Intraventricular haemorrhage in premature infants: the role of immature neuronal salt and water transport.

Intraventricular hemorrhage (IVH) is a common complication of premature birth. Survivors are often left with cerebral palsy, intellectual disability, and/or hydrocephalus. Animal models suggest that brain tissue shrinkage with subsequent vascular stretch and tear is an important step in the pathophysiology, but the cause of this shrinkage is unknown. Clinical risk factors for IVH are biomarkers of hypoxic-ischemic stress, which causes mature neurons to swell. However, immature neuronal volume might shift in the opposite direction under these conditions. This is because immature neurons express the chloride salt and water transporter NKCC1, which subserves regulatory volume increases in nonneural cells, whereas mature neurons express KCC2, which subserves regulatory volume decreases. When hypoxic ischemic conditions reduce active ion transport and increase the cytoplasmic membrane permeability, the effects of these transporters will be diminished. As a consequence, while mature neurons swell (cytotoxic edema) immature neurons might shrink. After hypoxic-ischemic stress, in vivo and in vitro multi-photon imaging of perinatal transgenic mice demonstrated shrinkage of viable immature neurons, bulk tissue shrinkage, and blood vessel displacement. Neuronal shrinkage was correlated with age-dependent membrane salt and water transporter expression using immunohistochemistry. Shrinkage of immature neurons was prevented by prior genetic or pharmacological inhibition of NKCC1 transport. These findings open new avenues of investigation for the detection of acute brain injury by neuroimaging, as well as prevention of neuronal shrinkage and the ensuing IVH, in premature infants.

Cellular resolution contributions to ictal population signals.

OBJECTIVE: The increased amplitude of ictal activity is a common feature of epileptic seizures, but the determinants of this amplitude have not been identified. Clinically, ictal amplitudes are measured electrographically (using, e.g., electroencephalography, electrocorticography, and depth electrodes), but these methods do not enable the assessment of the activity of individual neurons. Population signal may increase from three potential sources: (1) increased synchrony (i.e., more coactive neurons); (2) altered active state, from bursts of action potentials and/or paroxysmal depolarizing shifts in membrane potential; and (3) altered subthreshold state, which includes all lower levels of activity. Here, we quantify the fraction of ictal signal from each source. METHODS: To identify the cellular determinants of the ictal signal, we measured single cell and population electrical activity and neuronal calcium levels via optical imaging of the genetically encoded calcium indicator (GECI) GCaMP. Spontaneous seizure activity was assessed with microendoscopy in an APP/PS1 mouse with focal cortical injury and via widefield imaging in the organotypic hippocampal slice cultures (OHSCs) model of posttraumatic epilepsy. Single cell calcium signals were linked to a range of electrical activities by performing simultaneous GECI-based calcium imaging and whole-cell patch-clamp recordings in spontaneously seizing OHSCs. Neuronal resolution calcium imaging of spontaneous seizures was then used to quantify the cellular contributions to population-level ictal signal. RESULTS: The seizure onset signal was primarily driven by increased subthreshold activity, consistent with either barrages of excitatory postsynaptic potentials or sustained membrane depolarization. Unsurprisingly, more neurons entered the active state as seizure activity progressed. However, the increasing fraction of active cells was primarily driven by synchronous reactivation and not from continued recruitment of new populations of neurons into the seizure. SIGNIFICANCE: This work provides a critical link between single neuron activity and population measures of seizure activity.

Synthesis and Characterization of a Novel Concentration-Independent Fluorescent Chloride Indicator, ABP-Dextran, Optimized for Extracellular Chloride Measurement.

GABA, the primary inhibitory neurotransmitter, stimulates GABAA receptors (GABAARs) to increase the chloride conductance of the cytosolic membrane. The driving forces for membrane chloride currents are determined by the local differences between intracellular and extracellular chloride concentrations (Cli and Clo, respectively). While several strategies exist for the measurement of Cli, the field lacks tools for non-invasive measurement of Clo. We present the design and development of a fluorescent lifetime imaging (FLIM)-compatible small molecule, N(4-aminobutyl)phenanthridiunium (ABP) with the brightness, spectral features, sensitivity to chloride, and selectivity versus other anions to serve as a useful probe of Clo. ABP can be conjugated to dextran to ensure extracellular compartmentalization, and a second chloride-insensitive counter-label can be added for ratiometric imaging. We validate the utility of this novel sensor series in two sensor concentration-independent modes: FLIM or ratiometric intensity-based imaging.

