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.

Small-pore hydridic frameworks store densely packed hydrogen.

Nanoporous materials have attracted great attention for gas storage, but achieving high volumetric storage capacity remains a challenge. Here, by using neutron powder diffraction, volumetric gas adsorption, inelastic neutron scattering and first-principles calculations, we investigate a magnesium borohydride framework that has small pores and a partially negatively charged non-flat interior for hydrogen and nitrogen uptake. Hydrogen and nitrogen occupy distinctly different adsorption sites in the pores, with very different limiting capacities of 2.33 H2 and 0.66 N2 per Mg(BH4)2. Molecular hydrogen is packed extremely densely, with about twice the density of liquid hydrogen (144 g H2 per litre of pore volume). We found a penta-dihydrogen cluster where H2 molecules in one position have rotational freedom, whereas H2 molecules in another position have a well-defined orientation and a directional interaction with the framework. This study reveals that densely packed hydrogen can be stabilized in small-pore materials at ambient pressures.

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.

Potential Roles for the GluN2D NMDA Receptor Subunit in Schizophrenia.

Glutamate N-methyl-D-aspartate receptor (NMDAR) hypofunction has been proposed to underlie schizophrenia symptoms. This theory arose from the observation that administration of NMDAR antagonists, which are compounds that inhibit NMDAR activity, reproduces behavioural and molecular schizophrenia-like phenotypes, including hallucinations, delusions and cognitive impairments in healthy humans and animal models. However, the role of specific NMDAR subunits in these schizophrenia-relevant phenotypes is largely unknown. Mounting evidence implicates the GluN2D subunit of NMDAR in some of these symptoms and pathology. Firstly, genetic and post-mortem studies show changes in the GluN2D subunit in people with schizophrenia. Secondly, the psychosis-inducing effects of NMDAR antagonists are blunted in GluN2D-knockout mice, suggesting that the GluN2D subunit mediates NMDAR-antagonist-induced psychotomimetic effects. Thirdly, in the mature brain, the GluN2D subunit is relatively enriched in parvalbumin (PV)-containing interneurons, a cell type hypothesized to underlie the cognitive symptoms of schizophrenia. Lastly, the GluN2D subunit is widely and abundantly expressed early in development, which could be of importance considering schizophrenia is a disorder that has its origins in early neurodevelopment. The limitations of currently available therapies warrant further research into novel therapeutic targets such as the GluN2D subunit, which may help us better understand underlying disease mechanisms and develop novel and more effective treatment options.

Differential Effects of Chronic Methamphetamine Treatment on High-Frequency Oscillations and Responses to Acute Methamphetamine and NMDA Receptor Blockade in Conscious Mice.

Dysregulation of high-frequency neuronal oscillations has been implicated in the pathophysiology of schizophrenia. Chronic methamphetamine (METH) use can induce psychosis similar to paranoid schizophrenia. The current study in mice aimed to determine the effect of chronic METH treatment on ongoing and evoked neuronal oscillations. C57BL/6 mice were treated with METH or vehicle control for three weeks and implanted with extradural recording electrodes. Two weeks after the last METH injection, mice underwent three EEG recording sessions to measure ongoing and auditory-evoked gamma and beta oscillatory power in response to an acute challenge with METH (2 mg/kg), the NMDA receptor antagonist MK-801 (0.3 mg/kg), or saline control. A separate group of mice pretreated with METH showed significantly greater locomotor hyperactivity to an acute METH challenge, confirming long-term sensitisation. Chronic METH did not affect ongoing or evoked gamma or beta power. Acute MK-801 challenge reduced ongoing beta power whereas acute METH challenge significantly increased ongoing gamma power. Both MK-801 and METH challenge suppressed evoked gamma power. Chronic METH treatment did not modulate these acute drug effects. There were minor effects of chronic METH and acute METH and MK-801 on selected components of event-related potential (ERP) waves. In conclusion, chronic METH treatment did not exert neuroplastic effects on the regulation of cortical gamma oscillations in a manner consistent with schizophrenia, despite causing behavioural sensitisation.

