Screening of prototype antiseizure and anti-inflammatory compounds in the Theiler's murine encephalomyelitis virus model of epilepsy.

OBJECTIVE: Infection with Theiler's murine encephalomyelitis virus (TMEV) in C57Bl/6J mice results in handling-induced seizures and is useful for evaluating compounds effective against infection-induced seizures. However, to date only a few compounds have been evaluated in this model, and a comprehensive study of antiseizure medications (ASMs) has not yet been performed. Furthermore, as the TMEV infection produces marked neuroinflammation, an evaluation of prototype anti-inflammatory compounds is needed as well. METHODS: Male C57Bl/6J mice were inoculated with TMEV (day 0) followed by daily administrations of test compounds (day 3-7) and subsequent handling sessions (day 3-7). Doses of ASMs, comprising several mechanistic classes, were selected based on previously published data demonstrating the effect of these compounds in reducing seizures in the 6 Hz model of pharmacoresistant seizures. Doses of anti-inflammatory compounds, comprising several mechanistic classes, were selected based on published evidence of reduction of inflammation or inflammation-related endpoints. RESULTS: Several prototype ASMs reduced acute seizures following TMEV infection: lacosamide, phenytoin, ezogabine, phenobarbital, tiagabine, gabapentin, levetiracetam, topiramate, and sodium valproate. Of these, phenobarbital and sodium valproate had the greatest effect (>95% seizure burden reduction). Prototype anti-inflammatory drugs celecoxib, dexamethasone, and prednisone also moderately reduced seizure burden. SIGNIFICANCE: The TMEV model is utilized by the Epilepsy Therapy Screening Program (ETSP) as a tool for evaluation of novel compounds. Compounds reducing seizures in the TMEV comprise distinct mechanistic classes, some with mechanisms of action that extend beyond traditional ASMs.

Music-Enhanced Analgesia and Antiseizure Activities in Animal Models of Pain and Epilepsy: Toward Preclinical Studies Supporting Development of Digital Therapeutics and Their Combinations With Pharmaceutical Drugs.

Digital therapeutics (software as a medical device) and mobile health (mHealth) technologies offer a means to deliver behavioral, psychosocial, disease self-management and music-based interventions to improve therapy outcomes for chronic diseases, including pain and epilepsy. To explore new translational opportunities in developing digital therapeutics for neurological disorders, and their integration with pharmacotherapies, we examined analgesic and antiseizure effects of specific musical compositions in mouse models of pain and epilepsy. The music playlist was created based on the modular progression of Mozart compositions for which reduction of seizures and epileptiform discharges were previously reported in people with epilepsy. Our results indicated that music-treated mice exhibited significant analgesia and reduction of paw edema in the carrageenan model of inflammatory pain. Among analgesic drugs tested (ibuprofen, cannabidiol (CBD), levetiracetam, and the galanin analog NAX 5055), music intervention significantly decreased paw withdrawal latency difference in ibuprofen-treated mice and reduced paw edema in combination with CBD or NAX 5055. To the best of our knowledge, this is the first animal study on music-enhanced antinociceptive activity of analgesic drugs. In the plantar incision model of surgical pain, music-pretreated mice had significant reduction of mechanical allodynia. In the corneal kindling model of epilepsy, the cumulative seizure burden following kindling acquisition was lower in animals exposed to music. The music-treated group also exhibited significantly improved survival, warranting further research on music interventions for preventing Sudden Unexpected Death in Epilepsy (SUDEP). We propose a working model of how musical elements such as rhythm, sequences, phrases and punctuation found in K.448 and K.545 may exert responses via parasympathetic nervous system and the hypothalamic-pituitary-adrenal (HPA) axis. Based on our findings, we discuss: (1) how enriched environment (EE) can serve as a preclinical surrogate for testing combinations of non-pharmacological modalities and drugs for the treatment of pain and other chronic diseases, and (2) a new paradigm for preclinical and clinical development of therapies leading to drug-device combination products for neurological disorders, depression and cancer. In summary, our present results encourage translational research on integrating non-pharmacological and pharmacological interventions for pain and epilepsy using digital therapeutics.

Preclinical Comparison of Mechanistically Different Antiseizure, Antinociceptive, and/or Antidepressant Drugs in a Battery of Rodent Models of Nociceptive and Neuropathic Pain.

