Efficacy of Retigabine on Acute Limbic Seizures in Adult Rats

Abstract

Background and purpose: The efficacy of retigabine (RGB), a positive allosteric modulator of K+ channels indicated for adjunct treatment of partial seizures, was studied in two adult models of kainic acid (KA)-induced status epilepticus to determine it's toleratbility.

Methods: Retigabine was administered systemiclly at high (5 mg/kg) and low (1-2 mg/kg) doses either 30 min prior to or 2 hr after KA-induced status epilepticus. High (1 µg/µL) and low (0.25 µg/µL) concentrations of RGB were also delivered by intrahippocampal microinjection in the presence of KA.

Results: Dose-dependent effects of RGB were observed with both models. Lower doses increased seizure behavior latency and reduced the number of single spikes and synchronized burst events in the electroencephalogram (EEG). Higher doses worsened seizure behavior, produced severe ataxia, and increased spiking activity. Animals treated with RGB that were resistant to seizures did not exhibit significant injury or loss in GluR1 expression; however if stage 5-6 seizures were reached, typical hippocampal injury and depletion of GluR1 subunit protein in vulernable pyramidal fields occurred.

Conclusions: RGB was neuroprotective only if seizures were significantly attenuated. GluR1 was simultaneously suppressed in the resistant granule cell layer in presence of RGB which may weaken excitatory transmission. Biphasic effects observed herein suggest that the human dosage must be carefully scrutinized to produce the optimal clinical response.

Keywords: AMPA receptor; Anticonvulsant; Epilepsy; Hippocampus; Neurodegeneration; Retigabine.

Figure 1.

Biphasic effects of RGB before and after administration of KA. (A) RGB treated animal (5 mg/kg) is alert. (B) KA treated animal exhibiting a stage 6 seizure; foaming, rearing and forelimb clonus. (C) One treatment of RGB (5 mg/kg) 30 min prior to KA injection: Left rat has tonic convulsions without reaching stage 5–6; right rat is resistant to the seizure. (D) Two treatments of RGB (2 mg/kg) 2 hr post KA: both rats are calm and seizure free. (E) KA treatment prior to RGB injection exhibits arched back and postural control. (F) The same animl shown in panel E received a post-injection of RGB (5 mg/kg): This animal became ataxic and exhibited drooling with tongue held out, body tonus, and loss of postural control resembling a severe proconvulsant effect. (G) Graphical analysis of seizure scoring showed 2 prior treatments were more effective than one. (H) Seizure behavior onset and incidence of forelimb clonus (FLC) was significantly delayed or attenuated with two prior treatments of RGB. (I) RGB also reduced the percentage of animals with stage 5–6 seizure behavioral symptoms such as drooling, rearing, and FLC. 1×=one injection and 2×=two injections of RGB. KA, kainic acid; RGB, retigabine.

Figure 2.

EEG tracings after systemic or intrahippocampal KA ± RGB. Intraperitoneal treatment (IP): Left panels: Baseline activity; typical KA induced spikes and burst oscillations; pre-injection of RGB (5 mg/kg) reveals partial attenuation of spikes and bursts; post-injection of RGB (2 mg/kg) diminished synchronous spike and wave activity 2 hr after administration, anti-epileptic dosage; pro-convulsant and ataxic effect of RGB on KA seizures were found at a higher dose (5 mg/kg) when administered 2 hr after KA injection. Intrahippocampal treatment (IH): Right panels: Baseline activity; typical KA induced spikes and burst oscillations; RGB (0.25 μg/μl) reduced or prevented KA-induced epileptiform activity; RGB (1.0 μg/μl) enhanced KA-induced epileptiform activity; lower trace shows expansions of burst oscillations. EEG, electroencephalogram; KA, kainic acid; RGB, retigabine.

Figure 3.

Histological examination 28 days after KA ± RGB treatment at the level of the hippocampus. (A, D), Control NeuN labeling was intense and uniform throughout the hippocampal subfields; high magnification of CA1 labeled neurons showed they were round and healthy (400×). (B, E), After KA (15 mg/kg, i.p.), extensive cell loss was observed throughout the CA1, CA3, and hilar subfields (between arrows); the DG and CA2 were relatively resistant. High magnification revealed fewer, shrunken CA1 neurons throughout the layer; CA3 and hilar neurons were shrunken or absent. (C, F), Partial injury to CA1 and CA3 neurons was revealed after 2× RGB (5 mg/kg, i.p.) followed by KA; high magnification presented many healthy neurons. (G), Nissl staining of control animal labeled healthy, round CA1 neurons. (H), Nissl staining of KA animal revealed marked injury to CA1 neurons. (I), RGB+KA animals that were resistant to seizures displayed little or no injury, resembling the controls. (J, K), Graphic analyses of the number of injured vs. healthy appearing pyramidal cells at 3 and 28 days following systemic injections of both groups of animals that were either sensitive or resistant to seizures. Bars represent the mean averages ± SEM. ^*p<0.05; ^**p<0.01. One Way ANOVA. Scale=100 μm. KA, kainic acid; RGB, retigabine; DG, dentate gyrus; NeuN, neuronal nuclear antigen.

Figure 4.

GluR1 immunohistochemistry in presence and absence of RGB treatment illustrated at two magnifications (20×, 400×). (A, G) GluR1 control immunolabeling was intense and uniform. (B, H) GluR1 expression was stable after RGB treatment in the absence of KA. (C, I) At 24 hr after KA, GluR1 was steady in the CA1 and DG but reduced in the CA3 within areas of cell damage. (D, J) At 72 hr, GluR1 immunolabeling was depleted in the CA1/subiculum and hilus in areas of cell loss but spared in survival regions. (E, K) At 24 hr after RGB+KA treatment, GluR1 protein was stable. (F, L), At 72 hr, GluR1 was reduced in areas of injury, similar to the KA treatment and reduced GluR1 expression was observed within the GCL. Scale=100 μm. RGB, retigabine; KA, kainic acid; GCL, granule cell layer; ML, molecular layer; dentate gyrus.

Figure 5.

(A) GluR1 Densitometry measurements of both surviving and damaged regions show marked decreases in both CA1 and CA3 subregions at 3 days. (B) Within subject ratiometric analysis of GluR1 immunodenistometry of the spared regions is illustrated at 3 and 28 days after KA ± RGB treatment. Ratiometric mean averages were significantly decreased throughout the CA1 subfield at 3 days after KA ± RGB. Variable GluR1 expression was observed at 28 days; some had marked depletions that were commiserating with cell loss. Chronic RGB treatment did not alter CA1 expression. (C) GluR1 expression of the CA3 was less variable than CA1; ratiometric mean averages were significantly decreased throughout the CA3 subfield at 3 and 28 days after KA ± RGB. Chronic RGB treatment also reduced GluR1 expression of the CA3 in the absence of KA. (D) Within the GC, GluR1 expression significantly declined after RGB+KA and after chronic RGB treatment in the absence of KA. ^*p<0.05, One Way ANOVA. GLC, granule cell layer; DML, dorsal molecular layer; KA, kainic acid; RGB, retigabine; DG, dentate gyrus.