Ghrelin for Neuroprotection in Post-Cardiac Arrest Coma: A Randomized Clinical Trial.

Importance: Out-of-hospital cardiac arrest survival rates have markedly risen in the last decades, but neurological outcome only improved marginally. Despite research on more than 20 neuroprotective strategies involving patients in comas after cardiac arrest, none have demonstrated unequivocal evidence of efficacy; however, treatment with acyl-ghrelin has shown improved functional and histological brain recovery in experimental models of cardiac arrest and was safe in a wide variety of human study populations. Objective: To determine safety and potential efficacy of intravenous acyl-ghrelin to improve neurological outcome in patients in a coma after cardiac arrest. Design, Setting, and Participants: A phase 2, double-blind, placebo-controlled, multicenter, randomized clinical trial, Ghrelin Treatment of Comatose Patients After Cardiac Arrest: A Clinical Trial to Promote Cerebral Recovery (GRECO), was conducted between January 18, 2019, and October 17, 2022. Adult patients 18 years or older who were in a comatose state after cardiac arrest were assessed for eligibility; patients were from 3 intensive care units in the Netherlands. Expected death within 48 hours or unfeasibility of treatment initiation within 12 hours were exclusion criteria. Interventions: Patients were randomized to receive intravenous acyl-ghrelin, 600 µg (intervention group), or placebo (control group) within 12 hours after cardiac arrest, continued for 7 days, twice daily, in addition to standard care. Main Outcomes and Measures: Primary outcome was the score on the Cerebral Performance Categories (CPC) scale at 6 months. Safety outcomes included any serious adverse events. Secondary outcomes were mortality and neuron-specific enolase (NSE) levels on days 1 and 3. Results: A total of 783 adult patients in a coma after cardiac arrest were assessed for eligibility, and 160 patients (median [IQR] age, 68 [57-75] years; 120 male [75%]) were enrolled. A total of 81 patients (51%) were assigned to the intervention group, and 79 (49%) were assigned to the control group. The common odds ratio (OR) for any CPC improvement in the intervention group was 1.78 (95% CI, 0.98-3.22; P = .06). This was consistent over all CPC categories. Mean (SD) NSE levels on day 1 after cardiac arrest were significantly lower in the intervention group (34 [6] µg/L vs 56 [13] µg/L; P = .04) and on day 3 (28 [6] µg/L vs 52 [14] µg/L; P = .08). Serious adverse events were comparable in incidence and type between the groups. Mortality was 37% (30 of 81) in the intervention group vs 51% (40 of 79) in the control group (absolute risk reduction, 14%; 95% CI, -2% to 29%; P = .08). Conclusions and Relevance: In patients in a coma after cardiac arrest, intravenous treatment with acyl-ghrelin was safe and potentially effective to improve neurological outcome. Phase 3 trials are needed for conclusive evidence. Trial Registration: Clinicaltrialsregister.eu: EUCTR2018-000005-23-NL.

Prediction in cultured cortical neural networks.

Theory suggest that networks of neurons may predict their input. Prediction may underlie most aspects of information processing and is believed to be involved in motor and cognitive control and decision-making. Retinal cells have been shown to be capable of predicting visual stimuli, and there is some evidence for prediction of input in the visual cortex and hippocampus. However, there is no proof that the ability to predict is a generic feature of neural networks. We investigated whether random in vitro neuronal networks can predict stimulation, and how prediction is related to short- and long-term memory. To answer these questions, we applied two different stimulation modalities. Focal electrical stimulation has been shown to induce long-term memory traces, whereas global optogenetic stimulation did not. We used mutual information to quantify how much activity recorded from these networks reduces the uncertainty of upcoming stimuli (prediction) or recent past stimuli (short-term memory). Cortical neural networks did predict future stimuli, with the majority of all predictive information provided by the immediate network response to the stimulus. Interestingly, prediction strongly depended on short-term memory of recent sensory inputs during focal as well as global stimulation. However, prediction required less short-term memory during focal stimulation. Furthermore, the dependency on short-term memory decreased during 20 h of focal stimulation, when long-term connectivity changes were induced. These changes are fundamental for long-term memory formation, suggesting that besides short-term memory the formation of long-term memory traces may play a role in efficient prediction.

