Diet composition and sterilization modifies intestinal microbiome diversity and burden of Theiler's virus infection-induced acute seizures.

OBJECTIVE: Brain infection with Theiler's murine encephalomyelitis virus (TMEV) in C57BL/6J mice can induce acquired epileptogenesis. Diet alters acute seizure incidence in TMEV-infected mice; yet it is unclear whether intestinal dysbiosis may also impact acute or chronic behavioral comorbidities. This study thus assessed the impact of diet formulation and sterilization on acute seizure presentation, gut microbiome composition, and epilepsy-related chronic behavioral comorbidities. METHODS: Baseline fecal samples were collected from male C57BL/6J mice (4- to 5-weeks-old; Jackson Labs) upon facility arrival. Mice were randomized to either autoclaved (AC) or irradiated diet (IR) (Prolab RMH 3000) or IR (Picolab 5053). Three days later, mice underwent intracerebral TMEV or phosphate-buffered saline (PBS) injection. Fecal samples were collected from a subset of mice at infection (Day 0) and Day 7 post-infection. Epilepsy-related working memory deficits and seizure threshold were assessed 6 weeks post-infection. Gut microbiome diversity was determined by 16S rRNA amplicon sequencing of fecal samples. RESULTS: TMEV-infected mice displayed acute handling-induced seizures, regardless of diet: 28 of 57 IR Picolab 5053 (49.1%), 30 of 41 IR Prolab RMH 3000 (73.2%), and 47 of 77 AC Prolab RMH 3000 (61%) mice displayed seizures. The number of observed seizures differed significantly by diet: IR Picolab 5053 diet-fed mice had 2.2 ± 2.8 seizures (mean ± standard deviation), IR Prolab RMH 3000 diet-fed mice had 3.5 ± 2.9 seizures, and AC Prolab RMH 3000 diet-fed mice had 4.4 ± 3.8 seizures during the 7-day monitoring period. Gut microbiome composition differed significantly in TMEV-infected mice fed the AC Prolab RMH 3000 diet, with measured differences in gram-positive bacteria. These mice also displayed worsened long-term working memory deficits. SIGNIFICANCE: Diet-induced differences in intestinal dysbiosis in the TMEV model are associated with marked changes in acute seizure presentation, symptomatic recovery, and onset of chronic behavioral comorbidities of epilepsy. Our study reveals a novel disease-modifying impact of dietary manipulation on intestinal bacterial species after TMEV-induced acute seizures.

Chronic evoked seizures in young pre-symptomatic APP/PS1 mice induce serotonin changes and accelerate onset of Alzheimer's disease-related neuropathology.

OBJECTIVE: Hyperexcitability is intimately linked to Alzheimer's disease (AD) pathology, but the precise timing and contributions of neuronal hyperexcitability to disease progression is unclear. Seizure induction in rodent AD models can uncover new therapeutic targets. Further, investigator-evoked seizures can directly establish how hyperexcitability and AD-associated risk factors influence neuropathological hallmarks and disease course at presymptomatic stages. METHODS: Corneal kindling is a well-characterized preclinical epilepsy model that allows for precise control of seizure history to pair to subsequent behavioral assessments. 2-3-month-old APP/PS1, PSEN2-N141I, and transgenic control male and female mice were thus sham or corneal kindled for 2 weeks. Seizure-induced changes in glia, serotonin pathway proteins, and amyloid ß levels in hippocampus and prefrontal cortex were quantified. RESULTS: APP/PS1 females were more susceptible to corneal kindling. However, regardless of sex, APP/PS1 mice experienced extensive seizure-induced mortality versus kindled Tg- controls. PSEN2-N141I mice were not negatively affected by corneal kindling. Mortality correlated with a marked downregulation of hippocampal tryptophan hydroxylase 2 and monoamine oxidase A protein expression versus controls; these changes were not detected in PSEN2-N141I mice. Kindled APP/PS1 mice also exhibited soluble amyloid ß upregulation and glial reactivity without plaque deposition. SIGNIFICANCE: Evoked convulsive seizures and neuronal hyperexcitability in pre-symptomatic APP/PS1 mice promoted premature mortality without pathological Aß plaque deposition, whereas PSEN2-N141I mice were unaffected. Disruptions in serotonin pathway metabolism in APP/PS1 mice was associated with increased glial reactivity without Aß plaque deposition, demonstrating that neuronal hyperexcitability in early AD causes pathological Aß overexpression and worsens long-term outcomes through a serotonin-related mechanism.

