Transcriptomic alterations in cortical astrocytes following the development of post-traumatic epilepsy.

Post-traumatic epilepsy (PTE) stands as one of the numerous debilitating consequences that follow traumatic brain injury (TBI). Despite its impact on many individuals, the current landscape offers only a limited array of reliable treatment options, and our understanding of the underlying mechanisms and susceptibility factors remains incomplete. Among the potential contributors to epileptogenesis, astrocytes, a type of glial cell, have garnered substantial attention as they are believed to promote hyperexcitability and the development of seizures in the brain following TBI. The current study evaluated the transcriptomic changes in cortical astrocytes derived from animals that developed seizures as a result of severe focal TBI. Using RNA-Seq and ingenuity pathway analysis (IPA), we unveil a distinct gene expression profile in astrocytes, including alterations in genes supporting inflammation, early response modifiers, and neuropeptide-amidating enzymes. The findings underscore the complex molecular dynamics in astrocytes during PTE development, offering insights into therapeutic targets and avenues for further exploration.

Molecular mechanism analyses of post-traumatic epilepsy and hereditary epilepsy based on 10× single-cell transcriptome sequencing technology.

BACKGROUND: Single-cell RNA sequencing analysis has been usually conducted on post-traumatic epilepsy (PET) and hereditary epilepsy (HE) patients; however, the transcriptome of patients with traumatic temporal lobe epilepsy has rarely been studied. MATERIALS AND METHODS: Hippocampus tissues isolated from one patient with PTE and one patient with HE were used in the present study. Single cell isolates were prepared and captured using a 10× Genomics Chromium Single-Cell 3' kit (V3) according to the manufacturer's instructions. The libraries were sequenced on an Illumina NovaSeq 6000 sequencing system. Raw data were processed, and the cells were filtered and classified using the Seurat R package. Uniform Manifold Approximation and Projection was used for visualization. Differentially expressed genes (DEGs) were identified based on a p-value ≤0.01 and log fold change (FC) ≥0.25. Gene Ontology (GO, http://geneontology.org/) and KEGG (Kyoto Encyclopedia of Genes and Genomes, www.genome.jp/kegg) analyses were performed on the DEGs for enrichment analysis. RESULTS: The reads obtained from the 10× genomic platform for PTE and HE were 39.56 M and 30.08 M, respectively. The Q30 score of the RNA reads was >91.6%. After filtering, 7479 PTE cells and 9357 HE cells remained for further study. More than 96.4% of the reads were mapped to GRCh38/GRCm38. The cells were differentially distributed in two groups, with higher numbers of oligodendrocytes (6522 vs. 2532) and astrocytes (133 vs. 52), and lower numbers of microglial cells (2242 vs. 3811), and neurons (3 vs. 203) present in the HE group than in the PTE group. The DEGs in four cell clusters were identified, with 25 being in oligodendrocytes (13 upregulated and 12 downregulated), 87 in microglia cells (42 upregulated and 45 downregulated), 222 in astrocytes (115 upregulated and 107 downregulated), and 393 in neurons (305 upregulated and 88 downregulated). The genes MTND1P23 (downregulated), XIST (downregulated), and RPS4Y1 (upregulated) were commonly expressed in all four cell clusters. The DEGs in microglial cells and astrocytes were enriched in the IL-17 signaling pathway. CONCLUSION: Our study explored differences in cells found in a patient with PE compared to a patient with HE, and the transcriptome in the different cells was analyzed for the first time. Studying inflammatory and immune functions might be the best approach for investigating traumatic temporal lobe epilepsy in neurons.

Rapid volume pulsations of the extracellular space accompany epileptiform activity in trauma-injured neocortex and depend on the sodium-bicarbonate cotransporter NBCe1.

