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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. Supplementary Information The online version contains supplementary material available at 10.1007/s00429-021-02395-5.

Traumatic brain injury (TBI) causes 10–20% of structural epilepsy, with seizures typically originating in the cortex. Alterations in the neuronal microcircuits in the cortical epileptogenic zone, however, are poorly understood. Here, we assessed TBI-induced changes in perisomatic gamma aminobutyric acid (GABA)-ergic innervation in the perilesional cortex. We hypothesized that TBI will damage parvalbumin (PV)-immunoreactive inhibitory neurons and induce regulation of the associated GABAergic molecular interactome. TBI was induced in adult male Sprague-Dawley rats by lateral fluid-percussion injury. At 1-month post-TBI, the number of PV-positive somata was plotted on unfolded cortical maps and the distribution and density of immunopositive terminals analyzed. Qualitative analysis revealed either patchy microlesions of several hundred micrometers in diameter or diffuse neuronal loss. Quantitative analysis demonstrated a reduction in the number of PV-positive interneurons in patches down to 0% of that in sham-operated controls in the perilesional cortex. In the majority of patches, the cell numbers ranged from 71% to 90% that of the controls. The loss of PV-positive somata was accompanied by decreased axonal labeling. In situ hybridization revealed downregulated PV mRNA expression in the perilesional cortex. Gene Set Enrichment Analysis indicated a robustly downregulated expression profile of PV-related genes, which was confirmed by quantitative reverse transcriptase polymerase chain reaction. Specifically, we found that genes encoding postsynaptic GABA-A receptor genes, Gabrg2 and Gabrd, were downregulated in TBI animals compared with controls. Our data suggests that patchy reduction in PV-positive perisomatic inhibitory innervation contributes to the development of focal cortical inhibitory deficit after TBI.

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

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 relaxat...

Mechanisms initiated by traumatic brain injury (TBI), leading to the development of progressive secondary injury are poorly understood. MicroRNAs (miRNAs) have a proposed role in orchestrating the post-injury aftermath as a single miRNA can control the expression of several genes. We hypothesized that the post-injury level of circulating brain-enriched miR-124-3p explains the extent of post-TBI cortical lesion. Three separate cohorts of adult male Sprague-Dawley rats (total n = 57) were injured with lateral fluid-percussion-induced TBI. The miR-124-3p levels were measured in whole blood and/or plasma in cohorts 1 and 2 before TBI as well as at 2 d, 7 d, 2 months or 3 months post-TBI. The third cohort (22/57) was imaged with T2-weighted magnetic resonance imaging (MRI) at 2 months post-TBI to quantify cortical lesion area and perilesional T2-enhancement volume. Our data shows that miR-124-3p levels were elevated at 2 d post-TBI in both blood (FC 4.63, p 0.01) and plasma (FC 1.39, p 0.05) as compared to controls. Receiver operating curve (ROC) analysis indicated that plasma miR-124-3p level of 34 copies/µl or higher differentiated TBI animals from controls [area under curve (AUC) 0.815, p 0.05]. The data was validated in the third cohort (FC 1.68, p 0.05). T2-weighted MRI revealed inter-animal differences in cortical lesion area. Linear regression analysis revealed that higher the plasma miR-124-3p level at 2 d post-TBI, larger the lesion area at chronic time point (R

Traumatic brain injury (TBI) is defined as an alteration in brain function or other evidence of brain pathology caused by an external force. When epilepsy develops following TBI, it is known as post-traumatic epilepsy (PTE). PTE occurs in a subset of patients suffering from different types and severities of TBI, occurs more commonly following severe injury, and greatly impacts the quality of life for patients recovering from TBI. Similar to other types of epilepsy, PTE is often refractory to drug treatment with standard anti-seizure drugs. No therapeutic approaches have proven successful in the clinic to prevent the development of PTE. Therefore, novel treatment strategies are needed to stop the development of PTE and improve the quality of life for patients after TBI. Interestingly, TBI represents an excellent clinical opportunity for intervention to prevent epileptogenesis as typically the time of initiation of epileptogenesis (i.e., TBI) is known, the population of at-risk patients is large, and animal models for preclinical studies of mechanisms and treatment targets are available. If properly identified and treated, there is a true opportunity to prevent epileptogenesis after TBI and stop seizures from ever happening. With that goal in mind, here we review previous attempts to prevent PTE both in animal studies and in humans, we examine how biomarkers could enable better-targeted therapeutics, and we discuss how genetic variation may predispose individuals to PTE. Finally, we highlight exciting new advances in the field that suggest that there may be novel approaches to prevent PTE that should be considered for further clinical development. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13311-021-01119-1.

Disabilities resulting from traumatic brain injury (TBI) strongly correlate with the cytoarchitectonic part of the brain damaged, lesion area, and type of lesion. We developed a Web application to estimate the location of the lesion on mouse cerebral cortex caused by TBI induced by lateral fluid-percussion injury. The application unfolds user-determined TBI lesion measurements, e.g., from histologic sections to a reference template, and estimates the total lesion area, including the percentage of cortex damaged in different cytoarchitectural cortical regions. The resulting lesion can be visualized on a two-dimensional map of mouse cerebral cortex. The application also visualizes the development of the lesion over time when measurements from multiple time points are available. The web application was validated by comparing its performance to the manual method. The total area of the cortical lesion was similar between the manual (9.19 ± 0.66 mm2, range 4.25–14.93 mm2) and the automated analysis (9.27 ± 0.66 mm2, range 4.50–15.10 mm2) (p = 0.938). The results of the manual and automated analyses were strongly correlated (r = 0.999, p

A key event in the pathophysiology of traumatic brain injury (TBI) is the influx of substantial amounts of Ca2+ into neurons, particularly in the thalamus. Detection of this calcium influx in vivo would provide a window into the biochemical mechanisms of TBI with potentially significant clinical implications. In the present work, our central hypothesis was that the Ca2+ influx could be imaged in vivo with the relatively recent MRI technique of quantitative susceptibility mapping (QSM). Wistar rats were divided into five groups: naive controls, sham-operated experimental controls, single mild TBI, repeated mild TBI, and single severe TBI. We employed the lateral fluid percussion injury (FPI) model, which replicates clinical TBI without skull fracture, performed 9.4 Tesla MRI with a 3D multi-echo gradient-echo sequence at weeks 1 and 4 post-injury, computed susceptibility maps using V-SHARP and the QUASAR-HEIDI technique, and performed histology. Sham, experimental controls animals, and injured animals did not demonstrate calcifications at 1 week after the injury. At week 4, calcifications were found in the ipsilateral thalamus of 25–50% of animals after a single TBI and 83% of animals after repeated mild TBI. The location and appearance of calcifications on stained sections was consistent with the appearance on the in vivo susceptibility maps (correlation of volumes: r = 0.7). Our findings suggest that persistent calcium deposits represent a primary pathology of repeated injury and that FPI-QSM has the potential to become a sensitive tool for studying pathophysiology related to mild TBI in vivo.