Deciphering the code: Identifying true gamma neural oscillations.

Neural oscillatory activity occurring in the gamma frequency range (30-80 Hz) has been proposed to play essential roles in sensory and cognitive processing. Supporting this, abnormalities in gamma oscillations have been reported in patients with diverse neurological and neuropsychiatric disorders in which cognitive impairment is prominent. Understanding the mechanisms underpinning this relationship is the focus of extensive research. But while an increasing number of studies are investigating the intricate relationship between gamma oscillations and cognition, interpretation and generalisation of these studies is limited by the diverse, and at times questionable, methodologies used to analyse oscillatory activity. For example, a variety of different types of gamma oscillatory activity have been characterised, but all are generalised non-specifically as 'gamma oscillations'. This creates confusion, since distinct cellular and network mechanisms are likely responsible for generating these different types of rhythm. Moreover, in some instances, certain analytical measures of electrophysiological data are overinterpreted, with researchers pushing the boundaries of what would be considered rhythmic or oscillatory in nature. Here, we provide clarity on these issues, firstly presenting an overview of the different measures of gamma oscillatory activity, and describing common signal processing techniques used for analysis. Limitations of these techniques are discussed, and recommendations made on how future studies should optimise analyses, presentation and interpretation of gamma frequency oscillations. This is an essential progression in order to harmonise future studies, allowing us to gain a clearer understanding of the role of gamma oscillations in cognition, and in cognitive disorders.

Early life adversity accelerates epileptogenesis and enhances depression-like behaviors in rats.

OBJECTIVE: Early life stressors are well-established risk factors for psychiatric disorders, and evidence also suggests that these promote vulnerability to epilepsy. Given the high prevalence of psychiatric disorders in epilepsy, early life stress may represent a common driver for these comorbidities. We used animal modelling to investigate the effects of early life stress on epileptogenesis and depressive behaviors, also exploring HPA axis programming as a potential associative mechanism. METHODS: From post-natal day 2-9, Wistar rat dams (n = 3) and their offspring were exposed to the Limited Bedding and Nesting (LBN) model of early life adversity. Control dams (n = 3) were undisturbed. Maternal care was video-recorded, and behavior scored. As adults, rats (n = 7/group) underwent kainic acid-induced status epilepticus (SE), to trigger epilepsy development. Spontaneous seizures, depression-like behavior and HPA axis function were quantified. RESULTS: LBN significantly altered aspects of maternal care, including markedly reducing the consistency of care (p < 0.05), compared to control conditions. Following SE, LBN rats exhibited significantly accelerated epileptogenesis (p = 0.01) and greater disease severity (p = 0.001), compared to control rats. Anhedonia and behavioral despair were observed in epileptic rats exposed to LBN. LBN rats showed significantly dampened HPA axis responsivity, but epileptic rats showed greater corticosterone responses to CRH administration (all p < 0.05). SIGNIFICANCE: Early life adversity promotes a vulnerability to experimental epileptogenesis. These two 'hits' (early life stress and epilepsy) interact to create a depressive-like phenotype, but effects on HPA axis are complex and contrasting. This has implications for the mechanisms underpinning the increased prevalence of psychiatric disorders observed in people with epilepsy.

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.

Pediatric traumatic brain injury and a subsequent transient immune challenge independently influenced chronic outcomes in male mice.

