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.

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.

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.

Acute Hippocampal Damage as a Prognostic Biomarker for Cognitive Decline but Not for Epileptogenesis after Experimental Traumatic Brain Injury.

It is necessary to develop reliable biomarkers for epileptogenesis and cognitive impairment after traumatic brain injury when searching for novel antiepileptogenic and cognition-enhancing treatments. We hypothesized that a multiparametric magnetic resonance imaging (MRI) analysis along the septotemporal hippocampal axis could predict the development of post-traumatic epilepsy and cognitive impairment. We performed quantitative T2 and T2* MRIs at 2, 7 and 21 days, and diffusion tensor imaging at 7 and 21 days after lateral fluid-percussion injury in male rats. Morris water maze tests conducted between 35-39 days post-injury were used to diagnose cognitive impairment. One-month-long continuous video-electroencephalography monitoring during the 6th post-injury month was used to diagnose epilepsy. Single-parameter and regularized multiple linear regression models were able to differentiate between sham-operated and brain-injured rats. In the ipsilateral hippocampus, differentiation between the groups was achieved at most septotemporal locations (cross-validated area under the receiver operating characteristic curve (AUC) 1.0, 95% confidence interval 1.0-1.0). In the contralateral hippocampus, the highest differentiation was evident in the septal pole (AUC 0.92, 95% confidence interval 0.82-0.97). Logistic regression analysis of parameters imaged at 3.4 mm from the contralateral hippocampus's temporal end differentiated between the cognitively impaired rats and normal rats (AUC 0.72, 95% confidence interval 0.55-0.84). Neither single nor multiparametric approaches could identify the rats that would develop post-traumatic epilepsy. Multiparametric MRI analysis of the hippocampus can be used to identify cognitive impairment after an experimental traumatic brain injury. This information can be used to select subjects for preclinical trials of cognition-improving interventions.

MRI-Guided Electrode Implantation for Chronic Intracerebral Recordings in a Rat Model of Post-Traumatic Epilepsy-Challenges and Gains.

Brain atrophy induced by traumatic brain injury (TBI) progresses in parallel with epileptogenesis over time, and thus accurate placement of intracerebral electrodes to monitor seizure initiation and spread at the chronic postinjury phase is challenging. We evaluated in adult male Sprague Dawley rats whether adjusting atlas-based electrode coordinates on the basis of magnetic resonance imaging (MRI) increases electrode placement accuracy and the effect of chronic electrode implantations on TBI-induced brain atrophy. One group of rats (EEG cohort) was implanted with two intracortical (anterior and posterior) and a hippocampal electrode right after TBI to target coordinates calculated using a rat brain atlas. Another group (MRI cohort) was implanted with the same electrodes, but using T2-weighted MRI to adjust the planned atlas-based 3D coordinates of each electrode. Histological analysis revealed that the anterior cortical electrode was in the cortex in 83% (25% in targeted layer V) of the EEG cohort and 76% (31%) of the MRI cohort. The posterior cortical electrode was in the cortex in 40% of the EEG cohort and 60% of the MRI cohort. Without MRI-guided adjustment of electrode tip coordinates, 58% of the posterior cortical electrodes in the MRI cohort will be in the lesion cavity, as revealed by simulated electrode placement on histological images. The hippocampal electrode was accurately placed in 82% of the EEG cohort and 86% of the MRI cohort. Misplacement of intracortical electrodes related to their rostral shift due to TBI-induced cortical and hippocampal atrophy and caudal retraction of the brain, and was more severe ipsilaterally than contralaterally (p < 0.001). Total lesion area in cortical subfields targeted by the electrodes (primary somatosensory cortex, visual cortex) was similar between cohorts (p > 0.05). MRI-guided adjustment of coordinates for electrodes improved the success rate of intracortical electrode tip placement nearly to that at the acute postinjury phase (68% vs. 62%), particularly in the posterior brain, which exhibited the most severe postinjury atrophy. Overall, MRI-guided electrode implantation improved the quality and interpretation of the origin of EEG-recorded signals.