Seven large European Union (EU)-funded epilepsy-related research projects joined forces in May 2018 in Brussels, Belgium, in a unique community building event-the epiXchange conference. During this conference, 170 investigators from the projects DESIRE, EpimiRNA, EPISTOP, EpiTarget, EpiXchange, and EpiPGX as well as the European Reference Network EpiCARE, met up with key stakeholders including representatives of the European Commission, patient organizations, commercial partners, and other European and International groups. The epiXchange conference focused on sharing and reviewing the advances made by each project in the previous 5 years; describing the infrastructures generated; and discussing the innovations and commercial applications across five thematic areas: biomarkers, genetics, therapeutics, comorbidities, and biobanks and resources. These projects have, in fact, generated major breakthroughs including the discovery of biofluid-based molecules for diagnosis, elucidating new genetic causes of epilepsy, creating advanced new models of epilepsy, and the pre-clinical development of novel compounds. Workshop-style discussions focused on how to overcome scientific and clinical challenges for accelerating translation of research outcomes and how to increase synergies between the projects and stakeholders at a European level. The resulting advances would lead toward a measurable impact of epilepsy research through better diagnostics, treatments, and quality-of-life for persons with epilepsy. In addition, epiXchange provided a unique forum for examining how the different projects could build momentum for future novel groundbreaking epilepsy research in Europe and beyond. This report includes the main recommendations that resulted from these discussions.

We describe the infrastructure and functionality for a centralized preclinical and clinical data repository and analytic platform to support importing heterogeneous multi-modal data, automatically and manually linking data across modalities and sites, and searching content. We have developed and applied innovative image and electrophysiology processing methods to identify candidate biomarkers from MRI, EEG, and multi-modal data. Based on heterogeneous biomarkers, we present novel analytic tools designed to study epileptogenesis in animal model and human with the goal of tracking the probability of developing epilepsy over time.

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).

Onset of many forms of epilepsy occurs after an initial epileptogenic insult or as a result of an identified genetic defect. Given that the precipitating insult is known, these epilepsies are, in principle, amenable to secondary prevention. However, development of preventive treatments is difficult because only a subset of individuals will develop epilepsy and we cannot currently predict which individuals are at the highest risk. Biomarkers that enable identification of these individuals would facilitate clinical trials of potential anti-epileptogenic treatments, but no such prognostic biomarkers currently exist. Several putative molecular, imaging, electroencephalographic and behavioural biomarkers of epileptogenesis have been identified, but clinical translation has been hampered by fragmented and poorly coordinated efforts, issues with inter-model reproducibility, study design and statistical approaches, and difficulties with validation in patients. These challenges demand a strategic roadmap to facilitate the identification, characterization and clinical validation of biomarkers for epileptogenesis. In this Review, we summarize the state of the art with respect to biomarker research in epileptogenesis and propose a five-phase roadmap, adapted from those developed for cancer and Alzheimer disease, that provides a conceptual structure for biomarker research.

Post-traumatic epilepsy (PTE) is one of the clinical outcomes in about 16% of patients with traumatic brain injury (TBI). Patients develop PTE after several months to years following the initial brain injury. Over the years, there have been an increasing number of reports suggesting that post-traumatic inflammation, which is a major component of the pathophysiology of TBI, may be a key player in the development of PTE. The inflammatory process begins within minutes to hours after the primary injury and may proceed for several years after initial insult. Evidence suggest that the neurovascular unit (NVU) play a key role in the post-traumatic inflammatory cascade. Also, there is growing number of data supporting the hypothesis that anti-inflammatory treatments may improve the post-TBI clinical outcome. In this chapter we will give insights into how the different members of the NVU, including the blood-brain barrier, microglial cells, NG2-glial cells, pericytes, and astrocytes, contribute to the post-traumatic inflammation and the development of PTE.

There are no pharmacological interventions to prevent the development of epilepsy, although many promising compounds have been identified in the animal laboratory. Clinical trials to validate their effectiveness, however, would currently be prohibitively expensive due to the large subject population and duration of follow-up necessary. There is, therefore, the need to identify biomarkers of epileptogenesis that could identify patients at high risk for epilepsy following a potential epileptogenic insult to enrich the subject population, as well as biomarkers that could determine the effectiveness of therapeutic intervention without the need to wait for seizures to occur. Putative biomarkers under investigation for epileptogenesis and its treatment include genetic, molecular, cellular, imaging, and electrophysiological measures that might reliably predict the development or progression of an epileptic condition, the effects of antiepileptogenic treatment, or cure after surgery. To be clinically useful for most purposes, ideal biomarkers should be noninvasive, and it is anticipated that a profile of multiple biomarkers will likely be required. Ongoing animal research involves a number of experimental models of epileptogenesis, with traumatic brain injury, offering the best potential for translational clinical investigations. Collaborative and multicenter research efforts by multidisciplinary teams of basic and clinical neuroscientists with access to robust, well-defined animal models, extensive patient populations, standardized protocols, and cutting-edge analytical methodologies are likely to be most successful. Such biomarker research should also provide insights into fundamental neuronal mechanisms of epileptogenesis suggesting novel targets for antiepileptogenic treatments. This article is part of the special issue entitled 'New Epilepsy Therapies for the 21st Century - From Antiseizure Drugs to Prevention, Modification and Cure of Epilepsy'.

