ComBating inter-site differences in field strength: harmonizing preclinical traumatic brain injury MRI data.

Integrating datasets from multiple sites and scanners can increase statistical power for neuroimaging studies but can also introduce significant inter-site confounds. We evaluated the effectiveness of ComBat, an empirical Bayes approach, to combine longitudinal preclinical MRI data acquired at 4.7 or 9.4 T at two different sites in Australia. Male Sprague Dawley rats underwent MRI on Days 2, 9, 28, and 150 following moderate/severe traumatic brain injury (TBI) or sham injury as part of Project 1 of the NIH/NINDS-funded Centre Without Walls EpiBioS4Rx project. Diffusion-weighted and multiple-gradient-echo images were acquired, and outcomes included QSM, FA, and ADC. Acute injury measures including apnea and self-righting reflex were consistent between sites. Mixed-effect analysis of ipsilateral and contralateral corpus callosum (CC) summary values revealed a significant effect of site on FA and ADC values, which was removed following ComBat harmonization. Bland-Altman plots for each metric showed reduced variability across sites following ComBat harmonization, including for QSM, despite appearing to be largely unaffected by inter-site differences and no effect of site observed. Following harmonization, the combined inter-site data revealed significant differences in the imaging metrics consistent with previously reported outcomes. TBI resulted in significantly reduced FA and increased susceptibility in the ipsilateral CC, and significantly reduced FA in the contralateral CC compared with sham-injured rats. Additionally, TBI rats also exhibited a reversal in ipsilateral CC ADC values over time with significantly reduced ADC at Day 9, followed by increased ADC 150 days after injury. Our findings demonstrate the need for harmonizing multi-site preclinical MRI data and show that this can be successfully achieved using ComBat while preserving phenotypical changes due to TBI.

Tau Phosphorylation Patterns in the Rat Cerebral Cortex After Traumatic Brain Injury and Sodium Selenate Effects: An Epibios4rx Project 2 Study.

Sodium selenate (SS) activates protein phosphatase 2 (PP2A) and reduces phosphorylated tau (pTAU) and late post-traumatic seizures after lateral fluid percussion injury (LFPI). In EpiBioS4Rx Project 2, a multi-center international study for post-traumatic targets, biomarkers, and treatments, we tested the target relevance and modification by SS of pTAU forms and PP2A and in the LFPI model, at two sites: Einstein and Melbourne. In Experiment 1, adult male rats were assigned to LFPI and sham (both sites) and naïve controls (Einstein). Motor function was monitored by neuroscores. Brains were studied with immunohistochemistry (IHC), Western blots (WBs), or PP2A activity assay, from 2 days to 8 weeks post-operatively. In Experiment 2, LFPI rats received SS for 7 days (SS0.33: 0.33 mg/kg/day; SS1: 1 mg/kg/day, subcutaneously) or vehicle (Veh) post-LFPI and pTAU, PR55 expression, or PP2A activity were studied at 2 days and 1 week (on treatment), or 2 weeks (1 week off treatment). Plasma selenium and SS levels were measured. In Experiment 1 IHC, LFPI rats had higher cortical pTAU-Ser202/Thr205-immunoreactivity (AT8-ir) and pTAU-Ser199/202-ir at 2 days, and pTAU-Thr231-ir (AT180-ir) at 2 days, 2 weeks, and 8 weeks, ipsilaterally to LFPI, than controls. LFPI-2d rats also had higher AT8/total-TAU5-ir in cortical extracts ipsilateral to the lesion (WB). PP2A (PR55-ir) showed time- and region-dependent changes in IHC, but not in WB. PP2A activity was lower in LFPI-1wk than in sham rats. In Experiment 2, SS did not affect neuroscores or cellular AT8-ir, AT180-ir, or PR55-ir in IHC. In WB, total cortical AT8/total-TAU-ir was lower in SS0.33 and SS1 LFPI rats than in Veh rats (2 days, 1 week); total cortical PR55-ir (WB) and PP2A activity were higher in SS1 than Veh rats (2 days). SS dose dependently increased plasma selenium and SS levels. Concordant across-sites data confirm time and pTAU form-specific cortical increases ipsilateral to LFPI. The discordant SS effects may either suggest SS-induced reduction in the numbers of cells with increased pTAU-ir, need for longer treatment, or the involvement of other mechanisms of action.