Post-injury ventricular enlargement associates with iron in choroid plexus but not with seizure susceptibility nor lesion atrophy-6-month MRI follow-up after experimental traumatic brain injury.

Ventricular enlargement is one long-term consequence of a traumatic brain injury, and a risk factor for memory disorders and epilepsy. One underlying mechanisms of the chronic ventricular enlargement is disturbed cerebrospinal-fluid secretion or absorption by choroid plexus. We set out to characterize the different aspects of ventricular enlargement in lateral fluid percussion injury (FPI) rat model by magnetic resonance imaging (MRI) and discovered choroid plexus injury in rats that later developed hydrocephalus. We followed the brain pathology progression for 6 months and studied how the ventricular growth was associated with the choroid plexus injury, cortical lesion expansion, hemorrhagic load or blood perfusion deficits. We correlated MRI findings with the seizure susceptibility in pentylenetetrazol challenge and memory function in Morris water-maze. Choroid plexus injury was validated by ferric iron (Prussian blue) and cytoarchitecture (Nissl) stainings. We discovered choroid plexus injury that accumulates iron in 90% of FPI rats by MRI. The amount of the choroid plexus iron remained unaltered 1-, 3- and 6-month post-injury. During this time, the ventricles kept on growing bilaterally. Ventricular growth did not depend on the cortical lesion severity or the cortical hemorrhagic load suggesting a separate pathology. Instead, the results indicate choroidal injury as one driver of the post-traumatic hydrocephalus, since the higher the choroid plexus iron load the larger were the ventricles at 6 months. The ventricle size or the choroid plexus iron load did not associate with seizure susceptibility. Cortical hypoperfusion and memory deficits were worse in rats with greater ventricular growth.

Acute thalamic damage as a prognostic biomarker for post-traumatic epileptogenesis.

OBJECTIVE: To identify magnetic resonance imaging (MRI) biomarkers for post-traumatic epilepsy. METHODS: The EPITARGET (Targets and biomarkers for antiepileptogenesis, epitarget.eu) animal cohort completing T2 relaxation and diffusion tensor MRI follow-up and 1-month-long video-electroencephalography monitoring included 98 male Sprague-Dawley rats with traumatic brain injury and 18 controls. T2 imaging was performed on day (D) 2, D7, and D21 and diffusion tensor imaging (DTI) on D7 and D21 using a 7-Tesla Bruker PharmaScan MRI scanner. The mean and standard deviation (SD) of the T2 relaxation rate, multiple diffusivity measures, and diffusion anisotropy at each time-point within the ventroposterolateral and ventroposteromedial thalamus were used as predictor variables in multi-variable logistic regression models to distinguish rats with and without epilepsy. RESULTS: Twenty-nine percent (28/98) of the rats with traumatic brain injury (TBI) developed epilepsy. The best-performing logistic regression model utilized the D2 and D7 T2 relaxation time as well as the D7 diffusion tensor data. The model distinguished rats with and without epilepsy (Bonferroni-corrected p-value < .001) with a cross-validated concordance statistic of 0.74 (95% confidence interval [CI] 0.60-0.84). In a cross-validated classification test, the model exhibited 54% sensitivity and 91% specificity, enriching the epilepsy rate within the study population from the expected 29% to 71%. A model using the D2 T2 data only resulted in a 73% enriched epilepsy rate (regression p-value .007, cross-validated concordance 0.70, 95% CI 0.56-0.80, sensitivity 29%, specificity 96%). SIGNIFICANCE: An MRI parameter set reporting on acute and subacute neuropathologic changes common to experimental and human TBI presents a diagnostic biomarker for post-traumatic epileptogenesis. Significant enrichment of the study population was achieved even when using a single time-point measurement, producing an expected epilepsy rate of 73%.

