Seizure Forecasting Using a Novel Sub-Scalp Ultra-Long Term EEG Monitoring System.

Accurate identification of seizure activity, both clinical and subclinical, has important implications in the management of epilepsy. Accurate recognition of seizure activity is essential for diagnostic, management and forecasting purposes, but patient-reported seizures have been shown to be unreliable. Earlier work has revealed accurate capture of electrographic seizures and forecasting is possible with an implantable intracranial device, but less invasive electroencephalography (EEG) recording systems would be optimal. Here, we present preliminary results of seizure detection and forecasting with a minimally invasive sub-scalp device that continuously records EEG. Five participants with refractory epilepsy who experience at least two clinically identifiable seizures monthly have been implanted with sub-scalp devices (Minder®), providing two channels of data from both hemispheres of the brain. Data is continuously captured via a behind-the-ear system, which also powers the device, and transferred wirelessly to a mobile phone, from where it is accessible remotely via cloud storage. EEG recordings from the sub-scalp device were compared to data recorded from a conventional system during a 1-week ambulatory video-EEG monitoring session. Suspect epileptiform activity (EA) was detected using machine learning algorithms and reviewed by trained neurophysiologists. Seizure forecasting was demonstrated retrospectively by utilizing cycles in EA and previous seizure times. The procedures and devices were well-tolerated and no significant complications have been reported. Seizures were accurately identified on the sub-scalp system, as visually confirmed by periods of concurrent conventional scalp EEG recordings. The data acquired also allowed seizure forecasting to be successfully undertaken. The area under the receiver operating characteristic curve (AUC score) achieved (0.88), which is comparable to the best score in recent, state-of-the-art forecasting work using intracranial EEG.

Evaluation of the Biocompatibility of Polypyrrole Implanted Subdurally in GAERS.

This blinded controlled prospective randomized study investigates the biocompatibility of polypyrrole (PPy) polymer that will be used for intracranial triggered release of anti-epileptic drugs (AEDs). Three by three millimeters PPy are implanted subdurally in six adult female genetic absence epilepsy rats from Strasbourg. Each rat has a polymer implanted on one side of the cortex and a sham craniotomy performed on the other side. After a period of seven weeks, rats are euthanized and parallel series of coronal sections are cut throughout the implant site. Four series of 15 sections are histological (hematoxylin and eosin) and immunohistochemically (neuron-specific nuclear protein, glial fibrillary acidic protein, and anti-CD68 antibody) stained and evaluated by three investigators. The results show that implanted PPy mats do not induce obvious inflammation, trauma, gliosis, and neuronal toxicity. Therefore the authors conclude the PPy used offer good histocompatibility with central nervous system cells and that PPy sheets can be used as intracranial, AED delivery implant.

Injectable phenytoin loaded polymeric microspheres for the control of temporal lobe epilepsy in rats.

PURPOSE: Epilepsy is a prevalent neurological disorder with a high frequency of drug resistance. While significant advancements have been made in drug delivery systems to overcome anti-epileptic drug resistance, efficacies of materials in biological systems have been poorly studied. The purpose of the study was to evaluate the anti-epileptic effects of injectable poly(epsilon-caprolactone) (PCL) microspheres for controlled release of an anticonvulsant, phenytoin (PHT), in an animal model of epilepsy. METHODS: PHT-PCL and Blank-PCL microspheres formulated using an oil-in-water (O/W) emulsion solvent evaporation method were evaluated for particle size, encapsulation efficiency, surface morphology and in-vitro drug release profile. Microspheres with the most suitable morphology and release characteristics weresubsequently injected into the hippocampus of a rat tetanus toxin model of temporal lobe epilepsy. Electrocorticography (ECoG)from the cerebral cortex were recorded for all animals. The number of seizure events, severity of seizures, and seizure duration were then compared between the two treatment groups. RESULTS: We have shown that small injections of drug-loaded microspheres are biologically tolerated and released PHT can control seizures for the expected period of time that is in accord with in-vitro release data. CONCLUSION: The study demonstrated the feasibility of polymer-based delivery systems incontrolling focal seizures.

