Bedside interpretation of simplified continuous EEG after cardiac arrest.

BACKGROUND: Continuous EEG-monitoring (cEEG) in the ICU is recommended to assess prognosis and detect seizures after cardiac arrest but implementation is often limited by the lack of EEG-technicians and experts. The aim of the study was to assess ICU physicians ability to perform preliminary interpretations of a simplified cEEG in the post cardiac arrest setting. METHODS: Five ICU physicians received training in interpretation of simplified cEEG - total training duration 1 day. The ICU physicians then interpreted 71 simplified cEEG recordings from 37 comatose survivors of cardiac arrest. The cEEG included amplitude-integrated EEG trends and two channels with original EEG-signals. Basic EEG background patterns and presence of epileptiform discharges or seizure activity were assessed on 5-grade rank-ordered scales based on standardized EEG terminology. An EEG-expert was used as reference. RESULTS: There was substantial agreement (κ 0.69) for EEG background patterns and moderate agreement (κ 0.43) for epileptiform discharges between ICU physicians and the EEG-expert. Sensitivity for detecting seizure activity by ICU physicians was limited (50%), but with high specificity (87%). CONCLUSIONS: After cardiac arrest, preliminary bedside interpretations of simplified cEEGs by trained ICU physicians may allow earlier detection of clinically relevant cEEG changes, prompting changes in patient management as well as additional evaluation by an EEG-expert. This strategy requires awareness of limitations of both the simplified electrode montage and the cEEG interpretations performed by ICU physicians. cEEG evaluation by an expert should not be delayed.

Increased potassium conductance of brain mitochondria induces resistance to permeability transition by enhancing matrix volume.

Modulation of K(+) conductance of the inner mitochondrial membrane has been proposed to mediate preconditioning in ischemia-reperfusion injury. The mechanism is not entirely understood, but it has been linked to a decreased activation of mitochondrial permeability transition (mPT). In the present study K(+) channel activity was mimicked by picomolar concentrations of valinomycin. Isolated brain mitochondria were exposed to continuous infusions of calcium. Monitoring of extramitochondrial Ca(2+) and mitochondrial respiration provided a quantitative assay for mPT sensitivity by determining calcium retention capacity (CRC). Valinomycin and cyclophilin D inhibition separately and additively increased CRC. Comparable degrees of respiratory uncoupling induced by increased K(+) or H(+) conductance had opposite effects on mPT sensitivity. Protonophores dose-dependently decreased CRC, demonstrating that so-called mild uncoupling was not beneficial per se. The putative mitoK(ATP) channel opener diazoxide did not mimic the effect of valinomycin. An alkaline matrix pH was required for mitochondria to retain calcium, but increased K(+) conductance did not result in augmented DeltapH. The beneficial effect of valinomycin on CRC was not mediated by H(2)O(2)-induced protein kinase Cepsilon activation. Rather, increased K(+) conductance reduced H(2)O(2) generation during calcium infusion. Lowering the osmolarity of the buffer induced an increase in mitochondrial volume and improved CRC similar to valinomycin without inducing uncoupling or otherwise affecting respiration. We propose that increased potassium conductance in brain mitochondria may cause a direct physiological effect on matrix volume inducing resistance to pathological calcium challenges.

Uncoupling protein-2 prevents neuronal death and diminishes brain dysfunction after stroke and brain trauma.

Whereas uncoupling protein 1 (UCP-1) is clearly involved in thermogenesis, the role of UCP-2 is less clear. Using hybridization, cloning techniques and cDNA array analysis to identify inducible neuroprotective genes, we found that neuronal survival correlates with increased expression of Ucp2. In mice overexpressing human UCP-2, brain damage was diminished after experimental stroke and traumatic brain injury, and neurological recovery was enhanced. In cultured cortical neurons, UCP-2 reduced cell death and inhibited caspase-3 activation induced by oxygen and glucose deprivation. Mild mitochondrial uncoupling by 2,4-dinitrophenol (DNP) reduced neuronal death, and UCP-2 activity was enhanced by palmitic acid in isolated mitochondria. Also in isolated mitochondria, UCP-2 shifted the release of reactive oxygen species from the mitochondrial matrix to the extramitochondrial space. We propose that UCP-2 is an inducible protein that is neuroprotective by activating cellular redox signaling or by inducing mild mitochondrial uncoupling that prevents the release of apoptogenic proteins.

Effects on brain edema of crystalloid and albumin fluid resuscitation after brain trauma and hemorrhage in the rat.

