Modification of post-traumatic epilepsy by fecal microbiota transfer.

It has been well established that traumatic brain injury (TBI) modifies the composition of gut microbiome. Epilepsy, which represents one of the common sequelae of TBI, has been associated with dysbiosis. Earlier study showed that the risk of post-traumatic epilepsy (PTE) after lateral fluid percussion injury (LFPI) in rats can be stratified based on pre-existing (i.e., pre-TBI) gut microbiome profile. In the present study, we examined whether fecal microbiota transfer (FMT) from naïve rats with different prospective histories of PTE would affect the trajectory of PTE in recipients. Fecal samples were collected from naïve adult male Sprague-Dawley rats, followed by LFPI. Seven months later, upon four weeks of vide-EEG monitoring (vEEG), the rats were categorized as those with and without PTE. Recipients were subjected to LFPI, followed by FMT from donors with and without impending PTE. Control groups included auto-FMT and no-FMT subjects. Seven month after LFPI, recipients underwent four-week vEEG to detect spontaneous seizures. After completing vEEG, rats of all groups underwent kindling of basolateral amygdala. Fecal microbiota transfer from donors with impending PTE exerted mild-to-moderate pro-epileptic effects in recipients, evident as marginal increase in multiple spontaneous seizure incidence, and facilitation of kindling. Analysis of fecal samples in selected recipients and their respective donors confirmed that FMT modified microbiota in recipients along the donors' lines, albeit without full microbiome conversion. The findings provide further evidence that gut microbiome may actively modulate the susceptibility to epilepsy.

Susceptibility to epilepsy after traumatic brain injury is associated with preexistent gut microbiome profile.

OBJECTIVE: We examined whether posttraumatic epilepsy (PTE) is associated with measurable perturbations in gut microbiome. METHODS: Adult Sprague Dawley rats were subjected to lateral fluid percussion injury (LFPI). PTE was examined 7 months after LFPI, during 4-week continuous video-electroencephalographic monitoring. 16S ribosomal RNA gene sequencing was performed in fecal samples collected before LFPI/sham-LFPI and 1 week, 1 month, and 7 months thereafter. Longitudinal analyses of alpha diversity, beta diversity, and differential microbial abundance were performed. Short-chain fatty acids (SCFAs) were measured in fecal samples collected before LFPI by liquid chromatography with tandem mass spectrometry. RESULTS: Alpha diversity changed over time in both LFPI and sham-LFPI subjects; no association was observed between alpha diversity and LFPI, the severity of post-LFPI neuromotor impairments, and PTE. LFPI produced significant changes in beta diversity and selective changes in microbial abundances associated with the severity of neuromotor impairments. No association between LFPI-dependent microbial perturbations and PTE was detected. PTE was associated with beta diversity irrespective of timepoint vis-à-vis LFPI, including at baseline. Preexistent fecal microbial abundances of four amplicon sequence variants belonging to the Lachnospiraceae family (three enriched and one depleted) predicted the risk of PTE, with area under the curve (AUC) of .73. Global SCFA content was associated with the increased risk of PTE, with AUC of .722, and with 2-methylbutyric (depleted), valeric (depleted), isobutyric (enriched), and isovaleric (enriched) acids being the most important factors (AUC = .717). When the analyses of baseline microbial and SCFA compositions were combined, AUC to predict PTE increased to .78. SIGNIFICANCE: Whereas LFPI produces no perturbations in the gut microbiome that are associated with PTE, the risk of PTE can be stratified based on preexistent microbial abundances and SCFA content.

Diversity of kindling of limbic seizures after lateral fluid percussion injury in the rat.

Lateral fluid percussion injury (LFPI) in rats is used to model post-traumatic epilepsy (PTE), with spontaneous seizures occurring in up to ½ of the subjects. Using the kindling paradigm, we examined whether animals without detectable seizures had an altered seizure susceptibility. Male Sprague Dawley rats were subjected to LFPI. Seven-nine months later, spontaneous seizures were monitored for two weeks. Afterward, the animals underwent kindling of basolateral amygdala. For kindling outcomes, the animals were categorized based on the 95% confidence intervals of mean number trials to kindling (ie 3 consecutive stage 4-5 seizures). Spontaneous seizures were detected in 7 out of 24 rats. There was no correlation between the severity of LFPI and either baseline afterdischarge properties, or kindling rates. Six LFPI rats kindled at a rate comparable to those in sham-LFPI (n = 10) and in naïve (n = 7) subjects. Ten LFPI rats kindled faster and 8-slower than controls. None of slow-kindling rats had spontaneous seizures during the prekindling monitoring. During the same period, six fast-kindling and three normal-kindling rats had been seizure-free. Thus, kindling reveals a diversity to seizure susceptibility after LFPI beyond an overt seizure symptomatology, ranging from the increased susceptibility to the increased resistance.