Levetiracetam-loaded biodegradable polymer implants in the tetanus toxin model of temporal lobe epilepsy in rats.

Approximately one-third of people with epilepsy receive insufficient benefit from currently available anticonvulsant medication, and some evidence suggests that this may be due to a lack of effective penetration into brain parenchyma. The current study investigated the ability of biodegradable polymer implants loaded with levetiracetam to ameliorate seizures following implantation above the motor cortex in the tetanus toxin model of temporal lobe epilepsy in rats. The implants led to significantly shorter seizures and a trend towards fewer seizures for up to 1 week. The results of this study indicate that drug-eluting polymer implants represent a promising evolving treatment option for intractable epilepsy. Future research is warranted to investigate issues of device longevity and implantation site.

In vivo biocompatibility and in vitro characterization of poly-lactide-co-glycolide structures containing levetiracetam, for the treatment of epilepsy.

Epilepsy is a chronic neurological disorder characterized by recurrent seizures, and is highly resistant to medication with up to 40% of patients continuing to experience seizures whilst taking oral antiepileptic drugs. Recent research suggests that this may be due to abnormalities in the blood-brain barrier, which prevent the passage of therapeutic substances into the brain. We sought to develop a drug delivery material that could be implanted within the brain at the origin of the seizures to release antiepileptic drugs locally and avoid the blood brain barrier. We produced poly-lactide-co-glycolide drop-cast films and wet-spun fibers loaded with the novel antiepileptic drug Levetiracetam, and investigated their morphology, in vitro drug release characteristics, and brain biocompatibility in adult rats. The best performing structures released Levetiracetam constantly for at least 5 months in vitro, and were found to be highly brain biocompatible following month-long implantations in the motor cortex of adult rats. These results demonstrate the potential of polymer-based drug delivery devices in the treatment of epilepsy and warrant their investigation in animal models of focal epilepsy.

Exploring the tolerability of spatiotemporally complex electrical stimulation paradigms.

A modified cortical stimulation model was used to investigate the effects of varying the synchronicity and periodicity of electrical stimuli delivered to multiple pairs of electrodes on seizure initiation. In this model, electrical stimulation of the motor cortex of rats, along four pairs of a microwire electrode array, results in an observable seizure with quantifiable electrographic duration and behavioural severity. Periodic stimuli had a constant inter-stimulus intervals across the two-second stimulus duration, whilst synchronous stimuli consisted of singular biphasic, bipolar pulses delivered to the four pairs of electrodes at precisely the same time for the entire two second stimulation period. In this way four combinations of stimulation were possible; periodic/synchronous (P/S), periodic/asynchronous (P/As), aperiodic/synchronous (Ap/S) and aperiodic/asynchronous (Ap/As). All stimulation types were designed with equal pulse width, current intensity and mean frequency of stimulation (60 Hz), standardizing net charge transfer. It was expected that the periodicity of the stimulus would be the primary determinant of seizure initiation and therefore severity and electrographic duration. However, the results showed that significant differences in both severity and duration only occurred when the synchronicity was altered. For periodic stimuli, synchronous delivery increased median seizure duration from 5 s to 13 s and increased median Racine severity from 1 to 3. In the aperiodic case, synchronous stimulus delivery increased median duration from 5.5 s to 11s and resulted in seizures of median severity 3 vs. 0 in the asynchronous case. These findings may have implications for the design of future neurostimulation waveform designs as higher numbers of electrodes and stimulator output channels become available in next generation implants.

Closed-loop seizure control with very high frequency electrical stimulation at seizure onset in the GAERS model of absence epilepsy.