Post traumatic epilepsy (PTE) is a treatment-resistant consequence of traumatic brain injury (TBI). Recently, it has been revealed that epileptiform activity in acute chemoconvulsant seizure models is accompanied by transient shrinkages of extracellular space (ECS) called rapid volume pulsations (RVPs). Shrinkage of the ECS surrounding neurons and glia may contribute to ictogenic hyperexcitability and hypersynchrony during the chronic phase of TBI. Here, we identify the phenomenon of RVPs occurring spontaneously in rat neocortex at ≥ 3 weeks after injury in the controlled cortical impact (CCI) model for PTE. We further report that blocking the electrogenic action of the astrocytic cotransporter NBCe1 with 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) eliminates both RVPs and epileptiform activity in ex-vivo CCI neocortical brain slices. We conclude that NBCe1-mediated extracellular volume shrinkage may represent a new target for therapeutic intervention in PTE.

Applying Proteomics and Computational Approaches to Identify Novel Targets in Blast-Associated Post-Traumatic Epilepsy.

Traumatic brain injury (TBI) can lead to post-traumatic epilepsy (PTE). Blast TBI (bTBI) found in Veterans presents with several complications, including cognitive and behavioral disturbances and PTE; however, the underlying mechanisms that drive the long-term sequelae are not well understood. Using an unbiased proteomics approach in a mouse model of repeated bTBI (rbTBI), this study addresses this gap in the knowledge. After rbTBI, mice were monitored using continuous, uninterrupted video-EEG for up to four months. Following this period, we collected cortex and hippocampus tissues from three groups of mice: those with post-traumatic epilepsy (PTE+), those without epilepsy (PTE-), and the control group (sham). Hundreds of differentially expressed proteins were identified in the cortex and hippocampus of PTE+ and PTE- relative to sham. Focusing on protein pathways unique to PTE+, pathways related to mitochondrial function, post-translational modifications, and transport were disrupted. Computational metabolic modeling using dysregulated protein expression predicted mitochondrial proton pump dysregulation, suggesting electron transport chain dysregulation in the epileptic tissue relative to PTE-. Finally, data mining enabled the identification of several novel and previously validated TBI and epilepsy biomarkers in our data set, many of which were found to already be targeted by drugs in various phases of clinical testing. These findings highlight novel proteins and protein pathways that may drive the chronic PTE sequelae following rbTBI.

Post-traumatic seizures and factors associated among adult patients with depressed skull fractures at Mulago National Referral hospital; cross-sectional study.

INTRODUCTION: Post-traumatic seizures (PTS) are common among patients with depressed skull fractures (DSF). Understanding the burden of post traumatic seizures and the factors associated among adult patients with DSF is important to improve clinical care. OBJECTIVE: To determine the prevalence and factors associated with post-traumatic seizures among adult patients with DSF at Mulago National Referral hospital (MNRH). METHODS: A cross-sectional study was conducted among 333 study participants between March 2021 and February 2022. Socio-demographic, clinical laboratory factors and anti-seizure medications were collected using a study questionnaire. Data was analysed to determine the prevalence of PTS and factors associated with occurrence of PTS among patients with DSF. RESULTS: The mean age (±SD) of study participants was 31.2, (±10.5) years, with a male to female ratio of 10.4:1. Nearly half of the study participants had attained secondary level of education, while 31.6 % (105) were peasants (subsistence farmers). The overall prevalence of PTS among DSF study participants was 16.2 % (54participants). Late presentation of PTS was the highest at 9.0 % (30) followed by early PTS at 3.9 % [13] and immediate PTS at 3.3 % [11]. Moderate Glasgow coma score (GCS: 9-13), p < 0.015, severe traumatic brain injury (GCS: 3-8), p < 0.026 at the time of admission and midline brain shift (≥5mm), p < 0.009 were associated with PTS. Phenytoin (94.3 %) was the most commonly used ASM followed by phenobarbitone (1.4 %) and Valproate (1.1 %) among study participants. CONCLUSION: Patients with moderate and severe traumatic brain injury and midline brain shift were associated with post traumatic seizures. Early identification and intervention may reduce the burden of posttraumatic seizures in this category of patients.

Clinical characteristics and associated factors of posttraumatic epilepsy after traumatic brain injury in children: A retrospective case-control study.