Biomarkers for posttraumatic epilepsy.

A biomarker is a characteristic that can be objectively measured as an indicator of normal biologic processes, pathogenic processes, or responses to an exposure or intervention, including therapeutic interventions. Biomarker modalities include molecular, histologic, radiographic, or physiologic characteristics. To improve the understanding and use of biomarker terminology in biomedical research, clinical practice, and medical product development, the Food and Drug Administration (FDA)-National Institutes of Health (NIH) Joint Leadership Council developed the BEST Resource (Biomarkers, EndpointS, and other Tools). The seven BEST biomarker categories include the following: (a) susceptibility/risk biomarkers, (b) diagnostic biomarkers, (c) monitoring biomarkers, (d) prognostic biomarkers, (e) predictive biomarkers, (f) pharmacodynamic/response biomarkers, and (g) safety biomarkers. We hypothesize some potential overlap between the reported biomarkers of traumatic brain injury (TBI), epilepsy, and posttraumatic epilepsy (PTE). Here, we tested this hypothesis by reviewing studies focusing on biomarker discovery for posttraumatic epileptogenesis and epilepsy. The biomarker modalities reviewed here include plasma/serum and cerebrospinal fluid molecular biomarkers, imaging biomarkers, and electrophysiologic biomarkers. Most of the reported biomarkers have an area under the receiver operating characteristic curve greater than 0.800, suggesting both high sensitivity and high specificity. Our results revealed little overlap in the biomarker candidates between TBI, epilepsy, and PTE. In addition to using single parameters as biomarkers, machine learning approaches have highlighted the potential for utilizing patterns of markers as biomarkers. Although published data suggest the possibility of identifying biomarkers for PTE, we are still in the early phase of the development curve. Many of the seven biomarker categories lack PTE-related biomarkers. Thus, further exploration using proper, statistically powered, and standardized study designs with validation cohorts, and by developing and applying novel analytical methods, is needed for PTE biomarker discovery.

Postinjury weight rather than cognitive or behavioral impairment predicts development of posttraumatic epilepsy after lateral fluid-percussion injury in rats.

OBJECTIVE: To identify postinjury physiologic, behavioral, and cognitive biomarkers for posttraumatic epilepsy to enrich study populations for long-term antiepileptogenesis studies. METHODS: The EPITARGET cohort with behavioral follow-up and 1-month 24/7 video-electroencephalography (vEEG) monitoring included 115 adult male Sprague-Dawley rats with lateral fluid-percussion-induced traumatic brain injury (TBI), 23 sham-operated controls, and 13 naive rats. Animals underwent assessment of somatomotor performance (composite neuroscore), anxiety-like behavior (elevated plus maze, open field), spatial memory (Morris water maze), and depression-like behavior (Porsolt forced swim, sucrose preference). Impact force, postimpact apnea time, postimpact seizure-like behavior, and body weight were monitored. RESULTS: TBI rats were impaired in the composite neuroscore (P < .001) on days (D) 2-14 and in the spatial memory test (P < .001) on D35-39 post-TBI. Differences in the elevated plus-maze (D28 and D126) and in the open field (D29 and D127) between TBI rats and controls were meager. No differences were observed in the Porsolt forced swim and sucrose preference tests as compared with sham-operated controls. Epilepsy developed in 27% of rats by the end of the sixth month. None of the behavioral or cognitive outcome measures discriminated rats with or without epilepsy. The receiver-operating characteristic analysis indicated that a decrease in body weight between D0 and D4 differentiated TBI rats with epilepsy from TBI rats without epilepsy (48% sensitivity, 83% specificity, area under the curve [AUC] 0.679, confidence interval [CI] 95% 0.56-0.80, P < .01). A 16% body weight decrease during D0-D4 could be used as a biomarker to enrich the study population from 27% (observed) to 50%. SIGNIFICANCE: Single behavioral and cognitive outcome measures showed no power as prognostic/diagnostic biomarkers for posttraumatic epilepsy. A reduction in body weight during the first postinjury week showed some prognostic value for posttraumatic epileptogenesis and could serve as a subacute measure for selectively enriching the study population for long-term preclinical biomarker and therapy discovery studies of posttraumatic epileptogenesis.