A closed-loop system for the automated detection and control of epileptic seizures was created and tested in three Genetic Absence Epilepsy Rats from Strasbourg (GAERS) rats. In this preliminary study, a set of four EEG features were used to detect seizures and three different electrical stimulation strategies (standard (130 Hz), very high (500 Hz) and ultra high (1000 Hz)) were delivered to terminate seizures. Seizure durations were significantly shorter with all three stimulation strategies when compared to non-stimulated (control) seizures. We used mean seizure duration of epileptiform discharges persisting beyond the end of electrical stimulation as a measure of stimulus efficacy. When compared to the duration of seizures stimulated in the standard approach (7.0 s ± 10.1), both very high and ultra high frequency stimulation strategies were more effective at shortening seizure durations (1.3 ± 2.2 s and 3.5 ± 6.4 s respectively). Further studies are warranted to further understand the mechanisms by which this therapeutic effect may be conveyed, and which of the novel aspects of the very high and ultra high frequency stimulation strategies may have contributed to the improvement in seizure abatement performance when compared to standard electrical stimulation approaches.

Early hippocampal oxidative stress is a direct consequence of seizures in the rapid electrical amygdala kindling model.

Epilepsy is characterised by recurrent seizures, which are manifestations of aberrant cortical neuronal firing. It is unclear whether oxidative stress is a cause or consequence of seizure-related hippocampal neuronal loss or whether it occurs concomitantly with the initiation of cell death pathways. We utilised the rapid electrical amygdala kindling (REAK) model which does not induce cell death to examine early seizure-induced oxidative stress in wildtype and superoxide dismutase 2 (Sod2) +/- mice, which lack 50% of Sod2 activity and are therefore known to be more susceptible to mitochondrial oxidative stress. A significant increase in lipid peroxidation and superoxide production was noted in the hippocampi of wildtype mice and a more delayed response observed in Sod2 +/- mice at early time-points post-seizures, but protein carbonylation levels appeared unchanged. A 10-fold increase in superoxide production was seen in the Sod2 +/- CA2 neurons, indicating that Sod2 plays an important role in protecting the CA2 region of the hippocampus from seizure-induced free radical damage. Early hippocampal cell death was undetectable in wildtype or Sod2 +/- mice post-seizures. We were able to demonstrate that hippocampal oxidative stress occurred as a direct consequence of seizures rather than downstream of activation of cell death pathways. We were also able to show that this increase in oxidative stress was not sufficient to cause cell death within the time window investigated. Our data indicates that a possible upregulation of endogenous antioxidant activity might exist within selective hippocampal sectors in the Sod2 +/- mice that are as yet unknown.

Seizure severity and duration in the cortical stimulation model of experimental epilepsy in rats: a longitudinal study.

The aim of this study was to determine the current intensities necessary to elicit three levels of varying EEG and behavioural phenomena with electrical stimulation, and also to determine the consistency of the EEG and behavioural components of the triggered seizures over time. Electrical stimulation of the primary motor/somatosensory cortex was performed in 16 adult rats with multichannel microwire electrode arrays. Stimulation was delivered at a frequency of 60 Hz (1 ms pulse width), for 2 s duration, as biphasic rectangular pulses over four of the eight available electrode pairs. Current intensity thresholds for interruption of normal behaviour, epileptiform afterdischarge (EAD) longer than 5 s and motor seizures with Racine severity greater than 3 were not correlated to time post-surgery. The Racine threshold was shown to be negatively correlated to the EAD duration and Racine severity of seizures elicited in the following sessions. Seizures were reliably generated in rats through cortical stimulation with microwire electrode arrays and these seizures were not shown to be subject to any kindling type effects up to 53 days post-implantation. Both the electrographic duration and behavioural severity of stimulated seizures remained, on average, constant during this experimental period. Approximately one-third of stimulations did not cause observable motor seizures and of those that did result in seizures, forelimb clonus was the most common manifestation and the mean EAD duration was 18.5 s. No damage beyond that caused by surgical implantation of electrodes was observed in the histological analyses of stimulated and non-stimulated tissue. The consistency, duration and severity of seizures within this timeframe make this cortical stimulation model suitable for investigations into novel therapeutic interventions for epilepsy that require a known seizure focus.

Functional dentate gyrus neurogenesis in a rapid kindling seizure model.

