Ultra-Long-Term-EEG Monitoring (ULTEEM) Systems: Towards User-Friendly Out-of-Hospital Recordings of Electrical Brain Signals in Epilepsy.

Epilepsy is characterized by the occurrence of epileptic events, ranging from brief bursts of interictal epileptiform brain activity to their most dramatic manifestation as clinically overt bilateral tonic-clonic seizures. Epileptic events are often modulated in a patient-specific way, for example by sleep. But they also reveal temporal patterns not only on ultra- and circadian, but also on multidien scales. Thus, to accurately track the dynamics of epilepsy and to thereby enable and improve personalized diagnostics and therapies, user-friendly systems for long-term out-of-hospital recordings of electrical brain signals are needed. Here, we present two wearable devices, namely ULTEEM and ULTEEMNite, to address this unmet need. We demonstrate how the usability concerns of the patients and the signal quality requirements of the clinicians have been incorporated in the design. Upon testbench verification of the devices, ULTEEM was successfully benchmarked against a reference EEG device in a pilot clinical study. ULTEEMNite was shown to record typical macro- and micro-sleep EEG characteristics in a proof-of-concept study. We conclude by discussing how these devices can be further improved and become particularly useful for a better understanding of the relationships between sleep, epilepsy, and neurodegeneration.

On the relationships between epilepsy, sleep, and Alzheimer's disease: A narrative review.

Epilepsy, sleep, and Alzheimer's disease (AD) are tightly and potentially causally interconnected. The aim of our review was to investigate current research directions on these relationships. Our hope is that they may indicate preventive measures and new treatment options for early neurodegeneration. We included articles that assessed all three topics and were published during the last ten years. We found that this literature corroborates connections on various pathophysiological levels, including sleep-stage-related epileptiform activity in AD, the negative consequences of different sleep disorders on epilepsy and cognition, common biochemical pathways as well as network dysfunctions. Here we provide a detailed overview of these topics and we discuss promising diagnostic and therapeutic consequences.

Journal Club: Prehospital Detection of Large Vessel Occlusion Stroke With Electroencephalography: Results of the ELECTRA-STROKE Study.

The ELECTRA-STROKE study investigated the potential of EEG for prehospital triage of patients with ischemic stroke due to large vessel occlusion (LVO), in which fast triage to stroke centers for endovascular treatment is crucial. The study was conducted in 4 phases, and this Journal Club article focuses on the fourth phase in the prehospital setting with suspected stroke patients. An EEG cap with dry electrodes was used to measure brain activity. The main focus was on the diagnostic accuracy of the theta/alpha ratio, which yielded an area under the receiver operator characteristic curve (AUC) of 0.80. Secondary endpoints, particularly the Brain Symmetry Index (a quantified EEG interhemispheric cortical power asymmetry index) in the delta frequency band, showed an AUC of 0.91. Despite the convenient study design and user-friendly EEG device, limitations include a single-arm design, a relatively small sample size, and exclusions due to data quality issues. The usefulness of EEG in the detection of neuronal changes based on brain ischemia was highlighted, but uncertainties remain regarding its use in certain patient groups. The improvements in the Brain Symmetry Index from phase 3 to 4 of the study indicate the potential for further refinement and investigation of combined methods to improve diagnostic accuracy. The study provides insight into the role of EEG in prehospital stroke detection, recognizing both the strengths and limitations. Overall, the study contributes to understanding the promise of EEG in optimizing LVO stroke triage and urges further refinement and exploration of complementary diagnostic approaches.

Frequency and evolution of sleep-wake disturbances after ischemic stroke: A 2-year prospective study of 437 patients.

