, 10 mg/Kg, IP) for eight days, resulted in significant prolongation of latency times to the onsets of both myoclonic and clonic seizures induced by PTZ (85 mg/Kg, IP) in comparison to the vehicle group (P<0.001). (D) Sub-chronic pretreatment of mice with edaravone (12.5 mg/Kg, IP) plus L-NAME (5 mg/Kg, IP) or edaravone (12.5 mg/Kg, IP) plus 7-NI (60 mg/Kg, IP) for eight days, reduced the latency times to both myoclonic and clonic seizures induced by PTZ (85 mg/Kg, IP) in comparison to the administation of edaravone alone (P<0.001 for both co-administrations of LNAME plus edaravone or 7-NI plus edaravone). ***P≤0.001 represents significant differences with the vehicle group, ###P≤0.001 represents significant differences with edaravone. ED: edaravone; L-NAME: N (ω)-nitro-L-arginine methyl ester; 7-NI: 7-nitroindazole

Figure 2

The graphs shows the mean±SEM of clonic seizure threshold induced by intravenous infusion of PTZ in mice pretreated with acute or sub-chronic intraperitoneal infusion of edaravone (A) Acute pretreatment of mice with edaravone (7.5, 10 mg/Kg, IP) increased the seizure threshold more than the vehicle group (P<0.001). (B) Acute pretreatment of mice with concomitant injections of edaravone (10 mg/Kg) plus L-NAME (5 mg/Kg, IP) or 7-NI (60 mg/Kg, IP) decreased the seizure threshold in comparison to the edaravone alone (P<0.001 for both co-administrations of L-NAME plus edaravone or 7-NI plus edaravone). (C) Sub-chronic pretreatment of mice with edaravone (10 mg/Kg, IP) for eight days, significantly increased the seizure threshold as compared to the vehicle group (P<0.001). (D) Sub-chronic pretreatment of mice with concomitant injections of edaravone (10 mg/Kg) plus L-NAME (5 mg/Kg, IP) or 7-NI (60 mg/Kg, IP) for eight days decreased the seizure threshold in comparison to edaravone alone (P≤0.001 for both co-administrations of L-NAME plus edaravone or 7-NI plus edaravone). ***P≤0.001 represents significant differences with the vehicle group, ### P≤0.001 represents significant differences with edaravone. ED: Edaravone; L-NAME: N (ω)-nitro-L-arginine methyl ester; 7-NI: 7-nitroindazole

Figure 3

The graphs shows the mean±SEM of the duration of tonic hind-limb extension (THE) in mice pretreated with acute or sub-chronic intraperitoneal doses of edaravone: (A) Acute pretreatment of mice with edaravone (5, 10 mg/Kg, IP) decreased THE duration in comparison to the vehicle group (P<0.001). (B) Acute pretreatment of mice with concomitant injections of edaravone (5 mg/Kg, IP) plus L-NAME (5 mg/Kg, IP) or 7-NI (60 mg/Kg, IP) significantly increased THE duration in comparison to the administration of edaravone alone (P<0.001 for both co-administrations of LNAME plus edaravone or 7-NI plus edaravone). (C) Sub-chronic pretreatment of mice with edaravone (5, 10 mg/Kg, IP) for eight days, decreased THE duration as compared to the vehicle group (P<0.001). (D) Sub-chronic pretreatment of mice with concomitant injections of edaravone (5 mg/Kg, IP) plus L-NAME (5 mg/Kg, IP) or 7-NI (60 mg/Kg, IP) for eight days significantly increased THE duration in comparison to the administration of edaravone alone (P<0.001 for both co-administrations of LNAME plus edaravone or 7-NI plus edaravone). ***P≤0.001 represents significant differences with the vehicle group, ###P≤0.001 represents significant differences with edaravone. ED: Edaravone; L-NAME: N (ω)-nitro-L-arginine methyl ester; 7-NI: 7-nitroindazole