Early Increase in Cortical T2 Relaxation Is a Prognostic Biomarker for the Evolution of Severe Cortical Damage, but Not for Epileptogenesis, after Experimental Traumatic Brain Injury.

Prognostic biomarkers for post-injury outcome are necessary for the development of neuroprotective and antiepileptogenic treatments for traumatic brain injury (TBI). We hypothesized that T2 relaxation magnetic resonance imaging (MRI) predicts the progression of perilesional cortical pathology and epileptogenesis. The EPITARGET animal cohort used for MRI analysis included 120 adult male Sprague-Dawley rats with TBI induced by lateral fluid-percussion injury and 24 sham-operated controls. T2 MRI was performed at days 2, 7, and 21 post-TBI. The lesioned cortex was outlined, and the T2 value of each imaging voxel within the lesion area was scored using a five-grade pathology classification. Analysis of 1-month video-electroencephalography recordings initiated 5 months post-TBI indicated that 27% (31 of 114) of the animals with TBI developed epilepsy. Multiple linear regression analysis indicated that T2-based classification of lesion volume at day 2 and day 7 post-TBI explained the necrotic lesion volume with greatly increased T2 (>102 ms) at day 21 post-TBI (F(13,103) = 52.5; p < 0.001; R2 = 0.87; adjusted R2 = 0.85). The volume of moderately increased (78-102 ms) T2 at day 7 post-TBI predicted the evolution of large (>12 mm3) cortical lesions (area under the curve, 0.92; p < 0.001; cutoff, 1.9 mm3; false positive rate, 0.10; true positive rate, 0.62). Logistic regression analysis, however, showed that the different severities of T2 lesion volumes at days 2, 7, and 21 post-TBI did not explain the development of epilepsy (χ2(18,95) = 18.4; p = 0.427). In addition, the location of the T2 abnormality within the cortex did not correlate with epileptogenesis. A single measurement of T2 relaxation MRI in the acute post-TBI phase is useful for identifying post-TBI subjects at highest risk of developing large cortical lesions, and thus, in the greatest need of neuroprotective therapies after TBI, but not the development of post-traumatic epilepsy.

Harmonization of the pipeline for seizure detection to phenotype post-traumatic epilepsy in a preclinical multicenter study on post-traumatic epileptogenesis.

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.

Harmonization of pipeline for detection of HFOs in a rat model of post-traumatic epilepsy in preclinical multicenter study on post-traumatic epileptogenesis.

Studies of chronic epilepsy show pathological high frequency oscillations (HFOs) are associated with brain areas capable of generating epileptic seizures. Only a few of these studies have focused on HFOs during the development of epilepsy, but results suggest pathological HFOs could be a biomarker of epileptogenesis. The Epilepsy Bioinformatics Study for Antiepileptogenic Therapy" (EpiBioS4Rx) is a multi-center project designed to identify biomarkers of epileptogenesis after a traumatic brain injury (TBI) and evaluate treatments that could modify or prevent the development of post-traumatic epilepsy. One goal of the EpiBioS4Rx project is to assess whether HFOs could be a biomarker of post-traumatic epileptogenesis. The current study describes the work towards this goal, including the development of common surgical procedures and EEG protocols, an interim analysis of the EEG for HFOs, and identifying issues that need to be addressed for a robust biomarker analysis. At three participating sites - University of Eastern Finland (UEF), Monash University in Melbourne (Melbourne) and University of California, Los Angeles (UCLA) - TBI was induced in adult male Sprague-Dawley rats by lateral fluid-percussion injury. After injury and in sham-operated controls, rats were implanted with screw and microwire electrodes positioned in neocortex and hippocampus to record EEG. A separate group of rats had serial magnetic resonance imaging after injury and then implanted with electrodes at 6 months. Recordings 28 days post-injury were available from UEF and UCLA, but not Melbourne due to technical issues with their EEG files. Analysis of recordings from 4 rats - UEF and UCLA each had one TBI and one sham-operated control - showed EEG contained evidence of HFOs. Computer-automated algorithms detected a total of 1,819 putative HFOs and of these only 40 events (2%) were detected by all three sites. Manual review of all events verified 130 events as HFO and the remainder as false positives. Review of the 40 events detected by all three sites was associated with 88% agreement. This initial report from the EpiBioS4Rx Consortium demonstrates the standardization of EEG electrode placements, recording protocol and long-term EEG monitoring, and differences in detection algorithm HFO results between sites. Additional work on detection strategy, detection algorithm performance, and training in HFO review will be performed to establish a robust, preclinical evaluation of HFOs as a biomarker of post-traumatic epileptogenesis.