Traumatic brain injury (TBI) is a major contributor to death and disability worldwide. Children are at particularly high risk of both sustaining a TBI and experiencing serious long-term consequences, such as cognitive deficits, mental health problems and post-traumatic epilepsy. Severe TBI patients are highly susceptible to nosocomial infections, which are mostly acquired within the first week of hospitalization post-TBI. Yet the potential chronic impact of such acute infections following pediatric TBI remains unclear. In this study, we hypothesized that a peripheral immune challenge, such as lipopolysaccharide (LPS)-mimicking a hospital-acquired infection-would worsen inflammatory, neurobehavioral, and seizure outcomes after experimental pediatric TBI. To test this, three-week old male C57Bl/6J mice received a moderate controlled cortical impact or sham surgery, followed by 1 mg/kg i.p. LPS (or 0.9% saline vehicle) at 4 days TBI. Mice were randomized to four groups; sham-saline, sham-LPS, TBI-saline or TBI-LPS (n = 15/group). Reduced general activity and increased anxiety-like behavior were observed within 24 h in LPS-treated mice, indicating a transient sickness response. LPS-treated mice also exhibited a reduction in body weights, which persisted chronically. From 2 months post-injury, mice underwent a battery of tests for sensorimotor, cognitive, and psychosocial behaviors. TBI resulted in hyperactivity and spatial memory deficits, independent of LPS; whereas LPS resulted in subtle deficits in spatial memory retention. At 5 months post-injury, video-electroencephalographic recordings were obtained to evaluate both spontaneous seizure activity as well as the evoked seizure response to pentylenetetrazol (PTZ). TBI increased susceptibility to PTZ-evoked seizures; whereas LPS appeared to increase the incidence of spontaneous seizures. Post-mortem analyses found that TBI, but not LPS, resulted in robust glial reactivity and loss of cortical volume. A TBI × LPS interaction in hippocampal volume suggested that TBI-LPS mice had a subtle increase in ipsilateral hippocampus tissue loss; however, this was not reflected in neuronal cell counts. Both TBI and LPS independently had modest effects on chronic hippocampal gene expression. Together, contrary to our hypothesis, we observed minimal synergy between TBI and LPS. Instead, pediatric TBI and a subsequent transient immune challenge independently influenced chronic outcomes. These findings have implications for future preclinical modeling as well as acute post-injury patient management.

NMDA receptors on parvalbumin-positive interneurons and pyramidal neurons both contribute to MK-801 induced gamma oscillatory disturbances: Complex relationships with behaviour.

BACKGROUND: NMDAr antagonists induce disturbances to gamma frequency oscillations, including increasing ongoing gamma activity and reducing evoked gamma oscillations. We sought to investigate the role parvalbumin (PV+) neurons and CaMKIIα+ pyramidal cells in NMDAr antagonist-induced disturbances in gamma oscillatory activity and relate these to common behavioural consequences of these drugs by selectively deleting the obligatory GluN1 subunit from these cells in mice. METHODS: Adult mice (total n = 99) with GluN1 deleted from PV interneurons (PV:GluN1 KO) or CaMKIIα+ pyramidal cells (CaMKIIα:GluN1 KO), and WT littermates, were used. We assessed effects of the NMDAr antagonist MK-801 on prepulse inhibition (PPI) and locomotor behaviour. Then, mice were implanted with electrodes in the prefrontal cortex (mPFC) and hippocampus (dHPC), and the effects of MK-801 on gamma oscillations assessed. RESULTS: In WT mice, MK-801 increased ongoing gamma power, reduced evoked gamma power and increased gamma coherence. These changes were accompanied by hyperlocomotion and deficient PPI. The consequences of NMDAr antagonism were differentially regulated in the transgenic mice. The MK-801-induced increase in ongoing gamma power was significantly attenuated in both transgenic strains, but deficits to evoked gamma activity were unaffected by genotype. Deficient PPI was not affected by genotype, and only in PV:GluN1 KO mice was the hyperlocomotor phenotype of MK-801 attenuated. The emergence of abnormal gamma band hyperconnectivity between the mPFC and dHPC was absent in CaMKIIα:GluN1 KO mice. CONCLUSION: This study suggests that the effects of NMDAr antagonism on gamma band responses and behaviour have complex relationships, and rely on different populations of neurons.

The mGluR2/3 agonist LY379268 reverses NMDA receptor antagonist effects on cortical gamma oscillations and phase coherence, but not working memory impairments, in mice.