Diet composition and sterilization modifies intestinal microbiome diversity and burden of Theiler's virus infection-induced acute seizures.

Objective: Central nervous system infection with Theiler's murine encephalomyelitis virus (TMEV) in C57BL/6J mice can model acquired epileptogenesis. Diet alters the acute seizure incidence in TMEV-infected mice; yet it is unclear whether intestinal dysbiosis may also impact acute or chronic behavioral comorbidities. This study thus assessed the impact of diet sterilization in a specific pathogen-free vivarium on acute seizure presentation, the composition of the gut microbiome, and chronic behavioral comorbidities of epilepsy. Methods: Baseline fecal samples were collected from male C57BL/6J mice (4-5 weeks-old; Jackson Labs) upon arrival. Mice were randomized to either autoclaved (AC) or irradiated (IR) diet (Prolab RMH 3000 - UU diets) or IR (Picolab 5053 - UW IR diet). Mice then underwent intracerebral TMEV or PBS injection three days later. Fecal samples were collected from a subset of mice at infection (Day 0) and Day 7 post-infection. Epilepsy-related working memory deficits and seizure threshold were assessed 6 weeks post-infection. Gut microbiome diversity was determined by 16S rRNA amplicon sequencing of fecal samples. Results: TMEV-infected mice displayed acute handling-induced seizures, regardless of diet: 28/57 UW IR (49.1%), 30/41 UU IR (73.2%), and 47/77 UU AC (61%) mice displayed seizures. The number of observed seizures significantly differed: UW IR mice had 2.2±2.8 seizures (mean±standard deviation), UU IR mice had 3.5±2.9 seizures, and UU AC mice had 4.4±3.8 seizures during the 7-day monitoring period. The composition of the gut microbiome significantly differed in TMEV-infected mice fed the UU AC diet, with most measured differences occurring in Gram-positive bacteria. TMEV-infected mice fed the UU AC diet displayed worsened chronic working memory. Significance: Intestinal dysbiosis evokes stark differences in acute seizure presentation in the TMEV model and vastly influences the trajectory of post-TMEV infection-induced behavioral comorbidities of epilepsy. Our study reveals a novel disease-modifying contribution of intestinal bacterial species after TMEV-induced acute seizures.

Loss of normal Alzheimer's disease-associated Presenilin 2 function alters antiseizure medicine potency and tolerability in the 6-Hz focal seizure model.

Introduction: Patients with early-onset Alzheimer's disease (EOAD) experience seizures and subclinical epileptiform activity, which may accelerate cognitive and functional decline. Antiseizure medicines (ASMs) may be a tractable disease-modifying strategy; numerous ASMs are marketed with well-established safety. However, little information is available to guide ASM selection as few studies have rigorously quantified ASM potency and tolerability in traditional seizure models in rodents with EOAD-associated risk factors. Presenilin 2 (PSEN2) variants evoke EOAD, and these patients experience seizures. This study thus established the anticonvulsant profile of mechanistically distinct ASMs in the frontline 6-Hz limbic seizure test evoked in PSEN2-knockout (KO) mice to better inform seizure management in EOAD. Methods: The median effective dose (ED50) of prototype ASMs was quantified in the 6-Hz test in male and female PSEN2-KO and wild-type (WT) C57BL/6J mice (3-4 months old). Minimal motor impairment (MMI) was assessed to estimate a protective index (PI). Immunohistological detection of cFos established the extent to which 6-Hz stimulation activates discrete brain regions in KO vs. WT mice. Results: There were significant genotype-related differences in the potency and tolerability of several ASMs. Valproic acid and levetiracetam were significantly more potent in male KO than in WT mice. Additionally, high doses of valproic acid significantly worsened MMI in KO mice. Conversely, carbamazepine was significantly less potent in female KO vs. WT mice. In both male and female KO mice vs. WTs, perampanel and lamotrigine were equally potent. However, there were marked genotype-related shifts in PI of both carbamazepine and perampanel, with KO mice exhibiting less MMI at the highest doses tested. Gabapentin was ineffective against 6-Hz seizures in KO mice vs. WTs without MMI changes. Neuronal activation 90 min following 6-Hz stimulation was significantly increased in the posterior parietal association cortex overlying CA1 and in the piriform cortex of WT mice, while stimulation-induced increases in cFos immunoreactivity were absent in KO mice. Discussion: Acute ASM potency and tolerability in the high-throughput 6-Hz test may be significantly altered with loss of normal PSEN2 function. Seizures in discrete EOAD populations may benefit from precisely selected medicines optimized for primary ASM pharmacological mechanisms.