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.

E2730, an uncompetitive γ-aminobutyric acid transporter-1 inhibitor, suppresses epileptic seizures in a rat model of chronic mesial temporal lobe epilepsy.

OBJECTIVE: More than one third of mesial temporal lobe epilepsy (MTLE) patients are resistant to current antiseizure medications (ASMs), and half experience mild-to-moderate adverse effects of ASMs. There is therefore a strong need to develop and test novel ASMs. The objective of this work is to evaluate the pharmacokinetics and neurological toxicity of E2730, a novel uncompetitive inhibitor of γ-aminobutyric acid transporter-1, and to test its seizure suppression effects in a rat model of chronic MTLE. METHODS: We first examined plasma levels and adverse neurological effects of E2730 in healthy Wistar rats. Adult male rats were implanted with osmotic pumps delivering either 10, 20, or 100 mg/kg/day of E2730 subcutaneously for 1 week. Blood sampling and behavioral assessments were performed at several timepoints. We next examined whether E2730 suppressed seizures in rats with chronic MTLE. These rats were exposed to kainic acid-induced status epilepticus, and 9 weeks later, when chronic epilepsy was established, were assigned to receive one of the three doses of E2730 or vehicle for 1 week in a randomized crossover design. Continuous video-electroencephalographic monitoring was acquired during the treatment period to evaluate epileptic seizures. RESULTS: Plasma levels following continuous infusion of E2730 showed a clear dose-related increase in concentration. The drug was well tolerated at all doses, and any sedation or neuromotor impairment was mild and transient, resolving within 48 h of treatment initiation. Remarkably, E2730 treatment in chronically epileptic rats led to seizure suppression in a dose-dependent manner, with 65% of rats becoming seizure-free at the highest dose tested. Mean seizure class did not differ between the treatment groups. SIGNIFICANCE: This study shows that continuous subcutaneous infusion of E2730 over 7 days results in a marked, dose-dependent suppression of spontaneous recurrent seizures, with minimal adverse neurological effects, in a rat model of chronic MTLE. E2730 shows strong promise as an effective new ASM to be translated into clinical trials.

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.

Levetiracetam Pharmacokinetics and Brain Uptake in a Lateral Fluid Percussion Injury Rat Model.

Post-traumatic epilepsy (PTE) occurs in some patients after moderate/severe traumatic brain injury (TBI). Although there are no approved therapies to prevent epileptogenesis, levetiracetam (LEV) is commonly given for seizure prophylaxis due to its good safety profile. This led us to study LEV as part of the Epilepsy Bioinformatics Study for Antiepileptogenic Therapy (EpiBioS4Rx) Project. The objective of this work is to characterize the pharmacokinetics (PK) and brain uptake of LEV in naïve control rats and in the lateral fluid percussion injury (LFPI) rat model of TBI after either single intraperitoneal doses or a loading dose followed by a 7-day subcutaneous infusion. Sprague-Dawley rats were used as controls and for the LFPI model induced at the left parietal region using injury parameters optimized for moderate/severe TBI. Naïve and LFPI rats received either a bolus injection (intraperitoneal) or a bolus injection followed by subcutaneous infusion over 7 days. Blood and parietal cortical samples were collected at specified time points throughout the study. LEV concentrations in plasma and brain were measured using validated high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) methods. Noncompartmental analysis and a naive-pooled compartmental PK modeling approach were used. Brain-to-plasma ratios ranged from 0.54 to 1.4 to 1. LEV concentrations were well fit by one-compartment, first-order absorption PK models with a clearance of 112 ml/h per kg and volume of distribution of 293 ml/kg. The single-dose pharmacokinetic data were used to guide dose selection for the longer-term studies, and target drug exposures were confirmed. Obtaining LEV PK information early in the screening phase allowed us to guide optimal treatment protocols in EpiBioS4Rx. SIGNIFICANCE STATEMENT: The characterization of levetiracetam pharmacokinetics and brain uptake in an animal model of post-traumatic epilepsy is essential to identify target concentrations and guide optimal treatment for future studies.

Neuroinflammation: A Common Pathway in Alzheimer's Disease and Epilepsy.

