Late awakening, prognostic factors and long-term outcome in out-of-hospital cardiac arrest - results of the prospective Norwegian Cardio-Respiratory Arrest Study (NORCAST).

BACKGROUND: Outcome prediction after out-of-hospital cardiac arrest (OHCA) may lead to withdrawal of life-sustaining therapy if the prognosis is perceived negative. Single use of uncertain prognostic tools may lead to self-fulfilling prophecies and death. We evaluated prognostic tests, blinded to clinicians and without calls for hasty outcome prediction, in a prospective study. METHODS: Comatose, sedated TTM 33-treated OHCA patients of all causes were included. Clinical-neurological/-neurophysiological/-biochemical predictors were registered. Patients were dichotomized into good/poor outcome using cerebral performance category (CPC) six months and > four years post-arrest. Prognostic tools were evaluated using false positive rates (FPR). RESULTS: We included 259 patients; 49 % and 42 % had good outcome (CPC 1-2) after median six months and 5.1 years. Unwitnessed arrest, non-shockable rhythms, and no-bystander-CPR predicted poor outcome with FPR (CI) 0.05 (0.02-0.10), 0.13 (0.08-0.21), and 0.13 (0.07-0.20), respectively. Time to awakening was median 6 (0-25) days in good outcome patients. Among patients alive with sedation withdrawal >72 h, 49 % were unconscious, of whom 32 % still obtained good outcome. Only absence of pupillary light reflexes (PLR) -and N20-responses in somato-sensory evoked potentials (SSEP), as well as increased neuron-specific enolase (NSE) later than 24 h to >80 µg/L, had FPR 0. Malignant EEG (burst suppression/epileptic activity/flat) differentiated poor/good outcome with FPR 0.05 (0.01-0.15). CONCLUSION: Time to awakening was over six days in good outcome patients. Most clinical parameters had too high FPRs for prognostication, except for absent PLR and SSEP-responses >72 h after sedation withdrawal, and increased NSE later than 24 h to >80 µg/L.

Neuronal and glial DNA methylation and gene expression changes in early epileptogenesis.

BACKGROUND AND AIMS: Mesial Temporal Lobe Epilepsy is characterized by progressive changes of both neurons and glia, also referred to as epileptogenesis. No curative treatment options, apart from surgery, are available. DNA methylation (DNAm) is a potential upstream mechanism in epileptogenesis and may serve as a novel therapeutic target. To our knowledge, this is the first study to investigate epilepsy-related DNAm, gene expression (GE) and their relationship, in neurons and glia. METHODS: We used the intracortical kainic acid injection model to elicit status epilepticus. At 24 hours post injection, hippocampi from eight kainic acid- (KA) and eight saline-injected (SH) mice were extracted and shock frozen. Separation into neurons and glial nuclei was performed by flow cytometry. Changes in DNAm and gene expression were measured with reduced representation bisulfite sequencing (RRBS) and mRNA-sequencing (mRNAseq). Statistical analyses were performed in R with the edgeR package. RESULTS: We observed fulminant DNAm- and GE changes in both neurons and glia at 24 hours after initiation of status epilepticus. The vast majority of these changes were specific for either neurons or glia. At several epilepsy-related genes, like HDAC11, SPP1, GAL, DRD1 and SV2C, significant differential methylation and differential gene expression coincided. CONCLUSION: We found neuron- and glia-specific changes in DNAm and gene expression in early epileptogenesis. We detected single genetic loci in several epilepsy-related genes, where DNAm and GE changes coincide, worth further investigation. Further, our results may serve as an information source for neuronal and glial alterations in both DNAm and GE in early epileptogenesis.

Neurodegenerative Interplay of Cardiovascular Autonomic Dysregulation and the Retina in Early Multiple Sclerosis.