Frontline Sodium Channel-Blocking Antiseizure Medicine Use Promotes Future Onset of Drug-Resistant Chronic Seizures.

The mechanisms of treatment-resistant epilepsy remain unclear. We have previously shown that frontline administration of therapeutic doses of lamotrigine (LTG), which preferentially inhibits the fast-inactivation state of sodium channels, during corneal kindling of mice promotes cross-resistance to several other antiseizure medicines (ASMs). However, whether this phenomenon extends to monotherapy with ASMs that stabilize the slow inactivation state of sodium channels is unknown. Therefore, this study assessed whether lacosamide (LCM) monotherapy during corneal kindling would promote future development of drug-resistant focal seizures in mice. Male CF-1 mice (n = 40/group; 18-25 g) were administered an anticonvulsant dose of LCM (4.5 mg/kg, i.p.), LTG (8.5 mg/kg, i.p.), or vehicle (0.5% methylcellulose) twice daily for two weeks during kindling. A subset of mice (n = 10/group) were euthanized one day after kindling for immunohistochemical assessment of astrogliosis, neurogenesis, and neuropathology. The dose-related antiseizure efficacy of distinct ASMs, including LTG, LCM, carbamazepine, levetiracetam, gabapentin, perampanel, valproic acid, phenobarbital, and topiramate, was then assessed in the remaining kindled mice. Neither LCM nor LTG administration prevented kindling: 29/39 vehicle-exposed mice were kindled; 33/40 LTG-exposed mice were kindled; and 31/40 LCM-exposed mice were kindled. Mice administered LCM or LTG during kindling became resistant to escalating doses of LCM, LTG, and carbamazepine. Perampanel, valproic acid, and phenobarbital were less potent in LTG- and LCM-kindled mice, whereas levetiracetam and gabapentin retained equivalent potency across groups. Notable differences in reactive gliosis and neurogenesis were also appreciated. This study indicates that early, repeated administration of sodium channel-blocking ASMs, regardless of inactivation state preference, promotes pharmacoresistant chronic seizures. Inappropriate ASM monotherapy in newly diagnosed epilepsy may thus be one driver of future drug resistance, with resistance being highly ASM class specific.

Chronic evoked seizures in young pre-symptomatic APP/PS1 mice induce serotonin changes and accelerate onset on Alzheimer's disease-related neurpathology.

OBJECTIVE: People with early-onset Alzheimer's disease (AD) are at elevated seizure risk. Further, chronic seizures in pre-symptomatic stages may disrupt serotonin pathway-related protein expression, precipitating the onset of AD-related pathology and burden of neuropsychiatric comorbidities. METHODS: 2-3-month-old APP/PS1, PSEN2-N141I, and transgenic control mice were sham or corneal kindled for 2 weeks to model chronic seizures. Seizure-induced changes in glia, serotonin pathway proteins, and amyloid beta; levels in hippocampus and prefrontal cortex were quantified. RESULTS: APP/PS1 mice experienced worsened mortality versus kindled Tg- controls. APP/PS1 females were also more susceptible to chronic kindled seizures. These changes correlated with a marked downregulation of hippocampal tryptophan hydroxylase 2 and monoamine oxidase A protein expression compared to controls; these changes were not detected in PSEN2-N141I mice. Kindled APP/PS1 mice exhibited amyloid beta; overexpression and glial overactivity without plaque deposition. PSEN2 protein expression was AD model-dependent. SIGNIFICANCE: Seizures evoked in pre-symptomatic APP/PS1 mice promotes premature mortality in the absence of pathological amyloid deposition. Disruptions in serotonin pathway metabolism are associated with increased glial reactivity and PSEN2 downregulation without amyloid beta; deposition. This study provides the first direct evidence that seizures occurring prior to amyloid beta, plaque accumulation worsen disease burden in an AD genotype-specific manner.