Informatics tools to assess the success of procedural harmonization in preclinical multicenter biomarker discovery study on post-traumatic epileptogenesis.

The Epilepsy Bioinformatics Study for Antiepileptogenic Therapy (EpiBioS4Rx) is a National Institutes for Neurological Diseases and Stoke funded Centers-Without-Walls international multidisciplinary study aimed at preventing epileptogenesis. The preclinical biomarker discovery in EpiBios4Rx applies a multicenter study design to allow the number of animals that are required for adequate statistical power for the analysis to be studied in an efficient manner. Further, the use of multiple centers mimics the clinical trial situation, and therefore potentially the chance of successful clinical translation of the outcomes of the study. Its successful implementation requires harmonization of procedures and data analyses between the three contributing centers in Finland, Australia, and USA. The objective of the present analysis was to develop metrics for analysis of the success of harmonization of procedures to guide further data analyses and plan the future multicenter preclinical studies. The interim analysis of data is based on the analysis of data from 212 rats with lateral fluid-percussion injury or sham-operation included in the biomarker discovery by April 30, 2018. The details of protocols, including production of injury, post-injury follow-up, blood sampling, electroencephalogram recording, and magnetic resonance imaging have been presented in the accompanying manuscripts in this Supplement. Implementation of protocols in EpiBios4Rx project participant centers was visualized in 2D using t-distributed stochastic neighborhood embedding (t-SNE). The protocols applied to each rat were presented as feature vectors of procedure related variables (e.g., impact pressure, anesthesia time). The total number of protocol features linked to each rat was 112. The missing data was accounted in visualization by utilizing imputation and adding the number of missing values as a third dimension to 2D t-SNE plot, resulting in a 3D overview of protocol data. Intraclass correlation coefficient (ICC) using Euclidean distances and area under receiver operating characteristic curve (AUC) of k-nearest neighbor classifier (KNN) were utilized to quantify the degree of clustering by center. Both subsets of data with incomplete protocol vectors omitted and missing protocol data imputed were assessed. Our data show that a visible clustering by center was observed in all t-SNE plots, except for day 7 neuroscores. Both ICC and AUC indicated clustering by center in all protocol variable subsets, excluding unimputed day 7 neuroscores (ICC 0.04 and AUC 0.6). ICC for imputed set of all protocol related variables was 0.1 and KNN AUC 0.92. In conclusion, both ICC and AUC indicated differences in protocol between EpiBios4Rx participating centers, which needs to be taken into account in data analysis. Importantly, the majority of observed differences are recoverable as they relate to insufficient updates in record keeping. While AUC score of KNN is a more sensitive measure for protocol harmonization than ICC for data that displays complex splintered clustering, ICC and AUC provide complementary measures to assess the degree of procedural harmonization. This experience should be helpful for other groups planning such multicenter post-traumatic epileptogenesis studies in the future.