Oxygen gradient generator to improve in vitro modeling of ischemic stroke.

Introduction: In the core of a brain infarct, perfusion is severely impeded, and neuronal death occurs within minutes. In the penumbra, an area near the core with more remaining perfusion, cells initially remain viable, but activity is significantly reduced. In principle, the penumbra can be saved if reperfusion is established on time, making it a promising target for treatment. In vitro models with cultured neurons on microelectrode arrays (MEAs) provide a useful tool to investigate how ischemic stroke affects neuronal functioning. These models tend to be uniform, focusing on the isolated penumbra, and typically lack adjacent regions such as a core and unaffected regions (normal perfusion). However, processes in these regions may affect neuronal functioning and survival in the penumbra. Materials and methods: Here, we designed, fabricated, and characterized a cytocompatible device that generates an oxygen gradient across in vitro neuronal cultures to expose cells to hypoxia of various depths from near anoxia to near normoxia. This marks a step in the path to mimic core, penumbra, and healthy tissue, and will facilitate better in vitro modeling of ischemic stroke. Results: The generator forms a stable and reproducible gradient within 30 min. Oxygen concentrations at the extremes are adjustable in a physiologically relevant range. Application of the generator did not negatively affect electrophysiological recordings or the viability of cultures, thus confirming the cytocompatibility of the device. Discussion: The developed device is able to impose an oxygen gradient on neuronal cultures and may enrich in vitro stroke models.

Inducing oscillations in positive end-expiratory pressure improves assessment of cerebrovascular pressure reactivity in patients with traumatic brain injury.

The cerebral pressure reactivity index (PRx), through intracranial pressure (ICP) measurements, informs clinicians about the cerebral autoregulation (CA) status in adult-sedated patients with traumatic brain injury (TBI). Using PRx in clinical practice is currently limited by variability over shorter monitoring periods. We applied an innovative method to reduce the PRx variability by ventilator-induced slow (1/min) positive end-expiratory pressure (PEEP) oscillations. We hypothesized that, as seen in a previous animal model, the PRx variability would be reduced by inducing slow arterial blood pressure (ABP) and ICP oscillations without other clinically relevant physiological changes. Patients with TBI were ventilated with a static PEEP for 30 min (PRx period) followed by a 30-min period of slow [1/min (0.0167 Hz)] +5 cmH2O PEEP oscillations (induced (iPRx period). Ten patients with TBI were included. No clinical monitoring was discontinued and no additional interventions were required during the iPRx period. The PRx variability [measured as the standard deviation (SD) of PRx] decreased significantly during the iPRx period from 0.25 (0.22-0.30) to 0.14 (0.09-0.17) (P = 0.006). There was a power increase around the induced frequency (1/min) for both ABP and ICP (P = 0.002). In conclusion, 1/min PEEP-induced oscillations reduced the PRx variability in patients with TBI with ICP levels <22 mmHg. No other clinically relevant physiological changes were observed. Reduced PRx variability might improve CA-guided perfusion management by reducing the time to find "optimal" perfusion pressure targets. Larger studies with prolonged periods of PEEP-induced oscillations are required to take it to routine use.NEW & NOTEWORTHY Cerebral autoregulation assessment requires sufficient slow arterial blood pressure (ABP) waves. However, spontaneous ABP waves may be insufficient for reliable cerebral autoregulation estimations. Therefore, we applied a ventilator "sigh-function" to generate positive end-expiratory pressure oscillations that induce slow ABP waves. This method demonstrated a reduced variability of the pressure reactivity index, commonly used as continuous cerebral autoregulation measure in a traumatic brain injury population.

MEA-ToolBox: an Open Source Toolbox for Standardized Analysis of Multi-Electrode Array Data.