OBJECTIVE: In the absence of systematic and longitudinal data, this study prospectively assessed both frequency and evolution of sleep-wake disturbances (SWD) after stroke. METHODS: In 437 consecutively recruited patients with ischemic stroke or transient ischemic attack (TIA), stroke characteristics and outcome were assessed within the 1st week and 3.2 ± 0.3 years (M±SD) after the acute event. SWD were assessed by interview and questionnaires at 1 and 3 months as well as 1 and 2 years after the acute event. Sleep disordered breathing (SDB) was assessed by respirography in the acute phase and repeated in one fifth of the participants 3 months and 1 year later. RESULTS: Patients (63.8% male, 87% ischemic stroke and mean age 65.1 ± 13.0 years) presented with mean NIHSS-score of 3.5 ± 4.5 at admission. In the acute phase, respiratory event index was >15/h in 34% and >30/h in 15% of patients. Over the entire observation period, the frequencies of excessive daytime sleepiness (EDS), fatigue and insomnia varied between 10-14%, 22-28% and 20-28%, respectively. Mean insomnia and EDS scores decreased from acute to chronic stroke, whereas restless legs syndrome (RLS) percentages (6-9%) and mean fatigue scores remained similar. Mean self-reported sleep duration was enhanced at acute stroke (month 1: 07:54 ± 01:27h) and decreased at chronic stage (year 2: 07:43 ± 01:20h). CONCLUSIONS: This study documents a high frequency of SDB, insomnia, fatigue and a prolonged sleep duration after stroke/TIA, which can persist for years. Considering the negative effects of SWD on physical, brain and mental health these data suggest the need for a systematic assessment and management of post-stroke SWD.

Assaying embryotoxicity in the test tube: current limitations of the embryonic stem cell test (EST) challenging its applicability domain.

Testing for embryotoxicity in vitro is an attractive alternative to animal experimentation. The embryonic stem cell test (EST) is such a method, and it has been formally validated by the European Centre for the Validation of Alternative Methods. A number of recent studies have underscored the potential of this method. However, the EST performed well below the 78% accuracy expected from the validation study using a new set of chemicals and pharmaceutical compounds, and also of toxicity criteria, tested to enlarge the database of the validated EST as part of the Work Package III of the ReProTect Project funded within the 6th Framework Programme of the European Union. To assess the performance and applicability domain of the EST we present a detailed review of the substances and their effects in the EST being nitrofen, ochratoxin A, D-penicillamine, methylazoxymethanol, lovastatin, papaverine, warfarin, ß-aminopropionitrile, dinoseb, furosemide, doxylamine, pravastatin, and metoclopramide. By delineation of the molecular mechanisms of the substances we identify six categories of reasons for misclassifications. Some of these limitations might also affect other in vitro methods assessing embryotoxicity. Substances that fall into these categories need to be included in future validation sets and in validation guidelines for embryotoxicity testing. Most importantly, we suggest conceivable improvements and additions to the EST which will resolve most of the limitations.

Developmental toxicity testing in the 21st century: the sword of Damocles shattered by embryonic stem cell assays?

Modern society faces an inherent dilemma. In our globalized society, we are spoilt for choice by an ever-increasing number of products, many of which are made of new materials and compound mixtures. At the same time, as consumers we got accustomed to the idea of a life minimized for risk, including our own exposure to chemicals from the environment or to compounds present in and released from everyday products. Chemical safety testing bridges these obviously diverging interests, and the corresponding legislation has hence been tremendously extended (e.g., introduction of the European legislation REACH in 2007). However, the underlying regulatory toxicology still relies mainly on animal testing, which is relatively slow, expensive, and ethically arguable. Meanwhile, recent years have seen a surge in efforts to develop alternative testing systems and strategies. Expectations are particularly high for the applicability of stem cells as test systems especially for developmental toxicity testing in vitro. For the first time in history, test systems can be based on differentiating cells and tissue progenitors in culture, thus bringing the 'vision of toxicity testing in the 21st century' a step closer.

Alternatives to animal testing: current status and future perspectives.

On the occasion of the 20th anniversary of the Center for Alternative Methods to Animal Experiments (ZEBET), an international symposium was held at the German Federal Institute for Risk Assessment (BfR) in Berlin. At the same time, this symposium was meant to celebrate the 50th anniversary of the publication of the book "The Principles of Humane Experimental Technique" by Russell and Burch in 1959 in which the 3Rs principle (that is, Replacement, Reduction, and Refinement) has been coined and introduced to foster the development of alternative methods to animal testing. Another topic addressed by the symposium was the new vision on "Toxicology in the twenty-first Century", as proposed by the US-National Research Council, which aims at using human cells and tissues for toxicity testing in vitro rather than live animals. An overview of the achievements and current tasks, as well as a vision of the future to be addressed by ZEBET@BfR in the years to come is outlined in the present paper.