Effect of neonatal seizure burden and etiology on the long-term outcome: data from a randomized, controlled trial.

Background: Neonatal seizures are common, but the impact of neonatal seizures on long-term neurologic outcome remains unclear. We addressed this question by analyzing data from an early-phase controlled trial of bumetanide to treat neonatal seizures. Methods: Neonatal seizure burden was calculated from continuous video-EEG data. Neurologic outcome was determined by standardized developmental tests and post-neonatal seizure recurrence. Results: Of 111 enrolled neonates, 43 were randomized to treatment or control groups. There were no differences in neurologic outcome between treatment and control groups. A subgroup analysis was performed for 84 neonates with acute perinatal brain injury (57 HIE, 18 stroke, 9 ICH), most of whom (70%) had neonatal seizures. There was a significant negative correlation between seizure burden and developmental scores (p<0.01). Associations between seizure burden and developmental scores were stronger in HIE and stroke groups compared with ICH (p<0.05). Conclusion: Bumetanide showed no long-term beneficial or adverse effects, as expected based on treatment duration versus duration of neonatal seizures. For neonates with perinatal brain injury, higher neonatal seizure burden correlated significantly with worse developmental outcome, particularly for ischemic versus hemorrhagic brain injury. These data highlight the need for further investigation of the long-term effects of both neonatal seizure severity and etiology.

Can in vitro studies aid in the development and use of antiseizure therapies? A report of the ILAE/AES Joint Translational Task Force.

In vitro preparations (defined here as cultured cells, brain slices, and isolated whole brains) offer a variety of approaches to modeling various aspects of seizures and epilepsy. Such models are particularly amenable to the application of anti-seizure compounds, and consequently are a valuable tool to screen the mechanisms of epileptiform activity, mode of action of known anti-seizure medications (ASMs), and the potential efficacy of putative new anti-seizure compounds. Despite these applications, all disease models are a simplification of reality and are therefore subject to limitations. In this review, we summarize the main types of in vitro models that can be used in epilepsy research, describing key methodologies as well as notable advantages and disadvantages of each. We argue that a well-designed battery of in vitro models can form an effective and potentially high-throughput screening platform to predict the clinical usefulness of ASMs, and that in vitro models are particularly useful for interrogating mechanisms of ASMs. To conclude, we offer several key recommendations that maximize the potential value of in vitro models in ASM screening. This includes the use of multiple in vitro tests that can complement each other, carefully combined with in vivo studies, the use of tissues from chronically epileptic (rather than naïve wild-type) animals, and the integration of human cell/tissue-derived preparations.

2-Photon imaging of fluorescent proteins in living swine.

A common point of failure in translation of preclinical neurological research to successful clinical trials comes in the giant leap from rodent models to humans. Non-human primates are phylogenetically close to humans, but cost and ethical considerations prohibit their widespread usage in preclinical trials. Swine have large, gyrencencephalic brains, which are biofidelic to human brains. Their classification as livestock makes them a readily accessible model organism. However, their size has precluded experiments involving intravital imaging with cellular resolution. Here, we present a suite of techniques and tools for in vivo imaging of porcine brains with subcellular resolution. Specifically, we describe surgical techniques for implanting a synthetic, flexible, transparent dural window for chronic optical access to the neocortex. We detail optimized parameters and methods for injecting adeno-associated virus vectors through the cranial imaging window to express fluorescent proteins. We introduce a large-animal 2-photon microscope that was constructed with off-the shelf components, has a gantry design capable of accommodating animals > 80 kg, and is equipped with a high-speed digitizer for digital fluorescence lifetime imaging. Finally, we delineate strategies developed to mitigate the substantial motion artifact that complicates high resolution imaging in large animals, including heartbeat-triggered high-speed image stack acquisition. The effectiveness of this approach is demonstrated in sample images acquired from pigs transduced with the chloride-sensitive fluorescent protein SuperClomeleon.

2-Photon imaging of fluorescent proteins in living swine.