Neurogenesis in the adult mammalian hippocampus resulting in long-term persistence of new neurons with features of capacity for functional activation is recognized. Many stimuli are capable of increasing the rate of neurogenesis, including seizure activity. Whether these insults result in an increased number of new functionally active neurons over and above the baseline rate of neurogenesis is not known. The rapid electrical amygdala kindling (REAK) model of seizures isolates the effects of seizures alone in the absence of neuronal death and the resulting seizures induce expression of c-Fos in the vast majority of dentate gyrus (DG) granule cells. C57BL/6 mice were exposed to REAK then injected with bromodeoxyuridine (BrDU) to label dividing cells, then re-exposed to REAK after a delay period to allow detection of functional activation in new neurons by measurement c-Fos expression in response to seizures. Adult subgranular zone cells migrated into the DG granule cell layer (GCL), assumed a neuronal phenotype and demonstrated seizure-dependent responsiveness. Larger absolute numbers of new neurons demonstrating seizure-dependent activation were found in the GCL of previously kindled mice. Seizures are capable of increasing the number of new neurons with the capacity for functional activation laid down in the postseizure period and incorporated into seizure-activated circuitry.

A brightness-area-product-based protocol for the quantitative assessment of antigen abundance in fluorescent immunohistochemistry.

A problem frequently facing researchers examining abundance of expression of a given antigen is measurement. When the antigen is confined to the nucleus, absolute numbers of nuclei or a percentage of nuclei expressing the antigen in a given region can be estimated. When the antigen is localized to cytoplasm, cytoplasmic organelles or processes or membranes, the assessment becomes more difficult. In these settings, an observer/experimenter may assign a density score but intra- and inter-observer agreement using a three-tiered system, and finer resolution than this, is unlikely to be reproducible. Digital image analysis provides an opportunity to minimize observer bias in quantification of immunohistochemical staining. Previously, reported digital methods have mostly employed chromogen-staining methods and often report mean image brightness. We report a method for quantitatively assessing and expressing abundance of expression of an antigen in neural tissue stained with immunofluorescent methods by determining the brightness-area-product (BAP). The described protocol utilizes simple to use commercially available software and calculates BAP rather than mean brightness as a measure more representative of antigen abundance and visual interpretation. Accordingly, we propose this protocol as a useful adjunct to observer interpretation of fluorescent immunohistochemistry and its application to assessment of antigen abundance for varying patterns of antigen localization.

The influence of gender on the aggravation of absence seizures by carbamazepine in the low-dose pentylenetetrazol rat model.

OBJECTIVES: To determine whether carbamazepine (CBZ) aggravates absence seizures in the low-dose pentylenetetrazol (PTZ) rat model in both male and female animals, and investigate for gender differences. METHODS: Inbred Sprague-Dawley rats were implanted with EEG electrodes. Seven days later PTZ (20 mg/kg, i.p.) was administered following pre-treatment with vehicle or CBZ (20 mg/kg, i.p.) and the occurrence of spike-and-wave discharges (SWDs) on the EEG quantified. RESULTS: The cumulative SWD for 90-minute post-PTZ was higher in the CBZ versus vehicle pre-treatment arm for both female (mean 110 seconds vs. 62 seconds; P = 0.03) and male (mean 89 seconds vs. 60 seconds; P = 0.03) rats. The increase in SWD duration in the CBZ arm was greater in female rats for the first five 15-minute intervals, but none attained statistical significance (P > 0.05). CBZ pre-treatment resulted in reductions in both SWD frequency (Hz) (male, P = 0.003; female, P < 0.0001) and latency to onset of SWD (male, P = 0.002). The frequency of SWD in CBZ pre-treated rats was lower in females (5.8 Hz vs. 6.1 Hz, P = 0.002) as was the decrease in the SWD burst duration following CBZ versus vehicle pre-treatment (-0.05 seconds vs. -0.25 seconds, P = 0.046). CONCLUSIONS: CBZ consistently aggravates absence seizures in the low-dose PTZ model in both female and male rats. However, while some gender differences were found, the results failed to support the hypothesis that females are significantly more susceptible to aggravation of the number or duration of absence seizures by CBZ.