Drug-resistant epilepsy (DRE) affects approximately one-third of the patients with epilepsy. Based on experimental findings from animal models and brain tissue from patients with DRE, different hypotheses have been proposed to explain the cause(s) of drug resistance. One is the intrinsic severity hypothesis that posits that drug resistance is an inherent property of epilepsy related to disease severity. Seizure frequency is one measure of epilepsy severity, but frequency alone is an incomplete measure of severity and does not fully explain basic research and clinical studies on drug resistance; thus, other measures of epilepsy severity are needed. One such measure could be pathological high-frequency oscillations (HFOs), which are believed to reflect the neuronal disturbances responsible for the development of epilepsy and the generation of spontaneous seizures. In this manuscript, we will briefly review the intrinsic severity hypothesis, describe basic and clinical research on HFOs in the epileptic brain, and based on this evidence discuss whether HFOs could be a clinical measure of epilepsy severity. Understanding the mechanisms of DRE is critical for producing breakthroughs in the development and testing of novel strategies for treatment.

Post-traumatic epilepsy is the architype of acquired epilepsies, wherein a brain insult initiates an epileptogenic process culminating in an unprovoked seizure after weeks, months or years. Identifying biomarkers of such process is a prerequisite for developing and implementing targeted therapies aimed at preventing the development of epilepsy. Currently, there are no validated electrophysiological biomarkers of post-traumatic epileptogenesis. Experimental EEG studies using the lateral fluid percussion injury model have identified three candidate biomarkers of post-traumatic epileptogenesis: pathological high-frequency oscillations (HFOs, 80-300 Hz); repetitive HFOs and spikes (rHFOSs); and reduction in sleep spindle duration and dominant frequency at the transition from stage III to rapid eye movement sleep. EEG studies in humans have yielded conflicting data; recent evidence suggests that epileptiform abnormalities detected acutely after traumatic brain injury carry a significantly increased risk of subsequent epilepsy. Well-designed studies are required to validate these promising findings, and ultimately establish whether there are post-traumatic electrophysiological features which can guide the development of antiepileptogenic therapies.

Transcranial focal stimulation (TFS) is a non-invasive neuromodulation strategy with neuroprotective effects. On the other hand, 6-hidroxidopamine (6-OHDA) induces neurodegeneration of the nigrostriatal system producing modifications in the dopaminergic, serotoninergic, and histaminergic systems. The present study was conducted to test whether repetitive application of TFS avoids the biogenic amines changes induced by the intrastriatal injection of 6-OHDA. Experiments were designed to determine the tissue content of dopamine, serotonin, and histamine in the brain of animals injected with 6-OHDA and then receiving daily TFS for 21 days. Tissue content of biogenic amines was evaluated in the cerebral cortex, hippocampus, amygdala, and striatum, ipsi- and contralateral to the side of 6-OHDA injection. Results obtained were compared to animals with 6-OHDA, TFS alone, and a Sham group. The present study revealed that TFS did not avoid the changes in the tissue content of dopamine in striatum. However, TFS was able to avoid several of the changes induced by 6-OHDA in the tissue content of dopamine, serotonin, and histamine in the different brain areas evaluated. Interestingly, TFS alone did not induce significant changes in the different brain areas evaluated. The present study showed that repetitive TFS avoids the biogenic amines changes induced by 6-OHDA. TFS can represent a new therapeutic strategy to avoid the neurotoxicity induced by 6-OHDA.

Traumatic brain injury (TBI) is defined as a disturbance in brain functioning caused by an external force. The development of post traumatic epilepsy (PTE) is a serious risk associated with TBI. Indeed, other neurological impairments are also common following TBI. In this review, we analyze and discuss the most widely used and best validated rodent models of TBI, with a particular focus on their contribution to the understanding of the PTE development. Furthermore, we explore the importance of these models for the study of other neurobehavioral comorbidities associated with brain injury. The efficient and accurate diagnosis of epilepsy and other neurological comorbidities as a consequence of brain trauma should improve the survival and quality of life of patients after TBI.

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.

Objective. This study aims to develop and validate an end-to-end software platform, PyHFO, that streamlines the application of deep learning (DL) methodologies in detecting neurophysiological biomarkers for epileptogenic zones from EEG recordings.Approach. We introduced PyHFO, which enables time-efficient high-frequency oscillation (HFO) detection algorithms like short-term energy and Montreal Neurological Institute and Hospital detectors. It incorporates DL models for artifact and HFO with spike classification, designed to operate efficiently on standard computer hardware.Main results. The validation of PyHFO was conducted on three separate datasets: the first comprised solely of grid/strip electrodes, the second a combination of grid/strip and depth electrodes, and the third derived from rodent studies, which sampled the neocortex and hippocampus using depth electrodes. PyHFO demonstrated an ability to handle datasets efficiently, with optimization techniques enabling it to achieve speeds up to 50 times faster than traditional HFO detection applications. Users have the flexibility to employ our pre-trained DL model or use their EEG data for custom model training.Significance. PyHFO successfully bridges the computational challenge faced in applying DL techniques to EEG data analysis in epilepsy studies, presenting a feasible solution for both clinical and research settings. By offering a user-friendly and computationally efficient platform, PyHFO paves the way for broader adoption of advanced EEG data analysis tools in clinical practice and fosters potential for large-scale research collaborations.