BACKGROUND: Neuroinflammation is an innate immunological response of the central nervous system that may be induced by a brain insult and chronic neurodegenerative conditions. Recent research has shown that neuroinflammation may contribute to the initiation of Alzheimer's disease (AD) pathogenesis and associated epileptogenesis. OBJECTIVE: This systematic review aimed to investigate the available literature on the shared molecular mechanisms of neuroinflammation in AD and epilepsy. METHODS: The search included in this systematic review was obtained from 5 established databases. A total of 2,760 articles were screened according to inclusion criteria. Articles related to the modulation of the inflammatory biomarkers commonly associated with the progression of AD and epilepsy in all populations were included in this review. RESULTS: Only 7 articles met these criteria and were chosen for further analysis. Selected studies include both in vitro and in vivo research conducted on rodents. Several neuroinflammatory biomarkers were reported to be involved in the cross-talk between AD and epilepsy. CONCLUSION: Neuroinflammation was directly associated with the advancement of AD and epilepsy in populations compared to those with either AD or epilepsy. However, more studies focusing on common inflammatory biomarkers are required to develop standardized monitoring guidelines to prevent the manifestation of epilepsy and delay the progression of AD in patients.

Early preclinical plasma protein biomarkers of brain trauma are influenced by early seizures and levetiracetam.

OBJECTIVE: We used the lateral fluid percussion injury (LFPI) model of moderate-to-severe traumatic brain injury (TBI) to identify early plasma biomarkers predicting injury, early post-traumatic seizures or neuromotor functional recovery (neuroscores), considering the effect of levetiracetam, which is commonly given after severe TBI. METHODS: Adult male Sprague-Dawley rats underwent left parietal LFPI, received levetiracetam (200 mg/kg bolus, 200 mg/kg/day subcutaneously for 7 days [7d]) or vehicle post-LFPI, and were continuously video-EEG recorded (n = 14/group). Sham (craniotomy only, n = 6), and naïve controls (n = 10) were also used. Neuroscores and plasma collection were done at 2d or 7d post-LFPI or equivalent timepoints in sham/naïve. Plasma protein biomarker levels were determined by reverse phase protein microarray and classified according to injury severity (LFPI vs. sham/control), levetiracetam treatment, early seizures, and 2d-to-7d neuroscore recovery, using machine learning. RESULTS: Low 2d plasma levels of Thr231 -phosphorylated tau protein (pTAU-Thr231 ) and S100B combined (ROC AUC = 0.7790) predicted prior craniotomy surgery (diagnostic biomarker). Levetiracetam-treated LFPI rats were differentiated from vehicle treated by the 2d-HMGB1, 2d-pTAU-Thr231 , and 2d-UCHL1 plasma levels combined (ROC AUC = 0.9394) (pharmacodynamic biomarker). Levetiracetam prevented the seizure effects on two biomarkers that predicted early seizures only among vehicle-treated LFPI rats: pTAU-Thr231 (ROC AUC = 1) and UCHL1 (ROC AUC = 0.8333) (prognostic biomarker of early seizures among vehicle-treated LFPI rats). Levetiracetam-resistant early seizures were predicted by high 2d-IFNγ plasma levels (ROC AUC = 0.8750) (response biomarker). 2d-to-7d neuroscore recovery was best predicted by higher 2d-S100B, lower 2d-HMGB1, and 2d-to-7d increase in HMGB1 or decrease in TNF (P < 0.05) (prognostic biomarkers). SIGNIFICANCE: Antiseizure medications and early seizures need to be considered in the interpretation of early post-traumatic biomarkers.

Effective Preparation of [18F]Flumazenil Using Copper-Mediated Late-Stage Radiofluorination of a Stannyl Precursor.