The series of experiments herein evaluated prototype drugs representing different mechanisms of antiseizure, antinociceptive or antidepressant action in a battery of preclinical pain models in adult male CF#1 mice (formalin, writhing, and tail flick) and Sprague Dawley rats partial sciatic nerve ligation (PSNL). In the formalin assay, phenytoin (PHT, 6 mg/kg), sodium valproate (VPA, 300 mg/kg), amitriptyline (AMI, 7.5 and 15 mg/kg), gabapentin (GBP, 30 and 70 mg/kg), tiagabine (TGB, 5 and 15 mg/kg), and acetominophen (APAP, 250 and 500 mg/kg) reduced both phases of the formalin response to ≤ 25% of vehicle-treated mice. In the acetic acid induced writhing assay, VPA (300 mg/kg), ethosuximide (ETX, 300 mg/kg), morphine (MOR, 5 & 10 mg/kg), GBP (10, 30, and 60 mg/kg), TGB (15 mg/kg), levetiracetam (LEV, 300 mg/kg), felbamate (FBM, 80 mg/kg) and APAP (250 mg/kg) reduced writhing to ≤ 25% of vehicle-treated mice. In the tail flick test, MOR (1.25-5 mg/kg), AMI (15 mg/kg) and TGB (5 mg/kg) demonstrated significant antinociceptive effects. Finally, carbamazepine (CBZ, 20 and 50 mg/kg), VPA, MOR (2 and 4 mg/kg), AMI (12 mg/kg), TPM (100 mg/kg), lamotrigine (LTG, 40 mg/kg), GBP (60 mg/kg), TGB (15 mg/kg), FBM (35 mg/kg), and APAP (250 mg/kg) were effective in the PSNL model. Thus, TGB was the only prototype compound with significant analgesic effects in each of the four models, while AMI, GBP, APAP, and MOR each improved three of the four pain phenotypes. This study highlights the importance evaluating novel targets in a variety of pain phenotypes.

Validation of a Preclinical Drug Screening Platform for Pharmacoresistant Epilepsy.

The successful identification of promising investigational therapies for the treatment of epilepsy can be credited to the use of numerous animal models of seizure and epilepsy for over 80 years. In this time, the maximal electroshock test in mice and rats, the subcutaneous pentylenetetrazol test in mice and rats, and more recently the 6 Hz assay in mice, have been utilized as primary models of electrically or chemically-evoked seizures in neurologically intact rodents. In addition, rodent kindling models, in which chronic network hyperexcitability has developed, have been used to identify new agents. It is clear that this traditional screening approach has greatly expanded the number of marketed drugs available to manage the symptomatic seizures associated with epilepsy. In spite of the numerous antiseizure drugs (ASDs) on the market today, the fact remains that nearly 30% of patients are resistant to these currently available medications. To address this unmet medical need, the National Institute of Neurological Disorders and Stroke (NINDS) Epilepsy Therapy Screening Program (ETSP) revised its approach to the early evaluation of investigational agents for the treatment of epilepsy in 2015 to include a focus on preclinical approaches to model pharmacoresistant seizures. This present report highlights the in vivo and in vitro findings associated with the initial pharmacological validation of this testing approach using a number of mechanistically diverse, commercially available antiseizure drugs, as well as several probe compounds that are of potential mechanistic interest to the clinical management of epilepsy.

Acute treatment with minocycline, but not valproic acid, improves long-term behavioral outcomes in the Theiler's virus model of temporal lobe epilepsy.