Epilepsy is a debilitating neurological disorder that directly impacts millions of people and exerts a tremendous economic burden on society at large. While traumatic brain injury (TBI) is a common cause, there remain many open questions regarding its pathological mechanism. The goal of the Epilepsy Bioinformatics Study for Antiepilep-togenic Therapy (EpiBioS4Rx) is to identify epileptogenic biomarkers through a comprehensive project spanning multiple species, modalities, and research institutions; in particular, diffusion magnetic resonance imaging (MRI) is a critical component, as it probes tissue microstructure and structural connectivity. The project includes in vivo imaging of a rodent fluid-percussion model of TBI, and we developed a computational diffusion MRI framework for EpiBioS4Rx which employs advanced techniques for preprocessing, modeling, spatial normalization, region analysis, and tractography to derive imaging metrics at group and individual levels. We describe the system's design, present characteristic results from a longitudinal cohort, and discuss its role in biomarker discovery and further studies.

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

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.

Background Both the type and severity of functional impairments caused by damage to the cerebral cortex depend on the functional cortical areas and networks affected, as well as the lesion extent and type. New method To accelerate the laborious quantitative analysis of cortical lesion location and size, we created user-friendly software that generates two-dimensional unfolded cortical maps of the lesions in adult rats. The program imports and superimposes simple user-made measurements, e.g., from histologic sections, on a template. The software then quantifies the total lesion area and the area of damage in each cortical cytoarchitechtonic region. To validate the accuracy of the software, we compared computer-generated maps with manually created unfolded maps from 32 rats with lateral fluid-percussion-induced traumatic brain injury. Results The total area of the cortical lesions varied from 7.37 to 38.45 mm2 in the automated analysis and from 7.26 to 38.97 mm2 in the manual analysis (p > 0.05). The Pearson correlation coefficient between the automated and manual analyses was 0.998 (p Comparison with existing methods Compared with the manual method, the automated method accurately and quickly generates unfolded cortical maps of lesioned cortical areas. Conclusions Application provides a novel tool for accurately positioning the lesioned area on the cortical mantle and quantifying the lesion area in histologic sections or magnetic resonance images.

The major objective of preclinical translational epilepsy research is to advance laboratory findings toward clinical application by testing potential treatments in animal models of seizures and epilepsy. Recently there has been a focus on the failure of preclinical discoveries to translate reliably, or even to be reproduced in different laboratories. One potential cause is a lack of standardization in preclinical data collection. The resulting difficulties in comparing data across studies have led to high cost and missed opportunity, which in turn impede clinical trials and advances in medical care. Preclinical epilepsy research has successfully brought numerous antiseizure treatments into the clinical practice, yet the unmet clinical needs have prompted the reconsideration of research strategies to optimize epilepsy therapy development. In the field of clinical epilepsy there have been successful steps to improve such problems, such as generation of common data elements (CDEs) and case report forms (CRFs and standards of data collection and reporting) by a team of leaders in the field. Therefore, the Translational Task Force was appointed by the International League Against Epilepsy (ILAE) and the American Epilepsy Society (AES), in partnership with the National Institute of Neurological Disorders and Stroke (NINDS) and the National Institutes of Health (NIH) to define CDEs for animal epilepsy research studies and prepare guidelines for data collection and experimental procedures. If adopted, the preclinical CDEs could facilitate collaborative epilepsy research, comparisons of data across different laboratories, and promote rigor, transparency, and impact, particularly in therapy development.

Sleep disturbances commonly occur after traumatic brain injury (TBI) and may predispose patients to epileptic seizures. We hypothesized that unprovoked seizure occurrence post-TBI depends on the sleep-wake cycle, and that the electrographic characteristics of a given sleep stage provide biomarkers for post-traumatic epilepsy (PTE). We show, in a rat lateral fluid percussion model, that 92% of spontaneous generalized seizures occur during the transition from stage III to rapid eye movement sleep. Moreover, a reduction in spindle duration and dominant frequency during the transition stage present as specific and sensitive noninvasive biomarkers for experimentally induced PTE with generalized electrographic seizures.

Traumatic brain injury (TBI) causes damage to the hypothalamo-hypophyseal axis, leading to endocrine dysregulation in up to 40% of TBI patients. Hence, there is an urgent need to identify non-invasive biomarkers for TBI-associated hypothalamo-hypophyseal pathology. Sushi repeat-containing protein X-linked 2 (SRPX2) is a novel hypothalamic protein expressed in both rat and human brain. Our objective was to investigate the effect of acquired brain injury on plasma SRPX2 protein levels and SRPX2 expression in the brain. We induced severe lateral fluid-percussion injury in adult male rats and investigated changes in SRPX2 expression at 2 h, 6 h, 24 h, 48 h, 72 h, 5 days, 7 days, 14 days, 1 month, and 3 months post-injury. The plasma SRPX2 level was assessed by Western blot analysis. Hypothalamic SRPX2-immunoreactive neuronal numbers were estimated from immunostained preparations. At 2 h post-TBI, plasma SRPX2 levels were markedly decreased compared with the naïve group (area under the curve = 1.00

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.

BACKGROUND: Traumatic brain injury (TBI) causes 10-20% of acquired epilepsy, which typically develops within 2 years post-injury with poorly understood mechanisms. We investigated the location, severity, evolution and persistence of blood-brain barrier (BBB) dysfunction and associated neuroinflammation after TBI, and their contribution to post-traumatic seizure susceptibility.METHODS: TBI was induced with lateral fluid-percussion in adult male Sprague-Dawley rats (6 sham, 12 TBI). Permeability of the BBB was assessed using T1-weighted magnetic resonance imaging (MRI) with gadobutrol (Gd) contrast enhancement at 4 days, 2 weeks, 2 months, and 10 months post-injury and with intravenously administered fluorescein at 11 months post-TBI. Continuous (24/7) video-EEG monitoring was performed for 3 weeks at 11 months post-injury followed by the pentylenetetrazol (PTZ) seizure-susceptibility test. In the end, rats were perfused for histology to assess albumin extravasation, iron deposits, calcifications, reactive astrocytes, microglia and monocytes. To investigate the translational value of the data obtained, BBB dysfunction and neuroinflammation were investigated immunohistochemically in autopsy brain tissue from patients with TBI and PTE.RESULTS: MRI indicated persistent Gd leakage in the impacted cortex and thalamus of variable severity in all rats with TBI which correlated with fluorescein extravasation. In the impacted cortex BBB dysfunction was evident from 4 days post-injury onwards to the end of the 10-months follow-up. In the ipsilateral thalamus, leakage was evident at 2 and 10 months post-injury. The greater the BBB leakage in the perilesional cortex at 10 months after the injury, the greater the expression of the endothelial cell antigen RECA-1 (r = 0.734, p r = 0.699, p CONCLUSIONS: Rats and humans with TBI have long-lasting cortical BBB dysfunction and neuroinflammation. Focal Gd-enhancement matched with loci of neuroinflammation, particularly in the thalamus. Although BBB leakage did not associate with increased seizure susceptibility after TBI, our data suggest that treatments aimed to mitigate BBB damage and its secondary pathologies like chronic neuroinflammation, have a region-specific long-lasting therapeutic time window.