(1) Background: [18F]Flumazenil 1 ([18F]FMZ) is an established positron emission tomography (PET) radiotracer for the imaging of the gamma-aminobutyric acid (GABA) receptor subtype, GABAA in the brain. The production of [18F]FMZ 1 for its clinical use has proven to be challenging, requiring harsh radiochemical conditions, while affording low radiochemical yields. Fully characterized, new methods for the improved production of [18F]FMZ 1 are needed. (2) Methods: We investigate the use of late-stage copper-mediated radiofluorination of aryl stannanes to improve the production of [18F]FMZ 1 that is suitable for clinical use. Mass spectrometry was used to identify the chemical by-products that were produced under the reaction conditions. (3) Results: The radiosynthesis of [18F]FMZ 1 was fully automated using the iPhase FlexLab radiochemistry module, affording a 22.2 ± 2.7% (n = 5) decay-corrected yield after 80 min. [18F]FMZ 1 was obtained with a high radiochemical purity (>98%) and molar activity (247.9 ± 25.9 GBq/µmol). (4) Conclusions: The copper-mediated radiofluorination of the stannyl precursor is an effective strategy for the production of clinically suitable [18F]FMZ 1.

Characterising seizure development, behavioural comorbidities and neuroinflammation in a self-sustained electrical status epilepticus model of mesial temporal lobe epilepsy in C57BL/6J mice.

OBJECTIVE: Status epilepticus (SE) models in rodents are commonly used to research mesial temporal lobe epilepsy (mTLE) in translational epilepsy research. However, due to differences in susceptibility of mice strains to chemoconvulsants, developing this model in mice is challenging. Mice offer experimental advantages; in particular, the ability to use transgenic strains could provide novel insights about neurobiological mechanisms or ease of genetic modification to test potential therapeutic targets. This study aimed to characterise the neuroinflammation, epileptic seizures and behavioural comorbidities after self-sustained Electrical Status Epilepticus (SSSE) in C57BL/6J mice. METHODS: SSSE was induced in C57BL/6J mice via prolonged electrical stimulation through a bipolar electrode implanted in the ventral hippocampus. Video electroencephalography (vEEG) monitoring was then performed between 1st month (acute timepoint) and 4th month (chronic timepoint). Brain tissues were collected at two timepoints for gene expression and immunohistochemical analysis: 7-days and 16-weeks post-SE. Additionally, at the chronic timepoint, animals underwent a series of neurobehavioural tests. RESULTS: Sixty percent of animals that underwent SSSE developed spontaneous seizures within the first month, and an additional 25% developed seizures at the chronic timepoint. The number of seizures per week during the chronic period ranged from 0.2 to 15.7. Mortality rate was ~9% during or after SSSE. SSSE animals displayed significant spatial memory impairment and depression-like behaviour compared to sham animals. mRNA expression of inflammatory cytokines was upregulated at 7-days following SE, but equal to sham levels at 16-weeks. SIGNIFICANCE: This study provides evidence that SSSE in C57BL/6J mice induces epileptic seizures consistent with those seen in patients with mTLE, along with cognitive and behavioural comorbidities. This model therefore has the potential to be used experimentally to uncover mechanisms to target against epileptogenesis, or to test novel treatment approaches.

TSPO PET upregulation predicts epileptic phenotype at disease onset independently from chronic TSPO expression in a rat model of temporal lobe epilepsy.

Neuroinflammation is a key component of epileptogenesis, the process leading to acquired epilepsy. In recent years, with the development of non-invasive in vivo positron emission tomography (PET) imaging of translocator protein 18 kDa (TSPO), a marker of neuroinflammation, it has become possible to perform longitudinal studies to characterize neuroinflammation at different disease stages in animal models of epileptogenesis. This study aimed to utilize the prognostic capability of TSPO PET imaging at disease onset (2 weeks post-SE) to categorize epileptic rats with distinct seizure burden based on TSPO levels at disease onset and investigate their association to TSPO expression at the chronic epilepsy stage. Controls (n = 14) and kainic acid-induced status epilepticus (KASE) rats (n = 41) were scanned non-invasively with [18F]PBR111 PET imaging measuring TSPO expression. Animals were monitored using video-electroencephalography (vEEG) up to chronic disease (12 weeks post-SE), at which TSPO levels ([3H]PK11195) as well as other post-mortem abnormalities (namely synaptic density ([3H]UCB-J), neuronal loss (NeuN), and neurodegeneration (FjC)) were investigated. By applying multivariate analysis, TSPO PET imaging at disease onset identified three KASE groups with significantly different spontaneous recurrent seizures (SRS) burden (defined as rare SRS, sporadic SRS, and frequent SRS) (p = 0.003). Interestingly, TSPO levels were significantly different when comparing the three KASE groups (p < 0.0001), with the frequent SRS group characterized only by a limited focal TSPO increase at disease onset. On the contrary, TSPO measured during chronic epilepsy was found to be the highest in the frequent SRS group and correlated with seizure burden (r = 0.826, p < 0.0001). Importantly, early and chronic TSPO levels did not correlate (r = -0.05). Finally, significant pathological changes in neuronal loss, synaptic density, and neurodegeneration were found not only when compared to control animals (p < 0.01), but also between the three KASE rat categories in the hippocampus (p < 0.05). Early and chronic TSPO upregulation following epileptogenic insult appear to be driven by two superimposed dynamic processes. The former is associated with epileptogenesis as measured at disease onset, while the latter is related to seizure frequency as quantified during chronic epilepsy.

