Early treatment with rifaximin during epileptogenesis reverses gut alterations and reduces seizure duration in a mouse model of acquired epilepsy.

The gut microbiota is altered in epilepsy and is emerging as a potential target for new therapies. We studied the effects of rifaximin, a gastrointestinal tract-specific antibiotic, on seizures and neuropathology and on alterations in the gut and its microbiota in a mouse model of temporal lobe epilepsy (TLE). Epilepsy was induced by intra-amygdala kainate injection causing status epilepticus (SE) in C57Bl6 adult male mice. Sham mice were injected with vehicle. Two cohorts of SE mice were fed a rifaximin-supplemented diet for 21 days, starting either at 24 h post-SE (early disease stage) or at day 51 post-SE (chronic disease stage). Corresponding groups of SE mice (one each disease stage) were fed a standard (control) diet. Cortical ECoG recording was done at each disease stage (24/7) for 21 days in all SE mice to measure the number and duration of spontaneous seizures during either rifaximin treatment or control diet. Then, epileptic mice ± rifaximin and respective sham mice were sacrificed and brain, gut and feces collected. Biospecimens were used for: (i) quantitative histological analysis of the gut structural and cellular components; (ii) markers of gut inflammation and intestinal barrier integrity by RTqPCR; (iii) 16S rRNA metagenomics analysis in feces. Hippocampal neuronal cell loss was assessed in epileptic mice killed in the early disease phase. Rifaximin administered for 21 days post-SE (early disease stage) reduced seizure duration (p < 0.01) and prevented hilar mossy cells loss in the hippocampus compared to epileptic mice fed a control diet. Epileptic mice fed a control diet showed a reduction of both villus height and villus height/crypt depth ratio (p < 0.01) and a decreased number of goblet cells (p < 0.01) in the duodenum, as well as increased macrophage (Iba1)-immunostaining in the jejunum (p < 0.05), compared to respective sham mice. Rifaximin's effect on seizures was associated with a reversal of gut structural and cellular changes, except for goblet cells which remained reduced. Seizure duration in epileptic mice was negatively correlated with the number of mossy cells (p < 0.01) and with villus height/crypt depth ratio (p < 0.05). Rifaximin-treated epileptic mice also showed increased tight junctions (occludin and ZO-1, p < 0.01) and decreased TNF mRNA expression (p < 0.01) in the duodenum compared to epileptic mice fed a control diet. Rifaximin administered for 21 days in chronic epileptic mice (chronic disease stage) did not change the number or duration of seizures compared to epileptic mice fed a control diet. Chronic epileptic mice fed a control diet showed an increased crypt depth (p < 0.05) and reduced villus height/crypt depth ratio (p < 0.01) compared to respective sham mice. Rifaximin treatment did not affect these intestinal changes. At both disease stages, rifaximin modified α- and ß-diversity in epileptic and sham mice compared to respective mice fed a control diet. The microbiota composition in epileptic mice, as well as the effects of rifaximin at the phylum, family and genus levels, depended on the stage of the disease. During the early disease phase, the abundance of specific taxa was positively correlated with seizure duration in epileptic mice. In conclusion, gut-related alterations reflecting a dysfunctional state, occur during epilepsy development in a TLE mouse model. A short-term treatment with rifaximin during the early phase of the disease, reduced seizure duration and neuropathology, and reversed some intestinal changes, strengthening the therapeutic effects of gut-based therapies in epilepsy.

Identification of an epilepsy-linked gut microbiota signature in a pediatric rat model of acquired epilepsy.