BACKGROUND: Traumatic brain injury (TBI) affects approximately 69 million individuals annually, often resulting in well-documented complications such as epilepsy. Although numerous studies have been performed on posttraumatic epilepsy (PTE) in adults over the past decade, research on chronic consequences of TBI in children remains limited. Herein, we retrospectively assessed children who had experienced moderate to severe TBI to determine their clinical characteristics and identify associated factors associated with the development of PTE in the pediatric population. METHODS: The study population comprised children aged 0-18 years who had experienced moderate to severe TBI and underwent treatment at the Children's Hospital of Chongqing Medical University between 2011 and 2021. They were categorized into two groups: the PTE group, comprising individuals diagnosed with PTE within a one-year follow-up period, and the nPTE group, consisting of those who did not develop PTE during the same timeframe. The primary objective was to investigate the clinical characteristics and identify related associated factors. The relationship between various clinical factors and the incidence of PTE was assessed through univariate and multivariate logistic regression. RESULTS: A total of 132 patients were assessed. Most participants were male (65%) and the age distribution skewed towards younger children, with a median age of 41.0 months (interquartile range: 45.3). Upon their last clinical visit, 64 children (49%) were diagnosed with PTE. Notably, the first posttraumatic seizure predominantly occurred within the first week following the traumatic event. Further analyses revealed that increasing injury severity, as indicated by a lower Glasgow Coma Scale (GCS) score (odds ratio [OR]: 0.78, 95% confidence interval [CI]: 0.54-1.12, p= 0.018), a contusion load ≥3 (OR: 8.1, 95% CI: 2.3-28.9, p= 0.001), immediate posttraumatic seizures (IPTS) (OR: 8.9, 95% CI: 2.5-31.2, p < 0.001), and early posttraumatic seizures (EPTS) (OR: 54, 95% CI: 11-276, p < 0.001), were all significantly associated with a higher risk of developing PTE. CONCLUSION: This study highlights that the onset of PTE was associated with the markers of injury severity or PTS and identified GCS scores, contusion loads of ≥3, IPTS, and EPTS as independent associated factors significantly associated with the development of PTE.

Genetic diversity drives extreme responses to traumatic brain injury and post-traumatic epilepsy.

Traumatic brain injury (TBI) is a complex and heterogeneous condition that can cause wide-spectral neurological sequelae such as behavioral deficits, sleep abnormalities, and post-traumatic epilepsy (PTE). However, understanding the interaction of TBI phenome is challenging because few animal models can recapitulate the heterogeneity of TBI outcomes. We leveraged the genetically diverse recombinant inbred Collaborative Cross (CC) mice panel and systematically characterized TBI-related outcomes in males from 12 strains of CC and the reference C57BL/6J mice. We identified unprecedented extreme responses in multiple clinically relevant traits across CC strains, including weight change, mortality, locomotor activity, cognition, and sleep. Notably, we identified CC031 mouse strain as the first rodent model of PTE that exhibit frequent and progressive post-traumatic seizures after moderate TBI induced by lateral fluid percussion. Multivariate analysis pinpointed novel biological interactions and three principal components across TBI-related modalities. Estimate of the proportion of TBI phenotypic variability attributable to strain revealed large range of heritability, including >70% heritability of open arm entry time of elevated plus maze. Our work provides novel resources and models that can facilitate genetic mapping and the understanding of the pathobiology of TBI and PTE.

Brain abscess - A rare confounding factor for diagnosis of post-traumatic epilepsy after lateral fluid-percussion injury.