Chronic seizures induce sex-specific cognitive deficits with loss of presenilin 2 function.

Patients with early-onset Alzheimer's disease (EOAD) are at elevated risk for seizures, including patients with presenilin 2 (PSEN2) variants. Like people with epilepsy, uncontrolled seizures may worsen cognitive function in AD. While the relationship between seizures and amyloid beta accumulation has been more thoroughly investigated, the role of other drivers of seizure susceptibility in EOAD remain relatively understudied. We therefore sought to define the impact of loss of normal PSEN2 function and chronic seizures on cognitive function in the aged brain. Male and female PSEN2 KO and age- and sex-matched wild-type (WT) mice were sham or corneal kindled beginning at 6-months-old. Kindled and sham-kindled mice were then challenged up to 6 weeks later in a battery of cognitive tests: non-habituated open field (OF), T-maze spontaneous alternation (TM), and Barnes maze (BM), followed by immunohistochemistry for markers of neuroinflammation and neuroplasticity. PSEN2 KO mice required significantly more stimulations to kindle (males: p < 0.02; females: p < 0.02) versus WT. Across a range of behavioral tests, the cognitive performance of kindled female PSEN2 KO mice was most significantly impaired versus age-matched WT females. Male BM performance was generally worsened by seizures (p = 0.038), but loss of PSEN2 function did not itself worsen cognitive performance. Conversely, kindled PSEN2 KO females made the most BM errors (p = 0.007). Chronic seizures also significantly altered expression of hippocampal neuroinflammation and neuroplasticity markers in a sex-specific manner. Chronic seizures may thus significantly worsen hippocampus-dependent cognitive deficits in aged female, but not male, PSEN2 KO mice. Our work suggests that untreated focal seizures may worsen cognitive burden with loss of normal PSEN2 function in a sex-related manner.

Cannabidiol reveals a disruptive strategy for 21st century epilepsy drug discovery.

Over 30 antiseizure medicines (ASMs) have been uncovered in a diversity of preclinical seizure and epilepsy models, with several critical inflection points in the 20th century fundamentally transforming ASM discovery. This commentary aims to review the historical relevance of cannabidiol's (CBD; Epidiolex) approval for epilepsy in the context of other ASMs brought to market. Further, we highlight how CBD's approval may represent an inflection point for 21st century ASM discovery. CBD is one of the main phytocannabinoids of Cannabis sativa. Unlike its related phytocannabinoid, Δ9-tetrahydrocannabinol, CBD does not exert any euphorigenic, tolerance, or withdrawal effects at anticonvulsant doses. CBD also possess marked anti-inflammatory effects, offering the tantalizing potential of a new pharmacological approach in epilepsy. For decades, hints of the anticonvulsant profile of CBD had been suggested with a small handful of studies in rodent seizure models, yet difficulties in formulation, compounded by the social and regulatory pressures related to medical use of cannabis plant-derived agents constrained any clinical implementation. Nonetheless, CBD possesses a broad antiseizure profile in preclinical seizure and epilepsy models, but the transformative impact of CBD'-s approval came because of studies in a rodent model of the orphan disease Dravet syndrome (DS). DS is a pediatric developmental epileptic encephalopathy with high mortality, frequent spontaneous recurrent seizures, and marked resistance to conventional ASMs, such as phenytoin and carbamazepine. CBD was approved for DS by the US Food and Drug Administration in 2018 after convincing efficacy was established in randomized, placebo-controlled trials in children. Because of the clinical approval of CBD as a novel, cannabis plantderived ASM for DS, CBD has revealed a new strategy in ASM discovery to reignite 21st century therapeutic development for epilepsy. In this commentary, we review the major preclinical and clinical milestones of the late 20th century that made CBD, a compound historically subjected to regulatory restrictions, a key driver of a new discovery strategy for epilepsy in the 21st century.

Soticlestat, a novel cholesterol 24-hydroxylase inhibitor, modifies acute seizure burden and chronic epilepsy-related behavioral deficits following Theiler's virus infection in mice.