A common point of failure in translation of preclinical neurological research to successful clinical trials comes in the giant leap from rodent models to humans. Non-human primates are phylogenetically close to humans, but cost and ethical considerations prohibit their widespread usage in preclinical trials. Swine have large, gyrencencephalic brains, which are biofidelic to human brains. Their classification as livestock makes them a readily accessible model organism. However, their size has precluded experiments involving intravital imaging with cellular resolution. Here, we present a suite of techniques and tools for in vivo imaging of porcine brains with subcellular resolution. Specifically, we describe surgical techniques for implanting a synthetic, flexible, transparent dural window for chronic optical access to the neocortex. We detail optimized parameters and methods for injecting adeno-associated virus vectors through the cranial imaging window to express fluorescent proteins. We introduce a large-animal 2-photon microscope that was constructed with off-the shelf components, has a gantry design capable of accommodating animals > 80 kg, and is equipped with a high-speed digitizer for digital fluorescence lifetime imaging. Finally, we delineate strategies developed to mitigate the substantial motion artifact that complicates high resolution imaging in large animals, including heartbeat-triggered high-speed image stack acquisition. The effectiveness of this approach is demonstrated in sample images acquired from pigs transduced with the chloride-sensitive fluorescent protein SuperClomeleon.

Analysis of brain region-specific mRNA synthesis and stability by utilizing adult mouse brain slice culture.

Utilization of live animals for mechanistic study is challenging yet pivotal to elucidate pathogenesis of neurological diseases. Here, we present a protocol that employs cultured brain slices derived from adult mice to examine mRNA metabolism. We describe the preparation of acute brain slices and the treatments of RNA synthesis inhibitor and nucleotide analog to examine the effects of ataxin-1 loss-of-function on Bace1 mRNA stability and transcription in cortex. This protocol also includes electrophysiological recording of spontaneous neuronal activity in hippocampus. For complete details on the use and execution of this protocol, please refer to Suh et al. (2019).

Clarifications regarding bumetanide for neonatal seizures.

A recent Phase II randomized, controlled trial of bumetanide as an adjunctive treatment for neonatal seizures showed a robust efficacy signal and no evidence of toxicity. Concerns regarding bumetanide as an adjunctive anticonvulsant are addressed here. An adequately powered multi-institutional trial is needed to accurately determine efficacy.

In vitro ictogenesis is stochastic at the single neuron level.

Seizure initiation is the least understood and most disabling element of epilepsy. Studies of ictogenesis require high speed recordings at cellular resolution in the area of seizure onset. However, in vivo seizure onset areas cannot be determined at the level of resolution necessary to enable such studies. To circumvent these challenges, we used novel GCaMP7-based calcium imaging in the organotypic hippocampal slice culture model of post-traumatic epilepsy in mice. Organotypic hippocampal slice cultures generate spontaneous, recurrent seizures in a preparation in which it is feasible to image the activity of the entire network (with no unseen inputs existing). Chronic calcium imaging of the entire hippocampal network, with paired electrophysiology, revealed three patterns of seizure onset: (i) low amplitude fast activity; (ii) sentinel spike; and (iii) spike burst and low amplitude fast activity onset. These patterns recapitulate common features of human seizure onset, including low voltage fast activity and spike discharges. Weeks-long imaging of seizure activity showed a characteristic evolution in onset type and a refinement of the seizure onset zone. Longitudinal tracking of individual neurons revealed that seizure onset is stochastic at the single neuron level, suggesting that seizure initiation activates neurons in non-stereotyped sequences seizure to seizure. This study demonstrates for the first time that transitions to seizure are not initiated by a small number of neuronal 'bad actors' (such as overly connected hub cells), but rather by network changes which enable the onset of pathology among large populations of neurons.

Unique Actions of GABA Arising from Cytoplasmic Chloride Microdomains.