Protein biomarkers for in vitro testing of embryotoxicity.

There are new challenges for hazard and risk assessment in the chemical industry with regard to REACH legislation in Europe and related activities in the U.S. and Japan, which require the development of novel in vitro models for the molecular characterization of drug- or chemical-related effects replacing conventional animal testing. In the frame of a European FP6 project on reproductive toxicology ( www.reprotect.eu ), we prepared protein samples from mouse embryonic stem cells differentiated into contracting cardiomyocytes according to the validated embryonic stem cell test (EST) protocol, which had been exposed to toxic substances selected by an expert committee from different in vivo categories of embryotoxicity. Lysates were used to carry out the following investigations: (i) identify optimal dose range conditions in the EST that are suitable for (ii) performing a differential quantitative proteomic study of underlying molecular pathways, (iii) define classes of substances with similar proteomic response patterns, (iv) relate these classes to the traditional in vivo categories of embryotoxicity with (v) the final goal to identify novel surrogate protein biomarker candidates for embryo toxicity. We found two distinct classes of toxic substances (Dinoseb, Ochratoxin-A, and Nitrofen vs ß-aminoproprionitril, Metoclopramide, Doxylamine succinate, and d-penicillamine) with clear pathway-related differences in their proteomic patterns. Most notably, different responses to cluster 1 and cluster 2 substances were observed for Heat shock protein ß-1, Ras-GTPase-activating protein SH3-domain binding protein, Ran binding protein 5, and Calreticulin, Dihydropyrimidinase-like 2 (Ulip2 protein). On the other hand, Heat shock protein 8 and Fscn1 protein were down-regulated by all compounds from both clusters.

SAS Care 1: sleep-disordered breathing in acute stroke an transient ischaemic attack - prevalence, evolution and association with functional outcome at 3 months, a prospective observational polysomnography study.

Sleep-disordered breathing (SDB) is frequent in patients with acute stroke. Little is known, however about the evolution of SDB after stroke. Most of our knowledge stems from smaller cohort studies applying limited cardiopulmonary sleep recordings or from cross-sectional data collected in different populations. This study aims to determine prevalence, type and intra-individual evolution of SDB based on full-night polysomnography (PSG) in acute stroke and 3 months thereafter. Furthermore, we aimed to identify predictors of SDB in the acute and chronic phase and to evaluate associations between SDB and functional outcome at 3 months (M3). A total of 166 patients with acute cerebrovascular events were evaluated by full PSG at baseline and 105 again at M3. The baseline prevalence of SDB (apnoea-hypopnoea index (AHI)>5·h-1) was 80.5% and 25.4% of the patients had severe SDB (AHI>30·h-1). Obstructive sleep apnoea was more prevalent than central sleep apnoea (83.8% versus 13%). Mean±SD AHI was 21.4±17.6·h-1and decreased significantly at M3 (18±16.4·h-1; p=0.018). At M3, 91% of all patients with baseline SDB still had an AHI>5·h-1 and in 68.1% the predominant type of SDB remained unchanged (78.9% in obstructive sleep apnoea and 44.4% in central sleep apnoea). The only predictors of SDB at baseline were higher age and body mass index and in the chronic phase additionally baseline AHI. Baseline AHI was associated with functional outcome (modified Rankin score >3) at M3. The high prevalence of SDB in acute stroke, its persistence after 3 months, and the association with functional outcome supports the recommendation for a rapid SDB screening in stroke patients.

Prevalence of sleep-disordered breathing after stroke and TIA: A meta-analysis.

