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

doi:10.1016/j.eplepsyres.2019.03.008

Epilepsy Research

Volume 156, October 2019, 106110

https://doi.org/10.1016/j.eplepsyres.2019.03.008 Get rights and content

Highlights

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Harmonization of surgical techniques and standardization of epidural and intracerebral electrode placements.
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HFOs were detected from deep and cortical screw electrodes.
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The algorithm selected is one of the biggest sources of the high-variability in the detections.
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Progress towards the evaluation of HFOs as an electrophysiological biomarker of PTE in a multi-center design.

Abstract

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.

Introduction

Studies involving presurgical patients with epilepsy and rodent models of chronic epilepsy induced by status epilepticus show pathological high-frequency oscillations (HFOs; 80–500 Hz) are associated with epileptogenic tissue, and could play a role in generating seizures (for review see Jacobs et al., 2012; Jiruska et al., 2017). Results from studies that recorded EEG immediately after experimental status epilepticus (SE) suggest HFOs could also play a role in the development of epilepsy (Bragin et al., 2004, 2000; Lévesque et al., 2011). The work by Bragin et al. (2004) found little evidence of HFOs after status in rats that did not develop epilepsy, but prominent HFOs were detected in rats that later developed recurrent spontaneous seizures. Moreover, the sooner HFOs were detected, the sooner the first spontaneous seizure occurred (Bragin et al., 2004). These latter data support a hypothesis that pathological HFOs reflect progressive neuronal disturbances after an epileptogenic brain injury and could be a biomarker of epileptgoenesis.

Post-traumatic epilepsy (PTE) is a serious neurological sequela of traumatic brain injury (TBI) and develops in about 16% of cases of severe TBI (Annegers et al., 1998). Currently there are no biomarkers to predict who will develop PTE, which might not manifest until months or years after a TBI. The lack of biomarkers has hindered the development of new treatments that might modify or prevent PTE. However, recent work in a fluid-percussion injury (FPI) rat model of TBI detected pathological HFOs in the perilesional cortex of some, but not all, TBI rats (Bragin et al., 2007). No pathological HFOs were recorded in control rats. In rats that had long-term EEG recordings, rats that had pathological HFOs within two weeks of TBI later developed spontaneous seizures, and none of the rats without these events developed later seizures. Pathological HFOs, similar to those during epileptogenesis in status epilepticus models, might also reflect epileptogenesis in the FPI model.

The Epilepsy Bioinformatics Study for Antiepileptogenic Therapy (EpiBioS4Rx) is a NINDS-funded, international multi-center project designed to identify biomarkers of epileptogenesis and treatments that could modify the development of PTE. One of the goals of this project will be to determine whether HFOs are a biomarker of epileptogenesis in the rat FPI model. A significant aspect of this work involves the standardization of protocols for the FPI model, the electrodes, and the electrode placements; assessing EEG recording capabilities and algorithms for HFO detection and verification; and identifying issues and generating solutions to solve them, which is described in the current report.

Section snippets

Materials and methods

Three sites from the international NIH-funded Centre without Walls consortium, the Epilepsy Bioinformatics Study for Antiepileptogenic Therapy (EpiBioS4Rx) (http:// http://epibios.loni.usc.edu/) were involved in the harmonization for EEG recording and HFO analysis in the FPI model. These sites were The University of Eastern Finland (UEF), Monash University in Melbourne (Melbourne) and The University of California, Los Angeles (UCLA).

Surgery for impact and electrode implantation

Review of the surgical reports showed that at UEF 45 rats were randomized to TBI or sham-injury (37 TBI and 8 shams) with a 16% (6/37) post-impact mortality rate. The mean impact pressure was (2.79 ± 0.14 atm). At Melbourne randomization produced 32 TBI and 7 shams rats with a post-impact mortality rate of 59% (19/32). The mean impact pressure at Melbourne was 2.41 ± 0.21 atm. At UCLA randomization produced 32 TBI and 7 shams rats with a post-impact mortality of 59% (19/32). The mean impact

