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.

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.

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.

Comorbidity between epilepsy and depression: role of hippocampal interleukin-1beta.

Depression is a frequent comorbidity of temporal lobe epilepsy (TLE); however, its mechanisms remain poorly understood and effective therapies are lacking. Augmentation of hippocampal interleukin-1beta (IL-1beta) signaling may be a mechanistic factor of both TLE and clinical depression. We examined whether pharmacological blockade of hippocampal interleukin-1 receptor exerts antidepressant effects in an animal model of comorbidity between TLE and depression, which developed in Wistar rats following pilocarpine status epilepticus (SE). In post-SE animals, depression-like state was characterized by behavioral equivalents of anhedonia and despair; dysregulation of the hypothalamo-pituitary-adrenocortical axis; compromised raphe-hippocampal serotonergic transmission. Two-week long bilateral intrahippocampal infusion of human recombinant Interleukin-1 receptor antagonist (IL-1ra) improved all of the examined depressive impairments, without modifying spontaneous seizure frequency and without affecting normal parameters in naïve rats. These findings implicate hippocampal IL-1beta in epilepsy-associated depression and provide a rationale for the introduction of IL-1beta blockers in the treatment of depression in TLE.

Comorbidity between epilepsy and depression: experimental evidence for the involvement of serotonergic, glucocorticoid, and neuroinflammatory mechanisms.

Depression represents one of the most common comorbidities of temporal lobe epilepsy (TLE), and has profound negative impact on the quality of life of patients with TLE. However, causes and mechanisms of depression in TLE remain poorly understood, and effective therapies are lacking. We examined whether a commonly used model of TLE in rats could be used as a model of comorbidity between epilepsy and depression suitable for both mechanistic studies and for the development of mechanism-based antidepressant therapies. We established that animals that had been subjected to lithium chloride and pilocarpine status epilepticus (SE) and developed spontaneous recurrent seizures, exhibited a set of impairments congruent with a depressive state: behavioral equivalents of anhedonia and despair, dysregulation of the hypothalamus-pituitary-adrenal (HPA) axis, and compromised raphe-hippocampal serotonergic transmission. Pharmacologic studies have suggested that depressive impairments following SE develop as a result of enhanced interleukin-1beta signaling in the hippocampus, which leads to depression via inducing perturbations in the HPA axis and subsequent deficit in the raphe-hippocampal serotonergic transmission.

Elevated plasma corticosterone level and depressive behavior in experimental temporal lobe epilepsy.

Depression is frequently reported in epilepsy patients; however, mechanisms of co-morbidity between epilepsy and depression are poorly understood. An important mechanism of depression is disinhibition within the hypothalamo-pituitary-adrenocortical (HPA) axis. We examined the functional state of the HPA axis in a rat model of co-morbidity between temporal lobe epilepsy and depression. Epilepsy was accompanied by the interictal elevation of plasma corticosterone, and by the positively combined dexamethasone/corticotropin releasing hormone test. The extent of the HPA hyperactivity was independent of recurrent seizures, but positively correlated with the severity of depressive behavior. We suggest that the observed hyperactivity of the HPA axis may underlie co-morbidity between epilepsy and depression.

Seizure-induced neuronal death in the immature brain.

The response of the developing brain to epileptic seizures and to status epilepticus is highly age-specific. Neonates with their low cerebral metabolic rate and fragmentary neuronal networks can tolerate relatively prolonged seizures without suffering massive cell death, but severe seizures in experimental animals inhibit brain growth, modify neuronal circuits, and can lead to behavioral deficits and to increases in neuronal excitability. Past infancy, the developing brain is characterized by high metabolic rate, exuberant neuronal and synaptic networks and overexpression of receptors and enzymes involved in excitotxic mechanisms. The outcome of seizures is highly model-dependent. Status epilepticus may produce massive neuronal death, behavioral deficits, synaptic reorganization and chronic epilepsy in some models, little damage in others. Long-term consequences are also highly age- and model-dependent. However, we now have some models which reliably lead to spontaneous seizures and chronic epilepsy in the vast majority of animals, demonstrating that seizure-induced epileptogenesis can occur in the developing brain. The mode cell death from status epilepticus is largely (but not exclusively) necrotic in adults, while the incidence of apoptosis increases at younger ages. Seizure-induced necrosis has many of the biochemical features of apoptosis, with early cytochrome release from mitochondria and capase activation. We speculate that this form of necrosis is associated with seizure-induced energy failure.

Galanin and galanin receptors in epilepsy.