OBJECTIVE: To perform a systematic review and meta-analysis on the prevalence of sleep-disordered breathing (SDB) after stroke. METHODS: We searched PubMed, Embase (Ovid), the Cochrane Library, and CINAHL (from their commencements to April 7, 2017) for clinical studies reporting prevalence and/or severity of SDB after stroke or TIA. Only sleep apnea tests performed with full polysomnography and diagnostic devices of the American Academy of Sleep Medicine categories I-IV were included. We conducted random-effects meta-analysis. PROSPERO registration number: CRD42017072339. RESULTS: The initial search identified 5,211 publications. Eighty-nine studies (including 7,096 patients) met inclusion criteria. Fifty-four studies were performed in the acute phase after stroke (after less than 1 month), 23 studies in the subacute phase (after 1-3 months), and 12 studies in the chronic phase (after more than 3 months). Mean apnea-hypopnea index was 26.0/h (SD 21.7-31.2). Prevalence of SDB with apnea-hypopnea index greater than 5/h and greater than 30/h was found in 71% (95% confidence interval 66.6%-74.8%) and 30% (95% confidence interval 24.4%-35.5%) of patients, respectively. Severity and prevalence of SDB were similar in all examined phases after stroke, irrespective of the type of sleep apnea test performed. Heterogeneity between studies (I 2) was mostly high. CONCLUSION: The high prevalence of SDB after stroke and TIA, which persists over time, is important in light of recent studies reporting the (1) feasibility and (2) efficacy of SDB treatment in this clinical setting.

Electromyographic reactivity measured with scalp-EEG contributes to prognostication after cardiac arrest.

AIM: To assess whether stimulus-induced modifications of electromyographic activity observed on scalp EEG have a prognostic value in comatose patients after cardiac arrest. METHODS: 184 adult patients from a multi-centric prospective register who underwent an early EEG after cardiac arrest were included. Auditory and somatosensory stimulation was performed during EEG-recording. EEG reactivity (EEG-R) and EMG reactivity (EMG-R) were retrospectively assessed visually by board-certified electroencephalographers, and compared with clinical outcome (cerebral performance category, CPC) at three months. A favorable functional outcome was defined as CPC 1-2, an unfavorable outcome as CPC 3-5. RESULTS: Both EEG-R and EMG-R were predictors for good outcome (EEG-R accuracy 72% (95%-CI 66-79), sensitivity 86% (78-93), specificity 60% (50-69); EMG-R accuracy 65% (58-72), sensitivity 61% (51-75), specificity 69% (60-78)). When reactivity was defined as EEG-R and/or EMG-R, the accuracy was 73% (67-70), the sensitivity 94% (90-99), and the specificity 53% (43-63). CONCLUSION: Taking EMG into account when assessing reactivity of EEG seems to reduce false negative predictions for identifying patients with favorable outcome after cardiac arrest.

Post-transcriptional regulation of the let-7 microRNA during neural cell specification.

The let-7 miRNA regulates developmental timing in C. elegans and is an important paradigm for investigations of miRNA functions in mammalian development. We have examined the role of miRNA precursor processing in the temporal control and lineage specificity of the let-7 miRNA. In situ hybridization (ISH) in E9.5 mouse embryos revealed early induction of let-7 in the developing central nervous system. The expression pattern of three let-7 family members closely resembled that of the brain-enriched miRNAs mir-124, mir-125 and mir-128. Comparison of primary, precursor, and mature let-7 RNA levels during both embryonic brain development and neural differentiation of embryonic stem cells and embryocarcinoma (EC) cells suggest post-transcriptional regulation of let-7 accumulation. Reflecting these results, let-7 sensor constructs were strongly down-regulated during neural differentiation of EC cells and displayed lineage specificity in primary cells. Neural differentiation of EC cells was accompanied by an increase in let-7 precursor processing activity in vitro. Furthermore, undifferentiated and differentiated cells contained distinct precursor RNA binding complexes. A neuron-enhanced binding complex was shown by antibody challenge to contain the miRNA pathway proteins Argonaute1 and FMRP. Developmental regulation of the processing pathway correlates with differential localization of the proteins Argonaute, FMRP, MOV10, and TNRC6B in self-renewing stem cells and neurons.

Successful validation of in vitro methods in toxicology by ZEBET, the National Centre for Alternatives in Germany at the BfR (Federal Institute for Risk Assessment).

A short description of the history of the 3Rs concept is given, which was developed as the scientific concept to refine, reduce and replace animal experiments by Russel and Burch more than 40 years ago. In addition, the legal framework in Europe for developing alternatives to animal experiments is given and the current status of in vitro systems in pharmacology and toxicology is described including an update on metabolising systems. The decrease in experimental animal numbers during the past decade in Europe is illustrated by the situation in Germany and the contribution of international harmonisation of test guidelines on reducing animal numbers in regulatory testing is described. A review of the development of the principles of experimental validation is given and the 3T3 NRU in vitro phototoxicity test is used as an example for a successful validation study, which led to the acceptance of the first in vitro toxicity test for regulatory purposes by the OECD. Finally, the currently accepted alternative methods for standardisation and safety testing of drugs, biologicals and medical devices are summarised.