OBJECTIVE: To assess the prevalence of brain abscesses as a confounding factor for the diagnosis of post-traumatic epilepsy (PTE) in a rat model of lateral fluid-percussion-induced (FPI) traumatic brain injury (TBI). METHODS: This retrospective study included 583 rats from 3 study cohorts collected over 2009-2022 in a single laboratory. The rats had undergone sham-operation or TBI using lateral FPI. Rats were implanted with epidural and/or intracerebral electrodes for electroencephalogram recordings. Brains were processed for histology to screen for abscess(es). In abscess cases, (a) unfolded cortical maps were constructed to assess the cortical location and area of the abscess, (b) the abscess tissue was Gram stained to determine the presence of gram-positive and gram-negative bacteria, and (c) immunostaining was performed to detect infiltrating neutrophils, T-lymphocytes, and glial cells as tissue biomarkers of inflammation. In vivo and/or ex vivo magnetic resonance images available from a subcohort of animals were reviewed to evaluate the presence of abscesses. Plasma samples available from a subcohort of rats were used for enzyme-linked immunosorbent assays to determine the levels of lipopolysaccharide (LPS) as a circulating biomarker for gram-negative bacteria. RESULTS: Brain abscesses were detected in 2.6% (15/583) of the rats (6 sham, 9 TBI). In histology, brain abscesses were characterized as vascularized encapsulated lesions filled with neutrophils and surrounded by microglia/macrophages and astrocytes. The abscesses were mainly located under the screw electrodes, support screws, or craniectomy. Epilepsy was diagnosed in 60% (9/15) of rats with an abscess (4 sham, 5 TBI). Of these, 67% (6/9) had seizure clusters. The average seizure frequency in abscess cases was 0.436 ± 0.281 seizures/d. Plasma LPS levels were comparable between rats with and without abscesses (p > 0.05). SIGNIFICANCE: Although rare, a brain abscess is a potential confounding factor for epilepsy diagnosis in animal models of structural epilepsies following brain surgery and electrode implantation, particularly if seizures occur in sham-operated experimental controls and/or in clusters.

Successful harmonization in EpiBioS4Rx biomarker study on post-traumatic epilepsy paves the way towards powered preclinical multicenter studies.

OBJECTIVE: Project 1 of the Preclinical Multicenter Epilepsy Bioinformatics Study for Antiepileptogenic Therapy (EpiBioS4Rx) consortium aims to identify preclinical biomarkers for antiepileptogenic therapies following traumatic brain injury (TBI). The international participating centers in Finland, Australia, and the United States have made a concerted effort to ensure protocol harmonization. Here, we evaluate the success of harmonization process by assessing the timing, coverage, and performance between the study sites. METHOD: We collected data on animal housing conditions, lateral fluid-percussion injury model production, postoperative care, mortality, post-TBI physiological monitoring, timing of blood sampling and quality, MR imaging timing and protocols, and duration of video-electroencephalography (EEG) follow-up using common data elements. Learning effect in harmonization was assessed by comparing procedural accuracy between the early and late stages of the project. RESULTS: The animal housing conditions were comparable between the study sites but the postoperative care procedures varied. Impact pressure, duration of apnea, righting reflex, and acute mortality differed between the study sites (p < 0.001). The severity of TBI on D2 post TBI assessed using the composite neuroscore test was similar between the sites, but recovery of acute somato-motor deficits varied (p < 0.001). A total of 99% of rats included in the final cohort in UEF, 100% in Monash, and 79% in UCLA had blood samples taken at all time points. The timing of sampling differed on day (D)2 (p < 0.05) but not D9 (p > 0.05). Plasma quality was poor in 4% of the samples in UEF, 1% in Monash and 14% in UCLA. More than 97% of the final cohort were MR imaged at all timepoints in all study sites. The timing of imaging did not differ on D2 and D9 (p > 0.05), but varied at D30, 5 months, and ex vivo timepoints (p < 0.001). The percentage of rats that completed the monthly high-density video-EEG follow-up and the duration of video-EEG recording on the 7th post-injury month used for seizure detection for diagnosis of post-traumatic epilepsy differed between the sites (p < 0.001), yet the prevalence of PTE (UEF 21%, Monash 22%, UCLA 23%) was comparable between the sites (p > 0.05). A decrease in acute mortality and increase in plasma quality across time reflected a learning effect in the TBI production and blood sampling protocols. SIGNIFICANCE: Our study is the first demonstration of the feasibility of protocol harmonization for performing powered preclinical multi-center trials for biomarker and therapy discovery of post-traumatic epilepsy.

Epilepsy phenotype and its reproducibility after lateral fluid percussion-induced traumatic brain injury in rats: Multicenter EpiBioS4Rx study project 1.