The shift in the balance between the inhibition and the excitation in favor of the latter is a major mechanism of the evolvement of epileptic seizures. On the neurotransmitter level two major players contribute to such misbalance: an inhibitory transmitter gamma-aminobutyric acid, and an excitatory amino acid glutamate. Neuropeptides are powerful modulators of classical neurotransmitters, and thus represent an intriguing tool for restoring the balance between the inhibition and the excitation, through either blocking or activating peptide receptors depending on whether a peptide is pro- or anticonvulsant. Galanin, a 29-amino acid residues neuropeptide which inhibits glutamate release in the hippocampus, is a likely member of the anticonvulsant peptide family. During the past decade growing evidence has been suggesting that galanin is in fact a powerful inhibitor of seizure activity. This review summarizes the state of research of galanin in epilepsy, beginning with the first simple experiments which showed that central injection of galanin agonists inhibited seizures, and that seizures themselves affected galanin signaling in the hippocampus; exploring the impact of active manipulation with the expression of galanin and galanin receptors on seizures, using transgenic animals, antisense and peptide-expressing vector approaches; and concluding with the recent advances in pharmacology, which led to the synthesis of non-peptide galanin receptor agonists with anticonvulsant properties. We also address recently established functions of galanin in seizure-associated neuronal degeneration and neuronal plasticity.

Anticonvulsant effects of levetiracetam and levetiracetam-diazepam combinations in experimental status epilepticus.

Status epilepticus (SE) is a neurological emergency, with high mortality and high morbidity among survivors, and novel therapeutic agents are needed to improve this picture. We examined the effects of the antiepileptic drug levetiracetam (LEV) in an experimental model of self-sustaining status epilepticus (SSSE), induced in rats by electrical stimulation of the perforant path. LEV's unique spectrum of anticonvulsant activity, very high therapeutic index, and neuroprotective properties, make it a potentially interesting agent in the treatment of SE. Pretreatment with LEV intravenously reduced (30 mg/kg) or prevented (50-1000 mg/kg) the development of self-sustaining seizures. Treatment during the maintenance phase of SSSE diminished (at 200 mg/kg) or aborted seizures (in doses of 500 or 1000 mg/kg). Addition of LEV significantly enhanced the anticonvulsant effects of diazepam (DZP), even when both drugs where given in doses far below their therapeutic level. We conclude that LEV deserves further evaluation in the treatment of status epilepticus.

Anticonvulsant and antiepileptogenic effects of fluorofelbamate in experimental status epilepticus.

PURPOSE: To examine the seizure-protective properties of fluorofelbamate, a felbamate analog, on acute and chronic seizures in an experimental model of self-sustaining status epilepticus (SSSE). METHODS: SSSE was induced by stimulation of the perforant path for 30 min (PPS) through chronically implanted electrodes in free-running adult male Wistar rats. Fluorofelbamate was injected intravenously (i.v.) either 10 min, or 40 min after SSSE induction. Seizure and spike profiles were analyzed off-line. RESULTS: Fluorofelbamate injected during the early stages of SSSE (10 min after the end of PPS), shortened the duration of seizures in a dose-dependent manner. While a dose of 50 mg kg(-1) was ineffective, 100 and 200 mg kg(-1) reduced cumulative seizure time from 393 +/- 10 min to 15 +/- 8 min and 2.4 +/- 0.5 min respectively. Administration of fluorofelbamate (200 and 300 mg kg (-1)) at a late stage of SSSE, which is refractory to treatment with conventional anticonvulsants, also significantly attenuated seizures. Acute fluorofelbamate treatment (200 mg kg(-1) 10 min after PPS) significantly decreased the frequency of spontaneous seizures which follow SSSE after a 'latent' interval. Moreover, in contrast to control animals, fluorofelbamate-treated rats showed regression of spontaneous seizures, and an apparent remission of epilepsy within 2 months after SSSE. CONCLUSIONS: Acute treatment of SSSE with fluorofelbamate showed strong anticonvulsant effects even during the late stages of SSSE. In this model, it also displayed antiepileptogenic properties: it reduced the severity of chronic epilepsy after SSSE and lead to apparent remissions of that epilepsy.

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.

In vivo interaction between serotonin and galanin receptors types 1 and 2 in the dorsal raphe: implication for limbic seizures.

