Beneficial Effects of Selective Orexin-A Receptor Antagonist in 4-aminopyridine-induced Seizures in Male Rats

Abstract

Background: Orexins are excitatory neuropeptides which stimulate the central regulatory pathways. Orexins increase the penicillin-induced epileptic activity in rats. Orexin-A increases in different types of seizures and its elevated level is the characteristic feature in the epileptic children during polysomnography. Recently, the orexin receptor blockage has been reported to increase seizure threshold in mice; however, effect of the selective orexin-A receptor antagonist (SB-334867) on 4-aminopyridine (4-AP)-induced seizures has not been investigated.

Materials and methods: We used the intraperitoneal injection of 4-AP to induce seizure in male rats. Under urethane anesthesia, SB-334867 (50 and 100 nmol) was injected stereotaxically into the ventral hippocampal commissure. Using video recording, the effects of SB-334867 on electroencephalogram and tonic-clonic convulsions were compared to those that received diazepam or dimethyl sulfoxide (DMSO).

Results: SB-334867 significantly decreased the duration of spike trains compared to DMSO-treated rats (P < 0.001) and reduced the duration of convulsive seizures (P < 0.05). Seizure onset was increased significantly by SB-334867, 50 nmol, compared to DMSO (P < 0.05) and diazepam (P < 0.01) treated rats.

Conclusion: Antagonism of orexin-A receptor by a low-dose SB-334867 showed protective effects in 4-AP-induced seizure-like activities in anesthetized rats.

Keywords: 4-aminopyridine; electroencephalogram; microinjection; orexin-A receptor antagonist.

Figure 1

Duration of spike trains in treated groups with DMSO + normal saline, 4-aminopyridine + DMSO, SB-334867 (50 and 100 nmol) +4-aminopyridine, SB-334867 (100 nmol) + normal saline, and 4-aminopyridine + diazepam after intraperitoneal 4-aminopyridine injection. (a) *P < 0.05 versus DMSO + normal saline, ⁺P < 0.05 versus 4-aminopyridine + DMSO. (b) **P < 0.01 versus DMSO + normal saline, ⁺P < 0.05 versus SB 100 nmol + normal saline, ^xxP < 0.01 versus 4-aminopyridine + DMSO. (c) *P < 0.05 versus DMSO + normal saline, ⁺⁺P < 0.01 versus SB 100 nmol + normal saline, *P < 0.05 versus 4-aminopyridine + DMSO. Friedman test followed by Dunn's test. (d) ***P < 0.001 versus DMSO + normal saline, ^xxP < 0.01 versus SB 100 nmol + normal saline, and ⁺⁺⁺P < 0.001 versus 4-aminopyridine + SB 50 nmol. Kruskal–Wallis followed by Dunn's test. P <0.05 was considered as significant

Figure 2

Electroencephalogram patterns in different treated groups with DMSO, SB-334867 50 nmol (SB1), 100 nmol (SB2), and diazepam 30 min after intraperitoneal 4-aminopyridine injection. Calibration is 1 s on X axis, 1 mv on Y axis on each little square. ih = Intrahippocampal commissure, ip = Intraperitoneal

Figure 3

Duration of convulsive-like seizures in different treated groups with DMSO, SB-334867 (50 and 100 nmol), and diazepam after intraperitoneal 4-aminopyridine injection. One-way ANOVA followed by Bonferroni test. P < 0.05 was considered as significant. *P < 0.05: 4-aminopyridine + DMSO versus 4-aminopyridine + SB 50 and 100 nmol, ⁺P < 0.05: 4-aminopyridine + diazepam versus 4-aminopyridine + SB 50 nmol. DMSO + normal saline and SB 100 nmol + normal saline did not induce seizure behaviors, so they were neglected in data analysis

Figure 4

Seizure onset in different treated groups with DMSO, SB-334867 (50 and 100 nmol/rat), and diazepam after intraperitoneal 4-aminopyridine injection. One-way ANOVA followed by Bonferroni test. P < 0.05 was considered as significant. *P < 0.05: 4-aminopyridine + DMSO versus 4-aminopyridine + SB 50 nmol, ⁺⁺P < 0.01: 4-aminopyridine + diazepam versus 4-aminopyridine + SB 50 nmol. DMSO + normal saline and SB 100 nmol + normal saline did not induce seizure behaviors, so they were neglected in data analysis

Figure 5

Injection needle tip site through ventral hippocampal commissure. White arrow represents the site of injection needle. Hematoxylin eosin staining, (×100))