BACKGROUND: Abnormalities in neural oscillations that occur in the gamma frequency range (30-80 Hz) may underlie cognitive deficits in schizophrenia. Both cognitive impairments and gamma oscillatory disturbances can be induced in healthy people and rodents by administration of N-methyl-D-aspartate receptor (NMDAr) antagonists. AIMS: We studied relationships between cognitive impairment and gamma abnormalities following NMDAr antagonism, and attempted to reverse deficits with the metabotropic glutamate receptor type 2/3 (mGluR2/3) agonist LY379268. METHODS: C57/Bl6 mice were trained to perform the Trial-Unique Nonmatching to Location (TUNL) touchscreen test for working memory. They were then implanted with local field potential (LFP) recording electrodes in prefrontal cortex and dorsal hippocampus. Mice were administered either LY379268 (3 mg/kg) or vehicle followed by the NMDAr antagonist MK-801 (0.3 or 1 mg/kg) or vehicle prior to testing on the TUNL task, or recording LFPs during the presentation of an auditory stimulus. RESULTS: MK-801 impaired working memory and increased perseveration, but these behaviours were not improved by LY379268 treatment. MK-81 increased the power of ongoing gamma and high gamma (130-180 Hz) oscillations in both brain regions and regional coherence between regions, and these signatures were augmented by LY379268. However, auditory-evoked gamma oscillation deficits caused by MK-801 were not affected by LY379268 pretreatment. CONCLUSIONS: NMDA receptor antagonism impairs working memory in mice, but this is not reversed by stimulation of mGluR2/3. Since elevations in ongoing gamma power and regional coherence caused by MK-801 were improved by LY379268, it appears unlikely that these specific oscillatory abnormalities underlie the working memory impairment caused by NMDAr antagonism.

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.

Harmonization of lateral fluid-percussion injury model production and post-injury monitoring in a preclinical multicenter biomarker discovery study on post-traumatic epileptogenesis.

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 (<72 h post-injury), time to self-right after the impact, and severity of the injury (assessed with the neuroscore). The data was collected using Common Data Elements and Case Report Forms. The acute mortality was 15% (UEF), 50% (Melbourne) and 57% (UCLA) (p < 0.001). The sites differed in the duration of anesthesia, the shortest being at UEF < Melbourne < UCLA (p < 0.001). The impact pressure used also differed between the sites, the highest being in UEF > Melbourne > UCLA (p < 0.001). The impact pressure associated with the severity of the functional deficits (low neuroscore) (P < 0.05) only at UEF, but not at any of the other sites. Additionally, the sites differed in the duration of post-impact transient apnea (p < 0.001) and time to self-right (P < 0.001), the highest values in both parameters was registered in Melbourne. Post-impact seizure-like behavior was observed in 51% (UEF), 25% (Melbourne) and 2% (UCLA) of rats (p < 0.001). Despite the differences in means when all sites were compared there was significant overlap in injury parameters between the sites. The data reflects the technical difficulties in the production of lateral FPI across multiple sites. On the other hand, the data can be used to model the heterogeneity in human cohorts with closed-head injury. Our animal cohort will provide a good starting point to investigate the factors associated with epileptogenesis after lateral FPI.

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

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

High-Frequency Neuronal Oscillatory Abnormalities in the Phospholipase C-ß1 Knockout Mouse Model of Schizophrenia.