Temporal lobe epilepsy is the most common form of acquired epilepsy and can arise due to multiple inciting events, including central nervous system (CNS) infection. CNS infection with the Theiler's murine encephalomyelitis virus (TMEV) in male C57Bl/6J mice leads to acute, drug-resistant handling-induced seizures. Cholesterol 24-hydroxylase (CH24H) is a brain-specific enzyme that converts cholesterol into 24S-hydroxycholesterol; the primary mechanism of cholesterol catabolism in the brain. The novel CH24H inhibitor, soticlestat (SOT; or TAK-935), demonstrates the potential to restore excitatory/inhibitory balance in multiple preclinical models of hyperexcitability. This study thus sought to characterize the anticonvulsant potential of SOT in the TMEV model. Treatment with SOT (30 mg/kg, p.o.; n = 30) 0-7 days post-infection (DPI) reduced overall seizure burden and severity. SOT administration significantly delayed onset of infection-induced Racine stage 5 seizures, from 8.6 ± 0.6 (VEH-treated) to 10.8 ± 0.8 (SOT-treated) observation sessions. Infected mice were then allowed 36 days treatment-free recovery before assessing impact of earlier drug administration on epilepsy-related cognitive and behavioral comorbidities, including a non-habituated open field (OF) task. Total OF distance traveled was significantly less in SOT-treated mice compared to VEH-treated mice, suggesting attenuated TMEV-induced spatial memory deficits, or reduced chronic hyperexcitability. Mice with history of SOT treatment also spent significantly more time and traveled farther in the OF center, indicative of reduced epilepsy-induced anxiety-like behavior. These studies suggest that SOT is a mechanistically novel agent for symptomatic seizure control. Moreover, acute SOT administration during an epileptogenic insult may attenuate the resulting long-term behavioral comorbidities of epilepsy.

A companion to the preclinical common data elements for phenotyping seizures and epilepsy in rodent models. A report of the TASK3-WG1C: Phenotyping working group of the ILAE/AES joint translational task force.

Epilepsy is a heterogeneous disorder characterized by spontaneous seizures and behavioral comorbidities. The underlying mechanisms of seizures and epilepsy across various syndromes lead to diverse clinical presentation and features. Similarly, animal models of epilepsy arise from numerous dissimilar inciting events. Preclinical seizure and epilepsy models can be evoked through many different protocols, leaving the phenotypic reporting subject to diverse interpretations. Serendipity can also play an outsized role in uncovering novel drivers of seizures or epilepsy, with some investigators even stumbling into epilepsy research because of a new genetic cross or unintentional drug effect. The heightened emphasis on rigor and reproducibility in preclinical research, including that which is conducted for epilepsy, underscores the need for standardized phenotyping strategies. To address this goal as part of the TASK3-WG1C Working Group of the International League Against Epilepsy (ILAE)/American Epilepsy Society (AES) Joint Translational Task Force, we developed a case report form (CRF) to describe the common data elements (CDEs) necessary for the phenotyping of seizure-like behaviors in rodents. This companion manuscript describes the use of the proposed CDEs and CRF for the visual, behavioral phenotyping of seizure-like behaviors. These phenotyping CDEs and accompanying CRF can be used in parallel with video-electroencephalography (EEG) studies or as a first visual screen to determine whether a model manifests seizure-like behaviors before utilizing more specialized diagnostic tests, like video-EEG. Systematic logging of seizure-like behaviors may help identify models that could benefit from more specialized diagnostic tests to determine whether these are epileptic seizures, such as video-EEG.

A companion to the preclinical common data elements for rodent models of pediatric acquired epilepsy: A report of the TASK3-WG1B, Pediatric and Genetic Models Working Group of the ILAE/AES Joint Translational Task Force.