Discussion

This first report from the EpiBioS4Rx Consortium demonstrates the standardized placements of epidural and intracerebral electrodes used to record EEG in TBI and sham-injured rats. Twenty-eight days after electrode implantation, EEG files from 2 of the 3 centers contained bursts of HFOs similar to those found in status-epilepticus models of chronic epilepsy. Different computer-automated detection algorithms and small changes in detection parameters produced different results, and common review

Conclusions

Harmonizing the recording and detection of HFOs is crucial in the EpiBioS4Rx multi-center studies in order to establish robust, clinically translatable electrophysiological biomarkers of PTE. Our interim analysis found variability in the detection and review of HFOs that can be attributed to (1) the EEG signal quality, (2) the choice of algorithm and parameters used to detect HFOs, and (3) the manual review criteria for verifying HFOs. Reducing the variability between sites will involve

Acknowledgement

This research was supported by the National Institute of Neurological Disorders and Stroke (NINDS) Center without Walls of the National Institutes of Health (NIH) under Award Number U54NS100064 (EpiBioS4Rx).

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Successful harmonization in EpiBioS4Rx biomarker study on post-traumatic epilepsy paves the way towards powered preclinical multicenter studies
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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.
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.
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.
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.
Effects of the T-type calcium channel Ca<inf>V</inf>3.2 R1584P mutation on absence seizure susceptibility in GAERS and NEC congenic rats models
2023, Neurobiology of Disease
Low-voltage-activated or T-type Ca²⁺ channels play a key role in the generation of seizures in absence epilepsy. We have described a homozygous, gain of function substitution mutation (R1584P) in the Ca_V3.2 T-type Ca²⁺ channel gene (Cacna1h) in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS). The non-epileptic control (NEC) rats, derived from the same original Wistar strains as GAERS but selectively in-breed not to express seizures, are null for the R1584P mutation. To study the effects of this mutation in rats who otherwise have a GAERS or NEC genetic background, we bred congenic GAERS-Cacna1hNEC (GAERS null for R1584P mutation) and congenic NEC-Cacna1hGAERS (NEC homozygous for R1584P mutation) and evaluated the seizure and behavioral phenotype of these strains in comparison to the original GAERS and NEC strains.
To evaluate seizure expression in the congenic strains, EEG electrodes were implanted in NEC, GAERS, GAERS^-Cacna1hNEC without the R1584P mutation, and NEC^{-Cacna1hGAERS} with the R1584P mutation rats. In the first study, continuous EEG recordings were acquired from week 4 (when seizures begin to develop in GAERS) to week 14 of age (when GAERS display hundreds of seizures per day). In the second study, the seizure and behavioral phenotype of GAERS and NEC^{-Cacna1hGAERS} strains were evaluated during young age (6 weeks of age) and adulthood (16 weeks of age) of GAERS, NEC, GAERS^-Cacna1hNEC and NEC^{-Cacna1hGAERS}. The Open field test (OFT) and sucrose preference test (SPT) were performed to evaluate anxiety-like and depressive-like behavior, respectively. This was followed by EEG recordings at 18 weeks of age to quantify the seizures, and spike-wave discharge (SWD) cycle frequency. At the end of the study, the whole thalamus was collected for T-type calcium channel mRNA expression analysis.
GAERS had a significantly shorter latency to first seizures and an increased number of seizures per day compared to GAERS^-Cacna1hNEC. On the other hand, the presence of the R1584P mutation in the NEC^{-Cacna1hGAERS} was not enough to generate spontaneous seizures in their seizure-resistant background. 6 and 16-week-old GAERS and GAERS^-Cacna1hNEC rats showed anxiety-like behavior in the OFT, in contrast to NEC and NEC^{-Cacna1hGAERS}. Results from the SPT showed that the GAERS developed depressive-like in the SPT compared to GAERS^-Cacna1hNEC, NEC, and NEC^{-Cacna1hGAERS}. Analysis of the EEG at 18 weeks of age showed that the GAERS had an increased number of seizures per day, increased total seizure duration and a higher cycle frequency of SWD relative to GAERS^-Cacna1hNEC. However, the average seizure duration was not significantly different between strains. Quantitative real-time PCR showed that the T-type Ca²⁺ channel isoform Ca_V3.2 channel expression was significantly increased in GAERS compared to NEC, GAERS^-Cacna1hNEC and NEC^{-Cacna1hGAERS}. The presence of the R1584P mutation increased the total ratio of Ca_V3.2 + 25/−25 splice variants in GAERS and NEC^{-Cacna1hGAERS} compared to NEC and GAERS^-Cacna1hNEC.