Functional assessment of in vitro neuronal networks-of relevance for disease modelling and drug testing-can be performed using multi-electrode array (MEA) technology. However, the handling and processing of the large amount of data typically generated in MEA experiments remains a huge hurdle for researchers. Various software packages have been developed to tackle this issue, but to date, most are either not accessible through the links provided by the authors or only tackle parts of the analysis. Here, we present ''MEA-ToolBox'', a free open-source general MEA analytical toolbox that uses a variety of literature-based algorithms to process the data, detect spikes from raw recordings, and extract information at both the single-channel and array-wide network level. MEA-ToolBox extracts information about spike trains, burst-related analysis and connectivity metrics without the need of manual intervention. MEA-ToolBox is tailored for comparing different sets of measurements and will analyze data from multiple recorded files placed in the same folder sequentially, thus considerably streamlining the analysis pipeline. MEA-ToolBox is available with a graphic user interface (GUI) thus eliminating the need for any coding expertise while offering functionality to inspect, explore and post-process the data. As proof-of-concept, MEA-ToolBox was tested on earlier-published MEA recordings from neuronal networks derived from human induced pluripotent stem cells (hiPSCs) obtained from healthy subjects and patients with neurodevelopmental disorders. Neuronal networks derived from patient's hiPSCs showed a clear phenotype compared to those from healthy subjects, demonstrating that the toolbox could extract useful parameters and assess differences between normal and diseased profiles.

Maximum entropy models provide functional connectivity estimates in neural networks.

Tools to estimate brain connectivity offer the potential to enhance our understanding of brain functioning. The behavior of neuronal networks, including functional connectivity and induced connectivity changes by external stimuli, can be studied using models of cultured neurons. Cultured neurons tend to be active in groups, and pairs of neurons are said to be functionally connected when their firing patterns show significant synchronicity. Methods to infer functional connections are often based on pair-wise cross-correlation between activity patterns of (small groups of) neurons. However, these methods are not very sensitive to detect inhibitory connections, and they were not designed for use during stimulation. Maximum Entropy (MaxEnt) models may provide a conceptually different method to infer functional connectivity. They have the potential benefit to estimate functional connectivity during stimulation, and to infer excitatory as well as inhibitory connections. MaxEnt models do not involve pairwise comparison, but aim to capture probability distributions of sets of neurons that are synchronously active in discrete time bins. We used electrophysiological recordings from in vitro neuronal cultures on micro electrode arrays to investigate the ability of MaxEnt models to infer functional connectivity. Connectivity estimates provided by MaxEnt models correlated well with those obtained by conditional firing probabilities (CFP), an established cross-correlation based method. In addition, stimulus-induced connectivity changes were detected by MaxEnt models, and were of the same magnitude as those detected by CFP. Thus, MaxEnt models provide a potentially powerful new tool to study functional connectivity in neuronal networks.

The Association between Hypoxia-Induced Low Activity and Apoptosis Strongly Resembles That between TTX-Induced Silencing and Apoptosis.

In the penumbra of a brain infarct, neurons initially remain structurally intact, but perfusion is insufficient to maintain neuronal activity at physiological levels. Improving neuronal recovery in the penumbra has large potential to advance recovery of stroke patients, but penumbral pathology is incompletely understood, and treatments are scarce. We hypothesize that low activity in the penumbra is associated with apoptosis and thus contributes to irreversible neuronal damage. We explored the putative relationship between low neuronal activity and apoptosis in cultured neurons exposed to variable durations of hypoxia or TTX. We combined electrophysiology and live apoptosis staining in 42 cultures, and compared effects of hypoxia and TTX silencing in terms of network activity and apoptosis. Hypoxia rapidly reduced network activity, but cultures showed limited apoptosis during the first 12 h. After 24 h, widespread apoptosis had occurred. This was associated with full activity recovery observed upon reoxygenation within 12 h, but not after 24 h. Similarly, TTX exposure strongly reduced activity, with full recovery upon washout within 12 h, but not after 24 h. Mean temporal evolution of apoptosis in TTX-treated cultures was the same as in hypoxic cultures. These results suggest that prolonged low activity may be a common factor in the pathways towards apoptosis.

Consolidation of memory traces in cultured cortical networks requires low cholinergic tone, synchronized activity and high network excitability.