A dysfunctional gut microbiota-brain axis is emerging as a potential pathogenic mechanism in epilepsy, particularly in pediatric forms of epilepsy. To add new insights into gut-related changes in acquired epilepsy that develops early in life, we used a multi-omics approach in a rat model with a 56% incidence of epilepsy. The presence of spontaneous seizures was assessed in adult rats (n = 46) 5 months after status epilepticus induced by intra-amygdala kainate at postnatal day 13, by 2 weeks (24/7) ECoG monitoring. Twenty-six rats developed epilepsy (Epi) while the remaining 20 rats (No-Epi) did not show spontaneous seizures. At the end of ECoG monitoring, all rats and their sham controls (n = 20) were sacrificed for quantitative histopathological and immunohistochemical analyses of the gut structure, glia and macrophages, as well as RTqPCR analysis of inflammation/oxidative stress markers. By comparing Epi, No-Epi rats, and sham controls, we found structural, cellular, and molecular alterations reflecting a dysfunctional gut, which were specifically associated with epilepsy. In particular, the villus height-to-crypt depth ratio and number of Goblet cells were reduced in the duodenum of Epi rats vs both No-Epi rats and sham controls (p < 0.01). Villus height and crypt depth in the duodenum and jejunum (p < 0.01) were increased in No-Epi vs both Epi and sham controls. We also detected enhanced Iba1-positive macrophages, together with increased IL1b and NFE2L2 transcripts and TNF protein, in the small intestine of Epi vs both No-Epi and sham control rats (p < 0.01), denoting the presence of inflammation and oxidative stress. Astroglial GFAP-immunostaining was similar in all experimental groups. Metagenomic analysis in the feces collected 5 months after status epilepticus showed that the ratio of two dominant phyla (Bacteroidota-to-Firmicutes) was similarly increased in Epi and No-Epi rats vs sham control rats. Notably, the relative abundance of families, genera, and species associated with SCFA production differed in Epi vs No-Epi rats, describing a bacterial imprint associated with epilepsy. Furthermore, Epi rats showed a blood metabolic signature characterized by changes in lipid metabolism compared to both No-Epi and sham control rats. Our study provides new evidence of long-term gut alterations, along with microbiota-related metabolic changes, occurring specifically in rats that develop epilepsy after brain injury early in life.

mTOR and neuroinflammation in epilepsy: implications for disease progression and treatment.

Epilepsy remains a major health concern as anti-seizure medications frequently fail, and there is currently no treatment to stop or prevent epileptogenesis, the process underlying the onset and progression of epilepsy. The identification of the pathological processes underlying epileptogenesis is instrumental to the development of drugs that may prevent the generation of seizures or control pharmaco-resistant seizures, which affect about 30% of patients. mTOR signalling and neuroinflammation have been recognized as critical pathways that are activated in brain cells in epilepsy. They represent a potential node of biological convergence in structural epilepsies with either a genetic or an acquired aetiology. Interventional studies in animal models and clinical studies give strong support to the involvement of each pathway in epilepsy. In this Review, we focus on available knowledge about the pathophysiological features of mTOR signalling and the neuroinflammatory brain response, and their interactions, in epilepsy. We discuss mitigation strategies for each pathway that display therapeutic effects in experimental and clinical epilepsy. A deeper understanding of these interconnected molecular cascades could enhance our strategies for managing epilepsy. This could pave the way for new treatments to fill the gaps in the development of preventative or disease-modifying drugs, thus overcoming the limitations of current symptomatic medications.

ECoG spiking activity and signal dimension are early predictive measures of epileptogenesis in a translational mouse model of traumatic brain injury.

The latency between traumatic brain injury (TBI) and the onset of epilepsy (PTE) represents an opportunity for counteracting epileptogenesis. Antiepileptogenesis trials are hampered by the lack of sensitive biomarkers that allow to enrich patient's population at-risk for PTE. We aimed to assess whether specific ECoG signals predict PTE in a clinically relevant mouse model with ∼60% epilepsy incidence. TBI was provoked in adult CD1 male mice by controlled cortical impact on the left parieto-temporal cortex, then mice were implanted with two perilesional cortical screw electrodes and two similar electrodes in the hemisphere contralateral to the lesion site. Acute seizures and spikes/sharp waves were ECoG-recorded during 1 week post-TBI. These early ECoG events were analyzed according to PTE incidence as assessed by measuring spontaneous recurrent seizures (SRS) at 5 months post-TBI. We found that incidence, number and duration of acute seizures during 3 days post-TBI were similar in PTE mice and mice not developing epilepsy (No SRS mice). Control mice with cortical electrodes (naïve, n = 5) or with electrodes and craniotomy (sham, n = 5) exhibited acute seizures but did not develop epilepsy. The daily number of spikes/sharp waves at the perilesional electrodes was increased similarly in PTE (n = 15) and No SRS (n = 8) mice vs controls (p < 0.05, n = 10) from day 2 post-injury. Differently, the daily number of spikes/sharp waves at both contralateral electrodes showed a progressive increase in PTE mice vs No SRS and control mice. In particular, spikes number was higher in PTE vs No SRS mice (p < 0.05) at 6 and 7 days post-TBI, and this measure predicted epilepsy development with high accuracy (AUC = 0.77, p = 0.03; CI 0.5830-0.9670). The cut-off value was validated in an independent cohort of TBI mice (n = 12). The daily spike number at the contralateral electrodes showed a circadian distribution in PTE mice which was not observed in No SRS mice. Analysis of non-linear dynamics at each electrode site showed changes in dimensionality during 4 days post-TBI. This measure yielded the best discrimination between PTE and No SRS mice (p < 0.01) at the cortical electrodes contralateral to injury. Data show that epileptiform activity contralateral to the lesion site has the the highest predictive value for PTE in this model reinforcing the hypothesis that the hemisphere contralateral to the lesion core may drive epileptogenic networks after TBI.