The neuropeptide galanin suppresses seizure activity in the hippocampus by inhibiting glutamatergic neurotransmission. Galanin may also modulate limbic seizures through interaction with other neurotransmitters in neuronal populations that project to the hippocampus. We examined the role of galanin receptors types 1 and 2 in the dorsal raphe (DR) in the regulation of serotonergic transmission and limbic seizures. Infusion of a mixed agonist of galanin receptors types 1 and 2 [galanin (1-29)] into the DR augmented the severity of limbic seizures in both rats and wild-type mice and concurrently reduced serotonin concentration in the DR and hippocampus as measured by immunofluorescence or HPLC. In contrast, injection of the galanin receptor type 2 agonist galanin (2-11) mitigated the severity of seizures in both species and increased serotonin concentration in both areas. Injection of both galanin fragments into the DR of galanin receptor type 1 knockout mice exerted anticonvulsant effects. Both the proconvulsant activity of galanin (1-29) and seizure suppression by galanin (2-11) were abolished in serotonin-depleted animals. Our data indicate that, in the DR, galanin receptors types 1 and 2 modulate serotonergic transmission in a negative and a positive fashion, respectively, and that these effects translate into either facilitation or inhibition of limbic seizures.

Short-term plasticity of hippocampal neuropeptides and neuronal circuitry in experimental status epilepticus.

PURPOSE: We used a model of self-staining status epilepticus (SSSE), induced by brief intermittent stimulation of the perforant path in unanesthetized rats, to study the mechanism of initiation and of maintenance of SSSE and the role of neuropeptides in those processes. METHODS: The perforant path was stimulated intermittently for 7 min (ineffective stimulation) or 30 min (generating SSSE). Peptides and their agonists and antagonists were delivered either intraperitoneally, or directly into the hippocampus through a implanted cannula. Behavior and electroencephalogram (EEG) were recorded through a videotape-telemetry system with automatic spike and seizures detection programs, which were supplemented by manual review of the records to confirm the diagnosis. Immunocytochemistry and enzyme-linked immunosorbent assay followed published methods. RESULTS: Initiation of SSSE was blocked by many agonists of inhibitory neurotransmitters or neuromodulators, and by many antagonists of excitatory synapses, and was facilitated by agents with the opposite action, suggesting the activation of a complex circuit with multiple potential entry points. Once SSSE was established, however, only N-methyl-d-aspartate (NMDA)-receptor ligands and a few neuropeptides had major effects on its maintenance. Galanin and dynorphin had powerful anticonvulsant roles in the maintenance phase of SSSE, whereas somatostatin and neuropeptide Y suppressed seizures only transiently. SSSE seemed to induce maladaptive changes in neuropeptides: it depleted the hippocampus of the galanin- and dynorphin-immunoreactive (IR) fibers, which normally function as endogenous anticonvulsants; whereas it induced overexpression of the proconvulsant neuropeptides substance P and neurokinin B; however, late in the course of SSSE, galanin-IR interneurons appeared in the dentate hilus. CONCLUSIONS: Initiation of SSSE seems to involve a circuit with many points of entry, and blockage of any point along this circuit inhibits the development of SSSE. Far fewer agents alter the maintenance phase of SSSE. Galanin, dynorphin, somatostatin, and neuropeptide Y have anticonvulsant roles, matching the previous described convulsant role of substance P and neurokinin B. Galanin and dynorphin seem to undergo maladaptive changes, which appear to play an important role of the maintenance phase of SSSE. Later, the de novo expression of inhibitory neuropeptides in novel cells in hippocampus coincides with the waning of seizures and may play a role in their termination.

Anticonvulsant activity of a nonpeptide galanin receptor agonist.

Galanin is a neuropeptide with a wide variety of biological functions, including that of a strong endogenous anticonvulsant. No nonpeptide ligands, capable of activating galanin receptors, are available today. Based on known pharmacophores of galanin, a combinatorial library was designed, synthesized, and screened at the rat hippocampal galanin receptor. A low molecular weight galanin receptor agonist, 7-((9-fluorenylmethoxycarbonyl)cyclohexylalanyllysyl)amino-4-methylcoumarin (galnon) was found to displace (125)I-galanin with micromolar affinity at Bowes cellular and rat hippocampal membranes. Autoradiographic binding assay on rat spinal cord sections confirmed the ability of galnon to displace (125)I-galanin from its binding sites. Galnon inhibited adenylate cyclase activity, suggesting an agonist action at galanin receptors. When injected i.p. galnon reduced the severity and increased the latency of pentylenetetrazole-induced seizures in mice and reversed the proconvulsant effects of the galanin receptor antagonist M35, injected into a lateral ventricle. Intrahippocampal injection of galnon also shortened the duration of self-sustaining status epilepticus in rats, confirming its agonist properties in vivo. Pretreatment of rats with antisense peptide nucleic acid targeted to galanin receptor type 1 mRNA abolished the effect of galnon, suggesting mediation of its anticonvulsant properties through this receptor subtype. These findings introduce a systemically active nonpeptide galanin agonist anticonvulsant.