OBJECTIVE: Infection with Theiler's murine encephalomyelitis virus (TMEV) in C57Bl/6J mice induces acute seizures and development of spontaneous recurrent seizures and behavioral comorbidities weeks later. The present studies sought to determine whether acute therapeutic intervention with an anti-inflammatory-based approach could prevent or modify development of TMEV-induced long-term behavioral comorbidities. Valproic acid (VPA), in addition to its prototypical anticonvulsant properties, inhibits histone deacetylase (HDAC) activity, which may alter expression of the inflammasome. Minocycline (MIN) has previously demonstrated an antiseizure effect in the TMEV model via direct anti-inflammatory mechanisms, but the long-term effect of MIN treatment on the development of chronic behavioral comorbidities is unknown. METHODS: Mice infected with TMEV were acutely administered MIN (50 mg/kg, b.i.d. and q.d.) or VPA (100 mg/kg, q.d.) during the 7-day viral infection period. Animals were evaluated for acute seizure severity and subsequent development of chronic behavioral comorbidities and seizure threshold. RESULTS: Administration of VPA reduced the proportion of mice with seizures, delayed onset of symptomatic seizures, and reduced seizure burden during the acute infection. This was in contrast to the effects of administration of once-daily MIN, which did not affect the proportion of mice with seizures or delay onset of acute symptomatic seizures. However, VPA-treated mice were no different from vehicle (VEH)-treated mice in long-term behavioral outcomes, including open field activity and seizure threshold. Once-daily MIN treatment, despite no effect on the maximum observed Racine stage seizure severity, was associated with improved long-term behavioral outcomes and normalized seizure threshold. SIGNIFICANCE: Acute seizure control alone is insufficient to modify chronic disease comorbidities in the TMEV model. This work further supports the role of an inflammatory response in the development of chronic behavioral comorbidities and further highlights the utility of this platform for the development of mechanistically novel pharmacotherapies for epilepsy.

Acute cognitive impact of antiseizure drugs in naive rodents and corneal-kindled mice.

OBJECTIVE: Some antiseizure drugs (ASDs) are associated with cognitive liability in patients with epilepsy, thus ASDs without this risk would be preferred. Little comparative pharmacology exists with ASDs in preclinical models of cognition. Few pharmacologic studies exist on the acute effects in rodents with chronic seizures. Predicting risk for cognitive impact with preclinical models may supply valuable ASD differentiation data. METHODS: ASDs (phenytoin [PHT]; carbamazepine [CBZ]; valproic acid [VPA]; lamotrigine [LTG]; phenobarbital [PB]; tiagabine [TGB]; retigabine [RTG]; topiramate [TPM]; and levetiracetam [LEV]) were administered equivalent to maximal electroshock median effective dose ([ED50]; mice, rats), or median dose necessary to elicit minimal motor impairment (median toxic dose [TD50]; rats). Cognition models with naive adult rodents were novel object/place recognition (NOPR) task with CF-1 mice, and Morris water maze (MWM) with Sprague-Dawley rats. Selected ASDs were also administered to rats prior to testing in an open field. The effect of chronic seizures and ASD administration on cognitive performance in NOPR was also determined with corneal-kindled mice. Mice that did not achieve kindling criterion (partially kindled) were included to examine the effect of electrical stimulation on cognitive performance. Sham-kindled and age-matched mice were also tested. RESULTS: No ASD (ED50) affected latency to locate the MWM platform; TD50 of PB, RTG, TPM, and VPA reduced this latency. In naive mice, CBZ and VPA (ED50) reduced time with the novel object. Of interest, no ASD (ED50) affected performance of fully kindled mice in NOPR, whereas CBZ and LEV improved cognitive performance of partially kindled mice. SIGNIFICANCE: Standardized approaches to the preclinical evaluation of an ASD's potential cognitive impact are needed to inform drug development. This study demonstrated acute, dose- and model-dependent effects of therapeutically relevant doses of ASDs on cognitive performance of naive mice and rats, and corneal-kindled mice. This study highlights the challenge of predicting clinical adverse effects with preclinical models.

Evaluating an etiologically relevant platform for therapy development for temporal lobe epilepsy: effects of carbamazepine and valproic acid on acute seizures and chronic behavioral comorbidities in the Theiler's murine encephalomyelitis virus mouse model.

