Ethosuximide reduces epileptogenesis and behavioral comorbidity in the GAERS model of genetic generalized epilepsy

Abstract

Purpose: Ethosuximide (ESX) is a drug of choice for the symptomatic treatment of absence seizures. Chronic treatment with ESX has been reported to have disease-modifying antiepileptogenic activity in the WAG/Rij rat model of genetic generalized epilepsy (GGE) with absence seizures. Here we examined whether chronic treatment with ESX (1) possesses antiepileptogenic effects in the genetic absence epilepsy rats from Strasbourg (GAERS) model of GGE, (2) is associated with a mitigation of behavioral comorbidities, and (3) influences gene expression in the somatosensory cortex region where seizures are thought to originate.

Methods: GAERS and nonepileptic control (NEC) rats were chronically treated with ESX (in drinking water) or control (tap water) from 3 to 22 weeks of age. Subsequently, all animals received tap water only for another 12 weeks to assess enduring effects of treatment. Seizure frequency and anxiety-like behaviors were serially assessed throughout the experimental paradigm. Treatment effects on the expression of key components of the epigenetic molecular machinery, the DNA methyltransferase enzymes, were assessed using quantitative polymerase chain reaction (qPCR).

Key findings: ESX treatment significantly reduced seizures in GAERS during the treatment phase, and this effect was maintained during the 12-week posttreatment phase (p < 0.05). Furthermore, the anxiety-like behaviors present in GAERS were reduced by ESX treatment (p < 0.05). Molecular analysis revealed that ESX treatment was associated with increased expression of DNA methyltransferase enzyme messenger RNA (mRNA) in cortex.

Significance: Chronic ESX treatment has disease-modifying effects in the GAERS model of GGE, with antiepileptogenic effects against absence seizures and mitigation of behavioral comorbidities. The cellular mechanism for these effects may involve epigenetic modifications.

Figure 1. Study timeline and ethosuximide dosing

(A) Littermates were separated to receive ESX or control treatment at 3 weeks of age, which persisted until 22 weeks. EEG recordings were made at 20 and 34 weeks of age, as well as 1 day prior to, and following, treatment cessation in GAERS. Behavioural assessments were conducted at 7 and 34 weeks in all rats. (B) Over the course of the study, the concentration of ESX in the drinking water presented to GAERS and NEC rats was altered depending on their drinking habits. This stabilized for each rat at around 10 weeks of age, and thereafter, all animals consistently received 300mg/kg/day. (C) GAERS weigh significantly less than NEC rats across the study, and ESX treatment also results in lower weights. (D) Fluid intake was equal between GAERS and NEC, but reduced in ESX-treated rats. Once treatment was removed (ie: 22 weeks), drinking volumes returned to control levels, and actually rebounded above control levels in GAERS.

Figure 2. Ethosuximide reduces seizures during the treatment period, and this effect endures following treatment cessation

(A) An example of a recorded SWD from the EEG. GAERS exposed to ESX (n=5) show significantly reduced % time in seizure (B) and the number of seizures recorded (C) during the 24 hour recording period both during and after the treatment period, compared to control-treated GAERS (n=6). No differences were observed in seizure duration throughout the study period (D). * indicates significant (p<0.05) post-hoc comparison with control-treated GAERS. Data represent group mean + SEM.

Figure 3. Suppression of seizures is accompanied by improved behavioural outcome in GAERS

Representative traces of the path travelled by an NEC rat (A) and a GAERS (B) in the open field – the black square represents the trial start position. Note the reduced total distance and entry into the central area of the maze of the highly anxious GAERS. At 7 weeks, prior to the onset of seizures, GAERS displayed an anxiogenic phenotype compared with NEC rats, as evidenced by (C) significantly reduced distance travelled in the open field and (D) significantly reduced entries into the central area, and this phenotype was not influenced by ESX treatment. At 34 weeks however, this anxiogenic phenotype was ameliorated in ESX-treated GAERS, compared to control treated GAERS. * indicates significant (p<0.05) post-hoc comparison with relevant NEC group. # indicates significant (p<0.05) post-hoc comparison with control-treated GAERS. Data represent group mean + SEM. Sample sizes: GAERS treated with ESX - n=5, with water - n=6; NEC treated with ESX - n=7, with water - n=4.

Figure 4. DNA Methyltransferase (DNMT) mRNA expression is differentially altered in GAERS

Expression of DNMT1 (A) and DNMT3A (B) are elevated in chronically treated ESX-treated GAERS, whereas DNMT3B is elevated in GAERS irrespective of drug treatment (C). No gene expression changes were induced by acute treatment with ESX (D) * indicates significant (p<0.05) post-hoc comparison with relevant NEC group. ^& indicates significant (p<0.05) post-hoc comparison with relevant control-treated group. Data represent group mean + SEM. Sample sizes as for Figure 3.