Introduction: Autonomic nervous system (ANS) symptoms are prevalent in multiple sclerosis (MS) as is neurodegeneration. Our aim was to explore the occurrence of ANS symptoms and retinal neurodegeneration in a newly diagnosed MS population with tools available in a clinical setting. Methods: Forty-three MS patients and 44 healthy controls took part in the study. We employed a bedside cardiovascular ANS test battery together with classical pupillometry, optical coherence tomography (OCT) evaluation of retinal neurodegeneration in eyes without previous optic neuritis (MSNON) and patients' self-report forms on fatigue, orthostatic and ANS symptoms. Results: Half of the patients presented with ANS symptoms and a high level of fatigue. There was a significant difference in ganglion cell layer thickness (mean GCIPL) evaluated by OCT in MSNON compared to healthy control eyes. We found a negative linearity of mean GCIPL on group level with increasing disease duration. Three patients fulfilled the criteria of postural orthostatic tachycardia syndrome (POTS). Conclusion: Our results demonstrate retinal neurodegeneration in MSNON, a high frequency of fatigue and a high prevalence of ANS symptoms in newly diagnosed patients. Whether neurodegeneration precedes ANS dysfunction or vice versa is still open to debate, but as unveiled by the presence of POTS in this MS population, differences in stress-response regulation add to the understanding of variation in onset-time of ANS dysfunction in early MS.

Fatigue and cognition: Pupillary responses to problem-solving in early multiple sclerosis patients.

INTRODUCTION: In early multiple sclerosis (MS) patients, cognitive changes and fatigue are frequent and troublesome symptoms, probably related to both structural and functional brain changes. Whether there is a common cause of these symptoms in MS is unknown. In theory, an altered regulation of central neuropeptides can lead to changes in regulation of autonomic function, cognitive difficulties, and fatigue. Direct measurements of central neuropeptides are difficult to perform, but measurements of the eye pupil can be used as a reliable proxy of function. METHODS: This study assesses pupil size during problem-solving in early MS patients versus controls. A difference in pupil size to a cognitive challenge could signal altered activity within the autonomic system because of early functional brain changes associated with cognitive load. We recruited MS patients (mean disease duration: 2.6 years, N = 41) and age-matched healthy controls (N = 43) without eye pathology. Neurological impairment, magnetic resonance imaging, visual evoked potentials, depression, and fatigue were assessed in all of the patients. In both groups, we assessed processing speed and retinal imaging. Pupil size was recorded with an eye-tracker during playback of multiplication tasks. RESULTS: Both groups performed well on the cognitive test. The groups showed similar pupillary responses with a mean of 0.55 mm dilation in patients and 0.54 mm dilation in controls for all the tasks collapsed together. However, controls (N = 9) with low cognitive scores (LCS) had an increased pupillary response to cognitive tasks, whereas LCS MS patients (N = 6) did not (p < .05). There was a tendency toward a smaller pupillary response in patients with fatigue. CONCLUSIONS: This is the first study to investigate pupillary responses to cognitive tasks in MS patients. Our results suggest that MS-related changes in cognition and fatigue may be associated with changes in arousal and the autonomic regulation of task-related pupillary responses. This supports the theory of a link between cognition and fatigue in MS.

Eye and hand motor interactions with the Symbol Digit Modalities Test in early multiple sclerosis.

PURPOSE: Eye and hand motor dysfunction may be present early in the disease course of relapsing-remitting multiple sclerosis (RRMS), and can affect the results on visual and written cognitive tests. We aimed to test for differences in saccadic initiation time (SI time) between RRMS patients and healthy controls, and whether SI time and hand motor speed interacted with the written version of the Symbol Digit Modalities Test (wSDMT). METHODS: Patients with RRMS (N = 44, age 35.1 ± 7.3 years), time since diagnosis < 3 years and matched controls (N = 41, age 33.2 ± 6.8 years) were examined with ophthalmological, neurological and neuropsychological tests, as well as structural MRI (white matter lesion load (WMLL) and brainstem lesions), visual evoked potentials (VEP) and eye-tracker examinations of saccades. RESULTS: SI time was longer in RRMS than controls (p < 0.05). SI time was not related to the Paced Auditory Serial Addition Test (PASAT), WMLL or to the presence of brainstem lesions. 9 hole peg test (9HP) correlated significantly with WMLL (r = 0.58, p < 0.01). Both SI time and 9HP correlated negatively with the results of wSDMT (r = -0.32, p < 0.05, r = -0.47, p < 0.01), but none correlated with the results of PASAT. CONCLUSIONS: RRMS patients have an increased SI time compared to controls. Cognitive tests results, exemplified by the wSDMT, may be confounded by eye and hand motor function.