Epilepsy syndromes during the early years of life may be attributed to an acquired insult, such as hypoxic-ischemic injury, infection, status epilepticus, or brain trauma. These conditions are frequently modeled in experimental rodents to delineate mechanisms of epileptogenesis and investigate novel therapeutic strategies. However, heterogeneity and subsequent lack of reproducibility of such models across laboratories is an ongoing challenge to maintain scientific rigor and knowledge advancement. To address this, as part of the TASK3-WG1B Working Group of the International League Against Epilepsy/American Epilepsy Society Joint Translational Task Force, we have developed a series of case report forms (CRFs) to describe common data elements for pediatric acquired epilepsy models in rodents. The "Rodent Models of Pediatric Acquired Epilepsy" Core CRF was designed to capture cohort-general information; while two Specific CRFs encompass physical induction models and chemical induction models, respectively. This companion manuscript describes the key elements of these models and why they are important to be considered and reported consistently. Together, these CRFs provide investigators with the tools to systematically record critical information regarding their chosen model of acquired epilepsy during early life, for improved standardization and transparency across laboratories. These outcomes will support the ultimate goal of such research; that is, to understand the childhood onset-specific biology of epileptogenesis after acquired insults, and translate this knowledge into therapeutics to improve pediatric patient outcomes and minimize the lifetime burden of epilepsy.

A companion to the preclinical common data elements for rodent genetic epilepsy models. A report of the TASK3-WG1B: Paediatric and genetic models working group of the ILAE/AES joint translational TASK force.

Rodent models of epilepsy remain the cornerstone of research into the mechanisms underlying genetic epilepsy. Reproducibility of experiments using these rodent models, occurring across a diversity of laboratories and commercial vendors, remains an issue impacting the cost-effectiveness and scientific rigor of the studies performed. Here, we present two case report forms (CRFs) describing common data elements (CDE) for genetic rodent models, developed by the TASK3-WG1B Working Group of the International League Against Epilepsy (ILAE)/American Epilepsy Society (AES) Joint Translational Task Force. The first CRF relates to genetic rodent models that have been engineered based on variants described in epilepsy patients. The second CRF encompasses both spontaneous and inbred rodent models. This companion piece describes the elements and discusses the important factors to consider before documenting each required element. These CRFs provide tools that allow investigators to more uniformly describe core experimental data on different genetic models across laboratories, with the aim of improving experimental reproducibility and thus translational impact of such studies.

Higher susceptibility to 6 Hz corneal kindling and lower responsiveness to antiseizure drugs in mouse models of Alzheimer's disease.

OBJECTIVE: Epileptic spikes and seizures seem present early in the disease process of Alzheimer's disease (AD). However, it is unclear how soluble and insoluble amyloid beta (Aß) and tau proteins affect seizure development in vivo. We aim to contribute to this field by assessing the vulnerability to 6 Hz corneal kindling of young female mice from two well-characterized transgenic AD models and by testing their responsiveness to selected antiseizure drugs (ASDs). METHODS: We used 7-week-old triple transgenic (3xTg) mice that have both amyloid and tau mutations, and amyloid precursor protein Swedish/presenillin 1 dE9 (APP/PS1) mice, bearing only amyloid-related mutations. We assessed the absence of plaques via immunohistochemistry and analyzed the concentrations of both soluble and insoluble forms of Aß1-42 and total tau (t-tau) in brain hippocampal and prefrontal cortical tissue. Seven-week-old mice of the different genotypes were subjected to the 6 Hz corneal kindling model. After kindling acquisition, we tested the anticonvulsant effects of three marketed ASDs (levetiracetam, brivaracetam, and lamotrigine) in fully kindled mice. RESULTS: No Aß plaques were present in either genotype. Soluble Aß1-42 levels were increased in both AD genotypes, whereas insoluble Aß1-42 concentrations were only elevated in APP/PS1 mice compared with their respective controls. Soluble and insoluble forms of t-tau were increased in 3xTg mice only. 3xTg and APP/PS1 mice displayed more severe seizures induced by 6 Hz corneal kindling from the first stimulation onward and were more rapidly kindled compared with control mice. In fully kindled AD mice, ASDs had less-pronounced anticonvulsive effects compared with controls. SIGNIFICANCE: Mutations increasing Aß only or both Aß and tau in the brain enhance susceptibility for seizures and kindling in mice. The effect of ASDs on seizures measured by the Racine scale is less pronounced in both investigated AD models and suggests that seizures of young AD mice are more difficult to treat.

Can Old Animals Reveal New Targets? The Aging and Degenerating Brain as a New Precision Medicine Opportunity for Epilepsy.