Neuroimmunology of status epilepticus.

Status epilepticus (SE) is a very heterogeneous clinical condition often refractory to available treatment options. Evidence in animal models shows that neuroinflammation arises in the brain during SE due to the activation of innate immune mechanisms in brain parenchyma cells. Intervention studies in animal models support the involvement of neuroinflammation in SE onset, duration, and severity, refractoriness to treatments, and long-term neurological consequences. Clinical evidence shows that neuroinflammation occurs in patients with SE of diverse etiologies likely representing a common phenomenon, thus broadening the involvement of the immune system beyond the infective and autoimmune etiologies. There is urgent need for novel therapies for refractory SE that rely upon a better understanding of the basic mechanisms underlying this clinical condition. Preclinical and clinical evidence encourage consideration of specific anti-inflammatory treatments for controlling SE and its consequences in patients.

Cholesterol 24-hydroxylase is a novel pharmacological target for anti-ictogenic and disease modification effects in epilepsy.

Therapies for epilepsy mainly provide symptomatic control of seizures since most of the available drugs do not target disease mechanisms. Moreover, about one-third of patients fail to achieve seizure control. To address the clinical need for disease-modifying therapies, research should focus on targets which permit interventions finely balanced between optimal efficacy and safety. One potential candidate is the brain-specific enzyme cholesterol 24-hydroxylase. This enzyme converts cholesterol to 24S-hydroxycholesterol, a metabolite which among its biological roles modulates neuronal functions relevant for hyperexcitability underlying seizures. To study the role of cholesterol 24-hydroxylase in epileptogenesis, we administered soticlestat (TAK-935/OV935), a potent and selective brain-penetrant inhibitor of the enzyme, during the early disease phase in a mouse model of acquired epilepsy using a clinically relevant dose. During soticlestat treatment, the onset of epilepsy was delayed and the number of ensuing seizures was decreased by about 3-fold compared to vehicle-treated mice, as assessed by EEG monitoring. Notably, the therapeutic effect was maintained 6.5 weeks after drug wash-out when seizure number was reduced by about 4-fold and their duration by 2-fold. Soticlestat-treated mice showed neuroprotection of hippocampal CA1 neurons and hilar mossy cells as assessed by post-mortem brain histology. High throughput RNA-sequencing of hippocampal neurons and glia in mice treated with soticlestat during epileptogenesis showed that inhibition of cholesterol 24-hydroxylase did not directly affect the epileptogenic transcriptional network, but rather modulated a non-overlapping set of genes that might oppose the pathogenic mechanisms of the disease. In human temporal lobe epileptic foci, we determined that cholesterol 24-hydroxylase expression trends higher in neurons, similarly to epileptic mice, while the enzyme is ectopically induced in astrocytes compared to control specimens. Soticlestat reduced significantly the number of spontaneous seizures in chronic epileptic mice when was administered during established epilepsy. Data show that cholesterol 24-hydroxylase contributes to spontaneous seizures and is involved in disease progression, thus it represents a novel target for chronic seizures inhibition and disease-modification therapy in epilepsy.

A systems-level analysis highlights microglial activation as a modifying factor in common epilepsies.