Acute Non-Convulsive Status Epilepticus after Experimental Traumatic Brain Injury in Rats.

Severe traumatic brain injury (TBI) induces seizures or status epilepticus (SE) in 20-30% of patients during the acute phase. We hypothesized that severe TBI induced with lateral fluid-percussion injury (FPI) triggers post-impact SE. Adult Sprague-Dawley male rats were anesthetized with isoflurane and randomized into the sham-operated experimental control or lateral FPI-induced severe TBI groups. Electrodes were implanted right after impact or sham-operation, then video-electroencephalogram (EEG) monitoring was started. In addition, video-EEG was recorded from naïve rats. During the first 72 h post-TBI, injured rats had seizures that were intermingled with other epileptiform EEG patterns typical to non-convulsive SE, including occipital intermittent rhythmic delta activity, lateralized or generalized periodic discharges, spike-and-wave complexes, poly-spikes, poly-spike-and-wave complexes, generalized continuous spiking, burst suppression, or suppression. Almost all (98%) of the electrographic seizures were recorded during 0-72 h post-TBI (23.2 ± 17.4 seizures/rat). Mean latency from the impact to the first electrographic seizure was 18.4 ± 15.1 h. Mean seizure duration was 86 ± 57 sec. Analysis of high-resolution videos indicated that only 41% of electrographic seizures associated with behavioral abnormalities, which were typically subtle (Racine scale 1-2). Fifty-nine percent of electrographic seizures did not show any behavioral manifestations. In most of the rats, epileptiform EEG patterns began to decay spontaneously on Days 5-6 after TBI. Interestingly, also a few sham-operated and naïve rats had post-operation seizures, which were not associated with EEG background patterns typical to non-convulsive SE seen in TBI rats. To summarize, our data show that lateral FPI-induced TBI results in non-convulsive SE with subtle behavioral manifestations; this explains why it has remained undiagnosed until now. The lateral FPI model provides a novel platform for assessing the mechanisms of acute symptomatic non-convulsive SE and for testing treatments to prevent post-injury SE in a clinically relevant context.

Algorithm for automatic detection of spontaneous seizures in rats with post-traumatic epilepsy.

BACKGROUND: Labor intensive electroencephalogram (EEG) analysis is a major bottleneck to identifying anti-epileptogenic treatments in experimental models of post-traumatic epilepsy. We aimed to develop an algorithm for automated seizure detection in experimental post-traumatic epilepsy. NEW METHOD: Continuous (24/7) 1-month-long video-EEG monitoring with three epidural screw electrodes was started 154 d after lateral fluid-percussion induced traumatic brain injury (TBI; n = 97) or sham-injury (n = 29) in adult male Sprague-Dawley rats. First, an experienced researcher screened a total of 90,720 h of digitized recordings on a computer screen to annotate the occurrence of spontaneous seizures. The same files were then analyzed using an algorithm in Spike2 (ver.9), which searching for temporally linked power peaks (14-42 Hz) in all three EEG channels, and then positive events were marked as a probable seizures. Finally, an experienced researcher confirmed all seizure candidates visually on the computer screen. RESULTS: Visual analysis identified 197 seizures in 29 rats. Automatic detection identified 4346 seizure candidates in 109 rats, of which 202 in the same 29 rats were true positives, resulting in a false positive rate of 0.046/h or 1.10/d. The algorithm demonstrated 5% specificity and 100% sensitivity. The algorithm analyzed 1-month 3-channel EEG in 7 cohorts in 2 h, whereas analysis by an experienced technician took ∼500 h. COMPARISON WITH EXISTING METHODS: The algorithm had 100% sensitivity. It performed slightly better and was substantially faster than investigator-performed visual analysis. CONCLUSIONS: We present a novel seizure detection algorithm for automated detection of seizures in a rat model of post-traumatic epilepsy.