Binding of urokinase-type plasminogen activator receptor (uPAR) to its ligand uPA or to its plasma membrane partner, platelet-derived growth factor receptor β (PDGFRβ), promotes neuroprotection, cell proliferation, and angiogenesis. Following injury, single deficiency in uPA or uPAR leads in increased tissue loss and compromised vascular remodeling. We hypothesized that double-deficiency of uPAR (Plaur) and uPA (Plau) would result in increased lesion area and poor vascular integrity after traumatic brain injury (TBI). TBI was induced by lateral fluid-percussion injury in Plau/Plaur double-knockout (dKO) and wild-type (Wt) mice. The cortical lesion area was quantified in unfolded cortical maps prepared from thionin-stained sections at 4 d or 30 d post-TBI. The density of PDGFRβ+ pericytes and blood vessels was calculated from immunostained sections. Blood-brain barrier leakage was analyzed using ImageJ® from IgG-immunostained sections. Genotype had no effect on the total area of the cortical lesion at 4 d or 30 d post-TBI (p 0.05) or its progression as the overall lesion area was comparable at 4 d and 30 d post-TBI in both genotypes (p 0.05). Subfield analysis, however, indicated that damage to the visual cortex at 4 d post-TBI in dKO-TBI mice was 53 % of that in Wt-TBI mice (p 0.05). Both genotypes had a higher density of PDGFRβ-positive pericytes at 4 d than at 30 d post-TBI (p 0.05), but no genotype effect was detected between these time-points (p 0.05). TBI-induced increase in the density of PDGFRβ+ blood vessels at the region adjacent to the lesion core was comparable in both genotypes (p 0.05). Genotype had no effect on TBI-induced IgG leakage into the perilesional cortical parenchyma (p 0.05). Contrary to our expectations, Plau/Plaur double-deficiency did not aggravate TBI-related structural outcome.

Epilepsy is among the most common serious disabling disorders of the brain, and the global burden of epilepsy exerts a tremendous cost to society. Most people with epilepsy have acquired forms of the disorder, and the development of antiepileptogenic interventions could potentially prevent or cure epilepsy in many of them. However, the discovery of potential antiepileptogenic treatments and clinical validation would require a means to identify populations of patients at very high risk for epilepsy after a potential epileptogenic insult, to know when to treat and to document prevention or cure. A fundamental challenge in discovering biomarkers of epileptogenesis is that this process is likely multifactorial and crosses multiple modalities. Investigators must have access to a large number of high quality, well-curated data points and study subjects for biomarker signals to be detectable above the noise inherent in complex phenomena, such as epileptogenesis, traumatic brain injury (TBI), and conditions of data collection. Additionally, data generating and collecting sites are spread worldwide among different laboratories, clinical sites, heterogeneous data types, formats, and across multi-center preclinical trials. Before the data can even be analyzed, these data must be standardized. The Epilepsy Bioinformatics Study for Antiepileptogenic Therapy (EpiBioS4Rx) is a multi-center project with the overarching goal that epileptogenesis after TBI can be prevented with specific treatments. The identification of relevant biomarkers and performance of rigorous preclinical trials will permit the future design and performance of economically feasible full-scale clinical trials of antiepileptogenic therapies. We have been analyzing human data collected from UCLA and rat data collected from the University of Eastern Finland, both centers collecting data for EpiBioS4Rx, to identify biomarkers of epileptogenesis. Big data techniques and rigorous analysis are brought to longitudinal data collected from humans and an animal model of TBI, epilepsy, and their interaction. The prolonged continuous data streams of intracranial, cortical surface, and scalp EEG from humans and an animal model of epilepsy span months. By applying our innovative mathematical tools via supervised and unsupervised learning methods, we are able to subject a robust dataset to recently pioneered data analysis tools and visualize multivariable interactions with novel graphical methods.

Multi-center preclinical studies can facilitate the discovery of biomarkers of antiepileptogenesis and thus facilitate the diagnosis and treatment development of patients at risk of developing post-traumatic epilepsy. However, these studies are often limited by the difficulty in harmonizing experimental protocols between laboratories. Here, we assess whether the production of traumatic brain injury (TBI) using the lateral fluid-percussion injury (FPI) in adult male Sprague-Dawley rats (12 weeks at the time of injury) was harmonized between three laboratories - located in the University of Eastern Finland (UEF), Monash University in Melbourne, Australia (Melbourne) and The University of California, Los Angeles, USA (UCLA). These laboratories are part of the international multicenter-based project, the Epilepsy Bioinformatics Study for Antiepileptogenesis Therapy (EpiBioS4Rx). Lateral FPI was induced in adult male Sprague-Dawley rats. The success of methodological harmonization was assessed by performing inter-site comparison of injury parameters including duration of anesthesia during surgery, impact pressure, post-impact transient apnea, post-impact seizure-like behavior, acute mortality ( Melbourne > UCLA (p