OBJECTIVE: This study was undertaken to assess reproducibility of the epilepsy outcome and phenotype in a lateral fluid percussion model of posttraumatic epilepsy (PTE) across three study sites. METHODS: A total of 525 adult male Sprague Dawley rats were randomized to lateral fluid percussion-induced brain injury (FPI) or sham operation. Of these, 264 were assigned to magnetic resonance imaging (MRI cohort, 43 sham, 221 traumatic brain injury [TBI]) and 261 to electrophysiological follow-up (EEG cohort, 41 sham, 220 TBI). A major effort was made to harmonize the rats, materials, equipment, procedures, and monitoring systems. On the 7th post-TBI month, rats were video-EEG monitored for epilepsy diagnosis. RESULTS: A total of 245 rats were video-EEG phenotyped for epilepsy on the 7th postinjury month (121 in MRI cohort, 124 in EEG cohort). In the whole cohort (n = 245), the prevalence of PTE in rats with TBI was 22%, being 27% in the MRI and 18% in the EEG cohort (p > .05). Prevalence of PTE did not differ between the three study sites (p > .05). The average seizure frequency was .317 ± .725 seizures/day at University of Eastern Finland (UEF; Finland), .085 ± .067 at Monash University (Monash; Australia), and .299 ± .266 at University of California, Los Angeles (UCLA; USA; p < .01 as compared to Monash). The average seizure duration did not differ between UEF (104 ± 48 s), Monash (90 ± 33 s), and UCLA (105 ± 473 s; p > .05). Of the 219 seizures, 53% occurred as part of a seizure cluster (≥3 seizures/24 h; p >.05 between the study sites). Of the 209 seizures, 56% occurred during lights-on period and 44% during lights-off period (p > .05 between the study sites). SIGNIFICANCE: The PTE phenotype induced by lateral FPI is reproducible in a multicenter design. Our study supports the feasibility of performing preclinical multicenter trials in PTE to increase statistical power and experimental rigor to produce clinically translatable data to combat epileptogenesis after TBI.

Surgical Outcomes in Post-Traumatic Temporal Lobe Epilepsy: A Systematic Review and Meta-Analysis.

Epilepsy surgery provides excellent benefits in post-traumatic epilepsy of the temporal lobe (PTE-TL), but outcomes relative to non-traumatic epilepsy of the temporal lobe (NTE-TL) are less favorable. Large well-designed studies are recommended to further clarify the role of epilepsy surgery in PTE. It is unclear whether epilepsy surgery outcomes in PTE are as robust as described for drug resistant epilepsy (DRE) in general. Prior outcome studies in PTE are limited by small numbers, lack of a control group, or both. We performed a meta-analysis of studies in temporal lobe epilepsy (TLE) to evaluate post-surgical outcomes in those with PTE-TL and compare outcomes to those with NTE-TL. PubMed, EMBASE, and Web of Science databases were queried for studies reporting epilepsy surgery outcomes separately for PTE-TL and NTE-TL. Outcomes were divided into favorable (Engel Class I) or unfavorable (Engel Class II-IV) for comparison. Meta-analyses were performed to evaluate: 1) the proportion of Class I outcomes following epilepsy surgery in PTE-TL; and 2) calculate the odds of Class I surgical outcomes in PTE-TL compared with NTE-TL. Of 3669 articles that reported surgical outcomes in epilepsy, nine studies (n = 886) were identified that reported outcomes for both PTE-TL (n = 219) and NTE-TL (n = 667). The weighted proportion of favorable outcomes (Engel Class I) were high for both PTE-TL (70.1%, 95% CI 61.9%-78.3%) and NTE-TL (75.2%, 95% CI 69.4%-80.2%). Patients with PTE-TL were at greater risk of unfavorable (Engel Class II-IV) outcomes (relative risk 1.36, 95% CI 1.04-1.78) compared with NTE-TL.

Antiepileptic Drugs as Potential Dementia Prophylactics Following Traumatic Brain Injury.