BACKGROUND: It has been hypothesized that resuscitation with crystalloids after brain trauma increases brain edema compared with colloids, but previous studies on the subject have been inconclusive. To test this hypothesis, the authors compared groups resuscitated with either colloid or crystalloid. METHODS: After fluid percussion injury, rats were subjected to a controlled hemorrhage of 20 ml/kg and were randomized to 5% albumin at 20 ml/kg (A20), isotonic Ringer's acetate at 50 ml/kg (C50), or 90 ml/kg (C90). After 3 or 24 h, water content in the injured cortex was determined using a wet/dry weight method. Blood volume was calculated from plasma volume, measured by 125I-albumin dilution, and hematocrit. Oncotic pressure and osmolality were measured with osmometers. RESULTS: At 3 h, blood volume was equal in the A20 and C90 groups and lower in the C50 group. Oncotic pressure was reduced by 35-40% in the crystalloid groups and unchanged in the albumin group. Cortical water content in the A20 group was lower than in the C90 group (81.3 +/- 0.5% vs. 82.1 +/- 1.1%, P < 0.05), but it was not different from the C50 group (81.8 +/- 1.1%). At 24 h, oncotic pressure and blood volume were normalized in all groups, and cortical water content was significantly lower in the albumin group than in the crystalloid groups. Osmolality and arterial pressure were equal in all groups throughout the experiment. CONCLUSIONS: When given to the same intravascular volume expansion, isotonic crystalloids caused greater posttraumatic brain edema than 5% albumin at 3 and 24 h after trauma.

Overexpression of UCP2 protects thalamic neurons following global ischemia in the mouse.

Uncoupling protein 2 (UCP2) is upregulated in the brain after sublethal ischemia, and overexpression of UCP2 is neuroprotective in several models of neurodegenerative disease. We investigated if increased levels of UCP2 diminished neuronal damage after global brain ischemia by subjecting mice overexpressing UCP2 (UCP2/3tg) and wild-type littermates (wt) to a 12-min global ischemia. The histopathological outcome in the cortex, hippocampus, striatum, and thalamus was evaluated at 4 days of recovery, allowing maturation of the selective neuronal death. Global ischemia led to extensive cell death in the striatum, thalamus, and in the CA1 and CA2, and less-pronounced cell death in the CA3 and dentate gyrus (DG) hippocampal subfields. Histologic damage was significantly lower in the ventral posterolateral VPL and medial VPM thalamic nuclei in UCP2/3tg animals compared with wt. These thalamic regions showed a larger increase in UCP2 expression in UCP2/3tg compared with wt animals relative to the nonprotected DG. In the other regions studied, the histologic damage was lower or equal in UCP2/3tg animals compared with wt. Consequently, neuroprotection in the thalamus correlated with a high expression of UCP2, which is neuroprotective in a number of models of neurodegenerative diseases.

The emerging functions of UCP2 in health, disease, and therapeutics.

The uncoupling proteins (UCPs) are attracting an increased interest as potential therapeutic targets in a number of important diseases. UCP2 is expressed in several tissues, but its physiological functions as well as potential therapeutic applications are still unclear. Unlike UCP1, UCP2 does not seem to be important to thermogenesis or weight control, but appears to have an important role in the regulation of production of reactive oxygen species, inhibition of inflammation, and inhibition of cell death. These are central features in, for example, neurodegenerative and cardiovascular disease, and experimental evidence suggests that an increased expression and activity of UCP2 in models of these diseases has a beneficial effect on disease progression, implicating a potential therapeutic role for UCP2. UCP2 has an important role in the pathogenesis of type 2 diabetes by inhibiting insulin secretion in islet beta cells. At the same time, type 2 diabetes is associated with increased risk of cardiovascular disease and atherosclerosis where an increased expression of UCP2 appears to be beneficial. This illustrates that therapeutic applications involving UCP2 likely will have to regulate expression and activity in a tissue-specific manner.

Delayed neuromotor recovery and increased memory acquisition dysfunction following experimental brain trauma in mice lacking the DNA repair gene XPA.