Central nervous system infections can underlie the development of epilepsy, and Theiler's murine encephalomyelitis virus (TMEV) infection in C57BL/6J mice provides a novel model of infection-induced epilepsy. Approximately 50-65% of infected mice develop acute, handling-induced seizures during the infection. Brains display acute neuropathology, and a high number of mice develop spontaneous, recurrent seizures and behavioral comorbidities weeks later. This study characterized the utility of this model for drug testing by assessing whether antiseizure drug treatment during the acute infection period attenuates handling-induced seizures, and whether such treatment modifies associated comorbidities. Male C57BL/6J mice infected with TMEV received twice-daily valproic acid (VPA; 200 mg/kg), carbamazepine (CBZ; 20 mg/kg), or vehicle during the infection (days 0-7). Mice were assessed twice daily during the infection period for handling-induced seizures. Relative to vehicle-treated mice, more CBZ-treated mice presented with acute seizures; VPA conferred no change. In mice displaying seizures, VPA, but not CBZ, reduced seizure burden. Animals were then randomly assigned to acute and long-term follow-up. VPA was associated with significant elevations in acute (day 8) glial fibrillary acidic protein (astrocytes) immunoreactivity, but did not affect NeuN (neurons) immunoreactivity. Additionally, VPA-treated mice showed improved motor performance 15 days postinfection (DPI). At 36 DPI, CBZ-treated mice traveled significantly less distance through the center of an open field, indicative of anxiety-like behavior. CBZ-treated mice also presented with significant astrogliosis 36 DPI. Neither CBZ nor VPA prevented long-term reductions in NeuN immunoreactivity. The TMEV model thus provides an etiologically relevant platform to evaluate potential treatments for acute seizures and disease modification.

Rapamycin reverses status epilepticus-induced memory deficits and dendritic damage.

Cognitive impairments are prominent sequelae of prolonged continuous seizures (status epilepticus; SE) in humans and animal models. While often associated with dendritic injury, the underlying mechanisms remain elusive. The mammalian target of rapamycin complex 1 (mTORC1) pathway is hyperactivated following SE. This pathway modulates learning and memory and is associated with regulation of neuronal, dendritic, and glial properties. Thus, in the present study we tested the hypothesis that SE-induced mTORC1 hyperactivation is a candidate mechanism underlying cognitive deficits and dendritic pathology seen following SE. We examined the effects of rapamycin, an mTORC1 inhibitor, on the early hippocampal-dependent spatial learning and memory deficits associated with an episode of pilocarpine-induced SE. Rapamycin-treated SE rats performed significantly better than the vehicle-treated rats in two spatial memory tasks, the Morris water maze and the novel object recognition test. At the molecular level, we found that the SE-induced increase in mTORC1 signaling was localized in neurons and microglia. Rapamycin decreased the SE-induced mTOR activation and attenuated microgliosis which was mostly localized within the CA1 area. These findings paralleled a reversal of the SE-induced decreases in dendritic Map2 and ion channels levels as well as improved dendritic branching and spine density in area CA1 following rapamycin treatment. Taken together, these findings suggest that mTORC1 hyperactivity contributes to early hippocampal-dependent spatial learning and memory deficits and dendritic dysregulation associated with SE.

Inhibition of the mammalian target of rapamycin blocks epilepsy progression in NS-Pten conditional knockout mice.

PURPOSE: Increased activity of mTOR Complex 1 (mTORC1) has been demonstrated in cortical dysplasia and tuberous sclerosis complex, as well as in animal models of epilepsy. Recent studies in such models revealed that inhibiting mTORC1 with rapamycin effectively suppressed seizure activity. However, seizures can recur after treatment cessation, and continuous rapamycin exposure can adversely affect animal growth and health. Here, we evaluated the efficacy of an intermittent rapamycin treatment protocol on epilepsy progression using neuron subset-specific-Pten (NS-Pten) conditional knockout mice. METHODS: NS-Pten knockouts were treated with a single course of rapamycin during postnatal weeks 4 and 5, or intermittently over a period of 5 months. Epileptiform activity was monitored using video-electroencephalography (EEG) recordings, and mossy fiber sprouting was evaluated using Timm staining. Survival and body weight were assessed in parallel. KEY FINDINGS: NS-Pten knockouts treated with a single course of rapamycin had recurrence of epilepsy 4-7 weeks after treatment ended. In contrast, epileptiform activity remained suppressed, and survival increased if knockout mice received additional rapamycin during weeks 10-11 and 16-17. Aberrant mossy fiber sprouting, present by 4 weeks of age and progressing in parallel with epileptiform activity, was also blocked by rapamycin. SIGNIFICANCE: These findings demonstrate that a single course of rapamycin treatment suppresses epileptiform activity and mossy fiber sprouting for several weeks before epilepsy recurs. However, additional intermittent treatments with rapamycin prevented this recurrence and enhanced survival without compromising growth. Therefore, these studies add to the growing body of evidence implicating an important role for mTORC1 signaling in epilepsy.