Developmental, cellular, and subcellular variations in the direction of neuronal Cl- currents elicited by GABAA receptor activation have been frequently reported. We found a corresponding variance in the GABAA receptor reversal potential (EGABA) for synapses originating from individual interneurons onto a single pyramidal cell. These findings suggest a similar heterogeneity in the cytoplasmic intracellular concentration of chloride ([Cl-]i) in individual dendrites. We determined [Cl-]i in the murine hippocampus and cerebral cortex of both sexes by (1) two-photon imaging of the Cl--sensitive, ratiometric fluorescent protein SuperClomeleon; (2) Fluorescence Lifetime IMaging (FLIM) of the Cl--sensitive fluorophore MEQ (6-methoxy-N-ethylquinolinium); and (3) electrophysiological measurements of EGABA by pressure application of GABA and RuBi-GABA uncaging. Fluorometric and electrophysiological estimates of local [Cl-]i were highly correlated. [Cl-]i microdomains persisted after pharmacological inhibition of cation-chloride cotransporters, but were progressively modified after inhibiting the polymerization of the anionic biopolymer actin. These methods collectively demonstrated stable [Cl-]i microdomains in individual neurons in vitro and in vivo and the role of immobile anions in its stability. Our results highlight the existence of functionally significant neuronal Cl- microdomains that modify the impact of GABAergic inputs.SIGNIFICANCE STATEMENT Microdomains of varying chloride concentrations in the neuronal cytoplasm are a predictable consequence of the inhomogeneous distribution of anionic polymers such as actin, tubulin, and nucleic acids. Here, we demonstrate the existence and stability of these microdomains, as well as the consequence for GABAergic synaptic signaling: each interneuron produces a postsynaptic GABAA response with a unique reversal potential. In individual hippocampal pyramidal cells, the range of GABAA reversal potentials evoked by stimulating different interneurons was >20 mV. Some interneurons generated postsynaptic responses in pyramidal cells that reversed at potentials beyond what would be considered purely inhibitory. Cytoplasmic chloride microdomains enable each pyramidal cell to maintain a compendium of unique postsynaptic responses to the activity of individual interneurons.

KCC2 Chloride Transport Contributes to the Termination of Ictal Epileptiform Activity.

Recurrent seizures intensely activate GABAA receptors (GABAA-Rs), which induces transient neuronal chloride ([Cl-]i) elevations and depolarizing GABA responses that contribute to the failure of inhibition that engenders further seizures and anticonvulsant resistance. The K+-Cl- cotransporter KCC2 is responsible for Cl- extrusion and restoration of [Cl-]i equilibrium (ECl) after synaptic activity, but at the cost of increased extracellular potassium which may retard K+-Cl- extrusion, depolarize neurons, and potentiate seizures. Thus, KCC2 may either diminish or facilitate seizure activity, and both proconvulsant and anticonvulsant effects of KCC2 inhibition have been reported. It is now necessary to identify the loci of these divergent responses by assaying both the electrographic effects and the ionic effects of KCC2 manipulation. We therefore determined the net effects of KCC2 transport activity on cytoplasmic chloride elevation and Cl- extrusion rates during spontaneous recurrent ictal-like epileptiform discharges (ILDs) in organotypic hippocampal slices in vitro, as well as the correlation between ionic and electrographic effects. We found that the KCC2 antagonist VU0463271 reduced Cl- extrusion rates, increased ictal [Cl-]i elevation, increased ILD duration, and induced status epilepticus (SE). In contrast, the putative KCC2 upregulator CLP257 improved chloride homeostasis and reduced the duration and frequency of ILDs in a concentration-dependent manner. Our results demonstrate that measuring both the ionic and electrographic effects of KCC2 transport clarify the impact of KCC2 modulation in specific models of epileptiform activity. Anticonvulsant effects predominate when KCC2-mediated chloride transport rather than potassium buffering is the rate-limiting step in restoring ECl and the efficacy of GABAergic inhibition during recurrent ILDs.

A Pilot Randomized, Controlled, Double-Blind Trial of Bumetanide to Treat Neonatal Seizures.

