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.

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.

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.

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.

Galanin contributes to monoaminergic dysfunction and to dependent neurobehavioral comorbidities of epilepsy.

Status epilepticus (SE) in rats, along with chronic epilepsy, leads to the development of behavioral impairments resembling depressive disorder and/or attention deficit/hyperactivity disorder (ADHD), thus reflecting respective comorbidities in epilepsy patients. Suppressed neurotransmitter tone in the raphe nucleus (RN)-prefrontal cortex (PFC) serotonergic pathway and in the locus coeruleus (LC)-PFC noradrenergic pathway underlies depressive- and impulsive-like behavioral deficits respectively. We examined possible mechanisms leading to the monoamine dysfunction in brainstem efferents, namely modulatory effects of the neuropeptide galanin on serotonin (5-HT) and norepinephrine (NE) signaling. SE was induced in young adult male Wistar rats by LiCl and pilocarpine. Epileptic rats were categorized vis-à-vis behavioral deficits as not impaired, "depressed" and "impulsive". Depressive- and impulsive-like behaviors were examined in the forced swimming test (FST). The strength of serotonergic transmission in RN-PFC and of noradrenergic transmission in LC-PFC was analyzed using in vivo fast scan cyclic voltammetry. Galanin receptor type 1 (GalR1)/type 2 (GalR2) antagonist M40, and a preferential GalR2 antagonist M871 were administered over 3days locally into either RN or LC by means of ALZET osmotic minipumps connected to locally implanted infusion cannulas. Intra-RN injection of M40 improved serotonergic tone and depressive-like behavior in epileptic "depressed" rats. Intra-LC injection of M40 improved noradrenergic tone and impulsive-like behavior in epileptic "impulsive" rats. The effects of M40 were only observed in impaired subjects. The treatment did not modify neurotransmission and behavior in naïve and epileptic not impaired rats; in "depressed" rats the effects were limited to serotonergic transmission and immobility, while in "impulsive" rats - to noradrenergic transmission and struggling behavior. Intra-RN administration of M871 exacerbated depressive-like behavior, but had no effects on any other of the examined parameters in any category of animals. These findings suggest that endogenous galanin, acting through GalR1 may be involved in the pathophysiology of epilepsy-associated depression and ADHD via inhibiting RN-PFC serotonergic and LC-PFC noradrenergic transmissions respectively.

Effects of selective serotonin and norepinephrine reuptake inhibitors on depressive- and impulsive-like behaviors and on monoamine transmission in experimental temporal lobe epilepsy.

OBJECTIVE: Examine therapeutic potential of a selective serotonin reuptake inhibitor (SSRI) and a norepinephrine reuptake inhibitor (NERI) in an animal model of comorbidity between epilepsy, depression-like, and impulsive-like impairments. METHODS: Epilepsy was induced in male Wistar rats by LiCl and pilocarpine. An SSRI fluoxetine (FLX), and an NERI reboxetine (RBX) were administered either alone or as a combination over 1 week. Depressive-like and impulsive-like behaviors were examined using the forced swim test. Fast scan cyclic voltammetry was used to analyze serotonergic transmission in the raphe nucleus (RN)-prefrontal cortex (PFC) pathway, and noradrenergic transmission in locus coeruleus (LC)-PFC, and LC-RN projections. Monoamine levels in PFC were measured using high-performance liquid chromatography (HPLC). Functional capacities of 5-HT1A receptors and α2A adrenoreceptors in PFC were analyzed by autoradiography. RESULTS: Epileptic rats showed behavioral signs of depression and hyperimpulsivity, suppressed serotonergic and noradrenergic tones, decreased levels of serotonin (5-HT), and norepinephrine (NE); 5-HT1A receptor and α2A adrenoreceptors functions remained intact. FLX failed to improve behavioral deficits, but effectively raised 5-HT level and marginally improved RN-PFC serotonergic transmission. RBX reversed impulsive-like behavior, normalized content of NE and noradrenergic tone in LC-PFC and LC-RN. FLX-RBX combination fully reversed depressive-like behavior, and normalized RN-PFC serotonergic transmission. None of the treatment modified the function of 5-HT and NE receptors. SIGNIFICANCE: Depressive- and impulsive-like behaviors in the pilocarpine model of epilepsy stem respectively from dysfunctions of serotonergic and noradrenergic ascending pathways. At the same time, epilepsy-associated depression is SSRI resistant. The finding that an SSRI-NERI combination exerts antidepressant effect, along with RBX-induced improvement of LC-RN noradrenergic transmission point toward the involvement of LC-RN noradrenergic input in enabling therapeutic potential of FLX. Medications that improve serotonergic and noradrenergic transmission, such as serotonin-norepinephrine reuptake inhibitors may be effective in treating epilepsy-associated SSRI-resistant depression, as well as concurrent depression and attention-deficit/hyperactivity disorder (ADHD).