OBJECT: This study investigates the outcome after traumatic brain injury (TBI) in mice lacking the essential DNA repair gene xeroderma pigmentosum group A (XPA). As damage to DNA has been implicated in neuronal cell death in various models, the authors sought to elucidate whether the absence of an essential DNA repair factor would affect the outcome of TBI in an experimental setting. METHODS: Thirty-seven adult mice of either wild-type (n = 18) or XPA-deficient ("knock-out" [n = 19]) genotype were subjected to controlled cortical impact experimental brain trauma, which produced a focal brain injury. Sham-injured mice of both genotypes were used as controls (9 in each group). The mice were subjected to neurobehavoral tests evaluating learning/acquisition (Morris water maze) and motor dysfunction (Rotarod and composite neuroscore test), pre- and postinjury up to 4 weeks. The mice were killed after 1 or 4 weeks, and cortical lesion volume, as well as hippocampal and thalamic cell loss, was evaluated. Hippocampal staining with doublecortin antibody was used to evaluate neurogenesis after the insult. RESULTS: Brain-injured XPA(-/-) mice exhibited delayed recovery from impairment in neurological motor function, as well as pronounced cognitive dysfunction in a spatial learning task (Morris water maze), compared with injured XPA(+/+) mice (p < 0.05). No differences in cortical lesion volume, hippocampal damage, or thalamic cell loss were detected between XPA(+/+) and XPA(-/-) mice after brain injury. Also, no difference in the number of cells stained with doublecortin in the hippocampus was detected. CONCLUSIONS: The authors' results suggest that lack of the DNA repair factor XPA may delay neurobehavioral recovery after TBI, although they do not support the notion that this DNA repair deficiency results in increased cell or tissue death in the posttraumatic brain.

Hypothermia affects translocation of numerous cytoplasmic proteins following global cerebral ischemia.

Using a decapitation ischemia model, we studied translocation of proteins to and from the cytosol in normothermic (NT) and hypothermic (HT) rat brains. 2D gel analysis identified 74 proteins whose cytosolic level changed significantly after 15 min of ischemia. HT preserved the cytosolic levels of several glycolytic enzymes, as well as many plasticity related proteins, otherwise decreased following NT ischemia. The levels of redox-related proteins was lower in HT than in NT. Our results indicate that translocation of proteins to and from the cytosol is an important issue during ischemia.

The temperature dependence and involvement of mitochondria permeability transition and caspase activation in damage to organotypic hippocampal slices following in vitro ischemia.

The aggravating effect of hyperglycemia on ischemic brain injury can be mimicked in a model of in vitro ischemia (IVI) using murine hippocampal slice cultures. Using this model, we found that the damage in the CA1 region following IVI in the absence or presence of 40 mm glucose (hyperglycemia) is highly temperature dependent. Decreasing the temperature from 35 to 31 degrees C during IVI prevented cell death, whereas increasing the temperature by 2 degrees C markedly aggravated damage. As blockade of the mitochondrial permeability transition (MPT) is equally effective as hypothermia in preventing ischemic cell death in vivo, we investigated whether inhibition of MPT or of caspases was protective following IVI. In the absence of glucose, the MPT blockers cyclosporin A and MeIle4-CsA but not the immunosuppressive compound FK506 diminished cell death. In contrast, following hyperglycemic IVI, MPT blockade was ineffective. Also, the pan-caspase inhibitor Boc-Asp(OMe)fluoromethyl ketone did not decrease cell death in the CA1 region following IVI or hyperglycemic IVI. We conclude that cell death in the CA1 region of organotypic murine hippocampal slices following IVI is highly temperature dependent and involves MPT. In contrast, cell death following hyperglycemic IVI, although completely prevented by hypothermia, is not mediated by mechanisms that involve MPT or caspase activation.

Analysis of mitochondrial generation and release of reactive oxygen species.

BACKGROUND: Reactive oxygen species (ROS) are mainly produced in mitochondria and are important contributors to many forms of cell death. ROS also function as second messengers within the cell and may constitute a signaling pathway from mitochondria to the cytoplasm and nucleus. The aim of the present study was to develop a protocol to detect changes in intra- and extramitochondrial releases of ROS, which could be used to analyze the role of mitochondria in cell signaling and cell death. METHODS: Fluorescence-based assays were used to measure (a) total production of ROS, (b) intramitochondrial ROS, (c) extramitochondrial hydrogen peroxide, and (d) superoxide outside inverted (inside-out) submitochondrial particles. ROS generation in the samples was increased or decreased by the addition of different substrates, enzymes, and inhibitors of the electron transport chain. RESULTS: The individual assays used were sensitive to increased (e.g., after addition of antimycin A; increased signal) and decreased (ROS scavenging; decreased signal) levels of ROS. In combination, the assays provided information about mitochondrial ROS generation and release dynamics from small samples of isolated mitochondria. CONCLUSIONS: The combination of fluorescent techniques described is a useful tool to study the role of ROS in cell death and in cellular redox signaling.

Flow cytometric analysis of isolated liver mitochondria to detect changes relevant to cell death.