Alterations in Brain Inflammation, Synaptic Proteins, and Adult Hippocampal Neurogenesis during Epileptogenesis in Mice Lacking Synapsin2.

Synapsins are pre-synaptic vesicle-associated proteins linked to the pathogenesis of epilepsy through genetic association studies in humans. Deletion of synapsins causes an excitatory/inhibitory imbalance, exemplified by the epileptic phenotype of synapsin knockout mice. These mice develop handling-induced tonic-clonic seizures starting at the age of about 3 months. Hence, they provide an opportunity to study epileptogenic alterations in a temporally controlled manner. Here, we evaluated brain inflammation, synaptic protein expression, and adult hippocampal neurogenesis in the epileptogenic (1 and 2 months of age) and tonic-clonic (3.5-4 months) phase of synapsin 2 knockout mice using immunohistochemical and biochemical assays. In the epileptogenic phase, region-specific microglial activation was evident, accompanied by an increase in the chemokine receptor CX3CR1, interleukin-6, and tumor necrosis factor-α, and a decrease in chemokine keratinocyte chemoattractant/ growth-related oncogene. Both post-synaptic density-95 and gephyrin, scaffolding proteins at excitatory and inhibitory synapses, respectively, showed a significant up-regulation primarily in the cortex. Furthermore, we observed an increase in the inhibitory adhesion molecules neuroligin-2 and neurofascin and potassium chloride co-transporter KCC2. Decreased expression of γ-aminobutyric acid receptor-δ subunit and cholecystokinin was also evident. Surprisingly, hippocampal neurogenesis was reduced in the epileptogenic phase. Taken together, we report molecular alterations in brain inflammation and excitatory/inhibitory balance that could serve as potential targets for therapeutics and diagnostic biomarkers. In addition, the regional differences in brain inflammation and synaptic protein expression indicate an epileptogenic zone from where the generalized seizures in synapsin 2 knockout mice may be initiated or spread.

Sensitive and critical periods in the development of handling induced seizures in mice lacking synapsins: differences between synapsin I and synapsin II knockouts.

Mice lacking either synapsin I or synapsin II develop handling induced seizures from around two months of age. In mice lacking synapsin I (synapsin 1 knock-out mice, Syn1KO mice) such seizures can either consist of mild myoclonic jerks or of fully developed generalized tonic-clonic seizures, and the two seizure types are quite evenly distributed. In mice lacking synapsin II (synapsin 2 knock-out mice, Syn2KO mice) all seizures are in the form of generalized tonic-clonic seizures. Through the use of specialized animal rearing procedures whereby human-animal interaction was minimized (minimal handling procedures), this study investigated effects of handling also prior to the emergence of actual seizures. The effect of minimal handling procedures was significant in both genotypes, but most pronounced in Syn1KO mice. In this genotype, minimal handling reduced the frequency of mild seizures, and completely eliminated generalized tonic-clonic seizures when the animals were tested with regular handling at 4 1/2 months of age. Neither seizure frequency nor generalized tonic-clonic seizures could be re-established through regular handling from 4 1/2 to 8 months. This suggests that the period up to 4 1/2 months constitute a sensitive period for seizures in general, and a critical period for generalized tonic-clonic seizures in this genotype. In Syn2KO mice minimal handling did not remove generalized tonic-clonic seizures, as such seizures were present when handling was introduced at 4 1/2 months. We found an initial reduction of seizure frequency, but the seizure frequency eventually reached levels seen in mice kept under regular handling regimes. Thus, it is unlikely that the period up to 4 1/2 months is a sensitive period in the Syn2KO genotype.

Neuroethologically delineated differences in the seizure behavior of synapsin 1 and synapsin 2 knock-out mice.