Rapid eye movements sleep as a predictor of functional outcome after stroke: a translational study.

Study Objectives: Sleep disturbances are common in acute stroke patients and are linked with a negative stroke outcome. However, it is also unclear which and how such changes may be related to stroke outcome. To explore this link, we performed a sleep electroencephalogram (EEG) study in animals and humans after ischemic stroke. Methods: (1) Animal study: 12 male rats were assigned to two groups: ischemia (IS) and sham surgery (Sham). In both groups, sleep architecture was investigated 24 h before surgery and for the following 3 days. (2) Human study: 153 patients with ischemic stroke participating in the SAS-CARE prospective, multicenter cohort study had a polysomnography within 9 days after stroke onset. Functional stroke outcome was assessed by the modified Rankin Scale (mRS) at hospital discharge (short-term outcome) and at a 3-month follow-up (long-term outcome). Results: (1) Animal study: rapid eye movement (REM) sleep was significantly reduced in the IS group compared to the Sham group. (2) Human study: patients with poor short-term functional outcome had a reduction of REM sleep and prolonged REM latency during the acute phase of stroke. REM latency was the only sleep EEG variable found to be significantly related to short- and long-term functional impairment in a multiple linear regression analysis. Conclusions: Acute ischemic stroke is followed by a significant reduction of REM sleep in animals and humans. In humans, this reduction was linked with a bad stroke outcome; in addition, REM latency was found to be an independent predictor of stroke evolution. Potential explanations for this role of REM sleep in stroke are discussed. Clinical Trial Registration: http://clinicaltrials.gov. Unique identifier: NCT01097967.

CPAP as treatment of sleep apnea after stroke: A meta-analysis of randomized trials.

OBJECTIVE: To perform a systematic review and meta-analysis of randomized controlled trials (RCTs) examining the effectiveness of continuous positive airway pressure (CPAP) in stroke patients with sleep disordered breathing (SDB). METHODS: In a systematic literature search of electronic databases (MEDLINE, Embase, and the Cochrane Library) from 1980 to November 2016, we identified RCTs that assessed CPAP compared to standard care or sham CPAP in adult patients with stroke or TIA with SDB. Mean CPAP use, odds ratios (ORs), and standardized mean differences (SMDs) were calculated. The prespecified outcomes were adherence to CPAP, neurologic improvement, adverse events, new vascular events, and death. RESULTS: Ten RCTs (564 participants) with CPAP as intervention were included. Two studies compared CPAP with sham CPAP; 8 compared CPAP with usual care. Mean CPAP use across the trials was 4.53 hours per night (95% confidence interval [CI] 3.97-5.08). The OR of dropping out with CPAP was 1.83 (95% CI 1.05-3.21, p = 0.033). The combined analysis of the neurofunctional scales (NIH Stroke Scale and Canadian Neurological Scale) showed an overall neurofunctional improvement with CPAP (SMD 0.5406, 95% CI 0.0263-1.0548) but with a considerable heterogeneity (I2 = 78.9%, p = 0.0394) across the studies. Long-term survival was improved with CPAP in 1 trial. CONCLUSION: CPAP use after stroke is acceptable once the treatment is tolerated. The data indicate that CPAP might be beneficial for neurologic recovery, which justifies larger RCTs.

Publisher Correction: Personalized structural image analysis in patients with temporal lobe epilepsy.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Stroke causes a transient imbalance of interhemispheric information flow in EEG during non-REM sleep.