AIMS: The causes of distinct patterns of reduced cortical thickness in the common human epilepsies, detectable on neuroimaging and with important clinical consequences, are unknown. We investigated the underlying mechanisms of cortical thinning using a systems-level analysis. METHODS: Imaging-based cortical structural maps from a large-scale epilepsy neuroimaging study were overlaid with highly spatially resolved human brain gene expression data from the Allen Human Brain Atlas. Cell-type deconvolution, differential expression analysis and cell-type enrichment analyses were used to identify differences in cell-type distribution. These differences were followed up in post-mortem brain tissue from humans with epilepsy using Iba1 immunolabelling. Furthermore, to investigate a causal effect in cortical thinning, cell-type-specific depletion was used in a murine model of acquired epilepsy. RESULTS: We identified elevated fractions of microglia and endothelial cells in regions of reduced cortical thickness. Differentially expressed genes showed enrichment for microglial markers and, in particular, activated microglial states. Analysis of post-mortem brain tissue from humans with epilepsy confirmed excess activated microglia. In the murine model, transient depletion of activated microglia during the early phase of the disease development prevented cortical thinning and neuronal cell loss in the temporal cortex. Although the development of chronic seizures was unaffected, the epileptic mice with early depletion of activated microglia did not develop deficits in a non-spatial memory test seen in epileptic mice not depleted of microglia. CONCLUSIONS: These convergent data strongly implicate activated microglia in cortical thinning, representing a new dimension for concern and disease modification in the epilepsies, potentially distinct from seizure control.

CXCL1-CXCR1/2 signaling is induced in human temporal lobe epilepsy and contributes to seizures in a murine model of acquired epilepsy.

CXCL1, a functional murine orthologue of the human chemokine CXCL8 (IL-8), and its CXCR1 and CXCR2 receptors were investigated in a murine model of acquired epilepsy developing following status epilepticus (SE) induced by intra-amygdala kainate. CXCL8 and its receptors were also studied in human temporal lobe epilepsy (TLE). The functional involvement of the chemokine in seizure generation and neuronal cell loss was assessed in mice using reparixin (formerly referred to as repertaxin), a non-competitive allosteric inhibitor of CXCR1/2 receptors. We found a significant increase in hippocampal CXCL1 level within 24 h of SE onset that lasted for at least 1 week. No changes were measured in blood. In analogy with human TLE, immunohistochemistry in epileptic mice showed that CXCL1 and its two receptors were increased in hippocampal neuronal cells. Additional expression of these molecules was found in glia in human TLE. Mice were treated with reparixin or vehicle during SE and for additional 6 days thereafter, using subcutaneous osmotic minipumps. Drug-treated mice showed a faster SE decay, a reduced incidence of acute symptomatic seizures during 48 h post-SE, and a delayed time to spontaneous seizures onset compared to vehicle controls. Upon reparixin discontinuation, mice developed spontaneous seizures similar to vehicle mice, as shown by EEG monitoring at 14 days and 2.5 months post-SE. In the same epileptic mice, reparixin reduced neuronal cell loss in the hippocampus vs vehicle-injected mice, as assessed by Nissl staining at completion of EEG monitoring. Reparixin administration for 2 weeks in mice with established chronic seizures, reduced by 2-fold on average seizure number vs pre-treatment baseline, and this effect was reversible upon drug discontinuation. No significant changes in seizure number were measured in vehicle-injected epileptic mice that were EEG monitored in parallel. Data show that CXCL1-IL-8 signaling is activated in experimental and human epilepsy and contributes to acute and chronic seizures in mice, therefore representing a potential new target to attain anti-ictogenic effects.

Reorganization of Thalamic Inputs to Lesioned Cortex Following Experimental Traumatic Brain Injury.

Traumatic brain injury (TBI) disrupts thalamic and cortical integrity. The effect of post-injury reorganization and plasticity in thalamocortical pathways on the functional outcome remains unclear. We evaluated whether TBI causes structural changes in the thalamocortical axonal projection terminals in the primary somatosensory cortex (S1) that lead to hyperexcitability. TBI was induced in adult male Sprague Dawley rats with lateral fluid-percussion injury. A virus carrying the fluorescent-tagged opsin channel rhodopsin 2 transgene was injected into the ventroposterior thalamus. We then traced the thalamocortical pathways and analyzed the reorganization of their axonal terminals in S1. Next, we optogenetically stimulated the thalamocortical relays from the ventral posterior lateral and medial nuclei to assess the post-TBI functionality of the pathway. Immunohistochemical analysis revealed that TBI did not alter the spatial distribution or lamina-specific targeting of projection terminals in S1. TBI reduced the axon terminal density in the motor cortex by 44% and in S1 by 30%. A nematic tensor-based analysis revealed that in control rats, the axon terminals in layer V were orientated perpendicular to the pial surface (60.3°). In TBI rats their orientation was more parallel to the pial surface (5.43°, difference between the groups p < 0.05). Moreover, the level of anisotropy of the axon terminals was high in controls (0.063) compared with TBI rats (0.045, p < 0.05). Optical stimulation of the sensory thalamus increased alpha activity in electroencephalography by 312% in controls (p > 0.05) and 237% (p > 0.05) in TBI rats compared with the baseline. However, only TBI rats showed increased beta activity (33%) with harmonics at 5 Hz. Our findings indicate that TBI induces reorganization of thalamocortical axonal terminals in the perilesional cortex, which alters responses to thalamic stimulation.