In systems consolidation, encoded memories are replayed by the hippocampus during slow-wave sleep (SWS), and permanently stored in the neocortex. Declarative memory consolidation is believed to benefit from the oscillatory rhythms and low cholinergic tone observed in this sleep stage, but underlying mechanisms remain unclear. To clarify the role of cholinergic modulation and synchronized activity in memory consolidation, we applied repeated electrical stimulation in mature cultures of dissociated rat cortical neurons with high or low cholinergic tone, mimicking the cue replay observed during systems consolidation under distinct cholinergic concentrations. In the absence of cholinergic input, these cultures display activity patterns hallmarked by network bursts, synchronized events reminiscent of the low frequency oscillations observed during SWS. They display stable activity and connectivity, which mutually interact and achieve an equilibrium. Electrical stimulation reforms the equilibrium to include the stimulus response, a phenomenon interpreted as memory trace formation. Without cholinergic input, activity was burst-dominated. First application of a stimulus induced significant connectivity changes, while subsequent repetition no longer affected connectivity. Presenting a second stimulus at a different electrode had the same effect, whereas returning to the initial stimuli did not induce further connectivity alterations, indicating that the second stimulus did not erase the 'memory trace' of the first. Distinctively, cultures with high cholinergic tone displayed reduced network excitability and dispersed firing, and electrical stimulation did not induce significant connectivity changes. We conclude that low cholinergic tone facilitates memory formation and consolidation, possibly through enhanced network excitability. Network bursts or SWS oscillations may merely reflect high network excitability.

Neuroprotective Treatment of Postanoxic Encephalopathy: A Review of Clinical Evidence.

Postanoxic encephalopathy is the key determinant of death or disability after successful cardiopulmonary resuscitation. Animal studies have provided proof-of-principle evidence of efficacy of divergent classes of neuroprotective treatments to promote brain recovery. However, apart from targeted temperature management (TTM), neuroprotective treatments are not included in current care of patients with postanoxic encephalopathy after cardiac arrest. We aimed to review the clinical evidence of efficacy of neuroprotective strategies to improve recovery of comatose patients after cardiac arrest and to propose future directions. We performed a systematic search of the literature to identify prospective, comparative clinical trials on interventions to improve neurological outcome of comatose patients after cardiac arrest. We included 53 studies on 21 interventions. None showed unequivocal benefit. TTM at 33 or 36°C and adrenaline (epinephrine) are studied most, followed by xenon, erythropoietin, and calcium antagonists. Lack of efficacy is associated with heterogeneity of patient groups and limited specificity of outcome measures. Ongoing and future trials will benefit from systematic collection of measures of baseline encephalopathy and sufficiently powered predefined subgroup analyses. Outcome measurement should include comprehensive neuropsychological follow-up, to show treatment effects that are not detectable by gross measures of functional recovery. To enhance translation from animal models to patients, studies under experimental conditions should adhere to strict methodological and publication guidelines.

Neuroprotective effect of hypoxic preconditioning and neuronal activation in a in vitro human model of the ischemic penumbra.

OBJECTIVE: In ischemic stroke, treatments to protect neurons from irreversible damage are urgently needed. Studies in animal models have shown that neuroprotective treatments targeting neuronal silencing improve brain recovery, but in clinical trials none of these were effective in patients. This failure of translation poses doubts on the real efficacy of treatments tested and on the validity of animal models for human stroke. Here, we established a human neuronal model of the ischemic penumbra by using human induced pluripotent stem cells and we provided an in-depth characterization of neuronal responses to hypoxia and treatment strategies at the network level. APPROACH: We generated neurons from induced pluripotent stem cells derived from healthy donor and we cultured them on micro-electrode arrays. We measured the electrophysiological activity of human neuronal networks under controlled hypoxic conditions. We tested the effect of different treatment strategies on neuronal network functionality. MAIN RESULTS: Human neuronal networks are vulnerable to hypoxia reflected by a decrease in activity and synchronicity under low oxygen conditions. We observe that full, partial or absent recovery depend on the timing of re-oxygenation and we provide a critical time threshold that, if crossed, is associated with irreversible impairments. We found that hypoxic preconditioning improves resistance to a second hypoxic insult. Finally, in contrast to previously tested, ineffective treatments, we show that stimulatory treatments counteracting neuronal silencing during hypoxia, such as optogenetic stimulation, are neuroprotective. SIGNIFICANCE: We presented a human neuronal model of the ischemic penumbra and we provided insights that may offer the basis for novel therapeutic approaches for patients after stroke. The use of human neurons might improve drug discovery and translation of findings to patients and might open new perspectives for personalized investigations.

Mild stimulation improves neuronal survival in an in vitro model of the ischemic penumbra.

OBJECTIVE: In the core of a brain infarct, characterized by severely reduced blood supply, loss of neuronal function is rapidly followed by neuronal death. In peripheral areas of the infarct, the penumbra, damage is initially reversible, and neuronal activity is typically reduced due to ischemia-induced synaptic failure. There is limited understanding of factors governing neuronal recovery or the transition to irreversible damage. Neuronal activity has been shown to be crucial for survival. Consequently, hypoxia induced neuronal inactivity may contribute to cell death, and activation of penumbral neurons possibly improves survival. Adversely, activation increases ATP demand, and a balance should be found between the available energy and sufficient activity. APPROACH: We monitored activity and viability of neurons in an in vitro model of the penumbra, consisting of (rat) neuronal networks on micro electrode arrays (MEAs) under controlled hypoxic conditions. We tested effects of optogenetic and electrical activation during hypoxia. MAIN RESULTS: Mild stimulation yielded significantly better recovery of activity immediately after re-oxygenation, compared with no stimulation, and a 60%-70% higher survival rate after 5 d. Stronger stimulation was not associated with better recovery than no stimulation, suggesting that beneficial effects depend on a delicate balance between sufficient activity and available energy. SIGNIFICANCE: We show that mild activation during hypoxia/ischemia is beneficial for cell survival in an in vitro model of the penumbra. This finding opposes the current common belief that suppression of neuronal activity is the cornerstone of neuroprotection during cerebral ischemia, and may open new possibilities for the treatment of secondary brain damage after stroke.

Ischemic Stroke: Treatments to Improve Neuronal Functional Recovery in vitro.

The understanding of the neurophysiological processes that occur in the areas that surround the core of a brain infarct is crucial for the creation of new therapies and treatments to improve neuronal recovery. The present study aims to demonstrate that both rodent and human neuronal networks lose their activity under low oxygen conditions and that electrical stimulation can increase the probability of recovery. Hypoxia was induced in rodent and human neurons and the effects of electrical stimulation were assessed in the rat cultures. The results obtained show that neuronal activation, in the form of electrical stimulation, has the potential to maintain the networks at higher levels of activity and, therefore, to improve cell survival. This study will open the way for new treatment strategies based on brain-stimulation to enhance neuronal recovery and will be of large relevance for patients, families, and society.

In Vitro Models of Brain Disorders.

The brain is the most complex organ of the body, and many pathological processes underlying various brain disorders are poorly understood. Limited accessibility hinders observation of such processes in the in vivo brain, and experimental freedom is often insufficient to enable informative manipulations. In vitro preparations (brain slices or cultures of dissociated neurons) offer much better accessibility and reduced complexity and have yielded valuable new insights into various brain disorders. Both types of preparations have their advantages and limitations with regard to lifespan, preservation of in vivo brain structure, composition of cell types, and the link to behavioral outcome is often unclear in in vitro models. While these limitations hamper general usage of in vitro preparations to study, e.g., brain development, in vitro preparations are very useful to study neuronal and synaptic functioning under pathologic conditions. This chapter addresses several brain disorders, focusing on neuronal and synaptic functioning, as well as network aspects. Recent progress in the fields of brain circulation disorders, excitability disorders, and memory disorders will be discussed, as well as limitations of current in vitro models.

Evolution of Excitation-Inhibition Ratio in Cortical Cultures Exposed to Hypoxia.

In the core of a brain infarct, neuronal death occurs within minutes after loss of perfusion. In the penumbra, a surrounding area with some residual perfusion, neurons initially remain structurally intact, but hypoxia-induced synaptic failure impedes neuronal activity. Penumbral activity may recover or further deteriorate, reflecting cell death. Mechanisms leading to either outcome remain ill-understood, but may involve changes in the excitation to inhibition (E/I) ratio. The E/I ratio is determined by structural (relative densities of excitatory and inhibitory synapses) and functional factors (synaptic strengths). Clinical studies demonstrated excitability alterations in regions surrounding the infarct core. These may be related to structural E/I changes, but the effects of hypoxia /ischemia on structural connectivity have not yet been investigated, and the role of structural connectivity changes in excitability alterations remains unclear. We investigated the evolution of the structural E/I ratio and associated network excitability in cortical cultures exposed to severe hypoxia of varying duration. 6-12 h of hypoxia reduced the total synaptic density. In particular, the inhibitory synaptic density dropped significantly, resulting in an elevated E/I ratio. Initially, this does not lead to increased excitability due to hypoxia-induced synaptic failure. Increased excitability becomes apparent upon reoxygenation after 6 or 12 h, but not after 24 h. After 24 h of hypoxia, structural patterns of vesicular glutamate stainings change. This possibly reflects disassembly of excitatory synapses, and may account for the irreversible reduction of activity and stimulus responses seen after 24 h.

Loss and recovery of functional connectivity in cultured cortical networks exposed to hypoxia.

In the core of a brain infarct, loss of neuronal function is followed by neuronal death within minutes. In an area surrounding the core (penumbra), some perfusion remains. Here, neurons initially remain structurally intact, but massive synaptic failure strongly reduces neural activity. Activity in the penumbra may eventually recover or further deteriorate toward massive cell death. Besides activity recovery, return of brain functioning requires restoration of connectivity. However, low activity has been shown to initiate compensatory mechanisms that affect network connectivity. We investigated the effect of transient hypoxia and compensatory mechanisms on activity and functional connectivity using cultured cortical networks on multielectrode arrays. Networks were exposed to hypoxia of controlled depth (10-90% of normoxia) and duration (6-48 h). First, we determined how hypoxic depth and duration govern activity recovery. Then, we investigated connectivity changes during and after hypoxic incidents, mild enough for activity to recover. Shortly after hypoxia onset, activity and connectivity decreased. Following 4-6 h of ongoing hypoxia, we observed partial recovery. Only if the hypoxic burden was limited did connectivity show further recovery upon return to normoxia. Partial recovery during hypoxia was dominated by restored baseline connections, rather than newly formed ones. Baseline strengths of surviving (persisting or recovered) and lost connections did not differ nor did baseline activity at their "presynaptic" electrodes. However, "postsynaptic" electrodes of surviving connections were significantly more active during baseline than those of lost connections. This implies that recovery during hypoxia reflects an effective mechanism to restore network activity, which does not necessarily conserve prehypoxia connectivity.NEW & NOTEWORTHY Hypoxia reduced the firing rates of cultured neurons. Depending on hypoxic depth and duration, activity recovered during hypoxia and upon return to normoxia. Recovery (partial) during hypoxia was associated with restored baseline connections rather than newly formed ones. Predominantly, baseline connections with most active postsynaptic electrodes recovered, supporting the notion of effective activity homeostasis. This compensatory mechanism remained effective during ~20 h of hypoxia. Beyond 20 h of compensation, loss of activity and connectivity became irreversible.

Reduced Synaptic Vesicle Recycling during Hypoxia in Cultured Cortical Neurons.

Improvement of neuronal recovery in the ischemic penumbra, an area around the core of a brain infarct with some remaining perfusion, has a large potential for the development of therapy against acute ischemic stroke. However, mechanisms that lead to either recovery or secondary damage in the penumbra largely remain unclear. Recent studies in cultured networks of cortical neurons showed that failure of synaptic transmission (referred to as synaptic failure) is a critical factor in the penumbral area, but the mechanisms that lead to synaptic failure are still under investigation. Here we used a Styryl dye, FM1-43, to quantify endocytosis and exocytosis in cultures of rat cortical neurons under normoxic and hypoxic conditions. Hypoxia in cultured cortical networks rapidly depressed endocytosis and, to a lesser extent, exocytosis. These findings support electrophysiological findings that synaptic failure occurs quickly after the induction of hypoxia, and confirms that the failing processes are at least in part presynaptic.

Functionally different α-synuclein inclusions yield insight into Parkinson's disease pathology.

The formation of α-synuclein (α-S) amyloid aggregates, called Lewy bodies (LBs), is a hallmark of Parkinson's disease (PD). The function of LBs in the disease process is however still unclear; they have been associated with both neuroprotection and toxicity. To obtain insight into this contradiction, we induced the formation of α-S inclusions, using three different induction methods in SH-SY5Y cells and rat-derived primary neuronal cells. Using confocal and STED microscopy we observed induction-dependent differences in α-S inclusion morphology, location and function. The aggregation of α-S in functionally different compartments correlates with the toxicity of the induction method measured in viability assays. The most cytotoxic treatment largely correlates with the formation of proteasome-associated, juxta-nuclear inclusions. With less toxic methods cytosolic deposits that are not associated with the proteasome are more prevalent. The distribution of α-S over at least two different types of inclusions is not limited to cell models, but is also observed in primary neuronal cells and in human mesencephalon. The existence of functionally different LBs, in vivo and in vitro, gives important insights in the impact of Lewy Body formation on neuronal functioning and may thereby provide a platform for discovering therapeutics.

Progression of Neuronal Damage in an In Vitro Model of the Ischemic Penumbra.

Improvement of neuronal recovery in the ischemic penumbra around a brain infarct has a large potential to advance clinical recovery of patients with acute ischemic stroke. However, pathophysiological mechanisms leading to either recovery or secondary damage in the penumbra are not completely understood. We studied neuronal dynamics in a model system of the penumbra consisting of networks of cultured cortical neurons exposed to controlled levels and durations of hypoxia. Short periods of hypoxia (pO2≈20mmHg) reduced spontaneous activity, due to impeded synaptic function. After ≈6 hours, activity and connectivity partially recovered, even during continuing hypoxia. If the oxygen supply was restored within 12 hours, changes in network connectivity were completely reversible. For longer periods of hypoxia (12-30 h), activity levels initially increased, but eventually decreased and connectivity changes became partially irreversible. After ≈30 hours, all functional connections disappeared and no activity remained. Since this complete silence seemed unrelated to hypoxic depths, but always followed an extended period of low activity, we speculate that irreversible damage (at least partly) results from insufficient neuronal activation. This opens avenues for therapies to improve recovery by neuronal activation.

Acyl Ghrelin Improves Synapse Recovery in an In Vitro Model of Postanoxic Encephalopathy.

Comatose patients after cardiac arrest have a poor prognosis. Approximately half never awakes as a result of severe diffuse postanoxic encephalopathy. Several neuroprotective agents have been tested, however without significant effect. In the present study, we used cultured neuronal networks as a model system to study the general synaptic damage caused by temporary severe hypoxia and the possibility to restrict it by ghrelin treatment. Briefly, we applied hypoxia (pO2 lowered from 150 to 20 mmHg) during 6 h in 55 cultures. Three hours after restoration of normoxia, half of the cultures were treated with ghrelin for 24 h, while the other, non-supplemented, were used as a control. All cultures were processed immunocytochemically for detection of the synaptic marker synaptophysin. We observed that hypoxia led to drastic decline of the number of synapses, followed by some recovery after return to normoxia, but still below the prehypoxic level. Additionally, synaptic vulnerability was selective: large- and small-sized neurons were more susceptible to synaptic damage than the medium-sized ones. Ghrelin treatment significantly increased the synapse density, as compared with the non-treated controls or with the prehypoxic period. The effect was detected in all neuronal subtypes. In conclusion, exogenous ghrelin has a robust impact on the recovery of cortical synapses after hypoxia. It raises the possibility that ghrelin or its analogs may have a therapeutic potential for treatment of postanoxic encephalopathy.