The highly homologous nerve terminal phosphoproteins synapsin I and synapsin II have been linked to the pathogenesis of epilepsy through associations between synapsin gene mutations and epileptic disease in humans and to the observation of handling induced seizures in mice genetically depleted of one or both of these proteins. Whereas seizure behavior in mice lacking both synapsin I and synapsin II is well characterized, the seizure behavior in mice lacking either is less well studied. Through so called neuroethologically based analyses of fully established seizure behavior in Synapsin 1 and 2 knock-out mice (Syn1KO and Syn2KO mice) aged 4 1/2 months, this study reveals significant differences in the seizure behavior of the two genotypes: whereas Syn1KO mice show both partial and generalized forebrain seizure activity, Syn2KO mice show only fully generalized forebrain seizures. Analysis of seizure behavior at earlier stages shows that the mature seizure pattern in Syn2KO mice establishes rapidly from the age of ∼2 months, when Syn1KO partial seizures are rare, and Syn1KO generalized seizures are almost absent. The specific behavioral phenotypes of the two strains suggest that the slight differences in structure, function and expression of these highly related proteins could be important factors during seizure generating neural activity.

Electroencephalographic characterization of seizure activity in the synapsin I/II double knockout mouse.

We present a detailed comparison of the behavioral and electrophysiological development of seizure activity in mice genetically depleted of synapsin I and synapsin II (SynDKO mice), based on combined video and surface EEG recordings. SynDKO mice develop handling-induced epileptic seizures at the age of 2months. The seizures show a very regular behavioral pattern, where activity is initially dominated by truncal muscle contractions followed by various myoclonic elements. Whereas seizure behavior goes through clearly defined transitions, cortical activity as reflected by EEG recordings shows a more gradual development with respect to the emergence of different EEG components and the frequency of these components. No EEG pattern was seen to define a particular seizure behavior. However, myoclonic activity was characterized by more regular patterns of combined sharp waves and spikes. Where countable, the number of myoclonic jerks was significantly correlated to the number of such EEG complexes. Furthermore, some EEG recordings revealed epileptic regular discharges without clear behavioral seizure correlates. Our findings suggest that seizure behavior in SynDKO mice is not solely determined by cortical activity but rather reflects interplay between cortical activity and activity in other brain regions.

Seizure logging: A new approach to synchronized cable-free EEG and video recordings of seizure activity in mice.

We describe a new cable-free, non-telemetric method for synchronized electrophysiological and video recordings of seizure activity in freely moving mice. The electrophysiological recordings were made by a head-mounted 4-channel data-logging device, allowing the mouse to move freely in its cage, and even to be moved from cage to cage under ongoing recording. Seizures were studied in Synapsin I/II double knock-out (SynDKO) mice, a genetically engineered mouse line that shows seizures upon daily handling procedures such as tail lifting during cage changes, much in resemblance to the more studied El mouse. The ability to elicit seizures through daily handling in SynDKO mice undergoing electrophysiological recording is a significant improvement in comparison to the traditional cable-based set-up. Furthermore, with its four channels and a sample rate of up to 500Hz, the data-logging device opens for more varied electrophysiological studies than other available cable-free systems.

Seizure elements and seizure element transitions during tonic-clonic seizure activity in the synapsin I/II double knockout mouse: a neuroethological description.

Inactivation of genes for the synaptic terminal proteins synapsin I and synapsin II leads to development of epileptic seizures in mice (Syn-DKO mice) in which no other behavioral abnormalities or any gross anatomical brain deformities have been reported. In humans, mutated synapsin I is associated with epilepsy. Thus, the Syn-DKO mouse might model human seizure development. Here we describe a neuroethological analysis of behavioral elements and relationships between these elements during seizures in Syn-DKO mice. The seizure elements belong to one of three clusters each characterized by specific patterns of activity: truncus-dominated elements, myoclonic elements, and running-fit activity. The first two clusters, constituting the majority of seizural activity, evolve quite differently during ongoing seizure activity. Whereas truncus-dominated elements unfold in a strict sequence, the myoclonic elements wax and wane more independently, once myoclonic activity has started. These differences may point to neurobiological mechanisms relevant to both rodent and human epilepsies.