OBJECTIVE: Large-scale connectivity, especially interhemispheric connections, plays a crucial role for recovery after stroke. Here we used methods from information theory to characterize interhemispheric information flow in wake- and sleep-EEG after cerebral ischemia. METHODS: 34 patients with unilateral ischemic stroke were included. Symbolic Transfer Entropy (STE) was applied between bipolar EEG signals on the left and the right cerebral hemisphere during polysomnographic recordings in the acute phase and 3 months after stroke. RESULTS: In the acute phase, we found a sleep stage-dependent preferred interhemispheric asymmetry: during non-REM sleep the information flow was predominantly directed from the contralesional toward the ipsilesional hemisphere. This effect was greatly reduced in a follow-up recording 3 months after stroke onset. CONCLUSION: Our findings are consistent with functional imaging studies showing a transient hyperactivity of contralesional areas after stroke. We conclude that STE is a robust method for detecting post-stroke connectivity reorganizations, and that sleep stages have to be taken into account when assessing functional connectivity. SIGNIFICANCE: EEG is more widely available than functional MRI. Future studies will have to confirm whether EEG derived STE can be useful in a clinical setting during rehabilitation after stroke.

Periodic limb movements during sleep in stroke/TIA: Prevalence, course, and cardiovascular burden.

OBJECTIVE: To define the prevalence, time course, and associated factors of periodic limb movements during sleep (PLMS) in patients with ischemic stroke or TIA. METHODS: Patients enrolled in the prospective Sleep-Disordered Breathing in Transient Ischemia Attack (TIA)/Ischemic Stroke and Continuous Positive Airway Pressure (CPAP) Treatment Efficacy (SAS-CARE) study underwent a double polysomnographic investigation in the acute and chronic phases after stroke/TIA, together with a MRI brain scan and a 24-hour blood pressure evaluation. The prevalence of PLMS in patients was compared with that in a matched sample of randomly selected healthy controls from the HypnoLaus cohort. One hundred sixty-nine recordings were performed in the acute phase and 191 after 3 months (210 recordings were obtained from the same 105 patients in both phases) and were compared to those of 162 controls. RESULTS: The mean number of PLMS per hour and the percentage of participants with a PLMS index >10 and >15 per hour were similar between patients and controls. PLMS remained stable from the acute to the chronic phase after stroke. Factors positively associated with PLMS were age, body mass index, and history of hypertension. Blood pressure over 24 hours and the burden of cerebrovascular damage were similar between the groups with PLMS and without PLMS. CONCLUSIONS: PLMS are equally frequent in patients with stroke/TIA and the general population. The absence of higher blood pressure values and of a greater vascular brain damage found in patients with PLMS compared to those without PLMS might be due to a greater use of antihypertensive medication among patients with PLMS, which corresponds to a higher prevalence of previous diagnosis of hypertension in these patients.

The role of sleep in recovery following ischemic stroke: A review of human and animal data.

Despite advancements in understanding the pathophysiology of stroke and the state of the art in acute management of afflicted patients as well as in subsequent neurorehabilitation training, stroke remains the most common neurological cause of long-term disability in adulthood. To enhance stroke patients' independence and well-being it is necessary, therefore, to consider and develop new therapeutic strategies and approaches. We postulate that sleep might play a pivotal role in neurorehabilitation following stroke. Over the last two decades compelling evidence for a major function of sleep in neuroplasticity and neural network reorganization underlying learning and memory has evolved. Training and learning of new motor skills and knowledge can modulate the characteristics of subsequent sleep, which additionally can improve memory performance. While healthy sleep appears to support neuroplasticity resulting in improved learning and memory, disturbed sleep following stroke in animals and humans can impair stroke outcome. In addition, sleep disorders such as sleep disordered breathing, insomnia, and restless legs syndrome are frequent in stroke patients and associated with worse recovery outcomes. Studies investigating the evolution of post-stroke sleep changes suggest that these changes might also reflect neural network reorganization underlying functional recovery. Experimental and clinical studies provide evidence that pharmacological sleep promotion in rodents and treatment of sleep disorders in humans improves functional outcome following stroke. Taken together, there is accumulating evidence that sleep represents a "plasticity state" in the process of recovery following ischemic stroke. However, to test the key role of sleep and sleep disorders for stroke recovery and to better understand the underlying molecular mechanisms, experimental research and large-scale prospective studies in humans are necessary. The effects of hospital conditions, such as adjusting light conditions according to the patients' sleep-wake rhythms, or sleep promoting drugs and non-invasive brain stimulation to promote neuronal plasticity and recovery following stroke requires further investigation.