Disruption of intestinal barrier and endotoxemia after traumatic brain injury: Implications for post-traumatic epilepsy.

OBJECTIVE: Traumatic brain injury (TBI) may lead to the disruption of the intestinal barrier (IB), and to the escape of products of commensal gut bacteria, including lipopolysaccharide (LPS), into the bloodstream. We examined whether lateral fluid percussion injury (LFPI) and post-traumatic epilepsy (PTE) are associated with the increased intestinal permeability and endotoxemia, and whether these events in turn are associated with PTE. METHODS: LFPI was delivered to adult male Sprague-Dawley rats. Before, 1 week, and 7 months after LFPI, the IB permeability was examined by measuring plasma concentration of fluorescein isothiocyanate-labeled dextran (FD4) upon its enteral administration. Plasma LPS concentration was measured in the same animals, using enzyme-linked immunosorbent assay. PTE was examined 7 months after LFPI, with use of video-EEG (electroencephalography) monitoring. RESULTS: One week after LFPI, the IB disruption was detected in 14 of 17 and endotoxemia - in 10 of 17 rats, with a strong positive correlation between FD4 and LPS levels, and between plasma levels of each of the analytes and the severity of neuromotor deficit. Seven months after LFPI, IB disruption was detected in 13 of 15 and endotoxemia in 8 of 15 rats, with a strong positive correlation between plasma levels of the two analytes. Five of 15 LFPI rats developed PTE. Plasma levels of both FD4 and LPS were significantly higher in animals with PTE than among the animals without PTE. The analysis of seven rats, which were examined repeatedly at 1 week and at 7 months, confirmed that late IB disruption and endotoxemia were not due to lingering of impairments occurring shortly after LFPI. SIGNIFICANCE: LFPI leads to early and remote disruption of IB and a secondary endotoxemia. Early and late perturbations may occur in different subjects. Early changes reflect the severity of acute post-traumatic motor dysfunction, whereas late changes are associated with PTE.

Can we and should we use animal models to study neurobehavioral comorbidities of epilepsy?

Animal systems have been widely used to examine mechanisms of neurobehavioral comorbidities of epilepsy and to help in developing their effective therapies. Despite the progress made in the field, animal studies have their limitations stemming both from issues with modeling neuropsychiatric disorders in the laboratory and from drawbacks of animal models of epilepsy themselves. This review discusses advantages and weaknesses of experimental paradigms and approaches used to model and to analyze neurobehavioral comorbidities of epilepsy, from the perspectives of their needs, interpretation, ways of improvement, and clinical relevance. Developmental studies are required to adequately address age-specific aspects of the comorbidities. The deployment of preclinical Common Data Elements (pCDEs) for epilepsy research should facilitate the standardization and the harmonization of studies in question, while the application of Research Domain Criteria (RDoC) to characterize neurobehavioral disorders in animals with epilepsy should help in closing the bench-to-bedside gap. Special Issue: Epilepsy & Behavior's 20th Anniversary.

Melanotan-II reverses autistic features in a maternal immune activation mouse model of autism.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder characterized by impaired social interactions, difficulty with communication, and repetitive behavior patterns. In humans affected by ASD, there is a male pre-disposition towards the condition with a male to female ratio of 4:1. In part due to the complex etiology of ASD including genetic and environmental interplay, there are currently no available medical therapies to improve the social deficits of ASD. Studies in rodent models and humans have shown promising therapeutic effects of oxytocin in modulating social adaptation. One pharmacological approach to stimulating oxytocinergic activity is the melanocortin receptor 4 agonist Melanotan-II (MT-II). Notably the effects of oxytocin on environmental rodent autism models has not been investigated to date. We used a maternal immune activation (MIA) mouse model of autism to assess the therapeutic potential of MT-II on autism-like features in adult male mice. The male MIA mice exhibited autism-like features including impaired social behavioral metrics, diminished vocal communication, and increased repetitive behaviors. Continuous administration of MT-II to male MIA mice over a seven-day course resulted in rescue of social behavioral metrics. Normal background C57 male mice treated with MT-II showed no significant alteration in social behavioral metrics. Additionally, there was no change in anxiety-like or repetitive behaviors following MT-II treatment of normal C57 mice, though there was significant weight loss following subacute treatment. These data demonstrate MT-II as an effective agent for improving autism-like behavioral deficits in the adult male MIA mouse model of autism.