Cytokine-dependent bidirectional connection between impaired social behavior and susceptibility to seizures associated with maternal immune activation in mice.

Maternal immune activation (MIA) results in the development of autism in the offspring via hyperactivation of IL-6 signaling. Furthermore, experimental studies showed that the MIA-associated activation of interleukin-1ß (IL-1ß) concurrently with IL-6 increases the rate and the severity of hippocampal kindling in mice, thus, offering an explanation for autism-epilepsy comorbidity. We examined whether epileptic phenotype triggered by prenatal exposure to IL-6 and IL-1ß combination is restricted to kindling or whether it is reproducible in another model of epilepsy, whereby spontaneous seizures develop following kainic acid (KA)-induced status epilepticus. We also examined whether in mice prenatally exposed to IL-6 and IL-6+IL-1ß, the presence of spontaneous seizures would exacerbate autism-like features. Between days 12 and 16 of pregnancy, C57BL/6J mice received daily injections of IL-6, IL-1ß, or IL-6+IL-1ß combination. At postnatal day 40, male offspring were examined for the presence of social behavioral deficit, and status epilepticus was induced by intrahippocampal KA injection. After 6weeks of monitoring for spontaneous seizures, sociability was tested again. Both IL-6 and IL-6+IL-1ß offspring presented with social behavioral deficit. Prenatal exposure to IL-6 alleviated, while such exposure to IL-6+IL-1ß exacerbated, the severity of KA-induced epilepsy. Increased severity of epilepsy in the IL-6+IL-1ß mice correlated with the improvement of autism-like behavior. We conclude that complex and not necessarily agonistic relationships exist between epileptic and autism-like phenotypes in an animal model of MIA coupled with KA-induced epilepsy and that the nature of these relationships depends on components of MIA involved.

Autism-Like Behavior in BTBR Mice Is Improved by Electroconvulsive Therapy.

Autism is a developmental disorder characterized by impairments in social and communication abilities, as well as by restricted and repetitive behaviors. Incidence of autism is higher than earlier estimates, and treatments have limited efficacy and are costly. Limited clinical and experimental evidence suggest that patients with autism may benefit from electroconvulsive therapy (ECT). We examined the therapeutic potential of ECT in BTBR T+ tf/j mice, which represent a validated model of autism. A series of 13 electroconvulsive shocks (ECS) delivered twice a day over 7 days reversed core autism-like behavioral abnormalities-impaired sociability, social novelty, and repetitive behavior-when the animals were tested 24 h after the last ECS. The effect lasted up to 2 weeks after ECT. Neither single ECS nor a series of 6 ECS modified animals' behavior. Chronic infusion into the lateral brain ventricle of a preferential oxytocin receptor blocker (2S)-2-Amino-N-[(1S,2S,4R)-7,7-dimethyl-1-[[[4-(2-methylphenyl)-1-piperazinyl]sulfonyl]methyl]bicyclo[2.2.1]hept-2-yl]-4-(methylsulfonyl)butanamide hydrochloride abolished ECT-induced improvement of sociability and mitigated improvement of social novelty but did not affect ECT-induced reversal of repetitive behavior. These proof-of-principle experiments suggest that ECT may, indeed, be useful in the treatment of autism, and that its therapeutic effects may be mediated, in part, by central oxytocin signaling.

Behavioral impairments in rats with chronic epilepsy suggest comorbidity between epilepsy and attention deficit/hyperactivity disorder.

Attention deficit/hyperactivity disorder (ADHD) is encountered among patients with epilepsy at a significantly higher rate than in the general population. Mechanisms of epilepsy-ADHD comorbidity remain largely unknown. We investigated whether a model of chronic epilepsy in rats produces signs of ADHD, and thus, whether it can be used for studying mechanisms of this comorbidity. Epilepsy was induced in male Wistar rats via pilocarpine status epilepticus. Half of the animals exhibited chronic ADHD-like abnormalities, particularly increased impulsivity and diminished attention in the lateralized reaction-time task. These impairments correlated with the suppressed noradrenergic transmission in locus coeruleus outputs. The other half of animals exhibited depressive behavior in the forced swimming test congruently with the diminished serotonergic transmission in raphe nucleus outputs. Attention deficit/hyperactivity disorder and depressive behavior appeared mutually exclusive. Therefore, the pilocarpine model of epilepsy affords a system for reproducing and studying mechanisms of comorbidity between epilepsy and both ADHD and/or depression.

Structural basis for substrate specificity and mechanism of N-acetyl-D-neuraminic acid lyase from Pasteurella multocida.