Microglia proliferation plays distinct roles in acquired epilepsy depending on disease stages.

OBJECTIVE: Microgliosis occurs in animal models of acquired epilepsy and in patients. It includes cell proliferation that is associated with seizure frequency and decreased neuronal cells in human epilepsy. The role of microglia proliferation in the development of acquired epilepsy is unknown; thus, we examined its contribution to spontaneous seizure, neurodegeneration, and cognitive deficits in different disease phases. METHODS: We used a model of acquired epilepsy triggered by intra-amygdala kainic acid in C57BL6N adult male mice. Mice were electroencephalographically (EEG) monitored (24/7) during status epilepticus and in early and chronic disease. Microglia proliferation was blocked by GW2580, a selective CSF1 receptor inhibitor, supplemented in the diet for 21 days from status epilepticus onset. Then, mice were returned to placebo diet until experiment completion. Control mice were exposed to status epilepticus and fed with placebo diet. Experimental mice were tested in the novel object recognition test (NORT) and in Barnes maze, and compared to control and sham mice. At the end of the behavioral test, mice were killed for brain histopathological analysis. Additionally, seizure baseline was monitored in chronic epileptic mice, then mice were fed for 14 days with GW2580 or placebo diet under 24/7 EEG recording. RESULTS: GW2580 prevented microglia proliferation in mice undergoing epilepsy, whereas it did not affect microglia or basal excitatory neurotransmission in the hippocampus of naive mice. Mice with occluded microglia proliferation during early disease development underwent status epilepticus and subsequent epilepsy similar to placebo diet mice, and were similarly impaired in NORT, with improvement in Barnes maze. GW2580-treated mice displayed neuroprotection in the hippocampus. In contrast, blockade of microglia proliferation in chronic epileptic mice resulted in spontaneous seizure reduction versus placebo mice. SIGNIFICANCE: Microglia proliferation during early disease contributes to neurodegeneration, whereas in late chronic disease it contributes to seizures. Timely pharmacological interference with microglia proliferation may offer a potential target for improving disease outcomes.

In-depth characterization of a mouse model of post-traumatic epilepsy for biomarker and drug discovery.

Post-traumatic epilepsy (PTE) accounts for 5% of all epilepsies and 10-20% of the acquired forms. The latency between traumatic brain injury (TBI) and epilepsy onset in high-risk patients offers a therapeutic window for intervention to prevent or improve the disease course. However, progress towards effective treatments has been hampered by the lack of sensitive prognostic biomarkers of PTE, and of therapeutic targets. There is therefore a pressing clinical need for preclinical PTE models suitable for biomarker discovery and drug testing. We characterized in-depth a model of severe TBI induced by controlled cortical impact evolving into PTE in CD1 adult male mice. To identify sensitive measures predictive of PTE development and severity, TBI mice were longitudinally monitored by video-electrocorticography (ECoG), examined by MRI, and tested for sensorimotor and cognitive deficits and locomotor activity. At the end of the video-ECoG recording mice were killed for brain histological analysis. PTE occurred in 58% of mice with frequent motor seizures (one seizure every other day), as determined up to 5 months post-TBI. The weight loss of PTE mice in 1 week after TBI correlated with the number of spontaneous seizures at 5 months. Moreover, the recovery rate of the sensorimotor deficit detected by the SNAP test before the predicted time of epilepsy onset was significantly lower in PTE mice than in those without epilepsy. Neuroscore, beam walk and cognitive deficit were similar in all TBI mice. The increase in the contusion volume, the volume of forebrain regions contralateral to the lesioned hemisphere and white matter changes over time assessed by MRI were similar in PTE and no-PTE mice. However, brain histology showed a more pronounced neuronal cell loss in the cortex and hippocampus contralateral to the injured hemisphere in PTE than in no-PTE mice. The extensive functional and neuropathological characterization of this TBI model, provides a tool to identify sensitive measures of epilepsy development and severity clinically useful for increasing PTE prediction in high-risk TBI patients. The high PTE incidence and spontaneous seizures frequency in mice provide an ideal model for biomarker discovery and for testing new drugs.