OBJECTIVE: In the absence of controlled trials, treatment of neonatal seizures has changed minimally despite poor drug efficacy. We tested bumetanide added to phenobarbital to treat neonatal seizures in the first trial to include a standard-therapy control group. METHODS: A randomized, double-blind, dose-escalation design was employed. Neonates with postmenstrual age 33 to 44 weeks at risk of or with seizures were eligible. Subjects with electroencephalography (EEG)-confirmed seizures after ≥20 and <40mg/kg phenobarbital were randomized to receive additional phenobarbital with either placebo (control) or 0.1, 0.2, or 0.3mg/kg bumetanide (treatment). Continuous EEG monitoring data from ≥2 hours before to ≥48 hours after study drug administration (SDA) were analyzed for seizures. RESULTS: Subjects were randomized to treatment (n = 27) and control (n = 16) groups. Pharmacokinetics were highly variable among subjects and altered by hypothermia. The only statistically significant adverse event was diuresis in treated subjects (48% vs 13%, p = 0.02). One treated (4%) and 3 control subjects died (19%, p = 0.14). Among survivors, 2 of 26 treated subjects (8%) and 0 of 13 control subjects had hearing impairment, as did 1 nonrandomized subject. Total seizure burden varied widely, with much higher seizure burden in treatment versus control groups (median = 3.1 vs 1.2 min/h, p = 0.006). There was significantly greater reduction in seizure burden 0 to 4 hours and 2 to 4 hours post-SDA (both p < 0.01) compared with 2-hour baseline in treatment versus control groups with adjustment for seizure burden. INTERPRETATION: Although definitive proof of efficacy awaits an appropriately powered phase 3 trial, this randomized, controlled, multicenter trial demonstrated an additional reduction in seizure burden attributable to bumetanide over phenobarbital without increased serious adverse effects. Future trials of bumetanide and other drugs should include a control group and balance seizure severity. ANN NEUROL 2021;89:327-340.

Seven-Year Experience From the National Institute of Neurological Disorders and Stroke-Supported Network for Excellence in Neuroscience Clinical Trials.

Importance: One major advantage of developing large, federally funded networks for clinical research in neurology is the ability to have a trial-ready network that can efficiently conduct scientifically rigorous projects to improve the health of people with neurologic disorders. Observations: National Institute of Neurological Disorders and Stroke Network for Excellence in Neuroscience Clinical Trials (NeuroNEXT) was established in 2011 and renewed in 2018 with the goal of being an efficient network to test between 5 and 7 promising new agents in phase II clinical trials. A clinical coordinating center, data coordinating center, and 25 sites were competitively chosen. Common infrastructure was developed to accelerate timelines for clinical trials, including central institutional review board (a first for the National Institute of Neurological Disorders and Stroke), master clinical trial agreements, the use of common data elements, and experienced research sites and coordination centers. During the first 7 years, the network exceeded the goal of conducting 5 to 7 studies, with 9 funded. High interest was evident by receipt of 148 initial applications for potential studies in various neurologic disorders. Across the first 8 studies (the ninth study was funded at end of initial funding period), the central institutional review board approved the initial protocol in a mean (SD) of 59 (21) days, and additional sites were added a mean (SD) of 22 (18) days after submission. The median time from central institutional review board approval to first site activation was 47.5 days (mean, 102.1; range, 1-282) and from first site activation to first participant consent was 27 days (mean, 37.5; range, 0-96). The median time for database readiness was 3.5 months (mean, 4.0; range, 0-8) from funding receipt. In the 4 completed studies, enrollment met or exceeded expectations with 96% overall data accuracy across all sites. Nine peer-reviewed manuscripts were published, and 22 oral presentations or posters and 9 invited presentations were given at regional, national, and international meetings. Conclusions and Relevance: NeuroNEXT initiated 8 studies, successfully enrolled participants at or ahead of schedule, collected high-quality data, published primary results in high-impact journals, and provided mentorship, expert statistical, and trial management support to several new investigators. Partnerships were successfully created between government, academia, industry, foundations, and patient advocacy groups. Clinical trial consortia can efficiently and successfully address a range of important neurologic research and therapeutic questions.

Epilepsy Benchmarks Area III: Improved Treatment Options for Controlling Seizures and Epilepsy-Related Conditions Without Side Effects.