Diagnosis of ongoing epileptogenesis and associated hyperexcitability after brain injury is a major challenge. Given that increased neuronal activity in the brain triggers a blood oxygenation level-dependent (BOLD) response in functional magnetic resonance imaging (fMRI), we hypothesized that fMRI could be used to identify the brain area(s) with hyperexcitability during post-injury epileptogenesis. We applied fMRI to detect onset and spread of BOLD activation after pentylenetetrazol (PTZ)-induced seizures (PTZ, 30 mg/kg, intraperitoneally) in 16 adult male rats at 2 months after lateral fluid percussion (FPI)-induced traumatic brain injury (TBI). In sham-operated controls, onset of the PTZ-induced BOLD response was bilateral and first appeared in the cortex. After TBI, 5 of 9 (56%) rats exhibited ipsilateral perilesional cortical BOLD activation, followed by activation of the contralateral cortex. In 4 of 9 (44%) rats, onset of BOLD response was bilateral. Interestingly, latency from the PTZ injection to onset of the BOLD response increased in the following order: sham-operated controls (ipsilateral 132 ± 57 sec, contralateral 132 ± 57 sec; p 0.05)TBI with bilateral BOLD onset (ipsilateral 176 ± 54 sec, contralateral 178 ± 52 sec; p 0.05)TBI with ipsilateral BOLD onset (ipsilateral 406 ± 178 sec, contralateral 509 ± 140 sec; p 0.05). Cortical lesion area did not differ between rats with ipsilateral versus bilateral BOLD onset (p 0.05). In the group of rats with ipsilateral onset of PTZ-induced BOLD activation, none of the rats showed a robust bilateral thalamic BOLD response, only 1 of 5 rats had robust ipsilateral thalamic calcifications, and 4 of 5 rats had perilesional astrocytosis. These findings suggest the evolution of the epileptogenic zone in the perilesional cortex after TBI, which is sensitive to PTZ-induced hyperexcitability. Further studies are warranted to explore the evolution of thalamo-cortical pathology as a driver of epileptogenesis after lateral FPI.

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.

A biomarker is a characteristic that is 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. In 2015, the FDA-NIH Joint Leadership Council developed the BEST Resource (Biomarkers, EndpointS, and other Tools) to improve the understanding and use of biomarker terminology in biomedical research, clinical practice, and medical product development. The BEST biomarker categories include: (a) susceptibility/risk biomarkers, (b) diagnostic bio-markers, (c) monitoring biomarkers, (d) prognostic biomarkers, (e) predictive biomarkers, (f) pharmacodynamic/response biomarkers, and (g) safety biomarkers. Here we review 30 epilepsy biomarker studies that have identified (a) diagnostic biomarkers for epilepsy, epileptogenesis, epileptogenicity, drug-refractoriness, and status epilepticus - some of the epileptogenesis and epileptogenicity biomarkers can also be considered prognostic biomarkers for the development of epilepsy in subjects with a given brain insult, (b) predictive biomarkers for epilepsy surgery outcome, and (c) a response biomarker for therapy outcome. The biomarker modalities include plasma/serum/exosomal and cerebrospinal fluid molecular biomarkers, brain tissue molecular bio-markers, imaging biomarkers, electrophysiologic biomarkers, and behavioral/cognitive biomarkers. Both single and combinatory biomarkers have been described. Most of the reviewed biomarkers have an area under the curve > 0.800 in receiver operating characteristics analysis, suggesting high sensitivity and specificity. As discussed in this review, we are in the early phase of the learning curve in epilepsy biomarker discovery. Many of the seven biomarker categories lack epilepsy-related biomarkers. There is a need for epilepsy biomarker discovery using proper, statistically powered study designs with validation cohorts, and the development and use of novel analytical methods. A strategic roadmap to discuss the research priorities in epilepsy biomarker discovery, regulatory issues, and optimization of the use of resources, similar to those devised in the cancer and Alzheimer’s disease research areas, is also needed.

Purpose Subjects with Alzheimer's disease (AD) have been shown to be at a higher risk for epilepsy. The vast majority of the previous studies have not included a full neuropathological examination. Methods The objective of this study was to assess the prevalence of epilepsy and clinicopathological characteristics in a well-defined study group of 64 subjects with AD. We evaluated the clinicopathological findings in 64 subjects (mean age at death 85 ± 8.6 years) from a longitudi-nal study cohort of patients with dementia. Results Eleven out of the 64 subjects (17%) had a history of epilepsy, which is comparable to previous studies. The subjects with AD and epilepsy were significantly younger at the time of AD diagnosis and at the time of hospitalisation. In addition, their duration of AD was longer. Concomitant neuropathology in addition to AD was common in both groups and the ApoE genotypes did not differ significantly between the groups. Conclusion The strength of this study is a thorough neuropathological examination of all study subjects. Our findings support the previous literature regarding the prevalence of epilepsy in subjects with AD. We have shown that the subjects with AD and epilepsy differ significantly from the subjects without epilepsy.

Several components of the urokinase-type plasminogen activator receptor (uPAR)-interactome, including uPAR and its ligand sushi-repeat protein 2, X-linked (SRPX2), are linked to susceptibility to epileptogenesis in animal models and/or humans. Recent evidence indicates that urokinase-type plasminogen activator (uPA), a uPAR ligand with focal proteinase activity in the extracellular matrix, contributes to recovery-enhancing brain plasticity after various epileptogenic insults such as traumatic brain injury (TBI) and status epilepticus. Here, we examined whether deficiency of the uPA-encoding gene Plau augments epileptogenesis after TBI. Traumatic brain injury was induced by controlled cortical impact in the somatosensory cortex of adult male wild-type and Plau-deficient mice. Development of epilepsy and seizure susceptibility were assessed with a 3-week continuous video-electroencephalography monitoring and a pentylenetetrazol test, respectively. Traumatic brain injury-induced cortical or hippocampal pathology did not differ between genotypes. The pentylenetetrazol test revealed increased seizure susceptibility after TBI (p