BACKGROUND: Mitochondria are key players in many forms of cell death, and mitochondrial production of reactive oxygen species (ROS), membrane depolarization, permeability changes, and release of apoptogenic proteins are involved in these processes. Flow cytometric analysis of isolated mitochondria enables parallel analysis of mitochondrial structure and function in individual mitochondria, and small mitochondrial samples are sufficient for analysis. This article describes a well-characterized protocol for flow cytometric analysis of isolated liver mitochondria that can be used to detect mitochondrial alterations relevant to cell death. METHODS: Fluorescent probes were used to selectively stain mitochondria (nonyl acridine orange), and to measure membrane potential (tetramethylrhodamine-methyl-ester, 1,1',3,3,3',3'-hexamethylindodicarbocyanine-iodide), as well as production of ROS (2',7'-dichlorodihydrofluorescein-diacetate). Calcium-induced mitochondrial swelling was detected as a decrease in SSC. To ensure optimal concentrations of all probes, the effect on mitochondrial respiration was evaluated. RESULTS: This protocol can be used to determine the purity of the mitochondrial preparation, to detect calcium-induced morphological changes, small mitochondrial de- and hyperpolarizations, as well as physiological changes in ROS generation. CONCLUSIONS: Flow cytometry is a very useful tool to simultaneously analyze several mitochondrial parameters that are important in the induction of mitochondria-mediated cell death.

Flow cytometric analysis of mitochondria from CA1 and CA3 regions of rat hippocampus reveals differences in permeability transition pore activation.

Mitochondria are important in the pathophysiology of several neurodegenerative diseases, and mitochondrial production of reactive oxygen species (ROS), membrane depolarization, permeability changes and release of apoptogenic proteins are involved in these processes. Following brain insults, cell death often occurs in discrete regions of the brain, such as the subregions of the hippocampus. To analyse mitochondrial structure and function in such subregions, only small amounts of mitochondria are available. We developed a protocol for flow cytometric analysis of very small samples of isolated brain mitochondria, and analysed mitochondrial swelling and formation of ROS in mitochondria from the CA1 and CA3 regions of the hippocampus. Calcium-induced mitochondrial swelling was measured, and fluorescent probes were used to selectively stain mitochondria (nonyl acridine orange), to measure membrane potential (tetramethylrhodamine-methyl-ester, 1,1',3,3,3',3'-hexamethylindodicarbocyanine-iodide) and to measure production of ROS (2',7'-dichlorodihydrofluorescein-diacetate). We found that formation of ROS and mitochondrial permeability transition pore activation were higher in mitochondria from the CA1 than from the CA3 region, and propose that differences in mitochondrial properties partly underlie the selective vulnerability of the CA1 region to brain insults. We also conclude that flow cytometry is a useful tool to analyse the role of mitochondria in cell death processes.

Brain-derived respiring mitochondria exhibit homogeneous, complete and cyclosporin-sensitive permeability transition.

The mitochondrial permeability transition (mPT) is increasingly implicated in neuronal cell death. In the present study, isolated respiring brain mitochondria were examined for their ability to undergo calcium-induced mPT and their sensitivity to mPT inhibition by cyclosporin A (CsA). Previous studies have suggested a heterogeneous response to calcium, a limitation of CsA inhibition, and a relative resistance in the ability of respiring brain mitochondria to undergo mPT. Using fluorometric and electron microscopic analyses, we found that virtually the whole population of respiring brain mitochondria readily undergo mPT and swell upon calcium exposure. Further, brain mitochondria were highly sensitive to CsA which potentiated morphological recovery after transient swelling as well as completely blocked mPT induction in the presence of a low concentration of ADP. Using flow cytometry, which allows analysis of individual mitochondria, we demonstrate that both brain and liver mitochondria display homogeneous responses to calcium-induced mPT. We conclude that the mPT is one likely target for the broad in vivo neuroprotective effects displayed by CsA when allowed to penetrate the blood-brain barrier, and that development of compounds inhibiting mPT may prove beneficial for the treatment of severe brain disease.

The nonimmunosuppressive cyclosporin analogs NIM811 and UNIL025 display nanomolar potencies on permeability transition in brain-derived mitochondria.

Cyclosporin A (CsA) is highly neuroprotective in several animal models of acute neurological damage and neurodegenerative disease with inhibition of the mitochondrial permeability transition (mPT) having emerged as a possible mechanism for the observed neuroprotection. In the present study, we have evaluated two new nonimmunosuppressive cyclosporin analogs NIM811 (Novartis) and UNIL025 (Debiopharm) for their ability to inhibit mPT in rat brain-derived mitochondria. Both NIM811 and UNIL025 were found to be powerful inhibitors of calcium-induced mitochondrial swelling under energized and deenergized conditions, and the maximal effects were identical to those of native CsA. The potencies of mPT inhibition by NIM811 and UNIL025 were stronger, with almost one order of magnitude higher potency for UNIL025 compared to CsA, correlating to their respective inhibitory action of cyclophilin activity. These compounds will be instrumental in the evaluation of mPT as a central target for neuroprotection in vivo.