Bidirectional relations among common psychiatric and neurologic comorbidities and epilepsy: Do they have an impact on the course of the seizure disorder?

The treatment of epilepsy is not limited to the achievement of a seizure-free state. It must also incorporate the management of common psychiatric and neurologic comorbidities, affecting on average between 30 and 50% of patients with epilepsy, which have a significant impact on their lives at various levels, including quality of life and the prognosis of the seizure disorder. Mood and anxiety disorders are the most frequent psychiatric comorbidities, whereas stroke and migraine are among the more common neurologic comorbidities, migraine among the younger patients and stroke among the older patients. Not only do these psychiatric and neurologic comorbidities each have a bidirectional relation with epilepsy, but primary mood disorders have a bidirectional relation with these 2 neurologic disorders. Furthermore, depression and migraine have been each associated with a more severe epilepsy course, whereas depression has been associated with a more severe course of stroke and migraines. The purpose of this article is to review the clinical implications of the complex relations among epilepsy and these 3 comorbid disorders, and to identify any clinical and/or experimental evidence that may suggest that having more than one of these comorbid disorders may increase the risk of and course of epilepsy.

A companion to the preclinical common data elements on neurobehavioral comorbidities of epilepsy: a report of the TASK3 behavior working group of the ILAE/AES Joint Translational Task Force.

The provided companion has been developed by the Behavioral Working Group of the Joint Translational Task Force of the International League Against Epilepsy (ILAE) and the American Epilepsy Society (AES) with the purpose of assisting the implementation of Preclinical Common Data Elements (CDE) for studying and for reporting neurobehavioral comorbidities in rodent models of epilepsy. Case Report Forms (CRFs) are provided, which should be completed on a per animal/per test basis, whereas the CDEs are a compiled list of the elements that should be reported. This companion is not designed as a list of recommendations, or guidelines for how the tests should be run-rather, it describes the different types of assessments, and highlights the importance of rigorous data collection and transparency in this regard. The tests are divided into 7 categories for examining behavioral dysfunction on the syndrome level: deficits in learning and memory; depression; anxiety; autism; attention deficit/hyperactivity disorder; psychosis; and aggression. Correspondence and integration of these categories into the National Institute of Mental Health (NIMH) Research Domain Criteria (RDoC) is introduced. Developmental aspects are addressed through the introduction of developmental milestones. Discussion includes complexities, limitations, and biases associated with neurobehavioral testing, especially when performed in animals with epilepsy, as well as the importance of rigorous data collection and of transparent reporting. This represents, to our knowledge, the first such resource dedicated to preclinical CDEs for behavioral testing of rodents.

Facilitation of kindling epileptogenesis by chronic stress may be mediated by intestinal microbiome.

There has been growing interest in the role of intestinal microbiome in brain disorders. We examined whether dysbiosis can predispose to epilepsy. The study was performed in female and male Sprague-Dawley rats. To induce dysbiosis, the rats were subjected to chronic restraint stress (two 2-h long sessions per day, over 2 weeks). Cecal content from stressed and sham-stressed donors was transplanted via oral gavage to recipients, in which commensal microbiota had been depleted by the antibiotics. The study included the following groups: (1) Sham stress, no microbiota transplant; (2) Stress, no microbiota transplant; (3) Sham-stressed recipients transplanted with microbiota from sham-stressed donors; (4) Stressed recipients transplanted with microbiota from sham-stressed donors; (5) Sham-stressed recipients transplanted with microbiota from stressed donors; and (6) Stressed recipients transplanted with microbiota from stressed donors. After microbiota transplant, all animals were subjected to kindling of the basolateral amygdala. Both chronic stress and microbiome transplanted from stressed to sham-stressed subjects accelerated the progression and prolonged the duration of kindled seizures. Microbiome from sham-stressed animals transplanted to chronically stressed rats, counteracted proepileptic effects of restraint stress. These findings directly implicate perturbations in the gut microbiome, particularly those associated with chronic stress, in the increased susceptibility to epilepsy.

Kindling epileptogenesis and panic-like behavior: Their bidirectional connection and contribution to epilepsy-associated depression.