Global expression profiling of neurologic or psychiatric disorders has been confounded by variability among laboratories, animal models, tissues sampled, and experimental platforms, with the result being that few genes demonstrate consistent expression changes. We attempted to minimize these confounds by pooling dentate granule cell transcriptional profiles from 164 rats in seven laboratories, using three status epilepticus (SE) epilepsy models (pilocarpine, kainate, self-sustained SE), plus amygdala kindling. In each epilepsy model, RNA was harvested from laser-captured dentate granule cells from six rats at four time points early in the process of developing epilepsy, and data were collected from two independent laboratories in each rodent model except SSSE. Hierarchical clustering of differentially-expressed transcripts in the three SE models revealed complete separation between controls and SE rats isolated 1 day after SE. However, concordance of gene expression changes in the SE models was only 26–38% between laboratories, and 4.5% among models, validating the consortium approach. Transcripts with unusually highly variable control expression across laboratories provide a ‘red herring’ list for low-powered studies., published version, peerReviewed

The identification of mechanisms transforming normal to seizure-generating tissue after brain injury is key to developing new antiepileptogenic treatments. MicroRNAs (miRNAs) may act as regulators and potential treatment targets for epileptogenesis. Here, we undertook a meta-analysis of changes in miRNA expression in the hippocampal dentate gyrus (DG) following an epileptogenic insult in three epilepsy models. We identified 26 miRNAs significantly differentially expressed during epileptogenesis, and five differentially expressed in chronic epilepsy. Of these, 13 were not identified in any of the individual studies. To assess the role of these miRNAs, we predicted their mRNA targets and then filtered the list to include only target genes expressed in DG and negatively correlated with miRNA expression. Functional enrichment analysis of mRNA targets of miRNAs dysregulated during epileptogenesis suggested a role for molecular processes related to inflammation and synaptic function. Our results identify new miRNAs associated with epileptogenesis from existing data, highlighting the utility of meta-analysis in maximizing value from preclinical data.

Treatment of TBI remains a major unmet medical need, with 2.5 million new cases of traumatic brain injury (TBI) each year in Europe and 1.5 million in the USA. This single-center proof-of-concept preclinical study tested the hypothesis that pharmacologic neurostimulation with proconvulsants, either atipamezole, a selective α2-adrenoceptor antagonist, or the cannabinoid receptor 1 antagonist SR141716A, as monotherapy would improve functional recovery after TBI. A total of 404 adult Sprague-Dawley male rats were randomized into two groups: sham-injured or lateral fluid-percussion–induced TBI. The rats were treated with atipamezole (started at 30 min or 7 d after TBI) or SR141716A (2 min or 30 min post-TBI) for up to 9 wk. Total follow-up time was 14 wk after treatment initiation. Outcome measures included motor (composite neuroscore, beam-walking) and cognitive performance (Morris water-maze), seizure susceptibility, spontaneous seizures, and cortical and hippocampal pathology. All injured rats exhibited similar impairment in the neuroscore and beam-walking tests at 2 d post-TBI. Atipamezole treatment initiated at either 30 min or 7 d post-TBI and continued for 9 wk via subcutaneous osmotic minipumps improved performance in both the neuroscore and beam-walking tests, but not in the Morris water-maze spatial learning and memory test. Atipamezole treatment initiated at 7 d post-TBI also reduced seizure susceptibility in the pentylenetetrazol test 14 wk after treatment initiation, although it did not prevent the development of epilepsy. SR141716A administered as a single dose at 2 min post-TBI or initiated at 30 min post-TBI and continued for 9 wk had no recovery-enhancing or antiepileptogenic effects. Mechanistic studies to assess the α2-adrenoceptor subtype specificity of the disease-modifying effects of atipametzole revealed that genetic ablation of α2A-noradrenergic receptor function in Adra2A mice carrying an N79P point mutation had antiepileptogenic effects after TBI. On the other hand, blockade of α2C-adrenoceptors using the receptor subtype-specific antagonist ORM-12741 had no favorable effects on the post-TBI outcome. Finally, to assess whether regulation of the post-injury inflammatory response by atipametzole in glial cells contributed to a favorable outcome, we investigated the effect of atipamezole on spontaneous and/or lipopolysaccharide-stimulated astroglial or microglial cytokine release in vitro. We observed no effect. Our data demonstrate that a 9-wk administration of α2A-noradrenergic antagonist, atipamezole, is recovery-enhancing after TBI., final draft, peerReviewed

Alzheimer’s disease (AD), especially its rare familial forms, is associated with increased risk of epilepsy. Research during the past decade has revealed that the most important genetic mouse model of AD, mice expressing mutated human amyloid precursor protein (APP), alone or in combination with mutated human presenilin-1 transgene, express unprovoked recurrent seizures and frequent epileptic spiking as one essential component of their phenotype. Although all APP transgenic mice share common phenotypic features, the time course of their manifestation is highly variable between models. The cause of epilepsy in APP transgenic mice remains elusive, but is a consequence of accumulated APP breakdown products. Antiepileptic drugs show differential effect on epileptic EEG manifestations in APP transgenic mice, with the best-documented effect so far with levetiracetam and valproic acid. Both drugs also appear to decrease amyloid-β production and alleviate age-associated memory impairment in these mice.

According to the World Health Organization, 50 million people worldwide have epilepsy. Traumatic brain injury (TBI) is estimated to cause 10%–20% of all of symptomatic epilepsies, and 5% of all epilepsies. Over the past 15 years, several laboratories have reported spontaneous seizures after inducing TBI in rats and mice. The TBI models in which spontaneous seizures are detected include fluid percussion injury, and controlled cortical impact models. Increased seizure susceptibility has also been described in a weight-drop model. Recent studies identified some genotypes that predispose to post-TBI epileptogenesis or comorbidogenesis. The use of post-TBI models in antiseizure drug-discovery is rare, but they have been used in more than 10 proof-of-concept preclinical antiepileptogenesis trials in an attempt to find treatments to alleviate the development of post-TBI epilepsy and comorbidities. Despite significant progress in modeling posttraumatic epileptogenesis, the animal models still represent only a few of the injury types and pathologies of patients with TBI destined to develop epilepsy. Also, experimental studies investigating posttraumatic epileptogenesis in young or old animals, as well as in females, are lacking. Endophenotyping human epileptogenic TBI is critical for the advancement of preclinical model development.

