Development of an antiepileptogenesis drug screening platform: Effects of everolimus and phenobarbital.

OBJECTIVE: The kainic acid (KA)-induced status epilepticus (SE) model in rats is a well-defined model of epileptogenesis. This model closely recapitulates many of the clinical and pathological characteristics of human temporal lobe epilepsy (TLE) that arise following SE or another neurological insult. Spontaneous recurrent seizures (SRS) in TLE can present after a latent period following a neurological insult (traumatic brain injury, SE event, viral infection, etc.). Moreover, this model is suitable for preclinical studies to evaluate the long-term process of epileptogenesis and screen putative disease-modifying/antiepileptogenic agents. The burden of human TLE is highly variable, similar to the post-KA SE rat model. In this regard, this model may have broad translational relevance. This report thus details the pharmacological characterization and methodological refinement of a moderate-throughput drug screening program using the post-KA-induced SE model of epileptogenesis in male Sprague Dawley rats to identify potential agents that may prevent or modify the burden of SRS. Specifically, we sought to demonstrate whether our protocol could prevent the development of SRS or lead to a reduced frequency/severity of SRS. METHODS: Rats were administered either everolimus (2-3 mg/kg po) beginning 1, 2, or 24 h after SE onset, or phenobarbital (60 mg/kg ip) beginning 1 h after SE onset. All treatments were administered once/day for 5-7 days. Rats in all studies (n = 12/treatment dose/study) were then monitored intermittently by video-electroencephalography (2 weeks on, 2 weeks off, 2 weeks on epochs) to determine latency to onset of SRS and disease burden. RESULTS: Although no adverse side effects were observed in our studies, no treatment significantly modified disease or prevented the presentation of SRS by 6 weeks after SE onset. SIGNIFICANCE: Neither phenobarbital nor everolimus administered at several time points after SE onset prevented the development of SRS. Nonetheless, we demonstrate a practical and moderate-throughput screen for potential antiepileptogenic agents in a rat model of TLE.

Diurnal burden of spontaneous seizures in early epileptogenesis in the post-kainic acid rat model of epilepsy.

Patients with epilepsy can experience diurnal seizure patterns. However, few studies in rodent models of temporal lobe epilepsy (TLE) routinely quantify the diurnal pattern of spontaneous recurrent seizures (SRS), and those that have conducted such assessments used small groups. This study thus aimed to define whether there was a diurnal pattern of SRS in the early phases of epileptogenesis in a large cohort (n = 40) of post-kainic acid (KA)-induced status epilepticus (SE) male Sprague Dawley rats. Rats were monitored by continuous 24/7 video-EEG in two-week epochs up to 6 weeks post-KA-induced SE. The total number of SRS by 6 weeks post-SE correlated to body weight at the time of SE insult (R2  = .1465, P = .0143). The total number of spontaneous behavioral and electrographic seizures, seizure severity, and seizure burden was recorded during lights ON (light) or lights OFF (dark) phases. All measures significantly increased with time post-SE; we detected significantly more seizures during the lights OFF phase of the post-SE monitoring periods. Moreover, a subset of rats demonstrated marked seizure preference in the lights OFF phase. Our study confirms that a diurnal pattern of SRS is variably detectable in early epileptogenesis in this model of TLE.

Antiseizure drug efficacy and tolerability in established and novel drug discovery seizure models in outbred vs inbred mice.

OBJECTIVE: Initial identification of new investigational drugs for the treatment of epilepsy is commonly conducted in well-established mouse acute and chronic seizure models: for example, maximal electroshock (MES), 6 Hz, and corneal kindling. Comparison of the median effective dose (ED50) of approved antiseizure drugs (ASDs) vs investigational agents in these models provides evidence of their potential for clinical efficacy. Inbred and outbred mouse strains exhibit differential seizure susceptibility. However, few comparisons exist of the ED50 or median behaviorally impairing dose (TD50) of prototype ASDs in these models in inbred C57Bl/6 vs outbred CF-1 mice, both of which are often used for ASD discovery. METHODS: We defined the strain-related ED50s and TD50s of several mechanistically distinct ASDs across established acute seizure models (MES, 6 Hz, and corneal-kindled mouse). We further quantified the strain-related effect of the MES ED50 of each ASD on gross behavior in a locomotor activity assay. Finally, we describe a novel pharmacoresistant corneal-kindling protocol that is suitable for moderate-throughput ASD screening and demonstrates highly differentiated ASD sensitivity. RESULTS: We report significant strain-related differences in the MES ED50 of valproic acid (CF-1 ED50: 90 mg/kg [95% confidence interval (CI) 165-214] vs C57Bl/6: 276 mg/kg [226-366]), as well as significant differences in the ED50 of levetiracetam in the pharmacoresistant 6 Hz test (CF-1: 22.5 mg/kg [14.7-30.2] vs C57Bl/6: >500 mg/kg [CI not defined]). There were no differences in the calculated TD50 of these ASDs between strains. Furthermore, the MES ED50 of phenobarbital significantly enhanced locomotor activity of outbred CF-1, but not C57Bl/6, mice. SIGNIFICANCE: Altogether, this study provides strain-related information to differentiate investigational agents from ASD standards-of-care in commonly employed preclinical discovery models and describes a novel kindled seizure model to further explore the mechanisms of drug-resistant epilepsy.

