Pharmacoresponsiveness of spontaneous recurrent seizures and the comorbid sleep disorder of epileptic Kcna1-null mice

Abstract

Drug resistant epilepsy affects ∼30% of people with epilepsy and is associated with epilepsy syndromes with frequent and multiple types of seizures, lesions or cytoarchitectural abnormalities, increased risk of mortality and comorbidities such as cognitive impairment and sleep disorders. A limitation of current preclinical models is that spontaneous seizures with comorbidities take time to induce and test, thus making them low-throughput. Kcna1-null mice exhibit all the characteristics of drug resistant epilepsy with spontaneous seizures and comorbidities occurring naturally; thus, we aimed to determine whether they also demonstrate pharmacoresistanct seizures and the impact of medications on their sleep disorder comorbidity. In this exploratory study, Kcna1-null mice were treated with one of four conventional antiseizure medications, carbamazepine, levetiracetam, phenytoin, and phenobarbital using a moderate throughput protocol (vehicle for 2 days followed by 2 days of treatment with high therapeutic doses selected based on published data in the 6 Hz model of pharmacoresistant seizures). Spontaneous recurrent seizures and vigilance states were recorded with video-EEG/EMG. Carbamazepine, levetiracetam and phenytoin had partial efficacy (67%, 75% and 33% were seizure free, respectively), whereas phenobarbital was fully efficacious and conferred seizure freedom to all mice. Thus, seizures of Kcna1-null mice appear to be resistant to three of the drugs tested. Levetiracetam failed to affect sleep architecture, carbamazepine and phenytoin had moderate effects, and phenobarbital, as predicted, restored sleep architecture. Data suggest Kcna1-null mice may be a moderate throughput model of drug resistant epilepsy useful in determining mechanisms of pharmacoresistance and testing novel therapeutic strategies.

Keywords: Carbamazepine; Carbamazepine (PubChem CID: 2554); Chemical compounds studied in this article are phenobarbital (PubChem CID: 4763); Diurnal; Kv1.1; Levetiracetam; Levetiracetam (PubChem CID: 5284583); Phenobarbital; Phenytoin; Phenytoin (PubChem CID: 1775).

Figure 1.

Kcna1-null mice exhibit multiple types of generalized seizures. (A-E) Examples of EEG recordings and spectrograms of Types 2–6 generalized seizures. (F) The proportion of seizure types expressed in individual mice (n = 22).

Figure 2.

The diurnal relationship of seizures and sleep in Kcna1-null mice. (A) Seizures occurred with a diurnal rhythm with periodicity of 23.83 ± 0.35 hrs (non-linear regression: sine wave with non-zero baseline, wavelength constraint > 23 and phase shift constraint = −3.65). The zenith was at ZT8:00 and nadir at ZT20:00 (one-way repeated measures ANOVA, F = 2.285, p = 0.05; followed by Dunnett’s multiple comparisons test, *p < 0.05 vs ZT8:00; n =22 mice). (B) Overall, more seizures occurred during the light (rest) phase (paired t-test, ***p < 0.001; n = 22 mice). (C) Comparison of vigilance states between genotypes during the active (dark) phase (unpaired t-test, p > 0.05; n = 22 mice). (D) Comparison of vigilance states between genotypes during the rest (light) phase (unpaired t-test, ***p < 0.001; n = 22 mice). (E) Relationship of seizure types and vigilance states for all 226 seizures detected in 22 mice.

Figure 3.

Pharmacoresponsivess of Kcna1-null seizure frequency. (A) Cumulative seizure frequency over 24 hrs post-injection of vehicle or antiseizure medication presented in 4 hr bins. Two-way repeated measures ANOVA for each treatment vs. vehicle over time; carbamazepine (CBZ): F(5, 110) = 2.151, p = 0.065; time: F(5, 110) = 6.8, p < 0.0001, n = 6; phenytoin (PhT): F(5, 110) = 0.19, p = 0.97; time: F(5, 110) = 20.9, p < 0.0001, n = 6; levetiracetam (LEV): F(5, 70) = 0.91, p = 0.48; time: F(5, 70) = 12.8, p < 0.0001, n = 4; phenobarbital (PhB): F(5, 110) = 11.8, p < 0.0001; time: F(5, 110) = 11.9, p < 0.0001, n = 6; followed by Sidak’s multiple comparisons test, *p < 0.05, ***p < 0.001 vs. vehicle. Bottom bar represents light (empty)-dark (filled) phases associated with the hours post-injection. (B) Calculated area under the curve for graph in A (paired t-test, ***p< 0.001).

Figure 4.

Individual variation of drug effects. (A) Heat maps of seizure frequency of individual mice after injection of vehicle or drug. (B-D) The mean effect for all mice for each drug is depicted in by the squares (CBZ is purple, n = 6; PhT is green, n = 6; LEV is orange, n = 4). Stratification of antiseizure medication effects on seizure frequency normalized to vehicle for each 4 hr bin. Red triangles indicate seizures increased or did not change (CBZ, n = 3; PhT, n = 2; LEV, n = 1). Blue triangles indicate seizures were reduced (CBZ, n = 3; PhT, n = 4; LEV, n = 3). Two-way repeated measures ANOVA for each treatment vs. vehicle over time followed by Sidak’s multiple comparisons test, *p < 0.05, **, p < 0.01, ***p < 0.001. Bottom bar represents light-dark phases associated with the hours post-injection.

Figure 5.

Carbamazepine, phenytoin, levetiracetam effects on seizure type and burden. (A) Average proportion of seizure types for individual mice during vehicle and antiseizure medication treatment (paired t-tests, *p< 0.05). (B) Average seizure types and burdens during vehicle and antiseizure medication (circle symbols; CBZ is purple, PhT is green and LEV is orange, paired t-test, *p< 0.05). In addition, the responses of individual mice in which seizure frequency increased or did not change (red triangles: CBZ, n = 3; PhT, n = 2; LEV, n = 1) or was reduced (blue triangles: CBZ, n = 3; PhT, n = 4; LEV, n = 3) are also indicated.

Figure 6.

Pharmacoresponsivess of Kcna1-null sleep architecture. (A-D) In the first four hours post-antiseizure medication injection and in the normal rest (light) phase, phenobarbital, CBZ and PhT decreased wake and phenobarbital and CBZ significantly increased NREMS and REMS. (Ai) Phenobarbital achieved a strong effect on sleep architecture (left) by increasing the number of REMS bouts by 250% and reducing wake bouts by 40% and (right) by increasing the average time spent in a bout of NREMS and REMS by 250% and 270%, respectively. (Bi) Moderate effects on sleep by CBZ were achieved (left) by increasing the number of REMS bouts by 188%, and (right) by increasing the average length of NREMS and REMS bouts by 67% and 112%. (E-H) Twelve hrs later, in the active (dark) phase, no antiseizure medication affected wake, NREMS or REMS. One-way ANOVA for each vigilance state followed by Tukey’s multiple comparisons test, *p < 0.05, **, p < 0.01, ***p < 0.001 (PhB, n = 6; CBZ, n = 6; PhT, n = 6; LEV, n = 4).

Figure 7.

Antiseizure medication effects on the emergence of seizures from vigilance states. (A) phenobarbital prevented seizures in all vigilance states. (B) CBZ did not affect the proportion of seizures occurring in wake, NRMS or REMS. (C) PhT significantly increased the proportion of seizures occurring in NREMS. (D) There was a trend for LEV to prevent seizures from occurring in REMS in favor of NREMS. (A-D) Fisher’s exact test, *p < 0.05.