N-Acetylneuraminate lyases (NALs) or sialic acid aldolases catalyze the reversible aldol cleavage of N-acetylneuraminic acid (Neu5Ac, the most common form of sialic acid) to form pyruvate and N-acetyl-d-mannosamine. Although equilibrium favors sialic acid cleavage, these enzymes can be used for high-yield chemoenzymatic synthesis of structurally diverse sialic acids in the presence of excess pyruvate. Engineering these enzymes to synthesize structurally modified natural sialic acids and their non-natural derivatives holds promise in creating novel therapeutic agents. Atomic-resolution structures of these enzymes will greatly assist in guiding mutagenic and modeling studies to engineer enzymes with altered substrate specificity. We report here the crystal structures of wild-type Pasteurella multocida N-acetylneuraminate lyase and its K164A mutant. Like other bacterial lyases, it assembles into a homotetramer with each monomer folding into a classic (ß/α)8 TIM barrel. Two wild-type structures were determined, in the absence of substrates, and trapped in a Schiff base intermediate between Lys164 and pyruvate, respectively. Three structures of the K164A variant were determined: one in the absence of substrates and two binary complexes with N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Both sialic acids bind to the active site in the open-chain ketone form of the monosaccharide. The structures reveal that every hydroxyl group of the linear sugars makes hydrogen bond interactions with the enzyme, and the residues that determine specificity were identified. Additionally, the structures provide some clues for explaining the natural discrimination of sialic acid substrates between the P. multocida and Escherichia coli NALs.

Maternal immune activation promotes hippocampal kindling epileptogenesis in mice.

OBJECTIVE: Maternal immune activation (MIA) triggered by infections has been identified as a cause of autism in offspring. Considering the involvement of perturbations in innate immunity in epilepsy, we examined whether MIA represents a risk factor for epilepsy as well. The role of specific MIA components interleukin (IL)-6 and IL-1ß was also addressed. METHODS: MIA was induced in C57BL/6 mice by polyinosinic-polycytidylic acid (PIC) injected during embryonic days 12 to 16. Beginning from postnatal day 40, the propensity of the offspring to epilepsy was examined using hippocampal kindling; autismlike behavior was studied using the sociability test. The involvement of IL-6 and IL-1ß in PIC-induced effects was studied by the coadministration of the cytokine antibodies with PIC, and by delivering recombinant cytokines in lieu of PIC. RESULTS: The offspring of PIC-exposed mice exhibited increased hippocampal excitability, accelerated kindling rate, prolonged increase of seizure susceptibility after kindling, and diminished sociability. Epileptic impairments were abolished by antibodies to IL-6 or IL-1ß. Neither of the recombinant cytokines alone increased the propensity to seizures; however, when combined, they produced effects similar to those induced by PIC. PIC-induced behavioral deficits were abolished by IL-6 antibodies and were mimicked by recombinant IL-6; IL-1ß was not involved. INTERPRETATION: In addition to confirming the previously established critical role of IL-6 in the development of autismlike behavior following MIA, the present study shows that concurrent involvement of IL-6 and IL-1ß is required for priming the offspring for epilepsy. These data shed light on mechanisms of comorbidity between autism and epilepsy.

Neuroprotective and antiepileptogenic effects of combination of anti-inflammatory drugs in the immature brain.

BACKGROUND: Inflammatory signaling elicited by prolonged seizures can be contributory to neuronal injury as well as adverse plasticity leading to the development of spontaneous recurrent seizures (epilepsy) and associated co-morbidities. In this study, developing rat pups were subjected to lithium-pilocarpine status epilepticus (SE) at 2 and 3 weeks of age to study the effect of anti-inflammatory drugs (AID) on SE-induced hippocampal injury and the development of spontaneous seizures. FINDINGS: We selected AIDs directed against interleukin-1 receptors (IL-1ra), a cyclooxygenase-2 (COX-2) inhibitor (CAY 10404), and an antagonist of microglia activation of caspase-1 (minocycline). Acute injury after SE was studied in the 2-week-old rats 24 h after SE. Development of recurrent spontaneous seizures was studied in 3-week-old rats subjected to SE 4 months after the initial insult.None of those AIDs were effective in attenuating CA1 injury in the 2-week-old pups or in limiting the development of spontaneous seizures in 3-week-old pups when administered individually. When empiric binary combinations of these drugs were tried, the combined targeting of IL-1r and COX-2 resulted in attenuation of acute CA1 injury, as determined 24 h after SE, in those animals. The same combination administered for 10 days following SE in 3-week-old rats, reduced the development of spontaneous recurrent seizures and limited the extent of mossy fiber sprouting. CONCLUSIONS: Deployment of an empirically designed 'drug cocktail' targeting multiple inflammatory signaling pathways for a limited duration after an initial insult like SE may provide a practical approach to neuroprotection and anti-epileptogenic therapy.

Novel animal models of pediatric epilepsy.