The data from this study demonstrate that the R1584P mutation in isolation on a seizure-resistant NEC genetic background was insufficient to generate absence seizures, and that a GAERS genetic background can cause seizures even without the mutation. However, the study provides evidence that the R1584P mutation acts as a modulator of seizures development and expression, and depressive-like behavior in the SPT, but not the anxiety phenotype of the GAERS model of absence epilepsy.
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Preface - Practical and theoretical considerations for performing a multi-center preclinical biomarker discovery study of post-traumatic epileptogenesis: lessons learned from the EpiBioS4Rx consortium
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Citation Excerpt :
To move the field forward, we not only harmonized preclinical procedures for biomarker discovery in the three EpiBioS4Rx centers, but also performed a rigorous interim analysis of the success of procedural harmonization, which is reported in this virtual special issue. The analysis included success of harmonization of the production of animal model (Ekolle Ndode-Ekane et al., 2019), blood sampling (Kamnaksh et al., 2018), EEG analyses (seizures, high-frequency oscillations) (Casillas-Espinosa et al., 2019; Santana-Gomez et al., 2019), and MRI analysis (Immonen et al., 2019). We also present an informatics approach that developed parameters and applied visualization tools to assess the overall success of harmonization (Ciszek et al., 2018).
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
Harmonization of lateral fluid-percussion injury model production and post-injury monitoring in a preclinical multicenter biomarker discovery study on post-traumatic epileptogenesis
2019, Epilepsy Research
Citation Excerpt :
The EEG follow-up cohort received cortical and intracranial electrodes (EEG-group). Details of electrode implantation and EEG follow-up have been described in the papers, “Harmonization of pipeline for automated seizure detection for phenotyping of post-traumatic epilepsy in a preclinical multicenter study on post-traumatic epileptogenesis” (Casillas-Espinosa et al., 2019) and “Harmonization of pipeline for detection of HFOs in a rat model of post-traumatic epilepsy in preclinical multicenter study on post-traumatic epileptogenesis” (Santana Gomez et al., 2019). At UEF, 98 rats were used in the MRI-group.
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
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Citation Excerpt :
In particular, when considering the pre—project activities needed to train the personnel to achieve sufficient procedural harmonization and for collection of preliminary data to predict inter-center procedural variability to be included in power calculations. The detailed methodologies have been presented in accompanying articles by Ekolle Ndode-Ekane et al., (2019) for injury production and post-impact follow-up by Kamnaksh et al. (2018) for blood sampling, Casillas-Espinosa et al. (2019) for EEG analysis, and Santana Gomez et al. (2019) for HFO analysis. The outline of the study design in terms of variables included in this harmonization assessment is presented in Fig. 1.
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.

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^☆: This article is part of a special issue ‘Discovery of diagnostic biomarkers for post-traumatic epileptogenesis – an interim analysis of procedures in preclinical multicenter trial EpiBios4Rx’.

¹: Contributed equally to this manuscript.

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

Highlights

Abstract

Introduction

Section snippets

Materials and methods

Surgery for impact and electrode implantation

Discussion

Conclusions

Acknowledgement

Clin. Neurophysiol.

Prog. Neurobiol.

Prog. Neurobiol.

Neurobiol. Dis.

Clin. Neurophysiol.

Clin. Neurophysiol.

A population-based study of seizures after traumatic brain injuries

N. Engl. J. Med.

Chronic Epileptogenesis Requires Development of a Network of Pathologically Interconnected Neuron Clusters: A Hypothesis

Epilepsia