Objective: Urokinase-type plasminogen activator (uPA) and kallikrein-related peptidase 8 (KLK8) are serine proteases that contribute to extracellular matrix (ECM) remodeling after brain injury. They can be labelled with the novel radiotracer [In-111]MICA-401. As the first step in exploring the applicability of [In-111]MICA-401 in tracing the mechanisms of postinjury ECM reorganization in vivo, we performed in vitro and ex vivo studies, assessing [In-111]MICA-401 distribution in the brain in two animal models: kainic acid-induced status epilepticus (KASE) and controlled cortical impact (CCI)-induced traumatic brain injury (TBI). Methods: In the KASE model, in vitro autoradiography with [In-111]MICA-401 was performed at 7 days and 12 weeks post-SE. To assess seizure burden, rats were monitored using video-electroencephalography (EEG) for 1 month before the 12-week time point. In the CCI model, in vitro autoradiography was performed at 4 days and ex vivo autoradiography at 7 days post-TBI. Results: At 7 days post-SE, in vitro autoradiography revealed significantly decreased [In-111]MICA-401 binding in hippocampal CA3 subfield and extrahippocampal temporal lobe (ETL). In the chronic phase, when animals had developed spontaneous seizures, specific binding was decreased in CA3 and CA1/CA2 subfields of hippocampus, dentate gyrus, ETL, and parietal cortex. Of interest, KASE rats with the highest frequency of seizures had the lowest hippocampal [In-111]MICA-401 binding (r = -0.76, p

Traumatic brain injury (TBI) can result in several dentate gyrus-regulated disabilities. Almost nothing is known about the chronic molecular changes after TBI, and their potential as treatment targets. We hypothesized that chronic transcriptional alterations after TBI are under microRNA (miRNA) control. Expression of miRNAs and their targets in the dentate gyrus was analyzed using microarrays at 3 months after experimental TBI. Of 305 miRNAs present on the miRNA-array, 12 were downregulated (p, published version, peerReviewed

Models are representations that researchers use to create knowledge. There remains much to learn about seizures and epilepsy. Consequently, a wide variety of models could be helpful. As the contents of the 2nd Edition of Models of Seizures and Epilepsy show, we have a respectable roster of in silico, in vitro, as well as nonmammalian and mammalian in vivo models of seizures and epilepsy that can be used to investigate different aspects of the mechanisms of epileptic spikes, epileptiform discharges, epileptogenic networks, seizures, status epilepticus, epilepsy, and related comorbidities at different developmental ages and genders.

Traumatic brain injury (TBI) can cause a myriad of sequelae depending on its type, severity, and location of injured structures. These can include mood disorders, posttraumatic stress disorder and other anxiety disorders, personality disorders, aggressive disorders, cognitive changes, chronic pain, sleep problems, motor or sensory impairments, endocrine dysfunction, gastrointestinal disturbances, increased risk of infections, pulmonary disturbances, parkinsonism, posttraumatic epilepsy, or their combinations. The progression of individual pathologies leading to a given phenotype is variable, and some progress for months. Consequently, the different post-TBI phenotypes appear within different time windows. In parallel with morbidogenesis, spontaneous recovery occurs both in experimental models and in human TBI. A great challenge remains; how can we dissect the specific mechanisms that lead to the different endophenotypes, such as posttraumatic epileptogenesis, in order to identify treatment approaches that would not compromise recovery?

Post-traumatic epilepsy accounts for 10–20 % of symptomatic epilepsy in the general population and 5 % of all epilepsy. During the last decade, an increasing number of laboratories have investigated the molecular and cellular mechanisms of post-traumatic epileptogenesis in experimental models. However, identification of critical molecular, cellular, and network mechanisms that would be specific for post-traumatic epileptogenesis remains a challenge. Despite of that, 7 of 9 proof-of-concept antiepileptogenesis studies have demonstrated some effect on seizure susceptibility after experimental traumatic brain injury, even though none of them has progressed to clinic. Moreover, there has been some promise that new clinically translatable imaging approaches can identify biomarkers for post-traumatic epileptogenesis. Even though the progress in combating post-traumatic epileptogenesis happens in small steps, recent discoveries kindle hope for identification of treatment strategies to prevent post-traumatic epilepsy in at-risk patients.

The Epilepsy Bioinformatics Study for Antiepileptogenic Therapy (EpiBioS4Rx) is a NINDS funded Center-Without-Walls international study aimed at preventing epileptogenesis after traumatic brain injury (TBI). One objective of EpiBioS4Rx relates to preclinical biomarker discovery for post-traumatic epilepsy. In order to perform a statistically appropriately powered biomarker discovery study, EpiBioS4Rx has made a rigorous attempt to harmonize the preclinical procedures performed at the three EpiBioS4Rx centers, located in Finland, Australia, and the USA. Moreover, we have also performed a rigorous interim analysis of the success of procedural harmonization, which is reported in this virtual special issue. The analysis included harmonization of the production of animal model, blood sampling, electroencephalogram analyses (seizures, high-frequency oscillations) and magnetic resonance imaging analysis. Based on lessons learned, we propose a 3-stage protocol to facilitate the success of preclinical multicenter studies: preparation ⇨ testing ⇨ multicenter study. The need of funding for preparation and testing phases, which precede the actual multicenter study and are necessary for its success, should be taken into account in the design of funding schemes.

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.