The utilization of small non-mammals in traumatic brain injury research: A systematic review.

Traumatic brain injury (TBI) is the leading cause of death and disability worldwide and has complicated underlying pathophysiology. Numerous TBI animal models have been developed over the past decade to effectively mimic the human TBI pathophysiology. These models are of mostly mammalian origin including rodents and non-human primates. However, the mammalian models demanded higher costs and have lower throughput often limiting the progress in TBI research. Thus, this systematic review aims to discuss the potential benefits of non-mammalian TBI models in terms of their face validity in resembling human TBI. Three databases were searched as follows: PubMed, Scopus, and Embase, for original articles relating to non-mammalian TBI models, published between January 2010 and December 2019. A total of 29 articles were selected based on PRISMA model for critical appraisal. Zebrafish, both larvae and adult, was found to be the most utilized non-mammalian TBI model in the current literature, followed by the fruit fly and roundworm. In conclusion, non-mammalian TBI models have advantages over mammalian models especially for rapid, cost-effective, and reproducible screening of effective treatment strategies and provide an opportunity to expedite the advancement of TBI research.

Neurodegenerative pathways as targets for acquired epilepsy therapy development.

There is a growing body of clinical and experimental evidence that neurodegenerative diseases and epileptogenesis after an acquired brain insult may share common etiological mechanisms. Acquired epilepsy commonly develops as a comorbid condition in patients with neurodegenerative diseases such as Alzheimer's disease, although it is likely much under diagnosed in practice. Progressive neurodegeneration has also been described after traumatic brain injury, stroke, and other forms of brain insults. Moreover, recent evidence has shown that acquired epilepsy is often a progressive disorder that is associated with the development of drug resistance, cognitive decline, and worsening of other neuropsychiatric comorbidities. Therefore, new pharmacological therapies that target neurobiological pathways that underpin neurodegenerative diseases have potential to have both an anti-epileptogenic and disease-modifying effect on the seizures in patients with acquired epilepsy, and also mitigate the progressive neurocognitive and neuropsychiatric comorbidities. Here, we review the neurodegenerative pathways that are plausible targets for the development of novel therapies that could prevent the development or modify the progression of acquired epilepsy, and the supporting published experimental and clinical evidence.

Microglial polarization in posttraumatic epilepsy: Potential mechanism and treatment opportunity.

Owing to the complexity of the pathophysiological mechanisms driving epileptogenesis following traumatic brain injury (TBI), effective preventive treatment approaches are not yet available for posttraumatic epilepsy (PTE). Neuroinflammation appears to play a critical role in the pathogenesis of the acquired epilepsies, including PTE, but despite a large preclinical literature demonstrating the ability of anti-inflammatory treatments to suppress epileptogenesis and chronic seizures, no anti-inflammatory treatment approaches have been clinically proven to date. TBI triggers robust inflammatory cascades, suggesting that they may be relevant for the pathogenesis of PTE. A major cell type involved in such cascades is the microglial cells-brain-resident immune cells that become activated after brain injury. When activated, these cells can oscillate between different phenotypes, and such polarization states are associated with the release of various pro- and anti-inflammatory mediators that may influence brain repair processes, and also differentially contribute to the development of PTE. As the molecular mechanisms and key signaling molecules associated with microglial polarization in brain are discovered, strategies are now emerging that can modulate this polarization, promoting this as a potential therapeutic strategy for PTE. In this review, we discuss the relevant literature regarding the polarization of brain-resident immune cells following TBI and attempt to put into perspective a role in epilepsy pathogenesis. Finally, we explore potential strategies that could polarize microglia/macrophages toward a neuroprotective phenotype to mitigate PTE development.