Anxiety is one of the most common comorbidities of epilepsy, which has major detrimental effects on the quality of life. Generalized anxiety disorder (GAD) associated with epilepsy has been receiving most attention. However, several other forms of anxiety reportedly present in patients with epilepsy, including panic disorder (PD). In this study, using an animal model of limbic epilepsy, we examined the interplay between epilepsy and panic-like behavior (PLB). Further, considering the high degree of comorbidity between depression on the one hand, and both epilepsy and PD on the other hand, we studied whether and how the presence of PLB in animals with epilepsy would affect their performance in depression-relevant tests. Fifty-day-old male Wistar rats were subjected to repeated alternating electrical stimulations of the basolateral amygdala (BLA) to induce kindling of limbic seizures, and the dorsal periaqueductal gray (DPAG) to induce panic-like episodes. Seizure susceptibility and panic reaction threshold were examined before the first and 24h after the last stimulation. At the end of the stimulations, the rats were examined in depression-relevant tests: saccharin preference test (SPT) for anhedonia and forced swimming test (FST) for despair/hopelessness. With regard to kindling, BLA+DPAG stimulation induced more profound increase of seizure susceptibility than BLA stimulation alone (evident as the reduction of the afterdischarge threshold and the increase of the afterdischarge duration). With regard to PLB, the BLA+DPAG stimulation exacerbated the severity of panic-like episodes, as compared with the DPAG stimulation alone. Basolateral amygdala stimulation alone had no effects on panic-like reactions, and DPAG stimulation alone did not modify kindling epileptogenesis. Combined stimulation of BLA and DPAG induced depressive-like behavioral impairments. This is the first experimental study showing bidirectional, mutually exacerbating effect of epilepsy and PLB, and the precipitation of depressive-like state by the epilepsy-PLB comorbidity.

Neurobehavioral comorbidities of epilepsy: Role of inflammation.

Epilepsy is associated with a high incidence of comorbid neurologic and psychiatric disorders. This review focuses on the association of epilepsy with autism spectrum disorder (ASD) and depression. There is high concordance of these behavioral pathologies with epilepsy. We review data that unambiguously reveal that epilepsy, ASD, and depression are associated with elevated brain inflammatory markers and that these may interact with serotoninergic pathways. Interference with inflammatory pathways or actions can reduce the severity of seizures, depression, and ASD-like behavior. Inflammation in the brain can be induced by seizure activity as well as by behavioral, environmental, and physiologic stressors. Furthermore, induction of inflammation at an early time point during gestation and in early neonatal life can precipitate both an ASD-like phenotype as well as a more excitable brain. It appears likely that priming of the brain due to early inflammation could provide a means by which subsequent inflammatory processes associated with epilepsy, ASD, and depression may lead to comorbidity.

Inherent vulnerabilities in monoaminergic pathways predict the emergence of depressive impairments in an animal model of chronic epilepsy.

The objective was to determine whether the depression comorbid with epilepsy could be predicted based on inherent premorbid patterns of monoaminergic transmission. In male Wistar rats, despair-like and anhedonia-like behaviors were examined using forced swimming and taste preference tests, respectively. Serotonergic raphe nucleus (RN)-prefrontal cortex (PFC) and dopaminergic ventral tegmental area (VTA)-nucleus accumbens (NAcc) pathways were interrogated by fast scan cyclic voltammetry (FSCV). The assays were performed before and 2 months after pilocarpine status epilepticus. In a subset of naive rats, FSCV, coupled with the intensity-dependent stimulation paradigm, detected specific deviations in each pathway (six rats for RN-PFC and seven rats for VTA-NAcc, with overlap in two, of 19 total subjects) in the absence of behavioral impairments. During epilepsy, animals with preexisting deviations in RN-PFC invariably developed despair, and rats with deviations in VTA-NAcc developed anhedonia. Serotonergic and dopaminergic pathways, respectively, showed signs of explicit deterioration. We suggest that epilepsy triggers decompensations in the already vulnerable depression-relevant neuronal circuits, which culminate in depression. The established connection between the identified specific signatures in monoamine transmission in naive rats and specific symptoms of epilepsy-associated depression may help in understanding causes of comorbidity and in developing its early biomarkers.

Regulation of kindling epileptogenesis by hippocampal Toll-like receptors 2.