Reduced hippocampal GABAergic inhibition is acknowledged to be associated with epilepsy. However, there are no studies that had quantitatively compared the loss of various interneuron populations in different models of epilepsy. We tested a hypothesis that the more severe the loss of hippocampal interneurons, the more severe was the epilepsy. Epileptogenesis was triggered in adult rats by status epilepticus (SE) (56 SE, 24 controls) or by traumatic brain injury (TBI) (45 TBI, 23 controls). The total number of hippocampal parvalbumin (PARV), cholecystokinin (CCK), calretinin (CR), somatostatin (SOM), or neuropeptide Y (NPY) positive neurons was estimated using unbiased stereology at 1 or 6 months post-insult. The rats with TBI had no spontaneous seizures but showed increased seizure susceptibility. Eleven of the 28 rats (39 %) in the SE group had spontaneous seizures. The most affected hippocampal area after TBI was the ipsilateral dentate gyrus, where 62 % of PARV-immunoreactive (ir) (p < 0.001 compared to controls), 77 % of CR-ir (p < 0.05), 46 % of SOM-ir (p < 0.001), and 59 % of NPY-ir (p < 0.001) cells remained at 1 month after TBI. At 6 months post-TBI, only 35 % of PARV-ir (p < 0.001 compared to controls), 63 % of CCK-ir (p < 0.01), 74 % of CR-ir (p < 0.001), 55 % of SOM-ir (p < 0.001), and 51 % of NPY-ir (p < 0.001) cells were remaining. Moreover, the reduction in PARV-ir, CCK-ir, and CR-ir neurons was bilateral (all p < 0.05). Substantial reductions in different neuronal populations were also found in subfields of the CA3 and CA1. In rats with epilepsy after SE, the number of PARV-ir neurons was reduced in the ipsilateral CA1 (80 % remaining, p < 0.05) and the number of NPY-ir neurons bilaterally in the dentate gyrus (33-37 %, p < 0.01) and the CA3 (54-57 %, p < 0.05). Taken together, interneuron loss was substantially more severe, widespread, progressive, and included more interneuron subclasses after TBI than after SE. Interneurons responsible for perisomatic inhibition were more vulnerable to TBI than those providing dendritic inhibition. Unlike expected, we could not demonstrate any etiology-independent link between the severity of hippocampal interneuron loss and the overall risk of spontaneous seizures.

The search for new treatments for seizures, epilepsies, and their comorbidities faces considerable challenges. This is due in part to gaps in our understanding of the etiology and pathophysiology of most forms of epilepsy. An additional challenge is the difficulty in predicting the efficacy, tolerability, and impact of potential new treatments on epilepsies and comorbidities in humans, using the available resources. Herein we provide a summary of the discussions and proposals of the Working Group 2 as presented in the Joint American Epilepsy Society and International League Against Epilepsy Translational Workshop in London (September 2012). We propose methodologic and reporting practices that will enhance the uniformity, reliability, and reporting of early stage preclinical studies with animal seizure and epilepsy models that aim to develop and evaluate new therapies for seizures or epilepsies, using multidisciplinary approaches. The topics considered include the following: (1) implementation of better study design and reporting practices; (2) incorporation in the study design and analysis of covariants that may influence outcomes (including species, age, sex); (3) utilization of approaches to document target relevance, exposure, and engagement by the tested treatment; (4) utilization of clinically relevant treatment protocols; (5) optimization of the use of video-electroencephalography (EEG) recordings to best meet the study goals; and (6) inclusion of outcome measures that address the tolerability of the treatment or study end points apart from seizures. We further discuss the different expectations for studies aiming to meet regulatory requirements to obtain approval for clinical testing in humans. Implementation of the rigorous practices discussed in this report will require considerable investment in time, funds, and other research resources, which may create challenges for academic researchers seeking to contribute to epilepsy therapy discovery and development. We propose several infrastructure initiatives to overcome these barriers.

The present study was designed to test a hypothesis that functional magnetic resonance imaging (fMRI) can be used to monitor functional impairment and recovery after moderate experimental traumatic brain injury (TBI). Moderate TBI was induced by lateral fluid percussion injury in adult rats. The severity of brain damage and functional recovery in the primary somatosensory cortex (S1) was monitored for up to 56 days using fMRI, cerebral blood flow (CBF) by arterial spin labeling, local field potential measurements (LFP), behavioral assessment, and histology. All the rats had reduced blood-oxygen-level-dependent (BOLD) responses during the 1st week after trauma in the ipsilateral S1. Forty percent of these animals showed recovery of the BOLD response during the 56 day follow-up. Unexpectedly, no association was found between the recovery in BOLD response and the volume of the cortical lesion or thalamic neurodegeneration. Instead, the functional recovery occurred in rats with preserved myelinated fibers in layer VI of S1. This is, to our knowledge, the first study demonstrating that fMRI can be used to monitor post-TBI functional impairment and consequent spontaneous recovery. Moreover, the BOLD response was associated with the density of myelinated fibers in the S1, rather than with neurodegeneration. The present findings encourage exploration of the usefulness of fMRI as a noninvasive prognostic biomarker for human post-TBI outcomes and therapy responses.

We aimed to define the chronically altered gene expression signature of traumatic brain injury (TBI-sig) to discover novel treatments to reverse pathologic gene expression or reinforce the expression of recovery-related genes. Genome-wide RNA-sequencing was performed at 3 months post-TBI induced by lateral fluid-percussion injury in rats. We found 4964 regulated genes in the perilesional cortex and 1966 in the thalamus (FDR in vivo animal models of epilepsy. Other compounds revealed by the analysis were BRD-K91844626, BRD-A11009626, NO-ASA, BRD-K55260239, SDZ-NKT-343, STK-661558, BRD-K75971499, ionomycin and desmethylclomipramine. Network analysis of overlapping genes revealed the effects on tubulins (Tubb2a, Tubb3, Tubb4b), Nfe2l2, S100a4, Cd44 and Nfkb2, all of which are linked to TBI-relevant outcomes, including epileptogenesis and tissue repair. Desmethylclomipramine modulated most of the gene targets considered favorable for TBI outcome. Our data demonstrate long-lasting transcriptomics changes after TBI. LINCS analysis predicted that these changes could be modulated by various compounds, some of which are already in clinical use but never tested in TBI.

Animal models of epilepsy and seizures, mostly involving mice and rats, are used to understand the pathophysiology of the different forms of epilepsy and their comorbidities, to identify biomarkers, and to discover new antiepileptic drugs and treatments for comorbidities. Such models represent an important area for application of the 3Rs (replacement, reduction and refinement of animal use). This report provides background information and recommendations aimed at minimising pain, suffering and distress in rodent models of epilepsy and seizures in order to improve animal welfare and optimise the quality of studies in this area. The report includes practical guidance on principles of choosing a model, induction procedures, in vivo recordings, perioperative care, welfare assessment, humane endpoints, social housing, environmental enrichment, reporting of studies and data sharing. In addition, some model-specific welfare considerations are discussed, and data gaps and areas for further research are identified. The guidance is based upon a systematic review of the scientific literature, survey of the international epilepsy research community, consultation with veterinarians and animal care and welfare officers, and the expert opinion and practical experience of the members of a Working Group convened by the United Kingdom's National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs).