Prader-Willi Syndrome (PWS) is a complex genetic disorder with multiple cognitive, behavioral and endocrine dysfunctions. Sleep alterations and sleep disorders such as Sleep-disordered breathing and Central disorders of hypersomnolence are frequently recognized (either isolated or in comorbidity). The aim of the review is to highlight the pathophysiology and the clinical features of sleep disorders in PWS, providing the basis for early diagnosis and management. We reviewed the genetic features of the syndrome and the possible relationship with sleep alterations in animal models, and we described sleep phenotypes, diagnostic tools and therapeutic approaches in humans. Moreover, we performed a metaanalysis of cerebrospinal fluid orexin levels in patients with PWS; significantly lower levels of orexin were detected in PWS with respect to control subjects (although significantly higher than the ones of narcoleptic patients). Sleep disorders in humans with PWS are multifaceted and are often the result of different mechanisms. Since hypothalamic dysfunction seems to partially influence metabolic, respiratory and sleep/wake characteristics of this syndrome, additional studies are required in this framework. (C) 2021 Elsevier Ltd. All rights reserved.

Neuropeptides orexin A and B (OX‐A/B, also called hypocretin 1 and 2) are released selectively by a population of neurons which projects widely into the entire central nervous system but is localized in a restricted area of the tuberal region of the hypothalamus, caudal to the paraventricular nucleus. The OX system prominently targets brain structures involved in the regulation of wake–sleep state switching, and also orchestrates multiple physiological functions. The degeneration and dysregulation of the OX system promotes narcoleptic phenotypes both in humans and animals. Hence, this review begins with the already proven involvement of OX in narcolepsy, but it mainly discusses the new pre‐clinical and clinical insights of the role of OX in three major neurological disorders characterized by sleep impairment which have been recently associated with OX dysfunction, such as Alzheimer's disease, stroke and Prader Willi syndrome, and have been emerged over the past 10 years to be strongly associated with the OX dysfunction and should be more considered in the future. In the light of the impairment of the OX system in these neurological disorders, it is conceivable to speculate that the integrity of the OX system is necessary for a healthy functioning body.

Prader-Willi syndrome (PWS) is a neurodevelopmental disorder that is characterized by metabolic alteration and sleep abnormalities mostly related to rapid eye movement (REM) sleep disturbances. The disease is caused by genomic imprinting defects that are inherited through the paternal line. Among the genes located in the PWS region on chromosome 15 (15q11-q13), small nucleolar RNA 116 (Snord116) has been previously associated with intrusions of REM sleep into 0 wakefulness in humans and mice. Here, we further explore sleep regulation of PWS by reporting a study with PWScr(m+/P-) mouse line, which carries a paternal deletion of Snord116. We focused our study on both macrostructural electrophysiological components of sleep, distributed among REM5 and nonrapid eye movements. Of note, here, we study a novel electroencephalography (EEG) graphoelements of sleep for mouse studies, the well-known spindles. EEG biomarkers are often linked to the functional properties of cortical neurons and can be instrumental in translational studies. Thus, to better understand specific properties, we isolated and characterized the intrinsic activity of cortical neurons using in vitro microelectrode array. Our results confirm that the loss of Snord116 gene in mice influences specific properties of REM sleep, such as theta rhythms and, for the first time, the organization of REM episodes throughout sleep-wake cycles. Moreover, the analysis of sleep spindles present novel specific phenotype in PWS mice, indicating that a new catalog of sleep biomarkers can be informative in preclinical studies of PWS.

Background: Ischemic stroke causes hypoexcitability in the peri-infarct motor neocortex that stems from increased tonic γ-amino-butyric acid (GABA) activity in neurons. This hypoexcitability, while neuroprotective in the acute phase, may impair neuroplasticity and functional recovery in the subacute phase of stroke. The purpose of this study is to investigate the effect of delayed and prolonged administration of S44819, which is a potent and competitive selective antagonist of GABA A receptors, on the skilled reaching function in a rodent model of stroke. Methods: Male Sprague–Dawley rats ( n = 15) were subjected to permanent middle cerebral artery occlusion. Starting 3 days after stroke, a vehicle or S44819 (3 or 10 mg/kg, BID) was delivered orally twice a day for 28 days. All animals were euthanized 2 weeks later after the washout period. A single pellet reaching task (SPR) was performed before (baseline value) and after the ischemic surgery at several time points (3, 10, 17, 24, 31, 38, and 45 days) to assess the motor deficit. Infarct volume and body changes were also evaluated. Results: S44819, administered at 10 but not 3 mg/kg, significantly improves SPR results over the 45 days after the ischemic surgery. No effect was observed in the infarct size and in the body weight over time between the groups investigated. Conclusion: S44819 at 10 mg/kg significantly enhances motor recovery on a skilled reaching task after sensory-motor cortex lesion. Additionally, our study, in light of the results of the RESTORE BRAIN (Randomized Efficacy and Safety Trial of Oral GABA A α5 antagonist S44819 after Recent ischemic Event) trial, may help clinicians to design clinical studies and stratify variables and patients adequately.

STUDY QUESTION: Is the risk of imprinting disorders increased in children conceived after ART? SUMMARY ANSWER: We found an adjusted odds ratio (AOR) of 2.84 [95% CI: 1.34-6.01] for Beckwith-Wiedemann syndrome in ART children, while the risk of Prader-Willi syndrome, Silver-Russell syndrome or Angelman syndrome was not increased in children conceived after ART. WHAT IS KNOWN ALREADY: Earlier studies, most

Imprinted genes are highly expressed in the hypothalamus; however, whether specific imprinted genes affect hypothalamic neuromodulators and their functions is unknown. It has been suggested that Prader-Willi syndrome (PWS), a neurodevelopmental disorder caused by lack of paternal expression at chromosome 15q11-q13, is characterized by hypothalamic insufficiency. Here, we investigate the role of the paternally expressed Snord116 gene within the context of sleep and metabolic abnormalities of PWS, and we report a significant role of this imprinted gene in the function and organization of the 2 main neuromodulatory systems of the lateral hypothalamus (LH) - namely, the orexin (OX) and melanin concentrating hormone (MCH) - systems. We observed that the dynamics between neuronal discharge in the LH and the sleep-wake states of mice with paternal deletion of Snord116 (PWScr(m+/p-)) are compromised. This abnormal state-dependent neuronal activity is paralleled by a significant reduction in OX neurons in the LH of mutant mice. Therefore, we propose that an imbalance between OX- and MCH-expressing neurons in the LH of mutant mice reflects a series of deficits manifested in the PWS, such as dysregulation of rapid eye movement (REM) sleep, food intake, and temperature control.

Prader-Willi syndrome (PWS) is a neurodevelopmental disorder that is characterized by rapid eye movement (REM) sleep abnormalities. The disease is caused by genomic imprinting defects that are inherited through the paternal line. Among the genes located in the PWS region on chromosome 15 (15q11-q13), small nucleolar RNA 116 ( Snord116 ) has been previously associated with intrusions of REM sleep into wakefulness in both humans and mice. Here, we further explore the processes of sleep regulation by studying the PWScr m+/p- mouse line, which carries a paternal deletion of Snord116. We focused on microstructural electrophysiological components of sleep, such as REM sleep features and sleep spindles within NREM sleep. While the former are thought to contribute to neuronal network formation early in brain development, the latter are markers of thalamocortical processes. Both signals are often compromised in neurodevelopmental disorders and influence functional properties of cortical neurons. Thus, we isolated and characterized the intrinsic activity of cortical neurons using in vitro microelectrode array (MEA) studies. Our results indicate that the Snord116 gene in mice selectively influences REM sleep properties, such as theta rhythms and the organization of REM episodes throughout sleep-wake cycles. Moreover, sleep spindles present specific abnormalities in PWS model systems, indicating that these features of sleep may translate as potential biomarkers in human PWS. We observed abnormalities in the synchronization of cortical neuronal activity that are accounted for by high levels of norepinephrine. In conclusion, our results provide support for an important role of Snord116 in regulating brain activity during sleep and, in particular, cortical neuronal properties, thereby opening new avenues for developing interventions in PWS. Significance Statement We found that the Snord116 gene, a major player in Prader-Willi syndrome (PWS), significantly impacts REM sleep and its regulation. Additionally, we found that sleep spindles, a subtle electroencephalography (EEG) marker that occurs during NREM sleep, are dysregulated in PWS mice that carry a paternal deletion of the Snord116 gene. Using a combination of in vivo and in vitro experiments, we identified sleep features at the network and molecular level that suggest that Snord116 is fundamental in the synchronization of neuronal networks. Our study also provides a new pre-clinical tool to investigate the pathophysiology of sleep in PWS.

Background Ischemic stroke induces hypoexcitability of the peri-infarct cortex by increased tonic GABA activity, which impairs stroke recovery. The GABA A α5 antagonist S44819 has recently been shown to promote post-ischemic motor-coordination recovery after transient proximal middle cerebral artery occlusion (MCAO) in mice. The effects of S44819 on post-ischemic skilled limb movement recovery have so far not yet been assessed in rats. Methods Male Sprague-Dawley rats were subjected to permanent distal MCAO. Starting 3 days post-stroke, vehicle or S44819 (3 or 10 mg/kg) were delivered orally twice a day for 28 days. A single pellet reaching test was performed at baseline, before treatment onset and at weekly intervals thereafter. Animals were sacrificed at 45 days post-stroke (that is, at 42 days post-treatment onset) after 14 days of drug washout. Body weight was monitored, and infarct size was determined by histology. Results S44819, administered at 10 mg/kg but not 3 mg/kg significantly improved single pellet reaching performance over 45 days (F(2,96)=22.43, p<0.001). Body weight was not altered. S44819 had no effect on infarct size. Conclusion Our data indicate that post-acute administration of S44819 at 10 mg/kg promotes skilled forelimb movements. The effect was maintained after S44819 wash out.

Imprinted genes are highly expressed in the hypothalamus; however, whether specific imprinted genes affect hypothalamic neuromodulators and their functions is unknown. It has been suggested that Prader-Willi syndrome (PWS), a neurodevelopmental disorder caused by lack of paternal expression at chromosome 15q11-q13, is characterised by hypothalamic insufficiency. Here, we investigate the role of the paternally expressed Snord116 gene within the context of sleep and metabolic abnormalities of PWS, and we report a novel role of this imprinted gene in the function and organisation of the two main neuromodulatory systems of the lateral hypothalamus (LH), namely, the orexin (OX) and melanin concentrating hormone (MCH) systems. We observe that the dynamics between neuronal discharge in the LH and the sleep-wake states of mice with paternal deletion of Snord116 (PWScr m+/p− ) are compromised. This abnormal state-dependent neuronal activity is paralleled by a significant reduction in OX neurons in the LH of mutants. Therefore, we propose that an imbalance between OX- and MCH-expressing neurons in the LH of mutants reflects a series of deficits manifested in the PWS, such as dysregulation of rapid eye movement (REM) sleep, food intake and temperature control. Highlights Snord116 regulates neuronal activity in the lateral hypothalamus (LH), which is time-locked with cortical states of sleep. Loss of Snord116 reduces orexin neurons in the LH and affects sleep homeostasis and thermoregulation in mice. Snord116 and Peg3 independently control orexin expression in the LH. Paternally expressed alleles maximize the patrilineal effects in the control of REM sleep by the LH in mammals.

Event Abstract Back to Event Sleep- related electrophysiological activity of cortical cultures on MEA Ilaria Colombi1, 2*, Marta Pace1, Valter Tucci1 and Michela Chiappalone1 1 Fondazione Istituto Italiano di Technologia, Italy 2 Università di Genova, Italy Motivation Neuronal assemblies plated on MEAs show spontaneously synchronized, low frequency firing patterns, which could resemble the slow wave oscillations that characterize non-rapid eye movement (NREM) sleep in vivo [1]. The lack of the awake state counterpart limited the investigation of the main physiological aspects of sleep. To this end, in a previous work we stimulated our in vitro neuronal networks using the cholinergic agonist Carbachol (CCh, 20 µM) to suppress the sleep features. We observed for the first time that CCh treatment affected both the high and the low frequency components of the signal, causing a suppression of the classical sleep-like properties of activity [2]. Starting from this finding, we were then interested in understand how the electrophysiological signal changed in pathologies that affect sleep. Prader-Willi syndrome (PWS) is a rare neurodevelopmental disorder that is associated with a paternally-expressed genomic imprinting defects within the human chromosome region 15q11-13. One of the candidate genes is the small nucleolar ribonucleic acid-116 (SNORD116). PWS patients are often affected by sleep-wake disturbance associated with alteration during REM sleep, including abnormalities in theta waves. The same identical sleep/EEG defects were found using mutant mice (PWS) carrying a deletion in the gene Snord116 at the orthologous locus [3]. Then we wanted to answer one main question: can our model recapitulate the essential features in a pathological model of sleep? More specifically, it is possible to observe the same abnormalities in a simplified and accessible model in vitro? We performed preliminary experiments using cortical cultures of PWS mice and we applied Carbachol (CCh, 20 µM) to suppress the sleep waves. Interestingly, we found the same difference in the theta waves upon CCh administration, suggesting the idea that our model can be used to study and manipulate sleep properties in a very controlled way. Material and Methods Cell cultures prepared from embryonic mice at gestational day 18 were plated onto 60-channel MEAs (Multichannel Systems, MCS, Reutlingen, Germany) previously coated with poly-L-lysine in buffer borate to promote cell adhesion (final density around 1200 cells/mm2) (Figure 1A). We recorded the electrophysiological activity of four WT cultures and two PWS cultures. The experimental protocol adopted for these experiments included 2 hours of recording in culture solution defined as a basal condition. Then we chemically stimulated our cultures with CCh (20 µM) for 4 hours (Figure 2A). Starting from the raw data (i.e. wide band signal) we performed a dual process to analyse both MUA (Multi-Unit Activity, f>300 Hz) and LFP (Local Field Potential, f<300 Hz) (Figure 1B). In order to capture only MUA activity, we high pass filtered the raw signal. Once spikes (i.e. single over threshold peaks) and bursts (i.e. groups of tightly packed spikes) activity were detected from MUA recordings, we computed the following parameters: Mean Firing Rate, MFR [spike/sec], Inverse Burst Ratio, IBR, (percentage of spike outside the burst) and Burstiness Index, BI, (index of the burstiness level of the network) (Figure1 B, light blue boxes in the diagram) [4]. To select the LFP components, we low pass filtered the raw data between 1-300 Hz (Figure 1B, light grey boxes in the diagram). We then computed the power spectral density of the decimated signal (sampling frequency 1 kHz) (μV²/Hz), using the Welch method (Windows=5s, overlap=50%). We only considered the lower frequency bands of the signal, which are of particular interest for studying the sleep-wake cycle, in particular delta (1-4 Hz), theta (4-11 Hz), beta (11-30 Hz) and gamma (30-55 Hz) bands. Results We performed experiments using PWS cultures plated on MEA, according to the protocol depicted in Figure 2A. We normalized each experiment with respect to the mean of the selected parameter (i.e. MFR, IBR and BI) during the basal recording. Regarding the WT cultures (Figure 2B, blue line) the activity level, evaluated by means of the MFR, increased after CCh administration with respect to the basal condition. At the same time, CCh application caused an increased number of isolated spikes (i.e. a higher level of the IBR) and a decrease of the BI with respect to the basal phase, indicating a loss of bursting activity and then of synchronicity. Indeed, the results obtained on WT cultures treated with CCh are in line with the previous results obtained by using cortical cultures of embryonic rat coupled to MEA [5]. PWS cultures (Figure 2B, red line) showed a decrease of MFR only in the first hours after the CCh treatment. The number of isolated spikes (i.e. IBR) and BI did not show any variation upon CCh administration with respect to the their basal condition. When comparing the behaviour of WT vs PWS cultures, we observed the following. WT cultures showed higher values of MFR and IBR with respect to the PWS cultures during CCh administration. Conversely, the BI of WT cultures was lower compared to the PWS cultures. This suggests a reduced effect of CCh in PWS cultures, but more experiments and statistical analyses are necessary to confirm our preliminary results. The analysis of the LFP in WT and PWS mice revealed that CCh application caused a strong suppression of all main waves (i.e. delta, theta and, partly, beta), which characterize the classical sleep-wake-cycle. Interestingly, we found a difference between the two set of cultures (i.e. WT vs PWS) only in the theta waves during CCh application (Figure 2 C). These results are in line with a previous study that revealed abnormalities in theta waves during REM sleep [6]. Conclusion In this study, we presented electrophysiological evidence that primary cortical cultures, usually displaying synchronized low-frequency firing patterns under spontaneous conditions, are able to encompass some essential features of sleep also in a pathological model. We presented preliminary experiments and analyses suggesting that our results can replicate those obtained from in vivo animals and PWS patients. We need to increase the number of experiments in order to assess the reproducibility of the results. In conclusion, MEA recordings coupled to cortical cultures seem to represent a possible model to investigate the essential features of sleep in both physiological and Figure 1 Figure 2 References [1] Hinard, V., et al., Key electrophysiological, molecular, and metabolic signatures of sleep and wakefulness revealed in primary cortical cultures. J Neurosci, 2012. 32(36): p. 12506-17. [2] Colombi, I., et al., A simplified in vitro experimental model encompasses the essential features of sleep. Frontiers in Neuroscience, 2016. 10. [3] Lassi, G., et al., Deletion of the Snord116/SNORD116 alters sleep in mice and patients with Prader-Willi syndrome. Sleep, 2016. 39(3): p. 637- 644. [4] Wagenaar, D.A., et al., Controlling bursting in cortical cultures with closed-loop multi-electrode stimulation. J Neurosci, 2005. 25(3): p. 680-8. Keywords: Microelectrodes array, Carbachol, Sleep, Cortical cultures, Prader–Willi syndrome (PWS) Conference: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018. Presentation Type: Poster Presentation Topic: Stimulation strategies Citation: Colombi I, Pace M, Tucci V and Chiappalone M (2019). Sleep- related electrophysiological activity of cortical cultures on MEA. Conference Abstract: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays. doi: 10.3389/conf.fncel.2018.38.00043 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 18 Mar 2018; Published Online: 17 Jan 2019. * Correspondence: Dr. Ilaria Colombi, Fondazione Istituto Italiano di Technologia, Genoa, Italy, ilaria.colombi@iit.it Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Ilaria Colombi Marta Pace Valter Tucci Michela Chiappalone Google Ilaria Colombi Marta Pace Valter Tucci Michela Chiappalone Google Scholar Ilaria Colombi Marta Pace Valter Tucci Michela Chiappalone PubMed Ilaria Colombi Marta Pace Valter Tucci Michela Chiappalone Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

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.

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.

HomeStrokeVol. 48, No. 12Sleep, Preconditioning and Stroke Free AccessArticle CommentaryPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissionsDownload Articles + Supplements ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toSupplemental MaterialFree AccessArticle CommentaryPDF/EPUBSleep, Preconditioning and Stroke Alessandro Pincherle, MD, Marta Pace, PhD, Simone Sarasso, MD, PhD, Laura Facchin, MSc, Jens P. Dreier, MD, PhD and Claudio L. Bassetti, MD, PhD Alessandro PincherleAlessandro Pincherle From the Department of Clinical Neurosciences, Acute Neurorehabilitation Unit, University Hospital CHUV, Lausanne, Switzerland (A.P.); ZEN Department of Neurology, Bern University Hospital, Switzerland (M.P., L.F., C.L.B.); Department of Genetics and Epigenetics of Behavior, Istituto Italiano di Tecnologia, Genoa, Italy (M.P.); L. Sacco Department of Biomedical and Clinical Sciences, University of Milan, Italy (S.S.); and Department of Neurology (J.P.D.) and Department of Experimental Neurology (J.P.D.), Stroke Center, Charité University Medicine Berlin, Germany. , Marta PaceMarta Pace From the Department of Clinical Neurosciences, Acute Neurorehabilitation Unit, University Hospital CHUV, Lausanne, Switzerland (A.P.); ZEN Department of Neurology, Bern University Hospital, Switzerland (M.P., L.F., C.L.B.); Department of Genetics and Epigenetics of Behavior, Istituto Italiano di Tecnologia, Genoa, Italy (M.P.); L. Sacco Department of Biomedical and Clinical Sciences, University of Milan, Italy (S.S.); and Department of Neurology (J.P.D.) and Department of Experimental Neurology (J.P.D.), Stroke Center, Charité University Medicine Berlin, Germany. , Simone SarassoSimone Sarasso From the Department of Clinical Neurosciences, Acute Neurorehabilitation Unit, University Hospital CHUV, Lausanne, Switzerland (A.P.); ZEN Department of Neurology, Bern University Hospital, Switzerland (M.P., L.F., C.L.B.); Department of Genetics and Epigenetics of Behavior, Istituto Italiano di Tecnologia, Genoa, Italy (M.P.); L. Sacco Department of Biomedical and Clinical Sciences, University of Milan, Italy (S.S.); and Department of Neurology (J.P.D.) and Department of Experimental Neurology (J.P.D.), Stroke Center, Charité University Medicine Berlin, Germany. , Laura FacchinLaura Facchin From the Department of Clinical Neurosciences, Acute Neurorehabilitation Unit, University Hospital CHUV, Lausanne, Switzerland (A.P.); ZEN Department of Neurology, Bern University Hospital, Switzerland (M.P., L.F., C.L.B.); Department of Genetics and Epigenetics of Behavior, Istituto Italiano di Tecnologia, Genoa, Italy (M.P.); L. Sacco Department of Biomedical and Clinical Sciences, University of Milan, Italy (S.S.); and Department of Neurology (J.P.D.) and Department of Experimental Neurology (J.P.D.), Stroke Center, Charité University Medicine Berlin, Germany. , Jens P. DreierJens P. Dreier From the Department of Clinical Neurosciences, Acute Neurorehabilitation Unit, University Hospital CHUV, Lausanne, Switzerland (A.P.); ZEN Department of Neurology, Bern University Hospital, Switzerland (M.P., L.F., C.L.B.); Department of Genetics and Epigenetics of Behavior, Istituto Italiano di Tecnologia, Genoa, Italy (M.P.); L. Sacco Department of Biomedical and Clinical Sciences, University of Milan, Italy (S.S.); and Department of Neurology (J.P.D.) and Department of Experimental Neurology (J.P.D.), Stroke Center, Charité University Medicine Berlin, Germany. and Claudio L. BassettiClaudio L. Bassetti From the Department of Clinical Neurosciences, Acute Neurorehabilitation Unit, University Hospital CHUV, Lausanne, Switzerland (A.P.); ZEN Department of Neurology, Bern University Hospital, Switzerland (M.P., L.F., C.L.B.); Department of Genetics and Epigenetics of Behavior, Istituto Italiano di Tecnologia, Genoa, Italy (M.P.); L. Sacco Department of Biomedical and Clinical Sciences, University of Milan, Italy (S.S.); and Department of Neurology (J.P.D.) and Department of Experimental Neurology (J.P.D.), Stroke Center, Charité University Medicine Berlin, Germany. Originally published7 Nov 2017https://doi.org/10.1161/STROKEAHA.117.018796Stroke. 2017;48:3400–3407Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: January 1, 2017: Previous Version 1 Sleep is a complex physiological and behavioral state, which is needed for homeostasis at a cellular (neurons), organ (brain), and individual level, known to be fundamental for survival. Despite its importance, it is estimated that one third of the adult population is sleep deprived1 or complains about sleep disturbances.2Sleep disorders, classified into 6 major categories according to the recent International Classification of Sleep Disorders,3 determine sleep fragmentation, which in turn induces autonomic nervous system dysfunction, increases inflammation, alters coagulation, and induces oxidative stress responses.4,5 Sleep deprivation (SD)/fragmentation has been linked to several pathological conditions, including stroke (in the present review, the term stroke always refers to ischemic stroke).6,7 In the first part of this review, we address the role of sleep modulation in the pathophysiology of brain ischemia (Figure 1),8–12 and we briefly discuss the current epidemiological evidence linking sleep disorders to stroke risk. In the second part, we review the concept of ischemic preconditioning (IP), and we discuss the role of sleep as a potential preconditioning factor able to induce ischemic tolerance and neuroprotection.13Download figureDownload PowerPointFigure 1. The vicious and virtuous circles of sleep and stroke interaction. Sleep disruption after experimental stroke is detrimental, whereas promoting sleep after an ischemic event facilitates neuroplasticity and recovery in turn determining a better stroke outcome. REM indicates rapid eye movement; and SWS, slow-wave sleep.Does Sleep Play a Role in Stroke?Animal StudiesSleep may be artificially manipulated by inducing either SD or sleep enhancement before or after experimental stroke. Animal models have shown that increasing the length and repetition of SD after stroke is linked to detrimental effects affecting both stroke evolution and functional recovery.9,11,14 Additionally, it has been shown that the number of apoptotic cells is also significantly increased in the sleep-deprived animals9 and that neuroplasticity, axonal sprouting, and neurogenesis are significantly impaired.15Conversely, the pharmacological enhancement of sleep after stroke is associated with positive outcomes. Particularly, mice undergoing treatment with γ-hydroxybutyrate—a drug used to promote slow-wave sleep in humans—showed a faster recovery when compared with those treated with saline, and even a significant change of neuroplasticity-associated genes in the injured striatal region was also observed.10 Consistent with this finding, a recent study showed that chronic injections of Baclofen—a Gamma-aminobutyric acid B receptor agonist clinically used to promote sleep—positively influences both neuroplasticity and functional outcome.16 Neurogenesis and axonal sprouting were significantly increased after Baclofen injection16 and slow-wave sleep during the dark phase.16 The latter observation may be related to axonal sprouting, which is known to be associated to cortical synchronous low-frequency oscillations.17,18 However, it should be taken into account that γ-hydroxybutyrate19 and Baclofen20 are known to decrease spasticity and induce hypothermia, which may be linked to the beneficial effect observed after their injections independently from sleep modulation (for further information, please see the online-only Data Supplement, "Sleep, plasticity and stroke recovery").21Human Studies—Stroke RiskThe strong link between sleep disorders, especially sleep-disordered breathing (SDB), and cardiovascular disease and stroke risk, is supported by several epidemiological lines of evidence.6 Intermittent hypoxia, intrathoracic pressure changes, sympathetic activation, blood pressure swings, endothelial dysfunction, and proinflammatory state have been associated to SDB, predisposing to drug-resistant arterial hypertension, atherosclerosis, cardiac arrhythmia, and hypercoagulation.22,23 In the seminal observational cohort study from Yaggi et al,24 1022 patients were enrolled; the 697 (68%) diagnosed as having obstructive sleep apnea showed an increased risk of stroke and death from any cause independently from other risk factors, including hypertension. This risk association has been confirmed by a meta-analysis of prospective clinical and population-based studies suggesting that SDB is an independent stroke predictor (odds ratio, 2.24; confidence interval, 1.57–3.19), especially in man.25 Furthermore, a recent review evaluated the effect of SDB on stroke outcome in 1203 patients, showing a dose–response relationship between the severity of SDB and risk of recurrent events and all-cause mortality in stroke and patients with transient ischemic attacks (TIAs).26Continuous positive airways pressure—the gold standard to treat obstructive sleep apnea—is known be effective in reducing sleep fragmentation and arterial hypertension.27 Several randomized studies investigated the effect of continuous positive airways pressure in reducing cardiovascular and stroke risk leading to controversial results according to a recent meta-analysis.28 In particular, the recent SAVE study (Sleep Apnea Cardiovascular Endpoints) investigating 2717 patients with cardiovascular diseases (1183 with stroke) and obstructive sleep apnea concluded that continuous positive airways pressure intervention does not prevent the risk of further cardiovascular events in patients with moderate-to-severe obstructive sleep apnea.29 It should be noted, however, that a suboptimal nightly use of continuous positive airways pressure (3.3 hours in the latter study) might be insufficient to provide an efficacious cardiovascular protection effect.The risk of stroke related to other sleep disorders, such as insomnia or restless leg syndrome, should be cautiously considered (for further information, please see the online-only Data Supplement, "Stroke and cardiovascular risk: the role of sleep duration and shift work").6,30,31Preconditioning and StrokeAnimal StudiesCerebral ischemia activates a cascade of detrimental events but also triggers a coordinated response that attempts to counteract tissue damage. Several mechanisms of self-protection have been proposed going from autophagy and metabolism reduction at an early stage to epigenetic events at a later stage, including DNA methylation, histone modification, and microRNAs (small RNA molecules that can control their target gene expression post-transcriptionally).32,33The phenomenon of ischemic tolerance or preconditioning, defined as the application of a potentially harmful stimulus, near to but below the threshold of cell damage, which promotes the tolerance of the brain to subsequent ischemic injury, efficaciously demonstrates the potential for self-protection of the brain and allows dissecting the underling biological mechanisms.34 Several stimuli may be used to induce tolerance, such as hypoxia (IP), inflammatory mediators, metabolic blockers, anesthetics, cortical spreading depolarization (CSD), seizures,34 and SD.8,13 Most of the preconditioning treatments, including SD,13 reprogram the gene response to ischemia inducing a downregulated gene expression, particularly related to inflammation, metabolism, cell cycle regulation, and ion-channel activity.13,34 Genes involved in energy metabolism and protein turnover are also downregulated, pointing either to the reduction of energy expenditure or the improvement of the efficiency of energy metabolism.35 A concomitant upregulation of genes related to cell cycle checkpoints is induced, leading to a reversible cell cycle arrest to allow time for damage repair. Conversely, mitosis and other processes such as the G1/S transition, differentiation, proliferation, and cell death are downregulated.13 In addition, IP induces the expression of genes involved not only in neuroprotection but also in restorative mechanisms, such as neurogenesis36,37 and angiogenesis.38 IP may also antagonize one of the major pathogenic mechanisms of the ischemic cascade, that is, excitoxicity/CSD, characterized by an excessive synaptic release of glutamate. IP may induce tolerance either by lowering excessive glutamate release or by increasing glutamate uptake.34 Furthermore, IP may inhibit excitatory pathways through the downregulation of NMDA (N-methyl-d-aspartate) and AMPA (α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate) receptors, which result abnormally activated from ischemic events.39 It has been hypothesized that IP may underlie neuroprotection through 3 phases of molecular alterations.40 The priming phase occurs within the first few hours of preconditioning, inducing an increase of inflammatory serum cytokines and others genes that may finally mediate neuroprotection. After the priming phase, the system becomes refractive to subsequent injury for an extended period of time, from 1 to 7 days. Potential mediators of this phase are microRNA, epigenetic factors, and autophagy-related changes in metabolism.40 Finally, the third phase is the neuroprotective phase given by the reprogrammed response to the injury results.40 Several studies indicate that preconditioning suppresses damaging genes during ischemic injury and induces the expression of unique genes that are not normally expressed in ischemic animals alone.13Preconditioning may help to identify mechanisms for brain protection and regeneration, providing opportunity for clinical translation. One potential approach is to therapeutically exploit some of the mediators identified by IP. Currently, some of these mediators are being tested in clinical trials. For instance, erythropoietin,41 nitric oxide,42 and interleukin 1 receptor antagonist43 and finally, hypothermia44 have been already tested in humans to reduce ischemic injuries. Another clinical approach of IP in humans is the use of remote ischemic conditioning (RIC), classically consisting of short ischemic intervals to a limb/arm to protect target organs, such as heart and brain. RIC is by far one of the most studied approaches, likely because of its clinical applicability, its low-cost noninvasive nature.Human StudiesDespite the encouraging experimental evidence, the bench-to-bedside transition is limited by several factors. The most important is that in the clinical context, such as acute myocardial infarction or stroke, the unpredictable occurrence of the pathological event does not allow us to pretreat patients with a preconditioning agent/strategy nor to administer a preconditioning mimetic agent just before the infarction. However, preconditioning may be used to reduce the ischemic risk during planned cardiovascular procedures or in conditions where a delay in neurological deficits can be predicted or when the damage may be anticipated. For instance, 2 phase 1 trials showed the safety of RIC in aneurysmal subarachnoid hemorrhage (a condition characterized by a long clinical instability with a high rate of delayed ischemic neurological deficit), with no increase in the risk of deep venous thrombosis or in delayed ischemic neurological after RIC.45,46 A more recent prospective trial proved a better functional outcome in patients treated with RIC.47 Confirmatory results may come from ongoing trials, such as the RIPAT (Remote Ischemic Preconditioning in the Prevention of Ischemic Brain Damage During Intracranial Aneurysm Treatment) and the PreLIMBS trials (Preconditioning With Limb Ischemia for Subarachnoid Hemorrhage; NCT02162654 and NCT02411266 at www.clinicaltrials.gov).Given the unpredictable nature of stroke, IP is not applicable, but other strategies have been proposed to overcome this limitation. RIC is, indeed, known to provide protection not only when administered before ischemia, but also when applied during ischemia (remote ischemic perconditioning [RIPerC]).48 In a phase 3 clinical trial, RIPerC during transportation to hospital had no effect on penumbral salvage, infarct size, or infarct progression as measured by magnetic resonance imaging and 3-month clinical outcome; however, when adjusted for baseline severity of hypoperfusion, a voxel-by-voxel analysis demonstrated increased tissue survival after 1 month suggesting that prehospital RIPerC may be neuroprotective.49 A French multicenter randomized trial is ongoing to evaluate RIPerC early after stroke.50 Taken together, these data suggest the clinical applicability of RIPerC; however, the efficacy and safety of this technique need to be confirmed in adequately powered trials, based on solid and rigorous preclinical testing, with standardized conditioning protocols.Despite the lack of direct therapeutic implications, it is interesting to remember that several studies have aimed to test the hypothesis that an antecedent history of TIA or preinfarction angina induces ischemic tolerance with subsequent reduction in stroke/myocardial infarction severity similar to IP. For instance, 2 retrospective studies showed that the severity of subsequent stroke was reduced, and the outcome improved in patients with a previous TIA.51,52 A small neuroimaging study demonstrated a beneficial effect of TIAs on magnetic resonance imaging lesion size suggesting the existence of endogenous neuroprotection in the human brain,53 and a large German prospective cohort54 showed a lower stroke severity in patients with a preceding TIA. Another prospective study showed that preceding TIA was significantly associated with a lower probability for in-hospital case fatality after adjustment for potential confounds/pathogeneses.55 However, conflicting results have also been reported.56Sleep and PreconditioningWe have already presented the findings on the effect of pharmacological enhancement of sleep after ischemic stroke, supporting the idea that sleep is critical for recovery after cerebral ischemia.10,16 It has also been shown in different experimental settings that SD before ischemia is neuroprotective, improves functional performance, and reduces the signs of ischemic brain damage.8,12,57,58 In other terms, SD before ischemia acts as a form of preconditioning able to induce tolerance to ischemia. Sleep rebound59 is one of the mechanisms through which acute SD before ischemia might induce neuroprotection8,12,58 in line with the strong evidence that one of the main functions of nonrapid eye movement sleep is related to cortical plasticity.60 Interestingly, 2 independent experimental studies proved in rodents that tolerance induced by RIC is dependent on sleep gain after treatment61 and is effectively induced only when sleep rebound after stroke is observed8Preconditioning via SD induces a significant reduction in inflammatory response and apoptotic processes that strongly and negatively influence stroke outcome.13,57,58 A complex gene expression reorganization has been observed after SD: the protective pattern shares several similarities with other forms of preconditioning (Figure 2).13 Rats treated with SD preischemia for preconditioning display a reduction in apoptosis and suppression of neuronal death.13 Yet, inflammatory response has been observed to be downregulated in SD preischemia animals, probably caused by a preactivation of the immune system before stroke.13 Those animals also showed an upregulation of genes related to cell cycle checkpoints, rising to a sort of cell hibernation during which neurons could better resist the consequences of ischemia.35 As in the case of classic IP, not only attenuation of negative responses to ischemia but also increase in neurogenesis has been proposed to have a role in SD preconditioning,14 consistent with studies where pharmacological sleep promotion after stroke promotes neurogenesis in the peri-infarct region.16Download figureDownload PowerPointFigure 2. Mechanisms involved in preconditioning, and specific pathways activated by sleep-dependent preconditioning. SD indicates sleep deprivation.Two genes, MCH and OX, seem to be specifically upregulated exclusively in SD preconditioning, suggesting that these genes play a key role in such mechanism and may represent a future therapeutic target.13Another factor that may be relevant and specific for SD precondition is adenosine. Adenosine plays a role in sleep control, and in particular, the adenosine A1 receptor has been found to be increased after SD in humans62 and animals63 suggesting its role in SD preconditioning.64Role of CSDSpreading depolarization is a depolarization wave that propagates across the cortical mantle, basal ganglia, and other cerebral grey matter structures with a velocity between 2 to 9 mm/min. The hallmark of CSD is the near-complete breakdown of the neuronal ion gradients with dramatic loss in the free energy of neurons, whereas cellular death is characterized by the complete loss of active ion gradients and free energy. No state of neurons is thus closer to cell death than CSD, but neurons can recover from it under otherwise favorable circumstances.65 CSDs have been measured in patients with various brain disorders, including ischemic and hemorrhagic stroke and traumatic brain injury.66 In experimental stroke, CSD exacerbates neuronal injury through prolonged ionic breakdown and spreading ischemia. Interestingly, the reduction of the number of CSDs by using transcranial direct current stimulation significantly reduces the infarct size during the acute phase of ischemic stroke.67Despite numerous evidence for a detrimental role of CSD in infarct growth, cycling of depolarizations around the lesions might, on the contrary, also initiate upregulation of the neurobiological responses involved in repair and protection. The latter represent a sort of preconditioning property, and, as such, CSD can induce a tolerance effect on later ischemic events. Mechanisms underlying CSD-induced neuroprotection involve upregulation of cytokines and growth factors, downregulation of metabolism, altered neurotransmission, NO signaling, and cellular stress responses.68,69 BDNF (brain-derived neurotrophic factor), which represents one of the major mediators of neuroplasticity,70 has been described to play a prominent role in preconditioning by CSD. Indeed, BDNF-deficient mice preconditioned with CSD for 48 hours showed a reduced ischemic tolerance compared with wild-type mice that instead had a potent tolerance by showing 35% reduction of infarct volume.71CSD can be experimentally induced by the application of KCl to the neocortex and is known to reduce, under proper experimental settings, the occurrence of a subsequent CSD. It should be noted that at least part of the preconditioning effects attributed to CSD could also be a direct consequence of the lesion induced by the local potassium application. Indeed, it was shown that even a small cortical lesion, by itself, is sufficient in inducing tolerance to ischemia.72 It has been suggested that CSD may exert its neuroprotective effects when KCl is applied epidurally rather than intracortically;71 however, no systematic investigation has been conducted to date to determine whether the protective effects provided by CSD preconditioning differ depending on the induction method and timing.69Interesting results on the interplay between sleep and CSD might also contribute to the interpretation of the protective side of CSD. Although the effect of CSD on sleep (and vice versa) is still poorly understood and limited data are available, few studies in rats found that CSD increases nonrapid eye movement sleep,73 and a local and transitory increase of nonrapid eye movement–slow wave activity is observed after in vivo unilateral CSD.74 An increased expression of Arc (activity-regulated cytoskeleton-associated protein), BDNF, and NGFI-A (nerve growth factor-induced gene A) was also found in this latter experiment, suggesting that the observed slow-wave activity increase may be considered a marker of neuroplasticity. Interestingly, the same genes are involved in the neuroprotection cascade in the ischemic penumbra.75 In other terms, CSD, facilitating sleep, might be able to promote a local enhancement of cortical plasticity via slow-wave activity. It has also been shown that, 72 hours of, SD is able to increase CSD velocity in rats76 consistent with a facilitator effect of SD on CSD propagation.We can argue that CSD is like a 2-sided medal, with one good side (the neuroprotection properties shown by the preconditioning experiments) and a bad one (the evidence of a harmful role of CSD in metabolically compromised tissue in experimental ischemia and in several human conditions). One possibility is that CSD acts differently in early and late/chronic phases of stroke. Brain excitability, indeed, passes through different phases during the first weeks after stroke; acute ischemia triggers neuronal death via depolarizations and excitotoxicity, in contrast, in the chronic phase–enhanced tonic gamma-aminobutyric acid currents and synaptic glutamate signaling promote BDNF release.77,78Conclusions and Future DirectionsEpidemiological data converge to indicate that inadequate sleep duration or pathological sleep fragmentation (especially when secondary to SDB where other factors, such as intermittent hypoxemia, might concur) pose a substantial but still underscored2 hazard for cerebrovascular morbidity and mortality.6Sleep actively interferes with the cascade of harmful events in cerebral ischemia at different levels, including inflammatory responses, neurotransmitters activity, and gene expression.13 Preliminary data73,74 support the view that sleep may also influence the occurrence of CSD—a neurophysiological phenomenon known to exacerbate neuronal injury after stroke.SD in the acute phase of experimental stroke aggravates lesion evolution and negatively impacts the functional outcome, whereas sleep enhancement after experimental stroke is associated with a faster recovery and with a facilitation of neuroplasticity (Figure 3). In addition, poststroke sleep changes in humans seem to support poststroke neuronal plasticity underlying clinical recovery.7Download figureDownload PowerPointFigure 3. Sleep influence on the sequence of destructive and protective or restorative mechanisms in stroke. Adapted from Dirnagl et al83 with permission. Copyright © 2014, the American Heart Association.A strong theoretical and experimental framework supports the view that SD preconditioning positive effects are mediated by sleep rebound/increase in the early phase after stroke, so we argue that strategies aimed to enhance sleep after ischemia should be implemented. Noninvasive brain stimulation, either transcranial direct current stimulation or trancranial magnetic stimulation, has been successfully used to promote sleep (especially slow-wave activity)79,80; however, these methods might be impractical, and their safety after stroke, especially for chronic long-term exposure, is still unknown. Attention has been given to the possibility of enhancing slow waves by using more physiological stimuli, such as repeated acoustic stimulation during sleep.81 Sleep-modulating drugs, such as baclofen or γ-hydroxybutyrate, that proved to be promising in experimental stroke models may be translated into the clinical practice if their efficacy is confirmed after appropriate clinical trials.10,16,82IP proved to be clearly effective in experimental stroke models; however, only RIC proved to be a reasonable approach to date in the bench-to bed-side transfer. RIC is, indeed, an easy and safe way to induce tolerance to ischemic insult and has already been shown to be safe in cardiological settings and in aneurysmal subarachnoid hemorrhage. Promising data point that RIC may also be applied to the early phase of stroke, and, indeed, clinical trials are ongoing. TIA, given the high risk of subsequent stroke, might be seen as the ideal clinical condition to test potential preconditioning approaches, including RIC or sleep-enhancement therapies.To conclude, our literature review highlights the potential beneficial role of sleep promotion after an ischemic event and that good sleep (ie, optimal sleep duration and treatment of potential sleep disorders) is essential to prevent ischemic stroke and facilitate functional recovery after brain ischemia, given the prominent role of sleep in brain plasticity.AcknowledgmentsWe wish to acknowledge Prof Michael Chopp for his helpful reading and comments.Sources of FundingThis work was supported by grants from the Swiss National Science Foundation (320030-149752 and CRSII3-160803/1), the Deutsche Forschungsgemeinschaft (DR 323/5-1), and the German Federal Ministry of Education and Research (CSB 01 EO 0801).DisclosuresNone.Footnotes*Drs Pincherle and Pace contributed equally.The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.117.018796/-/DC1.Correspondence to Claudio L. Bassetti, MD, PhD, ZEN Department of Neurology, Inselspital–Bern University Hospital, 3010 Bern, Switzerland. E-mail [email protected]References1. Schoenborn CA, Adams PE. Health behaviors of adults: United states, 2005–2007.Vital Health Stat 10. 2010; 245:1–132.Google Scholar2. Léger D, Poursain B, Neubauer D, Uchiyama M. An international survey of sleeping problems in the general population.Curr Med Res Opin. 2008; 24:307–317. doi: 10.1185/030079907X253771.CrossrefMedlineGoogle Scholar3. American Academy of Sleep Medicine. International Cla

STUDY OBJECTIVESSleep deprivation (SDp) performed before stroke induces an ischemic tolerance state as observed in other forms of preconditioning. As the mechanisms underlying this effect are not well understood, we used DNA oligonucleotide microarray analysis to identify the genes and the gene-pathways underlying SDp preconditioning effects.DESIGNGene expression was analyzed 3 days after stroke in 4 experimental groups: (i) SDp performed before focal cerebral ischemia (IS) induction; (ii) SDp performed before sham surgery; (iii) IS without SDp; and (iv) sham surgery without SDp. SDp was performed by gentle handling during the last 6 h of the light period, and ischemia was induced immediately after.SETTINGSBasic sleep research laboratory.MEASUREMENTS AND RESULTSStroke induced a massive alteration in gene expression both in sleep deprived and non-sleep deprived animals. However, compared to animals that underwent ischemia alone, SDp induced a general reduction in transcriptional changes with a reduction in the upregulation of genes involved in cell cycle regulation and immune response. Moreover, an upregulation of a new neuroendocrine pathway which included melanin concentrating hormone, glycoprotein hormones-α-polypeptide and hypocretin was observed exclusively in rats sleep deprived before stroke.CONCLUSIONOur data indicate that sleep deprivation before stroke reprogrammed the signaling response to injury. The inhibition of cell cycle regulation and inflammation are neuroprotective mechanisms reported also for other forms of preconditioning treatment, whereas the implication of the neuroendocrine function is novel and has never been described before. These results therefore provide new insights into neuroprotective mechanisms involved in ischemic tolerance mechanisms.

SummaryThe validation of rodent models for restless legs syndrome (Willis–Ekbom disease) and periodic limb movements during sleep requires knowledge of physiological limb motor activity during sleep in rodents. This study aimed to determine the physiological time structure of tibialis anterior activity during sleep in mice and rats, and compare it with that of healthy humans. Wild‐type mice (n = 9) and rats (n = 8) were instrumented with electrodes for recording the electroencephalogram and electromyogram of neck muscles and both tibialis anterior muscles. Healthy human subjects (31 ± 1 years, n = 21) underwent overnight polysomnography. An algorithm for automatic scoring of tibialis anterior electromyogram events of mice and rats during non‐rapid eye movement sleep was developed and validated. Visual scoring assisted by this algorithm had inter‐rater sensitivity of 92–95% and false‐positive rates of 13–19% in mice and rats. The distribution of the time intervals between consecutive tibialis anterior electromyogram events during non‐rapid eye movement sleep had a single peak extending up to 10 s in mice, rats and human subjects. The tibialis anterior electromyogram events separated by intervals <10 s mainly occurred in series of two‐three events, their occurrence rate in humans being lower than in mice and similar to that in rats. In conclusion, this study proposes reliable rules for scoring tibialis anterior electromyogram events during non‐rapid eye movement sleep in mice and rats, demonstrating that their physiological time structure is similar to that of healthy young human subjects. These results strengthen the basis for translational rodent models of periodic limb movements during sleep and restless legs syndrome/Willis–Ekbom disease.

Levodopa-induced dyskinesia (LID) represents a major challenge for clinicians treating patients affected by Parkinson's disease (PD). Although levodopa is the most effective treatment for PD, the remodeling effects induced by disease progression and the pharmacologic treatment itself cause a narrowing of the therapeutic window because of the development of LID. Although animal models of PD provide strong evidence that striatal plasticity underlies the development of dyskinetic movements, the pathogenesis of LID is not entirely understood. In recent years, slow homeostatic adjustment of intrinsic excitability occurring during sleep has been considered fundamental for network stabilization by gradually modifying plasticity thresholds. So far, how sleep affects on LID has not been investigated. Therefore, we measured synaptic downscaling across sleep episodes in a parkinsonian animal model showing dyskinetic movements similar to LID. Our electrophysiological, molecular, and behavioral results are consistent with an impaired synaptic homeostasis during sleep in animals showing dyskinesia. Accordingly, sleep deprivation causes an anticipation and worsening of LID supporting a link between sleep and the development of LID.

Experimental focal brain ischemia generates in the penumbra recurrent depolarizations which spread across the injured cortex inducing infarct growth. Transcranial direct current stimulation can induce a lasting, polarity-specific, modulation of cortical excitability. To verify whether cathodal transcranial direct current stimulation could reduce the infarct size and the number of depolarizations, focal ischemia was induced in the rat by the 3 vessels occlusion technique. In the first experiment 12 ischemic rats received cathodal stimulation (alternating 15 min on and 15 min off) starting 45 min after middle cerebral artery occlusion and lasting 4 h. In the second experiment 12 ischemic rats received cathodal transcranial direct current stimulation with the same protocol but starting soon after middle cerebral artery occlusion and lasting 6 h. In both experiments controls were 12 ischemic rats not receiving stimulation. Cathodal stimulation reduced the infarct volume in the first experiment by 20% (p=0.002) and in the second by 30% (p=0.003). The area of cerebral infarction was smaller in animals receiving cathodal stimulation in both experiments (p=0.005). Cathodal stimulation reduced the number of depolarizations (p=0.023) and infarct volume correlated with the number of depolarizations (p=0.048). Our findings indicate that cathodal transcranial direct current stimulation exert a neuroprotective effect in the acute phase of stroke possibly decreasing the number of spreading depolarizations. These findings may have translational relevance and open a new avenue in neuroprotection of stroke in humans.

In the quest for susceptibility factors of inflammatory neuropathies, many genes implicated in the pathogenesis of autoimmune diseases have been investigated with negative or conflicting results. We studied, with a gene candidate approach, the CD1 system specialized in capturing and presenting glycolipids to antigen‐specific T cells, and the SH2D2A gene encoding for a T‐cell‐specific adapter protein implicated in control of early T‐cell activation. In Guillain–Barré syndrome, an initially positive association study with polymorphism of CD1A and CD1E genes was not confirmed. In chronic inflammatory demyelinating polyneuropathy, we did not find an association with CD1 genes, but we found an association with a homozygous genotype for a low repeat number of tandem GA in the SH2D2A gene. This genotype could result in defective control and elimination of autoreactive T cells. All the studies were performed on relatively small size populations. Confirmation in larger sized studies is required both for CD1 and SH2D2A genes. Considering the relative rarity of patients with inflammatory neuropathies, this can only be accomplished by international collaboration.

The SH2D2A gene encodes a T-cell-specific adapter protein involved in the negative control of T-cell activation. The genotype GA13-16 homozygote of the SH2D2A gene promoter has been associated with the susceptibility to develop multiple sclerosis. Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is an immune-mediated neuropathy sharing several pathogenetic mechanisms with multiple sclerosis. We genotyped the SH2D2A promoter region in 105 controls and 48 patients with CIDP. We found a significant association between CIDP and the genotype GA13-16 homozygote (OR 3.167; p 0.013). We hypothesize that this genotype is associated with the susceptibility to develop CIDP and may be implicated in the persistence of the disease.