The goals of Epilepsy Benchmark Area III involve identifying areas that are ripe for progress in terms of controlling seizures and patient symptoms in light of the most recent advances in both basic and clinical research. These goals were developed with an emphasis on potential new therapeutic strategies that will reduce seizure burden and improve quality of life for patients with epilepsy. In particular, we continue to support the proposition that a better understanding of how seizures are initiated, propagated, and terminated in different forms of epilepsy is central to enabling new approaches to treatment, including pharmacological as well as surgical and device-oriented approaches. The stubbornly high rate of treatment-resistant epilepsy-one-third of patients-emphasizes the urgent need for new therapeutic strategies, including pharmacological, procedural, device linked, and genetic. The development of new approaches can be advanced by better animal models of seizure initiation that represent salient features of human epilepsy, as well as humanized models such as induced pluripotent stem cells and organoids. The rapid advances in genetic understanding of a subset of epilepsies provide a path to new and direct patient-relevant cellular and animal models, which could catalyze conceptualization of new treatments that may be broadly applicable across multiple forms of epilepsies beyond those arising from variation in a single gene. Remarkable advances in machine learning algorithms and miniaturization of devices and increases in computational power together provide an enhanced opportunity to detect and mitigate seizures in real time via devices that interrupt electrical activity directly or administer effective pharmaceuticals. Each of these potential areas for advance will be discussed in turn.

Scalp recorded spike ripples predict seizure risk in childhood epilepsy better than spikes.

In the past decade, brief bursts of fast oscillations in the ripple range have been identified in the scalp EEG as a promising non-invasive biomarker for epilepsy. However, investigation and clinical application of this biomarker have been limited because standard approaches to identify these brief, low amplitude events are difficult, time consuming, and subjective. Recent studies have demonstrated that ripples co-occurring with epileptiform discharges ('spike ripple events') are easier to detect than ripples alone and have greater pathological significance. Here, we used objective techniques to quantify spike ripples and test whether this biomarker predicts seizure risk in childhood epilepsy. We evaluated spike ripples in scalp EEG recordings from a prospective cohort of children with a self-limited epilepsy syndrome, benign epilepsy with centrotemporal spikes, and healthy control children. We compared the rate of spike ripples between children with epilepsy and healthy controls, and between children with epilepsy during periods of active disease (active, within 1 year of seizure) and after a period of sustained seizure-freedom (seizure-free, >1 year without seizure), using semi-automated and automated detection techniques. Spike ripple rate was higher in subjects with active epilepsy compared to healthy controls (P = 0.0018) or subjects with epilepsy who were seizure-free ON or OFF medication (P = 0.0018). Among epilepsy subjects with spike ripples, each month seizure-free decreased the odds of a spike ripple by a factor of 0.66 [95% confidence interval (0.47, 0.91), P = 0.021]. Comparing the diagnostic accuracy of the presence of at least one spike ripple versus a classic spike event to identify group, we found comparable sensitivity and negative predictive value, but greater specificity and positive predictive value of spike ripples compared to spikes (P = 0.016 and P = 0.006, respectively). We found qualitatively consistent results using a fully automated spike ripple detector, including comparison with an automated spike detector. We conclude that scalp spike ripple events identify disease and track with seizure risk in this epilepsy population, using both semi-automated and fully automated detection methods, and that this biomarker outperforms analysis of spikes alone in categorizing seizure risk. These data provide evidence that spike ripples are a specific non-invasive biomarker for seizure risk in benign epilepsy with centrotemporal spikes and support future work to evaluate the utility of this biomarker to guide medication trials and tapers in these children and predict seizure risk in other at-risk populations.

A Proposed Mechanism for Spontaneous Transitions between Interictal and Ictal Activity.

Epileptic networks are characterized by two outputs: brief interictal spikes and rarer, more prolonged seizures. Although either output state is readily modeled in silico and induced experimentally, the transition mechanisms are unknown, in part because no models exhibit both output states spontaneously. In silico small-world neural networks were built using single-compartment neurons whose physiological parameters were derived from dual whole-cell recordings of pyramidal cells in organotypic hippocampal slice cultures that were generating spontaneous seizure-like activity. In silico, neurons were connected by abundant local synapses and rare long-distance synapses. Activity-dependent synaptic depression and gradual recovery delimited synchronous activity. Full synaptic recovery engendered interictal population spikes that spread via long-distance synapses. When synaptic recovery was incomplete, postsynaptic neurons required coincident activation of multiple presynaptic terminals to reach firing threshold. Only local connections were sufficiently dense to spread activity under these conditions. This coalesced network activity into traveling waves whose velocity varied with synaptic recovery. Seizures were comprised of sustained traveling waves that were similar to those recorded during experimental and human neocortical seizures. Sustained traveling waves occurred only when wave velocity, network dimensions, and the rate of synaptic recovery enabled wave reentry into previously depressed areas at precisely ictogenic levels of synaptic recovery. Wide-field, cellular-resolution GCamP7b calcium imaging demonstrated similar initial patterns of activation in the hippocampus, although the anatomical distribution of traveling waves of synaptic activation was altered by the pattern of synaptic connectivity in the organotypic hippocampal cultures.SIGNIFICANCE STATEMENT When computerized distributed neural network models are required to generate both features of epileptic networks (i.e., spontaneous interictal population spikes and seizures), the network structure is substantially constrained. These constraints provide important new hypotheses regarding the nature of epileptic networks and mechanisms of seizure onset.