Loss of presenilin 2 age-dependently alters susceptibility to acute seizures and kindling acquisition.

Patients with Alzheimer's disease (AD) experience seizures at higher rates than the general population of that age, suggesting an underexplored role of hyperexcitability in AD. Genetic variants in presenilin (PSEN) 1 and 2 genes lead to autosomal dominant early-onset AD (ADAD); patients with PSEN gene variants also report seizures. Pharmacological control of seizures in AD may be disease-modifying. Preclinical efficacy of FDA-approved antiseizure drugs (ASDs) is well defined in young adult rodents; however, the efficacy of ASDs in aged rodents with chronic seizures is less clear. The mechanism by which ADAD genes lead to AD remains unclear, and even less studied is the pathogenesis of epilepsy in AD. PSEN variants generally all result in a biochemical loss of function (De Strooper, 2007). We herein determined whether well-established models of acute and chronic seizure could be used to explore the relationship between AD genes and seizures through investigating whether loss of normal PSEN2 function age-dependently influenced susceptibility to seizures and/or corneal kindling acquisition. PSEN2 knockout (KO) and age-matched wild-type (WT) mice were screened from 2- to 10-months-old to establish age-dependent focal seizure threshold. Additionally, PSEN2 KO and WT mice aged 2- and 8-months-old underwent corneal kindling such that mice were aged 3- and 9-months old at the beginning of ASD efficacy testing. We then defined the dose-dependent efficacy of mechanistically distinct ASDs on kindled seizures of young versus aged mice to better understand the applicability of corneal kindling to real-world use for geriatric patients. PSEN2 KO mice demonstrated early-life reductions in seizure threshold. However, kindling acquisition was delayed in 2-month-old PSEN2 KO versus WT mice. Young male WT mice took 24.3 ± 1.3 (S.E.M.) stimulations to achieve kindling criterion, whereas age-matched PSEN2 KO male mice took 41.2 ± 1.1 stimulations (p < .0001). The rate of kindling acquisition of 8-month-old mice was no longer different from WT. This study demonstrates that loss of normal PSEN2 function is associated with age-dependent changes in the in vivo susceptibility to acute seizures and kindling. Loss of normal PSEN2 function may be an underexplored molecular contributor to seizures. The use of validated models of chronic seizures in aged rodents may uncover age-related changes in susceptibility to epileptogenesis and/or ASD efficacy in mice with AD-associated genotypes, which may benefit the management of seizures in AD.

Lamotrigine-resistant corneal-kindled mice: A model of pharmacoresistant partial epilepsy for moderate-throughput drug discovery.

OBJECTIVE: Despite numerous treatments for epilepsy, over 30% of patients remain resistant to available antiseizure drugs (ASDs). Thus, there is a strong need for more effective ASDs for these individuals. Early ASD discovery has historically relied on acute in vivo seizure models (maximal electroshock, subcutaneous pentylenetetrazol, 6 Hz), which lack the pathophysiology that defines chronic epilepsy. Etiologically relevant rodent models of pharmacoresistant epilepsy exist (eg, phenytoin (PHT)- and lamotrigine (LTG)-resistant amygdala-kindled rat and focal kainic acid mouse), but these models are resource- and labor-intensive and thus unsuitable for frontline ASD discovery. METHODS: We adapted the LTG-resistant amygdala-kindled rat protocol to the 60 Hz corneal-kindled mouse (CKM) to develop a medium-throughput model of pharmacoresistant chronic seizures. Male CF-1 mice were administered either vehicle (VEH; 0.5% methylcellulose) or LTG (8.5 mg/kg, ip) 30 minutes prior to each twice-daily corneal stimulation until mice achieved kindling criterion. Prototype ASDs were then evaluated in fully kindled mice. Compounds with specific mechanisms of action of interest to epilepsy (fluoxetine, minocycline, and celecoxib) were also evaluated. RESULTS: LTG did not modify kindling acquisition. A challenge dose of 17 mg/kg (ip) LTG did not block the secondarily generalized kindled seizure in LTG-kindled mice (mean seizure score [MSS] ± standard error of the mean: 5.67 ± 0.14), whereas VEH-treated mice were sensitive (MSS: 2.25 ± 0.30); confirming LTG-resistance. LTG-resistant CKM were also resistant to carbamazepine, retigabine, and valproic acid at doses that significantly reduced MSS in VEH-treated kindled mice. Fluoxetine, minocycline, and celecoxib were ineffective at the doses tested in either kindled cohort. Finally, the behavioral phenotype of LTG-resistant CKM was also characterized. CKM demonstrated exacerbated hyperexcitability and increased anxiety-like behavior in an open field relative to sham-kindled mice regardless of LTG sensitivity. SIGNIFICANCE: The pharmacoresistant LTG-resistant CKM provides an etiologically relevant moderate-throughput platform that is suitable for early compound discovery before advancing to more resource-intensive models of epilepsy.