Older people represent the fastest growing group with epilepsy diagnosis. For example, cerebrovascular disease may underlie roughly 30-50% of epilepsy in older adults and seizures are also an underrecognized comorbidity of Alzheimer's disease (AD). As a result, up to 10% of nursing home residents may take antiseizure medicines (ASMs). Despite the greater incidence of epilepsy in older individuals and increased risk of comorbid seizures in people with AD, aged animals with seizures are strikingly underrepresented in epilepsy drug discovery practice. Increased integration of aged animals into preclinical epilepsy drug discovery could better inform the potential tolerability and pharmacokinetic interactions in aged individuals as the global population becomes increasingly older. Quite simply, the ASMs on the market today were brought forth based on efficacy in young adult, neurologically intact rodents; preclinical information concerning the efficacy and safety of promising ASMs is not routinely evaluated in aged animals. Integrating aged animals more often into basic epilepsy research may also uncover novel treatments for hyperexcitability. For example, cannabidiol and fenfluramine demonstrated clear efficacy in syndrome-specific pediatric models that led to a paradigm shift in the perceived value of pediatric models for ASM discovery practice; aged rodents with seizures or rodents with aging-related neuropathology represent an untapped resource that could similarly change epilepsy drug discovery. This review, therefore, summarizes how aged rodent models have thus far been used for epilepsy research, what studies have been conducted to assess ASM efficacy in aged rodent seizure and epilepsy models, and lastly to identify remaining gaps to engage aging-related neurological disease models for ASM discovery, which may simultaneously reveal novel mechanisms associated with epilepsy.

Reductions in Hydrogen Sulfide and Changes in Mitochondrial Quality Control Proteins Are Evident in the Early Phases of the Corneally Kindled Mouse Model of Epilepsy.

Epilepsy is a heterogenous neurological disorder characterized by recurrent unprovoked seizures, mitochondrial stress, and neurodegeneration. Hydrogen sulfide (H2S) is a gasotransmitter that promotes mitochondrial function and biogenesis, elicits neuromodulation and neuroprotection, and may acutely suppress seizures. A major gap in knowledge remains in understanding the role of mitochondrial dysfunction and progressive changes in H2S levels following acute seizures or during epileptogenesis. We thus sought to quantify changes in H2S and its methylated metabolite (MeSH) via LC-MS/MS following acute maximal electroshock and 6 Hz 44 mA seizures in mice, as well as in the early phases of the corneally kindled mouse model of chronic seizures. Plasma H2S was acutely reduced after a maximal electroshock seizure. H2S or MeSH levels and expressions of related genes in whole brain homogenates from corneally kindled mice were not altered. However, plasma H2S levels were significantly lower during kindling, but not after established kindling. Moreover, we demonstrated a time-dependent increase in expression of mitochondrial membrane integrity-related proteins, OPA1, MFN2, Drp1, and Mff during kindling, which did not correlate with changes in gene expression. Taken together, short-term reductions in plasma H2S could be a novel biomarker for seizures. Future studies should further define the role of H2S and mitochondrial stress in epilepsy.

Continuous seizure emergency evoked in mice with pharmacological, electrographic, and pathological features distinct from status epilepticus.

OBJECTIVES: Benzodiazepines are the standard of care for the management of sustained seizure emergencies, including status epilepticus (SE) and seizure clusters. Seizure clusters are a variably defined seizure emergency wherein a patient has multiple seizures above a baseline rate, with intervening periods of recovery, distinguishing clusters from SE. Although these seizure emergencies are phenotypically distinct, the precise pathophysiological and mechanistic differences between SE and seizure clusters are understudied. Emergency-specific preclinical models may differentiate the behavioral and pathological mechanisms that are acutely associated with seizure emergencies and seizure termination to better manage these events. METHODS: Herein we characterize a novel model of sustained seizure emergency induced in CF-1 mice through the combined administration of high-dose phenytoin (PHT; 50 mg/kg, i.p.) and pentylenetetrazol (PTZ; 100 mg/kg, s.c.). RESULTS: We presently describe a mouse model of sustained seizure emergency that is pathologically, pharmacologically, and behaviorally distinct from SE. Acute administration of PHT 1 h prior to PTZ led to significantly more mice with unremitting continuous seizure activity (CSA; 73.4%) vs vehicle-pretreated mice (13.8%; p < .0001). CSA was sensitive to lorazepam and valproic acid when administered at seizure onset and 30 minutes later. Carbamazepine worsened seizure control and post-CSA survival. Mice in CSA exhibited electroencephalography (EEG) patterns distinct from kainic acid-induced SE and PTZ alone, clearly differentiating CSA from SE and PTZ-induced myoclonic seizures. Neuropathological assessment by Fluoro-Jade C staining of brains collected 24 h post-CSA revealed no neurodegeneration in any mouse that underwent CSA, whereas there was widespread neuronal death in brains from KA-SE mice. Finally, immunohistochemistry revealed acute seizure-induced astrogliosis (glial fibrillary acid protein; GFAP) in hippocampal structures, whereas hippocampal neuronal nuclei (NeuN) protein expression was only reduced in KA-SE mice. SIGNIFICANCE: We present a novel mouse model on which to further elucidate the mechanistic differences between sustained seizure emergencies (ie, SE and seizure clusters) to improve clinical interventions and define mechanisms of seizure termination.