Seizures and other forms of neurovolatility are emerging as druggable prodromal mechanisms that link traumatic brain injury (TBI) to the progression of later dementias. TBI neurotrauma has both acute and long-term impacts on health, and TBI is a leading risk factor for dementias, including chronic traumatic encephalopathy and Alzheimer's disease. Treatment of TBI already considers acute management of posttraumatic seizures and epilepsy, and impressive efforts have optimized regimens of antiepileptic drugs (AEDs) toward that goal. Here we consider that expanding these management strategies could determine which AED regimens best prevent dementia progression in TBI patients. Challenges with this prophylactic strategy include the potential consequences of prolonged AED treatment and that a large subset of patients are refractory to available AEDs. Addressing these challenges is warranted because the management of seizure activity following TBI offers a rare opportunity to prevent the onset or progression of devastating dementias.

Celecoxib treatment alleviates cerebral injury in a rat model of post-traumatic epilepsy.

Background: An important factor contributing to the development and occurrence of post-traumatic epilepsy (PTE) is neuroinflammation and oxidative stress. The effects of celecoxib include inhibiting inflammatory reactions and antioxidant stress and reducing seizures, making it a potential epilepsy treatment solution. Objective: To observe the effect of celecoxib on early epilepsy in post-traumatic epilepsy rats. Methods: Twenty-four adult healthy male Sprague-Dawley rats were randomly assigned to three groups: sham-operated, PTE, and celecoxib. A rat model of PTE was established by injecting ferrous chloride into the right frontal cortex. Afterward, the behavior of rats was observed and recorded. 3.0T superconducting magnetic resonance imaging (MRI) was used to describe the changes in ADC values of the brain. HE and Nissl staining were also used to detect the damage to frontal lobe neurons. Furthermore, the expression of COX-2 protein in the right frontal lobe was detected by Western blot. Moreover, the contents of IL-1 and TNF-α in the right frontal lobe were detected by enzyme-linked immunosorbent assay. Results: Compared with the PTE group, the degree of seizures in rats treated with celecoxib declined dramatically (P < 0.05). Celecoxib-treated rats had significant decreases in tissue structural damage and cell death in the brain. The results of the MRI showed that celecoxib reduced the peripheral edema zone and ADC value of the cortex around the damaged area of the right frontal lobe in the celecoxib-treatment group, which was significantly decreased compared with the PTE group (P < 0.05). Furthermore, celecoxib decreased the expression of COX-2, IL-1ß, and TNF-α in brain tissue (P < 0.05). Conclusions: In PTE rats, celecoxib significantly reduced brain damage and effectively reduced seizures. As a result of celecoxib's ability to inhibit inflammation, it can reduce the edema caused by injury in rat brain tissue.

Research Progress on the Immune-Inflammatory Mechanisms of Posttraumatic Epilepsy.

Posttraumatic epilepsy (PTE) is a severe complication arising from a traumatic brain injury caused by various violent actions on the brain. The underlying mechanisms for the pathogenesis of PTE are complex and have not been fully defined. Approximately, one-third of patients with PTE are resistant to antiepileptic therapy. Recent research evidence has shown that neuroinflammation is critical in the development of PTE. This article reviews the immune-inflammatory mechanisms regarding microglial activation, astrocyte proliferation, inflammatory signaling pathways, chronic neuroinflammation, and intestinal flora. These mechanisms offer novel insights into the pathophysiological mechanisms of PTE and have groundbreaking implications in the prevention and treatment of PTE. Immunoinflammatory cross-talk between glial cells and gut microbiota in posttraumatic epilepsy. This graphical abstract depicts the roles of microglia and astrocytes in posttraumatic epilepsy, highlighting the influence of the gut microbiota on their function. TBI traumatic brain injury, AQP4 aquaporin-4, Kir4.1 inward rectifying K channels.

Reduced total number of enlarged perivascular spaces in post-traumatic epilepsy patients with unilateral lesions - a feasibility study.