When mimicking epileptic processes in a laboratory setting, it is important to understand the differences between experimental models of seizures and epilepsy. Because human epilepsy is defined by the appearance of multiple spontaneous recurrent seizures, the induction of a single acute seizure without recurrence does not constitute an adequate epilepsy model. Animal models of epilepsy might be useful for various tasks. They allow for the investigation of pathophysiological mechanisms of the disease, the evaluation, or the development of new antiepileptic treatments, and the study of the consequences of recurrent seizures and neurological and psychiatric comorbidities. Although clinical relevance is always an issue, the development of models of pediatric epilepsies is particularly challenging due to the existence of several key differences in the dynamics of human and rodent brain maturation. Another important consideration in modeling pediatric epilepsy is that "children are not little adults," and therefore a mere application of models of adult epilepsies to the immature specimens is irrelevant. Herein, we review the models of pediatric epilepsy. First, we illustrate the differences between models of pediatric epilepsy and models of the adulthood consequences of a precipitating insult in early life. Next, we focus on new animal models of specific forms of epilepsies that occur in the developing brain. We conclude by emphasizing the deficiencies in the existing animal models and the need for several new models.

Interleukin-1ß causes fluoxetine resistance in an animal model of epilepsy-associated depression.

Depression represents a common comorbidity of epilepsy and is frequently resistant to selective serotonin reuptake inhibitors (SSRI). We tested the hypothesis that the SSRI resistance in epilepsy associated depression may be a result of a pathologically enhanced interleukin-1ß (IL1-ß) signaling, and consequently that the blockade of IL1-ß may restore the effectiveness of SSRI. Epilepsy and concurrent depression-like impairments were induced in Wistar rats by pilocarpine status epilepticus (SE). The effects of the 2-week long treatment with fluoxetine, interleukin-1 receptor antagonist (IL-1ra), and their combination were examined using behavioral, biochemical, neuroendocrine, and autoradiographic assays. In post-SE rats, depression-like impairments included behavioral deficits indicative of hopelessness and anhedonia; the hyperactivity of the hypothalamo-pituitary-adrenocortical axis; the diminished serotonin output from raphe nucleus; and the upregulation of presynaptic serotonin 1-A (5-HT1A) receptors. Fluoxetine monotherapy exerted no antidepressant effects, whereas the treatment with IL-1ra led to the complete reversal of anhedonia and to a partial improvement of all other depressive impairments. Combined administration of fluoxetine and IL-1ra completely abolished all hallmarks of epilepsy-associated depressive abnormalities, with the exception of the hyperactivity of the hypothalamo-pituitary-adrenocortical axis, the latter remaining only partially improved. We propose that in certain forms of depression, including but not limited to depression associated with epilepsy, the resistance to SSRI may be driven by the pathologically enhanced interleukin-1ß signaling and by the subsequent upregulation of presynaptic 5-HT1A receptors. In such forms of depression, the use of interleukin-1ß blockers in conjunction with SSRI may represent an effective therapeutic approach.

Plasticity of presynaptic and postsynaptic serotonin 1A receptors in an animal model of epilepsy-associated depression.

Depression is a common comorbidity of temporal lobe epilepsy and has highly negative impact on patients' quality of life. We previously established that pilocarpine-induced status epilepticus (SE) in rats, concurrently with chronic epilepsy leads to depressive impairments, and that the latter may stem from the dysregulation of hypothalamo-pituitary-adrenocortical (HPA) axis and/or diminished raphe-hippocampal serotonergic transmission. We examined possible involvement of presynaptic and postsynaptic serotonin 1A (5-HT1A) receptors in epilepsy-associated depression. Based on their performance in the forced swim test (FST), post-SE animals were classified as those with moderate and severe depressive impairments. In moderately impaired rats, the activity of the HPA axis (examined using plasma corticosterone radioimmunoassay) was higher than in naive subjects, but the functional capacity of presynaptic 5-HT1A receptors (measured in raphe using autoradiography) remained unaltered. In severely depressed animals, both the activity of the HPA axis and the function of presynaptic 5-HT1A receptors were increased as compared with naive and moderately depressed rats. Pharmacological uncoupling of the HPA axis from raphe nucleus exerted antidepressant effects in severely impaired rats, but did not modify behavior in both naive and moderately depressed animals. Further, the function of postsynaptic 5-HT1A receptors was diminished in the hippocampus of post-SE rats. Pharmacological activation of postsynaptic 5-HT1A receptors improved depressive deficits in epileptic animals. We suggest that under the conditions of chronic epilepsy, excessively hyperactive HPA axis activates presynaptic 5-HT1A receptors, thus shifting the regulation of serotonin release in favor of autoinhibition. Downregulation of postsynaptic 5-HT1A receptors may further exacerbate the severity of epilepsy-associated depression.