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.

Alzheimer's Disease and Epilepsy: A Perspective on the Opportunities for Overlapping Therapeutic Innovation.

Early-onset Alzheimer's disease (AD) is associated with variants in amyloid precursor protein (APP) and presenilin (PSEN) 1 and 2. It is increasingly recognized that patients with AD experience undiagnosed focal seizures. These AD patients with reported seizures may have worsened disease trajectory. Seizures in epilepsy can also lead to cognitive deficits, neuroinflammation, and neurodegeneration. Epilepsy is roughly three times more common in individuals aged 65 and older. Due to the numerous available antiseizure drugs (ASDs), treatment of seizures has been proposed to reduce the burden of AD. More work is needed to establish the functional impact of seizures in AD to determine whether ASDs could be a rational therapeutic strategy. The efficacy of ASDs in aged animals is not routinely studied, despite the fact that the elderly represents the fastest growing demographic with epilepsy. This leaves a particular gap in understanding the discrete pathophysiological overlap between hyperexcitability and aging, and AD more specifically. Most of our preclinical knowledge of hyperexcitability in AD has come from mouse models that overexpress APP. While these studies have been invaluable, other drivers underlie AD, e.g. PSEN2. A diversity of animal models should be more frequently integrated into the study of hyperexcitability in AD, which could be particularly beneficial to identify novel therapies. Specifically, AD-associated risk genes, in particular PSENs, altogether represent underexplored contributors to hyperexcitability. This review assesses the available studies of ASDs administration in clinical AD populations and preclinical studies with AD-associated models and offers a perspective on the opportunities for further therapeutic innovation.

A multiorganism pipeline for antiseizure drug discovery: Identification of chlorothymol as a novel γ-aminobutyric acidergic anticonvulsant.

OBJECTIVE: Current medicines are ineffective in approximately one-third of people with epilepsy. Therefore, new antiseizure drugs are urgently needed to address this problem of pharmacoresistance. However, traditional rodent seizure and epilepsy models are poorly suited to high-throughput compound screening. Furthermore, testing in a single species increases the chance that therapeutic compounds act on molecular targets that may not be conserved in humans. To address these issues, we developed a pipeline approach using four different organisms. METHODS: We sequentially employed compound library screening in the zebrafish, Danio rerio, chemical genetics in the worm, Caenorhabditis elegans, electrophysiological analysis in mouse and human brain slices, and preclinical validation in mouse seizure models to identify novel antiseizure drugs and their molecular mechanism of action. RESULTS: Initially, a library of 1690 compounds was screened in an acute pentylenetetrazol seizure model using D rerio. From this screen, the compound chlorothymol was identified as an effective anticonvulsant not only in fish, but also in worms. A subsequent genetic screen in C elegans revealed the molecular target of chlorothymol to be LGC-37, a worm γ-aminobutyric acid type A (GABAA ) receptor subunit. This GABAergic effect was confirmed using in vitro brain slice preparations from both mice and humans, as chlorothymol was shown to enhance tonic and phasic inhibition and this action was reversed by the GABAA receptor antagonist, bicuculline. Finally, chlorothymol exhibited in vivo anticonvulsant efficacy in several mouse seizure assays, including the 6-Hz 44-mA model of pharmacoresistant seizures. SIGNIFICANCE: These findings establish a multiorganism approach that can identify compounds with evolutionarily conserved molecular targets and translational potential, and so may be useful in drug discovery for epilepsy and possibly other conditions.