BACKGROUND: Schizophrenia is a complex neuropsychiatric disorder characterized by psychoses, socioaffective disturbances, and cognitive deficits. The phosphodiesterase enzyme phospholipase C-ß1 has been reported to be reduced in postmortem tissue of schizophrenia patients. Dysregulation of neuronal oscillations, particularly those in the higher frequency range such as beta (12-30 Hz) and gamma (30-80 Hz), are also associated with this disorder. We investigated the influence of phospholipase C-ß1 gene deletion on cortical oscillatory activity and sensorimotor gating behavior. METHODS: Adult phospholipase C-ß1 knockout and wild-type C57Bl/6J control mice (total n = 26) underwent surgical implantation of extradural electrodes to allow electrocorticography recordings. Electrocorticography was recorded during prepulse inhibition behavior sessions to measure ongoing and auditory-evoked electrophysiological responses. Mice were also pretreated with antipsychotic drugs haloperidol (0.25 mg/kg), clozapine (2.5 mg/kg), and olanzapine (5 mg/kg). RESULTS: Phospholipase C-ß1 knockout mice exhibited reduced prepulse inhibition and diminished power and phase synchrony of beta and gamma oscillatory responses to auditory stimuli as well as elevated ongoing beta oscillations. Reductions in prepulse inhibition were highly correlated with the power and phase synchrony of evoked oscillations. Clozapine and olanzapine ameliorated the prepulse inhibition deficit in phospholipase C-ß1 knockout mice, but not the electrophysiology abnormalities. CONCLUSIONS: Phospholipase C-ß1 reduction leads to disturbances to beta and gamma oscillatory dynamics and prepulse inhibition behavior. The strong relationships between these measures demonstrate the importance of event-related oscillatory activity to sensorimotor gating behavior. However, dissociation of these measures observed in the drug studies suggests that abnormalities in neuronal networks may not necessarily need to be corrected for behavioral improvement.

An experimental and computational study of CO2 adsorption in the sodalite-type M-BTT (M = Cr, Mn, Fe, Cu) metal-organic frameworks featuring open metal sites.

We present a comprehensive investigation of the CO2 adsorption properties of an isostructural series of metal-organic frameworks, M-BTT (M = Cr, Mn, Fe, Cu; BTT3- = 1,3,5-benzenetristetrazolate), which exhibit a high density of open metal sites capable of polarizing and binding guest molecules. Coupling gas adsorption measurements with in situ neutron and X-ray diffraction experiments provides molecular-level insight into the adsorption process and enables rationalization of the observed adsorption isotherms. In particular, structural data confirms that the high initial isosteric heats of CO2 adsorption for the series are directly correlated with the presence of open metal sites and further reveals the positions and orientations of as many as three additional adsorption sites. Density functional theory calculations that include van der Waals dispersion corrections quantitatively support the observed structural features associated with the primary and secondary CO2 binding sites, including CO2 positions and orientations, as well as the experimentally determined isosteric heats of CO2 adsorption.

Separation of Xylene Isomers through Multiple Metal Site Interactions in Metal-Organic Frameworks.

Purification of the C8 alkylaromatics o-xylene, m-xylene, p-xylene, and ethylbenzene remains among the most challenging industrial separations, due to the similar shapes, boiling points, and polarities of these molecules. Herein, we report the evaluation of the metal-organic frameworks Co2(dobdc) (dobdc4- = 2,5-dioxido-1,4-benzenedicarboxylate) and Co2( m-dobdc) ( m-dobdc4- = 4,6-dioxido-1,3-benzenedicarboxylate) for the separation of xylene isomers using single-component adsorption isotherms and multicomponent breakthrough measurements. Remarkably, Co2(dobdc) distinguishes among all four molecules, with binding affinities that follow the trend o-xylene > ethylbenzene > m-xylene > p-xylene. Multicomponent liquid-phase adsorption measurements further demonstrate that Co2(dobdc) maintains this selectivity over a wide range of concentrations. Structural characterization by single-crystal X-ray diffraction reveals that both frameworks facilitate the separation through the extent of interaction between each C8 guest molecule with two adjacent cobalt(II) centers, as well as the ability of each isomer to pack within the framework pores. Moreover, counter to the presumed rigidity of the M2(dobdc) structure, Co2(dobdc) exhibits an unexpected structural distortion in the presence of either o-xylene or ethylbenzene that enables the accommodation of additional guest molecules.