BACKGROUND: We investigated the value of automated enlarged perivascular spaces (ePVS) quantification to distinguish chronic traumatic brain injury (TBI) patients with post-traumatic epilepsy (PTE+) from chronic TBI patients without PTE (PTE-) in a feasibility study. METHODS: Patients with and without PTE were recruited and underwent an MRI post-TBI. Multimodal auto identification of ePVS algorithm was applied to T1-weighted MRIs to segment ePVS. The total number of ePVS was calculated and corrected for white matter volume, and an asymmetry index (AI) derived. RESULTS: PTE was diagnosed in 7 out of the 99 participants (male=69) after a median time of less than one year since injury (range 10-22). Brain lesions were observed in all 7 PTE+ cases (unilateral=4, 57%; bilateral=3, 43%) as compared to 40 PTE- cases (total 44%; unilateral=17, 42%; bilateral=23, 58%). There was a significant difference between PTE+ (M=1.21e-4, IQR [8.89e-5]) and PTE- cases (M=2.79e-4, IQR [6.25e-5]) in total corrected numbers of ePVS in patients with unilateral lesions (p=0.024). No differences in AI, trauma severity and lesion volume were seen between groups. CONCLUSION: This study has shown that automated quantification of ePVS is feasible and provided initial evidence that individuals with PTE with unilateral lesions may have fewer ePVS compared to TBI patients without epilepsy. Further studies with larger sample sizes should be conducted to determine the value of ePVS quantification as a PTE-biomarker.

A machine learning approach to seizure detection in a rat model of post-traumatic epilepsy.

Epilepsy is a common neurologic condition frequently investigated using rodent models, with seizures identified by electroencephalography (EEG). Given technological advances, large datasets of EEG are widespread and amenable to machine learning approaches for identification of seizures. While such approaches have been explored for human EEGs, machine learning approaches to identifying seizures in rodent EEG are limited. We utilized a predesigned deep convolutional neural network (DCNN), GoogLeNet, to classify images for seizure identification. Training images were generated through multiplexing spectral content (scalograms), kurtosis, and entropy for two-second EEG segments. Over 2200 h of EEG data were scored for the presence of seizures, with 95.6% of seizures identified by the DCNN and a false positive rate of 34.2% (1.52/h), as compared to visual scoring. Multiplexed images were superior to scalograms alone (scalogram-kurtosis-entropy 0.956 ± 0.010, scalogram 0.890 ± 0.028, t(7) = 3.54, p < 0.01) and a DCNN trained specifically for the individual animal was superior to using DCNNs across animals (intra-animal 0.960 ± 0.0094, inter-animal 0.811 ± 0.015, t(30) = 5.54, p < 0.01). For this dataset the DCNN approach is superior to a previously described algorithm utilizing longer local line lengths, calculated from wavelet-decomposition of EEG, to identify seizures. We demonstrate the novel use of a predesigned DCNN constructed to classify images, utilizing multiplexed images of EEG spectral content, kurtosis, and entropy, to rapidly and objectively identifies seizures in a large dataset of rat EEG with high sensitivity.

4-AP challenge reveals that early intervention with brivaracetam prevents posttraumatic epileptogenesis in rats.

PURPOSE: There are currently no clinical treatments to prevent posttraumatic epilepsy (PTE). Recently, our group has shown that administration of levetiracetam (LEV) or brivaracetam (BRV) shortly after cortical neurotrauma prevents the development of epileptiform activity in rats, as measured ex vivo in neocortical slices. Due to the low incidence of spontaneous seizures in rodent-based models of traumatic brain injury (TBI), chemoconvulsants have been used to test injured animals for seizure susceptibility. We used a low dose of the voltage-gated potassium channel blocker 4-aminopyridine (4-AP) to evaluate posttraumatic epileptogenesis after controlled cortical impact (CCI) injury. We then used this assessment to further investigate the efficacy of BRV as an antiepileptogenic treatment. METHODS: Sprague-Dawley rats aged P24-35 were subjected to severe CCI injury. Following trauma, one group received BRV-21 mg/kg (IP) at 0-2 min after injury and the other BRV-100 mg/kg (IP) at 30 min after injury. Four to eight weeks after injury, animals were given a single, low dose of 4-AP (3.0-3.5 mg/kg, IP) and then monitored up to 90 min for stage 4/5 seizures. RESULTS: The chemoconvulsant challenge revealed that within four to eight weeks, CCI injury led to a two-fold increase in percentage of rats with 4-AP induced stage 4-5 seizures relative to sham-injured controls. Administration of a single dose of BRV within 30 min after trauma significantly reduced injury-induced seizure susceptibility, bringing the proportion of CCI-rats that exhibited evoked seizures down to control levels. CONCLUSIONS: This study is the first to use a low dose of 4-AP as a chemoconvulsant challenge to test epileptogenicity within the first two months after CCI injury in rats. Our findings show that a single dose of BRV administered within 30 min after TBI prevents injury-induced increases in seizure susceptibility. This supports our hypothesis that early intervention with BRV may prevent PTE.