Epileptiform activity in traumatic brain injury predicts post-traumatic epilepsy.

We hypothesize that epileptiform abnormalities (EAs) in the electroencephalogram (EEG) during the acute period following traumatic brain injury (TBI) independently predict first-year post-traumatic epilepsy (PTE1 ). We analyze PTE1 risk factors in two cohorts matched for TBI severity and age (n = 50). EAs independently predict risk for PTE1 (odds ratio [OR], 3.16 [0.99, 11.68]); subdural hematoma is another independent risk factor (OR, 4.13 [1.18, 39.33]). Differences in EA rates are apparent within 5 days following TBI. Our results suggest that increased EA prevalence identifies patients at increased risk for PTE1 , and that EAs acutely post-TBI can identify patients most likely to benefit from antiepileptogenesis drug trials. Ann Neurol 2018;83:858-862.

Epileptogenesis in organotypic hippocampal cultures has limited dependence on culture medium composition.

Rodent organotypic hippocampal cultures spontaneously develop epileptiform activity after approximately 2 weeks in vitro and are increasingly used as a model of chronic post-traumatic epilepsy. However, organotypic cultures are maintained in an artificial environment (culture medium), which contains electrolytes, glucose, amino acids and other components that are not present at the same concentrations in cerebrospinal fluid (CSF). Therefore, it is possible that epileptogenesis in organotypic cultures is driven by these components. We examined the influence of medium composition on epileptogenesis. Epileptogenesis was evaluated by measurements of lactate and lactate dehydrogenase (LDH) levels (biomarkers of ictal activity and cell death, respectively) in spent culture media, immunohistochemistry and automated 3-D cell counts, and extracellular recordings from CA3 regions. Changes in culture medium components moderately influenced lactate and LDH levels as well as electrographic seizure burden and cell death. However, epileptogenesis occurred in any culture medium that was capable of supporting neural survival. We conclude that medium composition is unlikely to be the cause of epileptogenesis in the organotypic hippocampal culture model of chronic post-traumatic epilepsy.

A semi-automated method for rapid detection of ripple events on interictal voltage discharges in the scalp electroencephalogram.

BACKGROUND: High frequency oscillations are emerging as a clinically important indicator of epileptic networks. However, manual detection of these high frequency oscillations is difficult, time consuming, and subjective, especially in the scalp EEG, thus hindering further clinical exploration and application. Semi-automated detection methods augment manual detection by reducing inspection to a subset of time intervals. We propose a new method to detect high frequency oscillations that co-occur with interictal epileptiform discharges. NEW METHOD: The new method proceeds in two steps. The first step identifies candidate time intervals during which high frequency activity is increased. The second step computes a set of seven features for each candidate interval. These features require that the candidate event contain a high frequency oscillation approximately sinusoidal in shape, with at least three cycles, that co-occurs with a large amplitude discharge. Candidate events that satisfy these features are stored for validation through visual analysis. RESULTS: We evaluate the detector performance in simulation and on ten examples of scalp EEG data, and show that the proposed method successfully detects spike-ripple events, with high positive predictive value, low false positive rate, and high intra-rater reliability. COMPARISON WITH EXISTING METHOD: The proposed method is less sensitive than the existing method of visual inspection, but much faster and much more reliable. CONCLUSIONS: Accurate and rapid detection of high frequency activity increases the clinical viability of this rhythmic biomarker of epilepsy. The proposed spike-ripple detector rapidly identifies candidate spike-ripple events, thus making clinical analysis of prolonged, multielectrode scalp EEG recordings tractable.