Rationale

The Epilepsy Bioinformatics Study for Antiepileptogenic Therapy (EpiBioS4Rx) Centre without walls is an NIH funded multicenter consortium. One of EpiBioS4Rx projects is a preclinical post-traumatic epileptogenesis biomarker study that involves three study sites: The University of Eastern Finland, Monash University (Melbourne) and the University of California Los Angeles. Our objective is to create a platform for evaluating biomarkers and testing new antiepileptogenic treatments for post-traumatic epilepsy (PTE) using the lateral fluid percussion injury (FPI) model in rats. As only 30-50% of rats with severe lateral FPI develop PTE by 6 months post-injury, prolonged video-EEG monitoring is crucial to identify animals with PTE. Our objective is to harmonize the surgical and data collection procedures, equipment, and data analysis for chronic EEG recording in order to phenotype PTE in this rat model across the three study sites.

Methods

Traumatic brain injury (TBI) was induced using lateral FPI in adult male Sprague-Dawley rats aged 11-12 weeks. Animals were divided into two cohorts: a) the long-term video-EEG follow-up cohort (Specific Aim 1), which was implanted with EEG electrodes within 24 h after the injury; and b) the magnetic resonance imaging (MRI) follow-up cohort (Specific Aim 2), at 5 months after lateral FPI. Four cortical epidural screw electrodes (2 ipsilateral, 2 contralateral) and three intracerebral bipolar electrodes were implanted (septal CA1 and the dentate gyrus, layers II and VI of the perilesional cortex both anterior and posterior to the injury site). During the 7th post-TBI month, animals underwent 4 weeks of continuous video-EEG recordings to diagnose of PTE.

Results

All centers harmonized the induction of TBI and surgical procedures for the implantation of EEG recordings, utilizing 4 or more EEG recording channels to cover areas ipsilateral and contralateral to the brain injury, perilesional cortex and the hippocampus and dentate gyrus. Ground and reference screw electrodes were implanted. At all sites the minimum sampling rate was 512 Hz, utilizing a finite impulse response (FIR) and impedance below 10 KΩ through the entire recording. As part of the quality control criteria we avoided electrical noise, and monitoring changes in impedance over time and the appearance of noise on the recordings. To reduce electrical noise, we regularly checked the integrity of the cables, stability of the EEG recording cap and the appropriate connection of the electrodes with the cables. Following the pipeline presented in this article and after applying the quality control criteria to our EEG recordings all of the sites were successful to phenotype seizure in chronic EEG recordings of animals after TBI.

Discussion

Despite differences in video-EEG acquisition equipment used, the three centers were able to consistently phenotype seizures in the lateral fluid-percussion model applying the pipeline presented here. The harmonization of methodology will help to improve the rigor of preclinical research, improving reproducibility of pre-clinical research in the search of biomarkers and therapies to prevent antiepileptogenesis.

The Epilepsy Bioinformatics Study for Antiepileptogenic Therapy (EpiBioS4Rx) is an international, multicenter, multidisciplinary study aimed at preventing epileptogenesis (EpiBioS4Rx: https://epibios.loni.usc.edu/). One of the study's major objectives is the discovery of diagnostic, prognostic, and predictive plasma protein and microRNA (miRNA) biomarkers that are sensitive, specific, and translatable to the human condition. Epilepsy due to structural brain abnormalities, secondary to neurological insults such as traumatic brain injury (TBI), currently represents ∼50% of all epilepsy cases. In the preclinical EpiBioS4Rx study, TBI was induced in adult male Sprague Dawley rats using a standardized protocol for lateral fluid-percussion injury. Whole blood was collected from the tail vein at baseline and 2, 9 and 30 days post-injury and processed for plasma separation. Biomaterial properties, sample preparation and integrity, and choice of analysis platform can significantly impact measured marker levels and, in turn, interpretation with respect to injury and/or other variables. We present here the results of procedural harmonization for the first 320 rats included in the EpiBioS4Rx study study, from three international research centers, and preliminary proteomic and miRNA analyses. We also discuss experimental considerations for establishing rigorous quality controls with the goal of harmonizing operating procedures across study sites, and delivering high-quality specimens for preclinical biomarker discovery in a rat model of post-traumatic epilepsy (PTE).