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.

Image data harmonization tools for the analysis of post-traumatic epilepsy development in preclinical multisite MRI studies.

Preclinical MRI studies have been utilized for the discovery of biomarkers that predict post-traumatic epilepsy (PTE). However, these single site studies often lack statistical power due to limited and homogeneous datasets. Therefore, multisite studies, such as the Epilepsy Bioinformatics Study for Antiepileptogenic Therapy (EpiBioS4Rx), are developed to create large, heterogeneous datasets that can lead to more statistically significant results. EpiBioS4Rx collects preclinical data internationally across sites, including the United States, Finland, and Australia. However, in doing so, there are robust normalization and harmonization processes that are required to obtain statistically significant and generalizable results. This work describes the tools and procedures used to harmonize multisite, multimodal preclinical imaging data acquired by EpiBioS4Rx. There were four main harmonization processes that were utilized, including file format harmonization, naming convention harmonization, image coordinate system harmonization, and diffusion tensor imaging (DTI) metrics harmonization. By using Python tools and bash scripts, the file formats, file names, and image coordinate systems are harmonized across all the sites. To harmonize DTI metrics, values are estimated for each voxel in an image to generate a histogram representing the whole image. Then, the Quantitative Imaging Toolkit (QIT) modules are utilized to scale the mode to a value of one and depict the subsequent harmonized histogram. The standardization of file formats, naming conventions, coordinate systems, and DTI metrics are qualitatively assessed. The histograms of the DTI metrics were generated for all the individual rodents per site. For inter-site analysis, an average of the individual scans was calculated to create a histogram that represents each site. In order to ensure the analysis can be run at the level of individual animals, the sham and TBI cohort were analyzed separately, which depicted the same harmonization factor. The results demonstrate that these processes qualitatively standardize the file formats, naming conventions, coordinate systems, and DTI metrics of the data. This assists in the ability to share data across the study, as well as disseminate tools that can help other researchers to strengthen the statistical power of their studies and analyze data more cohesively.

Delayed treatment with ceftriaxone reverses the enhanced sensitivity of TBI mice to chemically-induced seizures.

The pathophysiological changes that occur after traumatic brain injury (TBI) can lead to the development of post-traumatic epilepsy, a life-long complication of brain trauma. The etiology of post-traumatic epilepsy remains unknown, but TBI brains exhibit an abnormal excitatory / inhibitory balance. In this study, we examine how brain injury alters susceptibility to chemically-induced seizures in C57Bl/6J mice, and if pharmacological enhancement of glutamate transporters can reduce chronic post-traumatic seizures. We found that controlled cortical impact (CCI) mice display delayed susceptibility to pentylenetetrazol (PTZ)-induced seizures. While CCI mice have no change in seizure susceptibility at 7d post-injury (dpi), at 70dpi they have reduced latency to PTZ-induced seizure onset, higher seizure frequency and longer seizure duration. Quantification of glutamate transporter mRNA showed that levels of Scl1a2 and Scl1a3 mRNA were increased at 7dpi, but significantly decreased at 70dpi. To test if increased levels of glutamate transporters can ameliorate delayed-onset seizure susceptibility in TBI mice, we exposed a new cohort of mice to CCI and administered ceftriaxone (200mg/kg/day) for 14d from 55-70dpi. We found that ceftriaxone significantly increased Scl1a2 and Scl1a3 in CCI mouse brain at 70dpi, and prevented the susceptibility of CCI mice to PTZ-induced seizures. This study demonstrates cortical impact can induce a delayed-onset seizure phenotype in mice. Delayed (55dpi) ceftriaxone treatment enhances glutamate transporter mRNA in the CCI brain, and reduces PTZ-induced seizures in CCI mice.