Cav 3.2 T-type calcium channel mutation influences kindling-induced thalamic neuronal firing patterns in Genetic Absence Epilepsy Rats From Strasbourg.

OBJECTIVE: Recent data indicate that amygdala kindling leads to significant changes in interictal neuronal firing patterns of thalamic reticular nucleus (TRN) neurons by decreasing the spontaneous firing rate and increasing burst firing in nonepileptic control (NEC) rats. Genetic Absence Epilepsy Rats From Strasbourg (GAERS) were resistant to these kindling-induced firing changes in TRN neurons, and are also resistant to the progression of kindling. We investigated whether a homozygous, missense, single nucleotide mutation (R1584P) in the Cav 3.2 T-type Ca2+ channel gene, which has been correlated with the expression of absence seizures in GAERS, influenced kindling progression and TRN firing patterns. METHODS: Double-crossed (GAERS vs NEC; F2) rats that were homozygous for the Cav 3.2 mutation (PP) and those negative for the mutation (RR) were implanted with a stimulating electrode in the amygdala. Rats received a total of 30 kindling stimulations at their afterdischarge threshold current twice daily, and kindling progression was evaluated. Thereafter, the extracellular neuronal activity of TRN neurons was recorded in vivo under neuroleptanesthesia to investigate the influence of Cav 3.2 mutation on TRN firing patterns. RESULTS: We found that the R1584P mutation did not affect kindling progression in F2 crosses (P = 0.78). However, it influenced kindling-induced neuronal firing of TRN neurons. After 30 stimulations, RR rats exhibited a lower firing rate and a higher percentage of burst firing compared to PP rats. The decrease in firing frequency was correlated with the increase in the amount of burst firing in RR rats (R2  = 0.497). SIGNIFICANCE: Our findings suggest that mutation in Cav 3.2 T-type Ca2+ channels may play a role in the resistance to kindling-induced changes in TRN neurons to a low-frequency and high-percentage bursting pattern seen in association with the convulsive stages of amygdala kindling, but is not in itself enough to explain the resistance to kindling progression observed in GAERS.

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.

Spatiotemporal expression and inhibition of prolyl oligopeptidase contradict its involvement in key pathologic mechanisms of kainic acid-induced temporal lobe epilepsy in rats.

OBJECTIVE: Prolyl oligopeptidase (PREP) has been implicated in neuroinflammatory processes and neuroplasticity and has been suggested as a target for the treatment of neurodegenerative disease. The aim of this investigation was to explore the involvement of PREP in the neuropathologic mechanisms relevant to temporal lobe epilepsy (TLE) using a PREP inhibitor in a well-established rat model. METHODS: PREP activity and expression was studied in Sprague-Dawley rats 2 and 12 weeks following kainic acid-induced status epilepticus (KASE). Continuous video-electroencephalography monitoring was performed for 2 weeks in the 12-week cohort to identify a relationship of PREP expression/activity with epileptic seizures. In addition, the animals included in the 2-week time point were treated with a specific inhibitor of PREP, KYP-2047, or saline continuously, starting immediately after SE. PREP activity and its expression were analyzed in rat brain by using enzyme kinetics and western blot. In addition, markers for microglial activation, astrogliosis, cell loss, and cell proliferation were evaluated. RESULTS: Enzymatic activity of PREP was unchanged following induction of SE after 2 and 12 weeks in rats. PREP activity in epileptic rats did not relate to the number of seizures/day at the 12-week time point. Moreover, continuous inhibition of PREP for 2 weeks after KASE did not alter the SE-mediated neuroinflammatory response, cell loss, or cell proliferation in the hippocampal subgranule zone measured at the 2-week time point. SIGNIFICANCE: PREP inhibition does not affect key pathologic mechanisms, including activation of glial cells, cell loss, and neural progenitor cell proliferation, in this KASE model of TLE. The results do not support a direct role of PREP in seizure burden during the chronic epilepsy period in this model.