This study examined whether Toll-like receptors 2 (TLR2) contribute to rapid kindling epileptogenesis. A TLR2 agonist, lipoteichoic acid (LTA), LTA antibody (LTA-A), or normal saline (control) was administered daily over 3 consecutive days, unilaterally into ventral hippocampus of adult male Wistar rats. Thirty minutes after the last injection, the animals were subjected to a rapid kindling procedure. The ictogenesis was gauged by comparing afterdischarge threshold (ADT) and afterdischarge duration (ADD) before the treatments, after the treatments prior to kindling, and 24 h after kindling. Kindling progression and retention were analyzed using video recording. The results showed that before kindling, LTA produced an ADT reduction. Neither LTA nor LTA-A affected baseline ADD. On kindling progression, LTA accelerated occurrence of generalized seizures, whereas LTA-A delayed this effect. Treatment with LTA-A reduced the number of secondary generalized complex partial seizures. Twenty-four hours after kindling, the rats of both the saline and LTA groups showed increased hippocampal excitability as compared with prekindling parameters. Administration of LTA-A prevented kindling-induced increase of hippocampal excitability. Immunostaining revealed that LTA-A attenuated the inflammatory response produced by seizures. These findings suggest that the activation of TLR2 in the hippocampus may facilitate limbic epileptogenesis.

Sex-Specific Life Course Changes in the Neuro-Metabolic Phenotype of Glut3 Null Heterozygous Mice: Ketogenic Diet Ameliorates Electroencephalographic Seizures and Improves Sociability.

We tested the hypothesis that exposure of glut3+/- mice to a ketogenic diet ameliorates autism-like features, which include aberrant behavior and electrographic seizures. We first investigated the life course sex-specific changes in basal plasma-cerebrospinal fluid (CSF)-brain metabolic profile, brain glucose transport/uptake, glucose and monocarboxylate transporter proteins, and adenosine triphosphate (ATP) in the presence or absence of systemic insulin administration. Glut3+/- male but not female mice (5 months of age) displayed reduced CSF glucose/lactate concentrations with no change in brain Glut1, Mct2, glucose uptake or ATP. Exogenous insulin-induced hypoglycemia increased brain glucose uptake in glut3+/- males alone. Higher plasma-CSF ketones (ß-hydroxybutyrate) and lower brain Glut3 in females vs males proved protective in the former while enhancing vulnerability in the latter. As a consequence, increased synaptic proteins (neuroligin4 and SAPAP1) with spontaneous excitatory postsynaptic activity subsequently reduced hippocampal glucose content and increased brain amyloid ß1-40 deposition in an age-dependent manner in glut3+/- males but not females (4 to 24 months of age). We then explored the protective effect of a ketogenic diet on ultrasonic vocalization, sociability, spatial learning and memory, and electroencephalogram seizures in male mice (7 days to 6 to 8 months of age) alone. A ketogenic diet partially restored sociability without affecting perturbed vocalization, spatial learning and memory, and reduced seizure events. We conclude that (1) sex-specific and age-dependent perturbations underlie the phenotype of glut3+/- mice, and (2) a ketogenic diet ameliorates seizures caused by increased cortical excitation and improves sociability, but fails to rescue vocalization and cognitive deficits in glut3+/- male mice.

WONOEP appraisal: Biomarkers of epilepsy-associated comorbidities.

Neurologic and psychiatric comorbidities are common in patients with epilepsy. Diagnostic, predictive, and pharmacodynamic biomarkers of such comorbidities do not exist. They may share pathogenetic mechanisms with epileptogenesis/ictogenesis, and as such are an unmet clinical need. The objectives of the subgroup on biomarkers of comorbidities at the XIII Workshop on the Neurobiology of Epilepsy (WONOEP) were to present the state-of-the-art recent research findings in the field that highlighting potential biomarkers for comorbidities in epilepsy. We review recent progress in the field, including molecular, imaging, and genetic biomarkers of comorbidities as discussed during the WONOEP meeting on August 31-September 4, 2015, in Heybeliada Island (Istanbul, Turkey). We further highlight new directions and concepts from studies on comorbidities and potential new biomarkers for the prediction, diagnosis, and treatment of epilepsy-associated comorbidities. The activation of various molecular signaling pathways such as the "Janus Kinase/Signal Transducer and Activator of Transcription," "mammalian Target of Rapamycin," and oxidative stress have been shown to correlate with the presence and severity of subsequent cognitive abnormalities. Furthermore, dysfunction in serotonergic transmission, hyperactivity of the hypothalamic-pituitary-adrenocortical axis, the role of the inflammatory cytokines, and the contributions of genetic factors have all recently been regarded as relevant for understanding epilepsy-associated depression and cognitive deficits. Recent evidence supports the utility of imaging studies as potential biomarkers. The role of such biomarker may be far beyond the diagnosis of comorbidities, as accumulating clinical data indicate that comorbidities can predict epilepsy outcomes. Future research is required to reveal whether molecular changes in specific signaling pathways or advanced imaging techniques could be detected in the clinical settings and correlate with epilepsy-associated comorbidities. A reliable biomarker will allow a more accurate diagnosis and improved treatment of epilepsy-associated comorbidities.